The purification and characterisation of the two forms of soluble starch synthase from developing pea embryos

Soluble starch synthase was purified 10000-fold from developing embryos of pea (Pisum sativum L.). The activity was resolved into two forms which together account for most if not all of the soluble starchsynthase activity in the embryo. The two isoforms differ in their molecular weights but are similar in many other respects. Their kinetic properties are similar, neither isoform is active in the absence of primer, and both are unstable at high temperatures, the activity being abolished by a 20-min incubation at 45° C. Both isoforms are recognised by antibodies raised to the granule-bound starch synthase of pea. Isoform II, which has the same molecular weight (77 kDa) as the granulebound enzyme, is recognised more strongly than Isoform I.     

Major differences in isoform composition of starch synthase between leaves and embryos of pea (Pisum sativum L.)

Isoforms of starch synthase (EC 2.4.1.21) in pea (Pisum sativum L.) leaves have been identified and compared with those in developing pea embryos. Purification and immunoprecipitation experiments show that most of the soluble starch synthase activity of the leaf is contributed by a novel isoform (SSIII) that is antigenically related to the major soluble isoform of the potato tuber. The major soluble isoform of the embryo (SSII) is also present in the leaf, but contributes only 15% of the soluble activity. Study of the leaf starch of lam mutant peas, which lack the abundant granule-bound isoform responsible for amylose synthesis in the embryo (GBSSI), indicates that GBSSI is not responsible for the synthesis of amylose-like material in the leaf. Leaves appear to contain a novel granule-bound isoform, antigenically related to GBSSI. The implications of the results for understanding of the role of isoforms of starch synthase are discussed. Received: 13 March 1997 / Accepted: 13 May 1997     

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.Abbreviations DEAE diethylaminoethyl - GBSS granule-bound starch synthase - NT nullisomictetrasomic We are grateful to the late John Hawker (University of Adelaide, Australia) and to John Snape (John Innes Centre, UK) for useful discussions during the course of this work, to John Snape and Catherine Chinoy (John Innes Centre, UK) for the gift of the NT lines and to Richard Batt (University of Adelaide, Australia) for technical assistance.     

Carbohydrates and carbohydrate enzymes in developing cotton ovules

Patterns of carbohydrates and carbohydrate enzymes were investigated in developing cotton ovules to establish which of these might be related to sink strength in developing bolls. Enzymatic analysis of extracted tissue indicated that beginning 1 week following anthesis, immature cotton seeds (Gossypium hirsutum L. cv. Coker 100A glandless) accumulated starch in the tissues which surround the embryo. Starting at 15 days post anthesis (DPA), this starch was depleted and starch simultaneously appeared in the embryo. Sucrose entering the tissues surrounding the embryo was rapidly degraded, apparently by sucrose synthase; the free hexose content of these tissues reached a peak at about 20 DPA. During the first few weeks of development these tissues contained substantial amounts of hexose but little sucrose; the reverse was true for cotton embryos. Embryo sucrose content rose sharply from the end of the first week until about 20 DPA; it then remained roughly constant during seed maturation. Galactinol synthase (EC 2.4.1.x) appeared in the embryos approximately 25 days after flowering. Subsequently, starch disappeared and the galactosides raffinose and stachyose appeared in the embryo. Except near maturity, sucrose synthase (EC 2.4.1.13) activity in the embryos predominated over that of both sucrose phosphate synthase (EC 2.4.1.14) and acid invertase (EC 3.2.1.26). Activities of the latter enzymes increased during the final stages of embryo maturation. The ratio of sucrose synthase to sucrose phosphate synthase was found to be high in young cotton embryos but the ratio reversed about 45 DPA, when developing ovules cease being assimilate sinks. Insoluble acid invertase was present in developing cotton embryos, but at very low activities; soluble acid invertase was present at significant activities only in nearly mature embryos. From these data it appears that sucrose synthase plays an important role in young cotton ovule carbohydrate partitioning and that sucrose phosphate synthase and the galactoside synthesizing enzymes assume the dominant roles in carbohydrate partitioning in nearly mature cotton seeds. Starch was found to be an important carbohydrate intermediate during the middle stages of cotton ovule development and raffinose and stachyose were found to be important carbohydrate pools in mature cotton seeds.     

Growth, sucrose synthase, and invertase activities of developing Phaseolus vulgaris L. fruits

Activities of the sucrose-cleaving enzymes, acid and neutral invertase and sucrose synthase, were measured in pods and seeds of developing snap bean (Phaseolus vulgaris L.) fruits, and compared with ¹⁴C-import, elongation and dry weight accumulation. During the first 10 d post-anthesis, pods elongated rapidly with pod dry weight increase lagging behind by several days. The temporal patterns of acid invertase activity and import coincided closely during the first part of pod development, consonant with a central role for this enzyme in converting imported sucrose during pod elongation and early dry weight accumulation. Later, sucrose synthase became the predominant enzyme of dry weight accumulation and was possibly associated with the development of phloem in pod walls. Sucrose synthase activity in seeds showed two peaks, corresponding to two phases of rapid import and dry weight accumulation; hence, sucrose synthase was associated with seed sink growth. Acid invertase activities in seeds were low and did not show a noticeable relationship with import or growth. All neutral invertase activities, during pod and seed development, were too low for it to have a dominant role in sucrose cleavage. Changes in activities of certain sucrose-cleaving enzymes appear to be correlated with certain sink functions, including import, storage of reserves, and biosynthetic activities. The data supports the association of specific sucrose-cleaving enzymes with the specific processes that occur in the developing pods and seeds of snap bean fruits; for example, acid invertase with pod elongation and sucrose synthase with fruit dry matter accumulation.     

Multiple, distinct isoforms of sucrose synthase in pea

Genes encoding three isoforms of sucrose synthase (Sus1, Sus2, and Sus3) have been cloned from pea (Pisum sativum). The genes have distinct patterns of expression in different organs of the plant, and during organ development. Studies of the isoforms expressed as recombinant proteins in Escherichia coli show that they differ in kinetic properties. Although not of great magnitude, the differences in properties are consistent with some differentiation of physiological function between the isoforms. Evidence for differentiation of function in vivo comes from the phenotypes of rug4 mutants of pea, which carry mutations in the gene encoding Sus1. One mutant line (rug4-c) lacks detectable Sus1 protein in both the soluble and membrane-associated fractions of the embryo, and Sus activity in the embryo is reduced by 95%. The starch content of the embryo is reduced by 30%, but the cellulose content is unaffected. The results imply that different isoforms of Sus may channel carbon from sucrose towards different metabolic fates within the cell.     

Starch synthases and starch branching enzymes from Pisum sativum

Concentrations of ADPglucose:α-1,4-glucan-4-glucosyltransferase (starch synthase) and α-1,4 glucan: α-1,4-glucan-6-glycosyltransferase (branching enzyme) from developing seeds of Pisum sativum were measured. Primed starch synthase activity increased from 8 to 14 days after anthesis and decreased by 50 % at 26 days. Citrate-stimulated starch synthase activity was highest at 10 days after anthesis decreasing to low levels by 22 days. Branching enzyme activity increased from 8 to 18 days after anthesis and decreased little by 26 days. Two fractions of starch synthase were recovered by gradient elution from DEAE-cellulose of extracts from 12- and 18-day-old seeds. The two fractions differed in primer specificity, K_m for ADPG and relative amounts of citrate-stimulated activity. A major and minor fraction of branching enzyme were observed in extracts from both 12- and 18-day-old seeds. Marked differences in the relative abilities ofthe two branching enzyme fractions to stimulate phosphorylase and to branch amylose as well as pH optima were found. Although the content of the starch synthase and branching enzyme fractions varied with seed age, little difference was seen in the properties of chromatographically similar fractions. Therefore, the changes in starch synthase and branching enzyme activity during pea seed development resulted from changes in the concentrations of a few enzyme forms, but not the appearance of different enzyme forms.     

云南野生稻籽粒淀粉合成关键酶活性测定

为研究云南3种野生稻直链淀粉合成机制并利用其直链淀粉含量较低的优良品质,以云南3种野生稻和4种当地栽培稻为材料,研究野生稻灌浆期籽粒4种淀粉合成关键酶(包括ADPG焦磷酸化酶、可溶性淀粉合成、颗粒凝结型淀粉合成酶、淀粉分支酶)活性变化。结果表明,野生稻中4种淀粉合成酶的变化趋势与栽培稻相似,但活性有较大差别。颗粒凝结型淀粉合成酶的活性与直链淀粉含量呈正相关,说明在野生稻中同样是由颗粒凝结型淀粉合成酶控制直链淀粉的合成。同时发现在同一灌浆期,同种材料中可溶性淀粉合成酶和淀粉分支酶的活性变化呈相反趋势,推测这两种酶之间可能在淀粉合成过程中存在某种反馈调节机制。     

Approaches to influence starch quantity and starch quality in transgenic plants

Starch plays a major role as a transitory and long-term storage compound in higher plants, and therefore is of prime importance for plant growth and development. Additionally, starch serves as a widely used material for a variety of industrial uses. The formation of starch can arbitrarily be divided into three types of event: (I) those leading to the supply of glucose-1-phosphate in the plastids; (II) the conversion of glucose-1-phosphate to ADP-glucose catalysed by the enzyme ADP-glucose pyrophosphorylase; and (III) the enzymatic reactions converting ADP-glucose to long-chain glucans (amylopectin, amylose). In recent years, numerous cDNA and genomic sequences encoding enzymes involved in starch metabolism have been identified. Some of these have been used to down-regulate enzyme activities via the antisense RNA technique. Additionally, bacterial genes have been ectopically expressed in transgenic plants in order to increase corresponding enzyme activities. By modulating the activity of ADP-glucose pyrophosphorylase in plastids, it was possible to decrease and increase, respectively, the starch content in source and sink organs of transgenic plants. In addition, down-regulation of granule-bound starch synthase (isoform I) resulted in the production of starch that was almost completely free of amylose. Further experiments aimed to modulate starch structure are currently underway and will briefly be discussed.     

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally. I am grateful to my colleagues Kay Denyer, Ian Dry (CSIRO, Adelaide, Australia), Rob Ireland (Mount Allison University, New Brunswick, Canada), Cathie Martin and Steve Rawsthorne for useful discussions during the course of this work, Cliff Hedley for the gift of pea seeds, and Ian Bedford for preparing pea starch and gels of starch-granule-bound proteins. This work was supported by the Agriculture and Food Research Council via a grant-in-aid to the John Innes Institute.     

禾谷类作物淀粉合酶

淀粉合酶是禾谷类作物淀粉合成所必需的一类酶.根据淀粉合酶家族成员的氨基酸序列的相似性,分别介绍了一个颗粒性淀粉合酶亚家族和四个可溶淀粉合酶亚家族的组成、基因结构和表达特点,并从转录、转录后和翻译后水平上对这些基因的表达调控做了概述.     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Suppression of sucrose synthase gene expression represses cotton fiber cell initiation,elongation, and seed development

Cotton is the most important textile crop as a result of its long cellulose-enriched mature fibers. These single-celled hairs initiate at anthesis from the ovule epidermis. To date, genes proven to be critical for fiber development have not been identified. Here, we examined the role of the sucrose synthase gene (Sus) in cotton fiber and seed by transforming cotton with Sus suppression constructs. We focused our analysis on 0 to 3 days after anthesis (DAA) for early fiber development and 25 DAA, when the fiber and seed are maximal in size. Suppression of Sus activity by 70% or more in the ovule epidermis led to a fiberless phenotype. The fiber initials in those ovules were fewer and shrunken or collapsed. The level of Sus suppression correlated strongly with the degree of inhibition of fiber initiation and elongation, probably as a result of the reduction of hexoses. By 25 DAA, a portion of the seeds in the fruit showed Sus suppression only in the seed coat fibers and transfer cells but not in the endosperm and embryo. These transgenic seeds were identical to wild-type seeds except for much reduced fiber growth. However, the remaining seeds in the fruit showed Sus suppression both in the seed coat and in the endosperm and embryo. These seeds were shrunken with loss of the transfer cells and were <5% of wild-type seed weight. These results demonstrate that Sus plays a rate-limiting role in the initiation and elongation of the single-celled fibers. These analyses also show that suppression of Sus only in the maternal seed tissue represses fiber development without affecting embryo development and seed size. Additional suppression in the endosperm and embryo inhibits their own development, which blocks the formation of adjacent seed coat transfer cells and arrests seed development entirely.     

Genetic evidence that the two isozymes of sucrose synthase present in developing maize endosperm are critical, one for cell wall integrity and the other for starch biosynthesis

In maize, two paralogous genes, Sh1 and Sus1, encode two biochemically similar isozymes of sucrose synthase, SS1 and SS2, respectively. Previous studies have attributed the mild starch deficiency of the shrunken1 (sh1) endosperm to the loss of the SS1 isozyme in the mutant. Here we describe the first mutation in the sucrose synthase1 (Sus1) gene, sus1-1, and the isolation of a double recessive genotype, sh1 sus1-1. Combined data from diverse studies, including Northern and Western analyses, RT-PCR and genomic PCR, cloning and sequencing data for the 3′ region, show that the mutant sus1-1 gene has a complex pattern of expression, albeit at much reduced levels as compared to the Sus1 gene. Endosperm sucrose synthase activity in sh1 sus1-1 was barely 0.5% of the total activity in the Sh1 Sus1 genotype. Significantly, comparative analyses of Sh1 Sus1, sh1 Sus1 and sh1 sus1-1 genotypes have, for the first time, allowed us to dissect the relative contributions of each isozyme to endosperm development. Starch contents in endosperm of the three related genotypes were 100, 78 and 53%, respectively. Anatomical analyses, which confirmed the previously described early cell degeneration phenotype unique to the sh1 Sus1 endosperm, revealed no detectable difference between the two sh1 genotypes. We conclude that the SS1 isozyme plays the dominant role in providing the substrate for cellulose biosynthesis, whereas the SS2 protein is needed mainly for generating precursors for starch biosynthesis. Received: 22 January 1998 / Accepted: 30 March 1998     

Sink development, sucrose metabolising enzymes and carbohydrate status in turnip (Brassica rapa L.)

Accumulation of 60–70 % of biomass in turnip root takes place between 49–56 days after sowing. To understand the phenomenon of rapid sink filling, the activities of sucrose metabolising enzymes and carbohydrate composition in leaf blades, petiole and root of turnip from 42–66 days of growth were determined. An increase (2–3 folds) in glucose and fructose contents of roots accompanied by an increase in activities of acid and alkaline invertases was observed during rapid biomass accumulating phase of roots. The observed decrease in the activities of acid and alkaline invertases along with sucrose synthase (cleavage) in petiole during this period could facilitate unrestricted transport of sucrose from leaves to the roots. During active root filling period, a decrease in sucrose synthase (cleavage) and alkaline invertase activities was also observed in leaf blades. A rapid decline in the starch content of leaf blades was observed during the phase of rapid sink filling. These metabolic changes in the turnip plant led to increase in hexose content (35–37 %) of total dry biomass of roots at maturity. High hexose content of the roots appears to be due to high acid invertase activity of the root.     

Changes in the activities of carbon metabolizing enzymes with pod development in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> L.)

Activities of some key enzymes of carbon metabolism sucrose synthase, acid and alkaline invertase, phosphoenol pyruvate carboxylase, malic enzyme and isocitrate dehydrogenase were investigated in relation to the carbohydrate status in lentil pods. Sucrose remained the dominant soluble sugar in the pod wall and seed, with hexoses (glucose and fructose) present at significantly lower levels. Sucrose synthase is the predominant sucrolytic enzyme in the developing seeds of lentil (Lens culinaris L.). Acid invertase was associated with pod elongation and showed little activity in seeds. Sucrose breakdown was dominated by alkaline invertase during the development of podwall, while both the sucrose synthase and alkaline invertase were active in the branch of inflorescence. A substantial increase of sucrolytic enzymes was observed at the time of maximum seed filling stage (10–20 DAF) in lentil seed. The pattern of activity of sucrose synthase highly paralleled the phase of rapid seed filling and therefore, can be correlated with seed sink strength. It seems likely that the fruiting structures of lentil utilize phosphoenol pyruvate carboxylase for recapturing respired carbon dioxide. Higher activities of isocitrate dehydrogenase and malic enzyme in the seed at the time of rapid seed filling could be effectively linked to the deposition of protein reserves.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.