Function Study Of The WD40-repeat Protein TAWD In Arabidopsis Thermotolerance

In previous study,the tawd(Thermo-tolerance Associated WD-40-repeat protein)mutant with increased luminescence compared to wild type was isolated through a mutant screening approach for altered expression of the luciferase reporter gene driven by the promoter of heat-inducible gene AtHSP18.2.The tawd mutant showed elevated luciferase activity in response to heat stress and was more sensitive to high temperature than the wild type.TAWD was identified by map-based cloning.This gene encodes a member of WD40 repeat protein(WDR)named TAWD in our work and expressing ubiquitously in Arabidopsis.WDRs act as a scaffold for biological molecules'interactions and are found in multitude of eukaryotic organisms.In Arabidopsis,members of WD40 proteins were reported to be involved in plant development processes and are recognized as key regulators of plant-specific events in development and signaling pathway during stress signaling.In this study,we found that the basal thermotolerance and acquired thermotolerance were reduced in tawd mutant plants than in wild type.Subcellular localization showed that TAWD is mainly localized in the nucleus and the localization is not affected upon heat stress.Under normal conditions,although the expression level of TAWD is low?and the expression is suppressed by high temperatures.However,the TAWD protein level under heat stress was higher than under the controls with longer duration as detected by quantitative Western Blot.Transcript levels of most heat stress responsive(HSR)genes including heat shock transcription factors(HSFs)and heat shock proteins(HSPs)were substantially reduced in tawd plants than in wild type under heat stress.ChIP(Chromatain Immunoprecipitation)assays detected the TAWD binding to the promoter cis-element region of HSP89.1.Together,these results indicate that TAWD is a key positive regulator of HSR gene expression and thermotolerance in plants.Furthermore,RIP(RNA Immunoprecipitation)detected the TAWD binding to mRNAs of HSP18.2 and HSP101,correlated with the reduced expression of HSPl8.2 and HSP101 in tawd than in wild type plants under heat stress.The intron retention was significantly enhanced among most of detected HSFs genes,such as HSFA7a,HSFA7b and HSFBI in tawd mutant plants under heat stress.Therefore,it was speculated that TAWD is involved in the regulations of mRNA stability and splicing.Finally,through IP-MS analysis,we identified several potential interacted proteins interacting with TAWD,including HSP70-1 and HSP90.2.As the physical interaction between AtHSP70-1 and AtBAG4 was reported previously.We subsequently performed Bimolecular Luciferase complementation(BiLC)and Co-IP(Coimmunoprecipitation)assays using tobacco leaves as materials.However,no interaction between TAWD and HSP70-1,BAG4 or HSP90.2 was detected by BiLC or CoIP.Furthermore,we constructed a series of combination of different mutants for phenotype analysis based on luminescence intensity and found that hsp70-1/tawd?bag4/tawd and hsp70-1/bag4/tawd plants displayed incresed LUC activity under heat stress,similar to the response of awd plants under heat stress.Interstingly,the tawd/hsp70-1 and tawd/bag4 plants displayed increased survival rates than tawd mutant plants,while the triple mutant hsp70/bag4/tawd showed increased thermotolerance compared with wild type plants under extreme temperature fluctuation.In brief,TAWD and HSP70-1 and BAG4 are together involved in plant thermotolerance regulations.In conclusion,our findings revealed that TAWD,as a heat-induced protein and heat stress responsive signaling molecules,is essential for the regulations of HSR genes and thermotolerance in plants.Firstly,TAWD regulates the transcription of HSR genes:TAWD binds to HSP89.1 cis-promoter element and modulates its expression.Secondly,TAWD might be involved in alternative splicing and modulating mRNA stability of heat inducible genes:the increased bindings between TAWD and mRNAs of HSPl8.2 or HSP101 was consistent with the reduced transcript levels of both genes in tawd under heat stress.Finally,TAWD.and HSP70-1 and BAG4 are all required for the plant's thermotolerance to cope with high temperature environments.Shortly,TAWD is involved in the regulations of plant thermotolerance through modulating the expression of HSR genes,and possibly cooperates with HSP70-1 and BAG4 to promote the plants adaptability under heat stress environments.