Molecular Mechanisms Underlying VvERF1B Promoting Oxalate Acid Secretion And Improving Alkaline Salt Tolerance In Grapevine Roots

Soil salinization and alkalization are a global environmental problem,posing significant threats to crop yield and quality.Particularly,alkali stress primarily caused by NaHCO3 and Na2CO3,subjects plants to triple stresses:osmotic stress,ionic toxicity,and p H stress.Hence,alkali stress is more destructive than salt stress.However,compared with the study of neutral salt tolerance,the mechanism of alkali tolerance in crops needs to be studied in depth.Previous research has shown that secretion of organic acids is a fundamental way to resist alkaline stress in plants,and plasma membrane H?-ATPase plays a central role in the process.However,it has not received enough attentions in fruit trees.Moreover,our previous research has identified a key role of ethylene in the secretion of organic acids,potentially involved in regulating the tolerance of plants to alkaline salt stress.Ethylene Response Factors(ERFs),plant-specific transcription factors,play a significant role in modulating plant responses to salt stress.However,the regulatory relationship and physiological function between ERFs and plasma membrane H?-ATPase are still unclear..Against this background,the’SA15’(alkaline salt-resistant cultivar)was used as the material to explore the role of organic acids in regulating NaHCO?stress and the mechanisms involved.It was discovered that the expression of VvERF1B(ethylene response factor 1B)was significantly activated by NaHCO?stress,which increased the activity of H?-ATPase through transcriptional regulation or post-transcriptional regulation and thereby promoted root acid secretion and enhancing plant tolerance to alkaline salt stress.Additionally,the molecular pathway of"VvERF1B-plasma membrane H?-ATPase-root acid exudation-alkaline salt resistance"was elucidated in detail.The specific research results are as follows:1.Oxalate acid enhances alkaline salt tolerance of grapevines.A total of six organic acids were identified,including oxalate,malate,tartrate,succinate,citrate and acetate.Oxalate was the mainorganic acid and accounted for 69.5–71.3%of the sum of the six organic acids in roots.Similar differences in organic acid concentrations were also found in root exudates;therefore,oxalate was the main organic acid synthesized and secreted by grapevine roots.Additionally,the content of oxalate acid significantly increased after NaHCO?treatment;compared to the control group,the oxalate acid content increased by 87.1%after 12 hours.The increased synthesis of oxalate promoted oxalate secretion and led to continuous accumulation in root exudates;the oxalate content in root exudates of the NaHCO3group was significantly higher than that of the control group.Hence,alkaline salt stress could induce the synthesis and exudation of oxalate acid in grape roots.Moreover,it was found that exogenous oxalate acid treatment alleviated NaHCO3 injury based on less withered leaves,lower MDA and higher root activity compared to the control.Taken together,ethylene increased NaHCO3 tolerance through promoting oxalate synthesis and secretion.2.The plasma membrane H?-ATPase plays a crucial role in regulating organic acid secretion of grapevine roots.Alkaline salt stress significantly enhanced the activity of the plasma membrane H?-ATPase in grape roots and thereby strengthened the efflux capacity of protons.The Non-invasive Micro-test Technology was used to measure the H?efflux rate,which further confirmed that alkaline salt treatment notably promotes the efflux of H~+.Additionally,to visually observe the process of oxalate acid secretion,bromocresol violet-containing medium was used to visualize the organic acid secretion in the roots.Compared to the control group,a distinct bright yellow coloration around the roots was observed at 0.5 days after NaHCO?treatment,indicating that alkaline salt stress induced the exudation of a large amount of acidic substances from the roots.Moreover,the H~+-ATPase inhibitor Na3VO4 almost completely inhibited the production of yellow color,suggesting that acid secretion is related to the PM H~+-ATPase activity.3.Ethylene promotes oxalate acid secretion and thereby enhances alkaline salts in grapevines.NaHCO?treatment significantly increased ethylene production.ACC application clearly alleviated NaHCO3 injury while 1-MCP led to more severe phenotype.Further investigation revealed that overexpression of the VvACS3(ACC Synthase 3)not only promoted the synthesis of ethylene but also significantly enhanced the growth of grape calli under NaHCO?stress,indicating that ethylene has a positive contribution to enhancing NaHCO?tolerance.In addition,the exogenous application of ACC significantly promoted the accumulation and secretion of oxalate acid in grapevine under alkaline salt stress,while the ethylene inhibitors completely blocked this process.Similarly,overexpression of VvACS3 also promoted the synthesis and secretion of oxalate acid.Therefore,ethylene plays an important role in regulating oxalate acid metabolism and improving alkaline salt tolerance.4.VvERF1B enhances oxalate acid secretion,thereby improving grape tolerance to alkaline salts.q RT-PCR analysis revealed that the expression of VvERF1B not only responds to changes in ethylene concentration and time but is also induced by NaHCO?.Therefore,VvERF1B is likely to be involved in the ethylene-mediated oxalate acid secretion.In this study,to reveal the function of VvERF1B in regulating NaHCO3 tolerance,the VvERF1B-overexpressing grape seedlings were obtained by genetic transformation system.Under NaHCO?stress,VvERF1B overexpression significantly reduced MDA accumulation,enhanced plasma membrane H?-ATPase activity,and promoted the secretion of H?and oxalate acid.Subsequently,the VvERF1B mutant transgenic calli were generated using CRISPR/Cas9.It was showed that the loss of VvERF1B function inhibited proton pump activity and oxalate acid secretion,resulting in higher sensitivity of calli to alkaline salt stress.Conversely,overexpression of VvERF1B significantly enhanced the alkaline salt tolerance of calli.These results indicate that VvERF1B is a key factor linking ethylene signaling with the regulation of grape tolerance to alkaline salts.5.The interaction between VvERF1B and VvMYC2 enhances the transcriptional activation of VvPMA10 and thereby improves proton pump activity.Overexpression of VvERF1B significantly induced the expression of VvPMA10,and overexpression of VvPMA10 significantly increased the levels of oxalate acid secretion and alkaline salt resistance in grape roots and calli.However,Y1H and EMSA experiments revealed that VvERF1B could not directly bind to the promoter of VvPMA10.Subsequently,using the promoter of VvPMA10 as bait in a screening library,the candidate transcription factor VvMYC2was obtained.Y1H,EMSA and dual-luciferase assays confirmed that VvMYC2 directly bound to the G-Box element of the VvPMA10 promoter.Functional analysis showed that VvMYC2promoted acid secretion,thereby positively regulating the alkaline salt tolerance of grapes.Moreover,VvERF1B interacted with VvMYC2.Further studies indicated that simultaneous overexpression of VvERF1B and VvMYC2 increased VvPMA10 expression,PMA activity,oxalate secretion and led to higher NaHCO3 tolerance,while VvERF1B overexpression and VvMYC2 mutation reduced VvERF1B-induced NaHCO3 tolerance.Thus,we revealed a molecular pathway by which"VvERF1B-VvMYC2-VvPMA10"enhances alkaline salt resistance.6.VvERF1B activates the expression of VvCIPK10,promotes the interaction VvCIPK10and VvPMA11 and increases NaHCO?tolerance.VvERF1B overexpression and mutation increased and inhibited the expression of VvCIPK10,respectively.And VvERF1B directly bound to the DRE element in the promoter region of VvCIPK10.VvCIPK10 promotes acid secretion and positively regulating alkaline salt tolerance.Additionally,membrane yeast two-hybrid,Bi FC,and dual-luciferase assays revealed that VvCIPK10 interacts with VvPMA11 and co-localize on the cell membrane,and the C-terminal 100 amino acids of VvPMA11 was the key region of protein-protein interaction.Subsequently,the contribution of VvPMA11 to alkaline salt tolerance was evaluated by gene editing technology,and the loss of VvPMA11 function inhibited oxalate acid secretion and reduced the tolerance of calli to NaHCO?,while overexpression of VvPMA11 significantly increased plasma membrane H?-ATPase activity and enhancd alkaline salt resistance.Therefore,we have elucidated a new pathway of"VvERF1B-VvCIPK10-VvPMA11"to regulate alkaline salt stress.In this study,we explored the coping mechanisms of grapes against alkaline salt stress,particularly focusing on the critical role of VvERF1B and it regulates the activity of VvPMAs and the secretion of organic acids.For the first time,we have revealed the VvERF1B-mediated alkaline salt tolerance function,and constructed an alkaline salt tolerance mechanism with"VvERF1B-VvPMAs-secretion"as the core.This discovery not only provides new insights into the molecular mechanisms of grape response to alkaline salt stress,but also holds significant practical value for the creation and improvement of grape germplasms resistant to alkaline salts.