Phytochelatin production in marine algae. 2. Induction by various metals

Phytochelatin has been quantified in Thalassiosira weissflogii, a marine diatom after exposure to a series of trace metals (Cd, Pb, Ni, Cu, Zn, Co, Ag, and Hg) at concentrations similar to those in the marine environment. Within the range of concentrations relevant to natural waters, Cd, and to a lesser extent Cu and Zn, are the most effective inducers of phytochelatins. The generality of this result was confirmed by short-term experiments with two other phytoplankton species. Quantification of intracellular Cd, Ni, and Zn shows that phytochelatin production does not follow a simple stoichiometric relationship to the metal quotas. The rapid formation of phytochelatin in T. weissflogii after Cd exposure and the fast elimination when metal exposure is alleviated reveal a dynamic pool of phytochelatin which is tightly regulated by the cell. Many trace metals have been shown to induce phytochelatin production in plants (Grill et al. 1987), although the concentration necessary to stimulate the response as well as the magnitude of the response depend on the particular metal. Although it is believed that production of this peptide is a general metal detoxification system, Cd has been found to be the most effective inducer of phytochelatin synthase (Grill et al. 1989). Our goal is to elucidate the factors that control phytochelatin production by phytoplankton in the laboratory in order to better understand what stimulates phytochelatin production in the field (Ahner et al. 1994). In a companion paper (Ahncr et al. 1995), we investigated phytochelatin production by several phytoplankton species in response to Cd. In this study we examine the response of Thalassiosira weissflogii to a variety of metals (Cd, Pb, Cu, Ni, Zn, Co, Ag, and Hg), all of which have been found to stimulate phytochelatin production in higher plants. As in our experiments with Cd, we tested free metal concentrations that would be encountered in natural seawater in order to evaluate which metals may stimulate this response in natural populations of algae. We performed short-term assays with two other phytoplankton species to compare the patterns of the phytochelatin response to various metals. Finally, to assess how changing environmental conditions might affect cellular concentrations of phytochelatin, we examined the kinetics of phytochelatin production and elimination upon changes in metal exposure. Materials and methods Cell preparation and HPLC chromatography T. weissflogii (Actin) was cultured in Aquil (Price et al. 199 1) Acknowledgments This work has been funded by grants from the National Science Foundation, Office of Naval Research, Environmental Protection Agency, MIT Sea Grant, and the Massachusetts Water Resource Authority. Special thanks to Robert Mason who provided cells and growth rates for the mercury cxperimcnts. as detailed by Ahner et al. 1995 but with the addition of different metals at several fret metal concentrations (pMe = -logEMe”+]). The metals were added in addition to the normal trace metals in the Aquil seawater medium containing either 10 or 100 PM ethylenediaminetetraacetic acid (EDTA). Most metal additions were made as MeEDTA complexes to achieve the various free ion (pMe) and total inorganic ion (Me’) concentrations (Table 1). Ag and Hg were added as their chloride salts, since they do not form strong complexes with EDTA. Methods outlining cell sample preparation and HPLC chromatographic methods are given by Ahncr et al. (1995). Metal quota experimentsT. weissflogii was cultured at four different concentrations each of Ni, Zn, and Cd (pMe = 12-9) as described above with the addition of carrier-free radiotracers (63Ni, 65Zn, lo9Cd) to each growth medium. Medium to which ‘j5Zn or lo9Cd was added was allowed to equilibrate for at least 24 h to ensure that the radiotraccrs were proportionally distributed between the fret ion and EDTA complex. For experiments involving 63Ni, 1 week of equilibration time is necessary because of the slow reaction rates of Ni (Price et al. 199 1). Near the end of exponential growth, cells were collected onto 25-mm 3-pm Poretics polycarbonate membrane filters with gentle filtration, incubated 15 min on the filter with 1 mM diethyltriaminepentaacetic acid (DTPA) dissolved in seawater to remove surface-bound lo9Cd and 65Zn, and then rinsed three times with filtered seawater (Ahner et al. 1995). Ni (63Ni)-containing cells were incubated first for 3 min with a 1 mM 8-hydroxyquinoline-5-sulfonate seawater solution and then rinsed with filtered seawater (Price and Morel 199 1). Isotope remaining in the cells on the filter was counted by liquid scintillation in a Beckman LS 1801. A calculated specific activity was then used to determine the metal quotas of the cells at the various levels of pMc. Radiolabeled cells were spiked with Lugol’s solution (Parsons et al. 1984) and counted microscopically with a hemacytometer.