Functional Identification And Gene Localization Of Female Sterile Mutants Of Arabidopsis Thaliana

Female sterility in plants was first discovered in the 1950 s and was observed in several crops.Female sterile mutants are helpful for us to unravel the molecular mechanism of female gametophyte and ovule development.The combination of female and male materials can produce hybrid varieties of crops,making it easier to improve the crop production.Female sterile mutant could not produce seeds,which provides a new approach for seedless fruit.What’s more,female sterile mutants can also be floral crops,perfectly avoiding the formation of fruits and seeds that reduce the life span of plants and accelerate the aging process by high-intensity flowering and seed bearing after fertilization.In recent years,a large number of female sterile mutants,with many traits,including megaspore mother cell development abnormalities,ovule abnormalities and embryo sac malformations,have been discovered successively by chemical mutagenesis,RNA interference,T-DNA or transposon insertion,and CRISPR/Cas9-based genome editing in Arabidopsis,rice,corn and so on.Here,female sterile mutants were screened out from mutagenesis library that was obtained by mutating Col-0 seeds with EMS,and their BC1F2 population after backcross was generated for map-based causal gene cloning.Genome re-sequencing and comparative genomic analysis of mutant pool and non-mutant pool were applied to identify of candidate genes,while the study of single mutant of candidate genes facilitated the cloning of causal genes of EMS mutants.The primary findings of this study were as follows:1)AP1G2 was identified to have a role in both megagametogenesis and microgametogenesis.AP1G2 is the γ subunit of the adapter protein complex(AP-1),and its functional deficiency resulted in the inability of functional megaspore to undergo the first mitosis,producing aborted female gametophytes.Meanwhile,the development of male gametophytes was also arrested in the phase of uninucleate microspore.And ap1g2 transgenic plants,carrying Pro AP1G2:AP1G2,all showed significantly higher seed set than themselves,respectively.AP1G2 expression in roots,stems,leaves and inflorescences was analyzed using q PCR,and the results showed that AP1G2 had the highest expression level in inflorescence.Vectors carrying GUS or GFP driven by AP1G2 promoter were transformed into wild-type Arabidopsis plants to analyze the temporal and spatial expression of AP1G2.We found that AP1G2 was highly expressed in developing female and male gametophytes,as well as in root tips and trichomes.2)Transcriptome analysis of the ovules of the ap1g2-1/ap1g2-3 and ap1g2-3P-/-P indicated that the expression of several CaP2+P related genes like CPK6(calcium dependent protein kinase 6)were down-regulated.So the CaP2+P distribution in the normal embryo sacs and defective ones were detected using FO3 staining and potassium antimonate.The results showed that CaP2+P concentration in aborted female gametophytes of ap1g2-3 were reduced.After the treatment of potassium pyroantimonate,anthers and pollens were observed by transmission electron microscopy.The results showed that abortive pollens were vacuolated before the first mitosis with cytoplasm gradually degraded,and the CaP2+P concentration was lower than that of the normally developed pollens.Therefore,we demonstrated that the deficiency of AP1G2 resulted in the reduce of CaP2+P concentration and down-regulated expression of related genes.3)TE(Transposable Element),which is located near centromere,also plays an important role in the development of female gametophyte.The female gametophyte abortion rate of teP+/-Preached nearly 70%.Most defective female gametophytes could not undergo the first mitosis but remained in the mononuclear phase,while a few could not undergo the second nuclear division and arrested at binuclear stage.In addition,microspore development of teP+/-P was also abnormal due to the inability to undergo mitosis.The findings of this study help us understand the function of AP1G2 and TE gene which is near the centromere of chromosonmeⅠ.This two genes would facilitate the establishment of female sterility gene network.Because the genes of Arabidopsis have similar functions with the corresponding genes of other flowering plants,this study also provides a reference for the germplasm resources of other cash crops with female abortion characteristics.