Mechanism Of Calcium Regulating Water Balance Under Osmotic Stress In Maize Seedlings

Drought stress seriously affects the growth and development of plants and seriously threatens the food security and sustainable development of agriculture in China.It is particularly important to explore the cultivation ways and breeding methods that can effectively improve the drought resistance of crops,which is the basis of realizing sustainable high and stable agricultural yield and ensuring food security.Moreover,as an important second messenger in plants,Ca²⁺is widely involved in the signal transduction process of plant growth and development and environmental stress response.The water balance of plants is the dynamic balance between the water absorption of the plant roots and the water dissipation of the canopy.Maintaining water balance is the basis of all metabolic activities of plants.Especially when plants are under drought stress,the ability to maintain water balance is an important basis for plants to resist drought stress.With the vigorous development of research on stress signal transduction such as ABA,ROS,NO,and Ca²⁺,more abstract molecular-level studies need to be integrated into the study of plant water physiology,and many such studies focus on stomata guard cells are used as models.However,there are few reports about the mechanism of short-term and long-term exogenous calcium addition to maize to adapt to osmotic stress.Whether ABA,NO,ROS and other signaling molecules are involved in the regulation of Ca²⁺on the overall water balance of plants and the hydraulic characteristics of roots is still unclear.Regarding the issue above,short-term（2 h）and long-term（10 d）exogenous Ca（Ca Cl2:10 m M）induced drought induced by PEG（10%PEG-6000:-0.2 MPa）,the effects of gas exchange parameters,water status,whole plant hydraulic conductivity,root water conductivity,root cell water conductivity,xylem SAP osmotic potential,cell osmotic potential,ABA content,NO content,and the expression of water pore protein gene,Ca signal and key genes of ABA signal pathway.The purpose of this study was to clarify the effects of short-term and long-term exogenous Ca application on improving drought resistance of maize and to analyze the mechanism of exogenous Ca application through regulating water balance of maize under drought stress.The main results are as follows:Osmotic stress decreased the relative water content and leaf water potential of maize leaves,and inhibited the stomatal conductance,transpiration rate and photosynthetic rate of maize.Short-term exogenous calcium application can alleviate the inhibitory effect of osmotic stress on plant growth by increasing leaf stomatal conductance,leaf transpiration and whole plant water conductance;long-term exogenous calcium application can reduce leaf stomatal conductance,leaf transpiration and whole plant water conductance,reduces water loss to increase plant tolerance to osmotic stress.It shows that maize seedlings under different exogenous calcium treatments adopt different response strategies to regulate water balance under osmotic stress.Exogenous calcium treatment can enhance the antioxidant system of maize to reduce oxidative damage caused by osmotic stress;especially long-term exogenous calcium treatment maintains a high level of ROS in maize and maintains the homeostasis of the redox system by enhancing the antioxidant system.Through the analysis of the water conductance of the whole plant,the osmotic potential of xylem sap and the water conductance of root cortex cells,it was found that short-term exogenous calcium application improved the transpiration water absorption and osmotic water absorption capacity of maize seedlings,and promoted the root system to take transpiration pull and osmotic gradient as the main driving force longitudinal transport of moisture;However,long-term exogenous calcium treatment improved the osmotic water absorption and active water absorption capacity of root cortex cells,and improved the lateral transport of water mainly through the apoplast-symplast pathway.It indicated that maize seedlings adopted different root hydraulic strategies to respond to osmotic stress under different exogenous calcium treatments.Subsequently,q RT-PCR was used to quantitatively analyze the expression of plasma membrane water poprotein gene PIPS,and it was found that under normal water condition,both short-term and long-term Ca treatment down-regulated water poprotein gene Zm PIP1s（Zm PIP1;1,Zm PIP1;2,Zm PIP1;3,Zm PIP1;4,Zm PIP1;5）Expression level,and up-regulated Zm PIP2s Expression level.Under osmotic stress,the expression of Zm PIP1s and Zm PIP2s genes in maize seedling roots was significantly down-regulated,while exogenous Ca treatment,especially long-term treatment,significantly up-regulated the expression of Zm PIP1s and Zm PIP2s.The results indicated that the effect of exogenous Ca on the improvement of root water conductivity under osmotic stress was partly due to the enhancement of the expression of aquaporin gene by exogenous Ca,thus increasing its activity.In this study,the effects of exogenous calcium treatment on Ca signal,ABA and NO signal were further analyzed.Under normal water condition,short-term Ca treatment had no effect on the ABA content in roots and leaves,while long-term Ca treatment increased the ABA content in roots and leaves.Under osmotic stress,the content of ABA in maize tissues was significantly increased,and the content of ABA in maize tissues was further increased under short-term or long-term Ca treatment,and the increase was greater under long-term Ca treatment.Short-term Ca treatment down-regulated the expression of Zm NCED,Zm ABA3and Zm AO2 genes related to ABA synthesis,while long-term Ca treatment significantly up-regulated the expression of these three genes.In addition,exogenous Ca also significantly affected the expression of Zm PP2C,Zm ABI1,Zm ABI2 and Zm Sn RKs,key genes in ABA signal transduction pathway under osmotic stress.These results indicated that exogenous Ca could significantly affect the ABA response of maize under osmotic stress,thus improving its drought resistance.After osmotic stress treatment,NO content in roots and leaves of maize seedlings increased significantly.Short-term exogenous Ca treatment increased NO content in roots but decreased NO content in leaves.Long-term Ca treatment significantly increased NO content in roots and leaves of maize.In conclusion,osmotic stress can cause obvious NO signal in maize seedlings,and exogenous Ca,especially long-term Ca treatment,can further enhance NO signal.Analysis of NOS activity in maize tissues showed that NOS activity was highly consistent with NO content,and the effect of osmotic stress or exogenous Ca treatment on NO signal was probably realized by changing NOS activity.The key factors of Ca signaling pathway calmodulin Ca M and the coding genes of Ca-dependent protein kinase CPK,Zm CAM1,Zm CPK1,Zm CPK4 and Zm CPK11,were quantitatively analyzed.It was found that short-term Ca treatment significantly down-regulated the expression of these key factors of Ca signaling pathway in maize seedling roots under osmotic stress.Long-term Ca treatment significantly upregulated the expression of key factors in these Ca signaling pathways.These results indicate that exogenous Ca treatment can significantly affect the Ca signaling pathway,but the mode of effect of short-term Ca treatment is different from that of long-term Ca treatment.In this study,exogenous Ca may enhance the osmotic regulation ability of root cells and enhance the activity of water pore protein by affecting Ca signal,ABA and NO signal pathways,thus improving the water absorption and transportation ability of maize roots,maintaining a high stomatal opening,and alleviating the inhibition of osmotic stress on photosynthesis of plants.This study reveals the internal mechanism of exogenous Ca application to enhance drought resistance of crops,and has important guiding value for reducing the adverse effects of drought on agricultural production through reasonable and effective Ca fertilizer in agricultural production.