Functional Study Of Receptor-Like Kinase RLKx And Transcription Factor WRKY47 In Al Resistance In Arabidopsis

Aluminum(Al)toxicity is one of the major factors that inhibit plant growth and development,leading to crop yield reduction in the acid soils.The physiological strategies of plants in response to Al toxicity include Al exclusion and internal tolerance mechanisms.Al-induced/activated organic acid secretion has been proposed as a major mechanism for Al exclusion,and the modification of cell wall properties is considered to be one of the determinants in Al tolerance of plants.Whereas,how these processes are regulated remains elusive.Here we focused on a kind of receptor-like kinases(RLKs)and transcription factors(TFs)that usually act upstream and downstream in the signaling pathways,respectively,aiming to understand how plants respond to Al and in turn initiate Al resistance strategies.For one thing,we performed mutant screen and got an Al hypersensitive mutant named rlkx,from more than 300 RLK T-DNA insertion mutant lines;For another,a previous work in our lab had identified a transcription factor mutant wrky47 that was sensitive to Al stress,which probably was resulted from altered cell wall modification as well as Al subcellular distribution.This thesis thus focused on functional study of RLKx and WRKY47 in Al resistance/tolerance by using genetical,physiological,biochemical and molecular strategies,thereby to illuminate Al resistance signaling pathway.The following are the main research results of this paper:1.RLKx is involved in Al resistancePlate phenotypic experiments showed that the Arabidopsis thaliana mutant rlkx exhibited an Al hypersensitive phenotype versus WT,while its overexpressing line 35Spro: RLKx(RLKxOE)was significantly more resistant to Al.Next,we examined the total root Al content and Al subcellular distribution in each genotype,and found that Al content in whole root,root protoplast and apoplast was all significantly increased in rlkx versus WT,while reduced in RLKxOE line,indicating that RLKx may be related to Al exclusion.The expression levels of gene ALMT1 and MATE and their respectively regulated organic acid secretion in the mutant rlkx were significantly down-regulated compared to WT,while the overexpressing lines were up-regulated.These data indicate that RLKx is involved in Al exclusion by regulating organic acid secretion.Next,by qPCR and GUS staining,we showed that RLKx could not respond to Al at transcriptional level.Nevertheless,the expression of P1,which encodes a peptide,a ligand of RLKx,was induced by low pH and Al treatment.Exogenous application of a synthetic P1 peptide could promote Al resistance in WT but not in rlkx mutant,implying that P1 might be involved in Al resistance.Meanwhile,we constructed a 35Spro:P1(P1-OE)overexpressing line and found that the P1-OE was more resistant to Al than WT,but such Al resistance was completely abolished under the genetic background of rlkx.Consistent with this,P1 overexpression reduced Al content in WT but not in rlkx mutant.In addition,P1 induces expression of ALMT1 and MATE as well as organic acid secretion in WT but not in rlkx mutant background,indicating that P1 is involved in Al exclusion through RLKx.Because Al-induced organic acid secretion is regulated by transcription factor STOP1,we then investigated the relationship between P1-RLKx module and STOP1.Based on genetic analysis,we showed that RLKxOE/stop1 line exhibited a similar Al sensitive phenotype to stop1,suggesting that STOP1 may be epistatic to RLKx.Moreover,the Al sensitive phenotype of the rlkx and stop1 double mutants was similar to that of the single mutant,which means that RLKx and STOP1 are located in one signaling pathway.Similarly,P1-OE/stop1 phenocopied stop1,suggesting that STOP1 acts downstream of P1.RT-qPCR and organic acid secretion measurement further showed that RLKx and P1 promoting the expression of ALMT1 and MATE and organic acid secretion was STOP1-dependent.Because STOP1 was not responsive to Al at transcriptional level,while its protein accumulation was obviously induced by Al,we then detected if P1-RLKx affects STOP1 protein level.By using a 35Spro:STOP1-GFP overexpressing line,we found that STOP1-GFP accumulation was reduced in rlkx versus WT upon Al treatment,while increased by P1 and RLKx overexpression.The yeast two-hybrid experiment further showed that RLKx could not interact with STOP1,implying that P1-RLKx module indirectly regulates STOP1 protein accumulation.Taken together,these results indicate that P1-RLKx is involved in Al resistance via regulation of STOP1 protein accumulation.2.WRKY47 confers Al tolerance by regulating cell wall modificationIn the previous study,we found that the transcription factor WRKY47 is involved in root growth and Al tolerance.The wrky47 mutant was very sensitive to Al stress,while the over-expressed line had significantly enhanced Al tolerance.We confirmed that the loss-of-function of WRKY47 significantly affected the subcellular distribution of Al in roots under Al stress,which was manifested by an increase in symplastic Al content and a decrease in apoplastic Al content.This was because the content of hemicellulose I in the mutant cell wall was reduced in the wrky47 mutant,resulting in a decrease of cell wall in Al binding capacity.Through gene chip technology,RT-qPCR and ChIP assays,we further demonstrated that WRKY47 directly regulated the expression of the genes responsible for cell wall modification,such as EXTENSIN-LIKE PROTEIN(ELP)and XYLOGLUCAN ENDOTRANSGLUCOSYLASE-HYDROLASES17(XTH17).Moreover,the increased expression of ELP and XTH17 partially restored wrky47 mutant root elongation defect and Al sensitive phenotypes.In summary,our results demonstrated that under both normal and Al stress conditions,WRKY47 regulated root growth by cell wall genes.In addition,the balance of Al content between root symplast and apoplast maintained by WRKY47 is important for plants to obtain Al tolerance.