Functional Analysis Of AJL1 And MtLAS In Compound Leaf Development In Medicago Truncatula

As a model species in legume,Meidcago truncatula is a diploid species with small and simple genome and has high genetic transformation efficiency.M.truncatula has compound leaves and can form nodules by interacting with rhizobium,so it is an ideal material for studying the development of compound leaves and the symbiosis between plants and rhizobia.Leaves are important vegetative organs of angiosperms.According to the number of leaves on the petiole,leaf can be divided into single leaf and compound leaf.The formation of compound leaves depends on the initiation of new leaflets in the formed leaf primordia.Therefor,the development of leaf primordia in simple leaf and compound leaves is different.Previous studies showed that the genetic networks controlling compound leaves in Solanum lycopersicum and cardamine hirsuta are different from M.truncatula and Pisum sativum.So,there is still a lot of work to be done to study the development mechanism of compound leaves.In this study,three projects were performed.I,through forward genetic screening,we identified the mutant additional juvenile leaf 1-1(ajl1-1)that shows the defects in the SAM maintenance and the initiation of juvenile leaf primordia during embryonic developmen.II,we identified the mutant with abnormal compound leaf-pattern through forward genetic screening and found the mutated gene was the homolog of LATERAL SUPPRESSOR(LAS)in Arabidopsis.We investigated the function of this gene and its homolog in compound leaf development,and the relationship between MtLASl and other compound leaf development genes.Ⅲ,under different gene regulatory networks and growth environments,homologe genes could have different functions in different species,so the function of specific genes in different species remains to be studied.The han mutant has flower developmental defects in all four whorls in Arabidopsis.However,the homologs of the HAN in M.truncatula are not involved in the development of flowers,but involved in nodule development in M.truncatula.The main findings of this paper are as follows:1.Regulation of juvenile leaf development by AJL1 in M.truncatulaBy screening the M.truncatula Tnt1-tagged population,a mutant with additional juvenile leaves were identified.Scanning electron microscope observations showed that the primordia of the juvenile leaf of the ajl1-1 mutant initiated at the embryonic development stage,and the mutant had defects in SAM maintenance,resulting in an enlarged SAM in seeds and embryos.Protein alignment and phylogenetic analysis showed that AJL1 is a homolog of Arabidopsis CLE family genes.Expression pattern analysis showed that AJL1 is specifically expressed in the central zone(CZ)of SAM.qRT-PCR and in situ hybridization showed that the expression of MtWUS is significantly up-regulated in the ajl1-1 mutant.The mtwus-1 mutant also displayed the defects in the initiation of the primordia of juvenile leaf.Genetic analysis showed that AJL1 regulates SAM maintenance and the initiation of juvenile leaf primordia during embryonic development by inhibiting the expression of MtWUS.At the same time,through in situ hybridization,we found that MtPIN1/SLM1 was specifically expressed in the position of juvenile leaf primordia,and the slm1 mutant in M.truncatula could not form juvenile leaf.We also found that,the mutation of SLM1 in the background of ajl1-1 can inhibit the initiation of juvenile leaf primordia during embryonic development.So,the initiation of increased juvenile leaf primordium in ajl1-1 depends on the maximization of auxin at this position mediated by SLM1.As a secreted peptide of the CLE family,AJL1 can participate in the maintenance of SAM through the MtCLV1 receptor.At the same time,we found that mutants of homolog of BARELY ANY MERISTEM(BAM)in M.truncatula,mtbam1-1 and mtbam2-1 have the same phenotype to the ajl1-1 mutant.This suggests that AJL1 may participate in the SAM maintenance and the initiation of juvenile leaf primordia through MtBAMl and MtBAM2.In addtiton,MtREV1 can participate in the initiation of juvenile leaf primordia by regulating the maintenance of SAM.The mutant form two young leaf primordia during embryonic development.Overexpression of MtREV1 in the qjl11 mutant can rescue the phenotype of the ajl1-1 mutant,indicating that the function of MtREV1 in regulate juvenile formation is epistatic to AJL1.This work expands our knowledge of the initiation of juvenile primordia during embryonic development.2.Regulation of compound leaf development by MtLASl in M truncatulaBy screening the M.truncatula Tnt1-tagged population,two mutants with abnormal compound leaf-pattern were identified.Wild type compound leaves usually consist of 3 leaflets.Compared with wild type,this mutant produces compound leaves containing two pairs of lateral leaves and one terminal leaf.Through Tnt1 transposon association analysis,it was found that the mutant gene is the homolog of Arabidopsis LAS in M.truncatula,and we named it MtLAS1.Phylogenetic analysis shows that MtLAS1 has another copy in M.truncatula and we named it MtLAS2.Through genetic cross,we constructed their double mutants.The results showed that the proportion of leaves with 5 leaflets in the double mutant increased significantly,indicating that there is a functional redundancy between the two genes.Scanning electron microscope observation revealed that the ecpotic leaf primordium are formed at the rachis between the first-initiated lateral leaf primordium and the terminal leaf primordium.Compared with the wild type,the leaf area,petiole and rachis length of the mtlas1-1 mtlas2-1 double mutant were increased.Expression pattern analysis in different organs showed that MtLAS1 has the highest expression level in shoot bud and flowers,and its expression level in other organs was relatively low.The expression of MtLAS2 is relatively high in the shoot bud,flowers and pods,and relatively low in other organs.Promoter-β-glucuronidase(promoter-GUS)results revealed that MtLASl is highly expressed in the root tips of germinated seeds,young leaves,stipules,and flowers,and has lower expression in mature leaves.And MtLAS1 is expressed in root tips of germinated seeds,young leaves,boundary between stem and lateral organs,and flowers,and its expression level is lower in mature leaves.In situ hybridization showed that MtLAS1 and MtLAS2 was specifically expressed in the boundary region between SAM and leaf primordium.By constructing overexpression transgenic lines,we found that the length of the petiole and rachis in 35S:MtLASl and 35S:MtLAS2 were shorter compared with that of wild type.By crossing with mutants involved in the development of compound leaves of M.truncatula,we found that MtNAM,FCL1 and SGL1 are epistatic to MtLAS1 and MtLAS2 during compound leaf development.This indicates that the initiation of the ectopic lateral leaflet primordia in mtlas1-1 mtlas2-1 double mutant depends on MtNAM,FCL1 and SGL1.At the same time,we found that knocking out of MtUFO can rescue the phenotype of the mtlas1-1 mtlas2-1.By overexpressing MtUFO in the wild type,the phenotype of the mtlas1-1 mtlas2-1 double mutant can be simulated.This indicates that MtLAS1 and MtLAS2 could regulate compound leaf development by regulating MtUFO.However,qRT-PCR showed that the expression of MtUFO did not change in the mtlas1-1 mtlas1-1 double mutant and 35S:MtLAS1 plant,indicating that MtLAS1 and MtLAS2 could not regulate the expression of MtUFO at the transcriptional level.At the same time,we found that overexpression of MtLAS1 in the 35S:MtUFO plant can repress the phenotype of 35S:MtUFO,indicating that MtLAS1 may regulate the function of MtUFO at the protein level.This work confirmed that MtUFO is involved in the compound leaf development of the inverted repeat lacking clade(IRLC)of Fabaceae.At the same time,it was found that MtLAS1 regulates the function of MtUFO at the protein level.This research provides references for studying the developmental mechanism of compound leaves of legumes and improving the quality of forages.3.The function of MtHANs in nodule development of Medicago truncatulaLegume plants can produce nodules with nitrogen-fixing function through interaction with rhizobia.Because the formation of nodules is an energy-consuming process,plants will precisely regulate the number of nodules through the autoregulation of nodulation(AON)pathway.High levels of nitrogen inhibits the initiation of nodules by activating the AON pathway.When the nitrogen concentration in the environment is high,the initiation and development of nodules will be affected.In Arabidopsis,HAN can regulate the chlorophyll content in response to nitrogen,and the han mutant has developmental defects for floral organs.Through protein sequence analysis and phylogenetic tree analysis,we found the homologs MtHAN1 and MtHAN2 of HAN in M.truncatula.Through reverse genetics,we screened the mutants of MtHAN1 and MtHAN2 and found that the mutants of mthanl and mthan2 did not affect the leaf and flower development in M.truncatula.Then,we constructed double mutant of mthan1 mthan2,and found that the double mutant still does not have the phenotype of flower development defects,suggesting that MtHAN1 and MtHAN2 have other functions in the development of M.truncatula.By analyzing the expression patterns of MtHANl and MtHAN2 in different organs,we found that MtHAN1 and MtHAN2 are expressed in roots.By constructing promoter-GUS vector,we found that MtHANl and MtHAN2 are expressed in the root tip,lateral root primordium and root vascular of M.truncatula.By detecting the expression of MtHAN1 and MtHAN2 in different developmental stages nodules,we found that the expression of MtHAN1 and MtHAN2 genes will change with the development of nodules.In order to study the functions of MtHAN1 and MtHAN2 in nodule development,we conducted nodule induction in single and double mutants.The results showed that the number of nodules in the single mutant of mthan2 and the double mutant of mthan1 mthan2 increased significantly.Also we found that the mthan1 mthan2 mutants did not affect the nodule morphology and nitrogenase activity.At the same time,we also constructed 35S:MtHAN1 and 35S:MtHAN2 transgenic lines,and found that the overexpression of MtHANs did not affect the development of leaves and flowers of the plants.The induction of nodules in overexpression plants showed that overexpression of MtHAN1 and MtHAN2 genes did not affect the morphology and nitrogenase activity of nodules.In order to study which aspects of MtHAN1 and MtHAN2 genes are involved in nodule development,we performed transcriptome analysis on mature nodules of wild type and mthan1 mthan2 double mutants.The results showed that many substance synthesis pathways and phytohormone signal transduction pathways related to the process of nodule formation were affected.At the same time,we found that in the nodules of mthan1 mthan2 double mutant,the expression of many peptidase and peptidase inhibitor genes related to polypeptide synthesis and processing changed.Further analysis showed that MtHANs can directly or indirectly regulate the expression of NCR genes including Medtr1g042940,Medtr5g072205 and Medtr5g072275.This work proves that MtHANs,the homologs of HAN in M.truncatula,has different functions in plant development.At the same time,it also provides a reference for studying the regulation of nodule formation in legumes.