NARROW AND ROLLED LEAF 2 regulates leaf shape,male fertility,and seed size in rice

Grain yield in rice(Oryza sativa L.) is closely related to leaf and flower development. Coordinative regulation of leaf, pollen, and seed development in rice as a critical biological and agricultural question should be addressed. Here we identified two allelic rice mutants with narrow and semirolled leaves, named narrow and rolled leaf 2-1(nrl2-1) and nrl2-2. Map-based molecular cloning revealed that NRL2 encodes a novel protein with unknown biochemical function. The mutation of NRL2 caused pleiotropic effects, including a reduction in the number of longitudinal veins, defective abaxial sclerenchymatous cell differentiation, abnormal tapetum degeneration and microspore development, and the formation of more slender seeds compared with the wild type(WT). The NRL2 protein interacted with Rolling-leaf(RL14),causing the leaves of the nrl2 mutants to have a highercellulose content and lower lignin content than the WT, which may have been related to sclerenchymatous cell differentiation and tapetum degeneration. Thus, this gene is an essential developmental regulator controlling fundamental cellular and developmental processes, serving as a potential breeding target for high-yielding rice cultivars.     

Role of ATP hydrolysis in the antirecombinase function of Saccharomyces cerevisiae Srs2 protein

Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.     

一个水稻内颖退化突变体的形态特征及基因的精确定位

水稻产量和品质受花器官发育的直接影响,因此对水稻颖花发育机理的研究将有助于水稻产量提高和品质的改良。文章利用60Coγ射线辐照亲本8PW33(籼稻背景)获得一个性状能稳定遗传的内颖退化突变体(编号:MU102),并对其农艺性状和花器官进行了观察和分析。结果显示,相对于野生型,该突变体的株高、每穗总粒数及剑叶宽均显著增加,而结实率则显著降低,差异均达显著水平。解剖镜下观察表明,该突变体内颖退化,外颖弯曲呈现镰刀状,其余器官与野生型表型基本一致。扫描电镜观察显示,突变体与野生型叶片维管束的结构组成以及外颖表皮细胞组成、排列均正常,没有明显差异;与野生型相比,突变体内颖表皮细胞排列较为紧密,推测可能是内颖收缩退化导致的。遗传分析显示该突变性状是由隐性单基因控制,并命名为pd2。利用实验室现有的SSR分子标记将PD2基因定位于水稻第9号染色体上,通过进一步扩大群体和开发新的Indel标记,将PD2基因定位在2个Indel标记之间,两者间的物理距离大约是82 kb。在该物理区间内有一个已经克隆的内颖发育基因REP1,经过测序和比对分析,推测REP1与PD2为等位基因。     

Deletion of the SRS2 gene suppresses elevated recombination and DNA damage sensitivity in rad5 and rad18 mutants of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genes RAD5, RAD18, and SRS2 are proposed to act in post-replicational repair of DNA damage. We have investigated the genetic interactions between mutations in these genes with respect to cell survival and ectopic gene conversion following treatment of logarithmic and early stationary cells with UV- and gamma-rays. We find that the genetic interaction between the rad5 and rad18 mutations depends on DNA damage type and position in the cell cycle at the time of treatment. Inactivation of SRS2 suppresses damage sensitivity both in rad5 and rad18 mutants, but only when treated in logarithmic phase. When irradiated in stationary phase, the srs2 mutation enhances the sensitivity of rad5 mutants, whereas it has no effect on rad18 mutants. Irrespective of the growth phase, the srs2 mutation reduces the frequency of damage-induced ectopic gene conversion in rad5 and rad18 mutants. In addition, we find that srs2 mutants exhibit reduced spontaneous and UV-induced sister chromatid recombination (SCR), whereas rad5 and rad18 mutants are proficient for SCR. We propose a model in which the Srs2 protein has pro-recombinogenic or anti-recombinogenic activity, depending on the context of the DNA damage.     

Molecular mapping of split rice spikelet mutantsrs-1 and analysis of its homeotic function in rice

srs-1, a new floral organ identity gene in rice, was mapped using RAPD and RFLP markers. Firstly, the cross was made between "ZhaiYeQing 8" (ZYQ8, indica) and split rice spikelet (SRS, japonica) mutant. The ratio of wild-type individuals and mutant plants in F2 population is 3:1, which indicates that the mutant characteristics are controlled by single recessive gene, srs-1. Consequently, BSA method was adopted and 520 random 10-mer primers were used to screen polymorphic bands between two bulks. Six primers could amplify polymorphic bands, of which S465 generates the most stable RAPD patterns. Then, S465 that cosegregates in F2 population has been converted into an RFLP marker successfully. Furthermore, srs-1 gene was mapped on chromosome 3 using DH mapping population. The effect of srs-1 gene results in the mutant of split rice spikelet. The mutant has longer and softer palea/lemma than those of wild-type, and two small palea/lemma-like organs between palea and lemma. In addition, there is a flower wit     

水稻短根突变体Osksr6的遗传分析和基因定位

该研究对从甲基磺酸乙酯(EMS)诱变的水稻突变体库中筛选到的一个短根突变体Osksr6(Oryza sativa kasalath short root 6)进行了表型鉴定、遗传分析与基因定位。结果表明:(1)生长7d的突变体Osksr6与野生型相比,株高与不定根数量差异不明显,但主根变短61.98%、不定根变短46.42%,侧根的发生与根毛的伸长也受不同程度抑制;成熟期的Osksr6分蘖数明显减少,总穗长与结实率均较野生型差。(2)遗传分析结果显示,突变体Osksr6的短根性状受1对隐性基因控制。(3)利用图位克隆技术,将突变基因OsKSR6定位于3号染色体InDel标记28420k和28880k之间,物理距离约460kb,该区间没有已报道的与根系发育相关的基因。该研究为进一步研究水稻根系生长的分子机理奠定了基础。     

Molecular mapping of split rice spikelet mutant srs-1 and analysis of its homeotic function in rice

srs-1, a new floral organ identity gene in rice, was mapped using RAPD and RFLP markers. Firstly, the cross was made between "ZhaiYeQing 8" (ZYQ8, indica) and split rice spikelet (SRS, japonica) mutant. The ratio of wild-type individuals and mutant plants in F2 population is 3:1, which indicates that the mutant characteristics are controlled by single recessive gene, srs-1. Consequently, BSA method was adopted and 520 random 10-mer primers were used to screen polymorphic bands between two bulks. Six primers could amplify polymorphic bands, of which S465 generates the most stable RAPD patterns. Then, S465 that cosegregates in F2 population has been converted into an RFLP marker successfully. Furthermore, srs-1 gene was mapped on chromosome 3 using DH mapping population. The effect of srs-1 gene results in the mutant of split rice spikelet. The mutant has longer and softer palea/lemma than those of wild-type, and two small palea/lemma-like organs between palea and lemma. In addition, there is a flower with three stamens and carpel in the axil of lemma. Thus, there are nine stamens and two carpels in the spikelet of mutant. srs-1 gene may belong to homeotic gene of class A according to the mutant characteristics and ABC model.     

Roles of the Arabidopsis G protein γ subunit AGG3 and its rice homologs GS3 and DEP1 in seed and organ size control

The size of seeds and organs is coordinately determined by cell proliferation and cell expansion, but the mechanisms that set final seed and organ size are largely unknown in plants. In a recent study, we have demonstrated that the plant specific G protein γ subunit (AGG3) promotes seed and organ growth by increasing the period of proliferative growth in Arabidopsis. AGG3 is localized in plasma membrane and interacts with the G protein β subunit (AGB1). Homologs of AGG3 in rice (GS3 and DEP1/qPE9–1) have been identified as important quantitative trait loci for seed size and yield. However, rice GS3 and DEP1 influence seed and organ growth by restricting cell proliferation. Here, we discuss the possible molecular mechanisms by which Arabidopsis AGG3 and its rice homologs GS3 and DEP1 control seed and organ size.     

直立密穗基因DEP2-1388的遗传分析及在杂交稻中的育种利用

通过EMS诱变籼型重穗恢复系蜀恢498,获得一个直立穗突变体R1338。与野生型相比,突变体表现为植株变矮、穗直立、穗长变短、一次枝梗变短、着粒密度增加、穗部抗弯曲力极显著增强、籽粒增宽增厚、粒长变短。组织细胞学分析发现,穗颈节直径、纤维素含量和木质素含量在穗部抗弯曲上发挥了重要的作用。遗传分析表明该直立穗表型受一对半显性核基因DEP2控制,通过重测序以及MutMap方法定位发现,在R1338突变体中,DEP2第7外显子有一个A到G的碱基置换,导致第928个精氨酸(AGG)被置换成甘氨酸(GGG),推测R1338直立穗性状可能由DEP2中该SNP导致。用R1338、野生型与不同穗型不育系分别配组,R1338与弯曲穗不育系所配组合穗部表现半直立,且保持较高的结实率和杂种优势,与DEP1直立穗不育系配组表现为基因累加效应的直立穗。本研究还讨论了直立穗突变体R1338在杂交水稻育种中的利用价值。     

水稻内颖畸形或缺失基因OsAPP1的图位克隆及表达分析

在育种基地材料中发现一株内颖畸形或缺失(abnormal or absent palea)突变体,将其命名为app1。该突变体在营养生长时期发育正常,但抽穗后突变体表现出内颖畸形(比外稃短导致颖壳不闭合,或者出现两个内稃)或缺失,其花粉育性为55.52%,结实率为6.48%,千粒重为10.811 g,种子发芽率为55.21%。以突变体app1与日本晴杂交构建了F1和F2群体,F1颖壳表型正常,F2群体出现内颖畸形和正常表型分离,内颖正常和突变表型分离比例为3∶1,表明app1内颖突变表型由单隐性核基因控制。以F2为分离群体,将app1精细定位于第3染色体上,位于分子标记ID4231和ID4246之间,遗传距离1.3 cM,对应物理距离为13.2 kb。该区段内完全包含1个开放阅读框,包含两个部分开放阅读框,经过测序分析发现候选基因LOC_Os03g11614启动子区发生点突变和245 bp缺失,qRT-PCR分析证实LOC_Os03g11614为OsAPP1基因。已有报道LOC_Os03g11614编码OsMADS1,是调控水稻花器官发育的重要明星基因,其不同位置的突变可以导致叶状颖壳和不育、以及控制籽粒大小。与3000份水稻种子资源SNP/Indel变异类型对比分析发现,突变体app1启动子的突变完全不同于现已OsMADS1研究报道突变类型,且与数据库中的自然突变类型多数不同。因此,本研究发现的app1突变体,是以往报道中从未出现的OsMADS1启动子发生突变的新型突变,且该类突变导致了其降低表达量,并产生了不同于前人研究的新表型,这为深入研究OsMADS1基因在水稻花器官发育中的功能提供了新的种质资源和思路。     

The short life span of Saccharomyces cerevisiae sgs1 and srs2 mutants is a composite of normal aging processes and mitotic arrest due to defective recombination

Evidence from many organisms indicates that the conserved RecQ helicases function in the maintenance of genomic stability. Mutation of SGS1 and WRN, which encode RecQ homologues in budding yeast and humans, respectively, results in phenotypes characteristic of premature aging. Mutation of SRS2, another DNA helicase, causes synthetic slow growth in an sgs1 background. In this work, we demonstrate that srs2 mutants have a shortened life span similar to sgs1 mutants. Further dissection of the sgs1 and srs2 survival curves reveals two distinct phenomena. A majority of sgs1 and srs2 cells stops dividing stochastically as large-budded cells. This mitotic cell cycle arrest is age independent and requires the RAD9-dependent DNA damage checkpoint. Late-generation sgs1 and srs2 cells senesce due to apparent premature aging, most likely involving the accumulation of extrachromosomal rDNA circles. Double sgs1 srs2 mutants are viable but have a high stochastic rate of terminal G2/M arrest. This arrest can be suppressed by mutations in RAD51, RAD52, and RAD57, suggesting that the cell cycle defect in sgs1 srs2 mutants results from inappropriate homologous recombination. Finally, mutation of RAD1 or RAD50 exacerbates the growth defect of sgs1 srs2 cells, indicating that sgs1 srs2 mutants may utilize single-strand annealing as an alternative repair pathway.     

Analysis of Mitotic and Meiotic Defects in Saccharomyces Cerevisiae Srs2 DNA Helicase Mutants

The hyper-gene conversion srs2-101 mutation of the SRS2 DNA helicase gene of Saccharomyces cerevisiae has been reported to suppress the UV sensitivity of rad18 mutants. New alleles of SRS2 were recovered using this suppressor phenotype. The alleles have been characterized with respect to suppression of rad18 UV sensitivity, hyperrecombination, reduction of meiotic viability, and definition of the mutational change within the SRS2 gene. Variability in the degree of rad18 suppression and hyperrecombination were found. The alleles that showed the severest effects were found to be missense mutations within the consensus domains of the DNA helicase family of proteins. The effect of mutations in domains I (ATP-binding) and V (proposed DNA binding) are reported. Some alleles of SRS2 reduce spore viability to 50% of wild-type levels. This phenotype is not bypassed by spo13 mutation. Although the srs2 homozygous diploids strains undergo normal commitment to meiotic recombination, this event is delayed by several hours in the mutant strains and the strains appear to stall in the progression from meiosis I to meiosis II.     

A recessive heterochronic mutation, plastochron1, shortens the plastochron and elongates the vegetative phase in rice

We describe two recessive alleles of a rice heterochronic gene, plastochron1-1 (pla1-1) and pla1-2, that reduce the length of the plastochron to approximately half that of the wild type. Because the onset of the reproductive phase in pla1 was not temporally affected, the number of leaves produced in the vegetative phase was nearly twice that produced in the wild type. Panicle development was severely disturbed in pla1 mutants. In pla1-1, many primordia of primary rachis branches were converted into vegetative shoots. These ectopic shoots repeated the initiation of panicle development and the conversion of primary rachis branches into shoots. In the weak allele pla1-2, however, only the basal one or two primordia developed as vegetative shoots, and the remaining primordia developed to produce a truncated panicle. These results indicate that both vegetative and reproductive programs are expressed simultaneously during the reproductive phase of pla1; however, the degree varied depending on the strength of the allele. Accordingly, pla1 is a heterochronic mutation that extends the vegetative period. The shoot apical meristem of pla1 was larger than that of the wild type, although the shape was not modified. An in situ hybridization experiment using the histone H4 gene as a probe revealed that cell divisions are accelerated in the pla1 meristem. The PLA1 gene is considered to regulate the duration of the vegetative phase by controlling the rate of leaf production in the meristem.     

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei, but not that of a meiosis-specific Dmc1. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and/or multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphases I and II, suggesting the absence of DNA damage-induced cell cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase I.

     

Fitness estimation through performance comparison of F1 hybrids with their parental species Oryza rufipogon and O. sativa

BACKGROUND AND AIMS: Introgression of crop genes into populations of wild relatives has important implications for germplasm conservation as well as for the persistence of novel transgenes in wild populations. Studies of hybrid fitness can be used to evaluate the potential for introgression to occur following episodes of interspecific hybridization. METHODS: This study estimated relative fitness of interspecific hybrids through performance comparison of F(1) hybrids with their parental species, a cultivated rice (Oryza sativa) Minghui-63 and perennial common wild rice (O. rufipogon) under the cultivation conditions. KEY RESULTS: Compared with their parents, the hybrids had the lowest values of seedling survival ability, pollen viability and seed production; intermediate values of seed germination, spikelet production and flag leaf areas; and the highest values of plant height, number of tillers and panicles. The hybrids performed poorly at the stage of sexual reproduction, although they had a slightly higher hybrid vigour at the vegetative growth stage and better tillering ability than their wild parent. There were no significant differences in composite fitness across the whole life-history between the hybrids and their wild parental species. CONCLUSIONS: Rice genes, including transgenes, might persist in wild rice populations through vegetative and sexual reproduction. Further studies are needed to examine whether the extent of gene flow from rice is sufficiently significant to influence genetic diversity in wild populations of O. rufipogon, a species that has become endangered in some regions of south-east Asia.     

转拟南芥P5CS1基因增强羽衣甘蓝的耐旱性

为提高羽衣甘蓝的耐旱性,本文将拟南芥Δ1-吡咯啉-5-羧酸合成酶（P5CS1）基因经农杆菌介导转入羽衣甘蓝植株中,检测转基因株系与野生型植株在干旱胁迫下P5CS1 mRNA表达量、幼苗脯氨酸含量、株系根系性状、整株干重、鲜重和整株存活率。结果表明,在15%PEG6000渗透胁迫下,转基因植株的P5CS1基因mRNA表达量明显增加,转基因植株脯氨酸含量是野生型的2.4倍;主根长、最长侧根长、侧根数目、整株干重和鲜重均高于野生型,干重/鲜重则低于野生型,转基因植株的平均存活率为78%,极显著高于野生型。数据显示,AtP5CS1基因在羽衣甘蓝中的表达明显改善了转基因植株的耐旱性。