Study On The Molecular Regulatory Mechanism Of Two Rtwrkys In Response To Salt Stress And Reproductive Development

WRKY transcription factor superfamily is one of the largest transcription factor families in plants.Numerous studies explored the role of WRKY proteins in plant growth and development processes,however,the studies about WRKY1 and WRKY23 protein remain few,and their regulatory mechanism in plant response to salt stress and reproductive development were unclear.Reaumuria trigyna?Reaumuria Linn genus,family Tamaricaceae?,an endangered dicotyledonous shrub with the features of a recretohalophyte,is endemic to the Eastern Alxa–Western Ordos area of China.R.trigyna has a strong tolerance to salinity desret and is an important candidate for sand fixation and soil improvement.Recently,the studies about R.trigyna mainly focused on the morphological and physiological characteristics of R.trigyna,however,the studies on the molecular mechanism of R.trigyna adapt to salinity desret were few.In present study,the expression patterns of two R.trigyna WRKY transcription factor genes RtWRKY1 and RtWRKY23 in response to diverse abiotic stresses and in different tissues were explored.RtWRKY1 and RtWRKY23 were transformed into Arabidopsis,and their biological functions and regulatory mechanisms in response to salt stress and reproductive development in plant were further studied.The data suggested that RtWRKY1 and RtWRKY23 all could improve the salt stress tolerance in plant via regulating ROS balance and osimotic balance.Additionally,RtWRKY23 also could regulate the reproductive development in plant.The results of this study as follow:1.RtWRKY1 and RtWRKY23 were mainly expressed in the stem,with lower expression levels in the leaf and almost undetectable expression in the root.RtWRKY1 was induced by salt,ABA,and cold treatments,and suppressed by heat treatment.RtWRKY23 was induced by salt,drought,cold,ultraviolet radiation and ABA treatments,and suppressed by heat treatment.2.Overexpression of RtWRKY1 and RtWRKY23 in Arabidopsis all improved the the tolerance of transgenic Arabidopsis to salt stress.Compared with wild type Arabidopsis,RtWRKY1 and RtWRKY23transgenic Arabidopsis had better root growth,fresh weight and chlorophyll content under salt stress.In addition,RtWRKY1 transgenic Arabidopsis had a better growth and fresh weight than wild type Arabidopsis under normal condition.3.RtWRKY1 transgenic Arabidopsis exhibited higher proline content,GSH-PX,POD,SOD,and CAT activities,and lower MDA content,Na⁺content,and Na⁺/K⁺ratio than wild type Arabidopsis under salt stress conditions.Salt stress affected the expression of ion transport,proline biosynthesis,and antioxidase related genes,including AtAPX1,AtCAT1,AtSOD1,AtP5CS1,AtP5CS2,AtPRODH1,AtPRODH2,and AtSOS1 in transgenic lines.4.The Microarray analysis of RtWRKY23 transgenic Arabidopsis shown that there were totally 449 differentially expressed genes between RtWRKY23 transgenic and wild type plants under 150 mM NaCl treatment.102 DEGs were up-regulated and others 347 DEGs were down-regulated.GO and KEGG pathway enrichment shown that the DEGs enriched in growth,development,plant hormone and environmental stress response related GO terms or KEGG pathway,including“Plant hormone signal transduction”,“Peroxisome”,and“Starch and sucrose metabolism”KEGG pathways and“Response to stress”,“Response to hormone”,and“Response to abiotic stimulus”GO terms.5.In the Microarray analysis,we found that the expression levels of three peroxidase?POD?genes and one proline degradation gene changed significantly under salt stress,including POD,POD22,POD23 and PRODH2.The qPCR analysis and yeast one-hybrid assay demonstrated that RtWRKY23 protein could directly regulate the expression levels of POD22,POD23 and PRODH2 by binding to the promoter region of POD22,POD23 and PRODH2 under 150 mM NaCl.Under salt stress,the O₂^-clearance rate,proline content and POD activity of RtWRKY23transgenic plants was significantly higher than that of wild type plants,and the H₂O₂ content of RtWRKY23 transgenic plants was significantly lower than that of wild type plants.6.In the Microarray analysis,three down-regulated DEGs respectively annotated as MAF5,HAT1 and ANT,which were reported as a regulator involving in plant reproductive development.The qPCR analysis and yeast one-hybrid assay indicated that RtWRKY23 could directly decrease the expression level of MAF5,HAT1 and ANT under normal condition.RtWRKY23 delayed the flowering in transgenic Arabidopsis.Meanwhile,the length and number of pod of RtWRKY23transgenic plants were significantly lower than that of WT plants,leading to a lesser seed production.