Functional Analysis Of Transcription Factor WRKY29 In Plant Drought Resistance

Drought is one of the major abiotic stresses affecting world agricultural production.After a long period of adaptation,plants survived in adversity for a long time have evolved a very unique mechanism of stress resistance,and contain rich genetic resources for stress resistance.Previously,many studies have been conducted on the Zygophyllum xanthoxylum in the northwest desert area,mainly focusing on physiological and related functional genes,such as ion transport.But there are few studies on its transcription regulation.By analyzing the transcriptome data of Z.xanthoxylum,it was found that the expression level of CL9880.Contig2_All was significantly up-regulated under salt and osmotic treatment,indicating that the gene may be involved in the process of Z.xanthoxylum in response to salt and osmotic stress.We cloned this gene,and it has the highest homology with Arabidopsis AtWRKY29.Thus,it is named ZxWRKY29.Previously research on AtWRKY29 mainly focuses on biotic stress,and the research related to drought stress has not been publicly reported.Therefore,we further studied the function and mechanism of WRKY29 in response to drought stress and obtained following main results:1.Expression pattern showed that the expression level of ZxWRKY29 in Z.xanthoxylum roots and leaves was significantly up-regulated under-0.5 MPa osmotic treatment and 50 mM NaCl treatment for 1 h and 6 h,consistent with the expression profile data.Further,ZxWRKY29 overexpression transgenic Arabidopsis was subjected to natural drought and 150 mM NaCl treatment.The results showed that ZxWRKY29 overexpression enhanced the salt and drought resistance of Arabidopsis.2.AtWRKY29 was significantly induced by osmotic and salt stresses.To further study its function,we ordered two wrky29 T-DNA insertion mutant lines,wrky29-1and wrky29-2,and obtained homozygous mutant lines via genotyping.Meanwhile,a number of AtWRKY29 overexpression lines were generated.Line OE5 with the middle expression level of AtWRKY29 and line OE7 with the higher expression level were selected and used as materials for subsequent experiments.3.Col-0,wrky29-1,wrky29-2,OE5,and OE7 were used as experimental materials.Phenotypic observation of 4-week-old seedlings showed that wrky29-1 and wrky29-2 with light green leaves were significantly larger than Col-0,while two overexpression lines OE5 and OE7 with dark green leaves were significantly smaller than Col-0.3-week-old seedling were performed for natural drought treatment.After14 d without watering,the wilting degree of the two loss of function mutants was greater than that of Col-0,while the overexpressing lines showed no obvious wilting.The results of analysis of chlorophyll a,chlorophyll b,net photosynthetic rate,water use efficiency,RWC,survival rate and dry/fresh weight of shoot indicated that dry and fresh weight of shoot was consistent with the phenotype of each line.However,there were no significant differences among these physiology indexes of each plant growth under normal condition.Compared with these of seedlings grown in normal condition,the corresponding indexes of wrky29-1 and wrky29-2 mutants under drought treatment decreased significantly,and the decline was much greater than that of Col-0,while the decline of OE5 and OE7 plants was significantly lower than that of Col-0.In addition,after drought treatment,the relative water content and survival rate of the two mutant lines were significantly lower than that of the wild type.Together,the results demostrated that AtWRKY29 plays a positive role in response to drought stress of Arabidopsis.4.A pairwise comparison analysis of transcriptome data of Col-0,wrky29 and OE5 showed that there are 1399 differentially expressed genes in the wrky29/Col-0group,including 1330 down-regulated genes and 69 up-regulated genes.There are449 differentially expressed genes in the group of OE5/Col-0,of which,379 genes are down-regulated and 70 genes are up-regulated.In OE5/wrky29 group,there are 496differentially expressed genes,including 51 down-regulated genes and 445up-regulated genes.GO clustering analysis result indicated that the differential expression genes are mainly enriched in cellular processes,metabolic pathways,response to stimuli,signal transduction,catalytic activity,etc.KEGG Pathway significant enrichment analysis showed that the differential expression genes caused by the AtWRKY29 gene deletion and overexpression mainly concentrated in the metabolic pathway,secondary metabolite metabolic pathway and flavonoid synthesis pathway.The analysis results of physiological indexes such as flavonoids content,leaves H₂O₂,DAB staining,active oxygen production rate,photosynthesis parameters,and relative plasma membrane permeability showed that AtWRKY29 can significantly increase the synthesis and accumulation of flavonoids,thereby slowing down the accumulation of active oxygen in plant cells,to prevent chlorophyll and other oxidative damage and protect the integrity of the cell membrane system,to help plants resist drought stress.This study evaluated the function of transcription factor WRKY29 in response to drought stress of plants.It also provided a theoretical basis for genetic engineering breeding of droght tolerant grass.