Functional Analysis Of MtKNOX4,MtLMI1s And MtBOPs In Compound Leaf Development In Medicago Truncatula

Legumes are the third largest families of angiosperms in the world which have been classified into six subfamilies.Legumes can supply grain,oil,forage,ornamental values and medicinal values for human beings.Therefore,it is of great significance to study the functions of leguminous species.Medicago truncatula functions as the model plant of leguminous species,it is a diploid species with small genome,short life cycle,high seed setting rate and high genetic transformation efficiency.In addition,the genome of M.truncatula has been sequenced and the mutant population has been available.So,M.truncatula is being extensively and deeply studied.Leaves function as the major photosynthetic organ in plants,the leaf development process includes three intertwined stages:leaf initiation in the shoot apical meristem(SAM),primary morphogenesis and secondary morphogenesis.Under the multiple regulation of genes,small RNAs and hormones,leaf primordia define the polatity along the the adaxial-abaxial,medio-lateral and proximal-distal axes.Later,the cells divide and differentiate in a specific direction and develop into a mature leaf with function.During the development process,the leaf veins,petioles,leaflets,serrations and stipules are also formed.In recent years,reports on compound leaf development of M.truncatula are increasing,but many mechanisms are still unknown,and the regulation network of compound leaf development needs to be improved.In this thesis,M.truncatula was used for studing the mechanism of MtKNOX4 in compround leaf patterning,the mechanism of MtLMI1a/b in leaf margin development,and the mechanism of MtBOPs in the regulation of stipules development.The main findings of this paper are as follows:1.Regulation of the compround leaf development by MtKNOX4We isolated and characterized a mutant with increased leaflets in M.truncatula,named mtknox4,by forward genetic screening.According to the MtKNOX4pro:GUS transgenic plants,MtKNOX4 was strongly expressed in young leaves as well as mature leaves.To gain a better spatial expression pattern of MtKNOX4,RNA in situ hybridization was performed.Strong MtKNOX4 transcripts were detected in leaf primordia and the developing leaves.These results evidence the MtKNOX4 functions in compround leaf development.Overexpressing the MtKNOX? genes could increase the leaf complexity which mimic the leaf phenotype of mtknox4.By qRT-PCR analysis,the expression level of MtKNOX2 was increased in mtknox4 mutant.Then,the mtknox2 mtknox4 double mutant was generated and the leaves were similar with mtknox4.Moreover,mtknox1/2/6 were introduced in the mtknox4 background,the leaves in the quadruple mutant were also similar with mtknox4,supporting that the leaf phenotype of mtknox4 is not due to ectopic activation of STMIBP-like MtKNOX?.To further test the function of MtKNOX4,MtKNOX4 was overexpressed in the wild type background,the trangetic plants showed shortened petioles and rachises,suggesting that MtKNOX4 may function in decreasing the leaf complexity.Both overexpressing the MtKNOX2 and MtKNOX4 in plant,the number of five leaves decreased greatly,implying thatthere exists an antagonism between MtKNOX2 and MtKNOX4.By exogenous spraying of CK and analysing the qRT-PCR data,MtKNOX4 may regulate CK signal transduction pathway.In addition,we demonstrated the inhibition between MtKNOX4 and FCL1 at the molecular level.Through the hybridization between mtknox4 and fcll,the inhibition between MtKNOX4 and FCL1 was also demonstrated at the genetic level.Overexpressing the FCL1 caused an extension in the petiole and rachis.Overexpressing both the FCLl and MtKNOX4 in plants,the lengths of petioles and rachises were between those in the parent plants,further demonstrating the inhibition relationship between MtKNOX4 and FCL1.Then,we found that MtKNOX4 regulated MtNAM and MtUFO at the transcriptional level.Finally,the knox3/5/9/10 mutants were isolated and characterized by reverse genetic screening.The quadruple mutant also showed normal leaflet number,suggesting the functional specificity of MtKNOX4.Overexpressing the MtKNOX5 showed shortened petiole and rachis which is similar with MtKNOX4,but is different from MtKNOX3,MtKNOX9 and MtKNOX10,indicating the diverse functions of KNOX? family.2.Regulation of the leaf margin development by MtLMI1sWe isolated and characterized the homologs of LATE MERISTEM IDENTITY1(LMI1)in M.truncatula,named MtLMI1a/b,by reverse genetic screening.MtLMI1s belong to the HD-Zip I family which is characterized by a DNA-binding homeodomain(HD)motif and one Leu Zip domain.MtLMI1s proteins are localized in the nucleus,which supports their roles as transcription factors.There also existed a physical interaction between MtLMIla and MtLMIlb,implying their potential capability to form heterodimers.After screening in the mutant population,we found no obvious defects in compound leaf pattern and leaf morphology of mtlmi1a or mtlmi1b,but the double mutant of mtlmi1a mtlmi1b showed a smooth leaf margin.This phenotype was also supported by the SEM analysis at the cellular level.We next analyzed the MtLMI1a/bpro:GUS plants and GUS signals were detected on leaf marginal teeth tips and veins.According to the RNA in situ hybridization,MtLMI1a transcripts were detected in leaf marginal teeth and leaf vein precursors,while MtLMI1b transcripts were accumulated in leaf sinus between the adjacent serrations.Therefore,MtLMI1a and MtLMI1b showed a near-complementary expression pattern during the initiation and development of leaf marginal serrations,implying that MtLMI1a and MtLMI1b may function cooperatively along the leaf margin.In addition,we improved the activities of MtLMI1s by the generation of 35Spro:MtLMI1s-GFP plants,the transgenic plants showed increased leaf serration numbers and curled leaves.By analysing the leaf margin of mtlmi1a mtlmi1b slml,we confirmed that MtLMI1s regulated SLM1/Auxin module.Later,by comparing the DR5:GUS and DR5:GFP signals in wild type and mtlmi1a mtlmi1b,we concluded that MtLMI1s regulated the distribution of auxin at the leaf margin to control the development of leaf serrations.In order to check the direct regulation between MtLMI1s and SLM1,we used the yeast one-hybrid,transient expression assay in protoplasts,ChIP-qPCR analysis and RNA in situ hybridization analysis,we demonstrated that MtLMI1a and MtLMI1b directly activate the transcription of SLM1 in leaf margin to regulate the formation of marginal serrations.MtAGO7 is involved in the trans-acting siRNA3(TAS3)pathway and the development of leaf serrations.Loss-of-function mtago 7 mutant displayed lobed leaves due to the ectopic expression ofA UXIN RESPONSE FACTOR3(MtARF3).We analysed the leaf serrations of mtlmi1a mtlmilb mtago 7 and mtlmi1a mtlmi1b MtARF3mut,demonstrated that MtLMI1s and MtAGO7-mediated TAS3 ta-siRNA pathway antagonistically functioned in controlling the formation of leaf marginal serrations.What's more,in M.truncatula,the mutation of boundary gene MtNAM exhibited relatively smooth leaf teeth,we generated the mtlmila mtlmi1b mtnam plants,and the leaf margin in the triple mutant became smoother than those in both mtnam and mtlmi1a mtlmi1b.This finding suggests that MtLMI1s and MtNAM play redundant roles in regulating the formation of leaf margin in M.truncatula.3.Regulation of the stipule development by MtBOPsThe diversity of stipule morphology is key to plant classification.Stipules are considerably diverse in size,function,and architecture,such as leaf-like stipules,spines,or tendrils.The function of the stipules determines their structural diversity.However,the molecular regulatory mechanism of stipule identification remains largely unknown.Here,we investigated the developmental process of stipules in M.truncatula.By forward genetic screening,we isolated a leaf-like stipules 1(lls1)mutant which displayed the abnormal stipules.LLS1 encodes the MtBOP1 in M.truncatula,and has one homolog MtBOP2.MtBOPs belong to the NPR1 family,and MtBOPs contain a BTB/POZ domain and four ANK repeat domains.The mtbop1 mtbop2 double mutant shows a higher proportion of transformation from stipules to leaf-like stipules compared with those of single mutants,suggesting that they redundantly determine stipule identity.In addition,the formation of stipules in wild type and mtbop1 mtbop2 double mutant were compared by SEM analysis.The results proved the stipule primordia in double mutant could initiate but appeared later than in wild type,which is consistent with the later appearance of stipule in double mutant.To further explore the effect of MtBOPs in leaves and stipule development,35Spro:MtBOP1-GFP and 35Spro:MtBOP2-GFP were introduced into wild type.No obvious abnormality was observed in leaves and stipules of transgenic plants,but the transgenic plants were compact.The phenotype of stipules in mtbop1 mtbop2 mutant could be partially rescued by 35Spro:MtBOPs-GFP.To understand the expression patterns of MtBOPs,MtBOP1 and MtBOP2 promoter-?-glucuronidase(GUS)transgenic plants were generated and GUS signals were detected in stipules.To gain a better spatial expression pattern,RNA in situ hybridization was performed.The results showed that MtBOPs expressed at the base of the leaf primordium where stipules formed,implying that MtBOPs function specifically in stipule primordium.Further investigations show that control stipule initiation together with SINGLE LEAFLET1(SGL1),which encodes a key factor in maintaining the indeterminacy of the leaf primordium to enable the development of lateral leaflets.But the the molecular mechanism needs to be further explored.