Effect Of Wood Vinegar On Wheat Seedling Growth And Analysis Of TaNAC4D Gene Cloning And Function

Wheat(Triticum aestivum L.)is one of the most important food crops in the world.As the world population continues to increase,the demand for wheat production is increasing.However,wheat growth and yield is seriously influenced by drought stress,most notably at the seedling,stem elongation,booting,anthesis,and grain formation stages.Wheat seedlings are more susceptible to drought stress because of their low biomass and incomplete protective structure.Roots are the initial receptors that signal a water deficit,followed by a series of responses at the morphological,physiological,and cellular levels.A well-developed root system is an important guarantee to improve the drought resistance of wheat.Many researches from a physiological standpoint indicated WV can promote the growth of plant.However,whether the WV can enhance the drought resistance ability of wheat and the molecular mechanism are still unknown.In this study,we analyzed systematically the effects of WV on the growth of wheat by the seed soaking and root irrigation treatments.Moreover,we investigate the proteome pattern of wheat roots following a WV seed presoaking treatment under drought stress to explore further the molecular mechanisms underlying WV induced drought tolerance.A new NAC transcription factor(TF)was cloned and analyzed the function during response to drought stress of wheat in this paper.The main research contents and results are as follows:(1)Different concentrations of wood vinegar were used for seed soaking treatment.The results indicated that the seed soaking treatment with 1:900 WV could significantly improve the germination potential of wheat seeds,increase the fresh and dry weight of shoots and roots,and promote the growth of wheat roots,including the root length,surface area,volume,and average diameter of roots.(2)Irrigation with different concentration of WV could promote the development of root hair,and 1:900 WV irrigation gave the most obvious promotion of root hairs.Root irrigation with 1:300 WV increased the accumulation of ROS in root;meanwhile,other concentration of WV(1:600,1:900,1:1200 and 1: 1500)can reduced the accumulation of ROS and 1:900 WV was most effectively to eliminate ROS.Moreover,root irrigation with1:900 WV can significatntly promote the photosynthetic rate and increase the content of chlorophyll,and to a certain extent,promote the photosynethesis of leaves.(3)Exogenous low concentrations of WV can cause slight oxidative stress in wheat roots but not oxidative damage.Results showed that O2– and H2O2 contents were higher in the WV treated group before drought stress than in the control group,whereas the content of MDA showed no significant difference between the two groups before drought stress.Moreover,the contents of O2–,H2O2,and MDA were significantly lower in the WV treated group than the control group after drought stress.Meanwhile,the activities of antioxidant enzymes were higher and related antioxidant genes were upregulated in the WV treated group under both drought and non-drought stress,which enabled them to cope better with continuous ROS production.(4)Results of LC-MS and Real-time PCR of Ta NECD indicated ABA content of shoots and roots were increased significantly in the WV treated group over those of the control group under both drought or non-drought stress.On the one hand,accumulation of ABA can regulate the stomatal apertures of leaves to prevent water loss;on the other hand,it can activate downstream antioxidative gene expression of shoots and roots to better resist subsequent drought stress.(5)Comparative proteomics profiles identified 138 differentially accumulated protein(DAP)spots representing 103 unique protein species responding to drought stress in wheat roots of the control and WV-treated groups.These DAPs are mostly involved in the stress response,carbohydrate metabolism,protein metabolism,and secondary metabolism.Proteome profiles showed the DAPs involved in carbohydrate metabolism,stress response,and secondary metabolism.Proteins interaction and biological processes network indicated WV soaking treatment promote the carbohydrate metabolism and energy metabolism,increase the abundance of anti-stress proteins,maintain the stability of the protein function and activate the secondary metabolites(JA and SA)mediated stress signal transduction pathway under drought stress.(6)A new NAC TF of wheat(Ta NAC4D)was mined and cloned from wheat.Bioinformatics analysis showed Ta NAC4 D contains a NAM conserved domain at N-terminal,with a large variation at the C-terminal by bioinformatics.Further analysis revealed that Ta NAC4 D belongs to the e subfamily of the NAC superfamily of wheat.Subcellular localization showed that Ta NAC4 D proteins was localized in the nucleus.The yeast self-activation experiment showed that Ta NAC4 D protein had transcriptional activation activity,and site of transcriptional activation was located at the C-terminal.Moreover,through the analysis of promoter of Ta NAC4 D gene,it was found that the promoter contained a large number of cis-acting elements of ABA response,Me JA response,light response,anaerobic response,low temperature response,biological and abiotic stress response.(7)Through the analysis of the expression pattern of Ta NAC4 D under different treatments,showed that Ta NAC4 D was up-regulated under ABA,PEG 6000,H2O2,Me JA and wood vinegar treatment.And Ta NAC4 D had a fastest response time to wood vinegar.This indicated that wood vinegar treatment could rapidly activate the expression of Ta NAC4 D.(8)Yeast experiments showed that overexpression of Ta NAC4 D had no toxic effect on yeast.Further,the yeast stress experiments(mannitol and H2O2 stress)showed that after overexpression of Ta NAC4 D,the growth rate of yeast was significantly faster than that of the positive control,indicating that the overexpression of Ta NAC4 D gene could significantly alleviate the inhibition of mannitol and H2O2 on the growth of yeast.(9)BMSV mediated gene silencing(VIGS)experiments show that,wheat plants were more sensitive to drought and presented more obvious stressed phenotype under drought stress after Ta NAC4 D gene silence.Further ananysis indicated ROS and MDA content in wheat plants of Ta NAC4 D konck-down were significantly higher than that of control and wheat seedlings suffered severe peroxidation and membrane lipid peroxidation damage injury.These results indicated Ta NAC4 D genes plays a positive regulatory role in drought stress in wheat.