Changes Of H₂O₂ And The Relationship Between H₂O₂ And Ca～(2+) In Wheat-leaf Rust Fungus Interaction

H2O2 burst in the defense responses of Wheat (Triticum aestivum) infected with compatible and incompatible leaf rust fungus races (Puccinia recondita f.sp. tritici) was investigated first. Cytofluorimetric analysis using the fluorescent probe DCFHDA scrutinized the generation of H2O2 in wheat primary leaves. It was observed that the fluorescence appeared 4h after inoculation in both compatible and incompatible combinations. Two transient increases of intracellular H2O2 production were detected in the incompatible interaction. The first transient increase occurred at 12 h after inoculation, and the fluorescence intensity of DCFHDA reached 10-fold higher than that of control treatment. Then, a second transient accumulation increased sharply 20h after inoculation. The fluorescence intensity was up by 1-fold over that of the first transient accumulation of H2O2. However, only the first transient increase occurred at 12h in the compatible interaction. The fluorescence intensity was 8 times as high as that of control treatment, and then it became as lower as that of control level till 48h after inoculation. The results indicated that H2O2 burst could be induced by either compatible or incompatible leaf rust fungus. However, the kinetics of H2O2 burst in incompatible interaction were biphasic, and the peak of phase II was much higher than that of phase I, whereas, there only existed the weak and transient phase I H2O2 accumulation in compatible interactions.In addition, pharmacological experiments were carried. Antioxidants(AsA, DTT), inhibitors of the mammalian neutrophil NADPH oxidase(DPI, imidazole) and calcium chelator(EGTA) as well as calcium channel blocker (LaC13) were separately injected into wheat primary leaves before inoculation to study their effects on hypersensitive reaction(HR) and H2O2 production in incompatible interaction while calcium ionophore A23187 was injected into wheat leaves without inoculation. The results were given as below.1 .Treatments of wheat leaves with AsA and DTT obviously suppressed the two peaks of intracellular H2O2 burst, whereas the second peak was inhibited much more, dropping of 50% from the control treatment. Moreover, these two kinds of reagents also reduced the area of dying cells caused by HR. The antioxidants' influence was more apparent 48h after inoculation, and the area of dying cells dropped down by 20% from that of the control level. These results suggested that H202 burst involved in HR caused by leaf rust invading.2.Treatments of wheat leaves with DPI and imidazole significantly reduced the intracellular H2O2 production. H2O2 generation on phase I and phaseâ…¡was inhibited by 60% and 80%, respectively A 40% inhibition of the area of dying cells caused by HR was observed 48 h after inoculation. The data indicated that Rboh was one of the sources of H2O2 burst during wheat responding to incompatible leaf rust fungus attack.3. The similar effects of antioxidants on HR and H2O2 production in wheat-leaf rust incompatible interaction were observed in treatments of wheat leaves with EGTA and LaCl3. Treatment with EGTA inhibited the second peak by 64% 12h after inoculation, and it diminished the area of dying cells by 40% 48h after inoculation. In addition, calcium ionophore A23187 was injected into wheat leaves without inoculation, and HR could be detected 24h after injection though the HR area in different time of sampling was much smaller than that of control. On the other hand, treatment with A23187 could also induce H2O2 burst with the same time course of the fluorescence intensity changes in control treatment. But the intensity of H2O2 burst was lower than that of control, for instance, the second peak accounted for 25% of the CK approximately, so intracellular Ca2+ appear to involve in signal transduction of wheat to resist the infection of leaf rust fungus by orchestrating the phase II H2O2 burst.Collectively, it is concluded that H2O2 production was associated with the formation of defensive barriers against leaf rust fungus in wheat. However there existed significant difference between compatible and incompatible combinations, indicating a role for H2O2 in the establishment of defenses. Pharmacological approaches also suggested that Rboh might mediate H2O2 generation during wheat-leaf rust fungus.