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.     

Two isoforms of the GBSSI class of granule-bound starch synthase are differentially expressed in the pea plant (Pisum sativum L.)

In addition to the GBSSI isoform of starch synthase described previously, the pea plant contains a second, granule-bound isoform, GBSSIb. GBSSI is abundant in pea embryos and Rhizobium root nodules, is present at low levels in pods and is absent from leaves. Mutations at the lam locus eliminate GBSSI from all of these organs. GBSSIb is present in pods, leaves and nodules and is unaffected by mutations at the lam locus. GBSSI and GBSSIb are very similar in molecular mass, primary sequence, activity and antigenic properties. GBSSIb, like GBSSI, can synthesize amylose in the presence of malto-oligosaccharides in isolated starch granules. However, its role in vivo is unclear. The lam mutation eliminates amylose from the starch of embryos but does not affect the relatively small amounts of amylose-like material in the starch of pods, leaves and nodules. The significance of these results for understanding of the regulation of amylose synthesis is discussed.     

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.     

A 56-kDa protein is a novel granule-bound starch synthase existing in the pericarps, aleurone layers, and embryos of immature seed in diploid wheat (Triticum monococcum L.)

A novel 56-kDa granule-bound starch synthase (GBSS; NDPglucose-starch glucosyltransferase, EC 2.4.1.21) responsible for amylose synthesis was found in the pericarps, aleurone layers and embryos of immature diploid wheat (Triticum monococcum L.). The GBSS and other proteins bound to starch granules of various tissues of immature normal and waxy diploid wheat seeds were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and their activities were examined. In the waxy mutant, the waxy protein (59.5 kDa, GBSSI) was absent, but amylose and GBSS activity were evident in all tissues except the endosperm. Of the proteins bound to starch granules, only the 56-kDa protein was associated with the presence of amylose and GBSS activities in the pericarps, aleurone layers and embryos. Mutations at the waxy locus did not affect the 56-kDa protein in these tissues. Changes in the amount of 56-kDa protein during the course of seed development, and the distribution of the 56-kDa protein in each tissue of immature seeds were quite different from those of the waxy protein. On the other hand, the N-terminal amino acid sequence of the 56-kDa protein had a 40–50% similarity to GBSSI of some other plant species and was antigenically related to the waxy protein. These results strongly suggest that the 56-kDa protein in diploid wheat is a GBSSI class enzyme and, hence, an isoform of the waxy protein. The waxy protein and 56-kDa protein, however, are expressed in different seed tissues and at different stages of seed development. Received: 15 May 1998 / Accepted: 18 June 1998     

Evidence that a 77-kilodalton protein from the starch of pea embryos is an isoform of starch synthase that is both soluble and granule bound.

In this paper we provide further evidence about the nature of a 77-kD starch synthase (SSII) that is both soluble and bound to the starch granules in developing pea (Pisum sativum L.) embryos. Mature SSII gives rise to starch synthase activity when expressed in a strain of Escherichia coli lacking glycogen synthase. In transgenic potatoes (Solanum tuberosum L.) expressing SSII, the protein is both soluble and bound to the starch granules. These results confirm that SSII is a starch synthase and indicate that partitioning between the soluble and granule-bound fraction of storage organs is an intrinsic property of the protein. A 60-kD isoform of starch synthase found both in the soluble and granule-bound fraction of the pea embryos is probably derived by the processing of SSII and is a different gene product from GBSSI, the exclusively granule-bound 59-kD isoform of starch synthase that is similar to starch synthases encoded by the waxy genes of cereals and the amf gene of potatoes. Consistent with this, expression in E. coli of an N-terminally truncated version of SSII gives rise to starch synthase activity.     

The relationship between the rate of starch synthesis, the adenosine 5′-diphosphoglucose concentration and the amylose content of starch in developing pea embryos

Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S _0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant embryos, based on the reduction in their ADPglucose contents and on the S _0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for the determination of starch composition are discussed. Received: 6 February 1999 / Accepted: 22 May 1999     

Characterization of cDNAs encoding two isoforms of granule-bound starch synthase which show differential expression in developing storage organs of pea and potato

We have isolated cDNA clones to two isoforms of granule-bound starch synthase (GBSS) from pea embryos and potato tubers. The sequences of both isoforms are related to that of glycogen synthase from E. coli and one, GBSSI, is very similar to the waxy protein of maize and other species. In pea, GBSSII carries a novel 203-amino-acid domain at its N-terminus. Genes encoding both proteins are expressed during pea embryo development, but GBSSII is most highly expressed earlier in development than GBSSI. Similarly, GBSSI and GBSSII are differentially expressed in developing potato tubers. Expression of both isoforms is much lower in other organs of pea than in embryos. GBSSII is expressed in every organ tested while GBSSI is not expressed in roots, stipules or flowers. The possible consequences of this differential use of GBSS isoforms are 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.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

冬小麦籽粒淀粉合成相关酶活性的日变化

蔗糖向淀粉的转化是决定小麦籽粒产量的重要因素.田间条件下研究了两个小麦(Triticum aestivum L.)品种"鲁麦22"和"鲁麦14"籽粒淀粉合成相关酶:蔗糖合酶(sucrose synthase,SS)、腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucose pyrophosphorylase,ADPGPPase)、可溶性淀粉合酶(soluble starch synthase,SSS)、束缚态淀粉合酶(starch granule-bound synthase,GBSS)的活性以及籽粒ATP含量的日变化.结果表明,上述酶活性呈现明显的昼夜变化特征,酶活性一般在白天较低,而在夜间呈现较高活性,而籽粒ATP含量趋势相反.相关分析表明,白天较低的酶活性可能与气温超过其适宜温度有关.对籽粒淀粉合成相关酶活性日变化的可能因子进行了讨论.     

The isolation and characterization of novel low-amylose mutants of Pisum sativum L.

Mutants of Pisum sativum L. with seeds containing low-amylose starch were isolated by screening a population derived from chemically mutagenized material. In all of the mutant lines selected, the low-amylose phenotype was caused by a recessive mutation at a single locus designated lam. In embryos of all but one mutant line, the 59 kDa granule-bound starch synthase (GBSSI) was absent or greatly reduced in amount. The granule-bound starch synthase activity in developing embryos of the mutants was reduced but not eliminated. These results provide further evidence that amylose synthesis is unique to GBSSI. Other granule-bound isoforms of starch synthase cannot substitute for this protein in amylose synthesis. Examination of iodine-stained starch granules from mutant embryos by light microscopy revealed large, blue-staining cores surrounded by a pale-staining periphery. In this respect, the low-amylose mutants of pea differ from those of other species. The differential staining may indicate that the structure of amylopectin varies between the core and peripheral regions.     

冬小麦籽粒淀粉合成相关酶活性的日变化

蔗糖向淀粉的转化是决定小麦籽粒产量的重要因素。田间条件下研究了两个小麦(TriticumaestivumL.)品种“鲁麦22”和“鲁麦14”籽粒淀粉合成相关酶:蔗糖合酶(sucrosesynthase,SS)、腺苷二磷酸葡萄糖焦磷酸化酶(ADP-glucosepyrophosphorylase,ADPGPPase)、可溶性淀粉合酶(solublestarchsynthase,SSS)、束缚态淀粉合酶(starchgranule-boundsynthase,GBSS)的活性以及籽粒ATP含量的日变化。结果表明,上述酶活性呈现明显的昼夜变化特征,酶活性一般在白天较低,而在夜间呈现较高活性,而籽粒ATP含量趋势相反。相关分析表明,白天较低的酶活性可能与气温超过其适宜温度有关。对籽粒淀粉合成相关酶活性日变化的可能因子进行了讨论。     

Evidence that amylose synthesis occurs within the matrix of the starch granule in potato tubers

Transgenic potatoes expressing reduced levels of granule-bound starch synthase I (GBSSI) have been used to investigate whether the synthesis of amylose occurs at the surface of the starch granule or within the matrix formed by the synthesis and organization of amylopectin. Amylose in these potatoes is wholly or largely confined to a central region of the granule. Consequently this core region stains blue with iodine whereas the peripheral zone stains red. By making extensive measurements of the relative sizes of the granules and their blue-staining cores in tubers over a range of stages of development, we have established that the blue core increases in size as the granule grows. The extent of the increase in size of the blue core is greater in potatoes with higher levels of GBSSI. These data show that amylose synthesis occurs within the matrix of the granule, and are consistent with the idea that the space available in the matrix may be an important determinant of the amylose content of storage starches.     

Contribution of sucrose synthase, ADP-glucose pyrophosphorylase and starch synthase to starch synthesis in developing pea seeds

Using genetic variability existing amongst nine pea genotypes (Pisum sativum L.), the biochemical basis of sink strength in developing pea seeds was investigated. Sink strength was considered to be reflected by the rate of starch synthesis (RSS) in the embryo, and sink activity in the seed was reflected by the relative rate of starch synthesis (RRSS). These rates were compared to the activities of three enzymes of the starch biosynthetic pathway [sucrose synthase (Sus), ADP-glucose pyrophosphorylase and starch synthase] at three developmental stages during seed filling (25, 50 and 75% of the dry seed weight). Complete sets of data collected during seed filling for the nine genotypes showed that, for all enzyme activities (expressed on a protein basis), only Sus in the embryo and seed coat was linearly and significantly correlated to RRSS. The contribution of the three enzyme activities to the variability in RSS and RRSS was evaluated by multiple regression analysis for the first two developmental stages. Only Sus activity in the embryo could explain, at least in part, the significant variability observed for both the RSS and the RRSS at each developmental stage. We conclude that Sus activity is a reliable marker of sink activity in developing pea seeds.     

Discrete forms of amylose are synthesized by isoforms of GBSSI in pea

Amyloses with distinct molecular masses are found in the starch of pea embryos compared with the starch of pea leaves. In pea embryos, a granule-bound starch synthase protein (GBSSIa) is required for the synthesis of a significant portion of the amylose. However, this protein seems to be insignificant in the synthesis of amylose in pea leaves. cDNA clones encoding a second isoform of GBSSI, GBSSIb, have been isolated from pea leaves. Comparison of GBSSIa and GBSSIb activities shows them to have distinct properties. These differences have been confirmed by the expression of GBSSIa and GBSSIb in the amylose-free mutant of potato. GBSSIa and GBSSIb make distinct forms of amylose that differ in their molecular mass. These differences in product specificity, coupled with differences in the tissues in which GBSSIa and GBSSIb are most active, explain the distinct forms of amylose found in different tissues of pea. The shorter form of amylose formed by GBSSIa confers less susceptibility to the retrogradation of starch pastes than the amylose formed by GBSSIb. The product specificity of GBSSIa could provide beneficial attributes to starches for food and nonfood uses.     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.