Functional Characterization Of A PtoWOX11/12a Gene In Stress Resistance Of Populus Tomentosa

When suffer from drought or salt,plant produce a series of physiological and biochemical variation including rebuilt ionic and signal balance in cell,repair damage and coordinate developmental regulation pathway.In this process,transcription factors play important roles in accepting and passing signal to downstream genes and then regulate their expression.WOX famliy is one of plant specific transcription factors which had reported play important roles in plant growth and development,such as plant morphogenesis,maintenance of stem cell division and differentiation,formation and enlongation of adventitious root.Many studies about WOX in model plant Arabidopsis,rice had been reported,but in woody plant there are little study about WOX in abiotic stress response.In this study,we characterized the roles of PtoWOX11/12 a from Populus tomentosa.An AP2 protein(PtoAP2)identified by yeast one hybrid system was specifically bound to DRE motif in the promoter of PtoWOX11/12 a and activated its expression.Transgenic poplar overexpressing PtoWOX11/12 a showed increased root system and biomass,and enhanced tolerance to salt and drought stresses.The main resluts are as follow:1.PtoWOX11/12 a expression was induced by salt and drought stresses.Real-time quantitative reverse transcription PCR(qRT-PCR)showed that gene expression level was significantly increased when exposed to salt or drought stress conditions.GUS staining result was consistented with qRT-PCR that ProPto WOX11/12a::GUS transgenic poplars leaves and roots appeared dark blue under salt and drought stresses.2.PtoWOX11/12 a protein was identified function as a transcriptional activator with transcriptional activation activity at C-terminus 129-255 aa using yeast two-hybrid system.3.An AP2 protein(PtoAP2)identified by yeast one hybrid system was specifically bound to DRE motif in the promoter of PtoWOX11/12 a and activated its expression.Meanwhile,Arabidopsis protoplast transient transformation was consistent with yeast one hybrid result.qRT-PCR analysis releaved that the expression of PtoAP2 and PtoWOX11/12 a genes share similar expression patterns under salt and drought stresses.4.Overexpression or suppression expression of PtoWOX11/12 a has effects on plant growth and development.The leaves width of overexpression PtoWOX11/12 a poplars was sharply larger than 84 K control.The leaves length and width of suppression expression PtoWOX11/12 a transgenic poplars were less than control,and the leaf margin had serrate incision.The height and diameter of overexpression and suppression expression PtoWOX11/12 a transgenic poplars were significantly less than 84 K control poplars.Anatomy analysis indicated that the xylem,the cambium cell layer number of overexpression PtoWOX11/12 a transgenic poplars was thinner than control and the xylem of the suppression expression of PtoWOX11/12 a was also thinner than control,but its wider than overexpression PtoWOX11/12 a transgenic poplars.qRT-PCR analysis showed that PtoWOX11/12 a was strongly induced and expressed in the stem during the process of adventitious roots development.The expression levels of auxin synthesis genes,YUCCA1 and YUCCA8,were increased at the third day.Furthermore,their expression levels were increased in overexpressed PtoWOX11/12 a transgenic poplars while decreased in suppressed PtoWOX11/12 a transgenic poplars.These results indicated that the PtoWOX11/12a-regulated grow and development of poplars is related with auxin synthesis genes.5.Transgenic poplar overexpressing PtoWOX11/12 a showed increased root system and biomass,and enhanced tolerance to salt and drought stresses,whereas PtoWOX11/12 a suppression expression transgenic poplars are senstitive to salt and drought.After salt and drought stresses,the “84K + OE” grafted poplars could grow well,but the “OE + 84K” grafted poplars suffered from stress.Thus,grafting experiment proved that stress tolerance was mainly owed to underground roots rather than aerial parts.Under stress conditions,the SOD,POD enzyme activities,proline,solube protein and total chlorophyll contents,and net photosynthetic rates were all higher than control.However,the electric conductivity,H2O2,O2·-and MDA contents in overexpressing transgenic poplar were lower than 84 K control.