Grain filling and starch synthesis in barley

Carbohydrate Reserves in Plants - Synthesis and Regulation A.K. Gupta andN. Kaur (Editors) © 2000 Elsevier Science B. V. All rights reserved.

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Grain Filling and Starch Synthesis in Barley Alan H. Schiilman*, 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 barley was later surpassed by rice, wheat, and maize a s major h u m a n staples, important niches such a s malt production nevertheless remain. Furthermore, barley h a s a wide physiological toleramce a n d is a major grain in marginal agricultural areas ranging from western Asia to near the Arctic circle. Barley yield is directly correlated with starch deposition in the developing grains, a process which occurs coordinately with the laying down of storage proteins. In this chapter, we shall consider primarily stgirch biosynthesis with respect to grain filling, and also shortly address protein biosynthesis, within the context of the ontogeny of the grain. Grain-filling from the developmental point of view will be examined first, followed by a n analysis of the biosynthesis and deposition of these two major components of the grain.

1. INTRODUCTION Barley was perhaps the most important cereal of the Classical world a n d h a s a history of cultivation extending back some 9000 years in the Near East (1). Although its u s e a s a staple for h u m a n consumption h a s declined in modern times, it continues to be the world's forth major cereal crop overgill a n d h a s important niche applications such a s the production of malt. Barley maintains its s t a t u s as a major crop in the countries of Northern Europe, a n d enjoys a uniquely broad distribution of cultivation, from the southern shores of the Mediterranean to the Himalayas, the deep s a n d s of Australia, a n d a s far north a s the Arctic circle in Scandinavia. In the 15 EU countries, barley is the second in total area planted a n d in to tad yield behind wheat. Virtually all u s e s of barley depend upon the grain, whether milled a s flour or germinated to produce malt. In turn, the characteristics of the foods, beverages, a n d non-food products made of the grain depend u p o n the grain's components, primarily starch, protein, lipid, a n d P-glucans. •Research by the authors reported herein was funded by Academy of Finland Grant 38053

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These accumulate during grain filling, the time following fertilization of the ovule when the storage compounds which will support the growth of the young seedling accumiolate. Grain yield, the key to sufficient food production a n d a long-time breeding goal, is merely the s u m of grainfilling activity until the point that the grain dries and ceases growth. Due to its importemce to yield and downstream applications, a full understanding of the mechanism and control of grain filling is essential. The dry weight of mature barley grains is comprised largely of carbohydrate £ind protein, a s seen in Table 1. A mature grain, in addition, contains about 15% water by weight following harvest. The thousandgrain weight for barley is approximately 50 g b u t varies with the cultivar a n d n u m b e r of its rows (6-row barley being lower t h a n 2-row). Yields in variety trials are well correlated with starch content (2) and, in the 15 countries of the EU, averaged 4.6 metric tons (range, 1-6.9) per hectare in 1998. Table 1 Components of the mature barley grain Component Carbohydrates starch p-D-glucan arabinoxylains 1 xylose

% of total by weight 78 —83 50 — 70 3—6 5 <1

fructans + raffinose

2

sucrose

2

other sugars

1

Protein Lipid

11 3

Mineral 2 Sources: L. Munck (2); R.J. Henry (3); C M Duffus and M.P. Cochrane (4). 2. GRAIN DEVELOPMENT Development of the grain and its concomitant filling commences following the fertilization of the ovule. A double fertilization initiates the

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process (5). The first fertilization, involving the fusion of the sperm cell nucleus a n d the egg cell nucleus, gives rise to the diploid embryo, whereas the second fertilization, between the second sperm cell nucleus a n d the two nuclei of the central cell, gives rise to the triploid endosperm (6, 7). The production of endosperm, a n event restricted to the angiosperms, is viewed a s critical to the success of angiosperms in evolution (8). In monocotyledonous plants or monocots, including barley a n d all other cereals, the endosperm is persistent amd comes to dominate the grain by weight a n d volume during grain maturation, ultimately comprising some 9 5 % of its total weight (9). The triple fertilization can be thought of, therefore, a s the starting point of grain filling. This situation differs from that of mgmy dicotyledonous plants or dicots such a s pea (castor bean being a well-known example of a dicot with a persistent endosperm), where the endosperm is eventually almost completely consumed to support the growth of the embryo. The embryonic leaves or cotyledons of dicots contain the greatest bulk of the storage compounds. In the monocots, the cellular products of the triple fertilization ultimately differentiate into two tissues, the aleurone layer a n d the starchy endosperm, seen in the grain cross-section of Figure 1. The aleurone layer is comprised of one to several cell layers a n d s u r r o u n d s the starchy endosperm. The aleurone is a primarily secretory tissue, and remains living following seed maturation. The proteins that accumulate in the aleurone during grain filling, and those that aire synthesized during germination, are primarily enzymes intended for mobilization of the storage reserves in the starchy endosperm. The starchy endosperm, in contrast, is the main site for the deposition of storage starch a n d storage proteins a n d dies at maturity. The development of the barley endosperm h a s attracted interest for more t h a n a century (10, 11). The cellular aspects of endosperm differentiation a n d development in barley have been most thoroughly studied by O-A Olsen and coworkers (11-20). The length of time required for endosperm differentiation is to some extent variety-dependent and increases with decreasing temperature. The stages described here are for cv. Bomi growing in a chamber with a diurnal temperature cycle of 15° / 10° C a s described earlier (13). During the first, or ''syncytial," stage of endosperm development, the triploid endosperm nucleus divides mitotically to produce a syncytium, a multinucleate structure lacking intervening cell divisions (14). The syncytial endosperm forms a hollow sphere appressed to the outer, maternal grain layers and surrounding a large central vacuole. Specific molecular markers have been identified for this stage (15), which lasts until about 6 days after pollination (DAP), and is followed by the commencement of cellularization. The production of anticlinal or "free-growing'' walls, growing inward toward the central vacuole, leads to subdivision of the endosperm. The cellularization also m a r k s the beginning of the accumulation of callose and (J-glucans a s part

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Figure 1. Scanning electron miprograph of a developing barley grain, cv. Bomi, 18 days after anthesis. In this cross section made mid-grain, the aleurone layer (A) amd pericarp (P) layers are visible surrounding the starchy endosperm (S). Within the starchy endosperm, starch granules can be seen at this stage. The grain is depicted ventral side down. Source: M. Jaaskelainen a n d A.H. Schulman of the forming walls. The various aspects of the cellularization process have been described in great detail (14). Cellularization is complete by 8 DAP and the central vacuole a s s u c h disappears when the advancing front of cell walls meet in the middle of the grain. However, cell divisions continue to subdivide the ceUs of the starchy endosperm until about 14 DAP, until about 70 000 cells are formed (20) . At about the same time that cellularization is completed, the aleurone differentiates from the starchy endosperm. Denser cytoplasm a n d multiple, small vacuoles form in the aleurone cells which become characterized by a distinct pattern of gene expression (17). CeU divisions in the aleurone layer continue until about 21 DAP, completing formation

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100

10

20 30 Days after anthesis

40

Figure 2. Grain filling in barley cv. Bomi. The increase in fresh weight (•) and dry weight (•) of grains from anthesis to maturity is shown. Source: M. Jaaskelainen and A.H. Schulman. of the cellular structure of the endosperm. This period between the completion of cellularization and the completion of cell division in the aleurone is referred to a s the differentiation stage (13). The final stage of endosperm development, maturation, is dominated by the acciimulation of storage products and ends with the drying of the grain, dormancy of the embryo, and death of the endosperm. Depending on the growth conditions and barley variety, this extends to approximately 40 DAA. In Figure 2, the increase in grain weight during development is depicted. Fresh weight peaks at about 25 DAA, and declines until maturity. After 30 DAA, little starch and protein biosynthesis and deposition have ended, leading to a plateau in dry weight. A careful study h a s been made of the sex (shrunken endosperm, xenic) m u t a n t s of barley (13). Although the final grain is reduced in yield of dry matter in each of these, they differ in their phenomenology. They are distinguished either by the n a t u r e of the blocks to development in each, which occurs at one of the four stages described above, or by am abnormal overall endosperm

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organization. Identification of the affected genes will be highly informative regarding the processes involved in the morphogenesis of the barley endosperm.

3 . SOURCE OF CARBON FOR GRAIN FILLING The filling of the starchy endosperm with storage products, which might properly be referred to a s "grain filling,'' commences before cell divisions in the endosperm cease. Carbohydrates may be synthesized either from stored reserves or from de novo fixation of carbon. The stored reserves include both assimilates accumulated prior to anthesis in vegetative organs a s well a s those present in the grain but then remobilized. In the early stages of grain filling, carbohydrate, predominantly starch, biosynthesis is fed by carbohydrates mobilized from the pre-existing starch granules, polymerized fructains, and free sugars that had accumulated in the maternal tissues, in particular the ovule a n d pericarp. Interest h a s been focused on photosynthate stored prior to anthesis a s a contributor to yield in poor growing seasons (21-23). Studies with wheat a n d barley indicate, however, that pre-anthesis assimilate contributes on a n average only 12% of the total yield under good conditions a n d 22% u n d e r drought stress (21). Barley, a s a temperate grass, cam accumulate fructans instead of starch a s a storage carbohydrate. Although accumulating primarily in the leaves, fructans can reach 1-2% dry weight in grains (24, 25). Once starch biosynthesis begins in developing endosperm, it replaces fructans a s the major storage carbohydrate. The accimaulated fructan is normally turned over to provide a n additional carbon source to support the starch biosynthesis (26). In mature, normal grains, fructans represent a minuscule proportion of the total stored carbohydrates, sdthough in the shx mutaint where starch biosynthesis is partially blocked the fructans are persistent (27). The major source of carbon for grain filling, however, comes from the flag leaf a n d from the awns in barley, where rates of photosynthesis are fairly high (28). Early work (29, 30), confirmed recently (31), indicated t h a t 17-30% of the CO2 fixed into the wheat grain a s carbohydrate derives from the ear, 10% comes from earlier reserves, and the rest from the flag leaf, 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, a s likewise aire chloroplasts a n d chromoplasts, from proplastids. Research on tobacco

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indicates t h a t the differentiation of proplastids into amyloplasts is controlled by the plant hormones auxin and cytokinin, cytokinin u p regulating the transcription of a suite of genes necessary for starch biosynthesis a n d concomitantly increasing starch accumulation itself. Amyloplasts are found not only in endosperm b u t also in other regions of starch accumulation such a s tubers. Starch is also synthesized for trgmsient assimilate storage in leaf chloroplasts. Starch a s it is synthesized acciunulates a s insoluble granules, the shape and size of which are characteristic for the tissue a n d plant species. In storage tissues, the granules grow to occupy the entire aimyloplast, eventuadly disrupting the plastid itself. In developing endosperm, starch granules first begin to appear within a day of the onset of the cellularization phase, discussed above in Section 1 (32). This involves the expression of a set of genes dedicated to starch biosynthesis, discussed below in more detail, which are induced before starch granules become visible. Starch biosynthesis in the leaves and storage organs of various species h a s been studied since the 1960's, so the biochemistry is fairly clear. In recent years, the enzymatic roles, localization, a n d expression pattern of the isozymes involved in the biosynthesis have begun to be clarified, greatly increasing our understanding of the overall process. 4 . 1 . Entry of photosynthate into the starch biosynthetic pathway A general outline of starch biosynthesis with its key enzymes a n d metabolites is presented in Figure 3. Photosynthate arrives to the endosperm in the form of sucrose via the phloem of the maternal tissues. Both source a n d sink strength are critical to ultimate starch yield in the developing endosperm. In some plants breakdown a n d resynthesis of sucrose appears necessary to maintain a sucrose gradient into the endosperm a n d t h u s sink strength. However, in barley no evidence h a s been found for this process (33). The sucrose taken into the endosperm is subsequently converted into UDPglucose by sucrose synthase (UDPglucose: D-fructose-2-glucosyltransferase EC 2.4.1.13) in the following reaction: Sucrose + UDP -^ UDPglucose + D-fructose This is a reversible reaction but, u n d e r the conditions found in storage tissues, the breakdown of sucrose is favored. In the many systems investigated, sucrose synthase activity appears to be important to overall sink strength and hence yield; this is therefore likely to hold for barley a s well. Pairticularly informative in this regard have been antisense reductions in sucrose synthase levels in transgenic plaints such as produced for tomato (34) a n d potato (35). Also

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

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Flag leaf

Amyloplast

Cytoplasm

Amylose A ylope tin PPi'c yrUDPglucv\-fruc UTP ATP

PPi - ^

ADPgluc

^ADPgluc^

T

Phloem

Figure 3. Schematic diagram of t±ie proposed pathway for starch biosynthesis in developing barley grains. Photosynthate is transported a s sucrose from the flag leaf through the phloem to the developing grain. The key enzymes directly on the pathway from sucrose to starch are: 1, sucrose synthase; 2, UDPglucose pyrophosphorylase; 3, ADPglucose pyrophosphorylase (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 9 5 % of AGP activity is cytoplasmic rather t h a n plastidic; in other cell types, particulgirly leaves, the AGP is localized in the aimyloplast £ind glucose-6phosphate or glucose-1-phosphate is translocated instead of ADPglucose. contributing to our understanding of the role of sucrose synthase h a s been the analyses of the shl a n d susl m u t a n t s of maize (36) a n d of other similar mutations in other plants. In many plants including barley (37), a n endosperm-specific form of sucrose synthase is found. In barley, a set of seg (shrunken endosperm genetic) mutations, segl, segS, seg6, and seg7, cause chalazal necrosis and thereby hinder sucrose flow into the grain a n d t h u s starch biosynthesis (33, 38, 39). The UDPglucose, produced by the sucrose synthase, is then converted to glucose-1-phosphate. This reaction is carried out by UDPglucose pyrophosphorylase (UTPglucose-1-phosphate uridylyltransferase, EC 2.7.7.9). The UDPglucose pyrophosphorylase enzyme h a s been purified (40) a n d the gene encoding it cloned (41) from bairley as well a s from other plants.

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Glucose-1-phosphate is further processed to ADPglucose, the specific nucleotide sugar which serves a s the substrate for the starch synthases. This is catalyzed by the enzyme ADPglucose pyrophosphorylase (AGP, glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) in the reaction: ATP + a-D-glucose-1-phosphate -^ Pyrophosphate + ADP-glucose 4 . 2 . ADPglucose pjrrophosphorylase and endosperm starch biosynthesis The conversion of glucose-1-phosphate to ADP glucose by AGP c a n be considered the first committed step of s t a r c h biosynthesis. The AGP in barley a n d elsewhere h a s been the m o s t extensively studied of t h e s t a r c h biosynthetic enzymes, m u c h of the work coming from the group of J . Preiss (Michigan State Univ.). Its properties have been extensively reviewed (42-44). The enzyme in both photosynthetic a n d storage o r g a n s is a h e t e r o t e t r a m e r comprising two regulatory (small) a n d two catalytic (large) s u b u n i t s (45). It is generally u n d e r allosteric regulation in p l a n t s , being activated by 3-phosphoglycerate b u t inhibited by o r t h o p h o s p h a t e . Due in p a r t to its allosteric regulation a n d also to the severely s h r u n k e n phenotype of AGP m u t a n t s in maize (46) a n d other p l a n t s , it h a s generally been viewed a s the gatekeeper for the flow of carbon into s t a r c h in p l a n t s a n d into glycogen elsewhere. However, flux a n a l y s e s indicate t h a t AGP does not strongly control t h e flow of carbon into s t a r c h (47, 48). In p h o t o s y n t h e t i c t i s s u e s , AGP is localized in the chloroplast, a s are all the enzymes catalyzing all s u b s e q u e n t steps in s t a r c h biosynthesis. Up u n t i l recently, it w a s accepted t h a t AGP in storage o r g a n s is also plastidial. However, several lines of evidence h a s forced a revision of t h a t view, a t least for barley a n d maize e n d o s p e r m (49). The brittle-1 (btl) m u t a t i o n of maize, which a c c u m u l a t e s more t h e n ten-fold higher t h a n n o r m a l levels of ADPglucose in developing kernels (50), h a s been shown to be a n adenylate translocator (51). Analysis of isolated a m y l o p l a s t s indicates t h a t some 9 5 % of AGP is cytoplasmic in maize (52). Differential splicing of barley AGP so t h a t the e n d o s p e r m form lacks a t r a n s i t peptide gives a consistent picture for barley (53, 54). A reasonable physiological explanation for the difference between chloroplasts a n d amyloplasts in AGP localization is b a s e d on chloroplasts being ATP s o u r c e s a n d amyloplasts ATP s i n k s . If AGP were plastidial in amyloplasts, the ATP would need to be imported where it would be converted to PPi a n d AGPglucose. This would be energetically less favorable t h a n movement of ADPglucose into the plastid a n d t r a n s p o r t of ADP outward in r e t u r n . An cytoplasmic AGP could also be linked with s u c r o s e s y n t h a s e in storage t i s s u e s to convert UDP-glucose to ADP-glucose via glucose-1-phosphate (49).

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4 . 3 . Synthesis of amylose Amylose is a polymer of glucose, linked primarily by a-1,4 bonds with occasional a-1,6 branches. The average chain length of bairley amylose h a s been estimated at 1800 glucose units (55). The amylose of barley grains generally comprises about 2 5 % of the total starch. The a-1,4 links in both amylose a n d amylopectin are formed by the starch synthases (EC 2.4.1.21) using ADPglucose a s the substrate, a s h a s been reviewed extensively (56-59). Recent evidence points to the presence of distinct starch synthases responsible for amylose biosynthesis respectively in the pericarp a n d in the endosperm (60). In the endosperm, amylose is primarily if not exclusively synthesized by starch synthase I (SSI), often referred to a s granule-bound starch synthase (GBSS or GBSSI). The role of SSI h a s been revealed through ansdyses of the waxy m u t a n t s of many plants. In these, amylose is almost completely eliminated b u t amylose levels are almost unaffected (61-63). The gene for SSI or GBSS h a s been cloned from barley (64). The enzyme a n d its gene is highly conserved among the cereals (65). The rare branches found in amylose are presiomably added not by SSI b u t instead by a starch branching enzyme (SBE), the activities of which are discussed in more detail below. 4 . 4 . Synthesis of amylopectin Amylopectin is a more complex molecule t h a n is amylose. It is comprised of linear, a-l,4-linked glucan chains frequently branched by a1,6 bonds. The average chain length (degree-of-polymerization, DP) in amylopectin is only 21 — 25 glucose residues, although by weight-average molecular weight (Mw) amylopectin molecules are some 300 times larger t h a n those of gimylose (66). The branch points are not randomly distributed in the molecule, b u t tend to be clustered. The chains of amylopectin are generadly classified a s the C-chain, the "core'' chain containing the only reducing glucose in the moleciile, the B-chains, branching from the C-chain, and the A-chain, the outermost branches which themselves are not branched. The B-chains aire distributed into several size classes, with DPs of 15 —20 present in the linear portions of clusters a n d chains of DP 45 —60 extending between clusters. The branching a n d chain-length distribution leads to concentric rings of amorphous regions contaiining branch points and crystalline arrays of the linear chains within the starch granule, which is 7 5 % amylopectin (6769). Biosynthesis of amylopectin involves the activities of the so-caUed soluble starch synthases (SSS), often now referred to a s SSII a n d SSIII, a n d the starch branching enzymes. The starch synthases synthesizing amylopectin have been referred to a s "soluble" because it was recognized from the 1960's onwards (70) that a n a-1,4 glucan -synthetic activity could be released easily a n d purified from endosperm tissue, whereas a second form, "granule-bound," (the GBSS or SSI) remained tightly

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associated with the starch fraction. Fractionation of the soluble starch synthases (EC 2.4.1.21) from the endosperm of maize (71), barley (72), £ind other cereals identified two forms of soluble starch synthase: Type I, active in vitro in the absence of exogenous glucan primer if particular additives, especially sodiimi citrate, were present in the reaction, a n d Type II, dependent on the added glucan primer. Type I is stimulated by sodium citrate to a greater extent t h a n Type II. In bairley, a to tad of six synthetically-active isoforms of soluble starch synthase, three of each type, were identified (73). Closer examination of the starch-bound proteins h a s established that the Wcuq/-encoded protein, GBSSI, is solely responsible for catalyzing formation of a-1,4 bonds of amylose a n d is highly disproportionately associated with the starch granules. The other starch synthases can be found both in the stromal portion of the amtyloplast a n d bound to the granule (74). Due to the multiple forms of starch synthase in barley, maize, potato, pea, a n d cassava, which are the most-investigated starch- storing crops, the nomenclature is fairly confused. The forms have generally been named in order of their chromatographic elution, which is not necessarily parallel for equivalent forms from different plants. A combination of analyses of m u t a n t s (75) and transgenics together with alignments of the encoded proteins from various plants (60) will eventually help sort the nomenclature out. In wheat a n d ostensibly barley endosperm, GBSSI is the primary amylose-synthetic enzyme, whereas GBSSII produces amylose in non-storage tissues (60). A consensus is, however, emerging to refer to GBSSI a s SSI, with most of the soluble starch synthases currently being identified a s SSII or SSIII. In wheat, SSII -type proteins of 100, 108, a n d 115 kD have been identified which are initially both soluble a n d granulebound, b u t later in endosperm development become increasingly partitioned onto the granule (76). In barley, both primer-independent a n d -dependent soluble starch synthase activities have been identified (73). A primer-independent form appears to be responsible for a block to starch synthesis, resulting in lower starch content and higher ADPglucose a n d soluble sugar content (27). It, however, causes no alterations in amylopectin structure (77). The various starch synthases all catalyze formation of the a-l,4-gluc£in bond a n d add a glucose residue from ADPglucose. However, they appear to play different roles in starch biosynthesis. This may be due to requirements or preferences for different primers a s well a s to the accessibility of their product for branching (see below). For example, evidence from m u t a n t s of the green alga Chlamydomonas at the locus for SSII indicate that this enzyme plays a role in elongating 8 — 50 -residue glucose chains, a n activity that cannot be replaced by other soluble starch synthase forms (78). Parallel experiments with antisense constructs in transgenic potatoes have been performed (79). These reduce the relative abundaince of chains of DP 18 — 50. The combination of a lack of effect

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on amylopectin structure a n d primer-independent activity of the shx m u t a n t of barley (73, 77) suggests that the S/zx-encoded soluble starch synthase may play a role in chain initiation rather t h a n extension. 4 . 5 . Branching of amylopectin In addition to the starch synthases, the starch branching enzyme (SBE, a-1,4 glucan, a-1,4 glucan-6-glucosyl transferase, EC 2.4.1.18, Q-enzyme) is crucial to the formation of amylopectin. The SBEs are transferases rather t h a n synthases, removing a n a-1,4 -linked oligoglucan from the end of a n amylopectin chain a n d transferring it into a n a-1,6 position elsewhere in the molecule. The SBE stimulates amylopectin biosynthesis by increasing the effective substrate concentration — the non-reducing aglucan ends — for the starch synthases. In many plants, a s for the starch synthases, several forms of SBE have been identified (80). In barley, protein fractionation h a s revealed four forms: SBE types I, Ila, lib and a low molecular weight form (81, 82). The genes for SBEIIa a n d SBEIIb have been isolated a n d sequenced (83); SBEIIa is expressed in all tissues, but SBEIIb is endosperm-specific. The level of transcripts for SBEIIb peaiks in the endosperm at 12 days after a n thesis, whereas the pool for SBEI reaches a maximium at 20 days (83). These peaks of expression are coincident with SSII and GBSSI respectively. The role of SBE in many plants h a s been clarified by a n anadysis of m u t a n t s . The general view is that SBEI transfers longer chains than does SBEII. Evidence from rice (84), maize (62), a n d other plants indicates that high-amylose, amyloseextender (ae) m u t a n t s are actually defective in amylopectin brsinching rather t h a n being over-producers of amylose. A similar m u t a n t of barley, amoly yields a high-amylose phenotype (85). One of the unanswered questions concerning amylopectin biosynthesis is how the SBE might produce the non-random distribution of branch points typical of all known amylopectins. Initial suggestions of a n answer came from the discovery that the sugary 1 m u t a n t of maize, which produces a highly branched amylopectin referred to a s phytoglycogen, lacks a debranching enzyme activity (86). A similar m u t a n t was later identified in Chlamydomonas (87). Recent work indicates t h a t the formation of amylopectin in barley endosperm a s well may require the activity of a debranching enzyme (88). The SBE a n d debranching enzymes have been proposed to function in discontinuous steps of synthetic a n d amylolytic activity (69), thereby avoiding a futile cycle of branch addition a n d removal. Whatever the details of the process itself, the role of debranching enzymes in amylopectin (and amylose?) biosynthesis is gaining widespread acceptance. 4.6. The starch granules of barley grains All plants, including barley, produce starch granules of characteristic shapes a n d typical size distributions. In the Triticeae, the cereal group

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that includes barley and wheat, mature gredns contain a bimodal distribution of large A-granules a n d small B-granules. These differ in the timing of their appeatrance in developing grains, in their shape, a n d in their properties (89, 90). At maturity, B-granules comprise 15% of the starch by volume but 8 5 % by number. The A-granules are generally lenticular or oblate whereas the B-granules are more spherical, often with faceted appressions. The A-granules appear earlier during endosperm development, a n d increase in both number and volume until near grain maturity, whereas the B-granules appear in the middle a n d later stages, increasing in nimiber b u t reaching a small maximum size , never growing into A-granules. At grain maturity, the A-granules in cv. Bomi have a m e a n diamieter of 13 p m and the B-granules a mean of 3.7 p m (89). The A- a n d B-granules are amylolytically digested during germination in different ways, indicating some underlying chemical or structural difference. Pinholes are formed in the A-granules, after which the granules are degraded from the inside out, whereas B-granules are degraded by surface erosion (91). These differences may be related to the greater lipid content of B-granules, associated especially with the amylose fraction (92). The formation of the A- and B-granules appears to be u n d e r separate genetic control. The shx mutation in barley reduces the size of A-granules in particular (89). The Ris0 29 m u t a n t contains larger B-granules but normal A-granules, Ris0 m u t a n t 527 h a s smaller A-granules and larger Bgranules, a n d Ris0 m u t a n t 16 h a s smaller A-granules b u t normal Bgranules (93). The enzymological nature of these differences remains unknown, although the modulation in starch synthase expression patterns in developing endosperm (73) suggests that particular starch synthase isozymes may play a role in formation of specific classes of granules.

5. DEPOSITION OF PROTEIN From a nutritional point of view, proteins maike u p the second major component (about 10% of the dry weight) of the barley grain, b u t contain little of the essential amino acids lysine, methionine, tryptophan a n d threonine. Although barley is the poor cousin of wheat when considering the baking value of its storage proteins, these proteins are nonetheless major contributors to the functional properties of the grain and influence its malting performance. The deposition of starch a n d storage proteins is in addition tightly linked during grain filling, so that mutations affecting either starch or protein biosynthesis have a pleiotropic effect on accumulation of the other component in the endosperm. For instance, the "high lysine" m u t a n t Ris0 1508, which affects a regulatory locus to abolish trypsin inhibitor CMe expression (94), develops fewer adeurone cells (17),

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prematurely ceases enlargement of the starchy endosperm cells (18), and also deposits larger b u t fewer B-granules of starch (93). Hence, we shall consider briefly what is known of protein accumulation in barley. 5 . 1 . Source of nitrogen for grain filling The developing endosperm is supplied with both carbohydrates and nitrogen by the maternal tissues. The nitrogen supplied by the maternal tissue is mobilized not only from nutrients taken u p from the soil, b u t also from proteins that are hydrolyzed in senescing leaves a n d in the degrading parts of the ovule (95). Changes in the proteinase complement during leaf senescence may be related to the regulation of nitrogen mobilization in barley (96); cysteine proteinases have been shown to be involved in leaf senescence in both monocotyledonous and dicotyledonous plants (97, 98). Two proteinases, nucellin (99) and nuceUain (100), have been identified to be also present during autolysis of the nuceUus in barley. Nucellin is a n aspartic proteinase, whereas nucellain, localized in the cell wall, shows homology to a n vacuolar-processing enzyme of castor bean (Ricinus communis]. The exact hydrolytic roles of these proteases, in leaves or nucella, have however not yet been determined. Therefore the regulatory mechanisms involved in the breaking down of a b u n d a n t proteins, such a s ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco), remain to be elucidated. During seed maturation, leaf senescence proceeds in a sequential m a n n e r from the lowermost leaves to the higher leaves, b u t detaiiled knowledge of the regulatory genes a n d steps is needed before a n understanding of this process can be reached. 5.2. Protein content of the endosperm Barley storage proteins are mainly prolamins, a polymorphic mixture of proteins with Mr values between 30 000 and 90 000 that are deposited into the starchy endosperm. The ones found in barley can, based on their amino acid sequences, be classified into three groups, the S-rich, the Spoor, a n d the high molecular weight (HMW) prolamins. The major (8090%) fraction of the prolamins belong to the S-rich class, which includes both polymeric a n d monomeric components and which constitutes two famines of storage proteins, the B and the y-hordeins. The C hordeins are S-poor prolamins, whereas the D hordeins are HMW prolamins (101). In developing grains, the hordeins are transported through the endoplasmic reticulum (ER) to the vacuole. Their synthesis is associated with the u p regulation of proteins involved in the maturation of secretory proteins within the ER-lumen (HSP70 and protein disulfide isomerase) a s well a s with the up-regulation of proteins that are involved in the transport of secretory proteins (Secl8p a n d Sarlp) from the ER to the ds-Golgi (102). In addition to the hordeins, the protein bodies of the starchy endosperm contain granular inclusions that do not react with antibodies to hordeins.

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These inclusions could correspond to the 12S 5-globulins that have been localized to the protein bodies of the starchy endosperm in wheat (103). Unlike the cells of the starchy endosperm, the cells of the aleurone layer do not contain starch or hordeins. Instead, the aleurone cells contain a s the major storage protein a 7S globulin (104), which become deposited into discrete protein storage vacuoles (101, 103). The transport mechanisms involved in targeting prolamins and globulins to the vacuoles may differ, b u t in barley there is no evidence for a pathway from the ER to the vacuole that 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, a n d bacteria in the resting seeds. Included in this second category of proteins localized in the endosperm are the chymotryptic inhibitors CI-1 a n d CI-2 that inhibit chymotrypsin, the trypsin/ a-amylase inhibitors that inhibit serine proteases a n d heterologous a-amylases, the hordothionins that interfere with redox systems, the endochitinases C and T that hydrolyze chitin, and also inhibitors of protein synthesis (103, 107, 108). Although the bulk of the hydrolytic enzymes needed for efficient mobilization of both the carbohydrate and the protein reserves of the grain is synthesized only upon germination, some hydrolytic enzymes accumulate already during seed maturation. Included in this group are carboxypeptidase II (109) and p-amylase, both of which accumulate in the starchy endosperm during seed maturation (110), a n d a n aspartic proteinase (111) which is deposited into protein storage vacuoles of the resting scuteUum and aleurone layer (112, 113). In addition, cysteine proteinases have been localized, in small amounts, to protein storage vacuoles before gibberellin treatment (113). The relative a m o u n t of these proteins is low in comparison to the hordeins. Nevertheless, a single enzyme, p-amylase, alone constitutes 1-2% of the total grain proteins (110). The activity of these enzymes is thought to be down-regulated until seed germination by mechanisms such a s covalent linkage to a n inactivating protein (in the case of p-amylase, the Hnkage is to protein Z), intracellular localization, or changes in pH (114, 115).

6. CONCLUSIONS AND FUTURE STRATEGIES Improvement in the grain filling of barley, together with the elimination of seed shattering, were perhaps the first breeding activities of mankind, presumably made by the preferential collection of plump grains remaining on the spike at maturity. The difference in protein and starch quantity between Hordeum spontaneum, the wild ancestor of cultivated barley, and all extant landraces and cultivars is great, and is due to h u m a n intervention. These chainges have been critically dependent on the components of the grain, the majority of which is starch and protein. A

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fuller understanding of grain filling in barley a n d biosynthesis of storage reserves will not only benefit current applications, b u t make mainy more possible. Two directions may be taken in the future: improvement of barley yield; improvement of barley starch or protein properties. Yield improvement per se, particularly under the agro-economic conditions of over-production s u c h a s obtained in Europe, is not being strongly emphasized at present. Nevertheless, yield stability, maintenance of yield u n d e r unfavorable growing conditions, remains a breeding target. Yield stability itself may have many components. One aspect is the accumulation of sufficient photosynthate a n d nitrogen early in the growing season to sustain grain filling should conditions later deteriorate. Disease resistance, pairticularly a s it affects the photosynthetic performance of the flag leaf, is extremely important. Drought resistance, important both for northern European conditions early in the growing season, a s well a s for other regions u n d e r dryland agricultural regimes, is valuable for maintaining yield. At least in wheat, water deficit h a s a direct effect on starch biosynthesis, reducing both the n u m b e r of B-granules a n d the size of A-granules (116). Cold a n d heat tolerance play a role in yield. Regarding barley starch biosynthesis a n d starch yield itself, there is considerable evidence that the soluble starch synthases in particular are heat sensitive (117-119). The temperature of the spike during grain filling is particularly critical. The long awns of most beirley varieties are effective heat sinks, although they require sufficient transpirationad water flow for this function (117). Hence, protein engineering of the starch synthases to increase their heat tolerance would be one goal, b u t would require transgenic barley in order to be implemented. Barley tremsformation is not at present compatible with stability of yield u n d e r "biodynamic" or "organic'' agricultural regimes, a major growth sector in the market driven by consumer preferences particularly in Western Europe a n d North America. Improvement of barley starch aind protein quality, in particular, h a s m u c h potential. Starch functional qualities are derived from the degree of starch crystallinity, granule size, lipid content, a n d amylose content. These in principal are all u n d e r genetic control. The degree a n d pattern of branching in amylopectin is critical to starch properties. Modification of starch through alteration of SEE, GBSS, a n d AGP h a s been xindertaken in potato (79, 120-122). The presence of multiple SBE forms which, though having different substrate affinities a n d chain-transfer preferences, may partially substitute for one another in knock-outs or knock-downs, considerably complicates the venture (79). Improvements through modulation of enzyme levels or activities will here, too, require transgenic modification of barley. Although barley can be transformed (123-125), it is a labor-intensive process compared with the transformation of rice or starch-producing dicotyledonous plants such a s potato. Nevertheless, the irreplaceable application niches that barley h a s , such a s malt production.

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