Functional Analysis Of The Arabidopsis AtAOG1 And MBD3 Genes In Gametophyte Development

Formation of female and male gametophytes is an important part of sexual reproduction in flowering plants.The female gametophytes are formed in the female organs,ovules,while the male gametophytes are formed in the male organs,anthers.The process is controlled by a complex genetic network.So far,little has been known about the genetic mechanisms underlying gametophytic formation in flowering plants.This study identified and characterized two Arabidopsis gametophytic mutants,ataogl and mbd3,and analyzed the roles of the corresponding genes in gametophytic formation.Genetic analysis showed that the genetic function of male and female ataogl was drastically affected,resulting in an obvious reduction in the genetic transmission through male and female gametophytes.The development of male ataogl gametophytes was disrupted at the uninucleate pollen stage,leading to degeneration of microspores.The development of female ataogl gametophytes was arrested at the uninucleate stage,resulting in defective embryo sacs.The defects in the male and female ataogl gametophytes dramatically affected the fertility of the gametophytes and caused a drastic reduction in seed set.AtAOGl encodes an unknown protein.It is expressed more highly in microspores,pollen grains and ovules than in the vegetative organs.The AtAOGl did not contain any known domain and was localized mainly in the nucleus.In addition,the ataogl mutant affected the expression of some genes which are involved in gametophyte development.The results indicated that AtAOGl might play important roles in male and female gametophyte development through influencing the expression of the gametophyte-related genes.The mbd3 mutant did not affect the female gametophyte development,but had a drastic impact on the male gametphyte development and pollen tube growth.Many microspores in mbd3 showed a defect at the uninucleate pollen stage.Only some of the microspores could develop into trinucleate pollen.Aniline blue assay showed that such the morphologically normal mutant pollen grains could germinate in vitro and in viva,but the resulting pollen tubes were shorter than the wild type pollen tubes,leading to a significant reduction in seed set.MBD3 encodes a predicted methylated DNA-binding protein(MBD),namely this MBD protein family may bind to the methylated DNA.The protein family includes 12 members that is MBD1-12.The MBD domain of MBD3 protein shares a high similarity in amino acid sequence with the other members from the MBD family,but the other parts of the protein did not.Similar to other members from the MBD family,MBD3 was expressed in many tissues.Its expression levels in inflorescences and siliques were higher than those in stems,roots and leaves.MBD3 protein was distributed throughout the cell.LCI assay indicated that MBD3 could form polymers not only by itself,but also through interaction with some other MBD proteins.Transcriptome comparative analysis of mbd3 and wild type inflorescences found that the mbd3 mutant could affect the expression of some genes involved in pollen development and pollen tube growth.These results indicated that MBD3 might play important roles in pollen development and pollen tube growth throught influencing the expression of downstream genes.