Molecular Mechanism Of CsSPL Regulating Reproductive Organ Development In Cucumber

Life continues through reproduction,spermatophyte transmits genetic material through seeds produced by sexual reproduction.In this process,the anther and ovule produce male gametophytes(pollen grains)and female gametophytes(mature embryo sac),respectively.Anther and ovule genesis preconditions crop fertilization and fruit production,and determines the survival,evolution and production of crops.In flowering plant Arabidopsis,many relative mutants were screened.Among,spl mutant showed male and female sterility,whose anther tapetum origination was inhibited and pollen sac couldn't produce sporocytes,while the ovule development was inhibited,and megasporocytes and embryo sacs were aborted in spl mutant.As a world cultivated vegetable crop with great economical value,cucumber(Cucumis satives L.)is a typical unisexual flower crop and its fruit is commodity organ as well as reproduction organ.The research of reproduction development in cucumber was focused on fruit quality and sex dertermination field.However,the research of anther and ovule,as the real reproductive tissue,is rare.Here,we utilized the methods and tools in developmental biology,phytophysiology and biochemistry to explore the function of the reproductive development gene SPOROCYTELESS(SPL)/NOZZLE(NZZ)in cucumber.In cucumber mango fruit mutant with severely defective anther and ovule development,CsSPL expression was severely inhibited.Futher,we cloned CsSPL and analyzed its function.(1)CsSPL,as a transcription factor,is expressed specifically in the developing anthers and ovules.Knockdown of CsSPL reduced male and female fertility with malformed pollen and suppressed ovule development.So,CsSPL has the conserved biological function.(2)Importantly,CsSPL directly interacted with CsWUS(WUSCHEL)in the nucellus and YABBY family genes in integuments,and positively regulated CsWUS expression,while HD-ZIP III gene CsPHB(PHABULOSA),expressed specifically in the nucellus,promoted CsSPL expression by binding to the CsSPL promoter.Thus,CsSPL acts as an adaptor to link CsPHB and CsWUS functioning,underpinning a previously unidentified regulatory pathway which is CsPHB-CsSPL-CsWUS orchestrating sex organ development in planta.(3)Auxin accumulation is reduced in male and female flower buds of CsSPL knockdown plants.Auxin plays an important role in plant reproduction development.Biochemical analyses further showed that CsSPL stimulated the expression of A UXIN RESPONSE FACTOR3(CsARF3)and was positively regulated by CsARF13 during reproductive organ development,indicating sequential interactions of CsSPL with auxin signaling components in orchestrating anther and ovule development.As such,this study dissected the biological function and regulatory mechanism of reproductive development gene CsSPL,uncovered a new regulatory pathway in a CsPHB(PHABULOSA)-CsSPL-CsWUS(WUSCHEL)manner during ovule development,and provided direct evidence between CsSPL and auxin signaling pathway in promoting the reproductive organ development.