Function Analysis Of MS1522Gene Involved In Arabidopsis Carpel Development And Genetic Mapping Of Arabidopsis Male Sterile Mutant Emsl227

Flowers are the reproductive organs of angiosperms, and they originate from celldifferentiation of the shoot apical meristem. The flower of dicotyledonous modelplant Arabidopisis thaliana is radially symmetrical, and it has four whorls of organs,from the outside to the inside: sepals, petals, stamens and carpels. Gynoecium consistsof two fused carpels, where pollination and seed development take place, so carpeldevelopment is the hotspot of plant reproductive biology. In this paper, we identifiedand analyzed an Arabidopsis mutant ms1522with polycarpellary pistils. Geneticsanalysis indicated that the phenotype of mutant including enlarged shoot apicalmeristem, increased carpel and other flower organs, was controlled by a singlerecessive nuclear gene. By using map-based molecular cloning method, MS1522genewas eventually mapped in a164kb region between molecular markers F10A5andT4O12on the Chromosome1. Bioinformatics analysis indicated that the intervalincludes a reported CLV1(CLAVATA1) gene which encodes a receptor protein kinase.The clv1mutant has similar phenotype with ms1522. The gene sequencing andallelism test showed that ms1522is an allelic mutant of clv1. Sequencing analysisindicated that ms1522mutant had a base-pair change at the2516bp locus in CLV1gene, leading to Ala839(GCT) change to Val839(GTT) in CLV1protein. Proteinsequence alignment showed that Ala839is very important for the function of receptorsserine/threonine protein kinase of CLV1protein. Cytology, scanning and transmissionelectron microscopy technology analyses of the carpel developmental process ofms1522mutant indicated that the septum in mutant gynoecium was unable to fusecompletely. By constructing double mutant with clv1and ufo, we found that the styleand stigma of double mutant gynoecia was significantly enlarged, the transmittingtract and vascular pattern were altered in the double mutant gynoecia. These dataindicated that CLV1and UFO genes played a common role in maintaining gynoeciumdevelopment. This study will be helpful to further study the ms1522mutated genefunction, and provide important clues to elucidate the molecular mechanism ofArabidopsis pistil development. In higher plants male sterility is a common phenomenon, which is an importantway to use heterosis of the crops. Abnormal anther and pollen development is one ofthe main causes of male sterility. Isolation of male sterile mutants and gene cloning isthe important method to study the molecular mechanisms of anther and pollendevelopment. This paper reported the functional analyses of the EMS1227gene inanther development by using Arabidopsis male sterile mutant ems1227. ems1227wasa male sterile mutant isolated from an EMS mutagenized Arabidopsis population.Compared with the wild-type, ems1227mutants have short siliques without seeds.Genetic analysis indicated that ems1227mutant was controlled by a single recessivenuclear gene. The mutant plant grows normally during the vegetative growth stage,but shows developmental defects at the reproductive growth stage. Alexander stainingand scanning electron microscopy showed that there was no pollen in most of themutant anthers, except that only few normal pollen grains observed in some mutantanthers. Cytology observation indicated that the ems1227mutant tapetum cells werehypertrophy and vacuolated, and tapetum layer was not separate from the middlelayer. Besides, the periclinal division of mutant tapetal cells appears abnormal,resulting in two cell layers, and the degradation of tapetum was delayed. The callosestaining method indicated that callose is not degraded in mutant plants. RT-PCRanalysis showed that the A6gene expression was significantly down regulated in themutant, and Real time-PCR experiment indicated that the expression level of A6genein the ems1227mutant was only6.41%of that in the wild-type. A map-based cloningapproach was used, and EMS1227gene was found to be linked to a molecular markerCIW11on chromosome3. Bioinformatics analysis revealed that there is a TDF1genenear the marker CIW11. Sequencing analysis indicated that ems1227mutant had aG459to A459base-pair change in the third exon of TDF1gene, which resulted in apremature termination mutation in this region. Allelism test indicated that EMS1227and TDF1belong to the same locus.