.............................................................. An antioxidant function for DMSP and DMS in marine algae W. Sunda*, D. J. Kieber , R. P. Kiene‡ & S. Huntsman* * Beaufort Laboratory, National Oceanic and Atmospheric Administration, Beaufort, North Carolina 28516, USA State University of New York, College of Environmental Science and Forestry, Chemistry Department, 1 Forestry Drive, Syracuse, New York 13210, USA ‡ University of South Alabama, Department of Marine Sciences, Mobile, Alabama 36688, USA ............................................................................................................................................................................. The algal osmolyte dimethylsulphoniopropionate (DMSP) and its enzymatic cleavage product dimethylsulphide (DMS) contrib- ute significantly to the global sulphur cycle 1–3 , yet their physio- logical functions are uncertain 4 . Here we report results that, together with those in the literature 5,6 , show that DMSP and its breakdown products (DMS, acrylate, dimethylsulphoxide, and methane sulphinic acid) readily scavenge hydroxyl radicals and other reactive oxygen species, and thus may serve as an anti- oxidant system, regulated in part by enzymatic cleavage of DMSP. In support of this hypothesis, we found that oxidative stressors, solar ultraviolet radiation 7 , CO 2 limitation 8 , Fe limitation, high Cu 21 (ref. 9) and H 2 O 2 substantially increased cellular DMSP and/or its lysis to DMS in marine algal cultures. Our results indicate direct links between such stressors and the dynamics of DMSP and DMS in marine phytoplankton, which probably influence the production of DMS and its release to the atmos- phere. As oxidation of DMS to sulphuric acid in the atmosphere provides a major source of sulphate aerosols and cloud conden- sation nuclei 3 , oxidative stressors—including solar radiation and Fe limitation—may be involved in complex ocean–atmosphere feedback loops that influence global climate and hydrological cycles 1,2 . DMSP occurs at high cellular concentrations (100–400 mmol l 21 ) in many marine algae, including prymnesiophytes and dinoflagel- lates, and thus functions as an osmolyte in these algae. DMSP has also been proposed to serve as a cryoprotectant in polar algae and as a grazing deterrent via its cleavage to acrylate, although its overall physiological functions remain unclear 4 . Its cellular concentration increases with light in many algal species 10,11 , an effect not readily explained by existing proposed functions. In laboratory exper- iments, we found that DMSP reacts rapidly with the hydroxyl radical ( · OH), and thus could serve as an effective cellular scavenger of this harmful radical (Table 1). DMSP’s enzymatic cleavage products, DMS and acrylate, are even more effective at scavenging · OH, as are the DMS oxidation products dimethylsulphoxide (DMSO) and methane sulphinic acid (MSNA) (Table 1). Calcu- lations suggest that, taken together, these molecules constitute an antioxidant system, which could be more effective at scavenging · OH in high-DMSP algae than other well-recognized antioxidants, such as ascorbate and glutathione (Table 1). An antioxidant func- tion would explain the observed increase in algal DMSP concen- trations at high light 10,11 , as · OH is produced as a by-product of photosynthesis. All five proposed antioxidant compounds—DMSP, acrylate, DMS, DMSO and MSNA (Table 1)—have unique chemical and physical properties that the cell can exploit. The metabolically compatible zwitterion DMSP is lysed to DMS and acrylate via the enzyme DMSP lyase, whose cellular function and regulation are not well understood 12 . Acrylate and DMS are ,20 and ,60 times more reactive towards · OH than is DMSP (Table 1), and DMS is highly reactive toward singlet oxygen 13 . Acrylate is charged at physiological pHs, and like DMSP, cannot penetrate into lipid membranes; but uncharged DMS molecules can readily diffuse into photosynthetic membranes, an important site of harmful lipid peroxidation reac- tions. Thus, DMSP lysis should substantially increase the antiox- idant protection in both aqueous and lipid membrane phases within the cell. This increased protection could be modulated by DMSP lyase, providing an important metabolic function for this enzyme. Much of the released DMS should oxidize to DMSO or other oxidized sulphur species, while the remainder would diffuse from the cell. The DMSO produced from · OH oxidation of DMS or DMSP (see Methods) is much more hydrophilic than DMS, retarding its loss across cell membranes and allowing it to accumu- late at high cellular concentrations 14–16 . These high concentrations, combined with a high reactivity toward · OH, makes DMSO an effective antioxidant (Table 1), as has been proposed previously 15 and has been directly observed in isolated chloroplasts 6 . DMSO oxidation by · OH produces the water-soluble antioxidant MSNA, which can further react with and scavenge · OH 17 (Table 1). Thus, enzymatic lysis of DMSP gives rise to four water- or lipid-soluble antioxidant scavengers of · OH radicals and at least one scavenger (DMS) of singlet oxygen. We suggest that this multiplicative effect combined with high cellular DMSP concentrations make DMSP and its lysis and oxidation products a highly effective antioxidant system. Organisms generally acclimate to oxidative stress by up-regu- lation of antioxidant systems 7–9 . Thus, under our hypothesis, phytoplankton should respond to higher levels of oxidative stress by increased DMSP synthesis and lysis to DMS. This indeed is what we observed for the diatom Thalassiosira pseudonana under CO 2 limitation, a known oxidative stressor 8 , and Fe limitation, shown here to also oxidatively stress cells. A nutrient-sufficient control culture had a high specific growth rate (1.45 d 21 ), high cellular chlorophyll a, low cellular DMSP (,1 mmol l 21 ), low DMS to cell- volume ratios, and low cellular activity of the antioxidant enzyme ascorbate peroxidase (APX; Table 2). But under CO 2 limitation and Fe limitation, we observed 20- to 60-fold increases in cellular DMSP, increased DMS/cell-volume ratios, and increased APX activities, in conjunction with large decreases in growth rate and chlorophyll a. APX is the main chloroplast enzyme for removal of H 2 O 2 , a reactive by-product of photosynthesis and a major precursor for · OH radical formation 18,19 . As antioxidant systems are up-regulated under increased oxidative stress, the significant increase in APX activity/ chlorophyll a ratios under CO 2 limitation (P , 0.001, t-test) and Fe limitation (P , 0.01) indicate that both increased oxidative stress within the chloroplast. The increase in APX activity is noteworthy as APX contains Fe, and would be expected to decrease under Fe limitation. But under Fe limitation, there is a decrease in Fe-containing components of the photosynthetic electron transport chain (for example, the cytochrome b 6 /f complex) relative to Table 1 Computed in vitro half-lives for ·OH radical Compound Concentration Rate constant* Ref. ·OH half-life (mmol l 21 ) (M 21 s 21 ) (ns) ............................................................................................................................................................................. DMSP 150–450 3 £ 10 8 This work 5–15 Acrylate ,1–40? 5.6 £ 10 9 Ref. 5 3–124 DMS ,1–40? 1.9 £ 10 10 Ref. 5 0.9–36 DMSO 30–90 6.6 £ 10 9 Ref. 5 1.2–3.5 MSNA ? 9 £ 10 9 This work ? Glutathione 2.4‡ 1.4 £ 10 10 Ref. 5 21 Ascorbate 1–6 § 1.1 £ 10 10 Ref. 5 11–63 ............................................................................................................................................................................. Data are shown for a range of concentrations of DMSP and its breakdown products, and of antioxidants (glutathione and ascorbate) in high-DMSP algae (for example, E. huxleyi). * Rate constant for reaction of each compound with ·OH at 25 8C: pH conditions are 7.5 for this work, and 7.0 for ref. 5. Approximate DMSP range that we observed in E. huxleyi. The cellular DMSO concentration was computed by dividing the cell DMSP concentration by the DMSP/DMSO ratio (,5) found in near- surface oceanic plankton 16 . ‡ Measured value in E. huxleyi (CCMP373) cultures 29 . § Estimated average range for phytoplankton based on the reported range per dry weight of algal cells (5–28 mmol g 21 : ref. 30), an assumed algal dry to wet weight ratio of 0.8, and a cell density of 1.03 g cm 23 (the value for sea water). letters to nature NATURE | VOL 418 | 18 JULY 2002 | www.nature.com/nature 317 © 2002 Nature Publishing Group