Physiological,Cellular And Molecular Mechanisms Of Responsing To Calcium Deprivation In Arabidopsis Thaliana And Leymus Chinensis Root

Calcium(Ca²⁺)is an essential mineral nutrient element for plant growth and development,and is involved in the structure of cell walls and cell membranes.Meanwhile,it is an crucial second messenger in the cell,participating in various signaling pathways which induced by environmental stimuli.Ecological studies have revealed significant decreases in Ca²⁺levels in surface of soil because of acid rain,and widely occurring physiological Ca²⁺deficits worldwide.These changes may cause decreases in plant diversity and increases in plant vulnerability to environmental stress,but the underlying cellular mechanism is not well understood.In this study,we first focusing on endocytosis and membrane lipid metabolism responsing to the Ca²⁺deprivation in Arabidopsis thaliana and Leymus chinensis roots,and lipids in Arabidopsis thaliana and Leymus chinensis roots were compared in detail,studied the physiological,cellular and molecular mechanisms of responsing to calcium deprivation in both of them.This study demonstrates the essential role of Ca²⁺in maintaining plasma membrane stability and the selectivity of plant roots cells,and it is expected to provide a cellular and molecular explanation for the difference in response of calcium deprivation in arabidopsis thaliana and leymus chinensis.The main results are as follow:1.Decreasing extracellular[Ca²⁺]enhances endocytosis in arabidopsis roots.There was no much endocytosis activity in the roots with full nutrition control?CK?medium.However,endocytosis was dramatically induced by treatment with Ca²⁺-free(0 mM Ca²⁺)medium,enhanced endocytosis was not observed with other mineral nutrition deprivation treatments,including Mg²⁺,Zn²⁺,Mn²⁺,NO^3-,PO₄^3-or K⁺deprivation.Ca²⁺deprivation induced endocytosis occurs through a conserved endocytotic pathway,including initial plasma membrane invagination,early endosome formation,endosome separate from plasma membrane,multi-vascular polymerization and fusion into the vacuole.2.Ca²⁺deprivation induced endocytosis is a non-selective fluid phase-based process in arabidopsis roots.Membrane-impermeable green fluorescent dye without biological activity Alexa Fluor 488 can be packaged inside roots cells under 0 mM Ca²⁺,indicate that Ca²⁺deprivation decreasing the selectivity of plasma membranes in arabidopsis roots;Ca²⁺deprivation induced endocytosis is not sensitive to the inhibitor of receptormediated endocytosis Tyr A23,indicate it is not mediated by receptor.At the same time,the Ca²⁺influx inhibitor GdCl₃ has no significant effect on Ca²⁺deprivation induced endocytosis,but calmodulin antagonist W7 has significantly increased endocytosis in arabidopsis roots;IP3K inhibitor Wortmannin has no significant effect on Ca²⁺deprivation induced endocytosis,but IP3 signaling pathway inhibitor Neomycin has effect on endocytosis in arabidopsis roots.3.Ca²⁺deprivation significantly alters membrane lipid biosynthesis in arabidopsis roots.Lipid profiling analysis measured 15 types,379 species of membrane and storage lipids in arabidopsis roots,the content of PC?PE?PI?TAG?LPC?LPE significantly promoted by Ca²⁺deprivation,and suppressed DAG and PA biosynthesis.Meanwhile,the PC:PE molar ratio increased significantly under Ca²⁺deprivation conditions.All those changes are considered to be a series of countermeasures to maintain membrane structural stability and membrane lipid balance under Ca²⁺deprivation in arabidopsis roots.4.Leymus chinensis seedlings do not exhibit a Ca²⁺dependent growth phenotype,and the endocytosis in Leymus chinensis is a selective cellular response that is not mediated by receptors and is not affected by Ca²⁺deprivation.In this study,we found that there was no significant difference in the growth of Leymus chinensis seedlings among different Ca²⁺concentration medium,but the accumulation of Ca in Leymus chinensis depended on the external Ca²⁺concentration.Cell biology studies have found that there are abundant endocytosis under CK condition in Leymus chinensis roots,and this endocytosis is selective,not affected by Ca²⁺deprivation.The endocytosis in Leymus chinensis roots is also not mediated by receptors,GdCl₃has no but W7 has significantly increased endocytosis in Leymus chinensis roots as well as arabidopsis roots.Subsequently,experiments show that Agropyron desertorum and Elymus sibiricicus roots had the same endocytosis response as that in Leymus chinensis,and were also not affected by Ca²⁺deprivation.5.Ca²⁺deprivation has no significantly effect on membrane lipid biosynthesis in Leymus chinensis roots.Lipid profiling analysis measured 16 types,352 species of membrane and storage lipids in Leymus chinensis roots,it was found that Ca²⁺deprivation only increase FFA content,but has less effect on other lipids.There are only 43 individual lipids changed by Ca²⁺deprivation in Leymus chinensis roots,and the PC:PE molar ratio did not changed.Four FFA drugs were used to treat Leymus chinensis seedlings.Among them,FFA18:0 and FFA18:1 inhibited the growth of Leymus chinensis roots,and FFA18:2 partially inhibited the growth of Leymus chinensis root hairs.6.Decreasing extracellular[Ca²⁺]increases SOD activity,decreases MDA content in Leymus chinensis.The effects of Ca²⁺deprivation on membrane lipids metabolite in Leymus chinensis and arabidopsis seedlings are inconsistent.Among them,the activity of SOD enzymes increase,while the content of MDA decrease in the leave and roots of Leymus chinensis;instead,the activity of SOD enzymes decrease,while the content of MDA increase in the leave and roots of arabidopsis.At the same time,Ca²⁺deprivation has no effect on the content of H₂O₂ in Leymus chinensis leaf,but it induces the accumulation of more H₂O₂ in Leymus chinensis roots.7.There are great difference in the membrane lipid composition between Leymus chinensis and arabidopsis roots.Except the proportion of PA has no,the other 14 lipids has significant difference in the two plant.Among them,the proportion of PC,PI,MGDG,PG,CL,DGDG and LPC in Leymus chinensis roots higher than arabidopsis,the proportion of PE,DAG,TAG,PS,LPA,LPE and LPS in Leymus chinensis roots lower than arabidopsis.A total of 31 lipids are contained in Leymus chinensis roots but not in arabidopsis roots;a total of 66 lipids are contained in arabidopsis roots but not in Leymus chinensis roots.In summary,conclusions drawn from the above studies as followed:In arabidopsis roots,Ca²⁺deprivation specifically enhanced non-specific fluid phase-based endocytosis,and significantly alters the synthesis and metabolism of membrane lipids;The physiological response to Ca²⁺deprivation is different between Leymus chinensis and arabidopsis,that Ca²⁺deprivation has no effect on endocytosis and membrane lipid synthesis and metabolism in Leymus chinensis roots.