Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the su1 mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, iso1, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.     

作物淀粉生物合成与转基因修饰研究进展

淀粉是高等植物中碳水化合物的主要贮藏形式 ,也是粮食作物产品的最主要成分。淀粉虽然都由直链淀粉和枝链淀粉组成 ,但在不同作物中两者的比例和枝链淀粉结构的存在很大差异。现已明确 ,直链淀粉是在颗粒结合淀粉合成酶 (granule boundstarchsynthase,GBSS)催化下合成的 ,而枝链淀粉是四种酶共同作用的结果 ,它们分别是腺嘌呤 -葡萄糖焦磷酸化酶 (ADP glucosepyrophosphorylase ,AGP) ,可溶性淀粉合成酶 (solublestarchsynthase ,SSS) ,淀粉分枝酶 (starchbranchingenzyme ,SBE)和脱分枝酶 (starchdebranchingenzyme ,DBE)。一方面 ,在不同作物中 ,这些酶本身存在多种形式 ,如在玉米胚乳中 ,AGP有大亚基和小亚基之分 ,SBE又可分BE1,BEIIa ,BEIIb 3种 ,SSS也可分为SSI和SSIII(或SSIIa)两种 ,而DBE也有异淀粉酶 (isoamylase)和限制性糊精酶 (pullu lanase)两种。另一方面 ,控制特定酶的基因 ,在不同作物甚至在同一种作物的不同品种中也可能存在不同的复等位基因 ,如籼稻和粳稻的GBSS分别由蜡质基因Wxa 和Wxb 控制 ,两者编码的GBSS活性差异显著。此外 ,环境条件也可通过影响基因的转录使酶的含量或催化性能发生变化。迄今 ,国内外已获得多种马铃薯和水稻的转基因材料 ,对淀粉合成进行修饰 ,试图培育优质品     

Class I beta-1,3-glucanase and chitinase are expressed in the micropylar endosperm of tomato seeds prior to radicle emergence.

beta-1,3-Glucanase (EC 3.2.1.39) and chitinase (EC 3.2.1.14) mRNAs, proteins, and enzyme activities were expressed specifically in the micropylar tissues of imbibed tomato (Lycopersicon esculentum Mill.) seeds prior to radicle emergence. RNA hybridization and immunoblotting demonstrated that both enzymes were class I basic isoforms. beta-1,3-Glucanase was expressed exclusively in the endosperm cap tissue, whereas chitinase localized to both endosperm cap and radicle tip tissues. beta-1,3-Glucanase and chitinase appeared in the micropylar tissues of gibberellin-deficient gib-1 tomato seeds only when supplied with gibberellin. Accumulation of beta-1,3-glucanase mRNA, protein and enzyme activity was reduced by 100 microM abscisic acid, which delayed or prevented radicle emergence but not endosperm cap weakening. In contrast, expression of chitinase mRNA, protein, and enzyme activity was not affected by abscisic acid. Neither of these enzymes significantly hydrolyzed isolated tomato endosperm cap cell walls. Although both beta-1,3-glucanase and chitinase were expressed in tomato endosperm cap tissue prior to radicle emergence, we found no evidence that they were directly involved in cell wall modification or tissue weakening. Possible functions of these hydrolases during tomato seed germination are discussed.     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

Diurnal oscillation of SBE expression in sorghum endosperm

Spatial and temporal expression patterns of the sorghum SBEI, SBEIIA and SBEIIB genes, encoding, respectively, starch branching enzyme (SBE) I, IIA and IIB, in the developing endosperm of sorghum (Sorghum bicolor) were studied. Full-length genomic and cDNA clones for sorghum were cloned, and the SBEIIA cDNA was used together with gene-specific probes for sorghum SBEIIB and SBEI. In contrast to sorghum SBEIIB, which was expressed primarily in endosperm and embryo, SBEIIA was also expressed in vegetative tissues. All three genes shared a similar temporal expression profile during endosperm development, with a maximum activity at 15-24 d after pollination. This differed from barley and maize, in which SBEI gene activity showed a significantly later onset compared to that of SBEIIA and SBEIIB. Expression of the three SBE genes in the sorghum endosperm exhibited a diurnal rhythm during a 24-h cycle.     

转基因技术改良稻米淀粉品质的研究进展

淀粉是稻米胚乳的主要组成成分, 而淀粉含量和性质的差异又直接决定稻米的品质。早期关于稻米淀粉的研究局限于其组成成分、品种间含量的差异以及稻米淀粉合成的经典遗传学方面。随着现代分子生物学和转基因技术的发展, 稻米品质、淀粉的生物合成及其分子调控成为研究热点。随着淀粉合成相关基因的克隆、分子特性及其表达调控的研究取得了较大进展, 人们也开始尝试利用现代基因工程技术, 高效地改良稻米品质, 如提高或降低淀粉含量及改变支链淀粉的结构。本文从综述稻米淀粉的组成、结构和淀粉合成相关酶的研究进展入手, 探讨了转基因技术改良稻米品质的研究进展和发展前景。     

An enzyme to degrade lettuce endosperm cell walls. Appearance of a mannanase following phytochrome- and gibberellin-induced germination

Summary Lettuce seeds (Lactuca sativa L. cv. Grand Rapids) stimulated to germinate by gibberellin and red light produce large amounts of endo--mannanase. This enzyme increases markedly following radicle emergence and is capable of degrading mannose-containing polysaccharides, which are the major components of the endosperm cell wall. Non-germinated seeds contain little enzyme and under conditions where gibberellin- or red light-stimulated germination is prevented (eg. by abscisic acid or prolonged far red light) enzyme levels remain low. Cycloheximide inhibits the increase in enzyme levels when supplied to germinating seeds, but the enzyme once produced is stable in vivo in the presence of this inhibitor for at least 24h. The majority of the extractable mannanase activity is located in the endosperm and we propose that the function of this enzyme is to mobilise the endosperm cell wall polysaccharides as a nutrient source for the growing embryo.Abbreviations ABA abscisic acid - BA benzyladenine - GA gibberellic acid     

Both subunits of ADP-glucose pyrophosphorylase are regulatory

The allosteric enzyme ADP-Glc pyrophosphorylase (AGPase) catalyzes the synthesis of ADP-Glc, a rate-limiting step in starch synthesis. Plant AGPases are heterotetramers, most of which are activated by 3-phosphoglyceric acid (3-PGA) and inhibited by phosphate. The objectives of these studies were to test a hypothesis concerning the relative roles of the two subunits and to identify regions in the subunits important in allosteric regulation. We exploited an Escherichia coli expression system and mosaic AGPases composed of potato (Solanum tuberosum) tuber and maize (Zea mays) endosperm subunit fragments to pursue this objective. Whereas potato and maize subunits have long been separated by speciation and evolution, they are sufficiently similar to form active mosaic enzymes. Potato tuber and maize endosperm AGPases exhibit radically different allosteric properties. Hence, comparing the kinetic properties of the mosaics to those of the maize endosperm and potato tuber AGPases has enabled us to identify regions important in regulation. The data herein conclusively show that both subunits are involved in the allosteric regulation of AGPase. Alterations in the small subunit condition drastically different allosteric properties. In addition, extent of 3-PGA activation and extent of 3-PGA affinity were found to be separate entities, mapping to different regions in both subunits.     

Comparison of soluble starch synthases and branching enzymes from leaves and kernels of normal andamylose-extender maize

Soluble starch synthases (SS) and branching enzymes (BE) from 20-day-old maize leaves and 22-day-old seeds of normal and amylose-extender (ae) were purified by DEAE-cellulose chromatography. Elution profiles of leaf extracts showed one major SS and two BE fractions from both genotypes. The SS fractions from normal and ae leaf extracts were capable of citrate-stimulated starch synthesis and had different reaction rates with various primers. The two BE fractions from normal leaf extracts differed significantly from each other but not when compared to the same BE from ae. Comparison of BE fractions from ae and normal leaves showed no differences based on chromatographic, kinetic, and immunological properties. Comparison of the leaf enzymes with endosperm enzymes showed major differences. Leaf extracts did not contain SSII or BEIIb observed in endosperm extracts. Developing ae endosperm lacks BEIIb activity and ae is the structural gene for BEIIb. The tissue specific expression of BEIIb in the endosperm provides the basis for explaining the tissue-specific expression of ae. We propose that as BEIIb is expressed in the endosperm, but not leaves, allelic substitution at the ae locus modifies only endosperm starch synthesis.     

Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non-photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.