Study Of Response Mechanism And Bioindication Of Bryophytes To Heavy Metal Pollution

With the interference of human activities on the environment growing badly,biomonitoring of the polluted environments became one of hot points for study ofenvironmental biology. The bryophytes as good biological indicators to investigatepollution and changes of the environments have been widely used in differentcountries in the world. However, only few researches and reports dealing with theresponse and mechanism of the bryophytes to environmental pollution were carriedout. In order to better understanding the response and mechanism of bryophytes toenvironment pollution, the experiments of stress of different heavy metals on the threemosses, Haplocladium microphylum, Brachthecium procumbens and Physcomitriumpatens, which were successfully tissue cultivated and regenerated from native plants,were carried out on vitro condition at present study. Using advanced technique ofsynchrotron radiation x-fluorescence （SRXRF） and methods of physiological andbiochemical analyses, the accumulation of heavy metal and nutrient elements as wellas physiological and biochemical responses in the plants of three mosses under heavymetal stress were analyzed and studied. The main results are as follows:（1） Based on numerous comparative experiments, the tissue cultivation andregeneration of the mosses Haplocladium microphyllum and Brachytheciumprocumbens were successfully completed and two kinds of systems of efficientartificial propagation of the gametophytes for the two mosses were established. Theresults showed the best conditions for cultivation of these two mosses are as follows:modified Knop’s basic medium add10g/L sucrose; pH value adjusted at5.8to6.0;culture bottle relative air humidity kept at75%to85the%; culture bottles to have amoderate permeability; culture temperature was20±2℃; illumination time was14hr; the illumination the strength for1500lux to2000lux. As the results, twoinvention patents were applied and obtained, and plants material for the experiments of heavy metal stress was gathered.（2） In the stress experiments under vitro condition, the different concentrationsof heavy metals ions including Pb, Cu, Cr, Fe in the plants of three mosses under thestress of single or combined condition of different heavy metals were analyzed bySRSRF. Under the stress of single heavy metal element, with increase of the pollutionconcentration, the cumulative amount of the same heavy metal ions had higher trendof significantly increasing in the plants. But, accumulation capacity and absorptionsensitivity of the heavy metals were different among three mosses. In comparison ofthe tested three mosses, Brachythecium procumbens had the highest content of Pb of1699.05147.14μg/g and152,9813.22μg/g in the plants under the highest Pb²⁺400mg/L pollution and lowest Pb²⁺50mg/L pollution, respectively. It indicated thatcumulative capacity and absorption sensitivity of Pb for Brachythecium procumbensare very strong and quantitative indicator of the Pb content in the plant is suitable forthe assessment of environmental Pb pollution levels. Same analysis showed thatHaplocladium microphyllum also had strong cumulative capacity and absorptionsensitivity of the Cr and Cu, so the content of the Cr and Cu elements in the plants issuitable indicators for Cr and Cu pollution. The combined stress of different elementssignificantly impact of the accumulation of heavy metals in the moss plants. Underthe three combined treatments of Fe²⁺20mg/L with Pb²⁺50mg/L, Cr⁶⁺50mg/L,Cu²⁺50mg/L, while Fe cumulative amount significantly increased, cumulativeamounts of Pb, Cr, Cu in the plants are significantly lower than those under the stressof single treatment with same element. This showed that the lower tconcentration ofFe²⁺had a strong sense of antagonism on Pb²⁺, Cr⁶⁺, Cu²⁺and can largely alleviatepoisoning effect of three heavy metals on the mosses. In addition, the scanningtechnology of U-SRXRF micro-probe analysis was used to show micro-distribution ofheavy metals on the stem and leaves of the mosses. The study reveals absorptionmechanism of accumulation of heavy metals in the tissue and cell levels in the mosses.At the same time, it is further confirmed that the tested three mosses have a strongaccumulation of heavy metals and sensitive biological response and can be used asindicator plants for biological monitoring of environmental heavy metal pollution.（3） The absorption capacity and changes of different nutrient elements including,P、K、Ca、S、Mn、Zn under different heavy metal stress were also analyzed. The resultsshowed that a certain concentration of heavy metal ions could have antagonistic effect on the absorption of the nutrient elements and inhibit absorption and utilization ofnutrient elements in the plants. With the increasing concentration of single heavymetal pollution, nutritive elements cumulative amount of the tested three mossesshowed two trends. The first one is that lower concentration of heavy metal promotedthe absorption of nutritional elements while excessive pollution concentrationsreduced the cumulative amount of the element. The second is the amount of nutrientaccumulation was directly downward with the increased pollution concentrations. Theabsorption and accumulation of nutrients were effected by different elements as wellas different mosses. Under stress treatment of Pb²⁺, Cu²⁺, Cr²⁺, Fe²⁺the lowestdecrease of P, K, Ca, S, Mn, and Zn nutrient elements in Haplocladium microphyllumwere high than in Physcomitrella patens. It implied that Haplocladium microphyllumhas strong tolerance to the four heavy metals pollution and is the most suitable forlong-term monitoring of the environmental heavy metal pollution. The results that thesignificant changes of P, K, Ca, S, Mn, and Zn nutrient elements in Brachytheciumprocumbens under the lowest concentration and shortest time treatment of Pb²⁺alsoindicated that Brachythecium procumbens responses to Pb pollution rapidly and canbe used as indicative plant to Pb pollution. Linear regression analysis of the resultsshowed that, between Pb²⁺pollution concentration of100mg/L to Pb²⁺pollutionconcentration of600mg/L, the content of P, K, Ca, S in the plants of Brachytheciumprocumbens and Pb²⁺pollution concentration presented a significant line regression. Itis recommended that the use of nutritional element content in the plants ofBrachythecium procumbens as additional indicators for monitoring of environmentalPb pollution.The impact of different kinds of combined heavy metals stress ondifferent mosses on nutrient absorption and accumulation capacity is obviouslydifferent. The impact could be divided into three types: a remission cumulativeinhibitory effect; enhanced cumulative inhibitory effect, and a significant promotionof cumulative effect.（4） The physiological response and changes of three species of mosses includingphotosynthetic pigment synthesis, peroxidase （POD） activity changes,malondialdehyde （MDA） content under heavy metal stress were measured andcompared. The fact that the biological responses of total chlorophyll content to heavymetal contamination in the mosses were very rapid indicated that the mosses aresensitive and suitable for monitoring heavy metal pollution. Pb²⁺pollution cause thethree mosses chlorophyll b decrease. The largest decline of chlorophyll b of Physcomitrella patens, Haplocladium microphyllum and Brachythecium procumbenswere58.55%,48.33%and46.17%, respectively. It implied that one of importantreasons of inhibiting plant photosynthesis is lower content of chlorophyll b caused byPb²⁺pollution. Under the different types of single heavy metal pollution stress, PODactivity response of three mosses showed two kinds of variation, a POD enzymeactivity gradually increased with increasing concentration of heavy metal pollution;and POD enzyme activity gradually enhanced in a concentration range of heavymetals polluting and then significantly lower under higher pollution concentrations.Significant dose-response relationship between POD activity and MDA content andconcentration of heavy metal pollution in the test three mosses fitted to the formationof significant functional equation of the curve regression relationship. Therefore, it isrecommended that the moss POD enzyme activity and MDA content can be used asauxiliary physiological indicators for monitoring environmental heavy metalpollution.In summary, the present study on the response analysis of three mosses to heavymetal pollution provided further evidence that bryophytes are sensitive and goodindicator plants to environmental pollution of heavy metals. The research resultsprovided scientific basis for further study and application of monitoringenvironmental pollution and changes by bryophytes.