Changes Of H₂O₂ And The Relationship Between H₂O₂ And Ca²⁺ In Wheat Suspension Cells-IWF Interaction

The IWF (Intercellular washing fluids)-260 of wheat (Triticum aestivum) which was infected with the incompatible leaf rust fungus races 260(Puccinia triticina) was used as elicitor to stimulate the suspension cells and the changes of H₂O₂ were detected by Chemiluminescence method.The fluorescent probe DCFHDA was used to detect the the generation of H₂O₂ in wheat suspension cells induced by IWF-260.The results showed that :IWF-CK could only induce a slow increase of (reactive oxygen species)ROS and disappear quickly. IWF-260 could induce a ROS outbreak in 1 h, when the ROS peak was about 0.1μmol / L at 30 min, and the highest fluorescence intensity was appeared at the same time.Pharmacological experiments were carried out. Antioxidants(CAT), inhibitors of the mammalian neutrophil NADPH oxidase(DPI, imidazole) and calcium chelator(EGTA) as well as calcium channel blocker (LaCl3) were separately added before IWF-260. And IWF-260 and Ca2 + carrier A23187 were added at the same time or only added A23187 to study the changes of H₂O₂ production and cell death condition. The main test results were as follows:1. Treated with CAT obviously suppressed the H₂O₂ burst, dropping of 20 % at 30 min. Moreover, it also reduced the number of dead cells to about 50 %. These results suggested that H₂O₂ burst might be involved in the death of cells caused by IWF-260.2. Treated with DPI or imidazole could also reduce the H₂O₂ production. Which was inhibited by 20 % and 10 % respectively. The corresponding number of death cells was been significantly reduced.The results showed that: the plasma membrane NADPH oxidase plays an important role in this process. In addition, we used the staining intensity of fluorescent probe HE (Hydroethidine) to study the plasma membrane NADPH oxidase activity level. The results showed that: NADPH oxidase have been activated at 10 min after the add of IWF-260. Noted the plasma membrane NADPH oxidase been activated before H₂O₂ accumalation.3. Treated with EGTA or LaCl3 could inhibit ROS by 30 % and 20 % at 30 min respectively. The number of death cells has declined markedly. A23187 caused a increase in H₂O₂ production and cell death.when A23187 added alone, the cells sustained performance for a long time to produce many ROS. The results showed that the influx of extracellular Ca2 + might regulates the H₂O₂ which was induced by IWF-260.The mode of A23187 and IWF-260 was obviously different.4. H₂O₂ might induce programmed cell death of wheat suspension cells.To sum up, IWF-260 could cause the outbreak of H₂O₂ in wheat suspension cultured cells. It played an important role in the programmed death of wheat suspension cells induced by IWF-260.The pharmacological experiments showed a further indication that: NADPH oxidase might be involved in the production of H₂O₂. The influx of extracellular Ca2 + could regulate the production of H₂O₂ .