REGULATION OF PRIMARY METABOLIC PATIIWAYS IN PLANTS

Proceedings of the Phytochemical Society of Europe

Volume 42

Regulation of Primary Metabolic Pathways in Plants

Edited by

Nicholas J. Kruger Steven A. Hin and

R. George Ratcliffe Department of Plant Sciences, University of Oxford, Oxford, u.K.

SPRINGER SCIENCE+BUSINESS MEDIA, B.V.

A C.I.P. Catalogue record for Ihis book is available from the library of Congress.

P,inled on acid-fiu paper

AII Righls Reserved (1 1999 Springer Science+Business Media Dordrecht Origina1ly published by Kluwer Academic Publishers in 1999 Softcovcr reprint ofthc hardcovcr Ist edition 1999

No pan of the material protected by Ihis copyright n()(ice may be reproduced or utilized in any form or by any means, electronic or mechanical, including pholocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

ISBN 978-94-010-6021-9 ISBN 978-94-011-4818-4 (eBook)

DOI 10.1007/978-94-011-4818-4

This book is dedicated to the memory of Professor Tom ap Rees, outstanding researcher, inspirational teacher and tireless champion of plant science.

TABLE OF CONTENTS

Preface Nicholas 1. Kruger, Steven A. Hill and R. George Ratcliffe

1. Rubisco: attempts to reform a promiscuous enzyme Martin A. 1. Parry, Alfred 1. Keys, Graeme Bainbridge, Steven P. Colliver, P. John Andralojc, Matthew J. Paul, Fiona M. Banks and Pippa J. Madgwick

2. Insights into the active site of the plant alternative oxidase and its relationship to function Charles Affourtit and Anthony L. Moore

3. The many-faceted function of phosphoenolpyruvate carboxykinase in plants Richard C. Leegood, Richard M. Acheson, Laszlo I. Tecsi and Robert P. Walker

4. Folate synthesis and compartmentation in higher plants Fabrice Rebeille and Roland Douce

5. Structure and function of plastid metabolite transporters U1f-Ingo Fliigge, Andreas Weber, Birgit Kammerer, Rainer E. Hausler and Karsten Fischer

6. Integration of metabolism within non-photosynthetic plastids, and with the cytosol Mike 1. Emes, Ian 1. Tetlow and Caroline G. Bowsher

7. Carbon flux to fatty acids in plastids Stephen Rawsthorne, Fan Kang and Peter 1. Eastmond

8. Compartmentation of metabolites between the subcellular compartments of leaves, the apoplast, the phloem and the storage tissue of different crop plants Gertrud Lohaus, D Heineke, Anne Kruse, Kirsten Leidreiter, Burgi Riens, David G. Robinson, Heike Winter, Thilo Winzer and Hans W. Heldt

9. Regulation of starch synthesis in storage organs Alison M. Smith

10. The integration of sucrose and fructan metabolism in temperate grasses and cereals Christopher J. Pollock, Andrew J. Cairns, Joseph Gallagher and Judith Harrison

11. Expression of fructosyltransferase genes in transgenic plants Irma Vijn, Anja van Dijken, Stefan Turk, Michel Ebskamp, Kees van Dun, Peter Weisbeck and Sjef Smeekens

12. The application of transgenic technology to the study of sink metabolism in potato . Richard N. Trethewey and Lothar WiIImitzer

13. Increasing the flux in a metabolic pathway: a metabolic control analysis perspective David A. Fell and Simon Thomas

14. Nitrate acts as a signal to control gene expression, metabolism and biomass allocation Mark Stitt and Wolf-Riidiger Scheible

Subject index

ix

17

37

53

101

117

137

159

173

195

227

239

257

275

307

PREFACE

Over the past decade, advances in molecular biology have provided the impetus for a resurgence of interest in plant metabolism. At a general level, the potential for modifying the quantity or quality of harvestable crop products through genetic manipulation has provided an agronomic rationale for seeking a greater understanding of primary plant metabolism and its regulation. Moreover, the now facile techniques for transformation of many plant species and the consequential capacity to manipulate the amounts of specific individual enzymes within specific cell types provides an exciting direct approach for studying metabolic problems. Such transgenic plants are also becoming invaluable tools in studies at the interface between metabolism and other sub-disciplines such as physiology and ecology. The interest generated in plant metabolism by these developments has also encouraged the re-introduction of more conventional biochemical techniques for metabolic analysis. Finally, in common with other areas of cell biology, the wealth of information that can be obtained at the nucleic acid level has provided the stimulus for identification and characterisation of metabolic processes in far greater detail than previously envisaged. The result of these advances it that researchers now have the confidence to address problems in plant metabolism at levels not previously attempted.

This book presents the proceedings of an international conference held on 9-11 January 1997 at St Hugh's College, Oxford under the auspices of the Phytochemical Society of Europe. The aim of the meeting was to provide a timely review of progress in the area of primary plant metabolism, and in particular to highlight the extent to which molecular techniques now influence the investigation and understanding of plant metabolism. We deliberately chose to limit the scope of the meeting to the processes related to the dominant pathways of carbohydrate production and utilisation. This was done in the belief that it would enable topics to be considered in sufficient detail to identify the emerging themes and ideas in the field. The book is arranged to reflect the present focus on three broadly overlapping areas of investigation. It starts with a consideration of the structure of several enzymes of primary metabolism. A detailed understanding of metabolic regulation will ultimately require a description of the molecular interactions that modulate enzyme activity. Currently several hundred protein structures are determined each year, yet very few of these proteins are from plant sources. The opening chapters illustrate how a consideration of protein structure at different levels can enhance our understanding of the metabolic roles of specific enzymes, and may serve to stimulate further

ix

x

interest in this approach. The second section of the book concentrates on integration of metabolism between organelles, cells, tissues and organs. Plant cells are both compartmented and differentiated. These features often define the unique organisation of metabolic processes and in turn determine the extent to which pathways and their intermediates may interact. The final section reviews attempts to define and manipulate some of the major pathways of carbohydrate metabolism, concluding with chapters considering theoretical difficulties associated with rational manipulation of metabolic flux, and the complex metabolic and developmental interactions that may arise as metabolism is perturbed.

The material in this book illustrates three general themes that emerged during the meeting. The first is the extent to which molecular techniques are being integrated into plant biochemistry, and in particular the degree to which transgenic plants are now being used to address metabolic problems (rather than being paraded as a late 20th century form of Victorian freak show). The second is our increasing appreciation of the inherent heterogeneity of metabolism, and the current awareness of the compartmentation of metabolic processes at both the cellular and subcellular level. The third feature is the progress that is being made towards fulfilling the promise of manipulating metabolism for beneficial or profitable purposes.

Nevertheless, we should not be too complacent about progress in this field. Although some of the changes that have been introduced in carbohydrate metabolism by genetic manipulation have been spectacular, in general they have resulted from conceptually simple alterations and have not been dependent on a profound understanding of regulation. Furthermore, as information accumulates, it is becoming increasingly apparent that metabolic processes vary between species (or even cell types). Thus, we cannot predict the precise pathways occurring in a particular tissue with any confidence. In addition, a common feature of plant metabolism is the degree to which individual enzyme activities or whole pathways are duplicated, often within different sub-cellular compartments. Such metabolic redundancy is often explained as a prerequisite for the flexibility needed by plants to regulate potentially conflicting pathways differentially in response to variable metabolic demands in a changing environment. Although this view is superficially attractive, we must be careful to guard against using it as a general explanation for the apparent duplication of metabolic processes, otherwise we will never seek a precise explanation for the function of individual isoforms, or the variable sub-cellular distribution of enzyme activities. As the results of the research described in this book illustrate, the task now facing researchers in this area is to understand the regulation

xi

of metabolism in specific cells within the context of the growth and development of the whole plant.

We conclude on a note of sadness. The meeting was over-shadowed by the memory of the untimely death of- Professor Tom ap Rees a few months before the conference. Tom was an inspirational research scientist and teacher, who influenced the work and careers of many of those attending the meeting. In addition, he was scheduled to present the concluding talk at the meeting and had agreed to contribute to this book. Thus, in recognition of his contribution to the field of plant metabolism and in grateful thanks for his unique influence on the lives of two of the editors (NJK and SAH) we are honoured to be able to dedicate this book to the memory of Tom ap Rees.

N.J. Kruger, S.A. Hill and R.G. Ratcliffe Department of Plant Sciences, University of Oxford

Chapter 1

Rubisco: attempts to reform a promiscuous enzyme

Martin A. J. Parry, Alfred J. Keys, Graeme Bainbridge, Steven P. Colliver, P. John Andralojc, Matthew J. Paul, Fiona M. Banks and Pippa J. Madgwick Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK

Key words: ribulose bisphosphate carboxylase; Rubisco; specificity factor.

Abstract: Despite its unique role in incorporating carbon from atmospheric CO2 into the organic substances of the biosphere, ribulose-I,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is an inefficient enzyme; it has a low turnover number and catalyses several competing reactions, including oxygenation of ribulose-I,5-bisphosphate (ribulose-P2), in addition to the carboxylation of ribulose-P2. Information on the relative specificity for CO2 and O2 and the turnover number for mutant and native Rubisco from diverse species complements the increasing knowledge of the 3-dimensional structure of Rubisco at atomic resolution. We report progress towards improving the catalytic function by protein engineering and consider future experimental objectives. In particular. we have focused on loop 6 of the large subunit a//3 barrel domain and its interaction with the C-terminus of the large subunit. Rubisco is a target of great agronomic importance and genetic engineering offers the prospect of increased net carbon assimilation by increasing the specificity factor. Whilst the technologies are available to achieve this, additional mutants and 3-dimensional structures are needed to distinguish the structural and ionic components that determine specific catalytic properties of Rubisco.

1. INTRODUcnON

Incorporation of carbon from atmospheric CO2 to organic carbon depends on the activity of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco catalyses the carboxylation of

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 1-16. © 1999 Kluwer Academic Publishers.

2 Chapter 1

ribulose-l,5-bisphosphate (ribulose-P2) to generate two molecules of 3 phosphoglycerate (3-P-glycerate). However, in spite of its fundamental importance, Rubisco is a grossly inefficient catalyst: it is slow and it also catalyses several wasteful alternative reactions, including the oxygenation of ribulose-P2• This oxygenation initiates photorespiratory metabolism in which, typically, more than 20% of fixed carbon is lost as CO2• The relative partitioning between the carboxylase and oxygenase reactions is not constant but differs considerably between Rubiscos isolated from diverse species (Parry et aI., 1987; Read and Tabita, 1994; Uemura et aI., 1994). The highest reported value for the Rubisco specificity factor (i.e. the ratio of Vc.KJVo.K) is 238, found in the red alga Galderia partita (Uemura et aI., 1997). This is almost 3-fold greater than the specificity factors reported for Rubisco from most crop plants and about 6-fold greater than those reported from the photosynthetic bacteria. Plants also require large amounts of Rubisco to photosynthesize rapidly because Rubisco has a low turnover number (e.g. 3 per sec for each wheat Rubisco catalytic site). Although an apparent negative correlation between specificity factor and turnover has been reported (Bainbridge et aI., 1995), (Figure 1), the linkage between these kinetic properties is not invariant since some point mutations have decreased kcat without increasing the specificity factor (Wildner et aI., 1996).

Genetic engineering offers the prospect of increased net carbon assimilation by increasing the specificity factor and/or the rate of turnover. The natural variation in kinetic properties of Rubisco from various species offers a key to understanding how differences in catalytic properties are determined by primary and tertiary structure. The genes encoding the large and small subunits of Rubisco, rbcL and rbcS, from a number of species have been cloned and expressed together in E. coli. The expression of both higher plant rbcL and rbcS in E. coli has not yet resulted in the production of functional enzyme. In contrast, expression of bacterial or cyanobacterial rbcL and rbcS in E. coli yield functional proteins which have been used extensively to investigate structure and function.

2. ENZYMESTRUCfURE

In most species Rubisco is a hexadecamer composed of 8 large (Mf

approximately 50-55,000) and 8 small (Mf 12-14,000) subunits. High resolution 3-dimensional structures have been reported for Rubisco from two higher plants, tobacco (Chapman et aI., 1987, 1988; Curmi et aI., 1992; Schreuder et aI., 1993) and spinach (Andersson, 1996; Knight et

1. Rubisco: attempts to reform a promiscuous enzyme

250

200 ,....

150 --

• • • G. partita

C. caldarium

P. cruenteum • T. aestivium

100 • • S. oleracea

- ·OJisthotscus

50 ,....

o o

I

2

N. tabacum

I

4

C. vinosum

• I I

6 8

3

Synechococcus

•

I

10 12

Figure 1. The specificity factor and Vc of Rubisco isolated from various natural sources. Data from (Jordan and Chollet, 1985; Jordan and Ogren, 1984; Jordan and Ogren, 1981; Parry et aI., 1989; Read and Tabita, 1994; Uemura et aI., 1997).

4 Chapter 1

ai., 1989, 1990; Taylor and Andersson, 1996), and for a cyanobacterium, Synechococcus (Newman and Gutteridge, 1990, 1993, 1994).

The large subunits are chloroplast-encoded, whilst the small subunits are nuclear-encoded by a small multi-gene family (of between 4-13 members) and targeted to the chloroplasts by a transit peptide. Not all the small subunits within the holoenzyme are identical, and in spinach Rubisco small subunits with different amino acid sequences have an orderly disposition within the hexadecamer (L8 SI4 SII4 structure) (Shibata et ai., 1996). The large subunits form a central core of 4 dimers. Each large subunit has two major domains; an N-terminal domain of mixed a helices and ~ sheets and a C-terminal a/~ barrel structure. Each dimer has two catalytic sites shared between the subunits, each made up from residues of the C-terminal domain of one subunit and the N-terminal domain of the other subunit within the dimer. Although the large subunits all have the same amino acid sequence, within the holoenzyme they exhibit heterogeneity in the positions of their side-chains (Y. Kai personal communication).

Comparison of the Synechococcus and spinach large subunit coordinates for activated Rubisco, to which the transition state analogue 2-carboxyarabinitol-bisphosphate (CABP) was bound, showed that there were no substantial differences in the number or disposition of any of the structural elements in the vicinity of the catalytic sites. Moreover, those residues that are in direct contact with the substrate analogue (those involved in carbamylation, metal co-ordination and bisphosphate binding) are essentially indistinguishable, although there may be differences in the conformation of the substrate analogue (Newman and Gutteridge, 1993). However, there are regions outside the primary sphere of catalytic residues that do show significant differences in the position of the Ca atoms; these regions are the C-terminal part of loop 6 and the C-terminal tail (Newman and Gutteridge, 1993).

3. CATALYSIS

Catalytic competence requires the initial carbamylation of the E­

amino group of an active site lysine (Lys-20P) and subsequent stabilization of the carbamate by a metal ion, normally Mg2+ (Lorimer et ai., 1976). With the substrate bound, the Mg2+ is coordinated to two adjacent acidic residues (Asp-203 and Glu-204) and to the C2 and C3 oxygen atoms of ribulose-P2 (Gutteridge and Gatenby, 1995). Both carboxylase and oxygenase reactions have the same initial step, which is

1 The spinach numbering of amino acid residues is used throughout this paper

1. Rubisco: attempts to reform a promiscuous enzyme 5

the formation of an enediol of ribulose-P2. The Mg2+ polarizes the carbonyl at C2 of the substrate reducing the pKa of C3 and the carbamate of Lys-20 1 accepts the C3 proton (Gutteridge et aI., 1984). The enediol intermediate is a potent nucleophile which can react with a range of electrophiles in addition to CO2. For example, in abortive side reactions this enediol intermediate is the subject of misprotonation either at C3 to generate xylulose bisphosphate or at C2 to give 3-keto-arabinitol bisphosphate (Edmonson et aI., 1990). Both these compounds are tight binding inhibitors (KD of about 0.2 IlM) and their release in vivo requires the influence of another protein, Rubisco activase and A TP (Robinson and Portis, 1989). Electrophilic attack by CO2 at C2 of the enediol results in the formation of a 2-carboxy, 3-keto intermediate (Lorimer et aI., 1986). This is hydrated at C3, perhaps simultaneously with the carboxylation, to form the gem diol (Jaworowski et aI., 1984; Schloss and Lorimer, 1982). Cleavage at the C2/C3 bond releases one molecule of 3-P-glycerate and a second enediol-like intermediate, the aci-carbanion form of 3-P-glycerate. Stereo-specific protonation of the aci-carbanion produces a 3-P-glycerate molecule (Saver and Knowles, 1982). The enediol intermediate is also susceptible to reaction with molecular oxygen to produce a 5-carbon hydroperoxy intermediate. This is attacked at position 3 by a hydroxyl ion to give one molecule of 2-P-glycolate and one of 3-P-glycerate (Hartman and Harpel, 1994).

Both activation and catalysis are accompanied by conformational changes, with movements of at least five loops and flexible elements of both N- and C-terminal regions of the large subunits. The movements are timed to specific steps in the catalytic cycle (Andersson, 1996; Gutteridge and Gatenby, 1995). Binding of ribulose-P2 induces the whole N-terminal domain to pivot 2° relative to the a/~ barrel and, in addition, causes the N-terminal regions around Thr-65 and loop 6 of the barrel to close over the substrate (Taylor and Andersson, 1996). These loops are held in place by another N-terminal loop around Lys-128 which forms hydrogen bonds with residues of loop 6 and the C-terminal tail. These movements ensure that the C2 and C3 oxygen atoms of ribulose-P2 are correctly coordinated to the active site Mg2+ ion, permitting the abstraction of the C3 proton to form the enediol intermediate. With the loops closed over the substrate only small molecules like CO2 and O2 can gain access to the enediol intermediate. With CO2 poised just above the C2 centre of the enediol intermediate Lys-334 is well positioned to polarize the two oxygens of CO2, thereby enhancing the electrophilic status of the carbon (Figure 2). Site-directed mutants containing substitutions for Lys-334 catalysed enediol formation but were unable to catalyse the reaction of the enediol with CO2 or to form a stable complex with CABP (Gutteridge et aI., 1993; Hartman and Lee, 1989; Soper et aI.,

6

I ~" Glu204

o e:

: 0 e

Chapter 1

Asp 203 e ~ ...... ~ H N-Lys 334 O . '2+ 3 'n-- ..... ~~(.:~g ........... \{ ..... O\H

A C--:--OH ~ 0 ,"-: \ /C3 HN e 6 ,1, §' 'CHOH CH OP02-'\ e -.............:'" ~ . 2 3

C\2 Ribulose-P 2 (enediol)

2-CHPP03

Lys 201

Figure 2. Electrophilic attack of COlon C2 of ribulose-Pl at the active site 0 f Rubisco, showing the involvement of catalytic site residues. Model based on the atomic coordinates kindly supplied by C-I Brlinden, UppsaJa Biomedical Centre, UppsaJa, Sweden.

1. Rubisco: attempts to reform apromiscuous enzyme 7

1988). Lys-334 also stabilizes the initial transition state intermediates for both the carboxylase and oxygenase reactions (Lorimer et aI., 1993). The cleavage of the 6 carbon intermediate signals the opening of the loops, the release of the first molecule of 3-P-glycerate and the rotation of the aci-carbanion intermediate before stereo-specific protonation and release of the second molecule of 3-P-glycerate. This leaves the active site loops open, ready to accept another molecule of ribulose-P2

(Gutteridge and Gatenby, 1995).

4. ATIEMPfSATREFORMATION

The identification of mutant forms of Rubisco from Chlamydomonas reinhardtii provided the first clue to the identity of residues that are involved in substrate specificity. Mutation of Val-331 to alanine reduced the specificity factor by almost 40% but could be partially compensated for by an additional mutation, V331A plus T3421 (Chen and Spreitzer, 1989). The same mutation in Synechococcus reduced Veto less than 10% but produced conflicting results for the specificity factor: little effect in one report (Parry et aI., 1992) but a 46% reduction in another (Gutteridge et aI., 1993). In spite of this it is commonly found that, with appropriate precautions, even small differences in Rubisco specificity factor can be determined accurately (Kane et aI., 1994; Demura et aI., 1994) while estimations of V c are more variable.

Attempts to engineer the specificity of Rubisco for CO2 have focused mainly on loop 6 with the objective of optimizing the position of Lys-334. Most of the amino acid residues in loop 6 are conserved between different species (Table 1) but the four residues (338-341) at the C­terminal end of the loop vary. Mutation of these four residues in Synechococcus Rubisco to the residues found in maize or tobacco resulted in 3-7% increase in the specificity factor. This was accompanied by a fall in V c of less than 10%, compared with the wild-type enzyme (Kane et aI., 1994; Parry et aI., 1992). In contrast, mutation to the spinach sequence did not significantly alter the specificity factor but reduced Vc by about 40% (Gutteridge et aI., 1993).

Substitution of individual amino acids had variable effects, both positive and negative, on the specificity factor (Table 2). In Synechococcus the mutation, A340E, led to a 17% decrease in the specificity factor with a fall in Vc of just over 10% (Parry et aI., 1992). In contrast, replacing the relatively small non-polar side-chain of this residue with much bulkier side-chains in mutants A340Y and A340H caused 12-13% increases in the specificity factor. This was accompanied

8 Chapter 1

Table 1. The percent increase or decrease in specificity factor (t) and rate (V J, relative to

wild-type, of L8S8 Rubisco in which residues within loop 6 and helix 6 have been altered. The

residue mnnber is preceded by the single-letter representation for the wild-type residue at that

position and followed by the single letter representation for the replacement. Data from: 1-

(Chen & Spreitzer 1989); 2- (Parry et al. 1992b); 3- (Gutteridge et at. 1993); 4- (Lee et al.

1993); 5-(Read & Tabita 1994); 6- P. Madgwick,personal corrnnunication; 7- (Zhu & Spreitzer

1996); 8- (Kane et al. 1994). nd = not detennined.

I Mutant

I Species

I 't%wt

I V.%

I Authors

I V331A C reinhardtii -37 -93 1

Synechococcus -2 -92 2

Synedwcoccus -46 -95 3

V331G Synedwcoccus -64 -99 3

V331I Syneclwcoccus nd -100 3

V331L Synechococcus -12 -95 3

V331M Synechococcus -71 -99 3

K334R SynecIwcoccus -99 -99 3

1335M Synechococcus -53 -65 4

13351 Synechococcus -67 -86 4

1332V Synechococcus -60 -61 4

1335T Synechococcus -35 -46 4

1335A Synechococcus -56 -70 4

D338E Synechococcus +4 -6 2

1. Rubisco: attempts to reform a promiscuous enzyme 9

K339R Synechococcus 0 0 2

K339P Synechococrus -2 -85 5

A340E SYllechococrus -17 -12 2

A340D Synechococrus +5 -33 6

A340G Synechococrus -6 -31 6

A340H Synechococms +13 -33 6

A340N Synechococrus +9 -29 6

A340R Synechococms +3 -4 6

A340Y Synechococcus +12 -25 6

A340L SYllechococrus -7 -34 5

S341I Synechococrus -I -10 2

S341M Synechococms +7 -I 5

V341I C reinhardtii -3 -4 7

1342A Synechococms nd -98 3

13421 Synechococrus -7 -70 3

-18 -45 5

1342V Synechococms -23 -48 5

1342L Synechococms -7 -60 3

TI42M Synechococrus -11 -94 3

DKAS338- Synechococms +7 -8 2

341EREl +5 nd 8

DKAS338- Synechococms +3 -40 3

341ERDI

Tab

le 2

. The

am

ino

acid

seq

uenc

e of

loop

6, (

with

som

e pr

eced

ing

and

follo

win

g re

sidu

es)

and

the

C-te

rmm

us o

f the

Rub

isco

larg

e su

buni

t in

diff

eren

t spe

cies

. (A

nder

son

& C

aton

1987

; Cur

tis &

Haz

elko

m 1

983;

Dro

n et

al.

1982

; Gin

gric

h &

Hal

lick

1985

; Har

diso

n et

al.

1992

; Hw

ang

& T

abita

199

1; M

cInt

osh

et a

l. 19

80; S

hino

zaki

& S

igiu

ra 1

982;

Shi

noza

ki

et a

l. 19

83;

Val

entin

~ Z

etsc

he 1

989;

Via

le e

t al.

1990

; Zu

raw

ski

et a

i. 19

81;

Zura

wsk

i et a

l. 19

86).

Res

idue

s id

entic

al to

thos

e of

SYl

lech

ococ

cus

are

show

n as

' .'

.

Syn

ech

oco

ccu

s

Zea

m

ays

Sp

ina

cea

o

lera

cea

Pis

um

sa

tivu

m

Nic

oti

an

a

tab

acu

m

Ch

lam

ydo

mo

na

s rein

ha

rd

tii

Eu

gle

na

g

ra

cil

is

An

ab

aen

a

Ch

rom

ati

um

vin

osu

m

Alc

ali

gen

es

eu

tro

ph

us

Po

rph

yri

diu

m

aeru

gin

eu

m

Cyli

nd

ro

theca

fu

sif

orm

is

Oli

sth

od

iscu

s

loop

6

v V

G K

LE

G

D K

A S

T

L

G F

ER

E

I

E

R

0 I

ER

E

I

E R

0

I

ER

E V

ER

E

V

ER

E

I M

R

P L

TV

Q

P L

M I

K

P L

M I

K

P L

M V

K

w

y

C-te

rmin

us

E F

E T

M D

K L

D G

F

K A

MD

T

I

P A

T

V

P A

T

N

A

V

V

D K

D T

I I

A

T V

o V

D

I

A H

K

N Y

T

P T

T

S

0 F

V

PT

A S

V A

N Y

T S

T

T

AD

F

V Q

T P

TA

N V

N Y

T S

T

TA

D

F A

A T

S T

AN

V

N Y

T S

T.

TA

D

F V

ET

A T

S N

P

-o Q

{3 ~

"l

......

1. Rubisco: attempts to reform a promiscuous enzyme 11

by a fall in Vc of less than 35%, compared with the wild-type enzyme. By analogy with triose phosphate isomerase, in which catalysis also involves a flexible loop in a alp barrel, the tip of the loop is expected to move as a rigid body with large movements only in the two hinge regions (Wierenga et aI., 1992). In the unactivated form of Rubisco (Schneider et aI., 1990) the N-terminal section of the loop forms an extra tum of helix 6 with a short extended region while in the activated form (Lundqvist and Schneider, 1991) the whole loop is extended, closing over the active site. An earlier model of the region around A340E, based on the spinach Rubisco structure (Parry et aI., 1992), suggested a novel hydrogen bond between the introduced glutamic acid and lysine 474. The mutant DKAS338-341EREI could possibly form a second hydrogen bond between glutamic acid at position 338 and threonine 471 thereby stabilizing the structure and protecting it from proteolysis (Parry et aI., 1992). It is possible that mutants A340H and A340Y can also form hydrogen bonds with the C-terminal region of the polypeptide and that the larger size of the mutant side-chains might constrain the movement of loop 6. The association of the transition state analogue CABP with native Rubisco is almost irreversible following the movement of loop 6 and other loops to occlude the active site. However, since the affinity of the A340H and A340Y Rubiscos for CABP was similar to the wild-type enzyme, the ability of loop 6 and other loops to close over the active site is not significantly impaired by these changes.

Apart from Lys-334 none of the loop 6 residues that have been altered interact directly with CABP and so their effect on the reactivity of the enediol intermediate must be indirect. In addition, interactions with other parts of the enzyme must be important. Mutations affecting loop 6 and the C-terminal tail in the Synechococcus large subunit have increased specificity factors. Modifying the Synechococcus gene to produce a polypeptide with a spinach loop 6 and C-terminus resulted in an enzyme with a 9% increase in specificity but a 40% fall in Vc (Gutteridge et aI., 1993). Removal of a single amino acid residue from the end of the C-terminus, with carboxypeptidase-A, reduced activity by 60-70% in spinach and C. reinhardtii but also resulted in 5% decrease in the specificity factor (Portis, 1990). In Synechococcus, substitution of Ala-340 with glutamic acid reduced the loss of activity on exposure to carboxypeptidase-A provided that ribulose-P2 was present during exposure (Parry et aI., 1993). The amino acid residues of the C-terminus are highly variable (Table 1) and their position poorly defined in some 3-dimensional structures (Taylor and Andersson, 1996). The construction and analysis of additional chimeric large subunits with modifications to loop 6 and the C-terminus may further enhance specificity.

12 Chapter 1

Although mutation of some other regions of the enzyme has increased specificity, Vc fell to less than 10% of the wild-type value; e.g. a mutant form of Rubisco from R. rubrum, S368A, had a 1.6-fold increase in specificity but Vc was only about 2% of that of the wild-type enzyme (Harpel and Hartman, 1992).

5. CONCLUSIONS

Considerable progress has been made m understanding the contribution of specific amino acids to catalysis. In addition, the specificity factor for mutant enzymes has been increased by up to 13% without catastrophic effects on Vc. However desirable, changes of this type have so far only been achieved for Synechococcus Rubisco. Equivalent changes in crop plants would have considerable agronomic importance. Recent experiments, although presently restricted to a single species, demonstrate that direct manipulation of higher plant rbcL is possible (Kanevski and Maliga, 1994; Svab et aI., 1990). Additional mutants and 3-dimensional structures will extend our understanding of the catalytic process. Thus, the ultimate goal of improving the efficiency of Rubisco in crop plants is gradually being realized.

ACKNOWLEDGEMENTS

IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.

REFERENCES

Anderson, K. and Caton, J. (1987). Sequence analysis of Alcaligenes eutrophus chromosomally encoded ribulose-l,5-bisphosphate carboxylase large and small subunit genes and their products. Journal of Bacteriology, 169, 4547-4558.

Andersson, I. (1996). Large structures at high resolution: the 1.6A crystal structure of spinach ribulose-l,5-bisphosphate carboxylase/oxygenase complexed with 2-carboxyarabinitol bisphosphate. Journal of Molecular Biology, 259, 160-174.

Bainbridge, G., Madgwick, P. J., Parmar, S., Mitchell, R., Paul, M. J., Pitts, J., Keys, A. 1. and Parry, M. A. J. (1995). Engineering Rubisco to change its catalytic properties. Journal of Experimental Botany, 46, 1269-1276.

Chapman, M., Suh, S., Cascio, D., Smith, W. and Eisenberg, D. (1987). Sliding-layer conformational change limited by the quaternary structure of plant Rubisco. Nature, 329, 354-356.

1. Rubisco: attempts to reform a promiscuous enzyme 13

Chapman. M. S., Suh, S. W., Curmi, P. M. G., Cascio, D., Smith, W. W. and Eisenberg, D. S. (1988). Tertiary structure of plant Rubisco: domains and their contacts. Science. 241, 71-74.

Chen, Z. and Spreitzer, R. 1. (1989). Chloroplast intragenic supression enhances the low CO2/02 specificity of mutant Ribulose-bisphosphate Carboxylase/Oxygenase. Journal of Biological Chemistry, 264, 3051-3053.

Curmi, P. M. G., Cascio, D., Sweet, R. M., Eisenberg, D. and Schreuder, H. (1992). Crystal structure of the unactivated form of ribulose-l,5-bisphosphate carboxylase/oxygenase from tobacco refined at 2.0-A resolution. Journal of Biological Chemistry, 267, 16980-16989.

Curtis, S. and Hazelkorn, R. (1983). Isolation and sequence of the gene for the large subunit of ribulose-I,5-bisphosphate carboxylase from the cyanobacterium Anabaena 7120. Proceedings of the National Academy of Sciences USA, 80, 1835-1839.

Dron, M., Rahire, M. and Rochaix, 1. (1982). Sequence of the chloroplast DNA region of Chlamydomonas reinhardtii containing the gene of ribulose-l,5-bisphosphate carboxylase and part of its flanking genes. Journal of Molecular Biology, 162, 775-793.

Edmonson, D. L., Kane, H. J. and Andrews, T. J. (1990). Substrate isomerization inhibits ribulosebisphosphate carboxylase-oxygenase during catalysis. FEBS Letters, 260, 62-66.

Gingrich, J. and Hallick, R. (1985). The Euglena gracilis chloroplast ribulose-l,5-bisphosphate carboxylase/oxygenase gene. Journal of Biological Chemistry, 260, 16162-16168.

Gutteridge, S. and Gatenby, A. A. (1995). Rubisco synthesis, assembly, mechanism, and regulation. The Plant Cell, 7, 809-819.

Gutteridge, S., Parry, M. A. J. and Schmidt, C. N. G. (1984). An investigation of ribulosebisphosphate carboxylase activity by high resolution IH NMR. FEBS Letters, 170, 355-359.

Gutteridge, S., Rhoades, D. F. and Herrmann, C. (1993). Site-specific mutations in a loop region of the C-terminal domain of the large subunit of ribulose bisphosphate carboxylase/oxygenase that influence substrate partitioning. Journal of Biological Chemistry, 268, 7818-7824.

Hardison, L., Boczar, B., Reynolds, A. and Cattolico, R. (1992). A description of the Rubisco large subunit gene and its transcripts in Olisthodiscus lute us. Plant Molecular Biology, 18, 595-599.

Harpel, M. and Hartman, F. C. (1992). Enhanced CO2/0 2 specificity of a site-directed mutant of ribulose-bisphosphate carboxylase/oxygenase. Journal of Biological Chemistry, 267, 6475-6478.

Hartman, F. and Lee, E. (1989). Examination of the function of active site lysine 329 of ribulose-I,5-bisphosphate carboxylase/oxygenase as revealed by proton exchange reactions. Journal of Biological Chemistry, 246, 11784-11789.

Hartman, F. C. and Harpel, M. R. (1994). Structure, function, regulation and assembly of o-Ribulose-l,5-bisphosphate carboxylase/oxygenase. Annual Review of Biochemistry, 63, 197-234.

Hwang, S-R. and Tabita, F. (1991). Cotranscription, deduced primary structure, and expression of the chloroplast encoded rbcL and rbcS genes of the marine diatom Cylindrotheca sp. strain NI. Journal of Biological Chemistry, 266, 6271-6279.

Jaworowski, A., Hartman, F. C. and Rose, I. A. (1984). Intermediates in the ribulose-l,5-bisphosphate carboxylase reaction. Journal of Biological Chemistry, 259, 6783-6789.

14 Chapter 1

Jordan, D. B. and Ogren, W. L. (1981). Species variation in the specificity of ribulose biphosphate carboxylase/oxygenase. Nature, 291, 513-515.

Jordan, D. B. and Ogren, W.L. (1984). The carbon doxide/oxygen specificity of ribulose-l,5-bisphosphate carboxylase/oxygenase. Planta, 161, 308-313.

Jordan, D. B. and Chollet, R. (1985). Subunit dissociation and reconstitution of ribulose-l,5-bisphosphate carboxylase from Chromatium vinosum. Archives of Biochemistry and Biophysics, 236, 487-496.

Kane, H. J., Viii, J., Entsch, B., Paul, K., Morell, M. K. and Andrews, T. J. (1994). An improved method for measuring C02/02 specificity of ribulose bisphosphate carboxylase-oxygenase. Australian Journal of Plant Physiology, 21, 449-461.

Kanevski, I. and Maliga, P. (1994). Relocation of the plastid rbcL gene to the nucleus yields functional ribulose-l,5-bisphosphate carboxylase in tobacco chloroplasts. Proceedings of the National Academy of Science USA, 91, 1969-1973.

Knight, S., Andersson, I. and Branden, c.-I. (1989). Reexamination of the three dimensional structure of the small subunit of Rubisco from higher plants. Science, 244, 702-705.

Knight, S., Andersson, I. and Branden, C.-I. (1990). Crystallographic analysis of ribulose-l,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Journal of Molecular Biology, 215, 113-160.

Lee, G. J., McDonald, K. A. and McFadden, B. A. (1993). Leucine 332 influences the C02/02 specificity factor of ribulose-l,5-bisphosphate carboxylase/oxygenase from Synechococcus. Protein Science, 2, 1147-1154.

Lorimer, G. H, Andrews, T. J., Pierce, J. and Schloss, J. (1986). 2'-carboxy-3-keto-D-arabinitol 1,5-bisphosphate the 6 carbon intermediate of the ribulose bisphosphate carboxylase reaction. Philosophical Transactions of the Royal Society of London, 313B, 397-407.

Lorimer, G. H., Badger, M. and Andrews, T. J. (1976). The activation of ribulose-l,5-bisphosphate carboxylase by carbon dioxide and magnesium ions. Biochemistry, 32, 9018-9024.

Lorimer, G. H., Chen, Y.-R. and Hartman, F. C. (1993). A role for the E-amino group of lysine-334 of ribulose-l,5-bisphosphate carboxylase in the addition of carbon dioxide to the 2,3-enediol(ate) of ribulose 1,5-bisphosphate. Biochemistry, 32, 9018-9024.

Lundqvist, T. and Schneider, G. (1991). Crystal structure of activated ribulose-l,5-bisphosphate carboxylase complexed with its substrate, ribulose-l,5-bisphosphate. Journal of Biological Chemistry, 266, 12604-12611.

McIntosh, L., Paulsen, C. and Bogorad, L. (1980). Chloroplast gene sequence for the large subunit of ribulose-l,5-bisphosphate carboxylase from maize. Nature, 288, 556-560.

Newman, J. and Gutteridge, S. (1990). The purification and preliminary X-ray diffraction studies of recombinant Synechococcus ribulose-l,5-bisphosphate carboxylase/oxygenase from Escherichia coli. Journal of Biological Chemistry, 265, 15154-15159.

Newman, J. and Gutteridge, S. (1993). The X-ray structure of Synechococcus ribulose-bisphosphate carboxylase/oxygenase-activated quaternary complex at 2.2-A resolution. Journal of Biological Chemistry, 268, 25876-25886.

Newman, J. and Gutteridge, S. (1994). Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate. Structure, 2, 495-502.

1. Rubisco: attempts to reform a promiscuous enzyme 15

Parry, M. A. J., Schmidt, C. N. G., Cornelius. M. J., Millard, B. N., Burton. S., Gutteridge. S., Dyer. T. A. and Keys, A. 1. (1987). Variations in properties of ribulose-l,5-bisphosphate carboxylase from various species related to differences in amino acid sequences. Journal of Experimental Botany, 38, 1260-1271.

Parry, M. A. 1., Keys, A. 1. and Gutteridge, S. (1989). Variation in the specificity factor of C3 higher plant Rubiscos determined by the total consumption of ribulose-Pz. Journal of Experimental Botany, 40, 317-320.

Parry, M., Madgwick, P., Parmar, S. and Keys, A. (1993). Changed COZ/02 specificity in mutants of Rubisco. In Murata, N. (Ed). Research in Photosynthesis (Proceedings of 9th International Congress on Photosynthesis) (Vol. 3, pp 609-612). Kluwer Academic Publishers, Dordrecht.

Parry, M. A. 1., Madgwick, P. J., Parmar, S., Cornelius, M. 1. and Keys, A. 1. (1992). Mutations in loop six of the large subunit of ribulose-l,5-bisphosphate carboxylase affect substrate specificity. Planta, 187, 109-112.

Portis, A. R. (1990). Partial reduction in ribulose 1,5-bisphosphate carboxylase/oxygenase activity by carboxypeptidase A. Archives of Biochemistry and Biophysics, 283, 397-400.

Read, B. and Tabita, F. R. (1994). High substrate specificity factor ribulose bisphosphate carboxylase/oxygenase from eukaryotic marine algae and properties of recombinant cyanobacterial Rubisco containing "algal" residue modifications. Archives of Biochemistry and Biophysics, 312, 210-218.

Robinson, S. R. and Portis, A. R. (1989). Ribulose-I,5-bisphosphate carboxylase/oxygenase activase protein prevents the in vitro decline in activity of ribulose-l,5-bisphosphate carboxylase/oxygenase. Plant Physiology, 90, 968-971.

Saver, B. G. and Knowles, J. R. (1982). Ribulose bisphosphate carboxylase: enzyme-catalysed appearance of solvent tritium at carbon 3 of ribulose-l,5-bisphosphate re-isolated after partial reaction. Biochemistry, 21, 5398-5403.

Schloss, J. V. and Lorimer, G. H. (1982). The stereochemical course of ribulose bisphosphate carboxylase. Journal of Biological Chemistry, 257, 4691-4694.

Schneider, G., Lindqvist, Y. and Lundqvist, T. (1990). Crystallographic refinement and structure of ribulose-l.5-bisphosphate carboxylase from Rhodospirillum rubrum at 1.7 A resolution. Journal of Molecular Biology, 211, 989-1008.

Schreuder, H. A., Knight, S., Curmi, P. M., Andersson, I., Cascio, D., Branden, c.-I. and Eisenberg, D. (1993). Formation of the active site of ribulose-I,5-bisphosphate carboxylase/oxygenase by a dis-order transition from the unactivated to the activated form. Proceedings of the National Academy of Sciences USA, 90, 9968-9972.

Shibata, N., Inoue, T., Fukuhara, K., Nagara, Y., Kitagawa, R., Harada, S., Kasai, N., Uemura, K, Kato, K, Yokota, A. and Kai, Y. (1996). Orderly disposition of heterogenous small subunits in D-ribulose-l,5-bisphosphate carboxylase/oxygenase from spinach. The Journal of Biological Chemistry, 271, 26449-26452.

Shinozaki, K. and Sigiura, M. (1982). The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-I,5-bisphosphate carboxylase/oxygenase. Gene, 20,91-102.

Shinozaki, K, Yamada, C., Takahata, N. and Masahiro, S. (1983). Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-I,5-bisphosphate carboxylase/oxygenase. Proccedings of the National Academy of Sciences USA, 80, 4050-4054.

Soper, T. S., Mural, R. 1., Larimer, F. W., Lee, E. H., Machanoff, R. and Hartman, F. C. (1988). Essentiality of Lys-329 of ribulose-I,5-bisphosphate carboxylase/oxygenase

16 Chapter 1

from Rhodospirillum rubrum as demonstrated by site-directed mutagenesis. Protein Engineering, 2, 39-44.

Svab, Z., Hajdukiewicz, P. and Maliga, P. (1990). Stable transformation of plastids in higher plants. Proceedings of National Academy of Sciences USA, 87, 8526-8530.

Taylor, T. and Andersson, 1. (1996). Structural transitions during activation and ligand binding in hexadecameric Rubisco inferred from the crystal structure of the activated unliganded spinach enzyme. Nature Structural Biology, 3, 95-101.

Uemura, K., Suzuki, Y., Shikanai, T., Wadano, A, Jensen, R.G., Chmara, W. and Yokota, A (1994). A rapid and sensitive method for determination of relative specificity of Rubisco from various species by anion exchange chromatography. Plant and Cell Physiology, 37, 325-331.

Uemura, K., Anwaruzzaman, Miyachi, S. and Yokota, A (1997). Ribulose-I,5-bisphosphate carboxylase/oxygenase from the thermophillic red algae with a strong specificity for C02 fixation. Biochemical and Biophysical Research Communications, 233, 568-571.

Valentin, K. and Zetsche, K. (1989). The genes for both subunits of ribulose-l,5-bisphosphate carboxylase constitute an operon on the plastome of red alga. Current Genetics, 16, 203-209.

Viale, A., Kobayashi, H. and Akazawa, T. (1990). Distinct properties of Escherichia coli products of plant-type ribulose-l,5-bisphosphate carboxylase/oxygenase directed by two sets of genes from the photosynthetic bacterium Chromatium vinosum. Journal of Biological Chemistry, 265, 18386-18392.

Wierenga, R. K., Borchert, T. V. and Noble, M. E. M. (1992). Crystallographic binding studies with triosephosphate isomerases: conformational changes induced by substrate and substrate-analogues. FEBS Letters, 307, 34-39.

Wildner, G. F., Schlitter, J. and Muller, M. (1996). Rubisco, an old challenge with new perspectives. Journal of Biosciences, 51, 263-276.

Zhu, G. and Spreitzer, R. J. (1996). Directed mutagenesis of chloroplast ribulose 1,5-bisphosphate carboxylase/oxygenase. Journal of Biological Chemistry, 271, 1894-1898.

Zurawski, G., Perrot, B., Bottomley, W. and Whitfeld, P. (1981). The structure of the gene for the large subunit of ribulose-l,5-bisphosphate carboxylase from spinach chloroplast DNA. Nucleic Acid Research, 9, 3251-3270.

Zurawski, G., Perrot, B. and Whitfe1d, P. (1986). Sequence for the gene for the large subunit of ribulose-l,5-bisphosphate carboxylase from pea chloroplasts. Nucleic Acid Research, 14, 3975.

Chapter 2

Insights into the active site of the plant alternative oxidase and its relationship to function

Charles Affourtit and Anthony L. Mooore Biochemistry Department, School of Biological Sciences, University of Sussex, Falmer, Brighton BN 1 9QG, UK

Key words: alternative oxidase; di-iron carboxylate proteins; mitochondria; molecular modelling; oxidative stress; oxygen scavenger; plant respiration.

Abstract: This review is focused upon our current understanding of the structure and function of the mitochondrial alternative oxidase. Molecular modelling techniques have been used to model the active site of the alternative oxidase using the 3D-coordinates of known di-iron carboxylate proteins (methane monooxygenase and ribonucleotide reductase). Results are discussed in terms of how the proposed structure of the alternative oxidase relates to its postulated function as a di-oxygen scavenger.

1. INTRODUCTION

Aerobic respiration is the primary source of the A TP that is required for cellular energy consuming reactions such as plant growth and development. The proton-pumping respiratory chain complexes generate the protonmotive force necessary to synthesise A TP and the primary features of these complexes are analogous to those found in other eukaryotic systems (Whitehouse and Moore, 1995). Plant respiration, however, does differ considerably from mammalian systems since some of its respiratory activity is insensitive to conventional respiratory inhibitors such as cyanide and antimycin A. The extent to which respiration is insensitive to these inhibitors varies from a few percent of the total oxygen consumption rate (as in the case of freshly isolated

17

N.l. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 17-36. © 1999 Kluwer Academic Publishers.

18 Chapter 2

potato tuber mitochondria) to 100% in the case of mitochondria isolated from thermogenic spadices of Arum maculatum or Sauromatum guttatum. The reason for this inhibitor-resistant respiratory activity is the possession, in addition to the conventional cytochrome c oxidase, of a cyanide- and antimycin-insensitive terminal oxidase ( Day et aI., 1995; McIntosh, 1994; Moore and Siedow, 1991; Siedowand Umbach, 1995; Wagner and Krab, 1995).

The alternative oxidase is an integral inner membrane protein (Moore and Siedow, 1991; Rasmusson et aI., 1990; Siedow et at, 1992) the activity of which does not generate a protonmotive force (Moore et aI., 1978; Whitehouse and Moore, 1995) and which reduces oxygen to water (Berthold and Siedow, 1993; Moore and Siedow, 1991). Since the alternative pathway branches from the main respiratory chain at the level of the ubiquinone pool plant mitochondrial electron transfer is bifurcated. Although it is not normally found in mammalian systems, the alternative oxidase is not exclusive to higher plants being found in fungi, yeasts, algae and several protista (see the following recent reviews Day et aI., 1995; McIntosh, 1994; Moore and Siedow, 1991; Moore et aI., 1995; Siedow and Umbach, 1995; Wagner and Krab, 1995).

The alternative oxidase harbours several interesting spectroscopic features since no electron paramagnetic resonance signals can be detected in either membrane bound (Moore and Siedow, 1991; Rich et aI., 1977a) or partially purified preparations of the alternative oxidase (Berthold and Siedow, 1993) and furthermore the partially purified enzyme does not exhibit any optical absorbance in the region above 350 nm (Berthold and Siedow, 1993). The lack of a fully purified alternative oxidase protein has hampered the identification of co-factors required for activity (see Moore and Siedow, 1991). The most conclusive results in this respect were obtained by Minagawa and co-workers who showed that iron is a prerequisite for alternative oxidase activity in Pichia stipitis (Minagawa et aI., 1990).

The alternative oxidase is subject to many regulatory mechanisms. Numerous extensive kinetic studies have revealed that activity of the enzyme is regulated by the reduction level of the ubiquinone pool ( Day et aI., 1991; Dry et aI., 1989; Moore et aI., 1988; Moore and Siedow, 1991; Siedowand Moore, 1993) and therefore indirectly by the activity of quinone-reducing enzymes (Van den Bergen et aI., 1994), the amount of alternative oxidase protein (Siedow and Moore, 1993), the mitochondrial concentration of a-keto acids, particularly pyruvate ( Hoefnagel et aI., 1995; Millar et aI., 1993), the redox status of the sulphydryl/disulphide system which determines whether the protein exists as a non-covalently or covalently bound dimer respectively (Umbach and Siedow, 1993, 1996; Umbach et aI., 1994) and the total amount of

2. Structure-function relationships of the alternative oxidase 19

ubiquinone (Ribas-Carbo et ai., 1995). Several models based on the idea that a central homogeneous ubiquinone-pool connects the different respiratory enzymes have been developed to describe and predict the kinetic behaviour of the alternative oxidase (reviewed by Krab, 1995).

The objective of this brief review is to focus on recent developments concerning the structure of the catalytic site of the alternative oxidase. In particular we consider how scrutiny of the primary structure of the alternative oxidase has improved our current understanding of the molecular nature of the enzyme. We have used molecular modelling techniques to gain further insight into the possible 3D-structure of the active site of the alternative oxidase and to determine whether the current model of the catalytic site (which is proposed to contain a coupled binuclear iron centre) (Moore et ai., 1995; Siedow et ai., 1995) is plausible. In addition we have used this information in a consideration of the relationship between the structure and the physiological function of the plant alternative oxidase.

2. STRUCTURE OF THE ALTERNATIVE OXIDASE

The majority of our current understanding of the structure of the plant alternative oxidase has arisen from amino-acid sequence comparisons derived from cDNAs encoding the oxidase. To date clones from six plant species (including soybean (Whelan et ai., 1995), mango (Cruz-Hernandez and Gomez-Lim, 1995), tobacco (Vanlerberghe and McIntosh, 1994), Arabidopsis (Kumar and Soli, 1992), Sauromatum (Rhoads and McIntosh, 1991) and potato (Hiser et ai., 1996)), Pichia (Sakajo et aI., 1993) Neurospora (Li et aI., 1996) and Trypanosoma (Chaudhuri and Hill, 1996) have been sequenced and in all cases the deduced amino-acid sequences of the mature protein are very highly conserved. All of the plant sequences include putative mitochondrial transit peptides at the N-terminus that vary in length. Secondary structure predictions of the deduced amino-acid sequences ( Day et ai., 1995; McIntosh, 1994; Moore and Siedow, 1991; Siedow and Umbach, 1995) indicate that three regions are hydrophobic and strongly a-helical and are located centrally within the body of the protein being flanked by Nand C-terminal hydrophilic domains which, according to limited proteolysis experiments (Rasmusson et ai., 1990; Siedow et ai., 1992), extend into the mitochondrial matrix. Hydropathy analysis of the deduced amino-acid sequence reveals that two of the a-helical regions are of such length and sufficiently hydrophobic to be membrane spanning ( McIntosh, 1994; Moore and Siedow, 1991; Siedow and Umbach, 1995).

20 Chapter 2

From a comparison of the hydrophobic plots for a number of the alternative oxidases (Figure 1) it is apparent that the plots are virtually identical and can now be considered to be a signature for the alternative oxidase. Interestingly the plot appears palindromic which is quite unusual for integral membrane proteins.

All of the alternative oxidase amino-acid sequences show a high degree of conservation particularly from the start of the first transmembrane helix continuing through the majority of the C-terminal hydrophilic domain. Even though the N-terminal region is much less conserved in all of the plant sequences, it contains two highly conserved cysteine residues (Day et aI., 1995; Siedow and Umbach, 1995). One of these is located at the start of the first transmembrane helix and has recently been postulated (Umbach and Siedow, 1996) to be the site of pyruvate action (Millar et aI., 1993) whilst the second cysteine residue, the more N­terminal of the two, may be the sulphydryl involved in redox regulation of oxidase activity (Umbach and Siedow, 1993; Umbach et aI., 1994).

More attention has been focused, however, upon the C-terminal domain since amino-acid sequence comparisons showed that all of the alternative oxidase sequences contain four copies of the primary motif (DIE-X-X-H). One of these motifs is located on the P-side of the inner mitochondrial membrane between the two transmembrane helices whereas the other three are located in the C-terminal hydrophilic domain. Interest in these motifs stems from the finding that such motifs act as the iron-binding sites of binuclear iron carboxylate proteins such as methane monoxygenase (MMO) and ribonucleotide reductase R2 (RNR R2) (Class I type according to Nordlund and Eklund, 1995). Interestingly MMO also shares other characteristics common to the alternative oxidase including the capability to reduce oxygen to water and the lack of any absorbance above 350 nm (Moore et aI., 1995; Siedowet aI., 1995). X-ray crystallography studies indicate the metal binding motifs in the class I type di-iron group of proteins are located within a four-helix bundle that acts as a scaffold to bind the iron atoms (Nordlund and Eklund, 1995).

With respect to which of the four copies of the metal-binding motif are present within the active site of the alternative oxidase, the first copy of the motif is located on the opposite of the inner membrane to the other three and therefore unlikely to play a role in providing ligands to bind the iron atoms and in the fourth copy (the more C-terminal copy) there is no conserved carboxylate in the Neurospora or Trypanosoma sequences (see Wagner and Moore, 1997). Using the other two highly conserved motifs, an iron-binding four-helical bundle can be constructed for the alternative oxidase which appears very similar to that

2. Structure-fimction relationships of the alternative oxidase 21

Figure 1. Hydropathy analysis of the unprocessed alternative oxidase amino-acid sequences. Nucleotide sequences encoding the structure of the unprocessed alternative oxidase proteins were downloaded from the Genbank database (http://www2.ncbi.nlm.nih.gov/cgi-bin/genbank). The DNA strider™ v1.2 application was used to deduce amino acid sequences and hydropathy analysis was carried out using the algorithm of Kyte and Doolittle. A window length of 19 was chosen for the calculation of the average hydropathy. A, Pichia stipitis; B, Mangifera indica; C, Trypanasoma brucei; D, Neurospora crassa; E, Arabidopsis thaliana; F, Sauromatum guttatum; G, Nicotiana tabacum; and H, Glycine max.

22 Chapter 2

observed in other di-iron carboxylate proteins (Moore et aI., 1995; Siedow et aI., 1995).

A hypothetical structure of the active site of the alternative oxidase, based upon the highly conserved residues within the four-helical bundle, has been proposed in which the ferric centre is co-ordinated by two histidines, one mono dentate and one bidentate glutamate (which is bridging), one aspartate and two water molecules. A bridging hydroxo atom was incorporated to account for the lack of absorbance above 350 nm. Conserved carboxylates are also found adjacent to each E-X-X-H motif which, similar to other di-iron carboxylates, probably hydrogen bond to the ligating histidine of the alternate E-X-X-H motif (Moore et aI., 1995; Siedow et aI., 1995). A comparison of the pattern of the primary ligation sphere of the di-iron centre of a number of di-iron carboxylate proteins with the one proposed for the alternative oxidase is shown in Figure 2. The similarity of the proposed active site of the alternative oxidase to the active sites of other di-iron proteins is striking suggesting that such structures have been conserved, to a large extent, during evolution. Furthermore it is apparent from Figure 2 that the proposed structure for the active site of the alternative oxidase is intermediate between MMO and RNR R2.

3. MOLECULAR MODELLING OF THE ACTIVE SITE OF THE ALTERNATIVE OXIDASE

Since the late 1950s, 3D-structures of hundreds of proteins have been deduced to atomic resolution by means of X-ray crystallography. In the absence of a fully purified active preparation of alternative oxidase protein, it has not been possible to date to perform crystallisation or NMR experiments and hence high resolution structural information of this protein is not currently available. As indicated in the previous section the carboxy-terminal hydrophilic domain of all alternative oxidase amino-acid sequences contain two highly conserved E-X-X-H motifs which, in MMO (3D-structure by Rosenzweig et aI., 1993) and RNR R2 (3D-structure by Nordlund et aI., 1990), provide coordination sites for the di-iron centres. The availability of both freely accessible databases containing the co-ordinates of published 3D-structures and computer software to visualise and manipulate these structures allows the 3D-structure of the active site of the plant alternative oxidase to be modelled.

In order to perform molecular modelling experiments, Protein Database (PDB) files of the hydroxylase protein of MMO (Immo) and

2. Structure-Junction relationships of the alternative oxidase

Asp-l06

Hi$.101

His·73 N

N~ I~N H"os·77

Fe (I)

Fe (2)

N~ ~N His-54

A

/\ His-118 N~ 1..___0

Fe (I)

/o/\~"' G .... 115 c~ 0

o~l/OH' Asp-2""

c

/\

Fe (2)

_2<1 N~I~OGlU-238 B 0

GIu-204

His-273 N "-... ~ 0

His447

GkJ-l1.41

o

Glu-270

N~ I~OH2 Fe (I)

Fe (2)

His-322

H.,-246 N -------I "-... 0 Glu-243

D 0 Glu-209

Fe (I)

Fe (2)

N~~O C 0 ___ 1

c 11$-179

Fe (I)

Fe (2)

His-26S N -------I ~OH. E

o

23

Figure 2. Schematic representation of the primary ligation sphere of the catalytic centre of di-iron carboxylate proteins. A, hemerythrin; B, RNR R2; C, alternative oxidase; D, MMO; and E, stearoyl-ACP 119

desaturase.

24 Chapter 2

RNR R2 (lrib) were downloaded from the Brookhaven Protein Database (http://www.pdb.bnI.gov/). The Macintosh-application Rasmac v2.6 (ftp://dcs.ed.ac.uk/pub/rasmol/) was used to display the 3D-active-site structure of both enzymes. In Figures 3 and 4 respectively, it can be seen that the di-iron centres in RNR R2 and MMO are both co-ordinated by side chains of amino-acids that reside within a 4-helix bundle. In di-iron carboxylate proteins this secondary structural motif is generally found to scaffold the iron atoms and to provide a buried hydrophobic environment suitable for reactive oxygen chemistry (Nordlund and Eklund, 1995).

To model the 3D-structure of the active site of the alternative oxidase the amino-acid sequence of the Sauromatum guttatum protein was aligned with the sequence of RNR R2 as previously described (Siedow et aI., 1995). The PDB-file of RNR R2 was subsequently used as a template for modelling procedures carried out within the Swiss PDB­viewer v2.0 application (http://expasy.hcuge.ch/swissmod/Swiss­PdbViewer/mainpage.html). Firstly, the sequence segments representing the four helices which harbour the di-iron centre in RNR R2 (Nordlund et aI., 1990) were truncated to the length of the segments representing the four proposed, relatively short, helices in the alternative oxidase. Secondly, the remaining amino-acid residues were appropriately mutated to the residues of the alternative oxidase sequence (see Moore et aI., 1995; Siedow et aI., 1995).

As stated previously, a characteristic feature of all known di-iron carboxylate proteins is that the active site is buried within a four a­helical bundle. It is therefore a prerequisite that the part of the primary alternative oxidase structure which is proposed to form the active site, folds into a-helices. In general, the secondary protein structure can be predicted adequately from the primary structure by investigating the possible conformations of the backbone of a protein. The backbone conformation of a protein can be described by pairs of dihedral angles, psi ('I') and phi (<1», with one pair per amino-acid residue. A repetition of similar '1'-<1> pairs along a protein chain produces regular elements of protein secondary structure and a Ramachandran plot in which the consecutive pairs of dihedral angles are graphically represented provides information with respect to the secondary protein structure. The Ramachandran plots shown in Figure 5 reveal that modelling the active site of the alternative oxidase based on the structure of RNR R2 results in a protein with a a-helical backbone conformation as the pairs of dihedral angles of the model are close to the theoretical a-helix combination (<1> = -5T, 'I' = -4T) and are indeed more or less identical to the comparable values of both RNR R2 and MMO. However, it should be noted that a a-helical backbone conformation does not necessarily result in a four-helix bundle.

2. Structure-jimction relationships of the alternative oxidase 25

___ H11B

"-... -..-H241

Figure 3. Three-dimensional view of the active site of RNR R2. The 4-helix bundle is shown in a backbone-representation. Amino acid residues providing coordination sites for the iron atoms are shown fully, i.e. backbone + side chain. The two iron atoms and bridging oxygen atom are shown as spheres with convenient radii which are smaller than the true Van der Waals radii. Coordinates for the 3D-structure as published by Nordlund et al. (1990) were obtained from the Brookhaven protein database (http://www .pdb.bnl.gov/). Visuali sation was achieved with the Rasmac v2.5 application which was run on a Macintosh Perform a 630 computer.

26 Chapter 2

Figure 4. Three-dimensional view of the active site of MMO. The 4-helix bundle is shown in a backbone-representation. Amino acid residues providing coordination sites for the iron atoms are shown fully, i.e. backbone + side chain. The two iron atoms are shown as spheres with convenient radii which are smaller than the true Van der Waals radii. Co-ordinates for the 3D-structure, as published by Rosenzweig et al. (1993), were obtained from the Brookhaven Protein Database (http ://www.pdb.bnl.gov/). Visualisation was achieved with the Rasmac v2.S application which was run on a Macintosh Performa 630 computer.

2. Structure-function relationships of the alternative oxidase 27

A B C

·180 ·120 .6<) 60 no 180 ·lHO ·120 ·60 60 121) 180 ·180 ·120 ·60 60 no 180 180 180

120 120

60 60

IJI 0

·60 . :.~. . :.~ .. ~ .

·60

·120 ·120

·180 ·180 ·180 ·120 ·60 60 120 180 ·180 ·120 ·60 60 120 180 ·180 ·120 ·60 60 120 180

$ $ $

Figure 5. Ramachandran plots showing pairs of dihedral angles ('V, 1jI) of the amino acid sequences forming 4-helix bundles The plots illustrate the pairs of dihedral angles of the amino-acid sequences forming 4-helix bundles in A, RNR R2; B, MMO; and C, the predicted (and modelled) 4-helix bundle of the alternative oxidase. Template for the model is the 3D-structure of RNR R2 as published by Nordlund et al. (1990) co-ordinates being obtained from the Brookhaven protein database (http://www.pdb.bnl.gov/). For modelling manipulations (see text for details) as well as for the production of the Ramachandran plots the Swiss-PDBViewer v2.0 application was used on a Macintosh Performa 630 computer.

28 Chapter 2

When the 3D-model of the active site of the alternative oxidase is visualised with Rasmac v2.6 (Figure 6) it is clear that the primary structure of the alternative oxidase can theoretically fold into a four­helical element of tertiary structure (turns are not shown). Interestingly, the model reveals that in this particular conformation the two iron atoms are co-ordinated by amino-acid residues from three helices only (helices 1, 2 and 4). Helix 3 (Moore et aI., 1995) appears not to be involved in ligating the iron-atoms as it appears to be too remote from the active site. However, this helix could still play an important role in the 'hydrophobic pocket' of the di-iron centre (Moore et aI., 1995; Siedowet aI., 1995). In Figures 6 and 7, in which a close-up of the modelled di-iron centre is presented, it can be seen that the two histidine residues (H273 and H322) and three carboxylates (E270, E319 and D283) are suitably orientated to co-ordinate the two iron atoms in this model. Further residues to co-ordinate the di-iron centre may be provided by water molecules (not shown in the model). This relatively open coordination of the alternative oxidase di-iron centre possibly contributes to the flexibility of the catalytic site which is also observed in other di-iron proteins (Nordlund and Eklund, 1995).

4. STRUCfURE-FUNCTION RELATIONSHIPS

From the preceding section on molecular modelling it is apparent that the conserved residues within the alternative oxidase sequence readily fit the model that the active site of the alternative oxidase contains a binuclear iron centre held within a four-helix bundle. Furthermore comparison with the active sites of MMO and RNR R2 indicates that the active site of the oxidase has many more free iron coordination sites available for di-oxygen binding than other di-iron carboxylates. For instance hemerythrin has only one free coordination site for di-oxygen binding (Holmes et aI., 1991) which, according to (Nordlund and Eklund, 1995), may explain why this protein carries di-oxygen rather than cleaving it. In contrast, both MMO and RNR R2 in the reduced forms have numerous free coordination sites available for di-oxygen binding (Nordlund and Eklund, 1995). Thus free coordination sites may be a prerequisite for di-oxygen cleavage. Although the X-ray crystal structures for both of these proteins are available (Nordlund et aI., 1990; Rosenzweig et aI., 1993), the mechanism of di-oxygen cleavage is still largely unknown although a number of models have been postulated. Whatever the mechanism may be (Nordlund and Eklund (1995), have suggested that the carboxylates within the active site play an important

2. Structure-function relationships of the alternative oxidase 29

D2B3

Figure 6. Modelled three-dimensional view of the active site of the alternative oxidase. The 4-helix bundle is shown in a backbone-representation. Amino acid residues providing coordination sites for the iron atoms are shown fully, i.e. backbone + sidechain. The two iron atoms and bridging oxygen atom are shown as spheres with convenient radii which are smaller than the true Van der Waals radii. Template for the model is the 3D-structure ofRNR R2 as published by Nordlund et al. (1990). Modelling manipulations (see text for details) were performed with Swiss-PDBViewer v2.0 and subsequent visualisation was achieved with Rasmac v2.5. Both applications were run on a Macintosh Performa 630 computer.

30 Chapter 2

Figure 7. Modelled three-dimensional view of the di-iron centre in the alternative oxidase. The amino acid residues providing coordination sites for the iron atoms are shown as sticks. The two iron atoms and bridging oxygen atom are shown as spheres with convenient radii which are smaller than the true Van der Waals radii. Template for the model is the 3D-structure of RNR R2 as published by Nordlund et al. (1990). Modelling manipulations (see text for details) were performed with Swiss-PDBViewer v2.0 and subsequent visualisation was achieved with Rasmac v2.5. Both applications were run on a Macintosh Performa 630 computer.

2. Structure-function relationships of the alternative oxidase 31

role in facilitating di-oxygen cleavage not only by causing the protonation and subsequent loss of the bridging oxo group (thereby increasing the available coordination sites) but also by stabilising high valent intermediates that are probably required for the cleavage reaction. Given that the alternative oxidase reduces oxygen through to water and on the basis that its active site contains a binuclear iron centre, it is probable that the reaction mechanism for the cleavage of di-oxygen will be similar to other di-iron carboxylate proteins. Insight into how this occurs in these proteins will provide clues as to the mechanism operating in the alternative oxidase.

Since oxygen reduction to water is efficiently achieved by haem containing proteins the question arises as to why the alternative oxidase is a di-iron carboxylate protein. Although the answer to this question is not immediately forthcoming it may be related to the physiological function of the alternative oxidase. In general, thermogenesis is considered to be one of the primary functions of the alternative oxidase. However, the dissipation of energy as heat is associated with specialised tissues for a specific purpose such as pollination. Since the alternative oxidase is widespread amongst the plant kingdom but the majority of plants are not thermogenic, the question arises as to what is its function in such plants (Day et ai., 1995; McIntosh, 1994; Moore and Siedow, 1991; Siedowand Umbach, 1995; Wagner and Krab, 1995). In general the amount of oxidase varies from plant species to plant species and also shows tissue specific expression (Kearns et ai., 1992). Alternative oxidase activity tends to be lower in non-green tissue and protein levels can be increased by stress conditions such as wounding, chilling, drought and osmotic stress. Its major function has generally been considered to allow TeA cycle activity to continue under conditions where the main respiratory chain is restricted (i.e. by adenylate control) (Palmer, 1976) thereby acting as an energy overflow mechanism (Lambers, 1985). More recently, however, it has been suggested that instead of energy overflow being one of its primary functions perhaps a more plausible function is in the prevention of oxidative stress (Day et ai., 1995; Purvis and Shewfelt, 1993; Purvis et ai., 1995; Wagner, 1995; Wagner and Moore, 1997). This is based upon the observation that situations that result in oxygen stress (such as wounding, chilling, osmotic stress) are, in the majority of cases accompanied by induction of alternative oxidase activity alongside restricted cytochrome pathway activity (Prasad et aI., 1994; Purvis and Shewfelt, 1993; Purvis et ai., 1995; Wagner and Moore, 1997). Respiratory engagement of the alternative oxidase would therefore maintain the ubiquinone pool (a major site of mitochondrial active oxygen species (Purvis et aI., 1995; Rich et aI., 1977b) relatively oxidised thereby decreasing superoxide production. Furthermore alternative

32 Chapter 2

oxidase activity would allow continued oxygen consumption to occur thereby maintaining the intracellular levels of this potentially dangerous toxin relatively low. A higher Km of the alternative oxidase for di-oxygen (Moore and Siedow, 1991), in comparison to cytochrome oxidase, also fits in with the idea of its function as a di-oxygen scavenger since this would suggest that, in general, engagement of the alternative pathway would only occur when the oxygen concentration rises to possibly harmful levels.

There are a number of observations within the literature that are consistent with a role of the oxidase in the oxygen defence mechanism such as induction of alternative oxidase gene expression upon treatment with H20 2 (Vanlerberghe and McIntosh, 1996; Wagner, 1995) or antimycin A (which increase the production of active-oxygen species) (Minagawa et aI., 1992; Vanlerberghe and McIntosh, 1992, 1994; Wagner et aI., 1992). The proposed structure and flexibility of the active site of the alternative oxidase fits well with its postulated role as a di­oxygen scavenger (Wagner and Moore, 1997) since such a structure (possessing multiple and flexible coordination sites to bind di-oxygen) is aptly suited to such role.

5. CONCLUSIONS

The model of the active site of the alternative oxidase has been postulated to contain a binuclear iron centre analogous to that found in other di-iron carboxylate proteins (Moore et aI., 1995; Siedow et aI., 1995) suggesting it is one of the newest members of this group of proteins. Within this class of proteins the active site is buried within a four-helix bundle with the majority of ligands being carboxylates. Ramachandran plots reveal that modelling the active site of the alternative oxidase based on the structure of either RNR R2 or MMO but using the plant alternative oxidase amino-acid sequence results in a protein with an a-helical backbone conformation comparable to that observed for the active site of RNR R2 or MMO. Such results are strongly indicative that the proposed model for the active site of the alternative oxidase is structurally viable. Modelling studies furthermore have revealed that the catalytic site of the alternative oxidase contains a high negative charge due to the carboxylate ligands and possesses a number of free coordination sites available for binding oxygen. Such a flexible structure would be advantageous to its postulated function as a di-oxygen scavenger since it would be able to bind oxygen under a variety of redox conditions.

2. Structure-function relationships of the alternative oxidase 33

ACKNOWLEDGEMENTS

The work described in this review was supported in part by the Biotechnology and Biological Sciences Research Council and the Royal Society. CA gratefully acknowledges a BBSRC studentship and ALM a Royal Society Leverhulme Trust Senior Research Fellowship (1995/96).

REFERENCES

Berthold, D.A and Siedow, J.N. (1993). Partial-purification of the cyanide-resistant alternative oxidase of skunk cabbage (Symplocarpus foetidus) mitochondria. Plant Physiology, 101, 113-119.

Chaudhuri, M. and Hill, G.C. (1996). Cloning, sequencing, and functional activity of the Trypanosoma brucei alternative oxidase. Molecular and Biochemical Parasitology, 83, 125-129.

Cruz-Hernandez, A and Gomez-Lim, M.A. (1995). Alternative oxidase from mango (Mangifera indica, L) is differentially regulated during fruit ripening. Planta, 197, 569-576.

Day, D.A., Dry, LB., Soole, K.L., Wiskich, 1T. and Moore, AL. (1991). Regulation of alternative pathway activity in plant-mitochondria - deviations from Q-pool behaviour during oxidation of NADH and quinols. Plant Physiology, 95, 948-953.

Day, D.A., Whelan, J., Millar, AH., Siedow, J.N. and Wiskich, J.T. (1995). Regulation of the alternative oxidase in plants and fungi. Australian Journal of Plant Physiology, 22, 497-509.

Dry, LB., Moore, AL., Day, D.A and Wiskich, J.T. (1989). Regulation of alternative pathway activity in plant-mitochondria - nonlinear relationship between electron flux and the redox poise of the quinone pool. Archives of Biochemistry and Biophysics, 273, 148-157.

Hiser, C., Kapranov, P. and McIntosh, L. (1996). Genetic-modification of respiratory capacity in potato. Plant Physiology, 110, 277-286.

Hoefuagel, M., Millar, AH., Wiskich, J.T. and Day, D.A. (1995). Cytochrome and alternative respiratory pathways compete for electrons in the presence of pyruvate in soybean mitochondria. Archives of Biochemistry and Biophysics, 318, 394-400.

Holmes, M.A, Letrong, I., Turley, S., Sieker, L.C. and Stenkamp, R.E. (1991). Structures of deoxy and oxy hemerythrin at 2.0-A resolution. Journal of Molecular Biology, 218, 583-593.

Keams, A, Whelan, 1, Young, S., Elthon, T.E. and Day, D.A (1992). Tissue-specific expression of the alternative oxidase in soybean and siratro. Plant Physiology, 99, 712-717.

Krab, K. (1995). Kinetic and regulatory aspects of the function of the alternative oxidase in plant respiration. Journal of Bioenergetics and Biomembranes, 27, 387-396.

Kumar, A.M. and Soli, D. (1992). Arabidopsis alternative oxidase sustains Escherichia coli respiration. Proceedings of the National Academy of Sciences USA, 89, 10842-10846.

Lambers, H. (1985). Respiration in intact plants and tissues: its regulation and dependence upon environmental factors, metabolism and invaded organisms. In:

34 Chapter 2

Douce, R. and Day, D.A (Eds). Encyclopedia of Plant Physiology, New Series (pp. 418-473). Springer-Verlag, Berlin.

Li, Q.H., Ritzel, R.G., Mclean, L., Mcintosh, L., Ko, T., Bertrand, H. and Nargang, F.E. (1996). Cloning and analysis of the alternative oxidase gene of Neurospora crassa. Genetics, 142, 129-140.

McIntosh, L. (1994). Molecular biology of the alternative oxidase. Plant Physiology, 105, 781-786.

Millar, AH., Wiskich, IT., Whelan, J. and Day, D.A. (1993). Organic acid activation of the alternative oxidase of plant mitochondria. FEBS Letters, 329, 259-262.

Minagawa, N., Sakajo, S., Komiyama, T. and Yoshimoto, A (1990). Essential role of ferrous iron in cyanide-resistant respiration in Hansenula anomala. FEBS Letters, 267, 114-116.

Moore, AL., Bonner, W.DJr and Rich, P.R. (1978). The determination of the protonmotive force during cyanide insensitive respiration in plant mitochondria. Archives of Biochemistry and Biophysics, 186, 298-306.

Moore, AL., Dry, I.B. and Wiskich, J.T. (1988). Measurement of the redox state of the Ubiquinone pool in plant mitochondria. FEBS Letters, 235, 76-80.

Moore, AL. and Siedow, J.N. (1991). The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria. Biochimica et Biophysica Acta, 1059, 121-140.

Moore, AL., Umbach, A.L. and Siedow, J.N. (1995). Structure-function relationships of the alternative oxidase of plant mitochondria - a model of the active site. Journal of Bioenergetics and Biomembranes, 27, 367-377.

Nordlund, P. and Eklund, H. (1995). Di-iron-carboxylate proteins. Current Opinion in Structural Biology, 5, 758-766.

Nordlund, P., Sjoberg, B.M. and Eklund, H. (1990). 3-dimensional structure of the free­radical protein of ribonucleotide reductase. Nature, 345, 593-598.

Palmer, J.M (1976) The organisation and regulation of electron transport in plant mitochondria. Annual Review of Plant Physiology, 27, 133-157.

Prasad, T.K., Anderson, M.D. and Stewart, C.R. (1994). Acclimation, hydrogen-peroxide, and abscisic-acid protect mitochondria against irreversible chilling injury in maize seedlings. Plant Physiology, 105, 619-627.

Purvis, AC. and Shewfelt, R.L. (1993). Does the alternative pathway ameliorate chilling injury in sensitive plant tissues? Physiologia Plantarum, 88, 712-718.

Purvis, AC., Shewfelt, R.L. and Gegogeine, lW. (1995). Superoxide production by mitochondria isolated from green bell pepper fruit. Physiologia Plantarum, 94, 743-749.

Rasmusson, AG., M0l1er, 1.M. and Palmer, J.M. (1990). Component of the alternative oxidase localized to the matrix surface of the inner membrane of plant mitochondria. FEBS Letters, 259, 311-314.

Rhoads, D.M. and McIntosh, L. (1991). Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (schott). Proceedings of the National Academy of Sciences USA, 88, 2122-2126.

Ribas-Carbo, M., Wiskich, IT., Berry, J.A. and Siedow, IN. (1995). Ubiquinone redox behaviour in plant mitochondria during electron transport. Archives of Biochemistry and Biophysics, 317, 156-160.

Rich, P.R., Moore, AL., Ingledew, l1. and Bonner, W.D. Jr (1977a). EPR studies of higher plant mitochondria. 1. Ubisemiquinone and its relation to alternative respiratory oxidations. Biochimica et Biophysica Acta, 462, 501-514.

2. Structure-function relationships of the alternative oxidase 35

Rich, P.R., Bonner, W.O. Jr and Moore, AL. (1977b). The possible site ofsuperoxide anion and hydrogen peroxide generation in mammalian and higher plant mitochondria. Biophysical Journal, 17, 253A

Rosenzweig, AC., Frederick, C.A, Lippard, S.J. and Nordlund, P. (1993). Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane. Nature, 366, 537-543.

Sakajo, S., Minagawa, N. and Yoshimoto, A (1993). Characterization of the alternative oxidase protein in the yeast Hansenula anomala. FEBS Letters, 318, 310-312.

Siedow, J.N., Whelan, J., Kearns, A, Wiskich, J.T. and Day, D.A. (1992). Topology of the alternative oxidase in soybean mitochondria. In: Lambers, H. and van der Plas, L.H.W. (Eds). Molecular, Biochemical and Physiological Aspects of Plant Respiration (pp., 19-27). SPB Academic Publishing bv, The Hague.

Siedow, J.N. and Moore, AL. (1993). A kinetic model for the regulation of electron transfer through the cyanide-resistant pathway in plant-mitochondria. Biochimica et Biophysica Acta, 1142, 165-174.

Siedow, J.N. and Umbach, A.L. (1995). Plant mitochondrial electron transfer and molecular-biology. Plant Cell, 7, 821-831.

Siedow, J.N., Umbach, AL. and Moore, AL. (1995). The active site of the cyanide­resistant oxidase from plant mitochondria contains a binuclear iron center. FEBS Letters, 362, 10-14.

Umbach, AL. and Siedow, J.N. (1993). Covalent and noncovalent dimers of the cyanide­resistant alternative oxidase protein in higher-plant mitochondria and their relationship to enzyme-activity. Plant Physiology, 103, 845-854.

Umbach, AL. and Siedow, J.N. (1996). The reaction of the soybean cotyledon mitochondrial cyanide-resistant oxidase with sulfuydryl reagents suggests that alpha­keto acid activation involves the formation of a thiohemiacetal. Journal of Biological Chemistry, 271, 25019-25026.

Umbach, AL., Wi skich, J.T. and Siedow, J.N. (1994). Regulation of alternative oxidase kinetics by pyruvate and intermolecular disulfide bond redox status in soybean seedling mitochondria. FEBS Letters, 348, 181-184.

Van den Bergen, C.W.M., Wagner, AM., Krab, K. and Moore, AL. (1994). The relationship between electron flux and the redox poise of the quinone pool in plant mitochondria - interplay between quinol- oxidizing and quinone-reducing pathways. European Journal of Biochemistry, 226, 1071-1078.

Vanlerberghe, G.C. and McIntosh, L. (1992). Coordinate regulation of cytochrome and alternative respiration in tobacco. Plant Physiology, 100, 1846-1851.

Vanlerberghe, G.c. and McIntosh, L. (1994). Mitochondrial electron transport regulation of nuclear gene expression - studies with the alternative oxidase gene of tobacco. Plant Physiology, 105, 867-874.

Vanlerberghe, G.C. and Mcintosh, L. (1996). Signals regUlating the expression of the nuclear gene encoding alternative oxidase of plant mitochondria. Plant Physiology, Ill, 589-595.

Wagner, AM. (1995). A role for active oxygen species as 2nd messengers in the induction of alternative oxidase gene expression in Petunia hybrida cells. FEBS Letters, 368, 339-342.

Wagner, AM. and Krab, K. (1995). The alternative respiration pathway in plants - role and regulation. Physiologia Plantarum, 95, 318-325.

Wagner, AM. and Moore, AL. (1997). Structure and function of the plant alternative oxidase: its putative role in the oxygen defence mechanism. Bioscience Reports, 17, 319-333.

36 Chapter 2

Whelan, J., Smith, M.K., Meijer, M., Yu, J.W., Badger, M.R., Price, G.D. and Day, D.A. (1995). Cloning of an additional cDNA for the alternative oxidase in tobacco. Plant Physiology, 107, 1469-1470.

Whitehouse, D.G. and Moore, A.L. (1995). Regulation of oxidative phosphorylation in plant mitochondria. In: Levings III, C.S. and Vasil, I.K. (Eds). The Molecular Biology of Plant Mitochondria (pp. 313-344). Kluwer Academic Publishers, Dordecht.

Chapter 3

The many-faceted function of phosphoenolpyruvate carboxykinase in plants

Richard C. Leegood, Richard M. Acheson, Laszlo I. Tecsi and Robert P. Walker Robert Hill Institute and Department of Animal and Plant Sciences, University of Sheffield, Sheffield SIO 2 TN, UK

Key words: C4 photosynthesis; Crassulacean Acid Metabolism; cucurbits; gluconeogenesis; phloem transport, phosphoenolpyruvate carboxykinase; protein phosphorylation.

Abstract: Phosphoenolpyruvate carboxykinase (PEPCK) undergoes light- or diurnally-regulated changes in phosphorylation in C3 and CAM plants but only in some C4 plants. The characteristics of this phosphorylation of PEPCK and its possible regulatory significance are discussed. The molecular mass of PEPCK from C4 plants is often smaller, and more variable, than the molecular mass of the enzyme in C3 and CAM plants. These differences probably reflect differences in the size of the N-terminal extension found in the enzyme from higher plants. This N-terminal extension contains the phosphorylation site and it is readily cleaved following extraction. PEPCK has four well-defined roles in plants: in photosynthesis in C4 and CAM plants, in the CO2-concentrating mechanism of certain algae and in gluconeogenesis following germination of fat-storing seedlings, but it has now also been located in the trichomes and in the phloem of some plants, such as cucurbits, suggesting a larger number of roles in plant metabolism than has hitherto been recognised. These possible roles are discussed, and in particular we discuss the general role of the phloem elements in cucurbits.

37

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 37-51. © 1999 Kluwer Academic Publishers.

38 ~~~3

1. ~ODUC1ITON

Although phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.49) catalyses a reversible reaction:

oxaloacetate + ATP H PEP + ADP + CO2

the direction of the reaction in vivo is generally the reverse of the reaction catalysed by PEP carboxylase, i.e. PEPCK usually acts as a decarboxylase (Ray and Black, 1976; Urbina and Avilan, 1989). Since, like PEP carboxylase, PEPCK is a cytosolic enzyme (Chapman and Hatch, 1981; Ku et aI., 1980; Watanabe et aI., 1984), the two enzymes constitute a potentially futile cycle which requires control if it is not simply to hydrolyse A TP. The importance of these two reactions in plants is that they playa key role in mediating the interconversion of glycolytic intermediates and organic acids and both may play anaplerotic roles in metabolism. In, 1983 Latzko and Kelly wrote a review entitled 'The many-faceted function of phosphoenolpyruvate carboxylase in plants' (Latzko and Kelly, 1983) which addressed the wide range of roles then proposed for PEP carboxylase in plant tissues. Recent research is revealing that PEPCK may similarly play a large number of different roles in plant tissues. For example, PEPCK is involved in C4

photosynthesis, in which it decarboxylates C4 acids in one sub-group of C4 plants (Hatch and Osmond, 1976), it is involved in Crassulacean Acid Metabolism, in which it decarboxylates C4 acids in some CAM plants (Leegood et aI., 1996), it is involved in gluconeogenesis in germinating seeds (Lee good and ap Rees, 1978) and it plays a role in the COz­concentrating mechanism of some algae (Reiskind and Bowes, 1991). Our own recent studies have also shown that PEPCK is present in appreciable amounts in the phloem and the trichomes of some plants, such as cucumber. Like PEP carboxylase and a number of other enzymes in plant tissues, PEPCK is also regulated by phosphorylation.

In this article we explore these multiple roles for PEPCK in plant tissues and review the possible significance of phosphorylation in regulating the activity of the enzyme.

2. FACTORSAFFECflNGPEPCKAFfER EXTRACTION

Many enzymes are susceptible to inactivation or modification during or after extraction from plant tissues, resulting in changes in regulatory

3. Phosphoenolpyntvate carboxykinase 39

properties. Although an appreciable number of studies of PEPCK have been made over the past 30 years, it is only recently that two particular problems have come to light which profoundly affect the activity and properties of PEPCK following extraction.

The first is that activity is rapidly lost unless thiols, such as dithiothreitol (OTT), are present in the extraction medium at high concentrations (a concentration as high as 50 mM OTT is desirable), although physiological concentrations of adenylates (A TP and AOP) can prevent loss of activity in the absence of thiols (Walker et aI., 1997). These observations suggest that loss of activity involves the oxidation of thiol and vicinal dithiol groups close to the active site, as has been observed in the yeast enzyme (which shows considerable homology with the plant enzyme) (Cardemil et aI., 1990) and in the enzyme from rat liver (in which the active site is similar to A TP-dependent PEPCKs (Carlson et aI., 1978; Kim and Smith, 1994». Thus it is possible that some previous estimates of the activity of the enzyme in crude extracts may be erroneous.

The second important feature is the rapid proteolytic cleavage of PEPCK which occurs in crude extracts. There are a number of examples of enzymes in plants, such as AOPglucose pyrophosphorylase (Kleczkowski et aI., 1993) and PEP carboxylase (Baur et aI., 1992), where proteolytic cleavage readily occurs following extraction. While such cleavage does not affect V max' it does affect regulatory properties such as sensitivity to effectors (e.g. malate sensitivity in the case of PEP carboxylase). Like these enzymes, PEPCK is rapidly cleaved in extracts of all higher plants studied (Walker et aI., 1995, 1997; Walker and Leegood, 1996b) but proteolysis of PEPCK does not appear to affect Vmax (Walker et aI., 1995; Walker and Leegood, 1996b). Amino acid sequencing has shown that proteolysis in extracts of leaves of the C4

plant, Urochloa panicoides involves cleavage of the N-terminal sequence (Finnegan and Burnell, 1995), which appears to be unique to PEPCK from higher plants, although the remaining part of the enzyme from cucumber and U panico ides is closely related to the enzymes from yeast, Rhizobium, Escherichia coli and, somewhat curiously, trypanosomes (Finnegan and Burnell, 1995; Kim and Smith, 1994; Walker and Leegood, 1995, 1996a).

The molecular mass of PEPCK varies between different plants. In gluconeogenic seedlings and most CAM plants the molecular mass is 74 kOa, but in some CAM plants (one group of Tillandsia) the molecular mass is 78 kDa (Walker and Leegood, 1996b), while in C4 plants, PEPCK is often smaller and the molecular mass ranges between 68 and 74 kOa (Walker and Leegood, 1996b; Walker et aI., 1997). For PEPCK from both cucumber and U pan ico ides, the difference in size between the

40 Chapter 3

intact and cleaved polypeptides is similar to the size of the N-terminal extension deduced from the cDNA sequence. In addition, proteolytic cleavage of PEPCK from a range of plants always yields a 62 kDa polypeptide. These observations suggest that the differences in molecular mass between PEPCK from different plants reflect the size of the N­terminal extension, which has presumably been modified in some plants to suit its particular role.

The phenomenon of rapid proteolysis of PEPCK during extraction means that it is difficult to purify the intact form of the enzyme from any higher plant. Thus a number of protease inhibitors were found to be ineffective in suppressing proteolysis of PEPCK in crude extracts of the leaves of C3 and C4 plants (results not shown), but alternative procedures have proved effective, notably extraction at alkaline pH (between pH 9.0 and pH 10.5) which appears to inactivate the protease. Protease activity also appears to be thiol-dependent (Walker et ai., 1997). Procedures based on extraction at alkaline pH have permitted purification of intact PEPCK from both cucumber cotyledons (Walker and Leegood, 1995) and from the leaves of the C4 plant, Panicum maximum (results not shown).

3. PHOSPHORYLATION OF PEPCK

PEPCK from higher plants was first cloned from cucumber by Kim and Smith (1994). The predicted amino acid sequence of PEPCK revealed a sequence in the N-terminal region (residues 64-68, Lys-Lys-Arg-Ser­Thr) which is recognised by cAMP-dependent protein kinases. The presence of this site raised the possibility that the intact form of PEPCK might be phosphorylated on either the serine or threonine residue in vivo and that phosphorylation might affect the, regulatory properties of the enzyme. Accordingly, incubation of the purified intact enzyme from cucumber with [y-32p]A TP and mammalian cAMP-dependent protein kinase led to incorporation of 32p into a part of the polypeptide which was cleaved during proteolysis and which was separate from the active site. This was reversed by incubation with protein phosphatase 2A. When experiments were done in vivo by supplying cucumber cotyledons with 32p, PEPCK was one of five major labelled polypeptides in darkened cotyledons and this labelling was reversed by illumination (Walker and Leegood, 1995). PEPCK was phosphorylated in a wide range of gluconeogenic seedlings studied and in leaves of all PEPCK-type CAM plants studied (Walker and Leegood, 1996b). In CAM plants, as in cucumber cotyledons, phosphorylation of PEPCK occurred at night and dephosphorylation occurred during the day, although it may be controlled

3. Phosphoenolpyruvate carboxykinase 41

by a circadian rhythm, rather than by light per se (Walker and Leegood, 1996b).

In C4 grasses the situation is more complex because the capacity to phosphorylate PEPCK appears to have been lost from many of them, with accompanying changes in the structure of the N-terminal sequence. The molecular mass of PEPCK varies considerably in C4 plants (Walker and Leegood, 1996b). PEPCK is reversibly phosphorylated during darkness in some C4 plants with PEPCK of a larger molecular mass, such as Panicum maximum or Spartina anglica (71 kDa), but it was not phosphorylated in the PEPCK-type C4 plants, Sporobolus pyramidalis (69 kDa) or Urochloa panicoides (68kDa) (Walker and Leegood, 1996b. Walker et ai., 1997). Moreover, the sequence of PEPCK in U. panico ides lacks the putative phosphorylation site present in cucumber (Walker and Leegood, 1996a).

These findings suggest that there has been a change in the structure of PEPCK and in the role of phosphorylation in its regulation during the evolution of C4 photosynthesis. It is known that C4 plants evolved separately many times from C3 plants and that they did so quite recently, perhaps no longer than 7-8 million years ago (Ehleringer et ai., 1993). The reason why PEPCK is not phosphorylated in these plants can only be speculative, but may be related to the fact that PEP carboxylase and PEPCK are located in different photosynthetic cell types, the mesophyll and bundle-sheath, respectively, whereas in other plants both enzymes are located in the cytosol of the same cells. There is, therefore, no possibility of a futile cycle with PEP carboxylase. On the other hand, it could be that the rate of phosphorylation and dephosphorylation exerts some constraint on C4 photosynthesis in, for example, fluctuating light environments, which may be disadvantageous.

There are a number of biochemical variants of C4 photosynthesis which are based on the major decarboxylase present (NADP-malic enzyme, NAD-malic enzyme and PEPCKlNAD-malic enzyme) (Hatch, 1987), but recent evidence suggests that there may be considerable variation in metabolism even within defined C4 sub-groups, particularly in regard to the presence of PEPCK. Gutierrez et al. (1974) detected PEPCK activity in a number of NADP-malic enzyme type plants. A survey of a wide range of C4 species for the presence of PEPCK using immunoblots showed that a 74 kDa form of PEPCK is present in the leaves of a number of NADP-malic enzyme plants, including maize, Echinochloa colona, Digitaria sanguinalis, Echinochloa crus-galli, and Paspalum notatum. However, PEPCK was not present in the leaves of certain other NADP-malic enzyme type plants (sorghum, sugar cane or Flaveria bidentis) or in any NAD-ME type plants studied (Amaranthus edulis, Panicum miliaceum, Amaranthus retroflexus) (Walker et ai.,

42 Chapter 3

1997). In leaf sections of maize, PEPCK was exclusively located in the bundle-sheath cells and the activity of PEPCK was about 45% of that in leaves of a PEPCK-type C4 plant, Panicum maximum (Walker et aI., 1997). Despite being a large molecular mass form (74 kDa), PEPCK from maize was not appreciably phosphorylated in vivo (Walker et aI., 1997). Since it is present in the bundle-sheath, PEPCK may playa role in C4 acid decarboxylation, perhaps in the decarboxylation of aspartate (Chapman and Hatch, 1981).

Although phosphorylation of PEPCK appears to be widespread in C3

and CAM plants, and also occurs in some C4 plants, its significance remains elusive. The occurrence of phosphorylation in diverse tissues makes it seem unlikely that it regulates protein turnover (e.g. Burlini et aI., 1989). By analogy with other enzymes, it seems more likely that the N-terminal extension confers regulatory properties on PEPCK and that these properties are modulated by phosphorylation. A number of other enzymes have regulatory properties conferred on them by phosphorylation of a residue contained within a sequence of amino acids that is absent from enzymes with otherwise very similar sequences. For example, mammals possess cell-specific isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. This enzyme contains a highly conserved central core to which C- and N-terminal extensions have been added in the various isoforms. Some of these extensions contain phosphorylation sites which regulate the activity of the enzyme (Pilkis et aI., 1995). PEP carboxylase from plants has regulatory properties conferred on it by phosphorylation/dephosphorylation of a serine residue contained in a sequence of amino acids at the N-terminus, which are lacking in the bacterial enzyme (Huber et aI., 1994). PEP carboxylase rapidly loses the N-terminal region, and much of its sensitivity to inhibition by malate, following proteolysis in crude extracts (Baur et aI., 1992). However, we have not yet been able to show any differential effects of metabolites on the kinetic properties of PEPCK isolated from illuminated or darkened cucumber cotyledons.

Comparatively few metabolites have large effects on the activity of PEPCK, although a number of phosphorylated intermediates modulate the activity from U. panicoides (Burnell, 1986). However, it must be remembered that these effectors have been tested on the proteolytically cleaved form of the enzyme. Adenylates and the A TPI ADP ratio are likely to play a significant role in regulating the activity of PEPCK entirely through the effects of mass-action, since PEPCK catalyses a freely reversible reaction (Walker et aI., 1997; Wood et aI., 1966). Recent work suggests that the ATP/ADP ratio in the cytosol of plants increases several-fold upon illumination (Gardestrom, 1993). A further feature of PEPCK which may be of considerable regulatory significance is

3. Phosphoenolpyruvate carboxykinase 43

its sensitivity to metal ions. First, PEPCK has an absolute requirement for Mn2+. Previous studies of PEPCK have involved assay at unphysiological concentrations of Mn2+ (> 0.5 mM), whereas the concentration of Mn2+ in the cytosol of maize roots (the only plant tissue in which it has been measured with any degree of accuracy) is submicromolar (Quiquampoix et ai., 1993). Second, PEPCK is strongly inhibited by Mg2+ (Burnell, 1986) at concentrations which are in the physiological range (i.e. a few millimolar). The activity of PEPCK at physiological concentrations of Mn2+ and Mg2+ can therefore be, at most, a few percent of the maximum catalytic activity measured in crude extracts. This raises the question as to how PEPCK operates at appreciable rates in plants, whether or not there are factors which influence the sensitivity of PEPCK to metal ions and what the concentrations of Mn2+ and Mg2+ are in the cytosol of illuminated and darkened leaves. It may be that phosphorylation of PEPCK alters its sensitivity to these metal ions. Here, comparisons may be drawn between PEPCK and the regulation of nitrate reductase, whose sensitivity to inhibition by Mg2+ is brought about by an inactivating protein, now known to be of the 14-3-3 type (Huber et ai., 1996). Interestingly, the putative phosphorylation site of PEPCK (Kim and Smith, 1994; Walker and Leegood, 1996a) is contained within a motif (Lys-Lys-Arg-Ser-Thr­Pro) which may be recognised by 14-3-3 proteins (Muslin et ai., 1996).

4. PEPCK IN OTHER TISSUES

Studies of cucumber leaves and roots have revealed that these tissues contain very low, but detectable activities of PEPCK. For example, in a cucumber leaf the activity of PEPCK is 8.1 ± 0.8 and in the root 2.1 ± 0.3 Jlmol h-1 g-l FW, compared with an activity of 353.2 ± 8.4 Jlmol h-1 g-l FW in a cucumber cotyledon at the peak of gluconeogenesis. The presence of PEPCK in these tissues has been confirmed in Western blots (Kim and Smith, 1994). This raises the question as to whether PEPCK is uniformly distributed in these tissues or whether it is confined to specific cell types. Immunolocalisation of PEPCK in cucumber leaves has revealed two specific locations, the trichomes and the phloem (Figure 1). PEPCK is also found in the trichomes of tobacco leaves (Figure 1). The function of PEPCK in the trichomes is open to speculation, but one possibility is that it may be involved in the synthesis of antimicrobial secondary metabolites, providing PEP for the biosynthesis of aromatic compounds in the shikimate pathway. PEPCK is also present in the cells that line the endocarp within grape berries (as well as in the phloem) and

44

Figure 1. Immunolocalisation of PEPCK in cucumber and tobacco.

Chapter 3

.¥ef

I ~X

,

50 Jlm I-----l ,..... ..

A. The trichome of a cucumber leaf. B. A glandular trichome of a tobacco leaf. C. A major vein in the stem of a cucumber leaf. PEPCK is confined to the extra-fascicular (ef) phloem elements, but is not detectable in the internal (int) or external (ext) phloem elements or in the xylem (x). D. A minor vein in a fully developed cucumber cotyledon. On the left-hand­side a transverse section through a vein shows that PEPCK is located in the adaxial phloem (indicated by the arrow). On the right-hand-side, a minor vein has been cut in a glancing longitudinal section, again showing the presence of PEPCK in the adaxial (upper) phloem element (indicated by the arrow).

3. Phosphoenolpyruvate carboxykinase 45

in cells lining the secretory ducts of the leaves of Clusia spp (data not shown), where it might have a similar function. In cucumber, PEPCK is present in the phloem in the leaves, stems and roots, but any attempt to understand its function in the phloem tissues requires a knowledge of the structure and function of the phloem.

5. PHLOEM STRUCfURE AND FUNCTION IN CUCUMBER

Cucurbits, such as cucumber, have bicollateral vascular bundles (Esau, 1965). In the minor veins, the inner phloem elements have symplastic connections and are abaxial (below the vascular bundle) while the outer phloem elements have apoplastic connections and are adaxial (above the vascular bundle) (Turgeon and Hepler, 1989; Shaffer et aI., 1996). In intermediate and major veins there is also an internal and external phloem, although here the internal phloem has apoplastic as well as symplastic connections (Turgeon et aI., 1975), but there are also extra­fascicular phloem elements (Esau, 1965) in the leaves, petioles and stems which lie outside the vascular bundles and which are also inter-connected via commissural elements into a network which sheaths, and communicates with, the vascular bundle. Immunolocalisation of PEPCK in cucumber leaves and stems show that it is associated with the phloem in the adaxial phloem of minor veins, in the internal phloem of intermediate veins and throughout the extra-fascicular phloem elements (Figure 1). The structural and functional relationships between these phloem elements of the minor and major veins have yet to be established, but in minor veins of another cucurbit, melon, it has recently been shown that the abaxial phloem is involved in the synthesis and transport of sucrose and oligosaccharides (Haritatos et aI., 1996), a conclusion which is supported by both histochemistry (Pristupa, 1983) and autoradiography (Schmitz et aI., 1987). However, the adaxial phloem in minor veins does not appear to transport carbohydrates and its function remains a matter for speculation. This then raises questions about the function of the adaxial phloem and the extra-fascicular phloem and the role that PEPCK might play.

There are, however, clues to the function of the adaxial phloem in cucurbits, including circumstantial evidence which supports the hypothesis that the adaxial phloem is involved in the transport of amino acids in minor veins. First, as mentioned above, both histochemistry (Pristupa, 1983) and autoradiography (Schmitz et aI., 1987) indicate that the adaxial phloem does not appear to be involved in the transport of

46 Chapter 3

carbohydrates. Secondly, in melon (CuGumis melo), the amino acid composition of the phloem sap is markedly different from the leaf sap at certain times of the day. For example, at mid-day the mesophyll sap is enriched in aspartate, whereas the phloem sap is enriched in glutamine (Mitchell et aI., 1992) (it should be noted that these measurements would underestimate the actual differences between the mesophyll contents and the phloem sap). This contrasts with some other plants which have a phloem sap which is similar in composition to the mesophyll sap, as in spinach, barley and sugar beet (Lohaus et aI., 1994; Riens et aI., 1991; Winter et aI., 1992). However Schobert and Komor (1989) showed that the phloem loading system in castor bean seedlings has a different transport specificity from that of the amino acid uptake system of the cotyledon and that it strongly discriminates between amino acids within the cotyledons. If the amino acid composition of the phloem sap is markedly different from the leaf sap, this requires the selective transport of amino acids, which could not occur across the symplastic boundary of the abaxial phloem. It also requires that the necessary metabolism of amino acids, such as the conversion of aspartate to glutamine, does not occur in the bulk of the leaf, but must occur within the phloem elements themselves, or in other types of cell associated with the phloem, such as companion cells or parenchyma cells, before apoplastic loading into the sieve elements. Amino acid transporters associated with the plasma membrane are known to be present in the plasma membranes of cucurbits (Hsiang and Bush, 1992). In addition, cucurbits possess a number of compounds which are specifically involved in transport, such as arginine and citrulline, which derive from glutamate and glutamine. It seems likely that, rather than being made throughout the mesophyll, these compounds may be made in or around the vasculature in cells adjacent to the site of export from the leaf. A final piece of evidence in support of amino acid transport in the adaxial phloem comes from studies with squash. In this species sugars are present at a lower concentration in the phloem than in an apoplastic loader, such as castor bean, but the difference in the concentration of amino acids is much smaller (Hall and Baker, 1972; Richardson et aI., 1982). This suggests that a mechanism might exist to concentrate amino acids in the phloem.

It seems possible that the association of PEPCK with the phloem in cucumber reflects an involvement in amino acid metabolism. Amino acids in the mesophyll, such as aspartate, would need to be converted into transported amino acids. This requires the net conversion of oxaloacetate to glutamate (Figure 2) and would require either malic enzyme and/or PEPCK to act in an anaplerotic capacity to supply acetyl CoA for the reaction catalysed by citrate synthase. In this regard it is important to note that NADP-malic enzyme in cucumber is strongly associated with

3. Phosphoenolpyruvate carboxykinase

sugars aromatics

47

~ iV\:~ PEP malate acetyl CoA

aspartate

glutamine

PEPC~ / ----------~.~ OAA

Krebs cycle

citrulline I I proline ~ glutamate -+- 2-oxoglutarate arginine

citrate

Figure 2. Possible roles of PEP carboxykinase (PEPCK) and NADP-malic enzyme (ME) in phloem associated metabolism. In cucurbits, aspartate predominates in the leaf sap whereas glutamate (and its derivatives) predominate in the phloem sap. Interconversion of these compounds would require PEPCK and/or malic enzyme working in an anaplerotic capacity. Alternatively, PEPCK could be involved in generating PEP from amino acids in the phloem for gluconeogenesis or for the formation of aromatic compounds in the shikimate pathway.

48 Chapter 3

the vasculature (data not shown). In addition, cucurbits are not the only plants which have phloem-associated PEPCK and NADP-malic enzyme, and we have evidence for a similar pattern in grapevine and Coleus, both of which are symplastic loaders of sugars.

In conclusion, it appears that PEPCK in plants plays at least seven different roles, in C4 and CAM photosynthesis and in algal CO2

concentrating mechanisms, in gluconeogenesis in germinating seeds, in the trichomes and phloem of some plants and in specialised cells other than the phloem in grape berries and in Clusia. It seems quite possible that it is present in other cell types in other plants. Elucidating its various roles and unravelling the manner in which it is regulated in all these tissues is a major challenge for the next few years.

ACKNOWLEDGEMENTS

This research was supported by research grants (C05229 and PG501590 (Biochemistry of Metabolic Regulation in Plants)), and a studentship to RMA, from the Biotechnology and Biological Sciences Research Council, UK.

REFERENCES

Baur, B., Dietz, K.J. and Winter, K. (1992). Regulatory protein phosphorylation of phosphoenolpyruvate carboxylase in the facultative Crassulacean-acid metabolism plant Mesembryanthemum crystallinum L.. European Journal of Biochemistry, 209, 95-101.

Burlini, N., Morandi, S., Pellegrini, R., Tortora, P. and Guerritore, A (1989). Studies on the degradative mechanism of phosphoenolpyruvate carboxykinase from the yeast Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 1014, 153-161.

Burnell, J.N. (1986). Purification and properties of phosphoenolpyruvate carboxykinase from C4 plants. Australian Journal of Plant Physiology, 13, 577-587.

Cardemil, E., Encinas, M.V. and Jabalquinto, AM. (1990). Reactive sulfhydryl groups in Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase. Biochimica et Biophysica Acta, 1040, 71-76.

Carlson, G.M., Colombo, G. and Lardy, H.A (1978). A vicinal dithiol containing an essential cysteine in phosphoenolpyruvate carboxykinase (guanosine triphosphate) from cytosol of rat liver. Biochemistry 17, 5329-5338.

Chapman, K.S.R. and Hatch, M.D. (1981). Aspartate decarboxylation in bundle sheath cells of Zea mays and its possible contribution to C4 photosynthesis. Australian Journal of Plant Physiology, 8, 237-248.

Ehleringer, JR., Sage, R.F., Flanagan, L.B. and Pearcy, R.W. (1993). Climate change and the evolution of C4 photosynthesis. Trends in Ecology and Evolution, 6, 95-99.

Esau, K. (1965). Plant Anatomy (p. 271). Wiley, New York.

3. Phosphoenolpyruvate carboxykinase 49

Finnegan, P.M. and Burnell, J.N. (1995). Isolation and sequence analysis of cDNAs encoding phosphoenolpyruvate carboxykinase from the PCK-type C4 grass Urochloa panicoides. Plant Molecular Biology, 27, 365-376.

Gardestrom, P. (1993). Metabolite levels in the chloroplast and extrachloroplast compartments of barley leaf protoplasts during the initial phase of photosynthetic induction. Biochimica et Biophysica Acta, 1183, 327-332.

Gutierrez, M., Gracen, V.E. and Edwards, G.E. (1974). Biochemical and cytological relationships in C4 plants. Planta, 119, 279-300.

Hall, S.M. and Baker, D.A. (1972) The chemical composition of Ricinus phloem exudate. Planta, 106, 131-140.

Haritatos, E., Keller, F. and Turgeon, R. (1996). Raffinose oligosaccharide concentrations measured in individual cell and tissue types in Cucumis melD L. leaves: implications for phloem loading. Planta, 198, 614-622.

Hatch, M.D. (1987). C4 photosynthesis: a unique blend of modified biochemistry, anatomy and ultrastructure. Biochimica et Biophysica Acta, 895, 81-106.

Hatch, M.D. and Osmond, e.B. (1976). Compartmentation and transport in C4 photosynthesis. In: Stocking, C.R. and Heber, U. (Eds). Encyclopedia of Plant Physiology (Vol. 3, pp 144-184). Springer-Verlag, Berlin.

Hsiang, B.C.H. and Bush, D.R. (1992). Stachyose, amino acid, and sucrose transport in plasma membrane vesicles isolated from zucchini. Plant Physiology, 99, 242S.

Huber, S.C., Huber, J.L. and McMichael, R.W. (1994). Control of plant enzyme activity by reversible protein phosphorylation. International Review of Cytology, 149, 47-98.

Huber, S.C., Bachmann, M. and Huber, J.L. (1996). Post-translational regulation of nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins. Trends in Plant Science, 1, 432-438.

Kim, D-J. and Smith, S.M. (1994). Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression. Plant Molecular Biology, 26, 423-434.

Kleczkowski, L.A., Villand, P., Luthi, E., Olsen, O.A. and Preiss, J. (1993). Insensitivity of barley endopserm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation. Plant Physiology, 101, 179-186.

Ku, M.S.B., Spalding, M.H. and Edwards, G.E. (1980). Intracellular localization of phosphoenolpyruvate carboxy kinase in leaves of C4 and CAM plants. Plant Science Letters, 19, 1-8.

Latzko, E. and Kelly, G.J. (1983). The many-faceted function of phosphoenolpyruvate carboxylase in C3 plants. Physiologie Vegetale, 21, 805-813.

Leegood, R.e., von Caemmerer, S. and Osmond, C.B. (1996) Metabolite transport and photosynthetic regulation in C4 and CAM plants. In: Dennis, D.T., Turpin, D.H., Layzell, D.D. and Lefebvre D.K. (Eds). Plant metabolism (pp. 341-369). Longman, London.

Leegood R.C. and ap Rees, T. (1978). Phosphoenolpyruvate carboxykinase and gluconeogenesis in cotyledons of Cucurbita pepo. Biochimica et Biophysica Acta, 524, 207-218.

Lohaus, G., Burba, M. and Heldt, H.W. (1994). Comparison of the contents of sucrose and amino acids in the leaves, phloem sap and taproots of high and low sugar-producing hybrids of sugar beet (Beta vulgaris L.). Journal of Experimental Botany, 45, 1097-1l0I.

Mitchell, D.E., Gadus, M.V. and Madore, M.A. (1992). Patterns of assimilate production and translocation in muskmelon (Cucumis melD L.). Plant Physiology, 99, 959-965.

50 Chapter 3

Muslin, A.J., Tanner, W.J., Allen, P.M. and Shaw, A.S. (1996) Interaction of 14-3-3 with signalling proteins is mediated by the recognition of phosphoserine. Cell, 84, 889-897.

Pilkis, S.1., Claus, T.H., Kurland, U. and Lange, A.1. (1995) 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: a metabolic signalling enzyme. Annual Review of Biochemistry, 64, 799-835.

Pristupa. N.A. (1983) Distribution of keto sugars among cells of conducting bundles of the Cucurbita pepo leaf. Fiziol Rastenii, 30, 492-498

Quiquampoix, H., Loughman, B.C. and Ratcliffe, R. G. (1993). A 31p_NMR study of the uptake and compartmentation of manganese by maize roots. Journal of Experimental Botany, 44, 1819-1827.

Ray, T.B. and Black, C.c. Jr. (1976) Characterization of phosphoenolpyruvate carboxykinase from Panicum maxicum. Plant Physiology, 58, 603-607.

Reiskind, J.B. and Bowes, G. (1991). The role of phosphoenolpyruvate carboxykinase in a marine macroalga with C4-like photosynthetic characteristics. Proceedings of the National Academy of Sciences USA, 88, 2883-2887.

Richardson, P.T., Baker, D.A. and Ho, L.c. (1982) The chemical composition of cucurbit vascular exudates. Journal of Experimental Botany, 33, 1239-1247.

Riens, B., Lohaus, G., Heineke, D. and Heldt, H. W. (1991). Amino acid and sucrose content determined in the cytosolic, chloroplastic and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiology, 97, 227-233.

Schaffer, A.A., Pharr, D.M. and Madore, M. (1996). Cucurbits. In Zamski, E. and Schaffer, A.A. (Eds). Photoassimilate distribution in plants and crops. Source-sink relationships (pp. 729-757). Marcel Dekker. Inc., New York.

Schmitz, K. Cuypers, B. and Moll, M. (1987). Pathway of assimilate transfer between mesophyll cells and minor veins in leaves of Cucumis melo L. Planta, 171, 19-29.

Schobert, C. and Komor, E. (1989). The differential transport of amino acids into the phloem of Ricinus communis L. seedlings as shown by the analysis of sieve-tube sap. Planta, 177, 342-349.

Turgeon, R. and Hepler, P.K. (1989). Symplastic continuity between mesophyll and companion cells in minor veins of mature Cucurbita pepo L. leaves. Planta, 179, 24-31.

Turgeon, R., Webb, J.A. and Evert, R.F. (1975) Ultrastructure of minor veins of Cucurbita pepo leaves. Protoplasma, 83, 217-232.

Urbina, J.A. and Avilan, L. (1989). The kinetic mechanism of phosphoenolpyruvate carboxykinase from Panicum maximum. Phytochemistry, 28, 1349-1353.

Walker, R.P. and Leegood, R.C. (1995). Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons. FEBS Letters, 362, 70-74.

Walker, R.P. and Leegood, R.C. (1996a). Regulation of phosphoenolpyruvate carboxykinase activity in plants. In Mathis, P. (Ed). Current Research in Photosynthesis (Proceedings of 10th International Congress on Photosynthesis) (Vol. 5, pp. 29-34), Kluwer Academic Publishers, Dordrecht.

Walker, R.P. and Leegood, R.C. (1996b). Phosphorylation of phosphoenolpyruvate carboxykinase in plants. Studies in plants with C4 photosynthesis and Crassulacean Acid Metabolism and in germinating seeds. Biochemical Journal, 317, 653-658.

Walker, R.P., Trevanion, S.1. and Leegood, R.C. (1995). Phosphoenolpyruvate carboxy kinase from higher plants: purification from cucumber and evidence of rapid proteolytic cleavage in extracts from a range of plant tissues. Planta, 195, 58-63.

3. Phosphoenolpyruvate carboxykinase 51

Walker, R.P., Acheson, R.M., Tecsi, L.I. and Leegood, R.C. (1997). Phosphoenolpyruvate carboxykinase in C4 plants: Its role and regulation. Australian Journal of Plant Physiology, 24, 459-468.

Watanabe, M., Ohnishi, J. and Kanai, R. (1984). Intracellular localization of phosphoenolpyruvate carboxykinase in bundle sheath cells of C4 plants. Plant and Cell Physiology, 25, 69-76.

Wood, H.G., Davis, J.J. and Lochmiiller, H. (1966). The equilibria of reactions catalyzed by carboxytransphosphorylase, carboxykinase and pyruvate carboxylase and the synthesis of phosphoenolpyruvate. Journal of Biological Chemistry, 241, 5692-5704.

Winter, H., Lohaus, G. and Heldt, H.W. (1992). Phloem transport of amino acids in relation to their cytosolic levels in barley leaves. Plant Physiology, 99, 996-1004.

Chapter 4

Folate synthesis and compartmentation in higher plants

Fabrice Rebeille and Roland Douce Laboratoire de Physiologie Cellulaire Vegetale, C.N.R.S. URA No 576, Departement de Biologie Moleculaire et Structurale, C.E.A.-Grenoble, F-38054 Grenoble Cedex 9, France

Key words: biosynthesis; mitochondria; one-carbon metabolism; tetrahydrofolate.

Abstract: Folate is a crucial intermediate for one-carbon metabolism. In higher plants, folate derivatives are found in the cytosol, mitochondria and, to a lesser extent, chloroplasts together with the enzymes serine hydroxymethyltransferase and methylenetetrahydrofolate reductase/cyclohydrolase required for the generation and interconversion of the one-carbon substituted folates. The sources of these one-carbon units include formate, glycine and serine. A defining feature of plant folate metabolism is that the synthesis of the tetrahydrofolate from 6-hydroxymethyldihydropterin and p-aminobenzoic acid, and its subsequent conversion to physiologically active polyglutamyl derivatives appears to be confined to the mitochondria. This leads to important questions regarding the intracellular transport of folate and its precursors, but for the moment the mechanisms involved are unknown.

1. INTRODUCTION

The synthesis of numerous biological compounds and the regulation of numerous metabolic processes require the addition or removal of one­carbon units (CI metabolism). These one-carbon transfer reactions are mediated by folate or S-adenosyl-methionine (SAM) coenzymes, the synthesis of the latter depending on the presence of the former. Thus, all the varied reactions involved in Cl metabolism rely, at one step or another, on the availability of folate coenzymes.

53

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 53-99. © 1999 Kluwer Academic Publishers.

54 Chapter 4

The characterization of folate coenzymes was initiated nearly 60 years ago. In the, 1940s it became apparent that vitamin U (Stokstad and Manning, 1938), vitamin Bc (Hogan and Parrott, 1940), folic acid (Mitchell et aI., 1941) and Lactobacillus casei factor (Day et aI., 1945) were related compounds containing pteroylglutamic acid (Angier et aI., 1946). The term folate originates from the latin name folium because this vitamin, also referred to today as vitamin B9, is relatively abundant in leaves. The structure of folate (or pteroylpolyglutamate) is represented in Figure I. The molecule is composed of three distinct parts: a pterin ring (the name pterin originates from the Greek word pteron (wing) because these compounds were found in the pigments of butterfly wings); a p-aminobenzoic acid; and a polyglutamate chain with up to 8 residues (Cossins, 1984; Kisliuk, 1981; McGuire and Coward, 1984). The folate molecule exists with various degrees of oxidation. Represented in Figure I shows 5,6,7,8-tetrahydropteroylpolyglutamate, but the pyrazine portion of the pterin ring also exists as 7,8-dihydro- or fully oxidized (Cossins, 1984; McGuire and Bertino, 1981). Only the tetra-reduced form of folate serves as coenzyme for one-carbon transfer reactions. In biological systems, one-carbon substituents are attached to tetrahydrofolate at positions 5, 10 or as a bridge between the two positions (Figure 1). These one-carbon substituents differ in their redox states, depending on the metabolic reactions in which they are utilized: 10-formyltetrahydrofolate and 5, 10-methenyltetrahydrofolate are involved in purine synthesis (Rowe, 1984); 5,10-methylenetetrahydrofolate is required for thymidylate synthesis (Santi and Danenberg, 1984) and glycine to serine conversion (Schirch, 1984); 5-formiminotetrahydrofolate is involved in catabolism of histidine (Shane and Stokstad, 1984); and 5-methyltetrahydrofolate is the methyl donor for the synthesis of methionine (Matthews, 1984). 5-formyltetrahydrofolate is not directly involved in one-carbon transfer reactions and its metabolic role is not yet fully understood (Stover and Schirch, 1993). As shown in Figure 2, one remarkable feature of folate metabolism is the possible interconversion of the one-carbon units between their various oxidation states (MacKenzie, 1984). Indeed, these interconversion reactions allow the biosynthesis of all the different substituted folate coenzymes from common sources of one-carbon units. Furthermore, all these coenzymes are part of a unique folate pool, which gives the whole of C 1 metabolism a great flexibility.

Some of the initial steps of folate synthesis are absent in animals and folate supply in these organisms is ensured by dietary intake. Folate deficiency may have severe repercussions on human health such as heart coronary desease (Hopkins, 1996) or nervous disorders (Kirke et aI., 1996). In contrast, microorganisms and plants are able to synthesize

4. Folate metabolism in plants

CHO

I CHNH

I CH CH3 / 2

I /CH\

\

CHO

I

~)H tCH,_~~ >-co_~_~:OH H 10'~ (CHZ)2 COOH

7( I I 8 H Co-N CH NH H ,

(CHZ)2

I +--------~~ Co-N

Pterin H

Pteroate

Folate

55

COOH I CH , (CH2h n, COOH

Figure 1: Structure of tetrahydrofolate showing the different positions of substituted one-carbon units.

56 Chapter 4

HCOOH ATP

ADP, Pi

~ ~ Glycine

G::~f: CO"NH, @ 5,lO-methylene H4F (,\ ~ 5-methyl H4F

NADP ~ NADPH NADP Q)

NADPH

5,lO-methenyl H4F

0)/ ~~DP,Pi /-NH3 CD~ATP

5-formimino H4F 5-formyl H4F

Histidine

Figure 2: Schematic representation of interconversion of one-carbon units. 1, serine hydroxymethyltransferase; 2, glycine decarboxylase; 3, formyltetrahydrofolate synthetase; 4, methylenetetrahydrofolate dehydrogenase; 5, cyclohydrolase; 6, methylenetetrahydrofolate reductase; 7, formiminotetrahydrofolate cyclodeaminase; 8, metheny Itetrahydrofolate synthetase. Note the dual function of the serine hydroxymethyltransferase which catalyzes the interconversion of glycine and serine, but also the irreversible conversion of 5, I 0-methenyl- into 5-formyltetrahydrofolate.

4. Folate metabolism in plants 57

tetrahydrofolate de novo. In animals and microorganisms considerable efforts have been focused on the characterization of folate-dependent enzymes because several steps of folate metabolism are of medical interest. This is the case, for example, for dihydrofolate reductase (DHFR), a target for anti-carcinogenous agents, (Blakley, 1984) and for dihydropteroate synthase (DHPS), a target for antimicrobial sulfonamide drugs in microorganisms (Allegra et aI., 1990; Shiota, 1984; Zhang and Meshnick, 1991). In plants, much information is now available regarding the nature of folates and the regulation of some of the enzymes involved in C 1 metabolism. However, there is much less data concerning the initial steps of tetrahydrofolate synthesis, despite the fact that they are potential targets for the design of novel herbicides. The aim of the present review is to summarize our recent knowledge about folate synthesis and compartmentation in plants.

2. FOLATE DISTRIBUTION AND FOLATE SYNTHESIS

2.1 The nature and distribution of folates in plants.

2.1.1 Folate content and the nature of folate derivatives

Folate content may greatly vary from one species to another and with the origin of the plant tissue. For example, wheat germ has a high level of folate (4.3 J..Ig g-' FW) and green vegetables generally contain more folate (0.5-1.5 Jlg g-' FW) than fresh fruits (0.1-0.3 J..Ig g-' FW) (Cossins, 1984). Taking into account that the most abundant form of folate in leaves is the pentaglutamate form (Imeson et aI., 1990), and assuming a cytoplasmic to vacuole ratio of about 8, it can be calculated that, in leaves the total folate concentration in the cytoplasm (cytosol plus organelles) is within the range 5-15 JlM. The nature of the different forms of substituted folate has also been investigated (Blondeau, 1973; Roos et aI., 1968; Roos and Cossins, 1971; Spronk and Cossins, 1972). In summary, it appears from these data that folates are largely dominated by the methyl (45-65% of the total pool) and the formyl (30-50% of the total pool) derivatives, the unsubstituted forms of folate (including the methy lene derivative) representing less than 10% of the total fo late pool. Similar results were also obtained with Euglena gracilis (Crosti and Bianchetti, 1983). Interestingly, the formyl pool of folate contained a large amount (20-40%) of the 5-formyl derivative (Spronk and Cossins,

58 Chapter 4

1972), a compound not directly involved in one-carbon transfer reactions. 5-formyltetrahydrofolate is formed during the irreversible hydrolysis of 5,1 O-methenyltetrahydrofolate, a reaction catalyzed by serine hydroxymethyl transferase (SHMT) (Stover and Schirch, 1992). The only enzyme that uses 5-formyltetrahydrofolate is the methenyltetrahydrofolate synthetase which catalyzes the A TP­dependent reverse conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate (Stover and Schirch, 1993). In animals, inhibition of this enzyme (Bertrand and Jolivet, 1989) resulted in an increase of the 5-formyltetrahydrofolate content, which was correlated with a decrease in both cell growth and purine synthesis. In addition, 5-formyltetrahydrofolate was also reported to be an inhibitor of several enzymes, including SHMT, AICAR formyltransferase, methionyl-tRNA formyltransferase, 5,1 O-methylene-tetrahydrofolate dehydrogenase and dihydrofolate reductase (Stover and Schirch, 1992). Although the metabolic role of 5-formyltetrahydrofolate is totally unknown in plants, one may speculate from the above information that this folate derivative serves either as a regulatory factor for Cl metabolism or as a form of folate storage (5-formyltetrahydrofolate is a stable compound). In agreement with this last hypothesis, it was reported that 85% of the tetrahydrofolate pool in conidiospores of Neurospora crassa was made up of 5-formyl compounds, but these derivatives were reduced to less than 10% of the total folate pool in twenty minutes after adding the spores to growth media (Kruschwitz et aI., 1994).

In photosynthetic tissues, the effect of light on folate content was also investigated. It was observed that green cotyledons have higher folate levels than etiolated tissues of similar ages (Spronk and Cossins, 1972). Likewise, the biosynthesis of folate compounds in pea seedlings during germination increased more rapidly in the light than in the dark (Okinaka and Iwai, 1970b). These results may suggest that Cl metabolism is more active in the light, presumably because of the photorespiratory activity. Indeed, photorespiration produces large amounts of glycine which is oxidized in mitochondria from photosynthetic tissues (Douce, 1985; Oliver et aI., 1990a, 1990b). Glycine oxidation relies on two folate dependent proteins, the T -protein of the glycine cleavage complex and SHMT (Bourguignon et aI., 1988). During this reaction, the glycine cleavage system catalyses the oxidation of glycine in the presence of NAD+ and tetrahydrofolate (H4PteGlun)

into CO2, NH3, NADH and methylenetetrahydrofolate (CHT H4PteGlun)

(Equation (1». The continuous operation of this reaction is ensured by NADH reoxidation through the mitochondrial electron transport chain and by recycling of CH2-H4PteGlun into H4PteGIun through a reversible reaction catalysed by SHMT (Equation (2».

4. Folate metabolism in plants 59

(2)

The glycine cleavage system and SHMT are light induced and are present in large amount in the matrix space of leaf mitochondria (Vauclare et aI., 1996). Considering the high rate of glycine oxidation (Oliver et aI., 1990a) it is thus possible that reactions (1) and (2) require an increased level of folate within mitochondria. In this connection, Spronk and Cossins (1972) observed that illuminated tissues contained significantly higher levels of un substituted forms of folate (CHr H4PteGlun was isolated as un substituted folate in these experiments) at the expense of the formyl derivatives.

2.1.2 Subcellular distribution of folates

There is now good evidence that folate is present in the various subcellular compartments. Okinaka and Iwai (1970b) reported that the largest pool of folate was found in a soluble (cytosolic) fraction, which contained the bulk of the methyl derivatives (Coffin and Cossins, 1986). Pools of folate dominated by formyl (10-formyl and 5-formyl) and, to a lesser extent, methyl derivatives, were also observed in mitochondria (Clandinin and Cossins, 1972; Coffin and Cossins, 1986) and chloroplasts (Cossins and Shah, 1972). The size of the mitochondrial folate pool was estimated by several authors. One analysis indicated that mitochondria from four-day-old pea cotyledons contained 3.5-4% of the total pool of folate (Clandinin and Cossins, 1972), but this level markedly increased with the time of growth (Coffin and Cossins, 1986). Another report indicated that the mitochondrial fraction obtained from one day old pea cotyledons contained up to 22% of the total folate pool (Okinaka and Iwai, 1970b). The subcellular distribution of folate in 2-week old pea leaves was investigated more recently using Percoll-purified mitochondria and chloroplasts, and a radioassay methodology for folate determination (Neuburger et aI., 1996). In mitochondria, the folate content was about 400 pmol mg-1 protein (i.e. about 400 J.lM, assuming a mitochondrial volume of 1J.lI mil protein) (Table 1). This value is in good agreement with previous measurements (390-1000 pmol mg- l protein) performed with a different method involving ZnlHCI treatments and HPLC separation of p-aminobenzoylpolyglutamate derivatives (Besson et aI., 1993). In contrast, the folate level in chloroplasts, on a protein basis, was approximately 150 times lower than in mitochondria (Table 1). In a

60 Chapter 4

Tablel

Folate pools in pea leaves, pea leaf mitochondria, pea leaf chloroplasts and potato tuber

mitochondria. The folate concentration was calculated assuming a volume of 1pl mg-1 protein

for the mitochondrial matrix and 1.5 pi mg-1 protein for the chloroplast stroma. In the leaf

extract, the amounts of protein originating from mitochondria and chloroplasts were estimated

through marker enzyme activities: fumarase for mitochondria and PRK for chloroplasts (from

Neuburger et al. 1996).

Folate content Folate Protein Folate

(pmol mg-1 prot.) concentration (%) distribution

(uM) (%)

Leaves 26:1: 1.4 100 100

Chloroplasts 25:1: 05 1.7 65 :1:10 6.3:1: 1

Mitochondria 400:1:50 400 3.5:1: 1 54:1: 15

(pea)

Mitochondria 200 :1:30 200

(potato)

4. Folate metabolism in plants 61

leaf extract, the amounts of protein originating from either mitochondria or chloroplasts could be determined through the activity of marker enzymes, fumarase for mitochondria and phosphoribulokinase (PRK) for chloroplasts (Table 1). According to these determinations, these authors (Neuburger et ai., 1996) calculated that approximately 50% of the total folate pool was associated with the mitochondria whereas only 6-7% was associated with chloroplasts, the remaining 40-45% of the folate pool being presumably associated with the cytosol and/or the nuclei. In order to determine if the high folate concentration of pea leaf mitochondria resulted from the presence of the glycine decarboxylase complex, the folate content of potato tuber mitochondria, which do not contain glycine decarboxylase activity, was also determined (Neuburger et ai., 1996). As shown in Table 1, the folate concentration in these mitochondria was still very high (200 IlM), although two times lower than in leaf mitochondria. Taken as a whole, these data indicate that folate in higher plant cells is mainly localized in the cytosol and the mitochondria, but, considering the small size of the mitochondrial compartment, these organelles most probably have the highest folate concentration.

2.1.3 The role of the polyglutamate chain

There is now strong evidence that the physiological forms of folate are y-glutamyl-linked polyglutamates (Imeson et ai., 1990; McGuire and Coward, 1984; Schirch and Strong, 1989). Differences in glutamyl chain length, ranging from one to eight, have been reported for a variety 0 f organisms, including plants (Chan et ai., 1986; Kisliuk, 1981; McGuire and Coward, 1984). In plants, there is considerable variation in the glutamate chain length, depending on the species (Zheng et aI., 1992). For example, cauliflower florets and tomato leaves contain a large proportion of highly conjugated folates (hexa- and heptaglutamates) whereas diglutamates are the predominant forms in carrot roots and 4-week old pea leaves. In pea seedlings, folylpolyglutamates were mainly tetra- and pentaglutamates (lmeson et ai., 1990) and these derivatives accounted respectively for 11 and 83% in cotyledons, 15 and 38% in leaves (the diglutamates accounted for 46%),42 and 21% in shoots, 44 and 40% in isolated chloroplasts. Likewise, the major polyglutamates recovered in the matrix space of pea leaf mitochondria were tetra- and pentaglutamates (Figure 3A) which accounted for 25 and 55% respectively of the total pool (Besson et ai., 1993). Considering the variability of the poly glutamate chain, one may question its physiological role. The conjugate nature of folates may have a role in the retention of these derivatives within a cell compartment. Indeed, in mammalian cells

62 Chapter 4

B

40

20

0~~~~1J o 2 3 4 5 6

OIL.l2QSJ!~1SZ'S2Is:;:z=EO:2lSZISZI...J 034 567

Number of glutamate residues Number of glutamate residues

5

Number of glutamate residues Time in air (Illn)

Figure 3: Effect of the polyglutamate chain of tetrahydrofolate on the GDC activity in pea leaf mitochondria. A, polyglutamate distribution in the matrix (from Besson et aI., 1993); B, effect of the number of glutamate residues on the Km of tetrahydrofolate for GDC (from Rebeille et aI., 1994); C, binding oftetrahydrofolate to GDC as a function of the number of glutamate residues; the binding was indirectly estimated by the ability of tetrahydrofolate to sustain glycine oxidation after removal of unbound tetrahydrofolate by filtration of the GDC complex plus folate mixture on a 10 kDa cut-off membrane (from Rebeille et aI., 1994); D, oxidative degradation oftetrahydrofolate pentaglutamate when either free in solution or bound to the GDC; the oxidative degradation of tetrahydrofolate was estimated indirectly by the ability of this cofactor to sustain glycine oxidation (from Rebeille, et aI., 1994).

4. Folate metabolism in plants 63

monoglutamyl folates are readily transported accross cellular membranes, but permeability to polyglutamyl folates is considerably less and these derivatives are usually retained by living cells (McGuire and Bertino, 1981). It is also clear from studies on microorganisms and animal tissues that a number of folate-dependent enzymes display greater affinity for the polyglutamate forms of their substrates than for the corresponding monoglutamates (McGuire and Coward, 1984; Schirch, 1984; Strong et aI., 1990). In this connection, it was observed in plant mitochondria that the affinity of tetrahydrofolate for the T -protein of the glycine cleavage system and SHMT increases considerably with the number of glutamate residues (Figure 3B) (Besson et aI., 1993). The maximal effect was obtained with three glutamates and longer polyglutamate chains did not significantly lower the Km value. However, an almost linear relationship was observed between the number of glutamate residues and the binding of tetrahydrofolate to these two proteins (Figure 3C). It was proposed that the tight binding of folate polyglutamates increases the efficiency of sequential folate-dependent proteins by enhancing the 'channelling' of intermediates between the active sites (MacKenzie, 1984; McGuire and Bertino, 1981; Schirch and Strong, 1989). This hypothesis was investigated in mitochondria from leaf tissues where two folate­dependent enzymes, the SHMT and the T-protein of the glycine cleavage system, interact through a common pool of polyglutamyltetrahydrofolates (Rebeille et aI., 1994). It was observed that methylenetetrahydrofolate, the product of the GDC reaction, accumulated in the bulk medium, indicating that in these in-vitro experiments, this cofactor was not channelled between T-protein and SHMT. However, as mentioned by these authors, the in-vivo situation might be different since the very high protein concentration in the matrix space (approximately 400 mg mr l ), together with the relatively low tetrahydrofolate level (0.4-1 mM), could lead to a situation where folate compounds are not released into the bulk medium. Indeed, there are apparently more protein binding sites for folate in the matrix space of plant leaf mitochondria than folate derivatives (Besson et aI., 1993). This hypothesis is further supported by recent NMR experiments showing that in vivo, methylenetetrahydrofolate does not accumulate in the matrix space of mitochondria (Prabhu et aI., 1996). Another consequence of the binding of folylpolyglutamates to folate-dependent enzymes is the protection of the molecule against oxidative degradation (Rebeille et aI., 1994). Indeed, tetrahydrofolate is a readily oxidizible compound which undergoes rapid oxidation when in contact with oxygen: in a well aerated buffer, 80-90% is degraded within 25 minutes (Rebeille et aI., 1994). In contrast, when tetrahydrofolate pentaglutamate is bound to folate-dependent proteins it is protected against oxidative degradation

64 Chapter 4

(Figure 3D). In conclusion, it appears that long polyglutamate chains play an important role in the efficiency of the reaction and in the protection of the folate molecule. However, it is not clear why there is such a variability in the polyglutamate chain length among all living organisms.

2.2 The synthesis of tetrahydrofolate.

The initial step of folate synthesis is the conversion of GTP to 7,8-dihydroneopterintriphosphate, a reaction catalyzed by the GTP cyclohydrolase (Brown, 1985). This compound is the precursor of the pterins, a family of molecules found in the pigments of some organisms and involved in several metabolic pathways such as metabolism of phenylalanine, tyrosine and tryptophan (Johnson and Rajagopalan, 1985; Kaufman and Kaufman, 1985; Kuhn and Lovenberg, 1985; Shiman, 1985). It is also the precursor of 6-hydroxymethyldihydropterin, which in tum is the source of tetrahydrofolate. The tetrahydrofolate synthesis pathway, from 6-hydroxymethyldihydropterin, requires the sequential operation of five enzymes: a dihydropterin pyrophosphokinase (HPPK); a dihydropteroate synthase (DHPS); a dihydrofolate synthetase (DHFS); a dihydrofolate reductase (DHFR) and a folylpolyglutamate synthetase (FPGS) (Figure 4). The three first steps of this pathway, that is from 6-hydroxymethyldihydropterin to dihydrofolate, are absent in animals. In plants, in contrast to bacteria, the study of the enzymes involved in folate synthesis is complicated by the presence of the different subcellular compartments, and cell fractionation experiments were often confused in their interpretations because of cross-contamination of the various subcellular fractions. In an attempt to solve this problem, the enzyme activities involved in tetrahydrofolate synthesis were determined in purified pea leaf mitochondria, in purified pea leaf chloroplasts and in a pea leaf cytosol-enriched fraction (Neuburger et ai., 1996). Surprisingly, these activities were detected only in mitochondria, suggesting that these organelles playa major role in this pathway (Table 2).

2.2.1 Dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS)

The two first steps of tetrahydrofolate synthesis are catalyzed by HPPK and DHPS. DHPS is the target of sulfonamide drugs. These chemicals are p-aminobenzoic acid (P-ABA) analogues that are recognized by DHPS as alternate substrates (Shiota, 1984). DHPS is therefore an attractive target for chemotherapy because, in contrast to

4. Folate metabolism in plants

CD Glu ATP

H,PteGluJ ~ HzPteroate ~ Mg++

ADP+Pi

NADPH

NADP

ADP+Pi Mg++ J

H,PteGlul n- H,PteGlu .... 1

ATP nGlu

CD

65

CD CD AMP ATP

~A H,PterinPPi ,\. ) H,Pterin

+ PPi

GTP

Figure 4: Tetrahydrofolate (HJ>teGlun) synthesis pathway from 6-hydroxymethyldihydropterin. 1, dihydropterin pyrophosphokinase; 2, dihydropteroate synthase; 3, synthetase; 4, dihydrofolate reductase; 5, folylpolyglutamate synthetase.

dihydrofolate

Table 2

Enzyme activities involved in tetrahydrofolate synthesis and interconversion of one-carbon

substituted folates in the different pea leaf cell compartments and in potato tuber mitochondria.

Fumarase, PRK and PEPc were marker enzymes respectively for mitochondria, chloroplasts

and cytosol. In the cytosol enriched fraction less than 1 % of the proteins were from

mitochondria and 30-45 % were from plastids. Although nuclei were purified on a Percoll

gradient, about 25 % of the proteins were from chloroplasts and 1 % were from cytosol (from

Neuburger et al. 1996). n.d. not detected.

PRJ(, phosphoribulokinase; PEPc, phosphoenolpyruvate carboxylase; HPPK, dihydropterin

pyrophosphokinase; DHPS, dihydropteroate synthase; DHFS, dihydrofolate synthetase;

DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; SHMf, serine

hydroxymethyltransferase; MTIIFDH, methylenetetrahydrofolate dehydrogenase;

Pea leaves Potato tuber

Mitochondria Chloroplasts Cytosol nuclei Mitochondria

Fumarase 51000 ±3000 n.d. 420± 120 n.d.

PRK n.d. 27000 ± 2400 10200±2400 7200± 1200

PEPc n.d. n.d. 6000 ± 800 6O±10

HPPK+ DHPS 1.8 ± 0.3 n.d. n.d. n.d. 2 ±OA

DHPS 16±3 n.d. 0.08±0.04 n.d. 9±2

DHFS 1.6±0.3 n.d. n.d. n.d. 2.1 ±0.4

DHFR 180 ±50 n.d. 1.5 ± 0.5 n.d. 500± 100

FPGS 3.5 ± 0.5 n.d. 0.04±0.03 n.d. 3.8±0.6

SHMT 2100±240 70±8 190 ±30 200±30 350±60

MTIIFDH 150 ± 20 200±50 180 ±40 230±50 85 ± 15

66 Chapter 4

dihydrofolate reductase a much studied target for antifolate agents, it has no mammalian counterparts and high level of selectivity could be achieved. Because of this medical interest, considerable efforts have been focused on the molecular characterization of DHPS enzymes from microorganisms (Dallas et aI., 1992; Lopez et aI., 1987; Triglia and Cowman, 1994; Volpe et aI., 1993). In prokaryotes, HPPK and DHPS are separate enzymes and their coding genes are part of a folic acid biosynthetic operon (Lacks et aI., 1995; Slock et aI., 1990; Talarico et ai., 1992). In eukaryotes, the related proteins studied so far were always multifunctional, containing HPPK and DHPS activities in Plasmodium Jalciparum and Toxoplasma gondii (Allegra et aI., 1990; Triglia and Cowman, 1994) and dihydroneopterin aldolase (DHNA), HPPK and DHPS activities in Pneumocystis carinii (Volpe et aI., 1993). DHNA catalyzes the conversion of 7,8-dihydroneopterin into 6-hydroxymethyl-7,8-dihydropterin, substrate of the HPPK activity. In higher plants, sulfonamide compounds are potential herbicides since they also block DHPS activity (Okinaka and Iwai, 1970a) and inhibit plant growth (lwai et ai., 1962). In plants, as in Plasmodium Jalciparum and Toxoplasma gondii, HPPK and DHPS activities are part of a bifunctional protein (Okinaka and Iwai, 1970a; Rebeille et aI., 1997). In an early work, Okinaka and Iwai (l970a), found that 75% of the HPPKlDHPS cellular activity was localized in mitochondria and 25% in a soluble (cytosolic) fraction. However, this study could not reject the possibility that the soluble fraction might be contaminated by the mitochondrial fraction. These authors also reported a native molecular weight of 180 kDa, but this value was not confirmed by the recent study of Rebeille et ai., (1997). According to these last authors, the activity was only found in mitochondria. The enzyme, purified from mitochondria, represents 0.06% of the matrix proteins, has a native molecular weight of 280-300 kDa and is possibly constituted of six identical subunits of 53 kDa. Km values of the plant bifunctional HPPKlDHPS for dihydropterin, A TP, dihydropterin pyrophosphate, and p-aminobenzoic acid are respectively 0.7 11M, 70 11M, 30 11M and 0.6 11M. Kinetic studies of the reaction catalyzed by the DHPS domain of the protein suggest a random bi­reactant system strongly retro- inhibited by dihydropteroate (Ki = 10 11M) (Rebeille et aI., 1997). The related cDNA encodes a polypeptide of 515 residues containing a 28 amino acid extension from the N-terminal end that resembles the import sequence of mitochondrial proteins. In addition, Southern blot experiments suggested that a single-copy gene codes for the enzyme. This last result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity is only detected in mitochondria, strongly supports the view that mitochondria are the unique site of 7,8-dihydropteroate synthesis in

4. Folate metabolism in plants 67

higher plant cells. When compared to other HPPK and DHPS, the plant enzyme shows highly conserved domains, possibly involved in catalysis and substrate binding, and has the appearance of a mere fusion of the two bacterial HPPK and DHPS enzymes. Little is known about bifunctional proteins and the biochemical properties of this interesting enzyme remain to be studied in more detail.

2.2.2 Dihydrofolate synthetase (DHFS)

The third step of tetrahydrofolate synthesis is catalysed by DHFS. Like HPPK and DHPS, DHFS is absent in animals and is, therefore, a potential target for hebicides. In bacteria, this enzyme is bifunctional, supporting also the folylpolyglutamate synthetase activity (Bognar et aI., 1987). In yeast, the enzyme is monofunctional and appears to be a monomeric protein of 52 kDa catalyzing a Mg2+ and K+ dependent reaction (McDonald et aI., 1995). Little is known about higher plant DHFS. The enzyme was purified from pea seedlings. It has a native molecular weight of 56 kDa and, like its yeast counterpart, requires Mg2+and K+for catalysis (Iwai and Ikeda, 1975). The Km values of DHFS for dihydropteroate, glutamate and A TP are respectively 1 /lM, 1.5 mM and 100 ~M. ADP is a feed-back inhibitor of the enzymatic reaction. In addition, analysis of the subcellular distribution of DHFS activity in plants indicated that the protein was mainly, if not only, localized III

mitochondria (Ikeda and Iwai, 1975; Neuburger et aI., 1996).

2.2.3 Dihydrofolate reductase (DHFR)

The fourth step of tetrahydrofolate synthesis is the reduction of dihydrofolate into tetrahydrofolate, a reaction catalyzed by DHFR. This enzyme is probably the most thoroughly studied folate enzyme because in animals it is an important target for chemotherapy in malignant deseases (Blakley, 1984). In bacteria and vertebrates the enzyme is a monomer of about 20 kDa. The DHFR of these two organisms have been crystallized and their structures have been solved (for a review, see Blakley, 1984), thus providing a good understanding of how ligands (substrates and inhibitors) and protein interact. In animal cells, DHFR is believed to be primarily localized in the cytosol (Brown et aI., 1965; Hurt et aI., 1984). In protozoa and higher plants, as well as in green algae, the situation is different because DHFR is part of a bifunctional protein, also containing thymidylate synthase (TS) activity, and appears in most studies as an homodimer of 50-60 kDa (Bachmann and Follmann, 1987; Beverley et aI., 1986; Cella et aI., 1988; F erone and Roland, 1980; Lazar et aI., 1993;

68 Chapter 4

Neuburger et aI., 1996). TS is involved in the synthesis of dTMP from dUMP according to the following equation (Equation 3).

(3)

In this reaction methylenetetrahydrofolate serves not only as one-carbon donor but also as an electron donor and the resulting dihydrofolate must be recycled back to methylenetetrahydrofolate to ensure the continuous operation of the reaction (see Figure 5). Compared to the monofunctional enzyme, the bifunctional protein exhibits distinct biochemical properties such as metabolic channelling from TS to DHFR (Meek et aI., 1985). Indeed, X-ray structure of DHFRlTS indicates that the transfer of dihydrofolate between the two active sites does not occur by a diffusional pathway but by an electrostatic channelling on the surface of the protein (Knighton et aI., 1994). The subcellular distribution of DHFRlTS is still a matter of debate. In the protozoan Leishmania major the DNA sequence coding for the bifunctional DHFRfTS predicts a mitochondrial transit-peptide (Beverley et aI., 1986), but immunogold experiments indicated a cytosolic distribution of the protein (Swafford et aI., 1990). In higher plants, isogenes of the bifunctional enzyme have been reported in Arabidopsis thaliana (Lazar et aI., 1993), but DHFRlTS was only found in mitochondria (Neuburger et aI., 1996) where it represents about 0.05% of the proteins. As previously observed with the bacterial monofunctional DHFR (Penner and Frieden, 1985), the reaction catalyzed by the bifunctional plant enzyme has a lag phase which is considerably shortened by preincubation in the presence of NADPH (Neuburger et aI., 1996), suggesting a possible allosteric activation. Interestingly, DHFR exhibits a high affinity for both the mono glutamate and the polyglutamate forms of dihydrofolate (Km for H2PteGIum 1 f.1M) whereas TS, as generally observed for most folate enzymes (McGuire and Bertino, 1981), only shows a high affinity for the polyglutamate forms of the cofactor (Km for CH2-H4PteGluJ, 22 f.1M; Km for CHr H4PteGlus, 3.5 f.1M) (Neuburger et aI., 1996). This illustrates the multiple roles of the mitochondrial DHFR. As schematized in Figure 5, the natural substrate of DHFR is the monoglutamate form of dihydrofolate when it is involved in tetrahydrofolate synthesis, and the polyglutamate form of dihydrofolate when it is coupled to TS for CH2-H4PteGlun recycling. Furthermore, DHFR might have a third important role in reducing the H2PteGIun produced by the non-enzymic oxidation of H4PteGIun (especially in leaves, during photosynthesis, where the internal concentration of O2 is very high).

In animal cells, TS is a monofunctional enzyme localized in the cytosol and/or nuclei (Brown et aI., 1965). Thus, the question arises

4. Folate metabolism in plants 69

1

dUMP

Glycine Serine

Figure 5: Schematic representation of the multiple roles of DHFRlTS in the matrix space of higher plant mitochondria. I, synthesis pathway of tetrahydrofolate from 6-hydroxymethyldihydropterin; 2, polyglutamylation of tetrahydrofolate; 3, nonenzymic oxidation of tetrahydrofolate into dihydrofolate; 4, conversion of tetrahydrofolate into methylenetetrahydrofolate in the SHMT catalyzed reaction.

70 Chapter 4

whether this monofunctional enzyme also exists in higher plant cells. However, as shown in Table 2 (Neuburger et aI., 1996), no activity could be detected in the different cell fractions other than mitochondria where, as described above, it is associated with the DHFR activity. This result suggests that mitochondria are also a major site for thymidylate synthesis, a conclusion that should be confirmed by measurements of thymidylate transport accross the mitochondrial inner membranes. Finally, the presence of NADPH-dependent enzymes in mitochondria raises the problem of the origin of the cofactor. Interestingly, it was observed by NMR studies that purified plant mitochondria contain a relatively large pool of NADP(H) (Roberts et aI., 1997), a result suggesting the presence, in these organelles, of a pyridine nucleotide kinase.

2.2.4 Folylpolyglutamate synthetase (FPGS)

The last step of tetrahydrofolate synthesis is the elongation of the polyglutamate chain by FPGS. Considerable attention has been given to this reaction because, as already stated, most folate enzymes display a high preference for polyglutamates over the corresponding monoglutamate substrates. In addition, many antifolate drugs are also converted into polyglutamate derivatives within the cell (Kim and Shane, 1994), a situation that increases their cell retention and thus their inhibitor activities. In bacteria, FPGS also displays DHFS activity and preliminary studies have suggested that the two synthetase activities are catalysed by separate sites (Shane, 1980b). The bacterial bifunctional protein is monomeric with a molecular weight of about 47-53 kDa (Bognar et aI., 1985; Shane, 1980a, 1980b). It catalyses an ordered Ter Ter mechanism with A TP binding first, tetrahydrofolate second and glutamate third (Shane, 1980c). In eukaryotes, FPGS is a monofunctional protein of 60 kDa in animals (Cichowicz and Shane, 1987; Garrowet aI., 1992) and 65-70 kDa in Neurospora crassa and plants (Chan et aI., 1991; Imeson and Cossins, 1991a; McDonald et aI., 1995; Neuburger et aI., 1996). The folate substrate specificity varies with the source of the enzyme. Indeed, although tetrahydrofolate monoglutamate is always an effective substrate, some species display a higher affinity for 10-formyltetrahydrofolate (E. coli) or 5,1 O-methylenetetrahydrofolate (Corynebacterium sp.; Neurospora crassa) (Chan and Cossins, 1991; McGuire and Coward, 1984). In higher plants, 5, I 0-methylenetetrahydrofolate is also an effective substrate for FPGS, displaying similar affinity to tetrahydrofolate (lmeson and Cossins, 1991b). In-vitro analysis of Neurospora and higher plant FPGS indicate that diglutamates are the major product of the reaction during the first

4. Folate metabolism in plants 71

two hours (Chan and Cossins, 1991; Imeson and Cossins, 1991b), but that longer incubation periods result in more highly conjugated derivatives. In mammalian cells, FPGS is mainly localized in the cytosol (McGuire and Coward, 1984) but the enzyme was also detected in purified mitochondria (Lin et at, 1993). In Neurospora crassa, approximately 50% of the FPGS activity was cytosolic and 50% mitochondrial (Cos sins and Chan, 1984). These two FPGS isoenzymes differed in their substrate specificity, the mitochondrial isoform catalyzing glutamate addition only with H4PteGlu2 as substrate and not with H4PteGlu\. In higher plants, FPGS was only detected in mitochondria (Neuburger et at, 1996), a situation which, if it holds true, raises the problem of folylpolyglutamate transport across the membranes of the different cell compartments. Indeed, the glutamate chain of folate derivatives is negatively charged, and, as mentioned above, highly conjugated folates are retained within the compartment where they are localized. Thus it is logical to think that folate derivatives must be deconjugated before being transported and then conjugated again after arriving at their final destination, a condition which requires the presence of FPGS in all the cell compartments. However, it was observed in animal cells that folylpolyglutamates formed within the mitochondria are released into the cytosol without prior hydrolysis (Kim and Shane, 1994), a situation that might also exist in higher plants where mitochondria appear to be the major site for folate synthesis (Neuburger et at, 1996). Clearly, the mechanisms involved in folate transport and folate traffic within the cell remain to be discovered.

3. ORIGIN AND INTERCONVERSION OF Cl COMPOUNDS

3.1 The origin ofCI compounds

As shown in Figure 2, formate and the amino acids serine, glycine and histidine are the main sources of one-carbon substituted tetrahydrofolate. Although the formation of 5-formiminotetrahydrofolate from histidine and its conversion to methenyltetrahydrofolate is well documented in mammals (Shane and Stokstad, 1984), it is not known in plants and will not be considered in this chapter.

3.1.1 Formate

3.1.1.1 The formyltetrahydrofolate synthetase Formate is a potential single-carbon source in higher plants and

several authors have shown that externally added formate is readily metabolized into glycine and serine (Prabhu et aI., 1996; Shingles et aI., 1984). The ATP-dependent synthesis of 10-formyltetrahydrofolate from formate (Figure 2) involves the enzyme formyltetrahydrofolate synthetase. In some organisms (yeast, animals) this activity is associated with two other activities, methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, to form a single dimeric trifunctional protein called C I-tetrahydrofolate synthase (MacKenzie, 1984; Pasternack et a!., 1992; Strong et aI., 1987). This multifunctional protein ranges in molecular weight from 150 to 218 kDa (Paukert et aI., 1977; Schirch, 1978), displays a greater affinity for the polyglutamate derivatives and, in yeast, is found in the mitochondria as well as in the cytosol (Shannon and Rabinowitz, 1986). The formyltetrahydrofolate synthetase is not present in Escherichia coli but is present as a monofunctional protein in the other prokaryotes examined (MacKenzie, 1984). Kinetic studies with the synthetases from Clostridia indicate that the equilibrium constant of the reaction favors 10-formyltetrahydrofolate (Curthoys and Rabinowitz, 1972) suggesting that this reaction has essentially a biosynthetic role. In higher plants, the synthetase has been purified from spinach leaves (Nour and Rabinowitz, 1991). The enzyme is a dimeric monofunctional protein with subunits of 67.7 kDa. It has greater homology with the synthetase domain of the mammalian multifunctional protein than with the monofunctional prokaryotic enzyme (Nour and Rabinowitz, 1992). The Km values reported for the different substrates were about 40 ~M for (6S)­tetrahydrofolate (monoglutamate), 40-100 ~ for ATP and 8-20 mM for formate (Kirk et aI., 1994; Nour and Rabinowitz, 1991). As for the other synthetases, the plant enzyme displays greater affinity for the folylpolyglutamate derivatives. Interestingly, it was also observed that binding of tetrahydrofolate pentaglutamate to the protein greatly reduced the value of Km for formate (from 8 mM to 35 /lM), suggesting a structural change of the enzyme (Kirk et aI., 1994). The plant formyltetrahydrofolate synthetase is mainly associated with the cytosolic fraction (Kirk et aI., 1994) but some activity was also detected in mitochondria (Clandinin and Cossins, 1972; Suzuki and Iwai, 1974). In chloroplasts, there is no direct evidence for the presence of this enzyme. However, in purified choloroplasts, relatively high levels of serine synthesis were observed in the presence of formate and glycine, which

4. Folate metabolism in plants 73

indirectly suggests that formyltetrahydrofolate synthetase is also present in this compartment (Shingles et aI., 1984).

3.1.1.2 The source of formate There are now several lines of evidence that higher plants do produce

formate. One observation which strongly supports this assumption is that higher plants have the enzymatic machinery required for formate degradation. Indeed, mitochondria from non-green tissues contain a substantial activity of formate dehydrogenase that catalyses the NAD+­dependent oxidation of formate to CO2 (Colas des Francs-Small et aI., 1993), thus indicating the presence of one or several pathways producing formate. However, these pathways, and the associated fluxes of carbon, are not well characterized. One possible route involves oxidative glyoxylate decarboxylation yielding formate and CO2 (Prather and Sisler, 1972). Two different processes are apparently involved in glyoxylate decarboxylation. The first process is a non-enzymatic oxidation of glyoxylate by H20 2 which is generated in leaf peroxisomes during the oxidation of glycolate to glyoxylate by glycolate oxidase (Halliwell and Butt, 1974). However, peroxisomes contain a large amount of catalase and H20 2 is very likely degraded as fast as it is produced which casts some doubts on the physiological significance of this reaction. A second process involves formate production from glyoxylate by an enzymically catalyzed reaction which requires the presence of thiamine pyrophosphate (Gifford and Cossins, 1982; Prather and Sisler, 1972). In addition to peroxisomes, chloroplasts and mitochondria were also shown to be capable of formate production from glyoxylate (Prather and Sisler, 1972; Zelitch, 1972). These last observations are intriguing because, as already stated, glyoxylate is produced in peroxisomes. There are two main pathways for glyoxylate production. The first one involves glycolate and glycolate oxidase. Although some glycolate oxidase activity is present in peroxisomes from non-photosynthetic tissues (Huang et aI., 1983), the source of glycolate remains unclear in these tissues. In photosynthetic tissues, glycolate oxidase activity is about 30-300 times higher than in non-photosynthetic tissues (Huang et aI., 1983) and glycolate is produced in large amounts during the course of the photorespiratory cycle (for a review, see Tolbert, 1981). The second pathway for glyoxylate production involves the conversion of isocitrate to succinate, a reaction catalyzed by the isocitrate lyase. This reaction is involved in gluconeogenesis from storage lipids and takes place in specialized peroxisomes (glyoxysomes) of oil storage tissues (Huang et aI., 1983). Glyoxylate decarboxylation and formate production have not been reported in these tissues, but H20 2 generated by the fatty acyl-CoA oxidase during ~-oxidation of fatty acids (Cooper and Beevers, 1969)

74 Chapter 4

might be responsible, if not totally degraded by catalase, for a non­enzymic oxidation of glyoxylate (Halliwell and Butt, 1974).

Two other routes are possibly involved in formate synthesis in higher plants. The first one is a direct reduction of CO2 into formate (Kent, 1972; Ramaswamy et aI., 1976). Evidence for this pathway relies on the observations that a large proportion of 14C02 was fixed into formate under conditions where photosynthesis was inhibited, and that CO2 and formate competed for assimilation (Kent, 1972). These authors proposed that ferredoxin, which has a comparable oxidation-reduction potential (-430 mY) to the oxidation-reduction potential of the formate-C02 couple (-420 m V), might play an important role in this redox process. However, it remains to establish whether this interesting pathway, which is subject to seasonal dependence (Kent, 1972), can significantly contribute to C 1 metabolism. Another interesting route is the oxidation of methanol in plants. Indeed, most plants produce and emit methanol, especially during the early stages of leaf expansion, (Fall and Benson, 1996; Nemecek-Marshall et aI., 1995) and this volatile organic compound exits leaves via stomata (Nemecek-Marshall et aI., 1995). The amount of methanol released to the atmosphere by the world vegetation is substantial, possibly higher than 100 million tons per year (for a review, see Fall and Benson, 1996). The proportion of methanol production which is recycled in plants is not known but it is obvious that plant tissues metabolize methanol. Indeed, it was observed that 14C_ methanol is converted to 14C02 by plants (Cossins, 1964) and the production of 14C-formate suggests stepwise oxidation of methanol into formaldehyde, formate and CO2. A likely source of methanol in leaves is pectin demethylation in the cell wall (for a review, see Fall and Benson, 1996). The precursors of pectin contain numerous galacturonate methyl esters which are demethylated during cell wall expansion, methanol being produced as a by-product. Other sources of methanol such as the protein repair pathway or lignin degradation have probably less contribution to methanol production in plants.

3.1.2 Glycine

3.1.2.1 The glycine decarboxylase complex Glycine breakdown is a potential source of

methylenetetrahydrofolate. This oxidative degradation is catalyzed by the glycine cleavage system (GDC), a multienzyme complex possibly present in the mitochondria of all tissues (Oliver, 1994). This complex is formed of four distinct proteins (Figure 6) (Bourguignon et aI., 1988; Neuburger et aI., 1986). The P-protein (a pyridoxal phosphate

4. Folate metabolism in plants

s- s

~ H

T

SHMT

H

~

75

NADH

~NAD L

HS SH

FGlu;>

Figure 6: Schematic representation of the GDC and SHMT coupled reactions. The lipoamide ann of the H protein interacts successively with the P, the T and the L proteins. Once charged in methylamine, the lipoamide arm rotates to come in contact with hydrophobic residues of a cavity opened at the surface of the protein, thus protecting the methylamine group against nucleophilic attack (Cohen-Addad et aI., \995). The rate of GDC is about three times higher than the rate of SHMT, thus maintaining a high level of methylenetetrahydrofolate which drives the SHMT reaction toward the production of serine (Rebeille et aI., 1994). Recycling oftetrahydrofolate through the SHMT reaction is the limiting step of the whole reaction (Bourguignon et aI., 1988; Rebeille et aI., 1994)

76 Chapter 4

containing protein) catalyzes the decarboxylation of glycine (Km for glycine, 6 mM) and the transfer of the remaining methylamine moiety on the oxidized lipoamide arm of the H-protein. The methylamine charged H-protein interacts thereafter with the T-protein (a tetrahydrofolate binding protein, Km for RtPteGlus, 0.5 11M) in a reaction leading to NH3 release and methylenetetrahydrofolate formation. Eventually, the resulting reduced lipoamide arm of the H-protein is recycled in its oxidized form by the L-protein (a flavin containing protein) with NADH production (Km for NAD+, 75 11M). All these proteins have been successfully purified from leaf mitochondria and their primary sequences have been determined (Bourguignon et aI., 1993; Macherel et aI., 1990; Turner et aI., 1992b, 1992c). In higher plants, this complex is present in large amounts in mitochondria from green tissues where it represents up to 40% of the soluble proteins (Bourguignon et aI., 1988). The accumulation of P, H and T proteins in leaf mitochondria is under the control of a light-dependent and tissue specific process (Vauclare et aI., 1996), which reflects the important role that GDC plays in photorespiration (Tolbert, 1980). In contrast, the expression of the L protein is not under tissue-specific control because this enzyme is a constituent of other complexes such as the pyruvate dehydrogenase complex and the 2-oxoglutarate dehydrogenase complex (Bourguignon et aI., 1996; Turner et aI., 1992b). The H-protein represents the mechanistic heart of the GDC since the P-, T - and L-proteins interact on its surface (Figure 6). The X-ray crystal structures of the oxidized form and methylamine charged form of the H-protein have been determined (Cohen-Addad et aI., 1995). The lipoate is attached to a specific lysine side chain located in the loop of a hairpin configuration. Interestingly, once charged with methylamine, the lipoate cofactor rotates to bind into a cleft at the surface of the protein and is, therefore, not free to move in the aqueous solvent. This explains how methylamine is protected against nucleophilic attack by water molecules. In this situation, the T -protein associated with tetrahydrofolate must be in close contact with the H­protein to facilitate the nucleophilic attack of the methylamine group by the N-5 atom of the pterin ring, leading to methylenetetrahydrofolate formation and NH3 release.

3.1.2.2 The source of glycine There are several routes for glycine formation in plants. First, glycine

can arise from protein degradation during protein tum-over. This is probably not an important route for the production of free glycine, except in particular situations such as cell autophagy (Aubert et aI., 1996). An important route leading to glycine synthesis is the serine to glycine conversion, catalyzed by SHMT, an enzyme presents in all the

4. Folate metabolism in plants 77

cell compartments (see below). This reaction is probably a significant source of glycine in non-photosynthetic tissues and results, as for glycine oxidation, in methylenetetrahydrofolate formation. If glycine is further metabolized through GDC, the coupling of these two reactions allows the formation of two C 1 units from one serine. An intriguing question is th e fate of methylenetetrahydrofolate when the SHMT reaction is aimed at glycine production. Indeed, methylenetetrahydrofolate must be recycled back to tetrahydrofolate to sustain the reaction. One might postulate that tetrahydrofolate recycling is made through C 1 metabolism. However, if glycine production exceeds the demand for Cl units, the methylene group of methylenetetrahydrofolate must be removed in another way to discharge the cofactor. In animal cells, the C 1 unit can be oxidized to CO2, probably by conversion of methylenetetrahydrofolate to methenyl- then 10-formyltetrahydrofolate, which is in tum the substrate for 10-formy ltetrahydrofolate dehydrogenase (MacKenzie, 1984). However, the presence of this last enzyme in higher plant cells remains, to be clearly established.

Another important pathway leading to glycine synthesis involves glyoxylate aminotransferases in peroxisomes (Keys, 1980). Thus, the origin of glycine in this pathway is connected with the origin of glyoxylate (see above, the source of formate). In non-green tissues, amination of glyoxylate seems to depend mainly on alanine and glutamate (Cossins and Sinha, 1965). In leaf peroxisomes, this transamination reaction is part of the photorespiratory cycle, and glutamate and serine are probably both involved as amino group donors (Tolbert, 1980). Serine-glyoxylate and glutamate-glyoxylate aminotransferases catalyze effectively irreversible reactions and the conversion of glyoxylate to glycine is thus unidirectional (Tolbert, 1980). In 'standard' (20°C; 175 W m-2; 330 ppm CO2) atmospheric conditions, the flux of carbon through photorespiration almost equals the flux through photosynthesis, thus making the photorespiratory cycle a major metabolite pathway in illuminated leaves. For example, assuming a rate of gross O2 evolution of 2 J.lmoles min-I mg-I chlorophyll and a photorespiratory O2 uptake of about 33% the rate of gross O2 evolution (Gerbaud and Andre, 1979), it can be calculated that the approximate rate of glycine production is 40 J.lmol h-I mg-I chlorophyll (that is about 4.5 g glycine h-I kg-I leaves). In comparison, assuming a net photosynthesis of about 55% the rate of gross O2 evolution (Gerbaud and Andre, 1979), a similar calculation indicates that the rate of sucrose synthesis is 6 J.lmol h-I mg-I chlorophyll (that is about 3 g sucrose h-I kg-I leaves). Clearly, the photorespiratory pathway produces large amounts of glycine that must be recycled back to glycerate (Waidyanatha et ai., 1975) in order to limit the draining of Benson-Calvin cycle intermediates. This recycling is

78 Chapter 4

ensured by the photorespiratory cycle, via serine and hydroxypyruvate (Tolbert, 1980). The conversion of glycine to serine is catalyzed by SHMT and requires the addition of one C 1 unit, which derives from the oxidation of a second molecule of glycine by GDC (Figure 6). Because of the high flux of glycine, the availability of tetrahydrofolate for the operation of the glycine cleavage system is a critical factor for glycine oxidation and appears as a limiting step for the whole system (Bourguignon et aI., 1988; Rebeille et aI., 1994). Glycine to serine conversion is not the thermodynamic direction of the reaction, which is continuously driven toward the production of serine as a result of the high methylenetetrahydrofolate concentration resulting from GDC activity (RebeilIe et aI., 1994). Taking into account these observations, one may question whether methylenetetrahydrofolate produced during the course of glycine oxidation is available for C 1 metabolism. Indeed, its withdrawal from glycine to serine conversion by SHMT would further limit glycine to glycerate recycling. This recycling is a requirement of the photorespiratory pathway and it was observed that chemical inhibition of either glycolate oxidase or SHMT resulted in a marked inhibition of the whole photosynthetic activity (Servaites and Ogren, 1977). From a dynamic point of view, it may be considered that each molecule of glycerate which is not recycled into the Benson-Calvin cycle must be replaced by a newly photosynthesized molecule, at the expense of sucrose synthesis. However, considering the high level of carbon fixation in illuminated leaves, it is very likely that in most conditions, photosynthesis could largely sustain the comparatively small requirements of Cl metabolism. The situation might be different in stress conditions, such as drought, where photosynthesis is low and photorespiration is high because of the closure of the stomata.

3.1.3 Serine

3.1.3.1 The serine hydroxymethyltransferase Serine is the major source of one-carbon units in most organisms

(Schirch, 1984). Its 3-carbon is transferred to tetrahydrofolate to generate methylenetetrahydrofolate and glycine in a reversible reaction catalyzed by SHMT (see above, equation 2). This enzyme has been purified from bacteria, plants and several mammalian sources (for a review, see Schirch, 1984). The plant enzyme, like its bacterial and mammalian counterparts, requires pyridoxal phosphate as coenzyme (Mazelis and Liu, 1967), has a native molecular weight of about 200 kDa and is a tetramer of identical subunits (Besson et aI., 1995; Bourguignon et aI., 1988; Turner et aI., 1992a). The role of

4. Folate metabolism in plants 79

methylenetetrahydrofolate (or tetrahydrofolate) is to release (or trap) formaldehyde at the active site (the one-carbon unit of methylenetetrahydrofolate and formaldehyde are at the same oxidation level). Although formaldehyde can be released from methylenetetrahydrofolate via a reversible non-enzymic reaction (equation 4) (Osborn at aI., 1960).

(4)

Free formaldehyde is apparently not an intermediate of the SHMT reaction. Indeed, detailed kinetic studies of the SHMT reaction suggest that transfer of formaldehyde from serine to tetrahydrofolate involves a mechanism in which formaldehyde is attached to a sulfhydryl group of the active site to form a thiohemiacetal intermediate (Schirch, 1984). Initial velocity patterns indicate a sequential random mechanism (Schirch et aI., 1977) and differential scanning calorimetry studies suggest that the enzyme exists as an equilibrium between an 'opened' and a 'closed' form (Schirch et aI., 1991): glycine and serine enter and leave the enzyme in the open form but catalysis occurs in the closed form. Although the interconversion of serine and glycine is fully reversible, the equilibrium distribution of the various substrates shows that the reaction favours serine to glycine conversion (Schirch et aI., 1977). In plants, the rate constant of serine to glycine conversion by SHMT is about 15 times higher than the rate constant of the reverse direction (Besson et aI., 1993). Tetrahydrofolate binds more tightly to SHMT than methylenetetrahydrofolate since the Kd value of the former compound is 14 times lower than the Kd value of the latter (Rebeille et aI., 1994). The Km value for serine is approximately 1-1.5 mM (Besson et aI., 1995), and glycine is a competitive inhibitor of serine (K j = 1.9 mM). As already stated, the plant enzyme, like SHMT from other sources, displays greater affinity for the polyglutamate forms of tetrahydrofolate than for the corresponding monoglutamate (Km for H4PteGlu], 37 flM; Km H4PteGlus, less than 3.5 flM) (Besson et aI., 1993).

The intracellular distribution of SHMT has also been investigated. In mammalian cells, the enzyme is present in both cytosol and mitochondria and the two isoenzymes have been purified (Strong et aI., 1990). In plants, the enzyme has also been purified from mitochondria (Besson et aI., 1995; Bourguignon et aI., 1988; Turner et aI., 1992a). Its presence in plastids has been suggested by several authors (Garde strom et aI., 1985; Shah and Cossins, 1969; Walton and Woolhouse, 1986) and the chloroplastic isoform was recently successfully purified (Besson et aI., 1995). Furthermore, using a gentle protoplast rupture technique and various marker enzymes to estimate the cross-contamination of the

80 Chapter 4

different cell compartments, these last authors demonstrated the presence of a cytosolic isoform. Thus, SHMT was present in the three main compartments of leaf cells (Table 2). In these tissues the mitochondrial activity represented about 50% of the cell activity whereas chloroplastic and cytosolic activity each represented 20-25% (Besson et aI., 1995). However it is likely that the asymmetric distribution observed in this tissue reflected the functional duality of the enzyme. Indeed, a high SHMT activity is required in the mitochondria from green leaf tissues in order to keep pace with the high glycine decarboxylase activity (Neuburger et aI., 1986; R6beill6 et aI., 1994). In non-green tissues the situation is probably different and it was repeatedly observed in purified mitochondria from etiolated or storage (potato tubers) tissues, which have almost no glycine cleavage system, that SHMT specific activity was low, about 10 to 20% of the activity found in green leaf mitochondria (Besson et aI., 1995). In non-green tissues it is likely that the mitochondrial SHMT accounts for only a minor part of the total cell activity, as is the case in animal cells where the cytosolic isoform predominates over its mitochondrial counterpart (Matthews et aI., 1982; Schirch and Peterson, 1980). The presence of SHMT in each cellular compartment reflects the physiological importance of this reaction. Indeed, serine is a main source of methylenetetrahydrofolate, which is later converted into methenyl and formyltetrahydrofolate. These one­carbon folates are an absolute requirement for nucleotide synthesis.

3.1.3.2 The source of serine Serine originates de novo from a 'glycolytic' or 'phosphorylated'

pathway, involving the conversion of 3-phosphoglycerate to serine via 3-phosphohydroxypyruvate and 3-phosphoserine. This is the main route in animals and yeast when glucose is available as a carbon source (Melcher and Entian, 1992; Snell, 1986; Ulane and Ogur, 1972). In higher plants, this pathway is also a main route for serine synthesis, especially in non photosynthetic tissues (germinating seeds, root apices) associated with rapid cell proliferation (Cheung et aI., 1968; Walton and Woolhouse, 1986). In photosynthetic tissues, the three enzymes required for the synthesis of serine from 3-phosphoglycerate are also present (Cheung etaI., 1968; Walton and Woolhouse, 1986). In these tissues this 'phosphorylated' pathway has been localized in chloroplasts, but a parallel pathway outside the chloroplasts also seems to be operative (Larsson and Albertsson, 1979). A second possible route for serine formation proceeds through non-phosphorylated intermediates via glyoxylate and glycine, the last step involving glycine to serine conversion by SHMT. Thus, the source of serine in this pathway is the same as the source of glycine (see above, the source of glycine). When

4. Folate metabolism in plants 81

serine is produced from glycine, it cannot generate net synthesis of one­carbon units since its formation requires the addition of one-carbon group. However, if glycine to serine and serine to glycine conversions take place in different subcellular compartments, the associated cyclic pathway would result in the transfer of one-carbon units from one compartment to another. During the photorespiratory process, serine is probably not a source of one-carbon units in mitochondria because the mitochondrial SHMT is continuously pushed toward the production of serine by high glycine and methylenetetrahydrofolate concentrations. Thus, to generate one-carbon units in photorespiratory conditions, serine must exit mitochondria to be converted into glycine and methylenetetrahydrofolate by the cytosolic or chloroplastic SHMT.

3.2 Interconversion of Cl units

3.2.1 The methylenetetrahydrofolate dehydrogenase­cyclohyd rolase

As shown in Figure 2, 5,1 O-methylenetetrahydrofolate, 5,10-methenyltetrahydrofolate and 10-formyltetrahydrofolate are readily interconvertible, thus providing an equilibrium between the pool of C 1 units at the formyl and methylene levels of oxidation. These interconversions are catalyzed by the methylenetetrahydrofolate dehydrogenase and the methenyltetrahydrofolate cyclohydrolase. In E. coli, the dehydrogenase and cyclohydrolase activities are associated (for a review, see MacKenzie, 1984) but this is not the case for all prokaryotes and in Clostridium cylindrosporum, for example, dehydrogenase and cyclohydrolase are separate proteins (Uyeda and Rabinowitz, 1967). The equilibrium constant of the dehydrogenase reaction was estimated by Uyeda and Rabinowitz, (1967) to be Keq = 0.14 (Keq = [5,10-CH­H4PteGluo][NADPH]/[5,10-CHrH4PteGluo][NADP+]). The equilibrium of the cyclohydrolase reaction lies in the direction of 10-formyltetrahydrofolate at neutral pH and proceeds both enzymically and non-enzymically (MacKenzie, 1984). In mammals and yeast, the NADP­dependent dehydrogenase and the cyclohydrolase activities are part of a trifunctional enzyme, also containing 10-formyltetrahydrofolate synthetase activity (Paukert and Rabinowitz, 1980) (see Figure 2). In these organisms, both mitochondria and cytosol possess this interesting trifunctional protein (McNeil et aI., 1996). This cellular distribution is consistent with the known roles of formyl-, methenyl- and methylenetetrahydrofolate in folate mediated one-carbon metabolism,

82 Chapter 4

such as the synthesis of purines and thymidylate and the synthesis of formylmethionyl-tRNA (Figure 7). The dehydrogenase domain of this trifunctional protein catalyzes an ordered reaction where NADP binds first and methenyltetrahydrofolate is released last (Cohen and MacKenzie, 1978). Methenyltetrahydrofolate, the product of the dehydrogenase reaction, is preferentially converted into 10-formyltetrahydrofolate rather than equilibrating with the bulk medium, suggesting a strong interaction between the dehydrogenase and cyclohydrolase catalytic sites (Cohen and MacKenzie, 1978). Interestingly, beside the cytosolic and mitochondrial trifunctional isoenzymes, there is also in S. cerevisiae an NAD-dependent methylenetetrahydrofolate dehydrogenase activity in the cytosol (West et aI., 1993, 1996). In contrast to the other methylenetetrahydrofolate dehydrogenases, this enzyme is a monofunctional homodimer with a subunit molecular mass of 36 kDa. Investigations into the metabolic role of the two cytosolic dehydrogenases suggested that they are probably not truly redundant. On the contrary, their presence in the same compartment possibly reflects the ability of the cell to respond to changing anabolic needs. The NAD+-dependent enzyme only plays a catalytic role in the oxidation of cytosolic one-carbon units, which are involved in de novo purine synthesis, whereas the NADP+ -dependent enzyme allows the interconversion of one-carbon units toward either the more oxidized form for purine synthesis or the more reduced form for methyl group generation and thymidylate synthesis (West et aI., 1996).

In plants, the interconversion between formyl- and methylenetetrahydrofolate is catalyzed by a bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, distinct from the formyltetrahydrofolate synthetase (Kirk et aI., 1995; Nour and Rabinowitz, 1991). This bifunctional enzyme was studied in detail by Kirk et al. (1995). SDS-PAGE analysis of the pea enzyme suggests that the protein is constituted of 38.5 kDa subunits. The reported apparent Km values of the dehydrogenase and cyclohydrolase reactions are 11 11M for NADP+, 21 11M for methylenetetrahydrofolate and 7 11M for methenyltetrahydrofolate. In contrast with most folate-dependent enzymes, the dehydrogenase reaction displays similar affinities for the mono and penta forms of the folate substrate. Interestingly, dihydrofolate appears as a competitive inhibitor of the folate substrates in both the dehydrogenase and the cyclohydrolase reactions and its effect increases with the length of the glutamate chain (Kirk et aI., 1995). This study also suggests that the active domains of the pea cotyledon bifunctional protein are kinetically interdependent and possibly share a common folate binding site, as already proposed for the eukaryotic trifunctional protein (MacKenzie,

4. Folate metabolism in plants

.. methionine

.........•

serine

thymidylate

... purines ..........

~.,

fonnylmethionyl-tRNA

... purines

83

Figure 7: Schematic representation showing the major utilization of one-carbon units in folate mediated reactions.

Pi + PPi ATP

~ ) . ® I . ~ methionine senne g ycme

SAM Q) t H4FGlun ~ CD CHr H4FGlun

SAH 0 CH3-H4FGlun r:: ~ homocysteine NADPH

t - NADP Q) adenosine

x

Figure 8: Schematic representation of the role of methionine in methylation reactions. I, methionine synthase; 2, methionine adenosyltransferase; 3, methyltransferases; 4, adenosylhomocysteinase; 5, methylenetetrahydrofolate reductase; 6, serine hydroxymethyltransferase.

84 Chapter 4

1984). The bifunctional enzyme was mainly found in the cytosolic compartment (Kirk et aI., 1995), but some activity was also localized in mitochondria (Neuburger et aI., 1996; Suzuki and Iwai, 1973, 1974) and chloroplasts (Neuburger et aI., 1996). The presence of this enzyme in the three main compartments of the plant cell matches the cellular distribution of SHMT. This is logical because as serine is probably the major source of one-carbon units, the combination of these various activities can supply each compartment with the one-carbon substituted folates required for their nucleotides and formylmethionyl-tRNA synthesis.

3.2.2 The methylenetetrahydrofolate reductase

Most of the biological methylation reactions require the methyl donor S-adenosylmethionine (SAM). This compound derives from methionine which is, in tum, synthesized from homocysteine (Figure 8). The methyl group for methylation of homocysteine comes from 5-methyltetrahydrofolate, a folate compound arising from reduction of 5,10-methylenetetrahydrofolate. Methyltetrahydrofolate has no other known metabolic fate. Reduction of methylenetetrahydrofolate to methyltetrahydrofolate is catalyzed by methylenetetrahydrofolate reductase (Figure 2). The mammalian and bacterial enzymes differ substantially in their properties. In the acetogenic bacteria Clostridium jormicoaceticum, the enzyme has a native molecular weight of about 237 kDa and consists of four each of two different subunits having the molecular weights of 26 and 35 kDa. This is an iron-sulfur flavoprotein that also contains zinc and needs ferredoxin or FAD as a source 0 f reducing equivalents (Clark and Ljungdahl, 1984). In animals, the enzyme is a homodimer of 77 kDa subunits (Daubner and Matthews, 1982). It is also a flavoprotein but, in contrast to bacteria, NADPH is the preferred substrate for reduction of methylenetetrahydrofolate. Each subunit consists of two spatially distinct domains: an N-terminal domain of 40 kDa and a C-terminal domain of 36 kDa (Matthews et aI., 1984). The N­terminal part of the protein is the regulatory domain containing a binding site for SAM, a potent allosteric inhibitor of the reaction (Sumner et aI., 1986). The catalytic reaction proceeds in two steps. First, the enzyme­bound flavin is reduced by NADPH, and because the redox potential of the enzyme-bound flavin is far above the one of NADPH, this reaction is almost irreversible (Daubner and Matthews, 1982) (Equation (5».

(5)

4. Folate metabolism in plants 85

Second, the reduced enzyme is oxidized by methylenetetrahydrofolate to give the methyl compound (Equation (6».

(6)

In contrast to the first step, oxidized and reduced forms of the enzyme­bound flavin are in equilibrium with the methylenetetrahydrofolate/ methyltetrahydrofolate couple, and this reaction is reversible. Thus, although the physiological reaction catalyzed by methylenetetrahydrofolate reductase is irreversible, this enzyme can catalyze the oxidation of methyltetrahydrofolate in the presence of a high potential acceptor like menadione. This property is utilized to measure the enzyme activity in crude extracts (Matthews, 1986). Like other folate-dependent enzymes, methylenetetrahydrofolate reductase presents a higher affinity for the polyglutamate forms of folate. Indeed, the K.n value for methylenetetrahydrofolate decreases from 7 to 0.1 !JM when the number of glutamate residues increases from 1 to 6 (Matthews, 1984). Interestingly, dihydrofolate is also a potent inhibitor of the reaction (Ki = 6.5 J.LM for the monoglutamate form) and the inhibition increases with the length of the glutamate chain. In mammalian tissues, this activity is mainly localized in the cytosol (Matthews, 1984).

Methylenetetrahydrofolate reductase activity was also detected in plants such as carrot (Fedec and Cos sins, 1976a, 1976b) and Lemna minor (Wong and Cossins, 1976). However, the catalytic properties of this enzyme were not studied, and it is not clear whether the plant enzyme is a bacterial type or a mammalian type enzyme. Likewise, the cellular distribution of this activity is not yet elucidated. Because methyltetrahydrofolate is found in all the cellular compartments (see above, subcellular distribution of folates) it is tempting to think that methylenetetrahydrofolate reductase is also present in all compartments. Taking into account that methyltetrahydrofolate is only associated with the synthesis of methionine, one may speculate that methylenetetrahydrofolate reductase and methionine synthase are closely associated. Apparently, plants do not contain cobalamin and methionine synthesis in these organisms is catalyzed by a cobalamin-independent methionine synthase. This enzyme was purified recently from Catharanthus roseus and immunoblot experiments indicated that it was localized in the cytosolic fraction (Eichel et aI., 1995). The cellular distribution was also examined in pea and barley leaf tissues (Wallsgrove et aI., 1983), and it was observed that most of the activity (84%) was in the cytosolic fraction. However, previous reports indicated that synthesis of methionine also occured in isolated mitochondria (Clandinin and Cossins, 1974) and chloroplasts (Shah and Cossins, 1970) from other

86 Chapter 4

plants. The situation is even more complex in Euglena gracilis, where a cobalamin-independent activity was found only in the cytosol, and a cobalamin-dependent activity was found in the three compartments (69% cytosolic; 18% chloroplastic; 10% mitochondrial) (Isegawa et al., 1994). The different nature of these enzymes, or their asymmetric cellular distribution, might reflect different physiological roles. Indeed, one must distinguish between the net synthesis of methionine involved in protein metabolism, and the recycling of homocysteine back to methionine following the methyl transfer reactions (Figure 8). The net synthesis of methionine, from cysteine and phospho-homoserine, depends on three reactions. The first reaction is catalyzed by the cystathionine y-synthase to generate cystathionine and is exclusively located in plastids (Ravanel et al., 1995; Wallsgrove et al., 1983). The second step, catalyzed by the cystathionine l3-lyase, results in homocysteine formation and is present in the chloroplasts and the cytosol (Droux et al., 1995). However, the kinetic properties of the cytosolic protein are very different from the ones of the plastidial isoenzyme (Ravanel et al., 1996). In particular, the Km of the cytosolic enzyme for cystathionine is very high, about 10 mM, which suggests that this isoenzyme might have a role other than methionine synthesis. Thus, it would be logical to think that the third and final step of net methionine synthesis is also localized in plastids and that chloroplasts play a major role in net methionine synthesis, as they do for the synthesis of other amino acids. In contrast, methyl transfer reactions are needed in each cellular compartment and it is likely that homocysteine is produced in these compartments. Recycling of homocysteine back to methionine is a requirement for the continuous operation of the methyl transfer reactions. Taking into account that homocysteine, like cysteine, is a rather unstable molecule, it is tempting to postulate that methionine synthase and methylenetetrahydrofolate reductase are also present in each cellular compartment. Clearly, the localizations of methyltetrahydrofolate synthesis and methionine synthesis require further investigation.

4. CONCLUSION

One-carbon substituted folates, with various degrees of oxidation, are required in the different cellular compartments for glycine, serine, purine, thymidylate, methionine and formylmethionyl-tRNA synthesis. In higher plants, all the cellular compartments contain folate derivatives and the enzymic machinery, that is SHMT and methylene-tetrahydrofolate dehydrogenase/cyclohydrolase, involved III the

4. Folate metabolism in plants 87

generation and interconversion of these one-carbon substituted folates. However, the intracellular localization of methylenetetrahydrofolate reductase and methionine synthesis remains unresolved. In contrast, synthesis of the backbone of the cofactor, that is tetrahydrofolate, from 6-hydroxymethyldihydropterin appears to be mainly restricted to mitochondria. This assumption is based on biochemical and molecular biology data for the two first steps of the pathway and on biochemical data only for the three remaining steps. If this assumption (summarized in Figure 9) holds true, this implies that tetrahydrofolate molecules synthesized within mitochondria are exported to the cytosol and the chloroplasts. Such a situation would be very different from that encountered in animal cells. In these cells, folates circulating in the blood vessels are taken up by specific folate receptors (Antony, 1996; Rijnboutt et aI., 1996) and transported into the cytosol. Once in the cytosol, the folate compounds are either polyglutamylated or directed toward the intracellular compartments (mitochondria). Several reports indicate that tetrahydrofolate is not transported through the mitochondrial membranes (Cybulski and Fisher, 1981; Home et aI., 1989), whereas folic acid or dihydrofolate are rapidly transported (Cybulski and Fisher, 1981). These results suggest that in animals, mitochondria receive folate in an oxidized form which must then be reduced in the matrix space. These compounds are thereafter polyglutamylated, to ensure their retention, and charged with one-carbon units (Appling, 1991; McGuire and Bertino, 1981). In higher plants the situation would be almost the opposite since tetrahydrofolate must exit mitochondia to supply cytosol and chloroplasts. Release of mitochondrial folylpolyglutamates in the cytosol has been observed in animal cells (Kim and Shane, 1994), but there is no evidence to date that in plants, mitochondrial tetrahydrofolate is transported toward the cytosol. It is certain that the demonstration of this process, and the comprehension of how the cell regulates its folate requirements between the cytosol, the mitochondria and the chloroplasts, are great challenges for future research in plant folate metabolism.

Finally, there is the question of the origin of the substrates specifically involved in folate synthesis such as 6-hydroxymethyldihydropterin and p-aminobenzoic acid. Indeed, the only known metabolic fate of p­aminobenzoic acid is folate synthesis. Thus, the pathway involved in the generation of this compound must be tightly controlled by folate metabolism. p-aminobenzoic is synthesized from chorismate, a branch point in the aromatic amino acid pathway which has been localized in plastids (Herrmann, 1995). However, the subcellular site of p­aminobenzoic acid formation remains to be determined. Likewise, the subcellular localization of 6-hydroxymethyldihydropterin synthesis is not

88 Chapter 4

CYTOSOL PLASTJDS

dTMP ..

r® .,/ ; ............ ~

dTMP • ....... :? \ ? \.J ?

H,}'lerin

/IJ.CHO-H'pleG/u • ...................... .......

... ,::~ ..... . .. / ..... PABA-:::::::::=:t-===-~::...

.......... / ..... .

"'"

CD CD .; AMP ATP ;

01" ATP pABA t .J....J ~) • H,PleGlu, '? ~ HJI'Ieroale ~ H,PrerlnPP/' M H,Pltrin

F.:~~~~=r {~~~.:~ - _.) H.PreGlu, ~ H.PreG/u. • ~ CH,-H.PteG/u.

ATP n 01" i ?"'\ t=. ADP CD ; Olycine CD co, + H, CD

,.... NADPH .... . ..... .

cFt -H.pleG/a.

!® MITOCHONDRIA IIJ.CHO-H.PltG/u.

Figure 9: Proposed subcellular distribution in higher plants of the enzymes involved in tetrahydrofolate synthesis and interconversion of one-carbon substituted folate coenzymes. 1, dihydropterin pyrophosphokinase; 2, dihydropteroate synthase; 3, dihydrofolate synthetase; 4, dihydrofolate reductase; 5, folylpolyglutamate synthetase; 6, serine hydroxymethyltransferase; 7, glycine decarboxylase; 8, thymidylate synthase; 9, methylenetetrahydrofolate dehydrogenase; 10, cyclohydrolase.

4. Folate metabolism in plants 89

known in higher plants. The synthesis of these metabolites and their eventual transport across the mitochondrial membranes are also possible factors of regulation for folate synthesis.

REFERENCES

Allegra, C.J, Boarman, D., Kovacs, J.A, Morrison, P., Beaver, J., Chabner, B.A. and Masur, H. (1990). Interaction of sulfonamide and sulfone compounds with Toxoplasma gondii dihydropteroate synthase. Journal of Clininical Investigation, 85, 371-379.

Angier, RB., Boothe, J.H., Hutchings, B.L., Mowat, J.H., Semb, J., Stokstad, E.L.R, Subbarow, Y., Walles, c., Cosulich, D.B., Fahrenbach, M.J., Hultquist, M.E., Kuh, E., Northey, E.H., Seeger, D.R, Sickels, J.P. and Smith, Jr, J.M. (1946). The stucture and synthesis of the liver L. casei factor. Science, 103, 667-669.

Antony, A.C. (1996). Folate receptors. Annual Review of Nutrition, 16,501-521. Appling, D.R. (1991). Compartmentation of folate-mediated one-carbon metabolism in

eukaryotes. FASEB Journal, 5, 2645-2651. Aubert, S., Gout, E., Bligny, R., Marty-Mazars, D. Barrieu, F., Alabouvette, J., Marty, F.

and Douce, R. (1996). Ultrastructural and biochemical characterization of autophagy in higher plant cells subjected to carbon deprivation: control by the supply of mitochondria with respiratory substrates. Journal of Cell Biology, 133, 1251-1263.

Bachmann, B. and Follmann, H. (1987). Deoxyribonucleotide biosynthesis in green algae: characterization of thymidylate synthase-dihydrofolate reductase in Scenedesmus obliquus. Archives of Biochemistry and Biophysics, 256, 244-252.

Besson, V., Neuburger, M., Rebeille, F. and Douce, R (1995). Evidence for three serine hydroxymethyltransferases in green leaf cells. Purification and characterization of the mitochondrial and chloroplastic isoforms. Plant Physiology and Biochemistry, 33, 665-673.

Besson, V., Rebeille, F., Neuburger, M., Douce, R and Cossins, E.A (1993). Effects of tetrahydrofolate polyglutamates on the kinetic parameters of serine hydroxymethyitransferase and glycine decarboxylase from pea leaf mitochondria. Biochemical Journal, 292, 425-430.

Bertrand, R. and Jolivet, J. (1989). Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-forrnyitetrahydrofolate polyglutamates. Journal of Biological Chemistry, 264, 8843-8846.

Beverley, S.M., Ellenberg, T.E. and Cordingley, J.S. (1986). Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proceedings of the National Academy of Sciences USA, 83, 2584-2588.

Blakley, RL. (1984). Dihydrofolate reductase. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 1 (pp., 191-253). Wiley (Interscience), New York.

Blondeau, R. (1973). Les derives pteroylglutamiques des plantules de Brassica napus oleifera. Planta, 114, 95-100.

Bognar, AL., Osborne, C. and Shane, B. (1987). Primary structure of the Escherichia coli folC gene and its folylpolyglutamate synthetase-dihydrofolate synthetase product and regulation by an upstream gene. Journal of Biological Chemistry, 262, 12337-12342.

90 Chapter 4

Bognar, AL., Osborne, C., Shane, B., Singer, S.C. and Ferone, R. (1985). Folylpoly-y­glutamate synthetase. Cloning and high expression of the Escherichia coli folC gene and purification and properties of the gene product. Journal of Biological Chemistry, 260, 5625-5630.

Bourguignon, J., Merand, V., Rawsthorne, S., Forest, E. and Douce, R. (1996). Glycine decarboxylase and pyruvate dehydrogenase complexes share the same dihydrolipoamide dehydrogenase in pea leaf mitochondria: evidence from mass spectrometry and primary-structure analysis. Biochemical Journal, 313, 229-234.

Bourguignon, J., Neuburger, M. and Douce, R. (1988). Resolution and characterization of the glycine-cleavage reaction in pea leaf mitochondria. Biochemical Journal, 255, 169-178.

Bourguignon, 1., Vauclare, P., Merand, V., Forest, E., Neuburger, M. and Douce, R. (1993). Glycine decarboxylase complex from higher plants. Molecular cloning, tissue distribution and mass spectrometry analyses of the T-protein. European Journal of Biochemistry, 217, 377-386.

Brown, G.M. (1985). Biosynthesis ofpterins. In: Blakley, R.L. and Benkovic, S.1., (Eds). Folates and pterins Vol. 1 (pp. 115-154). Wiley (Interscience), New York.

Brown, S.S., Neal, G.E. and Williams, D.C. (1965). Subcellular distribution of some folic acid-linked enzymes in rat liver. Biochemical Journal, 97, 34c-36c.

Cella, R., Nielsen, E. and Parisi, B. (1988). Daucus carota cells contain a dihydrofolate reductase: thymidylate synthase bifunctional polypeptide. Plant Molecular Biology, 10, 331-338.

Chan, P.Y., Coffin, J.W. and Cossins, E.A (1986). In-vitro synthesis ofpteroylpoly-y­glutamates by cotyledon extracts of Pisum sativum L. Plant and Cell Physiology, 27, 431-441.

Chan, P.Y., Dale, P.L. and Cossins, E.A (1991). Purification and properties of Neurospora folylpolyglutamate synthetase. Phytochemistry, 30, 3525-3531.

Cheung, G.P., Rosenblum, l.Y. and Sallach, H.1. (1968). Comparative studies of enzymes related to serine metabolism in higher plants. Plant Physiology, 43, 1813-1820.

Cichowicz, D.1. and Shane, B. (1987). Mammalian folylpoly-y-glutamate synthetase. 1. Purification and general properties of the hog liver enzyme. Biochemistry, 26, 504-512.

Clandinin, M.T. and Cossins, E.A (1972). Localization and interconversion of tetrahydropteroylpolyglutamates in isolated pea mitochondria. Biochemical Journal, 128, 29-40.

Clandinin, M.T. and Cossins, E.A. (1974). Methionine biosynthesis in isolated Pisum sativum mitochondria. Phytochemistry, 13, 585-591.

Clark, J.E. and Ljungdahl, L.G. (1984). Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum. Journal of Biological Chemistry, 259, 10845-10849.

Coffin, J.W. and Cos sins, E.A. (1986). Mitochondrial folates and methionyl-tRNA transformylase activity during germination and early growth of seeds. Phytochemistry, 25, 2481-2487.

Cohen, L. and MacKenzie, R.E. (1978). Methylenetetrahydrofolate dehydrogenase­methenyltetrahydrofolate cyclohydrolase- formyltetrahydrofolate synthetase from porcin liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme. Biochimica et Biophysica Acta, 522, 311-317.

Cohen-Addad, C., Pares, S., Sieker, L., Neuburger, M. and Douce, R. (1995). The lipoamide arm in the glycine decarboxylase complex is not freely swinging. Nature Structural Biology, 2, 63-68.

4. Folate metabolism in plants

Colas des Francs-Small, C., Ambard-Breteville, F., Small, l.D. and Remy, R. (1993). Identification of a major soluble protein in mitochondria from nonphotosynthetic tissues as NAD-dependent formate dehydrogenase. Plant Physiology, 102, 1171-1177.

Cooper, T.G. and Beevers, H. (1969). ~-oxidation in glyoxysomes from castor bean endosperm. Journal of Biological Chemistry, 244, 3514-3520.

91

Cossins, E.A. (1964). The utilization of carbon-1 compounds by plants. l. The metabolism of methanol-14C and its role in amino acid biosynthesis. Canadian Journal of Biochemistry, 42, 1793- I 802.

Cossins, E.A (1984). Folates in biological materials. In: Blakley, RL. and Benkovic, S.1., (Eds). Folates and pterins Vol. 1 (pp. 1-59). Wiley (lnterscience), New York.

Cossins, E.A and Chan, P.Y. (1984). Folypolyglutamate synthetase activities of Neurospora. Phytochemistry, 23, 965-971.

Cossins, E.A. and Shah, S.P.J. (1972). Pteroylglutamates of higher plant tissues. Phytochemistry, 11, 587-593.

Cossins, E.A and Sinha, S.K. (1965). Occurrence and properties of L-amino acid:2-glyoxylate aminotransferase in plants. Canadian Journal of Biochemistry, 43, 495-506.

Crosti, P. and Bianchetti, R (1983). Identification and cell level of folate derivatives from growing cultures of streptomycin-bleached Euglena gracilis. Plant Science Letters, 31, 205-214.

Curthoys, N. and Rabinowitz, J.C. (1972). FormyItetrahydrofolate synthase. Binding of folate substrates and kinetics of the reverse reaction. Journal of Biological Chemistry, 247, 1965-1971.

Cybulski, RL. and Fisher, R.R. (1981). Uptake of oxidized folates by rat liver mitochondria. Biochimica et Biophysica Acta, 646, 329-333.

Dallas, W.S., Gowen, J., Ray, P.H., Cox, J. and Dev, l.K. (1992). Cloning, sequencing, and enhanced expression of the dihydropteroate synthase gene of Escherichia coli MC4100. Journal of Bacteriology, 174, 5961-5970.

Daubner, S.C. and Matthews, R.G. (1982). Purification and properties of methylenetetrahydrofolate reductase from pig liver. Journal of Biological Chemistry, 257, 140-145.

Day, P.L., Mims, V., Totter, J.R, Stokstad, E.L.R., Hutchings, B.L. and Sloane N.H. (1945). The successful treatment of vitamin M deficiency in the monkey with highly purified Lactobacillus casei factor. Journal of Biological Chemistry, 157, 423-424.

Douce, R. (1985). Mitochondria in higher plants: structure, function and biogenesis. Academic Press, New-York.

Droux, M., Ravanel, S. and Douce, R. (1995). Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine ~-Iyase from spinach chloroplasts. Archives of Biochemistry Biophysics, 316, 585-595.

Eichel, J., Gonzales, J., Hotze, M., Matthews, R.G. and SchrOder, J. (1995). Vitamin B12-

independent methionine synthase from a higher plant (Catharanthus roseus). Molecular characterization, regulation, heterologous expression, and enzyme properties. European Journal of Biochemistry, 230, 1053-1058.

Fall, R and Benson, AA. (1996). Leafmethanol- the simplest natural product from plants. Trends in Plant Sciences, 1, 296-301.

Fedec, P. and Cossins, E.A. (1976a). Synthesis of folate derivatives in aerated storage tissue disks. Phytochemistry, 15, 359-362.

Fedec, P. and Cossins, E.A (1976b). Effects of giberellic acid and L-methionine on C1 metabolism in aerated carrot disks. Phytochemistry, 15, 1819-1823.

92 Chapter 4

Ferone, R. and Roland, S. (1980). Dihydrofolate reductase:thymidylate synthase, a bifunctional polypeptide from Crithidia Jasciculata. Proceedings of the National Academy of Sciences USA, 77, 5802-5806.

Gardestrtlm, P., Edwards, G.E., Henricson, D. and Ericson, I. (1985). The location of serine hydroxymethyltransferase in leaves of C3 and C4 species. Physiologia Plantarum, 64, 29-33.

Garrow, T.A, Admon, A. and Shane, B. (1992). Expression cloning of a human cDNA encoding folylpoly(y-glutamate) synthetase and determination of its primary structure. Proceedings of the National Academy of Sciences USA, 89, 9151-9155.

Gerbaud, A and Andre, M. (1979). Photosynthesis and photorespiration in whole plants of wheat. Plant Physiology, 64, 735-738.

Gifford, D.J. and Cossins, E.A (1982). Relationships between glycolate and formate metabolism in greening barley leaves. Phytochemistry, 21, 1485-1490.

Halliwell, B. and Butt, V.S. (1974). Oxidative decarboxylation of glycolate and glyoxylate by leaf peroxisomes. Biochemical Journal, 138, 217-224.

Hogan, AG. and Parrott, E.M. (1940). Anemia in chicks caused by a vitamin deficiency. Journal of Biological Chemistry, 132, 507-517.

Horne, D.W., Patterson, D. and Cook, R.I. (1989). Effect of nitrous oxide inactivation of vitamin B\2-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver. Archives of Biochemistry and Biophysics, 270, 729-733.

Herrmann, K.M. (1995). The shikimate pathway: early steps in the biosynthesis of aromatic compounds. The Plant Cell, 7, 907-919.

Hopkins, K.D. (1996). Coronary artery desease: the folate link. Lancet, 348, 464. Huang, AH.C., Trelease, R.N. and Moore, Jr, T.S. (1983). Plant peroxisomes. Academic

Press, New-York. Hurt, E., Pesold-Hurt, B. and Schatz, G. (1984). The amino-terminal region of an imported

mitochondrial precursor polypeptide can direct cytoplasmic dihydrofolate reductase into the mitochondrial matrix. EMBO Journal, 3, 3149-3156.

Imeson, H.C. and Cossins, E.A (1991a). Higher plant folylpolyglutamate synthetase. I. Purification, stability and reaction requirements of the enzyme from pea seedlings. Journal of Plant Physiology, 138, 476-482.

Imeson, H.C. and Cossins, E.A. (1991b). Higher plant folylpolyglutamate synthetase. II. Some major catalytic properties of the enzyme from Pisum sativum L. Journal of Plant Physiology, 138, 483-488.

Imeson, H.C., Zheng, L. and Cossins, E.A (1990). Folylpolyglutamate derivatives of Pisum sativum L. Determination of polyglutamate chain lengths by high performance liquid chromatography following conversion to p-aminobenzoylpolyglutamates. Plant and Cell Physiology, 31, 223-231.

Ikeda, M. and Iwai, K. (1975). The intracellular localization and stability of the dihydrofolate synthetase in pea seedlings. Journal of Nutrition Science and Vitaminology, 21, 1-6.

Isegawa, Y., Watanabe, F., Kitaoka, S. and Nakano, Y. (1994). Subcellular distribution of cobalamin-dependent methionine synthase in Euglena gracilis Z. Phytochemistry, 35, 59-61.

Iwai, K. and Ikeda, M. (1975). Purification and properties of the dihydrofolate synthetase from pea seedlings. Journal of Nutritional Science and Vitaminology, 21, 7-18.

Iwai, K., Nakagawa, S. and Okinaka, O. (1962). The growth inhibition of the germinating seeds by sulfonamides and its reversal by folic acid analogues. Journal of Vitaminology, 8, 20-29.

4. Folate metabolism in plants

Johnson, J.L. and Rajagopalan, K.V. (1985). Pterins in other metabolic pathways. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 2 (pp. 383-399). Wiley (Interscience), New York.

Kaufman, S. and Kaufinan, E.E. (1985). Tyrosine hydroxylase. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 2 (pp. 251-352). Wiley (Interscience), New York.

93

Kent, S.S. (1972). Photosynthesis in the higher plant Vicia faba. II. The non-Calvin cycle origin of acetate and its metabolic relationship to the photosynthetic origin of formate. Journal of Biological Chemistry, 247, 7293-7302.

Keys, A.J. (1980). Synthesis and interconversion of glycine and serine. In Mitlin, B.J., (Ed). The Biochemistry of plants Vol 5 (pp. 359-374). Academic Press, New York.

Kim, J-S. and Shane, B. (1994). Role of folylpolyglutamate synthetase in the metabolism and cytotoxicity of 5-deazacyclotetrahydrofolate, an antipurine drug. Journal of Biological Chemistry, 269, 9714-9720.

Kirk, C.D., Chen, L., Imeson, H.C., and Cossins, E.A. (1995). A 5,10-methylenetetrahydrofolate dehydrogenase: 5, I O-methenyltetrahydrofolate cyclohydrolase protein from Pisum sativum. Phytochemistry, 39, 1309-1317.

Kirk, C.D., Imeson, H.C., Zheng, L. and Cossins, E.A. (1994). The affinity of pea cotyledon IO-formyltetrahydrofolate synthetase for polyglutamate substrates. Phytochemistry, 35, 291-296.

Kirke, P.N., Daly, L.E., Molloy, A., Weir, D.G. and Scott, J.M. (1996). Maternal folate status and risk of neural tube defects. Lancet, 348, 67-68.

Kisliuk, R.L. (1981). Pteroylpolyglutamates. Molecular and Cellular Biochemistry, 39, 331-345.

Knighton, D.R., Kan, C.C., Howland, E., Janson, c.A., Hostomska, Z., Welsh, K.M. and Matthews, D.A. (1994). Structure of and kinetic channelling in bifunctional dihydrofolate reductase-thymidylate synthase. Nature Structural Biology, I, 186-194.

Kruschwitz, H.L., McDonald, D., Cossins, E.A. and Schirch, V. (1994). 5-formyltetrahydropteroylpolyglutamates are the major folate derivatives in Neurospora crassa conidiospores. Journal of Biological Chemistry, 269, 28757-28763.

Kuhn, D.M. and Lovenberg, W. (1985). Tryptophan hydroxylase. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 2 (pp. 115-154). Wiley (Interscience), New York.

Lacks, S.A., Greenberg, B. and Lopez, P. (1995). A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae. Journal of Bacteriology, 177,66-74.

Larsson, C. and Albertsson, E. (1979). Enzymes related to serine synthesis in spinach chloroplasts. Physiologia Plantarum, 45, 7-10.

Lazar, G., Zhang, H., and Goodman, H.M. (1993). The origin of the bifunctional dihydrofolate reductase-thymidylate synthase isogenes of Arabidopsis thaliana. Plant Journal, 3, 657-668.

Lin, B.F., Huang, R.F.S. and Shane, B. (1993). Regulation offolate and one-carbon metabolism in mammalian cells. III. Role of mitochondrial folylpolY-'Y-glutamate synthetase. Journal of Biological Chemistry, 268, 21674-21679.

Lopez, P., Espinosa, M., Greenberg, B. and Lacks, S.A. (1987). Sulfonamide resistance in Streptococcus pneumoniae: DNA sequence of the gene encoding dihydropteroate synthase and characterization of the enzyme. Journal of Bacteriology, 169, 4320-4326.

MacKenzie, R.E. (1984). Biogenesis and interconversion of substituted tetrahydrofolates. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. I (pp. 255-306). Wiley (Interscience), New York.

94 Chapter 4

Macherel, D, Lebrun, M., Gagnon, J., Neuburger, M. and Douce, R (1990). Primary structure and expression of H-protein, a component of the glycine cleavage system of pea leaf mitochondria. Biochemical Journal, 268, 783-789.

Matthews, R.G. (1984). Methionine biosynthesis. In: Blakley, R.L. and Benkovic, S.J. (Eds). Folates and pterins (Vol. 1, pp. 497-553). Wiley (Interscience), New York

Matthews, R.G. (1986). Methylenetetrahydrofolate reductase from pig liver. Methods in Enzymology, 122, 372-381.

Matthews, R.G., Ross, J., Baugh, C.M., Cook, J.D. and Davis, L. (1982). Interactions of pig liver serine hydroxymethyltransferase with methylene tetrahydropteroylpolyglutamate inhibitors and with tetrahydropteroylpolyglutamate substrates. Biochemistry, 21, 1230-1238.

Matthews, R.G., Vanoni, M.A., Hainfeld, IF. and Wall, J. (1984). Methylenetetrahydrofolate reductase. Evidence for spatially distinct subunit domains obtained by scanning transmission electron microscopy and limited proteolysis. Journal of Biological Chemistry, 259, 11647-11650.

Mazelis, M. and Liu, E.S. (1967). Serine transhydroxymethylase of cauliflower (Brassica oleracea var. botrytis L.): partial purification and properties. Plant Physiology, 42, 1763-1768.

McDonald, D., Atkinson, I.J., Cossins, E.A. and Shane, B. (1995). Isolation of dihydrofolate and folylpolyglutamate synthetase activities from Neurospora. Phytochemistry, 38, 327-33.

McGuire, J.J. and Bertino, J.R. (1981). Enzymatic synthesis and function of polyglutamates. Molecular and Cellular Biochemistry, 38, 19-48.

McGuire, J.J. and Coward, J.K. (1984). Pteroylpolyglutamates: Biosynthesis, degradation and function. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 1 (pp. 135-190). Wiley (Interscience), New York.

McNeil, J.B., Bognar, A.L. and Pearlman, R (1996). In vivo analysis of folate coenzymes and their compartmentation in Saccharomyces cerevisiae. Genetics, 142, 371-381.

Meek, T.D., Garvey, E.P. and Santi, D.V. (1985). Purification and characterization of the bifunctional thymidylate synthetase-dihydrofolate reductase from methotrexate­resistant Leishmania tropica. Biochemistry, 24, 678-686.

Melcher, K. and Entian, K-D. (1992). Genetic analysis of serine biosynthesis and glucose repression in yeast. Current Genetics, 21, 295-300.

Mitchell, H.K., Snell, E.E. and Williams, RJ. (1941). The concentration of «.folic acid ». Journal of the American Chemical Society, 63, 2284.

Nemecek-Marshall, M., MacDonald, R.C., Franzen, J.J., Wojciechowski, C.L. and Fall, R (1995). Methanol emission from leaves. Enzymatic detection of gas-phase methanol and relation of methanol fluxes to stomatal conductance and leaf development. Plant Physiology, 108, 1359-1368.

Neuburger, M., Bourguignon, J. and Douce, R. (1986). Isolation of a large complex from the matrix of pea leaf mitochondria involved in the rapid transformation of glycine into serine. FEBS Letters, 207, 18-22.

Neuburger, M., Rebeille, F., Jourdain, A., Nakamura, S. and Douce, R. (1996). Mitochondria are a major site for folate and thymidylate synthesis in plants. Journal of Biological Chemistry, 271, 9466-9472.

Nour, J.M. and Rabinowitz, J.C. (1991). Isolation, characterization and structural organization of lO-formyltetrahydrofolate synthetase from spinach leaves. Journal of Biological Chemistry, 266, 18363-18369.

4. Folate metabolism in plants 95

Nour, J.M. and Rabinowitz, J.e. (1992). Isolation and sequencing of the cDNA coding for spinach IO-formyltetrahydrofolate synthetase. Journal of Biological Chemistry, 267, 16292-16296.

Okinaka, O. and Iwai, K. (l970a). The biosynthesis of folic acid compounds in plants. IV. Purification and properties of the dihydropteroate-synthesizing enzyme from pea seedlings. Journal of Vitaminology, 16, 201-209.

Okinaka, o. and Iwai, K. (1970b). The biosynthesis of folic acid compounds in plants. III. Distribution of the dihydropteroate synthesizing enzyme in plants. Journal of Vitaminology, 16, 196-200.

Oliver, DJ. (1994). The glycine decarboxylase complex from plant mitochondria. Annual Review of Plant Physiology and Plant Molecular Biology, 45, 323-337.

Oliver, DJ., Neuburger, M., Bourguignon, J. and Douce, R. (1990a). Glycine metabolism by plant mitochondria. Physiologia Plantarum, 80, 487-491.

Oliver, DJ., Neuburger, M., Bourguignon, J. and Douce, R. (1990b). Interaction between the component enzymes of the glycine decarboxylase multi enzyme complex. Plant Physiology, 94, 833-839.

Osborn, M.J., Talbert, P.T. and Huennekens, F.M. (1960). The structure of active «formaldehyde» (N5,N IO-methylene tetrahydrofolic acid). Journal of the American Chemical Society, 82, 4921-4927.

Pasternack, L.B., Laude, Jr, D.A. and Appling, D.R. (1992). \JC NMR detection of folate­mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae. Biochemistry, 31, 8713-8719.

Paukert, J. and Rabinowitz, J.C. (1980). Formyl- methenyl- methylenetetrahydrofolate synthetase (combined): a multifunctional protein in eukaryotic folate metabolism. Methods in Enzymology, 66, 616-626.

Paukert, J.L., Williams, G.R. and Rabinowitz, J.C. (1977). Formyl-methenyl­methylenetetrahydrofolate synthetase (combined): correlation of enzymic activities with limited proteolytic degradation of the protein from yeast. Biochemical and Biophysical Research Communications, 77, 147-154.

Penner, M.H. and Frieden, e. (1985). Substrate-induced hysteresis in the activity of Escherichia coli dihydrofolate reductase. Journal of Biological Chemistry, 260, 5366-5369.

Prabhu, V., Chatson, K.B., Abrams, G.D. and King, J. (1996). \JC nuclear magnetic resonance detection of interactions of serine hydroxymethyltransferase with CI­tetrahydrofolate synthase and glycine decarboxylase complex activities in Arabidopsis. Plant Physiology, 112, 207-216.

Prather, C.W. and Sisler, E.C. (1972). Glycine and glyoxylate decarboxylation in Nicotiana rustica roots. Phytochemistry, II, 1637-1647.

Ramaswamy, N.K., Behere, A.G. and Nair, P.M. (1976). A novel pathway for the synthesis of solanidine in the isolated chloroplast from greening potatoes. European Journal of Biochemistry, 67, 275-282.

Ravanel, S., Droux, M. and Douce, R. (1995). Methionine biosynthesis in higher plants. I. Purification and characterization of cystathionine y-synthase from spinach chloroplasts. Archives of Biochemistry and Biophysics, 316, 572-584.

Ravanel, S., Job, D. and Douce, R. (1996). Purification and properties of cystathionine ~­lyase from Arabidopsis thaliana overexpressed in E. coli. Biochemical Journal, 320, 383-392.

Rebeille, F., Macherel, D., MouilIon, J.M., Garin, J. and Douce, R. (1997). Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-

96 Chapter 4

hydroxymethy 1-7,8-dihydropterin pyrophosphokinasel7 ,8-dihydropteroate synthase localized in mitochondria. EMBO Journal, 16,947-957.

Rebeille, F., Neuburger, M. and Douce, R. (I 994}. Interaction between glycine decarboxylase, serine hydroxymethyltransferase and tetrahydrofolate polyglutamates in pea leaf mitochondria. Biochemical Journal, 302, 223-228.

Rijnboutt, S., Jansen, G., Posthuma, G., Hynes, J.B. and Schomagel, J.H. (I 996}. Endocytosis of GPI-linked membrane folate receptor-ex. Journal of Cell Biology, 132, 35-47.

Roberts, J.K.M., Aubert, S., Gout, E., Bligny, R. and Douce, R. (1997). Cooperation and competition between adenyl ate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 3lP-nuclear magnetic resonance. Plant Physiology, 113, 191-199.

Roos, A.J. and Cossins, E.A. (1971). Pteroylglutamate derivatives in Pisum sativum L. Biosynthesis of cotyledonary tetrahydropteroylglutamates during germination. Biochemical Journal, 125, 17-26.

Roos, A.J., Spronk, A.M. and Cos sins, E.A. (1968). 5-methyltetrahydrofolic acid and other folate derivatives in germinating pea seedlings. Canadian Journal of Biochemistry, 46, 1533-1536.

Rowe, P.B. (I 984}. Folates in the biosynthesis and degradation of purines. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 1 (pp. 329-344). Wiley (Interscience), New York.

Santi, D.V. and Danenberg, P.V. (1984). Folates in pyrimidine nucleotide biosynthesis. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 1 (pp. 345-398). Wiley (lnterscience), New York.

Schirch, L.V. (1978). Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to lO-formyltetrahydrofolate. Archives of Biochemistry and Biophysics, 189, 283-290.

Schirch, L.V. (l984). Folates in glycine and serine metabolism. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. I (pp. 399-431). Wiley (lnterscience), New York.

Schirch, L. and Peterson, D. (I 980}. Purification and properties of mitochondrial serine hydroxymethyltransferase. Journal of Biological Chemistry, 255, 7801-7806.

Schirch, V., Shostak, K., Zamora, M. and Gautam-Bassak, M. (l99l). The origin of reaction specificity in serine hydroxymethyltransferase. Journal of Biological Chemistry, 266, 754-759.

Schirch, V. and Strong, W.B. (1989). Interaction of polyglutamates with enzymes in one­carbon metabolism. Archives of Biochemistry and Biophysics, 269, 371-380.

Schirch, L.V., Tatum, Jr, C.M. and Benkovic, S.J. (1977). Serine transhydroxymethylase: evidence for a sequential random mechanism. Biochemistry, 16,410-419.

Servaites, J.C. and Ogren, W.L. (1977). Chemical inhibition of the glycolate pathway in soybean leaf cells. Plant Physiology, 60, 461-466.

Shah, S.P.J. and Cossins, E.A. (l969). The biosynthesis of glycine and serine by isolated chloroplasts. Phytochemistry, 9, 1545-1551.

Shah, S.PJ. and Cossins, E.A. (1970). Pteroylglutamates and methionine biosynthesis in isolated chloroplasts. FEBS Letters, 7, 267-270.

Shane, B. (1980a). Pteroylpoly(y-glutamate) synthesis by Corynebacterium species. In vivo synthesis of folates. Journal of Biological Chemistry, 255, 5649-5654.

4. Folate metabolism in plants 97

Shane, B. (1980b). Pteroylpoly(y-glutamate) synthesis by Corynebacterium species. Purification and properties of folylpoly(y-glutamate) synthetase. Journal of Biological Chemistry, 255, 5655-5662.

Shane, B. (1980c). Pteroylpoly(y-glutamate) synthesis by Corynebacterium species. Studies on the mechanism of folylpoly(y-glutamate) synthetase. Journal of Biological Chemistry, 255, 5663-5667.

Shane, B. and Stokstad, E.L.R. (1984). Folates in the synthesis and catabolism of histidine. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. I (pp. 433-455). Wiley (lnterscience), New York.

Shannon, K.W. and Rabinowitz, J.C. (1986). Purification and characterization ofa mitochondrial isozyme of Cl-tetrahydrofolate synthetase from Saccharomyces cerevisiae. Journal of Biological Chemistry, 261, 12266-12271.

Shingles, R., Woodrow, L. and Grodzinski, B. (1984). Effects of glycolate pathway intermediates on glycine decarboxylation and serine synthesis in pea (Pisum sativum L). Plant Physiology, 74, 705-710.

Shiman, R. (1985). Phenylalanine hydroxylase and dihydropterin reductase. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 2 (pp. 115-154). Wiley (Interscience), New York.

Shiota, T. (1984). Biosynthesis of folate from pterin precursors. In: Blakley, R.L. and Benkovic, S.J., (Eds). Folates and pterins Vol. 1 (pp. 121-134). Wiley (Interscience), New York.

Slock, J., Stahly, D.P., Han, C., Six, E.W. and Crawford, l.P. (1990). An apparent Bacillus subtilis folic acid biosynthetic operon containing pab, an amphibolic trpG gene, a third gene required for synthesis of para-aminobenzoic acid, and the dihydropteroate synthase gene. Journal of Bacteriology, 172, 7211-7226.

Snell, K. (1986). The duality of pathways for serine biosynthesis is a fallacy. Trends in Biochemical Sciences, 11, 241-243.

Stokstad, E.L.R. and Manning, P.D.V. (1938). Evidence of a new growth factor required by chicks. Journal of Biological Chemistry, 125, 687-696.

Stover, P. and Schirch, V. (1992). Enzymatic mechanism for the hydrolysis of 5,10-methenyltetrahydropteroylglutamate to 5-formyltetrahydropteroylglutamate by serine hydroxymethyltransferase. Biochemistry, 31, 2155-2164.

Stover, P. and Schirch, V. (1993). The metabolic role of leucovorin. Trends in Biochemical Sciences, 18, 102-106

Strong, W., Joshi, G., Lura, R., Muthukumaraswamy, N. and Schirch, V. (1987). 10-formyltetrahydrofolate synthetase. Evidence for a conformational change in the enzyme upon binding of tetrahydropteroylpolyglutamates. Journal of Biological Chemistry, 262, 12519-12525.

Strong, W.B., Tendler, S.J., Seither, R.L., Goldman, l.D. and Schich, V. (1990). Purification and properties of serine hydroxymethyltransferase and Cdetrahydrofolate synthetase from Ll210 cells. Journal of Biological Chemistry, 265, 12149-12155.

Spronk, A.M. and Cossins, E.A. (1972). Folate derivatives of photosynthetic tissues. Phytochemistry, 11, 3157-3165.

Sumner, J.M., Jencks, D.A., Khani, S. and Matthews, R.G. (1986). Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine. Journal of Biological Chemistry, 261, 7697-7700.

Suzuki, N. and Iwai, K. (1973). The occurrence and properties of methenyltetrahydrofolate cyclohydrolase in plants. Plant and Cell Physiology, 14, 319-327.

98 Chapter 4

Suzuki, N. and Iwai, K. (1974). The occurrence and properties of methylene­tetrahydrofolate dehydrogenase in pea seedlings, and intracellular distribution of some folate-linked enzymes in the plant. Journal of Nutritional Science and Vitaminology, 20, 89-96.

Swafford, J.R., Beverley, S.M., Kan, C.C. and Caulfield, J.P. (1990). Distribution of dihydrofolate reductase-thymidylate synthetase polypeptide in methotrexate resistant and wild type Leishmania major: evidence for cytoplasmic distribution despite a hydrophilic leader sequence. In Peachey, L.D. and Williams, D.B. (Eds). Proceedings of the XIIth congress for electron microscopy, (pp. 610-611). San Fransico Press, San Francisco.

Talarico, T.L., Ray, P.H., Dev, I.K., Merill, B. and Dallas, W.S. (1992). Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase. Journal of Bacteriology, 174, 5971-5977.

Tolbert, N.E. (1980). Photorespiration. In Davies, D.D. (Ed). The Biochemistry of plants Vol 2 (pp. 487-523). Academic Press, New York.

Tolbert, N.E. (1981). Metabolic pathways in peroxisomes and glyoxysomes. Annual Review of Biochemistry, 50, 133-157.

Triglia, T. and Cowman, A.F. (1994). Primary structure and expression of the dihydropteroate synthase gene of Plasmodium falciparum. Proceedings of the National Academy of Sciences USA, 91, 7149-7153.

Turner, S.R., Ireland, R., Morgan, C. and Rawsthorne, S. (1992a). Identification and localisation of multiple forms of serine hydroxymethyltransferase in pea (Pisum sativum) and characterization of a cDNA encoding a mitochondrial isoform. Journal of Biological Chemistry, 267, 13528-13534.

Turner, S.R., Ireland, R. and Rawsthorne, S. (1992b). Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. Evidence for a single lipoamide dehydrogenase in plant mitochondria. Journal of Biological Chemistry, 267, 7745-7750.

Turner, S.R., Ireland, R., and Rawsthorne, S. (1992c). Cloning and characterization of the P subunit of glycine decarboxylase from pea (Pisum sativum). Journal of Biological Chemistry, 267, 5355-5360.

Ulane, R. and Ogur, M. (1972). Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces. Journal of Bacteriology, 109, 34-43.

Uyeda, K. and Rabinowitz, lC. (1967). Enzymes of clostridial purine fermentation. Methylene tetrahydrofolate dehydrogenase. Journal of Biological Chemistry, 242, 4378-4385.

Vauclare, P., Diallo, N., Bourguignon, J., Macherel, D. and Douce, R. (1996). Regulation of the expression of the glycine decarboxylase complex during pea leaf development. Plant Physiology, 112, 1523-1530.

Volpe, F., Ballantine, S.P. and Delves, CJ. (1993). The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. European Journal of Biochemistry, 216, 449-458.

Waidyanatha, U.P.deS., Keys, AJ. and Whittingham, C.P. (1975). Effects of oxygen on metabolism by the glycolate pathway in leaves. Journal of Experimental Botany, 26, 27-32.

Wallsgrove, R.M., Lea, PJ. and Miflin, B. (1983). Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves. Plant Physiology, 71, 780-784.

4. Folate metabolism in plants 99

Walton, N.J. and Woolhouse, H.W. (1986). Enzymes of serine and glycine metabolism in leaves and non-photosynthetic tissues of Pisum sativum L. Planta, 167, 119-128.

West, M.G., Barlowe, C.K. and Appling, D.R. (1993). Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD+ -dependent 5,10-methylenetetrahydrofolate dehydrogenase. Journal of Biological Chemistry, 268, 153-160.

West, M.G., Home, D.W. and Appling, D.R. (1996). Metabolic role of cytoplasmic isozymes of 5,1 O-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae. Biochemistry, 35, 3122-3132.

Wong, K. and Cossins, E.A. (1976). Control of methionine synthesis by lysine in Lemna minor. Phytochemistry, 15, 921-925.

Zelitch, 1. (1972). The photooxidation of glyoxylate by envelope-free spinach chloroplasts and its relation to photorespiration. Archives of Biochemistry and Biophysics, 150, 698-707.

Zhang, Y. and Meshnick, S.R. (1991). Inhibition of Plasmodium falciparum dihydropteroate synthetase and growth in vitro by sulfa drugs. Antimicrobial Agents and Chemotherapy, 35, 267-271.

Zheng, L.L., Lin, Y., Lin, S. and Cossins, E.A. (1992). The polyglutamate nature of plant folates. Phytochemistry, 31, 2277-2282.

Chapter 5

Structure and function of plastid metabolite transporters

Ulf-Ingo Fliigge, Andreas Weber, Birgit Kammerer, Rainer E. Hausler and Karsten Fischer Botanisches Institut der Universitiit zu K6in, Lehrstuhi II, Gyrhofstr. 15, D-50931, K6in, Germany

Key words: dicarboxylate trans locator; phosphate translocator; plastids.

Abstract: Plastids contain various transport proteins that mediate the exchange of metabolites between the plastids and the cytosol. These metabolite translocators reside in the inner envelope membrane and are involved in the translocation of photoassimilates in both photosynthetic and heterotrophic tissues. The characteristics of three of these translocators, all functioning as anti porters, are described in more detail. First, the chloroplast triose phosphate/phosphate translocator (cTPT) that exports the fixed carbon in form of triose phosphates and 3-phosphoglycerate from the chloroplasts in exchange for inorganic phosphate is described. Secondly, a phosphoenolpyruvate/phosphate translocator that is present in both photosynthetic and non-green tissues is considered. The main purpose of this transporter is presumably to supply the plastids with phosphoenolpyruvate as a substrate for the shikimate pathway. Finally, a dicarboxylate trans locator that imports carbon skeletons into chloroplasts in exchange with malate for ammonia assimilation is discussed. This nitrogen source is used for the formation of amino acids that are subsequently exported.

1. INTRODUCflON

Communication between chloroplasts and the surrounding cytosol occurs via the plastid envelope membrane. The inner envelope membrane contains various metabolite transporters that mediate the

101

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 101-116. © 1999 Kluwer Academic Publishers.

102 Chapter 5

exchange of metabolites between both compartments. The carbon fixed during the day can be exported from the chloroplasts via the chloroplast triose phosphate/phosphate translocator (cTPT) in the form of C3-compounds (triose phosphates and/or 3-phosphoglycerate) in exchange for inorganic phosphate (Fliege et aI., 1978). The exported carbon is mainly utilised for the biosynthesis of sucrose which is subsequently allocated to heterotrophic organs of the plant. When the rate of sucrose biosynthesis and export falls below that of CO2 assimilation, the fixed carbon is retained in the chloroplasts and directed into the biosynthesis of assimilatory starch. Remobilisation of starch during the following dark period and export of the starch breakdown products ensures a continuous supply of photosynthates to heterotrophic tissues in the dark. The T P T appears to be present only in photosynthetic tissues (Fliigge, 1995; Schulz et aI., 1993). Non-photosynthetic tissues rely therefore on the presence of other plastidic phosphate translocators. Transport experiments performed with intact non-green plastids from various plants as well as phosphate transport activities determined upon reconstitution of homogenates from non-green tissues into artificial membranes have shown that these plastids contain phosphate translocator(s) which enable the transport of hexose phosphates and/or phosphoenolpyruvate (PEP) (Borchert et aI., 1993; Fliigge and Weber, 1994; Neuhaus et aI., 1993).

The fixed carbon can also be directed into the biosynthesis of amino acids. These steps require the conversion of the exported C3-compounds into 2-oxoglutarate that is subsequently reimported into the chloroplasts for the assimilation of ammonia.

In this paper, we describe the characteristics of two different plastidic phosphate translocators, and of a dicarboxylate translocator that imports carbon skeletons into the plastids for the fixation of ammonia.

2. PLASTIDIC PHOSPHATE TRANSLOCATORS

2.1 The triose phosphate/phosphate translocator

The chloroplast TPT from spinach was the first plant transport system to be cloned (Fliigge et aI., 1989). Meanwhile, sequences from various plants are available, e.g. those from pea, potato, maize, Flaveria, and tobacco that all have a high similarity to each other (Fischer et aI., 1997; Knight and Gray, 1994). All TPTs are nuclear-encoded and possess N-terminal transit peptides that direct the adjacent protein correctly to

5. Metabolite translocators 103

the chloroplast and to the inner envelope membrane. The mature part of these transporters consisting of about 330 amino acid residues are highly hydrophobic and have 5-7 transmembrane helices that anchor the protein in the membrane. It has been shown that the functional T P T from spinach chloroplasts is composed of two identical subunits (Flilgge, 1985; Wagner et aI., 1989) and this holds presumably true for all TPTs. Thus, the chloroplast TPTs function as homodimers and belong to the group of transporters that possesses a 6+6 helix folding pattern as is the case for the mitochondrial carriers. No three-dimensional structure of any plant transport protein has been obtained to date, but a tentative model for the arrangement of the TPT in the membrane suggests that all the a-helices participate in the formation of a hydrophilic translocation channel through which the substrates could be transported across the membrane.

As outlined above, the TPT is an important link between the chloroplast and the cytosol. It is assumed that most (if not all) of the daily fixed carbon is exported from the chloroplast via this translocator. A reduction of the transport activity should therefore result in a marked perturbation of leaf metabolism. Antisense repression of the corresponding mRNA in transgenic potato plants indeed showed that these plants were limited in the capacity to export the fixed carbon from the chloroplasts in form of C3-compounds. Instead, about 90% of the fixed carbon was retained within the plastids leading to the accumulation of starch (Table 1; Heineke et aI., 1994; Riesmeier et aI., 1993). Thus, the starch content in transformants was increased by a factor of 2-5 compared to wild-type plants, both at the end of the day and end of the night. However, under ambient conditions, the photosynthetic rates were not affected in the transform ants and, consequently, these plants did not show any effect on plant growth and productivity. A detailed analysis of the transformants revealed that the major part of the daily accumulated carbon (about 90% in case of antisense TPT plant 39) could be efficiently mobilised and exported during the following night period. This is in contrast to wild-type plants which generally export the fixed carbon predominantly during ongoing photosynthesis (Table 1). The question arises how the mobilisation and export of photoassimilates is achieved in the antisense TPT plants during the night. Due to the reduced activity of the TPT, the mobilised carbon cannot be exported (at least not at high rates) from the chloroplasts as C3-compounds as end-products from phosphorylytic starch breakdown. Alternatively, starch can be hydrolytically degraded. This process yields glucose which can subsequently be exported from the chloroplasts via a hexose transport system. Presumably, the transformants follow this metabolic sequence. It could indeed be demonstrated that the transformants possess a higher

Tab

le 1

. Ant

isen

se r

epre

ssio

n of

the

TPT

in

tran

sgen

ic p

otat

o pl

ants

.

Eff

ect o

f an

tise

nse

repr

essi

on o

f th

e T

PT o

n C

O2

-as

sim

ilat

ion,

and

acc

umul

atio

n an

d ex

port

of

phot

oass

imil

ates

. TPT

39, a

ntis

ense

TPT

pot

ato

plan

t. D

ata

from

Hei

neke

et a

!. (1

994)

.

Rat

e of

net

C02

-as

sim

ilat

ion

Am

ount

of

assi

mil

ate

a) a

ssim

ilat

ed d

urin

g th

e da

y

b) a

ccum

ulat

ed d

urin

g th

e da

y

Rat

e of

ass

imil

ate

expo

rt

a) d

urin

g th

e da

y

b)

duri

ng th

e ni

ght

Pla

nts

Wil

d-ty

pe

TP

T39

90.4

81

.3

llm

ol C

02

/mg

Chl

. x

h

1085

97

5 ~atom C

/mg

Chl

.

466

(= 4

3%)

866

(=89

%)

~atom C

/mg

Chl

.

52 (

= 57

%)

39 (

= 4

3%)

9 (=

11 %

) ~atom C

/mg

ChI

. x h

72 (

= 8

9%)

~atom C

/mg

Chi

. x h

- o .j::..

Q

{5 ~

"'!

V,

5. Metabolite translocators 105

activity of the glucose trans locator compared to wild-type plants. It can be concluded that plants can compensate for their deficiency in T P T activity provided that a carbon sink (starch) can be generated during photosynthesis which can be mobilised during the following night period. If, in addition to the reduction of the TPT, starch formation is prevented during the day (e.g. by anti-sense repression of the ADPglucose pyrophosphorylase, AGPase), these transformants show a dramatic phenotype (Reineke et aI., 1995). On the one hand, they are not able to export sufficient amounts of fixed carbon during the day (due to antisense repression of the TPT) and on the other hand, they do not have a carbon store which they could use during the night period (due to antisense repression of the AGPase).

2.2 The phosphoenolpyruvate/phosphate translocator

In a search for other plastidic phosphate trans locators from non­green tissues, we recently purified a 31 kD protein from maize endosperm (Fischer et aI., 1997). Peptides obtained from enzymatic digestion of this protein allowed the synthesis of an oligonucleotide that was used to screen a cDNA library from maize endosperm. The cDNA fragment obtained was used to re-screen the cDNA library from maize endosperm and, in addition, cDNA libraries from maize roots, cauliflower inflorescences, tobacco leaves, and Arabidopsis leaves. From each of these libraries, we obtained independent clones that contained full-length cDNAs. The sequence homologies between selected phosphate transport proteins from spinach, maize, tobacco, cauliflower and Arabidopsis are shown in Table 2. Evidently, these phosphate trans locators belong to two distinct groups, the cTPTs and the PPTs. Within each group of transporters, the proteins are highly homologous to each other (precursor proteins, 62-92%; mature proteins, 73-96%). In contrast, the homologies between members of the cTPT family and the PPT family, respectively, are only 30-32% (precursor proteins) and 34-37% (mature proteins). The PPTs obviously represent a new class of plastidic phosphate translocators.

To study the transport characteristics of the cloned PPT proteins, the cDNA from the cauliflower PPT3 clone was fused to a DNA fragment encoding a tag of six consecutive histidine residues and was then sub cloned into the yeast expression vector pEVPll. This procedure enabled the one-step purification of the produced transporter to apparent homogeneity by metal-affinity chromatography for the subsequent analysis of the transport characteristics by reconstitution experiments (Loddenkotter et aI., 1993).

Tab

le 2

. Hom

olog

ies

betw

een

cTPT

an

d P

PT p

rote

ins

from

dif

fere

nt p

lant

s

Pre

curs

or/m

atur

e (%

/%)

cTPT

s fr

om

PPT

s fr

om

Spi

nach

C

auli

-M

aize

C

auli

-A

rabi

-T

obac

co

flow

er

flow

er

dops

is PP

T8

PPT

lO

PPT

3 P

PT

l2

Spi

nach

cT

PT

(100

/100

) 76

/88

72/8

5 30

/35

30/3

4 31

/35

31/3

5

Cau

lifl

ower

cT

PT

(100

/100

) 68

/81

30/3

5 30

/34

31/3

5 31

/36

Mai

ze c

TPT

(1

00/1

00)

30/3

4 30

/35

31/3

6 31

/36

Cau

lifl

ower

PPT

3 (1

00/1

00)

89/9

3 65

/80

67/7

7

Ara

bido

psis

PP

Tl2

(1

00/1

00)

64/7

6 66

/80

Tob

acco

PP

T8

(100

/100

) 78

/87

Tob

acco

PPT

lO

(100

/100

)

Mai

ze P

PT

l

Mai

ze P

PT4

Mai

ze

PP

Tl

PPT

4

31/3

5 32

/36

33/3

7 31

/35

32/3

5 32

/35

64/7

5 63

/75

64/7

3 62

/73

64/7

6 63

/74

65/7

6 64

/76

(100

/100

) 92

/96

(100

/100

)

.... o 0\ Q

{5 ~

"'! ~

5. Metabolite translocators 107

Figure 1 shows the substrate specificities of the purified PPT and cTPT, as determined by measuring C2p]phosphate transport into proteoliposomes that had been pre loaded with various counter-substrates. As representatives of C3-compounds phosphorylated at C-atom 3 or C­atom 2, triose phosphate (dihydroxyacetone phosphate, DAHP) and phosphoenolpyruvate (PEP), respectively, were used. The two phosphate trans locators differ greatly in their transport characteristics. The P P T protein transports inorganic phosphate preferentially in exchange with PEP. This is in sharp contrast to the transport characteristics of the cTPT. This transporter accepts either inorganic phosphate or C3-compounds that are phosphorylated at C-atom 3, i.e. triose phosphates and 3-phosphoglycerate (Fliege et aI., 1978). These substrates, however, are only poorly transported by the PPT protein. Both types of transporters rely strictly on the presence of an exchangeable substrate within the vesicles (Le. they function as antiport systems) and they do not transport hexose phosphates such as glucose 6-phosphate at all.

In order to analyse the expression of the PPT in comparison to the cTPT, Northern blots were performed. We have shown earlier (Flogge, 1995; Schulz et aI., 1993) that the TPT gene is expressed only in photosynthetically active tissues. In contrast, the PPT -specific transcripts can be detected in both leaves and non-green tissues although they are more abundant in non-green tissues (Fischer et aI., 1997).

2.2.1 Physiological role of the PPT

Do plastids rely on a supply of externally produced PEP or can PEP be generated inside the organelles? Work from several laboratories has shown that the conversion of hexose phosphates and/or triose phosphates via glycolysis cannot proceed further than to 3-phosphoglycerate due to the absence (or low activities) of phosphoglycerate mutase and/or enolase in most plastids (Borchert et aI., 1993; Journet and Douce, 1985; Miernyk and Dennis, 1992; Schulze­Siebert et aI., 1984; Stitt and apRees, 1979; Vander Straeten et aI., 1991). These plastids depend therefore on the provision of externally produced PEP. This fact has either been overlooked to date, or it has been assumed that the transport of PEP into chloroplasts is facilitated by the TPT. This, however, appears unlikely because PEP is only poorly accepted as a substrate by the TPT (Fliege et aI., 1978; Figure 1) and, under physiological conditions, it has to compete with inorganic phosphate, triose phosphates, and 3-phosphoglycerate for binding to the TPT. Hence, the presence of a PPT that bypasses the TPT is the most likely alternative for an efficient provision of the chloroplasts with PEP.

108

100 - r- r-.

F

;? 80 L

-c::

>--.; ;:; 60 (J -« t:: 0 c. f/j 40 c -cu ... I-

c:: 20 -

o ~ AR Pi TrioseP PEP

Substrates

Figure 1. Substrate specificities of the TPT and the PPT.

Glu6-P

Chapter 5

The cauliflower PPT3-His6 protein and the spinach TPT-His6 protein were expressed in Saccharomyces pombe cells and purified from these cells by metal affinity chromatography (Loddenkotter et aI., 1993). The recombinant proteins were reconstituted into liposomes that had been preloaded with with 25 mM substrates as indicated. e2p]phosphate transport activity was measured as described by Fischer et al. (1997) and is given as a percentage of the activity measured for proteoliposomes preloaded with inorganic phosphate. The 100% exchange activities (/lmol min·· mg·· protein) were 1.5 (PPT, dark grey) and 0.85 (TPT, light grey), respectively. Mean values of 3-5 different experiments. Pi, inorganic phosphate; TrioseP, dihydroxyacetone phosphate; Glu6-P, glucose 6-phosphate.

5. Metabolite trans locators 109

Several processes inside the plastids rely on PEP (see Figure 2). This compound is an immediate substrate for the shikimate pathway which leads to a large number of secondary compounds that are important in plant defence mechanisms and stress responses (for review see Herrmann, 1995). It has also been shown that the plastidically located fatty acid biosynthesis can be driven by externally added pyruvate (Liedvogel and Bauerle, 1986). Plastidic acetyl-CoA can subsequently be formed by the action of either acetyl-CoA synthetase or via the plastidic pyruvate dehydrogenase complex. In plastids missing a complete pathway from 3-phosphoglycerate to pyruvate, PEP could be imported into the organelles via the PPT and pyruvate kinase could subsequently convert PEP into pyruvate.

Figure 2 outlines the proposed physiological function of the PPT in chloroplasts. The fixed carbon is exported from the chloroplast by the TPT and partially converted into PEP in the cytosol. The PPT can then provide the chloroplast stroma with PEP for the shikimate pathway. In addition, PEP can be converted into pyruvate as a precursor for fatty acid biosynthesis, concomitantly providing the plastids with ATP. The latter issue is probably important in chloroplasts during night or in non­photosynthetic tissues. Because inorganic phosphate is used as a counter­substrate by both the TPT and the PPT, the combined action of both trans locators would result in an exchange of triose phosphate with PEP without net phosphate transport.

With the identification of a phosphate translocator specific for PEP, we propose that plastids contain a set of phosphate translocators with different structures but overlapping substrate specificities. Chloroplasts, or at least a subtype of chloroplasts, contain members of both the T P T and the PPT family. The TPT is obviously absent in non-green tissues. Nevertheless, these plastids have to contain a device for the transport of triose phosphates that are produced via the oxidative pentose phosphate pathway (Borchert et aI., 1993). In addition, these plastids have to contain an as yet unidentified phosphate trans locator that is capable of importing hexose phosphate as the precursor for starch biosynthesis. The presence of different phosphate trans locators with different substrate specificities would allow the uptake of particular phosphorylated substrates even in the presence of high concentrations of other phosphorylated metabolites, which would otherwise compete for the binding site of a single phosphate trans locator.

110 Chapter 5

SUCROSE r

t Chloroplast

I

:~ r- rPll

STARCIJ ... - - Fru6P 3·PCA --{ TPT TrioseP TrioseP .J j

~~ CD I

Cycle r PI PI I

Ery4P .. I (' PPT '\ PEP Pyruvate ,. 7' PEP

I .:-1

I ADP ~ J I IATP I PI I 1 DAHP I

+ ~ + I FATTY I I smKThDC ACroJ AMINO

ACro PATHWAY ACIDS

Aromatic compounds Cytosol

Figure 2. Proposed function of the PPT protein in photosynthetically active tissues. The combined action of the TPT and the PPT results in the supply of the organelle with PEP generated from photosynthetically fixed carbon. DAHP, 3-deoxY-D-arabino­heptulosonate-7-phosphate; Fru6P, fructose-6-phosphate; 3-PGA, 3-phosphoglycerate; RuBP, ribulose-l,5-bisphosphate; TrioseP, triose phosphates.

5. Metabolite trans/ocators 111

2.3 The dicarboxylate translocator

The photosynthetically fixed carbon (in the form of C3-compounds,) that is exported from the chloroplasts via the TPT as triose phosphates and 3-phosphoglycerate can also be used for the formation of carbon skeletons (2-oxoglutarate) via processes occurring in the cytosol and in the mitochondria. 2-oxoglutarate can then be reimported into the chloroplasts for the fixation of ammonia that derives from nitrate reduction or photorespiration. Fixation of ammonia is achieved via the glutamine synthetase/glutamate synthase cycle. The glutamate synthesized during this cycle is exported into the cytosol. Two different dicarboxylate antiport systems are involved in this process: the 2-oxoglutarate/malate trans locator (DiTl) transporting 2-oxoglutarate into the chloroplasts and a glutamate/malate trans locator (DiT2) exporting glutamate (Figure 3). Because both trans locators use malate as the substrate for counter-exchange, the resulting 2-oxoglutarate/glutamate transport proceeds without net malate transport (Woo et aI., 1987).

We have recently cloned the DiTl protein that provides the chloroplast with 2-oxoglutarate for ammonia assimilation (Weber et aI., 1995). Remarkably, DiTl does not possess any homology to its mitochondrial counterpart with similar transport characteristics (Runswick et aI., 1990). Moreover, the mitochondrial carrier belongs to the class of transporters with a 6+6 transmembrane helix pattern and consists of two monomers as is the case for the TPT (see above). In contrast, DiTI is the first example of an organellar translocator with a 12-helix transmembrane topology. The recently cloned plastidic ADP/ATP trans locator also belongs to this type of transporter (Kampfenkel et aI., 1995). Both transporters resemble the topology of plasmamembrane transporters from prokaryotes and eukaryotes that presumably all function as monomers. Expression of the DiTl full-length cDNA sequence in yeast cells revealed that the substrate specificities of the recombinant protein were almost identical to the translocator purified from envelope membranes. The activity of the recombinant protein is thus in accordance with its proposed function as a supplier of the chloroplasts with 2-oxoglutarate for ammonia assimilation.

As outlined above, the provision of carbon skeletons (2-oxoglutarate) via the DiTl for amino acid biosynthesis is essential for nitrate assimilation as well as for the re-assimilation of ammonia released during photorespiration. We have investigated the expression level of DiTl in leaves of tobacco as well as spinach using specific cDNA clones under a variety of environmental conditions. Our experiments show (Figure 4) that the level of DiTl-specific transcripts is substantially diminished

112 Chapter 4

CITOSOL TrioseP r NO,-

I I

• I R~;J NO,-

I co, Calvin

~ Cycle

! 2-0G Glutamine

Glutamate ~I X:~GOGi

Glu Glu I All' NH: I

~ CHLOROPLAST

I AMINO ACIDS I

Figure 3_ Two plastidic dicarboxylate translocators are involved in ammonia assimilation_ 2-0G, 2-oxoglutarate; DiTl, 2-oxoglutarate/malate translocator; DiT2, glutamate/malate translocator; Glu, glutamate; GS/GOGAT, glutamine synthetase/glutamate synthase; mal, malate; Pi, inorganic phosphate; RuBP, ribulose-l,5-bisphosphate; TrioseP, triose phosphates.

5. Metabolite trans locators

100

-:::e 0 - 80 "ii > ~ c: 60 0 'iii I/) CD ~ Q.

40 )( CD ,.... ~ i5

20

0 0 100 200 300

Time (min)

Recovery (ambient CO2)

t=12h

t=O.5h

113

Figure 4. Effect of a lowered atmospheric CO2 concentration of the expression level of DiTi. Detached spinach leaves were exposed to low CO2 (150 !ll.l0l) for up to 270 min in a Perspex chamber at an average photon flux density (PFD) of 200 !lmol m02 so' . Control leaves were kept in ambient CO2 (370 !ll.rl) for 270 min at an identical PFD (left panel). The recovery of the DiTl expression level as determined upon transfer of leaves from low to ambient C02 at the times indicated (right panel). Individual leaves were frozen in liquid nitrogen followed by extraction of total RNA. Northern blots were hybridized using the spinach DiT 1 cDNA as a probe.

114 Chapter 5

under conditions that increase photo respiratory fluxes relative to CO2

assimilation (i.e. low CO2), This decrease in the expression level is fast (about 20 min) and it substantially recovers within 12 h upon transfer to ambient conditions. Because the expression of DiTl is repressed under photorespiratory conditions, one has to assume that under these conditions, DiTl and DiT2 no longer work in a cascade-like manner, but that the exchange of amino acids with 2-oxoglutarate is exclusively mediated by DiT2. The latter translocator is able to transport both substrates. Work is now in progress to elucidate the role of DiTl in nitrate assimilation and photorespiration in more detail, e.g. by creating transgenic plants with an altered activity of DiTl.

ACKNOWLEDGEMENTS

This work was funded by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and by the BIOTECH Programme of the European Community, as part of the Project of Technological Priority, 1993-1996.

REFERENCES

Borchert, S., Harborth, J., SchUnemann, D., Hoferichter, P. and Heldt, H.W. (1993). Studies of the enzymatic capacities and transport properties of pea root plastids. Plant Physiology, 101, 303-312.

Fischer, K., Kammerer, B., Gutensohn, M., Arbinger, B., Weber, A., Hliusler, R. and Flllgge, U.1. (1997) A new class of plastidic phosphate translocators: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter. Plant Cell, 9, 453-462.

Fliege, R., FlUgge, U.I., Werdan, K. and Heldt, H.W. (1978). Specific transport of inorganic phosphate, 3-phosphoglycerate and triosephosphates across the inner membrane of the envelope in spinach chloroplasts. Biochimica et Biophysica Acta, 502, 232-247.

FlUgge, U.I. (1985). Hydrodynamic properties of the Triton X-IOO solubilized chloroplast phosphate translocator. Biochimica et Biophysica Acta, 815, 299-305.

FIUgge, U.I. (1995). Phosphate translocation in the regulation of photosynthesis. Journal of Experimental Botany, 46, 1317-1323.

FIUgge, U.I., Fischer, K., Gross, A., Sebald, W., Lottspeich, F. and Eckerskorn, C. (1989). The triose phosphate-3-phosphoglycerate-phosphate trans locator from spinach chloroplasts: Nucleotide sequence of a full-length cDNA clone and import of the in vitro synthesized precursor protein into chloroplasts. EMBO Journal, 8, 39-46.

FlUgge, U.I. and Weber, A. (1994). A rapid method for measuring organelle-specific substrate transport in homogenates from plant tissues. Planta, 194, 181-185.

Heineke, D., Kruse, A., FIUgge, U.I., Frommer, W.B., Riesmeier, J.W., Willmitzer, L and Heldt, H. W. (1994). Effect of antisense repression of the chloroplast triose phosphate

5. Metabolite translocators 115

translocator on photosynthetic metabolism in transgenic potato plants. Planta, 193, 174-180.

Heineke, D., Hattenbach, A. and Miiller-Rober, B. (1995). Adaption of potato plants to an antisense-inhibition of both ADPglucose pyrophosphorylase and the triose phosphate translocator. In: Mathis, P. (Ed). Photosynthesis: From Light to Biosphere (pp. 491-494). Kluwer Academic Publishers, Dordrecht.

Herrmann, K.M. (1995). The shikimate pathway: Early steps in the biosynthesis of aromatic compounds. Plant Cell, 7, 907-919.

Journet, E.P. and Douce, R. (1985). Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism. Plant Physiology, 79, 458-467.

Kampfenkel, K., Mohlmann., T., Batz, 0., Van Montagu, M., Inze, D. and Neuhaus, H.E. (1995). Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants. FEBS Letters, 374, 351-355.

Knight, J.S. and Gray, J.C. (1994) Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated but is repressed by sucrose. Molecular and General Genetics, 242, 586-594.

Liedvogel, B. and Bauerle, R. (1986). Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.) cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex. Planta, 169, 481-489.

Loddenkotter, B., Kammerer, B., Fischer, K. and Fliigge, U.1. (1993). Expression of the functional mature chloroplast triose phosphate translocator in yeast internal membranes and purification of the histidine-tagged protein by a single metal-affinity chromatography step. Proceedings of the National Academy of Sciences USA, 90, 2155-2159.

Miernyk, J.A. and Dennis, D.T. (1992) A developmental analysis of the enolase isoenzymes from Ricinus communis. Plant Physiology, 99, 748-750.

Neuhaus, H.E., Thorn, E., Batz, O. and Scheibe, R. (1993). Purification of highly intact plastids from various heterotrophic plant tissues. Analysis of enzyme equipment and precursor dependency for starch biosynthesis. Biochemical Journal, 296, 495-501.

Riesmeier, J.W., Fliigge, U.I., Schulz, B., Heineke, D., Heldt, H.W., Willmitzer, L. and Frommer, W.B. (1993). Antisense repression of the chloroplast triose phosphate translocator affects carbon partitioning in transgenic potato plants. Proceedings of the National Academy of Sciences USA, 90, 6160-6164.

Runswick, MJ., Walker, J.E., Bisaccia, F., Iacobazzi, V. and Palmieri, F. (1990). Sequence of the bovine 2-oxoglutarate-malate carrier protein: structural relationships to other mitochondrial transport proteins. Biochemistry, 29, 11033-11040.

Schulz, B., Frommer, W.B., Fliigge, U.I., Hummel, S., Fischer, K. and Willmitzer, L. (1993). Expression of the triose phosphate trans locator gene from potato is light dependent and restricted to green tissues. Molecular and General Genetics, 238, 357-361.

Schulze-Siebert, D., Heineke, D., Scharf, H. and Schulz, G. (1984). Pyruvate-derived amino acids in spinach chloroplasts: synthesis and regulation during photosynthetic carbon metabolism. Plant Physiology, 76, 465-471.

Stitt, M. and ap Rees, T. (1979). Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Phytochemistry, 18, 1905-1911.

Van Der Straeten, D., Rodrigues-Pousada, R.A., Goodman, H.M. and Van Montagu, M. (1991). Plant enolase: gene structure, expression and evolution. Plant Cell, 3, 719-735.

116 Chapter 5

Wagner, R., Apley, E.C., Gross, A. and FIllgge, U.I. (1989). The rotational diffusion of the chloroplast phosphate translocator and of lipid molecules in bilayer membranes. European Journal of Biochemistry, 182, 165-173.

Weber, A., Menzlaff, E., Arbinger, B., Gutensohn, M., Eckerskorn, C. and FIllgge, U.I. (1995). The 2-oxoglutarate/malate translocator of chloroplast envelope membranes: Molecular cloning of a transporter protein containing a 12-helix motif and expression of the functional protein in yeast cells. Biochemistry, 34, 2621-2627.

Woo, K.C., FIllgge, U.l. and Heldt, H.W. (1987). A two-translocator model for the transport of 2-oxoglutarate and glutamate in chloroplasts during ammonia assimilation in the light. Plant Physiology, 84, 624-632.

Chapter 6

Integration of metabolism within non-photosynthetic plastids, and with the cytosol

Mike J. Emes, Ian J. Tetlow and Caroline G. Bowsher University of Manchester, School of Biological Sciences, 3.614 Stopford Building, Oxford Road, Manchester MI3 9PT, UK

Key words: adenylates; carbohydrates; fatty acids; metabolism; nitrogen; plastids; reductant; roots; storage tissue; transport.

Abstract: Metabolism within non-photosynthetic plastids involves considerable interaction with that in the cytoplasm through the supply of intermediates which must cross the plastid envelope. This review considers our current knowledge of which intermediates may be transported, the potential for competition for substrates between intraplastidic pathways, and the integration of biosynthetic with oxidative metabolism within the organelle. There is considerable species and tissue diversity with respect to which carbohydrates may cross the plastid envelope including triose phosphates, glucose I-phosphate, glucose 6-phosphate, ADPglucose and phosphoenolpyruvate. Evidence from in vitro studies with purified organelles suggests that there is competition for pools of hexose phosphate, ATP and reductant between the pathways of carbohydrate oxidation, starch and fatty acid synthesis, and nitrogen assimilation and amino acid biosynthesis.

1. ~ODUC110N

The plastid is the unique compartment of photosynthetic eukaryotes, distinguishing such organisms from all other eukaryotes. As its name implies, this organelle is very plastic both in form and function and its role in photosynthesis has been extensively studied. Whilst the reduction of CO2 to fixed carbon is confined to chloroplasts of leaves, all plastids

117

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 117-136. © 1999 Kluwer Academic Publishers.

118 Chapter 6

are capable of supporting the biosynthesis of starch, fatty acids, amino acids and the assimilation of nitrite (Bowsher et ai., 1996). The degree to which these occur depends upon the tissue and species in which the plastid is found. Biosynthesis of these products requires a supply of carbon skeletons, reducing power and A TP. Unlike the situation in illuminated chloroplasts where many of these intermediates can be generated reductively, in non-photosynthetic plastids they are either imported direct from the cytosol or else have to be generated through oxidative metabolism within the organelle. This implies that there must be some co-ordination of metabolic pathways (a) within plastids, for instance through the turnover of, or competition for, a common intermediate, and (b) with the cytosolic milieu in which they sit, which ultimately is the source of precursors for those activities occurring within non­photo synthesising plastids.

These interactions are demonstrated in Figure 1, which illustrates the potential for competition for intermediary metabolites as well as the possible routes of entry of carbon and A TP. The aim of this chapter is to provide an overview of our present understanding of the regulation of intermediary metabolism within non-photosynthetic plastids, using appropriate examples to emphasise particular points. Some of the salient features will be discussed in more detail by other authors in this volume (see Chapters 5, 6 and 9) and, where appropriate, reference will be made to those aspects which have a bearing on the present discussion.

2. TRANSPORT OF CARBON AND ENERGY

All plastids are surrounded by a double envelope. The outer membrane contains porins which allow the free diffusion of hydrophilic molecules up to 10 kDa in molecular size (Fischer et ai., 1994). The outer envelope also provides the mechanism for the selective uptake of proteins into plastids which involves recognition, processing and import of precursor forms (Gray and Row, 1995). It is the inner envelope membrane which provides the selective barrier for small metabolites, movement of which is mediated by the operation of proteinaceous translocators. These transporters have been studied most intensively in chloroplasts, but there is now an increasing awareness of the important differences which exist between metabolite translocators of green and non-green plastids.

6. Integration of plastid metabolism

Hexo e pho phate CYTOSOL

I Hexose phosphate PLASTID

/~ Srarch p~, I , , , ,

\

Glycolysis Oxidative Pento e Pho phate Pathway

~

,s I Reductant I '"

", + assimilatioll

, ___ -j .. ~ a J ,Cz Unit } F tt - - -~ ATP Acid

119

Figure 1 Interaction between metabolic pathways in non-photosynthetic plastids.

120 Chapter 6

2.1 Phosphorylated intermediates

Since it had been known for some time that, in terms of phosphorylated intermediates, only triose phosphates and inorganic phosphate (Pi) cross the inner membrane of spinach envelopes (Fliigge and Heldt, 1984), it was assumed that the same would be true for non­photosynthetic plastids. This counter-exchange is catalysed by the triose phosphate translocator, TPT, (see Chapter 5). An analogous translocator was shown to exist in amy lop lasts from pea roots (Emes and Traska, 1987) which was subsequently shown also to transport glucose 6-phosphate (Glc6P), (Borchert et aI., 1989). The necessity to import hexose phosphate into amy lop lasts is a function not only of the oxidative metabolism which can occur within these organelles (see below) but also the observation that many tissues such as roots, some seeds and developing tubers, lack fructose bisphosphatase (Entwistle and ap Rees, 1988) thereby rendering it impossible to generate hexose phosphate gluconeogenically.

It is not yet clear whether a single plastid may possess both a triose phosphate translocator (TPT) and a hexose phosphate translocator (HPT) in the same membrane. Part of the uncertainty relates to overlapping substrate specificities. For example, kinetic studies of metabolite movement across pea root plastids had suggested that the same translocator is capable of transporting Glc6P, triose phosphates, Pi and phosphoenolpyruvate (PEP) (Borchert et aI., 1989). However, Fischer et ai. (1997) have recently shown that there is a distinct PEP/Pi transporter which is distinguishable from the TPT in a number of species, and that the expression of mRNA for the former is higher in non-green tissues than in leaves (see Chapter 5).

The nature of the hexose phosphate which crosses the envelope, and the mechanism by which it may enter, also seems to vary between species and organ. In a number of cases there is clear evidence that Glc6P is transported, and that it is exchanged with either inorganic phosphate or triose phosphate. This is true of roots (Borchert et aI., 1989; Hartwell et aI., 1996), some developing seeds (Hill and Smith, 1995; Kang and Rawsthome, 1994; Tetlow et aI., 1994), floral organs (Mohlmann et aI., 1995) and tubers (Schott et aI., 1995). Where there is some controversy is over the movement of glucose I-phosphate (GlclP). In some cases e.g. cauliflower bud (Mohlmann et aI., 1995) and maize endosperm (Batz et aI., 1993) it appears that GlclP does not involve counter-exchange with Pi. Glc 1 P has also been found to support starch synthesis in potato tuber amyloplasts (Naeem et aI., 1997) and soybean cell cultures (Coates and ap Rees, 1994) but it is not clear whether this involves counter-exchange

6. Integration of plastid metabolism 121

with Pi or is a uniport. In the case of the HPT from developing wheat endosperm there is unequivocal evidence that both G1c6P and G1clP can be transported in exchange for triose phosphate or Pi (Tetlow et aI., 1996). Envelope proteins from purified endosperm amyloplasts were reconstituted into proteoliposomes. Movement of G1clP, Glc6P, Pi or triose phosphates was absolutely dependent on the presence of an appropriate counter-ion (one of the same group) on the opposite side of the bilayer. Of particular note is the stoichiometric relationship between G1cIP and Pi counter-exchange. The data in Figure 2 (from Tetlow et aI., 1996) illustrate the strict I: I relationship between the movement of G1clP out of the proteoliposomes and transport of Pi into the proteoliposomes. Further, this counter-exchange was inhibited by G1c6P implying that, at least in this situation, the same transporter is responsible for the movement of both hexose phosphates. This has implications for the control of carbon metabolism both inside and outside these endosperm amyloplasts as discussed below.

First of all, the 1: 1 stoichiometric exchange between G1c 1 P (or Glc6P) and Pi gives rise, theoretically, to an anomaly. During starch synthesis, as a result of the action of ADPglucose pyrophosphorylase (AGPase) and alkaline pyrophosphatase (APPase), two moles of Pi are produced for each mole of hexose phosphate utilised. Consequently, there would be a continuous accumulation of Pi under these circumstances which patently does not occur in vivo. That this accumulation does not occur is illustrated by experiments in which the internal metabolite contents of wheat amyloplasts were monitored, under steady state, during starch synthesis in vitro. In Figure 3, it can be seen that the amyloplastic concentration of Pi is maintained below that of ADPglucose when GlclP and ATP are supplied to the organelles. It must therefore be concluded that some other mechanism exists to "dispose of' the additional Pi generated. Unidirectional transport of orthophosphate has been demonstrated from cauliflower bud amy lop lasts (Neuhaus and MaaB, 1996) which may account for this. This activity could be inhibited by DIDS and pyridoxal phosphate, inhibitors of both HPT and TPT, leaving open the possibility that the HPT could also be operating as a uniport. An alternative possibility is that there is substrate level phosphorylation of ADP within the organelle. This could occur for example via a triose phosphate shuttle (see section 2.2) or through the transport of PEP (see Chapter 5) and the activity of plastidial pyruvate kinase.

Secondly, in the case of amyloplasts from wheat endosperm (Tetlow et aI., 1994) and soybean cell suspensions (Coates and ap Rees, 1994) there appears to be a preference for GlclP over Glc6P in supporting starch synthesis, whilst the latter is utilised more effectively during carbohydrate oxidation within these organelles. The interconversion of

122 Chapter 6

100

--I r::: 80 CD -0 L.. c.. C)

60 E

'0 E c - 40

"'C CD r::: IV C)

I 20 a: N

'" o

o 20 40 60 80 100 120

14C-Glc 1 P lost (nmol. mg protein-I)

Figure 2 Stoichiometry of Glc1P/Pi exchange in reconstituted wheat endosperm amyloplast membranes at pH 8.0. Proteoliposomes were pre loaded with 10 mM C4C]Glc1P and incubated with 1 mM e2p]Pi at 20°C for different times. Simultaneous measurements of e2p]Pi uptake into and [14C]GlcIP loss from the proteoliposomes at different time intervals are shown as the means ± S.E.M. of at least three independent experiments. The line has a slope of 0.98 as determined by linear regression analysis indicating a 1: 1 exchange of hexose phosphate for Pi (from Tetlow et aI., 1996).

6. Integration of plastid metabolism 123

these two substrates is catalysed by phosphoglucomutase which is present in both cytosol and plastids (Entwistle and ap Rees, 1988), and which at equilibrium would bring about a 20-fold excess of Glc6P over GlclP (King, 1970). It follows from the above observation and the data in Figure 3 that this enzyme may not be catalysing an equilibrium reaction within amyloplasts. Further, its activity in the cytoplasm becomes equally significant if, in some tissues, glucose I-phosphate is imported into amyloplasts for starch synthesis. Estimates in whole extracts and non-aqueously isolated starch granules of maize endosperm indicate a 40-fold excess of Glc6P over GlclP in both compartments (Liu and Shannon, 1981). In potato, values of 15 nmol g-I fresh weight and 150 nmol g-I fresh weight for GlclP and Glc6P, respectively, have been determined (Burrell et aI., 1994) although there is no information on subcellular compartmentation of metabolites in this tissue. A similar situation is true for wheat endosperm (Glc6P = 230 nmol g-I fresh weight, GlclP = 14 nmol g-I fresh weight: U. Tetlow, unpublished results). If these are partitioned in similar proportions between cytoplasm and amyloplast it would be difficult to perceive how starch synthesis could continue utilising cytosolic GlclP as a substrate, uptake of which would be severely inhibited by the excess of Glc6P which, itself, appears unable to support starch synthesis. The reconciliation of this lies either in our incomplete understanding of intracellular metabolite contents and regulation of cytosolic phosphoglucomutase or in the possibility that there may be an alternative route for starch synthesis (see below).

2.2 Adenylates

The most obvious distinction between photosynthetic and non­photosynthetic plastids is the absence of photophosphorylation in the latter. It follows therefore that such heterotrophic plastids require mechanisms for the import of either ATP, or adenylates from which ATP can be generated within the organelle by substrate phosphorylation. Studies of A TP dependent processes in preparations of non­photosynthetic plastids, such as starch and fatty acid synthesis, have indicated a strict requirement for an exogenous supply of A TP (Hill and Smith, 1991; Kang and Rawsthorne, 1994, 1996; Mohlmann et aI., 1994; Tetlow et aI., 1994). However, an investigation of acetate dependent fatty acid synthesis in pea root plastids indicated that a triose phosphate shuttle in the presence of ADP could support rates of synthesis equivalent to those obtained with ATP alone (Kleppinger-Sparace et aI., 1992). This has not been studied in such detail in other systems and it therefore must remain an open possibility that substrate level

124 Chapter 6

3PGA

Pi

ATP rIJ ~ ADPG ..... ... -Q

UDPG .,Q

S ~ Glc1P :a

Fru6P

Glc6P

6PG

0 5 10 15

nmoles Figure 3 Metabolite pools in amyloplasts isolated from wheat endosperm. Intact amyloplasts were incubated with 1 mM ATP plus 5 mM GlclP for 30 min at 25°C prior to metabolite extraction. The amount of each metabolite measured is normalised to a constant 10-3 units of alkaline pyrophosphatase. Values for ruptured preparations at t = 0 and t = 30 min have been subtracted from the values for intact preparations. All values are the mean ± S.E.M. of at least three independent experiments.

6. Integration of plastid metabolism 125

phosphorylation can occur within such organelles at rates sufficient to support biosynthesis. Nonetheless, it is clear that whether ADP is phosphorylated within the organelle or enters as ATP, adenylates can be transported. An antiserum raised against the 29 kDa mitochondrial adenylate trans locator from Neurospora crassa was reported to cross­react with the plastidic equivalent in sycamore cells (Pozueta-Romero et aI., 1991). The same antibody did not, however, cross-react with membrane proteins from pea root plastids which were also much less sensitive to the classical mitochondrial inhibitors, bonkrekic acid and carboxyatractyloside (Schiinemann et aI., 1993). These observations suggest that the plastidic adenylate trans locator has a significantly different structure from that of its mitochondrial counterpart. This view is reinforced by the discovery of a new type of adenylate transporter in Arabidopsis thaliana (Kampfenkel et aI., 1995). These authors obtained a cDNA clone from Arabidopsis with some homology to an A TP transporter from the intracellular parasite Rickettsia prowazekii. The mRNA sequence for the plant protein codes for a protein of 68 kDa which reduces to 60 kDa after import into plastids (Neuhaus et aI., 1997). The transporter was overexpressed in E. coli and yeast, and when reconstituted into liposomes could catalyse the counter-exchange of A TP and ADP.

Recently, more attention has focused on the possibility of ADPglucose transport into amyloplasts. The driving force for this was the original suggestion by Akazawa and co-workers (Pozueta-Romero et aI., 1991) that this metabolite could be produced in the cytosol and imported into plastids. This possibility has taken on new significance with the demonstration that developing endosperm of maize (Denyer et aI., 1996) and barley (Thorbjemsen et at, 1996) possess an extraplastidial AGPase which is the major form of the enzyme in these tissues, although a plastidic form is still present. This is consistent with the observation that cDNAs cloned for the small subunit (Brittle2) of the enzyme from maize and barley endosperm apparently lack sequences for transit peptides which would be regarded as essential for targeting and import into plastids (Giroux and Hannah, 1994; Villand and Kleczkowski, 1994). In earlier work on wheat endosperm amyloplasts, Tetlow et ai. (1994) demonstrated that ADPglucose was by far the most effective precursor for starch synthesis when supplied to intact organelles, implying that it would be transported. Recently, M6hlmann et ai. (1997) have found that exogenous ADPglucose is also the most effective precursor for starch synthesis in maize endosperm amyloplasts. In agreement with this proposal is the observation that the Btl mutant of maize encodes a 44 kDa protein located in the plastidial envelope. Mutation at this locus not only reduces starch accumulation but also

126 Chapter 6

causes an increase in the ADPglucose content (Shannon et aI., 1996), though it has yet to be shown directly that Btl codes for an ADPglucose transporter. Interestingly Mohlmann et al. (1997) have found that ADPglucose transport via the carrier from maize endosperm amyloplasts involves counter-exchange with AMP not ADP when membrane proteins were reconstituted into proteoliposomes. We have recently found that there are two forms of AGPase small subunit in developing wheat endosperm, one of which is confined to amyloplasts (K. Vardy, unpublished data). These are expressed in roughly equal amounts at 10 days post-anthesis, though the larger of the two forms which represents the cytosolic form is either absent or relatively low in abundance prior to this stage (M.M. Burrell, unpublished data). There is therefore a case for believing that cereal endosperms possess the capacity to produce ADPglucose in either the cytoplasm or the amyloplast and that the balance between these two possibilities changes during development. The localisation of ADPglucose synthesis in the cytosol obviates the need to invoke novel mechanisms to dispose of Pi which, as argued earlier, would theoretically accumulate in amyloplasts, and is therefore attractive. It nonetheless leaves open the question of the role of the amyloplastic AGPase and APPase. Further, there are tissues such as potato and developing pea embryo where there is no evidence for extraplastidic AGPase (Hill and Smith, 1991; La Cognata et aI., 1995) and which appear to use hexose phosphate as the cytosolic substrate imported for starch synthesis. Interestingly Naeem et al. (1997) found that starch synthesis in intact potato amyloplasts could be supported by ADPglucose and was dependent on the intactness of the preparations. Nonetheless, the same report demonstrated that GlclP plus ATP supported equally high rates of starch synthesis, and localisation of enzymes was consistent with the view that the vast majority of AGPase was plastidial.

It would therefore appear that amyloplasts are capable of transporting a multiplicity of compounds related to carbohydrate metabolism. The operation of alternative routes for starch synthesis raises important questions about the transport of metabolites, whose trans locators may be under developmental control during the ontogeny of tissues such as endosperm, and further questions about the relationship between cytosolic and plastidic metabolism.

3. COORDINATION OF METABOLISM

By comparison with studies of chloroplasts there have been relatively few detailed investigations 0f metabolic fluxes and pathway interactions

6. Integration a/plastid metabolism 127

within non-photosynthetic plastids. Many reports have been confined to detennining which exogenous substrates are capable of supporting a particular biosynthetic process, e.g. starch synthesis, and therefore have tended to offer insight into transport rather than intermediary metabolism. However, there is an increasing number of examples where the interactions between metabolic pathways have been studied in such organelles by the use of radio1ablled precursors. An excellent example of this is reviewed by Rawsthome and co-workers (Chapter 7) who have carried out detailed studies of carbon fluxes between pathways of starch and fatty acid synthesis and carbohydrate oxidation in plastids of oil-seed rape embryos. Other examples will now be considered.

One of the best studied examples comes from work with amyloplasts from roots of Pisum sativum. It is possible to obtain relatively large quantities of the organelles in highly purified, intact fonn (Bowsher et a!., 1989; Emes and England, 1986) sufficient to study metabolism or as starting material for the purification of proteins within the plastid (Bowsher et a!., 1993a). A major function of these organelles is the assimilation of inorganic nitrite, produced in the cytoplasm, and the synthesis of amino acids. Both these activities require a source 0 f ferredoxin, which is reduced as a result of carbohydrate oxidation within the organelle. The entire sequence of reactions which comprise the oxidative pentose phosphate pathway (OPPP) have been found within pea root plastids (Emes and Fowler, 1979) and as indicated earlier there is ample evidence that GIc6P, the substrate for this pathway, can enter the organelles via a phosphate translocator. Flux through the plastidic OPPP can be followed by supplying [1-14C]-GIc6P, as CO2 is released specifically from C-l of 6-phosphogluconate during a decarboxylation reaction. Supplying Glc6P labelled in other carbon atoms indicates that no CO2 is lost from other carbon atoms (Bowsher et a!., 1992). Although these preparations contain most of the glycolytic sequence of reactions (Trimming and Emes, 1993) and an active pyruvate dehydrogenase (Qi et aI., 1996), there is no evidence of significant flux via this route. When either nitrite or the substrates for the glutamate synthase reaction are supplied there is a stimulation of 14C02 released from [1-14C]-GIc6P, consistent with the view that the OPPP is the primary source of reductant for these reactions. There is a tight-coupling between nitrite reductase (NiR), glutamate synthase and the OPPP as indicted in Figure 4. From the diagram it can be seen that dihydroxyacetone phosphate (DHAP) is exported in exchange for incoming Glc6P (Borchert et aI., 1993). Recent experiments in which the oxidation of Glc6P, export of triose phosphate (DHAP) and reduction of nitrite were monitored simultaneously have established the stoichiometry of this interaction to be 3: 1 :2, as illustrated in Figure 5 (Hartwell et aI., 1996). Further, by

128

Glc6P

DHAP

Chapter 6

Glc6P

.. :.-----------2 Glc6P+.------- 2 Fru6P

3 Glc6P / y--- 'ADP·~Fd".

( ~NADPH---'---rd •• Glc6P 36PG

DIlAP

Ga3P

~ AOp·~rd"d CO,-- .. ~ . D ,----,-

3 RuSP A PH rd ••

/"". 2XUSPXSP

c,':x= "" E~P Fru6P

'-----+Yru6P

Figure 4 Proposed interaction between the oxidative pentose phosphate pathway and nitrite reductase in pea root plastids.

6. Integration of plastid metabolism

600

-I C

CD -400 o "­Co

o o 20

129

40 60

TIME (min) Figure 5 Relationship between C02 evolution (T), DHAP production (e) and nitrite reduction (0) in intact p1astids incubated with 10 mM [1}4C]Glc6P and 1 mM nitrite (from Hartwell et aI., 1996).

130 Chapter 6

following the distribution of label and evolution of CO2 from carbon atom 2 it has been demonstrated that the OPPP operates cyclically within these organelles. Recently, Schnarrenberger et al. (1995) have suggested that in spinach leaf at least, the non-oxidative reactions of the OPPP are confined to plastids. Given the general importance of this pathway in supporting biosynthesis, this now needs to be investigated in non-photosynthetic tissue.

During the onset of nitrate assimilation in roots, which is an inducible process, there is up-regulation of NiR (Bowsher et aI., 1991), and an increase in the activity of plastidial glutamine synthetase (Emes and Fowler, 1983). This increase in flux from inorganic nitrogen to amino acids has to be sustained by carbohydrate oxidation. In recent times our view of what controls metabolism has moved away from a simple notion of looking for a "rate-limiting" step, towards a more sophisticated analysis based on the ideas of Kaeser (1987) and others who have established the theorem of flux control analysis. In the latter, flux is regarded as a shared property of all the steps in a pathway, with some exerting more control than others. A corollary of this theorem (see Chapter 13) is that to increase flux through a pathway will generally require up-regulation of several steps and not just one. For example, over-expression of a single enzyme in a transgenic plant seldom has the desired effect of increasing flux through the pathway in which it sits (Burrell et aI., 1994). It is therefore notable that, consistent with this view, during the onset of nitrate assimilation in roots there is an increase in several components related to meeting the elevated demands for reductant which the former would invoke. The activities of both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase increase in plastids during induction of nitrate assimilation (Emes and Fowler, 1983). These oxidative reactions produce NADPH, and the transfer of electrons to ferredoxin (required by NiR and glutamate synthase) is mediated by ferredoxin - NADP+ reductase (FNR). In roots, both these components are also induced during nitrate assimilation (Bowsher et aI., 1993b) although, as in the case of the dehydrogenases of the OPPP, there is residual activity even in the absence of nitrate which is necessary to sustain reactions such as those catalysed by glutamate synthase. These observations reinforce the view that integration of metabolism within non-green plastids at the level of the gene involves co-ordinate expression of several components in pathways which are proposed to interact.

It might be supposed from this, that the increased activity of all those steps involved in supporting NiR activity would meet the demand for reductant over and above that which is needed to support constitutive glutamate synthase activity. Consequently, reductant supply would not be

6. Integration of plastid metabolism 131

expected to be limiting either reaction. Surprisingly studies in vitro with purified root plastids suggest that NiR and glutamate synthase are competing for a limited pool of reductant despite the changes which occur during induction of nitrate assimilation (Bowsher et aI., 1996). Plastids, from roots in which nitrate assimilation had been induced, were supplied with the substrates for NiR and glutamate synthase independently and also together. The ability of G lc6P to support one reaction was diminished by the operation of the other at all concentrations of hexose phosphate used, including up to saturating levels. Measurement of CO2 released from C 1 of Glc6P (flux through the OPPP) in the presence of nitrite only, could not be further enhanced by the addition of substrates for glutamate synthase. Possible constraints on the ability to sustain both reductant requiring reactions optimally could lie at the point of entry (transport) of Glc6P into the organelle, or within the oxidative pathway itself. One approach to determine the importance of different steps in controlling metabolism is to determine their control coefficients (see Chapter 13). Dihyroxyacetone phosphate (DHAP) is a competitive inhibitor of Glc6P transport (Borchert et aI., 1989) which does not covalently modify the transporter and is not an alternative substrate for nitrite reduction. When pea root plastids were incubated with Glc6P and nitrite, uptake of Glc6P could be inhibited by up to 90% in the presence of DHAP with no significant effect on the rate of Glc6P-dependent nitrite reduction (Bowsher et aI., 1996). The data provide a value for a control coefficient for Glc6P transport over this process of less than 0.02 implying that control of this interaction, and the limitation on reductant supply, lies not with the translocator but in the oxidation of Glc6P within the organelle. If other pathways, requiring NADPH, such as fatty acid synthesis, are superimposed on this in vivo, then it seems likely that the OPPP plays an important role in regulating assimilatory and biosynthetic pathways.

The above example illustrates how two reactions in the same pathway (NiR and glutamate synthase) may compete with one another for a common substrate. There are also examples in the literature where opposing pathways in the same organelle may compete for a common metabolite. Cauliflower bud amyloplasts have been used to study the interdependence of starch synthesis and fatty acid synthesis on the availability of A TP (Mohlmann et aI., 1994) (see Figure 1). Acetate was supplied as the substrate for fatty acid synthesis and Glc6P as the precursor for starch synthesis. The latter activity is markedly dependent on the provision of phosphoglycerate (PGA) which is an activator of AGPase. When starch synthesis was stimulated by the addition of PGA, acetate-dependent fatty acid synthesis decreased even when A TP was supplied at concentrations which saturated each process independently,

132 Chapter 6

suggesting competition for energy within the organelle. A control coefficient of starch synthesis over fatty acid synthesis of -0.5 was calculated when A TP was limiting. Again, these results emphasise the point that the oxidative processes which generate A TP outside the plastid will have an enormous bearing on the flux of carbon within the plastid and could influence the nature of the end-product formed.

In other instances there may also be competition for intra-plastidic hexose phosphate (Figure 1). Purified wheat endosperm amy lop lasts are able to synthesise starch from GlclP, and can oxidise either GlclP or Glc6P through the OPPP (Tetlow et aI., 1994). When such preparations were also supplied with the substrates for glutamate synthase, the rate of starch synthesis from GlclP fell by 75% as hexose phosphate was diverted to the OPPP in order to generate reductant to support amino acid metabolism. To what extent such competition exists in vivo is impossible to establish at present, although Keeling and co-workers suggest that the loss of CO2 from the amyloplast OPPP in this tissue is substantial relative to the rate of starch synthesis (Keeling et aI., 1988).

4. CONCLUSIONS

Our knowledge of metabolism within non-photosynthetic plastids is gradually improving. With the potential for biotechnological modification of major end-products, such as starch and fatty acids which are produced in heterotrophic storage tissues, there is clearly a need for much greater emphasis on understanding coordination of metabolism within these organelles and with other subcellular compartments such as the cytosol. It is worth noting that as the number of studied examples of plastids from different sources has increased, so we have become aware of significant species differences. For example the pathway of starch synthesis, which previously would have been considered to be generically uniform, is now believed to be organised differently between cereals and dicotyledonous species. This has considerable implications for the movement of metabolites in and out of amy lop lasts and for the turnover of common metabolites, such as pyrophosphate, in different compartments.

In this review we have also attempted to highlight sites which could regulate intermediary metabolism including: (1) transport of metabolites across the plastid envelope; (2) the need to integrate biosynthetic demand with provision of ATP and NADPH; (3) the potential competition which exists between pathways which utilise hexose phosphate as a biosynthetic precursor, and those which oxidise it.

6. Integration of plastid metabolism 133

There are too few examples in the literature to draw any general conclusions but as Torn ap Rees once wrote "Thus we may proceed with optimism, provided it is tempered by the realization that the plasticity of plant metabolism and the sharing of control will often defeat us" (ap Rees, 1995).

ACKNOWLEDGEMENTS

The authors gratefully acknowledge financial support from BBSRC and the Royal Society, and they also thank M.M. Burrell, K. Vardy and E. Neuhaus for making unpublished data and papers in press available. We thank Ms A. Parker for her careful typing of the manuscript. Finally, like many of the contributors to this volume, we are indebted to the late Torn ap Rees for the many hours of enjoyable and stimulating discussion from which we benefited, and for his encouragement and friendship which was always given in full measure.

REFERENCES

ap Rees, T. (1995). Prospects of manipulating plant metabolism. Trends in Biotechnology, 13, 375-378.

Batz, 0., Scheibe, R. and Neuhaus, H.E. (1993). Identification of the putative hexose phosphate translocator of amyloplasts from cauliflower buds. Biochemical Journal, 294, 15-17.

Borchert, S., Grosse, H. and Heldt, H.W. (1989). Specific transport of phosphate, glucose 6-phosphate, dihyroxyacetone phosphate and 3-phosphoglycerate into amyloplasts from pea roots. FEBS Letters, 253, 183-186.

Borchert, S., Harborth, J., Schilnemann, D., Hoferichter, P. and Heldt, H.W. (1993). Studies of the enzymic capacities and transport properties of pea root plastids. Plant Physiology, 101, 303-312.

Bowsher, C.G., Boulton, E.L., Rose, J., Nayagam, S. and Emes, MJ. (1992). Reductant for glutamate synthase is generated by the oxidative pentose phosphate pathway in non­photosynthetic root plastids. Plant Journal, 2, 893-898.

Bowsher, C.G., Dunbar, B. and Emes, M.J. (1993a). The purification and properties of ferredoxin-NADP+-oxidoreductase from roots of Pisum sativum L. Protein Expression and Purification, 4, 512-518.

Bowsher, C.G., Hucklesby, D.P. and Emes, M.J. (1993b). Induction of ferredoxin-NADP+ oxidoreductase and ferredoxin synthesis in pea root plastids during nitrate assimilation. Plant Journal, 3, 463-467.

Bowsher, C.G., Hucklesby, D.P. and Emes, MJ. (1989). Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum sativum L. Planta, 177, 359-366.

134 Chapter 6

Bowsher, C.G., Long, D.M., Oaks, S.J. and Rothstein, S.J. (1991). The effect of light/dark cycles on expression of nitrate assimilatory genes in maize shoots and roots. Plant Physiology, 95, 281-285.

Bowsher, C.G., Tetlow, I.J., Lacey, AE., Hanke, G.T. and Emes, M.J. (1996). Integration of metabolism in non-photosynthetic plastids of higher plants. Comptes Rendus d'Academie Francaise des Sciences, 319, 853-60.

Burrell, M.M., Mooney, P.J., Blundy, M., Carter, D., Wilson, F., Green, J., Blundy, K.S. and ap Rees, T. (1994). Genetic manipulation of 6-phosphofructokinase in potato tubers. Planta, 194, 95-101.

Coates, S.A and ap Rees, T. (1994). Metabolism of glucose monophosphates by leucoplasts and amyloplasts from soybean suspension cultures. Phytochemistry, 35, 881-883.

Denyer, K., Dunlap, F., Thorbjornsen, T., Keeling, P. and Smith, A.M. (1996). The major form of ADP-glucose pyrophosphorylase in maize endosperm is extra-plastidial. Plant Physiology, 112, 779-785.

Emes, M.J. and England, S. (1986). Purification ofplastids from higher plant roots. Planta, 168, 161-166.

Emes, M.J. and Fowler, M.W. (1979). Intracellular interactions between the pathways of carbohydrate oxidation and nitrate assimilation in plant roots. Planta, 145, 287-292.

Emes, M.J. and Fowler, M.W. (1983). The supply of reducing power for nitrite reduction in plastids of seedling pea roots (Pisum sativum L.). Planta, 158,97-102.

Emes, M.J. and Traska, A. (1987). Uptake of inorganic phosphate by plastids purified from the roots of Pisum sativum L. Journal of Experimental Botany, 38,1781-1788.

Entwistle, G. and ap Rees, T. (1988). Enzymic capacities of amy lop lasts from wheat (Triticum aestivum) endosperm. Biochemical Journal, 255, 391-396.

Fischer, K., Kammerer, B., Gutensohn, M., Arbinger, B., Weber, A, Hausler, R. and FlOgge, U.-I. (1997). A new class of plastidic phosphate translocator: a putative link between primary and secondary metabolism by the phosphoenolpyruvate/phosphate antiporter. Plant Cell, 9, 453-462.

Fischer, K., Weber, A, Brink, S., Arbinger, B., Schuenemann, S., Borchert, S., Heldt, H.W., Popp, B., Benze, R., Link, T.A, Eckerskom, C. and FIOgge, U.-I. (1994). Porins from plants. Molecular characterization of two members of the porin family. Journal of Biological Chemistry, 269, 25754-25760.

FIOgge, U.-I. and Heldt, M.W. (1984). The phosphate-triose phosphate-translocator of the chloroplast. Trends in Biochemical Sciences, 9, 530-533.

Giroux, M.J. and Hannah, L.C. (1994). ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize. Molecular and General Genetics, 243, 400-408.

Gray, J.C. and Row, P.E. (1995). Protein translocation across chloroplast envelope membranes. Trends in Cell Biology, 5, 243-247.

Hartwell, J., Bowsher, C.G. and Emes, M.J. (1996). Recycling of carbon in the oxidative pentose phosphate pathway in non-photosynthetic plastids. Planta, 200, 107-112.

Hill, L.M. and Smith, AM. (1991). Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos. Planta, 185, 91-96.

Hill, L.M. and Smith, AM. (1995). Coupled movements of glucose 6-phosphate and triose phosphate across the envelopes of plastids from developing embryos of pea (Pisum sativum L.). Journal of Plant Physiology, 146,411-417.

Kacser, H. (1987). Control of metabolism. In: Davies, D.D. (Ed). The Biochemistry of Plants. Vol. 11. Biochemistry of Metabolism (pp 39-67). Academic Press, London.

6. Integration of plastid metabolism 135

Kampfenkel, K., M5hlmann, T., Batz, 0., van Montagu, M., Inze, D. and Neuhaus, H.E. (1995). Molecular characterisation of an Arabidopsis thaliana cDNA encoding for a novel putatitive adenylate transporter of higher plants. FEBS Letters, 374, 351-355.

Kang, F. and Rawsthorne, S. (1994). Starch and fatty acid synthesis in plastids from developing embryos of oil seed rape (Brassica napus L.). Plant Journal, 6, 795-805.

Kang, F. and Rawsthorne, S. (1996). Metabolism of glucose 6-phosphate and utilisation of multiple metabolites for fatty acid synthesis by plastids from developing oil seed rape embryos. Planta, 199,321-327.

Keeling, P.L., Wood, lR., Tyson, R.H. and Briggs, I.G. (1988). Starch synthesis in developing wheat grain. Plant Physiology, 87, 311-319.

King, J. (1970). Phosphoglucomutase. In: Bergmeyer, H.U. (Ed). Methoden der enzymatischen Katalyse (pp 764-785). Verlag Chern ie, Weinheim.

Kleppinger-Sparace, K.F., Stahl, R.I. and Sparace, S.A. (1992). Energy requirements for fatty acid and glycerolipid biosynthesis from acetate by isolated pea root plastids. Plant Physiology, 98, 723-727.

La Cognata, U., Willmitzer, L. and Miiller-R5ber, B. (1995). Molecular cloning and characterisation of novel isoforms of potato ADP-glucose pyrophosphorylase. Molecular and General Genetics, 246, 538-548.

Liu, T-T.Y. and Shannon, le. (1981). Measurement of metabolites associated with non­aqueously isolated starch granules from immature Zea mays L. endosperm. Plant Physiology, 67, 525-529.

M5hlmann, T., Batz, 0., MaaB, U. and Neuhaus, H.E. (1995). Analysis of carbohydrate transport across the envelope of isolated cauliflower bud amyloplasts. Biochemical Journal, 307, 521-526.

M5hlmann, T., Scheibe, R. and Neuhaus, H.E. (1994). Interaction between starch synthesis and fatty-acid synthesis in isolated cauliflower-bud amyloplasts. Planta, 194, 492-497.

M5hlmann, T., Tjaden, 1, Henrichs, G., Quick, W.P., H!iusler, R. and Neuhaus, H.E. (1997). ADPglucose drives starch synthesis in isolated maize-endosperm amyloplasts. Characterisation of starch synthesis and transport properties across the amyloplastidic envelope. Biochemical Journal, 324, 503-509.

Naeem, M., Tetlow, I.J. and Emes, M.J. (1997). Starch synthesis in amyloplasts purified from developing potato tubers. Plant Journal, II, 101-109.

Neuhaus, H.-E. and MaaB, U. (1996). Unidirectional transport of orthophosphate across the envelope of isolated cauliflower-bud amyloplasts. Planta, 198, 542-548.

Neuhaus, H,.E., Thorn, E., M5hlmann, T., Steup, M. and Kampfenkel, K. (1997). Characterisation of a novel ATP/ADP transporter from Arabidopsis thaliana L. Plant Journal, II, 73-82.

Pozueta-Romero, J., Frehner, M., Viale, A.M. and Akazawa, T. (1991). Direct transport of ADPglucose by an adenylate translocator is linked to starch biosynthesis in amyloplasts. Proceedings of the National Academy of Sciences USA, 88, 5769-5773.

Qi, Q., Trimming, B.A., Kleppinger-Sparace, K.F., Emes, M.I. and Sparace, S.A. (1996). Pyruvate dehydrogenase complex and acetyl-CoA carboxylase in pea root plastids: their characterisation and role in modulating glycolytic carbon flow to fatty acid biosynthesis. Journal of Experimental Botany, 47, 1889-1896.

Shannon, lC., Pien, F-M. and Lui, K-C (1996). Nucleotides and nucleotide sugars in developing maize endosperms. Plant Physiology, 110, 835-843.

Schnarrenberger, C., Flechner, A. and Martin, W. (1995). Enzymatic evidence for a complete oxidative pentose phosphate pathway in chloroplasts and an incomplete pathway in the cytosol of spinach leaves. Plant Physiology, 108, 609-614.

136 Chapter 6

Schott, K., Borchert, S., Milller-ROber B. and Heldt, H.W. (1995). Transport of inorganic phosphate and C3- and C6- sugar phosphates across the envelope membranes of potato tuber amyloplasts. Planta, 196, 647-652.

Schilnemann, D., Borchert, S., Flilgge, U.-I. and Heldt, H.W. (1993). ATP/ADP trans locator from pea-root plastids. Comparison with trans locators from spinach chloroplasts and pea-leaf mitochondria. Plant Physiology, 103, 131-137.

Tetlow, I.J., Blissett, K.J. and Emes, M.l. (1994). Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm. Planta, 194, 454-460.

Tetlow, I.J., Bowsher, C.G. and Emes, M.J. (1996). Reconstitution of the hexose phosphate trans locator from the envelope membranes of wheat endosperm amyloplasts. Biochemical Journal, 319, 717-723.

Trimming, B.A and Emes, MJ. (1993). Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots. Planta, 190, 439-445.

Thorbj0rnsen, T., Villand, P., Denyer, K., Olsen, O-A and Smith, AM. (1996). Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm. Plant Journal, 10, 243-250.

Villand, P. and Kleczkowski, L.A (1994). Is there an alternative pathway for starch biosynthesis in cereal seeds? Zeitschrift fur Naturforschung, 49, 225-219.

Chapter 7

Carbon flux to fatty acids in plastids

Stephen Rawsthorne, Fan Kang and Peter J. Eastmond Brassica and Oi/seeds Research Department, John Innes Centre, Cotney, Norwich NR4 7UH, UK

Key words: fatty acid synthesis; glucose 6-phosphate transport; oxidative pentose phosphate pathway; plastid; pyruvate transport; starch synthesis.

Abstract: This article considers our understanding of the pathways involved in the provision of precursors for de novo fatty acid synthesis in plastids. The characteristics of the plastidial enyzmes required for synthesis of acetyl­and malonyl-CoA are reviewed. The role of transporters in determining the metabolic routes by which carbon is imported into the plastids and the extent to which the different transporters are utilized for fatty acid synthesis are discussed. Interactions between metabolic pathways in plastids are illustrated by considering the partitioning of imported glucose 6-phosphate to starch, fatty acids and to C02 via the oxidative pentose phosphate pathway in plastids isolated from developing rapeseed embryos.

1. INTRODUCTION

In plants the de novo synthesis of fatty acids occurs primarily in the plastid (Harwood, 1988). These fatty acids are used for the synthesis of plastidial and other cellular membranes in all cells. In certain plant tissues, most notably in seeds, they are also used for the synthesis of storage oils (triacylglycerols). This aspect of lipid synthesis has attracted much attention recently because of the potential to produce feedstocks for the chemical industry in planta as components of storage oils. There are two routes to this goal. First, the development of plant species which synthesize novel oils into "alternative oilseed" crops. Second, the cloning

137

N. J. Kruger et at. (eds.), Regulation of Primary Metabolic Pathways in Plants, 137-157. © 1999 Kluwer Academic Publishers.

138 Chapter 7

of genes which determine the synthesis of these novels oils and introduction of these genes into existing crops such as oilseed rape (Brassica napus L.) through genetic transformation. Whichever approach is adopted we need to understand what controls the amount of storage oil accumulated in order to optimise yield. However, as yet we do not fully understand how the carbon is supplied for the synthesis of the fatty acids in oil storing tissues. Furthermore we know very little about how the flux of carbon to fatty acids and then oil is controlled, or indeed how the partitioning of carbon between oil and other storage products that are laid down simultaneously is determined.

In describing the present state of knowledge of fatty acid synthesis by plastids from oil storing tissues, which are mostly non-photosynthetic, it is important to consider what is known about fatty acid synthesis in chloroplasts and plastids from non-oil storing tissues. Acetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA to malonyl-CoA in what is considered to be the first committed step in fatty acid synthesis (Harwood, 1988). Since acetyl-CoA is not imported by plastids (Roughan et aI., 1979; Weaire and Kekwick, 1975) it must be generated by metabolism within the plastid. In chloroplasts photosynthesis provides an endogenous source of fixed carbon. Whether this fixed carbon can be utilized for the synthesis of acetyl-CoA depends upon the enzyme complement within the chloroplast and is discussed below. In the case of non-photosynthetic cells/organs the plastid is dependent upon import of metabolites from the cytosol in order to synthesize acetyl-CoA. This import process is likely to involve specific transporter proteins on the plastid envelope, as have been reported for non-photosynthetic plastids which carry out starch synthesis (e.g. Batz et aI., 1993; Borchert et aI., 1989; Hill and Smith, 1991; and see Chapter 5).

In this article we will begin by reviewing the literature regarding the plastidial enzymes that are required to synthesize acetyl- and malonyl­CoA. We will then describe which metabolites are known to be imported into plastids and used for fatty acid synthesis, and how this import occurs. Fatty acid synthesis requires A TP and reducing power and we will describe the current understanding as to how this requirement is met in photosynthetic and non-photosynthetic organs. In plastids from some organs/tissues, imported metabolites are used for more than one metabolic pathway, e.g. for starch and fatty acid synthesis, and the oxidative pentose phosphate pathway (OPPP) (Kang and Rawsthorne, 1994, 1996), or for starch synthesis and the OPPP (Tetlow et aI., 1994). This metabolic partitioning and the effects of development on carbon fluxes to fatty acid synthesis and other pathways will be presented in the

7. Carbon flux to/atty acids inplastids 139

context of our recent work which is using developing embryos of oilseed rape.

2. ENZl'MES WITlDNPLASTIDS

2.1 Synthesis of acetyl- and malonyl-Coenzyme A.

The synthesis of acetyl coenzyme-A (CoA) in plastids can occur through three separate routes. In the first, free acetate is activated to acetyl-CoA by acetyl-CoA synthetase (ACS) in an A TP-dependent reaction. The activity of ACS has been measured in chloroplasts of spinach, pea and maize (Kuhn et aI., 1981; Treede et aI., 1986) and is comparable to that of the other enzyme which might be involved in acetyl-CoA synthesis, the pyruvate dehydrogenase complex (POC) (see below). More recently, Zeiher and Randall (1991) have argued that the properties of the ACS purified from spinach chloroplasts are entirely consistent with it sustaining fatty acid synthesis in this organelle under physiological conditions.

As discussed below pyruvate can be produced by glycolytic activity within the plastid or by cytosolic glycolysis followed by import into the organelle. The pyruvate is converted to acetyl-CoA by POC in a reaction which also generates NAOH. In plant cells there are two forms of POC, one in the mitochondria and another in the plastid. Activity of POC has been reported in chloroplasts from several species (Hoppe et aI., 1993; Liedvogel, 1986; Williams and Randall, 1979). Plastids from castor endosperm, and developing pea and oilseed rape embryos contain POC activity which represents between 30 and 66% of the total cellular activity depending on the tissue studied (Oenyer and Smith, 1988; Kang and Rawsthome, 1994; Reid et aI., 1975, 1977).

A third possible route for intraplastidial synthesis of acetyl-CoA is through a plastidial camitine acetyltransferase reaction in which acetate is transferred from acetyl-camitine to CoA. It has been proposed that this activity represents part of a camitine-dependent acetyl/acyl transfer mechanism in the plant cell (Wood et aI., 1992). However, as we discuss later in considering the uptake and utilization of metabolites for fatty acid synthesis by isolated plastids, this proposed mechanism is controversial.

The synthesis of malonyl-CoA from acetyl-CoA is catalysed by acetyl-CoA carboxylase (ACCase) in an A TP-dependent step. The knowledge of the type of ACCase involved in this reaction in the plastid

140 Chapter 7

has developed considerably over the past three years. Two forms of ACCase are known to occur in plants: type I ACCase, which is a large multifunctional enzyme analogous to that found in yeast and mammals, and type II enzyme which is a multi subunit complex analogous to that found in prokaryotes. It is now known that the plastidial ACCase activity in most higher plants is due to a type II form of enzyme but in the Gramineae the activity is due to a type I enzyme (e.g. Alban et aI., 1994; Konishi et aI., 1996; Sasaki et aI., 1993). All higher plants possess an extraplastidial type I enzyme which is believed to be cytosolic (e.g. Egli et aI., 1993; Slabas and Hellyer, 1985). This picture has become more complicated still by the report of a type I enzyme protein in plastids purified from developing embryos of oilseed rape (Roesler et aI., 1997).

A great deal of interest has been focused on ACCase and the role that it might play in regulating the flux of carbon to fatty acids and so to storage oil. There are several experimental lines of evidence which suggest that ACCase does represent a regulatory step. Intermediates of fatty acid synthesis change during the transition to darkness in leaves and chloroplasts in a manner consistent with control at the level of ACCase (Post-Beittenmiller et aI., 1991, 1992). Page et aI. (1994) have reported that ACCase exerts strong flux control over fatty acid synthesis in isolated chloroplasts. More recently Shintani and Ohlrogge (1995) have suggested that ACCase activity can be regulated by feedback inhibition based upon experiments in which fatty acids were supplied to tobacco suspension cell cultures. Other indirect evidence comes from positive correlations between measurements of maximum catalytic activity of ACCase and the rate of lipid accumulation during seed development (Charles et aI., 1986; Simcox et aI., 1979) although this is not the case in oilseed rape (Kang et aI., 1994). Measurements of ACCase activity during seed development must now be re-examined in the light of multiple isoforms contributing to the total measurable activity in whole tissue homogenates. Of these isoforms it is only the plastidial activity which is important in de novo fatty acid synthesis. Recently Roesler et aI. (1997) targeted a type I ACCase to the plastids in developing oilseed rape embryos. They estimated that this increased total plastidial ACCase activity by up to two-fold which in tum resulted in an increase in fatty acid content of the seed of up to 5%. The elevated fatty acid phenotype in the transgenic plants was variable, and whether there was a significant increase was dependent upon the location in which the plants were grown. The extent to which ACCase or indeed any other plastidial enzyme step regulates fatty acid synthesis remains largely unresolved and more work is required.

7. Carbon jlLL'r to fatty acids in plastids 141

2.2 Synthesis of pyruvate.

If plastids are to utilise carbon from the level of hexose for the synthesis of pyruvate and so acetyl-CoA they require a complete glycolytic pathway. A complete glycolytic pathway has been reported to be present in non-photosynthetic plastids from pea and rapeseed embryos, cauliflower buds, and wheat and castor (Ricinus communis) seed endosperm (Denyer and Smith, 1988; Entwistle and ap Rees, 1988; Foster and Smith 1993; Journet and Douce, 1985; Kang and Rawsthorne, 1994; Simcox et aI., 1977). In several of these studies the activities of some of the glycolytic enzymes are reported to be low. In fact, phosphoglycerate mutase is reported to be absent from the plastids of pea roots and sycamore cells (Frehner et aI., 1990; Trimming and Emes, 1990). It is also uncertain if hexokinase and NAD-glyceraldehyde 3-phosphate dehydrogenase activities are present in the pea root plastids since conflicting evidence is presented by Trimming and Emes (1990) and Borchert et ai. (1993). Phosphoglycerate mutase and enolase have also been reported to be absent respectively from pea chloroplasts (Stitt and ap Rees, 1979) and castor seed leucoplasts (Miernyk and Dennis, 1982). Based upon measurements of the activities of glycolytic enzymes within the plastid it is clear that in some tissues, carbon sources at the level of hexose or triose could be metabolized to pyruvate in order to supply fatty acid synthesis. An additional route for the synthesis of pyruvate is through decarboxylation of imported malate by the plastidial NADP-dependent malic enzyme (NADP-ME). Activity of NADP-ME has been reported in plastids from oilseed rape embryos (Kang and Rawsthorne, 1994) and castor endosperm (Smith et aI., 1992).

3. UllLUATIONOFMffiTABOLITESFORFATTYACID SYNTHESIS BY ISOLATED PLASTIDS

Almost all of the research that has so far addressed the carbon source for plastidial fatty acid synthesis has relied on the isolation of intact plastids and the study of their ability to incorporate exogenously supplied metabolites into fatty acid products. This approach has led to a great deal of valuable information. However, it must be stated at the outset that such an approach will provide information which requires careful interpretation. First, consideration should be given to the possibility that the properties of the plastids may be altered during isolation. Secondly, for reasonable conclusions to be drawn, the plastids should be capable of rates of fatty acid synthesis that are comparable to the rates required for

142 Chapter 7

in vivo fatty acid synthesis. Thirdly, in vitro studies of isolated organelles are in general simplistic with single substrates supplied at saturating concentrations for fatty acid synthesis so that interpretation of the data is straightforward. This is a very different situation from that in vivo where multiple substrates are available for which the in vivo concentrations are not known, and where there is potential competition for the carriers and enzymes. Despite such reservations the studies to date have revealed that the metabolites that are taken up and utilized by plastids for fatty acid synthesis cover a broad spectrum (Figure 1) which can depend on the plant species, organ and stage of development.

In all studies reported so far acetate is taken up by isolated plastids and utilized for fatty acid synthesis. This includes studies made with chloroplasts (Roughan et aI., 1979; Springer and Heise, 1989), plastids from roots (Sparace et aI., 1988), seed tissues (Browse and Slack, 1985; Fuhrmann et aI., 1994; Kang and Rawsthome, 1994; Miemyk and Dennis, 1983), cauliflower buds (M6hlmann et aI., 1994), flowers (Liedvogel and Kleinig, 1979) and sycamore cell cultures (Alban et aI., 1989). Whether acetate is an in vivo substrate for fatty acid synthesis is impossible to address solely on the basis of in vitro studies with isolated plastids. Where studies have compared the utilization of acetate for fatty acid synthesis in vitro to that of other potential substrates the results have varied depending upon the tissue from which the plastids were extracted. For example, the rate of fatty acid synthesis from acetate is greater than that from pyruvate in spinach and pea chloroplasts (Roughan et aI., 1979; Springer and Heise, 1989), and cauliflower bud plastids (M6hlmann et aI., 1994). In contrast rates of fatty acid synthesis from acetate are less than those from pyruvate, malate, and/or Glc6P (depending on the tissue source and other substrates tested) for plastids from mustard cotyledons, castor endosperm, maize chloroplasts and developing oilseed rape embryos (Kang and Rawsthome, 1994; Liedvogel and Bauerle, 1986; Preiss et aI., 1994; Smith et aI., 1992). The studies on castor seed endosperm and developing oilseed rape embryos to date have demonstrated that plastids from these tissues utilize respectively malate and pyruvate as the most effective substrates for fatty acid synthesis (Kang and Rawsthome, 1994; Miemyk and Dennis, 1983; Smith et aI., 1992).

There has been debate in the literature as to whether acetyl-camitine represents a true in vivo substrate for plastidial fatty acid synthesis. The work of Masterson et ai. (1990a, 1990b) has suggested that this might be the case for chloroplasts isolated from pea leaves. In these experiments the rate of fatty acid synthesis from acetyl-camitine was more than four­fold greater than that from acetate. However, Roughan et ai. (1993) were unable to demonstrate incorporation of acetate from acetyl-camitine

7. Carbon flux to fatty acids in plastids 143

Cytosol

Starch ~ - ADP glucose

t Glucose 1-P --------{ }-------.,~ Glucose 1:P

H H Glucose 6-P --------{ )--------- Glucose 6-P

:! Y:

:! Y:

Triose P , Tri~se P ---------{

CO2 :

M··'~l }-------~~ Pyruvate

CoA + Py:!:' ~ -~, :: ~ 2 AceZ: CoA

A... r;;nitlne \ Carnitine' \- Acetyl carnitine-+l<,---........., __ Acetyl carnitine ,

CoA Fatty acids

Plastid

Figure 1. Pathways for the plastidial synthesis of starch and fatty acids from cytosolic precursors.

144 Chapter 7

into fatty acids by chloroplasts from spinach and pea leaves. Further work is required in order to resolve this debate.

Kang and Rawsthome (1994) were the first to demonstrate that a purified plastid fraction could incorporate carbon from imported Glc6P at high rates into fatty acids, thereby demonstrating that the entire glycolytic pathway was operating in these organelles. Incorporation of carbon from Glc6P into fatty acids had previously been shown to be undetectable or negligible by comparison with other substrates when it was supplied to plastids from developing pea embryos or castor seed endosperm (Foster and Smith, 1993; Miemyk and Dennis, 1983). Subsequently, Qi et aI., (1995) demonstrated that Glc6P and glucose were utilized for fatty acid synthesis by pea root plastids at rates which were comparable to those from pyruvate and acetate. This latter observation would suggest that in contrast to the data based on activities of plastidial enzymes (as discussed above and see Borchert et aI., 1993; Trimming and Emes, 1990), the glycolytic pathway is active in these pea root plastids.

Based upon such studies it is tempting to propose that the substrate which is utilized best in vitro is the most likely substrate in vivo. However, for the reasons given at the introduction to this section, caution should be applied when making such deductions from experiments carried out at a single time point in the ontogeny of the tissue. In some instances, for example during development, plastidial metabolism might change and so influence the rate at which plastids utilise exogenous substrates. In such cases the preferential utilization of a substrate, either at the level of import into the plastid or subsequent metabolism after import can be set into the context of other developmental changes in the tissue. For example an increasing ability of isolated plastids to utilize an exogenous metabolite might correlate positively with an increase in the in-vivo flux to a product that is derived from it. Whilst this improves the ability to make deductions about which metabolites might be imported into plastids for biosynthetic reactions, rigorous assessment at the in-vivo level is still ultimately required.

Changes in chloroplast metabolism with respect to import and utilization of metabolites have been reported to occur previously during development and also in response to: (i) artificially induced source to sink transitions in leaves; and (ii) salt stress imposed on the plant. As an example of the first case the utilization of NaH14C03 for isoprenoid and fatty acid synthesis by plastids (reflecting flux through the lower half of glycolysis and PDC) decreases during barley leaf development (Hoppe et aI., 1993). In the same leaves there is an increase in the utilization of acetate for isoprenoid and fatty acid synthesis. Earlier studies have shown that photosynthesis is repressed in detached spinach leaves which are supplied glucose through the transpiration stream and that the treated

7. Carbon flux, to fatty acids in plastids 145

leaves accumulate starch (Krapp et aI., 1991). More recently, Quick et aI. (1995) have shown that this accumulation of starch in the chloroplasts of these fed leaves is accompanied by an increase in the ability to import Glc6P. Exposure of maize plants to salt stress increases the ability of chloroplasts isolated from their leaves to utilise malate as a substrate for fatty acid synthesis (Preiss et aI., 1994). In this case the increase in utilization of malate is accompanied by an increase in the activity of the plastidial NADP-malic enzyme.

Since earlier studies had suggested that development could be an important factor in chloroplast metabolism we have recently determined how utilization of substrates for fatty acid synthesis by isolated plastids is affected by embryo age in oilseed rape (P.J. Eastmond and S. Rawsthome, unpublished). During development of oilseed rape embryos the storage product profile changes. At the earliest stages of cotyledon filling starch is accumulated (da Silva, 1993). This accumulation is transient and when oil accumulation is about one quarter completed (at around the early-mid cotyledon filling stage) the starch content of the embryo begins to decrease (da Silva, 1993). From about half way through oil accumulation the storage proteins are also deposited (Murphy and Cummins, 1989). With these developmental trends established we chose to isolate plastids from embryos at stages which represented early, early-mid and mid-late cotyledon filling (i.e. starch, starch and oil, and oil accumulating respectively). The plastids from embryos at the early cotyledon stage had properties that were similar in most respects to those from embryos at the early-mid stage (P.J. Eastmond and S. Rawsthome, unpublished). However there were distinct differences between the plastids from the two later stages.

The rate of fatty acid synthesis by plastids from the early-mid stage was greatest in the presence of Glc6P when compared to that from other substrates (Table 1; PJ. Eastmond and S. Rawsthome, unpublished). This confirmed our previous data on comparisons of Glc6P and pyruvate utilization for fatty acid synthesis by plastids from embryos at the early­mid cotyledon stage (Kang and Rawsthome, 1996). The rate of incorporation of carbon from Glc6P into fatty acids by plastids from embryos at the mid-late cotyledon stage was less than that at the early­mid stage (Table 1). In contrast there was a four-fold increase in the rate of pyruvate utilization for fatty acid synthesis over the same time period (Table 1). We are now focusing on understanding how these developmental changes in Glc6P and pyruvate utilization by isolated plastids are mediated.

146 Chapter 7

Table 1. The rate of incorporation of carbon from metabolites into fatty acids by plastids

isolated from oilseed rape embryos at different stages in development. The concentration of

14C-Iabelled metabolites was 1 mM. Incorporation into fatty acids was measured according

to Kang and Rawsthome (1996). A unit of NADP-glyceraldehyde 3-phosphate dehydrogenase

(NADP-GAPDH) activity is one Ilmol min-I. Each value is the mean ± SE of experiments

conducted on three separate plastid preparations.

Cotyledonary stage of

embryo development

Early-mid

Mid-late

Substrate

Glc6P Pyruvate

(nmol acetyl-CoA unirl GAPDH hoi)

442.3 ± 27.3

202.1 ± 17.4

292.5 ± 26.3

1279.5 ± 146

7. Carbon flux to fatty acids in plastids

4. IMPORT OF :METABOLITES FOR FATIY ACID SYNTHESIS BY PLASTIDS

147

Despite many years of investigation of plastidial fatty acid synthesis very little is known as to how exogenous substrates are imported into the plastids for this pathway. The movement of acetate into plastids is believed to occur via diffusion across the membranes of the plastidial envelope. An early study of pyruvate uptake suggested that if pyruvate was transported across the plastid envelope of pea chloroplasts then this activity was limited compared to the rate of passive diffusion (Proudlove and Thurmann, 1981). A role for the camitine/acetyl-camitine exchange transporter in moving acetate equivalents into the plastids has been proposed by Wood et ai. (1992), but this route for metabolic flux remains controversial as discussed above. The lack of information on the role of transporters in fatty acid synthesis is surprising given the interest received by fatty acid synthesis, and by plastid transporters associated with starch synthesis in non-photosynthetic plastids and photosynthesis in chloroplasts. We have begun to investigate which transporters might be involved in the supply of carbon for fatty acid and hence oil synthesis in oil storing tissues such as castor endosperm and developing oilseed rape embryos.

In keeping with the utilization of Glc6P by plastids of oilseed rape embryos (Kang and Rawsthome, 1994, 1996) we have been able to demonstrate the activity of a Glc6P transporter in the envelope of the plastids from this tissue (P.J. Eastmond and S. Rawsthome, unpublished). The activity of this transporter is in excess of the rate required to sustain Glc6P-dependent fatty acid synthesis by these plastids at all developmental stages that we have examined. The properties of this transporter are very similar to those reported for that from pea embryos and roots and cauliflower bud plastids (Batz et aI., 1993; Borchert et aI., 1989; Hill and Smith 1991). The activity of the Glc6P transporter in plastids from mid-late cotyledon embryos of oilseed rape is 30% lower than at the early cotyledon stage (P.J. Eastmond and S. Rawsthome, unpublished). This observation is in keeping with the changes in Glc6P utilization for fatty acid synthesis described earlier. However, the change in the Glc6P transporter activity is clearly not alone in accounting for the change in plastidial fatty acid synthesis as there are numerous metabolic steps involved in the conversion of Glc6P to pyruvate, many of which decline in activity during development (P.J. Eastmond and S. Rawsthome, unpublished).

Pyruvate has been demonstrated to be an effective substrate for plastidial fatty acid synthesis in both oilseed rape embryos and castor seed

148 Chapter 7

endosperm (Kang and Rawsthome, 1994, 1996; Miemyk and Dennis, 1983; Smith et at, 1992) but in neither case was it known how the metabolite crossed the plastid envelope. Our recent investigations (P.J. Eastmond and S. Rawsthome, unpublished) have revealed that pyruvate uptake across the envelope of rapeseed embryo plastids involves a carrier, the activity of which increases during embryo development. As was seen for G1c6P utilization, the developmental changes in the activity of this pyruvate carrier are correlated with those of plastidial fatty acid synthesis from pyruvate. In contrast, we have also shown that the kinetics of pyruvate uptake by plastids of castor seed endosperm are not consistent with the operation of a carrier, and that pyruvate uptake is more likely to be through passive diffusion (Eastmond et aI., 1997). The highest rates of fatty acid synthesis by plastids from castor endosperm are supported by exogenously supplied malate and not pyruvate (Smith et aI., 1992). Consistent with this observation we have identified a novel malate transport activity on the envelope of castor plastids (Eastmond et aI., 1997). This transporter activity appears to be via a phosphate counter-exchange mechanism, similar to that reported for mitochondria (Wi skich, 1974), and not the well characterized dicarboxylate exchange transporter of chloroplasts (Lehner and Heldt, 1978).

From our recent work we would argue that the activity of transporters can be important in determining: (i) the metabolic routes by which carbon is imported into the plastid and utilized for fatty acid synthesis; and (ii) the relative importance of these routes, at least in vitro. This approach will continue to identify what appear to be metabolic steps which are of potential importance in vivo. It is the manipulation of the activity of these transporters in vivo that will reveal their relative importance in determining how plastidial and cytosolic metabolism interact during fatty acid synthesis.

5. SOURCES OF ATP AND REDUONGPOWER FOR FATlY ACID SYNTHESIS

The synthesis of fatty acids requires both A TP and reducing power. In the case of the latter, NADPH, NADH and acetyl-CoA are required for each C2 addition to a growing acyl chain in the reactions catalyzed by fatty acid synthetase (Slabas and Fawcett, 1992). In chloroplasts, light energy can be used to provide the NADPH and A TP required for fatty acid synthesis. In heterotrophic tissues, the plastids need to import these cofactors or to generate them intraplastidially through carbohydrate oxidation or metabolite shuttles.

7. Carbon flux to jatty acids in plastids 149

Indirect evidence for A TP import comes from studies of fatty acid and starch synthesis in isolated plastids. For example, starch or fatty acid synthesis by plastids isolated from heterotrophic organs is widely reported to be dependent on the provision of exogenous A TP (e.g. Hill and Smith, 1991; Neuhaus et aI., 1993; Kleppinger-Sparace et aI., 1992; Smith et aI., 1992; Kang and Rawsthome, 1996). Direct evidence for import of A TP into plastids has been reported for plastids isolated from pea roots and sycamore cells, and in spinach chloroplasts (Ngemprasirtsiri et aI., 1989, Schunemann et aI., 1993). Very recently a cDNA has been cloned which encodes an ATP/ADP exchange translocator that is located on the plastid envelope in Arabidopsis thaliana (Neuhaus et aI., 1997).

Developing embryos of oilseed rape are photosynthetic (Eastmond et aI., 1996) and therefore have the potential to produce A TP within the plastid through photophosphorylation. We have recently assessed this potential by comparing rates of fatty acid and starch synthesis by plastids isolated from these embryos when incubations are made in the presence or absence of exogenous A TP and in the light or dark. These measurements revealed that incorporation of carbon from Glc6P into either product was strongly dependent on the provision of exogenous A TP (Table 2). Some incorporation of Glc6P was seen in the light alone but the rates were less than 20% of those sustained by exogenous A TP (Table 2). These results are similar to those reported previously for fatty acid synthesis from acetate by plastids from cotyledons of developing linseed (Browse and Slack, 1985).

Sparace and colleagues (Kleppinger-Sparace et aI., 1992, Qi et aI., 1994) have investigated whether the A TP required for fatty acid synthesis can be generated within the plastid through glycolysis. Their results have shown that this is possible. By setting up a triose phosphate shuttle (provision of 2 mM dihydroxyacetone phosphate, 2 mM oxaloacetic acid and 4 mM inorganic phosphate as defined in Kleppinger­Sparace et aI., 1992) in their incubations they demonstrated that rates of fatty acid synthesis from acetate were up to 44% of those obtained in the presence of exogenous A TP alone.

Two more recent studies would suggest that plastidial glycolytic metabolism is not capable of supporting fatty acid synthesis. Qi et ai. (1995) and Kang and Rawsthome (1996) have demonstrated that carbon from Glc6P can be incorporated into fatty acids by plastids isolated from pea roots and developing rapeseed embryos. In both cases this incorporation was strongly dependent upon exogenous A TP with rates reduced by at least 90% when ATP was excluded. It is reasonable to assume that the Glc6P was being metabolised by glycolysis within the plastid during these incubations. This should yield the ATP required to

150 Chapter 7

Table 2. The ATP- and light-dependence of the incorporation of carbon from G1c6P into fatty

acids and starch by plastids isolated from early-mid cotyledon oilseed rape embryos. The

concentration of I'C-labelled G1c6P was 1 mM. Incorporation into fatty acids and starch was

measured according to Kang and Rawsthorne (1996). Where present the concentration of ATP

was 4 mM and the photosynthetic photon flux density was 400 Ilmol m-2 S-I. This is the

estimated maximum PPFD that the embryo would recieve in vivo (Eastmond et al. 1996).

A unit of NADP-GAPDH activity is one Ilmol min-I. Each value is the mean ± SE of

experiments conducted on three separate plastid preparations. nd, not detectable.

Product Conditions

Dark Dark plus A TP Light

(nnwl acetyl-eoA unirl GAPDH hi)

Fatty acids nd 306.2 ± 25.1 63.5 ± 8.8

(nmol hexose unirl GAPDH h-I )

Starch nd 209.1 ± 11.4 31.4 ± 4.2

7. Carbon flux to fatty acids in plastids 151

drive the incorporation of acetyl-CoA into fatty acids and yet A TP had to be provided in order to see this. The problem with interpreting these experiments is that other metabolism with an A TP demand might be occurring simultaneously in the same plastids and so compete for A TP . This is certainly the case in plastids from developing rapeseed embryos which can synthesize starch and fatty acids simultaneously (Kang and Rawsthorne, 1996). Given the evidence to date it is very likely that fatty acid synthesis by plastids from heterotrophic organs is to a large extent dependent upon provision of A TP from the cytosol.

The source of reductant for fatty acid synthesis in heterotrophic tissues is not clear. The presence or absence of exogenous reduced pyridine nucleotides in incubations made with isolated plastids can give variable results. For example several studies have shown little effect of manipulating exogenous pyridine nucleotides (e.g. Kang and Rawsthorne, 1996; Smith et aI., 1992; Qi et aI., 1995). These results are usually associated with Glc6P-, malate-, or pyruvate-dependent fatty acid synthesis. In contrast, provision of reduced NAD(P) in incubations where the carbon source is acetate can lead to significant stimulation in the rate of fatty acid synthesis (Smith et aI., 1992; Stahl and Sparace, 1991). These data can be interpreted to suggest that certain substrates that are imported by the plastids can be metabolised to produce not only the acetyl-CoA for fatty acid synthesis but also the reducing equivalents. Smith et aI. (1992) have argued that the intraplastidial conversion of malate to acetyl-CoA is extremely efficient for fatty acid synthesis because the reactions catalysed by NADP-ME and PDC form acetyl-CoA, NADPH and NADH, which are all required for this pathway.

Sources of reducing power within the plastid are certainly capable of sustaining fatty acid synthesis. A TP-dependent incorporation of acetate can occur at measurable rates over incubations of one hour suggesting that reductant is available. Apart from malate and pyruvate metabolism another potential source of this reductant is the oxidative pentose phosphate pathway (OPPP). The OPPP has been shown to provide reductant for nitrogen assimilation in pea root plastids (Bowsher et aI., 1992 and see Chapter 6). Only two lines of evidence have been published which support the linkage between the OPPP and fatty acid synthesis and both are indirect. First, incorporation of pyruvate into fatty acids is increased in the presence of Glc6P, the latter metabolite being metabolised via the OPPP, and also to starch and fatty acids (Kang and Rawsthorne, 1996). Second, the flux through the OPPP in chloroplasts isolated from green pepper fruits is stimulated three-fold by inclusion of pyruvate in the incubations (Thorn and Neuhaus, 1995), pyruvate having been reported be an effective substrate for fatty acid synthesis by green pepper fruit chloroplasts (see Thorn and Neuhaus, 1995).

152 Chapter 7

Based upon the majority of the data available it is most likely that plastids in heterotrophic organs import metabolites and then oxidize them in order to generate the reductant that is required for fatty acid synthesis.

6. INTERACfION BETWEEN PATHWAYS WITIDN PLASTIDS

The interaction of pathways within plastids has received relatively little attention. This is perhaps not surprising since the experiments required to address these issues are inevitably more complex that those in which incorporation of carbon from a single metabolite into a single product are studied. Common approaches with respect to fatty acid synthesis are to provide a radio labelled substrate in the presence or absence of an unlabelled one and to look for interaction. This approach often reveals that one metabolite competes with another and they are therefore entering a common pathway. More recently, studies have been made which address how separate pathways (e.g. starch and fatty acid synthesis) interact or how single metabolites are utilized through more than one pathway.

The partitioning of imported Glc6P to starch and fatty acids, and to CO2 via the OPPP in plastids isolated from developing rapeseed embryos was investigated by Kang and Rawsthome (1996). These studies have shown that at the early-mid cotyledon stage of development, when the embryo is accumulating starch and oil, about 50% of the carbon from Glc6P is utilized for starch synthesis with the remainder divided equally between fatty acid synthesis and the OPPP. More recent work (P.J. Eastmond and S. Rawsthome, unpublished) has shown that as the embryo becomes more active in oil accumulation the fluxes of imported Glc6P to starch and fatty acids decrease while oxidation through the OPPP increases. This observation emphasizes the need to address developmental effects on plastidial metabolism (see above for carbon sources for fatty acid synthesis). Kang and Rawsthome (1996) also showed that total flux to fatty acids could be increased by provision of pyruvate and G1c6P simultaneously and that this did not affect flux of Glc6P to starch. This suggests that in the presence of exogenous A TP there is no competition between the two synthetic pathways for A TP within the plastid. In contrast, Mohlmann et al. (1994) have shown that increasing the rate of starch synthesis from Glc6P by isolated cauliflower bud amyloplasts reduces the rate of acetate-dependent fatty acid synthesis. They have interpreted this result to indicate that there is

7. Carbon flux to fatty acids in plastids 153

competition between the two synthetic pathways, presuming it to occur at the level of A TP availability within the plastid.

Interaction between pathways at the level of common metabolites is possible where there is access to the intermediates. Roughan and Ohlrogge (1996) have recently proposed that there is a "metabolon" in chloroplasts of spinach which channels acetate through to long chain fatty acids and involves acetyl-CoA synthetase, ACCase, and F AS. Whether such a "metabolon" exists in other plastids remains to be determined. Its presence would prevent interaction between different potential precursors for acetyl-CoA.

7. CONCLUSION

It is clear that our understanding of carbon flux to fatty acids has increased greatly in recent years. However, there are a number of questions to be resolved, particularly those relating to interactions that occur between metabolites within pathways and between pathways themselves. The use of isolated plastids continues to be a valuable tool in investigating fatty acid synthesis and for developing embryos of oilseed rape it is providing new insight into changes in metabolism. Ultimately we need to test hypotheses derived from such in vitro experimentation by manipulating, in vivo, the steps that are identified as important and examining the resultant phenotype.

ACKNOWLEDGEMENTS

We thank Dr L.M. Hill and Mr P. Johnson for their helpful comments on the manuscript.

REFERENCES

Alban, C., Baldet, P. and Douce, R. (1994). Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides. Biochemical Journal, 300, 557-565.

Alban, c., Joyard, J. and Douce, R. (1989). Comparison of glycerolipid biosynthesis in non-green plastids from sycamore (Acer pseudoplatanus) cells and cauliflower (Brassica oleracea) buds. Biochemical Journal, 259, 775-783.

Batz, 0., Scheibe, R. and Neuhaus, H.E. (1993). Identification of the putative hexose­phosphate translocator of amyloplasts from cauliflower buds. Biochemical Journal, 294, 15-17.

154 Chapter 7

Borchert, S., GroBe, H. and Heldt, H.W. (1989). Specific transport of inorganic phosphate, glucose-6-phosphate, dihydroxyacetone phosphate and 3-phosphoglycerate into amy lop lasts from pea roots. FEBS Letters, 253, 183-186.

Borchert, S., Harborth, J., Schiinemann, D., Hoferichter, P. and Heldt, H.W. (1993). Studies of the enzymic capacities and transport properties of pea root plastids. Plant Physiology, 101, 303-312.

Bowsher, e.G., Boulton, E.L., Rose, J., Nayagam, S. and Emes, M.J. (1992). Reductant for glutamate synthase is generated by the oxidative pentose phosphate pathway in non­photosynthetic root plastids. Plant Journal, 2, 893-898.

Browse, J. and Slack, C.R. (1985). Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons. Planta, 166, 74-80.

Charles, DJ., Hasegawa, P.M. and Cherry, J.H. (1986). Characterization of acetyl-CoA carboxylase in the seed of two soybean genotypes. Phytochemistry, 25, 55-59.

da Silva, P.M.F.R. (1993). Starch metabolism during embryo development of oilseed rape. PhD thesis, University of East Anglia, UK.

Denyer, K. and Smith, A.M. (1988). The capacity of plastids from developing pea cotyledons to synthesise acetyl-CoA. Planta, 173, 172-182.

Eastmond P.J., Dennis D.T. and Rawsthorne S. (1997). Evidence that a malate/inorganic phosphate exchange translocator imports carbon across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm. Plant Physiology, 114, 851-856.

Eastmond, P.J, Kohicmi, L. and Rawsthorne, S. (1996). Photosynthetic characteristics of developing embryos of oilseed rape (Brassica napus L.). Journal of Experimental Botany, 47, 1763-1769.

Egli, M.A., Gengenbach, B.G., Gronwald, J.W., Somers, D.A. and Wyse, D.L. (1993). Characterization of maize acetyl-coenzyme A carboxylase. Plant Physiology, 101, 499-506.

Entwistle, G. and ap Rees, T. (1988). Enzymic capacities of amy lop lasts from wheat (Triticum aestivum) endosperm. Biochemical Journal, 255, 391-396.

Foster, J.M. and Smith, A.M. (1993). Metabolism of glucose-6-phosphate by plastids from developing pea embryos. Planta, 190, 17-24.

Frehner, M., Pozueta-Romero, 1. and Akazawa, T. (1990). Enzyme sets of glycolysis, gluconeogenesis and oxidative pentose phosphate pathway are not complete in nongreen highly purified amyloplasts of sycamore (Acer pseudoplatanus L.) cell suspension cultures. Plant Physiology, 94, 538-544.

Fuhrmann, J., Johnen, T. and Heise, K.-P. (1994). Compartmentation of fatty acid metabolism in zygotic rape embryos. Journal of Plant Physiology, 143, 565-569.

Harwood, J.L. (1988). Fatty acid metabolism. Annual Review of Plant Physiology and Plant Molecular Biology, 39, 101-138.

Hill, L.M. and Smith, A.M. (1991). Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos. Planta, 185, 91-96.

Hoppe, P., Heintze, A., Riedel, A., Creuzer, C. and Schultz, G. (1993). The plastidic 3-phosphoglycerate acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids, plastidic isoprenoids and fatty acids during chloroplast development. Planta, 190, 253-262.

Journet, E.-P. and Douce, R. (1985). Enzymic capacities of purified cauliflower bud plastids for lipid synthesis and carbohydrate metabolism. Plant Physiology, 79, 458-467.

7. Carbon flux to fatty acids in piastids 155

Kang, F. and Rawsthorne, S. (1994). Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.). Plant Journal, 6, 795-805.

Kang, F. and Rawsthorne, S. (1996). Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by plastids from developing oilseed rape embryos. Planta, 199, 321-327.

Kang, F., Ridout, CJ., Morgan, C.L. and Rawsthorne, S. (1994). The activity of acetyl­CoA carboxylase is not correlated with the rate of lipid synthesis during development of oilseed rape (Brassica napus L.) embryos. Planta 193, 320-325.

Kleppinger-Sparace, K.F., Stahl, R. and Sparace, S.A. (1992). Energy requirements for fatty acid and glycerolipid biosynthesis from acetate by isolated pea root plastids. Plant Physiology, 98, 723-727.

Konishi, T. and Sasaki, Y. (1994). Compartmentalization of two forms of acetyl-Co A carboxylase in plants and the origin of their tolerance toward herbicides. Proceedings of the National Academy of Sciences USA, 91, 3598-3601.

Krapp, A., Quick, W.P. and Stitt, M. (1991). Ribulose 1,5-bisphosphate carboxylase­oxygenase, other photosynthetic enzymes and chlorophyll decrease when glucose is supplied to mature spinach leaves via the transpiration stream. Planta, 186, 58-69.

Kuhn, D.N., Knauf, M.J. and Stumpf, P.K. (1981). Subcellular localisation of acetyl-Co A synthetase in leaf protoplasts of Spinacia oleracea leaf cells. Archives of Biochemistry and Biophysics, 209, 441-450.

Lehner, K. and Heldt, H.W. (1978). Dicarboxylate transport across the inner membrane of the chloroplast envelope. Biochimica et Biophysica Acta, 501, 531-544.

Liedvogel, B. (1986). Acetyl coenzyme A and isopentenylpyrophosphate as lipid precursors in plant cells - biosynthesis and compartmentation. Journal of Plant Physiology, 124, 211-222.

Liedvogel, B. and Bauerle, R. (1986). Fatty-acid synthesis in chloroplasts from mustard (Sinapis alba L.). cotyledons: formation of acetyl coenzyme A by intraplastid glycolytic enzymes and a pyruvate dehydrogenase complex. Planta, 169, 481-489.

Liedvogel, B. and Kleinig, H. (1979). Galactolipid synthesis in chromoplasts in vitro. Specificities of acyl- and galactosyltransferases. Planta, 144, 467-471.

Masterson, C., Wood, C. and Thomas, D.R. (1990a). L-acetylcamitine, a substrate for chloroplast fatty acid synthesis. Plant Cell and Environment, 13, 755-765.

Masterson, C., Wood, C. and Thomas, D.R. (1990b). Inhibition studies on acetyl group incorporation into chloroplast fatty acids. Plant Cell and Environment, 13, 767-771.

Miernyk, lA. and Dennis, D.T. (1982). Isozymes of the glycolytic enzymes in endosperm from developing castor oil seeds. Plant Physiology, 69, 825-828.

Miernyk, J.A. and Dennis, D.T. (1983). The incorporation of glycolytic intermediates into lipids by plastids isolated from the developing endosperm of castor oil seeds (Ricinus communis L.) Journal of Experimental Botany, 34, 712-718.

M6hlmann, T., Scheibe, R. and Neuhaus, H.E. (1994). Interaction between fatty-acid and starch synthesis in isolated amyloplasts from cauliflower floral buds. Planta, 194, 492-497.

Murphy, DJ. and Cummins, I. (1989). Biosynthesis of storage products during embryogenesis in rapeseed. Journal of Plant Physiology, 135, 63-69.

Neuhaus, H.E., Batz, 0., Thorn, E. and Scheibe, R. (1993). Purification of highly intact plastids from various heterotrophic plant tissues: analysis of enzymic equipment and precursor dependency for starch biosynthesis. Biochemical Journal, 296, 395-401.

Neuhaus, H.E., Thorn, E., M6hlmann, T., Steup, M. and Kampfenkel, K. (1997). Characterization of a novel eukaryotic ATP/ADP translocator located in the plastid envelope of Arabidopsis thaliana L.. Plant Journal, 11, 73-82.

156 Chapter 7

Ngernprasirtsiri, J., Takabe, T. and Akazawa, T. (1989). Immunochemical analysis shows that an ATPI ADP-translocator is associated with the inner-envelope membranes of amyloplasts from Acer pseudoplatanus L. Plant Physiology, 89, 1024-1027.

Page, R., Okada, S. and Harwood, J.L. (1994). Acetyl-CoA carboxylase exerts strong flux control over lipid synthesis in plants. Biochimica et Biophysica Acta, 1210, 369-372.

Post-Beittenmiller, D., Jaworski, lG. and Ohlrogge, J.B. (1991). In vivo pools of free and acylated acyl carrier proteins in spinach. Evidence for sites of regulation of fatty acid biosynthesis. Journal of Biological Chemistry, 266, 1858-1865.

Post-Beittenmiller, D., Roughan, G. and Ohlrogge, J.B. (1992). Regulation of plant fatty acid biosynthesis. Plant Physiology, 100, 923-930.

Preiss, M., Koopmann, E., Meyer, G., Koyro, H.-W. and Schultz, G. (1994). Malate as additional substrate for fatty acid synthesis in a C4-plant type developed by salt stress from a C3-plant type maize. A screening for malate as substrate for fatty acid synthesis in chloroplasts. Journal of Plant Physiology, 143, 544-549.

Proudlove, M.O. and Thurmann, D.A. (1981). The uptake of 2-oxoglutarate and pyruvate by isolated pea chloroplasts. New Phytologist, 88, 255-264.

Qi, Q., Kleppinger-Sparace, K.F. and Sparace, SA (1994). The role of the triose­phosphate shuttle and glycolytic intermediates in fatty-acid and glyceroJipid biosynthesis in pea root plastids. Planta, 194, 193-199.

Qi, Q., Kleppinger-Sparace, K. F. and Sparace, S. A. (1995). The utilization of glycolytic intermediates as precursors for fatty acid biosynthesis by pea root plastids. Plant Physiology, 107, 413-419.

Quick, W.P., Scheibe, R. and Neuhaus, H.E. (1995). Induction of hexose-phosphate translocator activity in spinach chloroplasts. Plant Physiology, 109, 113-121.

Reid, E.E., Lyttle, C.R., Canvin, D.T. and Dennis, D.T. (1975). Pyruvate dehydrogenase complex activity in proplastids and mitochondria of developing castor bean endosperm. Biochemical and Biophysical Research Communications, 62, 42-47.

Reid, E.E., Thompson, P., Lyttle, C.R. and Dennis, D.T. (1977). Pyruvate dehydrogenase complex from higher plant mitochondria and proplastids. Plant Physiology, 59, 842-848.

Roesler, K., Shintani, D., Savage, L., Boddupalli, S. and Ohlrogge, J. (1997). Targetting of the Arabidopsis homomeric acetyl-Coenzyme A carboxylase to plastids of rapeseeds. Plant Physiology, 113, 75-81.

Roughan, P.G., Holland, R., Slack, C.R. and Mudd, J.B. (1979). Acetate is the preferred substrate for long-chain fatty acid synthesis in isolated spinach chloroplasts. Biochemical Journal, 184, 565-569.

Roughan, P.G. and Ohlrogge, J. (1996). Evidence that isolated chloroplasts contain an integrated lipid-synthesizing assembly that channels acetate into long-chain fatty­acids. Plant Physiology, 110, 1239-1247.

Roughan, G., Post-Beittenmiller, D., Ohlrogge, J. and Browse, J. (1993). Is acetylcamitine a substrate for fatty acid synthesis in plants? Plant Physisology, 101, 1157-1162.

Sasaki, Y., Hakamada, K., Suama, Y., Nagano, Y., Furusawa, I. and Matsuno, R. (1993). Chloroplast-encoded protein as a subunit of acetyl-CoA carboxylase in pea plant. Journal of Biological Chemistry, 268, 25118-25123.

SchUnemann, D., Borchert, S., FIUgge, U-1. and Heldt, H.W. (1993). ADP/ATP translocator from pea root plastids. Plant Physiology, 103, 131-13 7.

Shintani, D.K. and Ohlrogge, J. (1995). Feedback inhibition of fatty acid synthesis in tobacco suspension cell cultures. Plant Journal, 7, 577-587.

7. Carbon flux to fatty acids in plastids 157

Simcox, P.O., Reid, E.E. , Canvin, D.T. and Dennis, D.T. (1977). Enzymes of the glycolytic and pentose phosphate pathways in proplastids from the developing endosperm of Ricinus communis L. Plant Physiology, 59, 1128-1132.

Simcox, P.O., Garland, W., Deluca, Y., Canvin, D.T. and Dennis, D.T. (1979). Respiratory pathways and fat synthesis in the developing castor oil seed. Canadian Journal of Botany, 57, 1008-1014.

Slabas, A.R. and Hellyer, A. (1985). Rapid purification of a high molecular weight subunit polypeptide form of rape seed acetyl CoA carboxylase. Plant Science, 39, 177-182.

Slabas, A.R. and Fawcett, T. (1992). The biochemistry and molecular biology of plant lipid biosynthesis. Plant Molecular Biology, 19, 161-191.

Smith, R.G., Gauthier, D.A. , Dennis, D.T. and Turpin, D.H. (1992). Malate- and pyruvate­dependent fatty acid synthesis in leucoplasts from developing castor endosperm. Plant Physiology, 98, 1233-1238.

Sparace, S.A., Menassa, R. and Klcppinger-Sparace, K.F. (1988). A preliminary analysis of fatty acid synthesis in pea roots. Plant Physiology, 87, 134-137.

Springer, 1. and Heise, K.P. (1989). Comparison of acetate- and pyruvate-dependent fatty­acid synthesis by spinach chloroplasts. Planta, 177, 417-421.

Stahl, R.J. and Sparace, S.A. (1991). Characterization of fatty acid biosynthesis in isolated pea root plastids. Plant Physiology, 96, 602-608.

Stitt, M. and ap Rees, T. (1979). Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Phytochemistry, 18, 1905-1911.

Tetlow, U., Blisset, K.J. and Emes M.J. (1994). Starch synthesis and carbohydrate oxidation in amyloplasts from developing wheat endosperm. Planta, 194, 454-460.

Thorn, E. and Neuhaus, H.E. (1995). Oxidation of imported or endogenous carbohydrates by isolated chloroplasts from green pepper fruits . Plant Physiology, 109, 1421-1426.

Treede, H-J., Riens, B. and Heise, K-P. (1986). Species-specific differences in acetyl coenzyme A synthesis of chloroplasts. Zeischrift Naturforschung, 41 c, 733-740.

Trimming, B.A. and Emes, M.J. (1993). Glycolytic enzymes in non-photosynthetic plastids of pea (Pisum sativum L.) roots. Planta, 190, 439-445.

Weaire, B.P. and Kekwick, R.G.O. (1975). The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue. Biochcmical Journal, 146, 425-437.

Williams, M. and Randall, D.O. (1979). Pyruvate dehydrogenase complex from chloroplasts of Pisum sativum L. Plant Physiology, 64, 1099-1103.

Wiskich JT (1974). Substrate transport into plant mitochondria. Swelling studies. Australian Journal Plant Physiology, 1, 177-181.

Wood, c., Masterson, C. and Thomas, D.R. (1992). The role of carnitine in plant cell metabolism. In: Tobin, A.K. (Ed.). Plant Organelles (pp. 229-263). Cambridge University Press, Cambridge.

Zeiher, c.A. and Randall , D.O. (1991). Spinach leaf acetyl-coenzyme A synthetase: purification and characterization. Plant Physiology, 96, 382-389.

Chapter 8

Compartmentation of metabolites between the subcellular compartments of leaves, the apoplast, the phloem and the storage tissue of different crop plants

Gertrud Lohaus, Dieter Heineke, Anne Kruse, Kirsten Leidreiter, Berti Riens, David G. Robinson, Heike Winter, Thilo Winzer and Hans W. Heldt Illstitut for Biochemie der Pjlallze and Pjlanzenphysio!ogie Illstitut. Universitat G6ttingen. Untere Karspu!e 2 D37073 G6ttingen, Germany

Key words: amino acids; apoplast; cytosol; metabolite concentration; phloem sap; sucrose; vacuole.

Abstract: Non-aqueous fractionation of frozen, lyophilized leaves and morphometric analysis were used to determine metabolite concentrations in the cytosolic, chloroplastic and vacuolar fraction of leaves. In barley, potato, spinach and tobacco the concentrations of amino acids and sucrose are very high in the cytosol, but low in the vacuole. In contrast, hexoses are almost exclusively located in the vacuole in these species. The concentration of metabolites in the apoplast was measured in the solution obtained following vacuum infiltration of leaves. To complement these measurements, the concentrations of sucrose and amino acids in the phloem sap were determined in samples of exudate obtained from the severed sty lets of aphids feeding on the plants. Comparison of the concentrations of sucrose and amino acids in the cytosol, the apoplast and the phloem sap suggests that the efflux of these compounds from the cytosol to the apoplast is restricted, and that sucrose is transported into the sieve tubes in preference to amino acids. Differences in the phloem concentrations of sucrose and amino acids in sugar beet and fodder beet correlate with differences in the composition of the storage products in the tap roots of these two cultivars. This suggests that phloem loading may have a major influence on the quality of harvest products accumulated by non­photosynthetic storage tissues in plants.

159

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 159-172. © 1999 Kluwer Academic Publishers.

160 Chapter 8

1. INTRODUCTION

Compartmentation of metabolic pathways is fundamental for the regulation and control of metabolism. An understanding of metabolic regulation requires knowledge of in vivo concentrations of metabolites in the compartments involved, e.g. the subcellular compartments of mesophyll cells, (cytosol, chloroplast and vacuole), the aqueous compartments of the apoplast and the phloem, which take part in assimilate export, and storage compartments such as taproots. To obtain such information we have employed several analytical methods: a) A morphometric analysis to determine the volumes of the cytosolic,

chloroplastic and vacuolar compartments of leaves from spinach, barley and potato (Winter et aI., 1993).

b) A non-aqueous fractionation procedure for estimation of the metabolite concentrations in the three compartments listed above (Gerhardt and Heldt, 1984; Riens et aI., 1991).

c) A vacuum infiltration technique to obtain samples for metabolite analysis from the apoplast (Lohaus et aI., 1995; Speer and Kaiser, 1991 ).

d) Analysis of phloem sap obtained from laser severed aphid stylets (Barlow and McCully, 1972; Lohaus et aI., 1995).

e) Analysis of the total content of ions and metabolites in the taproots of sugar and fodder beet to give a measure of the content of the storage vacuoles of taproots (Winzer et aI., 1996). Here we present a summary of the results obtained with these methods

over the last few years for the leaves of spinach (Spinacia oleracea L.), barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), sugar and fodder beet (Beta vulgaris L.), all grown hydroponically, and for potato (Solanum tuberosum L.) grown on soil supplemented with nutrient solutions. The size of the cellular and subcellular compartments, as well as the concentrations of a range of ions and metabolites, are summarised; and as an example of the application of these measurements, it is shown that phloem loading is a decisive factor in sucrose accumulation in the taproot of sugar beet.

2. CELLULAR AND SUBCELLULAR VOLUMES

The sizes of the gas space and the apoplast differed greatly in the three species investigated: spinach, potato and barley (Table 1). The relative volumes occupied by the epidermal cells are also different, ranging from 4% of the total aqueous leaf volume in spinach leaves to

8. Compartmentalion of metabolites 161

Table 1. Volumes of cellular and subcellular compartments in leaves. Data from

Winter et al. (1993), Winter et al. (1994) Leidreiter et al. (1995).

Spinach Potato Barley

~LlmgChl

Gas space 381 100 204

Total aqueous space 810 472 698

MESOPHYLL CELL 688 394 379

Vacuole 545 300 278

Chloroplast 113 70 72

Stroma 65 32 35

Thylakoid 36 21 29

Starch 13 14 7

Plastoglobuli 0.5

Cytosol 24 20 25

Mitochondria 3.6 4.0 4.0

EPIDERMIS CELL 36 74 244

Vacuole 34 72 243

APOPLAST 60 21 41

162 Chapter 8

35% in barley leaves. However, common to all three species was the fact that the epidermal compartment was almost totally occupied by vacuoles. Although the absolute sizes of the subcellular compartments of the mesophyll cells differed, their relative sizes were remarkably similar (Table 2): the vacuole occupied 73-79% of the mesophyll cell volume and the chloroplasts 16-19%, of which the chloroplast stroma made up about 50%.

3. SUBCELLULAR METABOLITE CONCENTRATIONS

Subcellular metabolite contents assayed by non-aqueous fractionation (Gerhardt and Heldt, 1984) together with the volumes listed above enable an evaluation of metabolite concentrations in the stromal, vacuolar and cytosolic compartments to be made. Tables 3 and 4 show that the subcellular concentrations of various metabolites are quite similar in illuminated leaves of spinach and potato. Comparable results have also been obtained for barley leaves (Winter et aI., 1993). As expected phosphorylated intermediates are only found in the cytosol and the chloroplast stroma, which underlines the validity of the method. Malate, on the other hand, is found to be concentrated largely in the vacuole. Due to the comparatively large volume of the vacuole, the amount of malate in the cytosolic and stromal fraction is so low that it could not be accurately determined. The data given are the upper limits of the stromal and cytosolic concentrations.

In both spinach and potato the cytosolic sucrose concentration is much higher than in the vacuole as also observed for barley and tobacco leaves (Table 5). In the leaves where glucose and fructose were assayed, almost 99% of the total leaf content was found in the vacuolar fraction (Figure 4). Since the hexose concentrations given in the stroma and in the cytosol represent upper limits of detection, the actual concentration values may be somewhat lower. These results indicate that the transport of sucrose and hexoses into the vacuole proceeds by different modes: the transport of sucrose probably occurs passively, whereas active transport appears to be involved in the uptake of hexoses (see Heineke et aI., 1994).

In all the leaves investigated so far, the concentrations of the various amino acids in the chloroplast stroma are similar to those in the cytosol, and much higher than those in the vacuole (Tables 3, 4 and 5). The relatively low amino acid concentration in the vacuoles is not a result of the lack of amino acid trans locators, since there is clear evidence for specific amino acid permeases in leaf vacuoles (for ref. see Winter et aI.,

8. Compartmentation of metabolites

Table 2. Relative volumes of subceUular compartments in mesophyll cells. Calculated from data shown in Table 1.

Compartment Spinach

%

Total mesophyll cells = 100

Vacuole 79

Chloroplast 16

Mitochondria 0.5

Cytosol 3.4

Potato

%

= 100

76

18

1.0

5.1

Barley

%

= 100

73

19

1.1

6.6

163

Table 3. Subcellular metabolite concentrations in spinach leaves after 8.5 h illumination. Data from Winter et a1. (1994).

Metabolite Concentration of metabolite (ruM) Stroma Cytosol Vacuole

3-Phosphoglycerate 4.3 4.2 ,.,()

Dihydroxyacetone phosphate 0.21 0.57 :.::0 Glucose 6-phosphate 0.0 5.9 :.::0 UDPglucose 0.07 1.7 ,.,()

Sucrose <0.8 53 11

Malate 1.2 0.08 6.8

Aspartate 14 23 0.9 Glutamate 14 21 0.6 Glutamine 20 24 0.6 Alanine 5.5 3.9 0.2

Sum of amino acids 58 86 2.8

164 Chapter 8

Table 4. Subcellular metabolite concentrations in potato leaves after 3.5 h illumination. Data from Leidreiter et aI. (1995).

Metabolite Concentration of metabolite (mM) Stroma Cytosol Vacuole

3-Phosphoglycerate 2.0 1.7 ~

Dihydroxyacetone phosphate 0.32 0.21 ~ Glucose 6-phosphate 0.75 3.5 ~

Sucrose 3.1 23 4.4 Glucose <0.3 <0.4 3.1 Fructose <1.0 <1.0 9.3

Malate <2.0 <3.0 21

Aspartate 3.0 8.9 0.36 Glutamate 26 41 1.7 Glutamine 4.8 4.4 0.3 Alanine 1.0 3.4 0.36

Sum of amino acids 42 80 4.5

Table 5. Metabolite gradients between the cytosolic and vacuolar compartments in iUuminated leaves of various plants. The data have been calculated from the data of Figures 1 and 2 and of He in eke et aI., (1994), n.m., not measured

Spinach Potato Barley Tobacco

conc. cytosol / conc. vacuole

Hexoses n.m. <0.2 n.m. < 0.2

Sucrose 4.8 20 11 112

Amino acids 31 20 160 23

8. Compartmentation of metabolites 165

1993). The concentration gradients of amino acids, and also of sucrose, across the vacuolar membrane may be an osmotic response to the opposing concentration gradients of other substances, including malate, nitrate and hexoses, which are taken up into the vacuoles by active transport. That the low vacuolar concentrations of amino acids results from a dynamic process is supported by the observation that the amino acid concentrations in barley leaf vacuoles increase during the dark period (Winter et aI., 1993).

4. DISTRIBUTION OF METABOLITES BETWEEN THE CYTOSOLIC AND APOPLASTIC COMPARTMENT AND THE PHLOEM SAP

In most temperate crop plants, photoassimilates exported out of the source leaves must cross at least two membrane barriers: first from the cytosol of the mesophyll cells into the apoplast and then again from the apoplast into the sieve tubes. To elucidate these processes, the concentrations of sucrose, amino acids, and malate were determined in all three compartments (Table 6). The apoplastic concentrations of both sucrose and amino acids were very low compared to the cytosolic and phloem concentrations. This confirms that the transport of both sucrose and amino acids from the apoplast into the phloem are active processes, but also indicates that the downhill efflux from the cytosol into the apoplast is restricted. We have obtained similar results with barley, beet, maize, and rape leaves. The mechanism underlying the restriction of efflux from the mesophyll cells remains unresolved. The relatively low concentrations of sucrose and amino acids in the apoplast are not the result of a very rapid uptake into the phloem, since apoplastic metabolite concentrations also remain low when their export via the sieve tubes has been inhibited, e.g. by cold girdling (Lohaus et aI., 1995) or by a phloem­specific antisense inhibition of the proton-sucrose symporter which catalyzes the transport of sucrose from the apoplast into the sieve tubes (D. Heineke, unpublished). It may be noted that the content of the leaf apoplast may not be uniform (Canny, 1987) and that some workers have assumed that metabolite concentrations in a section of the apoplast at the site of phloem loading are higher than in the apoplastic compartment as a whole. But, until now no convincing evidence has been presented for the existence of a concentration gradient within the apoplast.

The export of amino acids from the cytosol via the apoplast into the phloem is not preferential for a particular amino acid (Table 7). The percentage of each amino acid from the total amino acid concentration

166 Chapter 8

Table 6. Metabolite concentrations in illuminates spinach leaves. Data from Lohaus et al. (1994).

Metabolite Concentration of metabolite (mM) Cytosol Apoplast Phloem

Sucrose 74 0.73 830

Nitrate <8 8.0 ~

Malate 0.8 0.5 10

Aspartate 23 0.45 24 Glutamate 21 1.03 65 Glutamine 24 0.41 18 Serine 7.5 0.13 16 Alanine 3.9 0.22 13

Sum of amino acids 86 2.9 192

Sucrose/Sum of amino acids 0.86 0.25 4.3

Table 7. Percentage of the concentration of each amino acid from total amino acid concentrations in the cytosol, apoplast and phloem sap of illuminated spinach leaves. Data from Lohaus et al. (1995).

Cytosol Apoplast Phloem

% % %

Aspartate 26 15 13

Asparagine 2 4 2

Glutamate 24 35 34

Glutamine 28 14 9

Serine 9 4 8

Glycine 2 4

Threonine 2 2 2

Alanine 5 7 7

Valine 3 3 4

8. Compartmentation of metabolites 167

in the phloem sap is similar to that in the cytosol of the mesophyll cells. This was also the case in barley, potato, beet and rape leaves. The amino acid concentration and the pattern of the amino acids in the cytosol and in the phloem sap varied depending on the plant species and the nutrient conditions, but in a similar manner for both compartments (Winzer et al., 1996). Whereas sucrose is the sole carbohydrate transported in the phloem in the crop plants discussed here, there is no special transport form for the products of nitrate assimilation, and all the amino acids are translocated in the phloem.

5. TRANSPORT FROM THE APOPLAST INTO THE SffiVETUBESGOVERNSTHEEXPORTOF PHOTOASSIMILATES

The ratio of sucrose to amino acids in the phloem sap is very much higher than in the cytosol and the apoplast (Table 8). The higher amount of sucrose translocated in the phloem compared to that of amino acids is obviously due to preferential transport from the apoplast into the sieve tubes. The sucrose concentration in the phloem sap is always higher than that of the amino acids, but the concentrations of both vary according to plant species or genotype. Thus the sucrose concentration in the phloem sap was found to vary between 0.6 and 1.5 M and the concentration of amino acids between 0.04 and 0.5 M (Lohaus et aI., 1995; Winzer et aI., 1996; G. Lohaus unpublished). The different phloem concentrations of sucrose and amino acids also influences the composition of the storage substances in storage tissues, e.g. sugar beet taproots differ from those of fodder beet in storing higher quantities of sucrose and lower quantities of amino acids (Table 9). In these two cultivars, the ratio of sucrose to amino acids in the cytosol and in the apoplast of the leaves is about the same, but it is much higher in the phloem sap of sugar beet than in fodder beet (Table 8). In sugar beet, the decrease in the transport of amino acids from the apoplast to the sieve tubes appears to play a decisive role in lowering the amino acid content in the taproots.

The ability to store high concentrations of sucrose in the taproots of sugar beet coincides with a very high sucrose concentration in the phloem sap. A passive uptake of sucrose has been observed with isolated beet root vacuoles at a high external sucrose concentration (Saftner et aI., 1983; Willenbrink et al., 1984). As the sucrose concentration in the phloem sap is about twice as high as in the taproot storage vacuoles (Table 10), it is possible that the transport of sucrose from the sieve tubes to the storage vacuoles proceeds passively. The high concentration of sucrose in

168 Chapter 8

Table 8. Ratio ofsucrose to the sum of amino acids in the cytosol, apoplast and

phloem sap of illuminated leaves.

The cytosolic concentrations in beet were evaluated from measurements of whole leaf

contents, assuming that the subcellular volumes and the distribution of metabolites in

beet are the same as in spinach. Data from Lohaus et al. (1995), Winzer e/ al. (1996).

sucrose / sum of amino acids

Cytosol Apoplast Phloem

Spinach 0.9 0.3 4.3

Barley 1.0 0.2 5.5

Fodder beet 0.5 0.9 7.4

Sugar beet 0.5 1.6 15.0

8. Compartmentation of metabolites 169

Table 9. Metabolite and ion contents in the tap roots of fodder beet and sugar beet. As the aqueous volume of the taproots mainly consists of vacuoles, and nearly all of the sucrose and ions are stored in the vacuole, the aqueous volume of the taproot was measured and the concentrations of the solutes were calculated from this. Data from Winzer et af. (1996).

Cl­

P043-

Nitrate

Malate

Citrate

Hexoses

Sum of amino acids

Sucrose

Sum of solutes

Sucrose / sum of amino acids

Osmolarity

Fodder beet

Peramono (mM)

190

10

4

3

85

28

11

2

6

2

39

212

592

5.4

(mOsmol)

500

Sugar beet

9EO 106 (mM)

125

12

5

3

32

34

2

5

16

508

742

31.0

(mOsmol)

706

170 Chapter 8

Table 10. Sucrose concentrations in the phloem sap and in the taproots offodder beet and sugar beet. Data from Winzer et at. (I996)

Phloem sap Taproot

Sucrose concentration (mM) Fodder beet Sugar beet

1095 1550 212 508

Figure 1. Scheme of sucrose transport in sugar beet from the mesophyll cells to the storage vacuoles of the taproot.

8. Compartmentation of metabolites 171

the storage vacuoles of the taproots may ultimately be caused by the proton-sucrose symporter involved in phloem loading (Figure 1).

On the other hand, the high sucrose concentration in the taproots of sugar beet as compared to fodder beet also appears to be governed by the active uptake of other osmotically active substances into the storage vacuoles. Wyse et al. (1986) found that the turgor of sugar beet storage tissue is an important factor in controlling sugar uptake. It seems likely that sucrose accumulation in the storage vacuoles is limited by the accumulation of other osmotically active solutes, mainly potassium and chloride, possibly in an indirect manner. The negative correlation between sucrose concentration and the concentration of other solutes in the storage vacuoles of the taproots of sugar and fodder beet also supports this conclusion (Table 9).

ACKNOWLEDGMENT

This work has been made possible by the Deutsche F orschungsgemeinschaft.

REFERENCES

Barlow, CA and McCully, M.E. (1972). The ruby laser instrument for cutting the sty lets offeeding aphids. Canadian Journal of Zoology, 50, 1497-1498.

Canny, M.J. (1987). Locating active proton extrusion in leaves. Plant Cell and Environment, 10, 271-274.

Gerhardt, R. and Heldt, H. W. (1984). Measurement of subcellular metabolite levels in leaves by fractionation of freeze-stopped material in nonaqueous media. Plant Physiology, 75, 542-547.

Heineke, D., Wildenberger, K., Sonnewald, U., Willmitzer, L. and Heldt, H.W. (1994). Accumulation of hexoses in leaf vacuoles: studies with transgenic tobacco plants expressing yeast derived invertase in the cytosol, vacuole or apoplast. Planta, 194, 29-33.

Leidreiter, K., Kruse, A., Heineke, D., Robinson, D.G. and Heldt, H.W. (1995). Subcellular volumes and metabolite concentrations in potato (Solanum tuberosum cv. Desiree) leaves. Botanica Acta, 108, 439-444.

Lohaus, G., Winter, H., Riens, B. and Heldt, H.W. (1995). Further studies of the phloem loading process in leaves of barley and spinach: comparison of metabolite concentrations in the apoplastic compartment with those in the cytosolic compartment and in the sieve tubes. Botanica Acta, 108, 270-275.

Riens, B., Lohaus, G., Heineke, D. and Heldt, H. W. (1991). Amino acid and sucrose determined in the cytosolic, chloroplastic and vacuolar compartments and in the phloem sap of spinach leaves. Plant Physiology, 97, 227-233.

Safiner, R.A., Daie, I. and Wyse R.E. (1983). Sucrose uptake and compartmentation in sugar beet tap root tissue. Plant Physiology, 72, 1-6.

172 Chapter 8

Speer, M. and Kaiser, W.M. (1991). Ion relations of symplastic and apoplastic space in leaves from Spinacia oleracea L. and Pisum sativum L. under salinity. Plant Physiology, 97, 990-997.

Willenbrink, J., Doll, S., Getz, H.P. and Meyer, S. (1984). Zuckeraufnahme in isolierten Vakuolen und Protoplasten aus dem Speichergewebe von Beta-Ruben. Berichte der Deutschen Botanischen Gesellshaft, 97, 27-39.

Winter, H., Robinson, D.G. and Heldt, H.W. (1993). Subcellular volumes and metabolite concentrations in barley leaves. Planta, 191, 180-190.

Winter, H., Robinson, D.G. and Heldt, H.W. (1994). Subcellular volumes and metabolite concentrations in spinach leaves. Planta, 193, 532-555.

Winzer, T., Lohaus, G. and Heldt, H.W. (1996). Influence of phloem transport, N­fertilization and ion accumulation on sucrose storage in the tap roots of fodder beet and sugar beet. Journal of Experimental Botany, 47, 863-870.

Wyse, R.E., Zamski, E. and Tomos, D. (1986). Turgor regulation of sucrose transport in sugar beet taproot tissue. Plant Physiology, 81, 478-481.

Chapter 9

Regulation of starch synthesis in storage organs

Alison M. Smith John Innes Centre, Colney Lane, Norwich NR4 7UH

Key words: ADPglucose pyrophosphorylase; amylopectin; amylose; plant mutants; plant storage organ; starch-branching enzyme; starch granule; starch synthase.

Abstract: This paper describes new information, derived from studies of mutant and transgenic plants, about the synthesis and organisation of the two polymers, amylose and amylopectin, that make up the starch granule.

The organisation of amylopectin molecules to form the matrix of the granule is made possible by the fact that the polymer has a polymodal distribution of branch lengths. It has been suggested that this distribution is brought about through the actions of two different isoforms of starch-branching enzyme with different properties, but data from mutant plants with only one isoform of starch-branching enzyme are not consistent with this idea. Studies of mutant and transgenic plants lacking specific isoforms of starch synthase indicate that individual isoforms of this enzyme play distinct roles in amylopectin synthesis. However, the contributions of particular classes of isoform appears to differ from one organ to another. Recent work on mutations that decrease debranching enzyme and lead to the production of highly-branched phytoglycogen has generated a new model for amylopectin synthesis. The validity of the model is discussed.

The synthesis of amylose is a function of a specific class of granule-bound starch synthases, but the mechanism of synthesis is unknown. New data indicate that amylose synthesis within the starch granule requires the presence of soluble malto-oligosaccharides. The generation of malto­oligosaccharides via a debranching enzyme putatively involved in amylopectin synthesis presents a means by which the synthesis of the two sorts of polymer might be integrated.

173

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 173-193. © 1999 Kluwer Academic Publishers.

174 Chapter 9

A further means by which amylose and amylopectin synthesis may interact is suggested by the relationship - observed across a range of mutant plants with lesions in the pathway from sucrose to ADPglucose - between the rate of starch synthesis and the amylose to amylopectin ratio of the starch. It seems likely that changes in ADPglucose concentration have different effects on the synthesis of the two polymers. Factors that control flux through the pathway of starch synthesis may therefore also affect starch structure. The control of flux through the pathway is not properly understood, but is likely to differ from one organ to another. This is highlighted by the recent discovery that ADPglucose pyrophosphorylase - a enzyme considered to be plastidic in most plant organs - is located primarily outside the plastid in the endosperms of some cereals.

1. INTRODUCTION

In this paper I shall discuss recent developments in our understanding of the synthesis of the starch granule. Studies of starch synthesis have tended to concentrate on the properties of individual enzymes, and have paid little attention to the ways in which the activities of several enzymes are integrated to allow the synthesis of the starch granule in vivo. An improved ability to create and to analyse mutant plants with altered starches, combined with an better understanding of the structure of the intact starch granule, is leading to new ideas and information about the ways in which starch polymers are synthesised and organised to form a granule. I shall first describe the structure of storage starch granules, and emphasise the features that we must explain if we are to understand their synthesis. I shall then discuss current ideas about the synthesis and organisation of the starch polymers.

2. THE SIRUCfURE OF THE GRANULE

The starch granule is not simply a random aggregate of polymerised glucose, but a complex and highly organised structure composed of two basic types of polymer, amylose and amylopectin. Amylose, an essentially linear polymer, makes up about 30% of storage starches (French, 1984). Amylopectin is a highly branched polymer, in which a.1,4-linked glucose chains are joined by a.1,6-linkages. Inside the granule, the branches are clustered along the long axis of the amylopectin molecule, at regular intervals of 9 nm. Adjacent branches form double helices, and these pack together in ordered arrays to form crystalline lamellae. The crystalline lamellae alternate with amorphous lamellae

9. Starch synthesis 175

formed by the regions between the clusters, in which branch-points occur (Jenkins et aI., 1993; Figure 1). It is important to note that this semi­crystalline organisation is possible because amylopectin has a polymodal distribution of branch lengths. Branches of about 12-20 glucose units lie within one cluster, branches of about 40 glucose units span two clusters, and so on (Hizukuri, 1986). Bacteria and animals make al,4-, al,6-linked glucans (glycogen), but these have an essentially unimodal distribution of branch lengths and do not form semi-crystalline granules.

The semi-crystalline zones composed of the crystalline and amorphous lamellae alternate within the granule with amorphous zones, in which the organisation of the amylopectin molecules is not understood. It is generally accepted that one semi-crystalline and one amorphous zone are laid down every day (a "growth ring") during the synthesis of storage starches, but almost nothing is known of the factors that determine this level of organisation (French, 1984). It seems likely that much of the amylose component of the starch may be located within the amorphous zones. However, beyond the fact that amylose is not essential for the formation of the granule as described above, and probably exists in a single-helical form within the granule, there is little direct information about its location (Gidley, 1992; Gidley and Bociek, 1988; Figure 1).

It seems likely that the organisation of the starch polymers to form a granule is largely determined by the way in which they are synthesised at the granule surface in vivo. The re-crystallisation of solubilised amylose and amylopectin in vitro does not result in a structure with the organisation of a starch granule.

3. THE COMPLEXITY OF THE POLYMER­SYNTHESISING ENZYMES

In theory, only two types of enzyme are required to synthesise al,4-, al,6-linked glucose polymers. Starch synthase transfers the glucosyl moiety of the sugar nucleotide ADPglucose to the non-reducing end of a growing chain of glucose units. Starch-branching enzyme transfers chains of glucosyl units from the non-reducing end of one chain to the side of another via a al,6-linkage, to form a branch. However, incubation of starch synthase, starch branching enzyme and ADPglucose in vitro results in a branched polymer that has the characteristics of neither amylose nor amylopectin, and does not become organised to form a granule. There must be some additional complexity in vivo that allows the synthesis of both amylose and amylopectin and their organisation into a granule. The

176

Amorphous

zone

Crystalline

lamella

Amorphous lamella

---- I

Figure 1 Organisation of amylopectin within the starch granule.

Chapter 9

The cluster structure believed to be adopted by amylopectin molecules within the starch granule is shown at the right. Packing of the double helices gives rise to crystalline lamellae, and these alternate with amorphous lamellae created by regions containing the branch points. Concentric zones of crystalline and amorphous lamellae (semicrystalline zones) alternate within the starch granule with zones in which the amylopectin is not organised in a crystalline manner (amorphous zones).

9. Starch synthesis 177

most obvious feature of starch synthesis in vivo that may provide this additional complexity is that both starch synthase and starch-branching enzyme exist as multiple isoforms, each with distinct properties.

The existence of multiple isoforms is illustrated by the situation in the developing pea embryo, a "model system" for which we have a good understanding of the starch-synthesising enzymes (Figure 2). Activity of starch-branching enzyme is accounted for by two isoforms, SBEI and SBEII (Bhattacharyya et aI., 1990; Burton et aI., 1995; Smith, 1988). Both are found primarily in the stroma of the amyloplast, but also become bound within the matrix of the starch granule as it grows (Denyer et aI., 1993). The two isoforms have different properties and different patterns of expression during the development of the embryo (Burton et aI., 1995). About 60-70% of the soluble (stromal) activity of starch synthase is accounted for by SSII, an isoform that is also bound into the matrix of the starch granule (Denyer and Smith, 1992; Denyer et aI., 1993; Dry et aI., 1992; Edwards et aI., 1996). Another isoform, granule­bound starch synthase I (GBSSI), is found exclusively within the matrix of the granule, and it accounts for most of the starch synthase activity measurable on intact, isolated starch granules (Denyer et aI., 1995a; Smith, 1990). A third isoform, ssm, appears to be exclusively soluble (1. Craig, John Innes Centre, personal communication), and there are probably other minor, exclusively soluble isoforms that we have not yet discovered.

4. THE SYNTHESIS AND ORGANISATION OF AMYLOPECfIN

4.1 The role of starch-branching enzyme

The existence of multiple isoforms of starch-branching enzyme has led to the suggestion that different isoforms might preferentially transfer branches of different lengths, giving rise to the polymodal distribution of branch lengths characteristic of amylopectin (Preiss and Sivak, 1996). Detailed studies of the enzyme seem to support this idea. First, all of the starch branching enzymes for which predicted amino-acid sequences are available can be grouped into two classes - A and B - on the basis of their primary sequences. The A and B classes differ in several obvious ways, for example the existence of a N-terminal domain on the A class which is absent from the B class. It appears that storage organs generally possess members of both classes. Multiple isoforms have been described from

178

SBEI

SSIII

stroma

starch granule

Chapter 9

Figure 2 The distribution of isoforms of starch synthase and starch-branching enzyme in the amyloplast of the developing pea embryo. SBE: starch-branching enzyme SS: starch synthase GBSS: granule-bound starch synthase

9. Starch synthesis 179

maize and rice endosperms, pea embryos and potato tubers, and each of these organs contains representatives of both A and B classes (Burton et aI., 1995). In the case of pea, SBEI and SBEII are A- and B-type isoforms respectively. Second, studies of the properties of the A and B isoforms of maize endosperm (maize SBEII and SBEI respectively) indicate that A and B isoforms preferentially transfer branches of different lengths. When allowed to branch amylose in vitro and when expressed in place of the endogenous glycogen branching enzyme in E. coli. the A isoform preferentially transfers shorter branches than the B isoform (Guan et aI., 1995; Guan and Preiss, 1993; Takeda et aI., 1993).

The different properties of the A and B isoforms of maize have led Jack Preiss and his colleagues to suggest that the two isoforms contribute branches of different lengths during the synthesis of amylopectin in vivo. They speculate that the A isoform may create the shorter branches that lie within clusters, and that the B isoform creates longer branches that span clusters (Preiss and Sivak, 1996; Takeda et aI., 1993).

This idea is potentially testable through the study of effects of mutations in maize, rice and pea that cause the specific loss of the A isoform of starch-branching enzyme. The mutation in pea - at the r locus - lies in the gene encoding SBEI and prevents the expression of SBEI protein. It causes a reduction in the activity of starch-branching enzyme throughout the development of the embryo, particularly at the early stages of development when activity in the mutant embryo is barely detectable (Bhattacharyya et aI., 1990; Smith, 1988). Comparison of the branching pattern of amylopectin of mutant embryos, containing only SBEII, with that of wild-type embryos should reveal whether both A and B isoforms are required to generate a polymodal distribution of branch lengths. However, the mutation has several secondary effects on the embryo which could themselves influence the structure of amylopectin. These include a reduction in the overall rate of starch synthesis, a change in the expression pattern of starch-branching enzyme through development, and a large change in the ratio of activities of starch synthase and starch-branching enzyme (Burton et aI., 1995; Smith, 1988). Several of these potential problems in interpretation are removed by studying the starch of leaves rather than embryos. The starch­branching enzyme activity of pea leaves is composed of the same isoforms as that of the embryo and the r mutation reduces activity by approximately 90% (Tomlinson et aI., 1997). The mutation has little effect on the rate of starch synthesis in leaves, and the analysis of amylopectin synthesised during a single photoperiod rules out any secondary effects of the mutation due to altered developmental pattern of branching enzyme activity.

180 Chapter 9

Analysis of enzymatically debranched amylopectin from embryos and leaves revealed that the mutation has no effect on the basic polymodal distribution of branch length in either organ. Analysis by gel permeation chromatography, which allows the full range of branch lengths to be studied, and high-performance anion-exchange chromatography (HP AEC), which allows detailed study of the shorter branch lengths, showed that although the mutation causes a general shift towards longer branch lengths in the embryo, the positions of maxima in the distribution are unchanged (Lloyd, 1995; Tomlinson et aI., 1997). The starch retains a structural periodicity of 9 nm, indicating that the amylopectin is organised into clusters (Jenkins and Donald, 1995). Amylopectin from leaves displays a strongly trimodal distribution of branch lengths, with maxima at 12, 15 and about 22 glucose units. The r mutation has no effect on the basic distribution (Tomlinson et aI., 1997).

The effects of the r mutation provide unambiguous evidence that the creation of a polymodal distribution of branch lengths within amylopectin does not require two distinct isoforms of starch branching enzyme. The factors that are responsible for the polymodal distribution must lie elsewhere in the starch-synthesising machinery. The existence of multiple isoforms of starch synthase makes it a candidate for this role.

4.2 The role of starch synthase

A lack of both information about the number and nature of starch synthase isoforms in storage organs and suitable mutants with which to study their roles has until recently made it impossible to assess whether different isoforms may make distinct contributions to the synthesis of amylopectin. However, study of a new pea mutant can potentially shed light on this problem. The mutant was isolated in a mutagenesis programme designed by Cliff Hedley and Trevor Wang at the John Innes Centre to generate novel variation in pea seed composition. Round­seeded, wild-type peas were mutagenised, and the mutant population was screened for lines with wrinkled seeds (Wang et aI., 1990; Wang and Hedley, 1991). Existing mutants with low starch contents - for example the r mutant described above - are wrinkled seeded. This is because accumulation of sucrose during development of the mutant embryo causes it to have a higher water content and hence a larger volume than the wild-type embryo. When the seed dries out at the end of development it becomes wrinkled as the larger loss of water is accommodated. The programme was successful in discovering mutations at three new loci that reduce the starch content of the embryo. Mutations at one of the loci -the rug5 locus - have a profound effect on the structure as well as the

9. Starch synthesis 181

amount of starch m the embryo. The starch granules are highly convoluted, and HPAEC of debranched amylopectin reveals that this is associated with major changes in branch length distribution (Lloyd, 1995; J. Lloyd, John Innes Centre, personal communication). The distribution of amylopectin branch lengths from wild-type embryos displays a maximum at about 15 glucose units, and branches of about 11 to 20 glucose units are the most abundant. In contrast, the maximum from the amylopectin of the mutant is at 10 glucose units, and the amylopectin is strongly enriched in branches of 7-10 glucose units at the expense of longer branches. This is a very important difference in structural terms because branches of less than 10 glucose units may be too short to form the double helices which pack to create the crystalline lamellae (Gidley and Bulpin, 1987). It is likely that the very different morphology of starch granules of the rug5 mutant is a consequence of a much-reduced crystallinity.

The effects of mutations at the rug5 locus suggested that activity of one of the polymer-synthesising enzymes must be affected, and this was confirmed by biochemical studies of the isoforms of starch synthase and starch-branching enzyme previously described from the embryo. The major soluble isoform of the embryo, SSII, was absent from the mutant embryo, and mapping experiments have confirmed that the SSII gene lies at the rug5 locus (Craig et aI., 1995; J. Craig, John Innes Centre, personal communication). It thus appears that SSII is specifically responsible for the elongation of short branches within the amylopectin - perhaps the primary products of starch-branching enzyme - to form the branches of 12-20 glucose units that lie within the clusters.

If SSII does indeed play this specific and important role in determining amylopectin structure in pea embryos, it might be expected that SSII-like isoforms would play a similar role in starch synthesis in other storage organs. We investigated whether this is the case in the potato tuber. The tuber contains an isoform of starch synthase very similar in primary sequence to pea SSIL Like pea SSII, this potato SSII is both soluble and granule-bound. However, immunoprecipitation experiments with an SSII antiserum showed that SSII accounts for only about 10% of the soluble activity of the tuber. This was confirmed by the expression of antisense RNA for SSII in potatoes: although SSII protein and activity were reduced to very low levels, soluble starch synthase activity was not significantly affected (Edwards et aI., 1995). Accordingly, we investigated the nature and role of other isoform(s) responsible for soluble activity in the tuber. Purification of soluble activity to homogeneity identified an isoform (SSm) which was not recognised by SSII antisera and which shows relatively little identity to SSII at the level of amino-acid sequence. Preliminary results indicate that potato ssm is related more closely to

182 Chapter 9

the minor ssm isoform of the pea embryo than to ssn isoforms. Immunoprecipitation experiments with an ssm antiserum showed that ssm accounts for about 85% of the soluble activity in the tuber, and plants expressing antisense RNA for ssm had starch synthase activities in their tubers which were up to 80% lower than those in untransformed tubers (Marshall et aI., 1996).

The large reductions in ssm activity in the tubers of the transgenic plants clearly have some effect on starch structure. The granule of these plants are deeply fissured, and sometimes break up to from "grape-bunch" clusters (Marshall et aI., 1996). However, preliminary analysis of the amylopectin shows that this change in morphology is not due to any major changes in average branch lengths in the range 6-20 glucose units. Overall, it appears that the contributions of SSII and ssm in amylopectin synthesis in the potato tuber may not be quantitatively or qualitatively equivalent to the contributions of the ssn and ssm isoforms in the pea embryo.

The conclusion that plant organs show considerable quantitative and qualitative variation in the nature and roles of their starch synthases is also supported by work on isoforms of starch synthase in cereal endosperms (Denyer et aI., 1995b; Hylton et aI., 1996) and pea leaves. Although we do not yet have a complete picture of the isoforms of starch synthase in the endosperm of any cereal, it is clear that the complements of isoforms differ from one species to another. The complement of isoforms in the pea leaf reveals that there is also variation between organs on the same plant. The major isoform in the leaf - accounting for about 80% of the activity - is closely related antigenically to the ssm isoforms of the embryo and the potato tuber, and is not recognised by ssn antisera (K. Tomlinson and J. Craig, John Innes Centre, personal communication).

4.3 A role for debranching enzyme?

So far I have discussed the structure of amylopectin as a function only of starch synthase and starch branching enzyme. There has been limited evidence for many years that debranching enzymes - enzymes that specifically cleave a.l,6-linkages - may also be important in determining amylopectin structure during its synthesis, and recent discoveries have led to a great resurgence of interest in this possibility.

Evidence for the involvement of debranching enzyme comes from studies of the effects of mutations at the sugary1 locus of maize and the sugary locus of rice. These mutations dramatically reduce starch synthesis in the endosperm, and cause the accumulation of

9. Starch synthesis 183

phytoglycogen, a soluble 0.1,4-, o.l,6-linked glucan that is more highly branched than amylopectin. Several enzymes involved in starch metabolism in the endosperm are affected by the mutations, but in both cases there are substantial reductions in the activity of debranching enzyme - measured by its ability to hydrolyse the o.l,6-linkages of the yeast glucan pullulan - during endosperm development (Nakamura et aI., 1996b; Pan and Nelson, 1984). This has led to the idea that the structure of amylopectin is somehow determined by the concerted actions of a branching enzyme and a debranching enzyme. Phytoglycogen is then the result of the action of starch branching enzyme alone, or in the presence of much reduced activities of debranching enzyme.

Further, direct evidence of the involvement of debranching enzyme in amylopectin synthesis has recently come from two sources. First, the gene at the sugary 1 locus of maize has been cloned by a transposon­tagging approach, and has been shown to encode a debranching enzyme (James et aI., 1995). Second, a phytoglycogen-accumulating mutant of the green alga Chlamydomonas has been identified. The mutation - at the sta7 locus - appears to lack specifically a debranching enzyme (Mouille et aI., 1996).

The clear association between debranching enzyme deficiencies and a failure to produce normal amylopectin has led Steven Ball and colleagues to propose a new model for the synthesis of amylopectin that involves the sequential actions of starch synthase, starch branching enzyme and debranching enzyme (Ball et al. 1996; Figure 3). Ball envisages that starch synthase elongates very short chains arising from a highly branched zone at the outer edge of the granule. Initially, the newly­synthesised chains are not branched because starch branching enzyme cannot act on short chains. The enzyme may actually act primarily or exclusively on double helices, in which case branching will occur only when the chains reach 10-12 glucose units and associate as double helices. At this point, unorganised synthesis and branching will occur, giving rise to an amorphous, phytoglycogen-like material at the periphery of the granule. Debranching enzyme then "trims" this structure, removing most of the newly synthesised branches. The remaining branches then form the highly branched zone from which the next round of chain elongation can occur.

It must be emphasised that this model is highly speculative. It makes several assumptions about the properties of the enzymes for which there is little evidence, and it does not provide roles for multiple isoforms of the enzymes. It is based on an understanding of the phytoglycogen­accumulating mutants that is still deficient in several respects. This problem is particularly acute in the case of the debranching enzymes. Remarkably little is known about the occurrence and properties of these

184

starch synthase

starch synthase +SBE

•

Chapter 9

debranching enzyme

Figure 3 The "trimming" model to explain the synthesis of amylopectin. The diagram shows the proposed stages in the synthesis of an amylopectin cluster at the periphery ofa growing starch granule, as proposed by Ball et aI. (1996). From left to right: starch synthase elongates short chains at the granule surface; the elongated chains are branched by starch-branching enzyme to form an unorganised structure ("preamy\opectin") at the surface; debranching enzyme trims the unorganised structure. Redrawn from Ball et al. (1996).

9. Starch synthesis 185

enzymes in higher plants, but evidence is now emerging that some -perhaps many - plant organs possess members of two distinct classes, with different substrate specificities. One class is related to the isoamylases of bacteria and can hydrolyse the a1,6-linkages of amylopectin but not of pullulan. The other is related to the pullulanases of bacteria, and can hydrolyse both amylopectin and pullulan (e.g. Doeh1ert and Knutson, 1991). It is not clear whether one or both of these classes of enzymes possesses the necessary specificity to participate in the trimming model, and current understanding of the effects of the sugary mutations confuse rather than clarify this point. The activity shown to be decreased in the endosperm of sugary1 (maize) and sugary (rice) mutants is a pullulanase, but the enzyme encoded by the sugary1 locus of maize is an isoamylase that is not active against pullulan when expressed in E coli (James et aI., 1995; A. Myers, Iowa State University, personal communication). It is also clear that the sugary locus of rice does not encode the pullulanase affected by the mutation, but the nature of the gene at this locus is not yet known (Nakamura et aI., 1996a).

In spite of these potential objections to the "trimming" model of amylopectin synthesis, it represents the first serious attempt to integrate information about the synthesis of amylopectin with information about its organisation in the granule. As such, it should stimulate much further thought and experimentation in the next few years.

5. THE SYNTHESIS OF AMYLOSE

It has been clear for many years that the synthesis of amylose is an exclusive function of a highly-conserved class of granule-bound starch synthases, granule-bound starch synthase I or GBSSI. Isoforms of this class have been found in all of the storage starches thus far examined. Evidence that they are responsible for amylose synthesis comes from study of cereals, potatoes and peas carrying mutations in the GBSSI gene, all of which lack amylose in their starch (Denyer et aI., 1995a; Smith et aI., 1995). Reductions in GBSSI activity brought about by expression of antisense RNA in potato tubers also result in a proportional reduction in amylose content (Visser et aI., 1991).

Although GBSSI is obviously responsible for the synthesis of amylose, the mechanism by which amylose is synthesised is much less clear. It has been proposed that the granule bound location of GBSSI "shelters" its product from the actions of starch-branching enzyme, allowing it to remain essentially unbranched. This explanation has been called into question by the discovery that the starches of most or all storage organs

186 Chapter 9

contain one or more isoforms of starch synthase in addition to GBSSI, and may also contain starch branching enzyme (Denyer et aI., 1993; Mu­Forster et aI., 1996). The GBSSI class of starch synthases must possess unique features that allow it to synthesise amylose.

As part of our efforts to define the unique features of GBSSI, we have been examining its activity in intact starch granules isolated from storage organs. The granule-bound starch synthases of such granules are capable of incorporating C4C] from ADPC4C]glucose into existing starch polymers within the granule. We thought that comparison of 14e incorporation by granules from wild-type organs and organs lacking GBSSI would provide a means of studying the properties of GBSSI. However, fractionation by gel permeation chromatography of the polymers from pea and potato starch granules incubated with ADPC4C]glucose revealed that GBSSI incorporated 14C not into amylose - as expected from its action in vivo - but into amylopectin (Denyer et aI., 1996a). Similar incorporation into amylopectin rather than amylose had been observed previously with starch granules isolated from sweet potatoes (Baba et aI., 1987) It seemed likely that the isolated granules lacked some component essential for amylose synthesis in vivo. Consistent with this idea, deproteinised, soluble extracts of potato tuber were able to promote amylose synthesis in isolated starch granules. Early studies of starch synthesis by isolated granules had shown that malto­oligosaccharides - maltose, maltotriose etc. - could promote the rate of starch synthesis (Leloir et aI., 1961). We tested whether these compounds were responsible for the stimulation of amylose synthesis by deproteinised extracts, by incubating the extracts with an a-glucosidase which hydrolyses malto-oligosaccharides to glucose. This treatment destroyed the ability of the extracts to promote amylose synthesis. An important role for malto-oligosaccharides in amylose synthesis was confirmed by addition of pure, linear malto-oligosaccharides, from maltose to maltohexose, to incubations of starch granules with ADPC4C]glucose. All of these compounds, but not glucose, could promote very high levels of amylose synthesis (Denyer et aI., 1996a). Preliminary results show that l4e is incorporated primarily into pre­existing but immature amylose molecules.

An understanding of the metabolism of malto-oligosaccharides is clearly important if we are to assess their importance in amylose synthesis in vivo. Several enzymes known to be present in the amy lop lasts of developing storage organs can influence the levels of malto-oligosaccharides, including amylases, phosphorylases, disproportionating enzyme and debranching enzyme. It is tempting to suggest that the trimming model of amylopectin synthesis (Ball et aI., 1996) provides a potential means of integrating amylose and

9. Starch synthesis 187

amylopectin synthesis through the metabolism of malto-oligosaccharides. The action of debranching enzyme during amylopectin synthesis in the trimming model generates malto-oligosaccharides which would then be available to promote amylose synthesis via GBSSI within the newly­formed matrix. It must be emphasised that this idea is purely speculative!

6. THERELATIONSIllP BETWEEN FLUX AND STRUCfURE

Although attempts to understand the factors that determine starch structure have concentrated on enzymes actually involved in the metabolism of starch polymers, there is increasing evidence that the overall flux through the pathway of starch synthesis may influence the structure of starch. This evidence comes from mutants of pea and Chlamydomonas with reduced rates of starch synthesis due to specific reductions in activities of enzymes that precede ADPglucose on the pathway of starch synthesis. In pea, mutations that reduce activities of sucrose synthase, plastidic phosphoglucomutase and ADPglucose pyrophosphorylase in the embryo all result in a lower ratio of amylose to amylopectin in the starch (Bogracheva et aI., 1995; Wang et al. 1997). In Chlamydomonas, mutations that reduce activities of plastidic phosphoglucomutase and ADPglucose pyrophosphorylase have the same effect (Van den Koornhuyse et aI., 1996). It appears that amylopectin synthesis is favoured over amylose synthesis as the overall rate of synthesis falls. The most likely explanation of this phenomenon is that decreases in flux through the pathway caused by these mutations result in decreases in the concentration of ADPglucose, and that this has a differential effect on the rate of synthesis of the two polymers because amylopectin synthesis has a higher affinity for ADPglucose than amylose synthesis.

Regardless of the actual mechanism, though, the influence of flux through the pathway of starch synthesis on starch structure shows that a full understanding of the control of structure requires information about the control of flux. The factors that are important in controlling flux are very poorly understood in starch-storing organs generally, but it is apparent that there are major differences in this respect both between organs and during organ development (Denyer et aI., 1995c; Smith et aI., 1997). These differences have recently been highlighted by the discovery that pathway of starch synthesis itself differs between storage organs, in that ADPglucose is synthesised in the plastid in many storage organs but in the cytosol in others.

188 Chapter 9

It has been widely assumed that ADPglucose pyrophosphorylase is a plastidic enzyme, and there are good experimental data to support this assumption for several starch-storing organs. However, recent studies of the subunits of the enzyme (a heterotetramer of distinct "large" and "small" subunits) from the endosperms of barley and maize have led to the suggestion that the enzyme might be extraplastidic in these organs. The subunits predicted from cDNAs derived from the endosperm apparently lack consensus transit peptides. The mature proteins visualised on immunoblots of endosperm extracts were of the same size as the products of the full length cDNAs synthesised in vitro or in expression systems, indicating that the subunits may not undergo a post­translational processing consistent with their import into a plastid (Giroux and Hannah, 1994; Thorbj0rnsen et aI., 1996a; Villand and Kleczkowski, 1994).

Although intriguing, these studies do not by themselves provide compelling evidence for an extraplastidic location of the enzyme (ap Rees, 1995). To provide quantitative information about the intracellular distribution of the enzyme, we isolated amyloplasts by mechanical methods from homogenates of developing endosperms of barley and maize (Denyer et aI., 1996b; Thorbj0rnsen et aI., 1996b). Measurements of enzymes widely accepted to be entirely plastidic (alkaline pyrophosphatase, soluble starch synthase) or cytosolic (pyrophosphate­dependent phosphofructokinase, alcohol dehydrogenase, sucrose synthase) in location showed that the yield of intact plastids was substantial, and the preparations had only very low levels of cytosolic contamination. The percentage of the endosperm activity of ADPglucose pyrophosphoryiase recovered in the plastid preparations was higher than that of cytosolic enzymes, but substantially lower than that of plastidic enzymes. Calculations revealed that only 15% of the activity was plastidic in barley, and less than 5% in maize. Immunoblotting experiments using antisera raised against the small subunit of ADPglucose pyrophosphorylase revealed that two immunoreactive proteins of slightly different molecular masses were present in crude homogenates. Plastid preparations were very strongly enriched in the smaller of the two proteins. This result indicates that the plastidic and extraplastidic activities of ADPglucose pyrophosphorylase are functions of different proteins.

Further support for the idea that the endosperms have distinct plastidic and extraplastidic forms of ADPglucose pyrophosphorylase comes from two sources. First, in both barley and maize two different cDNAs for both the large and the small subunits are expressed in the endosperm (Giroux and Hannah, 1994; Thorbj0rnsen et aI., 1996a). Second, we showed that the brittle2 mutant of maize, which lacks the

9. Starch synthesis 189

small subunit encoded by the major species of small-subunit cDNA, specifically lacks the cytosolic activity of the enzyme. Both plastidic activity and the plastid-located form of the small subunit are unaffected by the mutation. The endosperms of brittle2 mutants have lower starch contents than wild-type endosperms, indicating that the extraplastidic form of ADPglucose pyrophosphorylase is important in supplying ADPglucose for starch synthesis (Denyer et aI., 1996b).

The consequences of the cytosolic production of ADPglucose for the control of flux through the pathway of starch synthesis are not yet understood. It is obvious, however, that the factors that control flux in organs in which ADPglucose synthesis is largely cytosolic are likely to be very different from those in organs in which ADPglucose synthesis is plastidic. This means that the relationship between flux and starch structure is also likely to differ in these organs.

7. CONCLUSION

We are beginning to appreciate that starch-storing organs may differ very considerably from each other in the way in which the structure of the starch granule is determined. Some aspects of starch synthesis - for example the nature of the isoforms of starch branching enzyme and the mechanism of amylose synthesis - seem to be very similar wherever we look, and we may be justified in making generalisations in these cases. Other aspects - including the nature and role of the starch synthases responsible for amylose synthesis, the location of ADPglucose synthesis and probably the relationship between flux and structure - vary considerably. Generalisations about these aspects of starch synthesis, at least at our present state of knowledge, may be a hindrance rather than a help to future progress.

ACKNOWLEDGEMENTS

I would like to thank all of my colleagues and collaborators who have contributed to the work described above, and participated in lively discussions about it. I am particularly grateful to Cathie Martin and Kay Denyer for their support and criticism. Work carried out at the John Innes Centre has been supported by the Biotechnology and Biological Sciences Research Council (UK) through competitive grants and through grant-in-aid to the Centre, by the European Union and by Unilever pIc.

190 Chapter 9

REFERENCES

ap Rees, T. (1995). Where do plants make ADP-GIc? In: Pontis, H. G., Salerno, G. L. and Echeverria, E. 1. (Eds). Sucrose metabolism, biochemistry, physiology and molecular biology (pp. 143-155). American Society of Plant Physiologists, Rockville.

Baba, T., Yoshii, M. and Kainuma, K. (1987). Acceptor molecule of granular-bound starch synthase from sweet-potato roots. Starch, 39, 52-56.

Ball, S., Guan, H.-P., James, M., Myers, A., Keeling, P., Mouille, G., Buleon, A, Colonna, P. and Preiss, 1. (1996). From glycogen to amylopectin: a model explaining the biogenesis of the plant starch granule. Cell, 86, 349-352.

Bhattacharyya, M.K., Smith, AM., Ellis, T.H.N., Hedley, C. and Martin, C. (1990). The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch branching enzyme. Cell, 60, 115-122.

Bogracheva, T.Y., Davydova, N.!., Genin, Y.V. and Hedley, C.L. (1995). Mutant genes at the rand rb loci affect the structure and physico-chemical properties of pea seed starches. Journal of Experimental Botany, 46, 1905-1913.

Burton, R.A, Bewley, 1.D., Smith, AM., Bhattacharyya, M.K., Tatge, H., Ring, S., Bull, V., Hamilton, W.D.O. and Martin, C. (1995). Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development. Plant Journal, 7, 3-15.

Craig, 1., Smith, A, Wang, T.L., Lloyd, 1. and Hedley, C. (1995). Biochemistry of new wrinkled-seeded mutants of pea. In: Proc. 2nd European Conf. Grain Legumes, Copenhagen. Improving Production and Utilisation of Grain Legumes (p. 396). Association Europeenne de Recherche sur les Proteagineux, Paris.

Denyer, K., Barber, L.M., Burton, R., Hedley, c.L., Hylton, C.M., Johnson, S., Jones, D.A, Marshall, 1., Smith, AM., Tatge, H., Tomlinson, K. and Wang, T.L. (1995a). The isolation and characterization of novel low-amylose mutant of Pisum sativum. Plant Cell and Environment, 18, 1019-1026.

Denyer, K., Clarke, B., Hylton, C., Tatge, H. and Smith, AM. (1996a). The elongation of amylose and amylopectin chains in isolated starch granules. Plant Journal, 10, 1135-1143.

Denyer, K., Dunlap, F., Thorbjornsen, T., Keeling, P. and Smith, A.M. (1996b). The major form of ADP-glucose pyrophosphorylase in maize (Zea mays L.) endosperm is extra­plastidial. Plant Physiology, 112, 779-785.

Denyer, K., Foster, J. and Smith, AM. (l995c). The contributions of adenosine 5'­diphosphoglucose pyrophosphorylase and starch-branching enzyme to the control of starch synthesis in developing pea embryos. Planta, 97, 57-62.

Denyer, K., Hylton, C.M., Jenner, C. F. and Smith, AM. (1995b). Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm. Planta, 196, 256-265.

Denyer, K., Sidebottom, c., Hylton, C.M. and Smith, AM. (1993). Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos. Plant Journal, 4, 191-198.

Denyer, K. and Smith, AM. (1992). The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos. Planta, 186, 609-667.

Doehlert, D.C. and Knutson, C.A (1991). Two classes of starch debranching enzymes from developing maize kernels. Journal of Plant Physiology, 138, 566-572.

Dry, I., Smith, AM., Edwards, E.A., Bhattacharyya, M., Dunn, P. and Martin, C. (1992). Characterisation of cDNAs enr:oding two isoforms of granule-bound starch synthase

9. Starch synthesis

which show differential expression in developing storage organs. Plant Journal, 2, 193-202.

191

Edwards, A, Marshall, J., Denyer, K., Sidebottom, C., Visser, R.G.F., Smith AM. and Martin, C. (1996). Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound. Plant Physiology, 112, 89-97.

Edwards, A, Marshall, J., Sidebottom, C., Visser, R.G.F., Smith, AM. and Martin, C. (1995). Biochemical and molecular characterisation of a novel starch synthase from potato tubers. Plant Journal, 8, 283-294.

French, D. (1984). Organization of starch granules. In: Whistler, R.L., BeMiller, J.N. and Paschall, E.F. (Eds). Starch: Chemistry and Technology (pp. 183-247). Academic Press, Orlando.

Gidley, MJ. (1992). Structural order in starch granules and its loss during gelatinisation. In: Phillips, G.O., Williams, P.A. and Wedlock, D.l (Eds). Gums and Stabilisers for the Food Industry 6 (pp. 87-92). IRL Press, Oxford.

Gidley, MJ. and Bociek, S.M. (1988). \3C CPIMAS NMR studies of amylose inclusion complexes, cyclodextrins and the amorphous phase of starch granules: relationships between glycosidic linkage formation and solid-state \3C chemical shifts. Journal of the American Chemical Society, 110, 3820-3829.

Gidley, MJ. and Bulpin, P.V. (1987). Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices. Carbohydrate Research, 161, 291-300.

Giroux, M. and Hannah, M.C. (1994). ADP-glucose pyrophosphorylase in shrunken-2 and brittle-2 mutants of maize. Molecular and General Genetics, 243, 400-408.

Guan, H., Kuriki, T., Sivak, M. and Preiss, J. (1995). Maize branching enzyme catalyses synthesis of glycogen-like polysaccharide in glgb-deficient Escherichia coli, Proceedings of the National Academy of Sciences USA, 92, 964-967.

Guan, H.P. and Preiss, J. (1993). Differentiation of the properties of the branching isozymes from maize (Zea mays). Plant Physiology, 102, 1269-1273.

Hizukuri, S. (1986). Polymodal distribution of the chain lengths of amylopectin and its significance. Carbohydrate Research, 147, 342-347.

Hylton, C.M., Denyer, K., Keeling, P.L., Chang, M.-T and Smith, AM. (1996). The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms. Planta, 198, 230-237.

James, M.G., Robertson, D.S. and Myers, AM. (1995). Characterization of the maize gene sugary1, a determinant of starch composition in kernels. Plant Cell, 7, 417-429.

Jenkins, P.J., Cameron, R.E. and Donald, AM. (1993). A universal feature in the structure of starch granules from different botanical sources. Starch, 45, 417-420.

Jenkins, P.J. and Donald, A.M. (1995). The influence of amylose on starch granule structure. International Journal of Biological Macromolecules, 17, 315-321.

Leloir, L.F., De Fekete, M.A.R. and Cardini, C.E. (1961). Starch and oligosaccharide synthesis from uridine diphosphate glucose. Journal of Biological Chemistry, 236, 636-641.

Lloyd, lR. (1995). Effect and interactions of Rugosus genes on pea (Pisum sativum) seeds. PhD thesis. Univ. East Anglia, Norwich, UK

Marshall, l, Sidebottom, C., Debet, M., Martin, C., Smith, AM. and Edwards, A (1996). Identification of the major starch synthase in the soluble fraction of potato tubers. Plant Cell, 8, 1121-1135.

192 Chapter 9

Mouille, G., Maddelein, M.-L., Libessart, N., Tagala, P., Decq, A., Delrue, B. and Ball, S. (1996). Pre-amylopectin processing: a mandatory step for starch biosynthesis in plants. Plant Cell, 8, 1353-1356.

Mu-Forster, C., Huang, R., Powers, J.R, Harriman, RW., Knight, M., Singletary, G.W., Keeling, P.L. and Wasserman, B.P. (1996). Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Plant Physiology, 111, 821-829.

Nakamura, Y., Umemoto, T., Ogata, N., Kuboki, Y., Yano, M. and Sasaki, T. (1996a). Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene. Planta, 199, 209-18.

Nakamura, Y., Umemoto, T., Takahata, Y., Komae, K., Amano, E. and Satoh, H. (1996b). Changes in the structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis. Physiologia Plantarum, 97, 491-498.

Pan, D. and Nelson, O.E. (1984). A debranching enzyme deficiency in endosperms of the sugary-l mutants of maize. Plant Physiology, 74, 324-328.

Preiss, J. and Sivak, M. (1996). Starch synthesis in sinks and sources. In: Zamski, E. and Schaffer, A. A. (Eds). Photoassimilate Distribution in Plants and Crops (pp. 63-94). Marcel Dekker, New York.

Smith, A.M. (1988). Major differences in isoforms of starch-branching enzyme in embryos of round- and wrinkled-seeded peas (Pisum sativum L.). Planta, 175, 270-279.

Smith, A.M. (1990). Evidence that the "waxy" protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase. Planta, 182, 599-604.

Smith, A.M., Denyer, K. and Martin, C. (1995). What controls the amount and structure of starch in storage organs? Plant Physiology, 107, 673-677.

Smith, A.M., Denyer, K. and Martin, C. (1997). The synthesis of the starch granule. Annual. Review of Plant Physiology and Plant Molecular Biology, 48, 67-87.

Takeda, Y., Guan, H.-P and Preiss, J. (1993). Branching of amylose by the branching isoenzymes of maize endosperm. Carbohydrate Research, 240, 253-263.

Thorbj0l1lsen, T., Villand, P., Denyer, K., Olsen, a.-A. and Smith, A.M. (1996b). Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm. Plant Journal, 10, 243-250.

Thorbjernsen, T., Villand, P., Kleczkowski, L. and Olsen, a.-A. (1996a). A single gene encodes two different transcripts for the ADP-glucose pyrophosphorylase small subunit from barley (Hordeum vulgare). Biochemical Journal, 131, 149-154.

Tomlinson, K.L., Lloyd, J.R. and Smith, A.M. (1997). Importance ofisoforms of starch branching enzyme in determining the structure of starch in pea leaves. Plant Journal, 11, 101-113.

Van den Koornhuyse, N., Libessart, N., Delrue, B., Zabawinski, C., Decq, A., Iglesias, A., Carton, A., Preiss, J. and Ball, S. (1996). Control of starch composition and structure through substrate supply in the monocellular alga Chlamydomonas reinhardtii., Journal of Biological Chemistry, 271, 16281-16287.

Villand, P. and Kleczkowski, L. (1994). Is there an alternative pathway for starch biosynthesis in cereal seeds? Zeitschrift Naturforschung, 49c, 215-219.

Visser, RG.F., Somhorst, I., Kuipers, G.J., Ruys, N.J., Feenstra, W.J. and Jacobsen, E. (1991). Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Molecular and General Genetics, 225, 289-296.

9. Starch synthesis 193

Wang, T.L., Hadavizideh, A, Harwood, A., Welham, T J., Harwood, W.A, Faulks, R. and Hedley, C.L. (1990). An analysis of seed development in Pisum sativum. XIII The chemical induction of storage product mutants. Plant Breeding 105, 311-320.

Wang, T.L. and Hedley, C.L. (1991). Seed development in peas: knowing your three "r"s' (or four, or five) Seed Science Research, 1,3-14.

Wang, T.L., Barber, L., Craig, 1., Denyer, K., Harrison, C., Lloyd, J., MacLeod, M., Smith, A and Hedley, C.L. (1997). In: Richmond, P., Frazier, PJ. and Donald, AM. (Eds). Starch: structure and function (pp. 188-195). Royal Society of Chemistry, Cambridge.

Chapter 10

The integration of sucrose and fructan metabolism in temperate grasses and cereals

Christopher J. Pollock, Andrew J. Cairns, Joseph Gallagher and Judith Harrison Environmental Biology Department, Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Ceredigion Sf23 3EB, UK =

Key words: cereals; compartmentation; fructans; grasses; hydrolase; sinks; sources; sucrose; transferase; vacuole.

Abstract: The metabolism of fructose polymers (fructans) in temperate Gramineae provides an interesting and distinctive alternative to that of starch. This review concentrates upon the characterisation of novel elements of fructan metabolism. Fructan structures are shown to be both varied and complex, but to possess a consistency within species which argues for a biosynthetic mechanism with a high degree of specificity. The synthetic and degradative mechanisms currently thought to be involved are discussed, with particular reference to the presence of multifunctional enzymes and the strong effects of both substrate and enzyme concentration on the chemical nature of the products in vitro. The need to reconcile the behaviour of enzymes in vitro with the patterns of metabolism observed in vivo is stressed. Finally, the regulation of fructan synthesis is discussed in relation to the pivotal role of sucrose as the sole substrate and as a key element in the induction of fructan accumulation, apparently acting at the level of gene expression.

1. INTRODUCflON

A number of recent reviews have considered the metabolism and physiology of fructose polymers (fructans) in higher plants (Pollock et aI., 1996; Pollock and Kingston-Smith, 1997; Wiemken et aI., 1995).

195

N. J. Kruger et at. (eds.), Regulation of Primary Metabolic Pathways in Plants, 195-226. © 1999 Kluwer Academic Publishers.

196 Chapter 10

Although there has been increased interest in these compounds in terms of their agricultural or industrial potential (Fuchs, 1993a, 1993b), the metabolism of fructans remains of general scientific interest because of: (a) the discontinuous occurrence of the syndrome; (b) its unusual cellular location; (c) its distinctive mechanism of synthesis; and (d) its close structural and metabolic relationship with sucrose. Comparative biochemistry has often illuminated important elements in the control and limitations of both primary and secondary metabolic pathways and this review considers the extent to which the metabolism of fructans illuminates the more general factors which regulate sucrose metabolism in higher plants.

2. FRUCfAN STRUCTURE: "CONSISTENT DIVERSITY"

It has been estimated that about 10% of the higher plant flora possess the metabolic machinery to synthesise, store and utilise fructans (Hendry and Wallace, 1993). Most of the experimental work has concentrated upon the Asterales (e.g. Jerusalem artichoke and chicory), the Gramineae (mainly C3 temperate members of the Poales including wheat, barley and temperate forages such as rye-grass) and the Liliales (e.g. onion, leek and garlic). Within these families comparatively few species have been studied in great detail, but it has become very clear, following the development of modem separation and analytical techniques, that the diversity within fructan structures is very large. The most common form of fructan in higher plants is based upon the sequential addition of fructosyl residues to a sucrose motif, leading to a polydisperse, water soluble polymer with one glucose residue per molecule of fructan (Pollock et aI., 1996, and references therein). There have been reports of fructan series which do not contain glucose residues, but these form only a minor component in vivo (Ernst et aI., 1996).

The complexity of fructan structures arises in three ways: through variation in size, through variation in the linkage between adjacent fructan residues and through the position of the original sucrose motif. At its simplest, common in members of the Asterales, the polymer is a homologous series with the type structure G-l,2-F-l,2-(F)n with adjacent fructose residues linked in the ~ orientation (Edelman and Jefford, 1968). Where n = 1, the trisaccharide is known as l-kestose. This trisaccharide appears to be present in all species which accumulate fructans. In Jerusalem artichoke, every member of the series between n = 1 and n = c 40 can be isolated, and there is approximately the same mass of

10. Fructan metabolism 197

fructans in each separable oligosaccharide (i.e. molar abundance declines with increasing size). The fructan chain is linear and non-reducing (Edelman and Jefford, 1968).

In other species the situation is more complex. Two other trisaccharides have been isolated; 6-kestose (G-l,2-F-6,2-F) and neokestose (F-2,6-G-l,2-F) and chain elongation from both fructose residues with both linkage patterns is observed, leading to structures which are branched, mixed-linkage or contain an included glucose residue (Figure 1). The variation in mean size between species is very large (Table I) but tends to be consistent within species. Size may also be affected by the extent to which environmental conditions promote fructan accumulation. What is also consistent within species is the relative abundance of specific oligosaccharide isomers. Not all grasses accumulate all the possible structural variants and the "fingerprints" produced by thin-layer chromatography or high-performance anion-exchange chromatography are very distinctive (Chatterton et aI., 1993; Pollock and Cairns, 1991; Slaughter and Livingston, 1994). In temperate grasses, the high molecular weight polymer (which will not pass through dialysis membrane) is almost entirely a linear ~2,6-linked fructan with a terminal glucose (Pollock et aI., 1979), but analysis of the smaller fructan oligo saccharides in Lalium temulentum shows a progressive decline in the proportion of included glucose residues and 2: 1 linkages as the mean size of the oligosaccharides increases (Table 2).

Isotopic tracer studies (e.g. Cairns and Pollock, 1988a; Sims et aI., 1993) have demonstrated unequivocally that sucrose is the major donor of fructose residues to growing fructan chains and that l-kestose is the first labelled intermediate. Subsequently it appears that fructan residues are transferred to a range of oligosaccharide acceptors, most, if not all, of which are in isotopic equilibrium. The pattern of these acceptors is species-specific and changes radically with increasing size to give a much simpler "final" structure.

A fuller discussion of this complexity can be found in Pollock et al. (1996), but the brief outline given above raises a number of interesting issues in terms of the regulation of the process. Fructans appear unique among storage polymers in terms of the precision of the biosynthetic process within species and its variability between them. Any mechanistic hypothesis for the enzymology of fructan synthesis will need to explain these properties.

198 Chapter 10

Table 1 Variation in mean Molecular Size of Fructans from Different Species

Species

Gramineae Phleum pratense Phleum pratense Lolium perenne F estuca pratensis Dactylis glome rata Bromus inermis Liliales Allium sativa Asterales Helianthus tuberosus

Mean molecular Method employed size (kDa)

42 Size exclusion chromatography 46 End group analysis 12 Size exclusion chromatography 12 Size exclusion chromatography 20 Size exclusion chromatography 5 End group analysis

9 Size exclusion chromatography

5 End group analysis

Source: Pollock and Caims 1991.

10. rnlctan metabolism 199

Disaccharide GBF Sucrose(ubiquitous) Trisaccharides GBFIF FBGBF GBF

l-kestose neokestose 6 F

all accumulators Poales 6-kestose Liliales

Poales Liliales

Tetrasaccharides GBFIFlF FIFBGBF GBF GBF-IF 1,1 nystose 6 6

Asparagus F F all accumulators 6

FBGBF F Wheat 6 F Poales

L. temu/entum

Pentasaccharides GBFl(F)21F FBGBFIFIF GBF FIFBGBFIF 6

all accumulators (F)2 Asparagus 6

L. temu/entum F Oat

L. temulentum Wheat

GBFIFIF FBGBF FBGBF 6 6 6 6 F F F F

6 Wheat L. temulentum F

Oat Oat L. temlllentum

GBFIF GBF 6 6 GBFIF

FIF FIF 6 6 F

Wheat F 6 F

Wheat Wheat

Figure 1 The complexities of structure observed in tri-, tetra-, and pentasaccharides isolated from various monocot species. The "minimalist" structural conventions suggested by Pollock are adopted:

GHF = Sucrose; FHGHF = Neokestose; FIF = two adjacent residues linked ~2, I; F = two adjacent residues linked ~2,6 (from Lewis, 1993). 6 F

200 Chapter 10

Table 2 Linkage Analysis of Pooled High DP Fructans from Lolium temulentum Following Fractionation by GPC

Mean Degree ofPolymerisation (Dp)" Ratio of 2,6- to 2,1-linkagesb

Ratio of terminal to linkage G1c residuesc

a Mean DP was estimated from the G1c:Fru ratio.

A 7.2

2.4:1 2.6:1

Fraction B

12.8 12.6:1 7.4:1

c 30.1

40.1 :1 d

b The ratio of 2, 1- to 2,6-linkages was estimated from the relative peak intensities of rnJz = 161, 190 and 161,189, respectively.

c The ratio of terminal glucose to linkage glucose was estimated from relative peak areas following GC separation of partially methylated alditol acetates. d Linkage glucose peak too small for estimation. Source: Sims et al. ,1992

J O. Huetan metabolism 201

3. PHYSIOLOGY AND ENZYMOLOGY OF FRUCfAN MEfABOLISM IN GRASSES AND CEREALS

3.1 Fructan synthesis in leaves

Experimental perturbation of carbon partitioning in grass leaves is straightforward and has provided an excellent system for the study of fructan metabolism and its enzymology (Cairns and Pollock, 1988a, 1988b; Housley and Pollock, 1985; Simpson et aI., 1991; Wagner et aI., 1983). Leaves manipulated to contain low levels of photoassimilates fully degrade sucrose and fructan, and lose both the capacity for in vivo fructan synthesis and extractable fructosyl transferase activity (Cairns, 1992; Cairns and Pollock, 1988a, 1988b; Obenland et aI., 1991; Wagner et aI., 1986). When such tissues are subsequently placed in positive carbon balance, sucrose accumulates until a threshold concentration of 15-20 mg g-I FW is reached, at which point tissue sucrose concentration stabilises, conversion of sucrose into fructan is initiated and extractable fructosyl transferases are induced (the regulation of this process is discussed below). Synthesis of fructan occurs de novo, with sucrose as the major or sole precursor and with no apparent requirement for fructan primers. The energy conserved in the glycosidic bond of sucrose is nearly as high as that in uridine diphosphogucose (28 vs 32 kJ mor l ; Dey, 1980) and is sufficient to fuel the process. Fructan can accumulate at rates similar to those of photosynthetic sucrose synthesis (e.g. 1.6 mg g-I h-I at 250 JlE m-2 S-I) and can reach tissue concentrations of 20-40 mg g-I FW. Fructan synthesis thus represents a massive component of primary carbon metabolism, contributing substantially to resource allocation within the plant.

A previous review (Cairns, 1993) summarises earlier enzymological studies in grasses and introduces the current models and terminology used for the enzymes of fructan synthesis in plants and micro-organisms. Briefly, the current terminology for grasses is based on that of Edelman and Jefford (1968), which was developed to describe the synthesis of ~2,I-linked fructans (inulins) in roots of Helianthus tuberosus. This model designates two monofunctional enzymes: (i) sucrose: sucrose fructosyl transferase (SST), which forms l-kestose by fructosyl transfer from donor to acceptor sucrose; and (ii) fructan: fructan fructosyl transferase (FFT), which reversibly elongates acceptor fructans by the transfer of one fructosyl residue, at the expense of the equivalent shortening of a donor fructan.

202 Chapter 10

Most early enzymological studies in grasses detected l-kestose as the sole transferase product from sucrose. These data were interpreted as evidence for a monofunctional SST, and cited as evidence for the applicability of the SSTIFFT scheme in grasses, though little evidence was presented for further polymerisation by FFT or otherwise. By implication, the only route for carbon into grass fructan was through 1-kestose, consistent with the model of Edelman and Jefford (1968). However, the emerging enzymological evidence in grasses is equivocal in its support for this model since the isolated fructosyl transferase activities are generally multifunctional and/or also catalyse hydrolytic reactions. In addition, all the enzymes so far described have properties which are difficult to reconcile with the physiology of fructan synthesis in the source tissue. There are four areas in particular where behaviour in vivo conflicts with in vitro observations. First, fructosyl transferases from grasses (and other genera e.g. Koops and Jonker, 1996; Van den Ende and Van Laere, 1996) universally require high substrate concentrations for function in vitro. Their kinetic response generally approximates to linearity in the range 50-500 mM sucrose and concentrations for half maximal activity are in the range 100-300 mM. Secondly, in the few instances where direct comparisons have been made, the in vitro sucrose fructosyl transferase activity of grass leaves engaged in fructan synthesis are adequate to support the rates of flux of fructose into fructan in vivo (Cairns, 1992; Cairns and Pollock, 1988a, 1988b). However, the sucrose concentrations required to cause physiologically significant rates of fructosyl transfer markedly exceed those thought to exist in the vacuoles of grass mesophyll cells, the currently accepted site of fructan synthesis (Wagner et aI., 1983). Thirdly, the products of fructosyl transferases assayed in vitro are often different in linkage structure, and especially in size, from the native complement of fructans in the source tissue. Fourthly, polymerisation of the large ~2,6-linked fructans characteristic of grass tissues has been difficult to demonstrate in vitro, despite the magnitude of the process in vivo.

Reports of purified fructosyl transferases from grasses which have appeared since 1993 illustrate some of the above difficulties. For example, in barley, two enzymes have been isolated, both exhibiting a requirement for high sucrose concentration and neither fully saturating at 600 mM. The first, described as I-SST, synthesises l-kestose as the predominant product from sucrose, though higher oligosaccharides of degree of polymerisation (DP) ~ 5 were also formed (Simmen et aI., 1993). In contrast with the results of many studies on the SST from H. tuberosus, barley I-SST is at least bifunctional, having fructan-sucrase or FFT-like activity as well as SST activity. However, recent reports suggest that, under certain conditions, SST preparations from H tub eros us will

10. Fructan metabolism 203

also synthesise higher oligosaccharides (LUscher et aI., 1996). Therefore, the current terminology is not wholly consistent with the range of activities exhibited by this enzyme in different plant species. The second barley enzyme, termed 6-SFT, formerly 6-SST (Duchateau et aI., 1995; Simmen et aI., 1993), is also a multifunctional enzyme which, in the absence of l-kestose, synthesises 6-kestose directly from sucrose, providing an alternative route for carbon into fructan. 6-SFT also transfers single fructosyl residues from sucrose to carbon 6 of fructose residues in oligo saccharides of the inulin series, generating the branched fructans which occur naturally in barley leaf fructan. Independently, the partial reactions of I-SST and 6-SFT can explain the synthesis of barley oligofructans up to c. DP5, but there is no evidence that the rates are physiologically significant or that reconstitution of the two activities can generate oligosaccharides de novo. In addition, there is no direct evidence that they are involved in synthesis of the larger fructans which also occur naturally in barley. The third activity of the 6-SFT preparation is ~­fructofuranosidase (invertase). With 200 mM sucrose as sole substrate, this accounts for 78% of the total activity. In contrast to its low sucrose­affinity for trisaccharide synthesis, the Km for sucrose hydrolysis is c. 10 mM (Simmen et aI., 1993). The detailed physiological consequences of this high-affinity hydrolytic activity, particularly in the early stages of de novo synthesis, remain to be explored. The properties of barley I-SST and 6-SFT are clearly at variance with the SSTIFFT model, suggesting as they do, potential roles in grasses for: (i) multifunctional transferases; (ii) direct sucrase-type fructosyl transfer; and (iii) an alternative route for fructosyl residues into fructan, via 6-kestose.

An enzyme analogous to the FFT activity from H. tuberosus, which catalyses non-synthetic transfer between pre-existing primer inulins to generate oligosaccharides of DP :5: 6, has been isolated recently from L. rigidum (St. John et aI., 1997a). This enzyme did not saturate with increasing substrate concentration, even at 400 mM l-kestose. These authors note that this species accumulates predominantly high Mr fructan with ~2,6-linkages. The predominant affinity of the isolated FFT, by contrast, was for low Mn ~2,1-linked fructans. It showed no detectable donor or acceptor activity against ~2,6-linkages and could not, therefore, account for their synthesis in this tissue. When this FFT was incubated simultaneously with 100 mM sucrose and a partially purified, multifunctional preparation having SST, FFT and 6-kestose-forming activity, oligo saccharides of DP :5: 6 were synthesised (St. John et aI., 1997b). The only circumstances under which traces of larger fructans were generated (up to DP = 17) were in experiments where sucrose was periodically added to the assay to maintain a concentration of 100 mM over a 10 h reaction. These larger products contained both ~2,6- and

204 Chapter 10

B2, I-linkages, which is interesting in view of the absolute B2, I-specificity of the FFT when assayed alone and the B2,6-specificity of high Mr fructan synthesis in vivo. Clearly there is no simple relationship between enzymological and physiological data. The evidence from L. rigidum supports the Edelman and Jefford model to the extent that a fraction active against sucrose is separable from an FFT active against fructan only. However, because the structure of the FFT product differs from the native fructan and because the kinetic parameters and rates of product formation have not been compared with the source tissue, the physiological significance of these observations is not clear.

Since, for the reasons outlined above, it is often difficult to place incomplete enzymological data into a physiological context, we regard the demonstration of in vitro polymerisation of authentic grass fructan at physiological rates as a prerequisite for the understanding of the process in vivo. We have shown that this can be achieved using a combination of high concentrations of partially purified enzyme and high sucrose concentration (Cairns, 1992). The chromatographic fingerprints of the products are species-specific and strongly resemble the native fructan complement in the tissue used as the source of the enzyme (Cairns and Ashton, 1993). In the case of Phleum pratense we have established, using a combination of anion exchange chromatography-PAD and methylation/GC-MS analysis, the enzymatic synthesis of 2,6-linked polymers of high Mr (up to DP = c. 50, Figure 2). Because, (i) large fructans representative of the native polymers are synthesised de novo from sucrose at physiological rates, (ii) the products are species-specific, and (iii) the pattern of sucrose-regulation and its sensitivity to inhibitors of gene expression parallels that of the tissue (Cairns, 1992), we can be reasonably certain that the in vitro activities are involved in fructan synthesis in vivo.

The enzyme concentration required for this high fidelity synthesis in vitro, (c. 5.0 g FW cm-3; 10-14 nkat cm-3), is at least 10-fold greater than that generally employed in enzymological studies of fructan synthesis. The apparent Km for polymerisation in L. temulentum and P. pratense is in the range 200-300 mM sucrose, though the reactions typically tend to saturation only above 1.0 M sucrose. To emulate in vivo rates of accumulation of polymeric fructan, the substrate concentrations required for enzymatic synthesis are in the range 1.0-1.5 M. The pH optima for these polymerising reactions are 5.5-6.0 (Cairns and Ashton, 1994) and the polymerising fractions have Mr of 50-60kD based on size exclusion chromatography (Cairns et aI., 1997). KIm Mr and pH optima for polymerisation are similar to those generally reported for trisaccharide and oligosaccharide formation by fructosyl transferases from many plants.

10. Fructan metabolism

o ~ eo o .0

~IOO cl e ;;

e 80

'0 !! 50 . u c v <0 , .. 0 20

;; t- O

• • • • • • • • • • - • • • • • • • • · iii r'"

• t 4 • • ~ TlrM (hI

10

10

20 30

Time tm~nl

20

TIm. (min)

<0

--­..... :;,:t ·~; .....

c.o .. ~

Internl_ I s"ndud

30

50

40

1.0 g> ..

004 3

205

Figure 2 Polymerisation of fructan by a partially purified enzyme preparation from leaves of Phleum pratense L. a) Time course of fonnation of total fructan products (oligo- and poly saccharides) separated on TLC. Excess sucrose substrate was removed by HPLC prior to analysis. S, l-K, 6-K and DP> I 0 designate sucrose, l-kestose, 6-kestose and fructans of degree of polymerisation greater than 10. b) Analysis of polysaccharide products (DP> 10: ethanol precipitated) by anion exchange chromatography-pulsed amperometric detection. c) Structural determination of polysaccharide products. Fructan was methylated, hydrolysed, reduced with sodium borodeuteride and acetylated. Derivatives were separated by GC on a high polarity BPX70 column and their mass spectra determined. Deduced structures and predominant fragments are shown, indicative of terminal fructose, terminal glucose and 132,6-linked fructose (in order of elution). The internal standard was myo-inositol hexa-acetate.

206 Chapter 10

It is clear that fructosyl transferases assayed at a range of enzyme concentrations exhibit different product specificities, higher enzyme concentrations producing larger fructans over the same incubation period (Cairns, 1995). Given the strong similarity in general properties between extracts which produce large fructans and ones which will only synthesize oligosaccharides, it is possible to explain the absence of polymerisation by the latter, in terms of a requirement for high enzyme and substrate concentration. Enzymes assayed at low concentration and reported as "SST" and "6-SFT" may well polymerise larger fructans under different reaction conditions.

All reported enzymes of fructan synthesis exhibit peculiarities in specificity and/or kinetics which need to be considered against the conditions in vivo. Our polymerisation reactions are no exception. Whilst they make realistic fructans at realistic rates and the pattern of regulation parallels that in vivo, the physiological consequences of the in vitro properties of the polymerase demand closer examination. From the known in vivo and in vitro rates of synthesis in leaves of 1.6 mg g'l h'l (equivalent to 2.5 nkat g'l; Cairns and Ashton, 1994; Cairns and Pollock, 1988b), and estimates of mesophyll vacuolar volume at 441 mm3 i l FW (Cairns et aI., 1989), the concentration of synthetic activity in vivo could theoretically reach 5.7 nkat cm '3, half the concentration needed in vitro to sustain this rate of synthesis. But to achieve even this activity would require a substrate concentration of more than 1.0 M (Cairns and Ashton, 1994). By mensuration (Cairns et aI., 1989) and by direct measurement, (Koroleva et aI., 1997) the vacuolar sap of fructan­accumulating mesophyll cells contain sucrose at 100-200 mM, which would result in an activity of only 10-15 % of the necessary rates. If the properties of in vitro polymerisation reflect the situation in vivo, there is both insufficient enzyme and insufficient substrate in the vacuole to explain the rates of accumulation within the tissue. Kaeser (1983) reported micro-vesicular localisation of inulin synthesis in tubers of H tuberosus. It remains a possibility that high substrate and enzyme concentration could co-exist in such vesicles in grasses, satisfying the requirements for enzymatic polymerisation in vivo. This can be reconciled with findings localising fructans and their enzymes in isolated vacuoles (Cairns et aI., 1989; Wagner et aI., 1983) since presumably such vesicles would fuse with the tonoplast resulting in a final location of both enzyme and product in the vacuole.

10. Fructan metabolism 207

3.2 Fructan hydrolysis in leaves

In general, studies of fructan hydrolysis have concentrated on non­leaf tissue such as internodes, stem bases, leaf sheaths and stubble remaining after defoliation and these constitute the majority of available physiological and enzymological data. These earlier studies of fructan mobilisation and the enzymology of the process in gmsses were reviewed comprehensively by Simpson and Bonnett (1993). This section summarises the current state of understanding of fructan hydrolysis based on earlier work, emphasising the restricted number of studies of leaves and some more recent reports.

Briefly, the general view holds that both the net synthesis and breakdown of fructan is localised in the vacuole. These opposing processes are dynamic, the net direction depends on the carbon balance of the tissue and ultimately depends on the sink status of the whole plant. Leaves manipulated to be in negative carbon balance can completely hydrolyse any fructan and sucrose present and export the carbohydrate to sinks (Cairns and Pollock, 1988a; Simpson et aI., 1991). In general, sucrose and smaller fructans are degmded very quickly whilst higher Mr fructans tend to be more persistent. The tissue content of free fructose may increase slightly during mobilisation, (up to c. 4 mg g-I FW) though there is no concomitant accumulation or qualitative pattern change of oligosaccharides in the tissue. This indicates exohydrolysis of terminal fructose moieties rather than endo-cleavage of the polymers. This is confirmed by the retention of the non-reducing character of fructans during mobilisation. Endo-cleavage would release reducing end-groups (Pollock, 1982). The degradation products, fructose and a small amount of glucose (terminal sucrose will be hydrolysed by invertase), are thought to be transferred from the vacuolar site of hydrolysis to the cytoplasm where they are phosphorylated via hexokinase and exported after resynthesis into sucrose. This pathway requires 1.5 A TP equivalents per mole of hexose mobilised from fructan and resynthesised into sucrose.

Fructan hydro lases isolated from plants are universally exo-hydrolases (termed fructan exohydrolase; FEH), cleaving single terminal fructose residues. Hydrolysis of fructan in vitro parallels the process in vivo (there is a recent report of a hydrolase from L. rigidum which cleaves the terminal glucose). Exohydrolases from gmsses exhibit acid pH optima in the range 4.5-5.5 and have Mr of 40-69 kD (Simpson and Bonnett, 1993). There is emerging evidence that "FEH" is not one enzyme but a number of isoforms with differing molecular properties and specificities (Bonnett and Simpson, 1995)

208 Chapter 10

In common with synthesis, the enzymology of fructan hydrolysis has been difficult to reconcile with the known physiology of the process. The main areas of discrepancy relate to the kinetics, specificity, timing, and regulation of fructan breakdown. First, high substrate concentrations are required for in vitro function of FER. Reports of K.n vary widely between 0.22 and 89% w/v fructan but are generally c. 20 % w/v (Simpson and Bonnett, 1993). Given that maximal concentrations of fructan in leaf tissue are around 4 % w/v, these enzymes are likely to function at well below maximal rates in vivo. In addition, the rate of activity will be sensitive to substrate concentration and will be further limited as hydrolysis proceeds. Secondly, the majority of grass hydrolases are assayed with, and preferentially hydrolyse, P2,I-fructan, which is not the form predominantly accumulated in the source tissue. There have, however, been recent reports of activities with a preference for P2,6 bonds (Bonnett and Simpson, 1995; Henson and Livingston, 1996; Marx et aI., 1997). Thirdly, unlike the inducible enzymes of synthesis, leaf tissue contains substantial extractable, apparently constitutive FEH activity at all times, regardless of carbohydrate status. During fructan synthesis, there is, in theory, sufficient hydrolytic activity to counteract observed rates of fructan accumulation, particularly as the small oligofructans are especially susceptible to attack (Cairns et aI., 1997; Simpson et aI., 1991). Because synthesis, accumulation and hydrolysis of fructan are thought to be vacuolar, it is difficult to see how intermediate oligofructans persist for long enough to permit polymer building. Finally, during fructan mobilisation extractable FEH increases, though the increase occurs after substantial fructan breakdown has taken place (Simpson et aI., 1991). One explanation is that an increase in the absolute amount of enzyme will permit hydrolysis even at lower substrate concentrations because of the low affinity for the substrate (Simpson and Bonnett, 1993).

Because rates are commonly not quoted in physiologically relevant units, are frequently not compared with tissue characteristics and are determined (of necessity) using non-physiological substrates, it is difficult to place this problem into the physiological context. Since native grass fructans are not readily available for enzymological studies, commercial inulins of low (e.g. Neosugar) and high Mr are commonly used. This is problematic because grasses do not accumulate large inulins. In overview, studies of FEH are practically difficult and the results often difficult to interpret. Despite this, progress has been made with the recent purification of hydro lases with appropriate specificities for native grass fructan (Bonnett and Simpson, 1995; Henson and Livingston, 1996; Marx et aI., 1997). Because of the complexities of the breakdown process indicated above, it is going to be difficult to assign roles to fructan

10. Fructan metabolism 209

hydrolases in vivo unambiguously until specific activities can be abolished either by mutation or by antisense technology.

3.3 Fructan metabolism in stems

In terms of mass, the developing stem of cereal and grass influorescences accumulates more fructan than any other organism (Pollock and Jones, 1979) Although the upper internodes and the peduncle do contain chlorophyll, the majority of the carbon stored in the stems comes from leaf photosynthesis (Austin et aI., 1977). Photosynthate from lower leaves appears more likely to accumulate as fructan in stems, contrasting with photosynthate from flag leaves, most of which passes directly into the ear (Pearman et aI., 1978). Fructan concentrations can reach up to 40% of dry weight around the time of anthesis.

In annual cereals, up to 50% of stem dry weight is lost during grain maturation (Bonnett and Incoll, 1992) and fructan contents decline markedly. The proportion of mobilised fructan which promotes grain filling is not easy to determine and may depend upon both genotype and cultivation conditions (Schnyder, 1993). There are also indications that mobilisation of stem fructan is induced developmentally, rather than in direct response to increased demand for assimilate by the ears. (Bonnett and Incoll, 1992; Kiihbauch and Thome, 1989). In perennial grasses, fructan mobilisation from the stems during grain filling is not complete, and concentrations remain high in the lower two internodes. New tillers grow in the autumn from these buds and fructan is further mobilised at this stage (Pollock and Jones, 1979). These annual patterns of metabolism contrast dramatically with the short term fluctuations found in leaves. There have been few biochemical studies on stem fructan metabolism because of the recalcitrance of the tissue (Ban cal and Triboi, 1993; Dubois et aI., 1990; Yamamoto and Mino, 1989) but the a priori assumption is that significant changes in synthetic and degradative enzyme activity would be caused by changes in the amount of enzyme protein and would be under developmental control. The assumption is also that the site of storage would be similar to that in leaves i.e. in the vacuoles of the large parenchymatous cells within the stem. The development of in situ methods for localising specific enzymes and RNA sequences and for sampling and analysing vacuolar contents (Koroleva et aI., 1997; Tomos et aI., 1992a, 1992b) will, in due course, permit a more detailed analysis of the regulation of fructan metabolism in such tissues, but until then, the details remain unknown, despite the considerable physiological relevance of the process.

210 Chapter 10

3.4 Meristematic and extending tissues

The development by Silk (1984) of the continuity equation allowed measurements of composition and elemental growth rate to be used to calculate assimilate fluxes into tissues undergoing linear growth. This has led to the demonstration of a substantial involvement of fructan metabolism during the growth of grass and cereal leaves. The main contribution to studies of this kind has been by Schnyder, Nelson and co­workers (Schnyder, 1986; Schnyder and Nelson, 1987, 1989; Schnyder et aI., 1988). They demonstrated very large fluxes of material to fructan within the extension zone. As the segment of tissue aged, and moved further up the developing leaf, there was an equivalent flux out of fructan and into structural materials. Our own studies have shown that these fluxes are sensitive to changes in growth rate (associated with chilling) and assimilate abundance (caused by increased photoperiod or elevated CO2), but that fructans remain the major temporary sink within the extension zone. We have also demonstrated that elevated CO2 strongly stimulates hexose accumulation and that increased carbohydrate contents are associated with increased rates of respiration, regardless of growth rate (Table 3). Because of the extremely stable gradient of tissue development along the leaf, this experimental system has considerable potential for the study of metabolic regulation. Studies have been constrained by the small mass of tissue involved. However, the development of sensitive in situ methods (Marrison et aI., 1996) should lead to increased understanding of the factors regulating the very large changes in flux which occur during cell expansion.

3.5 Developing grains

Developing cereal grains also accumulate fructan. Final concentrations only reach 1 - 2% of the dry weight (MacLeod and McCorquodale, 1958) but the proportion in young developing grain is much higher (Escalada and Moss, 1976). Metabolic and enzymological measurements (Ho and Gifford, 1984; Housley and Daughtry, 1987) suggest that active fructan accumulation occurs very early in grain development, but that the fructan pool becomes progressively less accessible. During the main phase of starch biosynthesis, fructan forms a static pool making up a progressively declining proportion of total grain carbohydrate. It is not known how this process is regulated or what its physiological significance is, although it has been suggested that synthesis of fructan from sucrose would facilitate continued passive unloading from the phloem (Hendrix, 1983).

10. Fructan metabolism 211

Table 3

Respiration rate in extension zones of cereal leaves as related to growth treatment and soluble carbohydrate content.Jl A. Harrison and C. J. Pollock, unpublished observations.]

Growth treatment Respiration rate Carbohydrate content nmol O2 mg-! h-! mg g-! fresh wt

20°C:350ppm CO2 18 10.2

20°C:500ppm CO2 125 18.6

5°C:35Oppm CO2 163 20.3

212 Chapter 10

4. THE REGUlATION OF FRUCfANMETABOLISM

4.1 Fructan synthesis in leaves is inducible

If grass or cereal leaves are grown at low irradiance under a short photoperiod, they will not accumulate fructans. Excision and continuous illumination leads, as indicated above, to an increase in the concentration of sucrose and to the progressive appearance of fructans of increasing size. Treatment of leaves at the time of excision with inhibitors of gene expression blocks the conversion of sucrose into fructan without altering the total amount of soluble carbohydrate accumulated in the tissue (Cairns and Pollock, 1988b; Wagner and Wiemken, 1987; Table 4). Application of inhibitors at different times after excision indicated that the ability to convert sucrose into fructan was acquired fully within six hours of excision and illumination. After this, applications of cycloheximide or cordycepin had no effect (Winters et aI., 1994). These observations led to two significant conclusions. The first was that regulation of fructan metabolism occurs at the level of coarse control, i.e. changes in the amount of the enzymes which synthesise fructan or, less likely, the amount of a strong activator of existing enzyme. The second was that sucrose concentrations per se do not appear to affect carbon fixation in the short term. The rate of carbohydrate accumulation is constant over the first 54 hours of leaf excision (Housley and Pollock, 1985) and is insensitive to inhibitors which block the conversion of sucrose to fructan (Cairns and Pollock, 1988b). By this stage, soluble sugars can make up 40% of the dry weight of the leafl Feeding exogenous sugar in the dark to excised leaves also leads to a similar induction of fructan biosynthesis (Wagner and Wiemken, 1987; Table 5), leading to the conclusion that it is the rise in sucrose or in some related metabolite, which triggers the induction of fructan biosynthesis. (Wiemken et aI., 1995).

4.2 The regulation of fructan metabolism at the level of gene expression

As indicated above, the convenience of the excised leaf system has meant that almost all detailed studies on the biochemistry, enzymology and molecular biology of fructan metabolism in the Gramineae has been carried out using leaves. Studies on other tissues in grasses and cereals and on storage organs in the Liliales and the Asterales have not, however, suggested that any radically different mechanisms are operating

10. Fructan metabolism 213

Table 4 Quantitative Analysis of Water-Soluble Carbohydrate Fractions from Excised Leaves Illuminated in the Presence of Inhibitors of Gene Expression'

Inhibitor Concentration Total water-soluble carbohydrate Proportion with (f1M) accumulated in 24 h (mg g-t fresh DP>2 (%)

Water control L-MDMP D-MDMP Cycloheximide Cordycepin a-Amanitin

10 10 100

1000 1000

mass) 43.3 44.5 40.8 40.5 44.9 48.6

58.4 53.0 1.0 2.5 2.8 4.2

'Excised leaves of Lolium temulentum were stood in aqueous solutions of metabolic inhibitors for the initial 3 h of a 24-h illumination period. At the end of this period tissue was extracted and water-soluble carbohydrate analysed by high-performance liquid chromatography. Source: Cairns and Pollock 1988b.

214 Chapter 10

Table 5

The effects of exogenous sugars on fructan biosynthesis and fructosyl transferase activity in detached barley leaves kept in the dark.

Sugar Fructosyl transferase activity Fructan content nkatcm-3 mg g-l fresh mass

None 0.08 0.01 Sucrose 0.64 1.48 Maltose 0.99 2.07 Maltotrose 0.50 0.38 Trehalose 0.48 0.01 Fructose 0.47 1.08 Glucose 0.22 0.31 Cellobiose 0.33 0.43 Lactose 0.31 0.13

Sugars were supplied for 16 hr by standing leaf blades in 0.5M solutions. Data from Wagner, Wiemken and MatiIe 1986 .

10. Fructan metabolism 215

elsewhere, so the current assumption is that the regulatory factors operating in leaves are probably of general significance.

The regulation of fructan metabolism is intimately connected with the various roles of sucrose within the overall syndrome. Sucrose can act as both a fructosyl donor and a fructosyl acceptor. There is also evidence that sucrose prevents the inactivation of enzymes that synthesise fructans (Cairns and Ashton, 1994; Obenland et aI., 1991), and protects small fructan intermediates from the action of hydro lases during polymer building (Cairns et aI., 1997). In many systems sucrose is known to modulate carbon metabolism via fine control of existing enzyme activities and via its influence on the patterns of gene expression. It seems probable that all of the factors integrate to regulate the flow of carbon into fructans within leaves.

Newly synthesised proteins can be monitored very effectively in excised leaves by administering esS] methionine to the cut ends. Changes in mRNA can also be monitored by cell-free translation. In both cases, excised leaves fed sucrose in the dark showed a relatively small number of novel polypeptides which were synthesised up to about 8 hours after excision, suggesting that only a few genes were involved. Illuminated leaves showed larger changes in both protein synthesis and cell-free translation, suggesting that photosynthetic processes were affected even though this did not result in declining assimilation rates (Winters et aI., 1994). Differential screening of cDNA libraries from induced leaves using probes derived from induced and uninduced leaves revealed a number of clones exhibiting increased expression. One of these was analysed further and showed extremely strong sensitivity to both increases and decreases in sucrose concentration. This gene was also more highly expressed in lines of Lolium perenne which accumulate higher amounts of soluble carbohydrates (L. Skot, personal communication). Unfortunately, sequence analysis indicated that the DNA sequence coded for S-adenosyl methionine synthetase rather than for an enzyme capable of using sucrose as a substrate (Winters et aI., 1994). Subsequently, however, heterologous screening of a similar library with probes derived from maize root invertases has identified a gene sequence with close homology to the invertase family and to the barley kestose - sucrose fructosyl transferase (Sprenger et aI., 1995). This gene is strongly up-regulated in the presence of sucrose (J. Gallagher and C.J. Pollock, unpublished observations). This suggests that there are genes in fructan accumulating tissues which are up-regulated in the presence of sucrose and which code for enzymes which metabolise that sucrose, potentially, into fructan. The up-regulated clone from L. temulentum produces a protein which cleaves sucrose but which will also catalyse the synthesis of higher oligosaccharides (1. Gallagher and C.J. Pollock, unpublished observations).

216 Chapter 10

The clone isolated and sequenced by Sprenger et al. (1995) also codes for an enzyme which is capable of both hydrolysis and fructosyl transfer and which is more closely related to higher plant invertases than to bacterial fructosyl transferases. These authors argue that the fructan syndrome in plants has arisen polyphyletically through a range of modifications of various members of the acid P-fructofuranosidase (invertase) family (Wiemken et aI., 1995). Presumably such modifications would have been of selective advantage when they occurred in genes which were up­regulated by high levels of assimilate.

There is less direct evidence that the enzymes of fructan hydrolysis are regulated in a similar manner. Possible increases in assimilate abundance and fructan accumulation are associated with reductions in extractable fructan exohydrolase activity (Simpson et aI., 1991) and in some cases these changes are sensitive to inhibitors of gene expression (Wagner et aI., 1986). It is not known whether specific gene-products coding for enzymes which degrade fructans are down-regulated under such conditions, but the increasing availability of clones for enzymes of fructan synthesis and the likelihood that fructan hydro lases will also show sequence homology to invertases should lead to the development of specific probes to measure changes in message abundance, and of specific antibodies to estimate enzyme protein levels.

4.3 Fine control of fructan metabolism in leaves

As indicated above, the rate of sugar accumulation III excised, illuminated grass leaves remains constant until chlorophyll degradation sets in (Housley and Pollock, 1985). The rates are similar in magnitude to the maximum rate of leaf photosynthesis measured by gas exchange (Natr, 1969). If one assumes that sucrose concentrations are similar in the cytosol and vacuole and that the maximum sucrose content stabilises at around 20 mg g-l FW, this would equate to a uniform concentration change from zero to c. 60 mM during the course of the experiment. Any selectivity which would lead to high concentrations of sucrose in the cytosol (as suggested by Winter et aI., 1993) would, of course, amplify the effect. In plants where chloroplast starch forms the major leaf carbohydrate reserve, much smaller increases in sucrose concentration are thought to feed back, via elevated concentrations of the regulating fructose 2,6 bisphosphate, to slow export of triose phosphate from the chloroplast and stimulate starch biosynthesis by the activation of ADPG pyrophosphorylase (Stitt, 1996). This feedback does not apparently occur in grass leaves when sucrose accumulation is stimulated by chilling the sink tissue (Table 6).

10. Fructan metabolism 217

Table 6

Plant Leaf sucrose F2,6 BP Starch/sucrose (/lmol mg·t chi) (nmol mg- t chi) ratio

1 0.1 0.2 Spinach 5 0.2 0.4

10 0.3 0.6 5 0.1 0.1

Lolium 33 0.3 0.1 50 0.3 0.1

Contrasting effects of sucrose accumulation on starch synthesis and F2, 6BP accumulation in leaves of spinach and Lalium temulelltum (from Pollock et al. 1995)

218 Chapter 10

We investigated the possibility that the cytoplasmic fructose 1,6-bisphosphatase for L. temulentum was less sensitive to fructose 2,6-bisphosphate, but this was not the case (Collis and Pollock, 1991), suggesting that the inhibition observed in vitro is overcome in vivo, possibly via elevated triose phosphate concentrations. This "isolation" of chloroplast metabolism from large changes in cytosolic sucrose concentrations appears to be a significant element of the fructan syndrome in the Gramineae and may be part of its selective advantage. Many grasses evolved in an environment where, through shading, a perennial growth habit and herbivory, there would be large and rapid changes in the balance between supply of and demand for fixed carbon and thus large changes in the fluxes through primary carbon metabolism.

There is a further aspect of fine control of fructan metabolism which must be distinctive, although the evidence for its occurrence is circumstantial. The stoichiometry of fructan biosynthesis (via the accepted model of direct fructosyl transfer from sucrose) liberates one mole of glucose for every mole of fructose which is transferred. However, radiotracer experiments demonstrate unequivocally that almost all the radioactivity present in sucrose after feeding 14C02 is eventually accumulated in fructan (Pollock, 1979). Recycling of glucose must, therefore, occur, presumably via hexokinase and sucrose phosphate synthase. Such a recycling pathway would have two effects. Firstly, elevated flux through hexokinase could invoke the signalling responses associated with down-regulation of gene expression (Jang and Sheen, 1994) and secondly, the flux through sucrose phosphate synthase would increase dramatically in relation to that through cytoplasmic fructose 1,6-bisphosphatase (Collis and Pollock, 1992). Direct measurements of enhanced flux or of the activation state of sucrose phosphate synthase (Huber et aI., 1995) have not yet been made under these conditions. There have been suggestions that there may be direct transfer of fructose residues from UDPfructose to the growing fructan chain (Pontis, 1995). If this suggestion is substantiated, then it would reduce the flux through the pathways discussed above.

4.4 Regulation of fructan metabolism by compartmentation

4.4.1 Intracellular compartmentation

Sachs (1864) was the first researcher to propose a role for the vacuole in the storage of carbohydrates. By using ethanol to precipitate fructans,

10. Fructan metabolism 219

he observed the resulting sphaerocrystals in the vacuoles of members of the Asterales. For many years after that, only indirect evidence was available to support the hypothesis, but the ability to prepare isolated vacuoles permitted a direct examination of distribution (Pollock and Kingston-Smith, 1997). Measurements on enzymatically - released protoplasts and vacuoles indicate unequivocally that both the putative enzymes of fructan metabolism and the substrates and products can be found in vacuoles (Table 7). Concerns still exist over the disparity between the catalytic constants of putative enzymes measured in vitro and the apparent sucrose concentrations within vacuoles, but there seems little doubt that the vacuole is the major site of storage (Pollock and Kingston-Smith, 1997). It has been proposed, however, that fructan synthesis (as opposed to storage) may occur in small vesicles which subsequently fuse with the vacuole (Kaeser, 1983). Final resolution of these disparities will depend upon the purification of all the enzymes involved and the use of specific antibodies to localise these within the cell.

4.4.2 Intercellular compartmentation

Jellings and Leech (1982) estimated that photosynthetic mesophyll cells make up only 55% of the cell population in cereal leaves, whereas they would make up in excess of 90% of the cells used to prepare isolated vacuoles in the experiments described above. Histochemical analysis of starch in barley leaves has already demonstrated discontinuities in the distribution between mesophyll cells and the photosynthetic cells of the parenchymatous bundle sheath (Williams et aI., 1989) and there is no a priori reason for discounting such discontinuities in the metabolism of sucrose. Histochemical localisation of acid invertase has demonstrated high concentrations near the vasculature (Kingston-Smith and Pollock, 1996). Using the techniques of single-cell sampling (Tomos et aI., 1992a, 1992b) increases in fructan metabolism in mesophyll and bundle sheath have been shown to be clearly linked, with vacuolar invertase activity almost undetectable, leading to the conclusion that sucrose hydrolysis in leaves of temperate Gramineae is spatially separated from fructan biosynthesis (Koroleva et aI., 1997). Once again, histochemical location of relevant proteins and mRNA species will help to resolve the magnitude of gradients in primary carbon metabolism within leaves, but any tissue­level compartmentation would have very significant consequences for models of regulation based upon measurements of whole tissue extracts.

220 Chapter 10

Table 7

Sub-cellular distribution of enzymes and carbohydrates involved in fructan metabolism in leaves of barley

Metabolite/enzyme activity

Fructose Glucose Sucrose

Fructan (DP3) Fructan (DP>3)

SST FFT

Invertase FEH

Percentage in vacuoles from illuminated excised barley leaves

107 109 65 87 86 92 nd 81 94*

Vacuolar distribution was estimated using a-methyl mannosidase as a vacuolar marker and comparing activities in isolated protoplasts and the purified vacuoles liberated from such protoplasts.

nd: protoplasts prepared from leaves of whole seedlings undergoing fructan turnover. not determined.

(From Wagner et a11983; Wagner and Wiemken 1986.)

10. Fructan metabolism

5. CONCLUDING REMARKS - THE NEXT STAGE IN THE STUDY OF REGULATION.

221

The evidence presented in this review has, we feel, justified our view of fructan metabolism in temperate Gramineae as a distinctive extension of sucrose metabolism that illuminates the factors which may limit primary carbohydrate metabolism in other systems. However, much of the evidence to support the major hypotheses is circumstantial, since purified proteins and clones for higher plant fructan genes have only recently become available. There have been successful attempts to modify the sugar metabolism of other species by introducing both bacterial and higher plant genes associated with fructan metabolism (Caimi et aI., 1996; Pilon-Smits et aI., 1995a, 1995b, 1996; Sprenger et aI., 1997; Van der Meer et aI., 1994) and these experiments have generated plants with altered patterns of carbon partitioning and altered physiological responses. The value of such transgenics is, however, in terms of what they can tell us about the regulation of primary carbon metabolism in the recipient plants. Fructans are, in this case, only an alternative sink for carbon which may compete with existing metabolic pathways. What is needed for the study of fructan metabolism is fructan -accumulating plants that have an altered capacity to make fructans. We propose that such plants would be of the highest scientific value if leaf metabolism were altered, since the primary flux rates into sucrose and fructan are highest in these tissues. Now that transformation of temperate Gramineae is routine, such an approach is feasible, as is the screening of species for mutations that alter fructan synthesis. The integration of current studies on enzymology and tissue compartmentation with the availability of such material will, we believe, provide the next major advance in the study of the regulation of fructan metabolism.

ACKNOWLEDGEMENf

The work carried out in the authors' laboratory was funded by a series of grants from the Biotechnology and Biological Sciences Research Council.

222 Chapter 10

REFERENCES

Austin, R.B., Edrich, J.A, Ford, M.A. and Blackwell, R.D. (1977). The fate of the dry matter, carbohydrates and 14C lost from the leaves and stems of wheat during grain filling. Annals of Botany, 41, 1309-1321.

Bancal, P. and Triboi, E. (1993). Temperature effect on fructan oligomer contents and fructan-related enzyme activities in stems of wheat (Triticum aestivum L.) during grain filling. New Phytologist, 123, 247-253.

Bonnett, G.D. and Incoll, L.D. (1992). The potential pre-anthesis and post-anthesis contributions of stem internodes to grain yield in crops of winter barley. Annals of Botany, 69, 219-225.

Bonnett, G.D. and Simpson, RJ. (1993). Fructan hydrolase activities from Lolium rigidum Goudin. New Phytologist, 123, 443-451.

Bonnett, G.D. and Simpson, R.I. (1995). Fructan exohydrolase activities from Lolium rigidum that hydrolyze ~-2, I-glycosidic and ~-2,6-glycosidic linkages at different rates. New Phytologist, 131, 199-209.

Caimi, P.G., McCole, L.M., Klein, T.M. and Kerr, P.S. (1996). Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amylotiquefaciens sacB gene. Plant Physiology, 110, 355-363.

Cairns, A.I. (1992). Fructan biosynthesis in excised leaves of Lolium temulentum L. V. Enzymatic de novo synthesis of large fructans from sucrose. New Phytologist, 120, 463-473.

Cairns, AJ. (1993). Evidence for the de novo synthesis of fructan by enzymes from higher plants: a reappraisal of the SST/FFT model. New Phytologist, 123, 15-24.

Cairns, AJ. (1995). Effects of enzyme concentration of oligofructan synthesis from sucrose. Phytochemistry, 40, 705-708.

Cairns, A.I. and Ashton, J.E. (1993). Species-dependent patterns of fuctan synthesis by enzymes from excised leaves of oat, wheat, barley and timothy. New Phytologist, 124, 381-388.

Cairns, A.J. and Ashton, J.E. (1994). Fructan biosynthesis in excised leaves of Latium temulentum L. VI. Optimisation and stability of enzymatic fructan synthesis. New Phytologist, 126, 3-10.

Cairns, AJ., Bonnett, G.D., Gallagher, J.A., Simpson, R.J. and Pollock, C.J. (1997). Fructan biosynthesis in excised leaves of Lolium temulentum VII. Sucrose and fructan hydrolysis by a fructan-polymerising enzyme preparation. New Phytologist, 136, 61-72.

Cairns, AJ. and Pollock, CJ. (1988a). Fructan biosynthesis in excised leaves of Lolium temulentum L. I. Chromatographic characterisation of oligofructans and their labelling patterns following 14C02 feeding. New Phytologist, 109, 399-405.

Cairns, A.I. and Pollock, C.I. (1988b). Fructan biosynthesis in excised leaves of Lolium temulentum L. II. Changes in fructosyl transferase activity following excision and application of inhibitors of gene expression. New Phytologist, 109, 407-413.

Cairns, AJ., Winters, A and Pollock, CJ. (1989). Fructan biosynthesis in excised leaves of Lalium temulentum L. III. A comparison of the in vitro properties of fructosyl transferase activities with the characteristics of in vivo fructan accumulation. New Phytologist, 112, 343-352.

Chatterton, N.I., Harrison, P.A., Thornley, W.R. and Bennett, J.H. (1993). Structures of fructan oligomers in orchard grass (Dactylis glome rata L.) Journal of Plant Physiology, 142, 552-556.

10. Fructan metabolism 223

Collis, B.E. and Pollock, C.J. (1991). The control of sucrose synthesis in leaves of Lotium temulentum L., a fructan-accumulating grass. New Phytologist, 119, 483-489.

Collis, B.E. and Pollock, C.J. (1992). Cytoplasmic carbohydrate metabolism in leaf tissues undergoing fructan synthesis and breakdown. Journal of Plant Physiology, 140, 124-126.

Dey, P.M. (1980). Biochemistry of a-D-galactosidic linkages in the plant kingdom. Advances in Carbohydrate Chemistry and Biochemistry, 37, 283-372.

Dubois, D., Winzeler, M. and Nosberger, J. (1990). Fructan accumulation and sucrose:sucrose fructosyl transferase activity in stems of spring wheat genotypes. Crop Science, 30, 315-319.

Duchateau, N., Bortlik, K., Simmen, U., Wiemken, A, Bancal, P. (1995). Sucrose-fructan 6-fructosyl transferase: a key enzyme for diverting carbon from sucrose to fructan in barley leaves. Plant Physiology, 107, 1249-1255.

Edelman, J. and Jefford, T.G. (1968). The mechanism offructosan metabolism in higher plants as exemplified in Hetianthus tuberosus. New Phytologist, 67, 517-531.

Ernst, M., Chatterton, N.J. and Harrison, P.A (1996). Purification and characterisation of a new fructan series from species of Asteraceae. New Phytologist, 132, 63-66.

Escalada, J.A and Moss, D.N. (1976). Changes in the non-structural carbohydrate fractions of developing spring wheat kernels. Crop Science, 16, 627-631.

Fuchs, A. (1993a). Production and utilisation of inulin. Part 1. Utilisation of inulin. In: Suzuki, M. and Chatterton, N.J. (Eds) Science and Technology of Fructans (pp. 320-352). CRC Press, Boca Raton.

Fuchs, A (Ed) (1993b). Inulin and inulin-containing crops: Studies in plant science, 3. Elsevier, Amsterdam.

Hendrix, J.E. (1983). Phloem function: an integrated view. What's New in Plant Physiology, 14, 45-48.

Hendry, G.A.F. and Wallace, R.K. (1993). The origin, distribution and evolutionary significance of fructans. In: Suzuki, M. and Chatterton, N.J. (Eds). Science and Technology of Fructans (pp. 119-139). CRC Press, Boca Raton.

Henson, C.A and Livingston, D.P. (1996). Purification and characterization of an oat fructan exohydrolase that preferentially hydrolyzes beta-2,6-fructans. Plant Physiology, 110, 639-644.

Ho, L.C. and Gifford, R.M. (1984). Accumulation and conversion of sugars by developing wheat grains. V. The endosperm apoplast and apoplastic transport. Journal of Experimental Botany, 35, 58-73.

Housley, T.L. and Daughtry, C.S.T. (1987). Fructan content and fructosyl transferase activity during wheat seed growth. Plant Physiology, 83, 4-7.

Housley, T.L. and Pollock, C.J. (1985). Photosynthesis and carbohydrate metabolism in detached leaves of Lotium temulentum L. New Phytologist, 99, 499-502.

Huber, S.c., Bachmann, M., McMichael, R.W. and Huber, J.C. (1995). Regulation of sucrose phosphate synthase by reversible protein phosphorylation: manipulation of activation and inactivation in vivo. In: Pontis, H., Salerno, G. L. and Echeverria, E. J. (Eds). Sucrose Metabolism, Biochemistry, Physiology and Molecular Biology. (pp. 6-13). American Society of Plant Physiologists, Rockville.

Jang, J.C. and Sheen, J. (1994). Sugar sensing in higher plants. Plant Cell, 6, 1665-1679. Kaeser, W. (1983). Ultrastructure of storage cells in Jerusalem artichoke tubers

(Hetianthus tuberosus L.). Vesicle formation during inulin synthesis. Zeitschrift fUr Pflanzenphysiologie, 111, 253-260.

224 Chapter 10

Kingston-Smith, AH. and Pollock, C.J. (1996). Tissue level localisation of acid invertase in leaves: an hypothesis for the regulation of carbon export. New Phytologist, 134, 423-432.

Koops, AJ. and Jonker, H.H. (1996). Purification and characterisation of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus (Colombia). 2. Purification of sucrose-sucrose I-fructosyl transferase and reconstitution of fructan synthesis in vitro with purified sucrose-sucrose 1-fructosy I transferase and fructan-fructan 1-fructosy I transferase. Plant Physiology, 110, 1167-1175.

Koroleva, O.A, Farrar, J.F., Tomos, A.D. and Pollock, C.J. (1997). Patterns of solute in individual mesophyll, bundle sheath and epidermal cells of barley leaves induced to accumulate carbohydrate. New Phytologist, 136, 97-104.

Kuhbauch, W. and Thome, U. (1989). Nonstructural carbohydrates of wheat stems as influenced by source-sink manipulations. Journal of Plant Physiology, 134, 243-250.

Lewis, D.H. (1993). Nomenclature and diagrammatic representation of oligomeric fructans: a paper for discussion. New Phytologist, 124, 583-594.

Luscher, M., Erdin, C., Sprenger, N. Hochstrasser, n, Boller, T. and Wiemken, A (1996). Inulin synthesis by a combination of purified fructosyl transferases from tubers of Helianthus tuberosus. FEBS Letters, 385, 39-42.

MacLeod, AM. and McCorquodale, H. (1958). Water-soluble carbohydrates of seeds of the Gramineae. New Phytologist, 57, 168-182.

Marrison, lL., Schiinmann, P.H.D., Ougham, H.J. and Leech, R.M. (1996). Subcellular utilization of gene transcripts encoding key proteins of the chlorophyll accumulation process in developing chloroplasts. Plant Physiology, 110, 1089-1096.

Marx, S.P., Nosberger, l, Frehner, M. (1997). Hydrolysis offructan in grasses: A beta-(2-6)-linkage specific fructan-beta-fructosidase from stubble of Lolium perenne. New Phytologist, 135, 279-290.

Natr, L. (1969). Influence of assimilate accumulation on rate of photosynthesis of barley leaf segments. Photosynthetica, 3, 120-126.

Obenland, D.M., Simmen, U., Boller T. and Wienken A (1991). Regulation of sucrose­sucrose fructosyl transferase in barley leaves. Plant Physiology, 97, 811-813.

Pearman, 1., Thomas, S.M. and Thome, G.N. (1978). Effects of nitrogen fertiliser on the distribution of photosynthate during grain growth of spring wheat. Annals of Botany, 42, 91-99.

Pilon-Smits, E.AH., Ebskamp, MJ.M., Jeuken, MJ. W., Van der Meer, I.M., Visser, R.G.F., Weisbeek, PJ. and Smeekens, S.C.M. (1996). Microbial fructan production in transgenic potato plants and tubers. Industrial Crops and Products, 5, 35-46.

Pilon-Smits, E.A.H., Ebskamp, MJ.M., Paul, M.l, Jeuken, MJ.W., Weisbeek, PJ. and Smeekens, S.C.M. (1995a). Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology, 107, 125-130.

Pilon-Smits, E.A.H., Ebskamp, MJ., Weisbeek, PJ. and Smeekens, S.C.M. (1995b). Frucan-accumulation in trangenic plants: effect on growth, carbohydrate partitioning and stress resistance. In: Pontis, H.G., Salerno, G.O. and Echeverria, EJ. (Eds). Sucrose metabolism, biochemistry, physiology and molecular biology (pp. 88-99). American Society of Plant Physiologists, Rockville.

Pollock, CJ. (1979). Pathway of fructan synthesis in leaf bases of Dactylis glomerata. Phytochemistry, 18, 777-779.

Pollock, C.J. (1982). Patterns of turnover of fructans in leaves of Dactylis glomerata L. New Phytologist, 90, 645-650.

Pollock, CJ. and Cairns, AJ. (1991). Fructan metabolism in grasses and cereals. Annual Review of Plant Physiology and Plant Molecular Biology, 42, 77-101.

10. Fructan metabolism 225

Pollock, C.I., Cairns, A1., Sims, I.M. and Housley, T.L. (1996). Fructans as reserve carbohydrates in crop plants. In: Zamski, E. and Shaffer, A A (Eds). Photoassimilate distribution in plants and crops: source-sink relationships. (pp. 97-113). Marcel Dekker Inc, New York.

Pollock, C.I., Hall, M.A and Roberts, D.P. (1979). Structural analysis of fructose polymers by gas-liquid chromatography and gel filtration. Journal of Chromatography, 171, 411-415.

Pollock, C.J. and Jones, T. (1979). Seasonal patterns offructan metabolism in forage grasses. New Phytologist, 83, 8-15.

Pollock, C.J. and Kingston-Smith, AH. (1997). The vacuole and carbohydrate metabolism. In: Leigh, R.A and Sanders, D. (Eds). Advances in Botanical Research, 25, 195-215, Academic Press, London.

Pontis, H. (1995). A discussion on the present model of fructan biosynthesis. In: Pontis, H., Salerno, G.L. and Echeverria, E.I. (Eds). Sucrose Metabolism, Biochemistry, Physiology and Molecular Biology. (pp., 190-197). American Society of Plant Physiologists, Rockville, USA.

Sachs, 1. (1864). Uber die Spharokrystalle des Inulins und den mikroskopische Nachweisung in den Zellen. Botanische Zeitung 22, 77-81; 85-89.

Schnyder, H. (1986). Carbohydrate metabolism in the growth zone of tall fescue leaf blades. In: Randall, D. D., Miles, C. D., Nelson, C. 1., Blevins, D. G. and Miernyk, J. A (Eds). Current topics in plant biochemistry and physiology, (pp. 47-58). University of Missouri, Columbia.

Schnyder, H. (1993). The role of carbohydrate storage and redistribution in the source­sink relations of wheat and barley during grain filling - a review. New Phytologist, 123, 233-245.

Schnyder, H. and Nelson, C.J. (1987). Growth rates and carbohydrate fluxes within the elongation zone of tall fescue leaf blades at high and low irradiance. Plant Physiology, 85, 548-553.

Schnyder, H. and Nelson, C.J. (1989). Growth rates and assimilate partitioning in the elongation zone of tall fescue leaf blades at high and low irradiance. Plant Physiology, 90, 1201-1206.

Schnyder, H., Nelson, C.l. and Spollen, W.O. (1988). Diurnal growth of tall fescue leaf blades. II. Dry matter partitioning and carbohydrate metabolism in the elongation zone and adjacent expanded tissue. Plant Physiology, 86, 1077-1083.

Silk, W.K. (1984). Quantitative descriptions of development. Annual Review of Plant Physiology, 35, 479-518.

Simmen, U., Obenland, D., Boller, T. and Wiemken, A (1993). Fructan synthesis in excised barley leaves. Identification of two sucrose-sucrose fructosyl transferases induced by light and their separation from constitutive invertases. Plant Physiology, 101, 459-468.

Simpson, R.I. and Bonnett, G.D. (1993). Fructan exohydrolase from grasses. New Phytologist, 123, 453-469.

Simpson, R.I., Walker, R.P. and Pollock, C.I. (1991). Fructan exohydrolase in leaves of Lotium temu/entum L. New Phytologist, 119, 499-507.

Sims, I.M., Horgan, R. and Pollock, C.I. (1993). The kinetic analysis offructan biosynthesis in excised leaves of Lolium temu/entum L. New Phytologist, 123, 25-29.

Slaughter, L.H. and Livingston, D.P. (1994). Separation of fructan isomers by high -performance anion-exchange chromatography. Carbohydrate Research, 253, 287-29l.

Sprenger, N., Bortlik, K., Brandt, A, Boller, T. and Wiemken, A (1995). Purification, cloning and functional expression of sucrose-fructan 6-transferase, a key enzyme of

226 Chapter 10

fructan synthesis in barley. Proceedings of the National Academy of Sciences USA, 92, 11652-11656.

Sprenger, N., Schellenbaum, L., van Dun, K., Boller, T. and Wiemken, A (1997). Fructan synthesis in transgenic tobacco and chicory plants expressing barley sucrose:fructan 6-fructosyl transferase. FEBS Letters, 400, 355-358.

Stitt, M. (1996). Metabolic regulation of photosynthesis. In: Baker, N. R. (Ed). Photosynthesis and the environment (pp. 151-190). Kluwer Academic Publishers, Dordrecht.

St John, lA, Bonnett, G.D., Simpson, R.I. and Tanner, G.I. (1997a). A fructan:fructan fructosyl transferase activity from Lolium rigidum. New Phytologist, 135, 235-247.

St John, J.A., Sims, I.M., Bonnett, G.D. and Simpson R.I. (1997b). The identification of products formed by a fructan:fructan fructosyl transferase activity from Lotium rigidum. New Phytologist, 135, 249-257.

Tomos, AD., Leigh, R.A., Hinde, P., Richardson, P. and Williams, J.H.H. (1992a). Measuring water and soluble relations in single cells in situ. Current Topics in Plant Biochemistry and Physiology, 11, 168-177.

Tomos, AD., Leigh, R.A., Palta, J.A. and Williams, J.H.H. (1992b). Sucrose and cell water relations. In: Pollock, C.l, Farrar, J.F. and Gordon, A.I. (Eds). Carbon partitioning within and between organisms. (pp. 71-89). Bios, Oxford.

Van den Ende, W. and Van Laere, A (1996). De novo synthesis of fructans from sucrose in vitro by a combination of 2 purified enzymes (sucrose-sucrose 1-fructosy I transferase and fructan-fructan I-frutosyl transferase from chicory roots (Cichorium intybus) L. Planta, 200, 335-342.

Van der Meer, I.M., Ebskamp, M.I.M., Visser, R.G.F., Weisbeek, P.I. and Smeekens, S.C.M. (1994). Plant Cell, 6, 561-570.

Wagner, W., Keller, F. and Wiemken, A (1983). Fructan metabolism in cereals: induction in leaves and compartrnentation in protoplasts and vacuoles. Zeitschrift fUr Pflanzenphysiologie, 112, 359-372.

Wagner, W. and Wiemken, A (1987). Enzymology of fructan synthesis in grasses. Properties of sucrose-sucrose fructosyl transferase in barley leaves (Hordeum vulgare L. cv. Gerbel). Journal of Plant Physiology, 85, 706-710.

Wagner, W., Wiemken, A and Matile, P.H. (1986). Regulation offructan metabolism in leaves of barley (Hordeum vulgare L. cv. Gerbel). Journal of Plant Physiology, 81, 444-447.

Wiemken, A, Sprenger, N. and Boller, T. (1995). Fructan - an extension of sucrose by sucrose. In: Pontis, H G., Salerno, G.L. and Echeverria, E. 1 (Eds). Sucrose metabolism, biochemistry, physiology and molecular biology. (pp. 179-189.). American Society of Plant Physiologists, Rockville.

Williams, M.L., Farrar, J.F. and Pollock, C.I. (1989). Cell specialisation within the parenchymatous bundle sheath of barley. Plant, Cell and Environment, 12, 909-918.

Winter, H., Robinson, D.G. and Heldt, H.W. (1993). Subcellular volumes and metabolite concentrations in barley leaves. Planta, 191, 180-190.

Winters, AL., Williams, lH.H., Thomas, D.S. and Pollock, C.I. (1994). Changes in gene expression in response to sucrose accumulation in leaf tissue of Lolium temulentum L. New Phytologist, 128, 591-600.

Yamamoto, S. and Mino, Y. (1989). Mechanism of phleinase induction in the stem base of orchard grass after defoliation. Journal of Plant Physiology, 134, 258-260.

Chapter 11

Expression of frnctosyltransferase genes in transgenic plants

Irma VUn, Anja van Dijken, Stefan Turk, Michel Ebskamp, Kees van Dun*, Peter Weisbeck and Sjef Smeekens Department of Molecular Cell Biology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands * D. J. VanderHave B. v., PO Box 1, 4410 AA Rilland, The Netherlands

Key words: fructan; fructosyl transferases; potato; tobacco; transgenic plants.

Abstract: Fructans serve as a carbohydrate reserve in many plant species and are also synthesised by several microorganisms. Over the past decade interest in the use of fructans for food and non-food applications has increased exponentially. Our interest is to modify crops for the production of tailor­made fructans. Therefore we introduced genes encoding bacterial fructosyltransferases into several non-fructan storing plants, e.g. tobacco and potato. Different cellular targeting sequences were used for the expression of the bacterial levansucrases in transgenic tobacco and potato plants resulting in varying levels of fructan and often in changes in the phenotype.

Plant fructan biosynthetic genes have also been cloned and this greatly expands the opportunities for the production of tailor-made fructans in transgenic plants. Introduction of the onion gene encoding the enzyme fructan:fructan 6-glucosyl fructosyltransferase, a key enzyme in the formation of the inulin neoseries, into chicory enables this crop to synthesise the inulin neoseries in addition to linear inulin.

1. INTRODUCTION

Fructan (polyfructosylsucrose) is synthesised by several microorganisms and in about 15% of the flowering plant species (Hendry

227 N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 227-237. © 1999 Kluwer Academic Publishers.

228 Chapter 11

and Wallace, 1993). Among these plant species are important crops such as wheat and barley (Poales), chicory and Jerusalem artichoke (Asterales) and bulb-forming plants such as tulip and onion (Liliales). Besides being a carbohydrate reserve, fructan is considered to be important in drought and cold resistance (Hendry, 1993; Pilon-Smits et aI., 1995). The substrate for fructan synthesis is sucrose. Although fructan is synthesised by both microorganisms and plants, the structure of the produced fructan is very different. In plants five major classes of fructan can be distinguished: inulin consisting of linear 2, I-linked ~-D-fructosy I units (G-l,2-F-l,2-Fn), e.g. chicory (Cichorium intybus L.) (Bonnett et aI., 1994); linear levans composed of ~2,6-linked fructosyl units (G-l,2-F-6,2-Fn), e.g. timothy (Phleum pratense L.) (Suzuki and Pollock, 1986); mixed-linkage type levans composed of fructan containing both ~2,1- and ~2,6-linkages, e.g. wheat (Triticum aestivum L.) (Carpita et aI., 1989); the inulin neoseries, having a fructosyl residue on both the carbon 6 and carbon 1 of glucose producing a polymer with ~2,I-linked fructosyl residues on either end of the sucrose molecule (Fm-2,I-F-2,6-G-l,2-F-l,2-Fn), e.g. onion (Allium cepa L.) (Shiomi, 1989); and the levan neoseries having polymers with ~2,1- and ~2,6-linked fructosyl residues on either end of the sucrose molecule, e.g. oat (Avena sativa L.) (Livingstone III et aI., 1993). These fructan molecules are synthesised in the vacuole by the concerted action of several enzymes. The degree of polymerisation (DP) rarely reaches 100 fructosyl residues. Bacterial fructan is usually composed of /32,6-linked fructosyl units, which are only occasionally branched by a ~2,1-linked fructose residue, and DP may be over 100,000 fructose units. Furthermore, only one enzyme is sufficient for fructan synthesis.

Over the past decade the commercial interest in the use of fructans for food and non-food applications has increased dramatically (for review see: Fuchs, 1993; Suzuki and Chatterton, 1993). Major crops for the production of plant fructan are chicory and Jerusalem artichoke (Fontana et at, 1993; Fuchs, 1991). However, the isolated fructan has a varying DP due to the presence of fructan degrading enzymes in these plants, which break down the fructan in the roots or tubers upon harvesting and storage. This breakdown can be overcome by the production of fructan in plants which normally do not accumulate fructans and do not have the degrading enzymes. This can be achieved by the introduction of either a bacterial gene encoding a fructosyltransferase or by the introduction of a set of plant genes involved in fructan synthesis. Therefore we are isolating the plant genes involved in fructan synthesis in onion (Allium cepa L.). In onion fructan of the inulin neoseries is produced and most likely three enzymes are involved in the synthesis of this kind of fructan (Shiomi, 1981; Shiomi, 1989). In this paper we will give a brief overview

11. Heterologous expression of fructosyltransferases 229

of the results obtained so far by the introduction of the bacterial levansucrase (SacB) from Bacillus subtilis into tobacco and potato plants and further we will summarise the isolation, characterisation and expression of the onion fructan:fructan 6G-fructosyltransferase (6G­FFT) gene in transgenic tobacco and chicory plants.

2. ISOLATION OF ONION 6G-FFf

In plants producing linear inulin, a fructan consisting of linear 2,1-linked ~-D-fructosyl units, sucrose:sucrose I-fructosyltransferase (I-SST) initiates fructan synthesis by catalysing the formation of l-kestose (G-l,2-F-l,2-F) from two molecules of sucrose (G-l,2-F) and fructan:fructan 1-fructosyltransferase (1-FFT) elongates the fructose chain (G-l,2-F-l,2-Fn) (Koops and Jonker, 1996; LUscher et aI., 1996). In plants of the Liliales, like onion and asparagus, fructan of the inulin neoseries is produced. For the production of this kind of fructan an additional enzyme is required (Shiomi, 1981; Shiomi, 1989). This enzyme, called fructan:fructan 6G-fructosyltransferase (6G-FFT), catalyses the transfer of a fructosyl residue to the carbon 6 of the glucose moiety of sucrose resulting in the formation of the trisaccharide neokestose (F-2,6-G-l,2-F).

Recently the onion gene encoding 6G-FFT has been cloned (Vijn et aI., 1997). The amino acid sequence of this gene has a high homology (48%) to the sucrose:fructan 6-fructosyltransferase (6-SFT) polypeptide from barley (Sprenger et aI., 1995), which was used as a probe in screening the onion cDNA library (Figure I). The barley 6-SFT protein contains a putative signal peptide at the N-terminus, since the mature 6-SFT protein starts at the 68th codon (Sprenger et aI., 1995) (Figure 1). The amino acid comparison shows that the N-terminal part of the proteins are less conserved suggesting that 6G-FFT might also contain a signal peptide at the N-terminus (Figure 1). Striking was the even higher homology of the deduced amino acid sequence of 6G-FFT with acid invertases (over 50% identity) (Figure 1), supporting the idea that fructosyltransferases evolved from invertase (Sprenger et aI., 1995).

3. TRANSGENIC PLANTS EXPRESSING ONION 6G-FFf

Introduction of the onion 6G-FFT gene into tobacco plants and incubation of protein extracts of these plants with l-kestose and sucrose

230

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Ac6G-FFT Tglnv Hv6-SFT

Figure 1.

Chapter 11

MDAQDIESRHPLlGA---RPRRRALRSLSILLAAALLLGLVLFYA----- 42 MGGRDLESSTPLLHHEPYSPRKTITTIVSSIVAAALLLSLITLLNTKHEA 50 MGSHG---KPPL----PYAYKPLPSDAADGKRTGCMRWSACATVLTA--- 40

**

----------------NGTGSGTAVDPVRVDNEFPWTNDMLAWQRCGFHF 76 DHHPPDVAFPMSRGVFEGVSEKSTASLIGSAARFPWTDAMLEWQRTGFHF 100 ---SAMAVVVVGATLLAGLRMEQAVDEEAAAGGFPWSNEMLQWQRSGYHF 87

* *** **.*** *.** t

RTVRNYMNDPSGPMYYKGWYHLFYQHNKDFAYW~GHAVSRDLINW 125 QPEKNWMNDPDGPMFYKGWYHIFYQYNPVSAVWGN-ITWGHAVSRNLIHW 149 QTAKNYMSDPNGLMYYRGWYHMFYQYNPVGTDWDDGMEWGHAVSRNLVQW 137

* * **.* * * ****.***.* ******* *

QHLPVAVGPDHWYDISGVWTGSIIVVSEDRVVMLFTGGTKSF-DQSINLA 174 FHLPIAFVPDQWYDANGALTGSATFLPDGRIAMLYTGITTEF-VQVQCQV 198 RTLPIAMVADQWYDILGVLSGSMTVLPNGTVIMIYTGATNASAVEVQCIA 187

**.* * *** * **

EAADPSDPLLLKWIKYDNNPILWPPPGIVRDDFRDPNPIWYNASESTYHI 224 YPEDVDDPLLLKWFKSDANPILVPPPGIGSKDFRDPTTAWYDVAEASWKL 248 TPADPNDPLLRRWTKHPANPVIWSPPGVGTKDFRDPMTAWYDESDETWRT 237

* * ***** ** VVGSKND-SLQHTGIALVYLTKDFKKFDLLPTVLHSVDKVGMWECVEVYP 273 AIGSKDE---QHNGISLIYRTYDFVSYELLPILLHAVEGTGMWECVDFYP 295 LLGSKDDHDGHHDGIAMMYKTKDFLNYELIPGILHRVVRTGEWECIDFYP 287

*** * ** * * ** ** * * ***

VATTGPL-LHKAIDNFDVDRVLDRSTVKHVLKASMNDEWHDYYAIGTFDP 322 VLTNSTVGLDTSVPP--------GPGVRHVLKASLDDDKHDYYAIGTYDV 337 VGRRSSD---------------NSSEMLHVLKASMDDERHDY-SLGTYDS 321

.. *. **. *

IGNKWTPDDETVDVGIGLRYDWGKFYASRTFFDPLKQRRIIWGYIGEVDS 372 VSGTWIPDDVEADVGIGWRYDYGKFYASKTFFDWAKGRRVLFGFTGETDS 387 AANTWTPIDPELDLGIGLRYDWGKFYASTSFYDPAKNRRVLMGYVGEVDS 371

*.*** ***.****** * ** **.**

QKADIAKGWASLQGIPRSVLYDVKTGTNVLTWPIEEMEGLRMARKDFSGI 422 EQNNRLKGWASVLPIPRTILFDQKTGSNLLLWPVEEVERLRFNRQDFENI 436 KRADVVKGWASIQSVPRTVALDEKTRTNLLLWPVEEIETLRLNATELTDV 421

***** ** * ** * * **.**.* **

KIKKGSTVELSDFGDAFQIDlEAEFTISKEALEATlEADVGYNCSSSGGA 472 DIGIGAVVPL-DIGRAIQLDIVAEFEIDGATLEASVEADLGYNCSTSGGT 486 TINTGSVIHIPLRQGTHARHAEASFHLDASAVAALNEADVGYNCSSSGGA 471

*.* ***.*****.***

AIRGTLGPFGLLVLANQDL-TENTATYFYVSKGIDGSLITHFCQDETRSS 521 FGRGVLGPFGFLVLSDEDL-SEQTAIYFYVGRKVDGALQTFFCQDELRSS 535 VNRGALGPFGLLVLAAGDRRGEQTAVYFYVSRGLDGGLHTSFCQDELRSS 521

**.*****.*** * **.**** **.* * ***** ***

KANDIVKRVVGGTVPVLDGETFAVRILVDHSVIESFAMGGRTSATSRAYP 571 KADDLVKRVFGSIVPVLHGEILTMRILLDHSlVESFAQGGRTCITSRIYP 585 RAKDVTKRVIGSTVPVLDGEALSMRVLVDHSIVQGFDMGGRTTMTSRVYP 571

*** * ****.** * * *** .... * .• ***

TEAINSAARVFLFNNATGVDVlAESVKIWQMNSTYNDF-------Y---- 610 TKAFDGAARVFVFNNATGAKVTAKSIKIWR-------------------- 615 MESYQEA-RVYLFNNATGASVTAERLVVHEMDSAHNQLSNEDDGMYLHQV 620

---HF 612 615

LESRH 625

Comparison of the deduced amino acid sequence of onion 6G-FFT (Ac6G-FFT, EMBL acc. no. Y03878), with 6-SFT of barley (Hv6-SFT, EMBL acc. no. X83233) and vacuolar invertase of tulip (TgInv, EMBL acc. no. X95651). Identical amino acids are indicated by an asterisk, homologous amino acids by a dot. The seven putative glycosylation sites in 6G-FFT are underlined. The N-terminal amino acid of the barley 6SFT mature protein is printed in bold and indicated by an arrow

11. Heterologous expression offructosyltransferases 231

resulted in the formation of neokestose and higher DP sugars (Figure 2: lane 1 and 2). The sugar products with a DP higher than three do not all run at exactly the same position as the linear inulin sugars with DP4 or DP5 from jerusalem artichoke (Figure 2: lane H), but run at identical positions as sugar products of onion (Figure 2: lane 0), suggesting that these sugars are of the inulin neoseries as well. Analysis of these sugar products by HPLC and co-chromatography with an onion sugar extract confirmed these results. Furthermore this HPLC analysis showed that nystose, DP4 linear inulin, was formed.

These results showed that in these in vitro assays onion 6G-FFT is not only capable of transferring a fructosyl residue to the carbon 6 of the glucose moiety of sucrose and higher DP molecules, resulting in the formation of fructan of the inulin neoseries, but is also able to transfer a fructosyl residue to the carbon 1 of the fructose moiety of l-kestose resulting in the formation of nystose. This last reaction is by definition catalysed by a fructan:fructan I-fructosyltransferase (l-FFT), showing that onion 6G-FFT has I-FFT activity under the conditions used. Whether 6G-FFT will also have this activity in vivo is not known. It has been shown that the purified I-SST from Jerusalem artichoke also has some 1-FFT activity in vitro since it can produce nystose and the inulin pentamer (LUscher et aI., 1996).

Although the transgenic tobacco plants produced active protein, no fructan accumulation in the plants could be detected, which is most likely due to the absence of substrate, l-kestose, in the plants. This also shows that 6G-FFT has no I-SST activity in vivo to start fructan synthesis from sucrose. This problem can be overcome by the introduction of a gene encoding a I-SST. Currently we are isolating the onion I-SST gene.

Introduction of the onion 6G-FFT gene into chicory, a plant that normally accumulates linear inulin, resulted in the formation of inulin of the neoseries in addition to linear inulin. Neokestose was formed, as well as higher DP fructans of the inulin neoseries (Figure 3). These results show that it is possible to change the type of fructan produced in a plant by the introduction of a gene encoding a fructosyltransferase that catalyses a specific step in fructan biosynthesis, that is not present in the plant.

4. EXPRESSION OF ABACfERIAL FRUcrOSYLTRANSFERASE GENE IN PLANTS

Introduction of the bacterial fructosyltransferase (SacB) of Bacillus subtilis into tobacco and potato resulted in the accumulation of fructan in

232

F

G

s

N

1-K

Figure 2.

1 2 Wt 1-K 0 H

S

DP2

DP3

DP4

DP5

Chapter 11

Thin layer chromatography (TLC) of sugar products generated by protein extracts of transgenic tobacco plants harbouring 6G-FFT activity after incubation with 20 mM sucrose and 100 mM I-kestose. Protein extracts were incubated with the substrates at 27°e for 24 h. I, 2: Two independent transgenic tobacco plants harbouring 6G-FFT activity; Wt, wildtype control; Bl, sugar control, no protein extract added; 0, onion (Allium cepa) bulb extract; H, Jerusalem artichoke (Helianthus tuberosus) tuber extract; F, fructose; G, glucose; S, sucrose (G-I,2-F); N, neokestose (F-2,6-G-I,2-F); I-K, l-kestose (G-I,2-F-I,2-F).

11. Heterologous expression offructosyltransferases

F 0,6 G

~ Chicory

~c s

J-K t Nys

+ DP5

5 10 15 20

0.6 Chicory+Onion 6G-FFT

s J-K

Nys

~c DP5 6 7

o o 5 10 15 20

0.6 Onion

s J-K Nys

+ f~ DP5

* *!*

o o 5 10 15 20

minutes

Figure 3.

20 t 30 ,~~ ~ ~.~,A~." •• , ... t I:' ';", I • '.:':.,."",

25 30

233

A

35

B

·\""'i.i"" I ',".

\\i~;"';""""''''''~

25 30 35

c

25 30 35

HPLC analysis of sugar products extracted from (A) non-transformed chicory root, (B) transgenic chicory root harbouring the onion 6G-FFT and (C) onion bulbs. Peaks in the HPLC chromatograms: F, fructose; G, glucose; S, sucrose; l-K, l-kestose; N, neokestose; Nys, nystose; DPS-30, inulin fructan with degrees of polymerisation 5-30; *, fructan of the inulin neoseries.

234 Chapter 11

these plants. Up to 20% and 30% of dry weight could be accumulated in transgenic tobacco and potato plants, respectively (Smeekens et aI., 1996; Turk et aI., 1997; Van der Meer et aI., 1994).

The intracellular localisation of fructan accumulation in transgenic plants is important. Expression of the bacterial levansucrase into the cytosol of transgenic tobacco plants resulted in a changed visible phenotype. The plants showed necrotic leaf lesions once fructans accumulated to sufficiently high levels (Ebskamp, 1994; Smeekens et aI., 1996).

In fructan accumulating plants the fructans are synthesised in the vacuole. Transgenic tobacco plants expressing a vacuolar targeted bacterial levan sucrase show complex results. Until now two different targeting signals have been used, the vacuolar targeting sequence of the yeast carboxypeptidase Y (CPY) and the plant vacuolar targeting sequence of the sporamin protein from sweet potato (Ipomoea hatatas L.). Transgenic tobacco plants harbouring the CPY targeted levansucrase accumulated fructan up to 10% of dry weight without a visible change of their phenotype compared to wild type tobacco plants. However, we have not been able to show that fructan accumulation takes place in the vacuole of these plants and there are strong indications that the CPY­targeted levan sucrase does not end up in the vacuole, but is retained somewhere in the endomembrane system (ER-Golgi) (S. Turk and M. Ebskamp, pers. comm.).

The vacuolar targeting sequence of the sporamin protein is able to translocate the ~-glucuronidase protein (uidA from Escherchia coli) to the vacuole in transgenic tobacco plants and was therefore also used for targeting of the bacterial levansucrase to the vacuole. The transgenic tobacco plants harbouring the sporamin-Ievansucrase hybrid protein accumulated fructan up to 20% of their dry weight, which caused a deleterious phenotype (Turk et aI., 1997). The transgenic plants showed stunted growth, bleaching of the leaves, reduced root growth and an accumulation of glucose, fructose, sucrose and starch. Detailed analysis of the localisation of the sporamin-Ievansucrase hybrid protein showed that the protein is not translocated to the vacuole in the transgenic tobacco plants. The exact localisation of the hybrid sporamin-Ievansucrase protein is not known but it has been shown that the protein is glycosylated and that it is therefore probably retained somewhere in the endomembrane system (Turk et aI., 1997). It is not known why the sporamin-Ievansucrase harbouring transgenic plants show a changed phenotype and the CPY-Ievansucrase transgenic plants do not, while in both cases the hybrid protein seems to end up somewhere in the endomembrane system.

11. Heterologous expression of fructosyltransferases 235

The vacuolar targeting sequence of the sporamin protein from sweet potato has also been used to target the levan sucrase (sacB) gene of Bacillus amyloliquefaciens to the vacuole in transgenic maize. Although it has not been shown that fructan accumulation takes place in the vacuole in the transgenic maize plants, fructan is accumulated for up to 10% of dry weight of the mature seeds and the seeds did not show a changed phenotype (Caimi et aI., 1996). It will be interesting to know what the exact cellular localisation is of the different hybrid proteins and in which cell compartment fructan is accumulated. This might give more insight into why the sporamin-Ievansucrase transgenic tobacco plants do give a changed phenotype whereas the transgenic maize plants and the CPY-Ievansucrase harbouring tobacco plants do not.

The introduction of a CPY-Ievansucrase into transgenic potato plants resulted in an accumulation up to 30% of dry weight in the leaves and up to 13% of dry weight in the tubers (Smeekens et aI., 1996). The green tissue of the transgenic plants showed stunted growth and tuber yield varied from 20 to 50% compared to wild type, and the tubers of the transgenic line with the highest fructan accumulation showed a brown phenotype. Upon storage at room temperature some of the tubers, which looked normal shortly after harvesting developed a brown phenotype, indicating that fructan accumulation might continue after harvesting at the expense of other carbohydrates. The use of a tuber-specific promoter might overcome stunted growth and accumulation of fructan in the green tissues, resulting in an increase of fructan accumulation in the tubers.

5. CONCLUSIONS

The isolation of the onion 6G-FFT gene and the introduction of the gene into tobacco plants resulted in the expression of an active protein, but the transgenic plants do not accumulate fructan. The isolation of the onion I-SST gene will enable the accumulation of plant fructans in transgenic plants and the introduction of special combinations of genes encoding fructosyltransferases catalysing only a specific step in fructan synthesis will result in plants making only a specific type of fructan. Furthermore, the type of fructan made by a plant can be changed by the introduction of a specific fructosyltransferase, as was shown by the introduction of the onion 6G-FFT into chicory. This will open new ways for exploiting fructans in the food or non-food industry.

Introduction of the bacterial levansucrase (SacB) into tobacco and potato plants resulted in an accumulation of fructan in these plants. Depending on the intracellular targeting signal, the bacterial source and

236 Chapter 11

the plant species used, the level of accumulation varied extensively. The use of other targeting signals or other sources of the levansucrase might diminish the phenotypes developed upon fructan storage and fructan accumulation is expected to increase. In some cases it will be preferable to use tissue specific promoters, for example in potato, to prevent accumulation of fructan in non-storage organs. Currently we are testing these hypotheses.

ACKNOWLEDGEMENTS

This work was supported by a grant from the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Technology Foundation (STW) and the European Commission (BI02-CT93-0400).

REFERENCES

Bonnett, G.D., Sims, I.M., John, J.A.S. and Simpson, R.I. (1994). Purification and characterisation of fructans with ~-2, 1- and ~-2,6-glycosidic linkages suitable for enzyme studies. New Phytologist, 127, 261-269.

Caimi, P.G., McCole, L.M., Klein, T.M. and Kerr, P.S. (1996). Fructan accumulation and sucrose metabolism in transgenic maize endosperm expressing a Bacillus amyloliquefaciens SacB gene. Plant Physiology, 110, 355-363.

Carpita, N.C., Kanabus, J. and Housley, T.L. (1989). Linkage structure of fructans and fructan oligomers from Triticum aestivum and Festuca arundinaceae leaves. Journal of Plant Physiology, 134, 162-168.

Ebskamp, M.I .M. (1994) Fructan accumulation in transgenic plants. PhD Thesis, Utrecht University.

Fontana, A., Herman, B. and Guiraud, J. (1993). Production of high-fructose containing syrups from Jerusalem artichoke extracts with fructose enrichment through fermentation. In: Fuchs, A. (Ed). Inulin and inulin containing crops: Studies in plant science, 3 (pp 251-258). Elsevier, Amsterdam.

Fuchs, A. (1991). Current and potential food and non-food applications of fructans. Biochemical Society Transactions, 19, 555-560.

Fuchs, A. (1993). Inulin and inulin-containing crops: Studies in plant science, 3. Elsevier, Amsterdam.

Hendry, G.A.F. (1993). Evolutionary origins and natural functions of fructans - a climatological, biogeographic and mechanistic appraisal. New Phytologist, 123, 3-14.

Hendry, G.A.F. and Wallace, R.K. (1993). The origin, distribution, and evolutionary significance of fructans. In: Suzuki, M. and Chatterton, N.I. (Eds). Science and Technology of Fructans (pp. 119-139). CRC Press, Boca Raton, FL.

Koops, A.I. and Jonker, H.H. (1996) Purification and characterization of the enzymes of fructan biosynthesis in tubers of Helianthus tuberosus Colombia. II. Purification of sucrose:sucrose l-fructosyItransferase and reconstitution of fructan synthesis in vitro

11. Heterologous expression of fructosyltransferases

with purified sucrose:sucrose I-fructosyltransferase and fructan:fructan 1-fructosyltransferase. Plant Physiology, 110, 1167-1175.

237

Livingstone III, D.P., Chatterton, N.J. and Harrison, P.A (1993). Structure and quantity of fructan oligomers in oat (Avena spp.). New Phytologist, 123, 725-734.

LUscher, M., Erdin, C., Sprenger, N., Hochstrasser, U., Boller, T. and Wiemken, A (1996). Inulin synthesis by a combination of purified fructosyltransferases from tubers of Helianthus tuberosus. FEBS Letters, 385, 39-42.

Pilon-Smits, E.A.H., Ebskamp, MJ.M., Paul, MJ., Jeuken, M.J.W., Weisbeek, PJ. and Smeekens, S.C.M. (1995). Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiology, 107, 125-130.

Shiomi, N. (1981). Purification and characterisation of 6G-fructosyltransferase from the roots of asparagus (Asparagus officinalis L.). Carbohydrate Research, 96, 281-292.

Shiomi, N. (1989). Properties of fructosyltransferases involved in the synthesis of fructan in Liliaceous plants. Journal of Plant Physiology, 134, 151-155.

Smeekens, S., Pilon-Smits, E., Ebskamp, M. Turk, S. Visser, R. and Weisbeek, P. (1996). Transgenic fructan-accumulating tobacco and potato plants. In: Fuchs, A (ed). Proceedings of the Fifth Seminar on Inulin (pp 53-58). Carbohydrate Research Foundation, The Hague.

Sprenger, N., BortIik, K., Brandt, A, Boller, T. and Wiemken, A (1995). Purification, cloning, and functional expression of sucrose:fructan 6-fructosyltransferase, a key enzyme of fructan synthesis in barley. Proceedings of the National Academy of Sciences USA, 92, 11652-11656.

Suzuki, M. and Chatterton, NJ. (1993). Science and technology of fructans. CRC Press, Boca Raton.

Suzuki, M. and Pollock, C.J. (1986) Extraction and characterisation of the enzymes of fructan biosynthesis in timothy (Phleum pratense L.). Canadian Journal of Botany, 64, 1884-1887.

Turk, S., de Roos, K., Scotti, P.A, van Dun, K., Weisbeek, P. and Smeekens, S.C.M. (1997). The vacuolar sorting domain of sporamin transports GUS, but not levansucrase, to the plant vacuole. New Phytologist, 136, 29-38.

Van der Meer, 1.M., Ebskamp, M.J.M., Visser, R.G.F., Weisbeek, PJ. and Smeekens, S.C.M. (1994). Fructan as a new carbohydrate sink in transgenic potato plants. Plant Cell, 6, 561-570.

Vijn, 1., van Dijken, A, Sprenger, N., van Dun, K., Weisbeek, P., Wiemken, A and Smeekens, S. (1997). Fructan of the inulin neoseries is synthesized in transgenic chicory plants (Cichorium intybus L.) harbouring onion (Allium cepa L.) fructan:fructan 6Gfructosyltransferase. Plant Journal, II, 387-398.

Chapter 12

The application of transgenic technology to the study of sink metabolism in potato

Richard N. Trethewey and Lothar Willmitzer Max-Planck-Institut for Molekulare Pjlanzenphysiologie, Karl Liebknecht Str. 25, 14476, Golm, Germany

Key words: phloem; plant metabolism; potato tuber; Solanum tuberosum; starch; sucrose; transgenic plants.

Abstract: The ability to generate transgenic plants has been extensively applied in the study of potato tuber metabolism. All the major pathways, including many of the steps of carbohydrate metabolism have now been investigated by metabolic engineering strategies. The wide range of results that has been achieved, ranging from the insignificant to the dramatic, is reviewed in this article. It is concluded that a better knowledge of the interaction between metabolism and gene expression is required before transgenic approaches can be developed that lead to significant yield increases.

1. ~ODU~ON

The yield from crop plants has been subject to constant improvement through conventional breeding and refinements in agricultural methods. In the case of potato, which will be the main focus of this review article, the harvest index (the ratio of the dry weight of harvestable organs to the total plant dry weight) has improved from 0.09 in wild species up to 0.81 in modem cultivars (Inoue and Tanaka, 1978). Such improvements have been dramatic and revolutionary, although time consuming and slow. The development of technology for the genetic manipulation of plants offers the prospect of being able to improve the harvest index further by the application of strategies specifically targeted to increase

239

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 239-256. © 1999 Kluwer Academic Publishers.

240 Chapter 12

yield. In order for this to occur it is necessary to have a thorough understanding of how photoassimilate production is regulated in source tissues, the nature of the processes that determine the distribution 0 f photoassimilate throughout the plant, and the factors controlling metabolism and growth in sink tissues. There have been several good recent reviews that have covered the use of transgenic plants in the study of metabolism in source and phloem tissues that are recommended to the reader (e.g. Frommer and Sonnewald, 1995; Stitt, 1993; Stitt and Sonnewald, 1995). This article will summarise the strategies used for genetic manipulation of plant metabolism and then review their application in the study of sink metabolism in potato plants.

2. PRODUCTION OF TRANSGENIC PLANTS

The elucidation of the mechanisms whereby Agrobacterium tumefaciens induces crown gall disease in plants was the necessary prerequisite opening up the field of transgenic engineering (for a review see Hooykaas and Schilperoort, 1992). The subsequent development of binary transformation vectors with the phytohormone biosynthetic genes replaced by dominant selectable markers in the T-DNA region (Zambryski et aI., 1983), amenable multiple cloning sites, and the identification of promoter and terminator sequences has turned the genetic engineering of plants into a routine process (e.g. potato, Dietze et aI., 1995). Promoter sequences have now been identified and isolated which allow, for example, constitutive (35S; Franck et aI., 1980), tuber­specific (e.g. B33 patatin; Liu et aI., 1990), photosynthetic cell specific (e.g. L700; Stockhaus et aI., 1989), phloem specific (e.g. rol C, Schmulling et aI., 1989) and guard cell specific (Muller-Rober et aI., 1994) expression, thus facilitating the study of plant physiology at the organ level. The development of further promoters offering even finer spatial or temporal resolution can be expected in the next years. In terms of the chimeric gene constructs that have been introduced into plants, three basic strategies have been followed: overexpression, antisense and co-suppression. Overexpression has been used to introduce either new reactions into plant cells or to study the effect of bypassing endogenous regulatory mechanisms by introducing unregulated enzymes. The power of this approach has been greatly enhanced by the identification of target peptides that can be fused to heterologous proteins to direct them to specific compartments of the cell. Targeting of nuclear encoded proteins to the cytosol proceeds by default; further direction to mitochondria, plastids, vacuole or the secretory pathway requires a signal peptide which

12. Transgenic potato tubers 241

is normally at the N-terminus (summarised by Stitt and Sonnewald, 1995). The antisense approach, where a cDNA, or part of a cDNA, encoding an endogenous enzyme is introduced in reverse orientation, although still mysterious in exact mechanism, has been tremendously successful in reducing endogenous enzyme activities (reviewed by Kuipers et aI., 1995; Mol et aI., 1990). Antisense also offers the advantage of being able to generate lines with a range of activities from that of the wild type down to almost complete absence of activity, a property that is of great advantage for the application of metabolic control analysis (Hill and ap Rees, 1994). The third strategy of co-suppression is also poorly understood but it has been observed that if an endogenous gene is re­introduced in the sense orientation, then expression of both the new chimeric gene construct and the endogenous gene are suppressed, leading in some examples to complete elimination of the target enzyme activity. However, the results of co-suppression experiments can be very variable and dependent on factors such as developmental age (Boerjan et aI., 1994; Flipse et aI., 1996b). Examples, where all three strategies have been followed will be discussed in the following sections.

3. THESINK

The potato tuber is perhaps the sink organ that has been the most studied through transgenic experimentation. When considering the pathway from sucrose to starch, most of the individual steps have been subject to investigation through genetic manipulation and many combinatorial transgenics are currently being studied.

3.1 Cytosolic metabolism of sucrose

Sucrose arriving in the potato tuber probably arrives through the symplasm. This point has been much debated and remains under investigation (Oparka, 1990). In potato tubers there is a relatively high density of plasmodesmatal connections to the phloem (Oparka, 1986) and experiments with a fluorescent dye (Lucifer Yellow CH) indicate that compounds of low molecular weight can move symplastically through tuber tissue (Oparka and Prior, 1988). The question of whether apoplastic unloading also contributes to the flux into tubers has been re­opened following the cloning of the first potato sucrose transporter at the beginning of this decade. This sucrose transporter was shown to be only weakly expressed in tubers (Riesmeier et aI., 1993), however tuber specific antisense led to a decrease in tuber yields (Frommer and

242 Chapter 12

Sonnewald, 1995). The mechanism by which this decrease in yield occurs remains open for investigation: it could be that there is some apoplastic unloading in potato tubers, or that changes in osmotic relations following alteration of apoplastic sugar concentrations leads to influences on symplastic unloading (Oparka et al., 1992; Oparka and Wright, 1988).

The partitioning of incoming sucrose has been relatively poorly studied and a complete quantitative analysis has not been performed. Taking data from Oparka (1985) and Mares and Marschner (1980), ap Rees and Morrell (1990) concluded that some 50 to 70% of the carbon from sucrose is converted to starch and no more than 5-10% is deposited as structural polysaccharides. The remaining carbon is divided between respiration and storage. In terms of the steady state contents, Mares and Marschner (1980) found that 0.4-1.0% of the fresh weight of potato tubers is accounted for by soluble sugars, while starch contributes between 7 and 11 %. Therefore, despite the paucity of data, it is clear that the flux to starch is the major determinant of sink strength.

The metabolism of incoming sucrose proceeds via sucrose synthase which catalyses the production of UDPglucose and fructose (Morrell and ap Rees, 1986). This is in contrast to many other sink organs where invertases are the dominant enzyme responsible for cleaving sucrose. Fu and Park (1995) have shown that there are two differentially expressed classes of genes encoding sucrose synthase in potato which they named Sus3 and Sus4. Both classes were shown to contain 13 introns, including a particularly long leader intron, and the coding regions were found to be 87% identical at the nucleotide level. Sus3 is most highly expressed in stems and roots and using a Sus3-~-glucuronidase construct with 3.9 kb of 5' flanking sequence they found that the chimeric gene was expressed in the vascular tissues of leaves, stems, roots and tubers of transgenic plants (Fu et al., 1995b). This result strongly indicates that this class is important for energy provision in phloem tissues. In contrast, Sus4 genes are most strongly expressed in potato tubers and other sink tissues such as root tips and the basal tissues of shoots and axillary buds (Fu et al., 1995a). The Sus4 genes correspond to the cDNA for the T-type isoform first cloned by Salanoubat and Belliard (1987) and these workers also proposed that this isoform plays the dominant role in the metabolism of sucrose in potato tubers. The T-type isoform was subject to antisense inhibition using the 35S promoter, and a reduction in sucrose synthase activity was found only in the tubers (Zrenner et al., 1994). In tubers the activity was reduced by up to 95% and this led to a reduction in the starch and storage protein content of mature tubers although sucrose did not change. However, there was a significant accumulation of hexoses in the tubers which was supported by the observation that the soluble acid invertase activity increased by 40-fold whilst activity of hexokinases

12. Transgenic potato tubers 243

remained largely unchanged. The mechanisms whereby the invertase is induced as if to compensate for the reduction in sucrose synthase activity remain mysterious but further illustrates the flexibility of plant metabolism. Taken together the results of this work strongly indicate that sucrose synthase plays a significant role in determining potato tuber sink strength, at least when plants are studied with reduced activities. However, because the enzyme is likely to be close to equilibrium in vivo (Geigenberger and Stitt, 1993) it is unlikely that overexpression of the sucrose synthase would lead to increased sink strength.

The UDPglucose that is produced from sucrose synthase is an important metabolite that participates in several different pathways. As well as being converted to hexose phosphates to support starch synthesis, it is also the precursor for cellulose biosynthesis and for the formation of other sugar nucleotides important for cell wall biosynthesis (e.g. UDPxylose). The conversion to glucose I-phosphate is catalysed by UDPglucose pyrophosphorylase (UGPase) a reaction that could play a central role in the determination of sink strength. The UGPase is the most active enzyme of carbohydrate metabolism in potato tubers, and the maximum catalytic activity extractable from tubers is about 600 times greater than the estimated net flux to starch (ap Rees and Morrell, 1990). The reaction is also particularly noteworthy because it requires a supply of pyrophosphate (PPi). This step has been targeted in two ways: the antisense inhibition of the UGPase and the over-expression of an E. coli pyrophosphatase to reduce the supply of PPi for the reaction. The antisense inhibition was successful in the sense that more than 95% of the activity was removed, however there was no change in the growth and development of the transgenic potato plants (Zrenner et at., 1993). There was a change in the UDPglucose to glucose I-phosphate ratio indicating that the mass action ratio was shifted following the reduction in capacity at this step. This is perhaps a good example of how targeting steps with a large excess of capacity is unlikely to lead to a significant change in flux. More successful was the approach to inhibit the UGPase reaction by removing PPi by ectopic expression of an E. coli pyrophosphatase (Sonnewald, 1992). Here the lines with the highest activity of the new transgene were found to have dramatically increased sucrose contents along with a small reduction in starch (Sonnewald, 1992). A detailed metabolic analysis of growing tubers was undertaken by Jellito et at. (1992) and they found considerable shifts in the metabolite concentrations in the tubers. PPi contents were reduced from I to 0.4 nmol g-l FW and there was a corresponding 3-fold increase in the UDPglucose content. The interpretation of this work has to be cautious because the 35S promoter was used and in principle changes in the metabolism of other tissues such as leaves or phloem could influence the

244 Chapter 2

results in the tuber. However, it is a fine example of the changes that can be achieved by targeting the substrates of specific steps in metabolic engineering strategies.

The fructose that is produced by sucrose synthase must be phosphorylated to fructose 6-phosphate by hexokinase or fructokinase. It is known that fructose is capable of exerting feedback inhibition on sucrose synthase (Wolosiuk and Pontis, 1974) and it is therefore possible that this step is of some importance in regulating the flux of carbon to starch. In developing potato tubers, three hexokinase isoforms and three fructokinase isoforms have been reported (Renz et aI., 1993) which differ with respect to their kinetic properties and substrate specificities. The investigation of these reactions has been neglected at the molecular level: so far only one fructokinase (Smith et aI., 1993; Taylor et aI., 1994) and one hexokinase (R.N. Trethewey and L. Willmitzer, unpublished data) have been cloned from potato and no data from transgenic plants are yet available.

3.2 Import of carbon into amyloplasts

The route by which carbon enters the amyloplast is still under debate, largely because of the difficulty of investigating it experimentally (see also Chapters 5, 6 and 7). For a long time the discussion centred on the question of whether carbon entered as a C3 compound or as a C6 compound. This has now largely been resolved following the elegant work of Viola et aI. (1991) using l3C feeding experiments and nuclear magnetic resonance spectroscopy. They supplied l3C-glucose asymmetrically labelled at the I-position and deduced that if carbon enters the amyloplast as C6 units then the extent to which the 6-position of hexose units becomes labelled (randomisation) would be determined by the extent of triose phosphate recycling and would therfore be the same for both sucrose and starch. Conversely, if entry as C3 units is the major route by which starch synthesis is supported then there would be full randomisation of the label in the glucose units of starch. They found quite clearly the former result which indicates that starch biosynthesis is supported by entry of carbon as a C6 compound. However, this approach could not identify exactly which compound entered the amyloplast and this question has been the subject of some attention recently. The essential difficulty in studying this question is the isolation of good high quality amyloplasts from potato tubers: the high starch content is so disruptive during the preparation procedure that yields of amyloplasts are inevitably low and maybe biased towards specific developmental stages in the amyloplast population. Kosegarten and Mengel (1994) isolated

12. Transgenic potato tubers 245

amy lop lasts from suspension cell cultures and were able to obtain preparations that were up to 40% intact with only slight contamination by other cellular fractions. They demonstrated that glucose I-phosphate could be taken up by the amyloplasts, and that uptake was pH dependent (optimum pH 5.7) and showed Michaelis-Menten kinetics with an apparent Km of 0.5 mM. They found that uptake was not competitively reduced by the presence of external glucose 6-phosphate although they did not report whether glucose 6-phosphate was transported. Contrasting results were found by Schott et al. (1995) who used the imaginative approach of isolating amy lop lasts from transgenic potato plants with significantly reduced starch contents following antisense repression of ADPglucose pyrophosphorylase. They found that the synthesis of glutamate by the amyloplast preparations could be supported by external glucose 6-phosphate but not glucose I-phosphate implying that the former is taken up. Further, when they reconstituted the plastid membrane proteins into liposomes and determined the transport capacities of the liposomes they found that phosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, and glucose 6-phosphate were transported in a counter-exchange mode. They found virtually no transport of glucose I-phosphate into liposomes. Therefore, the question of which hexose phosphate enters the amyloplast remains largely unresolved at this stage. Two studies have given conflicting results, one using amy lop lasts prepared from suspension cell cultures and the other with amyloplasts purified from a transgenic line where the possibility of pleiotropic changes in metabolism has not been investigated. The question of which glucose phosphate enters amyloplasts and supports starch synthesis in normal wild type tuber tissue will require further experimentation, if not new approaches, to be resolved.

Phosphoglucomutase catalyses the interconversion of glucose 6-phosphate and glucose I-phosphate, a reaction that is expected to be near to equilibrium in potato tubers (ap Rees and Morrell, 1990). It is anticipated that there are two isoforms in potato tubers, a plastidic and a cytosolic form. The exact role of these two isoforms could be better interpreted when there is a resolution of the question of which glucose phosphate enters the amyloplast as the precursor for starch biosynthesis. If it is glucose 6-phosphate, then both isoforms will be necessary for the production of glucose I-phosphate in the amyloplast. However, if glucose I-phosphate is taken up then the role of these enzymes would be more complicated to interpret. The cytosolic form would then be necessary for the conversion of the fructose produced by sucrose synthase into glucose I-phosphate, whilst the amyloplast isoform would be superfluous to the starch biosynthetic flux. In both cases the enzymes could be important in determining the partitioning of hexose phosphates between the starch

246 Chapter 12

biosynthetic pathway and glycolysis in the cytosol and the plastid. Small changes in the ratio between the glucose phosphates might have a significant influence on this partitioning. It is not clear whether phosphoglucomutase is subject to regulatory mechanisms. Glucose 1,6-bisphosphate is required to activate the enzyme; it is the reaction intermediate and is present in potato tubers although the pathway of synthesis remains obscure.

3.3 Metabolic engineering of sucrose metabolism

As discussed above, the sucrose synthase pathway is the route by which sucrose is converted to the precursors of starch biosynthesis. However, by using transgenic technology, it is possible to attempt to metabolically engineer the potato to generate hexose phosphates from sucrose more efficiently. The first approach taken was to overexpress a yeast invertase, and given the uncertainty over phloem unloading, different lines of transgenic plants were generated where the invertase was expressed specifically in tubers, targeted either to the cytosol or the apoplast (Frommer and Sonnewald, 1995). It was possible to achieve a 60-fold increase in acid invertase activity in the tubers by both approaches and a decrease in sucrose content of up to 95% was found. Both transgenics also accumulated large amounts of glucose; there was fructose accumulation when the invertase was in the apoplast but no change in fructose content in tubers when the invertase was located in the cytosol. Analysis of the tubers with cytosolic expression showed dramatic changes in tuber morphology: the tubers were long, unevenly shaped, and had very large lenticels. Further there was a reduction in yield of about 30% and a reduction in starch content on a fresh weight basis of around 15%. Conversely when the invertase was expressed in the apoplast, the tubers become bigger and yield increased by up to 30% per plant. However, the starch content on a fresh weight basis was unchanged indicating that sink strength had increased at the whole plant level but not at the cellular level (U. Sonnewald and L. Willmitzer, pers. comm.). The reasons for these dramatically different results are currently under investigation. In general, this work shows the huge potential of phenotypic manipulation by the metabolic engineering of novel pathways in plants.

3.4 Starch biosynthesis in potato tuber amyloplasts

In potato tubers, starch is synthesised from glucose I-phosphate in amy lop lasts by the action of three enzymes: ADPglucose

12. Transgenic potato tubers 247

pyrophosphorylase, starch synthase, and branching enzyme. There are now known to be multiple isoforms of starch synthases and branching enzymes and the formation of ordered starch granules is a highly complex process involving the delicate interplay of all of these isoforms (see Chapter 9). The pathway of starch synthesis in potato tubers has been subject to intensive investigation using transgenic approaches and on the whole as many questions have been opened as have been answered.

Classical biochemical evidence indicates that the AGPase is the major site of regulation in the starch biosynthetic pathway (Preiss, 1991). This enzyme catalyses the formation of ADPglucose and PPi from A TP and glucose I-phosphate, and the reaction is believed to be effectively irreversible in vivo due to the rapid cleavage of PPi by alkaline pyrophosphatase. Despite recent new evidence that substantial AGPase activities are present in the cytosol of monocot endosperms (Thorbj0rnsen et aI., 1996), it remains clear that most if not all of the AGPase activity is located in the amyloplast of potato tubers. The enzyme is regulated by the 3-phosphoglycerate to phosphate ratio; the former is an activator and the latter an inhibitor of the enzyme (Preiss, 1991). Stark et ai. (1992) attempted to increase starch synthesis in potato tubers by taking a mutant form of the E. coli AGPase (glgc 16) which is insensitive to the 3-phosphoglycerate to phosphate ratio and expressing it in potato tubers. These authors reported an average 35% increase in starch content in the transgenic lines, however, no further biochemical characterisation was presented. The same approach was taken by an independent group and full results of this study have recently been reported (Sweetlove et al., 1996a, 1996b). These workers were able to achieve up to a 5-fold increase in the maximum catalytic activity of AGPase extractable from developing transgenic tubers. However, they did not find any increase in starch content in a range of different greenhouse and field experiments. They investigated this further by injecting radioactively labelled sucrose into tubers attached to the mother plant and monitoring its metabolism in the surrounding tissue. They found, remarkably, up to a 7-fold increase in the percentage of label incorporated into starch during a 3-hour incubation (pulse). When they followed metabolism during a subsequent thirteen-day chase (where the [U-14C]sucrose was replaced by unlabelled sucrose), they observed that the percentage of label incorporated into starch was significantly reduced in the transgenics, whereas the labelling of control starch remained broadly unchanged. The implication of these experiments is that although synthesis was indeed elevated, and a response coefficient for the AGPase of near 1 could be estimated, the degradation pathway was also activated in these plants. Sweetlove et al. (1996b) determined the activity of enzymes implicated in the process of starch degradation and

248 Chapter 12

found an increased ~-amylase activity, which they proposed to be responsible for the elevated degradation. The mechanism whereby increased capacity at the AGPase reaction leads to the induction of the degradative pathway remains unclear, but is a further example of how metabolic engineering approaches often run into problems as a consequence of endogenous signalling mechanisms. The reason for the discrepancy between the two studies in terms of starch yields may be explained by the use of different cultivars or different growth conditions, and serves as a warning that the success of a particular strategy may be greatly influenced by non-biochemical parameters.

The reverse approach, the antisense inhibition of AGPase activity, has been undertaken by MUller-Rober et al. (1992). These workers had previously. cloned cDNAs for both subunits of the enzyme using heterologous probes from maize (MUller-Rober et aI., 1990) and both were used in a chimeric antisense construct. Plants were selected with a 95-98% reduction in AGPase activity in the tubers, and a similar reduction in starch content was found, demonstrating the uniqueness of the AGPase pathway in starch biosynthesis. As might be expected with such a dramatic reduction in sink strength, there was an accumulation of soluble sugars to the extent that they contributed up to 40% of the dry weight of the potato tubers (in wild type tubers sugars normally account for just I % of the dry weight). A further indication of reduced sink strength was an increase in the number of tubers per plant from 8-15 to 70-90 in greenhouse grown plants.

The subsequent step of starch synthesis is the polymerisation reaction catalysed by starch synthases where the glucose moiety from ADPglucose is transferred to the non-reducing end of an al,4-g1ucan (Preiss, 1991). Starch consists of two fractions, linear amylose and branched amylopectin, and both can serve as substrates for the starch synthases. One isoform of starch synthase, granule-bound starch synthase I (GBSSI) is found only bound to the starch granule and has been extensively studied. A mutant of potato has been identified which is deficient in this activity and it has been shown to contain starch free of amylose (Hovenkamp-Hermelink et aI., 1987). Subsequently it was demonstrated that it is possible to complement this mutation by introducing the wild type gene into the mutant (Flipse et aI., 1996a, 1996b). The amylose content of potato tubers has also been reduced by antisense repression of GBSSI in wild type potato plants (Kuipers et aI., 1991, 1994, 1995; Visser et aI., 1991). Complete inhibition of GBSSI activity in tubers was achieved with both the 35S CaMV promoter and by using the tuber­specific GBSSI promoter, although the latter proved to provide less stable inhibition in field grown plants. No significant change was found in the

12. Transgenic potato tubers 249

total starch and sugar contents or in the agronomic characteristics of transgenic field-grown tubers.

In potato tubers there are also three soluble starch synthase isoforms (SSI, II and III) which have been relatively poorly studied to date. The 79.9 kDa SSII has been cloned from potato and following biochemical analysis and antisense experiments it was concluded that this isoform contributes up to 15% of the total starch synthase activity in the tuber (Edwards et aI., 1995). Not surprisingly, given the small contribution of SSII, no significant change in the starch content of developing tubers from antisense plants was found. Recently, two groups have independently reported the cloning of the major soluble starch synthase isoform, the 139.2 kDa ssm (Abel et aI., 1996; Marshall et aI., 1996). Again, the near complete removal of this isoform did not alter the total amount of starch that accumulates in the tubers, an observation that is more surprising given that this isoform accounts for the majority of the soluble starch synthase activity. However, changes were found in the structural properties of the starch: there is up to a 70% increase in the amount of phosphate covalently linked to starch and a change in the morphology of the starch granules. Therefore, the exact contribution of the individual starch synthases to the synthesis and physical properties of starch remains an active area of research.

The formation of a-l,6-linkages in starch is catalysed by branching enzyme. In potato tubers, for a long time only one isoform of branching enzyme had been cloned (BE I; KoBmann et aI., 1991). Almost complete inhibition of the BE I as determined by mRNA, protein and activity analysis did not lead to a change in amylose content, starch contents or growth (J. KoBmann and L. Willmitzer, pers. comm.). However, recently a second isoform (BE II) has been identified and this may account in part for the results found when BE I activity is reduced (Larsson et aI., 1996). The relative roles of the two branching enzymes are currently under investigation. The potential importance of manipulation at this step has been illustrated by the work of Kortstee et al. (1996) who were able to increase the degree of branching in amylopectin when they expressed an E. coli branching enzyme (glgB) in an amylose-free potato mutant.

3.5 Respiration in potato tubers

The conversion of sucrose into starch is an energy requiring process. For every molecule of sucrose that is metabolised to starch, one ATP is required to phosphorylate fructose, and two are required for the formation of two ADPglucose units. The UGPase reaction leads to the production of one UTP, although ATP may be needed for the synthesis

250 Chapter 12

of some of the PPi required for this step. Thus at least one A TP is needed for the incorporation of each glucose unit into starch. The supply of A TP is provided in potato tubers by the glycolytic and respiratory sequences. These reactions are relatively poorly studied in potato tubers and there have been only a few examples of manipulation of these pathways.

One of the most interesting studies was performed by Burrell et al. (1994) who introduced an unregulated phosphofructokinase (PFK) from E. coli into the cytosol of potato tubers. These authors were able to increase the extractable maximum catalytic activity of PFK by a factor of up to 21, without inducing changes in the activities of the other glycolytic enzymes. However, they found no evidence of an increase in glycolytic flux in either intact tubers or aged discs. Despite this there was a change in the balance of metabolic intermediates: hexose 6-monophosphates decreased by about 30% and the other glycolytic intermediates increased by between 2- and 8-fold, the most marked increase being in fructose 1,6-bisphosphate. Clearly, if an increase in glycolytic flux is desired it is insufficient to increase the activity at only one step, in this case PFK. However, changing the metabolite levels could have knock on effects on metabolism and unfortunately no data is presented on whether the increased 3-phosphoglycerate levels stimulate starch biosynthesis through an activation of the AGPase.

A further manipulation at this step was performed by Hajirezaei et al. (1994) who reduced the activity of the PPi-dependent: fructose 6-phosphate phosphotranferase (PFP). This cytosolic enzyme remains somewhat of an enigma in plant biochemistry (Stitt, 1990) as it is a potential bypass of one of the classical controlling enzymes of glycolysis, PFK. PFP could catalyse a net flux in the glycolytic or gluconeogenic directions, and in addition it may be important as a regulator of the PPi concentration in the cytosol. It might therefore be important for sink strength either in contributing to ATP production or in regulating the supply of PPi for the UGPase reaction. The a and ~ subunits have been cloned from potato (Carlisle et aI., 1990) and both were used to generate antisense plants in which a reduction in PFP activity of up to 99% in tubers was achieved (Hajirezaei et al., 1994). The plants developed normally although mature tubers contained up to 40% less starch than wild type tubers. No changes in PPi were found indicating that the restriction in starch synthesis is not created by a reduced capacity to metabolise UDPglucose. However, radioactive feeding experiments provided evidence that PFP catalyses a net flux in the glycolytic direction.

The only manipulation reported thus far in the tricarboxylic acid cycle (TCA cycle) has been reduction in the activity of citrate synthase

12. Transgenic potato tubers 251

by the antisense approach. LandschUtze et aI. (1995a) cloned a citrate synthase isoform from potato and used this cDNA to produce antisense transgenic plants (LandschUtze et aI., 1995b). Despite a reduction in activity to 6% of wild type, no visible phenotype was found: tuber initiation and formation was normal. However, a major change was seen on entering the generative phase. Flower buds formed up to 2 weeks later than in wild type plants, and these flower buds were not capable of maturing because of disintegration of the ovaries. It may be that the TeA cycle is particularly important in the transition from the vegetative to the generative phase, however it remains puzzling why no effects were seen in tubers. As with the PFP antisense work it may be that minimal residual activity is sufficient for the enzyme to fulfil its role under normal conditions, or it could be that the plasticity of plant metabolism allows the restriction at this step to be bypassed.

4. CONCLUSION

The development of transgenic technology has led to a large number of studies of plant metabolism through the manipulation of activities at specific steps. In particular the potato tuber has been extensively studied, and although one cannot directly extrapolate to other plants and tissues, the lessons learnt in the potato tuber are of general relevance to the study of plant metabolism. In some cases, steps which were proposed to be important and central for the determination of sink strength from classical studies have been confirmed as such by the antisense approach (e.g. sucrose synthase). However, in other cases the effects of manipulations at central steps in metabolism have been surprisingly small (e.g. PFK overexpression, citrate synthase and PFP antisense studies). In part this may be because of the plasticity of plant metabolism -redundancy allows pathways to withstand reductions in capacity at a particular point. It might also be because enzymes are often present in quantities in excess of the net flux. Whilst this gives flexibility, it might also ensure that antisense studies where incomplete inhibition is achieved will not give rise to phenotypes under normal conditions. In only one case, overexpression of AGPase by Stark et aI. (1991), has an increase in sink strength following genetic manipulation been reported. This result was not confirmed by another group (Sweetlove et aI., 1996a, 1996b) working in a different cultivar and using different growth conditions indicating that more factors than the simple genetic manipulation will be important for the targeted increase in sink strength. Indeed the work of Sweetlove et al. and the studies with the overexpression of invertase in

252 Chapter 12

the cytosol of tubers demonstrate that the heterologous overexpression of transgenes can give rise to complex changes in pathways beyond that being targeted. The elucidation of the signals and regulatory mechanisms leading to these pleiotropic responses will be a central challenge for plant biochemists in the future. A thorough understanding of the interaction between metabolism and gene expression would greatly aid the development of new strategies to improve the harvest index through targeted genetic manipulation.

REFERENCES

Abel, G.1.W., Springer, F., Willmitzer, L. and KoBmann, J. (1996). Cloning and functional analysis of a cDNA encoding a novel 139 kDa starch synthase from potato (Solanum tuberosum L.). Plant Journal, 10, 981-99l.

ap Rees, T. and Morrell, S. (1990). Carbohydrate metabolism in developing potatoes. American Potato Journal, 67, 835-847.

Boerjan, W., Bauw, G., Van Montagu, M. and Inze, D. (1994). Distinct phenotypes generated by overexpression and suppression of y-adenosyl-L-methionine synthetase reveals developmental patterns of gene silencing in tobacco. Plant Cell, 6, 1401-1414.

Burrell, M.M. Mooney, P.J. Blundy, M. Carter, D. Wilson, F. Green, J. Blundy, K.S. and ap Rees, T. (1994). Genetic manipulation of 6-phosphofructokinase in potato tubers. Planta, 194, 95-10l.

Carlisle, S.M., Blakeley, S.D., Hemmingsen, S.M., Trevanion, S.1., Hiyoschi, T. and Dennis, D.T. (1990). Pyrophosphate-dependent phosphofructokinase: conservation of protein sequence between alpha and beta subunits and with the ATP-dependent phosphofructokinase. Journal of Biological Chemistry, 265, 18366-1837l.

Dietze, J., Blau, A and Willmitzer, L. (1995). Agrobacterium-mediated transformation of potato (Solanum tuberosum). In: Potrykus, I. and Spangenberg, G. (Eds). Gene transfer to plants xxii (pp. 24-29). Springer-Verlag, Berlin.

Edwards, A, Marshall, J., Sidebottom, C., Visser, R.G.F., Smith, AM. and Martin, C. (1995). Biochemical and molecular characterisation of a novel starch synthase from potato tubers. Plant Journal, 8, 283-294.

Flipse, E., Schippers, M.G.M., Janssen, E.M., Jacobsen, E. and Visser, R.G.F. (1996a). Expression of wild-type GBSS transgenes in the offspring of partially and fully complemented amylose-free transformants of potato. Molecular Breeding, 2, 211-218.

Flipse, E., Straatmanengelen, I., Kuipers, AG.1., Jacobsen, E. and Visser, R.G.F. (1996b). GBSS T-DNA inserts giving partial complementation of the amylose free potato mutant can also cause co-suppression of the endogenous GBSS gene in a wild type background. Plant Molecular Biology, 31, 731-739.

Franck, A, Guilley, H., Jonard, G., Richards, K. and Hirth, L. (1980). Nucleotide sequence of Cauliflower Mosaic virus DNA Cell, 21, 285-294.

Frommer, W.B. and Sonnewald, U. (1995). Molecular analysis of carbon partitioning in solanaceous species. Journal of Experimental Botany, 46, 587-607.

Fu., H. and Park, W.D. (1995). Sink- and vascular-associated sucrose synthase functions are encoded by different gene classes in potato. Plant Cell, 7, 1369-1385.

12. Transgenic potato tubers

Fu., H., Kim, S.Y. and Park, W.D. (1995a). High-level tuber expression and sucrose inducibility of a potato sus4 sucrose synthase gene require 5' and 3' flanking sequences and the leader intron. Plant Cell, 7, 1387-1394.

253

Fu., H., Kim, S.Y. and Park, W.D. (1995b). A potato sus3 sucrose synthase gene contains a context-dependent 3' element and a leader intron with both positive and negative tissue-specific effects. Plant Cell, 7, 1395-1403.

Geigenberger, P. and Stitt, M. (1993). Sucrose synthase catalyses a readily reversible reaction in vivo in developing potato tubers and other plant tissues. Planta, 189, 329-339.

Hajirezaei, M., Sonnewald, U., Viola, R., Carlisle, S., Dennis, D. and Stitt, M. (1994). Transgenic potato plants with strongly decreased expression of pyrophosphate : fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta, 192, 16-30.

Hill, S.A and ap Rees, T. (1994). Metabolic control analysis of plant metabolism. Plant Cell and Environment, 17, 587-599.

Hooykas, PJJ. and Schilperoort, R.A (1992). Agrobacterium and plant genetic engineering. Plant Molecular Biology, 19, 15-38.

Hovenkamp-Hermelink, lH.M., Jacobsen, E., Ponstein, A.S., Visser, R.G.F., Vos­Scheperkeuter, G.H., Bijmolt, E.W., de Vries, IN., Witholt, B. and Feenstra, W.1. (1987). Isolation of an amylose-free starch mutant of the potato (Solanum tuberosum L.). Theoretical and Applied Genetics, 75, 217-221.

Inoue, H. and Tanaka, A (1978). Comparison of source and sink potentials between wild and cultivated potatoes. Journal of the Science of Soil Management of Japan 49, 321-327.

Jellito, T., Sonnewald, U., Willmitzer, L., Hajirezaei, M.R. and Stitt, M. (1992). Inorganic pyrophosphate content and metabolites in leaves and tubers of potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol: biochemical evidence that sucrose metabolism has been manipulated. Planta, 188, 238-244.

Kortstee, Al, Vermeesch, AM.S., de Vries, B.1., Jacobsen, E. and Visser, R.G.F. (1996). Expression of Escherichia coli branching enzyme in tubers of amylose-free transgenic potato leads to an increased branching degree of the amylopectin. Plant Journal, 10, 83-90.

Kosegarten, H. and Mengel, K. (1994). Evidence for a glucose I-phosphate translocator in storage tissue amyloplasts of potato (Solanum tuberosum) suspension-cultured cells. Physiologia Plantarum, 91, 111-120.

KoJ3mann, J., Visser, R.G.F., Muller-Rober, B. Willmitzer, L. and Sonnewald, U. (1991). Cloning and expression analysis of a potato cDNA that encodes branching enzyme: evidence for co-expression of starch biosynthetic genes. Molecular and General Genetics, 230, 39-44.

Kuipers, AG.1., Soppe, W.1.1., Jacobsen, E. and Visser, R.G.F. (1994). Field evaluation of transgenic potato plants expressing an antisense granule-bound starch synthase gene - increase of the antisense effect during tuber growth. Plant Molecular Biology, 26 1759-1773.

Kuipers, AGJ., Soppe, WJJ., Jacobsen, E. and Visser, R.G.F. (1995). Factors affecting the inhibition by antisense RNA of granule-bound starch synthase gene expression in potato. Molecular and General Genetics, 246, 745-755.

Kuipers, A.G.1., Vreem, IT.M., Meyer, H., Jacobsen, E., Feenstra, WJ. and Visser, R.G.F. (1991). Field evaluation of antisense RNA-mediated inhibition of GBSS gene expression in potato. Euphytica, 59, 83-91.

254 Chapter 12

LandschUtze, V., MUller-Rober, B. and Willmitzer, L. (1995a). Mitochondrial citrate synthase from potato: predominant expression in mature leaves and young flower buds. Planta, 196, 756-764.

LandschUtze, V., WiIImitzer, L. and MUller-Rober, B. (1995b). Inhibition of flower formation by antisense repression of mitochondrial citrate synthase in transgenic potato plants leads to a specific disintegration of the ovary tissues of flowers. EMBO Journal, 14, 660-666.

Larsson, C.-T., Hofvander, P., Khoshnoodi, J., Ek, B., Rask, L. and Larsson, H. (1996). Three isoforms of starch synthase and two isoforms of branching enzyme are present in potato tuber starch. Plant Science, 117, 9-16.

Liu, XJ., Prat, S., WiIImitzer, L. and Frommer W.B. (1990). Cis regulatory elements directing tuber-specific and sucrose-inducible expression of a chimeric class I patatin promoter-GUS-gene fusion. Molecular and General Genetics, 223, 401-406.

Mares, D.J. and Marschner, H. (1980). Assimilate conversion in potatoes in relation to starch deposition and cell growth. Berichte der Deutschen Botanischen Gesellschaft, 93, 299-313.

Marshall, J., Sidebottom, c., Debet, M., Martin, C., Smith, A.M. and Edwards, A. (1996). Identification of the major starch synthase in the soluble fraction of potato tubers. Plant Cell, 8, 1121-1135.

Mol, J., Van der Krol, A., Van Tunen, A., Van Blokland, R., de Lange, P. and Stuitje, A. (1990). Regulation of plant gene expression by antisense RNA. FEBS Letters, 268, 427-430.

Morrell, S. and ap Rees, T. (1986). Sugar metabolism in developing tubers of Solanum tuberosum. Phytochemistry, 25, 1579-1585.

MUller-Rober, B. KoBmann, J., Hannah, L.C., WiIImitzer, L. and Sonnewald, U. (1990). Only one of two different ADP-glucose pyrophosphorylase genes from potato responds strongly to elevated levels of sucrose. Molecular and General Genetics, 224, 136-146.

MUller-Rober, B., la Cognata, U., Sonnewald, U. and Willmitzer, L. (1994). A truncated version of an ADP-glucose pyrophosphorylase promoter from potato specifies guard cell-selective expression in transgenic plants. Plant Cell, 6, 601-612.

MUller-Rober, B. Sonnewald, U. and Willmitzer, L. (1992). Antisense inhibition of the ADP-glucose pyrophosphorylase in transgenic potato leads to sugar-storing tubers and influences tuber formation and expression of tuber storage protein genes. EMBO Journal, 11, 1229-1238.

Oparka, K.J. (1985). Changes in partitioning of current assimilate during tuber bulking in potato (Solanum tuberosum L.) cv Maris Piper. Annals of Botany, 55, 705-713.

Oparka, K.J. (1986). Phloem unloading in the potato Solanum tuberosum - tuber pathways and sites of ATPase. Protoplasma, 131,201-210.

Oparka, KJ. (1990). What is phloem unloading? Plant Physiol., 94, 393-396. Oparka, KJ. and Prior, D.A.M. (1988). Movement of Lucifer Yellow CH in potato tuber

storage tissues: a comparison of symplastic and apoplastic transport. Planta, 176, 533-540.

Oparka, KJ., Viola, R., Wright, K.M. and Prior, D.A.M. (1992). Sugar transport and metabolism in potato tuber. In: Farrar, J.F., Gordon, AJ. and Pollock, c.J. (Eds). Carbon partitioning within and between organisms (pp. 91-114). BIOS Scientific Publishers, Oxford.

Oparka, KJ. and Wright, K.M. (1988). Osmotic regulation of starch synthesis in potato tubers. Planta, 174, 123-126.

12. Transgenic potato tubers 255

Priess, J. (1991). Biology and molecular biology of starch synthesis and its regulation. In: Miflin, B.J. (Ed). Oxford Surveys of Plant Cellular and Molecular Biology (Vol. 7, pp. 59-114). Oxford University Press, Oxford.

Renz, A., Merlo, L. and Stitt, M. (1993). Partial purification from potato tubers of three fructokinases and three hexokinases which show differing organ and developmental specificity. Planta, 190, 156-165.

Riesmeier, J.W., Hirner, B. and Frommer, W.B. (1993). Potato sucrose transporter expression in minor veins indicates a role in phloem loading. Plant Cell, 5, 1591-1598.

Salanoubat, M. and Belliard, G. (1987). Molecular cloning and sequencing of sucrose synthase cDNA from potato (Solanum tuberosum L.): preliminary characterisation of sucrose synthase mRNA distribution. Gene, 60, 47-56.

Schmiilling, T., Schell, J. and Spena, A. (1989). Promoters of the rol A, B, and C genes of Agrobacterium rhizogenes are differentially regulated in plants. Plant Cell, 1, 665-670.

Schott, K., Borchert, S., Miiller-R6ber, B. and Heldt, H.W. (1995). Transport of inorganic phosphate and C3- and C6-sugar phosphates across the envelope membranes of potato tuber amyloplasts. Planta, 196, 647-652.

Smith, S.B., Taylor, M.A., Burch, L.R. and Davies, H.V. (1993). Primary structure and characterisation of a cDNA clone of fructokinase from potato Solanum tuberosum L. cv Record. Plant Physiology, 102, 1043.

Sonnewald, U. (1992). Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning. Plant Journal, 2, 571-581.

Stark, D.M., Timmermann, K.P., Barry. G.F., Preiss, J. and Kishore, G.M. (1992). Regulation of the amount of starch in plant tissues by ADP glucose pyrophosphorylase. Science, 258, 287-292.

Stitt, M. (1990). Fructose 2,6-bisphosphate as regulatory metabolite in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 41, 153-185.

Stitt, M. (1993). Control of photosynthetic carbon fixation and partitioning. How can use of genetically manipulated plants improve the nature and quality of information about regulation? Philosophical Transactions of the Royal Society of London Series B, 340, 225-233.

Stitt, M. and Sonnewald, U. (1995). Regulation of metabolism in transgenic plants. Annual Review of Plant Physiology and Plant Molecular Biology, 46, 341-368.

Stockhaus, J., Schell, J. and Willmitzer L. (1989). Identification of enhancer elements in the upstream region of the nuclear photosynthetic gene ST-LS1. Plant Cell, 1, 805-813.

Sweetlove, L.J., Burrell, M.M. and ap Rees, T. (1996a). Characterisation of transgenic potato (Solanum tuberosum) tubers with increased ADPglucose pyrophosphorylase. Biochemical Journal, 320, 487-492.

Sweetlove, LJ., Burrell, M.M. and ap Rees, T. (1996b). Starch metabolism in tubers of transgenic potato (Solanum tuberosum) with increased ADPglucose pyrophosphorylase. Biochemical Journal, 320, 493-498.

Taylor, M.A., Ross, H.A., Gardner, A. and Davies, H.V. (1994). Characterisation ofa cDNA encoding fructokinase from potato (Solanum tuberosum L.). Journal of Plant Physiology, 145, 253-256.

Thorbj0l1lsen, T., Viliand, P., Denyer, K, Olsen, O.-A. and Smith, A.M. (1996). Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm. Plant Journal, 10, 243-250.

256 Chapter 12

Viola, R., Davies, H.V. and Chudeck, A.R. (1991). Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.): elucidation by carbon-13 NMR spectroscopy. Planta, 183, 202-208.

Visser, R.G.F., Somhorst, I., Kuipers, G.J., Ruys, N.J., Feenstra, W.J. and Jacobsen, E. (1991). Inhibition of the expression of the gene for granule-bound starch synthase in potato by antisense constructs. Molecular and General Genetics, 225, 289-296.

Wolosiuk, R.A. and Pontis, H.G. (1974). Studies on sucrose synthetase. Archives of Biochemistry and Biophysics, 165, 140-145.

Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. and Schell, J. (1983). Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO Journal, 2, 2143-2150.

Zrenner, R., Salanoubat, M., Sonnewald, U. and Willmitzer, L. (1994). Evidence for the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.). Plant Journal, 7, 97-107.

Zrenner, R., Willmitzer, L. and Sonnewald, U. (1993). Analysis of the expression of potato uri dine diphosphate-glucose pyrophosphorylase and its inhibition by antisense RNA. Planta, 190, 247-252.

Chapter 13

Increasing the flux in a metabolic pathway: a metabolic control analysis perspective

David A. Fell and Simon Thomas School of Biological and Molecular Sciences, Oxford Brookes University, Headington, Oxford OX3 OBP, UK

Key words: metabolic control analysis; metabolic engineering; multisite modulation; Universal Method.

Abstract: Why is the solution to increasing the flux in a pathway not simply: (i) find the rate-limiting step; and (ii) amplify or activate it? There are theoretical and experimental grounds for expecting the above approach to fail:

• Control of flux is distributed; rarely does anyone enzyme have a large share of this control. This will be illustrated with results for ribulose­bisphosphate carboxylase and the control of the reductive pentose phosphate pathway.

•

•

Amplification or activation of a single enzyme will generally yield a limited flux response. Theory predicts this; practical examples include amplification of phosphofructokinase in potato tubers.

Large flux increases require coordinate changes in several/many enzyme activities - the method used in vivo. Again, this can be predicted theoretically and has been partially demonstrated in the engineering of yeast tryptophan synthesis. The in vivo examples include light activation of photosynthesis.

257

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 257-273. © 1999 Kluwer Academic Publishers.

258 Chapter 13

1. INTRODUCTION

There is a current concern to exploit the perceived potential of molecular biology to engineer the metabolism of plants. But the problems of making an intentional change in the flux through a chosen metabolic pathway have an intrinsic interest beyond the immediate goal. This is because living organisms are clearly able, at least to some degree, to make the changes that we find so elusive, and this therefore implies that there must be faults in our reasoning about how metabolism is controlled in vivo. In this paper we shall first review why the problem of increasing metabolic flux is not as simple as was once thought. Incidentally, the problems encountered in increasing and decreasing flux are not symmetrical; the latter is in some respects easier, but will not be considered in detail here. Then we shall draw on the theory of metabolic control analysis (Heinrich and Rapoport, 1974; Kacser and Burns, 1973) to examine the nature of the difficulty and to consider how metabolic engineers can and have avoided it. Finally we propose that this analysis has inevitable implications for how we interpret the control of metabolism in vivo.

2. METABOLIC CONTROL ANALYSIS CONFRONTS THE RATE-LIMITING STEP

According to the principles of metabolic control in vogue for much of this century, the solution to the problem of increasing the flux in a pathway ought to have been: find the 'pacemaker enzyme' (Krebs, 1946) or 'rate-limiting step' and increase its activity. The main points in the reasoning behind this proposal were (e.g. Newsholme and Start, 1973): 1. Flux is controlled by a rate-limiting step near the start of a pathway. 2. A rate-limiting step will be one of the non-equilibrium reactions. 3. Rate-limiting steps commonly show feedback inhibition. 4. Metabolic flux changes involve the action of effectors, covalent

modification or changes in enzyme level on the rate-limiting step of the pathway.

5. Non-rate-limiting steps (e.g. near-equilibrium enzymes) respond passively to metabolite changes to deliver required flux. Metabolic control analysis started by challenging the assumption that

there was necessarily a rate-limiting step in a pathway, initially on the basis of mainly theoretical arguments (Heinrich and Rapoport, 1974; Kaeser and Burns, 1973). Later, as metabolic control analysis became more widely employed in the design and analysis of experiments, it

13. Increasing metabolic flux 259

became clear that its criticisms were justified in practice (for a review see Fell, 1992). In essence, control analysis proposes that control can be distributed between the steps in a pathway, and the potential of any particular step to be an effective site for exercising control must be measured on a continuous scale between rate-limiting and completely non-rate-limiting. The steps in the argument can be illustrated by considering the effect of changes in Rubisco on photosynthetic flux as a typical example.

The change in photosynthetic flux as Rubisco activity is varied has been studied in some detail by Stitt and co-workers. A representative result is shown in Figure 1 taken from Figure 1 of Lauerer et al. (1993). This quasi-hyperbolic relationship, with normal in vivo levels of enzyme activity mapping onto the flatter regions of the curve, is typical of the results from similar experiments on various pathways from a range of organisms. (More examples are collected in Fell, 1996.) It follows that the response of the flux to a change in enzyme activity varies continuously, starting off as an almost proportional response at low levels of enzyme activity and gradually declining as the enzyme activity increases. For this reason alone, a dichotomous classification into rate­limiting and non-rate-limiting fails to capture an essential property of the system. In contrast, metabolic control analysis provides a measure of the strength of the response of flux to enzyme activity at each point on the curve.

Suppose we consider that, at a point on the flux-enzyme curve, a small change, 8Exase , is made in the amount of enzyme E xase' and that this produces a small change, 8Jydh , in the steady state pathway flux, J,

measured at the step catalyzed by ydh. If the change is made small enough, then the ratio 8Jydh/8Exase becomes equal to the slope of the

tangent to the curve of J ydh against Exase as shown in Figure 2. In

mathematical notation this tangent is represented as 8Jydh / liExase'

Obviously this represents the steepness of the response of the flux to the amount of enzyme, but has the disadvantage that its numerical value and its units will depend on the units used to measure the flux and the enzyme. This problem can be avoided if we compare the fractional changes in the enzyme and flux, i.e. 8Exase/ Exase and 8Jydh / Jydh ; since

the numerator and denominator of each fraction are measured in the same units, the result is dimensionless. (If multiplied by 100, each of these fractional changes can be regarded as percentage changes.) The flux

control coefficient C:ts~ is given by the ratio of these fractional changes as 8Exase/ Exase tends to zero:

260

~ ..-

en N' I

E 0

E -::i. ""'-'"

en en Cl)

...c +-c ~ en o

+-o

...c 0...

20

•

15 o o

10

5

o

•

750 U 300 U 100 U

Chapter 13

•

WT AntI

• 0 • 0 ~ ~

o 20 40 60 80 100 120

Rubisco activity -2 -1

Cumo/·m -s ) Figure 1: Variation of photosynthetic rate with Rubisco activity, Results of Lauerer et a1. (1993). Wild-type tobacco plants and transgenic plants expressing anti-sense rbcS to lower Rubisco activity were grown at three different light intensities.

13. Increasing metabolic flux 261

(1)

This can be rearranged so that the flux control coefficient is expressed as the tangent to a curve such as that in Figure 2 times a scaling factor Exase/ Jydh taken at the point e, j at which the control coefficient

is measured:

(2)

Figure 3 shows the typical values that the flux control coefficient takes at various points along the flux-enzyme curve.

Although the flux control coefficient has been defined in terms of the response of the flux to the variation of a single enzyme, it is nevertheless a property of the system as a whole and not a characteristic of the enzyme in isolation. The easiest way to illustrate this is with the flux summation theorem discovered by Kacser and Bums (1973). This states that the flux control coefficients of all the enzymes in a metabolic system that affect the flux in question sum to 1. Therefore, in Figure 3 when the enzyme activity is at the left end of the curve, the flux control coefficient is near 1 and it follows that the flux control coefficients of all the other enzymes must total only slightly above zero. (This is only strictly true for a linear pathway, but this does not affect the essence of the argument presented here.) On the other hand, when the enzyme activity has been increased to the right hand edge of the diagram, the situation is reversed and the sum of the control coefficients of the other enzymes is now near 1. Thus their flux control coefficients have increased even though these enzymes were held at constant activity. The same thing would happen if one of these enzymes were to have its level changed; this would affect the flux control coefficient of our original enzyme (by shifting the curve in Figure 2).

Now that there have been many measurements of flux control coefficients (see, e.g., Fell, 1992, 1996), it is becoming increasingly apparent that not only do enzymes traditionally regarded as rate-limiting have relatively small flux control coefficients, but also that values of flux control coefficients tend not to be as high as 1 when enzymes are present at wild-type levels in pathways that are not operating at the extremes of their range. As one illustration from many, we refer again to the results from Stitt's group on the flux control coefficient of Rubisco, Figure 4. The figure is typical in showing that the flux control coefficient of

262

. J

8 Jydh

a Exase " "

e

Concentration of enzyme, Exase

Figure 2: Definition of the flux control coefficient.

Chapter 13

8Jydh e 8 Exase 7

The flux control coefficient at a given level of activity, e, of an enzyme Exase given by the scaled slope of the flux-enzyme activity curve. See Equation (1) in the text.

13. Increasing metabolic flux 263

-X :::J

LL

~ 0.2

~ 0.5

Concentration of enzyme, Exase

Figure 3: Values of the flux control coefficient. Approximate values of the flux control coefficient are shown for various points on a quasi-hyperbolic flux-enzyme curve.

264

0 0 en :.c :::J ....

J

()

1.0

0.8

0.6

0.4

0.2

0.0 o

Chapter 13

o.

o· ....... 0

20 40 60 80 100

Rubisco (% wild-type level) Figure 4: Flux control coefficient of Rubisco on photosynthetic carbon assimilation. The flux control coefficients were measured at light levels the same as (3), and 3-fold higher than (2), those used for plant growth for wild-type and transgenic tobacco plants expressing anti-sense rbcS (Stitt et aI., 1991).

13. Increasing metabolic flux 265

Rubisco is low when measured at wild-type levels in the conditions in which the plants were raised.

To what then does the criticism of the rate-limiting step amount? One point of view would be that the concept of the rate-limiting step is too simplistic; that the distribution of the control of flux must be accepted; that this requires a quantitative measure of flux control; and that traditional methods of identifying rate-limiting steps are of mixed levels of reliability. This would imply that the first three items in the list at the start of this section are unreliable, but that the principles of the remaining two could be accepted provided that the actual words 'rate­limiting step' were replaced by 'enzyme with (significantly) non-zero flux control coefficient'. There has been little direct discussion of this point, but metabolic control analysts have not directly challenged these last two concepts. Indeed, the interpretation of allosteric control and covalent modification in terms of the response coefficient (Kacser and Bums, 1973) is an implicit acceptance of the approach. We believe however that recent developments in control analysis mean that the last two principles are also unreliable, at least when they are required to explain large increases in flux.

3. POTENTIAL STRATEGIES FOR INCREASING FLUX

The theory of metabolic control analysis we have described so far is exact, subject to certain provisos (see e.g. Fell, 1992), but deals only with the limit of very small changes in effector concentration or enzyme activity. Beyond the domain of small changes, the control coefficients of the components of the system start to change and can no longer be regarded as invariants of the system. For practical application to large changes in physiological flux, or large engineered changes in enzyme content, it would be useful if we could rely on the relatively small amounts of information about the system that the control coefficients represent, rather than on all the detailed and exact kinetic information needed to build a full and realistic simulation. However, there is an inevitable compromise: the more the amount of information is restricted, the less it is possible to be completely confident that any predictions are accurate. Nevertheless, Kacser and Small developed an approximate solution that is valid under certain conditions, namely those that cause the flux-enzyme relationship to be close to rectangular hyperbolic. Their finite change theory (Small and Kacser, 1993a, 1993b) states that if the flux control coefficient of an enzyme is C, then the relative change f in

266 Chapter 13

pathway flux brought about by an r-fold amplification of the enzyme activity is:

f= 1 1 r-l CJ --r- E

(3)

This function is plotted in Figure 5 where it is seen that the effects on the pathway flux from changing the amount of a single enzyme can be quite limited, unless its flux control coefficient is greater than 0.6.

In this context, the relatively small flux control coefficients obtained for Rubisco in many conditions (Lauerer et aI., 1993) suggest that the scope for achieving significant flux changes by activation of this enzyme could be limited. An example where the finite change approach seems to be applicable in plant metabolism is given by our recent analyses (Thomas et aI., 1997a, 1997b) of the experiments on the overexpression of phosphofructokinase in potato tubers (Burrell et aI., 1994; Mooney, 1994). This had no measurable effect on glycolytic flux, even with a 30-fold increase in the enzyme content, suggesting that the flux control coefficient is small. On the other hand, the concentrations of glycolytic metabolites were affected in broadly the manner expected from our control analysis of potato glycolysis and the finite change theory for metabolite concentrations (Small and Kacser, 1993b). The reason that the metabolite concentration changes downstream of phosphofructokinase do not translate into a change in flux in lower glycolysis is that they gradually die away along the length of the glycolytic pathway (Figure 6). Further, although phosphoenolpyruvate levels do rise slightly, it is a strong feedback inhibitor of plant phosphofructokinase and so is a major contributor to the suppression of the activity of the added enzyme. We demonstrated quantitatively in our control analysis of potato tuber glycolysis at in vivo levels of phosphofructokinase (Thomas et aI., 1997b) that the phosphoenolpyruvate inhibition was the major factor in causing the low flux control coefficient of the enzyme. In fact, this illustrates the argument put forward by Kacser and Bums (1973) that feedback inhibition of an enzyme diminishes its flux control coefficient and transfers the control of flux downstream of the feedback metabolite, and indeed, our model predicts that the control of tuber glycolysis mainly resides downstream from phosphoenolpyruvate.

In summarising the potential for increasing metabolic flux by activating a single enzyme, we conclude: • Even for a relatively large value of the flux control coefficient, the

increase in flux will be limited.

13. Increasing metabolicflux 267

10

8

-x 6 :::l ;;:

CI> > :;:::; n1 CI> 4 a::

2

o o 0.2 0.4 0.6 0.8 1

Flux control coefficient

Figure 5: The relative change of flux for large changes in enzyme amount. The increases in flux predicted by Equation (3) against the value of the flux control coefficient (Small and Kaeser, 1993a). The degree of amplification r of the enzyme amount is shown on each curve.

268

c 0 .~ ~ -c Q) () c 0 ()

Q) > .~

Q)

a:

12

10

8

6

4

2

o

.' .' .'

•....................................•

Chapter 13

o 5 1 0 15 20 25 30 35 40

Relative PFK activity

Figure 6: Metabolite changes in transgenic potato tubers. The results are from (Mooney, 1994).

13. Increasing metabolic flux 269

• Metabolic control analysis measurements have not found many enzymes with sufficiently large flux control coefficients to be capable of causing large flux changes.

• The reported results from overexpressing pathway enzymes are consistent with this analysis. Large changes in enzyme expression have been achieved, but have not greatly affected flux. For example, most of the glycolytic enzymes have been overexpressed in yeast without any change in glycolysis (Schaaff et aI., 1989).

One apparent solution to this dilemma would be to simultaneously activate a group of enzymes in a pathway whose flux control coefficients summed to a total close to 1. We know that activating all the enzymes of a metabolic system increases metabolic flux, since this is what happens when we increase the amount of biomass in a fermenter. The interesting question is whether there is some intermediate solution whereby arbitarily large flux increases could be obtained without needing to activate every enzyme. One class of solutions was proposed by Kacser and Acerenza (1993) as their Universal Method. Suppose it is intended to increase the flux to a metabolic end-product without disrupting or changing any other aspect of metabolism, in particular metabolite concentrations. Since metabolism is multiply branched, we can trace back from the end-product to the branch-point intermediate, S, at the last branch-point leading to it. If all the enzymes along this branch are increased by the same factor a then none of the intermediate concentrations between S and the end­product will be altered, but the flux should increase by a provided that S can be kept constant. This requires that the production of S before the branch-point should be increased by exactly the same amount as its consumption by the branch to the end-product. However, as the original flux towards S before the branch-point must have been greater than the flux in the branch to the end-product, the increase in production of Scan be achieved with a smaller factor increase in the flux. Thus if we trace metabolism back to the previous branch-point metabolite R, all the enzymes between R and S should be increased in activity by this same, smaller factor. As we trace backwards towards the nutrient inputs sustaining the metabolic flux, the degree of activation required in each previous branch might become sufficiently small to be negligible. Regardless of whether this technique is easily practicable with existing gene technology, the concept is that large increases in a metabolic flux, with no disturbance to the rest of metabolism, require sets of coordinated enzyme modulations.

Some experimental support for this proposal has come from activating tryptophan sysnthesis in yeast (Niederberger et aI., 1992) where 8-fold increases in tryptophan flux required the activation of at

270 Chapter 13

least 4 of the 5 steps from the branch-point metabolite chorismate. Significantly, abolishing the feedback inhibition of the first enzyme after the branch-point was largely ineffective on its own. Computer simulations (Cornish-Bowden et aI., 1995) have also supported the claim that the Universal Method is an effective way of obtaining large flux increases. The only other strategy that the simulations endorsed as effective was to activate consumption or export steps in pathways subject to strong feedback inhibition.

4. FLUX INCREASES IN VIVO

If it is difficult to engineer a large increase in metabolic flux, how do living organisms achieve this? In animal muscle, glycolytic flux increases of 100-fold or more are accompanied by negligible changes in glycolytic intermediates, which contrasts with the significant but localised changes produced by engineering changes in a single enzyme such as phosphofructokinase. From reviewing the evidence for a number of different pathways, we have concluded that large increases in flux are achieved by coordinate activation of many of the enzymes throughout the metabolic network, a phenomenon we termed multisite modulation (Fell and Thomas, 1995; Thomas and Fell, 1996). On long time scales, this comes from coordinate induction of enzyme synthesis; on shorter time scales, it seems likely that covalent modification of enzymes produces the response. Although attention has been focused on covalent modification of enzymes at the start of pathways, because of the rate­limiting step dogma, sites of covalent modification have been found to occur throughout a pathway.

In this respect, photosynthesis offers an excellent, but by no means isolated, example. (Others are given in Fell and Thomas, 1995 and Thomas and Fell, 1996.) In addition to the diurnal rhythm in photosynthesis there are much faster transients for many plants as light flecks move across leaves, perhaps causing lO-fold stimulation of photosynthetic flux within tens of seconds. In both cases a major mechanism is covalent modification; although Rubisco is light-activated by Rubisco activase, four other enzymes of the Calvin cycle (fructose bisphosphatase, sedoheptulose bisphosphatase, glyceraldehyde-3-phosphate dehydrogenase and ribulose-5-phosphate kinase) are known to be activated by the light-sensitive thioredoxin system (Anderson, 1986; Geiger and Servaites, 1994) in the chloroplast. Meanwhile in the cytoplasm, the light-dependent phosphorylation status of enzymes such

13. Increasing metabolicflux 271

as nitrate reductase and sucrose phosphate synthase produces coordinate modulations there.

5. CONCLUSION

Allosteric effectors and feedback inhibition have not featured in this account of how pathways are controlled. This is not an oversight; although there is not space to review this topic, there seems to be relatively little scope for allosteric effects to produce activation at many sites in a pathway. If this is indeed generally the case, then the role of allosteric effects, and in particular feedback inhibition, must primarily be to help ensure good metabolite homeostasis in the face of flux changes, as suggested by theoretical studies (Hofmeyr, 1995; Hofmeyr and Cornish-Bowden, 1991).

Taken together, the evidence from control analysis theory, the experience from over-expression of individual enzymes, and the examination of the mechanisms operating in known cases of large physiological flux changes all carry the same message: large increases in flux whilst maintaining good metabolite homeostasis cannot usually be generated by activation of single enzymes, but can be by activation at several sites along a pathway. This is an inevitable corollary of the distribution of control throughout a pathway. Hence despite the ability of molecular geneticists to drastically modify the genotype of organisms many, probably most, attempts to manipulate fluxes fail through applying incorrect strategies to identify steps for genetic overexpression. The route to new strategies for manipulation must be found in the light of new perceptions of metabolic control (Cornish Bowden et aI., 1995; Kacser and Acerenza, 1993). One point that does seem certain is that overexpression or other forms of activity engineering of a single enzyme in the supply pathway for a desired metabolite will produce disappointing results, except perhaps in the simplest of pathways.

REFERENCES

Anderson, L. E. (1986). Light/dark modulation of enzyme activity in plants. Advances in Botanical Research, 12, 1-46.

Burrell, M.M., Mooney, P.J., Blundy, M., Carter, D., Wilson, F., Green, J., Blundy, K.S., and ap Rees, T. (1994). Genetic manipulation of 6-phosphofructokinase in potato tubers. Planta, 194, 95-101.

Cornish-Bowden, A., Hofmeyr, J.-H.S., and Cardenas, M. L. (1995). Strategies for manipulating fluxes in biotechnology. Bioorganic Chemistry, 23, 439-449.

272 Chapter 13

Fell, D.A. (1992). Metabolic control analysis: a survey of its theoretical and experimental developments. Biochemical Journal, 286, 313-330.

Fell, D.A. (1996). Understanding the Control of Metabolism. Portland Press, London. Fell, D.A. and Thomas, S. (1995). Physiological control of flux: the requirement for

multisite modulation. Biochemical Journal, 311, 35-39. Geiger, D.R. and Servaites, J.C. (1994). Diurnal regulation of photosynthetic carbon

metabolism in C3 plants. Annual Review of Plant Physiology and Plant Molecular Biology, 45, 235-256.

Heinrich, R. and Rapoport, T.A. (1974). A linear steady-state treatment of enzymatic chains; general properties, control and effector strength. European Journal of Biochemistry, 42, 89-95.

Hofmeyr, J.-H.S. (1995). Metabolic regulation: a control analytic perspective. Journal of Bioenergetics and Biomembranes, 27, 479-489.

Hofmeyr, J.-H.S. and Cornish-Bowden, A. (1991). Quantitative assessment of regulation in metabolic systems. European Journal of Biochemistry, 200, 223-236.

Kacser, H. and Acerenza, L. (1993). A universal method for achieving increases in metabolite production. European Journal of Biochemistry, 216, 361-367.

Kacser, H. and Burns, J.A. (1973). The control of flux. Symposia of the Society for Experimental Biology, 27, 65-104. Reprinted in Biochemical Society Transactions 23, 341-366, 1995.

Krebs, H.A. (1946). Enzymologia, 12, 88-100. Lauerer, M., Saftic, D., Quick, W.P., Labate, c., Fichtner, K., Schulze, E.D., Rodermel, S.

R., Bogorad, L., and Stitt, M. (1993). Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with antisense rbcS. VI. Effect on photosynthesis in plants grown at different irradiance. Planta, 190, 332-345.

Mooney, PJ.F. (1994). Ph.D. thesis, University of London. Newsholme, E.A. and Start, C. (1973). Regulation in Metabolism. Wiley and Sons,

London. Niederberger, P., Prasad, R., Miozzari, G., and Kaeser, H. (1992). A strategy for increasing

an in vivo flux by genetic manipulation: the tryptophan system of yeast. Biochemical Journal, 287, 473-479.

Schaaff, I., Heinisch, J., and Zimmerman, F.K. (1989). Overproduction of glycolytic enzymes in yeast. Yeast, 5, 285-290.

Small, J.R. and Kacser, H. (1993a). Responses of metabolic sytems to large changes in enzyme activities and effectors. 1. The linear treatment of unbranched chains. European Journal of Biochemistry, 213, 613-624.

Small, J.R. and Kacser, H. (l993b). Responses of metabolic sytems to large changes in enzyme activities and effectors. 2. The linear treatment of branched pathways and metabolite concentrations. European Journal of Biochemistry, 213, 625-640.

Stitt, M., Quick, W.P., Schurr, U., Schulze, E.D., Rodermel, S.R., and Bogorad, L. (1991). Decreased ribulose 1,5-bisphosphate carboxylase/oxygenase in transgenic tobacco transformed with antisense rbcS. II. Flux control coefficients for photosynthesis in varying light, CO2 and air humidity. Planta, 183, 555-566.

Thomas, S. and Fell, D.A. (1996). Design of metabolic control for large flux changes. Journal of Theoretical Biology, 182, 285-298.

Thomas, S., Mooney, P.I.F., Burrell, M.M., and Fell, D.A. (1997a). Finite change analysis of lines of transgenic potato (Solanum tuberosum) overexpressing phosphofructokinase. Biochemical Journal, 322, 111-117.

Thomas, S., Mooney, PJ.F., Burrell, M.M., and Fell, D.A. (1997b). Metabolic control analysis of glycolysis in tuber tissue of potato (Solanum tuberosum): explanation for

13. Increasing metabolic flux 273

the low control coefficient of phosphofructokinase over respiratory flux. Biochemical Journal, 322, 119-127.

Chapter 14

Nitrate acts as a signal to control gene expression, metabolism and biomass allocation

Mark Stitt and Wolf-RUdiger Scheible Botanisches Institut der Universitiit, 1m Neuenheimer Feld 360, 69120 Heidelberg Germany

Key words: allocation; gene expression; nitrate; starch.

Abstract: Tobacco genotypes with decreased activity of nitrate reductase [NR] have been used to establish an in plant screen for processes that are regulated by nitrate. These genotypes resemble nitrate-limited wild-types when they are grown on low nitrate. However, the maximum daily rate of nitrate assimilation is restricted by the low activity of NR, and when the nitrate supply is increased these plants do not increase their rate of growth, their amino acid or protein content significantly. Instead, they accumulate large amounts of nitrate. This is accompanied by an increase of several transcripts (nia, nii, glnl, glu, icdhl, citrate synthase, cytosolic pyruvate kinase and ppc), by increased activity of the encoded enzymes, and by a dramatic accumulation of organic acids. The accumulation of nitrate also leads to repression of agpS2, a decrease of ADPglucose pyrophosphorylase activity, and a dramatic inhibition of starch synthesis. It is concluded that nitrate acts as a source of signals to initiate a coordinated and effective change in the expression of many genes whose products are required directly or indirectly during nitrate assimilation and use. Further, nitrate accumulation in the shoot results in a strong inhibition of root growth, that is primarily due to decreased formation of lateral roots, and is accompanied by changes in carbon allocation and use. It is concluded that nitrate in the shoot is monitored to provide information about the nitrogen status of the plant.

275

N. J. Kruger et al. (eds.), Regulation of Primary Metabolic Pathways in Plants, 275-306. © 1999 Kluwer Academic Publishers.

276 Chapter 14

1. INTRODUCfION

Nitrate is the major source of nitrogen for most higher plants (Marschner, 1995). However, in addition to supporting faster growth, nitrate application also leads to major changes in the metabolism, growth and phenology of higher plants. These include an increased capacity for nitrate uptake and assimilation (1m sande and Touraine, 1994; Shaner and Boyer, 1976), increased levels of amino acids and proteins (Marschner, 1995), accumulation of malate (Deng et aI., 1989; Martinoia and Rentsch, 1994), decreased starch (Hofstra et aI., 1985; Stitt and Schulze, 1994; Waring et aI., 1985), altered levels of phytohormones (Kuiper et aI., 1989; Wagner and Beck, 1993), decreased rates of root growth relative to shoot growth (Agren and Ingestad, 1987; Fichtner and Schulze, 1992; Lambers et aI., 1990; Marschner 1995; Wagner and Beck, 1993), and a delay of tuberisation (Burton, 1989) and flowering (Bernier et aI., 1993). Application of nitrate to a part of the root system leads to a local proliferation of lateral roots (Drew and Saker, 1975; Granato and Raper, 1989; Laine et aI., 1995). These far-reaching changes imply that nitrate or metabolites that are derived from nitrate assimilation are acting as signals to regulate many facets of plant metabolism and growth (Crawford, 1995; Redinbaugh and Campbell, 1991).

The regulation of nitrate uptake and assimilation and the interaction with cellular growth processes have been intensively studied in bacteria, fungi (Crawford and Arst, 1993; Marzluf, 1993) and Chlamydomonas (Fernandez and Cardenas, 1989). Genes that are required for nitrate uptake and nitrate assimilation are induced by nitrate and are repressed by metabolites that are formed during the assimilation of nitrate, in particular, glutamine. The feedback regulation is mediated by NIT2 in Neurospora and AREA in Aspergillus. These proteins contain cys2/cys2 type zinc finger motifs, and bind to promoter regions of nitrogen regulated genes to increase the expression of enzymes for nitrate assimilation in conditions of nitrogen limitation, specifically low glutamine. They also increase transcription of a large number of unlinked structural genes that specify nitrogen-catabolic enzymes.

It has been known for a long time that NR activity increases when nitrate is added to plants (Shaner and Boyer, 1976). This was originally seen as an interesting but unusual case of a microbe-like induction of an enzyme by its substrate in higher plants. Recent work has deepened and extended this picture. First, it is now clear that the increase of NR activity involves transcriptional regulation. Addition of nitrate leads to a rapid increase of transcript for nia (Cheng et aI., 1991; Pouteau et aI., 1989). This increase cannot be blocked with cycloheximide (Gowri et aI.,

14. Nitrate as a signal 277

1992), and also occurs in mutants where functional NR is not formed due to point mutations in nia or to mutations in the pathway for synthesis of pterin, which is an essential Mo-binding cofactor for NR (Vaucheret et ai., 1990) The increase of nia transcript is followed within 1 hour by an increase ofNR protein and NR activity (Scheible et ai., 1997c). Secondly, nia is repressed by glutamine (Hoff et aI., 1994), and the decrease of transcript is accompanied by a rapid decrease of NR protein and activity (Galangau et ai., 1988; Scheible et aI., 1997c). This is reminiscent of the response in fungi, but the molecular mechanism has not yet been defined in higher plants. Thirdly, nitrite reductase is regulated in a coordinated manner to NR (Faure et aI., 1991). It is also induced by nitrate and repressed by ammonium or products of ammonium assimilation (Kronenberger et aI., 1993; Wray, 1993). Fourthly, the high affinity nitrate transporter has recently been cloned from several higher plants. It is also induced by nitrate, and repressed by ammonium or products of ammonium assimilation (Quesada et aI., 1997; Trueman et aI., 1996).

Many further processes are also required during nitrate assimilation (Figure 1). These include the subsequent assimilation of ammonium, and modifications of fluxes of nitrate and other ions across cell membranes. Nitrate assimilation also requires a series of changes in carbon metabolism. Redox equivalents will be required to convert nitrate to ammonium and amino acids. Oxoglutarate is required to act as a primary acceptor for ammonium in the GOGA T pathway, a variety of further carbon skeletons will also be needed as acceptors in the amino acid biosynthesis pathways, and malate or other organic acids must be synthesised to prevent alkalinisation of the cytosol. This is especially important in aerial organs and in bulky tissues that are not in direct contact with the soil water, and therefore only have a limited ability to regulate their internal pH by proton exchange across membranes surrounding the cytoplasm. Changes in the synthesis and turnover of the major carbohydrate storage and transport pools like starch and sucrose will be necessary, in order to divert more carbon into organic acid and amino acid synthesis. We already know that nitrate assimilation is tightly controlled at the transcriptional (Hoff et aI., 1994; Vincentz et aI., 1993) and post-translational level (Kaiser and Huber, 1994) in response to the supply of sugar, and a reciprocal regulation of carbon metabolism by signals emanating from nitrogen metabolism is presumably also operative.

Studies of the regulation of the processes downstream of ammonium is complicated, however, because it is difficult to distinguish between regulation exerted by nitrate itself, and indirect effects that are triggered as a result of the conversion of nitrate to ammonium and other compounds. Nitrate addition leads to a rapid and cycloheximide-

278

CO2

3PGA t 1 \ Calvin 1

" c cle 1 ---I

~ 1 .... -GlulP

I

" starch IChloroplasd

!Cytosol!

~ 1\ cO'T-+ malate

1 \ \ (r.7':I ,Imltochondrionl

1!1 ~P ~ pyr -r :.C::: cit/ate c cle /

+-/

"'--tim 1

sucrose

II --IC~

co,

Chapter 14

1m NH'

A G>e.gy.~n aOG Glu

amino acids J Figure 1. Pathways of primary nitrogen and carbon metabolism in an autotrophic plant cell. The scheme shows the main enzymes (in filled boxes) including NR, NiR, GS, GOGAT, NADP-dependent isocitrate dehydrogenase (NADP-ICDH), citrate synthase (CS), pyruvate kinase (PK), PEPcase, SPS, and AGPase and key metabolites including glutamine (gIn), glutamate (glu), a-oxoglutarate (aOG), isocitrate (lC), pyruvate (pyr), phosphoenolpyruvate (PEP), glycerate-3-phosphate (3PGA), glucose I-phosphate (GluIP) and ribulose I,5-bisphosphate (RuBP). Enzymatic reactions are shown as thin black lines and transport processes as bold black lines. The figure is modified from Scheible et aI. (I997b) with permission.

14. Nitrate as a signal 279

insensitive increase of gln2 (encoding the plastidic glutamine synthetase [GS2]) (Redinbaugh and Campbell, 1993) and ferredoxin-NADP­oxidoreductase (Ritchie et aI., 1994) in maize roots. An analogous induction of gln2 was not seen in leaves. The de novo assimilation of nitrogen in leaves may occur via cytosolic GSI (Lam et aI., 1996), and addition of nitrate does induce one gln1 form in maize leaves (Sukanya et aI., 1994). Nitrate addition also leads to an increase of the transcript for ppc (which encodes phosphoenolpyruvate carboxylase [PEP case ]) in maize leaves (Sugiharto and Sugiyama, 1992) and an increase of the transcript for icdhl (which encodes the cytosolic NADP-dependent isocitrate dehydrogenase [NADP-ICDH] and is responsible for the synthesis of oxoglutarate during nitrogen assimilation) in potato leaves (Fieuw et aI., 1995). However, these changes could be indirect, and indeed, in the case of ppc it was shown that ammonium and glutamine were more effective than nitrate (Sugiharto et aI., 1992).

Identification of the compounds in nitrogen metabolism that are monitored to initiate the far-reaching changes in whole plant allocation, development and growth is even more difficult. Although some studies have found a correlation between total nitrogen and shoot-root allocation (Agren, 1985; Agren and Ingestad, 1987; Levin et aI., 1989), other studies have found that shoot-root allocation is correlated with the level of starch or sugars, or the ratio of amino acids to sugars (Buysse et aI., 1993; Chu et aI., 1992; Ericsson, 1995; Farrar, 1996; Huber, 1983; van der Werf and Nagel, 1996). Such correlations are to be expected, given that nitrate assimilation is likely to be tightly integrated with amino acid metabolism and carbon metabolism, and do not provide causal information about specific interactions.

In this chapter, we summarise recent experiments in which we have used tobacco plants with decreased expression of NR as a tool to dissect the nitrogen-signalling pathways in higher plants. In low-NR genotypes, the rate of nitrate assimilation is artificially restricted and the nitrate supply and the nitrate concentration in the plant can be varied without this leading to large changes in the rate of nitrate assimilation. This provides an in planta experimental screen, in which it is possible to identify processes that are directly regulated by nitrate, and to separate them from processes that are regulated by signals related to the levels of ammonium or amino acids, or processes that are affected very indirectly as a consequence of the increased rates of growth and changes in the levels of many other compounds in the plant.

280 Chapter 14

2. NITRATE REDUCfASE IS PRESENT IN LARGE EXCESS IN TOBACCO

The temporal changes in NR activity and nitrate assimilation in wild­type plants were investigated first, to provide background information to allow the most appropriate low-NR genotypes to be chosen, and the best sampling strategy to be planned. NR is present in excess in wild-type tobacco (see Scheible et aI., 1997c) and is subject to a sophisticated hierarchy of transcriptional, post-transcriptional and post-translational regulation, that allows NR activity to be continuously adjusted in response to the current availability of nitrate, the accumulation of glutamine, and the availability of carbon in the cell (Hoff et aI., 1994; Scheible et aI., 1997c). NR regulates the rate of nitrate assimilation by continually adjusting the rate of nitrate assimilation to the current environmental and physiological constraints, rather than by imposing a rigid bottleneck.

This can be illustrated by considering the diurnal changes of NR activity and nitrogen metabolism in wild-type plants growing in a day­neutral or long-day light regime in high nitrate. There is a maximum of the nia transcript at the beginning of the day, and NR protein and activity increase 2- to 3-fold during the first 2-4 hours of the photoperiod. As a result, nitrate is rapidly assimilated, nitrate declines in the leaf, glutamine accumulates, and feedback control of nitrate assimilation is initiated. The transcript for nia begins to decline early in the photoperiod, and NR protein and activity decline by 50% or more during the second part of the photoperiod (Galangau et aI., 1988; Scheible et aI., 1997c). Post-translational regulation leads to further, rapid, changes of NR activity that are superimposed on these slower changes. NR is activated after illumination, and deactivated after darkening (Kaiser and Huber, 1994). Inactivation involves phosphorylation followed by a magnesium-dependent binding of an inhibitory 14-3-3 protein (Bachmann et aI., 1996; Moorhead et aI., 1996). The post-translational regulation of NR is probably triggered by changes in sugars (Kaiser and Huber, 1994) and (see Scheible et aI., 1997c) by changes in glutamine or related compounds.

Nitrate assimilation is also a very dynamic process in plants growing on limiting nitrate. In this case, after each re-watering with a low concentration of nitrate, there is a rapid induction of NR and a brief period of rapid nitrate assimilation (Scheible et aI., 1997c).

14. Nitrate as a signal 281

3. NITRATE-REGULATED PROCESSES

3.1 Screening for nitrate-regulated processes

In order to restrict the rate of nitrate assimilation and override these highly flexible internal regulation systems, it was necessary to achieve a large and consistent inhibition of NR expression. Wild-type tobacco has 4 gene copies for NR at the loci nial and nia2. Homozygous single mutants with point mutations in either nial (F23) or nia2 (F22) and a double null mutant line (nial-, nial-, nia2-, nia2-) which exhibits no NR activity were originally isolated by MUller and co-workers (see MUller and Mendel, 1989). The mutated nia genes are still transcribed but a non­functional protein is produced (Vaucheret et aI., 1990). The double null mutant line Nia30 was transformed by Vaucheret et ai. (1990) with a 12 kb genomic sequence containing a Skb upstream promoter sequence, the entire structural sequence, and a 2 kb downstream section of the endogenous tobacco nia2 gene. The resulting lines Nia30(14S) and Nia30(461) have very low NR activity, equivalent to 1-3% of the activity in wild-type plants.

Our experiments could have been carried out with totally NR-deficient genotypes growing on ammonium supplemented with varying amounts of nitrate as a nitrogen source. However, growth on ammonium nitrate would have seriously complicated the design and interpretation of the experiments. In wild-type plants, changes in intracellular pH will be minimised because they are able to carry out parallel assimilation of nitrate and ammonium. In the transformants and mutants, however, the decreased rates of nitrate assimilation could lead to complicated changes in the cellular pH and their ability to utilise ammonium. The low level of NR activity in the transform ants Nia30(14S) and Nia30(461) was sufficient to allow slow growth on nitrate as the sole nitrogen source, thus avoiding potential complications in low-NR genotypes due to changes in pH and ammonium assimilation.

In these Nia30(14S) and Nia30(461) transformants, the diurnal changes of nia transcript were almost completely abolished (Scheible et aI., 1997c; Vaucheret et aI., 1990), and the dark inactivation of NR by post-translational modification was also abolished (Scheible et aI., 1997c). As a result, NR was present at a low but constant activity throughout the day and night (Scheible et aI., 1997c) that places a constant restriction on the rate of nitrate assimilation. When Nia30(14S) or Nia30(461) were grown on low (0.2 mM) nitrate they resembled nitrate-deficient wild-type plants with respect to their overall

282 Chapter 14

rates of growth and their content of protein and amino acids (Figure 2; see Scheible et aI., 1997a for more details). These plants maintain a low but constant rate of nitrate assimilation, whereas the wild-type has rapid but short-term bursts of nitrate assimilation after each re-watering (see above). When Nia30(145) was supplied with higher nitrate the plants remained, effectively, nitrogen-limited. The growth rate (Figure 2A) and their content of amino acids and protein (Figure 2B, 2C) only increased slightly, but they accumulated large amounts of nitrate (Figure 2D). The metabolism and growth phenology of the transformants growing on high nitrate should resemble that of nitrogen-deficient wild-types, except for processes which are responding directly to nitrate.

The transform ant Nia30(145) was also crossed with the single mutants F22 and F23 (see above) to generate lines with one functional copy of nial [F22xNia30] or one functional copy of nia2 [F23xNia30]. The single mutants were also crossed to generate plants with one functional copy of nial and one functional copy of nia2 [F22xF23]. These mutants with one or two instead of four functional nia gene copies had a small decrease in NR activity (50-80% of the wild-type NR activity when NR activity is measured at the daily maximum in the middle of the photoperiod, see Scheible et aI., 1997c), and also showed a strong dampening of the diurnal changes of NR activity and nitrate assimilation. In mutants with decreased NR activity, the decline of nitrate and increase of glutamine during the photoperiod occurs more slowly than in wild-type plants. As a result, the decline of NR activity and protein during the second part of the photoperiod is almost completely suppressed, and the dark inactivation of NR is reversed (Scheible et aI., 1997c), probably because this inactivation requires a combination of low sugar and high amino acids (Scheible et aI., 1997c; R. Morcuende and M. Stitt, unpublished).

These mutants with a small decrease in NR provide a less extreme system to check the conclusions drawn from studies of the very low-NR transformants. The mutants F22xNia30 and F23xNia30 grow at almost the same rate as wild-type plants, and also resemble wild-type plants with respect to their overall amino acid levels and protein content (Scheible et aI., 1997a). However, for most of the time they contain about 2-fold higher levels of nitrate and about 50% lower ammonium and glutamine than wild-type plants. These mutants should therefore respond like marginally nitrogen-limited wild-type plants, except for processes which are regulated by nitrate itself. Further, the modified diurnal changes of nitrate and glutamine provide a sensitive system to investigate whether small, short-term changes of nitrate or glutamine lead to significant changes of gene expression and metabolic activity.

14. Nitrate as a signal 283

0.4 Wild-type D Nia30(14S}

RGR A 0.3

.,~ 0.2 Ol

.9 0.1

0.0

~ 25

~ 20

Ol 15 Ol 10 g

5 0

~ 20

15 Ol (5 10 E 2: 5

0

-~ 200

100 Ol (5 40 E 2: 20

0

5 -~ 4 Ol 3 (5 2 E 2: 1

0 12 0.2 12 0.2

nitrate supply (mM)

Figure 2. Growth and composition of wild type plants and severely NR-deficient transform ants growing on high and low nitrate. Wild-type plants and Nia30(l45) were grown on nutrient medium containing 12 or 0.2 mM nitrate, and harvested at the rosette stage 32 days (wild-type plants on 12 mM nitrate), 62 days (wild-type plants on 0.2 roM nitrate and Nia30(\45) on 12 mM nitrate) or 85-90 days (Nia30(145) on 0.2 mM nitrate) after germination. Protein, amino acids and nitrate were measured in samples taken after 4 hours illumination in the first fully expanded leaf and are related to the fresh weight (FW) of the tissue. The relative growth rate (RGR) is given as the relative daily increase in dry weight (g g-l dol). The results are the mean ± SE of three experiments, each with four separate plants. The figure is modified from Scheible et al. (\ 997b) with permission.

284 Chapter 14

This plant set possessed a further feature that greatly aided the execution and interpretation of our experiments. In transformants where gene expression is inhibited by antisense or co-supression the magnitude of the inhibition of gene expression is typically rather variable, depending on the organ involved, the plant age or the conditions. In contrast, each low-NR genotype showed a remarkably constant depression of NR activity relative to the activity in the wild-type. For a given genotype, there was a similar inhibition of activity in the shoots and the roots (Scheible et aI., 1997a). The inhibition of NR activity in a given genotype (relative to wild-type activity) was also independent of the nitrate supply (Scheible et ai. 1997a), the presence of nitrate or ammonium nitrate (Lauerer, 1996; Scheible et aI., 1997a), daylength and the light intensity (P. Matt and M. Stitt, unpublished data). This greatly simplified the interpretation of our results, because it eliminated side­effects due to organ- or condition-specific fluctuations in the rate of nitrate assimilation in the mutants and transformants. The reproducible effect on NR activities is presumably a result of the nia genes being under the control of the same native promoter elements. In contrast, in antisense or cosupression transform ants, the inhibiting sequence is typically under the control of a different, and often non-native, promoter and the rates of transcription of the transgene and the target gene will vary independently, leading to a large varation in the extent of the inhibition.

The wild-type plants and the low-NR transformants were grown in controlled conditions in the presence of high or low nitrate. The first aim was to identify processes in Nia30(14S) and Nia30(461) grown on high nitrate which responded as if these low-NR plants were nitrogen-replete. We also investigated whether these changes could be reversed by growing these low-NR transformants on low nitrate, and if similar trends could be seen when F23xNia30 and F22xNia30 mutants with a small decrease in NR activity were compared with wild-type plants.

3.2 Gene expression

The midday levels of transcripts for several key genes in nitrogen and carbon metabolism are summarised in Figure 3. Growth of wild-type plants on low nitrate led to a decrease of the transcripts for nia, nii, gin I , glu and ppc, encoding NR, nitrite reductase, cytosolic glutamine synthetase, GOGAT and PEPcase, respectively. These transcripts were all dramatically increased in Nia30(14S) when it was grown on 12 mM nitrate, and therefore show the opposite response to that expected in a nitrogen-deficient plant. Nia30(14S) growing on high nitrate also has

14. Nitrate as a signal 285

high levels of the transcripts encoding the cytosolic pyruvate kinase, NADP-ICDH and citrate synthase (see Scheible et aI., 1997b).

When Nia30(14S) was grown on 0.2 mM nitrate the transcripts all decreased, although to a different extent. The transcripts for ppc and glu decreased to levels similar to those found in nitrate-limited wild-type plants, whereas the transcripts for nia, nii and gin 1 were still relatively high (Figure 2). Even in these conditions, there is a significant level of nitrate in Nia30(14S). The latter group of transcripts decreased to very low levels when transformants were grown on ammonium (see Scheible et aI., 1997b). The differing response of the two groups of genes may reflect differences in the sensitivity of their response to nitrate, or it may reflect a different interaction between signals derived from nitrate and from metabolites formed downstream of GS. It is known (see above) that low glutamine will sensitise nia and nii to nitrate induction. Further experiments are needed to investigate whether gin 1 is repressed and glu and ppc are induced by glutamine or related metabolites. For this purpose, the low-NR transformants will be invaluable, because they will allow ammonium and glutamine to be added without this automatically leading to changes in the rate of nitrate assimilation and the level of nitrate (which seriously interfere with the interpretation of such experiments in wild-type plants, R. Morcuende and M. Stitt, unpublished results).

The key enzymes for the regulation of carbohydrate synthesis are ADPglucose pyrophosphorylase (AGPase) for starch and sucrose phosphate synthase (SPS) and the cytosolic fructose-1,6-bisphosphatase for sucrose (Stitt, 1996). We investigated the transcripts for agpS2 (the regulatory subunit of AGPase) and sps. The transcript for agpS2 was high in nitrate-limited wild-type plants, was low in Nia30(14S) grown in the presence of 12 mM nitrate, and increased dramatically when Nia30(14S) was grown on low nitrate. In contrast, there were no marked changes of the sps transcript.

The transcripts measured in Figure 3 were prepared from plants that had been growing for several weeks at different nitrate supplies, and had large differences in their nitrate content.

Two further sets of experiments were therefore carried out to investigate whether (a) the transcripts change rapidly in response to short-term changes of the nitrate supply and (b) the transcripts for these genes change in response to small changes of nitrate.

In one approach, wild-type plants and Nia30(14S) were grown on low nitrate (which involved watering them each day 2 h into the light period with 0.2 mM nitrate) and then transferred to 12 mM nitrate. Supplying intact plants with 12 mM nitrate led, within 2 h, to a dramatic increase in the transcripts for nia and ppc in the leaves (Figure 4). This increase of transcript occurred before the nitrate level in the leaf had increased by

286 Chapter 14

wildtype Nia30(14S)

gene enzyme

acronyme 12 0.2

nia NR

nii NiR

GSl gln1

(cytosolic) •• -- --

gln2 GS2

glu •• ppc PEPcase -.

agpS2

SpS SPS

18S

Figure 3. Nitrate accumulation in severely NR-deficient transform ants induces genes encoding enzymes for nitrate and ammonium assimilation, induces PEPcase, and represses AGPase in source leaves. Transcripts for nia. nii. gin], gln2. glu. ppc. agpS2, sps and 18S as a control, are shown for RNA preparations from the youngest fully expanded leaves of two separate groups of plants. The plants were grown as in Figure 2 and samples were taken after 4 hours illumination. The figure is taken from Scheible et al. (1997b) with permission.

14. Nitrate as a signal 287

more than 2-fold over that in a nitrate-limited plant. The transcript increased before rapid nitrate assimilation commenced, malate and amino acids changed, or growth increased, and the transcripts anyway increased at a similar rate in wild-type plants and in NR-deficient lines (see Scheible et aI., 1997b for details). Nitrate also led to rapid changes of agpS2 transcript. This transcript showed a marked light-dependent increase at the start of the photoperiod. In the controls the subsequent addition of 0.2 mM nitrate led to a small and transient decrease of agpS2, and when 12 mM nitrate was added there was a dramatic and sustained decrease of agpS2 transcript (Figure 4). This decrease occurred at a similar rate in wild-type plants and Nia30(14S), was completed within 4 h of adding nitrate, and also preceded any changes in growth.

In a second approach, the diurnal changes of the transcripts for nia, nii, ppc, cytosolic pyruvate kinase, icdhl and citrate synthase were monitored at six time points during the day and night in wild-type plants and in F23xNia30 mutants with a small decrease in NR activity. The transcripts were all consistently increased in F23xNia30 (W.-R. Scheible and M. Stitt, data not shown). Further, each transcript showed a marked diurnal rhythm in wild-type plants, and the rhythm was modified in the mutants.

3.3 Organic acid formation

We next investigated whether the changes of transcript led to changes in the activities of the encoded enzymes and to changes in the fluxes in the pathways in which they operate. The results will be presented first for nitrogen and organic acid metabolism and then (see the next section) for starch metabolism.

Wild-type plants growing on low nitrate have rather low activities of NR, nitrite reductase and PEPcase. In contrast, Nia30(14S) growing on 12 mM nitrate had high levels ofNR protein (most of which of course is derived from the mutated gene copies in this transformed double null mutant), and very high nitrite reductase and PEPcase activity (Figure SA, SB, SD). These gene products increased by almost 10-fold compared to nitrate-limited wild-type plants, and were also 2 to 4-fold higher than in well-fertilised wild-type plants when the activities are compared on a protein basis. These results demonstrate that the changes of transcripts shown in Figures 3 and 4 lead to significant alterations in the activities of the encoded proteins. Marked increases in NR protein, nitrite reductase activity and PEPcase activity were also found in the roots of Nia30(14S) when it was grown on high nitrate.

288

time (hr) after

IrrigaHon

nia T

C

ppc T

C

agpS2 T

C

185 T

C

Chapter 14

NIA30(145) o 2 I 4 I 10 50

~-­~ L-__ ~~ ..... _~

Figure 4. Changes of nia, ppc and agpS2 transcript after re-supplying high nitrate to nitrate depleted wild-type plants and severely NR-deficient transformants. Plants had been grown on 0.2 mM nitrate provided each day after 2 hours illumination up to the day on which the experiment began. On this day, control plants were provided with 0.2 mM nitrate as usual (termed C in the panels) or were irrigated with 12 mM nitrate nutrient solution after 2 hours illumination (termed T). The figure is taken from Scheible et al. (l997b) with permission.

14. Nitrate as a signal 289

Overall GS activity (Figure 5C) did not increase in the leaves, but in this tissue most of the GS activity is attributable to the plastidic isoform GS2 (Lam et aI., 1996), and the transcript for this form was only slightly increased by nitrate (Figure 3). In roots, where the cytosolic en contributes most of the overall activity, there was a 3 to 5-fold increase of overall GS activity when Nia30(145) was grown on high nitrate (see Scheible et aI., 1997b).

PEPcase is also regulated by protein phosphorylation, leading to altered sensitivity to inhibition by malate (Chollet et aI., 1996; Li et aI., 1996). Such changes are found after illumination in C4 plants, and also after supplying nitrate to C3 leaves (Champigny and Foyer, 1992). Our results confirm that PEPcase is more sensitive to inhibition by malate in nitrate-limited wild-type plants (Figure 5G). They also reveal that there is a marked decrease in sensitivity to malate-inhibition when high nitrate is supplied to Nia30(145). This indicates that nitrate itself acts on the cascades that are responsible for the post-translational regulation of PEPcase.

The increase in PEPcase activity and activation in the leaves of Nia30(145) growing on high nitrate was accompanied by a large accumulation of organic acids including oxoglutarate, isocitrate and citrate, and a depletion of phosphoenolpyruvate and pyruvate (Figure 51-N). There was a similar or even larger accumulation of oxoglutarate, isocitrate, citrate and malate in the roots (see Scheible et aI., 1997b). These results show that the conversion of glycolytic precursors to organic acids is massively stimulated by nitrate. Nitrate therefore not only induces the expression of genes required for nitrate uptake and reduction, but also initiates a coordinated and highly effective increase in the expression of genes required for ammonium assimilation, and of genes that are required to synthesise organic acid acceptors.

3.4 Starch synthesis

Nitrate-limited wild-type plants have relatively high AGPase activity, and high levels of starch (Figure 5E, 5P). The low level of agpS2 transcript in Nia30(145) on high nitrate was accompanied by a low AGPase activity. When Nia30(145) was grown on low nitrate, the recovery of agpS2 transcript was accompanied by an increase of AGPase activity. Interestingly, this decrease in AGPase activity when nitrate accumulated was not accompanied by a decrease in sps transcript or SPS activity, indeed these even increased slightly (Figure 5F). This shows that nitrate is leading to a specific decrease of starch-synthesising enzymes, rather than a general inhibition of carbohydrate synthesis.

290

l;300

~200 JO

" ~ '00 ~

0

'00

200

0

300

200

' 00 0

-~ 75

- 50 " .,

25 e 3

7S

50

25

40

20

0

80

60

..- 40 0:.

40

20

0 12 0.2 12 0.2

-:- 800

iEooo co 400

1200

iE so:r J.-~L .. (..JI"""".-,-'--'-oJ.J1 i ~OO I 2~

12 0.2 12 0.2

1.0

0 .5

0.0 ....... ______ ..1-.LJ

0.31LJ]IOOCI1ta.o 0.2

-- 0.' ~ 0.0

I : LJel1to.,o

.0

a

:[J ~ ':W 1.00 N PEP

so

o

.. ~ 15 co Z '0

~ 5

12 0 .2 12

[ ,: .J..f-iL..JM~-' -'-n--'-'-n..LJ1 '0 5

~ o

12 0.2 12 0.2

nitrate supply (mM)

Chapter 14

Figure 5. Alterations of enzyme activities and of metabolites in the source leaves of wild­type plants (black bars) and Nia30(145) transformants (white bars) growing on 12 and 0.2 mM nitrate. All measurements were carried out on samples taken after 4 hours illumination from the youngest fully expanded leaf of the plants at the same time as samples were taken for the measurements of the transcripts shown in Figure 3. For plant age and sampling see legend to Figure 2. The results are related to fresh weight (FW) and are the mean ± SE of four to six separate plants (n.a. = not analyzed). The figure is modified from Scheible et at. (1997b) with permission.

14. Nitrate as a signal 291

AGPase is a highly regulated allosteric enzyme, that is activated by glycerate-3-phosphate and inhibited by inorganic phosphate (Preiss et aI., 1991). Glycerate-3-phosphate is relatively high in nitrate-limited wild­type plants (Figure SO), and this will reinforce the impact of the high expression of AGPase and favour starch synthesis in nitrogen-limited plants. When nitrate accumulates in Nia30(14S) there is a strong stimulation of organic acid synthesis (see above) and, as a result, the levels of phosphoenolpyruvate and linked metabolites including glycerate-3-phosphate decline (Figure SN, SO). Thus, nitrate also exerts an indirect effect on AGPase activity, via changes in the level of the allosteric activator.

The decreased expression of AGPase and decreased level of glycerate-3-P leads to a massive inhibition of starch synthesis when nitrate accumulates in Nia30(14S) (Figure S, see Scheible et aI., 1997a; 1997b for more results). Even though Nia30(14S) is severely deficient for organic nitrogen and is growing very slowly, the plants contain almost no starch in their leaves when they are grown on high nitrate. An even more striking result is obtained when wild-types and Nia30(14S) are grown on low nitrate, and then transferred to high nitrate. Both sets of plants contain high levels of starch when they are grown on low nitrate. When nitrate is added to wild-type plants, growth is stimulated after about 2 days, and the starch is almost completely re-mobilised within 4-S days. Starch is remobilised at almost the same rate after adding nitrate to Nia30(14S), even though the rate of growth is not significantly stimulated in these plants (see Scheible et aI., 1997b).

Nitrogen-deficient plants typically accumulate large amounts of starch (Fichtner and Schulze 1992; Hofstra et aI., 1985; Marschner, 1995; Waring et aI., 1985). It has been assumed that this just reflects the low rate of growth of the plants and the resulting excess of carbon. The results obtained with the Nia30(14S) plants, however, show that it is possible to completely break this correlation between starch accumulation and plant growth, and demonstrate that signals derived from nitrate play a major role in regulating the rate of starch synthesis. This co-ordinated feed-forward regulation of starch metabolism and nitrate assimilation will minimise imbalances due to the lags that would unavoidably develop if starch metabolism were regulated in a more indirect manner. It also explains why nitrogen-limited plants contain higher levels of starch even though the levels of phosphorylated metabolites and sugars are lower than in well-fertilised plants (Fichtner et aI., 1993; Lauerer, 1996; W.-R. Scheible and M. Stitt, unpublished data).

292 Chapter 14

3.5 Shoot:root ratio

The experiments described so far show that nitrate acts as a signal to regulate cellular gene expression and metabolism at the level of the cell. However, many of the responses to nitrogen occur at the whole plant level, involving changes in allocation, development and growth. To investigate whether nitrate also acts as a signal to modulate whole plant processes, we investigated shoot-root allocation. As outlined in the Section 1, low nitrate leads to a preferential stimulation of root growth, resulting in a decrease of the shoot root ratio. The ecological significance of this adaptation may be that it allows increased investment in root growth to improve nitrate acquisition from the soil. It will also decrease the amount of leaf biomass being formed, and hence decrease the requirement for nitrogen (Bloom et aI., 1985).

When wild-type tobacco plants were grown on low nitrate, the shoot root ratio decreased from about 3.2 on 12 mM nitrate to about 1.8 on 0.2 mM nitrate (Figure 6). Nia30(145) showed a similar shoot:root ratio to the wild-type on low nitrate, but when Nia30(145) was grown on 12 mM nitrate the shoot root ratio rose to 8 and more. Thus, although these plants were severely deficient in organic nitrogen, they show a more extreme response of shoot-root allocation than nitrogen-replete wild-type plants. The increase of the shoot root ratio in Nia30(145) in high nitrate was caused by a 70-80% inhibition of the rate of root growth, while the rate of shoot growth increased by 70-80% (Scheible et aI., 1997a). These results indicated that nitrate itself acts as a source of signals to regulate shoot-root allocation.

Two further sets of experiments were carried out to investigate whether small changes of nitrate lead to a change in shoot-root allocation. In one set of experiments, Nia30(14S) and wild-type plants were grown at a range of nitrate concentrations between 0.2 and 20 mM. Nitrate accumulated in Nia30(145) as the external nitrate was increased in the range between 0.2 and 1 mM nitrate, and this accumulation was accompanied by an inhibition of root growth and an increase of the shoot root ratio (Scheible et aI., 1997a). Nitrate accumulated in wild-type plants as the external concentration was increased in the range between 1.2 and 20 mM nitrate, and this was also accompanied by an inhibition of root growth and an increase of the shoot root ratio. In a second set of experiments, the shoot:root ratio and rates of root growth were compared in wild-type plants and in mutants with a small decrease of NR activity and increase of nitrate. In several replicate experiments a small but highly significant inhibition of root growth and increase of the shoot root ratio was found in the mutants. The results of these

14. Nitrate as a signal 293

experiments are summarised in Figure 6. There is a highly significant correlation between the nitrate content of the shoot and the shoot:root ratio. This correlation holds, irrespective of whether the nitrate content is changed by altering the nitrate supply to the plant, or by altering the NR activity in the plant.

This highly significant correlation indicated that the accumulation of nitrate in the shoot was responsible for the change in root growth and allocation. The changes of nitrate in the roots were less marked and did not correlate with the changes in allocation (data not shown). To provide more direct evidence that nitrate is sensed in the shoot, a split-root experiment was carried out. Plantlets were grown initially on 1 mM ammonium chloride, and the root systems were then divided into two halves and placed in separate pots. One half of the roots was subsequently fertilised with low (0.2 mM) nitrate and the other half was fertilised with high (12 mM) nitrate. The plants were harvested 3 and 6 weeks later. Control measurements (see Scheible et aI., 1997a) showed that the nitrate levels in the shoot of the split-root plants and in the high-nitrate root sector resembled those found in the shoot and roots of a control plant that was growing with an undivided root system in 12 mM nitrate. The low-nitrate root sector contained very low nitrate, as expected since nitrate is not effectively translocated in the phloem. In interpreting the following results, it is helpful to remember that application of nitrate to a small part of the root system leads to a local stimulation of root growth due to proliferation of lateral roots (see Introduction for references).

Growth was almost completely stopped in the root sector that received 0.2 mM nitrate (Figure 7). The impact of nitrate accumulation in the shoot on root growth is revealed by comparing (i) the rate of root growth in plants whose whole root system received 0.2 mM nitrate and (ii) the rate of growth of the root sector that received 0.2 mM nitrate in split-root plants. Both of these root systems are receiving the same local concentration of nitrate, but the former is attached to a plant with a low shoot nitrate content and the latter is attached to a plant with a high level of nitrate in the shoot. The root system grew 8-20 fold faster when the shoot contained low nitrate.

The sector of the split root plant that received 12 mM nitrate grew at a slightly higher rate than the root system of a plant whose entire root system was in 12 mM nitrate. This, plus the similar rates of growth of the shoot in control and split-root plants provides evidence that the split-root treatment has not altered the rate of growth of the plant. The faster growth of the 12 mM root sector compared to the 0.2 mM root sector is due to the local stimulatory effect of nitrate on root growth mentioned above. Our results show that this local stimulation of root growth by nitrate does not require high local rates of nitrate assimilation.

294 Chapter 14

12 12 • wlldtype IB 3mM NH: ~ ::Q • F22xF23 ~ 10 10 0 0.2 mM NO;

C • F23x145 0 0.4 mM NO, CI

~ 8 , 7 8 • F22x145 0 0.8 mM NO,

..!!! 15 0 Nia46 '\l 1.2 mM NO,'

0 6 c , 2

6 0 Nla34 /::;. 1.S mM NO,'

~ "}l] 0 Nia30(145) &. SmMNO; '0

38

e 4 36 4 0 Nia30(461) 0 12mM NO;

15 r = 0.978 34 Gl 20 mM NO;

0 2 32 2 .c 30 /II 200 400 600 800

0 0

0 500 1000 1500 2000 2500 3000 3500

shoot nitrate (llmol gOW')

Figure 6. Relation between shoot-root allocation and the nitrate content of the leaves. Results are shown for wild-types, for F22xF23, F23xNia30, F22xNia30 and Nia46 mutants with slightly or moderately decreased expression of nitrate reductase, and for Nia30(l45), Nia30(461) transformants and Nia34 mutants with strongly decreased expression of nitrate reductase (see Scheible et al. (1997a) for nitrate reductase expression). The plants were grown on 3 mM ammonium chloride, or on 0.2, 0.4, 0.8, 1.2, 1.6,6, 12 or 20 mM nitrate. The inserts show the results for plants growing on ammonium or low nitrate supply (top left hand comer) or for wild-type and mutants with slightly or moderately reduced NR activity growing on high nitrate (bottom right hand comer). The symbols for the genotype and nitrogen nutrition are given at the right side of the figure. The regression line was calculated by considering all plants with nitrate-pools higher than 100 !lmol g DW-1 and was extended to the axes. The figure is modified from Scheible et al. (1997a) with permission.

14. Nitrate as a signal

-,.... :a ~ C en 3: c en -

0.08

0.06

0.04

~ 0.02

o 0.00

split-root plants

0-3 weeks 3-6 weeks

295

controls

12 mM 0.2 mM

Figure 7. Comparison of the growth response of root sectors supplied with high and low nitrate in a split-root experiment. Plants were grown for seven weeks in 1 ruM ammonium chloride, before dividing their root systems into two equal halves, replanting in sand in pots with two compartments, one of which was watered daily to field capacity with nutrient solution containing 12 ruM nitrate, and the other with 0.2 ruM nitrate. Successive harvests were carried out after three and six weeks. The results are the mean ± SE (n= 4). Growth of the shoot (open), the root sector that received 12 ruM nitrate (hatched),and the root sector that received 0.2 ruM nitrate (solid) in the Nia30(l45) transforrnant, were calculated for the time intervals (after transfer to the split root system) of 0-3 weeks, and 3-6 weeks. The rates of shoot( open) and root growth of transformants growing with their whole root system in 12 (hatched) and 0.2 ruM (solid) nitrate are given for comparison. The results are from Scheible et al. (l997a) with permission.

296 Chapter 14

These experiments show that shoot nitrate plays a very important role in the control of shoot-root allocation. Shoot-root allocation may also be controlled by other signals further downstream in nitrogen metabolism (M.Stitt, G. Meyer zu Horste, A.Krapp, unpublished data; see also discussion in Scheible et aI., 1997a). However, nitrate can clearly act in an effective manner in the absence of signals derived from ammonia or amino acids. The inhibition of root growth by shoot nitrate could involve changes in the acquisition or transport of assimilates to the root, or it could involve a more direct signal that modulates growth processes in the roots. As will now be discussed, Nia30(145) provides a useful system to investigate the underlying mechanisms, because the changes in allocation have been uncoupled from the changes of nitrate assimilation and overall growth rate, which complicate the interpretation of experiments in wild­type plants.

One explanation for the decrease of the shoot root ratio in low pitrate postulates that the root NR assimilates an increased proportion of the incoming nitrate, and utilises it directly for root growth (Brouwer, 1962). Our results demonstrate that this model cannot explain the nitrate-mediated inhibition of root growth. When Nia30(145) was grown in the presence of high nitrate, the inhibition of root growth was accompanied by an increase of free amino acids and protein in the roots. The inhibition of root growth clearly cannot be explained by an inhibition of local nitrate assimilation, nor by an inhibition of re­translocation of nitrogenous compounds back to the roots from the shoot.

The inhibition of root growth in Nia30(145) grown on high nitrate was accompanied by an inhibition of starch synthesis in the leaves whereas sugar levels in the leaves remained relatively high (Scheible et aI., 1997a). There was a marked decrease of sugars in the roots, and the decrease in root sugar correlated with the inhibition of root growth (Scheible et aI., 1997a). These results provided correlative evidence that nitrate accumulation in the shoot may decrease carbon transport to the roots, and that this could contribute to the inhibition of root growth.

In recent experiments, this has been investigated more directly by growing wild-type and Nia30(145) in sterile conditions in vertically­oriented petri plates on nutrient agar containing various nitrate concentrations in the presence and absence of 2% sucrose (M.Stitt, unpublished results).The plantlets were harvested after about 3 weeks, and analysed for biomass allocation, and nitrate, amino acid, protein and sugar levels. Nia30(145) grew at a similar rate and contained similar levels of amino acids and protein to wild-type plants when it was grown on ammonium or low nitrate (data not shown). Whereas growth, amino acids and protein increased when wild-type plants were provided with

14. Nitrate as a signal 297

higher nitrate, they remained low in Nia30(l45) (data not shown). Nia30(l45) showed a higher shoot root ratio than wild-type plants when it was grown on high nitrate in the absence of sucrose (Figure 8C). The high shoot root ratio was mainly due to a marked inhibition of root growth (data not shown). When the relation between root growth in Nia30(l45) and the local sugar supply was investigated, two conclusions were reached. First, in contrast to older plants (see above and Scheible et aI., 1997a), Nia30(l45) growing on high nitrate contained higher levels of sucrose (Figure 8D), glucose and fructose (data not shown) than wild­type roots. This shows that the correlation between low root sugar and slow root growth that we have seen previously in older plants cannot be an essential component of the nitrate-mediated inhibition of root growth. Secondly, when 2% sucrose was added, it suppressed the inhibitory effect of high nitrate on root growth in Nia30(145) (Figure 8G). This indicates that one effect of the nitrate-mediated inhibition of root growth is that higher levels of sugars are needed in the root to achieve rapid root growth.

When the results from the studies in older plants and seedlings are compared, two tentative conclusions can be drawn. First, nitrate accumulation leads to several changes in carbon metabolism and allocation including an inhibition of starch synthesis, a restriction of carbon transport to the roots, and a shift in the relation between root sugar levels and the rate of root growth. The extent to which each of these effects can be observed, depends on the plant age and/or the growth conditions. Secondly, although all of these changes could contribute to the nitrate-mediated inhibition of root growth, they are unlikely to be an essential component of the transduction chain.

3.6 Lateral root formation

Visual inspection of Nia30(l45) growing at different nitrate levels revealed that the increased root growth was due to increased bushiness, rather than to increased root length (see Figure 4D in Scheible et aI., 1997a). This indicated that nitrate might alter lateral root formation. To test this possibility, lateral roots were scored in plantlets growing on vertical agar plates.

When Nia30(145) and wild-type plants were grown on ammonium or low nitrate, they contained a similar number of lateral roots per plant. The number of lateral roots per wild-type plant increased in parallel with the increase of plant size, root size and root length when the nitrate supply was increased (Figure 8B, SF). In contrast, in Nia30(l45) increased nitrate led to a decreased number of lateral roots per plant

298 Chapter 14

(Figure 8B, SF), even though root length increased in the same way as in wild-type plants (Figure SA, SE). Further, this inhibition of lateral root formation could not be reversed by adding exogenous sucrose to the roots (Figure SF). Taken together with the results of the split-root experiment in Scheible et ai. (1997a), these results show that the accumulation of nitrate in the shoot leads to an inhibition of lateral root formation.

Whereas high nitrate in the shoot leads to an inhibition of lateral root growth, local application of nitrate in the rooting zone leads to a localised increase of auxin (Sattelmacher and Thoms, 1995) and a stimulation of lateral root growth (Drew and Saker, 1975; Granato and Raper, 19S9; Laine et aI., 1995). The signals derived from shoot nitrate and local nitrate levels in the root system will provide different information for the plant. The nitrate pool in the shoot will provide information about the nitrogen status of the plant and allow the plant to modulate allocation between shoot and root growth at a coarse level, whereas monitoring of gradients of nitrate around the root system will provide information that allows preferential root growth at the sites where nitrate is available. An interaction between the shoot signal and the local signal would allow a sophisticated regulation of root growth, in response to the nitrate requirements of the plant and the distribution of nitrate in the environment. These signals from nitrate, of course, will have to be integrated with signals from nitrogen metabolism itself, and with signals that provide information about other nutrients to allow a flexible regulation of root growth in response to the nutrient status of the plant and the surrounding environment.

In this context, it is tempting to speculate about a possible interaction with phytohormones. It has long been known that exogenous addition of cytokinin and auxin represses and induces the formation of lateral roots, respectively (Webster and Radin, 1972). Studies of mutants have confirmed the importance of endogenous auxin (Celenza et aI., 1995; Hobbie and Estelle 1995; Simons et ai., 1995) and cytokinin (Boerjan et ai., 1995) in lateral root formation. It is also known that nitrate fertilisation leads to increased levels of cytokinins in the roots and root sap (Beck, 1996; Kuiper et ai., 19S9; Wagner and Beck, 1993), that cytokinin addition inhibits export of new photosynthate to the roots (Fetene and Beck, 1993), and that the induction of lateral root proliferation by nitrate is accompanied by a local increase of auxin (Sattelmacher and Thoms, 1995). Nia30(145) could provide a useful system to investigate whether nitrate-mediated signalling involves changes of hormones.

14. Nitrate as a signal

60

E 40 ()

20

o

A

15 B

10....

Q) 10 ..0 E :::l

Z 5

o ro 0::

o

10

5

o

Root length

Number of lateral roots per plant

0,5 1 4 10 N03 N03 N03 N03

299

300 Chapter 14

4

0 Root sucrose .,......

3 I

(9 LL 0>

2

0 E ::::l..

o

Figure 8. Shoot-root allocation and lateral root formation in wild-type plantlets and Nia30(I45) grown on vertical petri plates on nutrient agar containing 0.2 mM ammonium or 0.2, 0.5, I, 4 or 10 mM nitrate without sucrose (panels A-D) or with 2% sucrose (panels E-G). The plants were scored for lateral roots and the length of the longest root, and harvested for biomass analysis after about 3 weeks. Sugars were measured in the roots of plants that did not receive exogenous sucrose. The results are the mean ± SE of three batches of 6-8 plants (biomass analysis) or 13-18 individual plants (root length, lateral frequency). These data are unpublished.

14. Nitrate as a signal

60

E 40 U

20

o

E Root length

15 F Number of lateral roots

10....

Q) 10 ..0 E :::J

Z 5

o

10

o ~ a:

5

o

per plant

G Shoot Ratio ratio

0,2 0,2 0,5 1 4 10 NH4 N03 N03 N03 N03 N03

301

302 Chapter 14

4. PERSPECTTVES

Nitrate acts as an important source of signals to control cellular and whole plant processes, and to allow a coordinated response of metabolism and growth to changes in the availability of nitrate and the nitrogen status of the plant. In the future, it will be important to exploit this system to identify further processes that are also regulated by nitrate and other signals deriving from nitrogen metabolism, to investigate the sites and mechanisms of the receptor systems and transduction pathways, and to investigate how nitrate-signalling interacts with other internal and external signals in the control of plant metabolism and growth.

REFERENCES

Agren, G.!. (1985). Theory for growth of plants derived from the nitrogen productivity concept. Physiologia Plantarum, 64, 17-28.

Agren, G.!. and Ingestad, T. (1987). Rootshoot ratio is a balance between nitrogen productivity and photosynthesis. Plant Cell and Environment, 10, 579-586.

Bachmann, M., Huber, J.L., Liao, P.C., Gage, D.A. and Huber, S.c. (1996). The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14-3-3 protein. FEBS Letters, 387, 127-131.

Beck, E. (1996). Regulation of the shoot/root ratio by cytokinins in Urtica dioica. Plant and Soil, 185,3-12.

Bernier, G., Havelange, A., Houssa, C., Petitjean, A. and Lejeune, P. (1993). Physiological signals that induce flowering. Plant Cell, 5, 1147-1155.

Bloom, A.1., Chapin, F.S. and Mooney, H.A. (1985). Resource limitation in plants - an economic analogy. Annual Review of Ecology and Systematics, 16, 363-392.

Boerjan, W., Cervera, M.-T., Delarue, M., Beckman, T., Dewitte, W., Bellini, c., Caboche, M., Van Onckelen, H., Van Montagu, M. and Inze, D (1995). Superroot, a recessive mutation in Arabidopsis confers auxin overproduction. Plant Cell, 7, 1405-1419.

Brouwer, R. (1962). Nutrient influences on the distribution of the dry matter in the plant. Netherlands Journal of Agricultural Sciences, 10, 399-408.

Burton, W.G (1989). The Potato. Longman Scientific and Technical, Loughborough. Buysse, 1., Smolders, E. and Merkx, R. (1993). The role of free sugars and amino acids and

its regulation of biomass partitioning and plant growth. Plant and Soil, 156, 191-194. Celenza, J.L.Jr., Grisafi, P.L. and Fink, G.R. (1995). A pathway for lateral root formation in

Arabidopsis thaliana. Genes and Development, 9, 2131-2142. Champigny, M.L. and Foyer, C.H. (1992). Nitrate activation of cytosolic protein kinases

diverts photosynthetic carbon from sucrose to amino acid biosynthesis. Basis for a new concept. Plant Physiology, 100, 7-12.

Cheng, c.L., Acedo, G.N., Dewdney, 1., Goodman, H.M. and Conkling, M.A. (1991). Differential expression of two Arabidopsis nitrate reductase genes. Plant Physiology, 96, 275-279.

Chollet, R., Vidal, 1. and O'Leary, M.H. (1996). Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47, 273-298.

14. Nitrate as a signal 303

Chu, C.C., Coleman, J.S. and Mooney, H.A. (1992). Control of biomass partitioning between roots and shoot: atmospheric carbon dioxide enrichment and the acquisition and allocation of carbon and nitrogen in wild radish. Oecologia, 89, 580-587.

Crawford, N.M. (1995). Nitrate: nutrient and signal for plant growth. Plant Cell, 7, 859-868.

Crawford, N.M. and Arst Jr., H.N. (1993). The molecular genetics of nitrate assimilation in fungi and plants. Annual Review of Genetics, 27,115-146.

Deng, M.D., Moureaux, T. and Lamaze T. (1989). Diurnal and circadian fluctuations of malate levels and its close relationship to nitrate reduction in tobacco leaves. Plant Science, 65, 191-197.

Drew, M.C. and Saker, L.R. (1975). Nutrient supply and the growth of the seminal root system in barley. II. Localized compensatory changes in lateral root growth and the rates of nitrate uptake when nitrate is restricted to only one part of the root system. Journal of Experimental Botany, 26, 79-90.

Ericsson, T. (1995). Growth and shoot:root allocation of seedlings in relation to nutrient availablity. Plant and Soil, 168, 205-214.

Farrar, 1.F. (1996) Sinks - integral parts of a whole plant. Journal of Experimental Botany, 47, 1273-1279.

Faure J.D., Vincentz, M., Kronenberger, J. and Caboche M. (1991). Coregulated expression of nitrate and nitrite reductases. Plant Journal, 1, 107-113.

Fernandez, E. and Cardenas, 1. (1989). Genetics and regulatory aspects of nitrate assimilation in algae. In: Wray, J.L. and Kinghorn, J.L. (Eds). Molecular and Genetic Aspects of Nitrate Assimilation (pp 101-124). Oxford Science Publications, Oxford.

Fetene, M. and Beck, E. (1993). Reversal of sink-source relations in Urtica dioica L. plants by increasing cytokinin import into the shoot. Botanica Acta, 106, 235-240.

Fichtner, K., Quick, W.P., Schulze, E.-D., Mooney, H.A., Rodermel, S.R., Bogorad, L. and Stitt, M. (1993). Decreased ribulose-l,5-bisphosphate carboxylase-oxygenase in transgenic tobacco transformed with 'antisense' rbcS. V. Relationship between photosynthetic rate, storage strategy, biomass allocation and vegetative plant growth at three different nitrogen supplies. Planta, 190, 1-9.

Fichtner, K. and Schulze, E.-D. (1992). The effect of nitrogen nutrition on annuals originating from habitats of different nitrogen availability. Oecologia, 92, 236-241.

Fieuw, S., MUller-Rober, B., Galvez, S. and Willmitzer, L. (1995). Cloning and expression analysis of the cytosolic NADP+ -dependent isocitrate dehydrogenase from potato. Plant Physiology, 107, 905-913.

Galangau, F., Daniel-Vedele, F., Moureaux, T., Dorbe, M.F. Leydecker, M.T. and Caboche, M. (1988). Expression of leaf nitrate reductase gene from tomato and tobacco in relation to light dark regimes and nitrate supply. Plant Physiology, 88, 383-388.

Gowri, G., Ingemarsson, B., Redinbaugh, M.G. and Campbell, W.H. (1992). Nitrate reductase transcript is expressed in the primary response of maize to environmental nitrate. Plant Molecular Biology, 18, 55-64.

Granato, T.C. and Raper, C.D. Jr. (1989). Proliferation of maize roots in response to localized supply of nitrate. Journal of Experimental Botany, 40, 263-275.

Hobbie, L. and Estelle, M. (1995). The a.xr4 auxin-resistant mutants of Arabidopsis thai ian a define a gene important for root gravitropism and lateral root initiation. Plant Journal, 7, 211-220.

Hoff, T., Truong, H.-N. and Caboche, M. (1994). The use of mutants and transgenic plants to study nitrate assimilation. Plant Cell and Environment, 17, 489-506.

Hofstra, R., Lanting, L. and De Visser, R. (1985). Metabolism of Urtica dioica is dependent on the supply of mineral nutrients. Physiologia Plantarum, 63, 13-18.

304 Chapter 14

Huber, S.C. (1983). Relation between photosynthetic starch formation and dry-weight partitioning between the shoot and root. Canadian Journal of Botany, 61, 2709-2716.

Imsande,1. and Touraine, B. (1994). Nitrogen demand and the regulation of nitrate uptake. Plant Physiology, 105, 3-7.

Kaiser, W.M. and Huber, S.C. (1994). Posttranslational regulation of nitrate reductase in higher plants. Plant Physiology, 106, 817-821.

Kronenberger, J., Lepingle, A., Caboche, M. and Vaucheret, H. (1993). Cloning and expression of distinct nitrite reductases in tobacco leaves and roots. Molecular and General Genetics, 236, 203-208.

Kuiper, D., Kuiper, P.J.C., Lambers, H., Schuit, 1. and Staal, M. (1989). Cytokinin concentration in relation to mineral nutrition and benzyladenine treatment in Plantago major spp. pleiosperma. Physiologia Plantarum, 75, 511-517.

Laine, P., Ourry, A. and Boucaud, 1. (1995). Shoot control of nitrate uptake rates by roots of Brassica napus L.: effects of localized nitrate supply. Planta, 196, 77-83.

Lam, H.M., Coshigano, K., Oliveira, I., Melo-Oliveira, R. and Coruzzi, G. (1996). The molecular genetics of nitrogen assimilation into amino acids in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology, 47, 569-593.

Lambers, H., Cambridge, M.L., Konings, H. and Pons, T.L. (1990). Causes and consequences of variation in growth rate and productivity of higher plants. SPB Academic Publishing bv, The Hague.

Lauerer M. (1996). Wachstum, Kohlenstoff- und Stickstoffhaushalt von Nicotiana tabacum mit reduzierter Nitratreduktaseaktivitlit. Bayreuther Forum Okologie, Vol. 31, Bayreuther Institut fur Terrestrische Okosystemforschung, Bayreuth,

Levin, S.A., Mooney, H.A. and Field, C. (1989). The dependence of plant rootshoot ratios on internal nitrogen concentration. Annals of Botany, 64, 71-75.

Li, 8., Zhang X.-Q. and Chollet, R. (1996). Phosphoenolpyruvate carboxylase kinase in tobacco leaves is activated by light in a similar but not identical way as in maize. Plant Physiology, Ill, 497-505.

Marschner, M. (1995). Mineral Nutrition of Higher Plants, 2nd edition. Academic Press, London.

Martinoia, E. and Rentsch, D. (1994). Malate compartmentation - responses to a complex metabolism. Annual Review of Plant Physiology and Plant Molecular Biology, 45, 447-467.

Marzluf, G. A. (1993). Regulation of sulfur and nitrogen metabolism in filamentous fungi. Annual Review of Microbiology, 47, 31-55.

Moorhead, G., Douglas, P. and MacKintosh, C. (1996). Phosphorylated nitrate reductase from spinach leaves is inhibited by 14-3-3 proteins and activated by fusicoccin. Current Biology, 6, 1104-1113.

Muller, A. and Mendel, R. (1989). Biochemical and somatic cell genetics of nitrate reduction in Nicotiana. In: Wray, 1.L. and Kinghorn, J.L. (Eds). Molecular and Genetic Aspects of Nitrate Assimilation (pp 166-185). Oxford Science Publications, Oxford.

Pouteau, S., Cherel I., Vaucheret, H. and Caboche, M. (1989). Nitrate reductase mRNA regulation in Nicotiana plumbaginifolia nitrate reductase-deficient mutants. Plant Cell, 1, 1111-1120.

Preiss, J., Ball, K., Smith-White, B., Inglesias, A., Kakefuda, G. and Li, L. (1991). Starch biosynthesis and its regulation. Biochemical Society Transactions, 19, 539-547.

Quesada, A., Krapp, A., Trueman, L.J., Daniel-Vedele, F., Fernandez, E., Forde, B.G. and Caboche, M. (1997). PCR-identification of a Nicotiana plumbaginifolia cDNA

J 4. Nitrate as a signal

homologous to the high-affinity nitrate transporters of the crnA family. Plant Molecular Biology, 34, 265-274.

305

Redinbaugh, M.G. and Campbell, W.H. (1991). Higher plant responses to environmental nitrate. Physiologia Plantarum, 82, 640-650.

Redinbaugh, M.G. and Campbell, W.H. (1993). Glutamine synthetase and ferredoxin­dependent glutamate synthase expression in the maize (Zea mays) root: primary response to nitrate. Plant Physiology, 101, 1249-1255.

Ritchie, S.W., Redinbaugh, M.G., Shiraishi, N., Verba, J.M. and Campbell, W.H. (1994). Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP+ oxidoreductase. Plant Molecular Biology, 26, 679-690.

Sattelmacher, B. and Thoms, K. (1995). Morphology and physiology of the seminal root system of young maize plants as influenced by a locally restricted nitrate supply. Zeitschrift flir Pflanzenerniihrung und Bodenkunde, 158, 493-497.

Scheible, W.-R., Lauerer, M., Schulze, E.-D., Caboche, M. and Stitt, M. (1997a). Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco. Plant Journal, 11, 671-691.

Scheible, W.-R., Gonzales-Fontes, A, Lauerer, M., Miiller-Rober, B., Caboche, M. and Stitt, M. (1997b). Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell, 9, 783-798.

Scheible, W.-R., Gonzalez-Fontes, A, Morcuende, R., Lauerer, M., Geiger, M., Glaab, l, Gojon, A, Schulze, E.-D. and Stitt, M. (1997c). Tobacco mutants with a decreased number of functional nia-genes compensate by modifying the diurnal regulation of transcription, post-translational modification and turnover of nitrate reductase. Planta, 203, 304-319.

Shaner, D.L. and Boyer, J.S. (1976). Nitrate reductase activity in maize (Zea mays L.) leaves. I. Regulation by nitrate flux. Plant Physiology, 58, 499-504.

Simons, C., Migliaccio, F., Masson, P., Caspar, T. and Soli, D. (1995). A novel root gravitropism mutant of Arabidopsis thaliana exhibiting altered auxin physiology. Physiologia Plantarum, 93, 970-978.

Stitt, M. and Schulze, E.-D. (1994). Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology. Plant Cell and Environment, 17, 465-487.

Stitt, M. (1996). Metabolic regulation of photosynthesis. In: Baker, N.R. (Ed). Advances in Photosynthesis, Vol. 5, Photosynthesis and the Environment (pp. 151-190). Kluwer Academic Publishers, Dordrecht.

Sugiharto, B. and Sugiyama, T. (1992). Effects of nitrate and ammonium on gene expression of phosphoenolpyruvate carboxylase and nitrogen metabolism in maize leaf tissue during recovery from nitrogen stress. Plant Physiology, 98, 1403-1408.

Sugiharto, B., Suzuki, I., Burnell, IN. and Sugiyama, T. (1992). Glutamine induces the nitrogen-dependent accumulation of mRNAs encoding phosphoenolpyruvate carboxylase and carbonic anhydrase in detached maize leaf tissue. Plant Physiology, 100, 2066-2070.

Sukanya, R., Li, M.G. and Snustad, D.P. (1994). Root- and shoot-specific responses of individual glutamine synthetase genes of maize to nitrate and ammonia. Plant Molecular Biology, 26, 1935-1946.

Trueman, L.J., Richardson, A and Forde, B.G. (1996). Molecular cloning of higher plant homologues of the high affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans. Gene, 175, 223-231.

306 Chapter 14

Van der Werf, A and Nagel, O.W. (1996). Carbon allocation to shoots and roots in relation to nitrogen supply is mediated by cytokinins and sucrose: opinion. Plant and Soil, 185, 21-32.

Vaucheret, R., Chabaud, M., Kronenberger, J. and Caboche, M. (1990). Functional complementation of tobacco and Nicotiana plumbaginifolia nitrate reductase deficient mutants by transformation with the wild-type alleles of the tobacco structural genes. Molecular and General Genetics, 220, 468-474.

Vincentz, M., Moureaux, T., Leydecker, M.T., Vaucheret, R. and Caboche, M. (1993). Regulation of nitrate and nitrite reductase expression in Nicotiana plumbaginifolia leaves by nitrogen and carbon metabolites. Plant Journal, 3, 315-324.

Wagner, B.M. and Beck, E. (1993). Cytokinins in the perennial herb Urtica dioica L. as influenced by its nitrogen status. Planta, 190, 511-518.

Waring, R.H., McDonald, AJ.S., Larsson, S., Ericsson, T., Wiren, A, Arwidsson, E., Ericsson, A and Lohammar, T. (1985). Differences in chemical composition of plants grown at constant relative growth rates with stable mineral nutrition. Oecologia, 66, 157-160.

Wray, J.L. (1993). Molecular biology, genetics and regulation of nitrite reduction in higher plants. Physiologia Plantarum, 89, 607-612.

Webster, B.D. and Radin, J.W. (1972). Growth and development of cultured radish tips. American Journal of Botany, 59, 744-751.

SUBJECT INDEX

A

acetate in fatty acid synthesis, 123, 139, 142, 144, 149, 151, 153 uptake into plastids, 147

acetyl-CoA, 109, 138, 139, 141, 148, 151, 153

acetyl-CoA carboxylase (ACCase), 138-140, 153

adenylate translocator, Ill, 125, 149 ADPglucose, 121, 125, 126, 175, 187-

189,247-249 ADPglucose pyrophosphorylase

(AGPase), 39, 105, 121, 125, 126, 131,187-9,216,245-248,250,251, 285,289,291

ADPglucose translocator, 126 Allium cepa, 228 alternative oxidase

modelling of active site, 22-28 structure, 19-22 structure-function relationship 28-32

Amaranthus edulis, 41 Amaranthus retrojlexus, 41 p-aminobenzoic acid, 54, 64, 66, 87 amylopectin

in starch granule, 174, 175 synthesis and structure, 177-185,248, 249

amyloplast, 123, 126, 132, 177,244-246, 246-249

amylose in starch granule, 174, 175 synthesis, 185-187,248,249

antimycin A, 17,32 aphid stylet, 160 apoplasm, 45, 46, 160, 165, 167,241,

242,246 Arabidopsis thaliana, 19,68, 105, 125,

149 Arum maculatum, 18 Aspergillus nidulans, 276 ATP/ADP ratio, 42 auxin, 298 Avena sativa, 228

307

B

barley, see Hordeum vulgare Beta vulgaris, 160, 165, 167, 171 biomass, 269, 292, 296 Brassica napus, 127, 138-142, 145, 147,

149, 153, 165, 167 Brassica oleraeca, 61, 105, 120, 121,

131,141,142,147,152

C

C4 plants, 38-42, 289 Calvin cycle, 77, 270 carbon dioxide

assimilation by PEPCK, 38 assimilation by Rubisco, 1,5, 7, in formate metabolism, 73, 74, photorespiratory loss, 2, 58, 59, 114 release in oxidative pentose phosphate pathway, 127, 130-132, 152

2-carboxyarabinitoll,5-bisphosphate,4, 5,11

castor bean, see Ricinus communis catalase, 73, 74 Catharanthus rose us, 85 cauliflower, see Brassica oleracea Chlamydomonas reinhardtii, 7, 183,

187,276 chloroplast

fatty acid synthesis in, 138 translocators in, 102, 103, 109, 111 volume, 160-162

chorismate, 87, 270 Cichorium intybus, 228 circadian rhythm

in phosphorylation ofPEPCK, 41 citrate synthase, 46, 250, 251, 285, 287 Clostridium cylindrosporum, 72, 81 Clostridium formicoaceticum, 72, 84 Clusia spp., 45, 48 Coleus blumei, 48 control coefficient, 131, 132,258-265 Corynebacterium sp., 70 crassulacean acid metabolism, 38-40, 42,

48 Cucumis melo, 45, 46 Cucumis sativus, 38-46 cyanide, 17, 18 cytochrome c oxidase, 18 cytokinin, 298 cytosol

308

D

folate concentration in, 57, 61, volume of, 160-167

debranching enzyme, 182-187 dicarboxylate translocator, 111-114 Digitaria sanguinalis, 41 dihydrofolate reductase (DHFR), 57, 58,

64-70 dihydroneopterin aldolase (DHNA), 66 dihydropterin pyrophosphokinase

(HPPK),64-67 dihydropteroate synthase (DHPS), 57,

64-67 di-iron carboxylate proteins, 22, 24, 31,

32

E

Echinochloa colona, 41 Echinochloa crus-galli, 41 Escherichia coli, 2, 39, 70, 72, 81, 125,

179,185,234,243,247,249,250 Euglena gracilis, 57, 86 extra-fascicular phloem, 45

F FAD,84 fatty acid

synthesis in plastids, 109, 123, 127, 131,132,137-153 synthesis by isolated plastids, 141-146

fatty acid synthetase, 148, 153 ferredoxin, 74, 84, 127, 130 Flaveria bidentis, 41 flux control coefficient, 131, 132, 258-

265 fodder beet, see Beta vulgaris folate

distribution and synthesis, 57-71 originiinterconversion of C 1 compounds, 71-86

folylpolyglutamate synthetase (FPGS), 64, 70, 71

5-formyltetrahydrofolate, 72, 73, 80, 82 formyltetrahydrofolate synthetase, 72,

73,82 fructan

accumulation in transgenic tobacco, 227-236

hydrolysis in leaves, 207-209 regulation of metabolism, 212-220 structure, 196-200 synthesis in leaves, 201-206 metabolism in other tissues, 209-210

fructan: fructan fructosyl transferase (FFT), 201-204, 228-231

fructan hydrolase, 207-209 fructokinase, 244 fructose 1,6-bisphosphatase (FBPase),

218,285 fructose 2,6-bisphosphate, 218

G Galderia partita, 2 gluconeogenesis, 38, 43, 48, 73 glucose I-phosphate, 120, 123,243-247 glucose 6-phosphate, 107, 120, 130,245 glucose 6-phosphate dehydrogenase, 130 glucose translocator, 105 glutamine synthetase (GS), Ill, 130,

279,284,285,289 glutamine:2-oxoglutarate

aminotransferase (GOGAT), Ill, 127, 130, 131, 132,277,284

glycine decarboxylase complex (GDC); also glycine cleavage system, 58-63, 74,78,80

Glycine max, 19, 120, 121 glycolysis, 107, 139, 144, 149,246,250,

266,269 glyoxylate, 73, 74, 77, 80 glyoxysomes, 73 grapevine, see Vitis vinifora GTP, 64 GTP cyclohydrolase, 64

H

harvestindex,239,252 Helianthus tuberosus, 201 hexokinase, 141,207,218,244 hexose phosphate, 102, 107, 109, 120,

121, 126, 131, 132,243,245,246 hexose phosphate/phosphate translocator

(HPT), 120,121 Hordeum vulgare, 46, 85, 125, 144, 160,

162, 165, 167, 188, 196,202,203, 215,219,228,229

hydroxypyruvate, 78

I

invertase (~-fructofuranosidase), 203, 207,216,219,229,242,243,246, 251

Ipomoea batatas, 234 isoamylase, 185 isocitrate dehydrogenase (NADP­

dependent), 279, 285

J Jerusalem artichoke, see Helianthus

tuberosus

K 3-keto-arabinitoll,5-bisphosphate,5

L

Lactobacillus casei, 54 Leishmania major, 68 Lemna minor, 85 levansucrase, 229, 234, 235, 236 Lolium perenne, 215 Lolium rigidum, 203, 204, 207 Lolium temulentum, 197,204,215,218 lords and ladies, see Arum maculatum Lycopersicon esculentum, 61

M Madagascar periwinkle, see

Catharanthus rose us maize, see Zea mays malic enzyme (ME), 46, 141 malonyl-CoA, 138, 139 malto-oligosaccharide, 186-187 melon, see Cucumis melD metabolic control analysis, 241, 257-273 methylenetetrahydrofolate, 58, 59, 68,

79,85 methylenetetrahydrofolate

dehydrogenase/cyclohydrolase, 81-84 methylenetetrahydrofolate reductase, 84-

86 mitochondria

in folate metabolism, 58-87 volume, 59, 161

molecular modelling, 19,22,28

309

N

Neurospora crassa, 19,20,58,70,71, 125,276

Nicotiana tabacum, 2, 7, 19,43, 102, 105, lll, 140, 160, 162,229,231, 234,235,279,280,281,292

nitrate reductase (NR), 43, 271, 276-284, 287,292,293,296 role of 14-3-3 protein, 43, 280

nitrate-regulated processes, 281-301 nitrite, ll8, 131,277,284,287 nitrite reductase (NiR), 127, 130, 131 non-aqueous fractionation, 160, 162

o oat, see Avena sativa oilseed rape, see Brassica napus one-carbon metabolism

role offormate in, 72-74 role of glycine in, 74-78 role of methionine in, 84-87 role of serine in, 78-81

onion, see Alium cepa oxaloacetate, 38, 46 oxidative pentose phosphate pathway,

109, 127, 130-132, 138, 151, 152 oxidative stress, 31

p

Panicum maximum, 40-42 Panicum miliaceum, 41 Paspalum notatum, 41 pea, see Pisum sativum Phleum pratense, 204, 228 phloem,

analysis of sap, 160, 165-167 structure and function in cucumber, 45-48, transport of photosynthate, 241-246

phosphate translocator, see hexose phosphate/phosphate translocator, phosphoenolpyruvate/phosphate translocator, and triose­phosphate/phosphate translocator

phosphoenolpyruvate carboxykinase (PEPCK) properties of extracted enzyme, 38-40

phosphorylation, 40-43 phosphoenolpyruvate carboxylase

(PEPCase), 38-42, 279

310

phosphoenolpyruvate/phosphate translocator (PPT), 105-110, 120

phosphofructokinase (PFK), 250, 251, 266,270

phosphoglucomutase, 123, 187,246 phosphoribulokinase,61 photorespiration, 58, 76, 77, 78, 111,

114 photosynthesis, 38,41,48,68,74,77,

78,103,105,117,138,144,147, 209,216,270

phytoglycogen, 183 Pichia stipitis, 18 Pisum sativum, 58, 59, 61, 64, 67, 82,

85, 102, 120, 123, 125, 126, 127, 131,139,141,142,144,147,149, 151,177,179,180,181,182,186, 187

Plasmodiumfalciparum, 66 plastids

import of metabolites, 120-126, 147-152, 244-246

Pneumocystis carinii, 66 Poa pratense, 204 polyglutamates, 61-64, 68, 70-72, 79, 85 potato, see Solanum tuberosum promoter

35S, 215, 240, 242, 243,248 patatin, 240 sporamin, 234, 235

protein phosphorylation, 38-43,270, 280,289 role of 14-3-3 protein, 43, 280

proteolysis, 11, 19,39,40,42 pullulanase, 185 pyridoxal phosphate, 74, 78, 121 pyrophosphatase, inorganic, 121, 188,

243,247 pyrophosphate, inorganic, 66, 73, 132,

243,247,250 pyrophosphate:fructose 6-phosphate 1-

phosphotransferase (PFP), 188,250, 251

pyruvate dehydrogenase complex (PDC), 76,109,127,139,144,151

pyruvate kinase (PK), 109, 121,285,287

R

rbcL,2,12 rbcS,2 Rhizobium sp., 39 Rhodospirillum rubrum, 12

respiration in potato tubers, 249-251

ribulose 5-phosphate, 270 ribulose 1,5-bisphosphate

carboxylase/oxygenase (Rubisco), catalytic mechanism, 4-7 flux control analysis, 259-261, 265, 266,270 site-directed mutagenesis, 7-12 specificity factor, 2, 7, 11, 12 structure, 2-4

Ricinus communis, 46, 139, 141, 142, 144, 147, 148

Rickettsia prowazekii, 125 Rubisco, see ribulose 1,5-bisphosphate

carboxylase/oxygenase Rubisco activase, 5, 270

s SacB gene

from Bacillus amyloliquefaciens, 235 from Bacillus subtilis, 229, 231

Saccharomyces cerevisiae, 82 Saccharum officinarum, 41 S-adenosyl methionine (SAM), 53, 84,

215 Sauromatum gutta tum, 18, 24 serine hydroxymethyltransferase

(SHMT), 58, 59, 63, 76, 77, 78-80, 81,84,86

shikimic acid pathway, 43, 109 shoot root ratio

effect of nitrate, 292-297 sieve element, 46 Solanum tuberosum, 18, 19,61,80,102,

103, 120, 123, 126, 160, 162, 167, 179,181,182,185,186,229,231, 234,235,236,239,240,241,242, 243,244,245,246,247,248,249, 250,251,266,279

Sorghum bicolor, 41 soybean, see Glycine max Spartina anglica, 41 Spinacea oleracea, 2, 4, 7, 11,46, 72,

102, 103, 10~ Ill, 120, 130, 139, 142, 144, 149, 153, 160, 162

Sporobolus pyramidalis, 41 starch, see amylopectin and amylose

effect of nitrate, 289-291 structure of granule, 174-175 synthesis in amyloplasts, 246-249

starch branching enzyme (SBE), 175, 177-180,182-185,247,249

starch synthase granule-bound starch synthase (GBSS), 177, 185-187,248 soluble starch synthase (SSS), 180-182,188,249

stroma, 109, 162, 177 sucrose

manipulation of metabolism, 246 metabolism in cytosol, 241-244 phloem concentration, 167-171 subcellular concentration, 162-165

sucrose:fructan glucosyltransferase (SFT),203,206,229

sucrose phosphate synthase (SPS), 218, 271,285,289

sucrose synthase (SuSy), 187, 188,242, 243,244,245,246,251

sucrose: sucrose fructosyl transferase (SST), 201-203, 206

sugar beet, see Beta vulgaris sugarcane, see Saccharum officinarum sweet potato, see Ipomoea batatas Synechococcus sp., 4, 7, 11, 12

T

taproot, 160, 167 tetrahydrofolate, 54, 57, 58, 59, 63, 72,

76,77,78,79,87 sythesis, 64-71

thioredoxin, 270 thymidylate synthase (TS), see also

dihydrofolate reductase, 67, 68 Tillandsia sp., 39 tobacco, see Nicotiana tabacum tomato, see Lycopersicon esculentum Toxoplasma gondii, 66 tricarboxy lie acid cycle, 31, 250, 251 triose phosphate/phosphate translocator

(TPT), 102-105, 107, 109, Ill, 120, 121, 127

Triticum aestivum, 2, 57, 121, 123, 125, 126, 132, 141, 196,228

Trypanosoma brucei, 19,20

u UDPglucose, 242, 243, 250 UDPglucose pyrophosphorylase

(UGPase), 243, 249, 250 Universal method, 269, 270

Urochloapanicoides, 39, 41, 42

v vacuole, 57, 160-162,206,207,216,

218,219,228,234,235,240 Vitis vinifera, 43, 48

311

volume, subcellular, 59, 160- 162,206

w water

alternative oxidase, active site 22, 28 wrinkled seeds 180

wheat, see Triticum aestivum

x xylulose 1,5-bisphosphate, 5

y

yield, 2, 138, 149, 188,235,239,240, 242,246

z Zea mays, 7, 41, 42, 43, 102, 105, 120,

123, 125, 126, 139, 142, 145, 165, 179, 182, 183, 185, 188,215,235, 248,279

Proceedings of the Phytochemical Society of Europe

42. N.J. Kruger, S.A. Hill and R.G. Ratcliffe (eds.): Regulation of Primary Metabolic Pathways in Plants. 1999 ISBN 0-7923-5494-X

43. L. Bohlin and J.G. Bruhn (eds.): Bioassay Methods in Natural Product Research and Drug Development. 1999 ISBN 0-7923-5480-X

KLUWER ACADEMIC PUBLISHERS - DORDRECHT / BOSTON / LONDON