Mechanisms Of Apple Rootstocks Response To Saline-Alkali Stress And The Functional Analysis Of MdWRKY75d

[Objective]Apple is one of the most important economic fruit trees in China,with its area and output accounting for more than 50%of the world.Soil salinization limits both metabolic and physiological processes in plant,threating plant growth and sustainable development of apple industry.Grafting cultivars onto rootstocks capable of high saline-alkalinization tolerance is a promising way to improve apple production under specific regions with saline-alkali stress.However,studies on saline-alkali resistance of apple rootstock are limited to the mechanisms of physiological and molecular responses to neutral or alkaline salt stresses.The impacts of compound saline-alkali stress on the growth and development of apple rootstock and related physiological and molecular mechanisms are still unclear.The WRKY transcription factor plays an important role in plant growth and development and responses to stress.However,functional studies of WRKY in apple in response to saline-alkali stress are still limited.Therefore,under saline-alkali stress conditions,clarifying the above-and underground morphological,physiological,and molecular response mechanisms of apple rootstocks with various resistant tolerance,as well as the function of WRKY in this response process,will be helpful to reveal the saline-alkali response mechanism and tolerance of apple rootstocks.[Methods]（1）Tissue culture seedlings of eight apple rootstocks with various saline-alkali tolerance,‘Zumi’,‘Marubakaido’,‘Qingzhen 1’,‘M.9T337’,‘SH.6’,‘B.9’,‘M.26’’and‘Mark9’were selected and treated with75 m M Na Cl+Na HCO₃（molar ratio 1:1）compound saline-alkali stress with hydroponic system.The morphological,physiological and several key genes’responses were observed,and analysis of linear regression,principal component and cluster were conducted to distinguish the saline-alkali stress tolerance among eight rootstocks.（2）the morphological and physiological characteristics,and transcriptome of‘Qingzhen 1’apple rootstock that cultured with or without 75 m M Na Cl+Na HCO₃（molar ratio 1:1）was determined and analyzed after 0 h,1 h,6 h,12 h and 24 h,respectively,using hydroponic system.（3）The MdWRKY75d was cloned from apple rootstocks of‘Qingzhen 1’,‘M.9T337’,‘SH.6’,‘Mark9’and‘B.9’,respectively.Sequence of MdWRKY75d was analyzed using MEGA and DNAMAN software,and expression patterns of MdWRKY75d was determined by q RT-PCR.The subcellular location of MdWRKY75d was determined in tobacco leaves using agrobacterium tumefaciens-mediated transgenic method.Stable transformation in Arabidopsis and apple callus were conducted to elucidate the function of MdWRKY75d in response to saline-alkali stress and abscisic acid（ABA）.（4）Several target genes of MdWRKY75d were screened and verified by DAP-seq and yeast one-hybrid technology,and the function of target gene MdSAUR15 was analyzed by transgenic Arabidopsis thaliana.[Results]The main results of present study were as follows:（1）The saline-alkali stress tolerance of eight apple rootstocks was determined.In total,29 indexes that were related to morphological,physiological,and key genes in eight apple rootstocks were determined and significant linear relationships were detected among several indexes.Based on the selected 22 saline-alkali stresses indexes,the eight apple rootstocks were classified into three groups:high saline tolerance group:‘Zumi’and‘Qingzhen 1’;saline alkali tolerant group:‘Marubakaido’,‘M.9T337’,and‘SH.6’;saline-alkali sensitive group:‘B.9’,‘M.26’’and‘Mark9’.（2）The potential physiological and molecular mechanisms of‘Qingzhen 1’in response to saline-alkali stress were clarified.The results showed that‘Qingzhen 1’alleviated the decrease of root activity and photosynthetic capacity and oxidative damage caused by saline-alkali stress by increasing the activity of antioxidant enzymes（POD）and the content of endogenous ABA.Furthermore,the RNA-seq database of root response to saline-alkali stress of‘Qingzhen 1’was constructed,and the expression characteristics of differential genes related to ion transport,active oxygen scavenging,plant hormone pathway and transcription factors were determined.The results showed that saline-alkali stress significantly up-regulated the expression levels of most proton pumps,K⁺uptake,antioxidant enzymes,ABA and ethylene（Eth）related genes and down-regulated the expression levels of Na⁺/H⁺antiporter,indole acetic acid（IAA）,brassinosteroid（BR）and jasmonic acid（JA）-related genes in the roots of‘Qingzhen 1’compared to control.The genes related to transcription factor differences are mainly concentrated in WRKY,NAC,NAC,MYB,HSF and ERF families.According to the response characteristics and function,the key gene MdWRKY75d was selected.（3）The function of MdWRKY75d was analyzed under saline-alkali stress and exogenous ABA spraying treatments,respectively.The results showed that the amino acid sequence of MdWRKY75d was highly conserved in apple rootstocks and was evolutionarily close to Pb WRKY75.The protein was located in the nucleus and had no transcriptional activation activity.MdWRKY75d could be significantly induced by saline-alkali stress and exogenous ABA treatment,and its expression level was negatively correlated with the saline-alkali tolerance of rootstocks.Heterologous overexpression of MdWRKY75d increased the sensitivity of transgenic Arabidopsis to saline-alkali stress and ABA.Overexpression of MdWRKY75d in apple callus promoted the growth of apple callus and interference expression of MdWRKY75d inhibited the growth of apple callus under both saline-alkali and exogenous ABA treatments,respectively.（4）Several target genes of MdWRKY75d were selected and verified,the transcriptional regulation of MdSAUR15 by MdWRKY75d was clarified,and the function of MdSAUR15 was analyzed.DAP-seq analysis and yeast one-hybrid technique confirmed that MdWRKY75d could bind directly to the promoters of MdAKT1,MdSAUR15 and MdERF2.Furthermore,with the transgenic callus of apple as the background,the MdWRKY75d could inhibit the expression level of MdSAUR15.The protein sequence of apple MdSAUR15was highly conserved in Rosaceae plants and is evolutionarily close to Mb SAUR15.The protein of MdWRKY75 was located in the nucleus and cell membrane.Overexpression of MdSAUR15 can promote the root development of Arabidopsis and improve the saline-alkali tolerance of transgenic Arabidopsis.[Conclusion]The saline-alkali tolerance was varied among the studied eight apple rootstocks.The‘Qingzhen1’had better saline-alkali tolerance than other rootstocks and can be used as a candidate rootstock for apple production in specific areas and as ideal material for studies related to salt-alkali stress.The‘Qingzhen 1’up-regulated proton pump,K⁺uptake,antioxidant enzymes,ABA and Eth-related genes to response saline-alkali stress by participating in ion balance,active oxygen scavenging and endogenous hormone regulation.The apple MdWRKY75d gene has certain functions in saline-alkali stress and ABA response.Under 10 m M（Na Cl+Na HCO₃）saline-alkali stress and 10μM ABA treatment,overexpression of MdWRKY75d gene promotes the growth of apple calli.Overexpression of apple MdSAUR15 gene can promote Arabidopsis root development and improve salt tolerance,and MdSAUR15 can be used as a candidate gene for genetic improvement of apple rootstock resistance.