Functional Specialization And Regulatory Mechanism Of MtAGa And MtAGb In Determining Floral Organ Identity Of Medicago Truncatula

Flower is one of the most complex and diverse morphology organs of plants.The determination of floral organ identity is always a fundamental question in biological science.Papilionoideae plants,which exhibit the unique morphological structure and developmental characteristics in their floral organs,attracted more and more attention from developmental biologists.The C function gene AGAMOUS(AG)encodes for a MADS-box transcription factor required for floral organ identity and floral meristem determinacy in angiosperms.Unlike Arabidopsis,Medicago truncatula and most legume plants possess two AG homologues arose by an ancient genome duplication event prior to the speciation of legumes.The functional conservation and diversification of duplicated AG homologs in determining peculiar floral morphogenesis in legumes remains an open question.Whether the regulation mechanism of AG homologous similar with AG in Arabidopsis remains unkonwn.Here we characterized two AG-like genes MtAGa and MtAGb in the model legume Medicago truncatula by using forward and reverse genetics approaches to uncover their specific contribution to the control of petal and stamen identity,we investigated the role of duplicated MtAGa and MtAGb in M.truncatula flower development by isolating mtaga and mtagb loss-of function mutants.Lack MtAGa function in M.truncatula leads to serious defects in carpel fusion and ovule formation,resulting in split carpels and producing abnormal pods with reduced seed number.Loss-of-function of MtAGb leads to an incomplete fusion of staminal tubes with weak stamen-petaloid conversion.The mtaga mtagb double mutant analysis revealed that MtAGa and MtAGb together fulfill a full C-function activity,but the stamens and carpels are completely converted to numerous vexillum-like petals in mtaga mtagb.,likely by an effect from the ectopic expression of TCP genes MtCYCl-3.Interestingly,compared with the wild-type and the phenotype of single mutants,the mtaga mtagb/+,mtaga/+mtagb and mtaga mtagb plants show a dosage dependent effect on floral organ identity with peculiar floral phenotypes.Further realtime PCR analysis in different mtag mutants revealed that MtAGa and MtAGb can repress the expression of putative A and B function genes as well as MtWUS,but promote putative D function genes expression in M.truncatula.In addition,we found that the abnormal dorsal petal phenotype observed in the mtaga mtagb double mutant is associated with the upregulation of CYCLOIDEA(CYC)-like TCP genes.Our data suggest that the redundant MtAGa and MtAGb genes of M.truncatula employ a conserved mechanism of action similar to Arabidopsis in determining floral organ identity and floral meristem determinacy but may have evolved distinct function in regulating floral symmetry by coordinating with specific floral dorsoventral identity factors.In order to further analyze the genetic regulation pathway involved in MtAG gene in Medicago truncatula,mutants similar to MtAG double mutants were screened.Besides,we found a petals increased mutant pis which showed similar phenotype with the mtaga mtagb double mutant.we cloned the gene controlling the phenotype,sequence analysis showed this was a specific gene in dicotyledonous plant,there was no reports about its homologous function.MtAGa and MtAGb were downregulated obviously in pis,so we speculate PIS may regulate the C function genes to control floral developmemt.But the mechanism PIS regulating C class genes remains further study.In this study,we analyze the functional conservationand diversification of the M.truncatula duplicated AGAMOUS homolog genes in regulating floral development.Provide information for understanding the regulators in determining flower morphogenesis of Papilionoideae legumes.