Functional Study Of KANADI In Compound Leaf Development In Medicago Truncatula

Legumes are widely distributed angiosperms,containing about 751 genera and 19500 species.Most of the leguminous plants display typical compound leaves,while only a few plants display simple leaves.Compound leaves could improve the ability of plants to cope with harsh environments compared with the simple leaves.The development of compound leaves goes through three stages:the initiation and formation of leaf primordia,the formation of primary morphology,and the formation of secondary morphology.According to the different arrangement of leaflet in compound leaves,they are divided into pinnate compound leaves,palmate compound leaves,and unifoliate compound leaves.The morphological diversity of legumes makes it an important research material in the fields of evolutionary development,morphogenesis,and genetics.In addition,legumes are important economic and food source:proteins,oils,and vitamins in human food are inseparable from legumes.Medicago truncatula has typical ternate pinnate compound leaf,and it is diploid.The genome of M.truncatula has been sequenced and the Tnt1-tagged mutant population has been developed,so it has been widely explored and studied as a legume model plant.The formation of leaf polarity is an important prerequisite for leaf primordium to develop into leaf blade.So far,several transcription factors involved in leaf polarity development have been explored and a regulation network was formed to control the development of leaf polarity,so as to ensure the normal development of leaf.At present,most of the studies focus on the functions of polarity genes in the simple-leaf plants,but only a few polarity genes have been revealed in the compound-leaf plants.KANADI(KAN)is a key factor that determines the formation of abaxial polarity,but the research about KANADI mainly focus on the simple-leaf plants,such as maize,rice,and Arabidopsis.The function of KANADI in compound-leaf plants is still unclear.Therefore,in this study,we screened and identified the mutants of MtKANADI(MtKAN)from M.truncatula and carried out a series of experiments to explore their functions.In addition,our previous studies showed that the phosphorylation of PRE1 protein is important for plant growth and development in Arabidopsis,and Ser-67 is the key site for phosphorylation.In this study,we further refined the effect of phosphorylation on PRE1’s function,and revealed that Ser-67 site of the PRE family was conserved among species in M.truncatula and Arabidopsis.The main results of this research are summarized as follows:1.There are five MtKAN genes in M.tula.By comparing the sequences of KAN genes from Arabidopsis,we found that there are five MtKAN genes in M.atula genome(MtKAN1,MtKAN2,MtKAN3,MtKAN4,and MtKAN5).Among them,the evolutionary relationship between MtKAN4 and KAN4 is closer than other genes.In Arabidopsis,KAN4 is mainly involved in the development of ovule,while the other three genes are mainly responsible for the polarity development of leaves.In addition,some data in our laboratory have shown that MtKAN4 is also mainly involved in seed development.Therefore,we mainly focus on the functions of other four genes(MtKAN1,MtKAN2,MtKAN3,and MtKAN5)in the development of compound leaf in M.truncatula.Further analysis reveals that they all contain GARP domain which is responsible for DNA binding,and the subcellular location analysis also showed that both four proteins localized to the nucleus.The above results indicate that MtKAN perform their functions as nuclear location factors.2.The redundant functions between MtKAN genes.To further understand the functions of MtKAN1,MtKAN2,MtKAN3 and MtKAN5 in compound leaf development in M truncatula,we used the qRT-PCR to analyze their tissue expression patterns in the wild type plants.The results showed that they expressed in juvenile leaves,adult leaves,flowers and pods,suggesting that the function of MtKAN genes may be pleomorphic.We ordered Tnt1 insertion mutants of MtKAN from the M.truncatula mutant library.Phenotypic analysis showed that the mtkan1-1,mtkan2-1,mtkan3-1 and mtkan5-1 single mutants do not display obvious defects compared with those in wild type,and this may be due to functional redundancy among MtKAN genes.Further genetic analysis showed that mtkan1-1 mtkan3-1 double mutants displayed a variety of phenotypes,including leaf margin curled upward,leaves and lateral branches at the stem nodes increased,short and sterile plant,thinner stipules and dehiscent cotyledons.In addition,complementation experiments showed that both MtKAN1 and MtKAN3 can restore the phenotypes of mtkan1-1 mtkan3-1 double mutants.3.MtKAN control the development of abaxial polarity in M.truncatula.RNA in situ hybridization showed that MtKANl and MtKAN3 expressed on the abaxial side of leaf primordium,in addition,the expression of MtKAN1 is also detected in the SAM.GUS staining results also indicated that MtKAN1 and MtKAN3 showed similar expression patterns in M truncatula.In mtkan1-1 mtkan3-1 double mutants,the xylem was ectopically developed into the abaxial side of vascular bundle in the petiole and stem,leading to the adaxialized vascular bundle.In addition,the veins of mtkan1-1 mtkan3-1 double mutants were partially adaxialized.The loss-of-function of MtKAN1 and MtKAN3 also leads to elongated leaf epidermal cells.Moreover,mtkan1-1 mtkan2-1 mtkan3-1,mtkan1-1 mtkan3-1 mtkan5-1 and mtkanl-1 mtkan2-1 mtkan3-1 mtkan5-1 mutants all showed more obvious developmental defects of leaf polarity and morphological changes in leaves.In addition,the overexpression of MtKANl and MtKAN3 lead to serious defects in the development of leaf polarity and leaf morphology.4.MtKAN control the development of compound leaf pattern.In the early development stage of mtkan1-1 mtkan3-1 double mutants,an ectopic lateral leaflet(ELL)is formed on the abaxial domain of the junction between the two lateral leaflets.Furthermore,the scanning electron microscope results showed the formation of ectopic lateral leaflet primordium(ELLP)between the two lateral leaflet primordia.Meanwhile,the formation of ELL was observed in mtkan1-1 mtkan2-1 mtkan3-1,mtkan1-1 mtkan3-1 mtkan5-1 and mtkanl-1 mtkan2-1 mtkan3-1 mtkan5-1 mutants.mtkan1-1 mtkan3-1 double mutants showed significantly shorter petioles,and the vascular bundles of petiole in mtkan1-1 mtkan3-1 double mutants are partially adaxialized and the vascular bundles of petioles were increased.Morphology of ELL in mtkan1-1 mtkan3-1 double mutants tends to take on a trumpet-like shape,and the vascular bundles in ELL are partially adaxialized.5.The formation of ELL in mtkan1-1 mtkan3-1 double mutants is partly dependent on local auxin maximization.We introduced DR5rev:GFP into the wild type and mtkan1-1 mtkan3-1 double mutants by genetic hybridization,and the GFP signals were observed by confocal laser.Strong GFP signals were observed in the ELL primordia of mtkan1-1 mtkan3-1 mutant.Furthermore,treatment with auxin inhibitor NPA partially inhibit the formation of ELL in mtkan1-1 mtkan3-1 plant.This suggests that the initiation of ELL primordium in mtkan3-1 may be related to auxin transpotation and distribution.6.MtKAN and MtREV1 regulate compound leaf development antagonistically.In this study,we detect the increased expression level of MtREV1 in mtkan1-1 mtkan3-1 mutant by qRT-PCR.Moreover,studies have shown that ectopic expression of MtREV1 can also change the compound leaf pattern in M.tula.Meanwhile,heat map analysis shows that MtKAN and MtREV1 modulate the expression of genes involved in auxin synthesis and transport antagonistically.The above results indicate that MtKAN and MtREV1 may regulate the development of the compound leaf patterning antagonistically.7.The development of upward curled leaf margin in mtkan1-1 mtkan3-1 mutant dependent on MtPHAN.In this study,we observed that the leaf margin of mtkan1-1 mtkan3-1 mutant were curled toward the adaxial side,however,the leaf margin of mtphan mutant were curled towards the abaxial side.MtKAN and MtPHAN are the key factors controlling the development of abaxial polarity and adaxial polarity respectively.Meanwhile,we showed the up-regulation of MtKAN1 and MtKAN3 in mtphan mutant,and the up-regulation of MtPHAN in mtkan1-1 mtkan3-1 double mutants.In addition,the leaves of mtphan mtkan1-1 mtkan3-1 mutant were flat.In conclusion,we conclude that MtKAN and MtPHAN control the formation of leaf morphology by inhibiting each other.8.MtKAN negatively regulate the expression of SGL1.SGL1 is an important factor in control of the initiation of two lateral leaflet primordia in compound leaf development in M.truncatula.qRT-PCR results showed that the expression level of SGL1 was significantly increased in mtkan1-1 mtkan3-1 mutant,indicating that the ectopic expression of SGL1 may contribute to the formation of ELL in mutants.RNA in situ hybridization results also showed that SGL1 has ectopic expression in the ELL primordium in mtkan1-1 mtkan3-1 mutant.Meanwhile,the sgl1-2 mtkan1-1 mtkan3-1 mutant display a simple leaf.Furthermore,the transient expression assays demonstrated that MtKAN can inhibit the expression of SGL1 directly or indirectly.These results further indicate that SGL1 is downstream from MtKAN and is regulated by MtKAN negatively.9.Analysis of phosphorylation site and function of PRE1.Our previous studies showed that Ser-67 is a key site for phosphorylation of PRE1 protein.Phospho-mimicking mutation of Ser-67 to Glu-67 resulted in mPRE1 losing its ability to promote cell elongation.The expression of cell elongation related genes EXPANSIN1 and EXPANSIN8 were also inhibited.Moreover,ser-67 is the key site for PRE1 to interact with IBH1.In this study,we further verified it by rBiFC and Co-IP methods.Phospho-mimicking mutation of Ser-67 to Glu-67 inhibited the interaction between PRE1 and IBH1 exactly.Meanwhile,the conservativeness of Ser-67 in PRE1 homologous gene PRE2 was analyzed.Ser-68 in PRE2 is equivalent to the Ser-67 in PRE1.Y2H,β-gal,and BiFC experiments all proved that Ser-68 is the key site for PRE2 interacting with IBH1.In addition,we also verified Ser-67 as the key site for PRE1 to interact with other HLH proteins by Y2H.GST pull-down results showed that Ser-67 was the key site for PRE1 to inhibit the interaction between IBH1 and HBI1.By comparing the amino acid sequences,the same conserved amino acid sites of MtPRE family were found in M.truncatula,and the phospho-mimicking mutation of Ser-67 to Glu-67 reduced the function of MtPRE1.