Genome Wide Association Mapping of Grain Arsenic, Copper, Molybdenum and Zinc in Rice (Oryza sativa L.) Grown at Four International Field Sites

The mineral concentrations in cereals are important for human health, especially for individuals who consume a cereal subsistence diet. A number of elements, such as zinc, are required within the diet, while some elements are toxic to humans, for example arsenic. In this study we carry out genome-wide association (GWA) mapping of grain concentrations of arsenic, copper, molybdenum and zinc in brown rice using an established rice diversity panel of,300 accessions and 36.9 k single nucleotide polymorphisms (SNPs). The study was performed across five environments: one field site in Bangladesh, one in China and two in the US, with one of the US sites repeated over two years. GWA mapping on the whole dataset and on separate subpopulations of rice revealed a large number of loci significantly associated with variation in grain arsenic, copper, molybdenum and zinc. Seventeen of these loci were detected in data obtained from grain cultivated in more than one field location, and six co-localise with previously identified quantitative trait loci. Additionally, a number of candidate genes for the uptake or transport of these elements were located near significantly associated SNPs (within 200 kb, the estimated global linkage disequilibrium previously employed in this rice panel). This analysis highlights a number of genomic regions and candidate genes for further analysis as well as the challenges faced when mapping environmentally-variable traits in a highly genetically structured diversity panel.

水稻(Oryza sativa L. ssp japonica)幼苗根质膜蛋白质组分析

根质膜具有重要的生物学功能，它参与了根响应脱落酸（ABA）的一系列活动。尽管已经有很多有关ABA影响根的生长和发育的报道，但是在蛋白质组水平上研究参与ABA信号转导及相关活动的质膜蛋白质的报道还未见到。我们期望利用蛋白质组学技术平台研究外源ABA胁迫下水稻根质膜与ABA功能相关的蛋白质组的变化。 本论文通过双向电泳（2DE）结合质谱（MALDI-TOF MS 和 MALDI-TOF/TOF MS）分析的方法鉴定了102个质膜相关蛋白质。这些蛋白质功能涉及到跨膜运输（16.2%）、胁迫反应（14.3%）、物质运输（4.8%）、细胞骨架动态变化（5.7%）、细胞壁重建（3.8%）、碳代谢和能量循环（13.3%）、蛋白质代谢（14.3%）、信号转导（18.1%）和其他功能的蛋白质（4.8%），以及未知功能的蛋白质（2.9%）。其中大约30％的蛋白质以同工型的形式存在。在这些鉴定结果中，有10个斑点（代表10种蛋白质）已被报道为质膜特异的蛋白质;68个蛋白质斑点（代表58种蛋白质）是质膜相关蛋白质。其余54个蛋白质斑点（代表42种蛋白质）是首次在水稻根的质膜囊泡中被鉴定出来。 在ABA处理条件下，我们在2DE胶上发现了15个响应ABA调节的蛋白质斑点。9个上调的蛋白质斑点分别代表以下9种蛋白质：vacuolar proton-ATPase A subunit, vacuolar ATPase B subunit、patatin、 Salt-stress root protein RS1、谷氨酰氨合成酶（Glutamine synthetase，GS）、OSR40c1、H+-exporting ATPase （vacuolar ATPase E subunit）、甘油醛-3-磷酸脱氢酶I型（glyceraldehyde-3- phosphate dehydrogenase, type I，GADPH）和醛缩酶C-1（aldolase C-1）。6个下调的蛋白质斑点分别代表4种蛋白质：endosperm lumenal binding protein、remorin protein、富含脯氨酸蛋白质（glycine-rich protein，GRP）和蔗糖合成酶（sucrose synthase, SuSy）。其中，OSR40c1和endosperm lumenal binding protein与蛋白质合成相关，从它们与ABA的关系中可以看出，ABA可能抑制了细胞的蛋白质合成。而vacuolar proton-ATPase A subunit、vacuolar ATPase B subunit和 H+-exporting ATPase参与了细胞质pH的调控，ABA致使了细胞质pH的上升。甘油醛-3-磷酸脱氢酶I型、醛缩酶C-1和蔗糖合酶参与了细胞壁的生长发育，ABA的作用可能导致了细胞壁生长发育的延迟。ABA促使Patatin上升，其作用可能与质膜膜脂的降解有关。而ABA的刺激也使谷氨酰氨合成酶的表达显著上升，谷氨酰氨合成酶可以去除细胞内有害的游离NH+4。同时还有未知功能的富含脯氨酸蛋白质（glycine-rich protein，GRP）同样受到ABA的诱导，但具体的功能及其与ABA的关系还要进一步的实验证据。

Rastrojo de arroz (Oryza sativa L.) en sistemas de siembra directa : alternativas de manejo

Los objetivos principales del presente trabajo fueron: a) Estudiar cuál es el uso adecuado de las distintas herramientas para mejorar la mineralización del rastrojo de la cosecha de arroz (Oryza sativa L.) bajo siembra directa y b) Determinar, para períodos cortos, la cantidad de rastrojo depositado superficialmente y su tiempo de mineralización al utilizar las distintas técnicas de manejo del mismo. Los tratamientos fueron: cosechadora con desparramador deflector (T1DD), cosechadora sin desparramador triturador (T2SDT), cosechadora con desparramador centrífugo de caucho (T3DC) y cosechadora con desparramador metálico (T4DM). Alternativas implementadas: a) desmalezado b) rolo cuchilla y c) quemado de rastrojo. En las primeras dos alternativas, se aplicaron tres dosis de urea. Los resultados principales fueron: a) La T2SDT produjo, una mayor deposición de material en el centro de la máquina (21680 kg ha-1), b) la distribución de rastrojo de la (T3DC) fue más uniforme que para los otros tratamientos. Las principales conclusiones fueron: 1) Los desparramadores centrífugos de caucho y metálico produjeron mejor distribución del material, 2) El uso de desmalezadora o rolo cuchilla sobre rastrojo de los tratamientos T3DC y T4DM aceleró la mineralización de los mismos. 3) Las dosis de urea causó poco incremento en la mineralización del rastrojo.

Nitrogenase activity (acetylene reduction) associated with leaves of different cultivars of paddy (Oryza sativa L.)

An investigation was conducted to study the levels of nitrogen fixation on the leaf or sheath surfaces of four cultivars of paddy plants by using acetylene reduction technique. Varying levels of positive nitrogenase activity were observed on all the leaf surfaces. Sheath of IET 1991 cultivar showed a higher rate of fixation than the leaf surface. All the nitrogen-fixing organisms on the leaf or sheath surfaces belonged to the genus Beijerinckia. There was no correlation between the bacterial density and the level of fixation. Scanning electron microscopic data revealed that the upper surface of IET 1991 leaf was highly silicified and the microflora was either scanty or nil while the lower surface appeared quite different and harboured more micro-organisms. Similarly, the inner surface of sheath was devoid of silicification and showed the presence of micro-organisms.

Grain Accumulation of Selenium Species in Rice (Oryza sativa L.)

Efficient Se biofortification programs require a thorough understanding of the accumulation and distribution of Se species within the rice grain. Therefore, the translocation of Se species to the filling grain and their spatial unloading were investigated. Se species were supplied via cut flag leaves of intact plants and excised panicle stems subjected to a +/- stem-girdling treatment during grain fill. Total Se concentrations in the flag leaves and grain were quantified by inductively coupled plasma mass spectrometry. Spatial accumulation was investigated using synchrotron X-ray fluorescence microtomography. Selenomethionine (SeMet) and selenomethylcysteine (SeMeSeCys) were transported to the grain more efficiently than selenite and selenate. SeMet and SeMeSeCys were translocated exclusively via the phloem, while inorganic Se was transported via both the phloem and xylem. For SeMet- and SeMeSeCys-fed grain, Se dispersed throughout the external grain layers and into the endosperm and, for SeMeSeCys, into the embryo. Selenite was retained at the point of grain entry. These results demonstrate that the organic Se species SeMet and SeMeSeCys are rapidly loaded into the phloem and transported to the grain far more efficiently than inorganic species. Organic Se species are distributed more readily, and extensively, throughout the grain than selenite.

Variation in grain arsenic assessed in a diverse panel of rice (Oryza sativa) grown in multiple sites

• Inorganic arsenic (As(i) ) in rice (Oryza sativa) grains is a possible threat to human health, with risk being strongly linked to total dietary rice consumption and consumed rice As(i) content. This study aimed to identify the range and stability of genetic variation in grain arsenic (As) in rice. • Six field trials were conducted (one each in Bangladesh and China, two in Arkansas, USA over 2 yr, and two in Texas, USA comparing flooded and nonflood treatments) on a large number of common rice cultivars (c. 300) representing genetic diversity among international rice cultivars. • Within each field there was a 3-34 fold range in grain As concentration which varied between rice subpopulations. Importantly, As(i) correlated strongly with total As among a subset of 40 cultivars harvested in Bangladesh and China. • Genetic variation at all field sites was a large determining factor for grain As concentration, indicating that cultivars low in grain As could be developed through breeding. The temperate japonicas exhibited lower grain As compared with other subpopulations. Effects for year, location and flooding management were also statistically significant, suggesting that breeding strategies must take into account environmental factors.

Arsenic sequestration in iron plaque, its accumulation and speciation in mature rice plants (Oryza sativa L.)

A compartmented soil-glass bead culture system was used to investigate characteristics of iron plaque and arsenic accumulation and speciation in mature rice plants with different capacities of forming iron plaque on their roots. X-ray absorption near-edge structure spectra and extended X-ray absorption fine structure were utilized to identify the mineralogical characteristics of iron plaque and arsenic sequestration in plaque on the rice roots. Iron plaque was dominated by (oxyhydr)oxides, which were composed of ferrihydrite (81-100%), with a minor amount of goethite (19%) fitted in one of the samples. Sequential extraction and XANES data showed that arsenic in iron plaque was sequestered mainly with amorphous and crystalline iron (oxyhydr)oxides, and that arsenate was the predominant species. There was significant variation in iron plaque formation between genotypes, and the distribution of arsenic in different components of mature rice plants followed the following order: iron plaque > root > straw > husk > grain for all genotypes. Arsenic accumulation in grain differed significantly among genotypes. Inorganic arsenic and dimethylarsinic acid (DMA) were the main arsenic species in rice grain for six genotypes, and there were large genotypic differences in levels of DMA and inorganic arsenic in grain.

Iron plaque formation, arsenic uptake and speciation in different genotypes of mature rice plants (Oryza sativa L.)

A compartmented soil-glass bead culture system was used to investigate characteristics of iron plaque and arsenic accumulation and speciation in mature rice plants with different capacities of forming iron plaque on their roots. X-ray absorption near-edge structure spectra and extended X-ray absorption fine structure were utilized to identify the mineralogical characteristics of iron plaque and arsenic sequestration in plaque on the rice roots. Iron plaque was dominated by (oxyhydr)oxides, which were composed of ferrihydrite (81-100%), with a minor amount of goethite (19%) fitted in one of the samples. Sequential extraction and XANES data showed that arsenic in iron plaque was sequestered mainly with amorphous and crystalline iron (oxyhydr)oxides, and that arsenate was the predominant species. There was significant variation in iron plaque formation between genotypes, and the distribution of arsenic in different components of mature rice plants followed the following order:? iron plaque > root > straw > husk > grain for all genotypes. Arsenic accumulation in grain differed significantly among genotypes. Inorganic arsenic and dimethylarsinic acid (DMA) were the main arsenic species in rice grain for six genotypes, and there were large genotypic differences in levels of DMA and inorganic arsenic in grain. A compartmented soil-glass bead culture system was used to investigate characteristics of iron plaque and arsenic accumulation and speciation in mature rice plants with different capacities of forming iron plaque on their roots. X-ray absorption near-edge structure spectra and extended X-ray absorption fine structure were utilized to identify the mineralogical characteristics of iron plaque and arsenic sequestration in plaque on the rice roots. Iron plaque was dominated by (oxyhydr)oxides, which were composed of ferrihydrite (81-100%), with a minor amount of goethite (19%) fitted in one of the samples. Sequential extraction and XANES data showed that arsenic in iron plaque was sequestered mainly with amorphous and crystalline iron (oxyhydr)oxides, and that arsenate was the predominant species. There was significant variation in iron plaque formation between genotypes, and the distribution of arsenic in different components of mature rice plants followed the following order:? iron plaque > root > straw > husk > grain for all genotypes. Arsenic accumulation in grain differed significantly among genotypes. Inorganic arsenic and dimethylarsinic acid (DMA) were the main arsenic species in rice grain for six genotypes, and there were large genotypic differences in levels of DMA and inorganic arsenic in grain.

Phytochemical investigations on ‘black glumed’ Njavara (Oryza sativa L.), the medicinal rice, as compared to staple varieties and evaluation of their antioxidant, anti-inflammatory and anticancer effects

The study was planned to investigate the bioactive compounds in Njavara compared to staple varieties and their bioactivity to substantiate the medicinal properties. Results of the study on chemical indices, antioxidant activity and antiinflammatory activity (in vivo) of Njavara black rice bran and rice in comparison with non-medicinal varieties like Sujatha and Palakkadan Matta rice bran and rice are given. The phytochemical investigation and quantification of Njavara extracts in comparison with staple varieties are detailed in this study. The last chapter is divided in three sections (A, B and C). Section A comprises the antioxidant activity by in vitro assays like DPPH, superoxide anion radical and hydrogen peroxide scavenging activity of the compounds. Also, theoretical studies using DFT were carried out based on DPPH radical scavenging activity for understanding the radical stability and mechanism of antioxidant activity. Section B comprises the anti-inflammatory activity of the identified compounds namely tricin and two flavonolignans in both in vivo and in vitro models. Section C describes the cytotoxicity of the rare flavonolignans, tricin 4’-O-(erythro-β-guaiacylglyceryl) ether and tricin 4’-O-(threo-β-guaiacylglyceryl) ether towards multiple cancer cells belonging to colon, ovarian and breast tumours.

Genotype x culture media interaction effects on regeneration response of three indica rice cultivars

Interactive effects of genotypes with callus induction and regeneration media combinations on green plantlet regeneration response were studied for three indica rice (Oryza sativa L.) cultivars, IR-72, IR-54 and Karnal Local. Isolated mature-embryoswere used to derive scutellar callus and fifteen media combinations involvingMS, N6, R2, SK1 and some modifications were tested. Regeneration percentage as well as the shoot-bud induction frequency were influenced by genotype, callus induction medium, regeneration medium, interaction between genotype and the two media (callus induction and regeneration) as well the interaction between the callus induction medium and regeneration medium. Basal media combination of SK1m (callusing) and MS (regeneration) was found to be the best for cv. Karnal Local in which regeneration frequency of 88% and shoot-bud induction of 233% was observed. In IR-72, the highest regeneration frequency of 47.5% and shoot-bud induction frequency of 77% was obtained on MS-MS combination. In IR-54, highest regeneration frequency (25%) was recorded on MMS(N)-MMS(N) combination, whereas, highest frequency of shoot-bud induction (50%) was observed on MMS(S)-MS combination. Although genotype and the composition of the callus induction basal medium were the major determinants of regeneration response, an overall analysis of variation also revealed a significant interaction between the media used for de-differentiation (callusing) and re-differentiation (plantlet regeneration)

超高产杂交稻Oryza sativa L.光合作用和光抑制特性的研究

较系统地比较研究了超高产杂交稻两优培九培矮64S 93-11和华安3号X07S 紫恢100与多年来大规模推广种植的杂交稻品种汕优63珍汕97A 明恢63的光合生理特性结果表明 1 从苗期到抽穗期超高产杂交稻两优培九和华安3号的净光合速率Pn都比汕优63高而在苗期的午间强光条件下和分蘖期的早晨以及抽穗期的早晚相对弱光条件下其Pn的差别尤为显著说明超高产杂交稻两优培九和华安3号不仅有较高的Pn和较强的抗光抑制能力而且还能充分利用早晨和傍晚较弱的光照条件有效地进行光合作用 2 超高产杂交稻剑叶具有较高的光合色素含量和Chla/b比值同时也具有较高的水分利用效率WUE较高的Chla/b比值表明超高产杂交稻剑叶能够更有效地利用太阳能而较高的WUE则有利于后期节约稻田用水 3 两优培九和华安3号类囊体膜的77K荧光光谱在不同发育时期均高于对照汕优63对其进行Gaussan分析发现这两个超高产杂交稻的反应中心以及天线复合物的发射峰均优于汕优63表明超高产杂交稻具有更强光能吸收能力并且能够将所吸收的光能高效地应用于光合电子传递 4 超高产杂交稻在苗期和分蘖期的净光合速率Pn都明显高于对照可以为群体的扩大后期的生长发育和高产奠定坚实的物质基础 5 三个杂交稻品种抽穗期剑叶的净光合速率相差不大但两优培九和华安3号具有较汕优63高得多的表观量子效率和羧化效率即超高产杂交稻能够高效地利用光能和田间二氧化碳首次提出对光能和二氧化碳的高效利用是两优培九和华安3号高产地重要原因 在对杂交稻净光合速率日变化的研究中发现超高产杂交稻两优培九和华安3号在午间强光条件下具有较对照汕优63更高的净光合速率表明超高产杂交稻具有更强的抗光抑制能力为了研究其光保护机理进一步研究了杂交稻对光抑制的响应结果表明 1超高产杂交稻两优培九和华安3号较对照品种汕优63具有更强的抗光抑制及光保护能力同时在光抑制结束后又能够更迅速地恢复光合功能较强的抗光抑制能力和较高的恢复能力可能是其高产的重要生理原因之一 2光抑制过程中超高产杂交稻叶黄素循环玉米黄素积累速率和积累量都明显高于对照并且在其后的恢复过程中其恢复速率和恢复程度也明显高于汕优63发现叶黄素循环的脱环化作用在光抑制处理30min时即基本接近最大值并未随着光抑制的进一步加重而不断上升认为叶黄素循环在杂交稻光保护中的重要作用可能在于玉米黄素的快速积累对光保护作用的启动 3在对自然条件下光抑制的研究中发现汕优63比超高产杂交稻两优培九和华安3号更容易受到午间光抑制的伤害 4午间光抑制条件下叶黄素循环的玉米黄素Z和环氧玉米黄素A大量积累而叶黄素循环库则没有什么变化认为是叶黄素循环脱环化组分A和Z的积累而不是叶黄素循环库对水稻在中午强光条件下的光保护起重要作用 5在所研究水稻品种的午间光抑制实验中叶绿素荧光的非光化学猝灭系数和叶黄素循环的脱环化状态DES之间没有正比例关系进一步推论环式电子传递可能在杂交稻的光保护中起重要作用 6对用不同试剂处理的杂交稻叶片进行光抑制处理研究发现ASAVDE酶底物其含量可以有效地调节活性处理对杂交稻的抗光抑制能力并没有带来多大改善而DTTVDE酶的特异抑制剂处理也没有使其光抑制大大加重而用DBMIB环式电子传递抑制剂处理则使杂交稻受到比对照强得多的光抑制对qN解析的结果发现强光下qE并未上升反而下降而qT却在光抑制条件下表现出上升现象这些实验结果首次阐明叶黄素循环的热耗散在杂交稻的光保护中不起关键作用而环式电子传递则对于杂交稻的光保护起至关重要的作用其机理可能在于强光条件下环式磷酸化的加剧生成大量ATP用于光破坏的修复作用同时避免类囊体膜的过度酸化从而导致强光下qN的下降这也是光抑制条件下qE下降和qT上升的原因所在此外在研究中发现光抑制处理导致Chla/b比值的上升并且提出这种上升的原因可能在于强光条件下光合系统对LHCII需求减少从而导致对Chlb需求减少最终使得部分Chlb向Chla转化这种转化可能是杂交稻在光抑制条件下的一种保护性响应 7对超高产杂交稻华安3号冠层不同衰老程度叶片的光合功能比较研究的结果表明剑叶的光合功能最强第二叶次之第三叶具有一定的光合功能第四和第五叶则相当衰老基本上丧失光合能力而光合机构的衰老可能始于反应中心的衰老天线系统的衰老要迟于反应中心的衰老叶片衰老进程中Chla和Chlb同步降解但是Chlb先还原为Chla导致Chla/b比值的上升并且认为衰老过程中的这种Chlb的还原是Chlb降解的一个早期的和不可避免的步骤

Arsenite transport into paddy rice (Oryza sativa) roots

Here the mechanism of arsenite transport into paddy rice (Oryza sativa) roots, uptake of which is described by Michaelis-Menten kinetics, is reported. A recent study on yeast (Saccharomyces cerevisiae) showed that undissociated arsenite (its pK_a is 9.2) was transported across the plasma membrane via a glycerol transporting channel. To investigate whether the same mechanism of transport was involved for rice, competitive studies with glycerol, which is transported into cells via aquaporins, were performed. Glycerol competed with arsenite for transport in a dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were the same. Arsenate transport was unaffected by glycerol, confirming that arsenate and arsenite are taken up into cells by different mechanisms. Antimonite, an arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also competed with arsenite transport in a dose-dependent manner, providing further evidence that arsenite is transported into rice roots via glycerol transporting channels. Mercury (Hg²⁺) inhibited both arsenite and arsenate uptake, suggesting that inhibition of influx was due to general cellular stress rather than the specific action of Hg²⁺ on aquaporins. Arsenite uptake by pea (Pisum sativum) and wheat (Triticum aestivum) was also described by Michaelis-Menten kinetics.

Assistência de ar e volumes de aplicação na deposição de calda e no controle do arroz vermelho (Oryza sativa L.)

O trabalho teve como objetivo avaliar o efeito da assistência de ar junto à barra pulverizadora e de três volumes de pulverização na dessecação e deposição da calda em arroz vermelho, sob cultivo de nabo forrageiro, em áreas de recuperação de várzeas, utilizando o herbicida paraquat e o corante Azul Brilhante, respectivamente. Os volumes de pulverização foram 100; 200 e 300 L ha-1 da solução aquosa, contendo corante alimentício (1.500 mg L-1). Com ou sem a assistência de ar junto à barra, foram utilizadas pontas de pulverização de jato plano tipo AXI 110015 à pressão de 117,3 kPa, AXI 11002 e AXI 11003 a 276 kPa. A avaliação da deposição da pulverização deu-se em folhas de plantas de arroz vermelho. Os maiores volumes (200 e 300 L ha-1) pulverizados com a assistência de ar junto à barra pulverizadora proporcionaram maiores depósitos do corante em relação ao volume de 100 L ha-1. Não foram constatadas diferenças na deposição do corante para os volumes pulverizados, sem a assistência de ar junto à barra, tampouco entre os volumes de 200 e 300 L ha-1 com a assistência de ar junto à barra. As maiores percentagens de controle do arroz vermelho foram obtidas com a assistência de ar junto à barra, independentemente do volume pulverizado, equivalendo-se ao controle obtido com 300 L ha-1, sem o uso dessa tecnologia.

Ação alelopática de alguns extratos de plantas daninhas na germinação do arroz (Oryza sativa L. cv. IAC - 165)

O efeito do extrato aquoso de três plantas daninhas, Cynodon dactylon (l.)Pers., Cyperus rotundus l. e Sorghum halepense (l.) Pers. sobre a germinação e o crescimento de pl��ntulas de arroz (Oryza salina L. cv. IAC-165) foi estudado, com a finalidade de investigar a sensibilidade do arroz aos efeitos alelopáticos dessas plantas. Foram utilizados, na preparação dos extratos, raizes de Cynodon daclylon, tubérculos de Cyperus rolundus: e riomas de Sorglun halepense, os quais foram homoneizados em solução aquosa, submetidos a filtração e centrifugação. O sobrenadante foi aplicado no substrato de germinação das sementes, verificando-se, aos sete dias após a se meadura, que apenas os extratos de Cy-nodon daetylon e Sorghum halepense afetaram a porcentagem de germinação das sementes de arroz. Entretanto, o crescimento da radícula e da parte aérea das pl��ntulas foi prejudicado pelos três extratos testados, sendo o efeito mais drástico causado pelo extrato de Cyperus, retundus.