A novel male-sterile mutant of Arabidopsis thaliana,faceless pollen-1, produces pollen with a smooth surface and an acetolysis-sensitive exine

A mutant exhibiting conditional male sterility, in which fertility was restored under conditions of high humidity, was identified in T-DNA tagged lines of Arabidopsis thaliana. Scanning electron microscopy (SEM) demonstrated that the pollen surface was almost smooth and the reticulate pattern not prominent. Thus, the mutant was named faceless pollen-1 (flp1). Transmission electron microscopy (TEM) revealed that the smooth appearance was due to tryphine filling in the exine cavities and covering the pollen surface. The lipid droplets in the tryphine of mutant pollen were smaller and more numerous than those of the wild type. SEM analysis also demonstrated that pollen exine was easily damaged by acetolysis, suggesting that a component of exine, sporopollenin, was defective in the mutant. In addition, the stems and siliques had reduced amounts of wax crystals. A predicted amino acid sequence of the cDNA that corresponded to the tagged gene, fip1, showed sequence similarity to proteins involved in wax biosynthesis. The FLP1 protein is likely to play a role in the synthesis of the components of tryphine, sporopollenin of exine and the wax of stems and siliques.     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

A rice gene activation/knockout mutant resource for high throughput functional genomics

Using transfer DNA (T-DNA) with functions of gene trap and gene knockout and activation tagging, a mutant population containing 55,000 lines was generated. Approximately 81% of this population carries 1–2 T-DNA copies per line, and the retrotransposon Tos17 was mostly inactive in this population during tissue culture. A total of 11,992 flanking sequence tags (FSTs) have been obtained and assigned to the rice genome. T-DNA was preferentially (∼80%) integrated into genic regions. A total of 19,000 FSTs pooled from this and another T-DNA tagged population were analyzed and compared with 18,000 FSTs from a Tos17 tagged population. There was difference in preference for integrations into genic, coding, and flanking regions, as well as repetitive sequences and centromeric regions, between T-DNA and Tos17; however, T-DNA integration was more evenly distributed in the rice genome than Tos17. Our T-DNA contains an enhancer octamer next to the left border, expression of genes within genetics distances of 12.5 kb was enhanced. For example, the normal height of a severe dwarf mutant, with its gibberellin 2-oxidase (GA2ox) gene being activated by T-DNA, was restored upon GA treatment, indicating GA2ox was one of the key enzymes regulating the endogenous level of GA. Our T-DNA also contains a promoterless GUS gene next to the right border. GUS activity screening facilitated identification of genes responsive to various stresses and those regulated temporally and spatially in large scale with high frequency. Our mutant population offers a highly valuable resource for high throughput rice functional analyses using both forward and reverse genetic approaches. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users. Yue-Ie Hsing, Chyr-Guan Chern, and Ming-Jen Fan have contributed equally.     

Generation and flanking sequence analysis of a rice T-DNA tagged population

Insertional mutagenesis provides a rapid way to clone a mutated gene. Transfer DNA (T-DNA) of Agrobacterium tumefaciens has been proven to be a successful tool for gene discovery in Arabidopsis and rice (Oryza sativa L. ssp. japonica). Here, we report the generation of 5,200 independent T-DNA tagged rice lines. The T-DNA insertion pattern in the rice genome was investigated, and an initial database was constructed based on T-DNA flanking sequences amplified from randomly selected T-DNA tagged rice lines using Thermal Asymmetric Interlaced PCR (TAIL-PCR). Of 361 T-DNA flanking sequences, 92 showed long T-DNA integration (T-DNA together with non-T-DNA). Another 55 sequences showed complex integration of T-DNA into the rice genome. Besides direct integration, filler sequences and microhomology (one to several nucleotides of homology) were observed between the T-DNA right border and other portions of the vector pCAMBIA1301 in transgenic rice. Preferential insertion of T-DNA into protein-coding regions of the rice genome was detected. Insertion sites mapped onto rice chromosomes were scattered in the genome. Some phenotypic mutants were observed in the T₁ generation of the T-DNA tagged plants. Our mutant population will be useful for studying T-DNA integration patterns and for analyzing gene function in rice.Electronic Supplementary Material Supplementary material is available in the online version of this article at .Communicated by D. Mackill     

Induction of chromosomal inversion by integration of T-DNA in the rice genome

Transfer DNA(T-DNA) of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments,including inversions.However,there is very little information concerning the inversion.The present study reports a transgenic rice line selected from a T-DNA tagged population,which displays a semi-dwarf phenotype.Molecular analysis of this mutant indicated an insertion of two tandem copies of T-DNA into a locus on the rice genome in a head to tail mode.This inserti...     

Induction of chromosomal inversion by integration of T-DNA in the rice genome

Transfer DNA (T-DNA) of Agrobacterium tumefaciens integration in the plant genome may lead to rearrangements of host plant chromosomal fragments,including inversions.However,there is very little information concerning the inversion.The present study reports a transgenic rice line selected from a T-DNA tagged population,which displays a semi-dwarf phenotype.Molecular analysis of this mutant indicated an insertion of two tandem copies of T-DNA into a locus on the rice genome in a head to tail mode.This insertion of T-DNA resulted in the inversion of a 4.9 Mb chromosomal segment.Results of sequence analysis suggest that the chromosomal inversion resulted from the insertion of T-DNA with the help of sequence microhomology between insertion region of T-DNA and target sequence of the host plant.     

T-DNA插入产生的水稻迟抽穗突变体的遗传分析

在筛选和鉴定水稻T-DNA插入纯合体的过程中,观察到一个迟抽穗的突变体.对该突变体进行遗传分析的结果表明,分离群体后代中出现正常抽穗、迟抽穗和特迟抽穗3种类型,分离比率为1:2:1,符合一对基因的不完全显性遗传;对突变体及其后代分离群体做Basta抗性检测及PCR分子检测,证实该突变体是由T-DNA插入所引起的,突变性状与T-DNA共分离.该材料可用于插入座位的基因克隆.     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H₂O₂. The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenouslyapplied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H₂O₂. Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.     

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.     

水稻P0491E01基因调控花药发育的功能分析(简报)

高等植物的花药发育是包含基因不同程度相互作用的复杂发育过程,一般可分为两个阶段。第一个是花药的形态建成阶段,在这一阶段中细胞与组织发生分化,小孢子母细胞进行减数分裂形成四分     

OsEF3, a homologous gene of Arabidopsis ELF3, has pleiotropic effects in rice

Heading date is an important agronomic trait in rice. A rice mutant with a late heading date and no photoperiodic sensitivity in long or short day conditions was obtained from rice T-DNA insertion mutants in Zhonghua11 (ZH11). Through isolation and analysis of the flanking sequence of the T-NDA insertion site, the target sequence of insertion was obtained and found to locate in AP003296, the sequence accession number of rice chromosome 1 of RGP ( http://rgp.dna.affrc.go.jp ). The putative amino acid sequences of this target gene are homologous to the Arabidopsis protein ELF3 encoded by an early flowering gene. The rice target gene orthologous to Arabidopsis ELF3 is named OsEF3 ; this encodes a putative nematode responsive protein-like protein. OsEF3 has pleiotropic effects in rice that differ from the effects of Arabidopsis ELF3 , which only affects biological rhythms. OsEF3 regulates heading date by influencing the BVG stage and does not affect photoperiodic sensitivity, which suggests that the OsEF3 gene may be involved in an autonomous pathway in rice. OsEF3 may affect root development and kilo-grain weight by delaying cell division or cell elongation.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.     

Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system

We have previously generated a large pool of T-DNA insertional lines in rice. In this study, we screened those T-DNA pools for rice mutants that had defective chlorophylls. Among the 1,995 lines examined in the T2 generation, 189 showed a chlorophyll-deficient phenotype that segregated as a single recessive locus. Among the mutants, 10 lines were beta-glucuronidase (GUS)-positive in the leaves. Line 9-07117 has a T-DNA insertion into the gene that is highly homologous to XANTHA-F in barley and CHLH in ARABIDOPSIS: This OsCHLH gene encodes the largest subunit of the rice Mg-chelatase, a key enzyme in the chlorophyll branch of the tetrapyrrole biosynthetic pathway. In the T2 and T3 generations, the chlorina mutant phenotypes are co-segregated with the T-DNA. We have identified two additional chlorina mutants that have a Tos17 insertion in the OsCHLH gene. Those phenotypes were co-segregated with Tos17 in the progeny. GUS assays and RNA blot analysis showed that expression of the OsCHLH gene is light inducible, while TEM analysis revealed that the thylakoid membrane of the mutant chloroplasts is underdeveloped. The chlorophyll content was very low in the OschlH mutants. This is the first report that T-DNA insertional mutagenesis can be used for functional analysis of rice genes.     

水稻丝氨酸蛋白酶S8基因家族的分布及分析

水稻丝氨酸蛋白酶S8基因家族在水稻的生长发育过程中起着重要的调控作用。本研究利用公共数据库资源,分析水稻中丝氨酸蛋白酶S8基因家族,在水稻12条染色体上找到46个该类基因。通过其结构分析发现,每个基因的内含子数目从0到10各不相同,但氨基酸序列是非常保守的,都有催化活性位点和底物结合位点。系统进化树分析显示,这46个基因分为3个亚家族,S8-1亚家族最大。该家族基因的进化主要是通过基因重复复制的方式进行,其表达模式发生了变化,并且多个基因在穗部具有表达。     

A rice phenomics study—phenotype scoring and seed propagation of a T-DNA insertion-induced rice mutant population

With the completion of the rice genome sequencing project, the next major challenge is the large-scale determination of gene function. As an important crop and a model organism, rice provides major insights into gene functions important for crop growth or production. Phenomics with detailed information about tagged populations provides a good tool for functional genomics analysis. By a T-DNA insertional mutagenesis approach, we have generated a rice mutant population containing 55,000 promoter trap and gene activation or knockout lines. Approximately 20,000 of these lines have known integration sites. The T0 and T1 plants were grown in net “houses” for two cropping seasons each year since 2003, with the mutant phenotypes recorded. Detailed data describing growth and development of these plants, in 11 categories and 65 subcategories, over the entire four-month growing season are available in a searchable database, along with the genetic segregation information and flanking sequence data. With the detailed data from more than 20,000 T1 lines and 12 plants per line, we estimated the mutation rates of the T1 population, as well the frequency of the dominant T0 mutants. The correlations among different mutation phenotypes are also calculated. Together, the information about mutant lines, their integration sites, and the phenotypes make this collection, the Taiwan Rice Insertion Mutants (TRIM), a good resource for rice phenomics study. Ten T2 seeds per line can be distributed to researchers upon request. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chyr-Guan Chern, Ming-Jen Fan, and Su-May Yu have contributed equally to this work.     

The arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway

delayed dehiscence1 is an Arabidopsis T-DNA mutant in which anthers release pollen grains too late for pollination to occur. The delayed dehiscence1 defect is caused by a delay in the stomium degeneration program. The gene disrupted in delayed dehiscence1 encodes 12-oxophytodienoate reductase, an enzyme in the jasmonic acid biosynthesis pathway. We rescued the mutant phenotype by exogenous application of jasmonic acid and obtained seed set from previously male-sterile plants. In situ hybridization studies showed that during the early stages of floral development, DELAYED DEHISCENCE1 mRNA accumulated within all floral organs. Later, DELAYED DEHISCENCE1 mRNA accumulated specifically within the pistil, petals, and stamen filaments. DELAYED DEHISCENCE1 mRNA was not detected in the stomium and septum cells of the anther that are involved in pollen release. The T-DNA insertion in delayed dehiscence1 eliminated both DELAYED DEHISCENCE1 mRNA accumulation and 12-oxophytodienoate reductase activity. These experiments suggest that jasmonic acid signaling plays a role in controlling the time of anther dehiscence within the flower.