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Journal of Cereal Science 56 (2012) 67e80

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Journal of Cereal Science

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

Effects of environmental factors on cereal starch biosynthesis and composition

Maysaya Thitisaksakul a, Randi C. Jiménez a, Maria C. Arias b, Diane M. Beckles a,*

aDepartment of Plant Sciences, University of California, One Shields Avenue, Davis, CA 95616, USAbUnité de Glycobiologie Structurale et Fonctionnelle, Université des Sciences et Technologies de Lille, Unité Mixte de Recherche du Centre National de la Recherche Scientifiqueno. 8576, 59655 Villeneuve D’Ascq cedex, France

a r t i c l e i n f o

Article history:Received 16 September 2011Received in revised form3 April 2012Accepted 6 April 2012

Keywords:EndospermStarchEnvironmentStress

Abbreviations: ABA, Abscisic acid; AGPase, ADP-glAluminum; AMY, a-amylase; BAM, b-amylase; CO2, Caanthesis; FACE, Free Air CO2 Enrichment; GBSS, GraGWD, Glucan water dikinase; Kcat, Enzyme catalyconstant; LSU, Large subunit; N, Nitrogen; PUL, Pphosphate dikinase; QTL, Quantitative trait loci; Smorphism; SSU, Small subunit; NaCl, Sodium chlorenzymes; DBEs, Starch debranching enzymes; PHOStarch synthases; SuSy, Sucrose synthase; TF, Transcglucose pyrophosphorylase; Wx, Waxy.* Corresponding author. Tel.: þ1 530 754 4779; fax

E-mail address: dmbeckles@ucdavis.edu (D.M. Bec

0733-5210/$ e see front matter � 2012 Elsevier Ltd.doi:10.1016/j.jcs.2012.04.002

a b s t r a c t

The aim of this review is to examine how the quantity and quality of starch in cereal endosperm isaffected by abiotic stress. This is important because starch is the primary food source for humans, and itsaccumulation in cereal endosperm is a fundamental component of yield. Grain yield; however, is con-strained under environmental stress with negative ramifications for agricultural productivity andsustainability. This is a significant and likely to be growing problem given that weather patterns arepredicted to become increasingly extreme. In this review, we first describe starch structure andbiosynthesis in the developing endosperm. Next, we outline how starch biosynthesis, content andcomposition are altered in response to drought, temperature extremes, salinity, nitrogen deficiency,elevated carbon dioxide and acidity. Our focus will be on the enzymes involved in the conversion ofsucrose-to-starch, and how their activity is regulated at the transcriptional and post-translational level inresponse to certain stress. We then suggest experimental approaches for developing cereal germplasmthat maintains productivity and grain quality under sub-optimal conditions. Finally, we conclude thatthere is an urgent need to elucidate the regulatory mechanisms that modulate starch biosyntheticenzyme activity in response to environmental extremes.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Grain starch is the most important end-product of cereal growthand development. Approximately 70% (w/w) of the grain of rice,wheat, maize, barley, sorghum, millet, oat and rye consists ofstarch, which provides most of Mankind’s subsistence (Jung et al.,2008). Although dietary habits are constantly evolving, availabledata suggest that 50% of the calories humans consume is derivedfrom starch (Galliard, 1987; WHO, 2003), and this value may reach80% in impoverished countries (Burrell, 2003). The importance ofcereal starch is further reflected in the magnitude of its globalproduction e 70% of all arable land is devoted to cereals (Cooke,

ucose pyrophosphorylase; Al,rbon dioxide; DPA, Days postnule bound starch synthase;tic constant; Km, Michaelisullulanase; PPDK, PyruvateNP, Single nucleotide poly-ide; SBEs, Starch branching, Starch phosphorylase; SSs,ription factor; UGPase, UDP-

: þ1 530 752 9659.kles).

All rights reserved.

1977) and recent estimates put cereal starch output close to1 B tons (FAOSTAT, 2009). However, increasing the amount of starchproduced per acre is still necessary because of population growthand reduced resources for agriculture. This need is exacerbated bya constant threat of sub-optimal environmental conditions (Mittlerand Blumwald, 2010). If external factors reach or exceed the stressthresholds for cereal growth, there can often be catastrophicconsequences on yield with ensuing horrific economic andhumanitarian impacts (Shao and Chu, 2005). Understanding howstress modifies starch composition and accumulation in cerealendosperm can improve the predictions of grain and flour qualityand may also be useful to breeders when deploying new germ-plasm that can tolerate extremes of environment.

Starch is the primary carbon reserve of plants. It is insoluble,particulate and chemically inert, making it ideal for long-termstorage (Kossmann and Lloyd, 2000). The starch in cereal endo-sperm is synthesized to enhance plant survival in the next gener-ation (Preiss, 1991; Smith and Martin, 1993); however, humans aredependent on this storage starch for food. Current industrial use ofstarch as a biopolymer and biofuel takes advantage of its abun-dance, the diverse arrays of crystalline structures that occur innature and the ease with which it may be chemically modified anddegraded (Lynch et al., 2007; Muffler and Ulber, 2008).

M. Thitisaksakul et al. / Journal of Cereal Science 56 (2012) 67e8068

Starch consists of chains of a-(1,4)-linked glucose units that areoccasionally branched by a-(1,6)-linkages. These chains are orga-nized into two polymers called amylose and amylopectin that occurin a 30:70 ratio (w/w) in normal cereal endosperm (Morell et al.,2007; Tester et al., 2004). Amylopectin is larger than amylose(molecular weight 106e108 vs. 105e106 g/mol respectively) (Buleonet al., 1998) and has a higher frequency of branching (every 20 vs.100e10,000 glucoses respectively) (Tetlow, 2011). The regularity ofthe branching in amylopectin creates glucan chains of definedlengths which are arrayed into alternating crystalline and amor-phous regions (Fig. 1) (Bertoft, 1986; French, 1984; Jenkins et al.,1993; Robin et al., 1974).

The size, shape and number of starch granules vary amongdifferent cereal endosperms (Jane et al., 1994). Granules range from1 to 60 mm (Tester et al., 2004) and are classified into three mainmorphological groups: (i) simple individual granules maize,sorghum (ii) bi- or tri-modal distribution of granules that vary insize andmorphology, called Ae (10e35 mm), Be (<5e10 mm) and Ce (<5 mm) granules wheat, barley, rye or (iii) compounded granulesrice, oat (Evers et al., 1999; Shapter et al., 2009, 2008; Vandeputteand Delcour, 2004). The functional behavior of starch is intri-cately linked to its structure and morphology. Changes in glucanchain length distribution or degree of crystallinity can alter starchphysico-chemical characteristics (Copeland et al., 2009). Smallchanges in the proportion of amylose-to-amylopectin in particularhave been extensively documented to not only alter starch func-tionality (Lii et al., 1996; Zeng et al., 1997), but to also heavilyinfluence the nutritional characteristics of starchy foods (Akerberget al., 1998; Bird et al., 2006; Hazard et al., in press), and this traitremains a focal point of starch research.

2. The core starch biosynthetic pathway

The concerted action of four enzyme activities: ADP-glucosepyrophosphorylase (AGPase), starch synthases (SSs; GT5 CAZyfamily), starch branching enzymes (SBEs) and starch debranching

Fig. 1. Glucan chain distribution and arrangement in amylopectin. Chains of varyinglengths are arranged into alternating clusters of 9 nm whereby the branch points arepart of the amorphous region of the granule and the linear chains form the crystallineregions. The chains are classified as A and B1eB3 according to the chain length. A-chains are only attached to a single chain, while B1, B2 and B3 chains span 1, 2 and 3clusters respectively. There is a single C-chain, which has the reducing glucosemolecule. A-chains consist of 5e12 glucoses (5e12 dp), B1 (13e24 dp), B2 (25e36 dp)and B3 longer chains (37e54 bp). Adjacent chains (consisting of at least 10 glucoseunits) can form double helices. In cereals, these helices are arranged into A-typecrystallites. Amylose intercalates within the amylopectin molecule primarily in theamorphous region. Adapted from Tester et al. (2004); Umemoto et al. (1999);Vandeputte and Delcour, (2004).

enzymes (DBEs; GH13 CAZy family) leads to the synthesis of thestarch granule (Fig. 2). In addition, several other enzymes metab-olize sucrose to the carbon precursors for starch (Fig. 2), and otherssuch as starch phosphorylase (PHO) and pyruvate phosphate diki-nase (PPDK) are essential for normal endosperm starch accumu-lation (Jeon et al., 2010). The four primary enzymes will only bebriefly considered and the Reader is referred to (Jeon et al., 2010;Keeling and Myers, 2010; Tetlow, 2011) for recent, expandedreviews.

(i) ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) catalyzesADP- Glucose, the first committed step in starch biosynthesis.It is a heterotetramer of two small and two large subunits withdiverged functions. The small subunits (SSU) are primarilycatalytic and the large subunits (LSU) regulate responses toallosteric effectors; still, mutations that affect catalysis andregulation have been identified in both SUs (Cross et al., 2005,2004). In dicots AGPase is plastidial, but in cereal endospermmost of the activity is cytosolic (Beckles et al., 2001a, 2001b;Comparot-Moss and Denyer, 2009). AGPase activity is sensi-tive to allosteric effectors especially to the 3-phosphoglycericacid-to-phosphate ratio (Cross et al., 2004), and is also regu-lated by high temperature and redox mechanisms (Boehleinet al., 2008).

(ii) Starch synthases (SSs, EC 2.4.1.21) catalyze the transfer of theglucosyl moiety of ADP-Glucose to the non-reducing end ofa pre-existing a-(1,4)-linked glucan primer to synthesizeamylose and amylopectin. There are five main groups of SSs:SSIeSSIV and a Granule bound starch synthase (GBSSI) basedon sequence phylogeny (Leterrier et al., 2008). Most isoformselongate glucan chains of a defined length but there can befunctional overlap (Zhang et al., 2008). This redundancy in SSaction may influence the placement of branch points inamylopectin (Szydlowski et al., 2011). GBSSI alone makesamylose (Denyer et al., 1996), while SSIII and SSIV have a rolein granule initiation (Szydlowski et al., 2009).

(iii) Starch branching enzymes (SBEs; EC 2.4.1.18) catalyze theformation of the a-(1,6)-linkages within the polymer. Theyhydrolyze a-(1,4)-linkages and then transfer the cleaved chainto an acceptor chain via an a-(1,6)-branch point (Guan et al.,1997). There are two families: SBEI and SBEII, that can bedifferentiated based on the preference of glucan chain lengthfor transfer (Guan et al., 1997). SBEI may determine thespacing between branching points in cereal endosperm (Xiaet al., 2011), while the two isoforms of SBEII found in cereals,SBEIIa and SBEIIb, determine most of the branching (Reginaet al., 2010). SBEIIb is the main activity in rice and maize,and SBEIIa in wheat and barley (Regina et al., 2010). A newSBEIII family has been suggested (Han et al., 2007) and a novelisoform SBEIc appears to exist only in the Triticeae (Peng et al.,2000).

(iv) Starch debranching enzymes (DBEs) hydrolyze a-(1,6)-linkageswithin apolyglucans. This activity is required to regularize thebranching and maintain amylopectin crystallinity (Jeon et al.,2010; Mouille et al., 1996) There are two types of DBEs: iso-amylase (ISA, EC 3.2.1.68), and pullulanase (PUL or limit dex-trinase, EC 3.2.1.41), which vary in their preference of glucansubstrate (Zeeman et al., 2007). Three isoforms of ISA and oneisoform of PUL exist in cereals, with ISA2 having amajor role inglucan debranching and starch granule synthesis, while that ofPUL is relatively minor (Utsumi et al., 2011).

A central feature of starch biosynthesis is the multiplicity ofisoforms for the core enzymes. This allows for subfunctionalizationof enzyme activity whereby each isoform would vary somewhat in

Fig. 2. A simplified model of the starch biosynthetic pathway in a cereal endosperm cell. Enzymes are highlighted in boxes, and membrane transporters are circles. The legend is asfollows. INV e invertase; SuSy e Sucrose synthase; PGI e Phosphoglucoisomerase; PGM e Phosphoglucomutase; UGPase e UDP-glucose pyrophosphorylase; SPS e SucrosePhosphate Synthase; AGPase-S e Small subunit of ADPglucose pyrophosphorylase; AGPase-L e Large subunit of AGPase; ADPGT e ADPglucose transporter (Brittle-1 or Bt1); AATP e

plastidic ATP transporter; GTP e Glucose-6-Phosphate transporter; SS e Starch synthase; GBSSI e Granule bound starch synthase; SBE e Starch branching enzyme; Pul e Pul-lulanase; ISA e Isoamylase; PHO e Starch phosphorylase.

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the contribution made to the building of the starch granule (Dianet al., 2005; Leterrier et al., 2008). In cereals, the number of iso-forms is larger than in dicots due to an ancient genome duplicationso that there are at least two forms of most enzymes for whichthere is only one in dicots (Vandepoele et al., 2003). There are someexceptions, however. There is only one SSI in higher plants andmaize lacks an isoform of SSII and SSIV found in other cereals (Yanet al., 2009).

3. Starch biosynthesis in the developing endosperm

Starch deposition occurs synchronously with grain develop-ment (Altenbach et al., 2003; Singletary et al., 1994). Little starch isaccumulated during the ‘cell division’ or endosperm differentiationphase (w4e10 Days Post Anthesis; DPA), which defines the finalcell number of the grain (Altenbach et al., 2003). Starch accumu-lation is maximal between 12 and 35 DPA during ‘grain filling’ andthen ceases during ‘desiccation’ (w40e60 DPA) (Olsen, 2001; Olsenet al., 1999; Sabelli and Larkins, 2009; Sreenivasulu et al., 2010).Transcripts for the different isoforms of AGPase, SSs, SBEs and DBEsare present at distinct times during these phases (Hirose and Terao,2004; Ohdan et al., 2011; Shewry et al., 2009; Sreenivasulu et al.,2004; Stamova et al., 2009; Toyota et al., 2006; Zhu et al., 2003)and although they may be subject to post-transcriptional modifi-cations, transcript occurrence can still act as an indicator of thepossible activity of enzyme present. The unique set of enzymes inturn determines starch morphology and structure, and changes ineven one enzyme isoform can profoundly alter starch properties(Keeling and Myers, 2010). This is accentuated by the growingknowledge that starch biosynthetic enzymes form physicalcomplexes with one another and with the glucan substrate, andthat this interaction is essential for the proper architecture of thestarch granule (Hennen-Bierwagen et al., 2008; Lin et al., 2012;Tetlow et al., 2008, 2004).

4. Effect of environmental stress on starch accumulation

Plants exposed to sustained or episodic stress may show a widerange of complex and variable responses. The specific reactionmanifested is dependent on the severity of the perturbation andthe inherent sensitivity of that particular genotype to stress(Cramer et al., 2011). Stressed plants may be compromised in theirability to produce photosynthate for the sinks (Sicher et al., 1995;Stone et al., 1995). The grain developmental window is oftenshortened (Barnabas et al., 2008; Bernard and Habash, 2009;Dolferus et al., 2011; Hirel et al., 2007; Kim et al., 2003; Modhejaet al., 2008; Moradi and Ismail, 2007) and endosperm cellnumber reduced (Fabian et al., 2011; Nicolas et al., 1985), whichtogether lessen starch content in the mature grain. Stress also hasdirect effects on starch biosynthetic enzyme activity. This reducesthe amount, and often, changes the structure of the starch granulesat harvest (Kossmann and Lloyd, 2000; Raeker et al., 1998;Singletary et al., 1994). More importantly, changes in enzymeactivity also reduce sink capacity (Denyer et al., 1994; Yang andZhang, 2010).

There is now broad agreement that global climate change willhave negative effects on agriculture. Drought and heat stress posethe most serious limitations (Foulkes et al., 2007), and both canincrease soil salinity, which may be affecting 20% of all arable land(Yokoi et al., 2002) and could climb as high as 50% by 2050 (Smithet al., 2009). This scenario is particularly worrisome because overallcrop yields from the 1950s to 1990s, in the absence of such envi-ronmental challenges, had been relatively flat (Borlaug, 2007;Conway and Toenniessen, 1999). If the endgame is to produce high-yielding cereals under stress, then two breeding strategies may bedefined as such: either plants with higher yield, or plants withincreased tolerance to stress, must be developed to stabilize currentyields.

It is also generally accepted that amplifying grain sink strengthis a critical element in any effort to increase cereal yield (Reynolds

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et al., 2004). Since starch biosynthesis is the primary determinantof sink strength (Dale and Housley, 1986; Yoshida, 1975), targetingthis pathway to develop cereals that can maintain grain fillingunder stress may be fruitful. It has even been argued that theactivity of enzymes involved in the conversion of sucrose-to-starchis a major determinant of yield potential in rice (Yang and Zhang,2010) and this may be similarly suggested for heat-stressedwheat since sucrose availability was not limiting under theseconditions (Bhullar and Jenner, 1986; Denyer et al., 1994; Nicolaset al., 1984; Sofield et al., 1977). Multiple strategies are needed todevelop cereals that can maintain yield when the prevailingconditions are poor. In this context, we propose that engineeringthe starch pathway is a potentially viable option.

Here we review the effects of a variety of stresses on cerealendosperm starch biosynthesis to identify enzymes that are mostvulnerable to each perturbation. Such enzymes could be singled-out for genetic modification. Most experiments examined herewere done under controlled conditions where a single stress wasvaried. Each study presents a unique set of genotypes, treatmentsand environmental variables, so there may be discrepanciesbetween some reports. Most examples come from wheat and rice.Wheat provides about 20% of all calories eaten globally and is theonly cereal that can be used to make normal leavened bread andpasta products (Uhlmann and Beckles, 2010). Rice is a staple foodfor nearly half of the world’s population (IRRI, 2011). In addition,rice is consumed in a minimally processed form and changes instarch quality parameters due to environmental fluctuations oftenaffect palatability and appearance which are major concerns inAsian markets (Sun et al., 2011).

4.1. Drought

Water stress during grain development is the greatest impedi-ment to crop yields. Approximately 61% of all crop-production areashad precipitation lower than 500 mm in 2009 (Lichtfouse et al.,2009), an amount below the required threshold for productivedryland farming (Davis et al., 2006; Turton et al., 2003). Lower grainstarch is one of the leading causes of reduced yield due to drought.In a comprehensive study of 50 field-grown barley accessions,endosperm starch reductions ranged from 0 to 45% when waterwas withheld from 10 DPA until harvest, and changes in starchcontent correlated well (r2 ¼ 0.7) with yield (Worch et al., 2011).

In accordance with reduced yield, drought-stressed cerealsshow alterations in starch biosynthetic enzyme activity (Jenneret al., 1991). SSs are the most sensitive enzyme to drought, andchanges in SS activity may largely explain reduced starch content inwheat (Ahmadi and Baker, 2001). Interestingly, at the onset ofa water deficit, SS activity is sharply reduced, but under prolongeddrought, their activity remains relatively constant (Ahmadi andBaker, 2001; Caley et al., 1990). AGPase activity also declinesrapidly under water stress due to changes in osmotica, and underextreme water deficit, the loss of AGPase activity can exceed that ofSS leading to premature cessation of starch deposition (Ahmadi andBaker, 2001; Caley et al., 1990). Wheat GBSS activity is also affectedby drought, while SuSy and UGPase, which have lowered activity,are comparatively more stable (Ahmadi and Baker, 2001). It shouldbe noted that DNA sequence polymorphisms in SuSy 1 and 2 inindividuals among a large population of barley accessions parti-tioned according to the ability of the individual to maintain higherstarch accumulation under reduced watering regimes (Worch et al.,2011), pointing to an important role for SuSy in drought tolerance.

Surprisingly little is known about the mechanistic basis ofhormonal regulation of cereal endosperm starch under waterstress. Abscisic acid (ABA) modulates whole suites of genes inresponse to stress often in cross-talk with sugar signaling (Rook

et al., 2006), and changes in starch biosynthetic enzyme activitywould be an expected downstream response to an ABA-dependentstress-signaling cascade (Zheng et al., 2010). Such networks may beessential in wheat (Yang et al., 2003), rice (Zhang et al., 2012a) andbarley (Seiler et al., 2011) during grain filling because endogenousABA accumulates and correlates positively with higher grain starchin the moderately water-stressed grain; and, in the rice and wheatstudies, these changes were accompanied by increases in SuSy, SBEand SS activity (Yang et al., 2003; Zhang et al., 2012a). Furthermore,when 20 mM ABA was exogenously applied to the grain, thisincreased the activity of these enzymes (Yang et al., 2003; Zhanget al., 2012a). Notably, GBSS activity was not affected by ABA(Yang et al., 2003) indicating that changes in activity may bethrough ABA-independent pathways. The above examples werefrommoderately water-stressed plants. Under these circumstances,ABA is known to amplify the rate of starch biosynthesis duringgrain filling. This would presumablyminimize the negative effect ofthe shorter grain developmental window imposed by stress toensure maximal starch output for the next phase of the life cycle(Liu et al., 2005; Seiler et al., 2011). It is not clear how ABA affectsstarch biosynthetic enzyme activity under severe water deficit orwhen drought is experienced during cell division.

Reducing water supply to cereals alters endosperm starchgranule size distribution and amylose content. This is clearly shownin wheat where the size of the A-, B- and C-granules is reduced(Brooks et al., 1982; Fabian et al., 2011; Singh et al., 2008). However,the size and number of the B- and C-granules are disproportion-ately affected, thereby increasing the overall proportion of A-granules by volume (Brooks et al., 1982; Fabian et al., 2011; Singhet al., 2008). This result is also symptomatic of grain that experi-enced heat stress (see Section 4.2) andmay be explained by the factthat B- and C-granules are initiated later in grain development(10e16 and 18e21 DPA respectively) (Parker, 1985), and would besubjected to the cumulative effects of stress experienced earlier(Brooks et al., 1982). As may be expected, some genotypes vary intheir response to low soil moisture when tested side-by-side:certain varieties may have higher, lower or no change in theproportion of A- to B-granules (Zhang et al., 2010b). In barleyendosperm, starch particle size was perturbed only under long-term water stress and this was confined to the B- and C-typegranules only (Brooks et al., 1982). The starch from drought-treatedwheat (Fabian et al., 2011; Mall et al., 2011; Singh et al., 2008) andrice (Gunaratne, 2011; Gunaratne et al., 2011a; Liu et al., 2010) alsocontains lower amylose. In a large field trial, lower grain amylose inwater-stressed rice was accompanied by lower GBSSI expression,making it possible to attribute changes in amylose to transcrip-tional regulation of GBSS (Liu et al., 2010). It can also be envisionedthat a small reduction in GBSSI activity could significantly reducethe amylose-to-amylopectin ratio. This is because GBSS hasa higher Km (i.e. lower affinity) for ADP-glucose relative to thesoluble SSs (Clarke et al., 1999). Reduced carbon flux to the endo-sperm would excessively affect amylose biosynthesis (Clarke et al.,1999; Denyer et al., 2001) even though the measurable SS activitymay be lower than GBSS due to drought (Ahmadi and Baker, 2001).

4.2. Heat

Both temperate and subtropical cereals grow optimally at20e30 �C (Keeling et al., 1994). Wheat is particularly susceptible totemperatures above this range, resulting in losses of 10e15% ofyield in Australia and the United States (Blum et al., 1994; Wardlawand Wrigley, 1994). Some wheat varieties can lose 10e15% of yieldwith every 5 �C increase in temperature (Burrell, 2003). Althoughthere was a strong genotypic component, in controlled environ-ments, temperatures between 30e40 �C reduced barley endosperm

Table 1Changes in the relative expression of genes for enzymes involved in the conversionof sucrose-to-starch in rice, wheat and barley caryopses when exposed to heatstress. Gene symbols are as described in Fig. 2 legend. For rice and barley, thegreen boxes show genes that are down-regulated (<0.9), Yellow boxes show genesthat did not change (0.9e1.1) and red boxes show genes that are up-regulated(>1.1) in response to heat. Black boxes indicate that the gene was not studied.Rice data are from Yamakawa and Hakata (2010) and Yamakawa et al. (2007) with33 �C/28 �C (day/night) chamber temperature condition, the barley data are fromMangelsen et al. (2011) with 3, 6 and 12 h 42 �C chamber temperature conditionand both studies used microarrays. Wheat data is taken from Hurkman et al.(2003), which used RT-PCR on plants under 37 �C/17 �C (day/night) and37 �C/27 �C (day/night) chamber temperature regimes and was supplemented byresults for SuSy from Chauhan et al. (2011) with 2 h 37 �C and 42 �C chambertemperature condition. Additional key for genes: AGPS e ADPglucose pyrophos-phorylase small subunit; AGPL e ADPglucose pyrophosphorylase large subunit;AMY e a-amylase; BAM e b-amylase; GWD e Glucan water dikinase.

M. Thitisaksakul et al. / Journal of Cereal Science 56 (2012) 67e80 71

starch by 13e33% (Macleod and Duffus, 1988b; Savin and Nicolas,1996; Savin et al., 1997; Wallwork et al., 1998b), and wheat starchby 2e33% (Liu et al., 2011; Zhao et al., 2008). The most vulnerabledevelopmental window is 10e15 DPA when ‘grain filling’ is initi-ated and diminished yield at high temperature can almost alwaysbe ascribed to reduced starch biosynthetic enzyme activity (Jenner,1994; Keeling et al., 1993, 1994). In contrast, reductions in starchcontent were only 2e6% in tropical rice endosperm at temperatures30e40 �C (Cheng et al., 2005; Inukai and Hirayama, 2010).

Pioneering work by Keeling and coworkers showed that at hightemperature, reduced starch biosynthesis is due to the diminutionof SS activity (Keeling et al., 1993, 1994). Starch synthases intemperate cereals become largely non-functional at temperaturesabove 35 �C. For example, in wheat, SS activity is maximal between20 and 25 �C, but at 40 �C, 97% of activity is lost (Keeling et al., 1993).Nevertheless, most of this activity is recoverable if the SSs are re-exposed to optimal temperatures (Keeling et al., 1993). SS activitymay be lost due to protein denaturation and, in some species,reductions in the reaction velocity of the enzyme (Blum et al., 1994;Denyer et al., 1994; Jenner, 1994; Savin et al., 1997; Wallwork et al.,1998c).

GBSSI activity is also affected by heat although not to the sameextent as the SSs inwheat, barley (Jenner, 1994) or rice (Cheng et al.,2005). The response of GBSSI to temperature is defined by a singlenucleotide polymorphism (SNP) in the 50 leader intron of the GBSSIgene sequence in rice (Patron et al., 2002; Wang et al., 1995). Heatsensitive varieties such as Japonica have a GT in the leader sequence(AGGTATA), while more heat tolerant ones like Indica are TT(AGTTATA) (Isshiki et al., 1998; Sun et al., 2011; Wang et al., 1995).Interestingly, rice varieties with the GT motif have less GBSSItranscript at temperatures of 28 �C and higher so that GBSSI activitybecomes attenuated, while the TT-types show higher survivability(Cheng et al., 2005; Hirano and Sano, 1998; Inukai and Hirayama,2010; Jiang et al., 2003; Mikami et al., 2008; Tashiro andWardlaw, 1991b; Yamakawa et al., 2007).

SBEII enzyme activity in maize (Takeda et al., 1993) and rice(Ohdan et al., 2011) also appears to be heat labile. The optimumtemperature of SBEIIb activity in rice andmaize, which accounts forthe majority of SBE activity in these species, is 25 �C and 15e20 �Crespectively (Ohdan et al., 2011; Takeda et al., 1993). Some of thechanges in glucan chain length under heat stress may be attributedto changes in the activity of the SBEs in these species (Ohdan et al.,2011). What is not known is whether the loss of SBE activity intropical cereals limits starch biosynthesis in a comparable way tothe loss of SSs activity in temperate cereals at high temperatures.

Elevated temperature also affects the activity of endospermAGPase (Keeling et al., 1994). This enzyme has been best studied inmaize, where its thermal inactivation may supersede the role of SSsin limiting starch synthesis, offering a sharp contrast to observa-tions in barley and wheat (Ito et al., 2009; Jenner, 1994; Savin andNicolas, 1996, 1999; Singletary et al., 1994; Wallwork et al.,1998b). Maize AGPase is remarkably unstable at temperaturesabove 45 �C compared with dicots (Ballicora et al., 1995; Greeneand Hannah, 1998b). This heat liability is delimited to a motif inthe N-terminus of the SSU that reduces the Kcat and the stability ofheterotetramer at elevated temperatures compared with potato(Linebarger et al., 2005).

SuSy activity parallels the decreases in starch content after hightemperature treatment in rice and barley (Cheng et al., 2005;MacLeod and Duffus, 1988a). In barley, exposure to heat early indevelopment may irreversibly inactivate SuSy (MacLeod andDuffus, 1988a), while in rice, substantial losses of SuSy activityand a weakened catalytic capacity to cleave sucrose are cited asunderlying causes of lower starch content (Cheng et al., 2005).

Singletary et al. (1994) suggested that transcriptional regulationof gene expression may be a primary mechanism that restrictsmetabolism under heat stress. However, this restriction may alsodepend on whether the catalytic function of enzymes that exist inthe cell prior to the high temperature treatment is maintained(Singletary et al., 1994). Transcriptional profiling experimentsshowing the co-expression pattern of starch biosynthetic genes inrice (Yamakawa et al., 2007), barley (Mangelsen et al., 2011) andwheat endosperm (Hurkman et al., 2003) under heat stress supportthis notion (Table 1). Furthermore, cross-data analysis shows thatchanges in gene expression of isoforms of SuSy, SSs, GBSSI, SBEs andAGPase in different species were accompanied by similar direc-tional alterations in overall enzyme activity (Cheng et al., 2005;Jenner, 1994; Ohdan et al., 2011) (Table 1). The converse was seenfor a-amylase activity, which was unchanged inwheat and barley athigh temperature (Jenner, 1994), but the mRNA levels of severalisoforms are higher in barley and rice (Table 1). These data point to

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the potential importance of post-transcriptional regulation understress and the care required in interpreting gene expression data.

Elevated temperatures (>30 �C) alter starch composition intemperate cereals. In wheat, the proportion of amylose increases(Hurkman et al., 2003) and in addition, the proportion of shortamylopectin chains increases (Matsuki et al., 2003; Shi et al., 1994;Tester, 1997). The severity of the heat stress and the heat-susceptibility of the genotypes determine the effect on amylosecontent. Exposing 75 greenhouse-grown wheat cultivars to 40 �Cfor 3 days caused only mild stress, and instead of an increase inamylose, 64% accumulated less grain amylose, 33% showed nochange and 1% had increased amylose (Stone and Nicolas, 1995). Nosignificant changes in amylopectin fine structure were identified inbarley grain starch (Tester et al., 2004, 1991) although amylosecontent increased (Savin and Nicolas, 1999).

High temperatures also affect the number and size of the A- andB- type starch granules in wheat and to a lesser extent in barley.Generally, there is an increase in the proportion of A- to B-granulesin response to high temperatures, but the timing of application ofstress may also be critical (Savin and Nicolas, 1999). Hurkman et al.(2003) proposed that heat suppresses B-granule initiation and theavailable substrate may be diverted towards pre-existing A-gran-ules making them bigger and leading to a higher ratio of A-to B-granules by volume (Hurkman et al., 2003). This is borne out instudies where the stress was applied after anthesis in wheat(Bhullar and Jenner, 1985; Blumenthal et al., 1995; Hurkman et al.,2003) and barley (Macleod and Duffus, 1988b; Wallwork et al.,1998a). When wheat was held at 40 �C for 3 days before anthesis,the early initiated A-granules were overly affected, being lower innumber relative to B-granules and showing morphological defor-mation and fissuring (Liu et al., 2011), again illustrating theimportance of the developmental stage at which the stress occurs.

Higher growing temperature causes very different effects inmaize and rice compared with the temperate cereals. Heatdecreases amylose and increases the overall proportion of longeramylopectin chains in maize and rice starch (Asaoka et al., 1984; Luet al., 1996; Umemoto et al., 1999; Yamakawa et al., 2007), while ithas an opposite effect in wheat and barley. It has been suggestedthat this decrease in amylose in ricemay fully account for the loss ofgrain mass in response to heat (Inukai and Hirayama, 2010). Theincrease in frequency of longer chain lengths observed in rice at29 �C vs. 22 �Cmay be due to elevated SSI activity, the predominantSS in rice grains, which would preserve the elongation of the A andB chains of amylopectin, whereas the reduced activity of SBEIIb andSBEI, would lower the branching frequency of amylopectin (Chenget al., 2005; Jiang et al., 2003; Tashiro and Wardlaw, 1991b). Sincehigh temperatures reduce GBSSI activity, this, along with higher SSIactivity may explain the lower ratio of amylose-to-amylopectin inheat-stressed rice (Cheng et al., 2005). Elevated temperatures inrice produce chalky grains due to structural changes in amylopectin(Tashiro and Wardlaw, 1991a; Yamakawa, 2011; Yamakawa et al.,2007); in spite of this, the degree of chalkiness is not matched bycommensurate changes in amylopectin (Yamakawa et al., 2007).

4.3. Salinity

High salt concentrations cause osmotic stress after short-termexposure or ion toxicity after long-term exposure in plants(Munns and Tester, 2008; Sanchez-Diaz et al., 1982; Zeng, 2005).Sodium chloride (NaCl) is the salt of main concern due to its highsolubility (Munns and Tester, 2008) with 40 mM consideredproblematic for agriculture (Munns and Tester, 2008). Rice issensitive to salinity-stress while barley is more resistant (Munns,2002; Munns and Tester, 2008). Soil with 50 mM NaCl can cutyields in half in rice (Abdullah et al., 2001), while barley grown at

60e70 mM NaCl only has yield reductions of 20e36% (Katerji et al.,2006).

There is minimal information on changes in starch biosyntheticenzyme activity in grain under salinity-stress. Rice grown at 50mMNaCl, had a 44% (w/w) decline in yield (measured by total seedweight per plant) and was underscored by 3-fold reductions in SSactivity inw10 DPA grain (Abdullah et al., 2001). Meanwhile, directmeasurements of starch in field-grown varieties at w40 mM NaCl,showed modest reductions of 4e6% (w/w) in content, and at leastone genotype was unaltered (Siscar-Lee et al., 1990). Work per-formed on rice photosynthetic tissues suggests that salt can affectstarch biosynthetic enzyme activity, but caution must be applied inextrapolating this data to reproductive organs since their regula-tion could be different (Chen et al., 2008). In rice seedlings, starchcontent was reduced 3-fold at very high NaCl concentrations(200 mM) (Chen et al., 2008). GBSSI activity was heavily attenuatedwhile AGPase, SS and SBE activities were unaffected (Chen et al.,2008). No changes in a- and b-amylase activities were observed;therefore, low starch accumulation was due to defects in synthesis,rather than elevated degradation (Chen et al., 2008).

The suppression of GBSSI activity in salt-stressed rice leaf tissueis controlled by a transcriptional process or mRNA stability (Chenet al., 2008). Both GBSSIa and GBSSIb mRNA accumulation is sup-pressed by ionic but not osmotic stress, and the transcripts werereduced 4- and 5-fold respectively (Chen et al., 2008). The salt-stress signal transduction controlling GBSSI gene expressionmight operate through either an ABA-independent pathway ora combination of both ABA-dependent and ABA-independentsignals (Chen et al., 2008).

High salt reduces rice grain amylose content. The extent of thechange in amylose depended on rice genotype and the saltconcentration used, but not on the salt-sensitivity of the cultivar(Peiris et al., 1988; Siscar-Lee et al., 1990). For example, whenamylose was tested in both salt sensitive and tolerant cultivars ina controlled environment, it decreased only when the salt electricalconductancewas 8 dSm�1 or higher (w65mM for NaCl), regardlessof the salt-type used i.e. chloride-sulfate, sulfate-chloride or chlo-ride (Peiris et al., 1988). Under field conditions at w40 mM NaCl,amylose content decreased 7e11% (P< 0.05) in the grain of three ofthe four rice genotypes tested (Siscar-Lee et al., 1990).

4.4. Nitrogen deficiency

Nitrogen (N) is an indispensable nutrient for plants. In 2008,w99 M tonnes of N fertilizers were used to sustain global cropproduction (FAOSTAT, 2011). N-deficiency prior to or during earlyreproductive development severely limits crop yield (Bernard andHabash, 2009; Dolferus et al., 2011; Hirel et al., 2007; Kim et al.,2003; Modheja et al., 2008). Moderate reduction in N reducescrude protein (w12e22%) and leads to small increases (4%) instarch content inwheat and barley grain (Blacklow and Incoll, 1981;Hirel et al., 2007).

In maize, N-limitation decreased kernel starch accumulation(Cazetta et al., 1999; Demotes-Mainard and Jeuffroy, 2001;Seebauer et al., 2010) due to effects on carbohydrate biosyntheticenzyme activity (Seebauer et al., 2010; Singletary and Below, 1990;Singletary et al., 1990). These enzymes included: soluble- and cellwall invertase, aldolase, phosphoglucomutase, SuSy, phosphofruc-tokinase and hexokinase (Cazetta et al., 1999; Faleiros et al., 1996;Singletary and Below, 1990). Limited N also reduced the activity oftotal and soluble b-amylase in developing grain (Erbs et al., 2010;Howarth et al., 2008).

In rice, low N-soil increased amylose (Dong et al., 2007;Gunaratne et al., 2011b; Hao et al., 2007; Xiong et al., 2008) but didnot significantly change amylopectin content or structure

M. Thitisaksakul et al. / Journal of Cereal Science 56 (2012) 67e80 73

(Champagne et al., 2009; Gunaratne et al., 2011b; Xiong et al.,2008). Dong et al. (2007) stated that low N-soil leads to lowerSBEII activity in rice. This would reduce the proportion of highlybranched starch, and lead to a more unbranched amylose-likefraction, potentially explaining the higher amylose contentmeasured, but this should be investigated further. It should also benoted that other studies (Ahmad and Chughtai, 1982; Tamaki et al.,1989) failed to show differences in amylose content when Nfertilizer concentrations were varied. This difference in fertilizerimpact may depend on the crop nutritional needs (Champagneet al., 2009).

4.5. High carbon dioxide (CO2)

Atmospheric levels of carbon dioxide (CO2) have increased by35% since the Industrial Revolution (ca 1800) and are forecasted todouble by the end of the 21st century (Ainsworth et al., 2008;Bloom, 2010; DaMatta et al., 2010). Initial predictions suggestedthat this phenomenon would be a boon to crop productivity(Ainsworth and Long, 2004) and meta-analysis of several wheatstudies estimated average increases in yield of 31% if CO2 increasedfrom 350 to 700 ppm under optimal conditions (Amthor, 2001,2003). However, higher average temperatures (Ainsworth et al.,2008) and the need for supplemental N to achieve maximalyields (Bloom et al., 2010) have led to a more sober reassessment ofthe benefits of rising greenhouse gases on agriculture (Amthor,2001; Bloom, 2010; Lobell et al., 2011).

High CO2 would be expected to increase carbon allocation tograin resulting in higher starch accumulation. What is insteadobserved, is that CO2 at concentrations of 550 ppm or higherincreases yield in wheat through greater grain number only (Hogyand Fangmeier, 2008). There were no consistent changes in starchaccumulation and often supplemental N or phosphoruswas neededto achieve higher grain starch (Fangmeier et al., 1999) (See Table 2).In general, changes in starch morphology and composition wereunpredictable (Table 2). It appears that the mode of introducingCO2, the concentration and the genotype studied probably influ-ence the data (Rogers et al., 1998). For example, Blumenthal et al.(1995) observed a higher proportion of the large A-granules,while Rogers et al. (1998) found a higher proportion of the small B-granules in response to higher CO2 (Table 2).

Like wheat, rice yield at high CO2 increased because of highertillering (Seneweera et al., 1996; Terao et al., 2005; Yang et al.,2007). Growth at high CO2 led to higher setback viscosity ofstarch, whichmade the cooked grain firmer (Seneweera et al., 1996;Terao et al., 2005; Yang et al., 2007), but the effect on amylosecontent varied in these three studies, showing reduced, unchangedand higher levels respectively, probably due to the use of differentcultivars (Seneweera et al., 1996; Terao et al., 2005; Yang et al.,2007).

Table 2The effect of altered carbon dioxide (CO2) on wheat grain starch. FACE is (Free Air CO2 EnCO2 is w350 ppm.

Cultivar Method CO2 ppm Starch

Yecora Rojo FACE 550 IncreasedHartog Tunnel 700 No changeTRISO FACE 550 No changeMinaret ESPACE 700 IncreasedHartog Tunnel 900 No changeRosella 900 No change

280* No changeHereward Tunnel 700 Increased

e indicates that no measurements were taken.* Pre-Industrial Revolution atmospheric CO2 levels.

4.6. Cold

Lower temperatures tend not to devastate starch biosynthesis incereals as compared with higher temperatures. The effect of coldstress on starch biosynthesis have been examined inwheat (Cravenet al., 2007; Labuschagne et al., 2009; Singh et al., 2010), barley(Anker-Nilssen et al., 2006) and especially the subtropical speciesrice (Ahmed et al., 2008; Asaoka et al., 1984; Yan et al., 2006).

In rice, cold stress applied to the young grain slowed down andextended the ‘grain filling’ period, i.e. the timeframe of starchbiosynthesis, so that starch content was unaltered (Ahmed et al.,2008; Umemoto et al., 1995). This was marked by modestchanges in the activity of most carbohydrate biosynthetic enzymes(Ahmed et al., 2008). The catalytic activity of UGPase, SuSy, AGPase,SSs, PHO and SBEs at 12 �Cwas 78e107% compared to that grown at22 �C at an equivalent stage, while GBSSI activity was 278% higher(Ahmed et al., 2008). Umemoto et al. (1995) also found relativelyfew changes in AGPase, SBE and SS activity (69e102%) in rice grownat 15 �C compared with 25 �C, and they too showed that GBSSIactivity was significantly higher (331%) compared to the control.

As pointed out, low temperatures stimulate GBSSI activity, and,in accordance, grain amylose content is amplified leading toa higher ratio of amylose-to-amylopectin in rice (Asaoka et al.,1984; Umemoto et al., 1995). Lower temperatures also increasedamylose content in wheat (Labuschagne et al., 2009; Singh et al.,2010), and in maize, when grain was harvested from plantswhich experienced a higher number of cooler days, there wasa corresponding increase in amylose (Fergason and Zuber, 1962).

Rice GBSSI activity is regulated by temperature at the post-transcriptional level (Hirano and Sano, 1998). Temperaturesbetween 10 and 18 �C result in more efficient splicing of the GBSSIpre-mRNA, increasing the abundance of GBSSI transcript, proteinand enzyme activity (Hirano and Sano, 1998; Patron et al., 2002;Umemoto et al., 1995). The mechanism by which low temperaturesimprove mRNA processing is not known; however, there is aninteresting corollary between this phenomenon and the occurrenceof amylose accumulation in heat sensitive and tolerant rice varie-ties. Pre-mRNA of rice genotypes with alleles containing a TT-SNP(AGTTATA) in the leader sequence of the 50 intron of GBSSI is notproperly spliced, resulting in low GBSSI, and accordingly, low-amylose grain (Bligh et al., 1998; Cai et al., 1998; Hirano et al., 1998;Isshiki et al., 1998; Larkin and Park, 1999; Mikami et al., 1999;Wanget al., 1995). As temperatures decrease, GBSSI splicing is enhancedin these low-amylose TT-motif genotypes, which increases amylose(Hirano and Sano, 1998; Inukai and Hirayama, 2010; Larkin andPark, 1999). In contrast, the splicing of the GBSSI intron is inher-ently efficient in the GT-SNP (AGGTATA) genotypes, so they natu-rally accumulate intermediate to higher amylose (Hirano et al.,1998; Inukai and Hirayama, 2010; Larkin and Park, 1999; Mikamiet al., 2008). Lowering ambient temperatures in these genotypes

richment). ESPACE denotes meta-analysis of several high CO2 experiments. Ambient

Amylose B-Granule percentage Reference

e e (Porteaus et al., 2009)No change 17% decrease (Blumenthal et al., 1996)e e (Hogy et al., 2009)e e (Fangmeier et al., 1999)e 25% increase (Rogers et al., 1998)e 0%e 13% decreaseIncreased 0% (Tester et al., 1995)

M. Thitisaksakul et al. / Journal of Cereal Science 56 (2012) 67e8074

does not alter this pre-mRNA processing, so there are no ensuingchanges in GBSSI activity or amylose content (Hirano et al., 1998;Inukai and Hirayama, 2010; Larkin and Park, 1999; Mikami et al.,2008). As discussed under Section 4.2, generally, starch in biosyn-thesis in Japonica ecotypes is more affected by heat than Indicaecotypes. This heat sensitivity is demarcated by the presence of theGBSSI allele with the TT-motif in Japonica genotypes (Hirano andSano, 1998, 1991; Inukai and Hirayama, 2010; Mikami et al.,2008). This phenomenon among rice genotypes may be quasi-ubiquitous. As more GBSSI genes continue to be sequenced fromlarge and diverse rice populations, some exceptions to this generalrule may be found, but overall it is still generally true (Inukai andHirayama, 2010; Mikami et al., 2000, 2008; Sun et al., 2011).Furthermore, temperature changes in the starch-to-amylose ratiomay be the result of composite reductions in AGPase, SBE, SS andSuSy activity along with elevated GBSSI (Ahmed et al., 2008; Chenget al., 2005).

Lowering ambient temperature tends to reduce the relativeamylopectin chain length in rice (Asaoka et al., 1984; Suzuki et al.,2002, 2008; Umemoto et al., 1999). Almost no change in starchparameters was found in barley exposed to low temperatures(Anker-Nilssen et al., 2006). Interestingly, bread wheat varieties aremore sensitive to short periods of cold stress comparedwith durumwheat (Labuschagne et al., 2009), pointing to differences inendogenous tolerance mechanisms between species.

4.7. Acidity

Acidic soils have a pH of 5.0 or less and occur in greater than halfof the arable land worldwide. Acidity causes the release of thephytotoxic aluminum (Al3þ) into the soil solution, which inhibitsroot growth (Collins et al., 2008) and thereby grain production(Blamey, 2001; Collins et al., 2008). Acidic soil occurs commonly inthe tropics, where it drastically affects the productivity of rice, theprimary cereal species in which effects of acidity have been studied(Blamey, 2001).

Data on carbohydrate biosynthetic enzymes in cereals grownunder toxic levels of Al3þ are only available for rice vegetativetissues (Mishra and Dubey, 2008). Sugars and starch accumulate inshoots and to a lesser extent in roots, in response to high Al3þ ina time-dependent manner (Mishra and Dubey, 2008). Diminisheda- and b-amylase and increased SuSy activity respectively couldconceivably increase starch levels, while the augmentation in acidinvertase activity could promote higher sugars. The activities ofPHO and SPS also decreased, but how or if they contribute tochanges in carbohydrates is unknown (Mishra and Dubey, 2008).No other assayswere performed, so the effect of Al3þ on enzymes inthe committed pathway of starch biosynthesis remains unan-swered. While these alterations in enzyme activities caused byphytotoxic concentrations of Al3þmay promote survival in seed-lings by refashioning whole plant carbon partitioning, theymay notbehave similarly in the grain (Blamey, 2001; Mishra and Dubey,2008). They nonetheless suggest that these enzymes are induc-ible by Al3þ signals.

5. The regulation of starch biosynthesis

Changes in the external environment must be sensed andtransduced by plants to ensure the appropriate reprogramming ofphysiology to maximize survival of the current and future gener-ations. Long-term regulation involves changes in gene expression,and several starch biosynthetic genes in cereal endosperm aresubject to transcriptional regulation in response to stress(Mangelsen et al., 2010). Changes in gene expression require theactivation of transcription factors (TFs); however, very few TFs that

regulate starchmetabolic genes have been identified. These includea WRKY TF in the developing barley endosperm (Sun et al., 2003),an AP2/EREBP family TF in rice (Fu and Xue, 2010; Wuriyanghanet al., 2009), a bZIP TF in maize in rice (Kim and Guiltinan, 1999)and a MYC and EREBP TF regulate (Zhu et al., 2003). Several starchbiosynthetic genes that are regulated by ABA in rice and barley havebeen identified, exemplifying a pivotal convergence of hormonaland carbon signaling (Seiler et al., 2011; Tang et al., 2009; Zhanget al., 2012b; Zhu et al., 2011). A tentative ABA-signal trans-duction pathway mediated by SnRK2.6 and RCAR/PP2C (Hubbardet al., 2010), which in turn, are controlled by a supraregulatoryABA-responsive TF called ABF(Yoshida et al., 2010),was suggestedby Seiler et al. to modulate barley starch biosynthesis duringdrought (Seiler et al., 2011). Given the complexity of plant metab-olism and the need to regularly re-adjust carbon status in responseto environmental triggers, it is likely that there are several otherundiscovered TFs that regulate starch biosynthesis, and that someof these TFs may be activated by stress signals.

While transcriptional regulation would permit long-termreprogramming to endogenous and exogenous signals, post-translational modifications of the pre-existing starch biosyntheticenzymes would ensure an immediate response to stress(Geigenberger, 2011). Several enzymes in the cereal amyloplastincluding SBEs and DBEs are modulated by the thioredoxin/ferre-doxin system that may connect reductant availability with starchoutput (Balmer et al., 2006a, 2006b). Reversible protein phos-phorylation of starch biosynthetic enzymes, notably SBEs, SSs andPHO is a keymechanism for modulating their activity under normalconditions through multi-enzyme complex formation andenzymeeglucan interaction (Grimaud et al., 2008; Liu et al., 2012;Tetlow et al., 2008, 2004). Proteins from the 14-3-3 family mayhave regulatory roles in starch accumulation by phosphorylatingvarious enzymes (Diaz et al., 2011; Sehnke et al., 2001). Severalhave been found localized to barley amyloplasts (Alexander andMorris, 2006) and embedded in rice and maize starch granules(Koziol et al., 2012) which makes them potential players in starchbiosynthesis. Numerous phosphatases and kinases have also beensequenced from Arabidopsis chloroplasts, which may ostensiblyreflect the importance of protein phosphorylation to plastidmetabolism (Baginsky and Gruissem, 2009; Reiland et al., 2009;Schliebner et al., 2008). Still, whether or not these proteins exist incereal amyloplasts and how many regulate starch metabolicenzymes are not known as functional data is lacking. One plastidialkinase of note is MsK4, a glycogen synthase kinase-like proteinfrom Medicago sativa. MsK4 is starch granule bound with high-saltinducible activity (Kempa et al., 2007). Over-expression in Arabi-dopsis appeared to modulate starch metabolism and simulta-neously engendered plant tolerance to salt stress (Kempa et al.,2007).

6. Approaches for maintaining cereal yield and starchbiosynthesis under abiotic stress

The need to create cereal germplasm that can maintain starchquality and, more importantly, productivity under stress is clear.Here we describe both guided and ‘open’ genetic approaches forimproving cereal starch production under stress.

(i) QTL Mapping. The genetic characterization of segregatingpopulations of various cereals has allowed the identification ofQuantitative Trait Loci (QTL). QTLs are regions of the genomethat collectively control a trait and their identification does notrely on a priori knowledge of gene function, sincewe start withthe trait and thenwork ‘backwards’ to identify the genes. QTLsaffecting starch granule distribution in wheat and barley

M. Thitisaksakul et al. / Journal of Cereal Science 56 (2012) 67e80 75

(Borem et al., 1999; Howard et al., 2011; Igrejas et al., 2002),pasting properties of barley flour (Wang et al., 2010) andstarch synthesis and degradation under stress (Garcia-Suarezet al., 2010; Liu et al., 2008; Pelleschi et al., 2006; Worchet al., 2011) have been identified. The power of the QTLapproach may be further enhanced if the traits to be mappedare parameters from eco-physiological models (Reymondet al., 2003). If starch traits altered under stress conditions,such as amylose-to-amylopectin ratio and glucan chain lengthdistribution could be parameterized in these models, it shouldbe possible to simultaneously predict other starch traits thatwere not explicitly assayed, andmap them to the genome. Thisapproach was used successfully to investigate flowering inbarley (Yin et al., 2005) and carbohydrate metabolism intomato (Prudent et al., 2011) and could be very promising forpredicting cereal starch traits under different stresses. Traitsfor QTL mapping can also include gene expression values(Kerwin et al., 2011; Li and Burmeister, 2005), metabolitelevels (Keurentjes et al., 2008; Kliebenstein et al., 2008; Zhanget al., 2010a) and enzyme activities under stress (Cooksonet al., 2009; Zhang et al., 2010a), further enhancing theresolving power of QTLs for improving cereals.

(ii) Signal Transduction Cascades. Our knowledge of stress signaltransduction pathways and tolerance mechanisms, whilegrowing, is still fragmentary. In order to identify and dissectthe transcriptional networks that are part of a stressresponse, Weighted Gene Co-expression Network analysis(WGCNA) could be a very useful approach (Langfelder andHorvath, 2008; Zhang and Horvath, 2005). WGCNA is a bio-informatic tool for integrating transcriptomic with pheno-typic, i.e. physiological and biochemical data. It can thus beused to connect gene co-expression modules with environ-mental stress responses in plants (Weston et al., 2008). As anexample, if data such as starch content, particle size distri-bution and amylose-to-amylopectin ratio are included asinput, together with transcriptomic data of knock-outmutants for ABA biosynthetic genes exposed to environ-mental stress, then gene regulatory networks and theirhierarchical control underlying changes in starch under stressmay be constructed.

(iii) Enzyme Engineering. The activity of many starch biosyntheticenzymes is unstable under stress and flux through the starchpathway could be increased if enzyme variants are designedusing site-specific mutagenesis to increase their stability. Thisapproach has been particularly successful in AGPase wheremutated enzymes insensitive to feedback inhibition and heatliability, respectively, were transformed into cereals and thisled to increased sink strength and yield (Greene and Hannah,1998a, 1998b; Hannah and Greene, 1998; Hannah and James,2008). The SSs would be good targets for modulation giventheir extreme sensitivity to heat and high control over the fluxof the starch pathway in wheat (Smith, 2008). This idea isbuttressed when E. coli glycogen synthase was expressed inwheat, and it was observed that starch content increased atslightly higher temperatures in the transgenic lines (Burrell,2003). Very basic work will be needed to first (i) assay theenzymes extracted from plants grown under a range of stress,(ii) identify the target sites of regulation and (iii) discover howSS functioning in multi-enzyme complexes is altered understress. Eventually, examining the regulation of starch biosyn-thetic enzyme activity under multiple or combinatorialstresses will be crucial, to simulate real-world agronomicconditions. An important first step will be to developa complete systems-level view of changes in cereal grain inresponse to a single stress, as this data is still lacking.

7. Conclusions

Cereals have been bred to hyperaccumulate grain starch underoptimal conditions, but environmental stress often subverts yield inpart by altering the activity of starch biosynthetic enzymes. Theseenzymes may be modulated at the transcriptional, post-transcriptional level or both in response to stress, with devas-tating consequence for crop yield. Identifying targets for breedingwill require a thorough understanding of these different layers ofregulation and dissecting how they work both individually andcooperatively to control starch biosynthesis in response to envi-ronmental extremes. Temperate cereals and tropical grasses willrequire tailor-made strategies since the most vulnerable enzymesto different stresses vary among these types of cereals, sometimesshowing species-specific alterations. While we are not minimizingthe importance of other approaches to designing stress-tolerantcereals, or ignoring the limitations associated with geneticallymodifying single enzyme to change metabolic flux, it is reasonablethat one way to maintain yield-stability under stress is to amplifygrain sink strength through starch pathway engineering. There aresufficient data to support this. Relatively little is known about themechanistic basis by which starch biosynthetic enzymes areregulated in response to environmental perturbation. Therefore, itis imperative that we address this gap in our knowledge if we are tohave the broad base of tools to engineer cereals for the 21stCentury.

Acknowledgments

We wish to thank the Reviewers of this manuscript for theirinsightful comments. Work on cereal stress in DMB’s lab is sup-ported by California Hatch Project CA-D*-PLS-7198-H. MT thanksthe Royal Thai Government and the Henry A. Jastro GraduateResearch Award for Ph.D. funding. RCJ acknowledges GraduateStudent Research Fellowships from the UC Plant Sciences Depart-ment, UC Davis Horticulture & Agronomy Graduate Group and theNational Science Foundation. RCJ also thanks the California SeedAssociation and Henry A. Jastro Graduate for Research Awards. Theresearch collaboration between MCA and DMB is supported by theFrance Berkeley Fund.

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