Suppression of α‐amylase genes improves quality of rice grain ripened under high temperature

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism‐related genes. Here we report the involvement of a starch‐hydrolyzing enzyme, α‐amylase, in high temperature‐triggered grain chalkiness. In developing seeds, high temperature induced the expression of α‐amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α‐amylase activity, while it decreased an α‐amylase‐repressing plant hormone, ABA, suggesting starch to be degraded by α‐amylase in developing grains under elevated temperature. Furthermore, RNAi‐mediated suppression of α‐amylase genes in ripening seeds resulted in fewer chalky grains under high‐temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α‐amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

稻米品质性状对开放式空气二氧化碳浓度增高的响应

利用开放式空气CO2浓度增高(FACE)系统平台。研究大田栽培条件下粳稻武香粳14号稻米品质性状对CO2浓度增高200μmol·mol^-1的响应。结果表明．FACE处理稻谷的出糙率平均比CK高1．4个百分点,整精米率平均比CK低12．3个百分点,较低的供N水平有利于提高FACE条件下的出糙率．较高的供N水平有利于提高FACE条件下的整精米率;FACE处理的稻米垩白略有增加。垩白粒率平均比CK高11．9个百分点,垩白度平均比CK平均高2．8个百分点,较高的供N和供P水平有利于降低FACE条件下垩白大小、垩白粒率和垩白度;FACE处理稻米糊化温度平均比CK平均高0．52℃,胶稠度有提高的趋势,但对稻米直链淀粉含量影响较小,较高的供N和供P水平有利于降低FACE条件下稻米的直链淀粉含量,较低的供N和较高的供P水平有利于降低FACE条件下稻米胶稠度,较低的供N水平有利于降低FACE条件下稻米糊化温度;FACE处理使稻米蛋白质含量比CK平均低0．6个百分点,较低的供N和供P水平有利于降低FACE条件下稻米蛋白质含量。     

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.     

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.     

Tuber physiology and properties of starch from tubers of transgenic potato plants with altered plastidic adenylate transporter activity

We showed recently that antisense plants with decreased activity of the plastidic ATP/ADP-transporter protein exhibit drastically reduced levels of starch and a decreased amylose/amylopectin ratio, whereas sense plants with increased activity of the transporter possessed more starch than wild-type plants and an increased amylose/amylopectin ratio. In this paper we investigate the effect of altered plastidic ATP/ADP-transporter protein expression on primary metabolism and granule morphology in more detail. Tuber tissues from antisense and sense plants exhibited substantially increased respiratory activity compared with the wild type. Tubers from antisense plants contained markedly increased levels of free sugars, UDP-Glc, and hexose phosphates, whereas phosphoenolpyruvate, isocitrate, ATP, ADP, AMP, UTP, UDP, and inorganic pyrophosphate levels were slightly decreased. In contrast, tubers from sense plants revealed a slight increase in adenine and uridine nucleotides and in the levels of inorganic pyrophosphate, whereas no significant changes in the levels of soluble sugars and metabolites were observed. Antisense tubers contained 50% reduced levels of ADP-Glc, whereas sense tubers contained up to 2-fold increased levels of this sole precursor for starch biosynthesis. Microscopic examination of starch grain morphology revealed that the size of starch grains from antisense tubers was substantially smaller (50%) compared with the wild type. The large starch grains from sense tubers appeared of a more angular morphology, which differed to the more ellipsoid shape of wild type grains. The results suggest a close interaction between plastidial adenylate transport and starch biosynthesis, indicating that ADP-Glc pyrophosphorylase is ATP-limited in vivo and that changes in ADP-Glc concentration determine starch yield, as well as granule morphology. Possible factors linking starch synthesis and respiration are discussed.     

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 waxy ancestor. 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.     

Ethylene and ACC levels in developing grains are related to the poor appearance and milling quality of rice

The possible relationship between the levels of ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) in the grains and the quality of rice (Oryza sativa L.) were investigated by using 12 rice cultivars. The results showed that both the ethylene evolution rate and ACC content in grains during the grain filling period correlated negatively with head rice production and positively with chalky kernels, chalky size, and chalkiness. The levels of ethylene and ACC were not significantly correlated with alkali spreading value and amylose content. Application of ethephon, an ethylene-releasing agent, or ACC to panicles at the early grain filling stage significantly reduced the rates of brown rice, milled rice, and head rice, and significantly increased the percentage of chalky kernels, chalky size, and chalkiness. Application of aminoethoxyvinylglycine, an inhibitor of ACC synthase, had the opposite effect. Chalkiness appears to be a senescence related phenomenon which is stimulated by ethylene. The results suggest that ethylene and ACC in grains play an important role in regulating rice quality, and that grain appearance and milling quality would be improved though the reduction of ethylene and ACC in grains during grain filling.     

GBSS‐BINDING PROTEIN,encoding a CBM48 domain‐containing protein,affects rice quality and yield

The percentage of amylose in the endosperm of rice (Oryza sativa) largely determines grain cooking and eating qualities. Granule‐bound starch synthase I (GBSSI) and GBSSII are responsible for amylose biosynthesis in the endosperm and leaf, respectively. Here, we identified OsGBP, a rice GBSS‐binding protein that interacted with GBSSI and GBSSII in vitro and in vivo. The total starch and amylose contents in osgbp mutants were significantly lower than those of wild type in leaves and grains, resulting in reduced grain weight and quality. The carbohydrate‐binding module 48 (CBM48) domain present in the C‐terminus of OsGBP is crucial for OsGBP binding to starch. In the osgbp mutant, the extent of GBSSI and GBSSII binding to starch in the leaf and endosperm was significantly lower than wild type. Our data suggest that OsGBP plays an important role in leaf and endosperm starch biosynthesis by mediating the binding of GBSS proteins to developing starch granules. This elucidation of the function of OsGBP enhances our understanding of the molecular basis of starch biosynthesis in rice and contributes information that can be potentially used for the genetic improvement of yield and grain quality.     

遮光对小麦籽粒淀粉品质和花前贮存非结构碳水化合物转运的影响

以耐荫品种扬麦158和不耐荫品种扬麦11两个小麦品种为材料,研究了拔节至成熟期遮光对小麦籽粒产量、淀粉含量和淀粉糊化特性的影响,并分析了花前营养器官非结构碳水化合物转运及其与籽粒产量、淀粉含量和淀粉糊化特性的关系.结果表明：遮光条件下小麦花前营养器官中可溶性总糖转运量的下降是造成小麦籽粒产量降低的原因之一;小麦支链淀粉在遮光条件下显著降低而直链淀粉变化不明显,导致小麦籽粒总淀粉含量和支／直显著下降;遮光降低了两品种小麦籽粒淀粉峰值粘度;降低了不耐荫品种扬麦11的低谷粘度,提高了其糊化温度,但对耐荫品种扬麦158的低谷粘度和糊化温度无显著影响.     

Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis

In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein-protein interactions.     

粳稻穗角与稻米品质的相关性及稻米品质遗传分析

测定了粳稻直立穗品种丙8979与弯曲穗品种C堡杂交组合的P1、P2及其重组自交系349个株系的穗角和10个稻米品质性状, 分析了穗角与稻米品质性状之间的相关性, 并运用主基因+多基因混合遗传模型, 对稻米品质10个性状进行了遗传分析。结果表明,穗角与糙米率、整精米率、垩白粒率、垩白度、糊化温度、胶稠度和直链淀粉含量均无显著相关; 与精米率呈显著正相关(r=0.124*); 与粒长和长宽比均呈极显著正相关(相关系数分别为0.470**和0.241**)。糙米率、精米率和直链淀粉含量均受2对主基因+多基因控制, 2对主基因具有累加作用和加性×加性的上位性作用; 整精米率、粒长、长宽比和胶稠度受2对加性-上位性主基因+多基因控制;垩白粒率、垩白度和糊化温度均受3对加性-上位性主基因+多基因控制。糙米率、精米率、整精米率、垩白粒率、垩白度和糊化温度6个品质性状以主基因遗传为主,粒长、长宽比、胶稠度和直链淀粉含量4个性状以多基因遗传为主。     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

Gene expression of the biosynthetic enzymes and biosynthesis of starch during Rice Grain development

Amylose and amylopectin are determinants of the physicochemical properties for starch and grain quality in rice. Their biosynthesis is catalyzed by the interplay of ADP-glucose pyrophosphorylase (AGPase), granule-bound starch synthase (GBSS), soluble starch synthase (SSS), a starch branching enzyme (SBE), and a starch debranching enzyme (SDE). In this study, the genes for these enzymes were highly expressed 7 to 28 days after flowering during grain development, and their expression closely matched increases in both starch content and grain weight Among all the tested cultivars, amylose contents in the rice grains remained essentially constant throughout their development The AGPase gene was highly expressed in the high-yield cultivars of both glutinous and non-glutinous rice. The SSS gene was actively expressed when mature GBSS mRNA decreased. Genes responsible for amylopectin biosynthesis were simultaneously expressed in the late stage of grain development. We have now demonstrated that the expression patterns of starch biosynthetic genes differ between glutinous and non-glutinous rice, and between Tongil (a Japonica/ Indica hybrid) and Japonica types.     

淀粉含量不同的玉米自交系籽粒淀粉积累及其关键酶活性

以2个高淀粉和2个低淀粉玉米自交系为材料,分析了玉米籽粒淀粉的动态积累规律,同时对高低淀粉玉米籽粒灌浆过程中淀粉生物合成关键酶活性的动态变化及其与淀粉积累动态的相关性进行讨论分析。研究结果表明:灌浆过程中4个自交系淀粉含量变化趋势均呈sigmoid型曲线。灌浆过程中ADPG-PPase(腺苷二磷酸葡萄糖焦磷酸化酶)、SSS(可溶性淀粉合成酶)、GBSS(颗粒结合淀粉合成酶)活性均呈单峰曲线变化,峰值都出现在20～30DAP(授粉后天数)。2个高淀粉自交系的Q酶(淀粉分支酶)活性也呈单峰曲线变化,峰值也出现在20DAP,而2个低淀粉自交系的Q酶活性则呈双峰曲线变化,2个峰值分别出现在15～20DAP和30～35DAP。4个自交系籽粒淀粉的积累速率与各自交系ADPG-PPase、SSS和GBSS的活性变化呈极显著正相关。各自交系关键酶活性之间,ADPG-PPase、SSS和GBSS三者间活性变化呈极显著正相关,这3种酶活性变化与Q酶活性变化也呈不同程度的正相关。     

Waterlogging and simulated acid rain after anthesis deteriorate starch quality in wheat grain

The synthesis and accumulation of starch is greatly affected by environmental stress. Wheat grown in the downstream area of the Yangtze River is easily subjected to stress of waterlogging and acid rain. In order to probe the effect of waterlogging and acid rain on yield and starch characteristic, we used winter wheat cultivars of Yangmai 16 (more resistant) and Wennong 17 (relatively sensitive) to sole stress of acid rain or waterlogging and to their combinations during grain filling. The responses of grain yield, the physiochemical properties and morphology of starch granules in endosperm to the stresses were investigated. Compared with CT (control), grain yield in Wennong 17 were significantly decreased by both pH 4.0 acid rain and pH 2.5 acid rain, while in Yangmai 16 only by pH 2.5 acid rain. Waterlogging combined with acid rain significantly reduced grain weight and grain yield in both wheat cultivars. Acid rain, waterlogging and their combination depressed activities of ADP glucose pyrophosphorylase and soluble starch synthase (SSS) in grains of both cultivars. Acid rain and waterlogging damaged endosperm cell structure and caused abnormal starch granules. Starch granules at maturity became fragile and failed to keep in shape and some granules were even totally submerged in the protein matrix and other tissue under acid rain and waterlogging. Content of amylopectin and amylopectin/amylose ratio was also decreased, while content of amylose was increased under acid rain and waterlogging. As a result, swelling power and most viscosity parameters decreased, while the pasting temperature increased in both cultivars due to stresses. In sum, acid rain and waterlogging and their combination damaged cell structure and depressed synthesis of amylopectin, and led to the formation of abnormal fragile starch granules, and finally reduced grain weight and then yield, and deteriorated starch quality.     

Activities of key enzymes for starch synthesis in relation to growth of superior and inferior grains on winter wheat (Triticum aestivum L.) spike

Weight of individual grains is a major yield component in wheat. The non-uniform distribution of single grain weight on a wheat spike is assumed to be closely associated with starch synthesis in grains. The present study was undertaken to determine if the enzymes involved in starch synthesis cause the differences in single grain weight between superior and inferior grains on a wheat spike. Using two high-yield winter wheat (Triticum aestivum L.) varieties differing in grain weight and three nitrogen rates for one variety, the contents of amylose and amylopectin, and activities of enzymes involved in starch synthesis in both superior and inferior grains were investigated during the entire period of grain filling. Superior grains showed generally higher starch accumulation rates and activities of enzymes including SS (sucrose synthase), UDPGPPase (UDP-glucose pyrophosphorylase), ADPGPPase (ADP-glucose pyrophosphorylase), SSS (soluble starch synthase) and GBSS (starch granule bound starch synthase) and subsequently produced much higher single grain weight than inferior grains. Nitrogen increased enzyme activities and starch accumulation rates, and thus improved individual grain weight, especially for inferior grains. The SS, ADPGPPase and SSS were significantly correlated to amylopectin accumulation, while SS, ADPGPPase, SSS and GBSS were significantly correlated to amylose accumulation. This infers that SS, ADPGPPase and starch synthase play key roles in regulating starch accumulation and grain weight in superior and inferior grains on a wheat spike.     

籼型杂种稻米品质性状的数量遗传分析

应用新近提出的ｐ＾＋ｑ＾＋ｐｑ交配设计的种子性状遗传表达鉴别方法,分析了籼型水稻３个不育系和１０个恢复系配组的３０个杂种Ｆ１植株上的Ｆ２种子及其亲本的９个稻米品质性状。结果表明：（１）粒长、粒宽、粒重、糙米率和完整精米率５个性状在Ｆ２米粒间没有发生遗传分离,它们的遗传受二倍体母体基因型（Ｆ１植株）控制。（２）垩白率、直链淀粉含量、糊化温度和胶稠度４个性状则在Ｆ２米粒间有极显著的遗传分离,帮主要受三