Map-based Cloning And Functional Analysis Of Male-sterile Gene IPE1 In Maize

Maize(Zea mays)is an important food crop and industrial crop,and male-sterile lines are valuable for maize hybrid seed production.Anther cuticle and pollen exine are protective barriers for pollen grain formation.Despite that several regulators have been identified for anther cuticle and pollen exine development in rice(Oryza sativa)and Arabidopsis(Arabidopsis thaliana),few genes have been characterized in maize and the underling regulatory mechanism remains elusive.Through screening a Mutator mutant library,we obtained a complete male-sterile mutant ipel.Using map-based cloning and molecular biotechnology,we cloned the IPE1 gene and preliminarily illustrated molecular mechanism underlying the male-fertile formation mediated via IPE1 gene.The major results of our study are as follows:(1)Compared with the wild type,there were no obvious differences in ipel except the anther development.During the flowering stage,mutant anthers could not be spread from glumes,and became smaller,wilted with no mature pollen grains.(2)Cytological analysis revealed that the formation of anther cuticle and Ubisch bodies were disturbed in mutant anthers.The ipel meiosis was normal,while due to the bacula being undeveloped,sporopollenin deposition and morphology of pollen exine were defective,resulting in microspores degenerated started from the middle uninucleate microspore stage.Tapetal cells were degraded earlier than wild type did.(3)Using map-based cloning and allelism tests,we cloned the IPE1 gene,encoding a putative glucose-methanol-choline(GMC)oxidoreductase.RT-PCR,qRT-PCR and RNA in situ hybridization showed that the IPE1 gene was specifically expressed in young tassels,predominantly and strongly expressed in the tapetum at the tetrad and early uninucleate microspore stage.(4)Biochemical assays indicated that ipel anthers had a significant reduction of fatty acids.Consistently,RNA-Seq data displayed that genes implicated in wax and flavonoid metabolism,fatty acids synthesis and elongation were differentially expressed in ipel anthers.These results suggested IPE1 involves in lipidic synthesis required for anther cuticle and pollen exine development.(5)Phenotypic and expression analysis demostrated IPE1,MS26 and MS45 cooperatively regulate anther development in an independent manner.IPE1 and MS26 are involved in the formation of the anther cuticle and pollen exine,and MS45 serves in anther cuticle maturation and pollen exine thickening.(6)Analysis of the phylogenetic tree and T-DNA line of IPE1 orthologous gene in Arabidopsis showed that IPE1 and their orthologs have a partially conserved function in male organ development.Taken together,IPE1 participates in the putative oxidative pathway of co-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MS26 and MS45 in maize.Our results not only provide the key gene resource for maize seed production but also enrich genetic networks of lipidic synthesis in maize.