| Various environmental stresses can cause oxidative stress on plants,and it is of great significance to study the oxidative stress response mechanism in plants.Puccinellia tenuiflora is a salt-tolerant pasture that is widely distributed in the saline-alkali land of Northern China.The physiological and ecological mechanisms of salinity response in.tenuiflora have been extensively studied.Our previous studies have shown that.tenuiflora has specific signal and metabolic mechanisms in response to saline-alkali stress,but the molecular regulatory network in response to oxidative stress induced by saline-alkali stress is still not clear.In this study,the oxidative stress induced by salt-alkali stress was simulated by exogenous application of H2O2,and the molecular physiological mechanism of oxidative stress response in.tenuiflora was analyzed by using redox proteomics and molecular genetics approaches.Through analyses of the protein free sulfhydryl content,photosynthetic characteristics,chlorophyll fluorescence parameters,and reactive oxygen species(ROS)scavenging system,we found that oxidative stress led to oxidative damage in.tenuiflora,resulting in a decrease of protein free sulfhydryl content,inhibition of photosynthetic rate,and regulation of the activities of various antioxidant enzymes and the contents of non-enzymatic antioxidants.Redox proteomics studies showed that,under oxidative stress conditions(5 mM and 10 mM H2O2 treatments for 6 h),the abundances of 60 proteins were increased,and abundances of 58 proteins were decreased;70 peptides were more oxidized,and 51 peptides were reduced or over-oxidized.Meanwhile,the analysis of oxidative stress on isolated chloroplasts(5 mM and 10 mM H2O2 treatments for 10 min)indicated that,the abundances of 85 proteins were increased,and abundances of 58 proteins were decreased;20 peptides were more oxidized,and 20 peptides were reduced or over-oxidized.The change patterns of abundances and redox states of these proteins implied that Ca2+-mediated kinase signaling,membrane trafficking and cytoskeletal remodeling,ROS homeostasis,stress and defense,photosynthesis,gene expression and protein turnover,carbohydrate metabolism,amino acid metabolic pathways were pivotal to oxidative adaptation in.tenuiflora.In addition,our results showed that apoplastic germin like protein(PutGLP)was preferentially expressed in P.tenuiflora roots,and obviously induced when seedlings were exposed to saline-alkali and cadmium stresses.PutGLP possesses superoxide dismutase and oxalate oxidase activities and plays an important role in maintaining ROS homeostasis(O2·-and H2O2).Overexpression of PutGLP in wild-type Arabidopsis enhanced the root resistance to saline-alkali stresses.Moreover,our results indicated that PutGrxS10 belongs to the GrxC5/S12 subfamily of glutaredoxin(Grx)family,and has the conserved active motif "WCSYS" of GrxS12 subfamily.The redox state of PutGrxS 10 protein obtained by prokaryotic expression is regulated by reducing agents such as β-mercaptoethanol and DTT.Our redox proteomics results revealed that Cys78 in the conserved active motif of PutGrxS 10 was more oxidized under oxidative stress,suggesting that the redox regulated Cys78 may be involved in the modulation of its activity.PutGrxS 10 gene was expressed in roots,stems,leaves,flowers,sheaths,spikes and seeds of P.tenuiflora,and was induced significantly in response to H2O2,NaHCO3 and Na2CO3 stresses,respectively.This indicated that P.tenuiflora can modulate the ROS homeostasis in response to oxidative stress by regulating PutGrxS 10 transcription and post-translational modification.Based on the integration of physiological,redox proteomics and molecular genetics results,we hypothesized that P.tenuiflora applied several strategies to cope with oxidative stress,including:(1)modulation of Ca2+-mediated signal transduction by redox and reversible phosphorylation of important proteins,and thus regulating the expression of downstream oxidative stress-responsive genes;(2)regulation of membrane trafficking,protein transmembrane transport,lipid transport,and small molecules(water,ions,and metabolites)transport through modification of protein redox status;(3)dynamic remodeling of the cytoskeleton by regulating the cytoskeletal protein abundances;(4)maintaining intracellular ROS homeostasis by altering the abundances and redox states of important antioxidant enzymes;(5)regulation of the abundances and redox states of pathogenesis-related proteins in response to oxidative stress;(6)modulation of photosynthetic efficiency by regulating the abundances and redox levels of proteins involved in photo synthetic electron transport,ATP production and carbon assimilation;(7)induction of downstream response genes by increasing the abundances of proteins involved in transcription and protein synthesis,enhanced abundances of molecular chaperones to avoid protein oxidative damage,elevated abundances of protein degradation-related proteins to degrade oxidative damage proteins and thereby reduce their cytotoxicity;(8)regulation of the abundances and redox status of key enzymes that participate in carbohydrate metabolism,amino acid metabolism,and tetrapyrrole biosynthesis pathways to cope with oxidative stress.These results provide new clues for further study on the redox regulation network of plant saline-alkali stress response,and also provide important information for the breeding of salt-tolerant crops. |
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