Functional Analysis Of Wheat U-box Gene TaPUB26 In Brachypodium Distachyon Salt Tolerance

High salinity seriously affects the growth and development of plants,which leads to crop loss.It had great significance to strengthen the research on the mechanism of plant salt tolerance to improve the plant salt tolerance and yield of crops.U-box E3 ubiquitin ligase is a crucial enzyme in UPS and involved in many biological processes.Several U-box proteins have been reported to play important roles in the adaptation of plants to abiotic stress.We cloned a gene,TaPUB26,from wheat that encoded U-box protein.Sequence alignment and phylogenetic analysis of this gene were analyzed,and the expression patterns of this gene was detected.To understand the function of this gene in plant salt tolerance,transgenic Brachypodium distachyon plants with TaPUB26 overexpression were obtained.This study analyzed the functional mechanism of TaPUB26 in plant salt tolerance by transgenic Brachypodium distachyon plants.The main results are as follows:(1)We identified a highly up-regulated U-box gene TaPUB26(AK334574.1)by analysing the RNA-seq data.The full-length cDNA of this gene was 1703 bp,and it contained1338 bp that encoded 446 amino acids.Moreover,it contained a U-box domain at the N-terminus and ARM repeated sequences at the C-terminus.The TaPUB26 protein was found to localize in the nucleus.And the data of an in vitro ubiquitination assay demonstrated that TaPUB26 has E3 ubiquitin ligase activity,but its activity is dependent on E1 and E2.(2)We examined the expression patterns of TaPUB26 in different organs of wheat and in different abiotic stresses.The data showed that TaPUB26 mRNA was expressed in the leaves,stems and roots,and the transcript level in the leaves was highest.The transcript levels of this gene was affected to different degrees by NaCl,PEG,heat and multiple hormones.(3)We obtained transgenic Brachypodium distachyon plants and selected three TaPUB26-overexpressing lines(OE2,OE5 and OE8)for further study.We examined the salt tolerance of transgenic plants in seedling and mature stage.The results showed that the shoots and roots growth of transgenic plants were inhibited more severe compared with wide type(WT)plants in seedling stage.Moreover,the Chl content and photosynthetic capacity of transgenic plants was lower than that of the WT plants when exposed salt stress.These results suggested that overexpressing TaPUB26 reduced salt tolerance of transgenic Brachypodium distachyon plants.(4)We analyzed the accumulation of Na~+and K~+and ion flux in roots and leaves cell.The results suggested that transgenic lines had higher Na~+content but lower K~+content than WT under salt stress.As a result,the Na~+/K~+ratios were higher in transgenic plants when compared to WT plants.Then,the fluxes of Na~+,K~+and H~+were detected inside and outside of the cell membrane of root epidermis.According to the results of ion accumulation and flux,we speculated that TaPUB26 could decrease Na~+effluxes but increase K~+effluxes under salt stress.Therefore,the ion balance in the cell was disturbed and the tolerance to ion stress was decreased of transgenic lines.(5)The ROS accumulation and antioxidant capacity of transgenic lines was analyzed.The data showed that ROS content of transgenic plants was significantly higher than WT plants under salt stress.Furthermore,the MDA content and electrolyte leakage in transgenic plants were higher than in WT.But the transgenic plants had lower activities of various antioxidant enzymes compared with WT under salt stress.These results indicated that TaPUB26 reduced salt tolerance of transgenic plants may by decreased intracellular antioxidant enzymes activites and increased ROS accumulation resulting in enhancing oxidative stress.(6)We detected the genes expression level of transgenic plants under salt stress,including some genes encoding antioxidant enzymes,ion transporter-related genes and some salt-related genes.It was found that the expression level of most of the genes were lower in transgenic lines than in WT under salt stress.This result suggested that E3 ubiquitin ligase encoded by TaPUB26 may regulated the expression of stress-related genes in some ways.(7)We obtained an interaction protein TaRPT2a by Y2H and BiFC assay.We found that TaRPT2a may encode an ATPase subunit of the 26S proteasome complex.And we have found that overexpression of TaRPT2a increased the salt tolerance of transgenic Arabidopsis plants.In conclusion,TaPUB26 showed negative effects on salt tolerance of transgenic Brachypodium distachyon.In terms of physiologicalmechanism,TaPUB26 breaked ion homeostasis by regulated intracellular Na~+,K~+and H~+fluxes under salt stress.Meanwhile,TaPUB26 enhanced oxidative stress by reduced activities of antioxidant enzymes and increased ROS accumulation under salt stress.In terms of molecular mechanisms,TaPUB26may interacted with other proteins to regulate the expression of genes related to salt tolerance,such as TaRPT2a,resulting in affecting plant salt tolerance.