Purification and characterization of a XIP-type endoxylanase inhibitor from Rice (Oryza sativa)

A rice XIP-type inhibitor was purified by affinity chromatography with an immobilized Aspergillus aculeatus family 10 endoxylanase. Rice XIP is a monomeric protein, with a molecular mass of ca. 32?kDa and a pI of ca. 5.6. Its N-terminal amino acid sequence was identical to that of a rice chitinase homologue, demonstrating the difficulty when using sequence information to differentiate between endoxylanase inhibitors and (putative) chitinases in rice. Rice XIP inhibited different endoxylanases to a varying degree. In particular, it most strongly inhibited family 10 endoxylanases from A. niger and A. oryzae, while several family 11 enzymes from Bacillus subtilis, A. niger and Trichoderma sp. were not sensitive to inhibition. The above mentioned A. aculeatus endoxylanase was not inhibited either, although gel permeation chromatography revealed that it complexed rice XIP in a 1:1 molar stoichiometric ratio.     

Cloning and Characterization of Catalases from Rice,Oryza sativa L.

Catalase is the major H₂O₂-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)₆-tagged proteins in Escherichia coli. The recombinant (His)₆-polypeptides were enriched to apparent homogeneity and characterized. With H₂O₂ as substrate, the highest catalase k _cat value (20±1.71×10^?3 min^?1) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.     

Mutation of Oryza sativa CORONATINE INSENSITIVE 1b (OsCOI1b) delays leaf senescence

Jasmonic acid (JA) functions in plant development, including senescence and immunity. Arabidopsis thaliana CORONATINE INSENSITIVE 1 encodes a JA receptor and functions in the JA‐responsive signaling pathway. The Arabidopsis genome harbors a single COI gene, but the rice (Oryza sativa) genome harbors three COI homologs, OsCOI1a, OsCOI1b, and OsCOI2. Thus, it remains unclear whether each OsCOI has distinct, additive, synergistic, or redundant functions in development. Here, we use the oscoi1b‐1 knockout mutants to show that OsCOI1b mainly affects leaf senescence under senescence‐promoting conditions. oscoi1b‐1 mutants stayed green during dark‐induced and natural senescence, with substantial retention of chlorophylls and photosynthetic capacity. Furthermore, several senescence‐associated genes were downregulated in oscoi1b‐1 mutants, including homologs of Arabidopsis thaliana ETHYLENE INSENSITIVE 3 and ORESARA 1, important regulators of leaf senescence. These results suggest that crosstalk between JA signaling and ethylene signaling affects leaf senescence. The Arabidopsis coi1‐1 plants containing 35S:OsCOI1a or 35S:OsCOI1b rescued the delayed leaf senescence during dark incubation, suggesting that both OsCOI1a and OsCOI1b are required for promoting leaf senescence in rice. oscoi1b‐1 mutants showed significant decreases in spikelet fertility and grain weight, leading to severe reduction of grain yield, indicating that OsCOI1‐mediated JA signaling affects spikelet fertility and grain filling.     

逆境诱导水稻谷胱甘肽合成酶基因的表达研究

用RACE方法获得了全长的水稻谷胱甘肽合成酶(GS)基因的cDNA,并命名为OsGS(GeneBankaccession No.:AY453405)。该cDNA全长1 892 bp,编码一个由540个氨基酸组成的多肽,预测其氨基端(N端)含有一段定位叶绿体的信号肽。比较水稻基因组定位结果表明OsGS基因位于水稻12号染色体短臂上,转录区全长6 321 bp,由12个外显子和11个内含子组成。通过RT-PCR对OsGS在水稻正常生长条件和逆境条件下的表达进行了研究。结果表明,在正常生长条件下,OsGS在水稻幼苗的根和叶以及抽穗期水稻的根中表达;但不在抽穗期水稻的叶、茎和幼穗中表达,这显示OsGS在水稻中的表达具有发育和组织特异性。利用抽穗期水稻的叶片为材料,经高温、干旱和重金属逆境处理后,OsGS在抽穗期叶片中的转录被诱导表达;而在盐、低温、伤害逆境下则不被诱导。在轻度和中度干旱胁迫4 h后OsGS基因可被诱导表达。外源ABA处理也能够提高OsGS的转录水平,这显示OsGS可能是依赖ABA信号途径的环境胁迫诱导基因。     

Isolation and Characterization of Golgi Membranes from Suspension-cultured Cells of Rice (Oryza sativa L.)

The isolation of Golgi membranes from suspension-cultured cellsof rice (Oryza sativa L.) was attempted by linear glycerol densitygradient centrifugation. When "burst" membranes in the pelletobtaind after differential centrifugation at 100,000 ? g weresuspended in 20% (w/w) glycerol in 50 mM malate-NaOH (pH 6.0)and loaded onto a linear density gradient of glycerol, whichextended from 30 to 80% (w/w) in 1 mM EDTA in 50 mM glycylglycine-NaOH(pH 7.5), IDPase, a marker enzyme for Golgi membranes, was separatedfrom other membrane markers on the glycerol gradient. In addition,UDPase and GDPase activities overlapped with the peak fractionof IDPase activity. Furthermore, membrane glycoproteins in eachfraction were characterized by lectin-peroxidase staining. ConcanavalinA and lentil lectin, which have the ability to bind to the high-mannosetype of oligosaccharide, bound to glycoproteins distributedin ER membrane fractions, while wheat germ lectin, castor beanlectin, peanut lectin, and Ulex europaeus lectin-I which recognizethe complex type and/or the mucin type of oligosaccharides interactedwith glycoproteins in the Golgi membrane fractions but not withthose in the ER membrane. These results strongly suggest thatthe oligosaccharide structures of glycoproteins in the ER membraneare of the high-mannose type, whereas glycoproteins in the Golgimembrane have modified N-linked and/or O-linked oligosaccharidechains. (Received November 9, 1988; Accepted October 17, 1989)     

Photothermal Responses of Flowering in Rice (Oryza sativa)

Durations from sowing to panicle emergence in 16 diverse genotypesof rice (Oryza sativa L.) were recorded in 13 different photothermalregimes, comprising constant and diurnally alternating temperaturesbetween 16 and 32 °C and photoperiods between 10.5 and 15.0h d^–1—all provided by controlled-environment growthcabinets. In 11.5 h days and at sub-optimal temperatures, relationsbetween the rate of progress towards panicle emergence and meantemperature were linear in all genotypes, and amongst thesethe base temperature at that photoperiod varied between 6.6and 11.9 °C. In most cases progress was most rapid at 24–26°C, i.e. the optimum temperature was much cooler than expectedfrom previously published values of times to panicle emergencein a less extensive range of photothermal regimes. Only in threecultivars was it warmer than 28 °C, and in these there weresufficient data to establish that relations between rates ofprogress to panicle emergence and photoperiod in the diurnallyalternating temperature regime of 28–20 °C are alsolinear. Also, the responses of these three cultivars provideno evidence of any interaction between the effects of photoperiodand temperature. We conclude, then, that the model in whichrate of development is a linear function of both temperatureand photoperiod with no interaction, which has been shown tobe common to many other species, also applies to rice. Differencesamong genotypes in relative sensitivity of rate of progresstowards panicle emergence to both temperature and to photoperiodwere considerable; japonica cultivars tended to be more sensitiveto temperature and less sensitive to photoperiod than indicacultivars. Four indica cultivars bred and selected at The InternationalRice Research Institute (IRRI) in the Philippines did not differ(P > 0.10) in their relations between rate of progress towardspanicle emergence and sub-optimal temperatures in a daylengthof 11.5 h, but the optimum temperature for cv. IR 36 was appreciablywarmer than that for the cvs IR 5, IR 8 and IR 42. Oryza sativa, rice, flowering, temperature, photoperiod, photothermal responses     

水稻染色体G—带的研究

用改良的ASG法首次在籼稻(O.sativa subsp.indica)品种珍汕97和粳稻(O.subsp.iaponica)品种秀岭的有丝分裂染色体上显示了G-带,并作了相应的G-带核型分析。就同一材料来说,随着有丝分裂时期的推进,染色体上带纹数目逐渐减少。籼、粳亚种间相对应的同源染色体上G-带带纹特征彼此相似。讨论了水稻G-带带型与染色体不同区域分化的关系;G-带带型与籼、粳稻分歧的关系;以及G-显带的方法。     

Rice (Oryza sativa) as a Remediation Tool for Nutrient Runoff

Hypereutrophication of U.S. surface waters is one of the leading causes of impairment for water quality. With nutrient criteria development and total maximum daily load (TMDL) issues looming for regulators, agricultural research is focusing on practices aimed at decreasing nutrient contributions to receiving aquatic ecosystems. This study examined the use of rice (Oryza sativa) for luxury uptake of nitrogen and phosphorus components associated with agricultural storm runoff. Mesocosms (379 L) planted with rice were exposed to two concentrations (5 and 10 mg/L) of nitrate, ammonia, and orthophosphorus. Results from these mesocosms were compared to unvegetated controls (also amended with 5 or 10 mg/L nitrate, ammonia, and orthophosphorus) to determine efficiency of rice in remediating nutrient runoff. Statistically significant differences in ammonia and nitrate retention of vegetated mesocosms amended with 5 mg/L versus vegetated mesocosms amended with 10 mg/L were noted after the first exposure. Although rice is a nutrient-dependent aquatic plant, this study suggests that more efficient mitigation is possible at lower inflow concentrations as opposed to higher inflow concentrations.     

Organogenesis and Plantlet Formation from Organ- and Seedling-Derived Calli of Rice (Oryza sativa)

Callus was induced in different somatic organs of Oryza sativa L. Specific minimum 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations in the medium were necessary for the induction of callus from different organs while high levels of 2,4-D (6–10 mg/l) induced callus formation in each organ tested. The optimum 2,4-D concentration for callus induction and growth for root-derived calli was 2 mg/l and for leaf-derived 6 mg/l. Root and shoot organogenesis were induced in both root- and leaf-derived calli by sub-culturing to a medium lacking 2,4-D. Root organogenesis occurred at a higher frequency than shoot organogenesis. Shoot organogenesis rarely occurred in calli without differentiated roots. Increased age of callus cultures almost completely inhibited shoot development. The addition of the cytokinin 6-γ,γ-dimethylallyl-amino purine partially restored the potential for shoot organogenesis. Whole plants were easily recovered from the calli and grown to maturity with some plants exhibiting phenotypic abnormalities.     

3种水稻线粒体tRNA~(Trp)突变体的克隆和活力鉴定

 设计并完成了 3种水稻线粒体tRNATrp的突变 ,体外转录并用枯草杆菌和人色氨酰tRNA合成酶 (TrpRS)对tRNATrp及其突变体进行了活力测定 .3种突变体的氨酰化活力比野生型水稻线粒体tRNATrp分别上升了 1 8、1 5和 5倍 .说明A1 U72和G5 C68对于提高线粒体tRNATrp被细胞质TrpRS氨酰化能力的作用并不大 ,细胞质tRNATrp与细胞质TrpRS的识别方式并不适用于线粒体tRNATrp与细胞质TrpRS的相互识别 .研究结果对于了解线粒体tRNATrp和细胞质TrpRS的相互识别及药物设计有重要意义     

Molecular Cloning and Expression of a cDNA Encoding Lon Protease from rice (Oryza sativa)

The ATP-dependent Lon protease is a highly conserved enzyme that is present in archeae, eubacteria, and eukaryotes, and plays an important role in intracellular protein degradation. We have isolated a Lon protease gene, OsLon1, from Oryza sativa. The cDNA contained a 2,655 bp ORF. Comparative analysis showed that OsLon1 shared significant similarity with the previously reported Lon proteases from maize, Arabidopsis, human, and bacteria. Tissue expression pattern analysis revealed that OsLon1 was highly expressed in young leaves, mature leaves, and leaf sheaths but only weakly in young roots, mature roots, and young panicles. The OsLon1 gene was successfully expressed in E. coli and the detected protein size, about 120 kDa, matched the expected molecular mass of the His-tagged OsLon1 protein.