Salicylic Acid And Its Mediated Ca²⁺ Signaling Regulate The Response Mechanism Of Black Soybean Under Al Stress

In acid soils,aluminum?aluminum,Al?has become an important limiting factor affecting crop yield.The main hazard of Al³⁺toxicity is the inhibition of root elongation.Aluminum can also induce a large number of reactive oxygen species to cause membrane root peroxidation of plant roots.The root exudation of organic acids is the most important detoxification mechanism to alleviate aluminum toxicity.Salicylic acid?SA?,as an endogenous signal molecule in plants,plays an important role in regulating plant response to biotic and abiotic stresses.Studies have confirmed that SA can effectively alleviate the Al toxicity of soybean roots.As an important second messenger in plants,calcium ion(Ca²⁺)is involved in many environmental stresses.In this paper,the sensitive black soybean in Yunnan is used as the experimental material.The method used in the study included the liquid culture method,immunoprecipitation assay,fluorescence probe detection and real-time fluorescence quantitative technique.The effect of exogenous SA on physiological and biochemical effects and citric acid secretion of Al sensitive black soybean root under Al stress was systematically investigated.In addition,the effect of SA on the expression of Ca²⁺signaling pathway and its related genes under Al stress was preliminarily explored to reveal whether`SA could alleviate Al stress through Ca²⁺signaling pathway,so as to further enrich the mechanism of alleviating Al toxicity in plants.The main results are as follows:1.After 30?M AlCl₃ treatment,the inhibition rate of the root elongation of black soybean was reduced to 41.39%as compared with control.The intracellular Al³⁺,H₂O₂,O₂^·-and MDA contents were significantly increased under Al stress.The membrane lipid peroxidation and plasma membrane permeability were also increased under Al stress.The cell death in the root tips was serious,causing oxidative damage.100?M SA significantly alleviated the inhibition of30?M AlCl₃ on root elongation,reversed the above oxidative damage events caused by Al poison.2.75?M AMP?adenosine monophosphate,plasmalemma H⁺-ATPase and 14-3-3 protein interaction inhibitor?and 10?M Na₃VO₄,?sodium vanadate,plasmalemma H⁺-ATPase inhibitor?respectively were co-treated with after Al+SA.The alleviating effect of SA on Al-induced root elongation inhibition was significantly weakened.The contents of intracellular Al³⁺,H₂O₂,O₂^·-and MDA were significantly increased when compared with the Al+SA treatment.The membrane lipid peroxidation and plasma membrane permeability were also increased when compared with the Al+SA treatment.The cell death was serious,and the oxidative damage was aggravated.The effect of SA on alleviating the Al toxicity is reversed.3.Treated with Al+SA,the secretion of citric acid was significantly increased than those of Al treatment.After the addition of AMP and Na₃VO₄,the secretion of citric acid was significantly decreased as compared with those of free of AMP and Na₃VO₄.The H⁺-ATPase phosphorylation of plasma membrane and the level of its interaction with 14-3-3f prorein in SA+Al treatment were significantly higher than that of Al treatment.After AMP exposure,H⁺-ATPase phosphorylation and its interaction with 14-3-3f decreased.The activity of plasma membrane H⁺-ATPase and the capacity of pump H⁺in Al+SA treatment were significantly higher than that of Al treatment.The activity of plasma membrane H⁺-ATPase and pump H⁺in Al+SA treatment was significantly decreased by the addition of AMP and Na₃VO₄.The results indicated that SA mainly regulated the level of phosphorylation of ATPase in plasma membrane and the level of interaction with 14-3-3f protein and then improved the activity of plasma membrane H⁺-ATPase and H⁺pump,promoting the secretion of citric acid in the root system.4.90?M CaCl₂ treatment significantly alleviated the inhibition of Al-induced root elongation of black soybean.Inhibitory effect in root elongation in Al+SA+La³⁺and Al+SA+EGTA was more significant than that in SA+Al treatment.Those observations indicated that Ca²⁺participated in SA-mediated Al stress response.Under the SA+Al treatment,the expression level of antioxidant enzyme genes?Gu-ZnSOD1,Gu-ZnSOD2,Gu-ZnSOD3,CAT3,CAT4,CAT5,PRX2B,PRX4?was significantly higher than Al treatment.After adding La³⁺in SA+Al treatment,these antioxidant enzyme genes were down regulated compared with those free of La³⁺addition.Ca²⁺was involved in positive regulating the above genes related to antioxidative enzymes in SA-mediated Al stress response.Compared with the SA+Al treatment,there was no significant differencein the expression of ACO and CS between SA+Al and SA+Al+La³⁺treatments.However,the expression of AlCT in SA+Al was significantly decreased after the addition of La³⁺.Ca²⁺participated in the SA-induced the increase of citric acid secretion under Al stress by regulating the expression of AlCT.Al induced the expression of calmodulin related genes?CML38,CML41,CaM4,CBL9?significantly,However only the expression of CBL9 was significantly increased after the addition of Al under Al stress.SA may activate CBL9/Ca²⁺signaling pathway and alleviate Al toxicity in black beans.