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Journal of Cereal Science 56 (2012) 39e50

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Journal of Cereal Science

journal homepage: www.elsevier .com/locate/ jcs

Review

New insights into the effects of high temperature, drought and post-anthesisfertilizer on wheat grain development

Susan B. Altenbach*

USDA-ARS Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA

a r t i c l e i n f o

Article history:Received 20 September 2011Received in revised form9 December 2011Accepted 15 December 2011

Keywords:EnvironmentProteomicsTranscriptomicsWheat quality

Abbreviations: 2-DE/MS, two-dimensional gel eleetry; DPA, days post-anthesis; EST, expressed sequehyde-3-phosphate dehydrogenase; HMW-GS, highsubunit; HSP70, 70 kDa heat shock protein; LEA, laLMW-GS, low molecular weight glutenin subunit; LTmass spectrometry; MS/MS, tandem mass sgen:phosphorous:potassium fertilizer; PMF, peptide mquantitative reverse transcription polymerase chaitranscription polymerase chain reaction.* Tel.: þ1 510 559 5614; fax: þ1 510 559 5818.

E-mail address: susan.altenbach@ars.usda.gov.

0733-5210/$ e see front matter Published by Elsevierdoi:10.1016/j.jcs.2011.12.012

a b s t r a c t

Temperature, water and fertilizer have complex and interacting effects on wheat grain development,yield and flour quality. Transcript and protein profiling studies have provided insight into molecularprocesses in the grain and are now being used in conjunction with controlled growth experiments todecipher the effects of specific environmental variables on grain development. These studies arecomplicated because environmental treatments such as high temperature and drought shorten theduration of grain development and because effects of high temperature and drought on gene expressionand protein accumulation are superimposed upon those of post-anthesis fertilizer. The integration ofdata from recent proteomic and transcriptomic studies is an important step in identifying genes andproteins that respond to environment and affect yield and flour quality. Such information is needed todevelop wheat better able to adapt to global climate change.

Published by Elsevier Ltd.

1. Introduction

Climate model projections suggest that higher seasonaltemperatures will become commonplace in many parts of theworld by the end of the century (Battisti and Naylor, 2009).Periods of drought also are likely to become more frequent andmore severe. Wheat is one of the major crops grown in manydifferent environments worldwide and is an important source ofcalories for human nutrition. High temperatures and droughtalready limit wheat productivity in many parts of the world andrecent modeling studies indicate that increases in averagegrowing season temperatures are likely to cause further reduc-tions in wheat grain production, potentially threatening globalfood security (Asseng et al., 2011). Environmental factors alsoaffect wheat flour quality and thus are a major concern for end-users of wheat.

ctrophoresis/mass spectrom-nce tag; GADPH, glyceralde-molecular weight gluteninte embryogenesis abundant;P, lipid transfer protein; MS,pectrometry; NPK, nitro-ass fingerprinting; qRT-PCR,

n reaction; RT-PCR, reverse

Ltd.

Relationships between the environment and yield and quality ofwheat are complex (Triboï and Triboï-Blondel, 2002). In the field,unfavorable environmental conditions can occur at any time in thelife cycle of the wheat plant, can vary in intensity and duration, andcan affect diverse processes during either vegetative or reproduc-tive phases of the plant. The plant has devised numerous mecha-nisms to cope with environmental stress with the ultimate goal ofproducing a viable seed. These mechanisms involve changes at themolecular, cellular and physiological levels that vary with genotypeand are further influenced by the nutritional status of the plant.Ultimately, changes are manifested in the grain where they influ-ence the accumulation of starch and protein. Starch is a majorcomponent of the grain and is the most important factor for yieldwhile both the amount and composition of protein are critical forquality.

Several excellent reviews discuss the effects of high tempera-tures and drought onwheat and other cereals (Barnabás et al., 2008;Dupont and Altenbach, 2003; Yang and Zhang, 2006). Controlledenvironment studies have been essential for unraveling the effectsof individual environmental factors on wheat grain development.They also have made it possible to utilize proteomic and tran-scriptomic approaches to evaluate the effects of environment onproteins and genes that may be involved in determining yield andflour quality. However, each study provides only a small glimpse ofa complex picture.While other reviews in this issue provide updateson the effects of environment on starch production and the forma-tion of glutenin polymers in the grain, this review focuses on results

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e5040

from recent proteomic and transcriptomic studies aimed at uncov-eringmolecularmechanisms in the responseof thedeveloping grainto temperature, water and fertilizer.

2. Wheat grain development

The mature wheat grain consists of the endosperm that makesup about 89% of the grain, the embryo that comprises less than 3%,and various outer layers of dead maternal tissues that make upabout 9% of the grain (Evers and Millar, 2002). The endospermprovides most of the commercial value of the wheat grain andconsists of the starchy endosperm, in which starch granules areembedded in a matrix of protein, surrounded by the aleurone,a single cell layer. Wheat grain development is a complex processthat involves coordination among a number of different tissues(reviewed by Evers and Millar, 2002). Grain development is oftendescribed on the basis of morphological characteristics (Rogers andQuatrano,1983). However, to relate genes and proteins to processesthat occur during grain development, it is perhaps simpler toconsider wheat grain development in three stages: cell division,cell expansion/filling and desiccation. Grain development beginswith fertilization followed by a period of rapid cell division anddifferentiation into the endosperm, embryo and testa. The endo-sperm cells then expand and begin to accumulate large quantitiesof starch and protein. Finally, protein and starch deposition cease,the grains undergo a period of maturation and rapidly lose mois-ture, and the endosperm tissue undergoes apoptosis.

3. Effects of environment on the timing of grain development

Understanding how environmental treatments affect the timingof developmental processes in the grain provides an essentialframework for determining molecular changes that occur asa result of environment. Several recent controlled environmentstudies, each comparing a number of different environmentaltreatments, illustrate how high temperatures and drought accel-erate grain development.

Altenbach et al. (2003) evaluated grain development in thespring wheat cv. Butte 86 under three different temperature regi-mens. They defined physiological and molecular transition pointsthat allowed them to demonstrate that high temperatures and hightemperatures combined with drought advanced and compressed

Fig. 1. Duration of grain development under different temperature and water regimens. In edenote grain development under drought conditions. Temperature and drought treatmentsvarious 13-day periods during grain development. The times from anthesis to harvest maturipanel C. Data in panel A is summarized from Altenbach et al. (2003), B from Shah and Pau

the timing of key events during grain development. Under eachregimen, the onset and cessation of protein and starch accumula-tion, the time ofmaximumwater content,maximumdryweight andharvest maturity and the onset of apoptosis were used as develop-mental benchmarks. The time from anthesis to harvest maturityspanned 44 days under a 24/17 �C day/night regimen, 35 days undera 37/17 �C regimen and 26 days under a 37/28 �C regimen (Fig. 1A).The imposition of drought stress under a 37/17 �C regimen furthershortened thedurationof graindevelopment from35to28days. Theapplication of post-anthesis fertilizer affected the accumulation ofprotein in the grain, but did not influence the duration of graindevelopment under any of the temperature regimens.

Shah and Paulsen (2003) compared grain development in thespring wheat cv. Len under three temperature regimens with andwithout drought stress applied from one week after anthesis tomaturity. As observed in the previous study, increased tempera-tures decreased the duration of grain development. The time fromanthesis to harvest maturity was 47 days under a 15/10 �C regimen,31 days under a 25/20 �C regimen and 23 days under a 35/30 �Cregimen when plants were well watered (Fig. 1B). Drought alsoshortened grain development, but had the most dramatic effectunder the low temperature regimen. The duration of grain fillingwas reduced to 26 days under a 15/10 �C regimen, 24 days undera 25/20 �C regimen and 21 days under a 35/30 �C regimen underdrought conditions.

While the previous studies applied environmental treatmentsfrom anthesis to harvest maturity, Gooding et al. (2003) evaluatedthe effects of drought stress applied during specific stages of graindevelopment. The winter wheat cv. Hereward was subjected todrought stress from 1 to 14 days post-anthesis (DPA), 15 to 28 DPAand 29 to 42 DPA at 23/15 �C, corresponding roughly to the celldivision, cell expansion/grain filling and maturation phases,respectively, and the time to 37% moisture content was measured.The effects of drought on the timing of grain development weremost pronounced when the stress occurred during the first 14 daysafter anthesis (Fig. 1C). When drought was combined with a 28/20 �C heat treatment from 15 to 28 DPA, the effects were additive.High temperatures shortened the time to the end of grain filling by4.5 days relative to the 23/15 �C regimen andwater stress reduced itby another 1.1 days.

Changes in the timing of grain development in response toenvironmental factors have also been documented using

ach panel, solid lines denote grain development with adequate water and dashed lineswere applied from A) anthesis to maturity B) one week after anthesis to maturity or C)ty are shown in panels A and B while the times to 37% moisture content are indicated inlsen (2003), and C from Gooding et al. (2003).

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e50 41

microscopy. Hurkman andWood (2011) examined changes in grainmorphology that resulted from a high temperature treatment (37/28 �C) applied from anthesis to maturity. Light and scanning elec-tron micrographs demonstrated that endosperm cells at varioustime points under a 24/17 �C regimen were structurally similar tothose at much earlier time points under a 37/28 �C regimen.Morphological changes resulting from drought treatment werereported by Fábián et al. (2011) in drought-tolerant and drought-sensitive winter wheat varieties. Overall, a 5-day drought treat-ment between 5 and 9 DPA at 23/14 �C accelerated grain devel-opment by 10 days in both varieties. During drought stress andimmediately following, embryos were significantly greater in sizein drought-treated samples than in controls. However, due to theshortened duration of development, the embryos from drought-treated grain were actually smaller at maturity. The developmentof aleurone cells and the degradation of cell layers surrounding theovule also were accelerated in drought-treated kernels.

Alterations in the timing of grain development make it chal-lenging to detect changes in gene expression and protein accu-mulation that result from environmental factors. While thermaltime measured in degree-days sometimes is used rather thanchronological time to define equivalent developmental stagesunder different temperature regimens, similar methods are notavailable to account for developmental changes due to drought. It isthus necessary to assess effects of environmental treatments on thetiming of grain development in each new study.

4. Proteomics approaches for studying effects ofenvironment on grain development

Proteomics approaches such as two-dimensional gel electro-phoresis/mass spectrometry (2-DE/MS) have emerged as powerfulmethods to identify and quantify the large number of proteins inbiological samples (reviewed by Finnie et al., 2011; Neilson et al.,2010; Skylas et al., 2005). In short, proteins are extracted froma tissue, cell type, or sub-cellular compartment, separated by 2-DE,and individual protein spots are quantified using specialized soft-ware. Spots are excised from the gel, digested with trypsin oranother protease and analyzed by mass spectrometry. The identityof the protein in the spot is obtained by matching the spectra totheoretical data generated in silico from a database of proteinsequences.

The wheat grain proteins present certain challenges for pro-teomic analyses. First, there is a wide range of protein abundancesin the grain. The wheat gluten proteins, consisting of gliadins andglutenins, comprise 70e80% of the protein. Thus, in total proteinextracts, the more abundant gluten proteins often mask many ofthe less abundant proteins. One way to circumvent this problem isto fractionate proteins on the basis of solubility (reviewed byHurkman and Tanaka, 2007). While this approach allows theidentification of proteins that are minor components of the flour, itintroduces problems in protein quantification since protein frac-tions are rarely pure and environmental treatments can alter thepartitioning of proteins. The gluten proteins also present uniquechallenges for proteomic analyses. The gliadins and gluteninsconsist of many closely related proteins with similar molecularweights and pIs and therefore overlap considerably in 2-DE.Additionally, it is difficult to distinguish individual gluten proteinsby MS.

A number of proteomic studies have focused on identifying thearray of proteins in developing wheat grains and flour. Skylas et al.(2000) used N-terminal microsequencing to identify 177 endo-sperm proteins at 17 DPA and 45 DPA. Vensel et al. (2005) used MSto identify more than 250 proteins in an albumin/globulin fractionfrom endosperm at 10 and 36 DPA, Balmer et al. (2006) identified

289 amyloplast proteins from 10 DPA endosperm, Amiour et al.(2003) identified amphiphilic proteins in 143 spots from wholegrain flour, Dupont et al. (2011) identified more than 230 proteinsin milled white flour and Salt et al. (2005) identified 42 proteinsfrom dough liquor. Mak et al. (2006) identified 347 proteins inwheat germ, while Jerkovic et al. (2010) focused on proteins in theperipheral layers of the grain and identified proteins in 35 spotsfrom the epidermis and hypodermis,119 spots from cross cells, tubecells, testa and nucellar tissue and 672 spots from aleurone. Severalstudies also determined the accumulation profiles of proteinsthroughout development in endosperm (Hurkman et al., 2009),peripheral grain layers (Tasleem-Tahir et al., 2011) or whole grain(Gao et al., 2009; Nadaud et al., 2010). These studies provideinsights into molecular processes that take place in various parts ofthe grain during wheat grain development and establish a detailedpicture of flour protein composition that is critical for under-standing flour quality.

Recently, a number of groups have used 2-DE/MS to determinethe effects of environment on the accumulation of protein in thegrain (Table 1). Eight studies examined the effects of temperature(Dupont et al., 2006a,b; Hurkman et al., 2009; Laino et al., 2010;Majoul et al., 2003, 2004; Skylas et al., 2002; Yang et al., 2011),two studies addressed the effects of drought (Hajheidari et al.,2007; Yang et al., 2011), one study looked at the combined effectsof temperature and drought (Sancho et al., 2008) and four studiesexamined the effects of fertilizer (Altenbach et al., 2011; Dupontet al., 2006a,b; Grove et al., 2009). A number of different wheatvarieties were used in these studies. In some cases, varietiesconsidered tolerant to a stress were compared with those consid-ered to be susceptible to the stress (Hajheidari et al., 2007; Skylaset al., 2002). The growth conditions of the plants also variedconsiderably. In most temperature studies, daytime and nighttimetemperatures of the treatments were reported. Daytime tempera-tures ranged from 18 �C to 24 �C for control treatments and32 �Ce40 �C for high temperature treatments. The length of thetreatment and the timing during grain development also varied.In several studies, short periods of high temperature wereapplied early in grain development. Skylas et al. (2002) examineda 3-day heat treatment from 15 to 17 DPA, Laino et al. (2010) useda 5-day treatment from 5 to 10 DPA and Yang et al. (2011) evaluateda 10-day period of heat stress between anthesis and 10 DPA. Thehigh temperature treatments adopted by Hurkman et al. (2009)were more prolonged and extended from 10 DPA until maturitywhile the treatments used byMajoul et al. (2003, 2004) and Dupontet al. (2006a,b) were applied from anthesis to maturity. Sanchoet al. (2008) combined water deficits with elevated temperaturesfrom 15 DPA until maturity. Yang et al. (2011) applied awater deficitfrom anthesis and Hajheidari et al. (2007) limited the amount ofwater in field grown wheat from the booting stage throughoutgrain development. Grove et al. (2009) examined proteins thatchanged in response to fertilization with sulfur (S) while Dupontet al. (2006a,b) and Altenbach et al. (2011) focused on proteinsthat changed in response to nitrogen (N) fertilization. Most studiesassessed protein levels either late in development or at maturity.Only a few also compared accumulation profiles of proteinsthroughout grain development under the different treatments(Dupont et al., 2006a,b; Hurkman et al., 2009). Proteins fromwholegrain, endosperm, milled white flour or dough liquor wereanalyzed by 2-DE in the various studies. In addition, differentmethods of protein extraction were used. While some studiesanalyzed total protein fractions (Altenbach et al., 2011;Majoul et al.,2003), others focused on the albumin/globulin fraction (Hurkmanet al., 2009; Laino et al., 2010; Majoul et al., 2004; Sancho et al.,2008). Dupont et al. (2006a,b) focused on gluten proteins insol-uble in KCl, Grove et al. (2009) examined a glutenin fraction and

Table 1Summary of 2-DE/MS studies that identified proteins differentially accumulated as a result of specific treatments.

Reference Cultivara Treatment 1b Treatment 2b

Skylas et al., 2002 Wyuna, Fang 24 �C/18 �C 40 �C/25 �Cc

Majoul et al., 2003 Thesee 18 �C/10 �C 34 �C/10 �Cd

Majoul et al., 2004 Thesee 18 �C/10 �C 34 �C/10 �Cd

Dupont et al., 2006a,b Butte 86 24 �C/17 �C plus NPK fertilizationd,e 37 �C/28 �C plus NPK fertilizationd,e

Dupont et al., 2006a,b Butte 86 24 �C/17 �C minus NPK 37 �C/28 �Cd minus NPKSancho et al., 2008 Spark, Rialto, Soissons, Beaver 19.5 �C daily mean, wet 23.2 �C daily mean, dry (10% capacity)f

Hurkman et al., 2009 Butte 86 24 �C/17 �C plus NPK fertilizatione 37�C/28 �Cg plus NPK fertilizatione

Laino et al., 2010 T. durum ‘Svevo’ 20 �C/17 �Ch 37�C/17� Ch

Yang et al., 2011 Vinjett 20 �C/12 �Ci, then 24 �C/16 �Cj (cA-cB) 20�C/12 �Ci, then 32 �C/24 �Cj (cA-htB)Yang et al., 2011 Vinjett 20 �C/12 �Ci, then 24 �C/16 �Cj (cA-cB) 32 �C/24 �Ci, then 32 �C/24 �Cj (htA-htB)Yang et al., 2011 Vinjett 20 �C/12 �Ci, then 24 �C/16 �Cj (cA-cB) 20 �C/12 �Ci, then 24 �C/16 �C þ water deficitj (cA-wdB)Hajheidari et al., 2007 Arvand Field grown, irrigatedk Field grown, water deficitl

Hajheidari et al., 2007 Khazar-1 Field grown, irrigatedk Field grown, water deficitl

Hajheidari et al., 2007 Kelk Afghani Field grown, irrigatedk Field grown, water deficitl

Grove et al., 2009 Bastian Minus S fertilization Plus S fertilizationAltenbach et al., 2011 Butte 86 24 �C/17 �C minus NPK fertilizationd 24 �C/17 �C plus NPK fertilizationd,e

a T. aestivum unless otherwise noted.b Day/night temperatures are given unless otherwise noted.c From 15 DPA until 17 DPA.d From anthesis until maturity.e 300 mg/day 20-20-20 nitrogen:phosphorous:potassium (NPK) fertilizer.f From 14 DPA until maturity.g From 10 DPA until maturity.h From 5 to 10 DPA, then 25�C/20 �C until maturity.i At terminal spikelet.j From anthesis until 10 DPA.k Field temperatures, 75 þ/� 5 mm evaporation from boot stage.l Field temperatures, 150 þ/� 5 mm evaporation from boot stage.

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e5042

a Tris-soluble protein fraction and Yang et al. (2011) fractionatedproteins into albumins, globulins, gliadins and glutenins andanalyzed the albumin and gliadin fractions by 2-DE. Variousmethods were used for identification of proteins. Some of thestudies relied on peptide mass fingerprinting (PMF) for proteinidentification while others employed tandem mass spectrometry(MS/MS). The MS data may or may not be included with themanuscript. Databases used for the analysis of MS spectra alsodiffered among the studies, making it difficult to determinewhether the same protein or protein type was identified indifferent studies, particularly when spectra were matched tosequences from species other thanwheat or to expressed sequencetags (ESTs) or contigs from large EST databases. Although it ischallenging to compare data sets from proteomic studies that usedifferent cultivars, experimental treatments, source material andmethods, it is nonetheless valuable to consider the results of thedifferent studies together in hopes of discerning common threads.

4.1. Proteomic analysis of non-gluten proteins

The non-gluten proteins comprise only a small percentage of theprotein in the wheat grain, but many proteins within this diversegroup play critical roles in cellular metabolism, development andstress responses. Table 2 shows the identities of non-glutenproteins that increased or decreased in response to high tempera-tures, drought or fertilizer in the various 2-DE/MS studies.Hurkman et al. (2009) observed increases in individual proteinspots of up to 8.5-fold while Majoul et al. (2004) reported increasesas high as 19-fold when high temperatures were appliedthroughout much of grain development. In comparison, changesgenerally were less than 2.5-fold when high temperatures wereapplied early in grain development (Laino et al., 2010; Yang et al.,2011). Most changes in the levels of proteins in response todrought were less than 3-fold (Hajheidari et al., 2007; Yang et al.,2011) and changes in response to fertilizer generally were lessthan 4-fold (Altenbach et al., 2011). Of the 134 proteins noted in

Table 2, 46 showed changes in more than one study and 20 showedchanges in response to both temperature and drought.

It is perhaps not surprising that the majority of proteins thatshowed a response to high temperatures, drought or fertilizer areclassified as stress/defense proteins (Table 2). Of the 46 types ofstress/defense proteins that changed in these studies, 30 showeda response to high temperature, 17 showed a response to drought,18 responded to fertilizer and 15 were responsive to more than onetype of treatment. Monomeric and dimeric a-amylase inhibitors(WMAI, WDAI), 1-cys peroxiredoxin and multiple serpins showedresponses to high temperatures, drought and fertilizer, althoughresponses were not always the same to each stress. Dehy-droascorbate reductase, glyoxalase, several late embryogenesisabundant (LEA) proteins and xylanase inhibitor showed responsesto both high temperatures and drought. Tetrameric a-amylaseinhibitor WTAI-CM17, wheat a-amylase/subtilisin inhibitor (WASI),several chitinases, 27K lysosomal thiol reductase and 9 kDa lipidtransfer protein (LTP) showed responses to both high temperaturesand fertilizer and tetrameric a-amylase inhibitor WTAI-CM3showed a response to both drought and fertilizer. The a-amylaseinhibitors may help preserve grain starch, serpins may play roles ininhibiting proteases involved in storage protein breakdown, andLEA proteins are involved in seed desiccation. All are complexgroups of proteins. A number of other proteins, 1-cys peroxir-edoxin, dehydroascorbate reductase and 27K lysosomal thiolreductase, play roles in minimizing the effects of reactive oxygenspecies induced by stress.

Twenty proteins involved in carbohydrate metabolism alsoshowed significant changes (Table 2). Nineteen proteins showeda response to high temperature, six responded to drought and threeresponded to fertilizer. b-amylase as well as glucose and ribitoldehydrogenase showed responses to high temperatures, droughtand fertilizer. Both are involved in glucose degradation. Glyceral-dehyde-3-phosphate dehydrogenase (GADPH), involved in glycol-ysis, and granule-bound starch synthase responded to both hightemperatures and drought, and triosephosphate isomerase, also

Table 2Summary of non-gluten proteins identified in various 2-DE/MS studies that showed significant changes in relative abundance in response to temperature, drought or fertilizertreatments. þ indicates that spot increased, - indicates that spot decreased and þ/� indicates that one spot in a single study increased while another decreased. If more thanone spot of a particular protein type increased or decreased, the number of spots that showed the change is indicated. Proteins that showed changes inmore than one study arehighlighted in gray. Different treatments in Yang et al. (2011) and different genotypes in Hajheidari et al. (2007) are considered as separate studies.

Protein Typea Temperature Fertilizer

Drought

2-DE/MS Studyb

A B Cc D E F G H I J K L M

Stress/Defense proteinsa-amylase inhibitor, monomeric (WMAI) þ þ/� �2 e þ �2a-amylase inhibitor, dimeric (WDAI) þ þ þ2 þ �3a-amylase inhibitor, tetrameric

CM1 (WTAI-CM1)e þ e

a-amylase inhibitor, tetramericCM2 (WTAI-CM2)

þ �2

a-amylase inhibitor, tetramericCM3 (WTAI-CM3)

e �2 e þ2 þ2 �2

a-amylase inhibitor, tetramericCM16 (WTAI-CM16)

�2

a-amylase inhibitor, tetramericCM17 (WTAI-CM17)

þ þ �2

a-amylase/subtilisin inhibitor,endogenous (WASI)

þ þ e

Subtilisin/chymotrypsin inhibitor (WSCI) þChymotrypsin inhibitor (WCI) e

Protease inhibitor (CMx) �21-cys peroxiredoxin þ þ e þ e e þ27K protein, lysosomal thiol reductase þ e þ60 kDa jasmonate-induced protein þB15C protein þBarwin/pathogenesis-related protein

4/wheatwinþ

Calmodulin e

Catalase þChitinase þ3 �2Cinnamoyl-coA reductase þCold regulated protein e þ2Cystatin e

Dehydroascorbate reductase e e þFlavonoid 7-O-methyltransferase-like þGlutathione-S-transferase e e þGlycosyl hydrolase e

Glyoxalase I þ e e

Late embryogenesis abundant protein (LEA) þ þ2 þ2 þ �2Lipid transfer protein (LTP) þ e

Lipoxygenase e

Manganese superoxide dismutase þPeroxidase -(þ)Phosphoinositide-specific phospholipase C þPictinesterase þPuroindoline b e

Purple acid phosphatase e

RPM1-like protein þSelenium binding protein þSerpin �3 þ4 þ þ/� þ2 þ7Spermidine synthase 1 þThaumatin-like protein e

Thioredoxin h �2 �2 þ/�cd02947, thioredoxin family e

Translationally controlled tumor protein þTritin þ/�Xylanase inhibitor protein I þ e e

Carbohydrate metabolismPhosphoglycerate kinase e þ6-phosphogluconate dehydrogenase þADP-glucose pyrophosphorylase, LS �2(�3)Aldehyde dehydrogenase e

Aldolase e

Aldose reductase þ e

b-amylase þ þ þ/� e

Enolase þFructose-6-phosphate 1-phosphotransferase e

Fructose-6-phosphate-2-kinase þ(continued on next page)

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e50 43

Table 2 (continued )

Protein Typea Temperature Fertilizer

Drought

2-DE/MS Studyb

A B Cc D E F G H I J K L M

Glyceraldehyde-3-phosphatedehydrogenase (GAPDH)

þ þ5 þ þ e

Glucose and ribitol dehydrogenase þ/� þ3 e e

Glucose-1-phosphate adenyltransferase e e

Glucose-6-phosphate isomerasecytosolic B

þ

Granule-bound glycogen starch synthase þ þGranule-bound starch synthase þ þ e

Phosphopyruvate hydratase e

Pyruvate phosphate dikinase e

Triosephosphate isomerase (þ) e e

UDP-glucose pyrophosphorylase e

Protein synthesisHeat shock protein 17 þ (þ) þ4 þHeat shock protein 17.3 þHeat shock protein 26 þHeat shock 22 kDa protein þHeat shock 70 kDa protein þ �2(þ2) þ2 þ �4Heat shock associated protein þ/�Heat shock cognate protein 80 þHeat shock protein 80 kDa þ2Heat shock protein 81-1 þ2Heat shock protein 82 þChloroplast small heat shock protein þ þ340S ribosomal protein þ e

60S ribosomal protein L12 þElongation factor EF-TU þElongation factor 1-beta e

Elongation factor 2 e

Elongation factor EF1A e

Elongation factor-1-alpha 3 þ þEucaryotic translation initiation factor 4E þMethionine synthase e

Protein disulfide isomerase e e e �3Ribosomal protein S2 þSerine carboxypeptidase III e

Storage proteinsEmbryo globulin/globulin

Beg 1/globulin 3 (Glo 3)þ4 þ2,- �2 þ þ

Globulin-2 þ7 þ8,- e �3 �2Legumin-like protein -(þ) e

Seed globulin/19 kDa globulin/globulin 1 þ(þ4) þ þ e

Triticin (�)Farinin/avenin N9/avenin-like b e þ �6Purinin �6ATP interconversionATP synthase, b-chain �4 �2Vacuolar ATP synthase þ þ2Nucleoside diphosphate kinase (NDPK) þ þNitrogen metabolism2-isopropylmalate synthase þAcetohydroxyacid synthase e

Adenosyl-methionine synthetase 2 e

Alanine transaminase þFormyltetrahydrofolate synthetase e

Glutamate-cysteine ligase, chloroplastic e

Glutamine synthetase e

Ketol acid reductoisomerase e þMetabolismCarboxymethylene butenlidase þIsocitrate lyase þIsoflavone reductase e

Methylmalonate semialdehydedehydrogenase

e þ2 e

NADH dehydrogenase subunit J þPhospho-2-dehydro-3-deoxyheptonate

aldolaseþ

Phytochrome B þRibulose biphosphate carboxylase/

oxygenase activaseþ

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e5044

Table 2 (continued )

Protein Typea Temperature Fertilizer

Drought

2-DE/MS Studyb

A B Cc D E F G H I J K L M

Ribulose biphosphate carboxylaseLS binding protein

þ

Single-stranded nucleic acid-bindingprotein

e

Small Ras-related GTP-binding protein e

Succinyl-coA ligase b-chain e

Dihydroflavonol-4-reductase þ2Other14-3-3 homolog e e e

Guanine nucleotide-binding protein e

GTP-binding nuclear protein RNA 1B þ20S proteasome a-subunit þ/�26S proteasome e

Cell division control protein 4B þPoly(A)-binding protein �2Embryo-specific protein þ þ þ þHypothetical protein with enolase domain þIFR homolog þPuroindoline �2Reversibly glycosylated polypeptide þRPM1-like protein þSte20-related protein e

a May include proteins with more than one accession number.b Data are summarized from: A, Majoul et al., 2003; B, Majoul et al., 2004; C, Hurkman et al., 2009, 10 DPA treatment; D, Laino et al., 2010; E, Yang et al., 2011, cA-htB

treatment; F, Yang et al., 2011, htA-htB treatment; G, Sancho et al., 2008; H, Yang et al., 2011 cA-wdB treatment; I, Hajheidari et al., 2007, drought-sensitive genotypeKelk Afghani; J, Hajheidari et al., 2007, drought-sensitive genotype Arvand; K, Hajheidari et al., 2007, drought-tolerant genotype Khazar-1; L, Grove et al., 2009; M, Altenbachet al., 2011.

C Transient changes during grain development are indicated in parentheses.

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e50 45

involved in glycolysis, showed a response to both temperature andfertilizer.

Another important group of proteins that showed significantchanges in response to high temperatures and drought were theheat shock proteins which are molecular chaperones involved inprotein folding and assembly. Skylas et al. (2002) showed thata variety of isoforms of small heat shock proteins that range inmolecular mass from 15 to 30 kDa increased immediately followinga 3-day heat treatment from 15 to 17 DPA and that they weredifferentially expressed in the heat-susceptible cultivar Wyuna andthe heat-tolerant cultivar Fang. Hurkman et al. (2009) also reporteda transient increase in a small heat shock protein in response tohigh temperatures (Table 2). Small heat shock proteins alsoincreased in mature grain exposed to high temperatures (Lainoet al., 2010; Majoul et al., 2004) or drought (Hajheidari et al.,2007). In comparison, a 70 kDa heat shock protein (HSP70)showed a variable response to high temperatures. HSP70 showeda transient increase during grain development (Hurkman et al.,2009) and an increase in mature grain in three studies (Lainoet al., 2010; Majoul et al., 2003; Yang et al., 2011). However,decreases in two HSP70 spots in mature grain in response to hightemperatures (Hurkman et al., 2009) and four HSP70 spots inresponse to drought (Hajheidari et al., 2007) also were reported. Arange of other heat shock proteins of w80 kDa increased inresponse to high temperatures (Majoul et al., 2003, 2004). Anotherprotein involved in protein folding, protein disulfide isomerase,decreased in response to both high temperatures and drought(Hajheidari et al., 2007; Hurkman et al., 2009).

Although not previously thought to be involved in stressresponses, a variety of non-gluten storage proteins also showedresponses to the treatments (Table 2). Multiple spots on 2-DE gelswere identified as embryo globulins (also referred to as globulinBeg 1 or globulin 3) and globulin-2 proteins. These two types ofglobulins have distinct sequences but both contain barrel-like

regions referred to as cupin domains that are typical of the major7S and 11S seed storage proteins from dicotyledonous plants. Fourspots identified as embryo globulins increased in response to hightemperature in the Hurkman et al. (2009) study and two spotsincreased in the Laino et al. (2010) study. It is notable that theembryo globulins showed some of the largest changes in abun-dance in both studies. Embryo globulins also increased in responseto drought (Sancho et al., 2008; Yang et al., 2011), but showed anopposite response to temperature in the Yang et al. (2011) study.The closest wheat sequences to the embryo globulins are thoseencoded by the Glo 3 genes (Loit et al., 2009). Seven spots identifiedas globulin-2 proteins increased in response to high temperaturesin the Hurkman et al. (2009) study, while eight globulin-2 spotsincreased and one spot decreased in the Laino et al. (2010) study. Incomparison, Yang et al. (2011) reported decreases in one globulin-2spot in response to high temperatures and decreases in threeglobulin-2 spots in response to drought. Embryo globulins andglobulin-2 proteins are found in multiple spots in both starchyendosperm (Hurkman et al., 2009; Vensel et al., 2005) and milledwhite flour (Dupont et al., 2011). However, the picture is even morecomplex in the aleurone. Jerkovic et al. (2010) found that more thanhalf of the 606 spots identified in aleurone were globulins and thatthese represented nearly 75% of the aleurone protein. Tasleem-Tahir et al. (2011) also identified a large number of spots as glob-ulins in peripheral layers of the grain late in grain development. It isinteresting that immunolocalization experiments demonstratedthat globulins encoded by the Glo 3 gene accumulate in the embryoas well as the aleurone (Loit et al., 2009). Yet, globulins were notfound among the 347 spots identified in wheat germ (Mak et al.,2006). More than 40% of the proteins characterized by PMF in theMak et al. (2006) study did not yield valid identifications, so it maybe that good sequence matches were not present in the databasesused to analyze spectra. Another storage protein, referred to as seedglobulin or 19 kDa globulin, contains a domain typical of a-amylase

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e5046

inhibitors and 2S seed storage proteins, referred to as an AAI-SSdomain. The gene encoding this globulin was described by Guet al. (2006). One spot identified as seed globulin increased latein development in response to high temperatures in the Hurkmanet al. (2009) study while four spots showed transient increases. Thesame protein increased in response to drought in two drought-sensitive genotypes and decreased in a drought-tolerant geno-type (Hajheidari et al., 2007).

Other proteins that changed with the different treatmentsinclude 14-3-3 protein, a regulatory protein, and subunits of ATPsynthase (Table 2). A protein referred to as embryo-specific proteinthat has not been characterized in wheat but is similar to a riceprotein also increased in response to temperature (Hurkman et al.,2009; Laino et al., 2010) and to drought (Yang et al., 2011;Hajheidari et al., 2007). These proteins also may warrant furtherinvestigation.

It is interesting that a number of proteins that showed anincrease in response to high temperatures in the cultivar Butte 86showed a decrease in response to post-anthesis fertilizer in thesame cultivar (Altenbach et al., 2011; Hurkman et al., 2009). Theseinclude WASI, chitinase, LTP, globulin-2, glucose and ribitol dehy-drogenase, b-amylase and triosephosphate isomerase. By contrast,serpin decreased with high temperature in Butte 86 but increasedwith post-anthesis fertilizer. In the Hurkman et al. (2009) study,plants received 300 mg per day NPK fertilizer. These data supportthe notion that post-anthesis fertilizer might modulate some of theeffects of high temperatures and suggest that it is essential toconsider the nutritional status of the plant in assessing effects ofother stresses. In several of the studies (Sancho et al., 2008; Yanget al., 2011) fertilizer was applied only at sowing while the fertil-ization of the plants was not reported in other studies.

In the study of Hajheidari et al. (2007), five of the 12 proteinsthat increased with drought in a drought-tolerant genotype hadopposite responses in at least one drought-sensitive genotype.These included tetrameric a-amylase inhibitor WTAI-CM3, coldregulated protein, dehydroascorbate reductase, glutathione-S-transferase, and thioredoxin h. Five proteins that decreased withdrought in the tolerant genotype had opposite responses in at leastone of the sensitive ones, including 1-cys peroxiredoxin, 19 kDaglobulin, GAPDH, granule-bound starch synthase and methyl-malonate semialdehyde dehydrogenase. It is thus possible thatthese proteins play key roles in the drought response.

It is notable that many of the proteins identified in these studiesare likely allergens. Various a-amylase inhibitors, GADPH, tri-osephosphate isomerase and serpin are involved in the inhalationtype allergy referred to as bakers’ asthma while LTP, chitinase,WTAI-CM3, serpin, peroxidase and thaumatin-like protein aredesignated as food allergens in the Allergome database (http://www.allergome.org/). Globulin-2 and the pathogenesis-relatedprotein barwin/wheatwin also have substantial similarity toknown food allergens in other plants (Altenbach et al., 2008, 2009).Both globulin-2 and wheatwin recently were shown to react withsera from patients with food allergies (Larré et al., 2011; Sotkovskyet al., 2008, 2011). Embryo globulin/globulin 3 also has beenidentified as a potential food allergen (Larré et al., 2011) and hasbeen shown to be associated with type-1 diabetes (MacFarlaneet al., 2003). The data thus suggest that the growth environmentmight alter the allergenic potential of the flour.

A number of factors should be considered when interpretingproteomic studies of the wheat grain proteins. Several studies haveshown that the same protein sequence may be associated withmultiple 2-DE spots with different pIs and molecular masses(Dupont et al., 2011; Gao et al., 2009; Mamone et al., 2009; Yanget al., 2011). Indeed, 32 proteins in Table 2 were identified inmultiple spots in one or more of the studies. Multiple spots may

represent protein variants resulting from post-translational modi-fications, products of closely related genes that were not distin-guished by MS or charge trains of related proteins that differ onlyby small changes in net charge due to sample extraction or 2-DE(Dupont et al., 2011). Whatever the cause, it is important toconsider the combined responses of all spots associated witha single protein sequence to understand the effects of the treatmenton that protein. This was possible in the few studies wherecomprehensive 2-DEmapswere established (Altenbach et al., 2011;Hurkman et al., 2009). However, in most studies, only those spotsthat changed with a treatment were identified. As a result, the datarepresent only part of the picture.

In drawing conclusions from multiple proteomic studies, it alsois important to note that proteins in many spots that changed inresponse to environment were not identified, either because noattempt was made at MS analysis or because a valid identificationwas not obtained. For example, Laino et al. (2010) reported changesin the abundance of 132 spots with high temperature, but only 47were identified and Majoul et al. (2003) reported changes in 37spots, but only 26 were identified. Hajheidari et al. (2007) detectedchanges in 121 spots with drought, but only 57 were identified. Inaddition, 36 minor spots that changed with fertilizer were notidentified in the Altenbach et al. (2011) study. Some proteins aredifficult to identify by MS because they are of very low abundance.Other proteins may not cleave well with the proteases used for MSidentification or the resulting peptides may not have been detectedby the mass spectrometer. It is also possible that the correspondingprotein sequences were not present in the database searched withMS data. Fortunately, MSmethods are improving at a rapid rate andsequence databases are becoming more and more complete, so it islikely that future 2-DE/MS studies will yield a more completepicture of the proteins that change with environment.

4.2. Proteomic analysis of gluten proteins

The wheat gluten proteins are a complex group of abundantproteins that are themajor determinants of wheat flour quality. Thegluten proteins consist of the monomeric gliadins, separated into a/b-, g- andu-gliadins, and the polymeric glutenins, consisting of lowmolecular weight glutenin subunits (LMW-GS) linked by disulfidebonds to high molecular weight glutenin subunits (HMW-GS).Although a- and b-gliadins can be distinguished by acid electro-phoresis, both types of proteins fall into a single group on the basisof amino acid sequences and will be referred to here simply as a-gliadins. Only a few 2-DE/MS studies to date have considered theeffects of environment on the gluten proteins (Table 3). Majoul et al.(2003) identified three spots that increased 27.5-, 2.7- and 3.1-foldin response to high temperature as a-gliadins. Dupont et al.(2006a,b) studied the response of a sampling of proteins in eachgluten protein class during endosperm development and reportedincreases in HMW-GSs and a-gliadins and a decrease in a majorLMW-GS in response to temperature. Yang et al. (2011) reportedchanges in the relative amounts of 11 spots in a gliadin proteinfraction when high temperatures were applied from anthesis to 10DPA. Seven were identified as a-gliadins, one as a g-gliadin andthree as LMW-GSs. Ten of the spots increased from 1.5- to 1.9-foldwhile one spot identified as an a-gliadin decreased 2-fold. Whenhigh temperatures were applied from terminal spikelet to 10 DPA,eights spots associated with gluten protein sequences changedsignificantly. Four were identified as a-gliadins, two were g-glia-dins, one was an LMW-GS and one was an u-gliadin. One a-gliadinand an u-gliadin decreased while the other spots increased inresponse to the treatment. In the same study, when drought wasapplied, five spots identified as gluten proteins changed. One LMW-GS increased and one a-gliadin, one LMW-GS and two g-gliadins

Table 3Summary of gluten proteins that showed significant changes in relative abundance in response to temperature, drought or fertilizer treatments in 2-DE/MS studies.þ indicatesthat proteins increased, � indicates that proteins decreased, þ/� indicates that some proteins increased while other proteins in the class decreased.

High temperature Drought Fertilizer

Majoul et al., 2003 Dupont et al., 2006a,b Yang et al., 2011 Yang et al. 2011 Grove et al., 2009 Dupont et al., 2006a,b Altenbach et al., 2011

a-gliadins þ þ þ/� � þ þg-gliadins þ e þu-gliadins e þ þLMW-GS e þ þ/� þ e e

HMW-GS þ þ þ

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e50 47

decreased in response to the treatment. The identification of LMW-GSs in a gliadin fraction in the Yang et al. (2011) study illustrates thedifficulties in cleanly separating gliadins from glutenins. Dupontet al. (2006a,b) reported increases in u-gliadins, HMW-GS and a-gliadins and a decrease in a major LMW-GS in response to Nfertilization. Grove et al. (2009) reported increases in 13 spots anddecreases in seven spots from a glutenin fraction in response to Sfertilization, but only eight proteins yielded valid identifications. Ofthe proteins that increased, three were identified as LMW-GSs, fouras g-gliadins and one as avenin-like-b protein, more recentlyreferred to as farinin (Dupont et al., 2011). It is possible that certaing-gliadins partitioned into the glutenin fraction in this studybecause they contain an odd number of cysteine residues and thusfunction as LMW-GSs. However, it was not possible to associatespecific g-gliadin gene sequences with any of the proteins becauseMS coverages were very low, ranging from 6 to 12%. In fact, in all ofthe previous studies, identification of gliadins and LMW-GSs werebased on relatively few peptides. In addition, most gluten proteinsidentified in the Yang et al. (2011) study were associated withrelatively short ESTs that were either singletons or members ofcontigs that encoded incomplete proteins.

It is important to be able to distinguish individual glutenproteins because small changes in the sequences of proteins canalter their ability to assemble into large polymers and influenceflour quality. However, the wheat gliadins and glutenins areparticularly difficult to identify by MS. The proteins have highlyrepetitive sequences with an abundance of glutamine and prolineand yield few peptides when cleaved with trypsin, the enzymeused in most MS studies. Additionally, current sequence databasesdo not reflect the considerable heterogeneity that is found amongindividual proteins in different cultivars (Altenbach et al., 2010a,b;Mamone et al., 2005, 2009). Recently, Vensel et al. (2011) optimizedMS/MS methods for identification of gluten proteins by digestingeach protein with chymotrypsin and thermolysin in addition totrypsin, using improved search strategies for analysis of spectraldata, and including cultivar-specific sequences of gluten proteins indatabases searched with spectral data. These improvements madeit possible to significantly increase the MS/MS coverage of indi-vidual gluten proteins and to develop a 2-DE map of wheat flourproteins in the US wheat Butte 86 in which 22 LMW-GSs, 13 g-gliadins, and 23 a-gliadins were associated with specific genesequences (Dupont et al., 2011). For the a-gliadins, a particularlycomplex group of gluten proteins, MS coverages were as high as80%. The improved 2-DE map made it possible to evaluate theeffects of N fertilization on individual gluten proteins in flour(Altenbach et al., 2011). The study demonstrated that most u-gliadins and HMW-GS increased in response to post-anthesisfertilizer. Six of 23 a-gliadins also increased while significantdecreases were observed in three of 22 LMW-GSs, including oneLMW-GS that is the most abundant gluten protein in Butte 86 flour.Because N fertilization affects the accumulation of many glutenproteins, this study establishes an important baseline for futurework aimed at deciphering the effects of temperature and droughton the wheat gluten proteins.

5. Transcriptomic approaches for studying effects ofenvironment on grain development

Although the genome sequence of wheat is not yet complete,the availability of more than one million ESTs has made it possibleto use a variety of high-throughput methods to identify genes thatare differentially expressed during grain development (reviewed byLeader, 2005). Drea et al. (2005) surveyed the expression patternsof 888 genes in developing grain at 3, 6 and 9 DPA using in situhybridization. This study revealed genes involved in early stages ofgrain development and provided important information abouttheir tissue specificity. McIntosh et al. (2007) used serial analysis ofgene expression (SAGE) to identify differentially expressed genes atfive time points between 8 and 40 DPA and related the mostabundantly expressed genes to developmental processes.Laudencia-Chingcuanco et al. (2007) used cDNA microarrays toassess the expression of 7,835 genes at six time points between 3DPA and 35 DPA. Wan et al. (2008) developed detailed transcriptprofiles that included 10 time points between 6 and 42 DPA usingAffymetrix oligonucleotide arrays representing more than 55,000transcripts and Shewry et al. (2009) described transcript profiles forgliadin, glutenin and starch biosynthetic enzymes derived from theWan et al. (2008) array data. From these studies has emergeda picture of the molecular processes that take place during wheatgrain development. Understanding how environmental factors andagronomic inputs affect the expression of those genes is far moredifficult. Whereas most proteomic studies focused on the accu-mulation of proteins late in grain development or in flour, analysesof gene expression must be considered in a developmental context.This greatly increases the complexity of the analyses. Becauseenvironmental factors such as high temperature and drought alterthe timing of grain development, a major challenge of these studiesis to distinguish changes in gene expression due to the environ-mental treatment from effects on the timing of developmentalprocesses. To date, only a few groups have examined the effects ofhigh temperature or high temperature combined with drought onglobal gene expression (Table 4).

Altenbach and Kothari (2004) used semi-quantitative reversetranscription polymerase chain reaction (RT-PCR) to comparetranscript accumulation for 57 genes involved inmetabolism, signaltransduction, carbohydrate synthesis, protein synthesis, proteinturnover, and defense under two temperature regimens (Table 4).They determined the time at which protein and starch accumula-tion commenced and the times at which grains reached maximumwater contents, dry weights and harvest maturity under eachregimen and used these as developmental benchmarks to distin-guish changes in gene expression due to high temperature fromthose due to development. They showed that high temperatures(37/28 �C) applied from anthesis to maturity advanced andcompressed transcriptional programs within the grain. While thetiming of expression of most genes changed in a manner consistentwith developmental events, transcript profiles for a number ofgenes, including serpin, b-amylase, peroxidase, tritin and WTAI-CM3, were shifted under high temperatures.

Table 4Summary of studies that identified genes differentially expressed as a result of environmental treatments.

Reference Cultivara Treatment 1b Treatment 2b Analysis

Altenbach and Kothari 2004 Butte 86 24/17 �C 37/28 �Cc RT-PCRWan et al., 2008 Hereward 23/15 �C 28/20 �Cd 55k oligo array

23/15 �C 23/15 �C, water deficitd,e 55k oligo array23/15 �C 28/20 �C, water deficitd,e 55k oligo array

Szucs et al., 2010 Plainsman V 23/14 �C 23/14 �C, water deficitf 15k oligo array23/14 �C 34/24 �C, water deficitf 15k oligo array

Cappelle Desprez 23/14 �C 23/14 �C, water deficitf 15k oligo array23/14 �C 34/24 �C, water deficitf 15k oligo array

Chauhan et al., 2011 CPAN1676 20/20 �C 37 �C heat shockg Suppression subtractive hybridization20/20 �C 42 �C heat shockg Suppression subtractive hybridization

a T. aestivum.b Day/night temperatures are given unless otherwise noted.c From anthesis to maturity.d From 15 DPA until 28 DPA.e 44% field capacity.f From 1 to 5 DPA.g 2-h treatment at 7, 15, 21, 30 DPA.

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e5048

Wan et al. (2008) used 55k Affymetrix oligonucleotide arrays toevaluate the effects of high temperature (28/20 �C), drought, andhigh temperature combined with drought on gene expression(Table 4). In their experiments, treatments were applied from 15DPA until 28 DPA. Transcripts were assessed at only three timepoints for each environmental regimen, at 14 DPA prior to the stressand at 21 and 28 DPA. To identify genes affected by environmentaltreatments that were independent of general developmentaleffects, equivalent stages were determined by comparing tran-scriptomes from the three time points to those from a referencedata set of 14,550 differentially expressed transcripts. The referencedata set was developed by surveying gene expression at 10 timepoints from grain produced under an 18/15 �C regimen. Estimatesof developmental stages from transcriptomic data were in generalagreement with stages determined using the moisture content ofthe grain. All of the treatments accelerated development with hightemperatures combined with drought having the greatest effectand the drought treatment alone having the least effect. This studyrevealed surprisingly few genes that were significantly impacted bythe environmental treatments. Ribulose bisphosphate carboxylaseactivase was up-regulated in response to high temperature whilea cysteine proteinase, ethylene responsive transcriptional co-activator, heat shock protein 26.6 and histone were down-regulated in grain sampled at 21 DPA. Transcripts up-regulated at21 DPA due to drought included polygalacturonase, 1,3 b-glucanase,b-expansin, and nodulin while cytochrome P450 and Lt1.1 proteinwere down-regulated. While this study illustrates the validity ofthe approach, it is likely that more detailed transcript profilesincluding additional time points will be needed to identify otherdifferentially expressed genes.

A 15k oligonucleotide microarray was developed by Szucs et al.(2010) to investigate changes in gene expression that occurred inresponse to water deficit and water deficit combined with a 34/24 �C high temperature treatment during the first five days of graindevelopment in the drought-tolerant wheat Plainsman V and thedrought-sensitive wheat Cappelle Desprez (Table 4). At 5 DPA,water stress affected the expression of 69 and 71 of the genesexamined in the tolerant and sensitive genotypes, respectively,whereas 783 and 990 of the genes responded when plants weresubjected to both high temperatures and water stress. Few differ-ences were observed between the two genotypes. Genes that wereup-regulated under high temperatures plus drought included thoseencoding storage proteins and heat shock proteins, asparticproteases, tonoplast intrinsic proteins, and a range of proteinsinvolved in starch metabolism. Genes encoding several histonesand serine proteases were down-regulated. Twenty-two

transcription factors showed significant changes, but responseswere variable. However, most changes in gene expression wereconsistent with histological data that showed increased embryosize and accelerated accumulation of protein and starch in theendosperm in grain subjected to high temperatures and drought.Thus, it is essential to take changes in the developmental programinto account even when a short period of high temperature anddrought treatment is applied.

Chauhan et al. (2011) used subtractive hybridization coupledwith suppression subtractive hybridization to identify genes withaltered expression in developing grain following a short heat shocktreatment (Table 4). In their experiments, plants grown at 20 �C day/nightwere subjected to a 2-h heat shock treatment at either 37 �C or42 �C at 7, 15, 21 and 30 DPA. Many of the 95 up-regulated genesencoded proteins involved in stress responses, transcription, ormetabolic processes. A number of these genes correspond toproteins that showed changes late in grain development in proteo-mic studies. Among these were serpin, selenium binding protein,triosephosphate isomerase, translationally controlled tumorprotein, b-amylase, phosphofructokinase, elongation factor 1 alpha,several proteasome subunits, and a variety of heat shock proteins. Itis interesting that many of these genes remained up-regulated evenafter 1 or 4 days of recovery at 20 �C. Down-regulated genes wereinvolved in metabolic processes, particularly carbohydrate metab-olism. These included sucrose synthase, soluble starch synthase,phosphoglycerate kinase, starch branching enzyme, protein disul-fide isomerase, reversibly glycosylated polypeptide, and ADP ribo-sylation factor. Genes for several a-amylase inhibitors also weredown-regulated as were a number of genes for gluten proteins,including a-gliadins, LMW-GSs and a fewHMW-GSs and g-gliadins.The use of a heat shock treatment simplifies the analysis since geneexpression can be measured shortly after the stress. It may beinteresting to determine whether genes identified in this study alsorespond to more prolonged heat treatments.

Several other studies focused on specific groups of genes.Altenbach et al. (2002) and Dupont et al. (2006b) used hybridizationanalysis to determine the expression of genes encoding the majorgluten proteins. They demonstrated that transcripts from all of themajor familiesof glutenproteins accumulated ina coordinate fashionand that high temperatures (37/17 �C or 37/28 �C) and hightemperatures combinedwithdrought shifted the timingof transcriptaccumulation but did not affect the coordinate regulation of genesfrom different families. RT-PCR further showed that the timing oftranscript accumulation for individual genes within the HMW-GSand LMW-GS families was similar. However, more quantitativemethods are needed to assess the accumulation of transcripts for

S.B. Altenbach / Journal of Cereal Science 56 (2012) 39e50 49

individual genes within the complex gluten protein families in grainsubjected to different environmental treatments. This requiresa detailed understanding of the sequences of gluten protein genesexpressed in the cultivar (Altenbach et al., 2010a,b) so that shortgene-specific sequences can be identified for use as primers forquantitative RT-PCR (qRT-PCR) or for custom oligonucleotide arrays.

Many genes expressed in developing grain are part of multigenefamilies and further studies are required to identify individualmembers and to assess their expression under different environ-mental conditions. A few studies have provided more specificinformation about genes encoding proteins that responded to theenvironment in proteomic analyses. Ali-Benali et al. (2005) devel-oped primers that distinguished five genes encoding LEA proteinsand assessed their expression profiles in developing grain by qRT-PCR. Altenbach and Kothari (2007) used EST analysis to charac-terize families of genes encoding u-gliadins and examined theresponse of two types ofu-gliadins to post-anthesis fertilizer. Otherstudies focused on genes that encode LTPs,wheatwins and globulin-2 proteins and determined responses to high temperatures andpost-anthesis fertilizer (Altenbach et al., 2007, 2008, 2009). Thesestudies provide the foundation for functional analyses of specificgenes using transgenic approaches. Detailed knowledge about thegenes makes it possible to design RNA interference constructs tosilence the expression of specific genes in grain from transgenicplants (Fu et al., 2007) and to evaluate the importance of specificgenes in the response of the grain to environmental stress and theroles of the encoded proteins in flour quality.

6. Conclusions

Wheat adapts to temperature extremes anddroughtduring grainfilling byaccelerating and compressing thedevelopmental program,thereby maintaining the ability to produce a viable seed. However,thegrowthenvironment also influences yield aswell asflourquality.A number of studies within the last few years have provided newinsight into changes that occur in the proteome in response tovarious environmental factors. The picture that is emerging is thattemperature, drought and fertilizer cause numerous changes in theaccumulation of both non-gluten and gluten proteins, but most ofthe changes are relatively small. Among the non-gluten proteins,certain protein types, particularly those involved in stress/defenseand carbohydrate metabolism, were altered in several differentstudies. Heat shock proteins and globulins also changed innumerous studies. Until this point, it has generally been assumedthat the wheat globulins wereminor and relatively inconsequentialstorage proteins in the grain. However, the data suggest that theroles of the globulins should be studied further. Proteomic data onthe gluten proteins is more limited, in part because it has been verychallenging to identify these proteins by MS. However, recentimprovements in methods for the identification of gluten proteinsnow make it possible to identify changes in individual glutenproteins in response to temperature and drought and to relate themtochanges that result fromtheapplicationof post-anthesis fertilizer.Global analyses of gene expression have provided insight intomolecular processes that occur during grain development and areavaluable reference for studies aimed at uncovering changes in geneexpression that occur as a result of the growth environment.However, additional investments in transcriptomic studies areneeded to complement and extend the work summarized here.There is also a need to delve more deeply into the families of genesthat encode proteins identified in proteomic analyses and to inves-tigate their functions using transgenic approaches. This will equipscientists with new tools to develop cultivars better able to adapt toglobal climate change through conventional or marker-assistedbreeding or through biotechnology.

Acknowledgments

Thanks to Drs. William Hurkman and Frances Dupont for criticalreading of the manuscript. Mention of a specific product name bythe United States Department of Agriculture does not constitute anendorsement and does not imply a recommendation over othersuitable products. USDA is an equal opportunity provider andemployer.

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