Changes Of Glutathione REDOX Potential (E_GSH) In Arabidopsis Subcellular Structures Under Abiotic Stresses

Glutathione(GSH)is a low molecular weight mercaptan in eukaryotes.The REDOX state of glutathione plays an important role in regulating cell REDOX homeostasis.GSH not only acts as a ROS scavenger to eliminate the negative effects of oxidative stress,but also affects various aspects of Arabidopsis growth and development.In the intracellular environment,the glutathione redox state is also different in each organelle due to the constraints of membrane structure,and also plays an important role in Arabidopsis development.In this study,probes monitoring redox potential(E_GSH)in the cytoplasm,nucleus,chloroplast and mitochondria were constructed through gene coding technology using the redox properties of Grx1-RoGFP2,which can monitor the E_GSH in each subcellular structures of Arabidopsis thaliana cell under stresses.Through the Green Gate vector construction method,the plastid localization signal of tobacco Rubisco small subunit was fused to construct a plastid localization probe,35s:pt-Grx1-RoGFP2;the mitochondrial localization signal of yeast ScCOX4 was fused to construct a mitochondrial localization probe,35s:mt-Grx1-RoGFP2;fused with SV40 nuclear localization signal to construct a nuclear localization probe,35s:nls-Grx1-RoGFP2;without any signal before Grx1-RoGFP2,constructed a cytoplasmic localization probe,35s:Grx1-RoGFP2.The constructed probes were correctly localized to the specified subcellular regions revealed by transient expression in tobacco leaves.The four probes were transfected into Arabidopsis and obtained the transgenic lines.Using a microplate reader to detect the fluorescence excitation spectral characteristics,it was found that the four probes all have excitation peaks at 395 nm and 475 nm,which are consistent with the reported results.Next,in order to verify the validity of the probe,we treated the seedlings expressing cytoplasmic localized probe with different concentrations of H₂O₂,and found that E_GSH in Arabidopsis cells has a certain buffering effect.Only when the exogenous H₂O₂ reached a certain value,E_GSH changes were induced.However,exogenous application of H₂O₂ still inhibited root elongation even within the E_GSH buffer range.After treated the four probes with butthionine-sulfoximine(BSO),the elongation of Arabidopsis root was inhibited,but the application of GSH restored root development.Inhibition of glutathione synthesis by BSO turned E_GSH into an oxidized state in the nucleus,cytoplasm,mitochondria,and chloroplasts,especially in chloroplasts and mitochondria.To investigate the effect of oxidative stress on subcellular E_GSH,we treated the probe with methylviologen(MV),a drug capable of causing oxidative stress,and we found that MV could inhibit root elongation and cause oxidative stress.Chloroplast E_GSH was most sensitive to oxidative stress caused by MV;E_GSH in mitochondria and cytoplasm also affectedthe and E_GSH in nucleus was not affected and remained in a stable state.We treated the seedlings expressing probe with3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU),a drug that blocks photosynthesis,and rotenone,a drug that blocks respiration.We found that both drugs also inhibited root elongation.DCMU treatment decreased the contents of H₂O₂ and O_2·^-,and increased E_GSH in the cytoplasm,while other organelles remained unchanged.Rotenone treatment produced more H₂O₂ and O_2·^-,and the E_GSH in the cytoplasm was elevated,but not as obvious as DCMU treatment.We suggest that chloroplasts and mitochondria transfer GSSG into the cytoplasm in order to keep their own E_GSH at a very low level of reduction,and chloroplasts are the most sensitive to E_GSH.We treated the seedlings expressing probe with 2 mM NaHCO₃ and found that the root elongation of Arabidopsis was significantly inhibited.Additionally,the content of H₂O₂ increased in the meristem,and the content of O_2·^-decreased.All four subcellular E_GSH were elevated,and chloroplast was the most obvious.We then treated the probe with 150 mM NaCl to induce salt stress,and we found that salt stress inhibited root development and produced a large amount of ROS.Only E_GSH in the chloroplast and nucleus increased,and the others were unchanged.Thus,salt stress affects E_GSHhomeostasis in chloroplast and chloroplast function,and also affects the ability of the nucleus to drain excess GSSG into the cytoplasm.Finally,we treated the seedlings expressing probes with 200 mM mannitol to induce drought stress.We found that drought stress inhibited root development but did not increase ROS content.The E_GSHs of Chloroplast,nuclear and cytoplasmic were significantly increased compared with that of the control,while mitochondrial E_GSH was unchanged.We suggest that the elevated E_GSH in nuclear might because of the osmotic potential affecting the ability of the nucleus to transfer excess GSSG into the cytoplasm.