Study On The Molecular Mechanism Of Plant Polypeptide Hormones Regulating Meristem Activity

As a large group of deciduous trees,poplar is not only an excellent source of wood,but also plays important roles in sand fixation,water conservation and soil preservation.Peptide hormones are important intercellular signaling molecules and play crucial roles in regulation of plant growth and development.In this thesis,CLE(Clavata 3/Embryo Surrounding Region-Related,CLE)peptide hormone and its receptor kinase BAM(Barely Any Meristem,BAM)protein as well as xylogen glycosylated peptide hormone in poplar were studied as the research objects and their gene biological functions were analyzed by bioinformatics,biochemistry,molecular biology and genetics methods.The signal transduction pathway mediated by CLE peptide hormones is one of the most important mechanisms regulating stem cell division and differentiation in the apical and lateral meristems in plants.In this thesis,8 poplar CLE peptides with special amino acids composition were selected by multiple sequence alignment of their CLE motifs.The biological function of each chemically synthesized CLE peptide was compared and verified by in vitro application assay in Arabidopsis.The results showed that 8 Pt CLEp inhibited the meristematic activities of shoot and root apices of Arabidopsis to different degrees.Among them,Pt CLE9 p,Pt CLE17 p,Pt CLE18 p,Pt CLE40 p and Pt CLE49 p revealed significant inhibitory effects on the activity of both shoot and root apical meristems of Arabidopsis,while Pt CLE5 p and Pt CLE34 p mainly inhibited the activity of root apical meristem.As the main receptors of CLE peptides in plants,LRR-RLK(Leucine Rich RepeatReceptor Like Kinase,LRR-RLK)proteins play important roles in the recognization of CLE peptides as well as intercellular signal transduction.In this thesis,the BAM proteins with deleted different domains were constructed and their respecitve biological functions were analyzed.Our results showed that the BAM?SP protein with the signal peptide being deleted did not change its localization on the plasma membrane;however,its overexpression induced serious inhibitory effects on the apical meristems in transgenic Arabidopsis and such phenotypes were closely related to the increased expression level of CLV3 and the inhibited expression of WUS(Wuschel,WUS).Meanwhile,the root phenotypes of transgenic Arabidopsis were directly associated with the elevated expression of CLE40 and the decreases expression of WOX5(Wuschel-Related Homeobox 5,WOX5).This work demonstrated that transgenic plants with "dominant negative" phenotypes could be obtained by constructing mutants of LRR-RLK protein kinase with specific domains being deleted,which provided a new idea for overcoming the functional redundancy of gene family members in woody plants.In addition to CLE peptides,xylogen also plays an important role in regulating the activity of lateral meristem in plants.In this thesis,the gene expression patterns of Pt XYLP(Xylogen Like Protein,XYLP)gene family members were analyzed using bioinformatics analysis,which showed that the expression of Pt XYLP genes were closely related to the differentiation process of xylem cells.Second,transgenic plants in which the GUS gene was driven by Pt XYLP gene promoters were constructed and the Pt XYLP1pro:GUS was found to be mainly expressed in Arabidopsis leaf veins and roots.Overexpression of Pt XYLP1 gene caused interrupted cotyledon veins and reduced the expression level of a variety of genes involved in vascular development.This work demonstrated that the Pt XYLP gene may play an important role in the development of early vascular tissues in plants.Together,the biological functions of CLE peptide hormones,the receptor kinase BAM protein and xylogen peptide hormone of poplar trees were carried out by means of bioinformatics,reverse genetics,chemical synthesis and in vitro addition of peptides.This study laid a foundation for a more comprehensive understanding of the accurate and complex regulation networks by peptide hormones in meristems of woody plants and provided a theoretical knowledge for a more in-deepth study on its signaling pathways.