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.     

A method for screening copper-tolerant rice (Oryza sativa L.) cultivars based on hydroponic experiments and cluster analysis

A method for screening copper (Cu)-tolerant rice cultivars was studied by combining hydroponic experiments and cluster analysis, and the classification of cultivars in Cu stress tolerance was done. In the first hydroponic experiment, seedlings of Jiahe991 and Xiushui114 were planted in nutrient solution with different Cu²⁺ concentrations from 10 to 1800 μg/L. Results indicated that the toxic threshold of Cu concentration in solution ranged from 900 to 1200 μg/L, since SPAD (Soil and Plant Analyzer Development, SPAD-502, a portable chlorophyll meter, Minolta Camera Co. Ltd., Japan) values of leaves and seedlings biomass of the treatments with ≥900 and/or 1200 µg/L were significantly lower than the control. The second experiment was conducted with 16 local rice cultivars under three Cu treatments (10, 1000, and 1500 μg/L). The 16 cultivars were well classified into tolerant, normal, and sensitive groups as a result of cluster analysis based on the relative SPAD (Soil and Plant Analyzer Development, SPAD-502, a portable chlorophyll meter, Minolta Camera Co. Ltd., Japan) value, shoot and root dry weights, root length and root dehydrogenase activity, and oxidizing capacity and shoot Cu concentration. Xiushui123, Xiushui134, Jiahe991, and Xianghu301 belonged to the tolerant group; Xiushui137 belonged to the sensitive group. The cluster analysis based on hydroponic experiments is an effective method for identifying rice cultivars that are tolerant to Cu stress. In addition, four cultivars (Xiushui123, Xiushui134, Jiahe991, and Xianghu301) are recommended in local practice.     

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.     

Effects of Cadmium Stress on Growth,Morphology, and Protein Expression in Rhodobacter capsulatus B10

The effects of cadmium stress on growth, morphology, and protein expression were investigated in Rhodobacter capsulatus B10 using two-dimensional polyacrylamide gel electrophoresis and a scanning electron microscope with an energy dispersive X-ray spectrometer. The bacterium grew in the presence of 150 μM CdCl₂ and highly induced heat-shock proteins (GroEL and Dnak), S-adenosylmethionine synthetase, ribosomal protein S1, aspartate aminotransferase, and phosphoglycerate kinase. Interestingly, the ribosomal protein S1 was proportionally expressed as the amount of cadmium in the medium, suggesting that S1 may be required for the repair of cadmium-mediated cellular damage. On the other hand, we identified five cadmium-binding proteins: 2-methylcitrate dehydratase, phosphate peripalsmic binding protein, inosine-5′-monophosphate dehydrogenase/guanosine-5′-monophosphate reductase, inositol monophosphatase, and lytic murein transglycosylase. The cadmium-treated cells had a filamentous structure and contained less phosphorous than the untreated cells. We propose that these characteristics of the cadmium-treated cells may be due to the inactivation of the phosphate peripalsmic binding protein and lytic murein transglycosylase by cadmium.     

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.     

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).     

Discrimination of nitrogen isotopes during absorption of ammonium and nitrate at different nitrogen concentrations by rice (Oryza sativa L.) plants

Studies of uptake of ionic sources of N by two hydroponically grown rice (Oryza sativa L.) cultivars (paddy‐field‐adapted Koshihikari and dryland‐adapted Kanto 168) showed that the magnitude of the nitrogen isotope fractionation (?) for uptake of NH₄⁺ depended on the concentrations of NH₄⁺ and cultivar (averaging –6·1‰ for Koshihikari and –12·0‰ for Kanto 168 at concentrations from 40 to 200 mmol m^?3 and, respectively, –13·4 and –28·9‰ for the two cultivars at concentrations from 0·5 to 4 mol m^?3). In contrast, the ? for uptake of NO₃^? in similar experiments was almost insensitive to the N concentration, falling within a much narrower range (+3·2‰ to –0·9‰ for Koshihikari and –0·9‰ to –5·1‰ for Kanto 168 over NO₃^? concentrations from 0·04 to 2 mol m^?3). From longer term experiments in which Norin 8 and its nitrate‐reductase deficient mutant M819 were grown with 2 or 8 mol m^?3 NO₃^? for 30 d, it was concluded that the small concentration‐independent isotopic fractionation during absorption of this ion was not related to nitrate reductase activity.     

Effects of water stress on plant growth and root proteins in three cultivars of rice (Oryza sativa) with different levels of drought tolerance

Rice ( Oryza sativa L.) is considered a drought-sensitive crop species; however, within this species, there are considerable varietal differences in sensitivity to this environmental stress. In the present work, the effect of water stress on germination, plant growth and root proteins in three rice cultivars (Sinaloa, IR10120 and Chiapas) was analyzed. Seed germination and plant growth were found to be significantly inhibited by polyethylene glycol (PEG)-imposed water deficit in cv. Sinaloa; cvs IR10120 and Chiapas were more tolerant to water stress. Fluorographs of two-dimensional electropherograms of in vivo-labeled polypeptides were analyzed to identify changes in the root protein patterns that resulted when plants were grown in the presence of 10% PEG for 10 days. The treatment induced or increased the synthesis of eight polypeptides or groups of polypeptides in cv. Sinaloa, seven in cv, IR10120 and four in cv. Chiapas. The synthesis of several polypeptides was decreased by the PEG treatment in cv. Sinaloa and cv. IR10120. Most of these PEG-induced changes in the root protein patterns were cultivar-specific and only one 26-kDa protein with a pI of 6.0 was induced by water deficit in the two cultivars Sinaloa and IR10120.     

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.     

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。     

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。     

Suppression of phospholipase D genes improves chalky grain production by high temperature during the grain-filling stage in rice

High temperature (HT) during the grain developing stage causes deleterious effects on rice quality resulting in mature grains with a chalky appearance. Phospholipase D (PLD) plays an important role in plants, including responses to environmental stresses. OsPLDα1, α3 and β2-knockdown (KD) plants showed decreased production of chalky grains at HT. HT ripening increased H₂O₂ accumulated in the developing grains. However, the increase was canceled by the knockdown of OsPLDβ2. Expression levels of OsCATA which is one of three rice catalase genes, in developing grains of OsPLDβ2-KD plants at 10 DAF were increased compared with that in vector-controls in HT growth conditions. Overexpression of OsCATA markedly suppressed the production of chalky grains in HT growth conditions. These results suggested that OsPLDβ2 functions as a negative regulator of the induction of OsCATA and is involved in the production of chalky grains in HT growth conditions.     

Improved oxidative tolerance in suspension‐cultured cells of C_4-pepctransgenic rice by H_2O_2 and Ca~(2+)under PEG-6000

To understand the molecular responses of PC(Overexpressing the maize C4-pepc gene, which encodes phosphoenolpyruvate carboxylase(PEPC)), to drought stress at cell level, we analyzed changes in the levels of signaling molecules(hydrogen peroxide(H2O2), calcium ion(Ca2t), and nitric oxide(NO)) in suspension-cultured PC and wild-type(WT)rice(Oryza sativa L.) cell under drought stress induced by 20%polyethylene glycol 6000(PEG-6000). Results demonstrated that PC improved drought tolerance by enhancing antioxidant defense, retaining higher relative water content, survival percentages, and dry weight of cells. In addition, PEPC activity in PC under PEG treatment was strengthened by addition of H2O2 inhibitor, dimethylthiourea(DMTU) and NO synthesis inhibitor, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(c PTIO), respectively, while that in PC was weakened by addition of free calcium chelator, ethylene glycol-bis(b-aminoethylether)-N,N,N0,N0-tetraacetic acid(EGTA)t calcium channel outflow inhibitor, ruthenium red(RR) t plasma membrane channel blocker La(NO3)3, but EGTA t RR did Reseanot. Results also showed that NO and Ca2 twas lying downstream of H2O2 in drought-induced signaling. Calcium ion was also involved in the expression of C4-pepc in PC. These results suggested that PC could improve oxidative tolerance in suspension-cultured cells and the acquisition of this tolerance required downregulation of H2O2 and the entry of extracellular Ca2 tinto cells across the plasma membrane for regulation of PEPC activity and C4-pepc expression.     

施氮和肥料添加剂对水稻产量、氮素吸收转运及利用的影响

在苏南太湖地区开展田间试验,研究了施氮和肥料添加剂对水稻产量、氮素吸收转运及利用的影响.结果表明:施氮对水稻产量、各生育时期植株累积吸氮量、阶段氮累积量和花后氮素转运量具有显著的促进作用(P＜0.01),当施氮量高于200 kg·hm-2时,增施氮肥的增产效应不显著(P＞0.05);花后氮素转运率和氮肥利用率均随施氮量的增加而降低.施用肥料添加剂可进一步提高水稻产量、累积吸氮量、花后氮素转运量和氮肥利用率,且该效应在高施氮量( ≥200 kg·hm-2)条件下表现更明显.本试验条件下不施用肥料添加剂时,施氮150kg·hm-2可同时获得较高的产量和氮肥利用率.     

低钾胁迫对水稻(Oryza sativa L.)化感潜力变化的影响

研究以国际公认的化感水稻P1312777和非化感水稻Lemont为供体,稗草（Echinochloa cru-galli L.）为受体,采用稻／稗共培体系,研究低钾胁迫对水稻化感潜力变化的影响及其机制。受体稗草的形态指标分析结果表明,低钾胁迫促使化感水稻P1312777对共培稗草的根长、株高和干重的抑制率均升高,增幅远大于非化感水稻Lemont。受体稗草生理生化指标分析结果表明,低钾胁迫下化感与非化感水稻对受体稗草保护酶系（SOD、POD、CAT）及根系活力的抑制作用增强,但化感水稻P1312777比非化感水稻Lemont的抑制程度大,且达极显著差异。实时荧光定量PCR分析结果表明,低钾胁迫下,化感水稻P1312777根部与叶部中酚类代谢的关键酶——苯丙氨酸解氨酶、肉桂酸-4-羟化酶、羟化酶、O-甲基转移酶的基因均上调表达,而非化感水稻根部相应酶均下调表达,叶部除苯丙氨酸解氨酶上调,其余酶也下调表达。而萜类代谢途径关键酶——HMG—CoA还原酶、角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶的基因,在两种水稻根部中呈现出相同或相似的表达方式（上调或下调）,即HMG—CoA还原酶上调表达,角鲨烯合酶、单萜烯环化酶、倍半萜烯环化酶、二萜烯环化酶下调表达;而在水稻叶部,非化感水稻Lmont相应酶基因表达方式仍然不变,化感水稻P1312777除了角鲨烯合酶下调表达,其余4个酶均上调表达。水稻根系分泌物中酚类物质的HPLC分析结果表明,低钾胁迫下,化感水稻P1312777根系分泌物中,所检出的酚酸类物质总量是正常营养条件下的2．30倍,而非化感水稻Lemont则是正常营养条件下的0．91倍。综合分析认为低钾胁迫下,化感水稻P1312777抑草能力增强主要是由于酚类代谢途径关键酶基因表达上调,导致酚类代谢途径旺盛,分泌出更多的酚类物质,进而破坏受体稗草保护酶系统,抑制了稗草的正常生长。     

Effects of nitrogen nutrition on the relationships between the levels of rbcS and rbcL mRNAs and the amount of ribulose 1·5-bisphosphate carboxylase/oxygenase synthesized in the eighth leaves of rice from emergence through senescence

Soon after the emergence of the eighth leaf blades, rice plants were grown with two (1 and 4 mm ) levels of nitrogen (N) supply, and the relationships between the levels of rbcS and rbcL mRNAs, the amount of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) synthesized and the N influx were examined in the eighth leaf blades from emergence through senescence. The levels of both rbcS and rbcL mRNAs, the amount of Rubisco synthesized and the N influx were greater for the 4 mm N treatment than for the 1 mm N treatment throughout the experiment. The amount of Rubisco synthesized was well correlated with the levels of both mRNAs during leaf expansion, but not after the completion of leaf expansion in both N treatments. The ratio of the amount of Rubisco synthesized to the levels of both mRNAs dramatically declined after full expansion. On the other hand, the amount of Rubisco synthesized was well correlated with the N influx in both N treatments. These results indicate that the N influx, namely, N availability, rather than the levels of rbcS and rbcL mRNAs, is more closely related to the amount of Rubisco synthesized in the leaf blade of rice throughout the lifespan of a leaf.     

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.     

Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high-throughput method. Chemical derivatizations are a prerequisite for gas-chromatographic separation, and a large sample quantity is needed for liquid-chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis-mass spectrometry (CE-MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis-diode array detector (CE-DAD) system further enables almost all water-soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.     

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.