Study On The Molecular Mechanism Of Salt Tolerance In Arabidopsis Thaliana Ubiquitin Ligase PUB30 Mutant

Soil salinization has seriously affected the growth of various crops,thus limiting the development of agricultural production.Selecting and breeding salt tolerant cultivars and improving the adaptability to salinized land is an efficient way to utilize saline soil effectively.Therefore,it is important to understand the mechanism of salt tolerance in plants,collectively controlled by regulation of salt responsive genes,ion transport and signal transduction of plants under salt stress,and select new salt tolerant genes for the development of salt tolerant genes.Recently U-box E3 protein of the ubiquitin degradation pathway was found to play an important role in the regulation of plant physiological function.For example it is involved in the regulation of cell death,aging,disease resistance,flowering,hormone signaling and so on.Recently PUB30 has been reported to participate in the salt stress response as a negative regulator and the pub30 mutant was more tolerant to salt stress during seed germination,but the molecular mechanism remains to be elucidated.Here we explained the function of PUB30 gene in salt response and identified its protein interaction partner,the findings of the study were described as followed:?1?AtPUB30 encodes a total of 448 amino acid proteins,including a U-box domain at the N terminus,and 5 ARM-repeat domains at the C terminal.We ordered pub30 mutants salk₀12549?pub30-1?and Salk₀73739?pub30-3?from ABRC,and the pub31 mutant Salk₀54774?PUB30 and PUB31 have a homology of 81%at amino acid level?.Transcription of PUB30 and PUB31 could be detected in Col-0,and were close to 0 in pub30-1 and pub30-3 mutants.PUB31 transcription could hardly detected in the pub31 mutant,indicating that these three mutants are knockout mutants.?2?Under the condition of 150 mM NaCl,the pub30 mutant exhibited higher salt tolerance than the wild type,the pub31 mutant is a little sensitive to salt stress compared with the wild type,The double mutant pub30-1 pub31 behaved in between the pub30-1 and pub31 single mutant for salt stress response and resembled the pub31 mutant phenotype in radicle emergence rate,suggesting the epistatic effect of PUB31 in determining sensitivity to salt stress.The transgenic seeds harboring GFP-PUB30 in the pub30-1 mutant background showed sensitive phenotype on the MS medium containing 150 mM NaCl,implying that the GFP-PUB30 fusion protein are functional in complementing the salt-tolerant phenotype of the pub30-1 mutant.?3?Quantitive Real-time PCR was used to detect the expression of PUB30 in different tissues,including root,stem,cauline leaf,rosette leaf and silique.Higher relative expression level was found in root and stem,lower level was observed in cauline and rosette leaves.GFP-PUB30 and PUB30-YFP vectors were transiently expressed in tobacco leaves,and we found that these two vectors showed similar localization pattern where PUB30 was extensively localized in the cytoplasm,nucleus and plasma membrane.?4?To examine whether PUB30 is a functional E3 Ub ligase,we produced a full-length Arabidopsis PUB30 protein fused with maltose binding protein?MBP?in Escherichiacoli and subsequently affinity-purified from the soluble fraction.In vitro self-ubiquitination assays were performed in the presence of Arabidopsis El?UBA1?,Arabidopsis E2?UBC8?,and ubiquitin.Polyubiquitination was detected by Western blot which suggests that PUB30 possesses E3 Ub ligase activity in vitro.?5?Yeast two hybrid assay was used to screen proteins interacting with PUB30,BKI1?BRI1 KINASE INHIBITOR1?proteins of BR signaling pathway can interact with PUB30 specifically.BKI1 is the substrate of BRI1,which was the major component of BR signal.The BKI1 protein is localized on the plasma membrane,which can dissociate from the plasma membrane into the cytoplasm during the presence of exogenous BR.?6?GST-BKI1,MBP-PUB30 and MBP-PUB31 proteins were expressed in Escherichiacoli and purified.In vitro pull-down assays showed that GST-BKI1 was pulled down by MBP-PUB30 but not by MBP-PUB31,which indicated that PUB30 specifically interacts with BKI1.In vivo coimmunoprecipitation assays showed the same result as pull-down.Seedlings expressing Myc-BKI1 were treated with 50 ?M proteasome inhibitor MG 1.32 for different time,and the result showed that the level of Myc-BKI1 protein increased gradually as the incubation time with MG 132 was prolonged,which suggests that BKI1 protein could be degraded by the 26S proteasome.in vitro and in vivo degradation system were used to detect the degradation speed of BKI1 between wild-type and mutant.Immunoblot assays showed that degradation of the Myc-BKI1 protein was slowed down in pub30-1 mutant compared with the wild-type.All these result indicated that BKI1 was the target protein of PUB30.?7?Transgenic plants overexpressing BKI1 exhibited higher salt tolerance than the wild type,and the bkil mutant displayed salt-hypersensitive phenotype.The active form of brassinosteroid?brassinolide,BL?was employed to treat the BKI1-YFP transgenic seedlings and the signal was observed to transfers from membrane to the cytoplasm to form vesicles compared with the mock.When 50 mM NaCl was used,we found that BKI1 also dissociated from the plasma membrane and formed many vesicles in the cytoplasm.Therefore,similar to BL,salt can also induce the dissociation of BKI1 from plasma membrane.BR can promote the germination of Arabidopsis thaliana seeds,and when applied BR,the germination of pub30 mutant was faster than the wild type,indicating that the endogenous BR signal in pub30 mutant is enhanced.All these results indicate that PUB30 negatively regulates salt tolerance in Arabidopsis,BKI1 is the target protein of PUB30.BKI1 is a positive regulator in salt stress,PUB30 regulates salt tolerance through regulating the degradation of BKI1 and brassinosteroids signaling.