Mechanisms Of Cadmium Uptake And Tolerance In Phytolacca Americana L.

Heavy metals are toxic at excessive concentrations and may pollute the natural and man-made environment ecosystem. In recent years, heavy metal pollution of soil has received increasing attentions worldwide mainly because of the public awareness of environmental issues. Among all of the heavy metals, cadmium (Cd) is of most concern, for its great mobility and high toxicity in the soil environment. Meanwhile, large area of farmland is deeply contaminated by Cd, which leads to one of the major issues of agricultural product safety in China. Phytolacca americana L. (pokeweed) and Sedum alfredii Hance were reported to be Cd-tolerant plants or hyperaccumulators, which were benefit for phytoremediation of Cd polluted soil. However, the mechanisms of Cd uptake, accumulation, detoxification and tolerance were needed for further research. In the present work, we used hydroponically grown seedlings of pokeweed to find out the mechanisms in countering Cd stress in the plant cultures. We used inhibitors to study the Cd uptake way, investigated the impacts of Cd on plant physiology and biology characters and ultrastructure changes through biochemical analysis and transmission electron microscope (TEM) observation, examined cellular distribution of Cd with ultracentrifugation, TEM and energy disperse spectroscopy (EDX) analysis, isolated differential expression proteins from plant using SDS-PAGE and two dimensional electrophoresis (2-DE) technologies. The purpose of our study was to clarify the mechanisms of Cd uptake and tolerance in pokeweed, which could provide theoretical and scientific basis for phytoremediation of heavy metal contaminated soil. The main results were summarized as follows:Pokeweed is of high ability for accumulating Cd and this metal is mainly accumulated in plant root. With the hydraulic cultivation of 100μM Cd, the Cd contents were as high as 966.9 mg/kg in shoots and 8179.1 mg/kg in roots, respectively, and the plant showed a little toxicity symptoms. Meanwhile, pokeweed is still of high accumulating ability even under relatively low Cd concentration treatments. Both uncoupling agents 2,4-dinitrophenol (DNP) and Ca2+ channel inhibitor LaCl3 may inhibit the absorption of Cd into plant, suggesting that the uptake of Cd in pokeweed may be an active absorption process and it is closely related to Ca2+ channels. Furthermore, different Cd treatments have different effects on the accumulation of elements in plant. Pokeweed may have competitive absorption towards Cd and Mn. The uptake of Cd may have no/little relationship with Zn2+ channels while P and S may take part in the process of Cd tolerance and detoxification in pokeweed.Physiological and biochemical analysises showed that under 100μM Cd treatments, both chlorophyll and MDA (the peroxidating substance of plasma membrane) in pokeweed leaves were not significantly changed, indicating that the photosynthesis was not inhibited and plasma membrane was not injured. However, as the concentrations of Cd treatments continue to increase, all chlorophyll a, chlorophyll b and the total chlorophyll contents were declined while the MDA contents increased dramatically, suggesting chlorophyll synthesis, photosynthesis process and plant growth may all likely be inhibited by Cd. Besides, differences were noted in different antioxidase activities under Cd stress and antioxidase system may have certain extent to help relieve the toxicity of Cd in pokeweed. TEM results also illustrated varying degrees of damages in membrane system, chloroplast, mitochondria and other cellular organelles and these phenomenons may be due to the loss of cellular redox homeostasis and an increase of lipid peroxidation.Cd analysis at the subcellular level of plant tissue demonstrated that large proportion of Cd was stored in the soluble fraction (53.7%～68.3%) or bound to the cell wall fraction (23.4%～29.1%). Besides, Cd must be existed in different chemical forms among different tissues and Cd concentrations bound to each form in the plant increased in a concentration-dependent manner following Cd exposure. In roots, the majority of Cd was in inorganic form, while in stems and leaves most of the Cd was integrated with pectates and protein. Both the vacuoles and the cell walls might be involved in the Cd tolerance mechanisms to protect metabolically active cellular compartments from toxic Cd concentrationsDifferential expression proteins in pokeweed tissues under Cd stress were studied by SDS-PAGE and 2-DE techniques. SDS-PAGE results showed that the molecular weight of proteins isolated from roots and leaves of pokeweed is concentrated between 10～100 kDa, while in the 25 kDa and 50 kDa position scales there were two main protein bands. Compared with the control, root proteins did not change significantly after Cd treatment, while in leaves the intensity of protein bands decreased in the 60～150 kDa position scales and increased in the 25～50 kDa position scales, respectively. Moreover,2-DE results indicated that 26 proteins' expression changed in pokeweed roots after Cd treatment. Among them,8 proteins were disappeared or down-regulated, while 18 proteins were induced or up-regulated. In plant leaves,2-DE results indicated that a total of 46 proteins'abundance were altered, among them,21 proteins were two fold down-regulated and 25 proteins were two fold up-regulated These proteins may be related to the response of pokeweed to Cd stress, involved in the chelation of Cd and play an active role in the uptake and tolerance processes of Cd in pokeweed. Therefore, further studies with respect to protein identification and their functions should be carried out.