The Response Mechanisms Of Plasma Membrane H~+-ATPase In Masson Pine(Pinus Massoniana Lamb.) Roots To Acid Deposition

Acid deposition is a global environmental problem.Soil acidification and salt base cation leaching caused by acid deposition seriously damage the stability of terrestrial ecosystem and threaten forestry production.Masson pine（Pinus Massoniana Lamb.）is a perennial evergreen tree belonging to the genus Pinus,widely distributed in various provinces south of the Qinling-Huaihe River line in China,where acid deposition pollution is mainly concentrated in the regions south of the Yangtze River and east of the Yunnan-Guizhou Plateau.The plasma membrane（PM）H⁺-ATPase is a major functional protein widely present in plant cells.It pumps protons out of the cytoplasm to generate membrane potential and electrochemical H⁺gradients by consuming the energy produced by the hydrolysis of adenosine triphosphate（ATP）,thereby activating various ion channel proteins and secondary transporters to complete the transmembrane transport of substances,playing a key role in plant growth,development,and physiological activities.At present,there have been a lot of reports on the response mechanisms of plants to stresses,but the understanding of the tolerance and response mechanisms of Masson pine to acid deposition is not deep,especially the research on the role of PM H⁺-ATPase in Masson pine roots in the physiological regulation mechanism of self-tolerance under acid deposition is still blank.Therefore,further understanding the response and adaptability of Masson pine to acid deposition,clarifying the potential acid tolerance mechanisms of Masson pine related to root physiological processes,exploring the roles of root cellular signaling molecules,reactive oxygen species（ROS）and calcium ions(Ca²⁺),in the regulation of PM H⁺-ATPase activity during the response of Masson pine to acid stress,elucidating the physiological regulation mechanisms of acid resistance of Masson pine seedlings involving PM H⁺-ATPase in roots,providing a theoretical research basis for finding scientific methods to alleviate the loss of forestry production caused by acid deposition,and beneficial to the cultivation of Masson pine forests under acid deposition,is of great significance for the restoration of forest ecosystems eroded by acid deposition.This research focuses on the response of Masson pine seedlings to acid stress,using simulated acid rain（SAR）at different p H levels and chemical substances.The study examined the plant morphology,phenotypic traits,photosynthetic characteristics,and root physiological characteristics in response to acid stress.The key root physiological factors and the regulation of PM H⁺-ATPase activity during the response process were analyzed.The study also investigated the possible roles of root cellular ROS,cellular Ca²⁺,antioxidant enzymes,PM nicotinamide adenine dinucleotide phosphate（NADPH）oxidase,and PM Ca²⁺-ATPase in the defense mechanism regulated by root PM H⁺-ATPase during the response of Masson pine seedlings to acid stress and their signaling pathways,revealing the potential acid-tolerance mechanism involving the root PM H⁺-ATPase.The main results are as follows:（1）The effects of simulated acid deposition on the growth and physiological characteristics of Masson pine seedlings were related to the treatment acidity and the Masson pine family.Masson pine seedlings maintained good plant growth under SAR treatment at p H 4.5 and p H 5.6,but showed poor growth and a significant decrease in phenotypic parameters under SAR treatment at p H 3.5 and p H 2.5.In addition,SAR not only increased the membrane permeability of root PM in pine seedlings,but also induced the secretion of organic acids in the roots.Maintaining low permeability of root PM and high organic acid secretion under acid treatment helped Masson pine seedlings resist acid stress.Compared with the No.79 family Masson pine seedlings,the No.35family Masson pine seedlings maintained higher phenotypic parameters,chlorophyll content,and root vitality,as well as larger root absorption area,lower permeability of root PM,higher organic acid secretion,and rhizosphere soil p H under SAR treatment.（2）The PM H⁺-ATPase in Masson pine seedling roots played a regulatory role in the physiological defense process to acid deposition.Exposure to SAR and treatment with 0.1 m M Na₃VO₄（PM H⁺-ATPase inhibitor）increased the intracellular H⁺concentration in Masson pine roots.Compared to the control（p H 6.6）,SAR-treated Masson pine seedlings had higher primary root length,root dry weight,and number of root tips,while the addition of Na₃VO₄ led to significant growth inhibition and a decrease in phenotypic parameters.In addition,both the ATP hydrolysis and proton transport activities of root PM H⁺-ATPase in Masson pine seedlings were stimulated by SAR,and the maximum proton pump activity was observed in response to SAR exposure at p H 4.6.But Na₃VO₄ treatment had an inhibitory effect on PM H⁺-ATPase activity.Furthermore,SAR induced a relative increase in the expression levels of H⁺-ATPase subunits（Pmatp B,Pmatp E,Pmatp F,Pmatp H,and Pmatp I）in Masson pine roots,which promoted the stimulation of root PM proton pumping activity.（3）The cellular ROS and Ca²⁺were involved in the acid resistant physiological regulation of PM H⁺-ATPase in Masson pine roots as signaling molecules.Compared with the control,SAR treatment and the addition of exogenous H₂O₂ and Ca Cl₂ had little effect on the growth of Masson pine seedlings,and the phenotypic parameters remained stable.However,the application of Na₃VO₄,DMTU（1,3-dimethylthiourea,ROS scavenger）,DPI（diphenyleneiodonium chloride,PM NADPH oxidase inhibitor）,EGTA(ethylenebis（oxyethylenenitrilo）tetraacetic acid,Ca²⁺chelator),and La Cl₃(PM Ca²⁺-ATPase inhibitor)caused a substantial growth inhibition to Masson pine seedlings.SAR exposure and the addition of exogenous H₂O₂ or Na₃VO₄ increased the contents of cellular H₂O₂,O₂^·-,and malondialdehyde（MDA）,but those maintained at a relatively low levels with the use of DMTU and DPI.Additionally,the contents of H₂O₂ and Ca²⁺in the root cells of Masson pine seedlings significantly increased under SAR exposure and the addition of Ca Cl₂,but the contents of which were lower with the addition of EGTA or La Cl₃.Similarly,the enzymatic activities of antioxidants,PM NADPH oxidase,PM Ca²⁺-ATPase,and PM H⁺-ATPase in acid stressed Masson pine seedlings elevated with the increasing acidity,and a significant stimulation of these enzymatic activities obtained from SAR with exogenous H₂O₂ and Ca Cl₂ treatment was observed,whereas which decreased obviously with the addition of Na₃VO₄,DMTU,DPI,EGTA and La Cl₃.Moreover,a significant positive correlation was found between the PM H⁺-ATPase activity and the plant morphological attributes,cellular ROS contents,cellular Ca²⁺level,and the enzymatic activities of antioxidants,PM NADPH oxidase,and PM Ca²⁺-ATPase.The research further explained the response of the PM H⁺-ATPase in Masson pine roots to acid stress,clarified the acid-resistant physiological regulation mechanisms of Masson pine involving root PM H⁺-ATPase,deepened the understanding of the acid tolerance of Masson pine,and had extremely important theoretical and practical value for restoring forestry ecosystems and accelerating the construction of forestry ecological environment.