Molecular Mechanisms Of E3 Ubiquitin Ligases XBAT31/35 Regulating Thermomorphogenesis In Arabidopsis

Global warming and extreme high temperature have a great impact on plant growth and development,morphology and structure as well as yield and quality.Sessile plants evolve sophisticated regulatory mechanisms dealing with mild warm temperature.According to whether plants finish their life cycle under specific growth temperature conditions,environmental high temperature divides into mild warm temperature and high temperature stress.The normal temperature in model plant Arabidopsis thaliana is 22℃.Mild warm temperature refers to the range of 27℃-29℃.The phenomenon that plants changes morphology and structure under mild warm temperature is called thermomorphogenesis.The function of the core transcriptional factor PIF4(Phytochrome Interacting Factor 4)is enhanced at mild warm temperature,which promotes the expression of auxin synthesis and signal transduction related genes,and causes hypocotyl elongation.Liquid-liquid phase separation of ELF3 weakens the function to repress PIF4 protein due to higher ambient temperature.The previous research of our group shows that BBX18/23(Bbox 18/23)negatively regulate the protein stability of ELF3 under mild warm temperature.However,the ELF3 protein specific degradation mechanisms under mild warm temperature are still unclear.Our study focuses on Arabidopsis thaliana E3 ubiquitin ligase XBAT31(XB3 Ortholog 1 in Arabidopsis thaliana)induced by mild warm temperature(29℃),and its homologous gene XBAT35.The specific research results were as follows:1.XBAT31/35 promote thermoresponsive hypocotyl growth.The hypocotyl length of XBAT31/35 single mutants and double mutant was not obviously different from WT at normal temperature(22℃).The hypocotyl length of XBAT31/35 single mutants was obviously shorter than WT at mild warm temperature,and the hypocotyl length of XBAT31/35 double mutant was further shorter than each single mutant.The hypocotyl length of XBAT31 overexpression was obviously longer than WT at normal as well as mild warm temperature.The hypocotyl length of XBAT35 overexpression was not obviously different from WT at normal as well as mild warm temperature.2.PIF4 is epistatic to XBAT31/35.The hypocotyl phenotypes of PIF4ox-1xbat31-1 and PIF4ox-1 xbat35-3 were consistent with each PIF4ox-1 overexpression at normal as well as mild warm temperature.The hypocotyl phenotypes of pif4-101xbat31-1 double mutant and pif4-101 xbat35-3 double mutant were consistent with pif4-101 mutant at normal as well as mild warm temperature.Compared with the expression level of PIF4 and PIF4 downstream genes in WT,the transcriptional level of PIF4 in XBAT31/35 single mutants had no obviously difference than WT.However,the transcriptional level of PIF4 downstream genes in XBAT31/35 single mutants was obviously lower than WT at mild warm temperature.3.XBAT31/35 interact with ELF3 and regulate ELF3 accumulation at mild warm temperature through ubiquitination.The hypocotyl phenotypes of elf3-101xbat31-1 double mutant and elf3-101 xbat35-2 double mutant were consistent with elf3-101 mutant at normal as well as mild warm temperature.Yeast Two Hybrid(Y2H),Pull-Down,Luciferase Complementation Imaging(LCI)in tobacco,Bimolecular Fluorescence Complement(BiFC)in tobacco and CoImmunoprecipitation(Co-IP)in Arabidopsis assays proved that XBAT31/35 interacted with ELF3.Ubiquitination in vitro assays demonstrated that XBAT31/35 were active E3 ubiquitin ligases and directly ubiquitinated ELF3.Protein stability assays showed that the protein content of ELF3 in XBAT31/35 single mutants was obviously increased compared with WT at mild warm temperature.4.XBAT31/35 mediate ELF3 accumulation partly dependent on BBX18.The hypocotyl phenotype of bbx18-1 XBAT31ox-1 was more similar to bbx18-1 mutant at normal as well as mild warm temperature.The hypocotyl phenotype of bbx23-1XBAT31ox-1 material was consistent with XBAT31ox-1 overexpression at normal as well as mild warm temperature.Pull-Down,LCI in tobacco and BiFC in tobacco assays proved that XBAT31/35 interacted with BBX18.Protein stability assays showed that the protein content of ELF3 in bbx18-1 XBAT31ox-1 was obviously increased compared with XBAT31ox-1 overexpression at mild warm temperature.To sum up,E3 ubiquitin ligases XBAT31/35 are positive regulators in thermomorphogenesis of Arabidopsis.PIF4 and ELF3 are epistatic to XBAT31/35.XBAT31/35 interact with ELF3 in vitro and in vivo,ubiquitinate ELF3,and degrade ELF3 at mild warm temperature.In addition,XBAT31/35 interact with BBX18 in vitro and in vivo.XBAT31 negatively mediates ELF3 protein accumulation at mild warm temperature partially dependent on BBX18.This study revealed the important function of XBAT31/35 in regulating the thermomorphogenesis of Arabidopsis thaliana,clarified the regulation mechanism of temperature receptor ELF3 protein stability at mild warm temperature,and expanded the signal transduction and gene regulation network of plant response to ambient temperature rise.