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.     

Purification of two forms of starch branching enzyme (Q-enzyme) from developing rice endosperm

Two isoforms of starch branching enzyme (Q-enzyme), QEI and QEII, have been purified to honlogeneity from developing rice endosperm. QEI and QEII, with molecular weights of about 80 and 85 kDa, respectively, could be fully separated by anion-exchange or hydrophobic chromatography. The peptide maps obtained after V₈ proteinase digestion were quite different between the two enzymes. Antibodies prepared against QEI showed no immunological cross-reaction with the QEII protein in Western blot experiments, and anti-QEII serum did not react with the QEI protein. The data indicate that QEI and QEII are distinct proteins encoded by different genes in rice plants.     

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (M_r) of 102069 Da, similar in size to its M_r of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.     

The structure of starch can be manipulated by changing the expression levels of starch branching enzyme IIb in rice endosperm

When the starch branching enzyme IIb (BEIIb) gene was introduced into a BEIIb-defective mutant, the resulting transgenic rice plants showed a wide range of BEIIb activity and the fine structure of their amylopectins showed considerable variation despite having the two other BE isoforms, BEI and BEIIa, in their endosperm at the same levels as in the wild-type. The properties of the starch granules, such as their gelatinization behaviour, morphology and X-ray diffraction pattern, also changed dramatically depending on the level of BEIIb activity, even when this was either slightly lower or higher than that of the wild-type. The over-expression of BEIIb resulted in the accumulation of excessive branched, water-soluble polysaccharides instead of amylopectin. These results imply that the manipulation of BEIIb activity is an effective strategy for the generation of novel starches for use in foodstuffs and industrial applications.     

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.     

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion- exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SW_XL and G3000SW_XL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SW_XL and G4000SW_XL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 °C, respectively. The enzyme activity was completely inhibited by HgCl₂ and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995, Plant Cell 7: 417–429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al., 1988, J Biol Chem 263: 9271–9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type. Received: 29 October 1998 / Accepted: 10 December 1998     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine‐tuning of cooked rice texture

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine‐tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met‐723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica‐type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α‐amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.     

Correlation of cytokinin levels in the endosperms and roots with cell number and cell division activity during endosperm development in rice

Cell number and cell division activity in rice (Oryza sativa) endosperms are possibly regulated by cytokinin levels in the endosperm and its source in the roots. This study tried to find the possible correlations among them. Six rice genotypes were grown in nutrient solution. Two patterns of endosperm cell division, synchronous and asynchronous, were observed among the genotypes based on the cell division rate of superior and inferior spikelets. Contents of zeatin (Z) + zeatin riboside (ZR) were much higher than those of N6-isopentenyladenine (iP) and N6-isopentenyladenosine (iPR) in both endosperms and roots. Changes in Z + ZR levels in endosperms were significantly correlated with those in roots, and both were very significantly correlated with the cell division rate. Changes in iP + iPR contents in the roots were not significantly correlated with those in the endosperms and the cell division rate. When roots were treated with kinetin, endosperm cell number and grain weight were increased. Such enhancement was more significantly achieved by the root kinetin treatment than by spraying kinetin on leaves and panicles. The results suggest that the cell number and cell division activity in rice endosperms are regulated by cytokinin levels in the endosperm and that root-derived Z + ZR play a pivotal role.     

An analysis of soluble starch synthase isozymes from the developing grains of normal and shx cv. Bomi barley (Hordeum vulgare)

Soluble starch synthase (SSS, EC 2.4.1.21) catalyzes formation of the α-1,4 bonds of amylopectin. It occurs in multiple isozymes which are either type I, primer-independent in the presence of citrate, or type II. always primer-dependent. To analyze the enzyme. a sensitive native gel assay was developed, monitoring ADP-[¹⁴C]glucose incorporation into insoluble α-glucan in the presence of either sodium citrate or glycogen primer or both. Using this system, we observed multiple type I and type II forms in developing grains of barley ( Hordeum vulgare L.) cv. Bomi, the relative activities of which vary with seed development. At least one form comigrates in native gels with starch branching enzyme. Assays of the shx mutant, which is severely reduced in starch accumulation and in type I SSS activity, indicate that one type I isozyme becomes primer-dependent.     

免耕与秸秆高留茬还田对抛秧稻田土壤酶活性的影响

利用大田试验,研究了免耕+秸秆还田、免耕、常耕+秸秆还田、常耕4种耕作方式对稻田土壤剖面不同层次土壤酶活性的影响.结果表明:4种耕作方式的酶活性在土壤剖面上表现为上层高于下层,其中免耕+秸秆处理上、下土层间的差异大于其他处理.上层土壤的脲酶、酸性磷酸酶、蛋白酶和纤维素酶活性为免耕处理大干常耕处理,有秸秆还田处理大干无秸秆还田处理,以免耕+秸秆处理最高,常耕处理最低;下层土壤4种酶活性以常耕+秸秆处理最高,免耕+秸秆处理次之,免耕和常耕处理较低.水稻不同生育时期,脲酶和纤维素酶活性在分蘖期较低,增加至孕穗期达到峰值,至成熟时又降至低值;酸性磷酸酶活性以分蘖期较高,拔节期较低;蛋白酶活性在分蘖期和抽穗期分别出现峰值.     

水稻Ds插入淡绿叶突变体的鉴定和遗传分析

Ac/Ds插入突变是水稻基因功能鉴定的有力工具之一。文章从水稻中花11 Ds-T-DNA转化纯合体与Ac-T-DNA 转化纯合体的杂交群体中筛选到一个淡绿叶突变体。该突变体在三叶期由绿苗转为淡绿叶苗, 自然光照下突变体迅速焦枯, 但是在弱光照条件下, 突变体能缓慢生长至开花结实; 突变体光合作用特性研究表明该突变是典型的光抑制突变体。遗传分析表明该突变为Ds插入导致的隐性突变。     

FLOURY ENDOSPERM16 encoding a NAD‐dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.     

Characterization of ATPase activities in rice root plasmalemma vesicles isolated by two-phase partitioning

Plasma membrane vesicles were isolated from the roots of 7-day-old rice plants ( Oryza sativa L. cv. Bahía) by utilizing an aqueous polymer two-phase system with 6.2%:6.2% (w/w) Dextran T500 and polyethylene glycol 3350 (PEG) at pH 7.6. Plasmalemma vesicles of high purity were obtained as indicated by the vanadate-sensitive K⁺, Mg²⁺-ATPase activity that was 18 times higher in the upper (PEG-rich) phase than in the lower (Dextran-rich) phase and by specific staining with sodium silicotungstate. Two peaks of ATPase activity were found. One showed a pH optimum at 6.0 in the presence of 150 m M KCl and 3 m M ATP with apparent K_m (ATP) and V_max of 0.75 m M and 79 μmol (mg protein)⁻¹ h⁻¹, respectively. With 50 m M KCl and 7 m M ATP a pH optimum of 6.5, an apparent K_m (ATP) of 6.3 m M and V_max of 159 μmol (mg protein)⁻¹ h⁻¹ were determined. Both activities were specific for ATP, unspecific for monovalent cations, sensitive to sodium vanadate and Ca²⁺ but insensitive to azide and nitrate.     

早晚季不同耐光氧化特性水稻品种籽粒灌浆关键酶活性变化

选用光氧化敏感和不敏感的两大类型4个水稻品种研究早晚季耐光氧化反应特性与其稻米品质稳定性密切相关的生理生化原因.结果表明,耐光氧化反应特性强的适强光生态型水稻品种的平均灌浆速率(G_mean)、达最大灌浆速率时间(T_max.G)和达最大灌浆速率百粒重(W_max.G)等参数在早晚季灌浆结实期间保持相对稳定;而对光氧化敏感的弱光生态型品种佳禾早占以上参数的早晚季稳定性较差.比较早晚季籽粒灌浆充实过程中关键酶活性的变化结果可见,耐光氧化反应特性强的品种,灌浆速率高峰到灌浆终期ADPG-焦磷酸化酶和淀粉合成酶活性早晚季变异比耐光氧化反应特性弱的品种小,且耐光氧化反应特性强的品种灌浆高峰前后酶活性变异的幅度早晚季也较一致.     

Bottlenecks in carotenoid biosynthesis and accumulation in rice endosperm are influenced by the precursor–product balance

The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor–product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce β‐carotene in the presence of endogenous lycopene β‐cyclase. We combined this mini‐pathway with the Arabidopsis thaliana genes AtDXS (encoding 1‐deoxy‐D‐xylulose 5‐phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up‐regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles.     

水稻窄叶突变体zy17的遗传分析和候选基因鉴定

器官大小调控是一个基本的发育生物学过程,受细胞分裂和细胞扩展的影响。然而,植物器官大小调控的遗传和分子机理仍不清楚。为了进一步了解器官大小调控的分子机制,文章分离了一系列水稻叶子宽窄改变的突变体。其中,窄叶突变体zy17叶变窄,同时伴有植株矮化、穗子变小、枝梗数和穗粒数降低的表型。遗传分析表明该窄叶性状受1个隐性基因控制;细胞学分析表明该突变体叶子的细胞数目和维管束数目显著降低,表明ZY17影响了细胞分裂。基因组重测序进一步筛选出ZY17的3个候选基因：Os02g22390基因突变发生在内含子区,编码蛋白为逆转座蛋白;Os02g28280和Os02g29530基因突变都发生在外显子区,其中Os02g28280编码一个功能未知蛋白,该基因突变后,发生碱基置换,产生非同义突变;Os02g29530编码一个含糖基转移酶相关的PFAM结构域的蛋白,该基因突变后,出现两个碱基的缺失,从而导致其蛋白翻译提前终止。对候选基因的深入研究,将揭示水稻叶子大小调控的机制。