Physiological And Molecular Mechanisms Of Citrus Sinensis Seedlings In Response To PH-cu Interactions

As the concentration of Cu²⁺in soils decreases with the increase of soil pH,the toxicity of copper（Cu）to plants largely depends on the soil pH.With the widespread application of physiological acidic fertilizers and increased acid deposition,soil acidification has occurred in many regions of the world.Citrus are mostly planted on acidic soils such as red soil,yellow soil,or latosol in south China,and are highly susceptible to low pH stress in the soils.In some old Citrus orchards,excessive Cu has gradually become an important factor limiting Citrus yield and quality due to the long-term and extensive application of Bordeaux mixture to control leaf and fruit diseases and pests.So far,most studies have focused on the response of Citrus to low pH or Cu-toxicity,while there are few studies on how Citrus responds to pH-Cu interactions.Additionally,most of studies focus on the impacts of pH-Cu interactions on the uptake of nutrients and water in Citrus.This study used sand potted‘Xuegan’（C.sinensis）seedlings as experimental materials.Seedlings were irrigated six times weekly with nutrient solution at a pH 3,4,or 4.8 and a Cu concentration of 0.5（control or non-Cu-toxicity）or 300（Cu-toxicity）μM from Cu Cl₂.After17 weeks of treatment,the effects of pH-Cu interactions on the growth;leaf exchange;the concentrations of Cu,and the metabolisms of reactive oxygen species（ROS）and methylgyoxal（MG）in leaves and roots of C.sinensis seedlings were were investigated.On this basis,4 treatment combinations[2 Cu（0.5 and 300μM Cu）levels×2 pH（pH 3 and pH 4.8）levels]were used to examine the effects of pH-Cu interactions on levels of endogenous hormones,as well as the transcriptome,metabolome and related physiological levels[non-structural carbon hydration,cell wall materials（CWM）and cell wall components]in leaves and roots.The objective of this study is to systematically and deeply elucidate the molecular and physiological mechanisms of C.sinensis seddlings in response to pH Cu-interactions from the physiological,transcriptome and metabolome levels,and to provide a theoretical basis for high-yield and high-quality cultivation of Citrus.The main research results are as follows:1.Effects of pH-Cu interactions on growth and leaf photosynthesis,as well as Cu concentrations,and ROS and MG metabolisms in leaves and roots of C.sinensis seedlingsIncreased pH could alleviate Cu-toxicity-induced decreases in seedling growth and leaf photosynthesis,and increases in leaf and root Cu concentrations,thereby improving their Cu-tolerance.Cu-toxicity exacerbated the inhibitory effects of low pH on seedling growth and leaf photosynthesis.With Cu-toxicity,the concentrations of Cu in roots and leaves increased with the decrease of pH;Without Cu-toxicity,the concentration of Cu in roots slightly increased with a decrease in pH,while the concentration of Cu in leaves showed no significant change.Low pH broke the balance between ROS and MG production and removal,thereby causing oxidative damage in 300μM Cu-treated roots（RCu300）and 300μM Cu-treated leaves（LCu300）,but not in 0.5μM Cu-treated roots（RCK）and 0.5μM Cu-treated leaves（LCK）.The effects of low pH and Cu-toxicity on the production and removal of ROS and MG in leaves and roots displayed a significant synergistic effect.2.Effects of pH-Cu interactions on hormone levels in leaves and rootsWe used targeted metabolomics to investigate the effects of pH-Cu interactions on hormone levels in leaves and roots.The results indicated that the effects of low pH and Cu-toxicity on leaf and root hormone levels exhibited a significant synergistic effect.High pH-mediated decreases of cytokinins（CKs）,abscisic acid（ABA）,jasmonates（JAs）and gibberellins（GAs）,increases of（±）strigol（ST）and 1-aminocyclopropanecarboxylic acid（ACC）,and efficient maintenance of salicylates（SAs）and auxins（AUXs）homeostasis in RCu300;as well as efficient maintenance of hormone homeostasis in LCu300 might contribute to improved leaf and root growth.The upregulation of AUXs（IAA）,CKs,ABA and SAs in pH3.0+300μM Cu-treated leaves（P3CL）vs.pH 3.0+0.5μM Cu-treated leaves（P3L）and pH 3.0+300μM Cu-treated roots（P3CR）vs.pH 3.0+0.5μM Cu-treated roots（P3R）might be an adaptive response to Cu-toxicity,so as to cope with the increased need for ROS and Cu detoxification in LCu300 and RCu300.The increased accumulation of stress-related hormones（JAs and ABA）in leaves and roots might reduce photosynthesis and accumulation of dry matter,and trigger leaf and root senescence,thereby inhibiting their growth.3.Responses of transcriptome,metabolome and related physiological parameters in response to pH-Cu interactions in leavesThe results of this study demonstrated that increased pH reduced Cu-toxicity-induced leaf Cu accumulation and oxidative damage by reducing ROS production and maintaining the homeostasis of sulfur（S）-containing compounds（reduced glutathione,GSH）,ascorbate（ASC）,and cell redox,thus mitigating Cu toxic effects on leaf chlorophyll biosynthesis,photosynthesis,and metabolisms of carbohydrates,lipids,amino acids,and secondary metabolism.The increased pH mitigated Cu-toxicity-induced impairment of cell wall metabolism by reducing cell wall Cu concentration,thus improving leaf growth.Under low pH,C.sinensis leaves also displayed some adaptive responses to Cu-toxicity to meet the increased demand for the dissipation of excess light energy and the detoxification of Cu and ROS,including:（a）increased distribution of Cu in cell wall;（b）elevated photorespiration and thermal dissipation;（c）increased accumulation of nonstructural carbohydrates and upregulation of metabolism related to energy production;（d）downregulated phospholipid[Lyso PC 18:3（2n isomer）]and other phosphorus（P）-containing compounds（2’-deoxycytidine-5’-monophosphate and adenosine 5’-monophosphate）and upregulated of L-tryptophan metabolism and related amino acids（L-tryptophan and5-hydroxy-L-tryptophan）;and（e）increased accumulation of some secondary metabolites[SMs;total phenolics,lignin,alkaloids（3-indoleacrylic acid,N-acetyl-5-hydroxytryptamine and methyl nicotinate）,plumerane（indole and 3-indolepropionic acid）and coumarins（isoscopoletin,scopoletin,skimming and scopolin）].However,these adaptive responses could not protect low pH-treated leaves from Cu-toxicity.Cu-toxicity intensified the adverse effects of low pH on C.sinensis leaves.4.Responses of transcriptome,metabolome and related physiological parameters in response to pH-Cu interactions in rootsThe results of this study indicated that increased pH enhanced the capacity in RCu300 to maintain Cu homeostasis by reducing Cu uptake,possibly resulting from increased organic acid accumulation and secretion by roots and Cu translocation to young leaves,and lessened Cu-toxicity-triggered oxidative damage through diminishing ROS formation and free fatty acid abundances,and maintaining the S-containing compounds（GSH）and ASC and cell redox homeostasis,thereby lessening Cu-toxic impacts on the abundances of primary metabolites（PMs）and SMs in roots.Increased pH prevented root senescence and cell wall metabolism impairments caused by Cu-toxicity through lowering Cu levels in roots and root cell wall,thus improving root growth.Additionally,low pH-treated roots also exhibited adaptive responses to Cu-toxicity,including:（a）increased Cu fractions in roots and root cell wall;（b）increased accumulation of non-structural carbohydrates and synthesis of ATP;（c）decreased accumulation of nitrogen（N）-and P（phospholipid）-compounds due to increased degradation;and（d）enhanced accumulation of SMs,especially phenolic acid,alkaloids,lignin and coumarins.Cu-toxicity intensified low pH effects on primary and secondary metabolisms in roots.In conclusion,increased pH could alleviated Cu toxicity in various ways,including:（a）enhanced capacity to maintain Cu homeostasis by reducing Cu uptake,and increasing the chelation of Cu and distributions of Cu in roots and cell wall;（b）enhanced antioxidant and MG detoxification system,and capacity to maintain GSH,ASC and redox homeostasis;（c）enhanced capacity to maintain the homeostasis of hormones such as SAs and AUXs.Additionally,low pH-treated seedlings also exhibited adaptive responses to Cu-toxicity,including:（a）increased concentration of cell wall components and S-containing compounds;（b）increased accumulation of non-structural carbohydrates and production of energy;（c）upregulated biosynthesis and accumulation of SMs,and downregulated accumulation of N-and P-compounds;and（d）elevated levels of hormones such as JAs and ABA.This study revealed the molecular and physiological mechanisms of increased pH-mediated-alleviation of C.sinensis Cu-toxicity,improved researchers’understanding of the response of C.sinensis to pH-Cu interactions,provided ideas for the related research,and also provided a theoretical basis for the high-yield and high-quality cultivation of Citrus.