The Function Of FCNO1?NS1 And MtPHAN/MtAGO7 In Medicago Truncatula Compound Leaf Development

Legume plants are widely distributed in the world and are important source of protein,oil and vegetables of human food.Moreover,some leguminous species are excellent forages that provide large amounts of protein for livestock.Therefore,it is of great significance to study the gene function of leguminous species.Medicago truncutula is diploid species with short life cycle,high seed setting rate and high genetic transformation efficiency.In addition,the genome of M.truncutula has been sequenced and the Tnt1-tagged mutant population has been developed.Therefore,M.truncutula has been adopted as a model species for legume genomic studies.Leaves are the important photosynthesis organs of plants.Leaf primordia initiate from peripheral zone of the shoot apical meristem(SAM).Under the precise regulations by a series of genes and small RNAs,leaf primordia establish the polarity along adaxial-abaxial,proximal-distal,and medial-lateral axes.The established axes guide cells of leaf primordia to divide and differentiate at specifc directions and eventually to form leaves with certain shape and size.Recent molecular genetic studies have shown that several transcription factor genes,miRNA/tasiRNA,and cell cycle-related genes form a complex genetic regulation network to regulate the process of leaf polarity establishment.However,the mechanism of genetic regulation of compound leaf development remains unclear.In this study,by forword genetic screening the M.truncatula Tnt1-tagged mutants,we isolated and characterized flower can not open1(fcnol)and no stem1(nsl)mutants with defects in compound leaf development.In addition,we also studied the function of MtPHAN and MtAGO7 genes in compound leaf development.Finally,we have analyzed the function of the MtmiR319/MtTCPs regulatory module in nodule development.The main findings of this paper are as follows:1.Regulation of compound leaf development by FCNO1 in Medicago truncutula.By screening the M.truncutula Tnt1-tagged populations,four mutants with the same flower defects were identified.The flowers of mutants could not open normally,therefore,they were named flower can not openl(fcnol-1,fcnol-2,fcnol-3,and fcnol-4).The flower primordia of the fcnol initiated normally,but fcnol was infertile due to the defects of fertilization.Adhesion occurred between leaf and flower,leaf and leaf,and leaflets in fcno1 and the adhesion disappeared as plants grew older.The most prominent phenotype of the fcnol mutant is the increased leaflets number and pronounced leaf complexity.FCNO1 encoded a ?-ketoacyl-CoA synthetase,which is a rate-limiting enzyme in the very long-chain fatty acid(VLCFA)synthesis pathway,and is the ortholog of Arabidopsis FIDDLEHEAD(FDH/AtKCS10).FCNOl is specifically expressed in the outermost cell(L1 cell).Loss of function of FCNOl led to the decrease in the content of VLCFA components,resulting in defects in cuticle formation.Simultaneously,the reducted VLCFA content changed the integrity of the cell membrane,resulting in the disturbed auxin distribution mediated by the auxin efflux carrier protein SLM1/PIN1 in the outermost cell in SAM and leaf primordia.By screening the M.truncutula Tnt1-tagged populations,we dentified another two mutants with similar defects in flower development.The mutants were named flower can not open2(fcno2-1 and fcno2-2).The leaves of fcno2 also adhered,but the leaf complexity was not changed.FCN02 encoded a cytochrome P450 monooxygenase that is the ortholog of Arabidopsis thaliana ATT1(AtCYP86A2)and is involved in the catalytic oxidation of fatty acids.In addition,by reverse screening the Tnt1-tagged populations,we identified the mtkcsl2 mutant.The leaf development in the mtkcs12 was normal.In situ hybridization results indicated that the differences in the leaf development between fcno1,fcno2,and mtkcs12 were caused by the different gene expression patterns.FCNO1 is specifically expressed in the L1 cell,FCNO2 is expressed in the entire SAM and leaf primordia,and MtKCS12 is not expressed in leaves.Genetic evidence indicates that FCNO1 and FCN02 redundantly regulate leaf development.2.Regulation of compound leaf development by NS1 in Medicago truncutula.By forward screening the M.truncutula Tnt1-tagged populations,we dentified two mutants with defects in branch development.The mutants were named no stem1(ns1-1 and nsl-2).NS1 encodes the WUSCHEL-RELATED HOMEOBOX(WOX)family transcription factor and is the ortholog of Arabidopsis thaliana WUSCHEL(WUS).NS1 is expressed in organization center of the SAM and axillary meristem,flower,and leaf margin.Loss of function of NS1 led to the defects in SAM formation and maintenance,leaf and leaf margin development.Moreover,the leaf pattern of nsl was similar with that in slml.In situ hybridization results showed that the formation and maintenance of the SAM is required for the SLM1-mediated auxin distribution,which is critical for the leaf development.NS1 mainly acts as a transcriptional repressor and can physically interact with the co-repressor MtTPL/MtTPRs via its WUS domain and EAR motif to regulate SAM development.Genetic evidence suggested that NS1 indirectly regulates the auxin response at the leaf margin and regulates leaf margin formation in an auxin/SLM1-dependent manner.3.The function of MtPHAN and MtAG07 in Medicago truncutula compound leaf development.MtPHAN encoded the MYB transcription factor and is the ortho log of Arabidopsis thaliana ASYMMETRIC LEAVES1(AS1)and involved in the establishment of leaf polarity.MtPHAN is initially expressed in the entire SAM and leaf primordia and subsequently specifically expressed in the adaxial side of the leaf primordia.The leaves of mtphan are partially abaxialized.MtAGO7 is the ortho log of Arabidopsis thaliana ARGONAUTE7(AGO7)and is required for the biogenesis of a trans-acting short interfering RNA(ta-siRNA)to negatively regulate the expression of MtARF3/4a/4b in M.truncatula.The prominent phenotype of the mtago7 mutant is lobed leaf margins.Genetic analysis showed that the leaflets number of mtphan mtago7 double mutant was increased compared with both of the single mutants,which is similar with that in palm1 and OX-KNOXI transgenic plants.Further study found that MtPHAN and MtAGO7 redundantly repressed the expression of KNOXI and activated the expression of PALM1.Genetic evidence suggests that the PALM1 also inhibits the function of KNOXI.The content of Zeatin-R was reduced in 35S:PALM1 leaves,but significantly increased in,mtphan mtago 7 leaves,suggesting that PALM1 may inhibit the function of KNOXI by downregulation the expression of MtIPT.Moreover,PALM1 acted as a transcriptional repressor and could recruit MtTPL/MtTPRs to form a complex that inhibits the expression of MtIPT genes.Exogenous 6-BA and CKI treatment in mtphan mtago7 changed the leaflet number,further indicating that the changed leaf pattern in mtphan mtago7 is caused by the cytokinin content.4.MtmiR319-targeted MtTCPs involved in nodule development.TCP proteins,the plant-specific transcription factors,are involved in the regulation of multiple aspects of plant growth and development among different species.However,thus far,the roles of TCPs in legume,especially in nodulation are still not clear.In this study,a genome-wide analysis of TCP genes was carried out to discover their evolution and function in Medicago truncatula.In total,21 MtTCPs were identified and classified into class I and class II,and the class II MtTCPs were further divided into two subclasses,CIN and CYC/TB1.The expression profiles of MtTCPs are dramatically different.The universal expression of class I MtTCPs was detected in all organs.However,the MtTCPs in CIN subclass were highly expressed in leaf and most of the members in CYC/TB1 subclass were highly expressed in flower.In addition,most MtTCPs were downregulated during the nodule development,except for the putative MtmiR319 targets,MtTCP3,MtTCP4,and MtTCP10A.Overexpression of MtmiR319A significantly reduced the expression level of MtTCP3/4/10A/10B and resulted in the decreased nodule number,indicating the important roles of MtmiR319-targeted MtTCPs in nodulation.