Effect Of Exogenous Ca²⁺ On Plasma Membrane Composition, Ca²⁺Distribution And Transport In Rice Roots Under Acid Rain Stress

Acid rain is a global environmental problem,causing huge economic losses to agriculture and forestry.Facing the challenge of global population growth to food safety,it is urgent to reduce the harm of acid rain to plants.Calcium（Ca）is an important signal molecule that participates in the regulation of plants tolerance to abiotic stress.In particular,the Ca²⁺transport system regulates the concentration of Ca²⁺in the cytoplasm and plays an important role in protecting the structure and function of the plant plasma membrane.In order to clarify the regulation mechanism of exogenous Ca²⁺on the Ca²⁺environment in plant cytoplasm under acid rain stress,this study uses hydroponics to explore the effect of exogenous Ca²⁺on plasma membrane composition,calcium form,calcium distribution and Ca²⁺transport system in rice（Oryza sativa L.）roots under the simulated acid rain（SAR）stress.The main results are as follows:（1）Effect of exogenous Ca²⁺on plasma membrane composition and stability in rice roots under SAR stress showed that pH 4.5 SAR increased the contents of phosphatidylcholine（PC）,phosphatidylglycerol（PG）,phosphatidylethanolamine（PE）and membrane protein in plasma membrane of rice roots,but had no significant effect on membrane stability and growth of rice roots.SAR at pH 3.0 reduced the contents of PC,PG and membrane protein in the plasma membrane of rice roots,thereby destroying the stability of cell membranes and inhibiting growth of rice roots.The plasma membrane composition of rice roots treated with single Ca²⁺and pH 4.5+Ca²⁺had no different in that of the control.The content of phospholipid monolayer（PE）in the plasma membrane of rice roots treated with pH 3.0+Ca²⁺was still lower than that of the control,while the contents of phospholipid bilayer lipid and membrane protein were at the control level.This indicates that exogenous Ca²⁺can reduce the degradation of plasma membrane compotion in rice roots caused by SAR,thereby protecting the membrane stability and maintaining growth of rice roots.After a 5-d recovery,contents of phospholipid and membrane protein in rice roots treated with pH 3.0 SAR were still lower than the control.Exogenous Ca²⁺promoted the recovery of plasma membrane composition,the membrane stability and growth of rice roots.（2）Effect of exogenous Ca²⁺on calcium form and distribution in rice roots under SAR stress showed that SAR（pH 4.5 or 3.0）stimulated the increase of free Ca²⁺of water-soluble calcium in cytoplasm of rice roots.The free Ca²⁺concentration increased more and the Ca²⁺was unevenly distributed around the plasma membrane of rice roots treated with pH 3.0 SAR,and a large amount of non-water-soluble calcium（including calcium nitrate-calcium chloride,fruit calcium collates and calcium phosphate-calcium carbonate）was lost in the cell wall, cytoplasm and organelles.The single Ca²⁺treatment increased the content of water-soluble calcium in the cytoplasm and calcium oxalate in the organelles of rice roots.In the SAR（pH4.5 or 3.0）+Ca²⁺treatment group,the free Ca²⁺in the water-soluble calcium in the cytoplasm of rice roots was higher than that of the control,but lower than treated with SAR,and the content of non-water-soluble calcium in the cell wall and organelles was significantly higher than that of the control.It shows that exogenous Ca²⁺can promote the migration of water-soluble calcium in the cytoplasm of rice roots to the cell wall and organelles under SAR stress,thereby forming non-water-soluble calcium and alleviating the accumulation of cytoplasmic free Ca²⁺.It not only alleviated the damage of membrane stability caused by free Ca²⁺accumulation in cytoplasm,but also made up for the loss of calcium in cell wall and organelle.After a 5-d recovery,the calcium content in the cell wall,cytoplasm and organelles of rice roots treated with pH 3.0 SAR was still lower than that of the control,and exogenous Ca²⁺was beneficial to the recovery of various forms of calcium contents as well as calcium content in cell wall,cytoplasm and organelle in rice roots in rice roots.Correlation analysis between the plasma membrane composition and the form and distribution of calcium in rice roots treated with exogenous Ca²⁺and SAR revealed that the contents of total phospholipids,PC,PG and membrane protein was significantly negatively correlated with the content of water-soluble calcium,and significantly positively correlated with the contents of calcium pectate and calcium phosphate-calcium carbonate.Exogenous Ca²⁺promoted the conversion of water-soluble calcium in the cytoplasm of rice roots into calcium pectate and calcium phosphate-calcium carbonate under SAR,this may be an important reason for alleviating the damage of SAR to the plasma membrane composition of rice roots.（3）The effect of exogenous Ca²⁺on the Ca²⁺transport system of rice roots under SAR stress showed that pH 4.5 SAR up-regulated the expression of Ca²⁺channel genes（Os CNGC9,Os CNGC13,Os TPC1 and Os MAC1）to activate Ca²⁺channel,promoting the influx of extracellular Ca²⁺into the cytoplasm.Among them,pH 3.0 SAR decreased the expression of Ca²⁺-ATPase genes（Os ACA2,Os ACA4）and Ca M activity,and inhibited Ca²⁺-ATPase and Ca²⁺/H⁺transporters activity in plasma membrane,resulting in the inhibition of extracellular transport of Ca²⁺.At the same time,the activity of vacuolar Ca²⁺-ATPase treated with high-intensity SAR（pH 3.0）was decreased,and cytoplasmic Ca²⁺could not be transported to the vacuole in time,so a large amount of Ca²⁺accumulated in the cytoplasm of rice roots,causing damage to the plasma membrane.In the single Ca²⁺treatment,the expression of Os CNGC13in plasma membrane was up-regulated,and the intracellular Ca²⁺was increased.The increase of Ca M activity and Os ACA2 expression increased the activity of Ca²⁺-ATPase,and the Ca²⁺in cytoplasm was pumped out,thus avoiding the accumulation of Ca²⁺in cytoplasm.Therefore,the single Ca²⁺treatment maintained the intracellular Ca²⁺homeostasis,it may be an important reason for protecting the components and stability of plasma membrane in rice roots.SAR（pH 4.5 and 3.0）+Ca²⁺treatment increased the gene expression of plasma membrane Ca²⁺channel,further activated Ca²⁺channel,enhanced extracellular Ca²⁺influx.However,the increase of Ca M activity,Os ACA2 and Os ACA4 expression levels enhanced Ca²⁺-ATPase and Ca²⁺/H⁺transporter activities,promoted extracellular Ca²⁺transport.The increase of vacuolar Ca²⁺-ATPase activity accelerated cytoplasmic Ca²⁺transport to vacuoles,thus alleviated the accumulation of Ca²⁺in cytoplasm.After a 5-d recovery,the activity of Ca²⁺-ATPase and Ca²⁺/H⁺transporters in rice roots treated with pH 3.0 SAR was still low.The application of Ca²⁺promoted the recovery of Ca²⁺channel gene expression level as well as Ca²⁺-ATPase and Ca²⁺/H⁺transporter activities in rice roots to the control level,being conducive to maintaining cytoplasmic Ca²⁺homeostasis.In summary,exogenous Ca²⁺up-regulates the gene expression of plasma membrane Ca²⁺channel in rice roots under acid rain stress,promoting extracellular Ca²⁺influx.Then,exogenous Ca²⁺increased the Ca²⁺-ATPase activity in plasma membrane and vacuole membrane by increasing acid rain stress rice root Ca M activity and expression of Os ACA2and Os ACA4,increased the Ca²⁺/H⁺transporter activity,accelerating the transport of cytoplasmic Ca²⁺to the cell wall and organelles to form calcium pectate and calcium phosphate-calcium carbonate,and then alleviated the damage to the plasma membrane phospholipid bilayer lipid and membrane protein caused by excessive accumulation of Ca²⁺in the cytoplasm.,maintaining the stability of the cell membrane,,and finally be benefit fro reducing the inhibition of acid rain on rice roots growth and improving the tolerance of rice to acid.