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Research ArticleReceived: 2 January 2011 Revised: 19 May 2011 Accepted: 23 May 2011 Published online in Wiley Online Library: 4 August 2011

(wileyonlinelibrary.com) DOI 10.1002/jsfa.4548

Comparative proteome analysis of gluteninsynthesis and accumulation in developinggrains between superior and poor qualitybread wheat cultivarsWan Liu,a† Yanzhen Zhang,b† Xuan Gao,a† Ke Wang,a Shunli Wang,a

Yong Zhang,c Zhonghu He,c Wujun Mad∗ and Yueming Yana

Abstract

BACKGROUND: Wheat glutenins are the major determinants of wheat quality. In this study, grains at the development stagefrom three wheat cultivars (Jimai 20, Jin 411 and Zhoumai 16) with different bread-making quality were harvested based onthermal times from 150 ◦Cd to 750 ◦Cd, and were used to investigate glutenin accumulation patterns and their relationshipswith wheat quality.

RESULTS: High and low molecular weight glutenin subunits (HMW-GSs and LMW-GSs) were synthesised concurrently. Noobvious correlations between HMW/LMW glutenin ratios and dough property were observed. Accumulation levels of HMW-GSsand LMW-GSs as well as 1Bx13+1By16 and 1Dx4+1Dy12 subunits were higher in superior gluten quality cultivar Jimain 20 thanin poor quality cultivar Jing 411 and Zhoumai 16. According to the results of two-dimensional gel electrophoresis, six types ofaccumulation patterns in LMW-GSs were identified and classified. The possible relationships between individual LMW-GSs andgluten quality were established.

CONCLUSION: The high accumulation level of HMW-GSs and LMW-GSs as well as 1Bx13+1By16 and 1Dx4+1Dy12 subunitscontributed to the superior gluten quality of Jimai 20. Two highly expressed and 16 specifically expressed LMW gluteninsubunits in Jimain 20 had positive effects on dough quality, while 17 specifically expressed subunits in Zhoumai 16 and Jing411 appeared to have negative effects on gluten quality.c© 2011 Society of Chemical Industry

Keywords: glutenins; HMW-GS; LMW-GS; SDS-PAGE; RP-HPLC; 2-D gel electrophoresis

INTRODUCTIONWheat (Triticum aestivum L.) is one of the most important cerealcrops in the world. Based on sequential extraction and differentialsolubility, wheat grain proteins are traditionally classified intofour different groups: albumins, globulins, gliadins and glutenins.The glutenins, comprised of high and low molecular weightglutenin subunits (HMW-GS and LHW-GS, respectively) accountfor 40% of all wheat grain proteins, and are among the largestprotein molecules known, with molecular weights reaching over20 million.1 Glutenins, the major determinants of dough elasticityand bread-making properties,2,3 are classified as monomer andpolymer proteins on the basis of their degree of polymerisationand the biochemical characteristics of their polypeptide chains.

Based on their electrophoretic mobilities on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), gluteninscan be divided into four groups, A, B, C and D. The A groupcontains HMW-GS while the B, C and D groups include LMW-GS and some α/β-, γ - and ω-gliadins.4 Although HMW-GS areminor components among endosperm proteins and only makeup 10% of total seed storage protein,5,6 they play key roles in thedetermination of bread-making as well as other flour processing

qualities due to network formation in the dough from glutenpolymerisation. In contrast to HMW-GS, LMW-GS represent aboutone third of total seed proteins and about 60% of total glutenins.This has a pronounced effect on dough viscoelastic properties.

∗ Correspondence to: Wujun Ma, State Agriculture Biotechnology Centre,Murdoch University; Western Australian Department of Agriculture and Food,Perth, WA 6150, Australia. E-mail: w.ma@murdoch.edu.au

† Wan Liu, Yanzhen Zhang and Xuan Gao contributed equally to this work.

a Key Laboratory of Genetics and Biotechnology, College of Life Science, CapitalNormal University, 100048 Beijing, China

b College of Applied Sciences and Humanities of Beijing Union University, Beijing100083, China

c Institute of Crop Science/National Wheat Improvement Center, ChineseAcademy of Agricultural Sciences (CAAS) and International Maize and WheatImprovement Center (CIMMYT) China Office, c/o CAAS, Beijing 100081, China

d State Agriculture Biotechnology Centre, Murdoch University; Western AustralianDepartment of Agriculture and Food, Perth, WA 6150, Australia

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It has been reported that HMW-GS are encoded by two (x- andy-type) tightly linked genes at the Glu-A1, Glu-B1 and Glu-D1 locion the long arms of chromosomes 1A, 1B, and 1D, while LMW-GSare encoded by genes at the Glu-3 loci on the short arms ofchromosomes 1A, 1B, and 1D. In addition, some components ofLMW-GS are encoded by genes on the short arms of chromosomes6 and 7 D.7

The relationships between HMW-GS and bread-making qualityhave been studied extensively.4,5 Certain allelic subunits have beenshown to have differential impact upon bread-making quality.For instance, the subunit 1Dx5+1Dy10 is associated with gooddough quality while 1Dx2+1Dy12 are related to poor doughquality.8 In addition, some reports have shown that the ratioof HMW-GS/LMW-GS is tightly correlated with the percentageof unextractable polymers,2 which had a considerably higherratio of HMW-GS/LMW-GS than that associated with extractablepolymers.9 To date, however, the molecular mechanisms regardingthe effects of allelic variations at Glu-1 on gluten quality are stillnot clear.

LMW-GS are also important in the determination of the finaluse of wheat. Josephides et al.10 reported that LMW-GS, especiallysubunits encoded by loci on chromosome 1B, are involved in theend-use quality of durum wheat. LMW-2, an allelic form of typicalLMW-GS, was particularly related with the best pasta-makingproperties while LMW-1 was associated with poor pasta-makingcharacteristics.11 Compared to HMW-GS, the LMW-GS relationshipto dough quality are still under intensive research due to theirextensive allelic variation and complexity in the composition.The gene copy numbers of LMW-GS have been estimated tovary from 10–15 to 35–40 in hexaploid wheat.12,13 Therefore,advanced separation techniques are needed to investigate LMW-GS composition and their functions.

SDS-PAGE has been the most widely used method forisolating and characterising glutenin subunits. In recent years,a wide range of new separation methods, including reverse-phase (RP) and size-exclusion (SE) high-performance liquidchromatography (HPLC),14,15 two-dimensional gel electrophoresis(2-DE),16,17 high-performance capillary electrophoresis (HPCE)18

and matrix-assisted laser desorption/ionisation time-of-flightmass spectrometry (MALDI-TOF-MS),19 have been developedto better separate and characterise glutenin proteins.14,19 – 24

These techniques have provided powerful alternative tools forstudies of the synthesis, accumulation and functional propertiesof grain storage proteins.2,15,20 However, investigations of theexpression patterns of individual LMW-GS in developing grainsand their relationships with gluten quality have not been reportedso far.

In the present study, we focused on investigating thesynthesis and accumulation of HMW-GS and LMW-GS during graindevelopment among three bread wheat cultivars with differentgluten quality properties by combining RP-HPLC and 2-DE. Resultsobtained could be useful for further better understanding theexpression profiles and functional properties of glutenins.

MATERIALS AND METHODSPlant materialsBread wheat cultivars Jimai 20 with good bread-making quality,Jing 411 and Zhoumai 16 with poor gluten properties were plantedduring 2008–2009 at the experimental station of the ChineseAcademy of Agriculture Sciences (CAAS), Beijing. The majorgluten quality parameters of three wheat cultivars were tested

by wheat quality laboratory of the CAAS (Table 1) with methodsdescribed in Wang et al.20 Grain samples were harvested duringthe post-anthesis period based on thermal times25 correspondingto the following cumulative average temperatures (◦Cd): 150 ◦Cd,250 ◦Cd, 350 ◦Cd, 450 ◦Cd and 750 ◦Cd (fully ripened). Duplicatesamples of 80 caryopses collected from central parts of 10 headsat each stage.

Glutenin preparationGlutenin proteins were extracted from wheat grains using themethod described by Singh et al.26 with minor modifications.Grain samples were frozen with liquid nitrogen, freeze dried andground into fine powder using a mortar, and then stored at−20 ◦C for further use. A total of 30 mg of meal was extracted with1.0 mL 70% ethanol using 30 min of continuous vortexing. Aftercentrifuging for 10 min at 15700g, the supernatants were removedand the precipitates were mixed with 1.0 mL of 55% isopropanolwith complete vortexing. After incubation for 30 min at 65 ◦C in awater bath and centrifuging for 10 min at 15700g, the supernatantswere discarded. This step was repeated twice to allow for completeelimination of gliadins compared to the 2-DE pattern of gluteninsubunits.7 Subsequently, the glutenins were extracted with 100 µLextraction buffer (50% isopropanol, 80 mmol L−1 Tris-HCl, pH 8.0)with 1% dithiothreitol (DTT) incubated at 65 ◦C for 30 min, andthen alkylated by mixing with an equal volume of extraction buffercontaining 1.4% 4-vinylpyridine (v/v) instead of 1% DTT under thesame aqueous incubation conditions. After centrifugation (10 minat 15700g), the supernatants shifted to three new tubes withequal amount for SDS-PAGE analysis 2-DE analysis, and RT-HPLCanalysis.

Sodium dodecyl sulfate polyacrylamide gel electrophoresisSDS-PAGE was carried out by a modified method according toGupta et al.2 8 µL of sample buffer containing 2% SDS, 0.02%bromophenol blue, 0.08 mol L−1 Tris-HCl pH 8.0 and 40% glycerinwas mixed with the same volume of protein sample, and themixture was incubated at 90 ◦C for 5 min. After centrifugationat 15700g for 10 min, protein samples were electrophoresed at20 mA for 8 h, and the gels were stained with 1% Coomassiebrilliant blue for 1 h and then destained with solution containing10% ethanol and 10% acetic acid. Finally, the gels were scannedusing a GS-800 Calibrated Densitometer (Bio-Rad, Bio-Rad, Dallas,Texas, USA). The HMW-GS compositions of three cultivars wereidentified as described by Dong et al.14

Reversed-phase high-performance liquid chromatographyRP-HPLC was performed on three independent replicationsaccording to Dong et al.14 The mobile phases were ddH2O(containing 0.06% trifluoroacetic acid (TFA)) and acetonitrile (ACN)with 0.06% TFA, respectively, and HPLC grade solvents were usedin all cases. Glutenins were dissolved in solvent (50% ACN, 0.05%TFA) and 20 µL samples were injected and eluted by an Agilent1100 system with a linear gradient of 21% to 48% ACN (containing0.06% TFA). A ZORBAX 300SB-C18 column (300 Å pore size, 5 µmparticle size, 4.6×250 mm i.d.; Agilent, Santa Clara, California, USA)was used with a column temperature of 50 ◦C, and the proteinpeaks were detected by UV with absorbance areas at 210 nm. Thedata of peak areas (mAU s) were used to generate the proteinaccumulation profile.

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Tab

le1

.H

MW

-GS

com

po

siti

on

and

bre

ad-m

akin

gq

ual

ity

ofJ

ing

411,

Jim

ai20

and

Zh

ou

mai

16

Cu

ltiv

arG

lu-A

1G

lu-B

1G

lu-D

1G

P(%

)G

HG

HC

MT

PHEA

PHat

8m

inEA

at8

min

Jin

g41

1N

7+

82

+12

14.1

±0.

7523

21.9

±1.

3321

S1.

67±

0.31

2541

.2±

2.11

7856

.7±

2.55

6328

.4±

1.25

6326

4.9

±11

.234

5

Jim

ai20

113

+16

4+

1214

.1±

0.75

1168

.8±

2.54

63H

3.64

±0.

4416

45.1

±2.

3584

129.

3±

4.58

7636

.7±

1.75

6830

5.9

±14

.556

3

Zh

ou

mai

16N

7+

92

+12

13.3

±0.

7456

56.0

±2.

1245

H1.

75±

0.32

1143

.8±

2.34

1263

.0±

3.23

5730

.1±

1.57

8227

9.0

±12

.145

7

HM

W-G

Sw

ere

sho

wed

wit

hth

eire

nco

din

glo

ci.

GP,

gra

inp

rote

inco

nte

nt;

GH

,gra

inh

ard

nes

s;G

HC

,gra

inh

ard

nes

scl

ass;

MT,

mix

ing

tim

e;PH

,pea

kh

eig

ht;

EA,e

xten

sio

nar

ea.

Two-dimensional electrophoresisTwo-dimensional electrophoresis was performed on three inde-pendent replications according to methods described by Ikedaet al.27 with minor modifications. For the first dimension of 2-DE,isoelectric focusing (IEF) was carried out by EttanTM IPGphor IITM(Amersham Bioscience, Piscataway, New Jersey, USA) by usingimmobilised pH gradient (IPG) gel strips (Immobiline DryStrip, pH3–11, 13 cm; Amersham Bioscience), which had been rehydrated inrehydrating buffer (8 mol L−1 urea, 2% CHAPS, 0.002% bromophe-nol blue) about 12–15 h at room temperature. The protein samplewas diluted with IEF sample buffer (8 mol L−1 urea, 2 mol L−1

thiourea, 2% CHAPS, 20 mmol L−1 DTT, 0.5% IPG buffer pH 3–11,and 0.002% bromophenol blue) to a final volume of 100 µL andloading onto the loading cup placed close to the anodic end ofthe strip. Focusing conditions were as follows: 50 mA, 500 V, 1 h;4000 V, 1.5 h; 8000 V, 40 000 V h. Before SDS-PAGE, the IPG gelstrips were equilibrated twice in equilibrating buffer (1.5 mol L−1

Tris-HCL, pH 8.8, 6 mol L−1 urea, 2% SDS, 30% glycerin, 0.002%bromophenol blue). For the first equilibration, the equilibratingbuffer was mixed with 1% DTT; for the second equilibration, 1%DTT replaced by 4% iodoacetamide. The second dimension of2-DE was performed on an EttanTM DALT Six electrophoresis unit(13 cm; Amersham Bioscience) at 12 mA gel−1 for 1 h, and then20 mA gel−1 for about 7 h until 30 min after the bromophenolblue had run off the bottom of the gel. Finally, gels were stained,destained and scanned using Image MasterTM 2D Platinum soft-ware version 5.0 (Amersham Bioscience), and the expression levelsof protein spots on the 2-DE gels were determined by proteinabundance (vol%).

RESULTSHMW-GS compositions and bread-making quality propertiesof three cultivarsThe HMW-GS as well as LMW-GS compositions of three cultivars,Jimai 20, Zhoumai 16 and Jing411, separated by SDS-PAGE areshown in Fig. 1. The main parameters for bread-making qualityare listed in Table 1. Jimai 20 is a widely used bread wheat cultivarin the major wheat production area of China and has superiorgluten quality properties and high yield performance. Zhoumai16 and Jing 411 have poor bread-making quality but with highyield performance. Jimai 20 had HMW-GS 1Ax1, 1Bx13+1By16,1Dx4+1Dy12 while Zhoumai 16 and Jing 411 had null, 1Bx7+1By9,1Dx2+1Dy12 and null, 1Bx7+1By8, 1Dx2+1Dy12, respectively.The subunit 1Ax1 encoded by Glu-A1 and 1Bx13+1By16 encodedby Glu-B1 are considered to be good quality subunits28 whereas1Dx2+1Dy12 encoded by Glu-D1 are considered to be poor qualitysubunits.5 As shown in Table 1, the main gluten quality parametersdemonstrated that the values of GH, MT, PH, EA, PH at 8 min and EAat 8 min of Jimai 20 were much higher than those of Zhoumai 16and Jing 411, indicating that Jimai 20 had a superior gluten quality.

Synthesis and accumulation of glutenins during graindevelopmentThe accumulation patterns of HMW-GS and LMW-GS during graindevelopment in three cultivars detected by SDS-PAGE and RP-HPLC are shown in Figs 1–3. In general, deposition of HMW andLMW glutenin subunits began as early as 250 ◦Cd after anthesis,and progressed until maturity (Fig. 1). When separated by RP-HPLC,glutenin subunits in the three cultivars commenced at 250 ◦Cd.The amounts of glutenin subunits rapidly increased at 350 ◦Cd in

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1 235

7 891011

12 1314 15 16

17181920 21

2223

242526

146

8

Figure 1. SDS-PAGE of glutenins extracted from developing grainsharvested based on thermal times as the following cumulative averagetemperatures (◦Cd): 150 ◦Cd, 250 ◦Cd, 350 ◦Cd, 450 ◦Cd and 750 ◦Cd. Lines1–5: Jing 411; lines 6–10: Jimai 20; and lines 11–15: Zhoumai 16 from150 ◦Cd to 750 ◦Cd. Line 16: Chinese Spring (mature grain, Chinese Springserved as the marker cultivar to identify other cultivars).

Jimai 20, and gradually increased in both Jing 411 and Zhoumai16 (Fig. 2).

HMW and LMW glutenin subunits were synthesised concur-rently during grain development. All HMW-GS had similar accu-mulation patterns in the three cultivars, but LMW-GS synthesisappeared to be different. There existed three main accumulationpatterns for individual LMW glutenin subunits indicated in Fig. 1.The first group included 15 protein bands: 1, 3, 4, 5, 6, 7, 13,14, 16, 18, 19, 20, 22, 23 and 25. In general, their accumulationgradually increased during grain development. The second groupcontained 11 subunits (bands 2, 8, 9, 10, 11, 12, 15, 17, 21, 24 and26), and their synthesis mainly took place during the last period(750 ◦Cd) of grain development. The third group included bands1 and 6 in Jimai 20 and band 3 in Zhoumai 16, which graduallyincreased during first three periods but disappeared in the laststage (750 ◦Cd) in contrast to the behavior of the second group.

The accumulation curves for total amounts of HMW-GS andLMW-GS, HMW/LMW glutenin ratios, and individual HMW-GS inJimai 20, Zhoumai 16 and Jing 411 at five grain developmentalstages are shown in Fig. 3. During grain development, theexpression levels of HMW-GS in the three cultivars showed veryfew changes until 250 ◦Cd. However, the HMW-GS and LMW-GS inJimai 20 increased significantly from 250 ◦Cd to 350 ◦Cd, and thenshowed a rapid accumulation until maturity. However, HMW-GSand LMW-GS steadily accumulated from 250 ◦Cd to 450 ◦Cd andthen dramatically increased from 450 ◦Cd to 750 ◦Cd in Zhoumai16 and Jing 411. Eventually, the total amounts of HMW-GS of Jimai20 were between those of Jing 411 and Zhoumai 16 at maturity(750 ◦Cd), with Jing 411 being the highest (Fig. 3A,B). The HMW-GS/LMW-GS ratios gradually decreased from 150 ◦Cd to 750 ◦Cd in

Jimai 20 while they showed a slight increase at 450 ◦Cd in bothZhoumai 16 and Jing 411 (Fig. 3C).

For the accumulation patterns of individual HMW-GS, thereexisted clear expression differences between superior and poorgluten quality cultivars. All HMW-GS except for 1Ax1 in Jimai 20increased dramatically at 350 ◦Cd, and then gradually accumulateduntil 750 ◦Cd compared to those in Zhoumai 16 and Jing411 (Fig. 3D,F). The accumulation rates of HMW-GS in bothpoor quality cultivars remained low from 150 ◦Cd to 450 ◦Cd,but sharply increased from 450 ◦Cd to 750 ◦Cd (Fig. 3D–F).Higher accumulation rates of total glutenin amounts as well as1Bx13+1By16 and 1Dx4+1Dy12 subunits at the early developingstages in Jimai 20 might contribute to the superior gluten qualityproperties.

Synthesis and accumulation of individual LMW-GSs revealedby 2-DETwo-dimensional electrophoresis patterns of LMW gluteninsubunits in 750 ◦Cd of Jing 411, Jimai 20 and Zhoumai 16 are shownin Fig. 4A–C. Apparently, gliadins were completely eliminated onthe gels compared to the 2-DE pattern of glutenin subunitsreported by D’Ovidio and Masci.7 By comparing five developingstages among three cultivars, total of 83, 57 and 91 LMW gluteninprotein spots were identified and numbered in Jing 411, Jimai 20and Zhoumai 16, respectively (Fig. 4A–C). Of 147 protein spotsidentified, 42 LMW glutenin spots were present among all threecultivars, labeled by numbers from 11 to 52 (Table 2, Fig. 4A–C).

In order to investigate the expression patterns of individualLMW glutenin subunits in the three cultivars during five graindevelopmental stages, the 147 protein spots were classified intosix expression types according to the accumulation partern. Theseresults are summarised in Table 2 and Fig. 5. The proteins in patternI, the largest group including 79 protein spots, increased graduallyfrom 150 ◦Cd to 750 ◦Cd. The second largest group, pattern VI,included 71 protein spots and displayed a ‘∧’ shape accumulationpattern, which maintained a low level at 150 ◦Cd, reached a peakin a stage between 150 ◦Cd and 750 ◦Cd, and then decreased toanother minimum at 750 ◦Cd. Pattern IV, the third largest group,contained 58 protein spots. These proteins increased rapidly atthen early stages then decreased slowly, and finally increasedin amount up to 750 ◦Cd like an ‘N’ shape pattern. Pattern III,with 14 protein spots, and pattern V, with seven protein spots,accumulated like an ‘M’ and ‘V’, respectively. The smallest group,pattern II, only had two protein spots and their accumulationgradually decreased from 150 ◦Cd to 750 ◦Cd.

Given the synthesis and accumulation patterns of individualLMW glutenin subunits between superior and poor gluten qualitycultivars, some possible relationships between particular subunitsand gluten quality could be deduced. Among all the proteinspots shown in Table 2, 11 protein spots were present in all threecultivars, but their expression amounts (vol%) in mature grains(750 ◦Cd) were different. For instance, the amounts of the spots 40and 47 in Jimai 20 were much higher than those in Zhoumai 16and Jing 411. In contrast, the other nine spots 14, 19, 20, 21, 26, 28,29, 39 and 50 were expressed in much lower amounts in Jimai 20than in Zhoumai 16 and Jing 411. Additionally, 16, 4 and 13 specificprotein spots were found to express in Jimai 20 (g32, g34, g39,g49, g55, g56, g61, g70, g77, g81, g100, g109, g114, g115, g126,g136), Zhoumai 16 (z61, z70, z87, z123) and Jing 411 (j74, j76, j82,j90, j103, j108, j112, j114, j118, j132, j139, j170, j189), respectively.These results suggested that two identical and 16 specific spots inJimai 20 might be related to good gluten property whereas nine

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Figure 2. RP-HPLC patterns of glutenins at five stages (150 ◦Cd to 750 ◦Cd) of grain development in three cultivars: Jing 411, Jimai 20 and Zhoumai 16.The x axis is the elution time (min) and the y axis is the UV absorbance areas at 210 nm (mAU). HMW-GS area, LMW-GS area and subunit compositionswere indicated. (The identification methods of HMW-GS and LMW-GS are the same as described by Dong et al.14).

Figure 3. Expression characteristics of glutenins at five developmental stages in three cultivars were compared by the result of RP-HPLC. (A) HMWglutenins; (B) LMW glutenins; (C) HMW/LMW glutenin ratios; (D) HMW-GS 12; (E) HMW-GS 7; (F) HMW-GS 1, 13, 4 and 16, 2 and 8, 2 and 9 (the HMW-GS 4and 16, HMW-GS 2 and 8, HMW-GS 2 and 9 can not be separated, so they were calculated together). The x axis is cumulative average temperatures (◦Cd)and the y axis (except in panel C) is the UV absorbance areas at 210 nm (mAU). Each sample was separated three times.

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Figure 4. Proteome mapping of three cultivars at the cumulative average temperature of 750 ◦Cd. (A) Jing 411, (B) Jimai 20, and (C) Zhoumai 16. A totalof 42 similar protein spots shared by three cultivars were labelled by number from 11 to 52, and specific protein spots of each cultivar were numberedwith a prefix: Jimai 20 with ‘g’, Zhoumai 16 with ‘z’ and Jing 411 with ‘j’.

identical and 17 specific protein spots expressed in Zhoumai 16and Jing 411 are likely to associate with poor gluten quality.

DISCUSSIONIn the current research, SDS-PAGE, RP-HPLC and 2-DE werecombined to analyse the accumulation patterns of both HMW-GS and LMW-GS. SDS-PAGE offers sound separation of HMW-GSbut can not provide quantitative information of different subunits.Two-dimensional electrophoresis is more efficient in separatingLMW-GS compared to SDS-PAGE. HPLC can provide quantitativeinformation for the accumulation of glutenin subunits. Theinformation provided by the three listed methods work together

and are complementary to each other to provide a whole pictureof the accumulation pattern of wheat glutenin.

Over the past decades, a large number of studies have beencarried out to investigate HMW-GS and their relationship withbread-making quality.29 – 31 Attention had also been paid tostorage protein accumulation during grain development15,32,33

and the influences of environmental conditions such as tem-perature and fertiliser on the composition and accumulation ofglutenins.34,35 However, knowledge of the relationship betweenthe accumulation of glutenin during grain development and thebread-making quality remains limited. Studies have revealed thatdifferent combinations of HMW-GS are closely related to the qual-ity of gluten such as good quality subunits 1Dx5+1Dy10 and poorquality subunits 1Dx2+1Dy12 as well as 1Bx20.8,36

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Table 2. Classification of expression patterns of LMW-GS in three cultivars during five grain developmental stages identified by two-dimensionalelectrophoresis∗

Common protein spots Specific protein spots

Pattern Jing 411 Jimai 20 Zhoumai 16 Jing 411 Jimai 20 Zhoumai 16

I 12,15,19,20,21, 12,15,18,19,21, 14,15,16,17,18, j74,j76,j82,j90, g32,g34,g39,g49, z61,z70,z87,z123

24,26,28,31,41, 22,24,25,26,27, 20,21,23,26,28, j103,j108,j112,j114, g55,g56,g61,g70,

47,49,52 31,32,44,48,49,52 30,32,33,41,47, j118,j132,j139,j170, g77,g81,g100,g109,

48,50 j189 g114,g115,g126,g136

II – 36 – – g131 –

III 17,18,45,51 14,23,30 – j80,j85,j99,j126 g40,g122,g97 –

IV 13,22,25,27,29, 20,29,38,40,43, 12,13,19,22,25, j87,j113,j121,j141, g43,g73,g84,g85, z38,z41,z132,z135

32,33,35,38,44, 45,47,50,51 34,36,37,39,42, j160,j164,j165,j177 g117,g127,g128,g129,

50 43,44,45,46,49, g137

51,52

V – 17,34,39,42 – – g46,g135 z125

VI 11,14,16,23,30, 11,13,16,28,33, 11,24,27,29,31, j65,j66,j71,j72, g42,g44,g59,g78, z65,z107,z115,z122,

34,36,37,39,40, 35,37,41,46 35,38,40 j89,j93,j98,j100, g82,g86,g91,g95, z129,z134

42,43,46,48 j106,j128,j130,j147, g108,g133

j150,j159,j166,j169,

j180,j181,j185,j186,

j187,j190,j193,j195

∗ A total of 147 protein spots, including 42 common and 105 specific protein subunits among three cultivars were identified using Image Master 2DPlatinum software version 5.0, and they were classified into six patterns (I, II, III, IV, V, VI) according to their expression curves from 150 ◦Cd to 750 ◦Cd.

In the past few decades, considerable efforts have focused onthe biochemical and molecular mechanisms by which gluteninallelic variations affect gluten quality. It is clear that the cysteineresidues present in N-termini (normally three) and C-termini (one)of HMW-GS help to form intermolecular disulfide bonds and thuscontribute to the creation of large polymers, which is important toprovide good viscoelastic properties to dough. An extra cysteineresidue present in 1Dx5 subunit might responsible for its goodquality property.37 Meanwhile, the subunit 1Bx20 which has twocysteine residues substituted by two tyrosines in the N-terminusmay actually have a negative effect on dough strength becausethe number of cross-links among glutenin polymers is effectivelydecreased.36 Additionally, beta-turns in HMW-GS have a positiveeffect on dough quality.38 A higher proportion of repeats of theconsensus type present in subunit 1Dy10 than subunit 1Dy12could produce a more regular pattern of repetitive β-turns inthe polymers.39 The central repeated domain is a major reasonfor variations in size difference of HMW-GS, which adopts aβ-spiral structure that confers elasticity to the protein molecule.4

Additionally, Gupta et al.2 found that variation in the accumulationrate of HMW-GS could lead to their quality differences. For instance,1Dx5+1Dy10 accumulated larger polymers more quickly than1Dx2+1Dy12.

In the present study, comparison of the glutenin accumulationamong superior gluten quality cultivar (Jimai 20) and poor glutenquality cultivars (Zhoumai 16 and Jing 411) showed that totalamounts of HMW-GS and LMW-GS as well as 1Bx13+1By16 and1Dx4+1Dy12 subunits in Jimai 20 accumulated faster than thoseglutenins in Zhoumai 16 and Jing 411 at the early stage of graindevelopment, suggesting that a higher glutenin accumulation rateat the early stage of development might contribute to superiordough quality. It is reported that glutamine synthetase, whichplays a major role in assimilating ammonia during remobilisation

of nitrogen to the grain,40 is expressed to a a higher level at theearly period of grain development (250 ◦Cd) in the good bread-making quality cultivar Sunstate than in poor gluten cultivar Jing411.25 Furthermore, results from our laboratory (unpublishingdata) indicate that protein disulfide isomerase (PDI), which isinvolved in the assembly of wheat storage proteins,41 also hada much higher expression level in the first two stages (150 ◦Cd

and 250 ◦Cd) of grain development, and then decreased sharplythrough to grain maturity. Hurkman et al.42 also found that PDIwas very abundant at grain early stage. The high expression ofglutamine synthetase and PDI at early stage development couldfacilitate the folding of gluten proteins and help to bring about theformation of more regular glutenin polymers, which might resultin superior gluten quality.

Some reports have shown that HMW/LMW glutenin ratios playan important role in the determination of the dough strength.43,44

Nevertheless, results in the present study revealed that theHMW/LMW glutenin ratio in Jimai 20 was between Zhoumai16 and Jing 411, and suggested no significant correlation betweenthe HMW/LMW glutenin ratios and bread-making quality. Thisis in agreement with previous reports.45,46 The reason could bethat the ratio of HMW-GS/LMW-GS mainly affects the extensibility.In this study the parameters measured by the Mixograph werecomprehensive gluten quality. Therefore its effect on extensibilitytested by the Extensiograph can be observed. It is worth notingthat the solvent used in our study to remove the gliadins mayalso remove certain amount of LMW-GS.47 However, the HMW-GS/LMW-GS ratios of all samples will be altered systematically,which will not affect the comparative results.

It is clear that, in addition to HMW-GS, LMW-GS also affectsdough strength significantly in common wheat.16,48 For instance,Glu-B3 plays a more important part in the determination of doughproperties than Glu-A3. Of all the alleles identified, Glu-A3d and

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Figure 5. Six expression patterns of 22 typical low molecular weight glutenin during developing grains of Jimai 20, Zhoumai 16 and Jing 411.

Glu-B3d have a larger effect on quality than do other alleles.49 Theallelic form of LMW-2 in durum wheat has been shown to relatewith the wheat’s superior pasta-making properties.11 However,the relationship between synthesis and accumulation patterns ofindividual LMW glutenin subunits in developing grains and glutenquality are not clear.

As shown in this work (Table 2), 147 LMW glutenin proteinsfrom three bread wheat cultivars were separated by 2-DE and theycould be divided into six different expression patterns during graindevelopment, which might reflect some possible correlations withgluten quality. Particularly, 16 specific protein spots expressed aspattern I in Jimai 20 could have positive effects on dough quality.Conversely, four and 13 specific LMW-GS expressed as patternI in Zhoumai 16 and Jing 411, respectively, may have negativeeffects on gluten quality. In addition, the protein spots 40 and 47,whose expression amounts at 750 ◦Cd in Jimai 20 was twice asmuch as Zhoumai 16 and almost six times of Jing 411, might bea candidate subunit for gluten quality improvement. Conversely,spots 14, 19, 20, 21, 26, 28, 29, 39 and 50 were expressed atlow levels in Jimai 20 at 750 ◦Cd, but were abundant in bothZhoumai 16 and Jing 411. Therefore, they could be related withthe poor bread-making quality of these two lines. Some proteinspots, such as g131 in pattern II, g40, g122 and g97 in pattern III,

g43, g73, g84, g85, g117, g127, g128, g129 and g137 in patternIV, and g42, g44, g59, g78, g82, g86, g91, g95, g108 and g133 inpattern VI in Jimai 20, displayed a similar expression pattern to thatseen with glutamine synthetase and protein disulfide isomerasediscussed above at early stage, which could improve synthesis andfolding of these subunits, and benefit the formation of superiorgluten polymers. Thus, they might contribute to the good bread-making quality of Jimai 20. Additionally, some reports showedthat late nitrogen supply,50 sulfur limitation51 or salinity stress52

all have negative effects on bread-making quality. The proteinexpression characteristics, especially in patterns III, IV, V and VI,suggest that fertilisation, watering and cultivation at early graindevelopment stages such as at 250 ◦Cd or 350 ◦Cd, can improveglutenin synthesis and accumulation as well as the formation ofsuperior gluten quality.

The modern progress in proteome research technology hasmade this research possible. In the past, a great deal of effort hasbeen focused on the study the effects molecular weight or sizedistribution of the polymers and total protein before reduction topolypeptides or individual protein molecule on dough quality bymainly using size-exclusion HPLC technology. Parameters such aspercentage of polymeric protein, percentage of gliadins, glutenin-to-gliadin ratio, polymeric protein in the flour, and percentage of

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unextractable polymeric protein are usually obtained to evaluatewheat quality.53 – 56 In the current study, we focused on a differentangle by conducting a detailed expression profiles analysis of eachglutenin protein and their relationship with dough quality. Theindividual LMW-GS expression and their effects on dough qualityhave never been reported in the past. Further studies should becarried out by analysing a wider selection if genotypes to verifyour findings.

ACKNOWLEDGEMENTSThis research was financially supported by grants from theNational Natural Science Foundation of China (30830072), theChinese Ministry of Science and Technology (2009CB118303)and the Key Developmental Project of Science and Technology,Beijing Municipal Commission of Education (KZ200910028003).We would like to thank Dr James Siedow, professor from theBiology Department, Duke University, for all the help with revisionand advice on the manuscript.

REFERENCES1 Wrigley CW, Giant proteins with flour power. Nature 381:738–739

(1996).2 Gupta RB, Masci S, Lafiandra D, Bariana HS and MacRitchie F,

Accumulation of protein subunits and their polymers in developinggrains of hexaploid wheats. J Exp Bot 47:1377–1385 (1996).

3 Singh Nk, Donovan GR, Batey IL and MacRitchie F, Use of sonicationand size-exclusion high-performance liquid chromatography in thestudy of wheat flour proteins. I. Dissolution of total proteins in theabsence of reducing agents. Cereal Chem 67:150–161 (1990).

4 Gianibelli M, Larroque O, MacRitchie F and Wrigley CW, Biochemical,genetic, and molecular characterization of wheat glutenin and itscomponent subunits. Cereal Chem 78:635–646 (2001).

5 Payne P, Genetics of wheat storage proteins and the effect ofallelic variation on bread-making quality. Annu Rev Plant Physiol38:141–153 (1987).

6 Shewry P, Tatham A, Barro F, Barcelo P and Lazzeri P, Biotechnologyof breadmaking: Unraveling and manipulating the multi-proteingluten complex. Biotechnology 13:1185–1190 (1995).

7 D’Ovidio R and Masci S, The low-molecular-weight glutenin subunitsof wheat gluten. J Cereal Sci 39:321–339 (2004).

8 Payne PI, Holt LM and Law CN, Structural and genetical studies on thehigh-molecular-weight subunits of wheat glutenin. Part 1: Allelicvariation in subunits amongst varieties of wheat (Triticus aestivum).Theor Appl Genet 60:229–236 (1981).

9 Gupta RB, Khan K and MacRitchie F, Biochemical basis of flourproperties in bread wheats. I: Effects of variation in the quantity andsize distribution of polymeric protein. J Cereal Sci 18:23–41 (1993).

10 Josephides CM, Joppa LR and Youngs VL, Effect of chromosome 1B ongluten strength and other characteristics of durum wheat. Crop Sci27:212–216 (1987).

11 Pena RJ, Zarco-Hernandez J, Amaya-Celis A and Mujeeb-Kazi A,Relationships between chromosome 1B-encoded glutenin subunitcompositions and bread-making quality characteristics of somedurum wheat (Triticum turgidum) cultivars. J Cereal Sci 19:243–249(1994).

12 Harberd NP, Bartels D and Thompson R, Analysis of the gliadinmultigene loci in bread wheat using nullisomic–tetrasomic lines.Mol Gen Genet 198:234–242 (1985).

13 Huang XQ and Cloutier S, Molecular characterization and genomicorganization of low molecular weight glutenin subunit genes atthe Glu-3 loci in hexaploid wheat (Triticum aestivum L.). Theor ApplGenet 116:953–966 (2008).

14 Dong K, Hao CY, Wang AL, Cai MH and Yan YM, Characterizationof HMW glutenin subunits in bread and tetraploid wheats byreversed-phase high-performance liquid chromatography. CerealRes Commun 37:65–72 (2009).

15 Shewry PR, Underwood C, Wan YF, Lovegrove A, Bhandari D, Toole G,et al, Storage product synthesis and accumulation in developinggrains of wheat. J Cereal Sci 50:106–112 (2009).

16 Maruyama-Funatsuki W, Takata K, Nishio Z, Tabiki T, Yahata E, Kato A,et al, Identification of low-molecular weight glutenin subunitsof wheat associated with bread-making quality. Plant Breed123:355–360 (2004).

17 Payne PI, Holt LM, Burgess SR and Shewry PR, Characterisation bytwo-dimensional gel electrophoresis of the protein components ofprotein bodies, isolated from the developing endosperm of wheat(Triticum aestivum). J Cereal Sci 4:217–223 (1986).

18 Bean SR, Bietz JA and Lookhart GL, High-performance capillaryelectrophoresis of cereal proteins. JChromatogrA 814:25–41 (1998).

19 Wang AL, Appels R, Ma JH, Xia XC, Lan P, He ZH, et al, A MALDI-TOFbased analysis of high molecular weight glutenin subunits for wheatbreeding. J Cereal Sci 50:295–301 (2009).

20 Wang AL, Gao LY, Li XH, Zhang YZ, He ZH, Xia XC, et al,Characterization of two 1D-encoded omega-gliadin subunitsclosely related to dough strength and pan bread-making qualityin common wheat (Triticum aestivum L.). J Cereal Sci 47:528–535(2008).

21 Yan YM, Jiang Y, Sun MM, Yu JZ, Xiao YH, Zheng JG, et al, Rapididentification of HMW glutenin subunits from different hexaploidwheat species by acidic capillary electrophoresis. Cereal Chem81:561–566 (2004).

22 Yan YM, Hsam SLK, Yu JZ, Jiang Y, Ohtsuka I and Zeller FJ, HMW andLMW glutenin alleles among putative tetraploid and hexaploidEuropean spelt wheat (Triticum spelta L.) progenitors. Theor ApplGenet 107:1321–1330 (2003).

23 Yan YM, Yu J, Jiang Y, Hu YK, Cai MH, Hsam SLK, et al, Capillaryelectrophoresis separation of high molecular weight gluteninsubunits in bread wheat (Triticum aestivum) and related species withphosphate-based buffers. Electrophoresis 24:1429–1436 (2003).

24 Zhang Q, Dong YM, An XL, Wang AL, Zhang YZ, Li XH, et al,Characterization of HMW glutenin subunits in common wheatand related species by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS). J Cereal Sci47:252–261 (2008).

25 Gao LY, Wang AL, Li XH, Dong K, Wang K, Appels R, et al, Wheat qualityrelated differential expressions of albumins and globulins revealedby two-dimensional difference gel electrophoresis (2-D DIGE).J Proteomics 73:279–296 (2009).

26 Singh NK, Shepherd KW and Cornish GB, A simplified SDS-PAGEprocedure for separating LMW subunits of glutenin. J Cereal Sci14:203–208 (1991).

27 Ikeda TM, Araki E, Fujita Y and Yano H, Characterization of low-molecular-weight glutenin subunit genes and their proteinproducts in common wheats. Theor Appl Genet 112:327–334 (2006).

28 Pang BS and Zhang XY, Isolation and molecular characterization ofhigh molecular weight glutenin subunit genes 1Bx13 and 1By16from hexaploid wheat. J Integr Plant Biol 50:329–337 (2008).

29 Butow BJ, Ma W, Gale KR, Cornish GB, Rampling L, Larroque O, et al,Molecular discrimination of Bx7 alleles demonstrates that a highlyexpressed high-molecular-weight glutenin allele has a major impacton wheat flour dough strength. Theor Appl Genet 107:1524–1532(2003).

30 Ma WJ, Appels R, Bekes F, Larroque O, Morell MK and Gale KR, Geneticcharacterisation of dough rheological properties in a wheatdoubled haploid population: additive genetic effects and epistaticinteractions. Theor Appl Genet 111:410–422 (2005).

31 Payne PI, Corfield KG and Blackman JA, Identification of a high-molecular-weight subunit of glutenin whose presence correlateswith bread-making quality in wheats of related pedigree. Theor ApplGenet 55:153–159 (1979).

32 Flint D, Ayers GS and Ries SK, Synthesis of endosperm proteins inwheat seed during maturation. Plant Physiol 56:381–384 (1975).

33 Stenram U, Heneen WK and Olered R, The effect of nitrogen fertilizerson protein accumulation in wheat (Triticum aestivum L.). Swedish JAgric Res 20:105–114 (1990).

34 Dupont FM and Altenbach SB, Molecular and biochemical impacts ofenvironmental factors on wheat grain development and proteinsynthesis. J Cereal Sci 38:133–146 (2003).

35 Johansson E, Prieto-Linde ML, Svensson G and Jonsson JO, Influencesof cultivar, cultivation year and fertilizer rate on amount of proteingroups and amount and size distribution of mono- and polymericproteins in wheat. J Agric Sci 140:275–284 (2003).

36 Shewry PR, Gilbert SM, Savage AW, Tatham AS, Wan YF, Belton PS,et al, Sequence and properties of HMW subunit 1Bx20 from pasta

wileyonlinelibrary.com/jsfa c© 2011 Society of Chemical Industry J Sci Food Agric 2012; 92: 106–115

11

5

Proteome dynamics of wheat glutenins in developing grains www.soci.org

wheat (Triticun durum) which is associated with poor end useproperties. Theor Appl Genet 106:744–750 (2003).

37 Anderson OD and Greene FC, The characterization and comparativeanalysis of high-molecular-weight glutenin genes from genomes Aand B of a hexaploid bread wheat. Theor Appl Genet 77:689–700(1989).

38 Tatham AS, Miflin BG and Shewry PR, The beta-turn conformation inwheat gluten proteins: Relationship to gluten elasticity. Cereal Chem62:405–412 (1985).

39 Flavell RB, Goldsbrough AP, Robert LS, Schnick D and Thompson RD,Genetic variation in wheat HMW glutenin subunits andthe molecular basis of bread-making quality. Nat Biotechnol7:1281–1285 (1989).

40 Bernard SM, Møller AL, Dionisio G, Kichey T, Jahn TP, Dubois F, et al,Gene expression, cellular localisation and function of glutaminesynthetase isozymes in wheat (Triticum aestivum L.). Plant Mol Biol67:89–105 (2008).

41 Shimoni Y, Zhu XZ, Levanony H, Segal G and Galili G, Purification,characterization, and intracellular localization of glycosylatedprotein disulfide isomerase from wheat grains. Plant Physiol108:327–335 (1995).

42 Hurkman WJ, Vensel WH, Tanaka CK, Whitehand L and Altenbach SB,Effect of high temperature on albumin and globulin accumulationin the endosperm proteome of the developing wheat grain. J CerealSci 49:12–23 (2009).

43 Gupta RB and MacRitchie FA, A rapid one-step one-dimensional SDS-PAGE procedure for analysis of subunit composition of glutenin inwheat. J Cereal Sci 14:105–109 (1991).

44 Uthayakumaran S, Stoddard FL, Gras PW and Bekes F, Effects ofincorporated glutenins on functional properties of wheat dough.Cereal Chem 77:737–743 (2000).

45 Dupuis B, Bushuk W and Sapirstein HD, Characterization of acetic acidsoluble and insoluble fractions of glutenin of bread wheat. CerealChem 73:131–135 (1996).

46 Sadouki H, Cazalis R and Azzout B, Fractionation of Algeriancommon wheat proteins by HPLC and sodium dodecyl sulfate–

polyacrylamide gel electrophoresis; relationship with technologicalquality. LWT–Food Sci Technol 38:829–841 (2005).

47 Cinco-Moroyoqui FJ and MacRitchie F, Quantitation of LMW-GS toHMW-GS ratio in wheat flours. Cereal Chem 85:824–829 (2008).

48 Tanaka H, Shimizu R and Tsujimoto H, Genetical analysis ofcontribution of low-molecular-weight glutenin subunits to doughstrength in common wheat (Triticum aestivum L.). Euphytica141:157–162 (2005).

49 He ZH, Liu L, Xia XC, Liu JJ and Pena RJ, Composition of HMW andLMW glutenin subunits and their effects on dough properties, panbread, and noodle quality of Chinese bread wheats. Cereal Chem82:345–350 (2005).

50 Johansson E, Oscarson P, Heneen WK and Lundborg T, Differences inaccumulation of storage proteins between wheat cultivars duringdevelopment. J Sci Food Agric 64:305–313 (1994).

51 Fitzgerald M, Ugalde T and Anderson J, Sulphur nutrition affectsdelivery and metabolism of S in developing endosperms of wheat.J Exp Bot 52:1519–1526 (2001).

52 Garg M, Tanaka H, Ishikawa N, Takata K, Yanaka M and Tsujimoto H,Agropyron elongatum HMW-glutenins have a potential toimprove wheat end-product quality through targeted chromosomeintrogression. J Cereal Sci 50:358–363 (2009).

53 Batey IL, Gupta RB and MacRitchie F, Use of size-exclusion high-performance liquid chromatography in the study of wheat flourproteins: An improved chromatographic procedure. Cereal Chem68:207–209 (1991).

54 Bean SR, Lyne RK, Tilley KA, Chung OK and Lookhart GL, A rapidmethod for quantization of insoluble polymeric proteins in flour.Cereal Chem 75:374–379 (1998).

55 Huebner FR and Wall JS, Fractionation and quantitative differencesof glutenin from wheat varieties varying in baking quality. CerealChem 53:258–269 (1976).

56 Dachkevitch T and Autran JC, Prediction of baking quality of breadwheats in breeding programs by size-exclusion high-performanceliquid chromatography. Cereal Chem 66:448–456 (1989).

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