Molecular And Physiological Mechanisms Of Improving PH To Alleviate Aluminum Toxicity In Citrus Sinensis Seedlings Revealed By Multi-Omics

Aluminum（Al）toxicity is a global agricultural production problem that restricts crop yield in acidic soils.In acidic soils（pH<5.0）,Al mainly exists in the form of Al³⁺,which is toxic to plants,while in neutral and alkaline soils,Al mainly exists in the form of insoluble aluminosilicates or oxides.Therefore,soil pH had a great influence on the toxicity of Al to plants.So far,there have been few studies on the toxic effects of Al on plants at different pH levels,with a focus on herbaceous plants and less on woody plants.Citrus is a subtropical evergreen woody fruit tree,and the soil in the main production area is mostly acidic or strongly acidic red soil,yellow soil,or latosol,which is susceptible to high active Al and low pH.Previous studies have shown that increasing pH can alleviate Al toxicity in citrus,and the physiological mechanism of increasing pH to alleviate Al toxicity in citrus has been studied from the aspects such as photosynthesis,nutrient and water uptake,root organic acid secretion,as well as root and leaf carbohydrate,organic acid metabolism,production and detoxification of reactive oxygen species（ROS）and methylglyoxal.The molecular mechanism of increasing pH to alleviate Al toxicity in citrus has not yet been elucidated.Sand potted seedlings of‘Xuegan’（Citrus sinensis）were used as the materials.The seedlings were irrigated daily with nutrient solution at a pH of 3.0 or 4.0 and a concentration of 0 or 1 mmol/L Al Cl₃·6H₂O for 18weeks.Thereafter,the effects of Al toxicity on seedling growth and leaf pectin methylation;as well as Al,callose,cell wall materials（CWM）and its components,transcriptome,widely targeted metabolome,hormones（targeted metabolome）,and energy-rich compounds and their metabolites（ECMs;targeted metabolome）.The aims of this study were to reveal themolecular and physiological mechanisms of increasing pH to alleviate Al toxicity in‘Xuegan’seedlings from multi-omic levels,ultimately enhancing the adaptive ability of citrus to acidic soils,and providing a theoretical basis for the high-yield and high-quality cultivation of citrus and sustainable development of the citrus industry.The main research findings are as follows:1 Effects of Al toxicity on growth and Al contents in roots and leaves of‘Xuegan’seedlings under different pH levelsIncreasing pH alleviated the inhibitory effect of Al toxicity on the seedling growth,as well as the increase in root dry weight/shoot dry weight and Al contents in roots and leaves induced by Al toxicity.2 Molecular and physiological mechanisms of increasing pH to alleviate Al toxicity in‘Xuegan’roots revealed by transcriptome and metabolomeThe increase in pH from 3.0 to 4.0 reduced Al toxicity induced increase of callose content,and increased the number of Al toxicity responsive genes and metabolites in roots,indicating that an increase in pH enhanced the adaptability of roots to Al toxicity.The alleviation of root Al toxicity by increasing pH might be related to several factors,including（1）enhanced ability of roots to maintain phosphate and energy homeostasis,as well as ability to balance the generation and detoxification of ROS and aldehydes;and（2）increased accumulation of secondary metabolites（polyphenols,procyanidins,phenolamides,etc.）in roots and adaptability of root cell walls and plasma membranes to Al toxicity.3 Molecular and physiological mechanisms of increasing pH to alleviate Al toxicity in‘Xuegan’roots revealed by transcriptome and metabolomeThe increase in pH from 3.0 to 4.0 reduced（increased）the number of Al toxicity responsive genes（metabolites）in leaves,indicating that an increase in pH alleviated Al-toxic impacts on gene expression,but it enhanced the adaptability of metabolites to Al-toxicity.The alleviation of leaf Al toxicity by increasing pH might be related to several factors,including（1）decreased accumulation of callose and low methylesterified pectin,increased accumulation of high methylesterified pectin,and enhanced cell wall stability in leaves;（2）less downregulation of primary metabolites and alterations of lipid metabolism in leaves;（3）increased biosynthesis and accumulation of secondary metabolites in leaves;and（4）enhanced ability to maintain energy,phosphate,and cell redox homeostasis,as well as the ability to maintain the balance between reactive oxygen species and aldehyde generation and detoxification leaves.Some metabolic pathways such as glycolysis/gluconeogenesis and phenylpropanoid pathway;metabolites such as callose and phenolic compounds;andgenessuchasxyloglucan endotransglucosylase/hydrolases（XTHs）,purple acid phosphatases（PAPs）,phytochelatin synthase 3,and cysteine synthase,might involve in the raised pH-induced amelioration of leaf Al toxicity.4 The roles of hormones and ECMs in raised pH-mediated alleviation of Al toxicity in‘Xuegan’roots revealed by transcriptome and metabolomeThe content of total ECMs was reduced by Al toxicity in pH4.0-treated roots,but unaffected significantly in pH 3.0-treated roots.However,the content of most ECMs and total ECMs in Al-toxic roots was not lower at pH 4.0 than at pH 3.0.Additionally,Al-toxicity-induced reduction in the content of total phosphorus-containing ECMs was greater at pH 4.0 than at pH 3.0.These results indicated that the increase in pH improved the utilization efficiency of ECMs and the conversion of organic phosphorus in phosphorus-containing ECMs into available phosphate in Al-toxic roots,thereby enhancing the adaptability of root ECMs to Al-toxicity.The raised pH-mediated increase in the accumulation of jasmonic acid（JA）,methyl jasmonate（MEJA）,total jasmonates（JAs）,salicylic acid（SA）,and indole-3-acetic acid（IAA）and the decrease in the accumulation of total cytokinins（CKs）in Al-toxic roots might help to maintain the homeostasis of mineral nutrition and balance between the production and detoxification of ROS and MG,increase Al toxicity-induced secretion of citrate and malate and the compartment of Al in vacuoles,and reduce the uptake of Al,thereby reducing root Al toxicity.In summary,both ECMs and hormones are involved in the increased pH-mediated alleviation of root Al toxicity.5 The roles of hormones and ECMs in raised pH-mediated alleviation of Al toxicity in‘Xuegan’leaves revealed by transcriptome and metabolomeMore Al-toxicity-responsive hormones and ECMs were identified at pH 4.0-treated leaves than at pH 3.0,suggesting that increasing pH enhanced the adaptations of hormones and ECMs to Al-toxicity in leaves.The roles of hormones and ECMs in the increased pH-mediated mitigation of leaf Al toxicity might be related to the following aspects,including（1）improved leaf growth by upregulating the levels of jasmonoyl-L-isoleucine（JA-ILE）,6-benzyladenosine（BAPR）,N6-isopentenyladenosine（IPR）,cis-zeatin-O-glucoside riboside（c ZROG）,and auxins（AUXs）,preventing Al-toxicity-induced-reduction of gibberellin（GA）biosynthesis,and avoiding JA-mediated defense;（2）enhanced tryptophan（TRP）biosynthesis and accumulation,and subsequent upregulation of auxin,melatonin and secondary metabolite biosynthesis;（3）enhanced ability to maintain the homeostasis of energy（ATP）and phosphorus-containing ECMs;and（4）enhanced internal detoxification of Al due to increased organic acid and secondary metabolite accumulation and ability to detoxify ROS due to enhanced secondary metabolite accumulation.To conclude,the increased pH-mediated mitigation of citrus seedling Al toxicity might involve enhancing the ability to maintain the stability of cell wall and plasma membrane,the homeostasis of phosphorus and energy,and the balance between ROS and aldehyde production and scavenging,as well as increasing the accumulation of secondary metabolites in roots and leaves.Additionally,both ECMs and hormones are involved in the increased pH-mediated alleviation of citrus seedling Al toxicity.These findings increase our understanding of the molecular and physiological mechanisms of increasing pH to alleviate Al toxicity in plants,and assist in develop new approaches for reducing plant Al toxicity.