Physiological,Ionomic Characteristics And Metabolomic,Proteomics Analysis Of Malus Halliana Seedlings Under Saline-alkali Stress

The Northwest Loess Plateau is optimal ecological area for growing apples in China.The saline/alkali reaction of soil is a key limiting factor for apple growth and productivity.In the apple industry,the selection of saline-alkali-tolerant rootstocks is an effective strategy to minimize soil salinization and alkalinization problems.M.halliana is a higher saline-alkali resistance apple rootstock in northwest China.Therefore,it is important to understand the mechanisms response to saline-alkaline stress for the development of apple industry.Two-year-old of M.halliana were employed as experimental materials and three types of stress treatments,?i?salt stress?NaCl??SS?,?ii?alkali stress?NaHCO₃??AS?and?iii?saline-alkaline stress?1:1 M ratio of NaCl:NaHCO₃??MAS?,were simulated by watering Hogland nutrient solution.The aim of this study was to examine the effects of three stresses treatments on thephotosynthesis,Chl fluorescence and physiology?antioxidant system,solute accumulation?of M.halliana.Furthermore,the different physiological adaptive mechanism of plants under SS,AS and MAS were compared.Moreover,M.halliana seedlings was employed as experiment materials,which involved treatment with Hoagland's solution supplemented with NaCl+NaHCO₃?1:1 M ratio of NaCl:NaHCO₃??pH=8.2?.Ionomic,metabolic and integrated metabolomic and proteomic approach were used to analyze the plant pathways involved in the response to saline-alkali stress and their regulatory mechanisms.Main results are as follows:1.These results showed that SS,AS and MAS are three different stresses.The inhibitory effects of stresses on M.halliana growth and photosynthesis were AS>MAS>SS.Under SS,it was found that plants resisted stress by increasing WUE,improving the activity of SOD and POD,accumulating TSS and starting thermal dissipation protection mechanism.Under AS,the light protection mechanism and antioxidant enzyme system were destroyed,M.halliana plants mainly through the accumulation of Pro and OA to adjust osmotic balance and through xanthophyll cycle to dissipate excess energy.2.MAS had a certain synergistic effect,which plants not only kept the dynamic balance of photosynthetic system between injury and repair by starting heat dissipation mechanism and xanthophyll cycle,and osmotic regulationmianly depended on accumulating osmolytes and improving the activity antioxidant enzyme.3.Based on the changes of phenotypic and physiologic index in M.halliana,we selected the 4 d of exposed to saline-alkali stress for Omics analysis.Results showed that,the Na content accumulated significantly in M.halliana,which disrupted the uptake of K,Zn,Mg,Fe and Cu in roots,but did not affect Fe,Cu and Mn absorption in leaves.Based on the results that Na⁺/K⁺and Na⁺/Cl^-in leaves were significantly lower than that in roots,suggesting that M.halliana leaves have a greater capacity to maintain ion homeostasis under saline-alkali stress.4.The change of metabolism in M.halliana leaves are further analyzed through LC-MS.Importantly,140 differentially metabolites were identified and 35 pathway were enriched,mainly including alkaloid biosynthese,biosynthesis of phenylalanine,ATP-binding cassette?ABC?transporters,mineral absorption.Besides,the significantly increase of sucrose and amino acid,greater alkaloid biosynthesis,and the accumulation of flavonoid and carotenoids strongly improved the saline-alkali resistance by scavenging ROS.An increase of pheophorbide a content in porphyrin and chlorophyll metabolism lead to leaf senescence in M.halliana leaves,which contribute to reduce stress-induced injury.5.The results of integrated metabolomic and proteomic analysis showed that,In total,70 metabolic pathways exhibited changes and 16 enzymes were assigned to these pathways.Further analysis of these metabolic pathways suggested that these proteins and enzymes were involved mainly in energy metabolism as well as in dicarboxylate metabolism;secondary metabolite biosynthesis;alanine,aspartate and glutamate metabolism;phenylpropanoid biosynthesis;and Starch and sucrose metabolism.6.We found that 2 candidate proteins?PPD and PAO?associated with senescence and 6photosynthesis proteins,especially PSBO2,could act as regulators of photosystem II?PSII?repair.Moreover,sucrose acts a signaling molecule that directly mediates the accumulation of both D-phenylalanine and tryptophan and the expression of proteins related to aspartate and glutamate?ASP3,ASN1,NIT4 and GLN1-1?to regulate osmotic responses and to remove reactive oxygen species?ROS?.Additionally,sucrose induces the flavonoid biosynthesis by the upregulation of CYP75B1 and positively regulate alkaloid?vindoline and ecgonine?synthesis to controlled the homeostasis of ROS.7.The decrease in peroxidase superfamily protein?PER?and ALDH2C4 during lignin synthesis further triggered the plant saline-alkali response.In addition,sucrose signaling also promoted auxin signaling by activating the expression of T31B5₁70 to maintain M.halliana growth and development.