光敏色素B正调控水稻叶绿素合成和叶绿体的发育

水稻光敏色素基因家族包括3个成员,PHYA、PHYB和PHYC,它们在调控水稻去黄化、花期和育性等光形态建成中具有重要作用。我们比较了白光和红光条件下野生型、phyA、phyB和phyAphyB突变体中叶绿素含量。结果表明,phyB感受红光正调控水稻叶绿素合成,phyA的作用仅仅在phyB功能缺陷时才能表现出来。叶绿素合成相关基因表达模式分析结果表明,水稻光敏色素介导的光信号主要通过调控原叶绿素酸酯氧化还原酶基因(protochlorophyll oxidoreduc-tase A,PORA)的表达而影响叶绿素的合成。同时,还分析了phyB介导的红光信号对叶绿体发育的影响,结果表明,phyB介导的红光信号在调控叶绿体数目、基粒数目及叶绿体膜发育中具有重要作用。     

Phytochromes are Involved in Elongation of Seminal Roots and Accumulation of Dry Substances in Rice Seedlings

Phytochromes have been reported to play important roles in seedling de-etiolation and flowering in rice.To identify the roles of phytochromes in regulating root growth and accumulation of dry substances,the lengths of seminal roots and the dry weights of seedlings were measured in the wild type as well as the phytochrome A(phyA) and phytochrome B(phyB) mutants grown under different conditions.When the whole seedlings were exposed to white light,the elongation of the seminal roots was significantly photoinhibited in the wild type,whereas this inhibitory effect was clearly reduced in the phyA and phyB mutants.When the roots of the seedlings were blocked from white light,the phyA and phyB mutants exhibited significantly longer seminal roots than the wild type.These results suggest that both the root-localized and shoot-localized PHYA and PHYB are involved in the photoinhibition of seminal root elongation in rice seedlings.By measuring the dry weights of roots and shoots,it is revealed that PHYB positively regulates the accumulation of dry substances in shoots,however,PHYA exerts the contrary effects on the accumulation of dry substances in roots and shoots of rice seedlings.     

大麦阶段性低温诱导白化突变体的转录组分析

叶色突变体是研究叶绿素代谢、叶绿体发育和光合作用的重要材料。本研究从大麦鄂啤2号(野生型)⁷Li离子突变体库中筛选获得一份大麦阶段性低温诱导白化突变体(SLTW),该突变体受低温诱导后,于五叶期叶片开始出现白化现象,温度升高后于拔节期逐步恢复为绿色。与野生型相比,该突变体叶片中叶绿素a、叶绿素b和类胡萝卜素含量均显著下降。SLTW突变体与野生型的转录组比较分析表明,差异表达基因显著富集到类胡萝卜素合成、四吡咯结合、血红素结合、铁结合、氧化还原过程、光合作用等通路上。叶绿素合成基因(ID号为HORVU.MOREX.r2.2HG0174230)和类胡萝卜素代谢基因(ID号为HORVU.MOREX.r2.5HG0354290)在SLTW突变体与野生型中的表达量均差异显著,且均检测到SNP突变,推测这些基因可能与大麦阶段性白化有关。编码光合作用-天线蛋白、细胞色素P450、跨膜转运(ABC转运)蛋白及转录因子也可能是调控大麦阶段性白化的潜在候选基因。     

水稻白条纹突变体st13的表型分析及基因定位

【目的】叶色突变相关基因鉴定和克隆有助于研究光合作用,补充并完善叶绿体发育机理和色素合成代谢途径,为开展水稻的高光效育种提供理论依据。【方法】从粳稻品种Dongjin的组培后代中分离出一个白条纹突变体st13,成熟期测定野生型和st13的主要农艺性状,苗期测定色素含量并观察叶绿体的超微结构;将st13和Dongjin进行正反交,观察F_1植株表型,并对F_2表型分离进行卡方检验,对st13进行遗传分析;利用st13×南京11(籼稻品种)的F_2和F_(2:3)群体,对st13突变基因定位;采用qPCR分析叶绿体发育和叶绿素合成相关基因在st13与野生型相对表达量。【结果】与野生型Dongjin相比,该突变体的株高、单株有效穗数、穗长、结实率和千粒重等主要农艺性状显著下降。苗期的色素含量降低,分蘖期无差异。突变体的叶绿体中既有含丰富的类囊体膜结构的正常叶绿体,也存在无类囊体结构的叶绿体。遗传分析和基因定位结果表明,st13的突变表型受1对隐性核基因控制,突变基因位于第3条染色体长臂InDel(Insertion-Deletion)标记I3-21和I3-22之间。进一步在这两个标记之间设计了6对InDel标记,最终将基因定位在94kb区间内,此区间共有8个候选基因。【结论】这8个候选基因中,有5个假定的蛋白,其他三个都是有功能注释的蛋白,而这三个蛋白在水稻中均未见报道,因此,st13突变是由一个新的叶色基因突变引起的;同时st13中叶绿体发育、叶绿素合成和光合系统相关基因的表达也发生了显著改变,推测ST13可能是调控叶绿体发育的关键基因。     

Functions of Phytochrome in Rice Growth and Development

Phytochrome family mainly senses red and far-red light to regulate a range of developmental processes throughout the life cycle of plants. Rice phytochrome gene family is composed of three members known as PHYA, PHYB and PHYC. It has been elucidated that individual phytochromes display both unique and overlapping roles in rice photomorphogenesis by characterization of all rice phytochrome mutants including single mutants, all combinations of double mutants as well as triple mutants. Based on the published d...     

Photosynthetic Characteristics of Flag Leaves in Rice White Stripe Mutant 6001 During Senescence Process

Physiological, biochemical and electron microscopy analyses were used to investigate the photosynthetic performance of flag leaves in rice white stripe mutant 6001 during the senescence process. Results showed that the chlorophyll content at the heading and milk-ripe stages in rice mutant 6001 were about 34.78% and 3.00% less than those in wild type 6028, respectively. However, the chlorophyll content at the fully-ripe stage in rice mutant 6001 was higher than that in wild type 6028. At the heading stage, the net photosynthetic rate （Pn） in rice mutant 6001 was lower than that in wild type 6028. Rice mutant 6001 also exhibited a significantly slower decrease rate of Pn than wild type 6028 during the senescence progress, especially at the later stage. Furthermore, Ca2~-ATPase, Mg~~-ATPase and photophosphorylation activities exhibited the similar trends as the Po. During the senescence process, the 68 kDa polypeptide concentrations in the thylakoid membrane proteins exhibited a significant change, which was one of the critical factors that contributed to the observed change in photosynthesis. We also observed that the chloroplasts of rice mutant 6001 exhibited higher integrity than those of wild type 6028, and the chloroplast membrane of rice mutant 6001 disintegrated more slow during the senescence process. In general, rice mutant 6001 had a relatively slower senescence rate than wild type 6028, and exhibited anti-senescence properties.     

一个水稻低温移栽白条纹突变体wltt的鉴定和基因定位

【目的】叶色突变相关基因的鉴定与克隆为研究叶绿体发育、叶绿素合成和光合作用等分子机制提供理论基础。【方法】从常规粳稻镇糯19杂交后代中分离出一个低温移栽后叶色转成白条纹的自然变异突变体,命名为wltt (white stripe leaf after transplanting at low temperature)。成熟期测定野生型和wltt的主要农艺性状,分别在苗期、移栽后15 d和同时期直播条件下测定新生叶片的色素含量并观察叶绿体的超微结构;将wltt和野生型正反交进行遗传分析;用wltt与籼稻9311杂交产生的F_2作为定位群体进行基因定位;采用RT-qPCR分析叶绿体发育、叶绿素合成和光合作用相关基因在野生型和wltt中的表达水平。【结果】wltt突变体在苗期表现正常绿色,移栽15 d后心叶出现白条纹叶表型,至分蘖末期心叶叶色恢复;而不经移栽,突变体不会出现白条纹叶。人工模拟实验表明该表型是由低温条件下根损伤引起的。与野生型相比,wltt突变体移栽后的新生叶色素含量显著降低,光合速率下降;同时株高变矮,穗长、剑叶长和每穗粒数均显著降低。叶绿体的超微结构显示,突变体的叶肉细胞中,仅少数细胞含有正常的叶绿体,其余大部分叶肉细胞不含叶绿体。进一步研究发现,突变体中部分光合系统相关基因和叶绿体发育相关基因表达下调,叶绿素生物合成相关的14个基因表达也下调。遗传分析表明,该突变性状受一对隐性核基因控制。利用wltt突变体/9311的F_2群体,将该基因定位于水稻第2染色体着丝粒附近853kb区间内。目前,该区间内没有叶色相关基因的报道。【结论】WLTT是低温条件下移栽调控叶片转色的关键基因,在叶绿体发育过程中发挥重要作用。     

光敏色素在水稻生长发育中的作用

 光敏色素是植物体内的重要光受体,主要感受红光和远红光,调节着植物生命循环中许多重要发育过程。水稻光敏色素基因家族包括3个成员,即PHYA、PHYB 和PHYC。 对水稻光敏色素突变体的研究表明,3个水稻光敏色素基因在水稻光形态建成中既有独特作用又有交叉作用。根据已有报道和作者在该领域的研究工作,总结了光敏色素在水稻幼苗去黄化、根的向地性和延伸、株型、花期和育性等发育过程中的作用,并提出了水稻光敏色素研究领域急需解决的问题。     

白条纹水稻突变体的光合特性分析

从水稻组织培养突变体中发现可稳定遗传的白色条纹突变体,其生育期呈现白色和绿色条带相间的表型。电镜观察表明突变体绿色条带叶肉细胞具有正常的叶绿体发育,白色条带叶肉细胞则表现出叶绿体缺失。与野生型相比,突变体单位重量叶片叶绿素含量显著下降,但叶绿素光能吸收强度略有上升。突变体类囊体膜上的光系统II产生的激发能量降低,并将更多的激发能分配给光系统I。突变体保持叶片光合效率稳定的机制是通过提高胡萝卜素与叶绿素比值来补偿光合色素含量和激发能的降低引起的光合效率下降。     

Morphological Structure and Genetic Mapping of New Leaf-Color Mutant Gene in Rice (Oryza sativa)

Leaf-color mutations are a widely-observed class of mutations, playing an important role in the study of chlorophyll biosynthesis and plant chloroplast structure, function, genetics and development. A naturally-occurring leaf-color rice mutant, Baihuaidao 7, was analyzed. Mutant plants typically exhibited a green-white-green leaf-color progression, but this phenotype was only expressed in the presence of a stress signal induced by mechanical scarification such as transplantation. Prior to the appearance of white leaves, mutant plant growth, leaf color, chlorophyll content, and chloroplast ultrastructure appeared to be identical to those of the wild type. After the changeover to white leaf color, an examination of the mutated leaves revealed a decrease in total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content, a reduction in the number of chloroplast grana lamella and grana, and a gradual degradation of the thylakoid lamellas. At maturity, the mutant plant was etiolated and dwarfed compared with wild-type plants. Genetic analysis indicated that the leaf mutant character is controlled by a recessive nuclear gene. Genetic mapping of the mutant gene was performed using an F2 population derived from a Baihuaidao 7 × Jiangxi 1587 cross. The mutant gene was mapped to rice chromosome 11, positioned between InDel markers L59.2-7 and L64.8-11, which are separated by approximately 740.5 kb. The mutant gene is believed to be a new leaf-color mutant gene in rice, and is tentatively designated as gwgl.     

Nucleotide polymorphisms in the waxy gene of NaN3-induced waxy rice mutants

Spontaneous and induced waxy phenotype, associated with endosperm containing little or no amylose, has been recognized in rice (Oryza sativa L.). Mutation of a dominant gene Wx into a recessive gene wx, which causes the inactivation or absence of granule bound starch synthase, is believed to be responsible for the change in endosperm starch leading to the waxy grain. In the present study, the nucleotide polymorphism in the Wx gene of rice genotype Tainung 67 (wild type) and its 35 NaN₃-induced wx mutants were examined. Iodine staining confirmed that all the mutants had waxy grain trait. The G-to-T single base substitution analysis indicated that the wild type genotype Tainung 67 and its waxy mutants carried Wx^b allele. Moreover, 23-bp duplication in exon 2 was detected in all the waxy mutants. Microsatellite polymorphism (CT)_n was also detectable on the Wx gene of the tested genotypes and mutants, with at least 5 classes of (CT)_n microsatellites identified at the Wx locus. Electrophoretic analyses also confirmed the observed nucleotide polymorphsim. Thus, nucleotide polymorphsim exist among NaN₃-induced waxy mutants in rice. However, only the 23-bp duplication in exon 2 may be used as a molecular marker to characterize waxy grain trait in rice genotypes.     

Nucleus-Encoded Thylakoid Protein,OsY3IP1, Confers Enhanced Tolerance to Saline and Alkaline Stresses in Rice

Abiotic stress confers serious damage to the photosynthetic machinery, often resulting in plant growth inhibition. Hypothetical chloroplast open reading frame 3 (Ycf3)-interacting protein 1 (Y3IP1) is a nucleus-encoded thylakoid protein and plays an essential role in the assembly of photosystem I. The full-length cDNA over-expresser (FOX) gene-hunting system is an approach using systemically generated gain-of-function mutants. Among the FOX-rice lines, a line CE175 overexpressing rice Y3IP1 gene (OsY3IP1) displayed less inhibition of root growth under saline (NaCl) stress. The expression of OsY3IP1 was up-regulated under saline and alkaline (Na₂CO₃) stresses in the rice variety Kitaake. After saline and alkaline treatments, transgenic Kitaake overexpressing OsY3IP1-GFP (OsY3IP1-GFPox/Kit) displayed higher levels of chlorophyll content compared to Kitaake. Under the stress conditions, the maximum quantum yield of photosystem II photochemistry levels was higher in OsY3IP1-GFPox/Kit than in Kitaake. The increased tolerance conferred by OsY3IP1 overexpression correlated with reduced reactive oxygen species accumulation. Our data provide new insights into the possible role of OsY3IP1 in the pathway suppressing photooxidative damage under stress conditions. These features can be further exploited to improve saline and alkaline tolerances of rice plants in future.     

NRL3 Interacts with OsK4 to Regulate Heading Date in Rice

NRL3 is essential for the growth and development of rice leaves. In this study, we found that the loss function of NRL3 also delayed heading date under natural long daylight and short daylight conditions. The yeast two-hybrid and the bimolecular fluorescence complementation proved that NRL3 interacts with OsK4, a Snf1-related kinase. OsK4 localized to the nucleus and expressed in various rice tissues. The rhythmic expression pattern of OsK4 was similar to NRL3 under long daylight and short daylight conditions. Knock-out mutants of OsK4 exhibited early heading under long daylight conditions, indicating that it acts as a negative regulator of heading date in rice. Interestingly, the OsK4 mutant under the nrl3 mutant background rescued the late heading phenotype of nrl3 under long daylight conditions, suggesting that OsK4 functions downstream of NRL3. Moreover, both NRL3 and OsK4 controlled heading date through regulating the expression of Hd3a and RFT1 genes. These findings shed light on the heading date regulation in rice and provide a sound theoretical base to improve regional adaptability of rice.     

Genetic Analysis and Molecular Mapping of Novel White Striped Leaf Mutant Gene in Rice

A new white striped leaf mutant wsl1 was discovered from Nipponbare mutated by ethyl methanesulfonate. The mutant showed white striped leaves at the seedling stage and the leaves gradually turned green after the tillering stage. The chlorophyll content of wsl1 was significantly lower than that of wild-type during the fourth leaf stage, tillering stage and booting stage. The numbers of chloroplast, grana and grana lamella were reduced and the thylakoids were degenerated in wsl1 compared with wild type. Genetic analysis showed that the wsl1 was controlled by a single recessive gene. Molecular mapping of the wsl1 was performed using an F₂ population derived from wsl1/Nanjing 11. The wsl1 was finally mapped on the telomere region of chromosome 9 and positioned between simple sequence repeat markers RM23742 and RM23759 which are separated by approximately 486.5 kb. The results may facilitate map-based cloning of wsl1 and understanding of the molecular mechanism of the regulation of leaf-color by WSL1 in rice.     

Genetic interaction between rice PLASTOCHRON genes and the gibberellin pathway in leaf development

Background

The rice PLASTOCHRON (PLA) genes PLA1 and PLA2 regulate leaf maturation and the temporal pattern of leaf initiation. Although the function of PLA genes in the leaf initiation process has been analyzed, little is known about how they affect leaf growth. Previously, we suggested that PLA1 and PLA2 function downstream of the gibberellin (GA) signal transduction pathway. In the present study, we examined the phenotype of a double mutant of pla and slender rice 1 (slr1), which is a constitutive GA response mutant. By analyzing these double mutants, we discuss the relationship between PLA-related and GA-dependent pathways and the possible function of PLA genes in leaf growth.

Findings

Single slr1 and pla mutants exhibited elongated and dwarf phenotypes in the vegetative stage, respectively. The stature and leaf size of the pla1/slr1 and pla2/slr1 double mutants were intermediate between those of the pla and slr1 single mutants. However, the effects of slr1 on leaf elongation were markedly suppressed in the pla1 and pla2 mutant backgrounds. On the other hand, the change in cell length in the double mutants was almost the same as that in the single mutants. An expression analysis of genes involved in GA biosynthesis and catabolism indicated that feedback regulation functioned normally in the pla/slr1 double mutants.

Conclusions

Our genetic results confirm that PLA genes regulate leaf growth downstream of the GA pathway. Our findings also suggest that PLA1 and PLA2 are partly required for GA-dependent leaf elongation, mainly by affecting cellular proliferation.     

Expression Profiles and Protein Complexes of Starch Biosynthetic Enzymes from White-Core and Waxy Mutants Induced from High Amylose Indica Rice

Physicochemical properties of endosperm starches in milled rice determine its cooking and eating quality. Amylose is synthesized by granule-bound starch synthase I (GBSSI), whilst amylopectin is synthesized by the synergistic activities of starch synthases (SSs), branching enzymes (BEs) and debranching enzymes (DBEs). However, the complexes formed by starch biosynthetic enzymes are not well characterized. Gene expression profiles and protein complexes were determined in white-core (GM645) and waxy (GM077) mutants derived from a high amylose indica rice Guangluai 4 (GLA4). In GM645, genes including AGPS1, GBSSI, SSIIa, BEI, BEIIa, BEIIb, PUL, ISA1 and SP were significantly downregulated during seed development. In GM077, the expression levels of AGPL2, AGPS1, AGPS2b, SSIIIa, BEI, PUL and ISA1 were significantly upregulated. Co-immunoprecipitation assays revealed interactions of SSs-BEs, SSs-PUL and BEs-PUL in developing seeds. However, weak SSI-SSIIa interaction was detected in GM077, whilst SSI-PUL interaction was absent. Weak interaction signals for SSI-SSIIa, SSIIa-BEI, SSIIa-BEIIb, BEI-BEIIb and SSI-BEI were also observed in GM645. These results suggest that the protein-protein interactions for starch biosynthesis are modified in mutants, which provides insight into the mechanisms of starch biosynthesis, particularly in indica rice.     

Profiling of Polar Lipids in Marine Oleaginous Diatom Fistulifera solaris JPCC DA0580: Prediction of the Potential Mechanism for Eicosapentaenoic Acid-Incorporation into Triacylglycerol

The marine oleaginous diatom Fistulifera solaris JPCC DA0580 is a candidate for biodiesel production because of its high lipid productivity. However, the substantial eicosapentaenoic acid (EPA) content in this strain would affect the biodiesel quality. On the other hand, EPA is also known as the essential health supplement for humans. EPAs are mainly incorporated into glycerolipids in the microalgal cell instead of the presence as free fatty acids. Therefore, the understanding of the EPA biosynthesis including the incorporation of the EPA into glycerolipids especially triacylglycerol (TAG) is fundamental for regulating EPA content for different purposes. In this study, in order to identify the biosynthesis pathway for the EPA-containing TAG species, a lipidomic characterization of the EPA-enriched polar lipids was performed by using direct infusion electrospray ionization (ESI)-Q-TRAP-MS and MS/MS analyses. The determination of the fatty acid positional distribution showed that the sn-2 position of all the chloroplast lipids and part of phosphatidylcholine (PC) species was occupied by C16 fatty acids. This result suggested the critical role of the chloroplast on the lipid synthesis in F. solaris. Furthermore, the exclusive presence of C18 fatty acids in PC highly indicated the biosynthesis of EPA on PC. Finally, the PC-based acyl-editing and head group exchange processes were proposed to be essential for the incorporation of EPA into TAG and chloroplast lipids.     

Physiological Characters and Gene Mapping of a Yellow Leaf and Early-senescence Mutant osyes1 in Rice

【Objective】Leaf is a main photosynthetic organ and its color mutants of rice are ideal materials for the study of photosynthesis, chlorophyll biosynthesis and metabolism, and chloroplast development in plants. 【Method】The mutant osyes1 (Oryza sativa yellow-leaf and early senescence 1) was obtained through 60Co γ-radiation treatment of indica cultivar Zixuan 1. The seedlings of osyes1 and its wild type were treated with exogenous H2O2. The SOD and CAT activities, ROS level, MDA content, soluble protein contents, chlorophyll contents, the net photosynthetic rate and chloroplast ultrastructure were analyzed for osyes1 and its wild type leaves at heading stage. The main agronomic traits of osyes1 and its wild type plants were analyzed under field conditions at maturation stage in Hangzhou. The recessive individuals in F2 population derived from osyes1/02428 were used to locate the gene by the map cloning method.【Results】The yellow-leaf and early-senescence symptoms started at 3- or 4-leaf stage, and gradually spread to all of the leaves after heading. Due to the early-senescence of the leaves of the mutant osyes1, its major agronomic traits including plant height, panicle length, grain number per panicle and the seed setting rate were markedly reduced. Moreover, the mutant osyes1 exhibited hyper-sensitivity to exogenous H2O2. The physiological analysis indicated that the contents of chlorophyll and the activities POD and CAT in the third-top leaf was significantly lower than those in the flag leaf and the second-top leaf in wild-type (WT) plants, but all of them in the mutant plants were significantly lower than those in its WT. Compared with the WT plants, the contents of MDA, H2O2 and O2− followed a steady increasing trend in the flag, second-top and third-top leaves of the mutant, while their soluble protein levels were progressively dropping. Genetic analysis confirmed that osyes1 was controlled by a single recessive nuclear gene, which was mapped to a region of 708 kb flanked by two SSR markers (RM21353 and RM21384) on the short arm of chromosome 7. 【Conclusion】In this work, the main agricultural traits were significantly reduced in osyes1 for the yellow leaf and early senescence. OsYES1 was located in a 708 kb range between RM21353 and RM21384 by map-based cloning strategy.