Carbon Monoxide/Heme Oxygenase1Regulate Mercury Toxicity To Chlamydomonas Reinhardtii

Mercury (Hg) is one of the most toxic elements to plant cells and has become the main source of heavy metal pollutants. One of the the major toxic effects triggered by Hg is the generation of reactive oxygen species (ROS) and oxidative damage to plants. Carbon monoxide (CO), a molecular messenger, mediates many physiological responses induced by biotic and abiotic stresses. In this study we demonstrated that CO at40μM can attenuate the oxidative stress triggered by Hg in Chlamydomonas reinhardtii. Exogenous CO increased the cell number of C. reinhardtii. CO improved chlorophyll content and photosynthetic efficiency through increasing PSII electron transport chain rate. Treatment with CO decreased ROS levels and DNA fragments. Non-denaturing PAGE and Real time PCR assay indicated that both enzyme activities and transcripts of SOD, CAT and APX could be modified in the Hg-exposed cells. The CO-mediated alleviation of Hg toxicity was closely related to the less accumulation of Hg and peroxide.Heme oxygenase (HO) is a rate-limiting enzyme of the first step for heme degration and catalyzes heme into biliverdin (BV), concomitance with release of CO and ferrous ions. HO probably participated in the process of CO-regulated response to Hg stress. We examined the transcriptional level of HOI in the presence of CO. Exogenous bilirubin (BR) and hematin (Ht) decreased TBARS content in Hg-exposed cells. By contrast, addition of ZnPPIx, a specific inhibtor of HO, increased TBARS content and aggravated DNA laddering.To get insights into the regulatory role of HO, we constructed transgenic algae overexpressing HOI driven by HSP70A promoter and HO1::RNAi driven by35S promoter. Our analyses showed that transgenic cells carrying HSP70A::HO1showed very low quantity of ROS, whereas transformants with HO1::RNAi displayed higher levels of ROS when compared with the untransformants. These results suggest that expression of HO1could depress the generation of O2-and H2O2. Because overgeneration of ROS is closely associated with the oxidative damage to plant cells, we determined lipid peroxides, expressed as thiobarbituric acid reactive substances (TBARS). The transgenic algae overexpressing HOI showed significantly low levels of TBARS under the Hg exposure, with only76.7%of the WT. Also, the reduction of HOI transcripts resulted in significantly increased accumulation of peroxides.We further determined the Hg accumulation in the transgenic and untransformed cells under Hg exposure. The transgenic algae overexpressing HO1showed significantly lower Hg concentrations in cells compared to the untransformed algae exposed to Hg. Similarly, the concentration of Hg was significantly higher in the cells with HO1::RNAi. These results suggest that HOI is able to confer the alga tolerance to the heavy metal.