Research Collection

Doctoral Thesis

Impact of prolamin variation and 1BL.1RS translocation onbread-making quality parameters of wheat (Triticum aestivumL.)

Author(s): Gobaa, Samy

Publication Date: 2007

Permanent Link: https://doi.org/10.3929/ethz-a-005427073

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DISS. ETHNO. 17101

Impact of prolamin variation and 1BL.1RS translocation on bread-making

quality parameters of wheat {Triticum aestivum L.)

A dissertation submitted to

ETH ZURICH

for the degree of

Doctor of Sciences

presented by

SAMY GOBAA

DEA, Université Paul Sabatier (Toulouse III)

born 08.09.1977

citizen of

France

accepted on the recommendation of

Prof. Dr. Peter Stamp, examiner

Dr. GeertKleijer, co-examiner

Dr. Gérard Branlard, co-examiner

Zurich, 2007

Table of contents

Table of contents I

Summary Ill

Résumé V

List of abbreviations VIII

1 General introduction 1

Why wheat? 1

The wheat kernel 1

The prolamins 2

The 1BL. 1 RS translocation 3

The aim of the study 5

2 Ax2", a new high molecular weight glutenin subunit coded by Glu-Al: its predictedstructure and its impact on bread-making quality 7

2.1 Introduction 7

2.2 Material and methods 8

2.3 Results 9

2.4 Discussion 12

3 Effect of the IBL.IRS translocation and of the Glu-B3 variation on bread-making qualitytests in a doubled haploid population 15

3.1 Introduction 15

3.2 Materials and methods 16

3.2.1 Plant material and experimental design 16

3.2.2 Quality tests 17

3.2.3 SDS-PAGE and allelic variation 17

3.3 Results and discussion 18

3.3.1 Allelic variation in the DH population 18

3.3.2 Preliminary tests 20

3.3.3 Protein- and kernel-related traits 20

3.3.4 Starch-related tests 22

3.3.5 Rheology tests 22

3.3.6 Baking tests 24

3.4 Conclusions 24

4 Proteomic analysis of wheat recombinant inbred lines (Part I): effect of the IBL.IRS

translocation on the wheat grain proteome 26

4.1 Introduction 26

4.2 Material and methods 27

4.2.1 Plant material 27

4.2.2 Translocation mapping 27

4.2.3 1-D electrophoresis and allelic variation 27

4.2.4 2D SDS-PAGE and quantitative variation 28

4.2.5 Protein identification 29

4.3 Results 30

4.4 Discussion 39

5 Proteomic analysis of wheat recombinant inbred lines (Part II): variations in prolaminand dough rheology 43

I

5.1 Introduction 43

5.2 Material and methods 44

5.2.1 Plant material 44

5.2.2 Rheological tests and classification 44

5.2.3 Two-dimensional electrophoresis 45

5.2.4 Assignment of the prolamin fractions 45

5.2.5 Statistical analysis 46

5.2.6 Protein identification 47

5.3 Results 48

5.4 Discussion 55

6 Conclusions 60

7 References 64

Remerciements 72

Curriculum vitae 74

II

Summary

The suitability of wheat varieties for bread-making depends largely on prolamins. The amino acid

composition of these gluten building blocks as well as their quantitative regulation have a strong

influence on the rheology of the dough and, thus, on the suitability of the variety for bread-

making. The wheat/rye 1BL.1RS translocation was reported to drastically reduce rheological

properties. However, there are noticeable exceptions. This chromosomal rearrangement was

designed to transfer resistance to pathogens such as Puccinia recondita, Puccinia gramins,

Puccinia striiformis and Erysiphe graminis from rye to wheat. Today this resistance is overcome

but 1BL.1RS translocation remains interesting because of the improved yield it produces.

To better understand the impact of particular prolamins (Glu-Al 2 , Glu-BS j and Glu-DS c) on

quality and to gain better understanding on prolamin regulation in general, a population of

doubled haploid was created and sown in a two-year field experiment. The first part of this study

describes a new x-type high molecular weight glutenin subunit, encoded at the locus Glu-Al and

named 2.The statistical analysis demonstrated that the subunit 2 was as favorable for quality as

the subunit 2*. This is in accordance with the results showing that the 2 open reading frame had

the same number of cysteines as 2*. The small differences in the length of the central domain had

no detectable effect on the elasticity, tenacity and baking quality of the dough.

The effects of the rest of the polymorphic loci {Glu-BS and Glu-D3) also produced innovative

results; allele Glu-BS], which marks the 1BL.1RS translocation, was found to be associated with

lower rheology of the dough. The presence of this translocation resulted in less tenacity, less

extensibility, less strength and greater softening. Allele Glu-DS b was also detrimental to

rheology when compared to allele c. Its impact on the tenacity of the dough was important and

globally it affected quality to a similar extent as the 1BL.1RS translocation. The protein content

of the 1BL.1RS lines was significantly higher than that of the regular IB lines. This led to loaves

with a greater volume. Furthermore, the softening produced by the 1BL.1RS translocation and

Glu-DS b probably played a role in the greater bread volume by reducing the resistance to the

deformation of the dough.

The comparison of the 2-DE proteomic profiles of 16 doubled haploid lines, with or without the

1BL.1RS translocation, allowed an assessment of the impact of this event on the prolamins;

quantitative and qualitative proteic variations, induced by the 1BL.1RS translocation, were

III

reported. Eight spots were found only in lines with the 1BL.1RS translocation, 16 other spots

disappeared in the same lines. Twelve spots, present in both types of genotypes, met the criteria

for up- or down-regulation. In translocated genotypes, a y-gliadin-like LMW-GS, with nine

cysteine residues, was over-expressed by 801 %, suggesting that the lack of LMW-GS was

counterbalanced by an over-expression of relatively similar prolamins. Identification and

quantification of the prolamin fractions on the two-dimensional electrophoresis gels

demonstrated that the HMW-GS were up-regulated by 25 % in 1BL.1RS DH lines, even though

the corresponding genes were not located on the missing IBS chromosome. The y-gliadins were

also up-regulated by 36 %. Moreover, a significantly varying spot, identified as a dimeric alpha-

amylase inhibitor, may be considered as a valuable candidate to explain the starch gelatinization

defect observed in the 1BL.1RS lines of the DH population. To investigate the impact of the

1BL.1RS translocation on dough strength and to understand how 1BL.1RS genotypes overcome

the loss of Glu-BS and Gli-Bl, the same 16 DH lines were reclassified into high- or low-tenacity

and into high- or low-extensibility categories. The results showed that 32 spots, mainly prolamins,

were differentially expressed and that five others were specific to high-strength DH lines. The

polymeric prolamin fractions were also accumulating in high-tenacity lines and decreasing in

high-extensibility lines, confirming the role of the interchain disulfide bonds in the resistance to

deformation. In contrast, the monomeric fraction of a-gliadin, in particular the Gli-Al allele of

the parent Toronit, favored extensibility and decreased tenacity by higher accumulation (+12 %

of a-gliadins in high extensibility lines) when compared to the Gli-Al allele of parent 211.12014.

The results will help the establishment of efficient breeding strategies where translocated lines

are concerned. The accumulation of y-type HMW-GS and good-quality y-gliadin can improve

quality. These results also lead to a reconsideration of the mechanisms of prolamin evolution and

regulation.

IV

Résumé

Les prolamines déterminent en grande partie l'aptitude à la panification du blé. La séquence

d'acide aminé de ces dernières ainsi que leurs variations quantitatives déterminent, en grande

partie, les propriétés rhéologiques de la pâte. La translocation blé/seigle 1BL.1RS a été démontré

comme ayant un effet négatif sur ces paramètres. Cependant des exceptions notables existent et

certaines variétés de blé réussissent à atteindre un niveau satisfaisant de qualité malgré la

présence de cette translocation. Ce réarrangement chromosomique a été créé pour faire bénéficier

le blé de la résistance du seigle à des pathogènes tels que Puccinia recondita, Puccinia gramins,

Puccinia striiformis et Erysiphe graminis. Aujourd'hui ces résistances sont, pour la plupart,

surmontées mais la translocation 1BL.1RS reste employée pour les meilleurs rendements qu'elle

induit. Une population de lignée haploïdes doublés a été créée et évaluée sur deux années

d'expérimentation pour mieux saisir l'impact de certaines prolamines particulières (Glu-Al 2..,

Glu-BS j et Glu-DS c) et pour apporter de nouveaux éléments de compréhension sur le réseau de

régulation de ces protéines.

La première partie de cette étude rapporte l'existence d'une nouvelle gluténine de haut poids

moléculaire codée par Glu-Al et nommée 2 . L'analyse a démontré que cette sous unité gluténine

est aussi performante que la sous unité 2* en terme de rhéologie et de qualité boulangère. Ceci

s'explique par une composition en acide aminé très semblable entre les deux protéines. Seules

quelques petites différences existent au niveau de la longueur du domaine répété de ces deux

gluténines.

L'analyse des effets du reste des loci polymorphes {Glu-BS et Glu-D3) a aussi produit des

résultats originaux. L'allèle Glu-BS], qui signe la translocation, s'est montré défavorable sur tous

les tests rhéologiques réalisés. Les lignées portant cet allele ont eu de plus faibles ténacités, de

plus faibles extensibilités, de plus faibles forces et de plus forts ramollissements. L'allèle Glu-DS

b c'est lui aussi montré défavorable par rapport à l'allèle Glu-DS c. Cependant, il est à noter que

la variation à Glu-DS n'a eu d'effet que sur la résistance opposée à la déformation de la pâte

contrairement à la variation à Glu-BS. Globalement les variations sur Glu-BS et Glu-DS ont

produit des changements de rhéologie du même ordre de grandeur. Le taux de protéine contenu

dans les grains des lignées 1BL.1RS a été significativement plus important que celui des lignées

IB. Ceci a sûrement participé à l'obtention de volume de pain plus important chez les lignées

V

transloquées. De plus, les paramètres rhéologiques de toute la population ayant été élevés de

manière générale, la perte de force induite par les alleles Glu-BS j et Glu-DS b pourrait expliquer

en partie les meilleurs volumes de pain associé à ces derniers ceci en s'opposant moins

fermement à la déformation de la pâte que leurs pendants alléliques.

La comparaison des gels d'electrophoreses bidimensionnelles de seize lignées représentatives de

la population, ayant ou non la translocation 1BL.1RS, a permis d'observer les effets associés à ce

réarrangement chromosomique. Les variations qualitatives et quantitatives liées à cette

translocation ont pu être observées. Huit spots ont été retrouvés uniquement chez les lignées

1B1.1RS, seize autres n'y ont jamais été observés. Douze spots ont montré des variations

quantitatives significatives. Il a aussi été possible de démontrer qu'un spot identifié comme une

« y-gliadin LMW-like » avait subi une augmentation de volume de 801 % chez les lignées

1BL.1RS. Ceci suggère que, chez ces dernières, le manque de gluténines de faible poids

moléculaire a été contrebalancé par la surexpression de protéines relativement semblables

capables de remplir une mission similaire. La quantification des différentes fractions de

prolamine a permis d'établir que les gluténines de haut poids moléculaire et les y-gliadines

avaient augmenté respectivement de 25 et 26 % en réponse à la translocation 1BL.1RS. De plus il

a aussi été démontré que cette translocation induisait la perte d'un spot identifié comme un

inhibiteur dimeric d'alpha amylase. Cette protéine pourrait donc être considérée comme un

candidat intéressant expliquant le défaut de gélatinisation de l'amidon, lié à la translocation,

observé lors de l'évaluation au champ de la population. Pour établir l'impact de la translocation

sur la force de la pâte et pour comprendre comment certains génotypes arrivent à surmonter la

perte de Glu-BS et Gli-Bl, les mêmes seize lignées ont été « reclassifiées » en lignées de forte ou

faible ténacité et en lignées de forte ou faible extensibilité. Ceci a démontré que 32 spots,

majoritairement des prolamines, ont montré une variation quantitative significative. Cinq spots

n'ont été retrouvés que chez les lignées à fort W. il a aussi été démontré que les fractions de

prolamine polymérique augmentaient chez les lignées à forte ténacité et diminuaient chez les

lignées à forte extensibilité. Confirmant ainsi que les ponts disulfures jouent un rôle majeur dans

la résistance à la déformation de la pâte. La fraction monomérique des a-gliadines, plus

précisément 1'allele Gli-Al du parent Toronit, a été trouvé comme favorisant l'extensibilité via

une accumulation plus importante que celle produite par l'allèle Gli-Al du parent 211.12014.

VI

Les résultats obtenu aideront les sélectionneurs à encore améliorer la qualité des blés transloqués

en favorisant l'accumulation des gluténines de haut poids moléculaire de type y et celle des y-

gliadines de bonne qualité. Ces mêmes résultats aideront aussi à réévaluer la variabilité et la

régulation des prolamines.

VII

List of abbreviations

%Vol percentage of volume

1 -D mono dimensional

2-DE two dimensional gel electrophoresis

6-11NL non linear/?/ range of 6 to 11

aa amino acid.

A-PAGE acid Polyacrylamid gel electrophoresis

bp base pair

C crosslinking ratio

CHAPS 3 - [(3 -Cholamidopropyl)dimethylammonio] -1 -propanesulfonate

Chrs chromosome

CO2 carbon dioxide

CV coefficient of variation

DH doubled haploid.

DNA deoxyribonucleic acid

DTT dithiothreitol

Xgli sum of the volumes of the gliadin spots.

^Protamin sum of the volumes of the prolamin spots.

EST expressed sequence tag

Ext mixograph's extensibility

Fal falling number

GH grain hardness

H20 abs water absorption as measured with the Farinograph

HCL hierarchical clustering

HCl hydrochloric acid

HMW-GS high molecular weight glutenin subunits

HPLC high-performance liquid chromatography

ICC international association for cereal science and technology

le swelling index as measured by the Alveograph

IEF isoelectric focusing

IPG isoelectric point gradient

L Extensibility as measured by the Alveograph

LEA late embryogenesis abundant proteins

LMW-GS low molecular weight glutenin subumts

MALDI-TOF matrix-assisted laser desorption/ionization-time of flight

MS mass spectrometry

MS/MS tandem mass spectrometry

MW molecular weight

NanoLC nano-hquid chromatography

NCBI National Center for Bioinformatics Information

MRS near infrared reflectance spectroscopy

ns non significant

ORF open reading frame

P tenacity as measured by the Alveograph

PCR polymerase chain reaction

pH pondus hydrogenn I pouvoir hydrogène

pi isoelectric point

PR protein content

QTL quantitative trait loci

RES resistance as measured by the Fannograph

Rmax strength as measured by the Mixograph

RMT rapid mix test

SD standard deviation

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate ployacrylamid gel electrophoresis

SOF softening as measured by the Fannograph

T Polyacrylamid content (w/v)

TKW thousand kernels weight

Tns tnshydroxymethylaminomethane

v/v volume to volume

W strength as measured by the Alveograph

w/v weight to volume

IX

Zel Zeleny test

X

1 General introduction

Why wheat?

Wheat (Triticum aestivum L.) has become a major source of energy and protein for human beings

since agriculture developed in Mesopotamia. Such a success relied on multiple advantages. The

most decisive advantage was probably the non-dehiscence of the wheat caryopsis which enabled

the "human-assisted" multiplication and made selection easer. On the level of evolution, the loss

of wheat's natural ability for seed dispersal was counterbalanced by a total reliance on human

agriculture to ensure its survival. Today, modern wheat is sown on one of the largest surface

attributed to a crop and the worldwide wheat harvest ranks second in total production behind

maize, the third being rice (FAOSTAT 2004). Unique end-uses also played a crucial role in the

success of wheat. The leavening of the dough occurs after mixing flour, water and yeast enabling

the production of large-volume bread: one of the largest markets for wheat production today.

The wheat kernel

The endosperm of wheat consists mainly of starch. More than 70 % of the grain mass is

composed of this complex sugar. After degradation, starch is the unique source of carbohydrates

available to the embryo during germination. The protein content of the wheat kernel varies from

9 to 16 %. The wheat albumen contains three main groups of proteins: prolamins, globulins and

albumins. The latter two groups were found to play only a minor role in bread-making quality.

However, enzymes belonging to these two groups of protein, such as alpha- and beta-amylase,

may favor sprouting and, thus, lower the bread-making quality. Friabilin is a protein associated

with the starch granule in the endosperm of wheat. It determines the class of hardness of the

different wheat cultivars. The presence of friabilin is associated with a soft phenotype. Darlington

et al. (2000) demonstrated that this protein is controlled by the Ha locus on chromosome 5D.

Pentosanes have surface-active properties. These sugars were reported to influence the size of the

alveoli in the crumb. The wheat grain also contains 1.5 to 2 % of lipids but their role in bread-

making is limited.

1

The prolaminsProtamins exist in a large number of wild or cultivated grass species. Their initial role was to

provide the embryo with a source of amino acids during germination. Their structure was shaped

by their biological goal and, thus, they acquired the capacity to fold and polymerize in order to

stock up during grain filling. Human selection applied to primitive prolamins took advantage of

their natural propensity to polymerize and to withstand mechanical deformation, thus meeting the

requirement for human nutrition. Selection for high polymerization of prolamins and high-

strength gluten maximized the retention of CO2 during fermentation of the dough and resulted in

large volume bread. Variation in quality and in quantity of prolamin has a direct impact on bread-

making and on the rheology of the dough.

Prolamins account for 45 to 80 % of the total proteins in the grain of modern wheat cultivars. The

most widely accepted classification puts prolamins into three structural/functional classes: high

molecular weight gluten subunits (HMW-GS), low molecular weight glutenin subunits (LWM-

GS) and gliadins. The two types of glutenins are the only prolamins that are able to form

interchain disulfide bridges, which make up the gluten backbone. Gliadins produce only weak

hydrogen bonds with the other prolamins.

Anderson and Greene (1989) described the structure of HMW-GS. Three domains are present in

these proteins: the N- and C-terminal domains plus a central repetitive domain. The cysteines

tend to be located in the external domains and are responsible first for the globular conformation

of these domains and second for the cross-linking ability of HMW-GS. The number of cysteines

and the structure of the central domain differentiate between y-type and x-type HMW-GS.

HMW-GS are encoded by the three homeologous loci: the Glu-1 group. Each locus generally

controls the production of one x-type and one y-type subunit. However, because of the null

alleles, the average number HMW-GS subunits in the wheat cultivars is three to five. The

observed genetic variability of HMW-GS in wheat is limited to 20 subunits, i.e. 21 according to

this study. Regulation of HMW-GS accumulation is unclear. Guillaumie et al. (2004) reported

that a QTL controlling the amount of HMW-GS was co-localizing with the locus Glu-Bl

encoding HMW-GS. In an association study, Ravel et al. (2006) further demonstrated that the

amount of HMW-GS was allele-dependent. Allelic variability of HMW-GS and bread-making

quality was investigated in multiple studies and a quality index was established for the most

common alleles (Branlard et al., 1992). Quality can be explained in part by the variation in

2

HMW-GS and was found to be highly dependent on the structure of the HMW-GS alleles.

Anderson et al. (1989) reported the presence of an extra cysteine residue in the central domain of

subunit 1Dx5. This explained the positive impact of this subunit on quality and proved that cross-

linking is critical for dough rheology (Shewry et al., 1992).

LMW-GS are also important for bread-making. The presence of cysteine (for interchain cross-

linking), in even or uneven numbers, determines whether LMW-GS extend the gluten network or

acts as chain terminators. Multiple studies reported the impact of allelic variation ofLMW-GS on

a large number of quality parameters. Metakovsky et al, (1990) established a list of favorable

alleles for Rmax and W. Cornish et al. (1993) found that the combination of the allele b b b on

the three Glu-3 loci was favorable for extensibility. Gupta et al. (1994) concluded that the LMW-

GS alleles, encoded at the IB chromosome, are more important for Rmax than those encoded at

the 1A and ID chromosomes. Luo et al. (2001) established a list of favorable alleles for

Farinograph parameters, protein content, SDS sedimentation and Pelshenke tests. These findings

where often contradictory but they clearly established that LMW-GS allelic variation affects

quality and that the effects of the different alleles were mainly additive.

The structure of the gliadins is similar to that of the LMW-GS, but unlike LMW-GS, gliadins are

soluble in ethanol. This property is due to the absence of covalent cross-linking between the

gluten and the gliadins. The recent report that some gliadins are also able to produce interchain

disulfide bonds reduces the gap between these two classes of prolamins (Ferrante et al., 2006).

Gliadins are acknowledged to play the role of a solvent; their accumulation usually favors

extensibility (Branlard et al, 2001). Because the loci: Glu-3 (LMW-GS) and Gli-1 (gliadin) are

closely linked it is difficult to assess the effect of each kind of protein.

The 1BL.1RS translocation

The insertion of alien chromatin into the genetic background of wheat was designed to take

advantage of the rusticity of other species such as rye, the best example being the creation of

Triticale (x Triticosecale wittmack), which combines the A and B genomes of wheat with the R

genome of rye. Nevertheless, smaller-scale genetic rearrangements were created to limit the

disadvantages of the insertion of a full R genome. Zeller and Hsam (1983) traced the first

1BL.1RS wheat/rye chromosomal translocations to breeding programs using 1R (IB) substitution

lines in Germany. The most famous translocated wheat was probably the cultivar Kavkaz which

was used as the main source of the 1BL.1RS chromosome in the modern breeding programs.

3

Translocated cultivars were appreciated mainly for their resistance to powdery mildew, green bug,

wheat curl mite and stem rust (Zeller, 1973; Zeller and Hsam, 1983). Nevertheless, other

advantages, such as improved grain yield (Villareal, 1995; Kim 2004) and grain protein content

(Lee et al, 1995) were also important. Henry et al., (1993) also demonstrated that IBL.IRS

translocation greatly improved the in vitro regeneration of microspore-derived haploid embryos.

In contrast, IBL.IRS translocation was largely associated with end-use defects such as sticky

dough, poor rheology of the dough (strength, tenacity, and extensibility), low resistance to over-

mixing and small volume of the bread. Such attributes were explained first by the secalin

encoded by the 1RS chromosome, but it appeared that the quality defect associated with

IBL.IRS was due to the loss of the LMW-GS and gliadins encoded at Glu-BS and Gli-Bl

respectively (Figure 1.1). Since recombination is impossible between IBS and 1RS, the IBL.IRS

translocation is inherited as a block. Thus both its advantages and disadvantages are linked. To

break such an association, attempts to generate smaller insertions of rye material were tested with

the 1D.1R recombinant lines (Rogowsky et al, 1993). The other alternative would be to create a

genetic background in which the disadvantages of the IBL.IRS translocation are suppressed or at

least greatly reduced.

GH-B1 -11 Sec'Glu-B3 I

Glu-B1 -ü Glu-B1

Chromosome Chromosome

1B1BL. 1RS

Figure 1.1 "regular" IB chromosome (left) and 1BL 1RS

translocated chromosome (right) of wheat and their associated

prolamm loci The hatched material represents rye chromatin

4

The aim of the studyTo understand the basis of gluten rheology, it is necessary to determine the rheology of its

elementary component: the HMW-GS. The number of cysteine residues was proved to determine

the ability of each subunit to polymerize. Less is known about the determinants of extensibility.

The role of the helicoidal central domain was investigated unsuccessfully by evaluating

transgenic wheat lines, which carry a modified HMW-GS in the central domain. The goal of the

first part of the present study was to quantify the effects of small-sequence variations in HMW-

GS on the measured quality parameters. This approach was based on the comparable HMW-GS

alleles Glu-Al 2 and Glu-Al 2*, which were present in the studied DH population. Field

experiments were performed to analyze the impact of these alleles on a broad range of quality

traits. Furthermore, the population also had two other variant alleles of prolamin: Glu-BS and

Glu-D3. The presence of Glu-B3 j marked the presence of the 1BL.1RS translocation. The impact

of this chromosomal rearrangement is generally negative on the bread-making quality. However,

translocation does occur in some high-quality cultivars. Assessing how this translocation interacts

with the other prolamin loci and with the environment could lead to a better appreciation of the

quality profile of the 1BL.1RS genotypes. Analysis of the impact of the variation in the LMW-

GS of Glu-DS on quality was planned to address the relatively contradictory results published in

the literature. Establishing the effect on quality of the different alleles (at the polymorphic loci) of

HMW-GS and LMW-GS of the present population will provide valuable information for breeders.

Knowing the precise impact of these alleles on a broad range of quality traits will help in

achieving precise breeding objectives.

Fundamental insight will also be gained by observing how the wheat proteome copes with the

replacement of chromosome IBS by the chromosome 1RS (rye). This approach ought to prove

the existence of a complex prolamin regulation network. Previous studies suggested such

mechanisms: Dumur et al. (2004) demonstrated that, in monosomic lines of the cultivar Courtot,

the loss of prolamin loci boosted the production of other prolamins and even favored the

expression of prolamins that remained silent in the "wild-type" cultivar. Recent studies

demonstrated that HMW-GS accumulation is influenced by the nature of the HMW-GS (Ravel et

al, 2006). Nevertheless, no clear regulation scheme is available today for explaining the

regulation of prolamins.

5

As well as gaining a better understanding of the regulation networks of prolamins, the goal is to

determine how wheat is able to reactivate silenced prolamins when and if they are required. This

may lead to a re-evaluation of the variability available to breeders. Several prolamin-coding

sequences are present at each locus but few proteins are expressed and are accumulated in the

endosperm (Clarke and Appels 1999). Such observations lead to the question as to why such a

large pool of variability was conserved in the wheat genome and why it is kept silenced. It is

known that the accumulation of prolamins in the endosperm is vital for the persistence of the

species. Thus, robust mechanisms of compensation should exist to ensure the "packaging" and,

thus, the accumulation of prolamins in the endosperm even when a major locus is lost.

6

2 Ax2",a new high molecular weight glutenin subunit

coded by Glu-Al: its predicted structure and its

impact on bread-making quality.

Published in Plant Breading (2007), volume 126, issue 1, pages 1-4.

2.1 Introduction

By conferring visco-elasticity to the dough (for review Shewry et al, 1992), the high molecular

weight glutenin subunits (HMW-GS) determine the suitability of the wheat (Triticum aestivum

L.) for bread-making. The amino acid composition of these proteins explains their polymeric and

elastic behavior (Shewry and Tatham, 1997) and, consequently, determines the rheology and the

baking quality of the dough (Veraverbeke and Delcour, 2002).

The HMW-GS proteins are coded by the complex Glul loci on the long arm of the chromosomes

1A, IB and ID. Each locus codes for two structurally different kinds of proteins: the x-type and

the y-type HMW-GS. Due to silencing phenomena and/or pseudogenes, the total number of

HMW-GS in a cultivar ranges from three to five with some exceptions reported for Swedish

bread wheat cultivars which have six HMW-GS (Margiotta et al., 1996). To date, only three x-

type alleles at Glu-Al have been reported (Gianibelli et al, 2001).

The variability amongst the HMW-GS alleles has an impact on quality. Anderson et al. (1989)

reported the existence of an "extra" cysteine residue in the central domain of the HMW-GS Dx5

(allele Glu-Dld). In an in vitro system, Buoncore et al. (1998) demonstrated that this "extra"

cysteine residue is responsible for the good quality associated with this subunit. D'Ovidio et al.

(1996) established that the central domain of an HMW-GS coded by Glu-Dl shows an insertion

of a 187 amino acids. He et al. (2005) suggest that, in transformed plants, such an event may

increase the strength of the dough. No reports have been published yet, which discuss the impact

on quality of smaller changes occurring in the central repetitive domain.

The quantitative aspect also plays an important role in the bread-making ability. Thus, the over-

expressed Bx7 subunit in cultivars such as 'Glenlea' or 'CD87' leads to better quality (Marchylo

et al, 1992; Butow et al, 2003).

7

Here we report the sequence of a new HMW-GS x-type allele of Glu-Al and the impact of

limited changes in the sequence of its central repetitive domain on ten traits important for bread-

making.

2.2 Material and methods

Swiss genotypes of Triticum aestivum, from Agroscope Changins-Wädenswil (Nyon,

Switzerland), were used. The breeding line '211.12014' was utilized to clone and sequence the

allele 2 (at Glu-Al). As a control, we also used the cultivar 'Toronit' to clone and sequence the

allele 2* (at Glu-Al). A double haploid (DH) population was created by crossing the two

genotypes ('211.12014' X 'Toronit'). The microspores of Fi plants were isolated and cultivated

according to Kunz et al. (2000) to obtain 174 DH lines. This population was evaluated in field

experiments in 2004 and 2005 at Nyon (430m above sea level) on loamy cambisol soil. Average

precipitation was 1037mm in 2004 and 774mm in 2005. Fertilization was done to meet the

plant's needs.

The following measurements were carried out for each line: protein content (PR) and grain

hardness (GH) were measured by near infrared reflectance spectroscopy (MRS) according to the

ICC standard N°159, the Zeleny sedimentation test (ZEL) was done according to the ICC

standard N°l 16/1, the Alveograph parameters such as tenacity (P), elasticity (L) and strength (W),

according to the ICC standard N°121 and Farinograph parameters, such as water absorption

(H20), resistance (RES) and degree of softening (SOF) according to the ICC standard N° 115/1.

The Rapid Mix Test (RMT) was carried out as described by Pelshenke et al. (1970). The ICC

standards are referenced in Standard Methods of the International Association for Cereal Science

and Technology (1999).

The statistical analysis of the data was performed with the program SAS 8.2 (SAS Institut, Cary,

NC, USA). The distribution of the residuals from the analysis of each of the parameters was

evaluated by the Tukey-Anscombe and the QQ-plots methods. Since the assumptions of the

ANOVA (homoscedasticity, normality) were not met for PR, RES, SOF, P, W and GH, standard

data transformation for continuous (log), counts (square root) and percentages/proportions

(arcsines of the square root) were tested. The analysis was performed with the Mixed procedure.

Proteins were extracted from whole meal flour of 'Toronit', '211.12014' and all the DH lines

according to the protocol of Singh et al. (1991). The proteins were separated (230 minutes at

8

30mA) on 12 5% acrylamide SDS-PAGE gels (16X18 cm) The bands were revealed after

Coomassie blue staining to obtain the glutemn profile of the different genotypes

Genomic DNA was extracted from the leaves of young seedlings using the Clontech®

NucleoSpin® Plant Kit The polymerase chain reaction (PCR) of the Glu-Al gene was performed

with a Biometra® T3Thermocycler in 50ul volumes using the Qiagen HotStartTaq® The

primers PI (5'-TAGCCAACCTTCACAATCTCT-3') and P2 (5*-

ATAGCTAANGTGCATGCATGCC-3') were used to amplify the whole open reading frame

(ORF) of 2* and 2 , the primers P3 (5'-5-ATCAATCCCGCACATCCTCTC-3') and P4 (5*-

GCAAAAAGAACCAACCGCTTAGT-3') were used to amplify the upstream region of the 2

and 2* ORF The PCR products were cloned with the Quiagen PCR Cloning Kit® The obtained

Ax2 sequence was deposited in GenBank® (http //www ncbi nlm nih gov/Genbank/index html)

under the accession number DQ533690 The ORF detection was carried out with the ORF Finder

software at http //www ncbi nlm nih gov/gorf/gorf html The sequence alignment was performed

with Multahgn (Corpet, 1988) (http //prodes toulouse inra fr/multalin/multahn html) The

identification of the cis-acting elements in the upstream region of ORF was performed with the

PlantCARE software (http //bioinformatics psb ugent be/webtools/plantcare/html/)

2.3 Results

The SDS-PAGE experiment showed that the wheat genotype '211 12014' had an atypical x-type

Glu-Al allele In this cultivar, the HMW-GS coded by Glu-Al showed a small mobility shift

compared to a classical 2* subunit (NCBI reference N° m22208) as shown in Figure 2 1

The 2 ORF was detected with the ORF Finder program in NCBI This predicted ORF was 2475

bp long The cloned upstream region of the ORF was 2173 bp long

The sequence alignment of the upstream region of the 2 and 2* ORFs showed 17 single

nucleotide substitutions, three single-base insertions, two single-base deletions and one triple-

base deletion These sequences were analyzed with the PlantCARE program to identify

potentially different cis-elements that may play a role in the regulation of the expression of the

two alleles Compared to the 2* ORF upstream region, the 2 ORF upstream region did not show

a difference in the predicted cis-elements, which are known to be involved in prolamin

regulation, one prolamin box at -216 plus two CGN4-hke motifs (-568 and -547) were identical

in both promoters

9

The predicted protein sequence translated from the ORF of 2 had a theoretical molecular weight

of 89,47kDa (the theoretical mass predicted from the 2* sequence is 88,47kDa) and a length of

824 amino acids (2* is nine amino acids shorter). The predicted amino acid composition ofN and

C termini domains were identical in 2 and 2*. Thus, all the 2* cysteines were conserved in 2 .

No extra cysteine appeared in the central repetitive domain of 2 . Nevertheless, 21 single amino

acid substitutions, two insertions (six and nine amino acids) and one deletion (six amino acids)

were observed in the 2 central repetitive domain (Figure 2.2). The amino acid substitutions

occurred over the whole length of the repetitive domain. The insertions started at positions +134

and +507 respectively. The deletion occurred at the end of the repetitive domain (+715). These

mutations led to some changes in the tripeptide, hexapeptide and nonapeptide 2 central domain

composition compared with 2*. In 2 , 36 GQQ motifs were observed instead of 37 in 2*, 8

B

Glu-B1

GIU-D1

Figure 2.1 SDS-PAGE separation of the HMW-GS of '211 14014'

(A) in and 'Toromt'(B) The blanc arrowhead points to the new Glu-

Al allele 2 and the other one points to 2* The digits on the right

correspond to the HMW-GS of the Glu-Bl 7+9 and Glu-Dl 5+10

alleles (common to both genotypes).

10

PGQGQQ motifs instead of 6, and 8 GYYPTSPQQ motifs instead of 9. The two extra

hexapeptides were created by the two insertions at +134 and +507 and the missing nonapeptide

was the result of the substitution of Gly by an Arg at position +566.

The statistical analysis of the results of the quality measurements (Table 2.1) shows that the

impact of the year on the measured traits was highly significant (P < 0.001, except RMT P <

0.01). There were no significant variations (at 5 % error probability) due to 2 on the measured

traits in comparison to 2*, with the exception of the Zeleny sedimentation test showing that the 2

subunit induced an average increase of 3.46 % in 2004 and 6.98 % in 2005 compared to 2* (P <

0.05). The effect of the Glu-Al*Year was also non significant. It was also demonstrated, that 2

did not interact significantly with the other glutenin loci (data not shown). This implies that both

Glu-Al alleles, 2 and 2*, influenced the measured rheological parameters in an identical manner.

1 130

PBLLRlffLSVTSPOJVSYYPGaflS---

PflLLRRifLSVTSPQaVSYYPGaflSPQIl

131 260

Rx2* —SQRPGQGQQEYYLTSPQQSGQMQQrGQGQSGïyPTSPQQSGQKQPGYYPTBPMQPEQLQQPTQGQQRQQPGQGQQLRQGQQGQQSGOGQPRYyPTSSQQPGQLQQLnQGQQGQQPEKGOQGQQSGQÛ8x2" PGQGQ8PGQGQQEYYLTSPQQSGQUQQPGQGQSGYYPTSPQQ5GQEQPGYYPTSPMQP8QLQQPTQGQQRQQPGQGQQLRQGQQGQQSGQGQPRYYPTSSQQPGQLQQLRQGQQGQQPEÜGQQGQQSGQG

261 390

I I

11x2* QQLGQGQQGQQPGQKQQSGQGQQGYYPISPQQLGQGqQSGQGQLGYYPTSPQQSGQGQSGYYPTSHQQPGQLQQSTQEQQLGQEQQDQQSGQGRQGQQSGQRQQDQQSGQGQQPGQRQPGYYSTSPQQLG8x2" QQLGQGQQGQQPGQKQQSGQGQQGYYPISPQQLGQGQQSGQGQLGYYPTSPQQLGQGQSGYYPTSHQQPGQLQQSTQEQQLGQEQQDQDPGQGROGQQLGQROQDQQSGaGQQPGORQPGYYSTSPQQLG

391 520

I I

(1x2* QGQPRYYPTSPQQPGQEQQPRQLQQPEQGQQGQQPEQGQQGQQQRQGEQGQQPG0GQQG«QPGQGQPGYYPTSPQQSGQGQPGYYPTSPQQS6QLQQPRQGOQPGQEQQGQQPGQGQQ PGQ8x2" QGOPRYYPTSPQQPGQEQQPRQLQQPEQGQQGQQPEQGQQGQQPaQGEQGQQPGQGQQGKOPGQGQPGYYPTSPQOSGQGQPGYYPTSPeQSEQLBQPflQGQQPGQEQQGQQPGQGQQGQQPGOGQQPGO

521 G50

I I

11x2* OPPGYYPTSPQQSGQEQQLEQMQQSGQGQPGHYPTSPLQPGQGQPSYYPTSPQBIGQGQQPGQLQQPTQGQQGQQPGQGQQGQQPGEGCQGQQPGQGQQPGQOQPGYYPTSLQQSGQGQQPGqMQQPGCG8x2" RQPGYYPTSPQQSGQEQQLE0HQQSGQG0PGHYPTSPLQPGOGQPKYYPTSPQaiGQGQQPGQLQQPTQGQQ8QQPGQGQQGQQffGQGDQGQQPGt)GQQPGQGgPGYYPTSLQQSGQGQQPGQUQQPGQG

651 780

I I

flx2* QPGYYPTSSLQPEQGQQGYYPTSQQQPGQGPQPGQHQ0BGQGQQGYYPTSPQQ5GQGQQPG0ULQPGQHLQGGYYLTGPQQLGQGQQPRQULQPRQGQQGYYPTSPQQSGQGQQLGQGQQGYYPTGPQQS8x2" QPGYYPTSSL01GQGQQGYYPÏSQQQPGQGPQPGQMQQ1.GQGQQGYYPTSPQQSGQGQQPG0ULQ SGYYLTSPQQLGQGQQPROHLQPRQGQQGYYPTSPQQSGQGQQLGQGQQGYYPTSPQQS

781 830

I i

Rx2* GQGQQGYDSPYHVSREHQflflSLKVnKflQPLflflQLPflNCRtEGGDflttflS!!8x2-'GQGQQGYDSPYHVSflEHQHflSLKVRKRQQLflHQLPflrtCRLEGGDflLLflSi)Figure2.2AlignmentofthepredictedprimarystructureofAx2andAx2*HMW-GSThegrayshadingindicatesammoacidsubstitutionThepercentageofhomologybetweenthetwosequencesis945%(CLUSTALW18)11

2.4 Discussion

Shewry et al. (1992) and D'Ovidio et al. (1997) suggested that the repetitive ß-turns motives in

the central domain of HMW-GS explain their anomalously slow migration in SDS-PAGE. In our

SDS-PAGE experiments we also observed a difference between the subunits 2* and 2 , the

former migrating faster than the latter. This mobility shift is probably due to the combination of

two elements: the lkDa difference in the theoretical size of the two proteins, as well as

modifications in the protein's secondary structure produced by a different repetitive domain.

In the upstream region of the ORF, no differences in prolamin boxes, CGN4-like or other cis-

acting elements important for endosperm expression (Thomas and Flavell, 1990; Norre et al,

2002) were found between the two alleles. Such a high similarity between the two promoters is

not in favor of a differential level of expression between the two alleles. Moreover, the

rheological tests (discussion follows) do not show significant variation in quality due to one of

the alleles. The quantification of the transcripts would address this issue but we can already state

that we found no evidence in favor of a differential level of expression between the subunits 2*

and 2 after comparing their ORF upstream region.

Branlard et al. (2001) found that the alleles Axl and Ax2* have an identical impact on the

strength and extensibility of the dough as well as on the Pelshenke test. Other studies (Cornish et

al, 2001) confirmed a similar impact on other quality parameters. It was also demonstrated that

the null allele at the same locus has a negative effect on all the quality traits (Branlard et al,

1992; Cornish et al, 2001). The quality tests showed no significant differences between Ax2 and

Ax2* in our DH population with the exception of the Zeleny test (Table 2.1). This is in

accordance with the complete conservation of cysteines in the two alleles, indicating that the

patterns of intra- and inter-chain disulfide bonds are identical for both proteins. Indeed, visco-

elastic parameters of dough are reported to be affected strongly by additional cysteines (reviewed

by Shwery et al, 2000). Therefore it is coherent that the measured visco-elastic parameters in our

DH population are not significantly different between the two alleles. D'Ovidio et al. (1996)

reported a large insertion in the central domain of the Dx2.2* HMW-GS and He et al. (2005)

tried to demonstrate that larger repetitive domains may affect the strength of the dough. In this

study we demonstrated that small insertions (six plus nine amino acids) or deletions (six amino

acids) in the central domain -inducing minor modifications in the tri, hexa and nona-peptide

composition- do not produce a significant variation in the rheology of dough. The slightly higher

12

volume of protein sedimentation (ZEL) observed in genotypes carrying the Ax2 allele is

probably due to the same factors that explain the mobility shift; a small conformational

modification plus an increase of IkDa in mass. These differences might cause greater swelling of

the 2 subunit under the conditions of the Zeleny test (lactic acid solution). The fact that only the

glutenins are capable of swelling in the Zeleny test (Eckert et al. 1993) supports our observations.

Nevertheless, the differences observed in the Zeleny sedimentation test between subunits 2 and

2* are probably too small to produce differences in the other, more direct, quality parameters

such as RES, W or RMT.

By reporting a new (the fourth) x-type allele at Glu-Al we increased the variability available for

selection, even if the effects of the 2 subunit are indistinguishable from those of the 2* subunit

on quality parameters. Our study demonstrates that the alleles Ax2 , Ax2* (and so Axl) can be

considered as having the same positive impact on quality compared to the null allele (at Glu-Al).

Detecting the Ax2 allele in a breeding program predicts no detrimental impact of this glutenin on

quality.

13

Table

2.1Analysis

oftheimpactofthe

alle

licvariationon

qual

ityparameters

indoubledha

ploi

dlinescontaining

eitherAx2

orAx2*HMW-GS

PR

Kernel'spr

otei

ncontent,ZEL

Zeleny

test,H20

water

absorption,RES

resistance

ofdo

ugh,

SOF

softeningofdo

ugh,RMT

RapidMix

Test,P

tenacity,L

elasticity,W

strength,GH

gram

hard

ness

,BU

Brabender®

units,DF

degreesoffreedom

PR(%)

ZEL

(ml)

H20(%)

RES

(min)

SOF(BU)

RMT

(ml)

P(mmH20)

L(mm)

W(1

04J)

GH

(%)

Fixedeffects

Sourceofvariation

DF

Sign

ific

ance

Year

1***

***

***

***

***

***

***

***

***

***

Glu-Al

1ns

*ns

ns

ns

ns

Ns

ns

ns

ns

Year*Glu-Al

1ns

ns

ns

ns

ns

ns

Ns

ns

ns

ns

Random

effects

Covarianceparameters

Estimates

Geno

type

(G/w-^47)

068

542

171

046

795

1283

152

513

2628

106

Residual

021

209

122

092

302

588

44

1588

1470

279

Comparisonofmeans

Alleles

2004

Ax2

1562±0

10

6156±096

6027±044

647±0

14

8813±368

6233±78

5736±156

1687±3

92480±7

32209±023

Ax2*

1558±0

11

5950±098

6031±046

631±0

14

8912±377 2005

6325±72

5747±1

58

1566±40

2416±7

52200±024

Ax2

1386±0

10

5041±091

5917±040

491±0

13

1123±3

56066±46

6393±147

1141±3

51972±68

2020±021

Ax2*

1380±0

10

4712±092

5856±040

478±0

13

1144±3

56150±47

6480±148

1125±36

1974±68

1987±021

Signific

ant

atP<005,P<001andP<0001

respectively, ns

non

sign

ific

ant

14

3 Effect of the 1BL.1RS translocation and of the Glu-

B3 variation on bread-making quality tests in a

doubled haploid population.

Submitted for publication to Journal ofCereal Science

3.1 Introduction

Wheat (Triticum aestivum) is the most suitable cereal for bread-making. This unique feature is

determined by the gluten polymer that gives its rheological property to the dough. The gluten

enables the retention of CO2 in the dough during fermentation, giving its volume to the bread.

The gluten polymer is an assemblage of different prolamin subunits, namely the high molecular

weight glutenin subunits (HMW-GS), the low molecular weight glutenin subunits (LMW-GS)

and the gliadins. Polymerization of the different subunits is achieved through interchain disulfide

or hydrogen bonds (Veraverbeke and Delcour, 2002). The nature and the amount of these

elements in the endosperm, as well as their allelic variations, determine most of the variation in

the bread-making quality (Branlard et al, 2001). Several rheological studies demonstrated that

glutenins play a large role in resistance to deformation (for review, see Shewry et al, 2002) due

to the interchain disulfide bonds that they produce. Gliadins mainly affect extensibility by acting

as 'solvent' agents (Wieser and Kieffer, 2001). In an effort to summarize the known effects of

HMW-GS and LMW-GS, quality indexes were created and the most common alleles were

scored (Branlard et al, 1992).

The substitution of the chromosome arm IBS of wheat by the 1RS arm of rye (1BL.1RS

translocation) was designed to improve the resistance of wheat to several pathogens such as

Puccinia recondita, Puccinia graminis, Puccinia striiformis and Blumeria graminis (Zeller,

1973; Zeller and Hsam, 1983). This chromosomal rearrangement had also important impact on

yield and rheology. The loss of Glu-BS and Gli-Bl loci, encoding LMW-GS and gliadins

respectively, was shown to be detrimental to parameters related to end-use quality (Burnett et al.,

1995a; Wieser et al. 2000). However, the persistence of the 1BL.1RS translocation in the

breeding material is certainly due to its positive impact on yield (Villareal et al, 1991; Kim et al,

2004).

15

Evaluation of bread-making quality is achieved by performing direct baking tests (Pelshenke et

al, 1970). Nevertheless, rheology tests (Alveograph and Farinograph) and analysis of the grain

constituents (protein content, grain hardness) provide more detailed information about the

different quality components and, thus, are used to predict bread-making quality in breeding

programs. In the present study 15 highly informative quality parameters, covering the whole

range of the bread-making process, were investigated to achieve a better understanding of the

impact of 1BL. 1RS translocation and of the allelic variation at Glu-DS. The doubled haploid

(DH) population used here shows rather limited prolamin polymorphism. Analysing this

population in a two-year experiment enabled a thorough quantification of the contribution to the

quality of the 1BL.1RS translocation and of the b and c alleles encoded at Glu-DS to quality. The

results should allow a better understanding of wheat bread-making quality and should be of

interest to breeders.

3.2 Materials and methods

3.2.1 Plant material and experimental design

The doubled haploid population was created by crossing two genotypes of the Swiss spring

wheat breeding program: line 211.12014 and cultivar Toronit (Brabant et al, 2006). The

microspores of the Fi plants were isolated and cultivated according to Kunz et al. (2000). The

population was multiplied in the green house by selfing the DH lines. For the quality assessment

tests, the population was sown in the field at Nyon in 2004 and 2005 (430 m above sea level) on

loamy cambisol soil. The sums of the daily mean temperature and precipitation were measured

during the vegetative period. According to the local recommended practice, 110 and 120 kg/ha of

nitrogen were applied to the fields in 2004 and 2005 respectively. Assessment of phosphate,

potassium and magnesium were performed before seedling to ensure that sufficient amounts of

these elements were available to the plants.

The experiments were performed in fully randomized blocks. Because of the limited amount of

seeds obtained after multiplication in 2004, 138 DH lines were sown in one to five micro-plots of

1.5 m2. In 2005 174 DH lines were sown in four micro-plots of 4.77 m2. A total of 126 lines were

used in the trials in both years. To asses the homogeneity of the experimental field in 2004, both

parents were sown on 60 micro-plots and were randomly distributed over the experimental field.

16

In 2005, no different number of replications biased the results because all the DH lines were

sown on four micro-plots.

3.2.2 Quality tests

Protein content (PR) and grain hardness (GH) were measured on intact kernels by means of a

Foss® Feed & Forage Analyzer (FOSS Analytical A/S, Hilleroed, Denmark) near infrared

reflectance spectroscopy (MRS) system according to the ICC standard N°159. Thousand kernel

weight (TKW) was calculated by counting the number of kernels in a representative 10 g sample.

For the Zeleny sedimentation test (ZEL) seeds were round in a Quadrumat® Junior grinder

(Brabender, Duisburg, Germany). The Zeleny test was performed according to the ICC standard

N° 116/1. The falling number (FN) was measured on the wholemeal of grains ground in a

Perten® grinder (Perten Instruments AB, Huddinge, Sweden). The test was performed according

to ICC standard N° 107/1. To test the rest of the quality parameters, flour was obtained by

grinding the wheat grains in a MLU-202 grinder (Bühler, Uzwil, Switzerland). The ash content

was adjusted to approximately 0.55 % by adding the corresponding higher ash fraction if

necessary. Farinograph parameters, such as water absorption (H20), resistance (RES) and degree

of softening (SOF), were measured on 50 g of flour according to the ICC standard N° 115/1.

Chopin's Alveograph (CHOPIN Technologies, Villeneuve-la-Garenne, France) parameters were

measured according to the ICC standard N°121; 250 g of flour were used for each test. The

amount of water added was adjusted according to the water content of the flour. The

representative Alveograph curve of each sample was deduced from the best four of five curves.

Tenacity (P), extensibility (L) and strength (W), elasticity index (le) and P/L were evaluated in

this test. All the ICC standards cited here are referenced in Standard Methods of the International

Association for Cereal Science and Technology (1999). To evaluate the baking behaviour of

each DH line in a direct test, the rapid mix test (RMT) was carried out as described by Pelshenke

et al. (1970). The amount of water and flour used for this test were adjusted for each DH line

according to the water absorption measured with the Farinograph and to the water content of the

flour measured by MRS.

3.2.3 SDS-PAGE and allelic variation

Proteins were extracted from single grains of the cultivar Toronit, 211.12014 and from all the

DH lines according to the protocol of Singh et al. (1991). The embryos were excised from the

17

kernels that were used to establish the prolamin profiles The extracted glutenins were separated

on 12 5 %T 1-D SDS-PAGE (18X16 cm) gels (3 h 30 min at 35 mA) The ro-ghadins were

separated on 10 %T 1-D SDS-PAGE gels (3 h at 35 mA) The bands were visible after staining

with Coomassie blue The HMW-GS, LMW-GS and co-ghadin profiles were established for all

the genotypes in the experiment based on the obtained gels

3 2 4 Statistic analysis

The statistical analysis of the data was performed with the SAS 8 2 program (SAS Institute, Cary,

NC, USA) The distribution of the residuals of the analysis of each of the parameters was

evaluated by the Tukey-Anscombe and the QQ-plots methods Since the assumptions of the

ANOVA (homoscedasticity, normality) were not met for PR, GH, RES, SOF, P and W, standard

data transformation for continuous (log) counts (square root) and percentages/proportions

(arcsines of the square root) were tested The analysis was performed with the MIXED procedure

The allelic variation nested inside the genotypes were considered to be random factors The best

fit for the models was determined with the stepAIC procedure in R 2 0 1 (http //www r-

project org/)

3.3 Results and discussion

3 3 1 Allelic variation in the DH population

SDS-PAGE analysis of the two parents and of the DH offspring demonstrated that the prolamin

variation was limited to four loci Glu-Al, Glu-B3, Glu-D3 and Gh-Bl (Table 3 1) The HMW-

GSs of the DH lines were similar with the exception of those encoded at Glu-Al with either

allele 2* or 2 was observed Both have the same impact on the rheology of the dough and on

bread-making quality (Gobaa et al, 2007a) The presence of the LMW-GS null allele j at Glu-BS

indicated the presence of the IBL 1RS translocation, it was always associated with the secahn

allele 1 at Gh-Bl The % test demonstrated that the IBL 1RS translocation was inherited in a 1 1

ratio (at P < 0 001) No recombination was found between the alleles of Glu-BS and Gh-Bl,

suggesting that the IBL 1RS translocation was inherited as a block Additional tests, based on

four PCR markers, confirmed that the IBL 1RS translocation of parent Toronit replaced the

whole IB short arm of wheat by the 1R short arm of rye (Gobaa et al, 2007b)

18

Table 3.1 Prolamm allelic profiles (as revealed by SDS-PAGE) and

means of 15 quality parameters of the parental lines used to generate

the doubled haploid lines, as measured in 2004 and 2005

Traits 211.12014 Toronit

HMW-GS

Glu-Al / Glu-Bl / Glu-Dl 2 / 7-9/5-10 2*/7-9/5-10

LMW-GS

Glu-A3 / Glu-B3 / Glu-D3 a/c/c a/j/b

co-gliadinsGh-Al /Gh-Bl /Gh-Dl a/b/b a/l/b

Protein and kernels

Zeleny (mL) 60 5±148 53 75±10 2

PC (%) 14 43±1 33 13 96±1 22

GH (%) 19 96±3 20 42±1 16

TKW (g) 39 8±0 57 39 4±1 27

Starch

Amylo (BU) 1265±117 798±96

Fal (s) 397±52 353±37

Rheology

H2Oabs (%) 61 5±0 57 58±2 55

RES (mm) 5 2±1 77 5 3±2 05

SOF (BU) 97±23 3 65±46 0

P (mmH20) 79±22 6 48±1 4

L (mm) 126±60 153±14

P/L 0 75±0 54 0 31±0 01

W (10"4J) 233±33 94 185±25 46

Baking test

RMT(mL) 627±34 3 573±53 7

BU Brabender units, PC protein content, GH grain hardness, TKW thousand

kernels weight, FN falling number, H20 abs water absorption, RES resistance,SOF softening, P tenacity, L extensibility, W strength, le swelling index, RMT

rapid mix test

19

3.3.2 Preliminary tests

In 2004 the DH lines were grown in a different number of replications. To estimate the bias due

to the pool of a different number of replications, the protein content was measured of the 120

parental lines sown in micro-plots and randomly distributed across the five replications of the

experiment. The effect of replication was not found significant (at P < 0.05) for the protein

content of the kernels (data not shown). This observation proved that the environmental

conditions tended to be homogeneous in 2004 (data not shown). In 2005, all the DH lines were

sown in four replications. Thus, no bias due to the pool of a different number of repetitions is

possible. In a separate comparison of the results of 2004 and 2005, a comparison of the means

proved that the variation in the 15 quality parameters was always concurrent for all the

polymorphic loci, although there was some degree of difference in the range of the variation

(data not shown).

3.3.3 Protein- and kernel-related traits

All the protein- and kernel-related traits (Table 3.2) were highly influenced by year. The higher

temperature in July 2004 may explain the higher amount of protein and, thus, the greater Zeleny

values and grain hardness in that year (Spiertz et al, 2006). Nevertheless, the 1BL.1RS

translocation increased the protein content of the endosperm by an average of 4.5 % across both

years of the experiment. This increase was not due to a difference in grain size because the

variation in TKW was not significant (Table 3.2). According to Ehdaie et al. (2003), 1BL.1RS

genotypes mature later and, thus, allow longer photosynthate accumulation and produce higher

yield. Since the accumulation of protein riches a plateau in the late grain filling stage (Carceller

and Aussenac, 1999), delayed maturity probably favoured the accumulation of protein over the

accumulation of starch. The variation in kernel hardness was influenced mainly by year and less

by the chromosome IB status; the variation was always within the boundaries of the hard wheat

class of both parental lines. Thus, identical settings of the grinder were used to produce the flour

used in the experiments. The allele Glu-Al 2 induced slightly higher Zeleny values than the

allele Glu-Al 2* (Gobaa et al, 2007a). Moreover, 1BL.1RS DH lines had significantly lower

sedimentation volumes. Allele b of Glu-DS was also associated with an average decrease of

11 %when compared to allele c of the same locus predicting on average better quality parameters

for the c alleles encoded at Glu-B3 and Glu-D3.

20

Table3.2

Adjusted meanscomparisonbetweenthepolymorphic

alle

lesofthedoubledha

ploi

dpo

pula

tion

forfifteenqualityparameters

QualityTraits

Glu-Al

2Variation

Glu-B3

jVariation

Glu-D3

bVariation

2004

Year

2005

Variation

Number

ofDH

lines

153

159

162

150

135

177

138

174

Proteinandkernels

Zeleny(mL)

540±058

565±058

+480%

b599±058

506±059

+155%"

585+063

520+054

-111%"

610+052

495+047

+233%"

Proteincontent(%)

146+008

147±009

-143±009

15±009

-46%"

147+009

146+008

-156+007

138+006

+113%"

Grainhardness(%)

208±02

211±02

-206±02

213±02

-37%b

21+02

209+02

-22+02

20+0

1+93%a

TKW

(g)

388±037

377±038

-290%c

383±037

382±038

ns

381+04

384+035

ns

3801+0

3385+0

3ns

Starch

Amylograph

(BU)

1017±487

860±49

0-1550%c

1117±48

1761±496

+319%"

960+52

917+45

ns

959+38

918+36

ns

Fallingnumber

(s)

342±6

6323±6

6-585%c

352±6

6313±67

+113%a

329+7

1335+62

ns

339+5

6326+52

+42%c

Rheology

H20

abso

rpti

on(%)

597±022

597±022

ns

592±022

602±022

-17%b

597+024

598+0

21

ns

603+0

18

592+0

17

+19%a

Resistance(mm)

541±0

1560±0

1-

538±0

1563±0

11

579+0

11

522+009

-98%"

631+0

11

48+007

+239%a

Soft

enin

g(BU)

98±2

3953±2

3-

807±2

1114±2

5-412%"

878+24

1059+22

+206%"

837+1

81105+19

-32%"

Tenacity(mmH20)

604±1

2604±1

2-

625±1

3583±1

2+68%b

648+14

562+1

-132%a

57+09

639+1

-121%"

Extensibility(mm)

137±27

143±27

ns

151±27

129±27

+149%"

144+29

137+2

5ns

163+243

118+217

+381%"

P/L

049±002

049±002

ns

046±002

052±002

-12%c

--

-037+002

06+002

-384%"

Strength

(lO^

J)2182±47

224±4

8-

2458±5

1977±46

+196%"

2541+5

51903+4

1-251%"

2447+4

51986+3

6189%"

le

491±048

495±049

ns

506±048

480±049

+51%"

523+052

463+045

-115%"

523+043

463+038

+131%"

Baking

test

RMT

(mL)

624±4

8612±5

1ns

606±44

630±4

8-4%

"

608+5

628+42

+33%

b627+5

1609+3

1+29%

"

a,bandc

sign

ific

ant

atP<0001,P<001andP<005respectively,TKW

thousandkernelweight

,P/L

tenacity/extensibility,le

elasticity

inde

x,

RMT

rapid mix

test,nsnon

signif

ican

t,BU

Brabender

units,

-

non

avai

labl

e,theco

rres

pond

ingfactorswereeliminatedfromthemodelbythe

steptAIC

proc

edur

ensnon

sign

ific

ant

AllelesGlu-Al

2*,Glu-B3

c,Glu-D3

candyear2005werereferencesinthecalculationofthevariations

Standarderrorsforpr

otei

ncontent,gramhardness,resistance,softeningtenacity

and

strength wereap

prox

imat

edformback-transformedvalues

21

3.3.4 Starch-related tests

With respect to the gelatinization potential of the flour, 1BL.1RS lines were clearly at a

disadvantage with regard to the quality of starch. The Amylograph and falling number values of

these lines were 31 % and 11 % lower than those of the IB lines, respectively (Table 3.2). Year

did not have an effect in either FN or Amylograph test. Pentosanes assessment was performed on

the flour samples of 2004. No significant effects of the 1BL.1RS translocation or of other

polymorphic loci were observed (data not shown). Similar observations were made by Burnett et

al. (1995b), who demonstrated that starch from 1BL.1RS genotypes was less viscous and more

soluble than starch from "regular" IB wheat. These results demonstrated that the 1BL.1RS lines

produce functionally different starch. The proteomic analysis of 16 DH lines representative of

the present population revealed the absence of a dimeric alpha-amylase inhibitor in the 1BL.1RS

lines (ExPASy database: Q4U1A2) suggesting a higher alpha-amylase activity (Gobaa et al.,

2007b). Whether this different quality of starch is involved in the dough stickiness phenotype,

associated with the 1BL.1RS genotypes, is under investigation.

Subunit Ax2* of Glu-Al was also associated with slightly higher viscosity (Table 3.2). As a

direct involvement of the glutenin subunits in the Amylograph viscosity is unlikely, the observed

effect was probably due to unidentified elements located on the long arm of chromosome 1A.

This is supported by the recent mapping of a QTL, in the vicinity of Glu-Al, for starch quality

(McCartney et al, 2006).

3.3.5 Rheology tests

Table 3.2 shows that translocated DH lines had significantly lower P, L, W and le values and

higher SOF and P/L values. This decrease of the rheological parameters is certainly the

consequence of the loss of the LMW-GS encoded at Glu-BS and located on the short arm of

chromosome IB (Graybosch, 2001; Gobaa et al., 2007c). Such loss of polymeric glutenin

subunits was reported to produce weaker gluten and thus reduced Rnax (Farinograph) and

extensibility (Wieser et al., 2000).

In the Farinograph test, necessary hydration to reach 500 Brabender® units of strength was

significantly higher in 1BL.1RS lines. This was probably due to the poorer starch gelatinization

of translocated lines discussed above. Variation at Glu-DS did not produce significant changes in

this trait.

22

DH lines with the allele Glu-D3 b had significantly lower RES, P, W and le values and higher

SOF values than DH lines with the allele Glu-DS c. However, unlike 1BL.1RS translocation,

Glu-D3 din not have a direct impact on extensibility (L and P/L). The variation at Glu-D3

seemed to preferentially affect resistance to deformation. These results contradict those of

Branlard et al. (2001), who found that Glu-D3 c and Glu-D3 b have exactly the same positive

effect on W. The presented results contradict also with those of Gupta et al. (1994), who

demonstrated that the c and b alleles in Australian cultivars were equivalent for Rmax but that

higher extensibility was associated with allele b rather than allele c. However, correlations

between particular allelic forms of LMW-GS and quality parameters of bread wheat can differ,

possibly due to the genetic background, gene interactions and environmental effects (D'Ovidio

and Masci, 2004). According to Xu et al. (2006) an LMW-GS encoded at Glu-D3 with extra

cysteine residues could improve the rheological parameters of the dough. Whether or not the

allele Glu-D3 c of parent 211.12014 had such this LMW-GS was not investigated.

When the unweighted mean variation in the seven rheology parameters were measured, both

alleles Glu-B3 j and Glu-D3 b decrease quality by an average of 16 %. This demonstrates the

equal importance of the variation at Glu-B3 and Glu-D3 for the rheological parameters.

Interactions of glutenins and year, of variable significance, were also observed for the parameters

Zeleny, SOF, RES, L, W, le and RMT (Table 3.3). The association of the two detrimental alleles

(Glu-B3 j and Glu-D3 b) had, of course, a strong negative effect on the quality parameters.

Martin et al. (2001) reported a comparable effect when 1BL.1RS was associated with the allele

Glu-Dl 2+12. Therefore, the production of high-quality cultivars combining the 1BL. 1RS

translocation with a detrimental glutenin is unlikely. The interaction of Glu-Al with the other

prolamins was limited; the Glu-Al 2 IGlu-D3 c combination had better W and le values than the

other combinations. The interaction between Glu-B3 and year was highly significant for the

Zeleny test, RES and W. In 2005 the lower protein content associated with the 1BL.1RS

translocation seemed to drastically reduce the strength of the dough. None of the other prolamin

loci showed a similar Environment X Genotype interaction; effects of the glutenin alleles

encoded at Glu-Al and Glu-D3 were stable in both years for all of the measured quality

parameters.

23

Table 3.3 F values for the observed significant interactions between the studied sources of variation

Glu-A1*YR ns

Glu-B3*YR 20 59*** 12 84***

Glu-D3*YR ns ns

Glu-A1*Glu-B3 3 97* 451* ns

Glu-A1*Glu-D3 ns ns ns

Glu-B3*Glu-D3 7 71** 6 16* 14 56***

Source Zeleny Resistance Softening Extensibility Strength le RMT

ns ns

9 91**

ns

ns 5 2* ns

412* 4 2* 4 25*

5_8£ 5 05* 8 25** nsThe empty cases correspond to the factors eliminated from the model by the stepAIC procedure ns non

significant, le elasticity index, *, **, *** significant at P < 0 05, P < 0 01 and P < 0 001 respectively

3.3.6 Baking tests

The volumes of the loaves, measured by the RMT test, were significantly lower in regular IB

and in Glu-DS c lines, despite better results in the rheology tests (Table 3.2). This apparent

contradiction could be due to several factors. First, the amount of water used for the RMT test is

adjusted according to the water absorption as measured by the Farinograph (Pelshenke et al.,

1970). Since H20 absorption is significantly higher in 1BL.1RS lines, the greater amount of

water used in the RMT test for the 1BL.1RS lines may explain a small part of the larger volumes

associated with IBLIRS lines. Second, loaf volume is highly correlated with the protein content

of the flour (Wieser and Kieffer, 2001) but only weakly correlated with other rheological

parameters such as the Rmax of the Mixograph (Kieffer et al, 1998). In the present study, the

average protein content was quite high (14 %) and it was significantly higher (P < 0.001) in

1BL. 1RS lines. Seemingly, the resistance to deformation opposed to the dough during

fermentation was limiting the volume of the loaves above a certain value of P. These results can

be compared to those obtained in experiments with transgenic wheat genotypes over-expressing

a glutenin subunit (1Dx5). Such lines exhibited very high tenacity and, thus, abnormal

rheological behaviour (Rooke et al, 1999; Popineau et al, 2001).

3.4 Conclusions

The study of DH lines contrasting for three alleles of prolamins, with and without the 1BL.1RS

translocation, determined their impact on 15 important quality traits. For the first time an

important defect in the gelatinization of starch, revealed by the Amylograph test, was associated

with the 1BL.1RS translocation in a DH population. However, the mechanisms leading to such a

24

defect remain unknown. More investigations are needed to determine whether IBL.IRS wheat

had a naturally "sprouted-like" starch or whether the defect in gelatinization was caused by an

original starch branching.

The negative effect of IBL.IRS translocation on rheology was confirmed by the present study,

but it was limited. IBL.IRS translocation produced a change in rheology comparable to that

produced by "regular" polymorphism at another LMW-GS-encoding locus. Allelic variation at

Glu-DS caused even greater variation in tenacity and strength. This indicates, that in breeding

programs, the lower quality predicted by the detection of the IBL.IRS translocation can be

counterbalanced by accumulating favourable alleles at Glu-AS and Glu-DS and/or by selecting

genotypes with a high protein content. However, care should be taken translocated material is

included in breeding programs, because the importance of the impact of IBL.IRS translocation

on rheology seemed to be modulated by the environmental factor. The final baking test

demonstrated that, in the present case, lowering rheological parameters can lead to an increase in

the volume of the loaves. It is important to consider that this occurred in a population that

accumulates the beneficial alleles for quality at Glu-Al, Glu-Bl and Glu-Dl and in which the

mean protein content was high. The observation that very high-strength genotypes may produce

lower bread volumes and that they need a high energy input during mixing can seriously

handicap their direct use in bakery. They may be more suitable for improving the quality, in

mixture with poor quality genotypes.

25

4 Proteomic analysis of wheat recombinant inbred

lines (Part I): effect of the 1BL.1RS translocation

on the wheat grain proteome.

Proteomics (2007), in press.

4.1 Introduction

In Triticum aestivum cultivars, the replacement of the IB short-arm chromosome by the 1R short-

arm chromosome of rye was originally designed to contribute to resistance to diseases such as

leaf rust, stem rust, stripe rust and powdery mildew (Zeller, 1973; Zeller and Hsam, 1983). Even

though, the corresponding pathogens (Puccinia recondita, Puccinia gramins, Puccinia striiformis

and Blumeria graminis respectively) have since overcome this resistance, 1BL.1RS translocation

is still used in breeding programs, mainly for its beneficial effect on grain yield (Villareal et al.,

1995; Kim et al, 2004). Nevertheless, severe end-use defects (sticky dough, low tolerance to

over-mixing, dough tenacity, low SDS-sedimentation volume and low specific loaf volume) of

flour obtained from translocated genotypes have been reported (reviewed by Graybosch, 2001).

Storage proteins, mainly prolamin (glutenin and gliadins), play a major role in baking quality and

in the rheology of dough through their allelic or quantitative variation (Shewry et al, 1992;

Veraverbeke and Delcour, 2002). It has been suggested that the impact of 1BL.1RS translocation

on quality can be explained in part by the loss of the Glu-BS locus, coding for one third of the

low molecular weight glutenin subunits (LMW-GS), and by the substitution of the co-gliadins

(encoded at Gli-Bl) by co-secalins (encoded at Sec-1). These changes drastically reduce the

polymeric fraction of gluten and increase the monomeric fraction. However, it is not understood

how the 1BL.1RS translocation acts on the proteome of the wheat grain.

Previous studies, on the proteome of wheat grain, demonstrated that the presence of one or two

doses of chromosomes 1 A, IB or ID or the lack thereof has an impact on the amount and nature

of several proteins that are not even located on the pair of chromosomes affected by the

aneuploidy (Dumur et al., 2004). This demonstrated that storage proteins are under the control of

complex regulation mechanisms and that compensation may occur when loci coding for

prolamins are lost. The effects of heat stress (Majoul et al, 2003) and nitrogen fertilization

26

(Bahrman et al, 2004) were also analyzed. However, to the best of our knowledge, no reports

exist on the impact of fragments of rye chromosomes on the wheat grain proteome.

The goal of this study was to report on the effects of 1BL.1RS translocation on the mature grain

proteome in contrasting doubled haploid (DH) lines of Triticum aestivum. The comparison

between proteomic profiles characterizing translocated genotypes and proteic profile

characterizing regular IB genotypes can contribute to a better understanding of the mechanisms

that are affected by 1BL.1RS translocation, such as the synthesis and accumulation of storage

proteins and the metabolism of starch.

4.2 Material and methods

4.2.1 Plant material

A doubled haploid population was produced by crossing two Swiss genotypes of Triticum

aestivum, one carrying the 1BL.1RS translocation (Toronit) and the other having a regular IB

chromosome (the breeding line 211.12014). The microspores of Fi plants were isolated and

cultivated according to Kunz et al. (2000) to obtain 174 DH lines. These lines were grown on

loamy Cambisol soil at Agroscope Changins-Wädenswil (430m above sea level, Nyon,

Switzerland) in 2004. The average precipitation was 1037 mm. Fertilization was performed

according to the local recommended practice (110 kg of nitrogen per hectare).

4.2.2 Translocation mapping

Genomic DNA was extracted from the leaves of young seedlings using the Clontech®

NucleoSpin® Plant Kit (Clontech Laboratories Inc., Mountain View, CA, USA). To estimate the

size of the 1RS material in the genome of the cultivar Toronit and the breeding line 211.12014, a

polymerase chain reaction (PCR) was performed with four markers: 5S, RIS, TEL and NOR

(Koebner, 1995). The annealing temperature was set at 65°C. PCRs were performed with a

Biometra® T3Thermocycler (Biometra GmbH, Goettingen, Germany) in 50 \A volumes and with

Qiagen HotStartTaq® (Quiagen GmbH, Hilden, Germany). The amplification products were

visualized on 1 % w/v standard agarose gels and co-migrated with the Bio-Rad® 20 bp

Molecular Ruler (Bio-Rad Laboratoires, Hercules, CA, USA) to determine their sizes.

4.2.3 1-D electrophoresis and allelic variation

Proteins were extracted from single grains of the cultivar Toronit, 211.12014 and all the DH lines

according to the protocol of Singh et al. (1991). The glutenins were separated on 12.5 %T 1-D

27

SDS-PAGE (18X16 cm) gels (3 h 30 min at 35 mA) The ro-ghadins were separated on 10 %T 1-

D SDS-PAGE gels (3 h at 35 mA) After Coomassie blue staining the bands became visible,

giving the glutenin and co-ghadin profiles of the different genotypes

Eight DH lines carrying the IBL 1RS chromosome and eight DH lines carrying the regular IB

chromosome were selected from the 174 available DH lines The DH lines were selected on the

basis of the glutenin and ghadin profiles established by 1-D SDS-PAGE

4 2 4 2D SDS-PAGE and quantitative variation

The 2-D SDS-PAGE experiment was performed on 16 selected DH lines Wheat kernels, from

which the embryo had been excised, were ground in a Cyclotec 14920 grinder (Hillerod,

Denmark) The proteins were extracted from 100 mg of flour according to Branlard and Bancel

(2006) IPG buffer 6-11 (2% v v) from GE Healthcare (Uppsala, Sweden) was used as ampholyte

carrier and DTT 20 mM was added in the solubilization solution The concentration in protein of

the extracts was assayed with a PlusOne 2-D Quant® Kit (GE Healthcare) in a 5 \il aliquot of the

supernatant collected after the first centnfugation 120 ug of extracted proteins were dissolved in

250 (il of extraction solution to passively réhydrate immobilized pH gradient strips (Immobihne

DryStrip®, pH 6-11, 13 cm) Isoelectric focusing (IEF) and 2-DE migration were performed

according to Dumur et al (2004) Fixation and Coomassie blue staining were done according to

the method described by Neuhoff et al (1988) The resulting gels were scanned with an

ImageScanner II (GE Healthcare) and the images were analyzed with the ImageMaster 2D

Platinum v5 0 software (GE Healthcare) The percentage of volume (%Vol) was measured for

each spot The obtained protein profiles of each selected DH line were the result of duplicated

protein extractions used in four 2-DE replicates The spots were considered only if they were

detected in, at least, three out of the four replicates

The statistical analysis was performed with the SAS v8 2 Software (SAS Institut, Cary, NC,

USA) The GLM procedure was used to detect the spots showing a significant quantitative

variation (P < 0 001) in their %Vol between the conditions "DH lines with IBL 1RS" and "DH

lines with the normal IB" Spots considered to be up- or down-regulated showed at least a

twofold variation in their mean %Vol when both sets of experimental conditions were compared

28

4 2 5 Protein identification

After image and statistical analyses, the relevant spots were cut out and transferred to Eppendorf

low-binding micro tubes for mass spectrometry First, Coomassie blue was eliminated with

ammonium bicarbonate-acetomtnle buffers The spots were incubated for 30 mm in 100 ul of

buffer 1 containing 25 mM NH4HCO3 and 5 % v/v acetonitnle at room temperature Then the

spots were washed twice with 100 ul of buffer 2 containing 25 mM NH4HCO3 and 50 % v/v

acetonitnle (30 min incubation at room temperature) Finally, the spots were washed for 10 mm

with 200 ul of pure acetonitnle The tubes were dried in a Heto-Vac system (VR-1 Jovan Nordic,

Allerod, Denmark) For in-gel digestion with trypsin plus chymotrypsin digestion, 15 ul of

trypsin (V5111, Promega, Madison, WI, USA), 10 ng/ul in 25 mM NH4HCO3, 15 ul of

chymotrypsin (C6423, Sigma, St Louis, MO, USA), 10 ng/uL in 50 mM NH4HCO3 and 10 mM

CaCl2 were added to the dry gel Digestion was performed overnight at 37°C The pieces of gel

were centnfuged and 10 ul acetonitnle were added to extract the peptides The mixture was

sonicated for 5 mm and centnfuged at 5,000 g for 5 mm For MALDI-TOF MS analysis, 1 (il of

supernatant was loaded directly onto the MALDI target One microliter of the matrix solution (5

mg ml"1 a-cyano-4-hydroxycinnamic acid in 50% acetonitnle / 0 1% tnfluoroacetic acid) was

added immediately and the mixture was left to dry at room temperature A Voyager DE-Pro

model of the MALDI-TOF mass spectrometer (Perseptive BioSystems, Farmingham, MA, USA)

was used in positive-ion reflector mode for peptide mass fingerprinting External calibration was

performed with a standard peptide solution (Proteomix, LaserBio Labs, Sophia-Antipohs,

France) Monoisotopic peptide masses were assigned and used from NCBI or from a local

version of poaceae uniprot or from EST database searches with the Mascot and ProFound

softwares (http //www matrrxscience com and http //prowl rockefeller edu) Matches to protein

sequences from the Viridiplantae taxon were considered acceptable if at least four peptide masses

from the PMF matched and a Z score of 1 00 or higher was obtained from ProFound or a

significant score was obtained from MASCOT, which rates scores as significant if they are above

the 95 % significance threshold (P < 0 05)

For NanoLC-MS/MS analysis, FIPLC was performed with an Ultimate LC system combined with

a Famos autosample and a Switchos II microcolumn switching for preconcentration (LC

Packings, Amsterdam, The Netherlands) The samples after hydrolysis were loaded onto the

column (PEPMAP CI8, 5 urn, 75 urn ID, 15 cm, LC Packings) using a preconcentration step in a

29

microprecolumn cartridge (300 \im ID, 1 mm) The sample (6 (iL) was loaded onto the pre-

column at 40 uL/min After 3 mm, the pre-column was connected to the separating column and

the gradient was started at 200 nL/min The solvents with 0 5 % formic acid contained 95 %

water / 5 % acetomtnle (A) and 95 % acetomtnle / 5 % water (B) A linear gradient of 10 to 90 %

B was applied for 45 mm For ion trap-MS, a LCQ deca with a nano electrospray interface

(ThermoElectron, Les Ulis, France) was used Ionization (18 kV ionization potential) was

performed with a liquid junction and an uncoated capillary probe (New Objective, Cambridge,

MA, USA) Peptide ions were analyzed by the data-dependent "triple-play" method as follows

(l) full MS scan (m/z 400-2000), (n) ZoomScan (scan of the major ion with higher resolution),

(in) MS/MS of the later ion Mass data, collected during a LC-MS