CHAPTER I

INTRODUCTION

Rice (Oryza sativa L.) is an important worldwide cereal crop and is the

principal staple food of the major population in the world. As a result of increased

demands on food production from escalating population growth and environmental

degradation, interest in improving the productivity and quality traits of cereal crops

has become crucially important. Starch forms the major content of cereal grains

accounting for approximately 70% of cereal seed weight. Progress in cereal starch

production is especially important because these starches comprise 55 to 75% of daily

human food intake. Cereal starch production forms the basis of subsistence for much

of the world's human and domesticated animal populations.

Starch and modified starches have a broad range of applications even in the

non-food sectors. The largest users of starch are the paper, cardboard and corrugating

industries. Other important fields of starch application are textiles, cosmetics,

pharmaceuticals, construction and paints. In the long run, starch will play an

increasing role in the field of "renewable raw materials" for the production of

biodegradable plastics, packaging material and molds. Therefore it is important to

improve both the quality as well as quantity of starch to meet the future demands. The

common applications for modified and native starches in the food and non-food based

industry are varied and numerous, of which, some are described in Table 1.1 and 1.2.

Starch concentration and composition in cereal grain are controlled by many

genes. The concentration and composition of starch together contribute to grain

weight in cereals finally affecting the yield of the cereal crop. Thus, identifying and

studying those genes and alleles that control these traits in cereals is an important step

in meeting the future goals ofboth agriculture and industry. There are various factors,

such as seed starch biosynthetic enzymes, affecting the crop yield. Figure 1.1 below

shows some of the major factors affecting crop yield.

1.1 Rice grain and cereal grain development

The dehulled rice gram, or brown rice, consists of the outer covering (the

pericarp, seed coat, nucellar layer, and aleurone layer), the starchy endosperm and the

embryo (Figure 1.2A and B). Brown rice has a greater food value than white, since

1

Table 1.1

S.No.

1.

2.

3.

4.

5.

6.

Table 1.2

S.No.

1.

2.

3.

4. 5.

6.

Common applications for modified and native starches in the food based industry

Industry Applications (Food based)

Cereals and snacks Hot extruded snacks, chips etc; Extruded and fried foods, pretzels and ready-to-eat cereals

Bakery Pies, tarts, fillings, glazes, custards and icings, cakes, donuts, Danish and icing sugar

Batters and breadings Coated fried foods, frozen battered, vegetables, fish and meat and dry mix coatings

Dressings, soups and sauces Pourable salad dressings (high shear), spoonable dressings, instant dry salad dressing mixes, low-fat dressing, canned gravies and sauces, frozen gravies and sauces, soups and chowders

Confectionery Dusting powder, licorice, jelly gums, hard gums,

panned candies, confectioners sugar Cooked meat binder Water binder for formed meat, smoked meats, low-fat

meats, pet foods (dried and canned)

Common applications for modified and native starches in the non-food based industry

Industry (Non-food based)

Textiles Industry

Paper Industry

Cosmetic and Pharmaceutical Industry

Explosives Industry Adhesives

Construction Industry

Applications

Warp sizing, fabric finishing, printing

Internal sizing, filler retention, surface sizing, paper

coating (regular and colour), carbonless paper stilt material, disposable diapers, feminine products

Dusting powder, make-up, soap filler/extender, face

creams, pill coating, dusting agent, tablet binder/ dispersing agent Wide range binding agent, match-head binder

Hot-melt glues, stamps, bookbinding, envelopes, labels

(regular and waterproof), wood adhesives, laminations, automotive, engineering, pressure sensitive adhesives, corrugation, paper sacks

Concrete block binder, asbestos, clay/limestone binder,

fire-resistant wallboard, plywood/chipboard adhesive, gypsum board binder, paint filler

t Environmental factors Genetic factors

t

Enzymes of starch synthesis

Figure 1.1 Major factors affecting crop yield

(A) Rice grain (B) Awn - [

Hull .'!! - Perlcarp "'

~ - Tegmen 0 c:-

PeD3p ~leurone layer "' ~ Starchy ~

E~ endosperm a:

Scutellum}~ c: ~

Epiblast :o :::>

Enilryo Plumule ~ cO

Rachllla

Radlde Sterile lemmas

Figure 1.2 Structure of the rice grain (A) Outer layer and internal structures of a rice grain (B)

the outer brown coatings contains proteins and minerals; the white endosperm is

chiefly carbohydrate. As a food, rice is low in fat and protein content in comparison to

other cereal grains.

Rice, like other cereal grains, stores starch, proteins and lipids, which are

assembled separately in discrete starch granules, protein bodies (PBs) and oil bodies

respectively, as reserves for use in germination. The endosperm storage proteins are

found in two types of PBs, termed PB-I and PB-II. PB-I is spherical with a concentric

ring structure and is the site of deposition of prolamins, whereas PB-II lacks the ring

structure and is rich in glutelins and globulins. Rice oil bodies, on the other hand, are

found in the embryo and aleurone layer as well as the endosperm. Unique structural

proteins known as oleosins are found on the surface of oil bodies of the embryo and

the aleurone layer, but not those of the starchy endosperm. A detailed account of

proteins expressed in cereal grains is discussed later.

Seed development commences after fertilization of the ovule, which, in the

angiosperms, involves the participation of two male nuclei. One nucleus fuses with the

egg forming a diploid zygote and gives rise to the embryo, the other fuses with two

polar nuclei to produce a triploid nucleus, further division of which produces the

endosperm.

The endospem1, the largest organ in the seed consists of two tissues, the

interior starch filled endosperm surrounded by an outer layer of cells, called the

aleurone layer. The embryo is comprised of the embryonic axis (incorporating the

embryonic root and the hypocotyl), a single cotyledon, containing the first true leaves,

and the scutellum. Development of the endosperm is orchestrated by the coordinated

activities of a large number of genes that encode metabolic and regulatory enzymes

and other proteins (Becraft et a!., 2000; Olsen, 2001; Olsen, 2004; Lid and Olsen,

2004).

Cereal gram development can be divided into two main stages, grain

enlargement and grain filling. The first stage, grain enlargement, involves early,

rapid division of the zygote and triploid nucleus. Cell division is followed by the

influx of water, which drives cell extension. This stage occurs at approximately 3±20

2

days post-anthesis (dpa) i.e. post flower opening. During the second stage, grain

filling, cell division slows and then ceases and storage products (starch) are

accumulated, beginning at around 10 dpa until maturity when the grain serves its

function as a carbohydrate store (Briarty et al., 1979; Bewley and Black, 1994).

Briarty et al have carried out an extensive morphological analysis of changes,

which occur during cereal grain development (Briarty et al., 1979). The data are based

on a stereological analysis of the volumes occupied by each component within the

cells of the grain. Whilst they cannot be related to one another in terms of dry matter,

and will be influenced by water content, which changes during development, the data

give a good indication of the major changes, which are occurring, and their timing.

Amyloplasts, involved in starch synthesis and storage, also increases in number per

cell. Indeed, the volume of the cells occupied by starch increases throughout

development, reaching 65% at grain maturation (36 dpa).

The starch of cereal grain is composed of two types of granules (A- and B­

type granules), the synthesis of which appears to be developmentally regulated. The

larger, A-type granules, appear approximately four days earlier in development than

the smaller, B-type granules ( ~22 dpa), and are contained in A-type and B-type

amyloplasts, respectively (Langeveld et al., 2000; Jane et al, 2003). The rough

endoplasmic reticulum (RER), involved in the synthesis of proteins, was shown to

increase in surface area per cell, and surface±volume ratio, between 10±12 dpa, at

about the time that protein deposits are observed (Bechtel et al., 1982). Interestingly,

Briarty et al also observed that the mitochondrial number per cell increases during

development, but that the individual mitochondrial volume decreases, as does the area

of the inner membrane per unit volume of total cytoplasm (Briarty et al, 1979; Emes et

a!., 2003).

Grain filling is the final stage of growth in cereals. Grain filling is a process of

active metabolism of carbohydrate and starch accumulation in kernels. Its duration

and rate determine the final grain weight, a key component of the total yield. In

today's crop production systems with their high yield outputs, improvement in grain

filling has become more challenging than ever (Saini and Westgate, 2000; Zahedi and

3

Jenner, 2003). Grain filling in cereals depends on carbon from two sources: current

assimilates transferred directly to the grain and assimilates redistributed from reserve

pools in vegetative tissues either pre- or post-anthesis. Reserve pools provide the

substrate needed to maintain transport and supply of assimilate to grains during the

dark period of the diurnal cycle and during the later grain-filling period, when the

photosynthetic apparatus is senescing and the rate of dry matter accumulation of

grains exceeds the rate of dry matter accumulation of the total crop (Schnyder, 1993).

1.2 Grain filling- The 'Source' and the 'Sink' concept

'Source' organs are usually photosynthetically active and are defined as net

exporters of photoassirnilates, represented mainly by mature leaves, and 'sink' refers

to the organs that are photosynthetically inactive and are net importers of fixed carbon.

Sinks can be further divided into at least two different classes: utilization and

storage sinks. Utilization sinks are highly metabolically active, rapidly growing

tissues such as rneristerns and mature leaves, while storage sinks are the organs like

tubers, seeds and roots, where the imported carbohydrates are deposited in the form of

storage compounds (e.g. starch, sucrose, fatty acids, or proteins). The storage sinks

are usually specialized for other essential processes such as mineral acquisition (roots)

or reproduction (seeds, fruits and potato tubers). Sink or source status of a particular

organ is under developmental control e.g. immature leaves are metabolic sinks while

after maturation leaves become photosynthetically active sources. Growing potato

tubers are storage sinks, however during sprouting they tum into source organs where

the stored compounds are mobilized to provide transportable organic nutrients for the

growth ofbuds. The other storage sinks incude roots, sterns, fruits and seeds.

Warren Wilson introduced the term 'sink strength' to define the ability of the

sink to obtain assimilates in a competing system [sink strength = sink size x sink

activity]. The term "sink strength" describes the overall effects that sinks exerts on the

transport of photoassimilates towards them. Flow of metabolites into a specific sink is

determined by the interactions among the source, alternative sinks and the sink itself.

4

It is the effect that sinks exert on sugar allocation in the whole plant system (Marcelis

et al., 1995; Wolswinkel, 2004; Herbers, 1998).

The sink cells regulate the steepness of the sucrose gradient along the

translocation path, stimulate withdrawal of osmotically active photoassirnilates from

the translocation path, and affect the turgor potential in the translocation path. This

implies that once photoassirnilate enters the translocation path at the source leaf, its

destination is controlled by events occurring at the sink, rather than the source. Since

sink to some extent represents an "end station" for assimilates, they can influence

osmotic concentration in the phloem by (a) assimilate utilization, either by

consumption (respiration) or by chemical alteration of the translocate (sucrose

hydrolysis or polysaccharide formation) and (b) cornpartrnentation (starch in plastids,

fructan and soluble sugars in vacuoles and polyrnannans in the cell wall).

The relationship between sink strength and assimilate supply is specific to each

sink and is governed by supply and demand. When there is a demand in a specific

sink, the developing photoassirnilate shortage causes a steep osmotic gradient between

source and sink, which ensures an increase in supply until a new equilibrium is

reached. Presence of active fruit sinks causes an increase in the rate of photosynthesis

and export from the source leaf. Alterations in sink demand cause a rapid change in

the distribution of photosynthates, whereas sink removal and stern girdling cause a

rapid decrease in photosynthetic rates.

The evolution of sink and source organs in higher plants generates the need of

resource allocation between sink and source organs, which is a major determinant of

plant growth and productivity. Accordingly, two general strategies can be envisaged to

improve crop yields. One strategy is to improve source capacity namely the rate of

carbon export. This can be achieved by targeting (a) the rate of photosynthesis, (b) the

rate of carbon translocation via specific carrier proteins, (c) partitioning of

photoassirnilates between anabolism and catabolism, and (d) the rate of sucrose

synthesis.

5

As an alternative to the above strategy, enhancing the sink strength has

attracted some attention as a technique to increase the yield potential, since it has been

suggested that cereal grain weight is limited by the capacity of developing seed

endosperm to convert photoassimilates into starch (Choi et al., 1998). One way to

achieve this in cereals is by targeting the enzymes of the starch biosynthetic pathway.

An increase in the sink strength or rapid production of new sinks (for instance,

flowering and fruit set) cause a concomitant increase in the demand for assimilates as

well as for other nutrients thereby causing an even greater demand for sugars. All this

data suggest that yield is sink limited and not source limited. Understanding the

regulation of enzymes of starch biosynthetic pathway would therefore be useful.

1.3 Starch biosynthesis

The pathway of starch synthesis in the cereal grain is unique, and requires

several enzymes that are not present in other cereal tissues or non-cereal plants. The

key components of the starch metabolic pathways are shown in Figure 1.3.

Sites of starch synthesis

The starch synthesized in the leaves is called the transitory starch where it is

transient and later it is transferred to the sink organs for storage. However, the starch

synthesized in the sink organs is called the storage starch.

Starch is synthesized in the plastid compartment. Starch is synthesized in

plastids that have other specialized functions, such as chloroplasts (photosynthetic

carbon fixation), plastids of oilseed (fatty acid biosynthesis), and chromoplasts of

roots such as carrot (carotenoid biosynthesis). In the storage organs, the plastids are

called amyloplasts. In some cases, for example, in the storage cotyledons of some

legumes, amyloplasts in storage organs develop from chloroplasts. These amyloplasts

may maintain considerable amounts of stacked lamellar material from the thylakoids

6

ATP

+

+

.urc:6 .,. ....

---~ ...,....(U&J•

..... u • ..,.._(DI&J

ADP

Figure 1.3. General pathway of starch synthesis in plants

+ ..

and, in cells receiving sufficient light, may undertake some photosynthetic carbon

fixation for use in starch.

Enzymes involved in starch biosynthesis

ADP-glucose pyrophosphorylase (AGPase)- AGPase (AGPase; EC

2. 7. 7 .27) is responsible for the production of ADP- Glucose, the soluble precursor and

substrate for starch synthases by using glucose-1-phosphate (glucose-1-P) and ATP.

The AGPase reaction is the first committed step in the biosynthesis of both transient

starch in chloroplasts/chromoplasts, and storage starch in amyloplasts. AGPase from

higher plants is heterotetrameric, consisting of two large (AGP-L) subunits and two

small (AGP-S) catalytic subunits encoded by at least two different genes (Preiss and

Sivak, 1996). A detailed account of AGPase, its isoforms, location and regulation is

described later.

Starch Synthases (SS) (elongation of the glucan chain)- The starch

synthases (SS, EC 2.4.1.21) catalyse the transfer of the glucosyl moiety of the soluble

precursor ADP-Glc to the reducing end of a pre-existing a- (1 ,4)-linked glucan primer

to synthesize the insoluble glucan polymers amylose and amylopectin. Plants possess

multiple isoforms of SSs. The major classes of SS genes can be broadly split into two

groups, the first group primarily involved in amylose synthesis, and the second group

principally confined to amylopectin biosynthesis (Ping Wu et al., 2004; Nakamura et

al., 2000).

Granule-bound starch synthases (GBSS) (amylose biosynthesis)- The

first group of SS genes contains the granule-bound starch synthases (GBSS), and

includes GBSSI and GBSSII. GBSSI is encoded by the Waxy locus in cereals,

functioning specifically to elongate amylose (de Fekete et al., 1960; Nelson and Rines,

1962) and is found, essentially, completely within the granule matrix (one of the so­

called granule-associated proteins). In addition to its role in amylose biosynthesis,

GBSSI was also found to be responsible for the extension of long glucans within the

amylopectin fraction in both in vitro and in vivo experiments (Delrue et al., 1992;

Maddelein et al., 1994; van de Wal et al., 1998). Expression of GBSSI appears to be

7

mostly confined to storage tissues, and a second form of GBSS (GBSSII), which is

encoded by a separate gene, is thought to be responsible for amylose synthesis in

leaves and other non-storage tissues, which accumulate transient starch (Nakamura et

al., 1998; Fujita and Taira, 1998; Vrinten and Nakamura, 2000).

An interesting aspect of the control of polymer (amylose) elongation has been

observed in the leaves of sweet potato (Ipomoea batatas) where GBSSI transcript

abundance and protein levels were shown to be under circadian control, in addition to

being modulated by sucrose levels (Wang et al., 2001).

Soluble starch synthases (amylopectin biosynthesis)- The second group

of SS genes (designated SSI, SSII, SSIII, and SSIV) are exclusively involved in

amylopectin biosynthesis, and their distribution within the plastid between the stroma

and starch granules varies between the species, tissue, and developmental stages. The

individual SS isoforms from this group probably play unique roles in amylopectin

biosynthesis. The study of SS mutants in a number of systems has been helpful in the

assignment of in vivo functions/roles for the soluble and granule-associated SS

isoforms in amylopectin synthesis. Valuable information about the roles of the SS

isoforms in vivo is being derived from mutants lacking specific isoforms.

Biochemical evidence suggests that SSI is primarily responsible for the

synthesis of the shortest glucan chains, i.e. those with a degree of polymerization (DP)

of 10 glucosyl units or less (Commuri and Keeling, 2001), and further extension of

longer chains is achieved by the activities of SSII and SSIII isoforms, each of which

act on progressively longer glucan chains.

Two classes of SSII genes are found in monocots: SSIIa and SSIIb. The role of

the latter in starch biosynthesis is unknown, as no mutants have been identified to

date. In vitro studies of the two SSII forms from maize reveal different substrate

specificities and kinetic properties (Imparl-Radosevich et al., 2003). SSIIa

predominates in cereal endosperms, whilst SSIIb is mostly confined to photosynthetic

tissues. Loss of SSIIa (in monocots) and SSII (in dicots) results in reduced starch

content, reduced amylopectin chain-length distribution, altered granule morphology,

8

and reduced crystallinity, suggesting that the SSII forms have similar roles in starch

biosynthesis across different species boundaries. In monocots, SSIIa (Morell et al.,

2003) plays a specific role in the synthesis of the intermediate-size glucan chains of

DP 12-24 by elongating short chains of DP <10, and its loss/down-regulation has a

dramatic impact on both the amount and composition of starch, despite the fact that

SSIIa is a minor contributor to the total SS activities in cereal endosperms, as opposed

to SSI and SSIII (Smith, 2001 ).

Studies with plants lacking SSIII suggest that the primary role of this enzyme

in amylopectin synthesis, although the impact of loss of SSIII appears to differ with

the genetic background. Antisense suppression of SSIII in potato has a major impact

on the synthesis of amylopectin, resulting in amylopectin with modified chain length

distribution and decreased starch synthesis (Edwards et a!., 1999). However,

mutations in maize eliminating SSIII ( du1) lead to a subtle phenotype, which is only

conspicuous in Waxy backgrounds (Gao eta!., 1998). Sequences for SSIV appear in a

wide range of higher plants in EST databases although, to date, no mutants have been

isolated with lesions in this gene and no role has been assigned for this class of SS in

the process of starch biosynthesis.

Starch branching enzymes (SBE) (branching of the glucan chain)­

Starch branching enzymes (SBEs, EC 2.4.1.18) generate a- (1 ,6)-linkages by cleaving

internal a- (1,4) bonds and transferring the released reducing ends to C6 hydroxyls to

form the branched structure of the amylopectin molecule. As with the elongation of

glucan chains by SSs, SBE activity is also a function of multiple isoforms, some of

which are tissue and/or developmental stage specific in their expression patterns.

Analysis of the primary amino acid sequences of higher plant SBEs reveals two major

classes- SBEI (also known as SBE B) and SBEII (also known as SBE A). The two

classes of SBE differ in terms of the length of the glucan chain transferred in vitro and

their substrate specificities; SBEII proteins transfer shorter chains and show a higher

affinity towards amylopectin than their SBEI counterparts, which show higher rates of

branching with amylose (Takeda et al., 1993; Guan and Preiss, 1993).

9

The construction of chimeric forms of maize SBEI and SBEII and analysis of

their catalytic properties by Kuriki et al indicated that the N- and C-termini of these

proteins play important roles in determining substrate preference, catalytic capacity,

and chain length transfer (Kuriki et al., 1997). In monocots, the SBEII class is made

up of two closely related but discrete gene products, SBEIIa and SBEIIb (Rahman et

al., 2001). To date, only mutations in SBEII isoforms give clear phenotypes, and in

monocots this is confined to SBEIIb mutants (James et al., 2003).

Down-regulation or elimination of SBEI activity in both monocots and dicots

appears to have minimal effects on starch synthesis and composition in photosynthetic

and non-photosynthetic tissues (Blauth et al., 2002; Satoh et al., 2003; Flipse et al.,

1996). Recent analysis of a maize SBEIIa mutant showed a clear phenotype in the leaf

starch, but showed no apparent alterations in the storage starch of the endosperm

(Blauth et al., 2001 ). This observation suggests a primary role for SBEIIa in leaf

(transient) starch synthesis, and either no critical role for SBEIIa in amylopectin

biosynthesis in the endosperm, or else a role that can easily be compensated for by

other SBEs in its absence.

SBEII isoforms are also partitioned between the plastid stroma and the starch

granules. As with the granule-associated SSs (above), the factors/mechanisms

involved in partitioning the SBE proteins to the starch granules remain undetermined.

The ability of proteins to become granule-associated may be a function of the relative

affinities of their active sites for the glucan polymer, although it has recently been

suggested that alternative splicing of a SBEII form in Phaseolus vulgaris causes an

alteration in the properties of the enzyme, and partitioning within the starch granule

(Hamada et al., 2002).

Debranching (DBEs) enzymes in starch synthesis- The analysis of

low-starch mutants that accumulate a water-soluble polysaccharide termed

phytoglycogen have been described in a wide range of higher plants, including

Arabidopsis and maize, as well as the unicellular alga Chlamydomonas (Zeeman et al.,

1998; James et al., 1995; Mouille et al., 1996) and indicate that starch synthesis

involves isoamylases, also termed de branching enzymes, DBEs, (DBEs, EC 3 .2.1.41,

and EC 3.2.1.68) in addition to SSs and SBEs. (Morell and Myres, 2005)

10

Two groups of DBEs exist in plants: isoamylase-type, and pullulanase-type

(also known as limit- dextrinases), which efficiently hydrolyse (debranch) a- (1,6)­

linkages in amylopectin and pullulan (a fungal polymer of maltotriose residues),

respectively, and are part of the a-amylase 'super-family' of enzymes. The

Arabidopsis genome contains three isoamylase-type DBEs (isa-1, isa-2, and isa-3) and

one pullulanase-type DBE. Both groups of DBEs in higher plants share a common N­

terminal domain whose function is yet to be elucidated. The decrease/loss of either

isa-1 or isa-2 isoamylase-type DBE activities is thought to be responsible for the

accumulation of phytoglycogen rather than starch in mutant/transgenic plants (Bustos

et al., 2004) and algae (Mouille et al., 1996), and it is thought that, in rice endosperm,

residual pullulanase-type DBE activity modulates these phenotypic effects (Nakamura

et al., 1998).

In maize endosperm, the pullulanase-type DBE activity is thought to have a

bifunctional role, assisting in both starch synthesis and degradation (Dinges et al.,

2003). In wheat, the expression of a eDNA for the isoform of an isoamylase-type DBE

(Iso-1) is highest in developing endosperm and undetectable in mature grains, which

suggests a biosynthetic role for isoamylase in this tissue. The precise roles for the

isoamylase-type and pullulanase-type DBEs in starch biosynthesis are not yet known.

Two models have been proposed which could define a role for the DBEs in starch

synthesis and phytoglycogen accumulation. The 'glucan-trimming' (pre-amylopectin

trimming) model proposes that glucan trimming is required for amylopectin

aggregation into an insoluble granular structure (Ball et al., 1996; Myers et al., 2000).

DBE activity would be responsible for the removal of inappropriately positioned

branches (pre-amylopectin) generated at the surface of the growing starch granules,

which would otherwise prevent crystallization. As such, the debranched structure

would favour the formation of parallel double helices, leading to polysaccharide

aggregation. Recent observations, which show that the surface of the immature

granules contains numerous short chains, are consistent with this model (Nielsen eta!.,

2002) (Figure 1.4).

11

granule surface (soluble)

D-enzyme

MOS

+r--- sucrose

ADPGic + primer + ss

linear precursor

ss

~ rE

pre-Ap BE DBE D-enzyme

'----r--.---'

I

·-----------• PG SS,BE

Ap +-~-------' granule interior crystallization

(insoluble)

Figure 1.4. Combined glucan trimming and clearing models of starch deposition.

ADP-Glc-ADP glucose, SS-Starch synthase, BE- Branching enzyme, DBE- Debranching enzyme, D-enzyme- Disproportionate enzyme, MOS- Maltooligosaccharides

Adapted from Myers eta!., Plant physiology, 2000

An alternative to the glucan-trimming model proposes that the DBEs function

in starch synthesis indirectly in a 'clearing' role, removing soluble glucan that is not

attached to the granule from the stroma. This removes a pool of substrates for the

amylopectin synthesizing enzymes (SSs and SBEs), and thereby, prevents the

random/futile synthesis of glucan polymers by these enzymes, which could cause the

accumulation of phytoglycogen, ultimately leading to a reduction in the rate of starch

synthesis. This model could also explain the accumulation of phytoglycogen at the

expense of amylopectin observed in DBE mutants (Zeeman et al., 1998).

Starch phosphorylase (SP)- Starch phosphorylase (SP, EC 2.4.1.1)

catalyses the reversible transfer of glucosyl unit from glucose-1-P to the non-reducing

end of a a-1, 4-linked glucan chains and may be driven in either a synthetic or a

degradative direction by the relative concentrations ofthe soluble substrates.

However, the role of SP in higher plant starch metabolism is unclear. Plastidial

SP (referred to as Phol or the L-form) is characterized by higher affinity for

amylopectin than glycogen. Kinetic analysis of the maize endosperm Phol showed

that the phosphorolytic reaction is favoured over the synthetic reaction in the presence

of maltooligosaccharides (MOS) (Mu et al., 2001 ). Although the precise role of Pho 1

in starch metabolism is unclear, available experimental evidence indicates that Pho 1

probably contributes to starch synthesis, as a number of studies have found that

SP/Pho1 gene expression and activity measurements both correlate with starch

biosynthesis (Duwenig et al., 1997; van Berkel et al., 1991; Yu et al., 2001). Studies

with sweet potato roots have shown that the activity of the plastidial isozyme (L-form)

of SP may be regulated by proteolysis of a 78-amino acid peptide (L 78). Removal of

L 78 by an endogenous protease increased the catalytic activity of SP in the

phosphorolytic direction (Chen et al., 2002). One possible role for Pho1 would be in

controlling the availability of MOS, which are required for amylose synthesis, and

acting in a 'clearing' role similar and complementary to that proposed for the DBEs.

Figure 1.5 shows how starch phosphorylase is regulated by phosphorylation resulting

in the formation of a multiprotein complex.

12

Figure 1.5.

A. Amyloplast

ATP protein kinase(s)

• protein phosphatase(s)

B. Amyloplast/Chloroplast ATP

protein kinasc(s)

)I .... protein phosphatase(s)

Model of phosphorylation-dependent protein complex formation involved in storage starch biosynthesis.

Activation of SBEIIa (in chloroplasts and amyloplasts, A and B), and activation and complex formation involving SBEI, SBEIIb, and SP by protein phosphorylation in the amyloplast stroma (A) stimulates mylopectin biosynthesis. The functional relationships between the different components of the putative protein complex are unclear. It is notable that in mutants lacking SSIIa, that starch granules are also observed to be devoid of SST, SHEila, and SBEIIb, suggesting that these components may also be capable of forming a complex under in vivo conditions.

Adapted from Tetlow eta/., J Exp Botany, 2004

Disproportionating enzymes- It has been suggested that

disproportionating enzymes (D-enzymes) may work in conjunction with SP,

contributing to starch synthesis via the phosphorolytic SP reaction (Takaha et al.,

1998). According to this model, which is based on the 'glucan-trimming' model

proposed by Ball et al, short chain MOS liberated in the trimming reaction by DBEs

are converted to longer-chain glucans by D-enzyme, which in tum are available for

phosphorolysis by SP, liberating G1ucose-1-P used to synthesize ADP-Glucose by

plastidial AGPase (Ballet al., 1996). Indeed, the phosphorolytic SP reaction has been

shown to be stimulated by the presence ofD-enzyme (Colleoni et al., 1999). Table 1.3

shows the localization of these various enzymes of the starch biosynthetic pathway of

the monocots and the dicots.

1.4 Factors influencing starch biosynthesis and grain filling

Carbohydrate partitioning

Carbon fixed during photosynthesis has also to be partitioned between starch

synthesis in the chloroplast for temporary storage and sucrose synthesis in the cytosol

for export within the mesophyll cell. Triose phosphates synthesized in the chloroplast

during photosynthesis can either be transported into the cytosol by the inner

chloroplast membrane localized triose-phosphate translocator (TPT) to mainly feed

into sucrose synthesis (Lunn, 2007; Ulf-Ingo Flugge, 1999) or they can be retained

within the chloroplast and used for starch synthesis. These two pathways are highly

coordinated in source leaves. During the course of photosynthesis rising sucrose levels

lead to posttranslational inactivation of sucrosephosphate synthase (SPS) (Javier et al.,

2005) one of the regulatory enzymes in the pathway of sucrose synthesis. When

sucrose accumulates during the day, feedback control of SPS in tum leads to inhibition

of sucrose synthesis and consequently results in the accumulation of phosphorylated

intermediates and depletion of Pi (Figure 1.6).

The rising 3-PGA to Pi ratio due to the feed back inhibition of sucrose

synthase eventually activates AGPase, leading to a stimulation of starch synthesis

(Stitt et al., 1987). Down regulation of TPT and cFBPase in transgenic plants leads to

the inhibition of sucrose synthesis and concomitantly to a stimulation of starch

13

Table 1.3 Localization of enzymes constituting the known core pathway of starch

biosynthesis in higher plants

Enzyme Monocotyledon Dico!Yledon

Endosperm Leaf Granule Embryo/tuber Leaf granule

AGPase ( cytosolic) + s AGPase(plastidial) + + s + + s GBSSI + G + G

GBSSII + G + G

SSI + + G/S G/S

SSIIa + G/S

SSIIb + ?

SSII + + G/S

SSIII + + s + + s SSIV ? ? ? ? ? ?

SBEib + + s + + s SBEicc + G

SBEIIa + + G/S

SBEIIb + G/S

SBEII + + G/S

Isoamylase-TypeDBE(Iso-1) + + s + + s Iso-2 ? ? ? ? ? ?

Iso-3 ? ? ? ? ? ?

Pullulanase Type DBE + + s + + s SPd + + s + + s D-enzymed + + s + + s RlProtein ? + G/S + + G/S

a-Amylased + + s + + s 0-amylased + + s + + s

8 G. granule associated; S. soluble. Granule associated proteins are defined as those proteins remaining attached/ associated with the starch mutants following extensive washing treatments with buffers/ SDS and acetone. bNot all plant tissues appear to possess an SBEI gene ; for example, there is no SBEI homologue in the Arabidopsis genome. cSBEic appears to be restricted to the A-type granules of storage starches from the endosperms of Triticum sp.,Hordeum sp.,and Secalesp.(Baga et al.,2000;Peng et al2000); these species(Festucoideae) show a bimodal granule size distribution. dEnzymes possessing both plastidial and extraplastidial forms; the latter are not presumed to have any role in starch metabolism within the plastid and are not shown in the table.

Figure 1.6.

I ught I

Fatty acids

i Triose-P

1 Starch Hexose-P

1 Sucrose

Schematic representation of photosynthetic C02 assimilation and carbon partitioning in leaves.

In the presence of light, Calvin cycle converts inorganic C02 to phosphorylated intermediates which are used for starch and fatty acid synthesis in the plastid or sucrose synthesis in the cytosol. The transitory starch that accumulates during the day is degraded in the following night to maltose and glucose, which are exported to support cytosolic metabolism. Fatty acids are used to synthesize lipids via different routes that are located inside or outside the plastid

Adapted from Geigenberger et a!., J Exp Botany, 2005

synthesis via accumulation of 3-PGA (Zrenner et al., 1996; Strand et al., 2000)

providing additional evidences in support of this biochemical model.

Sucrose synthesis in source leaves

In most of the higher plants, sucrose is the mam form of carbohydrates

transported from source to sink tissues. During photosynthesis, triose phosphates are

exported into the cytosol and subsequently converted into sucrose photosynthetic

sucrose synthesis in source leaves is assumed to be regulated at the two-enzymatic

steps catalyzed by cytosolic fructose-1, 6 bisphosphatase ( cFBPase) and sucrose­

phosphate synthase (SPS) respectively (Figure 1.7).

cFBPase represents the first regulatory step in sucrose synthesis. It converts

fructose-1 ,6-bisphosphate ( fructose-1 ,6-P) into fructose-6-phosphate (fructose-6-P).

This enzyme is subject to allosteric control by the signal metabolite fructosse-2,6-

bisphosphate that acts as a potent inhibitor of enzymatic activity. Early studies in

transgenic potato and Arabidopsis plants with antisense repression of cFBPase or a

mutant line of Flaveria linearis, respectively, showed that a reduction of this enzyme

strongly impaired the photosynthetic sucrose synthesis, thus demonstrating its

importance in the control ofthe pathway (Zrenner et al., 1996; Strand et al., 2000).

SPS represents the second regulatory in the sucrose synthesis pathway and

forms sucrose-6-phosphate from UDP-glucose and fructose-6-P. SPS is known to be

regulated by a hierarchy of several interacting mechanisms. Firstly, transcription is

under developmental control or responds to changes in irradiance. Secondly, SPS

activation is modulated by reversible protein phosphorylation at multiple sites. In

spinach leaves phosphorylation of two distinct seryl residues, Ser-158 and Ser-424, is

required for inactivation of the enzyme in the dark and activation by water stress.

Thirdly, SPS activity is subject to allosteric modulation by metabolic effectors with

glucose-6-P as an activator and Pi as an inhibitor. Moreover, glucose-6-P and Pi can

also inhibit SPS-Kinase and SPS protein phosphatase, respectively thus affecting the

regulation of SPS by phosphorylation/ dephosphorylation. Lastly, SPS can interact

with 14-3-3 proteins, a group of small acidic regulatory proteins, in a phosphorylation

14

CYIDSOL IX\

Rul,5-P~ 3-PGA 3-PGA

5

Figure 1.7.

2 t l~p «=="'~ .;

t 8

~ ';~ ~Jl;j.. __ · .. -... :. --::./;. /V ""-

9 \ ..... .. >:-· 11

Frup-6-P #" ,~c-6-P ~ l3 •·

.

14 'f T

SUCROSE

Schematic representation source leaves

of sucrose

Gcl-P

'fl2 UIPglc

synthesis

Reactions shown are catalysed by the following enzymes (note in some instances multiple reactions are represented by a single arrow):

I, Rubisco; 2, chloroplastic PGK and chloroplastic TPI; 3, chloroplastic Fru-1-6-P2 aldolase; 4, chloroplastic FBPase; 5, transketolase, sedoheptolase-1 ,7 -bisphosphatase aldolase, sedoheptolase-1, 7 -bisphosphatase, phosphopentoepimerase, phosphoriboisomerase and phosphoribulokinase; 6, triose phosphate transporter; 7, cytoslic PGK and cytosolic TPI; 8, cytosolic Fru-l-6-P2 aldolase; 9, cytosolic FBPase; 10, cytosolic PGI; 11, cytosolic PGM, 12, UGPase, 13, SPS, 14, sucrose phosphatase.

m

dependent way although the biochemical implication of this protein protein interaction

is not clear yet as both inactivation and activation of the enzyme have been observed.

Sucrose-6-phosphate phosphatase (SPP, EC3 .1.3 .24) catalyzes the final step in

the pathway of sucrose synthesis, hydrolyzing sucrose-6-P to sucrose and Pi. The

reaction catalyzed by SPP is essentially irreversible and displaces the reversible SPS

reaction from equilibrium into the direction of net sucrose synthesis.

Transport of sucrose to the sink tissues

The evolution of sink and source tissue systems generated a need for long

distance transport between these specialized tissues, which is carried out by the

phloem cells of the plant's vascular system. To accomplish long distance transport

from source to sink, sucrose has to be loaded into the phloem in source tissues.

In most plant species such as Arabidopsis, tobacco and potato, evidences

suggest sucrose to be loaded into the phloem via an apoplastic route (Kuhn et al.,

1997; Gottwald et al., 2000). In the apoplastic phloem-loading route, sucrose is

released into the extracellular space somewhere in the proximity of the phloem, where

it diffuses through the cell wall milieu. After reaching the sieve element and

companion cell (SE/CC) complex, sucrose is then loaded against a concentration

gradient into the phloem via the well-characterized plasmalemma bound sucrose/

proton symporter (Kuhn et al., 1997; Gottwald et al., 2000). However, in some species

like Coleus blumei and Cucumis melo, evidences suggest sucrose loading into the

phloem might be symplastic (Ayre et al., 2003).

In symplastic phloem loaders, sucrose is supposed to be transported from cell

to cell via plasmodesmata-nanopores lined by plasma membrane that bridge the

cytoplasm of most plant cells to their neighbors, and eventually released into the

SE/CC. In both loading routes, once sucrose is loaded into sieve tubes in the source

organs, water is drawn in osmotically across the cell membranes, leading to a building

up of physical pressure. The hydrostatic pressure then drives mass flow transport to

less pressurized regions of the plant (sink organs) following the sieve tubes.

15

Sucrose unloading and metabolism in sink organs

After reaching the sink tissues, water and sucrose move out from the phloem to

the surrounding cells, where sucrose is utilized. Evidences suggest that symplastic

unloading of sucrose from the phloem to heterotrophic sink tissues through

plasmodesmata connections constitutes the principal unloading route.

Although the symplastic unloading route is predominant, apoplastic route is

inevitably required in some cases e.g. unloading to the filial tissue in developing seeds

that has no symplastic connection with the maternal tissue. In the apoplastic unloading

pathway, sucrose released into the apoplast can either directly enter the heterotrophic

cells via a plasma membrane-bound sucrose transporter or be hydrolyzed into glucose

and fructose by cell wall bound invertase that are subsequently imported into the cells

via a hexose transporter located in the plasmamembrane. In some organs, sucrose

phloem-unloading mode is also under developmental control e.g. in potato tubers,

during the early stages of tuberization apoplastic unloading of sucrose into swelling

stolons is predominant, followed by cleavage of sucrose into glucose and fructose with

the action of cell wall invertase (Appeldoom et al., 2001). In storage sinks such as

tubers, a large proportion of the imported sucrose is metabolized and converted to

storage compounds e.g. starch. Inevitably, sucrose metabolism in the sink organs plays

an important role in determining sink strength. Potato tubers are ideal storage sinks to

study sink strength and hence have been subject to intensive studies for a long time.

As mentioned above, apoplastic unloading of sucrose is predominant in swelling

stolons and the sucrose is then cleaved into glucose and fructose by the action of cell

wall invertase (Viola et al., 2001). In the growing tuber, sucrose is mainly unloaded

from the phloem via plasmodesmata and subsequently cleaved within the cytosol

mainly by sucrose synthase (Susy) leading to the formation of uridine diphosphate

glucose (UDPG) and fructose. UDPG can be further converted into glucose-1-P via

UGPase and fructose is readily phosphorylated into fructose-6-P by fructokinase. The

resulting hexose phosphates are equilibrated by the action of cytosolic isoforms of

phosphoglucoisomerase and phosphoglucomutase, and Glucose-6-P is subsequently

transported into amyloplast and used for starch synthesis (Tauberger et al., 2000).

16

Transport of metabolites for starch synthesis into amyloplasts

The possibility exists, therefore, that adenosine diphosphate glucose (ADPG)

may be synthesized in both the cytosol and the amyloplast of wheat/cereal endosperm.

Figure 1.8 shows the major metabolites and enzymes involved in the conversion of

sucrose to starch in storage organs. Synthesis within the organelle requires a supply of

hexose phosphate and ATP. FluEgge and co-workers have cloned a 'hexose

phosphate:Pi exchanging carrier' from a number of species and were able to express a

functional protein, heterologously, corresponding to that found in the envelopes of pea

root plastids (Kammerer et al., 1998). Whilst this protein was able to bind glucose-6-

P, it was unable to transport glucose-1-P. However, the reconstitution of envelope

proteins from wheat endosperm amyloplasts into proteoliposomes demonstrated the

presence of a protein, which was able to catalyse the counter exchange of either

glucose-6-P or glucose-1-P with orthophosphate (Tetlow et al., 1996).

The hexose phosphates were competitive inhibitors with each other, suggesting

a common carrier and leaving open the possibility that either could be imported from

the cytosol to support starch synthesis. Studies of starch synthesis in isolated

amyloplasts, suggested that glucose-1-P is a better precursor for starch synthesis than

glucose-6-P (Tetlow et al., 1994), although this observation must be mitigated by the

relative in vivo concentrations of the two hexosephosphates, where glucose-6-P tends

to be in 10±20 fold excess of glucose-1-P because of the equilibrium constant of

phosphoglucomutase. Whichever hexosephosphate is transported, there is a

requirement for ATP within the amyloplast. Whilst this could be generated by

oxidative metabolism within the organelle (Plaxton, 1996), studies with potato tubers

suggest that a plastidic ATP/ ADP transporter protein (AATP) is more significant in

regulating starch synthesis when ADPG is synthesized within amyloplasts

(Kampfenkel et al., 1995; Tjaden et al., 1998; Geigenberger et al., 2001). Neuhaus

and his coworkers have demonstrated that this protein is distinct from its

mitochondrial counterpart, and has a profound effect on starch synthesis.

Decreasing AA TP activity in potato tuber by antisense technology caused a

reduction in starch synthesis, whilst increased activity (caused by heterologous

expression of AA TP from Arabidopsis thaliana) led to an increase in starch synthesis

17

Figure 1.8.

CYTOSOL SUCROSE

i~~~~:~~~ug4 sucrose synthase

fructose + UDP glucose

1 UOP glucose pyrophosphorylase

ADPglucose

I 1 h h t • AOP glucose g ucose- -p osp a e rb' pyrophosphorylase

! glucose-1-phosphate phosphoglucomutase •

rug3 ' phosphoglucomutase G6P

glucose-6-fhosphate -----l~glucose-6-phosphate trans locator

phosphoglucoisomerase

fructose-6-phosphate

Major metabolites and enzymes involved in the conversion of sucrose to starch in storage organs

Enzymes are Sucrose synthase; UDP-glucose pyrophosphorylase; ADP-glucose pyrophosphorylase; phosphoglucomutase; Starch synthase; Starch branching enzyme; Glucose-6-P translocater. Selected starch mutants are placed at the point in the pathway where they are known or hypothesized to exert their effects.

and tuber yield. Further, the activity of AA TP also influenced the ratio of

amylose:amylopectin by altering the content of ADPG. Amylose synthesis is relatively

more favoured at higher ADPG concentrations, as the affnity of the granule bound

starch synthase, which is responsible for amylose synthesis, is about 10-fold lower

than that of the soluble starch synthases which contribute to amylopectin synthesis

(Smith et al., 1997). Thus, in transgenic potato tubers, as the activity of AATP

increased, so also did the content of ADPG and the ratio of amylose:amylopectin

(Tjaden et al., 1998; Geigenberger et al., 2001 ). There are no published reports of this

transporter in monocots, though a partial eDNA with a high degree of homology to the

Arabidopsis protein has been obtained from wheat endosperm (HENeuhaus, personal

communication).

There is also a requirement to transport ADPG into the amyloplast, which

cannot be met by the AATP referred to previously, as this is highly specific for ATP

and ADP and shows no affinity towards nucleotide sugars. The Btl locus of maize

encodes a protein of 38±42 kDa localized in amyloplast membranes (Sullivan and

Kaneko, 1995) and is believed to play a role in the transport of ADPG (Shannon et

al., 1998). Recent studies have demonstrated the presence of an ADPG transporter in

wheat endosperm amyloplasts (Emes et al., 2001). The protein is able to catalyse the

counter exchange of ADPG with AMP, ADP or ATP, but does not bind UDPG or

other uridylates (M. Emes, unpublished observations). The ADPG transporter can be

identified in wheat endosperm by covalent cross-linking to radiolabelled azido-ADPG.

The data suggest that it is not readily detected until 10 dpa and that content increases

subsequently, coinciding with the major period of grain filling and consistent with the

changes observed in the expression of cytosolic AGPase in barley endosperm (Doan et

al., 1999).

Since this protein is possibly the major route for the import of the soluble

substrate of starch synthesis, ADPG, it is likely that its activity will also have an

impact on the balance of amylose and amylopectin synthesis in the same way as has

been observed for the potato AA TP.

18

1.5 AGPase - The key enzyme in starch biosynthesis in cereal

grains

Starch is an important end product of carbon fixation in plants and it is the

storage product in cereal grains contributing to the economic yield potential of the

plants. Starch synthesis is regulated by a set of key enzymes of starch metabolism.

ADP-glucose pyrophosphorylase (AGPase) is one such key enzyme which catalyses

the formation of ADP-glucose, the rate limiting step in starch biosynthesis. Therefore,

AGPase and its regulation would be an important starting point to study in some

representative rice varieties.

Historically, AGPase has received considerable attention because of its

allosteric properties and its position as the first unique step in starch synthesis. The

first definitive evidence for its rate-limiting role in a plant system came with the work

of Stark et al (Stark et al., 1992). Those investigators increased starch content 30% by

expressing an allosterically altered bacterial AGPase in the potato tuber. Subsequently,

Giroux et al modified the allosteric properties of the maize endosperm AGPase and

increased maize seed weight approximately 15% (Giroux et al., 1996). Hence,

AGPase represents an important target for genetic manipulation.

Evolution of plant AGPases

The higher plant AGPase is believed to have evolved from homotetrameric

forms similar to those seen in enteric bacteria today, where the enzyme is activated by

fructose 1 ,6-bisphosphate and inhibited by Pi. A next step in AGPase evolution

appears to have been an adoption to different allosteric activators such as 3-

phosphoglyceric acid (3-PGA) and inorganic phosphate (Pi), the effectors of the

cyanobacterial AGPase, although the enzyme is active as a homotetramer.

The heterotetrameric forms of the enzyme are believed to have evolved

through duplication and divergence of the ancestral AGPase gene, facilitating the

evolvement of an interaction between a main catalytic (SS) subunit and a regulatory

(LS) subunit. Interestingly, the SS of higher plants such as potato (Ballicora et al.,

19

1995) and barley has not lost its activity as a homotetramer, an activity that, similar to

the ancestral enzyme, is responsive to 3-PGA and Pi.

Location of AGPase- plastidial, cytosolic or both

For many years, it was generally accepted that AGPase is exclusively located

in plastids of one type or another, be they chloroplasts or amyloplasts. There is now

good reason for believing that this does not hold true in cereal endosperm. In the

developing endosperms of maize and barley, there is a cytosolic isoform (Javier et al.,

2005) which accounts for 85± 95% of the total activity (Denyer et al., 1996;

Thorbjurnsen et al., 1996). The enzyme is a heterotetramer, consisting of large, AGP­

L, and small, AGP-S, subunits (the latter bearing the catalytic activity). Doan et a!

have expressed both barley endosperm subunits in baculovirus, separately and in

combination (Doan et al., 1999). Whilst AGP-S was sensitive to activation by PGA,

when AGP-S was expressed alongside AGP-L, this sensitivity was lost. Further, this

PGA-insensitive form of the enzyme has been shown to be extraplastidic, lacking any

identifiable transit peptide necessary for targeting to amyloplasts.

Expression of the barley endosperm cytosolic AGPase subunits appears to be

developmentally regulated, with both large and small subunit mRNAs appearing at

around 10±12 dpa (Doan et al., 1999). The equivalent subunits have also been cloned

from wheat endosperm (Ainsworth et al., 1993, Ainsworth et al., 1995) with AGP-S

being expressed earlier in endosperm development than AGP-L. Although then

cDNAs cloned by Ainsworth et al. were originally thought to code for plastidic

subunits, alignment of the sequences with the barley subunits indicates that they are

virtually identical.

Wheat endosperm (Tetlow et al., 2003), like barley and maize, appears to

contain AGPase activity in both the cytosol and amyloplast (Vardy et al., 2002).

Beckles et al reasoned that because of the presence of UDP-glucose (UDPG)

pyrophosphorylase in the cytosol, the ratio of ADPG to UDPG would be higher in

tissues expressing a cytosolic AGPase, compared with species where UGPase and

AGPase are in discrete subcellular compartments (Beckles et a!., 2001 ). A thorough

analysis of a large number of mono-and dicotyledonous species showed that the ratio

20

of ADPG: UDPG was generally an order of magnitude higher in cereal endosperms

compared to other species/organs (Beckles et al., 2001), and also concluded that there

was no evidence for a cytosolic AGPase in nongraminaceous species. The dual

location of AGPase in different subcellular compartments has implications for both the

regulation of starch synthesis, and movement of metabolites into amyloplasts.

AGPase protein and its isoforms

AGPase has been purified from E. coli and several plant sources (Ghosh and

Preiss, 1966; Ozaki and Preiss, 1972; Fuch and Smith, 1979; Plaxton and Preiss, 1987;

Green et al., 1998). Although the bacterial and plant enzymes catalyze the same

reaction they have different structures. The bacterial AGPase is composed of a single

sub-unit, encoded by the glgC gene, which oligomerizes to form a homotetramer. The

wheat endosperm AGPase is also a homotetramer of sub unit molecular weight of

about 51,000 daltons (Sivak and Preiss, 1994 ).

On the other hand, AGPases from many other plant species are

heterotetramers. In these cases, the enzyme is composed of two related but distinct

subunits [large subunit (LS) and small subunit (SS)], which form an LS2 SS2

heterotetrameric protein (Okita et al 1990; Cognata et al., 1995; Denyer et al., 1996;

Thorbjomsen et al., 1996; Bae and Liu, 1997; Chen and Janes, 1997). The enzyme

from these plants has been extensively studied by western blotting, 2-D

electrophoretic gels or by comparative analysis of the deduced amino acid sequences

from eDNA clones. Table 1.4 describes the properties of the plant and bacterial

AGPases.

Recently, multiple forms of both the small and the large subunits have been

found in several plants. Several isoforms of the large subunit were observed when the

purified potato (Solanum tuberosum L.) tuber AGPase was subjected to high

resolution 2-D PAGE (Okita et al., 1990). The identification of three AGP large

subunit cDNAs from potato tuber suggests that multiple polypeptides are not the result

of proteolytic degradation or posttranslational modification (Cognata et al., 1995).

58 2.." l :so 41Cf2..4 D49_~_ .St

582.13041 TH 0494 St

21 111111111111! 1m 11111111111111 TH16527

Table 1.4 Plant and bacterial AGPases

Origin Subunit structure Genes Activator Inhibitor

E. coli Homotetramer e glgC gene Fructose- I, 6-biphosphate AMP

Large subunit gene

Plants Heterotetramer 3-PGA Pi

Small subunit gene

Similarly, multiple AGPase polypeptides have been detected in pea (Pisum

sativum L.) and rice endosperm (Hylton and Smith, 1992; Nakamura and Kawaguchi,

1992). Multiple eDNA clones for AGPase have also been isolated from barley

(Hordeum vulgare L.; Villand et al., 1992), Arabidopsis (Villand et al., 1993), maize

(Zea mays L.; Giroux and Hannah, 1994), broad bean (Weber et al., 1995), pea

(Burgess et al., 1997), and sweet potato (Bae and Liu, 1997).

Whereas the presence of isoforms seems to be a common feature of plant

AGPases, the significance of their occurrence is not presently known. One possible

explanation is that individual isoforms could have different intracellular locations. It

was proposed that AGPase of cereal endosperm exists in the cytoplasm as well as in

the amyloplasts (Hannah et al., 1993; Villand and Kleczkowski, 1994).

The enzyme exhibits presence of organ-specific isoenzyme pattern, which

originates from the subunit forms that assemble into the holoenzyme (Ainsworth et al.,

1993 ). Recently Park and Chunq cloned three isoforms of LS of AGPase from tomato

plants (Park and Chunq, 1998). The expression of these isoforms was found to be

organ specific with respect to leaves, roots and fruits. Isoforms are reported in potato

also (Cognata et al., 1995). There may also be distinct plastidial and cytosolic forms of

AGPase (Denyer et al., 1996; Kleczkowski, 1996).

The existence of isoforms particularly with respect to the large subunit which

controls the catalytic properties of the enzyme resident in the small sub unit and the

tissue specific expression of the isoforms adds an important element of the structure­

function relationships of this protein which may be of significance for its regulation.

The existence of isoforms of the plant enzyme is of regulatory significance in the

context of its allosteric behavior. The precise composition of the holoenzyme could

vary with, say, different stages of tuber development or other physiological/ metabolic

conditions of the tissue. Existence of different enzyme forms may also be related to

different intracellular locations such as chloroplasts, amyloplasts and cytosol.

Alternatively, the various AGPase peptides may contribute to cell-specific isozyme

patterns. Such cell specificity could be achieved by driving expression of different

genes in sub set of cells giving rise to different forms of the enzyme. The dual location

22

of AGPase in different subcellular compartments has implications for both the

regulation of starch synthesis, and movement of metabolites into amyloplasts.

Regulatory regions of AGPase

Random mutagenesis of potato tuber AGPase, via PCR, hydroxylamine, or

gene shuffling and PCR combined, identified three regions involved in regulation in

the large subunit and two regions in the small subunit (Preiss et al., 2006; Kavakli et

al., 2001; Kavakli et al, 2001; Salamone et al., 2000; Salamone et al, 2001).

Regulatory regions in the small subunit consisted of 50 amino acids in the N terminus

and the last 150 amino acids of the processed subunit. Regulatory regions in the large

subunit consisted of 50 amino acids in the N terminus, a middle region and the last

100 residues of the processed subunit. Since several mutants exhibited complex

phenotypes, or resulted from mutagenesis on small subunit homotetramers,

conformational modifications were hard to separate from changes in the regulator

binding sites. However, pyridoxal 5'-phosphate (PLP) labeling experiments supply

further information.

PLP-labeling of spinach leaves decreased activation by 3-PGA by 6-fold

(Morell et al., 1988) and was hindered by 3-PGA and phosphate. Hence, PLP target

sites are identical or close to those of Pi and 3-PGA binding. The substrates had no

effect on PLP-labeling, which points to different catalytic and regulatory sites.

AGPase treatment with radioactive PLP-labeled three sites in the large subunit and

one in the small subunit (Ball and Preiss, 1994). Lysine 3, the C terminal lysine

modified in the large subunit, is conserved in all AGPase sequences available in both

the large and small subunit. It corresponds to the site modified in the small subunit.

The second C-terminal lysine, lysine-2, and the N-terminal lysine, lysine-1, are not

conserved. For instance, endosperm AGPases only possesses lysine-3 in the large and

small subunit (Ball and Preiss, 1994). PLP-labeling pointed to a partial overlap of

inhibitor and activator sites as Pi only protected modification of lysine-2 while 3-PGA

protected all three lysines. PLP-labeling experiments confirm the role of the C-termini

in regulation.

23

AGPase and Allosteric behaviour

The catalytic activity of AGPase is allosterically regulated by small effector

molecules whose levels control the rate of starch biosynthesis (Preiss 1982). Plant

AGPase is activated by 3-PGA and inhibited by Pi while the E. coli AGPase is

activated by fructose- I ,6-bisphosphate and inhibited by AMP. However, barley

endosperm AGPase has been reported to be relatively insensitive to 3-PGA/Pi

regulation (Kleczkowski et al. 1993) indicating some degree of heterogeneity among

the plant enzymes. Phosphate decreases 3-PGA activation and shifts the activation

curve from a hyperbola to a sigmoidal curve. Likewise, 3-PGA decreases phosphate

inhibition. Sensitivity to the regulators varies depending on the tissue and cell location

of the enzyme and divides AGPases into three groups.

Leaf AGPases are activated 10 to 15-fold by 3-PGA. Barley leaf is less

sensitive. Spinach leaf AGPase activated 58-fold by 3-PGA is an exception (Ghosh

and Preiss, 1966; Kleczkowski et al., 1993; Sanwal et al., 1968; Singh et al., 1984).

Half saturation values for 3-PGA (Ka) are low at less than 0.5 mM. Substrate Kms are

decreased 8 to 10 fold for ATP and 3 fold for glucose-1-P by 3-PGA. In plants with

crassulacean acid metabolism, 3-PGA increases glucose-1-P affinity more than that of

A TP. Leaf AGPases also respond to low levels of phosphate as the I 0.5 in the absence

of 3-PGA is less than 0.5 mM and activity at saturating amounts of phosphate

decreases to negligible levels. In conclusion, in leaves regulation affects activity as

well as substrate Kms and involves regulatory concentrations of less than 1 mM.

Anabaena AGPase, often used as a model, also exhibits a leaf type regulation (Iglesias

et al., 1991).

In tomato fruit and potato tuber AGPases, 3-PGA is almost required for

activity (Chen and Janes, 1997; Sowokinos and Preiss, 1982). The activator changes

substrate Km two fold at most in potato tuber AGPases to values comparable to those

of leaf AGPases. Kms for 3-PGA are below 0.5 mM. Tomato fruit and potato tuber

AGPases are extremely sensitive to phosphate inhibition. Iso values are below 1 mM

and activity levels are non detectable in saturating amounts of phosphate. In

24

conclusion, in potato tubers and tomato fruits regulation affects activity greatly,

substrate Kms only slightly and involves effector concentrations lower than 1 mM.

Endosperm tissues display more complex regulation patterns. In all studied

endosperm AGPases, 3-PGA shifts the ATP curve from a sigmoid to a hyperbola.

Barley and wheat are insensitive to phosphate inhibition and 3-PGA activation, being

activated less than two fold at 3-PGA concentrations of several millimolar

(Kleczkowski eta!., 1993; Olive et al., 1989). The activator does not change substrate

Km values noticeably. Comparatively, maize endosperm AGPase retains some

sensitivity to 3-PGA (Dickinson and Preiss, 1969) with an activation of3 to 6-fold for

a Ka of 2.2 mM. Activator influence on Km is limited to a less than two fold decrease

of the glucose-1-P and ATP Kms. These results were contradicted when Plaxton and

Preiss (1987) measured high activation on a maize endosperm purified in a different

manner. Degradation was proposed as being responsible for the results reported by

Dickinson and Preiss (1969) results. However, the results of Plaxton and Preiss (1987)

were not reproducible even on degraded enzyme (Plaxton and Preiss, 1987; Hannah et

al., 1995). As a consequence, maize endosperm AGPase is considered as being

slightly sensitive to 3-PGA activation. In conclusion, regulation in endosperm

AGPases only slightly modifies activity and substrate Kms while involving millimolar

amounts of regulators.

Physiological relevance of AGPase regulation in storage organs­

Leaf AGPases responds to photosynthesis and sucrose synthesis. Indeed, the activator

3-PGA is produced during the Calvin cycle, while the inhibitor phosphate is

synthesized during sucrose synthesis and transports into the amyloplast. Activation

increases substrate affinities of AGPase and enzyme activity. Phosphate inhibition

decreases AGPase activity and 3-PGA affinity. Hence the ratio 3-PGA/phosphate will

determine AGPase activity and substrate affinities. Activity varies most when the 3-

PGA to phosphate ratio is less than 1.5 (Kleczkowski, 1999).

In storage tissues, the physiology of regulation is not so understandable. Given

the low sensitivity of most endosperm AGPases to 3-PGA and phosphate, it was

earlier suggested that their sensitivity to 3-PGA and phosphate had been inherited

25

during earlier stages of evolution and is now progressively disappearing. Another

observation is that endosperm AGPases are localized in the cytoplasm as opposed to

the amyloplast or chloroplast. This different location would modify the environment

and hence the concentrations of regulators available. Rice endosperm AGPase though

highly activated was localized in the cytoplasm by amyloplast separation. However,

the results are uncertain because of a high cytoplasmic contamination (Sikka et al.,

2001).

AGPase gene expression and regulation

Three mechanisms are known to regulate AGPase activity 1) Transcriptional

regulation; 2) Allosteric regulation, with glycerate-3-phosphate (activator) and

inorganic phosphate (inhibitor); and 3) post-translational redox modification m

response to sugars (Muller-Rober et al., 1990). AGPase genes are known to

accumulate transcript in response to sugars and to decrease transcript in response to

nitrate (Ham et al., 2000) and phosphate exposure (Nielson et al., 1998).

It appears that the primary mode of regulation of AGPase expression in leaves

IS different than in non-photosynthetic tissues such as storage organs. In leaves,

allosteric regulation plays the major regulatory role followed by post transcriptional

and transcriptional control. The allosteric behaviour of AGPase is previously

described (Sowokinos and Preiss, 1982). On the other hand, in starch-storing organs

such as potato tubers, the predominant level of regulation seems to be transcriptional

or mRNA stability as revealed by a strong correlation between levels of transcripts,

the peptides and AGPase activity. The high steady state level of the transcripts may be

required in storage organs to meet high demands of starch synthesis. Also, this will

facilitate lower dependence on allosteric control since the concentrations of the

effector molecules (3-PGA and Pi) do not vary significantly in these tissues. In potato

tubers, coordinated expression of both small and large subunit is seen at the transcript

and polypeptide level. The levels of both increased steadily during tuber development

(Nakata and Okita, 1995).

Similar transcript accumulation profiles have been observed during

development of starch containing sink organs of other plant species like wheat

26

(Anderson et al., 1991; Reeves et al., 1986). In studies on the expression of AGPase

in maize during grain filling (Prioul et al., 1994), the time course of AGPase activity

and of starch accumulation rate was measured in developing grain. Variation of the

activity paralleled the contents of both the subunits and also the level of corresponding

mRNA suggesting a transcriptional control of regulation. The steady state levels of an

mRNA are generally controlled by a balance between the rate of transcription and the

rate of its degradation. AGPase mRNA was found to undergo rapid degradation

during seed maturation in oat (Johnson, et al., 1999); but the underlying mechanism is

not understood. Role of csrA gene product in regulation of the stability of AGPase

mRNA has been shown in E. coli (Liu et al., 1995). However, the question of

regulation of AGPase gene in plants at the level of mRNA stability needs to be

investigated more (not much is known about other plant mRNA degradation either).

While the amount of the AGPase polypeptide is controlled by steady state

levels of the transcripts (transcriptional or rnRNA stability control), there are

additional post transcriptional mechanisms which regulate tissue or organ-specific

expression of the type of the particular sub unit species discussed above and hence the

final form and properties of the enzyme.

In barley, a single gene has been shown to encode two different transcripts,

bepsFJ and blpsl4, for the small sub unit of AGPase (Thorbjornson et al., 1996).

One transcript was found to be specific for starchy endosperm and other for leaves.

Sequence of the two was about 90% identical with difference at 5' region. The unique

5' end of the two transcripts differs with respect to the sequence motif encoding a

putative transit peptide characteristic of a plastid-targeted protein. Characterization of

the gene from genomic library revealed alternative exons encoding different 5' ends of

the transcripts. Thus two transcripts destined for distinct spatial localization seem to

originate from the same gene by alternative splicing- a process not very common in

plants. Other possible regulatory modes are also documented.

In potato, the enzyme is a homotetramer in early tuber development although

both the transcripts are present. This may imply that either only one mRNA is

translated into protein or only one sub unit protein is stable to form active AGPase

27

(Muller-Rober et al., 1990). When accumulation of cDNAs and peptide levels in

leaves and during tuber development was compared, the results indicated that even in

leaves there might be regulation at the level of translation (Nakata et al., 1994;

Nakata and Okita, 1995). Luo et al recently studied processing of SS homologues of

AGPase from barley leaves and endosperm by using E. coli expression system and

over production of proteins derived from either full length or truncated cDNAs (Luo et

al., 1997). The data suggests that the seed protein does not undergo any

posttranslational proteolytic processing whereas the leaf homologue is processed to a

smaller protein, which results in allosteric property. From this and other work

discussed in the earlier section (Wu and Preiss, 1998), protein processing seems to

play regulatory role in the activity and allosteric property of these two enzyme

proteins.

Recent studies revealed another mechanism involving the posttranslational

regulation of AGPase in both photosynthetic and non-photosynthetic tissues in a range

of plant species (Fu et al., 1998, Tiessen et al., 2002; Hendriks et al., 2003). This

mechanism involves the redox regulated, reversible formation of an intermolecular

Cys bridge between the AGPB subunits in the AGPase heterotetramer.

Monomerization in response to light or rising sugar levels leads to activation of the

enzyme, which allows starch synthesis to be increased without any necessary changes

in the levels of 3-PGA and Pi (Hendriks et al., 2003). The presence of this redox

regulatory mechanism increases the flexibility of the regulatory network involving the

control of photosynthetic carbon partitioning (Figure 1.9 and 1.10).

Redox modulation of AGPase provides a novel mechanism that combines with

known mechanisms to coordinate AGPase activity in a network that allows starch

synthesis to respond across a range of time scales to a variety of physiological and

environmental stimuli (Teisson et al., 2002, Hendriks et al., 2003). Allosteric control

by 3-PGA and Pi operates in a time frame of seconds to adjust the rate of starch

synthesis to the balance between sucrose breakdown and respiration. Post-translational

redox modulation leads to changes in AGPase activity in a time frame of 30 to 60 min.

Activation occurs in response to factors directly or indirectly related to increased

sucrose availability and leads to stimulation of starch synthesis and decreased

28

Redox­Modulation

Figure 1.9.

Reduction .. Thioredoxin

1111

AGPB-Dimer Oxidation

AGPB-Monomers

ADP-glucose pyrophosphorylase (AGPase), catalysing the first committed step of starch synthesis in the plastid, is subject to post-translational redox modification

The redox regulation of potato tuber AGPase by thioredoxin involves reduction of a disulphide bond between the Cys82 of the two AGPB subunits of the tetrameric protein, which leads to a change in the kinetic properties of the enzyme

Carbon status oftbe tuber

(Sucrose)

., . Sucrose ---- • (i) m"t11bolism .

Adapted.fi"om Geigenberger eta!. , J Exp Botany, 2005

AR

?

~'n .-.Bl--1 lS I 1-.!

Tnonscrlptlon•J Regul•tion

~ GGPS I-

1

1 AGPase Ho1Mn7) mc

J

~.· .;,,~.~ T"luor<do•"' • _R_fll_o-,---·n rfll. """"""" 0 '":.... Rao. or

~ tarch Glycol. is+ 3PGA -----1-~ synthe is

Pi llosteric Regu luion

Resptrat ion

Figure 1.10. Redox regulation of AGPase in potato tubers

Adapted from Tiessen eta!. , The plant cell, 2002

glycolytic metabolite levels. The signaling components leading to redox modulation of

AGPase are unknown and may involve thioredoxins as well as putative sugar sensors.

Transcriptional regulation in response to changes of sucrose allows more gradual

changes in AGPase activity, which can require days to develop.

Turnover of ADP glucose- Evidence has recently been provided for a further

component, which may regulate the ADPG content of starch synthesizing tissues.

Pozueta-Romero and coworkers (RodroAguez-LoApez et al., 2000) have provided

evidence for the existence of an enzyme, which specifically breaks down ADPG,

ADP-glucose pyrophosphatase (AGPPase ), catalysing the reaction:

ADP-Glucose-----. Glucose-1-P +AMP

Unlike AGPase, AGPPase is a 35 kDa monomeric protein which is inhibited

by PGA as well as Pi, PPi, A TP, and ADP. The enzyme was found in both the cytosol

and plastids of barley endosperm with about 30% of the total activity being accounted

for inside amyloplasts. The activity of AGPPase was highest just after pollination (8

dap ), and had decreased to a minimal level by 20 dap. The enzyme was also found in

several other species including wheat leaves and endosperm, tomato leaves, sycamore

cell cultures, potato tubers and Arabidopsis leaves.

The presence of such an enzyme in endosperm tissue suggests that the

regulation of ADPG content in either the cytosol or amyloplast may include a further

level of complexity. The observation that AGPPase activity declines markedly during

endosperm development suggests that it is regulated developmentally to a level

consistent with the role of this tissue as a storage organ. This shows that AGPPase also

plays an important role in regulation of enzymes of starch synthesis.

AGPase and transgenics

Transgenic plants provide great opportunities both for understanding

fundamental processes in plant molecular biology and for introducing agronomically

important traits in the crop plants. Methods for gene transfer have become increasingly

successful and sophisticated in recent years. New protocols of Agrobacterium-

29

mediated transformation, applicable to monocots are now available and are being used

for rice. There are many genes, which have been introduced in cereal plants to develop

improved plant qualities, and the variety of promoters, which can properly control the

expression of the transgenes, is on the increase (Komari et al., 2000). A transgenic

variety, which can make beta-carotene in the endosperm, has been developed (Ye et

al., 2000).

The starch synthesizing ability of plants, particularly cereals, can also be

modified by genetic engineering approaches (Vissier, 1993). Barry et al produced

transgenic plants by introducing AGPase genes in several plant species, which resulted

in production of seeds having reduced oil content as well as elevated levels of starch

(Barry et al., 1994). In another study, AGPase from E. coli was introduced into potato

and strong effect was observed with respect to starch content (Stark et al., 1992). The

transformed potato tubers showed 25-60% higher starch content than the normal

potato. Thus transgenic varieties of potato incorporating genetically altered/

manipulated AGPase have already been developed. Rolletschek et al developed

transgenic Vicia plants with decreased AGPase activity by expressing antisense

AGPase mRNA. Antisense inhibition of AGPase resulted in moderately decreased

starch (Rolletschek et al., 2002).

Recently, Smidansky et al transformed rice plants with modified mmze

AGPase large subunit that has decreased sensitivity to allosteric inhibition (Smidansky

et al., 2003). The transgenic plants had enhanced AGPase activity and showed 20%

increased seed and biomass yield. Together, these studies support the possible

applicability of the genetic engineering approach for manipulation of this enzyme and

starch biosynthesis in general.

1.6 Other proteins and their role in cereal grains

Cereal grains contain relatively little protein compared to legume seeds, with

an average of about 10-12% of the dry weight. Nevertheless, they provide over 200

metric tones (mt) of protein for the nutrition of humans and livestock, which is about

three times the amount derived from the more protein-rich (20-40%) legume seeds. In

addition to their nutritional importance, cereal seed proteins also influence the

30

utilization of the grain in food processing. Therefore, cereal seed proteins have been a

major topic of research for many years, with the aim of understanding their structures,

control of synthesis and role in grain utilization (Shewry et al., 2002).

Cereal seed storage proteins

The scientific study of cereal grain proteins extends back for over 250 years,

with the isolation of wheat gluten first being described in 1745 (Beccari, 1745). Since

then more systematic studies have been carried out, notably by T. B. Osborne ( 1859-

1929), who is regarded as the father of plant protein chemistry. Osborne developed a

classification of plant proteins based on their solubility in a series of solvents, for

example, albumins in water, and globulins in dilute saline. Although 'Osborne

fractionation' is still widely used, it is more usual today to classify seed proteins into

three groups: storage proteins, structural and metabolic proteins, and protective

proteins. Seed storage proteins fall into three different Osborne fractions and occur in

three different tissues of the grain (Figure 1.11 ).

Storage globulins 7S- The embryo and outer aleurone layer of the

endosperm contain globulin storage proteins, and those from maize embryos have

been characterized in some. These proteins are readily soluble in dilute salt solution

and have sedimentation coefficients of about 7. They have limited sequence similarity

with, and may be homologous to, the 7S vicilins of legumes and other dicotyledonous

plants; they also have similar structures and properties (Kriz, 1999). Related proteins

have been found in embryos anduor aleurone layers of wheat, barley and oats (Burgess

and Shewry, 1986; Yupsanis et al., 1990; Heck et al., 1993). 7S globulins from rice

embryos have also been characterized (Horikoshi and Morita, 1975), but their

relationships to other plant 7S globulins have not been established. The 7S globulins

are stored in protein bodies and appear to function solely as storage proteins.

However, they do not appear to be absolutely required for normal seed function, at

least in maize, where a null mutant behaves normally in terms of development and

germination.

Storage globulins 11-12S- Storage globulins of 11-12S, located in the

starchy endosperm, are also present in at least some cereal grains. In fact, in oats and

31

Globulins 115 75

Soluble in dilute satt

I Storage protein families I

/ J Albumins

25

Soluble in water

"" Prolamins

Soluble in alcohol­water mixtures

Figure 1.11 Classification of seed proteins on the basis of their solubility

rice these proteins form the major endosperm storage protein fraction, accounting for

about 70-80% of the total protein. It is now known that these proteins are related to

the widely distributed 'legumin' type globulins, which occur in most dicotyledonous

species (Casey, 1999). The rice proteins are not readily soluble in dilute salt solutions

and hence are classically defined as glutelins, but they clearly belong to the 11-12S

globulin family. They comprise subunits of Mr approx. 55000 that are post­

translationally cleaved to give acidic (Mr approx. 33000 daltons in oats, 28-31000 in

rice) and basic (Mr approx. 23000 and 20-22000, respectively) polypeptide chains

linked by a single disulphide bond (Shotwell, 1999; Takaiwa et al., 1999). The oat

globulin also resembles the legumins in forming a hexameric structure with a

sedimentation coefficient of about 12. Proteins related to legumins, called 'triticins',

are present in starchy endosperm of wheat, although they account only for about 5% of

the total seed protein (Singh et al., 1988). Triticins consist oflarge (Mr about 40000)

and small (Mr about 22-23000) polypeptide chains, but the subunits appear to form

dimeric structures rather than the typical legumin hexamers.

Prolamin storage proteins- With the exceptions of oats and rice, the

major endosperm storage proteins of all cereal grains are prolamins. This name was

originally based on the observation that they are generally rich in proline and amide

nitrogen derived from glutamine, but it is now known that the combined proportions

of these amino acids actually vary from about 30-70% of the total among different

cereals and protein groups. Similarly, although prolamins were originally defined as

soluble in alcohol:water mixtures (e.g. 60-70% (v/v) ethanol, 50-55% (v/v) propan-1-

ol or propan-2-ol), some occur in alcohol-insoluble polymers. Nevertheless, all

individual prolamin polypeptides are alcohol-soluble in the reduced state. The

prolamins vary greatly, from about 10 000 to almost 100 000, in their molecular

masses.

The new system of classification assigns all of the prolamins to three broad

groups: sulphur-rich (S-rich), sulphur-poor (S-poor) and high molecular weight

(HMW) prolamins, with several subgroups within the S-rich group (Table 1.5). These

groups do not correspond directly to the polymeric and monomeric fractions in wheat

(glutenins and gliadins, respectively) recognized by cereal chemists, as both

monomeric and polymeric forms of S-rich and S-poor prolamins occur.

32

Table 1.5 Summary of the types and characteristics of prolamins

Components Mr(%total) Polymers or monomer Partialaminoacid composition(mol%)

HMW Prolamins

HMW subunits of glutenin

S-rich Prolamins

y- gliadins

P -gliadins

B and C type LMW

S-poor prolamins

c0 -gliadins

D-type LMW

65-90,000

30-45000

(70-80%)

30-75000

(10-20%)

Polymers

Monoml Monomers

Polymers

Monome}s

Polymers

30-35% Gly, 10-16% Pro, 15-20% Gly,

0.5-1.5% Cys, 0.7-1.4% Lys

30-40% Gin, 15-20% Pro, 2-3%Cys,

<1.0%Lys

40-50% Gin, 20-30% Pro, 8-9% Phe,

0.05% Lys, <0.5% Cys

C-type LMW subunits are essentially polymeric forms of a and y-gliadins and D-type LMW subunits

polymeric wgliadins. The B-type LMW subunits constitute a discrete group of S-rich prolamins. Cys is

present in D type LMW subunits, but not gliadins.

LMW- Low molecular weight

HMW- High molecular weight

The prolamins of other panicoid cereals (e.g. mmze, sorghum and many

millets) are comprised of one major group of proteins (a-zeins) and several minor

groups(~, y and 8-zeins) (Coleman and Larkins, 1999; Leite et al., 1999). Amino acid

sequence comparisons demonstrate that the ~' y and 8-zeins are all members of the

prolamin superfamily, but only the y -zeins contain repeated amino acid sequences

(either two or eight tandem repeats ofPro-Pro-Pro-Val-His- Leu). The~ -zeins and 8-

zeins are both rich in methionine. By contrast, the a -zeins do not appear to be related

to any other prolamins except the a -type prolamins of other panicoid cereals. They

consist of two major subclasses called the 19K and 22K zeins based on their Mr

determined by SDS-PAGE although they have true molecular masses of 23-24 000

and 26 500-27 000, respectively. Both subclasses contain degenerate repeats of about

20 aminoacid residues, with nine such blocks present in the Z 19 and ten in the Z22

zeins. The a -zeins contain only one or two cysteine residues per molecule and are

present in the grain as monomers or oligomers, while the ~' y and 8 -zeins are all

richer in cysteine and form polymers.

Non-specific lipid transfer proteins (LTP's)

The LTP family is made up of low molecular weight (7-9 kDa) monomeric

proteins, which are very basic. They are able to catalyze the transfer of lipids between

vesicles and membranes in vitro and there is increasing evidence that their role in vivo

may be in cutin biosynthesis. The proteins are made up of a bundle of four a -helices

with a lipid-binding cavity in the center. There are indications that bound lipid

increases resistance to proteolysis; these proteins also survive thermal treatments, and

can refold to their native structure on cooling.

Structural and metabolic proteins

These include a wide range of proteins, which play roles in the structure and

function of plant cells. Many may be regarded as "housekeeping" proteins, since they

are essential for the structure and function of all cells, while others are synthesized

only in specific cell types or tissues at a specific developmental stage or in response to

specific signals. They include enzymes involved in biosynthesis and catabolism,

33

structural proteins present in membranes and cell walls, transporters, and components

of signaling cascades. For example, analysis of the proteins encoded by the

Arabidopsis genome (a model plant species related to crucifers such as cabbage and

oilseed rape) predicts that about 22.5% of the genes encode enzymes of primary and

secondary metabolism and 11% encode proteins involved in defence, while the

remainder encode proteins involved in growth and division, energy generation, signal

transduction, transcription, protein synthesis, and cellular trafficking and transport

(The Arabidopsis Genome Initiative, 2000).

Most structural and metabolic proteins are present m very small amounts,

sometimes too small to be identified by electrophoresis of total protein fractions (for

example, components of signaling pathways). However, this is not the case for some

other components, such as photosynthetic enzymes and associated proteins in green

tissues. The most abundant of these proteins, ribulose-!, 5-bisphosphate carboxylase/

oxygenase (usually abbreviated to Rubisco), accounts for about 30--40% of total leaf

protein in most species.

Protective pathogenesis related proteins

Plant tissues contain a range of proteins and peptides that have been

demonstrated to inhibit the growth of pathogenic microorganisms (fungi, bacteria,

viruses) or the feeding or infection by invertebrate pests (e.g. nematodes, molluscs,

insects), either in vivo (e.g. in transgenic plants) or in vitro (e.g. in feeding tests).

These proteins are widely considered to play a natural role in plant resistance,

although this is difficult to establish conclusively. They are also considered to have

potential for exploitation to confer resistance in transgenic crops and a number of

putative protective proteins have been demonstrated to have such effects when tested

in model crop plants or under controlled growth conditions (Shewry and Lucas, 1997).

However, despite the fact that this effect was first described over a decade ago (Hilder

et al., 1987), commercial crops expressing plant-derived protective proteins have yet

to be produced. This contrasts with the success achieved using the endotoxins derived

from the bacterium Bacillus thuringiensis (Bt toxins) in crops such as maize (com)

and cotton.

34

Presently, 17 different classes of pathogenesis related (PR) proteins have been

defined, as summarized in Table 1.6. These clearly vary widely in their biological

properties, but four types of activity are particularly noteworthy. (1) Enzyme

inhibition, inhibitors of proteinases and amylases are widespread, but inhibitors of

polygalacturonase (an enzyme secreted by plant pathogenic fungi) and other enzymes

also occur. (2) Hydrolysis of polysaccharides or proteins including lysozyme, which

hydrolyzes bacterial cell walls, a1,3- glucanases and endochitinases, which hydrolyze

fungal hyphae and/or insect cuticles. (3) Destabilization of membranes- resulting in

leakage, including thionins, 2S albumins, thaumatin-related proteins lipid transfer

proteins, and some defensins ( 4) Binding to chitin (an a-1, 4-linked polymer of N­

acetyl-glucosomine present in fungal cell walls and insect cuticles), lectins, hevein,

endochitinases, and some defensins.

Cysteine proteases

Cysteine pro teases of the C 1, or papain-like, family are part of a much larger

family of cysteine proteases that were originally characterized by their having a

cysteine residue as part of their catalytic site. The C1 family was identified as having

conserved Gin, Cys, His, and Asn residues at the active site and includes many

endopeptidases, aminopeptidases, and dipeptidyl peptidases, some enzymes having

both both exo- and endopeptidase activities (Rawlings and Barrett, 1993). While some

family members share sequence homology, they may not have protease activity, a

notable example being the P34 protein from soybean, where the active site cysteine

residue has been replaced with a glycine (Kalinski et al., 1990). Members of the

papain family are widespread in nature, having been found in baculovirus, eubacteria,

yeasts, and practically all protozoa, plants, and mammals. The proteins are generally

lysosomal or secreted, being synthesized as a proprotein, proteolytic cleavage of the

propeptide is required for enzyme activation. Many members are also glycosylated,

with the N-linked glycan moiety of the cysteine protease of pineapple, bromelain,

having been determined to be Man-a-D-Man (1-6)-[(3_-D-Xyl-(1-2)]-13-D-Man-(1-4)­

~-D-GlcNAc-(1-4)-[_a-L-Fuc-(1-3)] ~-D-GlcNAc- (1-N) (Ishihara et al., 1979).

One important role for cysteine proteases in eukaryotic systems that has

emerged in recent years is their critical role in the programmed cell death ( apoptosis)

35

Table 1.6 The family of pathogenesis related (PR) proteins

Family Biological properties

PR-1 Unknown

PR-2 P-1 ,3-glucanase

PR-3 Typel,II.IV,V,VI,VII chitinases

PR-4 Typel,II chitinases

PR-5 Thaumatin like

PR-6 Proteinase inhibitor

PR-7 Endoproteinase

PR-8 Type III Chitinase

PR-9 Peroxidase

PR-10 "Ribonuclease -like"

PR-11 Type! chitinase

PR-12 Defensin

PR-13 Thionin

PR-14 Lipid transfer protein

PR-15 Oxalate oxidase

PR-16 "Oxalate oxidase- like"

PR-17 "Proteinase like"

Based on VanLoon and Van Strein (1999) and Christensen et al (2002)

pathway of animal cells (Martin and Green, 1995). Such a role is now emerging in

plants (Solomon et al., 1999) and is associated with apoptopic events that accompany

plant responses to pathogen attack and environmental stress.

36

1. 7 Open questions and rationale of the study: AGPase, its

regulation and factors affecting starch synthesis and grain

weight

Grain size, grain number, number of panicles and grain filling in crop plants

are complex traits determined by various genetic and environmental factors. They

collectively contribute to the "yield" of crop plants. The process of grain filling

involves synthesis of storage material comprising of starch, proteins and other

metabolites, which are used during seed germination. Grain starch and grain protein

form the major proportion of the total grain constituents and the discussion in the

preceding section elaborates how starch synthesis and its accumulation in the grain is

one of the most factors involved in determining grain weight. Therefore a clear

understanding of the regulation of enzymes of the starch synthesis pathway would be

useful.

Although, there have been many studies on the enzymes of the pathway and

their regulation, the regulation of AGPase (which catalyses the formation of ADP­

glucose, the precursor for starch synthesis and a key enzyme of the pathway) is yet to

be completely understood. Regulation of plant AGPase expression seems to be

complex and a variety of regulatory mechanisms seem to operate. Adding to this

complexity is the influence of developmental and environmental signals (Lalonde et

al., 1997) on these regulatory modes. Understanding how these diverse regulatory

elements are interlinked to constitute the overall regulation of AGPase still remains a

challenge.

Proteins of the cereal grams form the second important constituents and

include storage proteins, enzymes required for metabolic activities, protective proteins

such as protease inhibitors and stress proteins. They are important in determining the

quality and end use properties of the grain but may also have a yet unidentified role in

determining a molecular environment that may influence the enzyme activities in the

grain. Understanding these proteins, their expression and levels would thus be useful.

The studies included in this thesis are thus an attempt to understand the regulation of

AGPase expression and activity in developing rice grain. Availability of rice varieties

37

with varymg patterns of starch contents offers a useful system to explore the

biochemical and regulatory aspects of AGPase and gain insight into the characteristics

of this enzyme in rice. The specific objectives are therefore structured to address the

above broad questions:

Our specific objectives are:

1. Determination of grain weight, starch and protein content in the grains of rice

varieties during grain development.

2. Study of AGPase enzyme activity m the gram protein extracts from nee

varieties during grain development.

3. Determination of AGPase gene sequences and comparative sequence analysis

to check for the presence of any significant sequence variations in the allosteric

and substrate binding sites of the enzyme which may be responsible for the

activity variations among the rice varieties.

4. Study of AGPase transcript levels and their correlation to activity variations.

5. Study of AGPase protein levels in relation to activity.

6. Study of differential proteins expressed during grain development to identify

the specific proteins, which may have plausible role in starch synthesis or

effect on AGPase.

A broad objective of these studies is also to integrate these results and identify

AGPase gene or cellular condition which may be exploited to develop a strategy for

better efficiency in starch synthesis and its accumulation in rice grains.

38

1.8 Experimental approach to meet the objectives

For Objective 1

For determination of the grain weight, 1 OOmg of grains were weighed and the weight

of single grain was calculated. The starch and the protein content for 1 OOmg grains of

each variety during development were also estimated by the standard procedure and

the content per grain was calculated.

For Objective 2

Activity assays- The activity assays were performed in the synthesis direction by

measuring the amount of ADP-glucose synthesized using the standardized procedure

ofPreiss and Ghosh, 1966.

For Objective 3

AGPase gene sequence studies- To understand the variations m the activity m

different varieties, the complete AGPase eDNA for small and large subunit were

amplified by RT-PCR using gene specific primers (designed by using the japonica

sequences A Y028314 and A Y028315 of NCBI database as reference sequences) and

sequenced. The sequences were then compared by using the multiple alignment

software CLUSTAL W to look for variations in the allosteric and substrate binding

sites of the enzyme (PGA and Pi-activator and inhibitor binding sites, ATP and

glucose-1-P the substrate binding sites) which may be responsible for variation in

activity.

For Objective 4

AGPase transcript analysis- To study AGPase transcript levels, semiquantitative

RT-PCR approach was used. Total RNA (free of genomic DNA) was isolated from the

grains of each variety at various days after anthesis. The method was standardized and

PCR was performed in the linear range to allow the detection and quantification of

PCR products by densitometric quantification.

39

For Objective 5

AGPase protein levels- Total gram protein from each rice variety during gram

development was extracted and an equal amount of protein (15 ).lg) was subjected to

SDS-PAGE followed by immunoblotting with antisera raised against an antigenic

synthetic peptide of AGPase small subunit. The band developed corresponded to the

AGPase small subunit (based on molecular weight) and it was quantified at all stages

in each rice variety by densitometric quantification.

For Objective 6

Study of protein profiles of the rice seeds- Total grain protein extract was made for

each variety at each daa and TCA precipitated. About 600 ).lg of the protein sample

was subjected to 2-D gel electrophoresis by our standardized procedure. The gels were

stained by the fast coomassie staining method and images were acquired using Flour-S

Multilmager. Annotation of rice proteome for one of the rice varieties (Jaya at 15 daa)

was done. Then, comparative analysis of 2-D gel images at the day (daa) with

maximum AGPase activity within and among the varieties was carried out using PD­

Quest software. Differentially expressed proteins were identified by peptide mass

fingerprint (PMF) search after mass spectrometric analysis [matrix-assisted laser

desorpti onlionizati on-time of flight (MALD I-TO F)].

1.9 Thesis organization

This thesis has four chapters in a logical progression.

Chapter I provides the relevant literature and introduces adequate background and

rationale for concepts pertaining to this study. Chapter II gives a detailed description

of various methodologies and reagents used during the course of the study.

Chapter III describes the results of various experiments done to understand the

regulation of AGPase during grain development in rice. These experiments include

determination of grain weight, starch and protein content in the grains of rice varieties

40

during gram development, AGPase activity assays (specific and total activity of

AGPase) in the grains of rice varieties during grain development, AGPase gene

sequence studies in rice varieties, AGPase transcript level analysis and AGPase

protein level studies in the grains of rice varieties during grain development. This

chapter also focuses on the study of the developing grain proteome by the 2-DE MS

proteomics approach. The grain proteins identified were subjected to functional

mapping and were assessed for their plausible involvement in starch synthesis and/or

grain filling. Chapter IV focuses on the analysis and discussion of the results

obtained in chapter III.

41