In the present study, we evaluated the protective effect of exogenous heme oxygenase-1 （HO-1 EC 1.14.99.3） inducer hematin against mercury-induced oxidative damage in the roots of Alfalfa （Medicago sativa L.）. Plants exposed to mercury （HgCl2） exhibited a significant increase of lipid peroxidation, as well as inhibition of root elongation. However, hematin （50 μM） supplementation to HgCl2 （100 μmol/L） treated plants effectively reduced the lipid peroxidation and partially increased the root elongation. These responses were mimicked by the application of aqueous solution of carbon monoxide （CO） with 50% saturation. Also, treatment with hematin could result in the potent induction of HO-1 transcript in the root tissues, as detected 12h following treatment. Moreover, the activation of anti-oxidant enzyme, including glutathione reductase, monodehydroascorbate reductase and superoxide dismutase activities, and the decrease of lipoxygenase activity, were induced by hematin at 12h or 24h, which was further confirmed by histochemical staining for the detection of lipid peroxidation and loss of membrane integrity. Whereas, ascorbate peroxidase and guaiacol peroxidase isozyme activities or their transcripts were reduced, respectively, indicating that hydrogen peroxide might act as a signal to mediate Hg- tolerance at the beginning of treatment. The ameliorating effects of hematin were specific, since the CO scavenger hemoglobin differentially reversed the above actions. Taken together, our results suggested that hematin exhibits a vital role in protecting the plant against Hg-induced oxidative damage.
Yi Han Wei Xuan Tian Yu Wen-Bing Fang Tian-Ling Lou Yin Gao Xiao-Yue Chen XiaoXiao Wen-Biao Shen
Pretreatment of lower H2O2 doses （0.05, 0.5 and 5 mM） for 24 h was able to dose-dependently attenuate lipid peroxidation in wheat seedling leaves mediated by further oxidative damage elicited by higher dose of H2O2 （150 mM） for 6 h, with 0.5 mM H2O2 being the most effective concentrations. Further results illustrated that 0.5 mM H2O2 pretreatment triggered the biphasic production of H2O2 during a 24 h period. We also noticed that only peak Ⅰ （0.25 h） rather than peak Ⅱ （4 h） was approximately consistent with the enhancement of heme oxygenase （HO） activity, HO-1 gene expression. Meanwhile, enhanced superoxide dismutase （SOD） activity, Mn-SOD and Cu, Zn-SOD transcripts might be a potential source of peak Ⅰ of endogenous H2O2. Further results confirmed that 0.5 mM H2O2 treatment for 0.5 h was able to upregulate HO gene expression, which was detected by enzyme activity determination, semi-quantitative reverse transcription-polymerase chain reaction and western blotting. Meanwhile, the application of N,N＇-dimethylthiourea, a trap for endogenous H2O2, not only blocked the upregulation of HO, but also reversed the corresponding oxidation attenuation. Together, the above results suggest that endogenous H2O2 production （peak Ⅰ） plays a positive role in the induction of HO by enhancing its mRNA level and protein expression, thus leading to the acclimation to oxidative stress.
Carbon monoxide （CO）, an endogenous signaling molecule in animals, also provides potent cytoprotective effects including attenuation of lung lipid peroxidation induced by oxidant in the mouse. Our recent work demonstrated that 0.01 μmol/L hematin （a CO donor） treatment of wheat plants alleviated salt-induced oxidative damage in seedling leaves. In this report, we further discovered that hematin pretreatment （≤ 0.1 μmol/L） could delay wheat leaf chlorophyll loss mediated by further treatment of H202 and paraquat, two reactive oxygen species （ROS） sources, in dose-and even time-dependent manners. Also, compared with the control samples, seedling leaves pretreated with 0.01 or 0.1 μmol/L hematin for 24 h exhibited lower levels of H2O2 and lipid peroxidation, as well as higher contents of chlorophyll and activities of antioxidant enzymes. Such beneficial effects exerted by hematin were mimicked by the pretreatment of antioxidant butylated hydroxytoluene （BHT）, and differentially reversed when CO scavenger hemoglobin （Hb）, or CO specific synthetic inhibitor ZnPPIX was added, respectively. Taken together, the results presented In this paper directly illustrate for the first time that CO is able to strongly protect plants from oxidative damage caused by the overproduction of ROS, and strengthens the evidence that CO is a potent antioxidant in various abiotic and biotic stresses, as similar results have been shown in animal tissues.
