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.     

Response of four rice varieties to Rhodobacter capsulatus at seedling stage

The phototrophic purple nonsulphur bacterium (PPNSB) Rhodobacter capsulatus was used to inoculate seedlings of four rice varieties Giza 159, Giza 171, Giza 176 and Giza 181, grown in hydroponic culture with or without nitrogen. After three weeks the seedling growth parameters were measured. Inoculation with R. capsulatus enhanced seedling growth of all rice varieties tested. The response to inoculation as compared to control plants (no nitrogen, no R. capsulatus) were 52% to 75% for shoot height, 47% to 100% for aerial part dry weight, 45% to 78% for aerial part N content, –37% to –9% for maximum root length, –4% to 8% for root system dry weight and 50% to 62% for root N content.     

灌浆期高温对水稻光合特性、内源激素和稻米品质的影响

以2个籼稻品种:温度钝感型品种K30和温度敏感型品种R21为材料,利用人工气候室控温,在水稻灌浆期设置高温(日均温度34.9℃)和适温(日均温度28.0℃)处理,测定不同灌浆时期(5,10,15,20,25d和30d)光合特性、内源激素含量及稻米品质的变化。结果表明,高温增强了K30的光合能力,K30高温处理净光合速率(Pn)在整个灌浆期都明显高于对照;R21在灌浆前期(5、10d和15d)高温处理与适温处理Pn差异不明显,在灌浆后期(20、25d和30d)高温处理Pn下降。高温增加了K30叶片和籽粒脱落酸(ABA)含量;而R21高温处理和适温处理叶片与籽粒ABA含量的对比不明显。高温处理对两个水稻品种叶片和籽粒赤霉素(GA3)与生长素(IAA)含量也有不同程度影响。高温胁迫降低稻米品质,但K30比R21表现出更强的温度钝感特性。     

NRT1.1B improves selenium concentrations in rice grains by facilitating selenomethinone translocation

Selenium (Se) is an essential trace element for humans and other animals, yet approximately one billion people worldwide suffer from Se deficiency. Rice is a staple food for over half of the world's population that is a major dietary source of Se. In paddy soils, rice roots mainly take up selenite. Se speciation analysis indicated that most of the selenite absorbed by rice is predominantly transformed into selenomethinone (SeMet) and retained in roots. However, the mechanism by which SeMet is transported in plants remains largely unknown. In this study, SeMet uptake was found to be an energy‐dependent symport process involving H⁺ transport, with neutral amino acids strongly inhibiting SeMet uptake. We further revealed that NRT1.1B, a member of rice peptide transporter (PTR) family which plays an important role in nitrate uptake and transport in rice, displays SeMet transport activity in yeast and Xenopus oocyte. The uptake rate of SeMet in the roots and its accumulation rate in the shoots of nrt1.1b mutant were significantly repressed. Conversely, the overexpression of NRT1.1B in rice significantly promoted SeMet translocation from roots to shoots, resulting in increased Se concentrations in shoots and rice grains. With vascular‐specific expression of NRT1.1B, the grain Se concentration was 1.83‐fold higher than that of wild type. These results strongly demonstrate that NRT1.1B holds great potential for the improvement of Se concentrations in grains by facilitating SeMet translocation, and the findings provide novel insight into breeding of Se‐enriched rice varieties.     

Golgi/plastid‐type manganese superoxide dismutase involved in heat‐stress tolerance during grain filling of rice

Superoxide dismutase (SOD) is widely assumed to play a role in the detoxification of reactive oxygen species caused by environmental stresses. We found a characteristic expression of manganese SOD 1 (MSD1) in a heat‐stress‐tolerant cultivar of rice (Oryza sativa). The deduced amino acid sequence contains a signal sequence and an N‐glycosylation site. Confocal imaging analysis of rice and onion cells transiently expressing MSD1‐YFP showed MSD1‐YFP in the Golgi apparatus and plastids, indicating that MSD1 is a unique Golgi/plastid‐type SOD. To evaluate the involvement of MSD1 in heat‐stress tolerance, we generated transgenic rice plants with either constitutive high expression or suppression of MSD1. The grain quality of rice with constitutive high expression of MSD1 grown at 33/28 °C, 12/12 h, was significantly better than that of the wild type. In contrast, MSD1‐knock‐down rice was markedly susceptible to heat stress. Quantitative shotgun proteomic analysis indicated that the overexpression of MSD1 up‐regulated reactive oxygen scavenging, chaperone and quality control systems in rice grains under heat stress. We propose that the Golgi/plastid MSD1 plays an important role in adaptation to heat stress.     

控制水稻胚乳淀粉合成代谢若干关键酶基因对花后高温的响应表达

以稻米品质温度敏感型的早籼稻品种嘉早935为材料,利用人工气候箱控温试验和实时荧光定量PCR技术,探讨了不同灌浆温度(日均温分别为22和32 ℃)处理下胚乳淀粉分支酶(SBE)、淀粉去分支酶(DBE)和淀粉合酶(SS)的10个同工型基因(sbe1、sbe3、sbe4、pul、isa1、isa2、isa3、Wx、sss1和sss2a)的相对表达量差异及动态变化特征.结果表明: 淀粉合成相关功能基因对水稻灌浆期高温胁迫的响应表达方式存在明显差异,而且因同工型的类型而不同.在高温处理下,sbe1和sbe3的相对表达量显著下降,二者属于SBE类基因中对高温胁迫较敏感的主要同工型;DBE基因中,pul属于高表达的同工型,而且其对高温胁迫响应比isa1、isa2和isa3敏感;在Wx、sss1和sss2a中,sss2a的相对表达量显著低于sss1和Wx, 但sss2a和sss1对高温胁迫响应比Wx敏感,因此二者可能也是高温胁迫对胚乳淀粉结构进行调控的重要位点,尤其在水稻灌浆的中后期发挥重要作用.     

FLOURY ENDOSPERM11‐2 encodes plastid HSP70‐2 involved with the temperature‐dependent chalkiness of rice (Oryza sativa L.) grains

The frequent occurrence of chalky rice (Oryza sativa L.) grains becomes a serious problem as a result of climate change. The molecular mechanism underlying chalkiness is largely unknown, however. In this study, the temperature‐sensitive floury endosperm11‐2 (flo11‐2) mutant was isolated from ion beam‐irradiated rice of 1116 lines. The flo11‐2 mutant showed significantly higher chalkiness than the wild type grown under a mean temperature of 28°C, but similar levels of chalkiness to the wild type grown under a mean temperature of 24°C. Whole‐exome sequencing of the flo11‐2 mutant showed three causal gene candidates, including Os12g0244100, which encodes the plastid‐localized 70‐kDa heat shock protein 2 (cpHSP70‐2). The cpHSP70‐2 of the flo11‐2 mutant has an amino acid substitution on the 259th aspartic acid with valine (D259V) in the conserved Motif 5 of the ATPase domain. Transgenic flo11‐2 mutants that express the wild‐type cpHSP70‐2 showed significantly lower chalkiness than the flo11‐2 mutant. Moreover, the accumulation level of cpHSP70‐2 was negatively correlated with the chalky ratio, indicating that cpHSP70‐2 is a causal gene for the chalkiness of the flo11‐2 mutant. The intrinsic ATPase activity of recombinant cpHSP70‐2 was lower by 23% at V_max for the flo11‐2 mutant than for the wild type. The growth of DnaK‐defective Escherichia coli cells complemented with DnaK with the D201V mutation (equivalent to the D259V mutation) was severely reduced at 37°C, but not in the wild‐type DnaK. The results indicate that the lowered cpHSP70‐2 function is involved with the chalkiness of the flo11‐2 mutant.     

Suppression of OsMADS7 in rice endosperm stabilizes amylose content under high temperature stress

High temperature significantly alters the amylose content of rice, resulting in mature grains with poor eating quality. However, only few genes and/or quantitative trait loci involved in this process have been isolated and the molecular mechanisms of this effect remain unclear. Here, we describe a floral organ identity gene, OsMADS7, involved in stabilizing rice amylose content at high temperature. OsMADS7 is greatly induced by high temperature at the early filling stage. Constitutive suppression of OsMADS7 stabilizes amylose content under high temperature stress but results in low spikelet fertility. However, rice plants with both stable amylose content at high temperature and normal spikelet fertility can be obtained by specifically suppressing OsMADS7 in endosperm. GBSSI is the major enzyme responsible for amylose biosynthesis. A low filling rate and high expression of GBSSI were detected in OsMADS7 RNAi plants at high temperature, which may be correlated with stabilized amylose content in these transgenic seeds under high temperature. Thus, specific suppression of OsMADS7 in endosperm could improve the stability of rice amylose content at high temperature, and such transgenic materials may be a valuable genetic resource for breeding rice with elite thermal resilience.     

不同生育阶段夜温升高对双季水稻物质生产与养分吸收的影响

利用2间玻璃室内夜间不同的温度条件,研究了水稻生长期间夜温升高对双季早、晚稻物质生产和养分积累的影响.结果表明:播种-幼穗分化期夜温升高增加早稻的干物质积累,而减少晚稻的干物质积累;幼穗分化期-抽穗的夜温升高对早、晚稻的干物质积累均有一定的负作用;抽穗后的夜温升高对早稻物质生产影响较小,但降低其茎鞘物质的转运,而晚稻的干物质生产和茎鞘物质的转运均提高.在早稻幼穗分化前夜温适度升高有利于提高根系活力,促进植株养分的积累,但不利于晚稻植株养分的积累;幼穗分化-抽穗期间的夜温升高均不利于早、晚稻植株的养分积累;灌浆结实期的夜温升高会造成早稻根系早衰,不利于养分吸收,但有利于保持晚稻根系较高活力,促进养分吸收.试验证明,不同生育阶段的夜温升高对双季水稻不同生育阶段的干物质生产和养分吸收的影响是不同的.     

生育前期遮光对水稻后期功能叶生长及稻米品质的影响

以2个不同直链淀粉含量的水稻品种为材料,研究了生育前期(插秧至幼穗分化期)遮光对水稻后期功能叶生长及稻米品质的影响。结果表明,前期遮光处理后,水稻剑叶面积明显增大,功能叶的碳、氮代谢水平提高;2个品种的籽粒直链淀粉积累过程受到的影响不同,而蛋白质积累受到的影响均一致(即百分含量下降幅度增大);糙米率有所降低,但未达到显著水平;精米率和垩白率极显著下降;整精米率和直链淀粉含量显著或极显著升高;粒长、粒宽和蛋白质含量无显著性变化,但蛋白质含量表现为下降趋势;2个品种间存在着差异。     

Does increased zinc uptake enhance grain zinc mass concentration in rice?

Rice (Oryza sativa) is the worlds’ most important cereal and potentially an important source of zinc (Zn) for people who eat mainly rice. To improve Zn delivery by rice, plant Zn uptake and internal allocation need to be better understood. This study reports on within‐plant allocation and potential Zn accumulation in the rice grain in four so‐called aerobic rice cultivars (Handao297, K150, Handao502 and Baxiludao). Two controlled‐condition experiments were carried out, one with a wide range of constant Zn concentrations in the medium and one with a range of plant growth rate‐related supply rates. In both experiments, increased Zn supply induced increased plant Zn uptake rate throughout crop development, when expressed as daily Zn uptake (μg day^?1) or as daily Zn uptake per gram of plant dry matter (μg g^?1). Zinc mass concentration (ZnMC) in all plant organs increased with an increase in Zn supply but to various degrees. At higher uptake levels, the ZnMC in stems increased most, while the ZnMC in hulled grains (brown rice) increased least. The increase in leaf ZnMC was generally small, but at toxic levels in the medium, leaf ZnMC increased significantly. It appears that regulation of grain Zn loading differs from regulation of Zn loading to other organs. A milling test on seeds of Baxiludao and Handao502 showed that when ZnMC in brown rice increased from 13 to 45 mg kg^?1, ZnMC in polished rice grains (endosperm) also increased from 9 to 37 mg kg^?1 but remained three to five times lower than that in the bran. Irrespective of the ZnMC in the brown rice, around 75% of total grain Zn was present in the endosperm. In both cultivars, there was a major difference in ZnMC between bran and endosperm (120 and 37 mg kg^?1, respectively), suggesting a barrier for Zn transport between the two tissues. There seems to be a second barrier between stem and rachis, as their ZnMCs also differed greatly (300 and 100 mg kg^?1, respectively) in both cultivars at higher plant ZnMC. It is concluded that there is too little scope from a human nutrition perspective to enhance ZnMC in rice endosperm by simply increasing the Zn supply to rice plants because Zn allocation to the endosperm is limited, while observed genotypic differences indicate scope for improvement through breeding.     

Distribution and characterization of over 1000 T-DNA tags in rice genome

We generated T-DNA insertions throughout the rice genome for saturation mutagenesis. More than 1,000 flanking sequences were mapped on 12 rice chromosomes. Our results showed that T-DNA tags were not randomly spread on rice chromosomes and were preferentially inserted in gene-rich regions. Few insertions (2.4%) were found in repetitive regions. T-DNA insertions in genic (58.1%) and intergenic regions (41.9%) showed a good correlation with the predicted size distribution of these sequences in the rice genome. Whereas, obvious biases were found for the insertions in the 5'- and 3'-regulatory regions outside the coding regions both at 500-bp size and in introns rather than in exons. Such distribution patterns and biases for T-DNA integration in rice are similar to that of the previous report in Arabidopsis, which may result from T-DNA integration mechanism itself. Rice will require approximately the same number of T-DNA insertions for saturation mutagenesis as will Arabidopsis. A database of the T-DNA insertion sites in rice is publicly available at our web site (http://www.genomics.zju.edu.cn/ricetdna).     

Distribution of T-DNA carrying a Ds element on rice chromosomes

Over 3000 rice plants with T-DNA carrying a Ds element were constructed by Agrobacterium tumefaciens mediation. Using inverse PCR methodology, 590 unique right flanking sequences of T-DNA (Ds) were retrieved from independent transformants and classified into six main types on the basis of the origin of filler DNA between the right border of T-DNA and flanking sequence of rice genome. Type I sequences were the most common and showed canonical integration that T-DNA right border was followed by rice genome sequence with or without filler DNA of no more than 50 bp, while type II sequences displayed a vector-genome combination that T-DNA right border was followed by a vector fragment and then connected with rice genome sequence. The location and distribution of 340 type I and II flanking sequences on the rice chromosome were determined using BLAST analysis. The 340 Ds insertions at an average interval of 0.8 megabase (Mb) constructed a basic framework of Ds starter points on whole rice chromosomes. The frequency of T-DNA (Ds) inserted into the exons of predicted genes on chromosome one was 21%. Knowledge of T-DNA (Ds) locations on chromosomes will prove to be a useful resource for isolating rice genes by Ds transposon tagging as these Ds insertions can be used as starting lines for further mutagenesis.     

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.     

Metabolic engineering and profiling of rice with increased lysine

Lysine (Lys) is the first limiting essential amino acid in rice, a stable food for half of the world population. Efforts, including genetic engineering, have not achieved a desirable level of Lys in rice. Here, we genetically engineered rice to increase Lys levels by expressing bacterial lysine feedback‐insensitive aspartate kinase (AK) and dihydrodipicolinate synthase (DHPS) to enhance Lys biosynthesis; through RNA interference of rice lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) to down‐regulate its catabolism; and by combined expression of AK and DHPS and interference of LKR/SDH to achieve both metabolic effects. In these transgenic plants, free Lys levels increased up to ~12‐fold in leaves and ~60‐fold in seeds, substantially greater than the 2.5‐fold increase in transgenic rice seeds reported by the only previous related study. To better understand the metabolic regulation of Lys accumulation in rice, metabolomic methods were employed to analyse the changes in metabolites of the Lys biosynthesis and catabolism pathways in leaves and seeds at different stages. Free Lys accumulation was mainly regulated by its biosynthesis in leaves and to a greater extent by catabolism in seeds. The transgenic plants did not show observable changes in plant growth and seed germination nor large changes in levels of asparagine (Asn) and glutamine (Gln) in leaves, which are the major amino acids transported into seeds. Although Lys was highly accumulated in leaves of certain transgenic lines, a corresponding higher Lys accumulation was not observed in seeds, suggesting that free Lys transport from leaves into seeds did not occur.     

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.     

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。     

灌浆期气温与源库强度对稻米品质的影响及其生理分析

利用自然条件下的早、晚季种植作为温度处理,选择米质低、中、优等级的6个水稻品种（组合）作为供试材料,研究了早、晚季种植下的稻米品质、淀粉分支酶活性变化,以及温度处理间的差异.结果表明,在籽粒灌浆初期,高温环境下灌浆充实的籽粒,其稻米的垩白增加、透明度变差、整精米率下降,对出糙率及精米率没有明显影响.与早季种植的稻米相比,晚季种植稻米的胶稠度、整精米率、透明度增加.早、晚季种植对稻米直链淀粉含量、蛋白质含量等没有显著影响.剪叶限源处理显著降低稻米质量,疏粒减库处理显著提高稻米品质.     

野生型和ospk1突变体水稻幼苗根对外源糖分的吸收和响应

植物根系如何响应环境因子变化是植物发育和营养吸收研究的重要科学问题。丙酮酸激酶OsPK1在根部的表达主要在根尖成熟区和根毛区,其表达水平变化有可能影响水稻对外源糖分的吸收。采用日本晴和水稻突变体ospk1,通过改变1/2 MS培养基中蔗糖含量,探索水稻幼苗对外源糖分的吸收和响应。通过GC-MS的方法检测了水稻幼苗叶片、叶鞘和根中蔗糖、葡萄糖、果糖和半乳糖的含量。发现根与培养基中糖分接触能明显提高幼苗中的糖含量。并且这些幼苗的根系长度大于那些不加蔗糖的培养基培养的幼苗,表明外源糖分被吸收后能促进根的伸长。OsPK1表达下调影响了糖代谢和外源糖分的吸收。半定量RT-PCR结果显示,幼苗根与糖分的直接接触明显上调根中OsPIP2;4,OsPIP2;5和OsTIP2;1三个水孔蛋白基因的表达。