Functional Analysis Of MtWOX3 And WFM In Flora Development In Medicago Truncatula

Floral development is one of the model systems for investigating the mechanisms underlying organogenesis in plants.The flowers among different plant species displayed the distinct color and morphologies.The color of flower refers to the color of all petal structures in plant flower organs,and what we usually call is the color of petals.The pigments in plants mainly include flavonoids,carotenoids and alkaloids,which are the main factor affect plant flower color.Carotenoids are a general term for natural pigments,which are normally found in plants,animals,and fungi with yellow or orange pigments.In the plant,carotenoids are secondary metabolites,which are synthetic precursors of abscisic acid ABA and strigolactone in plants.At the same time,the content of carotenoids in plants also effect the color change of plant flowers and fruits.Carotenoids play an important role in plant growth and development,photosynthesis,and attracting insects.At the same time,carotenoids also have a good protective effect on visual system and the skin tissue in human.Medicago truncatula is the model legume species.The Tht1-tagged mutant population in M.truncatula has been applied in different research area in legume.This thesis includes two parts.In the first part,we screened a mutant line with an abnormal flower color by forward genetics.The color of the petal of the wild type was yellow,but the color of the mutant changed to white.Thus,the mutant named wfm.The flower organs in mutant were dissected,and we found that the floral organs structure in mutant was normal.Moreover,other organs in mutant,such as leaves,pods,seeds,etc.,had no obvious defects.Then,we found that this mutant was caused by the Tntl insertion in the WFM gene.WFM belongs to the MYB gene family.Tissue-specific expression analysis revealed that it has the highest expression in flowers.Subcellular localization shows that WFM mainly functions in the nucleus.Transcriptional activities by protoplast transformation experiments showed that WFM mainly plays a transcriptional repressing function.Compared with wild type,the content of carotenoids in the mutant was dramatically decreased.The qRT-PCR results showed that the expression of the related genes in the carotenoid synthesis pathway was decreased,but the genes of the degradation pathway did not change significantly.The analysis of RNA-seq results found that the 45 genes of carotenoid synthesis pathway had down-regulated.In the further analysis,several candidate genes will be selected for the possible regulation by WFM in the carotenoid metabolism pathway in petals of M.truncatula.In the second part of thesis,a flower mutant mtwox3 which was reported previously was used for the study of formation of petal conical epidermal cells.WOX3 belongs to the WUS branch of the WOX gene family.In M.truncatula,the mtwox3 mutant exhibits the separated petals including alae,keel and the two keels,and the base of the sepal is split.A series of researches have found that MtWOX3 has the highest expression in young flowers,and it is mainly expressed at the boundary of organ fusion:MtWOX3 can interact with the co-repression transcription factor TOPLESS and inhibit transcription of downstream genes.In this study,we found that the petal conical epidermal cells in mtwox3 mutant became flat.compared with those in wild type by scanning electron microscopy.Thus,we proposed a hypothesis that the expansion of petal conical epidermal cells is defective in mutants.OBP4 belongs to the DOF transcription factor family,which is a negative regulator of cell expansion in Arabidopsis.Its direct downstream gene is the cell wall amplification gene XTHs.In the mtwox3 mutant,the expression levels of MtOBP4a and MtOBP4b were up-regulated,while the expression levels of MtXTH9a and MtXTH9b were down-regulated.To verify our hypothesis,the MtOBP4a was overexpressed in wild type and the transgenic plant was obtained.We also isolated the knock-out mutant of MtOBP4a by reverse genetic screening.In the further analysis,we will check the conical epidermal cells of petals in these plants,and identify the possible relationship between MtWOX3 and MtOBP4a in flower development.