Kay Denyer

Starch synthesis in the cereal endosperm

The pathway of starch synthesis in the cereal endosperm is unique, and requires enzyme isoforms that are not present in other cereal tissues or non-cereal plants. Recent information on the functions of individual enzyme isoforms has provided insight into how the linear chains and branch linkages in cereal starch are synthesized and distributed. Genetic analyses have led to the formulation of models for the roles of de-branching enzymes in cereal starch production, and reveal pleiotropic effects that suggest that certain enzymes may be physically associated. For the first time, tools for global analyses of starch biosynthesis are available for cereal crops, and are heralded by the draft sequence of the rice genome.

The Major Form of ADP-Glucose Pyrophosphorylase in Maize Endosperm Is Extra-Plastidial

Preparations enriched in plastids were used to investigate the location of ADP-glucose pyrophosphorylase (AGPase) in the developing endosperm of maize (Zea mays L.). These preparations contained more than 25% of the total activity of the plastid marker enzymes alkaline pyrophosphatase and soluble starch synthase, less than 2% of the cytosolic marker enzymes alcohol dehydrogenase and pyrophosphate, fructose 6-phosphate 1-phosphotransferase, and approximately 3% of the AGPase activity. Comparison with the marker enzyme distribution suggests that more than 95% of the activity of AGPase in maize endosperm is extra-plastidial. Two proteins were recognized by antibodies to the small subunit of AGPase from maize endosperm Brittle-2 (Bt2). The larger of the two proteins was the major small subunit in homogenates of maize endosperm, and the smaller, less abundant of the two proteins was enriched in preparations containing plastids. These results suggest that there are distinct plastidial and cytosolic forms of AGPase, which are composed of different subunits. Consistent with this was the finding that the bt2 mutation specifically eliminated the extra-plastidial AGPase activity and the larger of the two proteins recognized by the antibody to the Bt2 subunit.

Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm

We have investigated the nature and locations of isoforms of starch synthase in the developing endosperm of wheat (Triticum aestivum L.). There are three distinct granule-bound isoforms of 60 kDa (the Waxy gene product), 77 kDa and 100–105 kDa. One of these isoforms, the 77-kDa protein, is also present in the soluble fraction of the endosperm but it contributes only a small proportion of the total soluble activity. Most of the soluble activity is contributed by isoforms which are apparently not also granule-bound. The 60-kDa and 77kDa isoforms of wheat are antigenically related to isoforms of very similar size in the developing pea embryo, but the other isoforms in the endosperm appear to have no counterparts in the pea embryo. The significance of these results in terms of the diversity of isoforms of starch synthase and their locations is discussed.

The control of amylose synthesis

Summary Starch granules are composed of two types of glucose polymer, amylose and amylopectin, that differ in size and structure. One of the most intriguing challenges in understanding starch synthesis is to explain the apparently simultaneous synthesis of two such different polymers. One isoform of starch synthase, GBSSI, is responsible for amylose synthesis but can also contribute to amylopectin synthesis. The factors which determine the partitioning of GBSSI activity between these two processes are largely unknown. Understanding the properties of GBSSI and how these differ from the properties of the amylopectin-synthesising isoforms of starch synthase are important to the understanding of the control of amylose synthesis. In this review, we will describe how the synthesis of amylose and amylopectin are integrated and what factors may determine the relative amounts of these two polymers.

The Altered Pattern of Amylose Accumulation in the Endosperm of Low-Amylose Barley Cultivars Is Attributable to a Single Mutant Allele of Granule-Bound Starch Synthase I with a Deletion in the 5′-Non-Coding Region

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5′-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxyancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.

The lys5 Mutations of Barley Reveal the Nature and Importance of Plastidial ADP-Glc Transporters for Starch Synthesis in Cereal Endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.

A Low-Starch Barley Mutant, Risø 16, Lacking the Cytosolic Small Subunit of ADP-Glucose Pyrophosphorylase, Reveals the Importance of the Cytosolic Isoform and the Identity of the Plastidial Small Subunit

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A- and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.

What Controls the Amount and Structure of Starch in Storage Organs

The regulation of starch synthesis in the starch-storing organs of higher plants-organs such as tubers, the embryos of grain legumes, and the endosperms of cereals, in which starch constitutes 50 to 80% of the dry weight at maturity-is complex and poorly understood. Our ignorance of this process has been highlighted by the recent upsurge of interest in starch synthesis, fueled largely by the possibility of producing novel starches by genetic modification. Details of the metabolic pathway itself remain the subject of controversy, and remarkably little is known of the factors that determine either the rate of synthesis or the structure of the starch in storage organs. Some widely accepted views about the regulation of starch synthesis are no longer tenable or require revision in the light of recent findings. We shall discuss briefly the nature of the pathway of starch synthesis, then examine critically the current ideas about factors that determine the flux through the pathway and two important aspects of starch structure: the ratio of amylose to amylopectin and the branching of amylopectin.

Characterization of the Genes Encoding the Cytosolic and Plastidial Forms of ADP-Glucose Pyrophosphorylase in Wheat Endosperm

In most species, the synthesis of ADP-glucose (Glc) by the enzyme ADP-Glc pyrophosphorylase (AGPase) occurs entirely within the plastids in all tissues so far examined. However, in the endosperm of many, if not all grasses, a second form of AGPase synthesizes ADP-Glc outside the plastid, presumably in the cytosol. In this paper, we show that in the endosperm of wheat (Triticum aestivum), the cytosolic form accounts for most of the AGPase activity. Using a combination of molecular and biochemical approaches to identify the cytosolic and plastidial protein components of wheat endosperm AGPase we show that the large and small subunits of the cytosolic enzyme are encoded by genes previously thought to encode plastidial subunits, and that a gene, Ta.AGP.S.1, which encodes the small subunit of the cytosolic form of AGPase, also gives rise to a second transcript by the use of an alternate first exon. This second transcript encodes an AGPase small subunit with a transit peptide. However, we could not find a plastidial small subunit protein corresponding to this transcript. The protein sequence of the purified plastidial small subunit does not match precisely to that encoded by Ta.AGP.S.1 or to the predicted sequences of any other known gene from wheat or barley (Hordeum vulgare). Instead, the protein sequence is most similar to those of the plastidial small subunits from chickpea (Cicer arietinum) and maize (Zea mays) and rice (Oryza sativa) seeds. These data suggest that the gene encoding the major plastidial small subunit of AGPase in wheat endosperm has yet to be identified.

The evolution of the starch biosynthetic pathway in cereals and other grasses

In most species, the precursor for starch synthesis, ADPglucose, is made exclusively in the plastids by the enzyme ADPglucose pyrophosphorylase (AGPase). However, in the endosperm of grasses, including the economically important cereals, ADPglucose is also made in the cytosol via a cytosolic form of AGPase. Cytosolic ADPglucose is imported into plastids for starch synthesis via an ADPglucose/ADP antiporter (ADPglucose transporter) in the plastid envelope. The genes encoding the two subunits of cytosolic AGPase and the ADPglucose transporter are unique to grasses. In this review, the evolutionary origins of this unique endosperm pathway of ADPglucose synthesis and its functional significance are discussed. It is proposed that the genes encoding the pathway originated from a whole-genome-duplication event in an early ancestor of the grasses.