Grain Filling and Starch Synthesis in Barley...Grain ﬁlling and starch synthesis in barley 149 process (5). The ﬁrst fertilization, involving the fusion of the sperm cell nucleus

Carbohydrate Reserves in Plants — Synthesis and RegulationAK. Gupta and N Kaur (Editors)© 2000 Elsevier Science B. V. All rights reserved. 147

Grain Filling and Starch Synthesis in Barley

Alan H. Schulman*, Pia Runeberg-Roos, and Marko Jaaskelainen

Institute of Biotechnology, University of Helsinki, Viikki Biocentre, P.O.Box 56, FIN-00014 Helsinki, Finland

Barley is one of the oldest cultivated crops in the world. Although barleywas later surpassed by rice, wheat, and maize as major human staples,important niches such as malt production nevertheless remain.Furthermore, barley has a wide physiological tolerance and is a majorgrain in marginal agricultural areas ranging from western Asia to near theArctic circle. Barley yield is directly correlated with starch deposition inthe developing grains, a process which occurs coordinately with the layingdown of storage proteins. In this chapter, we shall consider primarilystarch biosynthesis with respect to grain filling, and also shortly addressprotein biosynthesis, within the context of the ontogeny of the grain.Grain—filling from the developmental point of view will be examined first,followed by an analysis of the biosynthesis and deposition of these twomajor components of the grain.

1 . INTRODUCTION

Barley was perhaps the most important cereal of the Classical worldand has a history of cultivation extending back some 9000 years in theNear East (1). Although its use as a staple for human consumption hasdeclined in modern times, it continues to be the world’s forth major cerealcrop overall and has important niche applications such as the productionof malt. Barley maintains its status as a major crop in the countries ofNorthern Europe, and enjoys a uniquely broad distribution of cultivation,from the southern shores of the Mediterranean to the Himalayas, the deepsands of Australia, and as far north as the Arctic circle in Scandinavia. Inthe 15 EU countries, barley is the second in total area planted and in totalyield behind wheat.

Virtually all uses of barley depend upon the grain, whether milled asflour or germinated to produce malt. In turn, the characteristics of thefoods, beverages, and non-food products made of the grain depend uponthe grain’s components, primarily starch, protein, lipid, and B—glucans.

*Research by the authors reported herein was funded by Academy ofFinland Grant 38053

148 AH. Schulman, P. Runeberg-Roos and M. Jfifiskelfiinen

These accumulate during grain filling, the time following fertilization of theovule when the storage compounds which will support the growth of theyoung seedling accumulate. Grain yield, the key to sufficient foodproduction and a long—time breeding goal, is merely the sum of grain-filling activity until the point that the grain dries and ceases growth. Dueto its importance to yield and downstream applications, a fullunderstanding of the mechanism and control of grain filling is essential.

The dry weight of mature barley grains is comprised largely ofcarbohydrate and protein, as seen in Table 1. A mature grain, in addition,contains about 15% water by weight following harvest. The thousand—grain weight for barley is approximately 50 g but varies with the cultivarand number of its rows (6-row barley being lower than 2—row). Yields invariety trials are well correlated with starch content (2) and, in the 15countries of the EU, averaged 4.6 metric tons (range, 1—6.9) per hectare in1998.

Table 1Components of the mature barley grain

Component % of total byweight

Carbohydrates 78 —83

starch 50 — 70

B—D-glucan 3 — 6

arabinoxylans 5

xylose < 1

fructans + raff'mose 2

sucrose 2

other sugars 1

Protein 1 1

Lipid 3

Mineral 2Sources: L. Munck (2); R.J. Henry (3); CM Duffus and M.P. Cochrane (4).

2. GRAIN DEVELOPMENT

Development of the grain and its concomitant filling commencesfollowing the fertilization of the ovule. A double fertilization initiates the

Grain filling and starch synthesis in barley 149

process (5). The first fertilization, involving the fusion of the sperm cellnucleus and the egg cell nucleus, gives rise to the diploid embryo, whereasthe second fertilization, between the second sperm cell nucleus and thetwo nuclei of the central cell, gives rise to the triploid endosperm (6, 7).The production of endosperm, an event restricted to the angiosperms, isviewed as critical to the success of angiosperms in evolution (8). Inmonocotyledonous plants or monocots, including barley and all othercereals, the endosperm is persistent and comes to dominate the grain byweight and volume during grain maturation, ultimately comprising some95% of its total weight (9). The triple fertilization can be thought of,therefore, as the starting point of grain filling. This situation differs fromthat of many dicotyledonous plants or dicots such as pea (castor beanbeing a well—known example of a dicot with a persistent endosperm),where the endosperm is eventually almost completely consumed tosupport the growth of the embryo. The embryonic leaves or cotyledons ofdicots contain the greatest bulk of the storage compounds.

In the monocots, the cellular products of the triple fertilizationultimately differentiate into two tissues, the aleurone layer and thestarchy endosperm, seen in the grain cross-section of Figure l. Thealeurone layer is comprised of one to several cell layers and surrounds thestarchy endosperm. The aleurone is a primarily secretory tissue, andremains living following seed maturation. The proteins that accumulate inthe aleurone during grain filling, and those that are synthesized duringgermination, are primarily enzymes intended for mobilization of thestorage reserves in the starchy endosperm. The starchy endosperm, incontrast, is the main site for the deposition of storage starch and storageproteins and dies at maturity.

The development of the barley endosperm has attracted interest formore than a century (10, 11). The cellular aspects of endospermdifferentiation and development in barley have been most thoroughlystudied by O-A Olsen and coworkers (1 1-20). The length of time requiredfor endosperm differentiation is to some extent variety—dependent andincreases with decreasing temperature. The stages described here are forcv. Bomi gron in a chamber with a diurnal temperature cycle of 15° /10° C as described earlier (13). During the first, or “syncytial,” stage ofendosperm development, the triploid endosperm nucleus dividesmitotically to produce a syncytium, a multinucleate structure lackingintervening cell divisions (14). The syncytial endosperm forms a hollowsphere appressed to the outer, maternal grain layers and surrounding alarge central vacuole. Specific molecular markers have been identified forthis stage (15), which lasts until about 6 days after pollination (DAP), andis followed by the commencement of cellularization. The production ofanticlinal or “free—growing” walls, growing inward toward the centralvacuole, leads to subdivision of the endosperm. The cellularization alsomarks the beginning of the accumulation of callose and B-glucans as part

A

150 A.H. Schulman, P. Runeberg-Roos and M. Jéifiskeléiinen

Figure 1. Scanning electron micrograph of a developing barley grain, cv.Bomi, 18 days after anthesis. In this cross section made mid-grain, thealeurone layer (A) and pericarp (P) layers are visible surrounding thestarchy endosperm (S). Within the starchy endosperm, starch granulescan be seen at this stage. The grain is depicted ventral side down.Source: M. Jaéiskelainen and A.H. Schulman

of the forming walls. The various aspects of the cellularization processhave been described in great detail (14).

Cellularization is complete by 8 DAP and the central vacuole as suchdisappears when the advancing front of cell walls meet in the middle ofthe grain. However, cell divisions continue to subdivide the cells of thestarchy endosperm until about 14 DAP, until about 70 000 cells areformed (20) . At about the same time that cellularization is completed, thealeurone differentiates from the starchy endosperm. Denser cytoplasmand multiple, small vacuoles form in the aleurone cells which becomecharacterized by a distinct pattern of gene expression (17). Cell divisionsin the aleurone layer continue until about 21 DAP, completing formation


100

I

O) O I

Seed

weight

(mg)

AO i

Days after anthesis

Figure 2. Grain filling in barley cv. Bomi. The increase in fresh weight (I)and dry weight (D) of grains from anthesis to maturity is shown.Source: M. Jéiéiskeléjnen and AH. Schulman.

of the cellular structure of the endosperm. This period between thecompletion of cellularization and the completion of cell division in thealeurone is referred to as the differentiation stage (13).

The final stage of endosperm development, maturation, is dominated bythe accumulation of storage products and ends with the drying of thegrain, dormancy of the embryo, and death of the endosperm. Dependingon the growth conditions and barley variety, this extends to approximately40 DAA. In Figure 2, the increase in grain weight during development isdepicted. Fresh weight peaks at about 25 DAA, and declines untilmaturity. After 30 DAA, little starch and protein biosynthesis anddeposition have ended, leading to a plateau in dry weight. A careful studyhas been made of the sex (shrunken endosperm, genie) mutants of barley(13). Although the final grain is reduced in yield of dry matter in each ofthese, they differ in their phenomenology. They are distinguished eitherby the nature of the blocks to development in each, which occurs at one ofthe four stages described above, or by an abnormal overall endosperm

152 AH. Schulman, P. Runeberg—Roos and M. Jéiiiskeliiinen

organization. Identification of the affected genes will be highly informativeregarding the processes involved in the morphogenesis of the barleyendosperm.

3. SOURCE OF CARBON FOR GRAIN FILLING

The filling of the starchy endosperm with storage products, which mightproperly be referred to as “grain filling,” commences before cell divisions inthe endosperm cease. Carbohydrates may be synthesized either fromstored reserves or from de novo fixation of carbon. The stored reservesinclude both assimilates accumulated prior to anthesis in vegetativeorgans as well as those present in the grain but then remobilized. In theearly stages of grain filling, carbohydrate, predominantly starch,biosynthesis is fed by carbohydrates mobilized from the pre-existingstarch granules, polymerized fructans, and free sugars that hadaccumulated in the maternal tissues, in particular the ovule and pericarp.Interest has been focused on photosynthate stored prior to anthesis as acontributor to yield in poor growing seasons (21-23). Studies with wheatand barley indicate, however, that pre—anthesis assimilate contributes onan average only 12% of the total yield under good conditions and 22%under drought stress (21).

Barley, as a temperate grass, can accumulate fructans instead of starchas a storage carbohydrate. Although accumulating primarily in the leaves,fructans can reach 1-2% dry weight in grains (24, 25). Once starchbiosynthesis begins in developing endosperm, it replaces fructans as themajor storage carbohydrate. The accumulated fructan is normally turnedover to provide an additional carbon source to support the starchbiosynthesis (26). In mature, normal grains, fructans represent aminuscule proportion of the total stored carbohydrates, although in theshx mutant where starch biosynthesis is partially blocked the fructans arepersistent (27).

The major source of carbon for grain filling, however, comes from theflag leaf and from the awns in barley, where rates of photosynthesis arefairly high (28). Early work (29, 30), confirmed recently (31), indicatedthat 17—30% of the C02 fixed into the wheat grain as carbohydrate derivesfrom the ear, 10% comes from earlier reserves, and the rest from the flagleaf, whereas in barley 50% is from the ear due to the longer awns.

4. DEPOSITION OF STARCH

Starch biosynthesis takes place in the living cells of the endosperm,within amyloplasts which are specialized plastids derived, as likewise arechloroplasts and chromoplasts, from proplastids. Research on tobacco


indicates that the differentiation of proplastids into amyloplasts iscontrolled by the plant hormones auxin and cytokinin, cytokinin up—regulating the transcription of a suite of genes necessary for starchbiosynthesis and concomitantly increasing starch accumulation itself.Amyloplasts are found not only in endosperm but also in other regions ofstarch accumulation such as tubers. Starch is also synthesized fortransient assimilate storage in leaf chloroplasts. Starch as it issynthesized accumulates as insoluble granules, the shape and size ofwhich are characteristic for the tissue and plant species. In storagetissues, the granules grow to occupy the entire amyloplast, eventuallydisrupting the plastid itself.

In developing endosperm, starch granules first begin to appear within aday of the onset of the cellularization phase, discussed above in Section 1(32). This involves the expression of a set of genes dedicated to starchbiosynthesis, discussed below in more detail, which are induced beforestarch granules become visible. Starch biosynthesis in the leaves andstorage organs of various species has been studied since the 1960's, sothe biochemistry is fairly clear. In recent years, the enzymatic roles,localization, and expression pattern of the isozymes involved in thebiosynthesis have begun to be clarified, greatly increasing ourunderstanding of the overall process.

4.1. Entry of photosynthate into the starch biosynthetic pathwayA general outline of starch biosynthesis with its key enzymes and

metabolites is presented in Figure 3. Photosynthate arrives to theendosperm in the form of sucrose via the phloem of the maternal tissues.Both source and sink strength are critical to ultimate starch yield in thedeveloping endosperm. In some plants breakdown and resynthesis ofsucrose appears necessary to maintain a sucrose gradient into theendosperm and thus sink strength. However, in barley no evidence hasbeen found for this process (33). The sucrose taken into the endospermis subsequently converted into UDPglucose by sucrose synthase(UDPglucose: D—fructose-2-glucosy1transferase EC 2.4.1.13) in thefollowing reaction:

Sucrose + UDP —> UDPglucose + D-fructose

This is a reversible reaction but, under the conditions found in storagetissues, the breakdown of sucrose is favored.

In the many systems investigated, sucrose synthase activity appears tobe important to overall sink strength and hence yield; this is thereforelikely to hold for barley as well. Particularly informative in this regardhave been antisense reductions in sucrose synthase levels in transgenicplants such as produced for tomato (34) and potato (35). Also

154 AH. Schulman, P. Runeberg-Roos and M. Jfifiskelfiinen

Flag leaf

Amyloplast CytoplasmSUC

AmyloseAmylope tin

UDPX' . fruc

PPi>CD1PgluclUTPATP

IX\F-6- P

PP ADPgluc

SUC

SUC

frucitans

VacuolePhloem

Figure 3. Schematic diagram of the proposed pathway for starchbiosynthesis in developing barley grains. Photosynthate is transported assucrose from the flag leaf through the phloem to the developing grain. Thekey enzymes directly on the pathway from sucrose to starch are: 1,sucrose synthase; 2, UDPglucose pyrophosphorylase; 3, ADPglucosepyrophosphorylase (AGP); 4, granule—bound starch synthase (GBSS; SSI);5, soluble starch synthase; 6, starch branching enzyme (SBE); 7,debranching enzyme. This scheme is based on current evidence that 95%of AGP activity is cytoplasmic rather than plastidic; in other cell types,particularly leaves, the AGP is localized in the amyloplast and glucose-6—phosphate or glucose-l-phosphate is translocated instead of ADPglucose.

contributing to our understanding of the role of sucrose synthase hasbeen the analyses of the shl and susl mutants of maize (36) and of othersimilar mutations in other plants. In many plants including barley (37),an endosperm—specific form of sucrose synthase is found. In barley, a setof seg (shrunken endosperm genetic) mutations, segl, 3693, 5e96, andseg7, cause chalazal necrosis and thereby hinder sucrose flow into thegrain and thus starch biosynthesis (33, 38, 39).

The UDPglucose, produced by the sucrose synthase, is then convertedto glucose—l—phosphate. This reaction is carried out by UDPglucosepyrophosphorylase (UTPglucose—l—phosphate uridylyltransferase, EC2.7.7.9).

The UDPglucose pyrophosphorylase enzyme has been purified (40) andthe gene encoding it cloned (41) from barley as well as from other plants.


Glucose—l—phosphate is further processed to ADPglucose, the specificnucleotide sugar which serves as the substrate for the starch synthases.This is catalyzed by the enzyme ADPglucose pyrophosphorylase (AGP,glucose—l—phosphate adenylyltransferase, EC 2.7.7.27) in the reaction:

ATP + a-D—glucose—l-phosphate —> Pyrophosphate + ADP—glucose

4.2. ADPglucose pyrophosphorylase and endosperm starchbiosynthesis

The conversion of glucose—l—phosphate to ADP glucose by AGP canbe considered the first committed step of starch biosynthesis. TheAGP in barley and elsewhere has been the most extensively studied ofthe starch biosynthetic enzymes, much of the work coming from thegroup of J. Preiss (Michigan State Univ.). Its properties have beenextensively reviewed (42-44). The enzyme in both photosynthetic andstorage organs is a heterotetramer comprising two regulatory (small)and two catalytic (large) subunits (45). It is generally under allostericregulation in plants, being activated by 3—phosphoglycerate butinhibited by orthophosphate. Due in part to its allosteric regulationand also to the severely shrunken phenotype of AGP mutants in maize(46) and other plants, it has generally been viewed as the gatekeeperfor the flow of carbon into starch in plants and into glycogenelsewhere. However, flux analyses indicate that AGP does not stronglycontrol the flow of carbon into starch (47, 48). In photosynthetictissues, AGP is localized in the chloroplast, as are all the enzymescatalyzing all subsequent steps in starch biosynthesis.

Up until recently, it was accepted that AGP in storage organs is alsoplastidial. However, several lines of evidence has forced a revision ofthat View, at least for barley and maize endosperm (49). The brittle—1(btl) mutation of maize, which accumulates more then ten—fold higherthan normal levels of ADPglucose in developing kernels (50), has beenshown to be an adenylate translocator (51). Analysis of isolatedamyloplasts indicates that some 95% of AGP is cytoplasmic in maize(52). Differential splicing of barley AGP so that the endosperm formlacks a transit peptide gives a consistent picture for barley (53, 54). Areasonable physiological explanation for the difference betweenchloroplasts and amyloplasts in AGP localization is based onchloroplasts being ATP sources and amyloplasts ATP sinks. If AGPwere plastidial in amyloplasts, the ATP would need to be importedwhere it would be converted to PPi and AGPglucose. This would beenergetically less favorable than movement of ADPglucose into theplastid and transport of ADP outward in return. An cytoplasmic AGPcould also be linked with sucrose synthase in storage tissues toconvert UDP—glucose to ADP-glucose Via glucose— 1—phosphate (49).

156 AH. Schulman, P. Runeberg-Roos and M. Jfiaskelainen

4.3. Synthesis of amylaseAmylose is a polymer of glucose, linked primarily by a-1,4 bonds with

occasional a—1,6 branches. The average chain length of barley amylose hasbeen estimated at 1800 glucose units (55). The amylose of barley grainsgenerally comprises about 25% of the total starch. The oc-1,4 links in bothamylose and amylopectin are formed by the starch synthases (EC2.4.1.21) using ADPglucose as the substrate, as has been reviewedextensively (56—59). Recent evidence points to the presence of distinctstarch synthases responsible for amylose biosynthesis respectively in thepericarp and in the endosperm (60). In the endosperm, amylose isprimarily if not exclusively synthesized by starch synthase I (SSI), oftenreferred to as granule-bound starch synthase (GBSS or GBSSI). The roleof SSI has been revealed through analyses of the waxy mutants of manyplants. In these, amylose is almost completely eliminated but amyloselevels are almost unaffected (61-63). The gene for SSI or GBSS has beencloned from barley (64). The enzyme and its gene is highly conservedamong the cereals (65). The rare branches found in amylose arepresumably added not by 881 but instead by a starch branching enzyme(SBE), the activities of which are discussed in more detail below.

4.4. Synthesis of amylopectinAmylopectin is a more complex molecule than is amylose. It is

comprised of linear, oc—1,4—linked glucan chains frequently branched by oc-l,6 bonds. The average chain length (degree—of—polymerization, DP) inamylopectin is only 21 — 25 glucose residues, although by weight-averagemolecular weight (Mw) amylopectin molecules are some 300 times largerthan those of amylose (66). The branch points are not randomlydistributed in the molecule, but tend to be clustered. The chains ofamylopectin are generally classified as the C—chain, the “core” chaincontaining the only reducing glucose in the molecule, the B—chains,branching from the C-chain, and the A—chain, the outermost brancheswhich themselves are not branched. The B-chains are distributed intoseveral size classes, with DPs of 15 —20 present in the linear portions ofclusters and chains of DP 45 —60 extending between clusters. Thebranching and chain-length distribution leads to concentric rings ofamorphous regions containing branch points and crystalline arrays of thelinear chains within the starch granule, which is 75% amylopectin (67—69).

Biosynthesis of amylopectin involves the activities of the so—calledsoluble starch synthases (SSS), often now referred to as 8811 and 85111,and the starch branching enzymes. The starch synthases synthesizingamylopectin have been referred to as “soluble” because it was recognizedfrom the 1960's onwards (70) that an oc-l,4 glucan —synthetic activitycould be released easily and purified from endosperm tissue, whereas asecond form, “granule—bound,” (the GBSS or 881) remained tightly


associated with the starch fraction. Fractionation of the soluble starchsynthases (EC 2.4.1.21) from the endosperm of maize (71), barley (72),and other cereals identified two forms of soluble starch synthase: Type 1,active in vitro in the absence of exogenous glucan primer if particularadditives, especially sodium citrate, were present in the reaction, and TypeII, dependent on the added glucan primer. Type I is stimulated by sodiumcitrate to a greater extent than Type II. In barley, a total of sixsynthetically—active isoforms of soluble starch synthase, three of eachtype, were identified (73). Closer examination of the starch—boundproteins has established that the Waxy—encoded protein, GBSSI, is solelyresponsible for catalyzing formation of oc—1,4 bonds of amylose and ishighly disproportionately associated with the starch granules. The otherstarch synthases can be found both in the stromal portion of the

_ amyloplast and bound to the granule (74).Due to the multiple forms of starch synthase in barley, maize, potato,

pea, and cassava, which are the most—investigated starch- storing crops,the nomenclature is fairly confused. The forms have generally beennamed in order of their chromatographic elution, which is not necessarilyparallel for equivalent forms from different plants. A combination ofanalyses of mutants (75) and transgenics together with alignments of theencoded proteins from various plants (60) will eventually help sort thenomenclature out. In wheat and ostensibly barley endosperm, GBSSI isthe primary amylose-synthetic enzyme, whereas GBSSII produces amylosein non—storage tissues (60). A consensus is, however, emerging to refer toGBSSI as 881, with most of the soluble starch synthases currently beingidentified as 8811 or 88111. In wheat, SSII ~type proteins of 100, 108, and115 kD have been identified which are initially both soluble and granule—bound, but later in endosperm development become increasinglypartitioned onto the granule (76). In barley, both primer-independent and-dependent soluble starch synthase activities have been identified (73). Aprimer-independent form appears to be responsible for a block to starchsynthesis, resulting in lower starch content and higher ADPglucose andsoluble sugar content (27). It, however, causes no alterations inamylopectin structure (77).

The various starch synthases all catalyze formation of the a-1,4—g1ucanbond and add a glucose residue from ADPglucose. However, they appearto play different roles in starch biosynthesis. This may be due torequirements or preferences for different primers as well as to theaccessibility of their product for branching (see below). For example,evidence from mutants of the green alga Chlamydomonas at the locus for8811 indicate that this enzyme plays a role in elongating 8 — 50 —residueglucose chains, an activity that cannot be replaced by other soluble starchsynthase forms (78). Parallel experiments with antisense constructs intransgenic potatoes have been performed (79). These reduce the relativeabundance of chains of DP 18 — 50. The combination of a lack of effect

158 AH. Schulman, P. Runeberg—Roos and M. Jafiskelainen

on amylopectin structure and primer-independent activity of the shxmutant of barley (73, 77) suggests that the t—encoded soluble starchsynthase may play a role in chain initiation rather than extension.

4.5. Branching of amylopectinIn addition to the starch synthases, the starch branching enzyme (SBE,

oc—1,4 glucan, a—1,4 g1ucan—6—g1ucosyl transferase, EC 2.4.1.18, Q—enzyme)is crucial to the formation of amylopectin. The SBEs are transferasesrather than synthases, removing an oc- 1,4 —linked oligoglucan from the endof an amylopectin chain and transferring it into an oc-l,6 positionelsewhere in the molecule. The SBE stimulates amylopectin biosynthesisby increasing the effective substrate concentration — the non-reducing a—glucan ends — for the starch synthases. In many plants, as for the starchsynthases, several forms of SBE have been identified (80). In barley,protein fractionation has revealed four forms: SBE types I, Ila, IIb and alow molecular weight form (81, 82). The genes for SBEIIa and SBEIIb havebeen isolated and sequenced (83); SBElIa is expressed in all tissues, butSBEIIb is endosperm-specific. The level of transcripts for SBEIIb peaks inthe endosperm at 12 days after anthesis, Whereas the pool for SBEIreaches a maximum at 20 days (83). These peaks of expression arecoincident with SSII and GBSSI respectively. The role of SBE in manyplants has been clarified by an analysis of mutants. The general view isthat SBEI transfers longer chains than does SBEII. Evidence from rice(84), maize (62), and other plants indicates that high-amylose, amylase-extender (ae) mutants are actually defective in amylopectin branchingrather than being over-producers of amylose. A similar mutant of barley,amol, yields a high-amylose phenotype (85).

One of the unanswered questions concerning amylopectin biosynthesisis how the SBE might produce the non—random distribution of branchpoints typical of all known amylopectins. Initial suggestions of an answercame from the discovery that the sugary] mutant of maize, whichproduces a highly branched amylopectin referred to as phytoglycogen,lacks a debranching enzyme activity (86). A similar mutant was lateridentified in Chlamydomonas (87). Recent work indicates that theformation of amylopectin in barley endosperm as well may require theactivity of a debranching enzyme (88). The SBE and debranching enzymeshave been proposed to function in discontinuous steps of synthetic andamylolytic activity (69), thereby avoiding a futile cycle of branch additionand removal. Whatever the details of the process itself, the role ofdebranching enzymes in amylopectin (and amylose?) biosynthesis isgaining widespread acceptance.

4.6. The starch granules of barley grainsAll plants, including barley, produce starch granules of characteristic

shapes and typical size distributions. In the Triticeae, the cereal group


that includes barley and wheat, mature grains contain a bimodaldistribution of large A—granules and small B—granules. These differ in thetiming of their appearance in developing grains, in their shape, and intheir properties (89, 90). At maturity, B-granules comprise 15% of thestarch by volume but 85% by number. The A-granules are generallylenticular or oblate whereas the B—granules are more spherical, often withfaceted appressions. The A-granules appear earlier during endospermdevelopment, and increase in both number and volume until near grainmaturity, whereas the B—granules appear in the middle and later stages,increasing in number but reaching a small maximum size , never groninto A—granules. At grain maturity, the A-granules in cv. Bomi have amean diameter of 13 um and the B—granules a mean of 3.7 pm (89). TheA- and B-granules are amylolytically digested during germination indifferent ways, indicating some underlying chemical or structuraldifference. Pinholes are formed in the A-granules, after which thegranules are degraded from the inside out, whereas B—granules aredegraded by surface erosion (91). These differences may be related to thegreater lipid content of B—granules, associated especially with the amylosefraction (92).

The formation of the A— and B—granules appears to be under separategenetic control. The shx mutation in barley reduces the size of A—granulesin particular (89). The Risa 29 mutant contains larger B-granules butnormal A-granules, Risa mutant 527 has smaller A—granules and larger B-granules, and Risa mutant 16 has smaller A—granules but normal B—granules (93). The enzymological nature of these differences remainsunknown, although the modulation in starch synthase expressionpatterns in developing endosperm (73) suggests that particular starchsynthase isozymes may play a role in formation of specific classes ofgranules.

5. DEPOSITION OF PROTEIN

From a nutritional point of View, proteins make up the second majorcomponent (about 10% of the dry weight) of the barley grain, but containlittle of the essential amino acids lysine, methionine, tryptophan andthreonine. Although barley is the poor cousin of wheat when consideringthe baking value of its storage proteins, these proteins are nonethelessmajor contributors to the functional properties of the grain and influenceits melting performance. The deposition of starch and storage proteins isin addition tightly linked during grain filling, so that mutations affectingeither starch or protein biosynthesis have a pleiotropic effect onaccumulation of the other component in the endosperm. For instance, the“high lysine” mutant Risa 1508, which affects a regulatory locus to abolishtrypsin inhibitor CMe expression (94), develops fewer aleurone cells (17),

160 AH. Schulman, P. Runeberg—Roos and M. Jfifiskelfiinen

prematurely ceases enlargement of the starchy endosperm cells (18), andalso deposits larger but fewer B-granules of starch (93). Hence, we shallconsider briefly what is known of protein accumulation in barley.

5.1. Source of nitrogen for grain fillingThe developing endosperm is supplied with both carbohydrates and

nitrogen by the maternal tissues. The nitrogen supplied by the maternaltissue is mobilized not only from nutrients taken up from the soil, but alsofrom proteins that are hydrolyzed in senescing leaves and in the degradingparts of the ovule (95). Changes in the proteinase complement during leafsenescence may be related to the regulation of nitrogen mobilization inbarley (96); cysteine proteinases have been shown to be involved in leafsenescence in both monocotyledonous and dicotyledonous plants (97, 98).Two proteinases, nucellin (99) and nucellain (100), have been identified tobe also present during autolysis of the nucellus in barley. Nucellin is anaspartic proteinase, whereas nucellain, localized in the cell wall, showshomology to an vacuolar-processing enzyme of castor bean (Ricinuscommunis). The exact hydrolytic roles of these proteases, in leaves ornucella, have however not yet been determined. Therefore the regulatorymechanisms involved in the breaking down of abundant proteins, such asribulose—l,5—bisphosphate carboxylase/oxygenase (Rubisco), remain to beelucidated. During seed maturation, leaf senescence proceeds in asequential manner from the lowermost leaves to the higher leaves, butdetailed knowledge of the regulatory genes and steps is needed before anunderstanding of this process can be reached.

5.2. Protein content of the endospermBarley storage proteins are mainly prolamins, a polymorphic mixture of

proteins with Mr values between 30 000 and 90 000 that are deposited intothe starchy endosperm. The ones found in barley can, based on theiramino acid sequences, be classified into three groups, the S—rich, the S—poor, and the high molecular weight (HMW) prolamins. The major (80-90%) fraction of the prolamins belong to the S—rich class, which includesboth polymeric and monomeric components and which constitutes twofamilies of storage proteins, the B and the y-hordeins. The C hordeins areS—poor prolamins, whereas the D hordeins are HMW prolamins (101). Indeveloping grains, the hordeins are transported through the endoplasmicreticulum (ER) to the vacuole. Their synthesis is associated with the up-regulation of proteins involved in the maturation of secretory proteinswithin the ER—lumen (HSP70 and protein disulfide isomerase) as well aswith the up—regulation of proteins that are involved in the transport ofsecretory proteins (SeclSp and Sarlp) from the ER to the cis—Golgi (102).In addition to the hordeins, the protein bodies of the starchy endospermcontain granular inclusions that do not react with antibodies to hordeins.


These inclusions could correspond to the 128 6—globulins that have beenlocalized to the protein bodies of the starchy endosperm in wheat (103).

Unlike the cells of the starchy endosperm, the cells of the aleurone layerdo not contain starch or hordeins. Instead, the aleurone cells contain asthe major storage protein a 78 globulin (104), which become deposited intodiscrete protein storage vacuoles (101, 103). The transport mechanismsinvolved in targeting prolamins and globulins to the vacuoles may differ,but in barley there is no evidence for a pathway from the ER to the vacuolethat would not involve the Golgi (105, 106).

In addition to the major storage proteins, other proteins are present.These seem to play a protective role against insects, fungi, and bacteria inthe resting seeds. Included in this second category of proteins localized inthe endosperm are the chymotryptic inhibitors CH and CI-2 that inhibitchymotrypsin, the trypsin/ cit-amylase inhibitors that inhibit serineproteases and heterologous oc-amylases, the hordothionins that interferewith redox systems, the endochitinases C and T that hydrolyze chitin, andalso inhibitors of protein synthesis (103, 107, 108).

Although the bulk of the hydrolytic enzymes needed for efficientmobilization of both the carbohydrate and the protein reserves of the grainis synthesized only upon germination, some hydrolytic enzymesaccumulate already during seed maturation. Included in this group arecarboxypeptidase II (109) and (B-arnylase, both of which accumulate in thestarchy endosperm during seed maturation (110), and an asparticproteinase (111) which is deposited into protein storage vacuoles of theresting scutellum and aleurone layer (112, 113). In addition, cysteineproteinases have been localized, in small amounts, to protein storagevacuoles before gibberellin treatment (113). The relative amount of theseproteins is low in comparison to the hordeins, Nevertheless, a singleenzyme, B-amylase, alone constitutes 1—2% of the total grain proteins(1 10). The activity of these enzymes is thought to be down—regulated untilseed germination by mechanisms such as covalent linkage to aninactivating protein (in the case of B-amylase, the linkage is to protein Z),intracellular localization, or changes in pH (1 14, l 15).

6. CONCLUSIONS AND FUTURE STRATEGIES

Improvement in the grain filling of barley, together with the eliminationof seed shattering, were perhaps the first breeding activities of mankind,presumably made by the preferential collection of plump grains remainingon the spike at maturity. The difference in protein and starch quantitybetween Hordeum spontaneum, the Wild ancestor of cultivated barley, andall extant landraces and cultivars is great, and is due to humanintervention. These changes have been critically dependent on thecomponents of the grain, the majority of which is starch and protein. A

162 AH. Schulman, P. Runeberg-Roos and M. Jafiskeléiinen

fuller understanding of grain filling in barley and biosynthesis of storagereserves will not only benefit current applications, but make many morepossible.

Two directions may be taken in the future: improvement of barley yield;improvement of barley starch or protein properties. Yield improvement perse, particularly under the agro—economic conditions of over-productionsuch as obtained in Europe, is not being strongly emphasized at present.Nevertheless, yield stability, maintenance of yield under unfavorablegrowing conditions, remains a breeding target. Yield stability itself mayhave many components. One aspect is the accumulation of sufficientphotosynthate and nitrogen early in the gron season to sustain grainfilling should conditions later deteriorate. Disease resistance, particularlyas it affects the photosynthetic performance of the flag leaf, is extremelyimportant. Drought resistance, important both for northern Europeanconditions early in the growing season, as well as for other regions underdryland agricultural regimes, is valuable for maintaining yield. At least inwheat, water deficit has a direct effect on starch biosynthesis, reducingboth the number of B—granules and the size of A—granules (1 16).

Cold and heat tolerance play a role in yield. Regarding barley starchbiosynthesis and starch yield itself, there is considerable evidence that thesoluble starch synthases in particular are heat sensitive (117-119). Thetemperature of the spike during grain filling is particularly critical. Thelong awns of most barley varieties are effective heat sinks, although theyrequire sufficient transpirational water flow for this function (1 17). Hence,protein engineering of the starch synthases to increase their heat tolerancewould be one goal, but would require transgenic barley in order to beimplemented. Barley transformation is not at present compatible withstability of yield under “biodynarnic” or “organic” agricultural regimes, amajor growth sector in the market driven by consumer preferencesparticularly in Western Europe and North America.

Improvement of barley starch and protein quality, in particular, hasmuch potential. Starch functional qualities are derived from the degree ofstarch crystallinity, granule size, lipid content, and amylose content.These in principal are all under genetic control. The degree and pattern ofbranching in amylopectin is critical to starch properties. Modification ofstarch through alteration of SBE, GBSS, and AGP has been undertaken inpotato (79, 120-122). The presence of multiple SBE forms which, thoughhaving different substrate affinities and chain-transfer preferences, may

' partially substitute for one another in knock—outs or knock-downs,considerably complicates the venture (79). Improvements throughmodulation of enzyme levels or activities will here, too, require transgenicmodification of barley. Although barley can be transformed (123—125), it isa labor-intensive process compared with the transformation of rice orstarch—producing dicotyledonous plants such as potato. Nevertheless, theirreplaceable application niches that barley has, such as malt production,


and its major importance as a grain crop in many parts of the world, arguein favor of making this effort in the future.

REFERENCES

1. R.K. Newman and C.W. Newman, Cereal Foods World, 36 (1991) 800.2. L. Munck, in P. R. Shewry, (ed.), Barley: Genetics, Biochemistry,

Molecular Biology and Biotechnology, CAB International,Wallingford, UK, 1992 p. 573.

3. R.J. Henry, J. Inst. Brew., 94 (1988) 71.4. C.M. Duffus and M.P. Cochrane, in A. W. MacGregor and R. S.

Bhatty (eds), Barley: Chemistry and Technology, AmericanAssociation of Cereal Chemists, Inc., St. Paul, MN, 1993 p. 31.

5. DD. Cass and WA. Jensen, Am. J. Bot., 47 (1970) 62.6. TA. Steeves and I.M. Sussex, Patterns in Plant Development,

Cambridge University Press, Cambridge, UK, ed. 2, 1989.7. C. Dumas and BL. Morgensen, Plant Cell, 5 (1993) 1337.8. G.L. Stebbins, Flowering Plants: Evolution Above the Species Leve1.,

Harvard University Press, Cambridge, MA, USA, 1974.9. A. Tallberg, Hereditas, 96 (1982) 229.10. W. Johannsen, Meddelilser fra Carlsberglaboratoriet, Copenhagen,

1884, vol. 2, p. 103.11. O.—A. Olsen, Plant Cell, 10 (1998) 485.12. M. Bosnes, E. Harris, L. Aigeltinger and O.—A. Olsen, Theor. Appl.

Genet, 74 (1987) 177.13. M. Bosnes, F. Weideman and O.—A. Olsen, Plant J., 2 (1992) 661.14. RC. Brown, B.E. Lemmon and O.-A. Olsen, Plant Cell, 6 (1994)

1241.15. D.N. Doan, C. Linnestad and O.—A. Olsen, Plant M01. Biol., 31 (1996)

877.16. SS. Klemsdal, A. Kvaale and O.-A. Olsen, Physiol. Plant, 67 (1986)

453.17. SS. Klemsdal, W. Hughes, A. Lonneborg, R.B. Aalen and O.-A.

Olsen, Mol. Gen. Genet, 228 (1991) 9.18. O.-A. Olsen and T. Krekling, Hereditas, 93 (1980) 147.19. O.-A. Olsen, R.H. Potter and R. Kalla, Seed Sci. Res., 2 (1992) 117.20. A. Kvaale and O.—A. Olsen, Ann. Bot., 57 (1986) 829.21. F. Bidinger, R.B. Musgrave and RA. Fischer, Nature, 270 (1977) 431.22. G.D. Bonnett and L.D. Incoll, J. Exp. Bot., 44 (1993) 83.23. RE. Gaunt and AC. Wright, Plant Pathol., 41 (1992) 688.24. A.M. MacLeod and H. McCorquodale, New Phytol., 57 (1958) 168.25. A.M. MacLeod, J. Inst. Brew., 59 (1953) 462.

164

26.

27.

28.

29.30.31.32.

33.

34.

35.

36.

37.

38.39.40.41.

42.43.

44.

45.46.47.48.

49.50.

51.

52.

53.

A.H. Schulman, P. Runeberg-Roos and M. Jfifiskelfiinen

26. C.J. Pollock and A.J. Cairns, Annu. Rev. Plant Physiol. PlantM01. Biol., 42 (1991) 77.J. Tyynelé, M. Stitt, A. Lonneborg, S. Smeekens and A.H. Schulman,Physiol. Plant, 93 (1995) 77.J .D. Bewley and M. Black, Physiology and Biochemistry of Seeds,Springer-Verlag GMBH, Berlin, , 1978.L.T. Evans and HM. Rawson, Aust. J. Biol. Sci., 23 (1970) 245.DJ. Carr and I.F. Wardlaw, Aust. J. Biol. Sci., 18 (1965) 711.T. Gebbing and H. Schnyder, Plant Physiol., 121 (1999) 871.M.D. Bennett, J.B. Smith and I. Barclay, Phil. Trans. R. Soc. Lond.B, 272 (1975) 199.F.C. Felker, D.M. Peterson and O.E. Nelson, Plant Physiol., 74 (1984)43.M.—A. D'Aousta, S. Yellea and B. Nguyen-Quoc, Plant Cell, 11 (1999)2407.R. Zrenner, M. Salanoubat, L. Willmitzer and U. Sonnewald, Plant J.,7 (1995) 97.RS. Chourey, E.W. Taliercio, S.J. Carlson and Y.L. Ruan, Mol. Gen.Genet., 259 (1998) 88.O. Martinez de Ilarduya, J. Vicente-Carbajosa, P. Sanchez de la H02and P. Carbonero, FEBS Lett., 320 (1993) 177.A.J. Jarvi and RF. Eslick, Crop Sci., 15 (1975) 363.RT. Ramage and L. Crandall, Barley Genet. Newsl., 11 (1981) 34.L. Elling and MR. Kula, J. Biotechnol., 34 (1994) 157.K. Eimert, P. Villand, A. Kilian and LA. Kleczkowski, Gene, 170(1996) 227.J. Preiss, Biotechnol. Annu. Rev., 2 (1996) 259.L. Kleczkowski, P. Villand, A. Lonneborg, O.—A. Olsen and E. Lfithi, Z.Naturforsch., 46c (1991) 605.J. Preiss and T. Romeo, Prog. Nucleic Acid Res. Mol. Biol., 47 (1994)299.T.W. Greene and L.C. Hannah, Plant Cell, 10 (1998) 1295.M.J. Giroux and L.C. Hannah, Mol. Gen. Genet., 242 (1994) 400.K. Denyer, J. Foster and A.M. Smith, Planta, 197 (1995) 57.G.W. Singletary, R. Banisadr and PL. Keeling, Plant Physiol., 113(1997) 293.LA. Kleczkowski, Trends Plant Sci., 1 (1996) 363.J.C. Shannon, F.-M. Pien and K.~C.-. Liu, Plant Physiol., 110 (1996)835.J.C. Shannon, F.M. Pien, H. Cao and KC. Liu, Plant Physiol., 117(1998) 1235.K. Denyer, F. Dunlap, T. Thorbjarnsen, P. Keeling and A.M. Smith,Plant Physiol., 112 (1996) 779.T. Thorbjzrnsen, P. Villand, L.A. Kleczkowski and O.—A. Olsen,Biochem. J., 313 (1996) 133.

164

26.

27.

28.

29.30.31.32.

33.

34.

35.

36.

37.

38.39.40.41.

42.43.

44.

45.46.47.48.

49.50.

51.

52.

53.


26. C.J. Pollock and A.J. Cairns, Annu. Rev. Plant Physiol. PlantM01. Biol., 42 (1991) 77.J. Tyynelé, M. Stitt, A. Lonneborg, S. Smeekens and A.H. Schulman,Physiol. Plant, 93 (1995) 77.J .D. Bewley and M. Black, Physiology and Biochemistry of Seeds,Springer-Verlag GMBH, Berlin, , 1978.L.T. Evans and HM. Rawson, Aust. J. Biol. Sci., 23 (1970) 245.DJ. Carr and I.F. Wardlaw, Aust. J. Biol. Sci., 18 (1965) 711.T. Gebbing and H. Schnyder, Plant Physiol., 121 (1999) 871.M.D. Bennett, J.B. Smith and I. Barclay, Phil. Trans. R. Soc. Lond.B, 272 (1975) 199.F.C. Felker, D.M. Peterson and O.E. Nelson, Plant Physiol., 74 (1984)43.M.—A. D'Aousta, S. Yellea and B. Nguyen-Quoc, Plant Cell, 11 (1999)2407.R. Zrenner, M. Salanoubat, L. Willmitzer and U. Sonnewald, Plant J.,7 (1995) 97.RS. Chourey, E.W. Taliercio, S.J. Carlson and Y.L. Ruan, Mol. Gen.Genet., 259 (1998) 88.O. Martinez de Ilarduya, J. Vicente-Carbajosa, P. Sanchez de la H02and P. Carbonero, FEBS Lett., 320 (1993) 177.A.J. Jarvi and RF. Eslick, Crop Sci., 15 (1975) 363.RT. Ramage and L. Crandall, Barley Genet. Newsl., 11 (1981) 34.L. Elling and MR. Kula, J. Biotechnol., 34 (1994) 157.K. Eimert, P. Villand, A. Kilian and LA. Kleczkowski, Gene, 170(1996) 227.J. Preiss, Biotechnol. Annu. Rev., 2 (1996) 259.L. Kleczkowski, P. Villand, A. Lonneborg, O.—A. Olsen and E. Lfithi, Z.Naturforsch., 46c (1991) 605.J. Preiss and T. Romeo, Prog. Nucleic Acid Res. Mol. Biol., 47 (1994)299.T.W. Greene and L.C. Hannah, Plant Cell, 10 (1998) 1295.M.J. Giroux and L.C. Hannah, Mol. Gen. Genet., 242 (1994) 400.K. Denyer, J. Foster and A.M. Smith, Planta, 197 (1995) 57.G.W. Singletary, R. Banisadr and PL. Keeling, Plant Physiol., 113(1997) 293.LA. Kleczkowski, Trends Plant Sci., 1 (1996) 363.J.C. Shannon, F.-M. Pien and K.~C.-. Liu, Plant Physiol., 110 (1996)835.J.C. Shannon, F.M. Pien, H. Cao and KC. Liu, Plant Physiol., 117(1998) 1235.K. Denyer, F. Dunlap, T. Thorbjarnsen, P. Keeling and A.M. Smith,Plant Physiol., 112 (1996) 779.T. Thorbjzrnsen, P. Villand, L.A. Kleczkowski and O.—A. Olsen,Biochem. J., 313 (1996) 133.


54. T. Thorbjornsen, P. Villand, K. Denyer, O.—A. Olsen and A.M. Smith,

55.

56.57.58.

59.

60.61.

62.

63.

64.

65.

66.

67.68.69.

70.

71.72.73.74.

75.

76.

77.

78.

Plant J., 10 (1996) 243.Y. Kano, N. Kunitake, T. Karakawa, H. Taniguchi and M. Nakamura,Agric. Biol. Chem., 45 (1981) 1969.A.M. Smith, Curr. Opin. Plant Biol., 2 (1999) 223.J. Preiss and MN. Sivak, Genet. Eng. (N Y), 20 (1998) 177.J. Preiss, K. Ball, B. Smith-White, A. Iglesias, G. Kakefuda and L. Li,Biochem. Soc. Trans., 19 (1991) 539.A.M. Smith, K. Denyer and CR. Martin, Plant Physiol., 107 (1995)673.P.L. Vrinten and T. Nakamura, Plant Physiol., 122 (2000) 255.Z.Y. Wang, F.Q. Zheng, G.Z. Shen, J.P. Gao, D.P. Snustad, M.G. Li,J.L. Zhang and M.M. Hong, Plant J., 7 (1995) 613.Y.-J. Wang, P. White, L. Pollak and J. Jane, Cereal Chem., 70 (1993)171.T. Nakamura, M. Yamamori, H. Hirano, S. Hidaka and T. Nagamine,M01. Gen. Genet, 248 (1995) 253.W. Rhode, D. Becker and F. Salamini, Nucl. Acids Res., 16 (1988)7185.T. Taira, N. F‘ujita, K. Takaoka, M. Uematsu, A. Wadano, S. Kozakiand S. Okabe, Biochem. Genet, 33 (1995) 269.A.W. MacGregor and GB. Fincher, in A. W. MacGregor and R. S.Bhatty (eds.), Barley: Chemistry and Technology, AmericanAssociation of Cereal Chemists, Inc., St. Paul, MN, 1993 p. 73.S. Hizukuri, Carbohydr. Res., 141 (1985) 295.S. Hizukuri and Y. Maehara, Carbohydr. Res., 206 (1990) 145.S. Ball, H.-P. Guan, M. James, A. Myers, P. Keeling, G. Mouille, A.Buléon, P. Colonna and J. Preiss, Cell, 86 (1996) 349.KB. Frydman and CE. Cardini, Biochem. Biophys. Res. Comm., 14(1964) 353.C. Pollock and J. Preiss, Arch. Biochem. Biophys., 204 (1980) 578.AH. Schulman and H. Ahokas, Physiol. Plant, 78 (1990) 583.J. ’I‘yynela and AH. Schulman, Physiol. Plant, 89 (1993) 835.K. Denyer, C. Sidebottom, C.M. Hylton and A.M. Smith, Plant J., 4(1993) 191.J. Craig, J .R. Lloyd, K. Tomlinson, L. Barber, A. Edwards, T.L. Wang,C. Martin, C.L. Hedley and A.M. Smith, Plant Cell, 10 (1998) 413.Z. Li, X. Chu, G. Mouille, L. Yan, B. Kosar-Hashemi, S. Hey, J.Napier, P. Shewry, B. Clarke, R. Appels, M.K. Morell and S. Rahman,Plant Physiol., 120 (1999) 1147.A.H. Schulman, S. Tomooka, A. Suzuki, P. Myllarinen and S.Hizukuri, Carbohydr. Res., 275 (1995) 361.T. Fontaine, C. D'Hulst, M.L. Maddelein, F. Routier, T.M. Pepin, A.Decq, J.M. Wieruszeski, B. Delrue, N. Van den Koornhuyse, J.-P.Bossu, B. Fournet and S. Ball, J. Biol. Chem., 268 (1993) 16223.

166

79.

80.81.

82.

83.

84.

85.86.

87.

88.

89.

90.

91.92.

93.

94.

95.

96.

97.

98.99100.

101.102.103.


J .R. Lloyd, V. Landschutze and J. Kossmann, Biochem. J ., 338(1999) 515.C. Martin and A.M. Smith, Plant Cell, 7 (1995) 971.C. Sun, P. Sathish, B. Ek, A. Deiber and C. Jansson, Physiol. Plant.,96 (1996) 473.C. Sun, P. Sathish, S. Ahlandsberg, A. Deiber and C. Jansson, NewPhytol., 137 (1997).C. Sun, P. Sathish, S. Ahlandsberg and C. Jansson, Plant Physiol.,118 (1998) 37.K. Mizuno, T. Kawasaki, H. Shimada, H. Satoh, E. Kobayashi, S.Okumura, Y. Arai and T. Baba, J. Biol. Chem., 268 (1993) 19084.A.—C. Salomonsson and B. Sundberg, Starch/Starke, 46 (1994) 325.M.G. James, D.S. Robertson and A.M. Myers, Plant Cell, 7 (1995)417.G. Mouille, M.L. Maddelein, N. Libessart, P. Talaga, A. Decq, B.Delrue and S. Ball, Plant Cell, 8 (1996) 1353.C. Sun, P. Sathish, S. Ahlandsberg and C. Jansson, Plant Mol. Biol.,40 (1999) 431.A.H. Schulman, R.F. Tester, H. Ahokas and W.R. Morrison, J. CerealSci., 19 (1994) 49.A.M.L. McDonald, J.R. Stark, W.R. Morrison and RP. Ellis, J. CerealSci., 13 (1991) 93.A.W. MacGregor and D.L. Ballance, Cereal Chem., 57 (1980) 397.P. Myllarinen, K. Autio, A.H. Schulman and K. Poutanen, J. Inst.Brew., 104 (1998) 343.R.F. Tester, W.R. Morrison and A.H. Schulman, J. Cereal Sci., 17(1993) 1. _P. Rodriguez—Palenzuela, J. Royo, L. Gémez, R. Sénchez—Monge, G.Salcedo, J .L. Molina—Cano, F. Garcia-Olmedo and P. Carbonero, Mol.Gen. Genet, 219 (1989) 474.R.J. Simpson, H. Lambers and M.J. Dalling, Plant Physiol., 71 (1983)7.J. Kervinen, M. Kontturi and J. Mikola, Cereal Res. Comm., 18(1990) 191.C.M. Griffiths, S.E. Hosken, D. Oliver, J. Chojecki and H. Thomas,Plant M01. Biol., 34 (1997) 815.F.X. Xu and M.L. Chye, Plant J., 17 (1999) 321.F. Chen and MR. Foolad, Plant Mol. Biol., 35 (1997) 821.C. Linnestad, D.N. Doan, R.C. Brown, B.E. Lemmon, D.J. Meyer, R.Jung and O.-A. Olsen, Plant Physiol., 118 (1998) 1169.RR. Shewry, Biol. ReV., 70 (1995) 375.S. Mogelsvang and D.J. Simpson, Plant M01. Biol., 36 ( 1998) 541.RR. Shewry, in A. W. MacGregor and R. S. Bhatty (eds), Barley:Chemistry and Technology, American Associaton of Cereal Chemists,Inc., St. Paul, Minnesota, USA, 1993 p. 131.


104.

105.106.107.

108.

109.

110.

111.

112.

113.

114.115.116.

117.118.119.

120.

121.

122.

123.

124.125.

T. Yupsanis, S.R. Burgess, P.J. Jackson and RR. Shewry, J. Exp.Bot., 41 (1990) 385.K. Mfintz, Plant Mol. Biol., 38 (1998) 77.J.M. Neuhaus and J.C. Rogers, Plant M01. Biol., 38 (1998) 127.F. Garcia-Olmedo, G. salcedo, R. Sanchez-Monge, C. Hernandez—lucas, M.J. Carmona, J.J . Lopez—Fando, J .A. Fernandez, L. Gomez, J.Royo, F. Garcia-Maroto, A. Castagnaro and P. Carbonero, Trypsin/cr—amylase inhibitors and thionins: Possible defence proteins frombarley. P. R. Shewry, (ed., Barley: Genetics, Biochemistry, MolecularBiology and Biotechnology, The Alden Press, Ltd., Oxford, UK, ,1992.M. Kreis and PR. Shewry, in P. R. Shewry (ed.), Barley: Genetics,Biochemistry, Molecular Biology, and Biotechnology, CAB.International, Wallingford, UK, 1992 p. 319.F. DelDagan, A. Rocher, V. Cameron-Mills and D. Wettstein, Proc.Natl. Acad. Sci. USA, 91 (1994) 8209.M. Kreis, M. Williamson, B. Buxton, J. well, J. Hejgaard and I.Svendsen, Eur. J. Biochem., 169 (1987) 517.P. Runeberg-Roos, J. Kervinen, V. Kovaleva, N. Raikhel and S. Gal,Plant Physiol., 105 (1994) 321.S. Marttjla, B.L. Jones and A. Mikkonen, Physiol. Plant., 93 (1995)317.RC. Bethke, S. Hillmer and R.L. Jones, Plant Physiol., 110 (1996)521.J. Hejgaard, J. Inst. Brew., 84 (1978) 43.S.J. Swanson, P.C. Bethke and R.L. Jones, Plant Cell, 10 (1998) 685.A. Brooks, C.F. Jenner and D. Aspinall, Aust. J. Plant Physiol., 9(1982) 423.R. Savin and ME. Nicolas, Aust. J. Plant Physiol., 23 (1996) 201.A.H.G.C. Rijven, Plant Physiol., 81 (1986) 448.M.A.B. Wallwork, S.J. Logue, L.C. MacLeod and CF. Jenner, Aust. J.Plant Physiol., 25 (1998) 173.J .R. Lloyd, F. Springer, A. Buleon, B. Muller—Rober, L. Willmitzer andJ. Kossmann, Planta, 209 (1999) 230.A.J. Kortstee, A. Vermeesch, M., B.J. de Vries, E. Jacobsen and R.G.Visser, Plant J., 10 (1996) 83.A.G. Kuipers, W.J. Soppe, E. Jacobsen and R.G. Visser, Plant Mol.Biol., 26 (1994) 1759.M.J. Cho, J.H. Wong, C. Marx, W. Jiang, P.G. Lemaux and BB.Buchanan, Proc. Natl. Acad. Sci. USA, 96 (1999) 14641.Y. Wan and P.G. Lemaux, Plant Physiol., 104 (1994) 37.A. Ritala, K. Aspegren, U. Kurtén, M. Sakmenkallio-Marttila, L.Mannonen, R. Hannus, V. Kauppinen, T.H. Teeri and T.—M. Enari,Plant M01. Biol., 24 (1994) 317.

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Grain Filling and Starch Synthesis in Barley...Grain ﬁlling and starch synthesis in barley 149 process (5). The ﬁrst fertilization, involving the fusion of the sperm cell nucleus