The Physiological Mechanism Of Na~+ Transport And Compartmentalization In Nitraria Sibirica Pall. Seedlings Under Salt Stress

Soil salinization is one of the important limiting factor affecting the sustainable development of forestry and the construction ecological environment in the world.The damage of salt stress to plants is mainly caused by the high concentration of salt ions?especially Na⁺?in the soil,and excessive Na⁺enters plants results in ion imbalance and toxicity.Maintaining the homeostasis of K⁺/Na⁺ratio and low absolute concentration of Na⁺in cells are the key factors for plant salt tolerance.Therefore,in-depth analysis of plant Na⁺absorption,transport and distribution rules is an important basis for revealing the ion balance regulation and salt adaptation mechanism under salt stress.Nitraria sibirica Pall.has strong salt resistance ability,the study of its regulation mechanism of Na⁺absorption,transport and compartmentalization under salt stress,is of great significance for revealing the mechanism of plants salt-tolerance,excavating salt-tolerant genes and cultivating new salt tolerant germplasm.This study intents to use hydroponic Nitraria sibirica seedling treated with NaCl as the research material.Physiological,pharmacological and molecular biological test methods were used to study the growth response,Na⁺and K⁺distribution,compartmentalization characteristics at levels of tissue and cell level and root dynamic ion exchange characteristics of Nitraria sibirica seedlings under NaCl stress.Comprehensive analysis of Na⁺absorption and compartmentalization characteristics and physiological regulation mechanism of Nitraria sibirica seedlings.The main finding as follows:?1?Nitraria sibirica has strong salt tolerance.The growth of Nitraria sibirica seedling under 100-200 mmol·L^-1NaCl treatment was significantly higher than that under control and high concentration(?400 mmol·L^-1)NaCl treatment.The plant height,ground diameter,the growth of root and leaf,the biomass accumulation,root to shoot ratio and relative biomass of Nitraria sibirica seedlings increased first and then decreased with the increase of NaCl concentration,and the maximal values were at 200 mmol·L^-1NaCl treatment compared with the control.However,high concentration NaCl(?400 mmol·L^-1)treatment significantly reduced the growth of Nitraria sibirica seedlings,and the salt tolerance index?STI?was decreased.?2?The Nitraria sibirica seedlings enhances the adaptability of salt stress by maintaining the relative balance of ions in the root and enhancing the selective transport and distribution of ions to stems and leaves.The content of Na⁺and Cl^-in roots,stems and leaves were significantly increased with the increase of NaCl treatment,and Na⁺and Cl^-was mainly accumulated in leaves;The content of K⁺in roots were significantly increased with the increase of NaCl treatment;The content of Ca²⁺and Mg²⁺in roots,stems and leaves were stay steady or rise under 200 mmol·L^-1 NaCl treatment whereas decreased under 400 mmol·L^-1NaCl treatment.With the increase of NaCl concentration,the K⁺/Na⁺,Ca²⁺/Na⁺,Mg²⁺/Na⁺ratio in different organs of seedlings were decreased,and the K⁺/Na⁺,Ca²⁺/Na⁺,Mg²⁺/Na⁺ratio in root was always higher than that of stem and leaves.The root-stem selective transport capacity SK,Na,SCa,Na,SMg,Na and stem-leaf selective transport capacity SK,Na,SCa,Na,SMg,Na were remained stable or elevated under NaCl treatment,and stem-leaf selective transport capacity SCa,Na,SMg,Na were higher than root-stem selective transport capacity SCa,Na,SMg,Na.?3?The Nitraria sibirica seedlings can improve the abapt ability of salt stress by enhancing the ability of retain K⁺and Na⁺exclusion to maintenance the relative balance of K⁺/Na⁺in root.Non-invasive Micro-test Technology?NMT?data shows that NaCl stress significantly increased the steady Na⁺efflux from the Nitraria sibirica seedlings roots.Steady K⁺effluxes were measured in the control roots?without NaCl?and in the roots treated with 200mmol·L^-1 NaCl,and no significant differences were observed between the two treatments.While the steady K⁺efflux from roots treated with 400 mmol·L^-1 NaCl decreased gradually.Pharmacological experiments showed that amiloride and sodium vanadate significantly inhibited the Na⁺efflux and H⁺influx,suggesting that the Na⁺efflux was mediated by a Na⁺/H⁺antiporter using energy provided by plasma membrane H⁺-ATPase.The NaCl-induced root K⁺efflux was inhibited by the K⁺channel inhibitor tetraethylammonium chloride?TEA?,and it was significantly increased by the H⁺-ATPase inhibitor sodium vanadate,suggesting that NaCl-induced K⁺efflux was mediated by depolarization-activated outward-rectifying K⁺channels and nonselective cation channels.Under salt stress,Nitraria sibirica seedlings showed increased H⁺efflux due to increased plasma membrane H⁺-ATPase and Na⁺/H⁺antiporter activity.High H⁺pump activity not only restricts the Na⁺influx but also limits K⁺leakage,leading to maintenance of the K⁺/Na⁺balance and higher salt tolerance.?4?The Nitraria sibirica seedlings increases the adaptability of salt stress by accumulating Na⁺in the old leaves and retaining K⁺in the young leaves to protect the growth of young leaves.With the increase of NaCl concentration and the prolonged of stress time,the Na⁺content in old,mature and new leaves increased significantly,while the K⁺content decreased significantly.Among them,the Na⁺content in the old leaves was the highest,the Na⁺content in the young leaves was the lowest,while the K⁺content were the highest in the young leaves,and the lowest in the old leaves.With the increase of NaCl concentration and the prolonged of stress time,the K⁺/Na⁺ratio in old leaf decreased significantly,while there was no significant difference in the K⁺/Na⁺ratio in the young leaves of the mature leaves under the treatment of200 and 400 mmol·L^-1 NaCl,and the K⁺/Na⁺ratio in the young leaves was significantly higher than that in the mature and old leaves.?5?The Nitraria sibirica seedlings improve salt tolerance of leaf tissue by Na⁺vacuolar compartmentalization.With the increase of NaCl concentration and the prolonged of stress time,Coro Na Green specific fluorescence was significantly enhanced in the vacuoles of leaf palisade tissue.Meanwhile,NMT results also showed that the net Na⁺flux in mesophyll cells was significantly reduced with the increase of NaCl treatment concentration,indicating that Na⁺in leaves was mainly isolated into the vacuoles of palisade tissue cells.At the same time,the activity of plasma membrane?PM?and tonoplast H⁺-ATPase and tonoplast H⁺-PPase of Nitraria sibirica leaves were significantly enhanced,and the expression of tonoplast Na⁺/H⁺antiporter?Ns NHX1?,tonoplast H⁺-PPase?Ns VP1?and plasma membrane Na⁺/H⁺antiporter?Ns SOS1?were increased.But the activity of tonoplast H⁺-ATPase and H⁺-PPase was significantly higher than plasma membrane H⁺-ATPase activity,the expression of Ns NHX1and Ns VP1 was significantly higher than Ns SOS1,suggesting that Nitraria sibirica mainly through enhancing Na⁺compartmentation in vacuolar to regulate ionic blance in cytoplasm.The variation trend of Ns NHX1 and Ns VP1 gene expression was the same as that of the activity of tonoplast H⁺-ATPase and H⁺-PPase.This indicates that the proton pumpa activity of the tonoplast and the Na⁺/H⁺antiporter synergistically transport Na⁺into the vacuole to regulate the ion homeostasis in the cells to adapt salt stress.In conclusion,Nitraria sibirica seedlings in the root by increasing the activity of Na⁺/H⁺antiporter and proton pump to increase Na⁺efflux and K⁺retention,thereby maintaining the relative balance of root K⁺/Na⁺,while transporting a large amount of Na⁺to the leaf and compartmentalizing in the vacuole,which improved the osmotic regulation ability while maintaining the low Na⁺content in the cytoplasmic solution,so as to maintain normal physiological activities under salt stress.Nitraria sibirica adopts different salt tolerance strategies in roots and leaves,showing obvious tissue specificity salt tolerance,and can highly coordinate these physiological characteristics to make Nitraria sibirica have higher salt tolerance.