Biochemical And Molecular Mechanisms Of H₂S Inducing Salt Tolerance Of Malus Hupehensis

Malus hupehensis is a perennial tree belonging to the Malus in the Rosaceae,and has high ornamental value.Malus hupehensis young leaves can be made into tea drinks,which are rich in tea polyphenols,flavonoids and trace elements.Therefore,Malus hupehensis also has high medical and health effects.The growth of Malus hupehensis is uniform,so Malus hupehensis is an excellent rootstock for apple.The cultivated area of Malus hupehensis is large in Shandong Province,but its growing area overlaps with the saline-alkali soil in Shandong Province.Therefore,the growth of Malus hupehensis is seriously affected by soil salinization.Salt stress is one of the common abiotic stresses in plants.Salt stress affects plant physiological processes in all aspects,including ionic toxicity,inhibition of plant growth,mineral nutrient distribution,osmotic stress,disturbance of cell membrane permeability,and reduction of leaf photosynthetic efficiency,etc.H₂S is a toxic gas molecule,and is the third gas signaling molecule ranking after carbon monoxide and nitric oxide.H₂S is involved in a variety of plant physiological processes,including seed germination,stomatal movement,photosynthesis,root growth,and stress resistance.Currently,the regulating of H₂S in plant stress resistance is a research hotspot,but most studies have been performed on physiological level.Compared with genes,the correlation between metabolism and plant stress resistance is more direct.The core regulation mechanism of metabolism in plant salt tolerance mechanism is not well understood.Therefore,we investigated the key mechanism in metabolic regulation of plant salt tolerance through NaHS（a donor of H₂S）induction.In this study,through phenotype analyses,¹³CO₂ transient labeling,targeted metabolome,transcriptome and heterologous expression of targeted genes in Arabidopsis,we found that H₂S activated organic acid metabolism in respiratory metabolic pathway,induced MhALMT4and MhCS expression and increased CO₂ assimilation rate.H₂S also promoted the flow of malate and other organic acids in the tricarboxylic acid cycle,promoted the top-down transportation of substances.Correspondlingly,H₂S induced the salt tolerance of Malus hupeensis.The results were as follows.1.NaHS largely restored the aboveground phenotype of salt-stressed Malus hupeensis.The net photosynthetic rate and growth of SHS0.5 treatment（0.5m M NaHS after salt treatment）almost recovered to the control levels.The relative electrical conductivity of leaves in SHS0.5decreased significantly.The ratio of GSH/GSS in SHS0.5 also increased significantly.Through principal component analysis（PCA）and clustering analysis of physiological parameters such as aboveground and underground dry and fresh weight,net photosynthetic rate,chlorophyll content,root growth,plant height and leaf area,we found that SHS0.5treatment mainly played a role in promoting the recovery of aboveground physiological phenotype.PCA and cluster analysis of underground parameters showed that the performance of SHS0.5 was more similar to salt stress,indicating that the improvement of underground parameters was not obvious in SHS0.5.2.NaHS induced salt tolerance of Malus hupeensis in a top-down manner.When 0.5m M NaHS was applied only to the aboveground part of Malus hupeensis,the aboveground phenotype of plants could be alleviated significantly,and the root growth was better than that in salt stress.However,when 0.5m M NaHS was applied only to the underground part of Malus hupeensis,it could not effectively restore the stress phenotype in the aboveground part of the plant,and the root growth was inferior to that in salt stress.Therefore,we determined that root application of NaHS induced salt tolerance of Malus Hupeiensis seedlings by affecting the top-down material transport.3.NaHS induced salt tolerance of Malus hupeensis by promoting Calvin cycle and CO₂assimilation rate.¹³C labeling experiment showed that the labeling rate of plants in control was fast.The labeling degree of plants was high in control when ¹³C labeling curve reached stable state.The labeling curve of salt stressed plants reached stable state quickly and the labeling degree was very low.The labeling degree of 3-PGA and RUBP was significantly increased in SHS0.5 treatment.Based on mass isotopomer distributions（MIDs）,we found that[M]⁺and[M+1]⁺of metabolites in salt-stressed plants appeared at the early stage of labeling,but their relative contents did not decrease significantly.The results indicated that¹³C distribution in the photosynthetic pathway of salt stressed plants was very slow and photosynthetic efficiency of salt stressed plants was very low.In the SHS0.5,[M]⁺and[M+1]⁺appeared at the early stage of labeling and increased slowly,and[M+3]⁺appeared.The results indicated that SHS0.5 treatment had higher photosynthetic efficiency.In addition,compared with that in salt stress,the net photosynthetic rate of SHS0.5 treatment was significantly increased,even reaching the plants in control level.4.NaHS induced salt tolerance of Malus hupeensis by increasing respiration rate,promoting tricarboxylic acid circulation,reducing accumulation of organic acids and hexose,and reducing photosynthetic feedback inhibition.After salt stress,the contents of organic acids such as citrate and pyruvate increased.After NaHS treatment,the contents of organic acids such as citrate and pyruvate decreased.Transcriptome date showed that the expression levels of key enzymes in the tricarboxylic acid cycle such as NAD/NADP malic enzyme（NAD/NADP-ME）decreased after salt stress.The expression levels of NAD/NADP-ME increased after NaHS treatment.Salt stress decreased the respiration rate of Malus hupeensis,while NaHS treatment increased the respiration rate of Malus hupeensis.5.Malus with different salt tolerance had different turnover capacity of organic acids.Malus robusta had strong salt tolerance,and Malus hupeensis was salt sensitivity.The contents of organic acid of Malus robusta were compared with that of Malus hupeensis after salt stress.After salt stress,a large amount of organic acids significantly accumulated in Malus hupeensis seedlings,while organic acids did not excessively accumulated in Malus robusta seedlings.In addition,NAD/NADP-ME was an important gene in response to salt stress.The expression level of NAD/NADP-ME in Malus robusta were significantly higher than that in Malus hupeensis under normal conditions.The results indicated that Malus robusta had a more active metabolic capacity of organic acids,and the efficient turnover of organic acids enhanced its salt tolerance.6.NaHS induced salt tolerance of Malus hupeensis by inducing MhALMT4 and MhCS expression.Malus hupeensis aluminum induced malate transporter 4（MhALMT4）increased the synergic efflux of malate and GABA,and promoted the flow of organic acids in the tricarboxylic acid cycle under salt stress.MhALMT4 also avoided the excessive accumulation of organic acids and the excessive degradation of starch.Therefore,MhALMT4 induced the salt tolerance of Malus hupeensis.In addition,overexpression of MhALMT4 in Arabidopsis increased salt tolerance.