Molecular Mechanism Of PagUPB1 Gene Regulating Tree Growth Rate

There is plasticity in plant growth.As immobile photoautotrophic organisms,plants can spontaneously adjust their development in response to the changes in ambient environment.However,the exact mechanism underlying the regulation of cellular activities for adjusting growth and development in plants is yet not clear.Wood is the most important natural source of energy and also plays critical function in protecting ecological environment.Considering the significant role of wood in the world,in this study,we looked for the valuable genes for regulating wood growth by using integration of molecular methods and other biological technology and analyzed genes' function.Main obtained results were as followed:(1)Proteomics results of poplar primary and secondary growth-young stem showed that a total of 10.316 proteins were identified in these growing tissues,and among them.3,106 proteins were found to be significantly differentially expressed between primary growth and secondary growth.These differentially expressed proteins were participants of various biological pathways.In addition to signalling,phytohormone,cell wall synthesis and degradation,secondary metabolism,carbohydrate and energy metabolism,as well as protein syrnthesis and degradation,cell cycle,cell division and redox metabolism also played important roles in the transition from primary to secondary growth process.(2)Previous experimental results indicated that two bHLH family transcription factors were found in Populus alba X P.gland.ulosa.Alignment of amino acid sequence showed that these two are the homologous proteins of Arabidopsis UPB1 in poplar(with low homology).They were named PagUPB1-1 and PagUPB1-2.Quantitative real-time PCR(qRT-PCR)results showed that PagUPB1-1 and PagUPB1-2 shared the same expression patterns,both of them were highly expressed in apical buds,primary growth-stems and root tips;however,they were lowly expressed in mature tussues undergoing robust secondary growth.(3)In situ hybridization results showed that PagUPBl expression was localized in the tissues and cells where vascular genesis took place,including leaf primordium,stem apical meristem,procambium,primary phloem,primary xylem,metaphloem,metaxylem,phloem cells,epidermal cells in root tips,rootpericycle and vascular cells in lateral roots.(4)Five plant expressed vectors,including four PagUPBl over-expressed vectors and one amiRNA vector for PcagUPBl,were constructed in this study.And they were transformed to Populus alba XP.glandulosa via agrobacteriurm.Plenty of transformant poplar plants with up-regulated or down-regulated PagUPBl expression were acquired.(5)Physiological analysis results showd that plants height,internode number,internodal length,intenode diameter,main roots number,lateral roots number and dry weight of non-transgenic plants were lower than those in the amiRNA-PagUPBl plants,but higher than those in the PagUPBl over-expressed plants.These results indicate that the up-regulation of PagUPBl expression inhibits plant growth,while the down-regulation of PagUPBl expression promotes plant growth.Meanwhile,long-term cultivation and observation of the selected transformant poplar lines suggested none malformation caused by manipulation of PagUPBl expression.(6)Hand-free and semi-thin section results showed that in the wamiRNA-PagUPB1 poplar there were evidently more cells in the shoot apical meristem and the internodes initiate secondary growth at an earlier stage compared with non-transgenic poplar.On the contrary,in PagUPB1 over-expressed poplar,there were fewer cells in the shoot apical meristem and the secondary growth of the internodes was largely retarded compared with non-transgenic poplar.These results suggest that the expression level of PagUPB1 gene has influence on differentiation rate of meristem cells,procambial cells,and cambial cells as well as the cell division rate of the mother cells of secondary xylem.(7)DAB and NBT staining results showed that hydrogen peroxide and superoxide anion accumulated in shoot apical meristem,leaf primordium,procambium,primary phloem and primary xylem,which coincides with the location of PagUPBl expression.Moreover,it was revealed that environmental stimuli such as drought or osmotic stress induced by PEG could also induce PagUPBl expression in poplar in this study.Combining all the results,it can be inferred that the UPB1 gene is a result of evolution in vascular plants for the monitoring of reactive oxygen level induced by internal signal or external stimuli.