The Role Of Nitric Oxide In Antioxidant Defense Induced By Water Stress And Abscisic Acid In Leaves Of Maize Plants

Abscisic acid(ABA) can regulate a variety of physiological and molecular responses for plant against stresses,including drought,chilling,salinity,and so forth.Nitric oxide(NO) is a multifunctional bioactive molecule and as a signal is involved in gene activation,protein expression and activity regulation of enzymes response to stresses in plants.Recent work showed that ABA can cause the generation of NO and hydrogen peroxide(H₂O₂),and the increases in the levels of calcium([Ca²⁺]_i) and calmodulin(CAM) in maize leaves,which induce the expression of antioxidant genes,and enhance the capacity of antioxidant defense systems.NO is an important intermediate component in the ABA signaling.However,the detailed mechanism about how NO mediates ABA signaling in plant cells remains to be determined.In the present study,the role of NO and the relationship between H₂O₂,Ca²⁺/CaM and NO in ABA-and water stress-induced antioxidant defense in leaves of maize(Zea mays L.) plants were investigated.The results are as follows:In order to investigate the production of NO in leaves of maize plants exposed to CaCl₂ treatment,the leaf segments were loaded with the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate(DAF-2DA) and confocal laser scanning microscopy (CLSM) was used to monitor changes in NO-induced fluorescence,10 mM CaCl₂ led to a rapid increase of NO.The generation of NO maximized at 1 h and remained high for 2 h after CaCl₂ treatment,and then decreased after 2 h of CaCl₂ treatment.After the 1 h of CaCl₂ treatment,the activity of NOS reached the maximum values.Treatment with CaCl₂ for 1 h enhanced the activity of NOS in cytosolic and microsomal fractions by 92.7%and 148%, respectively,compared with the control values.To establish a link between NO,Ca²⁺/CaM and antioxidant defense in ABA signaling,the detached plants were pretreated with Ca²⁺ inhibitors or CaM antagonists,respectively,and then exposed to ABA,H₂O₂ or CaCl₂ treatment.Experimental results showed that pretreatments with Ca²⁺ inhibitors or CaM antagonists nearly fully arrested ABA-,H₂O₂- or CaCl₂-induced increases in the generation of NO and the activity of NOS,and also blocked the activities of the chloroplastic and cytosolic antioxidant enzymes SOD,APX,GR and the expression of the genes cAPX,GRl and SOD4 induced by SNP treatment in leaves of maize plants.These results indicate that the NOS, which mediates ABA- and H₂O₂-induced NO production,is Ca²⁺/CaM-dependent,and Ca²⁺/CaM is required for NO-induced subcellular antioxidant defense.On the other hand,our results showed also that exogenous NO led to significant increases in the levels of Ca²⁺/CaM. ABA-induced the increases in levels of Ca²⁺/CaM were partially blocked by pretreatments with the NO scavenger c-PTIO and the NOS inhibitor _L-NAME,suggesting that NO is involved in ABA-induced increases in the levels of Ca²⁺/CaM.These results suggest that Ca²⁺/CaM functions both upstream and downstream of NO production,which is mainly from NOS,in ABA- and H₂O₂-induced antioxidant defense in leaves of maize plants.Water stress induced the increases in the generation of NO in mesophyll of maize leaves. NO was detectable as early as 1 h after water stress treatment and maximized at 4 h,and then declined.Water stress induced the increases in the activity of NOS in maize leaves.The activity of NOS maintained at a continuously ascending trend during 4-h of water stress treatment.After 4 h of water stress treatment,the activity of NOS in cytosolic and microsomal fractions reached maximum values,which were 4.3-fold and 7.2-fold, respectively,higher than those in the controls.These results suggest that the activity of NOS in cytosolic and microsomal fractions is remarkably induced by water stress,and the activity of NOS in microsomal fraction was higher and more susceptible to water stress than that in cytosolic fraction.Pretreatments with _L-NAME and PBITU completely blocked the increases in the activity of NOS in cytosolic and microsomal fractions induced by water stress treatment. Pretreatments with c-PTIO,KCN and NaN₃ inhibited the generation of NO but hardly affected the activity of NOS in cytosolic and microsomal fractions induced by water stress treatment.These results suggest that NOS and NR are involved in water stress-induced NO production and NOS is the major source of NO.To investigate whether water stress-induced H₂O₂ production is regulated by NO,the effect of SNP on water stress-induced the activities of subcellular antioxidant enzymes and the accumulation of H₂O₂ were examined,by using the methods of histochemistry with 3,3-diamino benzidine(DAB) staining,spectrophotometry in leaf extracts,and cytochemistry with CeCl₃ staining and transmission electron microscopy,respectively.Experimental results showed that pretreatment with SNP resulted in remarkable increases in the activities of water stress-induced the chloroplastic and cytosolic antioxidant enzymes superoxide dismutase (SOD),ascorbate peroxidase(APX),and glutathione reductase(GR),which decrease the accumulation of H₂O₂ induced by water stress treatment.However,pretreatment with Fe(â…¢)CN had a very little effect on the accumulation of H₂O₂,and reduced the activities of chloroplastic and cytosolic antioxidant enzymes GR,SOD and APX induced by water stress treatment.Pretreatments with NO scavenger,NOS inhibitors and NR inhibitors markedly blocked the increases in the activities of the chloroplastic and cytosolic antioxidant enzymes SOD,APX,GR and the expression of the genes cAPX,GRl and SOD4 induced by H₂O₂ or water stress,indicating that NO is involved in H₂O₂-and water stress-induced subcellular antioxidant defense.These results suggest that the potential ability of NO to scavenge H₂O₂ is, at least in part,due to the induction of subcellular antioxidant defense.The relationship between ABA,Ca²⁺/CaM and NO in water stress-induced antioxidant defense in leaves of maize plants were investigated.The application of ABA synthesis inhibitor blocked the generation of NO and the increase in the activity of NOS induced by water stress treatment,indicating that ABA is a key inducer of the induction of NO and the activation of NOS under water stress.NO is involved in water tress-induced accumulation of ABA and increases in the levels of Ca²⁺/CaM.On the other hand,pretreatments with Ca²⁺ inhibitors or CaM antagonists nearly fully arrested water stress-induced increases in the generation of NO and the activity of NOS,indicating that Ca²⁺/CaM mediates NO generation in leaves of maize plants exposed to water stress.In addition,our results showed also that water stress induced NO production in isolated chloroplasts and mitochondria of maize leaves. Pretreatments with Ca²⁺ chelator and CaM antagonist almost fully blocked water stress-induced NO production in mitochondria,but did not affect NO production in chloroplasts.These results suggest that there exists different species of NOS in chloroplasts and mitochondria of maize leaves.Taken together,our results suggest that the increases in the levels of Ca²⁺/CaM trigger NO production by activation of NOS,which then affects the levels of Ca²⁺/CaM,thus resulting in up-regulation in the activities of antioxidant enzymes and the expression of related genes in different subcellular compartments,and a crosstalk between NO and Ca²⁺/CaM plays a pivotal role in the ABA signaling transduction.