Development of the grain and its concomitant filling commences following the fertilization of the ovule. A double fertilization initiates the process (5). The first fertilization, involving the fusion of the sperm cell nucleus and the egg cell nucleus, gives rise to the diploid embryo, whereas the second fertilization, between the second sperm cell nucleus and the two nuclei of the centred cell, gives rise to the triploid endosperm (6, 7). The production of endosperm, an event restricted to the angiosperms, is viewed as critical to the success of angiosperms in evolution (8). In monocotyledonous plants or monocots, including barley and all other cereals, the endosperm is persistent and comes to dominate the grain by weight and volume during grain maturation, ultimately comprising some 95% of its total weight (9). The triple fertilization can be thought of, therefore, as the starting point of grain filling. This situation differs from that of many dicotyledonous plants or dicots such as 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 and the starchy endosperm, seen in the grain cross-section of Figure 1. The aleurone layer is comprised of one to several cell layers and surrounds 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 are 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 and storage proteins and dies at maturity.
The development of the barley endosperm has attracted interest for more than a century (10, 11). The cellular aspects of endosperm differentiation and 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 as 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 marks the beginning of the accumulation of callose and p-glucans as part
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) and 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 and A.H. Schulman
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) and 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 and 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 as such disappears when the advancing front of cell walls meet in the middle of the grain. However, cell divisions continue to subdivide the cells 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 and multiple, small vacuoles form in the aleurone cells which become characterized by a distinct pattern of gene expression (17). Cell divisions in the aleurone layer continue until about 21 DAP, completing formation
Figure 2. Grain filling in barley cv. Bomi. The increase in fresh weight (■) and diy weight (□) of grains from anthesis to maturity is shown. Source: M. Jaaskelainen and A.H. Schulman.
Figure 2. Grain filling in barley cv. Bomi. The increase in fresh weight (■) and diy 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 as the differentiation stage (13).
The final stage of endosperm development, maturation, is dominated by the accumulation 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 has been made of the sex (shrunken endosperm, xenic) mutants 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 nature of the blocks to development in each, which occurs at one of the four stages described above, or by an abnormal overall endosperm organization. Identification of the affected genes will be highly informative regarding the processes involved in the morphogenesis of the barley endosperm.
The filling of the starchy endosperm with storage products, which might properly be referred to as "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 as well as 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 fructans, and free sugars that had accumulated in the maternal tissues, in particular the ovule and pericarp. Interest has been focused on photosynthate stored prior to anthesis as a contributor to yield in poor growing seasons (21-23). Studies with wheat and barley indicate, however, that pre-anthesis assimilate contributes on an 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 starch as 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 as the major storage carbohydrate. The accumulated fructan is normally turned over to provide an additional carbon source to support the starch biosynthesis (26). In mature, normal grains, fructans represent a minuscule proportion of the total stored carbohydrates, although in the shx mutant 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 and from the awns in barley, where rates of photosynthesis are fairly high (28). Early work (29, 30), confirmed recently (31), indicated that 17-30% of the CO2 fixed into the wheat grain as 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.
Starch biosynthesis takes place in the living cells of the endosperm, within amyloplasts which are specialized plastids derived, as likewise are chloroplasts and chromoplasts, from proplastids. Research on tobacco indicates that the differentiation of proplastids into amyloplasts is controlled by the plant hormones auxin and cytokinin, cytokinin up-regulating the transcription of a suite of genes necessary for starch biosynthesis and concomitantly increasing starch accumulation itself. Amyloplasts are found not only in endosperm but also in other regions of starch accumulation such as tubers. Starch is also synthesized for transient assimilate storage in leaf chloroplasts. Starch as it is synthesized accumulates as insoluble granules, the shape and size of which are characteristic for the tissue and plant species. In storage tissues, the granules grow to occupy the entire amyloplast, eventually 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 has been studied since the 1960's, so the biochemistry is fairly clear. In recent years, the enzymatic roles, localization, and 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 and 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 and sink strength are critical to ultimate starch yield in the developing endosperm. In some plants breakdown and resynthesis of sucrose appears necessary to maintain a sucrose gradient into the endosperm and thus sink strength. However, in barley no evidence has 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 220.127.116.11) in the following reaction:
Sucrose + UDP -> UDPglucose + D-fructose
This is a reversible reaction but, under 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 as well. Particularly informative in this regard have been antisense reductions in sucrose synthase levels in transgenic plants such as produced for tomato (34) and potato (35). Also
Figure 3. Schematic diagram of the proposed pathway for starch biosynthesis in developing barley grains. Photosynthate is transported as 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 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-1-phosphate is translocated instead of ADPglucose.
contributing to our understanding of the role of sucrose synthase has been the analyses of the shl and susl mutants of maize (36) and of other similar mutations in other plants. In many plants including barley (37), an endosperm-specific form of sucrose synthase is found. In barley, a set of seg (shrunken endosperm genetic) mutations, seg 1, seg3, seg6, and seg7, cause chalazal necrosis and thereby hinder sucrose flow into the grain and thus 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 18.104.22.168).
The UDPglucose pyrophosphorylase enzyme has been purified (40) and the gene encoding it cloned (41) from barley as well as from other plants.
Glucose-1-phosphate is further processed to ADPglucose, the specific nucleotide sugar which serves as the substrate for the starch synthases. This is catalyzed by the enzyme ADPglucose pyrophosphorylase (AGP, glucose-1-phosphate adenylyltransferase, EC 22.214.171.124) in the reaction:
ATP + a-D-glucose-1-phosphate -» Pyrophosphate + ADP-glucose
4.2. ADPglucose pyrophosphorylase and endosperm starch biosynthesis
The conversion of glucose-1-phosphate to ADP glucose by AGP can be considered the first committed step of starch biosynthesis. The AGP in barley and elsewhere has been the most extensively studied of the starch biosynthetic enzymes, much 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 and storage organs is a heterotetramer comprising two regulatory (small) and two catalytic (large) subunits (45). It is generally under allosteric regulation in plants, being activated by 3-phosphoglycerate but inhibited by orthophosphate. Due in part to its allosteric regulation and also to the severely shrunken phenotype of AGP mutants in maize (46) and other plants, it has generally been viewed as the gatekeeper for the flow of carbon into starch in plants and into glycogen elsewhere. However, flux analyses indicate that AGP does not strongly control the flow of carbon into starch (47, 48). In photosynthetic tissues, AGP is localized in the chloroplast, as are all the enzymes catalyzing all subsequent steps in starch biosynthesis.
Up until recently, it was accepted that AGP in storage organs is also plastidial. However, several lines of evidence has forced a revision of that view, at least for barley and maize endosperm (49). The brittle-1 (btl) mutation of maize, which accumulates more then ten-fold higher than normal levels of ADPglucose in developing kernels (50), has been shown to be an adenylate translocator (51). Analysis of isolated amyloplasts indicates that some 95% of AGP is cytoplasmic in maize (52). Differential splicing of barley AGP so that the endosperm form lacks a transit peptide gives a consistent picture for barley (53, 54). A reasonable physiological explanation for the difference between chloroplasts and amyloplasts in AGP localization is based on chloroplasts being ATP sources and amyloplasts ATP sinks. If AGP were plastidial in amyloplasts, the ATP would need to be imported where it would be converted to PPi and AGPglucose. This would be energetically less favorable than movement of ADPglucose into the plastid and transport of ADP outward in return. An cytoplasmic AGP could also be linked with sucrose synthase in storage tissues to convert UDP-glucose to ADP-glucose via glucose-1-phosphate (49).
Amylose 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 has been estimated at 1800 glucose units (55). The amylose of barley grains generally comprises about 25% of the total starch. The a-1,4 links in both amylose and amylopectin are formed by the starch synthases (EC 126.96.36.199) using ADPglucose as the substrate, as has been reviewed extensively (56-59). Recent evidence points to the presence of distinct starch synthases responsible for amylose biosynthesis respectively in the pericarp and in the endosperm (60). In the endosperm, amylose is primarily if not exclusively synthesized by starch synthase I (SSI), often referred to as granule-bound starch synthase (GBSS or GBSSI). The role of SSI has been revealed through analyses of the waxy mutants of many plants. In these, amylose is almost completely eliminated but amylose levels are almost unaffected (61-63). The gene for SSI or GBSS has been cloned from barley (64). The enzyme and its gene is highly conserved among the cereals (65). The rare branches found in amylose are presumably added not by SSI but instead by a starch branching enzyme (SBE), the activities of which are discussed in more detail below.
Amylopectin is a more complex molecule than is amylose. It is comprised of linear, a-1,4-linked glucan chains frequently branched by a-1,6 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 than those of amylose (66). The branch points are not randomly distributed in the molecule, but tend to be clustered. The chains of amylopectin are generally classified as the C-chain, the "core" chain containing the only reducing glucose in the molecule, the B-chains, branching from the C-chain, and the A-chain, the outermost branches which themselves are not branched. The B-chains are distributed into several size classes, with DPs of 15 —20 present in the linear portions of clusters and chains of DP 45 —60 extending between clusters. The branching and chain-length distribution leads to concentric rings of amorphous regions containing branch points and crystalline arrays of the linear chains within the starch granule, which is 75% amylopectin (6769).
Biosynthesis of amylopectin involves the activities of the so-called soluble starch synthases (SSS), often now referred to as SSII and SSIII, and the starch branching enzymes. The starch synthases synthesizing amylopectin have been referred to as "soluble" because it was recognized from the 1960's onwards (70) that an a-1,4 glucan -synthetic activity could be released easily and purified from endosperm tissue, whereas a second form, "granule-bound," (the GBSS or SSI) remained tightly associated with the starch fraction. Fractionation of the soluble starch synthases (EC 188.8.131.52) from the endosperm of maize (71), barley (72), and 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 sodium citrate, were present in the reaction, and Type II, dependent on the added glucan primer. Type I is stimulated by sodium citrate to a greater extent than Type II. In barley, a total of six synthetically-active isoforms of soluble starch synthase, three of each type, were identified (73). Closer examination of the starch-bound proteins has established that the Waxy-encoded protein, GBSSI, is solely responsible for catalyzing formation of a-1,4 bonds of amy lose and is highly disproportionately associated with the starch granules. The other starch 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 been named in order of their chromatographic elution, which is not necessarily parallel for equivalent forms from different plants. A combination of analyses of mutants (75) and transgenics together with alignments of the encoded proteins from various plants (60) will eventually help sort the nomenclature out. In wheat and 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 as SSI, with most of the soluble starch synthases currently being identified as SSII or SSIII. In wheat, SSII -type proteins of 100, 108, and 115 kD have been identified which are initially both soluble and granule-bound, but later in endosperm development become increasingly partitioned onto the granule (76). In barley, both primer-independent and -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 and 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-glucan bond and 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 as well as to the accessibility of their product for branching (see below). For example, evidence from mutants of the green alga Chlamydomonas at the locus for SSII indicate that this enzyme plays a role in elongating 8 — 50 -residue glucose chains, an 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 abundance of chains of DP 18 — 50. The combination of a lack of effect on amylopectin structure and primer-independent activity of the shx mutant of barley (73, 77) suggests that the Shx-encoded soluble starch synthase may play a role in chain initiation rather than extension.
In addition to the starch synthases, the starch branching enzyme (SBE, a-1,4 glucan, a-1,4 glucan-6-glucosyl transferase, EC 184.108.40.206, Q-enzyme) is crucial to the formation of amylopectin. The SBEs are transferases rather than synthases, removing an a-1,4 -linked oligoglucan from the end of an amylopectin chain and transferring it into an a-1,6 position elsewhere in the molecule. The SBE stimulates amylopectin biosynthesis by increasing the effective substrate concentration — the non-reducing a-glucan ends — for the starch synthases. In many plants, as for the starch synthases, several forms of SBE have been identified (80). In barley, protein fractionation has revealed four forms: SBE types I, Ila, lib and a low molecular weight form (81, 82). The genes for SBEIIa and SBEIIb have been isolated and sequenced (83); SBEIIa is expressed in all tissues, but SBEIIb is endosperm-specific. The level of transcripts for SBEIIb peaks in the endosperm at 12 days after anthesis, whereas the pool for SBEI reaches a maximum at 20 days (83). These peaks of expression are coincident with SSII and GBSSI respectively. The role of SBE in many plants has been clarified by an analysis of mutants. The general view is that SBEI transfers longer chains than does SBEII. Evidence from rice (84), maize (62), and other plants indicates that high-amylose, amylose-extender (ae) mutants are actually defective in amylopectin branching rather 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 biosynthesis is how the SBE might produce the non-random distribution of branch points typical of all known amylopectins. Initial suggestions of an answer came from the discovery that the sugary 1 mutant of maize, which produces a highly branched amylopectin referred to as phytoglycogen, lacks a debranching enzyme activity (86). A similar mutant was later identified in Chlamydomonas (87). Recent work indicates that the formation of amylopectin in barley endosperm as well may require the activity of a debranching enzyme (88). The SBE and debranching enzymes have been proposed to function in discontinuous steps of synthetic and amylolytic activity (69), thereby avoiding a futile cycle of branch addition and removal. Whatever the details of the process itself, the role of debranching enzymes in amylopectin (and amylose?) biosynthesis is gaining widespread acceptance.
All 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 bimodal distribution of large A-granules and small B-granules. These differ in the timing of their appearance in developing grains, in their shape, and in their properties (89, 90). At maturity, B-granules comprise 15% of the starch by volume but 85% 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, and increase in both number and volume until near grain maturity, whereas the B-granules appear in the middle and later stages, increasing in number but reaching a small maximum size , never growing into A-granules. At grain maturity, the A-granules in cv. Bomi have a mean diameter of 13 ]im and the B-granules a mean of 3.7 jim (89). The A- and 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 under separate genetic control. The shx mutation in barley reduces the size of A-granules in particular (89). The Riso 29 mutant contains larger B-granules but normal A-granules, Riso mutant 527 has smaller A-granules and larger B-granules, and Riso mutant 16 has smaller A-granules but normal B-granules (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.
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