Mechanism Study Of The Regulation Of FtRMA1 Ubiquitination On FtPIP1;3 Response To Drought Stress In Tartary Buckwheat

Plasma membrane intrinsic proteins(PIPs)are water channel proteins located on the plasma membrane of plants,which regulate water and small molecules transport in and out of cells and participate in plant stress response processes.Previous studies have shown that PIPs have different functions in plant stress resistance.Overexpression of Bj PIP1 in Arabidopsis improves drought tolerance,while overexpression of Go PIP1 in Arabidopsis makes it more sensitive to drought.Tartary buckwheat is a typical high stress-resistant medicinal and edible crop in western China.Previous studies have found that FtPIP1;3 in tartary buckwheat interacts physically with the E3 ubiquitin ligase FtRMA1(homologous to human RING membrane-anchor E3 ubiquitin ligase),but its regulatory function in stress resistance has not been reported.Based on further confirmation of the interaction between FtPIP1;3 and FtRMA1 and the transcriptional expression levels under drought conditions,an in vitro ubiquitination system was constructed to analyze the ubiquitination function of FtRMA1 on FtPIP1;3.The function of FtRMA1 in tartary buckwheat stress resistance was preliminarily analyzed using Arabidopsis pip1;3 mutants and FtRMA1 overexpression lines,and the mechanism of FtRMA1 ubiquitination regulation of FtPIP1;3 was explored.The main results obtained in this study are as follows:(1)Yeast two-hybrid assay showed that the Tartary buckwheat ubiquitin ligase FtRMA1 physically interacted with the plasma membrane intrinsic protein FtPIP1;3.This interaction was further confirmed by His-Pulldown and bimolecular fluorescence complementation assays.Bioinformatics analysis revealed that FtRMA1 had a typical RING-finger domain and belongs to the single-subunit ubiquitin ligase.(2)Real-time fluorescence quantitative PCR analysis showed that FtRMA1 and FtPIP1;3 were expressed in various tissues and organs throughout the entire growth period of Tartary buckwheat.The expression level of FtRMA1 was highest in mature roots and lower in stem during seedling stage,while that of FtPIP1;3 was highest in flowers during grain filling stage and lower in roots during leafy stage.Drought and ABA treatment significantly induced the expression of FtRMA1 and FtPIP1;3 in Tartary buckwheat seedlings.(3)Using three compatible dual expression frames vectors,an in vitro ubiquitination system was constructed in E.coli BL21Star(DE3)for co-expression of ubiquitin,ubiquitin-activating enzyme,ubiquitin-conjugating enzyme,FtRMA1 and substrate FtPIP1;3.To analyze the ubiquitination effect of FtPIP1;3,the FtUb gene was constructed into p RSFDuet-1,FtPIP1;3 and ubiquitin-activating enzyme At UBA1 were co-constructed into p CDFDuet-1,and FtRMA1 and ubiquitin-conjugating enzyme At UBC8 were co-constructed into p ACYCDuet-1.Immunoblotting using Ub monoclonal antibody showed that FtRMA1 had typical self-ubiquitination activity.Immunoblotting using Ub and MBP monoclonal antibodies showed that FtPIP1;3 could be ubiquitinated by FtRMA1 in the co-expression system.The construction of this in vitro ubiquitination system provides a basis for further analysis of the ubiquitination modification site of FtPIP1;3 and other related studies.(4)Prediction results of cis-acting elements in the promoters of FtRMA1 and FtPIP1;3showed that there were numerous light-responsive elements,multiple MYB-responsive elements,hormone-responsive elements,etc.on both gene promoters,and there were 2ABA-responsive elements on the FtRMA1 promoter and 4 ABA-responsive elements on the FtPIP1;3 promoter,suggesting that they might have similar expression patterns under drought stress.The promoter of FtRMA1 was constructed into the p CAMBIA3301-Gus vector,transformed and screened for homozygous Arabidopsis plants.Under mannitol-induced drought and ABA treatment,the Gus enzyme activity in Arabidopsis increased,indicating that the activity of the FtRMA1 promoter was induced by drought and ABA signals.(5)Construct the plant overexpression vector p CAMBIA3301-FtRMA1,transform and screen homozygous Arabidopsis;screen pip1;3 homozygous Arabidopsis mutants by tri-primer method,and conduct natural drought-rewater analysis.The results showed that the drought tolerance of Arabidopsis overexpressing FtRMA1 was significantly improved.Under drought conditions,the chlorophyll content and SOD activity of overexpressing plants increased compared with the wild type,and the content of malondialdehyde decreased;pip1;3 Arabidopsis showed sensitivity to drought.The above research results suggested that FtRMA1 mignt have multiple target substrates of plant drought resistance or FtRMA1 responds to drought stress by ubiquitination regulating the processing and transport of FtPIP1;3.Its intrinsic mechanism needs further analysis.The above studies have shown that FtRMA1 can ubiquitinate FtPIP1;3,and FtRMA1 and FtPIP1;3 played a positive role in drought stress,providing a theoretical basis for elucidating the response and function of the ubiquitin system under stress.