Analysis Of Alternative Respiratory Pathway And Origin, Function Of H₂O₂ In Interaction Systems Between Plants And Xanthomonas

In this paper, a monoclonal antibody to the terminal oxidase of the alternative pathway from Sauromatum guttatum, the inhibitor of alternative oxidase and the scavenger of reactive oxygen species (ROS) were used to detect the expression of alternative oxidase protein, the physiological roles and the signals of hydrogen peroxide in compatible and incompatible interaction system between monocot plants rice (Oryza sativa L.) and Xanthomonas oryzae pv. Oryzae and with incompatible interaction system between cowpea (Vigna unguiculata L. cv. Longdouwang 1) and Xanthomonas campestris pv. phaseoli. Furthermore, we analyzed the origin of hydrogen peroxide and photosynthetic functional state in incompatible interaction system. The main results and conclusions obtained are listed as follows:We used rifampicin treatment to kill off the Xanthomonas campestris pv. phaseoli in the plant tissues and established experimental mode of interaction between cowpea and Xanthomonas campestris pv. phaseoli. Optimal concentration and duration of the rifampicin treatment to eradicate the bacteria in plants were determined. Rifampicin treatment reduced H2O2 levels in Xpw-infected plants more than it did in XpahpC-infected plants, the reduced level of H2O2 seen in this system could be produced in Xpw. H2O2 accumulation was also significantly decreased with the increasing concentration of rifampicin in rice inoculated with Xoo. According to these results, we speculated that the observed change in H2O2 concentration was caused by bacteria. The H2O2 generated by phytopathogenic bacteria in plants will lead us to better understand plant pathogen interactional processes.H2O2 concentration was markedly increased in cowpea inoculated with wild-type Xanthomonas campestris pv. phaseoli, and AOX was rapidly induced in this system. Using histochemical analysis under electron microscopy, we found that accumulated H2O2 was mainly located in chloroplast of plants. The induction of AOX was associated with the destruction of chloroplast structure, the photosynthetic rate parameters were significantly decreased. Almost effects of AOX induction were not observed in cowpea inoculated with XpahpC. After treatment with rifampicin, reduced H2O2 accumulation was detected in Xpw and cowpea systems. Intriguingly, the expression of AOX was also reduced. H2O2 generated by bacteria could induce AOX expression. Correlations exist in photosynthetic rate parameters and H2O2. These results indicate that H2O2 generated by bacteria may be involved in the regulation of plant photosynthesis and alternative respiratory pathway during plant pathogen interaction.During initial interaction between rice seedlings and Xoo (12h), that alternative oxidase (AOX) activity increased suggested the induction of AOX in both systems. However, the activity of alternative pathway was higher in the incompatible interaction system than in the compatible interaction system. The treatment of AOX inhibitors salicylhydroxamic acid (SHAM) decreased markedly the activity of AOX. After interaction for about 60 hours, AOX activity increased more significantly in incompatible interaction system than that in the compatible interaction system, whereas a reverse result in compatible interaction system after treatment with SHAM was noticed. Intestresting, the concentration of hydrogen peroxide in this system was higher than compatible interaction system. This result indicated that AOX has an important role in inocompatible interaction system. In interaction system of cowpea with XpahpC and Xpw, a decrease of total activity of alternative oxidase was found in interaction of cowpea with XpahpC, which indicated that hydrogen peroxide was involved in the regulation of the alternative pathway and the expression of alternative oxidase. The rice seedling were treated with N, N’-Dimethylthiourea (DMTU) or 4,5-dihydroxy-1,3-benzene disulfonic acid (Trion,) after Xoo inoculation. Trion treatment did not inhibit the expression of alternative oxidase, while a reverse result in DMTU treatment indicates the H2O2 is an up-regulation signal in the expression of alternative oxidase. The effects of H2O2 not superoxide anion on the activity of alternative pathway are correlated with AOX proteins.