Studies On The Ecophysiological Effects Of Microcystin-RR Against Higher Plants

Toxic cyanobacterial blooms in fresh water bodies has been becoming a kind of ecological disaster worldwide. One of the most harmful effects of cyanobacteria blooms is typically from the toxins produced by the algae, which present a major threat to the health of human, livestock and wildlife. Of all the algal toxins, microcystins are the most toxic and abundant species. Most investigations into the toxicity of microcystins are focused on animals. In this paper, the ecophysiological effects of microcystins on higher plants and its possible mechanism were studied. The main results are as follows: 1. Microcystins from Microcystis bloom in Lake Dianchi were extracted and purified with high performance liquid chromatography (HPLC). MC-RR with the purity over 90% was finally obtained by preparative HPLC. 2. Accumulation of MC-RR in the roots and leaves of Vallisneria natans were evaluated by ELISA. Results indicated that microcystin-RR could accumulate differentially in these two organs of V. natans seedlings. The accumulation of toxin in the roots and leaves was time and dose dependent, with higher uptake detected in the roots. The BCF value of the roots and leaves was low, and at the same time, decreased with the increase of MC-RR concentration. 3. V. natans seeds and seedlings were exposed to different concentrations of microcystin-RR. The results indicated that at the concentration of 0.0001-10 mg/L, seed germinaton rate, cotyledon length, leaf length, root length and root hairs declined, the fresh mass of plants, the root and leaf formation were inhibited. V. natans was relatively insensitive to microcystin-RR at concentrations ranging from 0.0001 to 0.01 mg/L. However, when the toxin concentration was over 0.01 mg/L, both the fresh weight and the longest leaf length of seedlings were significantly reduced after 30 d treatment. When the seedlings were treated with 10 mg/L toxin, the root growth and leaf formation were significantly inhibited and the root hairs disappeared, and the new leaf turned yellow. It can be concluded that MC is harmful to Vallisneria natans. 4. Arabidopsis thaliana suspension cells were exposed to a range dose of microcystin-RR. Lipid peroxidation, a main manifestation of oxidative damage, was studied and a time-and dose-dependent increase in malondiadehyde was observed. In contrast, glutathione (GSH) levels in the cells decreased after 48 h treatment with 1 and 5 mg/L of microcystin-RR. The activities of superoxide dismutase (SOD) and catalase (CAT) increased significantly after 48 h exposure to 1 and 5 mg/L of microcystin-RR, but glutathione S-transferase (GST) activity showed no difference compared with the control. These results clearly indicate that microcystin-RR is able to cause oxidative damage in A. thaliana suspension cells. Decrease of GSH content and increases of SOD and CAT activities reveal that the antioxidant system may play an important role in eliminating or alleviating the toxicity of microcystin-RR. 5. Comparative studies of MC-RR absorption by tobacoo BY-2 suspention cells and isolated carp hepatecytes showed that carp hepatecytes absorb MC-RR very quickly. And the absorption peaked after 4 h's exposure to the toxin. The tobacoo BY-2 suspention cells absorbed MC-RR very slowly and its absorption was time and dose dependent. With 0.1 mg/L MC-RR treatment for 4 h and 24 h, MC-RR taken up by tobacco BY-2 cells was 1/300 and 1/60 of that absorbed by carp liver cells respectively. After 108 h's treatment with 50 mg/L MC-RR, MC-RR content in tobacco BY-2 cells only reached 344.61±71.75 ng/mg Prot, which just equal to the amount taken up by carp liver cells after 4 h with 0.1mg/L MC-RR treatment. 6. Tobacco BY-2 suspension cell line was applied to examine the effects of microcystin-RR on plant cells. Cell viability as well as six biochemical parameters, i.e., reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPX), peroxide dismutase (POD) and glutathione (GSH), were investigated when the cells were exposed to 0.1 and 50mg/L microcystin-RR respectively. Results showed that 0.1 microcystin-RR had no significant effects oncell viability while 50 mg/L microcystin-RR evoked decline of cell viability to approximately 80% after 144 h of treatment. The significant increase in ROS levels and POD and GPX activities of the treated cells were detected with a time and dose dependent manner. Changes of SOD and CAT activities were also detected in BY-2 cells when exposed to 50 mg/L microcystin-RR. After 168 h's recovery treatment, ROS contents, POD and GPX activities returned to normal levels. These results suggested that the toxicity of microcystin-RR caused the increase of ROS contents in the in vitro cultured plant cells and these changes led to oxidant shock, at the same time, the plant cells would improve their antioxidant abilities to combat mirocysin-RR induced oxidative injury. Our recovery experiment showed that microcystin-RR induced injury is reversible. 7. Apoptosis-like morphological changes such as chromatin condensation and plasmolysis were observed in MC treated Tobacco BY-2 suspension cells. The inhibition of cell division and inducing of cell apoptosis were detected by flow cytometry. Apoptotic peak was detected after 8 d'MC treatment and the apoptosis rate reached 17.43%, which is significantly higher than that of the control.