Identification And Analysis Of The Genes Related To TPD1-EMS1 Signaling Pathway In Arabidopsis

Pollen formation is important to sexual reproduction in flowering plant and closely related to agricultural production.Pollen formation relies on both microsporocyte development and substantial support from tapetum.Defect in tapetum may cause male sterility.Previous studies showed that the TPD1,which may function as the ligand of the receptor protein kinase EMS1,is an important component of the TPD1-EMS1 signaling pathway and plays important roles in tapetum development.Overexpression of TPD1(OE-TPD1)in wild type Arabidopsis can increase the size of siliques,indicating that the function of TPD1 is related to cell growth and development.So far,however,little has been known about the molecular mechanism of TPD1 underlying tapetum and silique development.This thesis attemped to identify and characterize the downstream genes of TPD1 and EMS1,by analyzing the impact of tpdl mutant,emsl mutant and OE-TPD1 on the expression of the downstream genes influenced by TPD1 and EMS1 to further study the mechanism of TPD1-EMS1 signaling pathway that controls development of the tapetal cells and siliques.Microarray assays showed that compared to that in wild type flower buds,the expression of many genes was significantly affected in the flower buds of tpdl,OE-TPD1 and ems1 plants.The results could be summarized as followings.(1)Four genes,namely AT5G61605,AT1G73220(ATOCT1),AT5G07540(ATGRP16)and AT3G25050(XTH3),were significantly upregulated in OE-TPD1 flower buds,but downregulated in tpd1 and ems1 mutant flower buds.This result was further confirmed by simi-quatitative RT-PCR assay.(2)Many genes,which were significantly downregulated in tpdl and emsl flower buds,are involved in lipid transport,lipid storage,sugar metabolism,protein metabolism and sporopollenin biosynthesis,indicating that the genes affected by the mutants are related to the physiological functions of tapetal cells.(3)Many transcription factor genes which play important roles in tapetum development,for examples,AT5G62320(ATMYB99),AT2G16910(AMS),AT2G31210(ATbHLH091),AT1G06170(bHLH089)and AT3G28470(TDF1)were significantly downregulated in tpdl and ems1 mutant flower buds.(4)Several genes which are related to phytohormone signaling,such as AT2G19500(ATCKX2),AT2G40670(ARR16),AT1G30840(ATPUP4),AT1G80440(KFB20)and AT3G59940(KFB50)were also significantly downregulated in tpdl and ems1 mutant flower buds.All these results indicated that the genes affected by overexpression or functional defects of TPD1 were related to cell development and physiological functions of tapetal cells.Among the genes affected in tpd1 mutant,AT5G61605 encodes a small cysteine-rich secretory peptide.The AT5G61605 protein shares a higher homology with AT2G16505 from MEG family.Moreover,homology analysis showed that AT5G61605,AT2G16505 and AT2G16535 share a higher similarity of amino acid sequence.Real-time PCR assays showed that the three genes all were highly expressed in flower buds.Furthermore,the expression levels of the three genes were significantly downregulated in tpd1 mutant flower buds and upregulated in the TPD1-overexpressing flower buds,compared to that in wild type flower buds.Further promoter activity assay showed that AT5G61605 was expressed in tapetal cells during the whole process of tapetum development.Phenotypic analysis showed that RNAi-AT5G61605 plants and the mutants of AT2G16505 and AT2G16535 genes did not exhibit any phenotype.The siliques of RNAi-AT5G61605-transgenic OE-TPD1 plants had a shape similar to that of wild type siliques.RT-PCR assay showed that the expression levels of both AT5G61605 and TPD1 were reduced in the RNAi-AT5G61605-transgenic OE-TPD1 plants,in which the wide-silique phenotype was reduced or disappeared.In addition,the expression levels of AT5G61605 and TPD1 gene were correlated to reduction in wide-silique phenotype of RNAi-AT5G61605-transgenic OE-TPD1 plants.Thus,AT5G61605 may be also involved in silique development of OE-TPD1 plants,similar to TPD1.In summary,this study indentified and characterized some of downstream genes influenced by TPD1 gene,among which the candidate gene AT5G61605 is expressed in the developing tapetal cells,implying that it might be related to tapetum development.Down-regulation of AT5G61605 expression could restore the wide-silique phenotype of OE-TPD1 plants,indicating that the function of AT5G61605 is related to cell development and growth.The results could provide a new clue and research materials for further study of molecular mechanisms underlying tapetum and silique development.