Mosses As Biomonitors Of Atmospheric Heavy Metal Pollution And Their Response To Copper Stress

Mosses can accumulate large quantities of heavy metals from dry and wet deposition. This has popularized their use in the biomonitoring of heavy metals. Human economic activities result in a progressive build-up of Cu in the environment. Cu is a ubiquitous metal present in the environment and is the most common contaminant of industrial effluents such as those produced by mining and metal processing. Mosses can be used as a model for physiological responses of plants to elevated concentrations of heavy metals. In this study, we choose appropriate moss species to biomonitor the atmospheric trace metal deposition in Nanjing on the base of the investigation six sites of China. Furthermore, copper accumulation and distribution, copper toxicity to moss species were also studied using solution treatments in the laboratory.At first, we investigated the atmospheric trace metal deposition of six sites of China using several mosses species. The results showed that the concentration of heavy metal in the bryophytes had obviously difference among different sites and species. The copper concentration was much higher in Tongling (reached 100.5 mg kg-1) than that of Huangshan (12.24 mg kg-1). The adsorption abilities to heavy metal of H. microphyllum, B. campylothallum and H. plumaeforme were much higher than that of L.juniperoideum. In addition, the relationship of different mosses at same site reached above 0.73 except L.juniperoideum. The atmospheric trace metal deposition was much higher as compared to European countries.On the base of the research mentioned above, the atmospheric trace metal deposition in Nanjing was been indicated using the mosses P. cuspidatum and B. campylothallum. The concentrations of Cu, Cd, Pb and Zn in the mosses collected in Mt. Zijin were much higher than that in the Mt. Huangshan. L.juniperoideum was not suitable to monitor the atmospheric trace metal deposition. We measured the Cu, Pb, Cd and Zn contents in P. cuspidatum and corresponding soils at monitoring sites of Nanjing City, and analyzed the relationships between plant heavy metals contents and environment. The results indicated that these 9 monitoring sites could be divided into three groups of different degree Heavy metals pollution, i. e., seriously polluted, polluted and basically unpolluted. The heavy metals contents in P. cuspidatum plants had some correlations with corresponding soils, and there were some other factors besides soil affecting the heavy metals contents in P. cuspidatum. It was suggested that bryophytes as biological indicators could be used to indicate the heavy metals pollution in Nanjing City.Moreover, copper accumulation and distribution in different moss species were studied at copper solutions supply. As the copper concentrations increasing, the content of Cu in different moss species increased significantly, and most of Cu was accumulated in the cell walls. The concentration of Cu in H. plumaeforme was significantly higher than that of P. cuspidatum. H. plumaeforme collected from different sites contained the same concentration of Cu, but H. plumaeforme collected from copper mine could accumulation more extracellular Cu to reduce the damage to the plants.Responses of three terrestrial moss species, P. cuspidatum、B. campylothallum and H. plumaeforme, were studied in order to characterize the physiological background of the metal response. In addition, copper adaption and detoxicity mechanisms of three terrestrial moss species were investigated under solution supplied with different concentrations (0,5, 10,50μM) of Cu for 4 d. It was found that 50μM Cu supply for 4 d significantly decreased the chlorophyll a content, but had no significant effect on chlorophyll b content. Cu-treated moss material showed pronounced toxic symptoms at concentrations and time extension, the apex, the leaf blade cells accumulated more Cu ions than the middle cells and the adjacent cells, which result to more serous damages than the middle cells. The leaf cells of bryophytes were damaged, such as chloroplasts browned and forming polygon, contracted or breached, cell wall thickened and darkened, and somewhat chloroplasts congealed to massive groups, protoplasts of cell disintegrated, and total loss of green color in cells, as shown by analytical electron microscopy. Moreover, Cu at 50μM significantly decreased the free amino acid contents of mosses. The contents of GSSG and GSSG+GSH were significantly increased in all three moss species. In addition, the concentrations of reduced glutathione (GSH) in P. cuspidatum and B. campylothallum were significantly increased but that of H. plumaeforme were decreased.50μM Cu increased the non-protein thiols (NPT) and total thiols contents of H. plumaeforme. Two UV-absorbing peaks could be eluted out through gel filtration chromatography on Sephadex G-50. A large amount of Cu was detected in the UV-absorbing peaks in 95-125 ml elution fractions of the moss plant extract. The results suggested that the adaptive Cu detoxification mechanism in three moss species might not involve in the free amino acid, the non-protein thiols (NPT) might have part role with no evidence for the participation of phytochelatins. These results also indicate that GSH may play an essential role in heavy metal detoxification of H. plumaeforme but not P. cuspidatum and B. campylothallum.Time-course of Cu-induced oxidative stress in P. cuspidatum was studied in this paper. The accumulation of H2O2 in leaves of P. cuspidatum was observed with Cu treatment time extension. Our results showed that Cu induced production of H2O2 at 4 h did not cause membrane damage and cell death, because the increased APX and POD activity could be attributed to remove a part of H2O2. As time extension, Cu toxicity resulted in more H2O2 which in turn caused membrane damage and cell death.Using both histochemical and cytochemical methods, we investigated the effects of excess copper (Cu) on the production of hydrogen peroxide (H2O2) and superoxide anion (O2·-) in the leaves of the moss P. cuspidatum. Excess Cu significantly increased the contents of total thiobarbituric acid-reactive substances (TB ARS) and H2O2, as well as the activity of guaiacol peroxidase (GPOD) and superoxide dismutase (SOD). Native PAGE detected all three forms of SOD (Mn-SOD, Fe-SOD and CuZn-SOD) in P. cuspidatum, and the increase in the total SOD activity appeared to be mainly caused by an increase in CuZn-SOD activity. According to cytochemical results, H2O2-dependent CeCl3 precipitates were primarily localized in the plasma membranes and cell walls, and O2·- was chiefly localized on the inner side of the plasma membrane and in the cytoplasm surrounding the chloroplasts. Experiments using imidazole as an inhibitor of NADPH oxidase, N-N-diethyldithiocarbamate as an inhibitor of CuZn-SOD, and 1,2-dihydroxybenzene-3,5-disulphonic acid as an O2·- scavenger indicated that a partial source of H2O2 in the cell walls may be NADPH oxidase. NADPH oxidase can use cytosolic NADPH to produce O2·-, which quickly dismutates to H2O2 via SOD. These results also demonstrated that peroxidase (POD) is involved in the detoxification of H2O2. Increased POD activity induced by Cu may remove excess H2O2 caused by Cu and may thus serve a detoxifying role.