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10 September 2017, Volume 31 Issue 5 Previous Issue    Next Issue
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Nicotinamide Adenine Dinucleotide(NAD) Biosynthesis Pathway and Leaf Senescence in Rice Ping CHEN, Liwen WU, Zhongwei WANG, Yu ZHANG, Longbiao GUO 2017, 31(5): 447-456.  DOI: 10.16819/j.1001-7216.2017.7028 447 Abstract ( )   HTML ( )   PDF (64362KB) ( )   Leaf is an important photosynthetic organ in rice, and its senescence is initiated by the endogenous genetic and developmental signals and the external environmental stress. Regulation of leaf senescence is a very complicated and delicate process in plants. NAD (nicotinamide adenine dinucleotide), a coenzyme of dehydrogenase, plays an irreplaceable role in glycolysis, gluconeogenesis, Krebs cycle, respiratory chain and other metabolism. The latest researches showed that the NAD biosynthesis in rice is involved in the regulation of the enzyme activity of silencing gene Sirtuins, histone H3K9 deacetylation, plant hormone jasmonic acid (JA) and leaf senescence. This paper highlights cell physiological characteristics, enzyme activity of Sirtuins, NAD biosynthesis of plant leaf senescence, and the OsSRT1-NAD and OsSRT1-MeOH-JA regulation pathways of leaf senescence in rice. It is beneficial to elucidation of the molecular mechanism and regulation pathways of leaf senescence, and application of high yield breeding in rice.

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Function Analyses of Rice Nitrate Transporter Gene OsNPF7.9 in Nitrogen Accumulation and Transport Huimin FENG, Hong LU, Hanqing WANG, Xinyue LI 2017, 31(5): 457-465.  DOI: 10.16819/j.1001-7216.2017.7031 457 Abstract ( )   HTML ( )   PDF (15777KB) ( )   【Objective】 NPF family in plants can transport nitrate and peptide, etc. Functional analyses of OsNPF7.9 can lay a theoretical basis for studying molecular mechanism of high nitrogen use-efficiency.【Method】 Bioinformatics of OsNPF7.9 was predicted with different biological softwares. The OsNPF7.9 promoter-GUS transgenic rice was obtained for analyzing the spatial expression pattern of OsNPF7.9. And the expression patterns of OsNPF7.9 under different N supply levels were observed using semi-quantitative RT-PCR and pOsNPF7.9::GUS transgenic rice. OsNPF7.9 overexpressing rice was obtained, and their different physiological indexes were measured. 【Result】 OsNPF7.9 is localized on plasma membrane with 12 transmembrane domains and a big hydrophilic loop between the 6th and the 7th transmembrane domain. Tissue expression pattern showed that OsNPF7.9 was expressed in roots, leaves, root-shoot junction and flowers. Both RT-PCR and GUS staining of pOsNPF7.9::GUS transgenic rice indicated that the expression of OsNPF7.9 was not affected by N concentration and form. Overexpression of OsNPF7.9 could significantly increase not only nitrate concentration in shoots and roots-shoots nitrate ratio; but also total nitrogen concentration and accumulation in shoots, and roots-shoots total nitrogen ratio. 【Conclusion】OsNPF7.9 participated nitrate transport from roots to shoots, and overexpression of OsNPF7.9 could increase N accumulation and transport from roots to shoots.

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Map-Based Cloning of OsCAD2 Regulating Golden Hull and Internode in Rice Hong WANG, Yingxin ZHANG, Lianping SUN, Shuai MENG, Peng XU, Weixun WU, Shihua CHENG, Liyong CAO, Xihong SHEN 2017, 31(5): 465-474.  DOI: 10.16819/j.1001-7216.2017.6170 465 Abstract ( )   HTML ( )   PDF (11349KB) ( )   【Objective】Rice pigments not only have important physiological effects on their own development, but also have been widely used in rice breeding, by-product improvement, and so on. Phenotypic analysis and gene mapping of pigment-related genes could lay the foundation for the further study of rice pigments metabolism and regulation mechanism. 【Method】A golden hull and internode rice mutant, gh881, was isolated from an elite japonica cultivar Changlijing (CLJ) using ethyl methane sulphonate(EMS) mutagenesis strategy;the main agricultural traits of gh881 and its wild type(WT) were measured at mature stage; we crossed gh881 with WT and ZH8015, respectively, followed by observation of F1 and BC1F1 plants phenotype, and we made genetic analysis using Chi-square test in BC1F2 population and gene mapping using map-based cloning in F2 population; the related-genes expression of young panicles, internodes and flag leaf sheaths of gh881 and WT at various stages were studied by qPCR. 【Result】Compared with wild type, gh881 exhibited golden hull and internode at the mature stage. Except for no significant difference in the number of panicles per plant, there were significant reduction in plant height, the seed-setting rate, the number of spikelets per panicle, the number of grains per panicle and 1000-grain weight between WT and gh881. Genetic analysis and gene fine-mapping results suggested that gh881 was controlled by a single recessive gene and the mutant gene was mapped to a region of 33.2 kb in which four open reading flames (ORFs) existed on the short arm of chromosome 2 between markers FH-13 and RH-25. 【Conclusion】Sequencing analysis revealed that a single base mutation (G to A) occurred at the site 3563 bp of OsCAD2 (Os02g0187800) encoding a Cinnamyl-Alcohol Dehydrogenase (CAD), which led to a G297D mutation (codon GGC to GAC). This implied that gh881 might carry a novel allele of OsCAD2. The quantitative real-time PCR analysis showed that the relative expression level of OsCAD2 decreased significantly in the internode, while it almost increased significantly in the flag leaf sheath and panicle, and the related-genes also almost changed significantly. These results demonstrated that OsCAD2 is an important gene involved in lignin metabolic pathway, and may regulate other related-genes expression feedback.

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Preparation and Application of Centromere-Specific Protein CENH3 Antibody in Rice Chao XUE, Rong ZHANG, Rui GUO, Shuai LIU, Xiaoyu LIU, Mingchen SHEN, Shifeng DENG, Zhiyun GONG 2017, 31(5): 475-482.  DOI: 10.16819/j.1001-7216.2017.7036 475 Abstract ( )   HTML ( )   PDF (656KB) ( )   【Objective】 The centromeres of eukaryotic chromosomes are essential for cell division and inheritance of genetic information. The centromere is a complex composed of centromeric proteins and DNA motifs. In particular, a centromere-specific histone 3 variant, referred to CENH3, is a key centromere-specific protein for centromeric chromatin. So the preparation of CENH3 antibody is necessary for the research of centromeric structure and function. 【Method】We designed a short-peptide for the rabbit immune experiment and prepared CENH3 antibody in rice. ELISA and protein immunofluorescence(IF) assay were performed to detect the effectiveness and availability of CENH3 antibody. 【Result】 The effective dilution of CENH3 antibody was detected at 1:400 000. Through IF analysis, obvious signals were detected in the centromeric region of each chromosome in rice and maize. DNA associated with CENH3 was selectively immunoprecipitated in chromatin immunoprecipitation assay(ChIP). After PCR amplification and FISH(fluorescence in situ hybridization) analysis, the results showed ChIP-DNA was located in functional centromere in rice. 【Conclusion】 These results demonstrated that CENH3 antibody prepared in this study can meet the requirement of related experiments in the research of centromere, and the antibody was prepared successfully.

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Heterologous Expression and Oligomeric Identification of a Small Heat Shock Protein (SHSP) from Oryza sativa Nianjun CAI, Liuming GUO, Jing LI, Congying XIANG, Jian YANG, Jianping CHEN, Hengmu ZHANG 2017, 31(5): 483-488.  DOI: 10.16819/j.1001-7216.2017.7010 483 Abstract ( )   HTML ( )   PDF (739KB) ( )   【Objective】In our previous study, a small heat shock protein gene OsSHSP17.6 was cloned from Oryza sativa and its expression was shown to be significantly up-regulated by heat shock or viral infection, suggesting that the OsSHSP17.6 could play an important role in both biotic and abiotic stress responses. In this study, our objective is to further identify the characteristics of OsSHSP17.6. 【Method】 The OsSHSP17.6 gene was sub-cloned into the plasmid pET-32a, a prokaryotic expression vector, and transformed into Escherichia coli BL21(DE3)pLysS for inducible expression. Then the recombinant protein was purified with affinity chromatography and used for native PAGE and Western-blotting assays. 【Result】Its heterologous expression appeared to alleviate the poisonous effect of IPTG on the host E. coli. Native PAGE and Western-blotting assays showed that the purified recombinant OsSHSP17.6 could form homological dimers and oligomer in vitro. 【Conclusion】 Taken together, these findings supported the hypothesis that the protein should be functional molecular chaperone in vivo and indicated that OsSHPS17.6 could be involved in the stress response by its homological oligomerization, which could contribute to the functional identification of OsSHSP17.6.

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Identification and Fine Mapping of Pale Green Leaf PGL11 in Rice Zhengjun TU, Guoxing ZOU, Lichao HUANG, Long CHEN, Liping DAI, Yihong GAO, Yujia LENG, Li ZHU, Guangheng ZHANG, Jiang HU, Deyong REN, Zhenyu GAO, Guojun DONG, Guang CHEN, Longbiao GUO, Qian QIAN, Dali ZENG 2017, 31(5): 489-499.  DOI: 10.16819/j.1001-7216.2017.7043 489 Abstract ( )   HTML ( )   PDF (7268KB) ( )   【Objective】 Leaf is the main site of photosynthesis in rice. The change of leaf color is directly related to the growth and development of rice. The research of leaf color mutants is an important genetic basis for the study of rice functional genomics. 【Method】We identified a pale green leaf mutant termed pgl11(pale green leaf 11) from japonica cultivar Nipponbare by ethyl methylsulfonate (EMS) treatment. The chlorophyll content of wild type(WT) and mutant was measured at different growth stages. At the seedling stage, the chloroplast structure of the leaves of the WT and the mutant were observed with a transmission electron microscopy. At the tillering stage, the photosynthetic parameters of WT and pgl11 were measured and the stomatal structure was observed. At the mature stage, the main agronomic traits of WT and pgl11 were determined. F2 population derived from pgl11/Nanjing 6 was used to map this gene by position cloning approach.【Result】At seedling stage, every new leaf of the mutant pgl11 was pale green. As the leaves matured, the leaf color gradually turned green. Compared with wild type, the chlorophyll content in pgl11 decreased at seedling stage. However, there was no significant difference at heading stage. In addition, the photosynthetic rate and stomatal conductance of pgl11 were significantly decreased, but the intercellular CO2 concentration was apparently increased. The stomata was abnormal in pgl11. The agronomic traits including plant height, flag leaf width, secondary rachis branch number, grain number per panicle, grain length, grain width, 1000-grain weight and seed setting rate decreased significantly in pgl11 compared with wild type. The expression of genes associated with chloroplast transcription and translation were upregulated in pgl11, while the expression of chlorophyll synthesis and photosynthesis related genes were downregulated. Genetic analysis showed that the mutant phenotype was controlled by a single recessive nuclear gene, and PGL11 was mapped to a 110kb region between the markers C6 and C8 on the short arm of chromosome 1. 【Conclusion】It was suggested that PGL11 gene would be a putative novel pale green leaf gene.

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Effects of Water-Nitrogen Management on Root Traits, Nitrogen Accumulation and Utilization and Grain Yield in Rice with Different Nitrogen Use Efficiency Na LI, Zhiyuan YANG, Zou DAI, Yongjian SUN, Hui XU, Yan HE, Mingjin JIANG, Tianrong YAN, Changchun GUO, Jun MA 2017, 31(5): 500-512.  DOI: 10.16819/j.1001-7216.2017.6145 Abstract ( )   HTML ( )   PDF (2181KB) ( )   【Objective】 The objective of the experiment is to study the effects of water-nitrogen management on root traits, nitrogen accumulation and utilization and grain yield in rice with different N use efficiencies, and the relationship between root traits and nitrogen accumulation and utilization and yield.【Method】In this study, two different nitrogen use efficiency (NUE) rice varieties Dexiang 4103 (high NUE) and Yixiang 3724 (low NUE) were used as the trial material to determine the effects of water-nitrogen management on root traits and grain yield in rice with different NUEs, conventional irrigation and controlled alternative irrigation coupled with SPAD-diagnosis N management, optimal N management and farmer’s usual N management were established as treatment factors. During this progress, the relationship between root traits, nitrogen accumulation and utilization and grain yield were analyzed by ridge regression analysis.【Result】There were significant genotypic differences in root traits at heading stage, grain yield, spikelet number per panicle, 1000-grain weight and total spikelet number. The high NUE variety (Dexiang 4103) had more spikelet number per panicle and larger population capacity, with the yield being 0.24%–11.31% higher than that of low NUE variety (Yixiang 3724). Compared with conventional irrigation, the 1000-grain weight under controlled alternative irrigation was significantly larger, while the former was more favorable for improving panicle number, spikelet number per panicle and population spikele number. SPAD-diagnosis-based N management and optimal N management could increase the panicle number and spikelet number per panicle, which increased the population spikelet number, ensuring the grain yield advantage over farmer’s usual N management. Because of the water-nitrogen interaction effect, the gap in 1000-grain weight between N application and zero N treatments under controlled alternative irrigation narrowed considerably compared with that of conventional irrigation, leading to better effect of N application on grain yield under controlled alternative irrigation. The nitrogen use efficiency of Dexiang 4103 was 8.69% higher than that of Yixiang 3724, the nitrogen accumulation of rice under conventional irrigation was higher, as well as the nitrogen recovery efficiency, agronomic efficiency, physiological efficiency under controlled alternative irrigation. SPAD-diagnosis-based N management and optimal N management were more favorable for imroving nitrogen accumulation and utilization efficiency of rice than farmer’s usual N management.The determination coefficient of ridge regression equation between root traits and grain yield of rice at jointing stage, heading stage and maturity stage ranged from 0.4198 to 0.9028. The relationship between root traits at heading stage and grain yield was the most closely related, and the determination coefficients of high NUE and low NUE varieties all exceeded 0.9. At the jointing stage, the rice fine lateral root length had the greatest effect on grain yield. At the heading stage, there was difference between the high NUE and low NUE varieties, and the coarse lateral root length of the former and the fine lateral root surface area of the latter had the greatest effect on grain yield, respectively. In maturity stage, the relationship between adventitious root length and grain yield was the most important. Rice root morphology at heading stage had close relation with the change of nitrogen accumulation, and the determination coefficient of ridge regression equation was close to 0.7. In terms of water and nitrogen management practice, SPAD-diagnosis N management coupled with conventional irrigation or optimal N management combined with alternative irrigation should be applied to achieve grain yield improvement for both high NUE and low NUE varieties.【Conclusion】The root morphology characters of rice at heading stage was closely related to yield and nitrogen accumulation, and the yield and nitrogen accumulation and utilization efficiency of rice could be improved by reasonable water-nitrogen management.

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Effects of Interaction Between Irrigation Regimes and Nitrogen Rates on Rice Yield and Water and Nitrogen Use Efficiencies Guang CHU, Tingting CHEN, Song CHEN, Chunmei XU, Danying WANG, Xiufu ZHANG 2017, 31(5): 513-523.  DOI: 10.16819/j.1001-7216.2017.7048 513 Abstract ( )   HTML ( )   PDF (1024KB) ( )   【Objective】In order to lay a scientific basis for high yield and high water and nitrogen use efficiencies in rice production, we estimated the effects of the interaction between the alternate wetting and drying (AWD) regime and different nitrogen rates on grain yield, water and nitrogen use efficiencies.【Method】The field experiments were conducted with two irrigation regimes, AWD and conventional irrigation (CI) and three N rates, 80 (low amount, LN), 160 (medium amount, MN) and 240 kg/hm2 (high amount, HN) in Fuyang, Zhejiang Province in 2015 and 2016. The local high-yielding rice cultivar Tianyouhuazhan (three-line indica hybrid combination) was used as experimental material. 【Result】There was an obvious interaction between irrigation regimes and N rates. When compared with CI regime, AWD could reduce redundant vegetative growth and control the number of rice tillers, improve the percentage of productive tillers by 8.1%–10.7%; increase the leaf area duration (LAD) and crop growth rate (CGR) from heading to maturity; increase root dry weight at a depth of 10-20 cm at heading by 24.4%–32.3%, and the root oxidation activity (ROA) during the re-watering period after heading; AWD could promote the remobilization of non-structural carbohydrates (NSC) in culms and sheaths for grain filling. The grain yield, water and nitrogen use efficiencies were the highest in the combination of AWD and MN due to the improved population quality and grain filling, and the combination of AWD and MN was the best water-nitrogen management model in this study. 【Conclusion】The aim of high grain yield and high water and nitrogen use efficiencies could be achieved through integrating AWD with the medium amount of nitrogen application in rice production.

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Effects of Elevated Atmospheric CO2 and Temperature on Dynamics of Leaf Chlorophyll Contents and SPAD Value of Rice in Open-Air Field Conditions Ning ZHOU, Liquan JING, Yunxia WANG, Jianguo ZHU, Lianxin YANG, Yulong WANG 2017, 31(5): 524-532.  DOI: 10.16819/j.1001-7216.2017.7022 Abstract ( )   HTML ( )   PDF (1121KB) ( )   【Objcetive】In view of the potential impacts of increasing atmospheric carbon dioxide (CO2) concentration and air temperature, the dynamic effects of these two important environmental factors and their interaction on leaf chlorophyll contents and SPAD values of rice in field were studied.【Method】 By using T-FACE(Temperature-Free Air CO2 Enrichment) facility, a high yield and excellent-quality japonica rice (Oryza sativa L.) Wuyunjing 23 was grown at two levels of CO2 (ambient and elevated CO2 concentration) and two temperature regimes (ambient and elevated temperature) in a field experiment. We measured leaf chlorophyll contents and SPAD values during the whole growth period of rice plants.【Result】Elevated CO2 concentration increased chlorophyll a, b, a+b contents of rice on 41, 77 and 94 days after transplanting (DAT) , with the maximum increase of 6.4%. On the contrary, they were decreased by elevated CO2 concentration on 110 and 119 DAT, with the maximum decrease of 5.4%. Due to the greater responses of chlorophyll b to CO2 concentration than that of chlorophyll a, elevated CO2 concentration decreased chlorophyll a/b ratio on 41, 77 and 94 DAT by 4.7%, 2.3% and 0.9%, but increased it on 110 and 119 DAT by 1.9% and 5.3%, respectively. No obvious effect of elevated CO2 concentration on leaf SPAD values was detected in the early and middle growth stages. But elevated CO2 concentration decreased leaf SPAD values by 3.5% (P=0.1) and 19.1% (P<0.01) on 110 and 119 DAT. 1℃increase in temperature on average had positive effects on chlorophyll a, b, a+b contents in each growth stage of rice, but negative effects were found on chlorophyll a/b ratio. In general, the magnitudes of variation induced by temperature were less than those by elevated CO2 concentration. No obvious effect of temperature elevation on leaf SPAD values was detected in the early and middle growth stages. But temperature elevation deceased leaf SPAD values by 7.1% (P<0.01) and 14.8% (P<0.01) on 110 and 119 DAT, respectively. No CO2-temperature interaction was detected for most of measured parameters, but significant CO2- or temperature-growth stage interactions were found.【Conclusion】The results indicated that elevated CO2 concentration favored the leaf chlorophyll formation of rice in the early and middle growth stages. But in the late growth stage, leaf chlorophyll contents and SPAD value declined, meanwhile chlorophyll a/b ration increased significantly. Such phenomenon of quick-leaf-senescence induced by elevated CO2 concentration was identical under the two temperature regimes.

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Induced Expression Profiles of Hsp70s in Brown Planthoppers, Nilaparvata lugens, Under Different Temperatures Dan SHAN, Lihua WANG, Yueliang ZHANG, Yangchun HAN, Hongtao NIU, Lei PAN, Jichao FANG 2017, 31(5): 533-541.  DOI: 10.16819/j.1001-7216.2017.6171 533 Abstract ( )   HTML ( )   PDF (2136KB) ( )   【Objective】The brown planthopper (BPH), Nilaparvata lugens, is one of the major insect pests on rice in China. In recent years, the overwintering area of BPH expanded with global warming. In order to explore the response mechanisms of BPH to temperature, the hsp70s were cloned and the induced expression profiles of these genes were analyzed. 【Method】 The nucleotide sequences of hsp70s were gotten from transcriptome sequencing and GenBank database, the phylogenetic tree of HSP70 was based on neighbor-joining method by MEGA4.1, and the induced expression profiles were analyzed by real-time quantitative PCR. 【Result】 It was found that fifteen hsp70 homologous genes with complete open reading frame were gotten in BPH, and 8 out of 15 genes were expressed in normal conditions or temperature stress. These eight hsp70s were distributed in the cytoplasm/nucleus, endoplasmic reticulum or mitochondria, and had significantly different expression in normal conditions. After 30-44°C high temperature treatment, the expression levels of KX976471, KX976473, KX976475, KX976476, KX976477 and KX976478 were up-regulated, and the maximum increased times was 1.72-245.33. However, only the expression level of KX976475 increased by 2.38 times, the other seven were not changed or even significantly decreased in expression level after 0-22°C temperature treatment.【Conclusion】It was found that eight hsp70s were expressed in normal conditions or temperature stress in the cytoplasm/nucleus, endoplasmic reticulum or mitochondria. These hsp70s might play an important role in high temperature adaptation, but a limited role in cold adaptation in BPH.

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Functional Analysis of MoMET3 in Growth, Development and Pathogenicity of Magnaporthe oryzae Xiangyang FENG, Zhen ZHANG, Rongyao CHAI, Haiping QIU, Jiaoyu WANG, Xueqin MAO, Yanli WANG, Guochang SUN 2017, 31(5): 542-550.  DOI: 10.16819/j.1001-7216.2017.7018 Abstract ( )   HTML ( )   PDF (4760KB) ( )   【Objective】 Sulfur is generally made biologically available by activation with ATP in a reaction catalyzed by ATP sulfurylase in fungi. The function of ATP sulfurylase in the growth and pathogenecity of Magnaporthe oryzae was analyzed to lay a foundation for target determination for rice blast prevention.【Method】The knockout vector was constructed by homologous recombination method, and the phenotype of the mutant was analyzed.【Result】MoMET3 was required for vegetative growth and conidiation, but not a virulence factor. The growth rate and conidiation of MoMET3- deleted mutants were significantly decreased in complete medium. Loss of MoMET3 did not affect conidial germination and appressiorial formation, but the growth of secondary hyphae was inhibited in the minimal medium. After complementation of MoMET3 gene, the mutant returned to normal growth and can utilize sulfate. 【Conclusion】These evidences revealed that the reduction of inorganic sulfur is not necessary for the process of conidial germination and appressiorial formation. The endogenous reduced sulfur in conidia was sufficient to meet the sulfur needs in the process of conidial germination and appressiorial formation, and utilization of host-derived reduced sulfur played an important role in the hypha expansion of the blast fungus in hosts.

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A Primary Study on the Mechanism Behind Resistance to Bacterial Panicle Blight of Rice Lu LI, Yihua XU, Mengqi LIANG, Ling WANG, Lianmeng LIU, Yuxuan HOU, Qiqin LI, Shiwen HUANG 2017, 31(5): 551-558.  DOI: 10.16819/j.1001-7216.2017.6167 551 Abstract ( )   HTML ( )   PDF (1362KB) ( )   【Objective】Rice is more susceptible to bacterial panicle blight at the booting stage than seedling stage. The objective of the study is to understand the resistant mechanism in different growing stage of rice and lay a basis for breeding rice varieties with high resistance.【Method】The spraying and injection method were used to inoculate seedlings and panicles of resistant and susceptible rice with Burkholderia glumae, respectively. The activities of three antioxidant enzymes (peroxidase, catalase and superoxide dismutase) of treatments and control were investigated. Real-time PCR was used to detect the expression of five defense response genes (PR1a, PR10b, Rcht, LOX and PAL).【Result】The infection of B. glumae induced the accumulation of reactive oxygen species, the increase of antioxidant enzymes activities and the higher expression of some defense genes. However, the defense response was different at the seedling stage and booting stage of rice. At the booting stage, the activities of antioxidant enzymes (peroxidase, catalase and superoxide dismutase) and the expression of PR10b, Rcht and PAL were higher, but the expression levels of PR1a and LOX were lower than those at the seedling stage.【Conclusion】B. glumae could induce more defense response of rice in booting stage, salicylic acid signaling pathway played an important role during the defense.