Effects Of Hydrogcn Sulfide On Induction Of Alternative Oxidase Expression And Changes Of Redox State In Cell Suspcnsion Culture Of Rice

Embryogenic calluses and suspension cells of Oryza sativa L. were established. Suspension cells of Oryza sativa L were treated with H2S in different concentration. The expression of alternative oxidase, the changes of antioxidant enzymatic (SOD, POD, CAT, APX) activities, the redox state and the cell viability were tested. The results were as follows:1. H2S enhanced the activity of the AP in cell suspension cultures of rice. After4hour of H2S treatment, the AP activity increased dramatically and achieved the peak value at a concentration of2mM NaHS. Then decreased at5-10mM NaHS and maintained a steady level.2. Semi-Q-PCR was used to analyze the regulation of expression of Aoxla, Aoxlb, and Aoxlc of alternative oxidase gene family in different H2S concentration. Results shown that the Aoxl gene transcript level parallel the change of the AP activity. Interestingly, Different NaHS concentrations had different effects on the transcription of Aoxla, Aoxlb, and Aoxlc. In the control, the transcript level of AOXla and AOXlb were detected but AOXlc was not detected. During NaHS incubation, the AOX1a transcript level increased quickly at the concentration of0.5mM and then remained at a stable level. The AOXlb transcript level increased and achieved the peak value at2mM in2-fold than that of the beginning. The different expression of Aoxla, b, c revealed H2S up-regulate the transcription of Aoxl gene and this regulation can control the AP activity in rice.3. The content of H2O2and antioxidant enzymes activities of rice suspension cells was changed under different doses of NaHS treatment. Under low doses of NaHS treatment (≦2mM), the activities of CAT, APX increased dramatically and the content of H2O2reached the rock bottom within2hours. The activities of SOD significantly increased and the content of H2O2remain a low level under different doses of NaHS treatment within4hours. The antioxidant enzymes activities of CAT, under high doses of NaHS treatment for long time was higher than that of the control cells whereas the activities of APX, SOD, POD was lower than that of the control cell suspension cultures. At the same time, the content of H2O2was increased significantly. These results suggest that H2S regulate the activities of antioxidant enzymes by the content of H2O2.At the same time, the content of GSH and GSH/GSSG of suspension cell in different doses of NaHS were also changed. The content of GSH was increased under the low dose of NaHS (≦2mM). The content of GSH was the highest in2mM of NaHS. GSH/GSSG was increased and then decreased as the treatment time was prolonged. The radio of GSH/GSSG was the highest at2hour treatment. GSH/GSSG was decreased under high dose of treatment. According to our previous investigation, we suppose that H2S can regulate the radio of GSH/GSSG by regulating the content of H2O2.4. The viability of rice suspension cells under different concentrations of NaHS was analyzed by three independent methods:triphenyltetrazolium chloride (TTC) reduction assay, Evans blue uptake and fluorescence microscope. The results indicate NaHS at the concentrations ranging from0.5to2mM for4h did not cause a decrease in cell viability. The viability of rice suspension cells reached the peak value in2mM NaHS. High dose of NaHS treatment decreased viability of rice suspension cell and resulted in the death of cells. This result was coincident with the change of GSH/GSSG. So we can presume that the increase of GSH/GSSG would results in the increasing of cell viability.