The Function Mechanism Characterization Of GbWRKY1 In Cotton Phosphate Homeostasis And Multiple Resistance

During the adaptation to complex environmental conditions,plants have evolved sophisticated signaling networks to regulate multiple stresses and development.WRKY transcription factors are one of plant specific transcription factor families which play important roles in plant development and stress responses.GbWRKY1 was identified as a defense responsive gene from cotton after Verticillium dahliae inoculation in our early study.The previous results showed that overexpression of GbWRKY1 in Arabidopsis positively regulates phosphate deficiency response.Further functional study in cotton demonstrated that GbWRKY1 can bind the promoter of JAZ1 and activate its expression,which leads to the coordination regulation between plant defense response and development.Here,based on previous research results,we characterize the function of GbWRKY1 in cotton phosphate deficiency response and explore the effect of phosphate deficiency on cotton disease resistance and the potential mechanism.In addition,we also characterize the regulation role of GbWRKY1 in salt and drought stress responses.The main findings of this study are as follows: 1)The effect of GbWRKY1 in regulating cotton phosphate deficiency response is not obviousWe performed phosphate deficiency treatments of wild type and GbWRKY1 transgenic cotton lines using pot and hydroponic methods.However,no regular variation tendency was found between wild type and GbWRKY1 transgenic cotton lines with regard to Pi contents,fresh weight,plant height and hormone levels.This may be due to the intrinsic high tolerance of cotton to phosphate deficiency condition.2)Phosphate deficiency can activate JA signaling and phenylpropanoid metabolic pathway and significantly increase cotton resistance to Verticillium dahliaeWe characterized the disease resistance to Verticillium dahliae of cotton grown under normal and phosphate deficiency treatment for three weeks.The results indicated that phosphate deficiency treatment greatly enhances cotton resistance to Verticillium dahliae V991.The results of disease index statistic,relative fungi biomass detection and pathogen recovery experiments were all consistent with the phenotype.Moreover,JA and JA-Ile content in cotton leaves are significantly increased and JA related gene expression was activated under phosphate deficiency condition.Transcriptomic analysis using leaf sample of cotton plants grown under control and low phosphate conditions for three weeks has identified about 2700 differentially expressed genes.KEGG pathway enrichment analysis of upregulated genes under LP condition revealed that the important branch of phenylpropanoid metabolic pathwayflavonoids biosynthesis is significantly activated after LP treatment.Further global analysis of DEGs involved in phenylpropanoid metabolic pathway suggested that three key enzymes(PAL,C4 H and 4CL),located in the main upstream position,are all significantly upregulated under LP condition,and most DEGs involved in lignin biosynthesis also upregulate under LP condition.To further confirm the metabolic changes caused by LP treatment,metabolomic analysis was further conducted.Among the 240 compounds that can be exactly characterized,we identify 69 differentially accumulated metabolites(DAMs)which significantly increased or decreased under LP condition.Further compound classification suggests that 28% of 69 DAMs are flavonoids and all of them display increased contents under LP condition.KEGG enrichment analysis of these 69 metabolites also shows that flavonoids biosynthesis is significantly enriched.These data demonstrated that phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating flavonoid and lignin biosynthesis.3)Overexpression of GbWRKY1 impairs salt and drought tolerance through suppressing ABA signaling pathwayThe expression of GbWRKY1 is significantly induced by Na Cl,PEG and ABA treatments.Sensitivity test of seed germination in Arabidopsis indicates that GbWRKY1 overexpression Arabidopsis are more sensitive to salt and osmotic stress.Stress treatments of adult plants suggest that GbWRKY1 overexpression Arabidopsis are less tolerant to salt and drought.The GbWRKY1 overexpression cotton plants also display reduced tolerance to drought.Expression analysis indicates that the induced level of two ABA responsive genes-RAB18 and RD29 A is significantly reduced in GbWRKY1 overexpression lines under Na Cl,mannitol and ABA treatment.In addition,ABA treatment assays in Arabidopsis reveal that GbWRKY1 overexpression lines are significantly insensitive to ABA inhibition on seed germination and root elongation.Knockout of JAZ1 in AOV9 can restore the ABA insensitive phenotype,and overexpression of JAZ1 also displays similar ABA insensitive phenotype.Further yeast two hybrid assay indicates that JAZ1 interacts with ABI1.The data suggest that GbWRKY1 negatively regulates ABA signaling through JAZ1 and ABI1 to affect salt and drought tolerance.