Function Characterization Of PtoGT55 In Regulating Salt Tolerance Of Populus Tomentosa

As the phenomenon of soil salinization becomes more and more serious,salt stress is one of the the most important and common abiotic factors that restricting plant growth and crop yields.Improving the the survival rate of plants on saline-alkali ground is urgent in agriculture and forestry production.Compared with other tree species,poplars are more salt-tolerant,and are usually used as afforestation tree species in saline-alkali soils.Therefore,identification and functional analysis of genes related to salt-tolerance is the key for molecular breeding in tree species.In this study,we selected PtoGT55 as the candidate salt-tolerance gene,which has a conserved Trihelix domain and belongs to the SIP1 subfamily.The function of PtoGT55in salt stress is not clear.To understand the role of PtoGT55,transgenic lines with overexpression or RNAi were obtained.The responses of transgenic plants to salt stress were examined.Meanwhile,the related physiological indicators and gene expression were also detected.The main results are as follows:（1）To identify the genes related to salt stress in Populus tomentosa,a FOX library containing multi-tissue full-length CDS of Populus tomentosa was constructed.Then the FOX library was genetically transformed into Arabidopsis thaliana,and the FOX line with enhanced salt-tolerance under 120 m M Na Cl were screened.The gene overexpressing from salt-tolerance FOX line was cloned and sequenced,and PtoGT55gene was found.（2）In order to understand the role of this gene,we obtained transgenic lines with overexpression of PtoGT55 and RNAi knockdown transgenic lines in Populus tomentosa and Arabidopsis.Compared with WT,the salt tolerance of PtoGT55 overexpressing transgenic poplar and Arabidopsis was significantly increased,while the RNAi knockdown transgenic Populus tomentosa lines were obviously sensitive to salt,indicating that PtoGT55 positively regulate the salt tolerance.（3）Transient expression of a PtoGT55-GFP fusion gene revealed that PtoGT55 is a nucleus-localized protein.Self-activation assays in yeast showed that PtoGT55 has transcriptional activating activity.Taking this together,PtoGT55 exhibits as a Trihelix transcription factor with transcriptional activating activity.（4）The results of real-time quantitative PCR detection in wild-type Populus tomentosa showed that the PtoGT55 expression was induced by 150 m M Na Cl salt stress,1%H₂O₂,200 m M ABA and 350 m M mannitol.Among them,the degree of PtoGT55 in response to 150 m M Na Cl salt stress was the most significant,and its expression abundance was increased about 4 times when treated for 6 h,which implied that PtoGT55was involved in salt stress in Populus tomentosa.GUS staining of pro PtoGT55:GUS transgenic poplar plants showed that PtoGT55 was most abundantly expressed in roots,then in vascular tissues of leaves.The expression of pro PtoGT55:GUS was significantly induced in pro PtoGT55:GUS transgenic plants after treated with 150 m M Na Cl,indicating that at the transcription level,PtoGT55 is significantly upregulated.（5）RNA-seq analysis was performed on wild-type and PtoGT55-OE transgenic lines.The data showed the genes in plant-pathogen interaction pathways,MAPK signaling cascade pathways,plant hormone signaling pathways were all significantly up-regulated in PtoGT55-OE line,compared with WT.The increased expression of receptor proteins,calmodulin,CDPK,MAPKs,NCED3（ABA key synthase encoding gene）,Rboh F/D（reactive oxygen burst homologue）,WRKY transcription factor,and b HLH transcription factor were futher conformed by RT-q PCR.In addition,part of this genes were induced by salt stress,as showing in the RNA-seq data of wild-type poplar treated by salt stress.These results indicates that PtoGT55 may enhance the salt tolerance of poplar by promoting the transcription of these early salt stress signaling and responses genes.（6）Flag-PtoGT55 was transiently expressed in tobacco leaves,and the results of western blotting showed that,part of the PtoGT55 protein showed changes in mobility under salt stress.Bioinformatics analysis showed that there is a serine phosphorylation site recognized by MPKs at position 247 of the PtoGT55 protein sequence.We further showed that PtoGT55 interacts with Pto MPK3.1,Pto MPK4.1 and Pto MPK6.1 through yeast two-hybrid screening.Bi FC and Co-IP analyses were further conducted to validate the interaction between PtoGT55 and Pto MPK in vivo.Co-expression of PtoGT55 fused to the amino-terminal half of yellow florescent protein（n YFP-PtoGT55）and Pto MPKs fused to the carboxy-terminal half of YFP（Pto MPK3.1/4.1/6.1-c YFP）led to visible fluorescence in the nucleus of co-transformed tobacco leaves.Co-IP results revealed that Flag-PtoGT55 was co-immunoprecipitated by the anti-HA antibody when they were co-expressed with HA-Pto MPK3.1.GST-pull down results shows there was a protein interaction in vitro.To verify that 247 amino acid site is indeed a phosphorylation site,PCR site-directed mutagenesis stategy was utilized to construct the expression vectors of constitutive phosphorylation state of PtoGT55 by mutating Ser²⁴⁷to Asp²⁴⁷.The overexpression vector was constructed to transform Populus tomentosa.The results showed that under 100 m M Na Cl treatment,35:Flag-PtoGT55^S247D（constitutive phosphorylation）has a stronger salt tolerance than Flag-PtoGT55^WT.The salt tolerance of 35:Flag-PtoGT55^S247D suggests that this phosphorylation of PtoGT55 may play an important role in the response of Populus tomentosa to salt stress.In summary,this study screened a new salt stress related gene PtoGT55,which positively regulate plant salt-tolerance.PtoGT55 may act as the phosphorylation substrate of Pto MPKs to play a role in MAPK signaling pathway under salt stress,and PtoGT55may positively regulate the transcription of early salt stress signaling and responses genes to enhance plant salt stress resistance.With both biochemical and genetic evidences,we figured out the role and the regulation mechanism of PtoGT55 in salt stress,which provides a genetic strategy for salt tolerance improvement in woods.