The Regulatory Mechanism Of Calcium On The Hydraulic And Nonstructural Carbohydrate Characteristics Of Pinus Sylvestris Var. Mongolica Under Drought

Drought-induced forest decline is a serious threat to global carbon balance and ecological security,which urgently requires a deeper mechanistic understanding of the drought-induced tree mortality process.There are also many other factors that may act to accelerate the tree decline and death.Calcium is a necessary nutrient for ecosystem structure and function.In ad-dition,calcium is a signal substance that regulates stomatal movement response to drought,which in turn affects transpiration and carbon assimilation.Plants mainly absorb calcium through the extracellular pathway with almost no reabsorption occurring,resulting in weak compensatory capacity for calcium.Therefore,exploring the regulatory role of calcium in forest drought physiology can provide an important theoretical basis for alleviating forest drought death.In this study,8-year-old Mongolian pine（Pinus sylvestris var.mongolica）were selected as the research object.Under two drought types（mild drought and severe drought）,calcium addition(0,5 and 10 m M Ca²⁺)experiments were carried out.The photosynthetic characteris-tics,xylem anatomy,hydrodynamic parameters,and non-structural carbohydrate（NSC）content of Mongolian pine under water stress were investigated using physiological and biochemical,scanning electron microscopy and transcriptomics techniques in response to exogenous calcium addition.And the regulatory effect of calcium on NSC content and hydraulic characteristics of Mongolian pine during drought was explored to enrich the understanding of physiological mechanisms of tree drought death,in order to improve the ability to predict extensive forest mortality in the context of global climate change.The main results of this study are as follows:（1）With or without the addition of exogenous calcium,sustained mild drought had no effect on NSC content in all organs.At the end of the drought,although 10 m M Ca²⁺signifi-cantly reduced the NSC content in the phloem of one-year-old branches,the NSC content in the xylem significantly were increased by 24.91%and 29.35%.Differential genes related to exog-enous calcium-regulated glucose entry into cells,sugar transport proteins,and carbohydrate transport biological processes were up-regulated under mild drought.（2）Under mild water stress,exogenous calcium balanced water transport and carbon as-similation by regulating stomatal movement,and maintains photosynthesis through rational en-ergy allocation.Exogenous calcium significantly reduced stomatal aperture by 25.94%and24.39%at the beginning of water stress（S1）to avoid hydraulic failure,while stomatal aperture significantly increased by 52.39%and 29.61%after calcium addition at the end of drought（S5）to increase net photosynthetic rate and stomatal conductance to ensure carbon supply.Mean-while,stomatal density was significantly increased by 17.82%and 21.34%after calcium addi-tion at the beginning of water stress.In addition,exogenous calcium increased DIo/CS to en-hance photosynthesis by avoiding photoinhibition.（3）Exogenous calcium reduces the risk of hydraulic failure in Mongolian pine under mild drought by regulating tubular cell structure and osmoregulation.Under water stress,calcium addition significantly increased predawn and midday water potential and hydraulic conductivity and decreased hydraulic conductivity loss rate.The exogenous calcium under water stress in S6significantly reduced the tracheid diameter by 16.63%and 33.16%,thickened the tracheid wall thickness by 26.50%and 10.4%and thickness span ratio（（t/b）²）by 74.63%and 350.27%,re-spectively,and increased the tracheid density by 124.36%and 230.53%,respectively.Differ-ential gene expression of exogenous calcium-regulated cellulose degradation-related enzymes was down-regulated and differentially expressed genes regulating lignin biosynthesis were up-regulated,which was consistent with the results of thickened tubular cell walls and dense wood density.Correlation analysis showed that xylem calcium concentration was significantly and positively correlated with xylem soluble sugars,starch,NSC content and tracheid wall thick-ness under mild drought,and significantly and negatively correlated with hydraulic conductiv-ity loss rate and tracheid diameter,indicating that exogenous calcium could increase xylem soluble sugars content and form a safer cell structure to reduce hydraulic failure.（4）During severe drought mortality,exogenous calcium prolonged the survival time of Mongolian pine and the longest survival time of 118 days was observed in 10 m M Ca²⁺treated.The soluble sugar,starch and NSC contents of each organ showed a decreasing trend with the prolongation of drought,but none of them was depleted.10 m M Ca²⁺treatment significantly increased soluble sugar and NSC contents of each organ,and the ratio of soluble sugar to starch in xylem was increased,which might be the reason for the longer survival time of Mongolian pine in drought.During drought,exogenous calcium significantly increased predawn and mid-day water potential and hydraulic conductivity and decreased hydraulic conductivity loss rate.LA/SA showed a decreasing trend with drought duration and drought increased LA/SA in 0m M Ca²⁺and 10 m M Ca²⁺treatments.LA/SA in 10 m M Ca²⁺treatment was increased at the beginning of drought and decreased significantly at the end,avoiding hydraulic failure.（5）During severe drought,exogenous calcium regulated stomata while maintaining pho-tosystem I electron transfer for carbon assimilation and safe water transport.At the early stage of drought mortality,exogenous calcium significantly reduced stomatal aperture of Mongolian pine by 12.11%and 20.64%,respectively;at the later stage stomatal aperture significantly in-creased by 25.95%and 13.29%.Drought resulted in higher Fo and Mo and lower Fm and Sm;lowerψPo,δRo andφRo.Exogenous calcium addition significantly increased Sm and Fm,and significantly decreased V_I and Mo.Ca10 also increasedψPo,δRo andφRo,improving PSⅠelectron transfer efficiency.In summary,this study clarified the regulatory effects of calcium on stomata,tracheid cell structure and photosynthetic physiology,demonstrated that calcium could minimize the risk of hydraulic failure under water stress while not increasing the risk of carbon starvation,enriched our understanding of the mechanisms underlying drought mortality,provided a theoretical foun-dation for predicting the risk of drought mortality in trees,and provided a theoretical basis for studying feasible methods to mitigate drought mortality in Mongolian pine.