Study On The Function Of BdWAR1 Between Auxin And Aba Signaling Pathway In Brachypodium Distachyon

Auxin and abscisic acid are two very important plant hormones,they can work independentiy or coordinately to regulate the seed germination,vegetative growth,seed maturity and dormancy and various processes of plant growth,development and environmental adaptation.When auxin is increased inside the plants,AUX/IAAs are ubiquitinated by the auxin receptor SCF^TIR1/AFBs,and then degraded by the 26S proteasome,thereby ARFs released and further regulated the expression of downstream auxin-related genes.ABA is a small molecule that function to inhibit plant growth and to regulate stress responses.When the ABA level is increased,the phosphatase activity of PP2Cs is reduced,and the activity of SnRK2s phosphokinase is increased,thereby activating the expression of downstream ABA related transcription forctor such as ABI3,ABI5.Recently,the crosstalk between auxin and ABA signaling pathways has become hot for research in developmental biology.However,in the process of ABA and auxin signal transduction,whether there is an interaction between the pathways especially after the signals being accepted by the receptor and before the activation of downstream transcription factors,there is little research so far.The purpose of this study was to examine new ways of interaction in grasses to futher explore the complexity of ABA and auxin coordination in regulating plant development.We obtained a mutant of Brachypodium distachyon which is weakly sensitive to auxin through the screening its mutant library.The results from research on this mutation are as follows:?1?Through the screening of B.distachyon T-DNA insertion mutant library,we obtained a mutant with weak response to auxin,and named it bdwar1?Brachypodium distachyon weak auxin response 1?.We confirmed that the T-DNA insertion was located at 262bp upstream of ATG of Bradi4g39520 and caused the down regulation of gene expression for 4 times.?2?Due to the BdWAR1 mutation,the phenotypic defects of the plant are short meristem,dense root hair at the root tip,short plant,narrow and short seed.At the same time,we constructing pBdWAR1:BdWAR1 connected to the vector of pPZP211,then transformed the mutant plants.We found that the transformed plants can complementarily rescue the phenotype seed size in mutant.In addition,the Ubi::BdWAR1-GFP transformed wild-type plants had enhanced phenotypes on WT,which proves the mutant phenotype is indeed caused by the down-regulation of BdWAR1 gene expression.?3?qRT-PCR indicated that the expression level of wild-type BdWAR1 gene changed after hormone and stress treatments,which proved that BdWAR1 responded to both hormone and stress.?4?Sequence analysis reveals obvious nuclear localization signals in the gene.So we constructed 35S::BdWAR1-GFP and 35S::BdWAR1??NLS?-GFP without the nuclear localization signal which were transiently transformed into tobacco.The results indicated that full-length BdWAR1 is expressed only in the nucleus,while without the nuclear localization signal,it is not only expressed in the nucleus,but also in the cell cytoplasm.?5?Through tissue sampling in the time course for expression study,we found that the expression level of BdWAR1 was higher in tissues with high cell division activity like the roots and leaves of seedlings,young stem and spikes.?6?In Arabidopsis lines over-expressing BdWAR1,germination and greening experiments showed that BdWAR1 has a positive regulatory effect on the ABA response in Arabidopsis.?7?The leaf senescence experiments in Brachypodium mutants and over-expressing plants,as well as drought stress and germination experiments,showed that BdWAR1 has a negative regulatory effect on ABA.?8?Both yeast two-hybrid and luciferase experiments showed that BdWAR1 interacted with BdTPL and BdABI3.Our preliminary research,shows that BdWAR1,as a new transcription factor,responds to plant hormones and stress signals by interacting with BdTPL and BdABI3,thereby inhibits ABA signal transduction and promotes auxin signal transduction,hence plays a role in the synergistic regulation of ABA and auxin on plant growth and development.