Functional Study Of JAGGED And ARP/KNOXI Regulatior Module In Compound Leaf Development Of Medicago Truncatula

Leaves are the main organs of plant photosynthesis and transpiration.Leaf development basically includes three steps:initiation of leaf primordia,establishment of leaf polarity and further differentiation and development.Elucidation of molecular mechanism in leaf development can not only give us a deeper understanding of leaf morphology,but also help us to reform the leaf type and the plant type by biological approaches.As a model species of legume,Medicago truncatula has the simplest compound leaf form,which provides an advantage for the study of compound leaf development.The function of JAGGED and ARP/KNOXI regulation module in compound leaf development of M.truncatula was studied respectively in this thesis.Part one:JAGGED(JAG)is a key gene in leaf development and there are two homologous genes MtJAG1 and MtJAG2 in M.truncatula.We screened the mutants of two genes through reverse genetic means from Tnt1 retrotransponson-tagged mutant population of M.truncatula,however,both of single mutants did not show obvious defects.MtJAG1 and MtJAG2 are adjacent to each other on the chromosome,therefore,it is difficult to obtain double mutants by sexual hybridization.We used the CRISPR/cas9 editing technique to knock out the MtJAG2 in the background of mtjagl(-/-)mutants.The T0 generation transgenic plants of mtjag1(-/-)mtjag2(+/-)showed pronounced leaf serration and loss or degeneration of the lateral leaflet in some cases at the late stage of development,implying the dosage effect of MtJAGs.Then,the mtjag1 mtjag2 double mutant was successfully isolated at T1 generation.mtjag1 mtjag2 has severe developmental defects,such as dwarfish plants,lobbed leaf margin,needle-shaped leaves and loss of petiole.The scanning electron microscopy analysis showed that the abnormal initiation of the lateral leaf primordia resulted in the simplification of the compound leaves.Further analysis showed that the leaf polarity of mtjag1 mtjag2 was disordered.The epidermal cells of the needle-shaped leaves changed significantly,which is similar with the vein cells of wild-type.In addition,the lateral leaves of mtjag1 mtjag2 converged upwardly and could not carry out normal leaf movement,indicating the abnormal pulvinus.The wild-type pulvinus was an abaxialized organ,but the abaxial epidermal cells of pulvinus in the double mutant were elongated,and its adaxial phloem of vascular bundle was absent,which suggested the abaxialization degree of mtjag1 mtjag2 pulvinus was weakened.The floral organs of mtjag1 mtjag2 was narrow,which is similar with the flower of jagged in Arabidopsis thaliana.Moreover,the degree of fusion between the petals in mtjag1 mtjag2 was decreased,and both male and female organs were damaged and sterile.Developmental abnormalities in leaves and floral organs suggested that JAG is conserved in the development of plant lateral organs,and MtJAG plays a crucial role in the establishment of adaxial-abaxial polarity.The molecular mechanism of mtjag1 mtjag2 remains to be further explored.Part two:Our previous studies have shown that the petiole length in the mtphan mutants is almost the same as that in the wild type at the vegetative stage,but it become significantly shorter at the reproductive stage.Thus,we proposed a hypothesis:This phenomenon may be related to the heterotopic expression of MtKNOXI at petiole,and this expression pattern is regulated by age-related genes.We found that the expression levels of MtKNOXI genes in rachis and petiole of mtphan were up-regulated along with the development.Moreover,GUS staining was performed on the leaves of proMtKNOXI:GUS transgenic plants of wild type and mtphan.The results showed that MtKNOXI was ectopic expressed in the compound leaf unit in mtphan,especially in the petiole.Furthermore,the petiole length of mtphan mtknox7 and mtphan mtknox1 mtknox2 mtknox6 mtknox7 were partially recovered in comparison with that of mtphan and mtphan mtknox1 mtknox2 mtknox6 at reproductive stage.This genetic evidence indicated that up-regulation of MtKNOX7 mainly contributes to the defects in the petiole development of mtphan.miR156 is the only known molecular marker of age.Its expression levels decreases with plant growth,and the expression levels of miR156-targeted SPLs increase gradually.In 35S:MtMIR156 transgenic plants,the length of petiole was similar to that of wild-type at reproductive stage,and the expression levels of MtKNOX and MtPHAN were not dramatically changed.However,the length of petiole in 35S:MIM156 transgenic plants became shorter,and the expression level of MtSPL3 was up-regulated significantly,implying that increased MtSPL3 may result in the shorter petiole length.Mutants with the same defective phenotypes often imply the same regulatory pathway interrupted.The petiole development is abnormal in sgll and fcl1.However,our data showed that the regulation of SGL1,FLC1 and MtPHAN on petiole development belong to different regulatory pathways.In the further,we will continue to explore the mechanism on regulating the petiole development of M.truncatula.