Redox Function Analysis Of PutGRXS12 From Puccinellia Tenuiflora

The Glutaredoxin（GRX）family is a ubiquitous small oxidoreductase,which belongs to the thioredoxin superfamily.In plants,GRXs mediate the reversible reduction of disulfide bond in substrate proteins depended on GSH,thereby regulating the redox state of cells.Besides,they work as an iron-sulfur cluster scaffold protein for the synthesis and transmission of iron-sulfur clusters.Therefore,GRXs play an important role in plant development and stress response.Puccinellia tenuiflora is a perennial gramineous grass with strong salt-alkali tolerance,and is taken as an ideal model for the investigation of plant stress response.Our previous redox proteomics have revealed that PutGRXS12 is critical for P.tenuiflora in response to oxidation and salt-alkali stresses.In this study,we conducted a phylogenetic tree and analyzed the amino acid sequence of homologous proteins of the GRX family,which indicated that PutGRXS12belongs to the GRXC5/S12 subfamily of class I GRX,and has the closest homology relationship with rice Os GRXS12.Chloroplast-localized PutGRXS12 contains two Cys-contained signal peptide,a"WCSYS"conserved active motif at the N-terminus with two conserved Cys,and a"GGC"conserved domain at the C-terminus.The predicted three-dimensional structure of PutGRXS12 shows that the two conserved Cys sites do not form intramolecular disulfide bond.When the Cys78 and/or Cys136 of PutGRXS12 were replaced by serine（Ser）,its redox activity was changed.The PutGRXS12 protein separated by SDS-PAGE was detected and identified by Western blot and mass spectrometry.The PutGRXS12-6His fusion protein expressed from prokaryotic cells formed in monomer（14k Da）,dimer（28k Da）,and multimer under natural condition.However,the proteins with C78S or C136S only have monomer and dimer,but no multimers,the protein with C78S and C136S was hard to resolved in extraction buffer.These indicated that PutGRXS12would form multimers by intermolecular disulfide bonds between Cys78 and Cys136.HED enzyme activity detection and redox sensitivity analysis revealed that the Cys78and Cys136 in PutGRXS12 were key sites for the deglutathionylation or disulfide bond reduction of its target proteins.Using the A.thaliana protoplast and tobacco transient expression system,we found that PutGRXS12 localized in chloroplasts,and the Cys mutation does not affect its subcellular localization.Furthermore,we analyzed the phenotypes of A.thaliana wild-type,mutant atgrxs12,PutGRXS12 overexpression plants（i.e.,normal,C78S,C136S,and C78SC136S）,and PutGRXS12 replenishment atgrxs12 plants（i.e.,normal,C78S,C136S,and C78SC136S）.Overexpression of PutGRXS12 promotes the vegetative growth of rosette leaves,plant bolting,branches,and grain yield.Under oxidative and salt stresses,the root growth rate and seed germination rate of overexpression PutGRXS12 were increased,but the chlorophyll accumulation were decreased.In addition,the results of NBT staining showed that O₂^·-accumulation was obviously reduced in leaves from overexpression of PutGRXS12 plants,and less in PutGRXS12 replenishment atgrxs12 plants than that in WT.All these implied that PutGRXS12 could enhance the plant salt and oxidation tolerance by regulating cell redox homeostasis.We used the Cys mutation capture target protein technology to catch the target proteins that interact with PutGRXS12 in P.tenuiflora leaves,and identified 107candidate target proteins using tims TOF high-resolution mass spectrometer.Our results implied that Cys78 in PutGRXS12 is very important for its binding ability.The PutGRXS12 target proteins are mainly involved in amino acid biosynthesis,cofactor metabolism,carbon metabolism,light capture,cadmium ion response,temperature stimulation,protein complex aggregation,as well as water shortage response.In summary,chloroplast-localized PutGRXS12 mediate diverse metabolic pathways by regulating the redox state of its target proteins in chloroplasts,which is critical for regulating plant vegetative growth,bolting,flowering,branches,grain yield,seed germination,as well as saline-alkali stress response.These results provide important information for in-depth understanding of the redox regulatory networks during plant development and stress response processes,and lay an vital foundation for the molecular design breeding.