Some properties of starch branching enzyme from indica rice endosperm (Oryza sativa L.)

Starch branching enzyme (α-1,4-glucan: α-1,4-glucan-6-glycosyl transferase; EC 2.4.1.18) catalyzes the formation of the α-1,6-bond in branched starch molecules such as amylopectin. Some characteristics of starch branching enzyme in rice endosperm (Oryza sativa L.) were determined because of the importance of starch structure for rice quality. Two or three peaks of starch branching enzyme activity were resolved by anion-exchange chromatography of extracts from high amylose rice. The properties of rice starch branching enzyme were similar to those found for the enzyme from other plant sources except for a much lower molecular weight. Rice branching enzyme had an apparent molecular weight of 40 000 as estimated by gel permeation chromatography. Multiple forms of starch branching enzyme could also be resolved in milled rice, suggesting that relationships between starch quality and characteristics of starch branching enzyme could be examined in the mature grain after harvest.     

淀粉分支酶3存在于水稻胚乳的淀粉粒之中

对水稻胚乳淀粉颗粒结合的淀粉分支酶进行了研究.酶活性分析表明水稻胚乳中存在着与淀粉颗粒结合的淀粉分支酶.氨基酸测序分析结果表明结合于水稻胚乳淀粉粒的淀粉分支酶是分子量为84 kD的淀粉分支酶3(rice starch branching enzyme 3; RBE3).从开花后5 d到种子成熟,淀粉颗粒结合的RBE3蛋白都保持较为稳定的含量.Northern 分析表明水稻胚乳发育过程中RBE4最先表达而RBE3和RBE1的表达滞后.综合以上研究结果说明RBE3存在于水稻胚乳的淀粉之中是由于RBE3与淀粉葡聚糖链具有较高亲和性而难以和葡聚糖链解离,进而随着淀粉粒的增长而被其包裹.     

Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.     

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.     

Activity of starch synthase and the amylose content in rice endosperm

The content of amylose in endosperm of non-waxy japonica rice (Oryza sativa cv Akitakomachi) was increased by lowering the growth temperature from 25° to 15° during the ripening period. The activities of sucrose synthase, ADPglucose pyrophosphorylase, starch branching enzyme (Q-enzyme) and soluble starch synthase in endosperm developed at 15° were lower than or similar to those at 25°, when compared on a endosperm basis at the similar ripening stage. In contrast, the activity of starch granule-bound starch synthase, which is considered to be indispensable for amylose synthesis, was higher by 3–3.5-fold in the endosperm developed at the low temperature than that at the high ambient temperature. The results suggest that the low temperature specifically accelerates the expression of the bound starch synthase gene (waxy gene) in rice endosperm, which resulted in elevated amylose biosynthesis in the endosperm when developed at lower temperatures.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Starch Synthesis and Programmed Cell Death during Endosperm Development in Triticale(× Triticosecale Wittmack)

Triticale(× Triticosecale Wittmack) grains synthesize and accumulate starch as their main energy source.Starch accumulation rate and synthesis activities of ADP-glucose pyrophosphorylase,soluble starch synthases,granule-bound starch synthase and starch-branching enzyme showed similar pattern of unimodal curves during endosperm development.There was no significant difference in activity of the starch granule-bound protein isolated from total and separated starch granules at different developmental stages after anthesis in triticale.Evans Blue staining and analysis of DNA fragmentation indicated that cells of triticale endosperm undergo programmed cell death during its development.Dead cells within the endosperm were detected at 6 d post anthesis(DPA),and evidence of DNA fragmentation was first observed at 21 DPA.The period between initial detection of PCD to its rapid increase overlapped with the key stages of rapid starch accumulation during endosperm development.Cell death occurred stochastically throughout the whole endosperm,meanwhile,the activities of starch biosynthetic enzymes and the starch accumulation rate decreased in the late stages of grain filling.These results suggested that the timing and progression of PCD in triticale endosperm may interfere with starch synthesis and accumulation.     

Fine mapping and candidate gene analysis of the floury endosperm gene, FLO(a), in rice

In addition to its role as an energy source for plants, animals and humans, starch is also an environmentally friendly alternative to fossil fuels. In rice, the eating and cooking quality of the grain is determined by its starch properties. The floury endosperm of rice has been explored as an agronomical trait in breeding and genetics studies. In the present study, we characterized a floury endosperm mutant, flo(a), derived from treatment of Oryza sativa ssp. japonica cultivar Hwacheong with MNU. The innermost endosperm of the flo(a) mutant exhibited floury characteristics while the outer layer of the endosperm appeared normal. Starch granules in the flo(a) mutant formed a loosely-packed crystalline structure and X-ray diffraction revealed that the overall crystallinity of the starch was decreased compared to wild-type. The FLO(a) gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domaincontaining protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs. Taken together, our map-based cloning approach pinpointed an OsTPR as a strong candidate of FLO(a), and the proteins that contain TPR motifs might play a significant role in rice starch biosynthetic pathways.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

Starch characteristics in cultures of normal and mutant maize endosperm

Summary Vigorously growing suspension cultures of normal, amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with jagged edges were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.Supported in part by the United States Department of Agriculture Competitive Grant 85-CRCR-1-1740. Contribution No. 94, Department of Horticulture. The Pennsylvania State University. Authorized for publication as paper No. 7373 in the journal series of the Pennsylvania Agricultural Experiment Station     

Purification and some properties of starch branching enzyme (Q-enzyme) from tuberous root of sweet potato

The pattern of isoforms of starch branching enzyme II or Q-enzyme II in the tuberous root of sweet potato was distinct from those of other organs; altogether 7 isoforms of QEII were contained in the sweet potato plant. The QEIIf isoform, one of the two major QEII isoforms in the tuberous root, was purified to homogeneity by using a variety of HPLC columns. The purified QEIIf was a monomeric protein with a molecular mass of about 85 kDa. Western blot analysis showed that the polyclonal antibodies raised against the purified QEIIf was significantly reactive to the rice endosperm QEI, but not to the rice endosperm QEIIa. Furthermore, the sweet potato QEIIf reacted with the antiserum raised against the rice endosperm QEI, but not with that against the rice endosperm QEIIa. The results suggest that the sweet potato QEIIf is more similar to the rice endosperm QEI than to the rice endosperm QEIIa.     

Characterization of plastidial starch phosphorylase in Triticum aestivum L. endosperm

Starch phosphorylase (Pho) catalyses the reversible transfer of glucosyl units from glucose1-phosphate to the non-reducing end of an α-1,4-linked glucan chain. Two major isoforms of Pho exist in the plastid (Pho1) and cytosol (Pho2). In this paper it is proposed that Pho1 may play an important role in recycling glucosyl units from malto-oligosaccharides back into starch synthesis in the developing wheat endosperm. Pho activity was observed in highly purified amyloplast extracts prepared from developing wheat endosperms, representing the first direct evidence of plastidial Pho activity in this tissue. A full-length cDNA clone encoding a plastidial Pho isoform, designated TaPho1, was also isolated from a wheat endosperm cDNA library. The TaPho1 protein and Pho1 enzyme activity levels were shown to increase throughout the period of starch synthesis. These observations add to the growing body of evidence which indicates that this enzyme class has a role in starch synthesis in wheat endosperm and indeed all starch storing tissues.     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases

Soluble starch synthase and starch-branching enzymes in extracts from kernels of four maize genotypes were compared. Extracts from normal (nonmutant) maize were found to contain two starch synthases and three branching enzyme fractions. The different fractions could be distinguished by chromatographic properties and kinetic properties under various assay conditions. Kernels homozygous for the recessive amylose-extender (ae) allele were missing branching enzyme IIb. In addition, the citrate-stimulated activity of starch synthase I was reduced. This activity could be regenerated by the addition of branching enzyme to this fraction. No other starch synthase fractions were different from normal enzymes. Extracts from kernels homozygous for the recessive dull (du) allele were found to contain lower branching enzyme IIa and starch synthase II activities. Other fractions were not different from the normal enzymes. Analysis of extracts from kernels of the double mutant ae du indicated that the two mutants act independently. Branching enzyme IIb was absent and the citrate-stimulated reaction of starch synthase I was reduced but could be regenerated by the addition of branching enzyme (ae properties) and both branching enzyme IIa and starch synthase II were greatly reduced (du properties). Starch from ae and du endosperms contains higher amylose (66 and 42%, respectively) than normal endosperm (26%). In addition, the amylopectin fraction of ae starch is less highly branched than amylopectin from normal or du starch. The above observations suggest that the alterations of the starch may be accounted for by changes in the soluble synthase and branching enzyme fractions.     

水稻淀粉胚乳程序性细胞死亡中的去核化

对水稻品种中籼8836淀粉胚乳细胞的去核化发育阶段的细胞超微结构变化和同期籽粒灌浆速率及相关酶活性的动态进行了观察和分析。开花受精后约在第3天胚乳完成细胞化,花后第5天少数淀粉胚乳细胞启动去核发育过程。核消亡是淀粉胚乳细胞程序性细胞死亡(PCD)的第一步。同一籽粒淀粉胚乳细胞的去核进程是不同步的。花后第13天所有淀粉胚乳细胞都已完成去核过程。在去核过程中,胚乳核的形态变化特征既有动植物PCD的共性又有其特殊性。伴随核降解过程,一部分线粒体解体,表明去核化与线粒体解体有一定联系。在去核化发育阶段,与PCD有关的酶类,如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性非常高;与淀粉合成有关的酶类,如ADPG焦磷酸化酶、可溶性淀粉合成酶(SSS酶)、淀粉分支酶(或Q酶)也表现出很高的活性。去核化发育阶段籽粒灌浆速率最高,籽粒增重亦最快。淀粉胚乳细胞去核之后,细胞并未立即死亡,这些无核的细胞仍维持正常有序的代谢活动,继续进行淀粉和贮藏蛋白的合成与积累,但上述酶类的活性明显降低,灌浆速率也明显趋缓。淀粉胚乳细胞最终被贮藏物质充满时成为死细胞,完成其程序性死亡过程。Evan‘s blue染色鉴定表明淀粉胚乳细胞死亡不同步,细胞死亡在淀粉胚乳组织中是随机发生的。     

复合调控Wx与SBE3基因对水稻籽粒直链淀粉含量的影响

颗粒淀粉合成酶（GBSS）和淀粉分支酶3（SBE3）是淀粉合成过程中的两个关键酶,这两个酶主要由耽和SBE3两个基因分别控制,它们的表达量直接影响直链淀粉和支链淀粉的含量比例。为了探讨水稻淀粉关键酶基因耽过量与SBE3干涉复合表达对直链淀粉含量的影响,构建了Wx过量表达与SBE3干涉结合的多基因表达载体,并通过农杆菌介导的方法将其导入日本晴水稻中。经过PCR检测分析获得了65株转基因阳性植株,半定量RT—PCR检测表明转基因株系中Wx基因表达量明显增加,而SBE3基因表达量显著减少。转基因株系籽粒透明度明显降低,直链淀粉含量比野生型的平均高45％,但是千粒重变化不大,与野生型相当。遗传分析表明这些转基因株系多数可稳定遗传。     

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