The Role And Mechanism Of Growth Regulators GRF12a And GEF12b On The Growth And Development Of Poplar

Growth regulating factors(GRFs)are a kind of plant specific transcription factors,which play an important role in the growth and development of plants.At present,the research on GRF gene function is active in Arabidopsis,rice and maize;the researchers found that GRF plays an important role in leaf size control,root development,stem radial growth and other growth and development.The regulation of GRF is complex,it is not only regulated by mi R396,but also interacts with GIF(GRF interacting factor)to regulate the expression of downstream genes in a modular way.In the secondary growth of woody plants,the study of GRF function is just beginning.In our previous study,we found that GRF is involved in the regulation of leaf size and secondary growth,but the regulating mechanism of GRF needs further study.In view of the important role of GRF in the regulation of growth and development of woody plants,here,GRF12 a and GRF12 b of Populus alba × P.glandula(84K poplar)were used as the research object to explore the regulatory role and mechanism of PagGRF12 a and PagGRF12 b in the growth and development of Poplar through the analysis of expression pattern,transgenic analysis,interaction assay,downstream target gene screen and domain exchange analysis.The main results of this study are as follows:1.According to the results of bioingormatics analysis,degradome analysis and transient transcriptional activity experiments,it was found that Ptr GRF12 a and Ptr GRF12 b are a pair of genes from genome / gene doubling.However,Ptr GRF12 a is regulated by mi R396 while Ptr GRF12 b not.2.The leaves of PagGRF12 a and PagGRF12 b overexpressed transgenic plants were larger than that in the control.The results of Histocytology showed that the number of cells in leaves increased and the size of cells decreased.Subsequently,the gene expression alteration analysis showed that the expression of cell proliferation marker genes increased significantly in these overexpressed plants,while the expression of cell expansion marker genes decreased significantly in these overexpressed plants.The above results showed that PagGRF12 a and PagGRF12 b affected leaf development through regulating cell proliferation as well as cell expansion,and mainly affected leaf development by regulating cell proliferation and participated in the control of leaf size..3.The height and radial diameter of PagGRF12 a overexpressed poplar showed a decrease trend compared with that of the control,while that of PagGRF12 a represssed poplar was the opposite.Internode cross section analysis showed that the ratio of xylem to internode diameter of PagGRF12 a overexpressed poplar was significantly lower than that of the control,while that of PagGRF12 a represssed poplar was the opposite,indicating that PagGRF12 a could inhibit the development of xylem.Later,through phenotype and anatomy analysis,quantitative expression analysis,Ch IP-PCR analysis,transcriptional regulation analysis,yeast two hybrid and luciferase complementary experiment,we found that PagGRF12 a can interact with PagGIF1 b to form a complex,to activate the expression of xylem NAC domain 1(XND1a),and thus inhibit the development of xylem.4.The height and radial diameter of PagGRF12 b overpresssed poplar showed an increase trend when compared with that of the control.Internode cross section analysis showed that the ratio of xylem to internode diameter ratio of PagGRF12 b overpresssed poplar was significantly larger than that of the control,indicating that PagGRF12 a could promote the development of xylem.Then,through phenotype and anatomy analysis,quantitative expression analysis,transcriptional regulation analysis and luciferase complementary experiment,we found that PagGRF12 b can interact with PagGIF1 a to form a complex,and to inhibit the expression of XND1 a,and thus promote the development of xylem.5.To further study the mechanism underlying PagGRF12 a and PagGRF12 b in xylem development,the QLQ and WRC domains of PagGRF12 a and PagGRF12 b were exchanged,and PagGRF12 a Q12b W and PagGRF12 b Q12a W overexpressed transgenic plants were generated and analyzied.Phenotypic observation showed that the xylem width of PagGRF12 a Q12b W overexpressed plants was narrowed,while the xylem width of PagGRF12 b Q12a W overexpressed plants was widened,indicating that QLQ domain is the key domain of PagGRF12 a and PagGRF12 b in xylem development.In this study,the role of PagGRF12 a and PagGRF12 b in poplar growth and development,especially in secondary growth was studied.It is revealed that PagGRF12 a and PagGRF12 b can regulate cell proliferation and cell expansion to involve in leaf size control and regulate the expression of XND1 a to invovle in xylem development.These studies provide an important basis for revealing the regulation mechanism of leaf development and wood formation,analysis the function of GRF gene in growth and development of poplar,and provides potential target genes for the improvement of woody plants.