Studies On Biomarkers And Response Mechanisms Of Mosses As Environmental Bioindicators To The Stress Induced By Combined Pb And Ni

Mosses have been widely used in environmental biomonitoring in recent years, but, little was known about the mechanisms of biomonitoring and oxidative stress and molecular damage in moss under metal pollution. In this paper, the response mechanisms of antioxidative systems in three mosses - Hypnum plumaeforme (HP), Thuidium cymbifolium (TC) and Brachythecium piligerum (BP) to the stress caused by combined Pb and Ni have been investigated. The investigated parameters were i) the changes in morphology of mosses: fading, etiolating, browing and dying; ii) the accumulation and depletion of radical oxygen species (ROS) including superoxide radicals (·O2-) and hydrogen peroxide (H2O2); iii) the parameters reflecting the extent of the cells being injured: chlorophyll (Chl), malondialdehyde (MDA) and proline (Pro); iv) the response of antioxidants included ascorbate (AsA); v) the response of antioxidative enzymes included superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and polyphenol oxidase (PPO). The aims were i) to compare the response of 3 mosses to the single or combined stress of Pb and/or Ni; ii) to disclose the combined effect of Pb and Ni on the antioxidants in the moss, and iii) to validate physiological parameters in the moss as biomarkers of heavy metal pollution. It will highlight a better understanding of the mechanisms adopted by these moss species in response to toxic concentration of heavy metals, and could be the first step in determining physiological parameters as toxicity biomarkers in mosses biomonitoring. The main results were showed as follows:1. Sensitivities of mosses to Pb and Ni stress were varied among moss species. The sensitivities of the 3 mosses to Pb and Ni stress were ordered as: BP > HP > TC, which was related to the morphologies and cell structures in mosses. Morphologically, there are pleats on leaves of HP and BP, which makes them have larger leaf-surfaces than TC and as a result facilates the entering of heavy metal ions into moss cells. As for the cell structures of mosses, the 1-2 warts in cells of TC could be one of the reasons for its'less sensitivity to Pb and Ni stress; on the other hand, the thicker cell wall in HP might prevent the entering of metal ions into the cells, and was one of the reasons for its'less sensitive to Pb and Ni stress than BP, which was thin and crystal in cell wall.2. Under single Pb or Ni stress, chlorophyll were initiated at lower metal concentration but inhibited at higher concentration. Pb and Ni stress induced the dose-dependent accumulation of radical oxygen species (ROS) including superoxide radicals (·O2-) and hydrogen peroxide (H2O2), malondialdehyde (MDA), proline (Pro) and ascorbate (AsA) content. For antioxidative enzymes, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) activities decreased but peroxidase (POD) activity increased under Pb and Ni stress. A high POD activity than CAT, APX and PPO activity in this study suggested that POD can serve as a better intrinsic defense tool to resist the oxidative stress in moss caused by Pb and Ni.3. The effects of combined stress of Pb and Ni on mosses were influenced by the treatment regimes and concentrations. When the Pb was combined with lower than 0.01 mM of Ni, the combined effects of Pb and Ni were exhibited as the alleviation of Ni to Pb toxicity; when Pb was combined with higher than 0.1 mM of Ni, the toxicity of Pb was strengthened. 4. With the increase of the duration of Pb and Ni stress, the morphologies of mosses were changed from fading, etiolatiing, browing to dying. When the duration was lower than 2 d, the Chl content was decreased appreciably, and no injury was observed in moss leaves, but there were accumulation of·O2-, H2O2 and MDA; Similarly, SOD, CAT and APX activities were appreciably decreased. When the duration was 2-4 d, the Chl content decreased further, the·O2-, H2O2 and MDA were accumulated more rapidly and SOD, CAT, APX activities were decreased more dramatically. Meanwhile, fading and etiolating was observed in moss leaves, indicating moss was injured by Pb and Ni stress. When the duration was 4-8 d, Chl content decreased dramatically, the accumulation of·O2-, H2O2 and MDA and the decreases of SOD, CAT, APX, were slowed down. Morphologically, the leaves were etiolating completely, indicating mosses were severely injured. When the duration was more than 8 d, Chl content decreased dramatically, the values of all the physiological parameters were decreased, and the leaves of mosses were browning and ultimately dead.5. Ecotoxcological effects of Pb and Ni stress and the response mechanisms of mosses were: Pb and Ni stress induced the accumulation of radical oxidative species (ROS) and as result induced the oxidative stress in mosses. In order to keep the balance between the production and depletion of ROS, mosses induced two kind of antioxidative systems, i.e., nonenzymic antioxidants including AsA and antioxidative enzymes including SOD, CAT, POD and APX, etc., they could scavenge the excessive ROS, keep the balance in metabolism, and consequently prevent the cell from injury. Under the stress of lower Pb or Ni concentrations, the ROS induced by Pb and Ni could be scavenged by the antioxidantive systems in mosses. But, with the increase of duration and concentration of metal stress, the gradually accumulation of ROS was observed. When the accumulation of ROS was out the range of the capacities of antioxidative system, the ROS may react with lipids, proteins, nucleic acids, etc., and resulted in the decrease of antioxidative enzyme activities, the accumulation of proline, the lipid peroxidation of membrane, the degradation of chlorophyll and the destroy of cell structure.6. The parameters, MDA and Pro contents as well as CAT and POD activities were increased or decreased dose-dependently with Pb and Ni concentration, therefore could be used as biomarkers in atmospheric biomonitoring.7. Moss bags technique and moss biomarkers—the two kinds of biomonitoring methods, both of them could reflect the atmospheric heavy metal pollution. But the former was mainly reflected the gross contents of pollutants, the latter was concentrated on the biological effects of pollutants on organisms, it was more sensitive to pollutants and therefore was significant in predicting the changes of environment quality, and preventing environmental pollution.