Functional Analysis Of The HIR Genes And The Characterization Of The Atnop10 Mutants

In flowering plants,the male gametophytes,which also are called pollen,are produced in the anther of the stamen.The mature pollen grain is a tricellular unit that consists of two smaller sperm cells enclosed in a bigger vegetative cell.The female gametophyte,which also is called embryo sac,is formed in the ovule of the pistil.In Arabidopsrs,the mature embryo sac is a tetracellular unit consisting of a central cell,an egg cell and two synergid cells.During anthesis,the pollen grain is transferred to the stigma of the pistil.It is hydrolated and germinates a pollen tube after recognition with the stigmatic cells.The pollen tube undergoes polar growth,enters the embryo sac and releases the two sperm cells for double fertilization.The male and female gametophyte formation and fertilization is an important step in plant sexual reproduction and is closely related to agricultural production.Therefore,study of gametophyte formation,pollen tube growth and fertilization is important for understanding of genetic mechanisms underlying plant sexual production,which also could provide theorectical references to the crop breeding and production.Our previous study indicated that the Arabidopsis transcription factor gene AtTFIIB1 plays important roles in pollen tube growth and endosperm development.This study identified and characterized the down stream genes involved in pollen tube growth using the attfiib1-2 mutant.The microarray analyses using the the attfiib1-2 pollen grains showed that in the attfiib1-2 pollen,1048 genes were downregulated two folds and 2139 genes were upregulated two folds,compared to their expression levels in wild type pollen grains.Real-time PCR was applied to further comfirm the impacts on the expression of the genes affected in the attfiibl-2.Furthermore,predicted functions of the affected genes identified five gene families that are expressed highly in pollen and may be related to pollen tube growth.The HIR family genes were selected for further functional characterization.In Arabidopsis,the HIR family has four members.They share higher similarities in amino acid sequences and structures.The promoter activity assays showed that all four HIR members were expressed in pollen.The HIR proteins were located in plasma membranes of pollen tubes.In the heterozygous hir1(hir1/+),the mutant siliques were shorter and had lower seed set,compared to the wild type siliques.Part of the pollen grains from the hur1/+ plant were aborted.Part of the ovules in the the hir1/+ plant exhibited defect in embryo sac development with disruption of the first gametophytic division and absence of the central vacuole.However,the homozygous hir1 plant did not exhibit the phenotypes.The further assays for the self-pollination of hur1/+;hir2/+ double mutant indicated that only the plants with the heterozygous hirl genotype had the gametophyte-defective phenotype.In addition,the assays for the prociprocal crosses of the hirl and wild type plants also showed that only the plants with the heterozygous hirl genotype exhibited the gametophyte-defective phenotypes.Complentation using the full length genomic sequence of HIR1 gene demonstrtated that the gametophyte-defective phenotype in the heterozyougs hirl plant was caused by the mutation in the HIR1 gene.The Y2H and IP assays showed that any two of the four HIR proteins could interact with each other.The IP-MS analyses identified the protein RBSC3B and Lhb1B2 that could interact with HIR1.BiFC and LCI assays further demonstrated that HIR1 could interact with RBSC3B or Lhb1B2.The primary genetic analysis showed that the heterzyous rbsc3b plant exhibited gametophyte-defective phenotype,similar to that of the hirl mutant.Taken together,the results indicated that HIR1 is expressed in pollen and pollen tube,could interact with the RBSC3B and may be involved in gametophyte development.Meanwhile,this study also characterized the gametophyte-defective atnoplO and the AtNOP10 gene.The atnop10-1 was generated using the Ac/Ds gene trap and enhacer trap technology and had shorted siliques with lower seed set.The atnop10-2 mutant was produced by CRISPR/Cas9 and exhibited a phenotype similar to that of atnop10-1.The cell biological observation showed that the polar nucleates did not fuse in the atnop10-1,leading to abortion of embryo sac development,but male gametophyte development was only slightly affected.These results suggest that the AtNOP10 plays important roles in female gametophyte formation by involvement in the polar nuclear fusion.