The Modulatory Role Of Heme Oxygenase/Carbon Monoxide Signal System In Mercury And Cadmium-Induced Oxidative Stress In Medicago Sativa L.

Heme oxygenase(HO)/carbon monoxide(CO) has recently proven to be an important signaling system in mammalian cells.However,little information has been investigated in plants.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 the significant increase of lipid peroxidation as well as the inhibition of root elongation.However,hematin(50μM) supplementation to HgCl₂(100μM) 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 12 h following treatment.Moreover,the activation of anti-oxidant enzyme,including glutathione reductase(GR), monodehydroascorbate reductase(MDHAR) and superoxide dismutase(SOD) activities,and the decrease of lipoxygenase(LOX) activity,were induced by hematin at 12 h or 24 h,which was further confirmed by the histochemical staining for the detection of lipid peroxidation and loss of membrane integrity.Whereas,ascorbate peroxidase(APX) and guaiocol peroxidase(POD) isozyme activities or their transcripts were reduced,respectively,indicating that H₂O₂ might act as a signal to mediate Hg-tolerance at the beginning period of treatment.The ameliorating effects of hematin were specific,since the CO scavenger hemoglobin(Hb) differentially reversed above actions.Taken together,our results preliminarily suggested that CO produced by HO exhibits a vital role in protecting the plant against Hg-induced oxidative damage.The effect of HO/CO signal system on heavy metal-induced oxidative stress in the roots of Alfalfa was further investigated.The cadmium(Cd) treatments(100,200 and 500μM CdCl₂) showed a dose-dependent enhancement in lipid peroxidation evaluated as thiobarbituric acid reactive substances(TBARS) formation.Interestingly, under 100 and 200μM CdCl₂ treatments,CO releasing monitored by gas chromatography and mass spectrometry(GC/MS) were increased and approximately consistent with the changes of heme oxygenase(HO) activity and HO-1 transcript,an important CO synthetic enzyme both in animals and plants.Further results showed that 100μM Cd exposure increased CO releasing in a time-dependent manner. Whereas,treatment with 500μM CdCl₂ provoked a significant decrease in HO-1 transcript,also CO releasing and HO activity could not be easily detected. Interestingly,based on the above fact,we deduced that CO,an important endogenous signaling molecule,produced by HO,is involved in Cd-induced oxidative stress.100μM Cd exposure enhanced NPT formation,the oxidation of ascorbate(AsA), and reduced glutathione(GSH) to oxidized glutathione(GSSG),another clear indication of oxidative stress.Interestingly,the effect of Cd on GSH and GSSG content was potentiated by the pretreatment of CO scavenger hemoglobin(Hb), TBARS formation was also enhanced.Plants pretreated for 6 h with 50% CO-saturated aqueous solution,which could induce the rapid endogenous CO releasing followed by a gradual decrease when subsequently exposed to 100μM CdC₂ for 72 h,effectively reduced plant growth inhibition,decreased the Cd accumulation and oxidative damage as compared with the plants without CO pretreatment. Meanwhile,CO pretreatment modulated several enzymes responsible for GSH metabolism.The activities or transcripts of glutathione reductase(GR) and glutathione S-transferase(GST),and the transcripts of glutamylcysteine synthetase (ECS) and glutathione synthetase(GS) were induced,ascorbate peroxidase(APX) expression was down-regulated,thus resulting in the partial restoration of GSH/GSSG and higher level of AsA.CO-alone pretreatment did not change above investigated parameters.However,both CO-induced restoration of GSH/GSSG and alleviation of lipid peroxidation were significantly blocked by Hb.To further understand the putative role of CO in protecting against Cd-induced oxidative damage,we measured the activities of several antioxidant enzymes and observed that Cd-alone treatment caused a decrease in SOD and POD activities. However,CO pretreatment induced increases in SOD and POD activity under Cd stress.In this case,CO probably reduced the oxidative injury by enhancing POD activity for removal of H₂O₂.We also measured the activity of lipoxygenase(LOX) and found that treatment with Cd caused an increase in LOX activity.However, pretreatment with CO had a negative effect on the activity.These results indicated that CO inhibited Cd-induced LOX activity and reduced lipid peroxidation catalyzed by LOX,and thereby,reduced oxidative damage,which was further confirmed by the histochemical staining for the detection of lipid peroxidation and loss of membrane integrity.Taken together,these results are suggestive of a role for HO/CO signal system for the alleviation of Cd-induced oxidative damage in the roots of alfalfa plants by modulating glutathione metabolism.Thus,we further deduced that CO as an important endogenous signaling is involved in responses to abiotic stresses,and CO production induced by HO in plants might rapidly increase in response to various abiotic stresses.Moreover,ABA,H₂O₂ and CO signals could contribute to more integrated communication system among cell compartments for abiotic stress tolerance.