Genome Wide Analysis Of PtGRF Gene Family And Functional Characterization Of PtGRF1/2d

Growth-regulating factors(GRFs) have been demonstrated to play significant roles in regulation of plant growth and development. The expression of most GRFs is regulated by microRNA miR396 and miR396-GRF pathway is considered as one of the two main ways for organ size control. Although GRFs have been studied vigorously in herbal plants such as Arabidopsis, rice and maize, few studies on GRFs in woody plants with perennial growth and an unique carbon allocation. Therefore it is both theoretically and economically important to investigate the roles of GRFs in growth and development in woody plants. In this study, we performed a genome-wide identification and a comprehensive analysis of the expression of the GRF gene family in Populus, and their effects on the development of leaf, vascular tissues and adventitious roots using transgenic approach were also accessed. This provides an important information for revealing the molecular mechanisms of GRFs in growth and development,which could be used to develop theoretical and technological means in tree genetic improvement.Through genome-wide analysis, nineteen candidate GRF sequences were identified in the genome of Populus trichocarpa. The phylogenetic analysis revealed that 19 PtGRFs were phylogenetically clustered into six groups. The gene structure and motif composition were relatively conserved in each paralogous pairs. The transient expression assay showed that PtGRFs were localized in the nucleus and the expression of 18 PtGRFs were regulated by mi R396, except for PtGRF12 b. Expression analysis indicated that most Pt GRFs were expressed in leaves, stems, roots and cambial zone in Populus. PtGRF1/2d was the one with the highest expression among all PtGRFs genes, suggesting its important roles in poplar growth and development.PtGRF1/2d with the highest expression in different tissues was selected for further study the regulation of growth and development of GRFs in Populus. The expression analysis was done by staining the plants trasformed with the GUS gene driven by PtGRF1/2d promotor fromhybrid poplar(Populus alba × P. glandulosa). GUS signals were presented in leaf, stem and root, with the highest signal in young leaves and stems. The signals in root was concentrated in the pericycle and root apical. The transient expression assay showed that the expression of PtGRF1/2d was regulated by miR396. MiR396-resistant version of mGRF1/2d, containing silent mutations within the mi R396-complementary domain, were used to generate mPtGRF1/2d and mPtGRF1/2d-GFP overexpression transgenic poplars, and PtGRF1/2dSRDX dominant repressed transgenic poplars were also obtained. The PtGRF1/2d overexpression transgenic poplars showed a series of phenotypic changes, including dwarf plant, larger leaf, thicker xylem layer, reduced cambium zone, delayed adventitious root appearance, reduced number and length of adventitious roots. PtGRF1/2d-SRDX dominant repressed transgenic poplars had conflicting phenotypes, however. qRT-PCR analysis indicated that PtGRF1/2d regulated leaf development through the promoting of cell expansion, and inhibited the development of adventitious root through down-regulating the expression of transcript factors involved in early stage of adventitious rooting, including PtSCR, PtAIL9,PtBBM2, PtPLT1.2 and PtWOX11 b. In addition, transverse sections of stem showed that PtGRF1/2d promoted the development of xylem.Here, we have systematically analyzed the GRF gene family in populus, and revealed the diversified and dynamic expression patterns, which provides important clues for further revealing the roles of GRFs underlying growth and development of poplar. In addition, we also show that Pt GRF1/2d causes enlasrged leaf size through promoting cell expansion, accelerates the development of xylem through promoting cambium differentiation, and inhibites formation and reduced number of adventitious roots through down-regulating the trascription factors related to AR formation. These results provide the basis for revealing the mechanisms of developmental regulation of GRFs and for the genetic improvement through manipulating of GRFs in woody plants.