Populus Euphratica Transcription Factor PeWRKYl Mediates Signaling Network Conferring Ionic Homeostasis Under Salt Stress

Soil salinity and secondary salinization cause serious ecological and environmental problems worldwide.Populus euphratica is the only woody plant that can form forest in the desert and semi-desert regions of northwest China.This poplar species serves as a model for elucidating physiological and molecular mechanisms of stress tolerance in tree species.Under high salt stress,P.euphratica can efficiently extrude Na~+from plants,and reduces the salt-elicited K~+efflux,thus maintaining K~+/Na~+homeostasis and alleviating salt damage to plants.Transcription factors,for example,WRKY1,play a crucial role in P.euphratica to tolerate the saline environment.Previous studies have shown that P.euphratica Pe WRKY1 promoted Na~+efflux in transgenic tobacco under salt stress.More importantly,the transgenic tobacco overexpressing Pe WRKY1 could promote the H~+pumping activity under salt stress.However,the regulatory mechanism of plasma membrane(PM)H~+-ATPase is unknown.The objective of this study was to screen Pe WRKY1-interacting genes,aiming at elucidating the molecular mechanism of signaling pathways in the salt response of P.euphratica.In this study,in vitro protein purification system was used to purify the Pe WRKY1 recombinant protein.DNA affinity purification sequencing(DAP-Seq)revealed that Pe WRKY1 protein targeted the downstream genes encoding(PM)H~+-ATPase(Pe HA1)and more axillary branches2(Pe MAX2).Moreover,a yeast two-hybrid library was established to screen the protein Pe GRP2,glycine-rich proteins(GRPs),interacting with P.euphratica Pe MAX2.The gene function of Pe WRKY1,Pe MAX2,and Pe GRP2 was respectively determined by overexpressing these genes in model plants,tobacco and Arabidopsis.The interaction network between transcription factor Pe WRKY1 and target genes,Pe HA1,Pe MAX2,and protein interaction between Pe MAX2 and Pe GRP2,were investigated in the present study.Consequently,a signalling pathway,Pe WRKY1-Pe HA1/Pe MAX2-Pe GRP2,mediating K~+/Na~+homeostasis in P.euphratica was put forward and discussed.The main results obtained in this study are listed as follows:1.High levels of Na Cl(200 m M)induced expression of Pe WRKY1 and Pe HA1 in P.euphratica roots and leaves.Notably,salt-elicited Pe WRKY1 showed a trend similar to Pe HA1,but with an earlier increase at 1 h in both roots and leaves.To determine the importance of the upstream regulatory region for salt-induced Pe HA1 expression,a 2022-bp promoter fragment upstream of the translational start of Pe HA1 was isolated from P.euphratica based on P.euphratica genome on NCBI website.Pe HA1promoter regions harbored cis-acting W-box,implying that transcription factor WRKY1 mediates Pe HA1 expression.The promoter-reporter construct Pe HA1-pro::GUS was transferred to tobacco plants,demonstrating thatβ-glucuronidase activities increased in root tips,leaf mesophyll and veins,and vascular tissues of the stem under salt stress.Therefore,the salt-enhanced expression of Pe HA1 in P.euphratica was shown to be due to promoter activity.DAP-Seq revealed that Pe WRKY1 protein targeted the Pe HA1 gene.The salt-induced transcriptional response of Pe WRKY1 and its interaction with Pe HA1 in P.euphratica were assessed.Pe WRKY1 binding to the Pe HA1 W-box in promoter region was verified by a yeast one-hybrid assay,electrophoretic mobility shift assay,and luciferase reporter assay.Virus-induced gene silencing showed that the expression of Pe HA1 in leaves significantly decreased when Pe WRKY1 was silenced in salinized P.euphratica.Pe WRKY1-ransgenic tobacco increased Na~+extrusion but reduced K~+loss under salt stress.As a result,the K~+/Na~+homeostasis was retained in salinized plants.The increased capacity to maintain ionic homeostasis was due to the H~+pumping activity in the PM.Overexpression of Pe WRKY1 in tobacco promoted proton-pumping activity in both in vivo and in vitro assays.Under salinity stress,transgenic tobacco exhibited a more negative membrane potential in roots than the WT and VC plants.The upward-regulated H~+-pumps sustained an H~+gradient to drive the Na~+/H~+antiport across the PM;and preserved a less-depolarized membrane potential,thus restricting the K~+efflux through depolarization-activated outward rectifying K~+channels(DA-KORCs)and depolarization-activated non-selective cation channels(DA-NSCCs).The increased H~+pumping activity in the PM was,at least in part,resulted from the upward regulation of Nt HAs.Transgenic tobacco plants overexpressing Pe WRKY1 had improved expression of PM H~+-ATPase genes,Nt HA2 and Nt HA4.It is noting that Nt HA4 expression was enhanced in transgenic tobacco plants,irrespective of control and salt conditions.The promoter fragments upstream of the translational start of Nt HA2(1963-bp)and Nt HA4(1912-bp)were isolated from tobacco plants.The regulatory region of Nt HA4 has a cis-acting W-box for the binding of Pe WRKY transcription factor.It is concluded that salt stress upregulated Nt HA4transcription due to the binding of Pe WRKY1 to the W-box in the promoter region of Nt HA4.Thus,the enhanced H~+pumping activity enabled salt-stressed plants to retain Na~+homeostasis.2.P.euphratica Pe MAX2 is the targeted gene of Pe WRKY1 protein.Using P.euphratica c DNA as a template,the full-length CDS region of Pe MAX2 gene was obtained by PCR cloning.Pe MAX2contained a 2106-bp coding sequence encoding a putative protein of 701 amino acids.Multiple sequence alignment analysis showed that the Pe MAX2 protein contained the F-box domain.Pe MAX2-GFP protein is mainly localizated in the nucleus,which is consistent with the localization of the transcription factor Pe WRKY1 protein.Fluorescence of GFP alone was detected almost uniformly throughout the cytoplasm and nucleus.Pe MAX2 was overexpressed in Arabidopsis to testify its role in salt tolerance.Pe MAX2 overexpression alleviated the inhibitory effect of high Na Cl on root growth of Arabidopsis thaliana,and reduced the damage of high salt on cell membrane.Pe MAX2 maintained K~+/Na~+balance by promoting the Na~+efflux and reducing K~+loss in transgenic A.thaliana under salt stress,thereby reducing the excessive accumulation of toxic Na~+in the plants.To determine the importance of the upstream regulatory region of Pe MAX2,a 1902-bp promoter fragment was sequenced and analyzed.The DNA sequence of the promoter contained a W-box cis-acting element.The promoter-reporter construct Pe MAX2-pro::GUS was transferred to tobacco plants to demonstrating the upstream regulatory region for salt-induced Pe MAX2 expression.VIGS showed that the salt-induced expression of Pe MAX2 significantly decreased in Pe WRKY1-silenced P.euphratica leaves.Pe WRKY1binding to the Pe MAX2 W-box in promoter region was further verified by an Y1H assay.Therefore,P.euphratica Pe WRKY1 interacted with the promoter of the Pe MAX2 and increased its expression under salt stress.The upregulated Pe MAX2 promoted the efflux of Na~+and reduced K~+loss,thus enabled salinized plants to maintain K~+/Na~+homeostasis.Noteworthy,the protein-protein interaction analysis revealed that the benifical effects of Pe MAX2 on salt tolerance resulted from its inhibition on glycine-rich protein Pe GRP2,which facilitated Na~+entry under salt stress.3.Pe GRP2,the Pe MAX2-interacting protein,was obtained by screening a dual-hybrid library of P.euphratica.The gene encoding Pe GRP2 was cloned from P.euphratica.The full-length Pe GRP2contained a 405-bp open reading frame(ORF)encoding a putative protein of 134 amino acids,which is heterologous to the model plant,Arabidopsis thaliana.Pe GRP2 plays a negative regulatory role in Arabidopsis response to salt stress.After being subjected to Na Cl,the efflux of Na~+in Pe GRP2-transgenic Arabidopsis was significantly lower than that of wild-type and vector control.As a result,excess Na~+accumulation caused salt damage to plants.Pe GRP2-GFP protein colocalized with the nucleus marker DAPI,which is consistent with the localization of the interaction protein Pe MAX2.The protein interaction between Pe MAX2 and Pe GRP2 was further testified with yeast two-hybrid(Y2H),bimolecular fluorescence complementation(Bi FC)assay,and bimolecular luciferase(Bi LUC)assay.Pe MAX2 and Pe GRP2 recombinant proteins were prokaryotic expressed in Escherichia coli and purified for in vitro proteasome degradation experiments.The in vitro degradation assay showed that Pe MAX2 promoted the degradation of Pe GRP2,while treatment with proteasome inhibitor MG132inhibited the degradation of Pe GRP2,indicating that P.euphratica Pe MAX2 participated in the degradation of the negative salt tolerance regulator,Pe GRP2.This would limit the Pe GRP2-facilitated Na~+entry,thus assisting the P.euphratica plants adapting to saline environments.In conclusion,the data revealed that Pe WRKY1 activated Pe HA1 and Pe MAX2 expression by binding to the cis-acting W-box of the promoter in Pe HA1 and Pe MAX2.Ionic homeostasis in salt-stressed P.euphratica was maintained through the two Pe WRKY1-mediated signaling pathways:(i)Pe WRKY1 activated Pe HA1 transcription by binding to the promoter region,conferring H~+-pumping under salt stress.Proton pumps in P.euphratica sustained an H~+gradient to drive the Na~+/H~+antiport across the PM,and preserved a less-depolarized membrane potential to restrict the K~+efflux through DA-KORCs and DA-NSCCs.(ii)Pe WRKY1 activated Pe MAX2 transcription by binding to the promoter region.The activated Pe MAX2 supressed Pe GRP2 via proteasome degradation,thus subsequently restricted the Pe GRP2-facilitated Na~+entry under salt stress.The less Na~+buildup in plants would reduce the toxic effect of salt ions.Based on these results,we propose a signalling pathway mediating K~+/Na~+homeostasis in P.euphratica,i.e.,Pe WRKY1-Pe HA1/Pe MAX2-Pe GRP2.This hypothetical signaling pathway increases our understanding of salt response of the model species,P.euphratica.