Phaeocystis pouchetii» in the waters of McMurdo
A H202
chemicalmodels that incorporate depth-dependent light processes. We thank our graduate students (B.M. Brown, J.G. Qian, and B.H. Yocis) and postdoctoral investigator (S.R. Konduru). We also thank the chief scientist, Patrick Neale, and the captain and crew of the R/V Nathaniel B. Palmer for their support throughout the cruise. Thanks are also extended to the Antarctic Research Associates and the Chilean agents (Agunsa) for their logistical support. We gratefully acknowledge the financial support from National Science Foundation grants OPP 93-12767 to David J. Kieber and OPP 92-21598 to Kenneth Mopper.
30 25 20 15 10 >..
a
0 C
5 0
C C.,
30
-D
25 20
0
CL
References
B OH-
0
.4-
Cooper, W.J., R.G. Zika, R.G. Petasne, and A.M. Fischer. 1989. Sunlight-induced photochemistry of humic substances in natural waters: Major reactive species. In I.H. Suffet and P. MacCarthy (Eds), Aquatic humic substances. Washington, D.C.: American Chemical Society. Kieber, R.J., X. Zhou, and K. Mopper. 1990. Formation of carbonyl compounds from UV-induced photodegradation of humic substances in natural waters: Fate of riverine carbon in the sea. Limnology and Oceanography, 35(7), 1503-1515. Kirchman, D.L. 1990. Limitation of bacterial growth by dissolved organic matter in the subarctic pacific. Marine Ecology Progress Series, 62, 47-54. Miller, W.L, and D.R. Kester. 1988. Hydrogen peroxide measurement in seawater by (p-hydroxyphenyl) acetic acid dimerization. Analytical Chemistry, 60(24), 2711-2715. Mopper, K., and X. Zhou. 1990. Hydroxyl radical photoproduction in the sea and its potential impact on marine processes. Science, 250, 661-664. Vastano, S.E., P.J. Mime, W.L. Stahovec, and K. Mopper. 1987. Determination of picomolar levels of flavins in natural waters by solid phase ion-pair extraction and liquid chromatography. Analytica ChimicaActa, 201,127-133. Zepp, R.G., M.M. Gumz, D.J. Bertino, and W.L. Miller. 1992. Use of valerophenone as an ultraviolet-B actinometer for environmental
15 10 5 0
0
2
4
6 10 15 20
Depth (m) Figure 3. In situ photochemical production rates of (A) hydrogen peroxide and (B) the OH radical as a function of depth in the water column. Error bars denote the 95 percent Cl.
Perhaps the most exciting aspect of this study was that we were able to determine in situ photochemical production rates for hydrogen peroxide and the OH radical. The buoy study also provided evidence for photochemical destruction and/or decreased production of hydrogen peroxide in surface waters. In situ measurements will form the basis for photo-
studies. American Chemical Society Environmental Chemistry Symposium Volume (203rd National Meeting of the American
Chemical Society, San Francisco, 1992). Washington, D.C.: Ameri can Chemical Society.
Analysis of dimethylsulfoniopropionate from Phaeocystis pouchetii in the waters of McMurdo Sound, Antarctica MICHELE K. NISHIGucHI, Department of Biology, University of California, Santa Cruz, Santa Cruz, California 95064 BRIAN DUVAL, Department of Microbiology, University of Massachusetts, Amherst, Amherst, Massachusetts 01003 DuANE P. MOSER, Center for Great Lakes Studies, Milwaukee, Wisconsin 53204
haeocystis pouchetii is a marine microalga that forms P globular, gelatinous colonies up to several millimeters in diameter. Phaeocystis also produces dimethyl sulfide (DMS), a sulfur-based compound that is released into the atmosphere and is transformed into sulfate aerosols (Stefels and van Boekel 1993). From these aerosols, cloud condensation nuclei
(CCN) are formed and are the basis of cloud cover and possibly global climate change (Sieburth 1979; Andreae 1991). The importance of this solute has global implications; DMS contributes approximately 30 percent of the total sulfur found on the Earth today (Andreae 1991; Andreae and Raemdonck 1983). Certain species of phytoplankton are responsible for
ANTARCTIC JOURNAL - REVIEW 1994 102
1 percent of the measured irradiation at the surface (3,100 microeinsteins) was recorded in the water column below 1.0-1.5m. Chlorophyll-a concentrations were determined from 1-, 3-, 6-, 9-, 12-, and 15-m depths. Water samples were collected with a Wildco Alpha Bottle and transferred into light impermeable Nalgene containers. Samples were kept on ice until filtration, usually within 2 hours of collection. Filters were treated in 90 percent acetone, vortexed, and kept cold for a period of 15 hours. Samples were shaken 1 hour prior to taking fluorometric measurements using a Turner Designs model 10-Au Fluorometer. All manipulations and measurements were performed in the dark. Readings were timed and calibrated against a standard curve to determine chlorophylla concentrations in micrograms per liter (tgIL), R2=0.997. Vertical plankton tows were made to a depth of 18-19 m, seining through approximately 65,000 L of water column and concentrating the samples in 1 -L bottles. DMS concentrations in the community fraction were determined via DMSP hydrolysis using 10-molar (Al) sodium hydroxide (NaOH) (Karsten et al. 1994). Three replicate samples along with filtered sea-water controls were analyzed by gas chromatography with a Hewlitt Packard Gas Chromatograph fitted with a Flame Photometric Detector (FPD). Known- concentration DMSP samples were measured during each run to calculate a standard curve. Most of the isolation techniques for DMPT dethiomethylase were taken from Nishiguchi and Goff (in preparation). Because of the nature of most Chrysophyte algae, a french press was used to break open cell walls. Purification of the enzyme involved differential centrifugation, two different column chromatography methods (gel sieve and affinity), and activity assays monitored via FPD GC. Western blot analysis was performed from rabbit polyclonal antibodies made from DMPT dethiomethylase from Polysiphonia paniculata (Nishiguchi and Goff in preparation). Western blot analysis and Km studies were completed by Nishiguchi following many runs of the isolation protocol. From the CTD profiles, average water temperature at Tulip Hut increased from -1.5°C in early January to -0.4°C by the end of the month, perhaps due to the southerly current from the open-water regions. The temperature profile of the deeper waters bordering the ice shelf ranged from -1.4°C near the surface to -1.9°C at 470 m (figure 1). Salinity was between 34.2 and 34.3 parts per thousand (ppt) at Tulip Hut and from 34.3 to 34.7 ppt at the ice barrier. Secchi disk readings were recorded daily to note any changes in water-column productivity due to turbidity. Water clarity remained relatively constant throughout the study period. Light transmittance was 0.84 percent at 9 m and 0.40 percent at 18 m. Chlorophyll-a and phytoplankton decreased with depth but at a lesser rate than light extinction. Chlorophyll-a concentrations at Tulip Hut ranged from 0.060 to 0.172 .tg/L. These chlorophyll-a values are an order of magnitude lower than most of the Research on Antarctic Coastal Ecosystem Rates (RACER) field data from the peninsular coastal region (Karl 1993, pp. 1-63) and probably represent an
the large amounts of DMS produced in the ocean (Keller 1989; Keller et al. 1989a, pp. 167-182; Keller et al. 1989b, pp. 101-115), but clearly no mechanism for explaining this efflux of DMS in a homeostatic environment has been proven in vivo.
The process of conversion is accelerated by an enzyme called dimethyipropiothetin dethiomethylase (DMPT dethiomethylase), or better known as dimethylsulfoniopropionate lyase (DMSP lyase) (Nishiguchi and Goff in preparation). The substrate DMSP is found in some species of macroalgae and phytoplankton (Cantoni and Anderson 1956; Reed 1983; Dickson, Wyn Jones, and Davenport 1982; Edwards et al. 1987) and has been extensively studied for its properties as an osmoregulatory solute (Vairavamurthy, Andreae, and Iverson 1985; Kadota and Ishida 1968). DMSP production in macro- and microalgae has been associated with numerous variables in marine habitats including fluctuations in salinity (Vairavamurthy et al. 1985; Dickson et al. 1982; Dickson and Kirst 1986), light intensity (Karsten, Wiencke, and Kirst 1990, 1991), and nitrate concentration (Turner et al. 1988; Grone and Kirst 1992). These previous studies indicate that abiotic factors have a substantial influence on the production of DMSP, but little evidence about the mechanisms which control the accumulation and subsequent conversion of DMSP into DMS is available. Although the rates of production and accumulation of DMSP in algae have been studied extensively, little research on the identification, characterization, and isolation of DMSP lyase has been done. Cantoni and Anderson (1956) were the first to isolate and partially characterize this enzyme. They isolated proteins from the red alga Polysiphonia lanosa (Ceramiales, Rhodomelaceae), and obtained a crude extract containing DMSP lyase activity. The enzyme occurred only in an insoluble protein fraction and contained sulffiydryl groups bound to a membrane-intact system. The sulfydryl compounds are necessary for enzyme activity. Nishiguchi and Goff (in preparation) have recently punfled this enzyme from the northeast Pacific taxon, Polysiphonia paniculata, and have characterized the molecular weight, p1, Km, and the active site that binds with a number of protease inhibitors and is affected by calcium ions in solution. By comparison, we wanted to measure DMSP and DMS concentrations around McMurdo Sound, Antarctica, and correlate them with the amount of Phaeocystis in the water column. We also isolated the DMSP lyase enzyme from P. pouchetii from our sampling stations and compared the enzymatic affinities and activity of this protein. Although the preliminary study was not completed on a purified culture, further studies on pure cultures in vitro have been planned. Conductivity-temperature-depth (CTD) profiles were taken from a depth of 1 meter (m) to a maximum depth of 19 mat Tulip Hut (20 m to bottom). Percentage of light penetration was determined at Tulip Hut using a LI-COR spherical SPQA bulb and recorded on a LI- 1000 data logger. Solar irradiation and percentage of light penetration was measured at the surface and through the water column at 1-m intervals to 18 m. Due to light attenuation by snow-covered ice, less than
ANTARCTIC JOURNAL - REVIEW 1994 103
oligotrophic condition (Kottmeier personal communication; Dieckmann, Arrigo, and Sullivan 1992). Sea water at the 1-m depth was 35-45 percent more productive (per chlorophyll-a) than water at the 15-rn depth at Tulip Hut (figure 2). Palmisano et al. (1986) report higher amounts of chlorophylla, 12 tg/mL, during period of Phaeocystis bloom. From the vertical plankton tows, DMS concentrations ranged between 1.3 and 64.0 nanomoles per liter (nmol/L) sea water. These values are lower than normal DMS levels. In previous blooms, DMS concentrations resulting from Phaeocystis blooms averaged approximately 290 nmol/L (Gibson et al. 1990). Andreae and Raemdonck (1983) report a global weighted-average of 102 nMIL DMS in surface sea water. The observation that coastal sea-water samples (Tulip Hut) had higher DMSP concentrations than water collected from either the marginal ice or the ice-shelf zones may be due to a richer phytoplankton community found in these coastal waters (McTaggert and Burton 1992). Results of the DMSP lyase isolation are shown in figure 3. Apparently another type of sulfur product is being produced by the organism, and that other type is also a sulfur-related compound (figure 3A). Although earlier elution times indicate a less organic-type sulfur product, we had no standards with which to compare this for estimating and evaluating the product. The likelihood of this product being either carbonyl sulfide, dimethyldisulfide, or dimethylsulfoxide is highly possible. From the SDS-PAGE analysis, three proteins that had activity on the FPD GC were isolated from the glutathione column. The first has a molecular weight greater than 100 kD. This weight may be due to some of the membrane-bound protein not being solubilized and running only partially into the gel matrix. Although solubiization of the enzyme was performed overnight, the polymeric structure of this protein may be composed of many subunits that allow the enzyme to function due to osmotic changes or solute control inside the cell. This band is visualized by Western analysis from the Polysiphonia antibody. The second band isolated runs approximately 80 kD and is also positive with the Polysiphonia antibody as well. A third band runs just below band 2 (this has an approximate molecular weight of 72 1W) and does not have a positive reaction in the Western Blot analysis. Because this protein was eluted on the glutathione affinity column, it may be one that is responsible for the production of the other sulfur compound on the GC. The Michaelis-Menton constant (K m) of DMPT dethiomethylase was also evaluated using the enzyme from the Phaeocystis consortia preparation (figure 3B). The assay displayed biphasic kinetics, a characteristic that is different from the Km produced from the Polysiphonia enzyme. Although this study was on the mixed consortia of Phaeocystis, the possibility of other dinofiagellates in the water column producing this enzyme could show another type of binding affinity for DMSP. We report low Phaeocystis numbers in the water off McMurdo station and vicinity for January 1994. Few Phaeocystis blooms were observed in other parts of the Ross Sea (Dunbar personal communication). Because this organism is
IBZ3JN.C141): Ice barrier, hole 4. 23 Ju, 1994, 7752 0125s,166 513452
aI IIn
588.888
Figure 1. CTD profile at the ice shelf barrier site. Salinity (fainter line) increases with depth as temperature decreases 0.5°C through a thermocline of 100 m. (PSU denotes practical salinity units.)
Chlorophyll a in Water column, Tulip Hut
01/25/94 depth (m)
0 03
01 /24/94
• 12 • 15
01/22/94
0.0
0.2
Figure 2. Chlorophyll-a concentrations in the water column at Tulip Hut, McMurdo Station. A 35-45 percent decrease was noted from 1to 15-rn depths. (mg/m3 denotes milligrams per cubic meter.) a major component of the antarctic marine ecosystem, its population cycles (i.e., blooms and population declines) may affect the marine life around McMurdo Sound and could have major control of the amount of sulfate aerosols being produced from antarctic coastal waters. This work was undertaken as part of the McMurdo Biology Course, Biological Adaptations in Extreme Environments, supported by National Science Foundation grant OPP 9317696 to the University of Southern California. We thank Rich Bartlett and Rob Edwards of the McMurdo Dry Valley Long-Term Ecological Research (LTER) project and Lynda J. Goff, who was the algal project group leader, for their assistance.
ANTARCTIC JOURNAL - REVIEW 1994 104
0.1
CHL a (mg/m3)
Dunbar, R. Personal communication. Edwards, D.M., R.H. Reed, J.A. Chudek, R. Foster, and W.D.P. Stewart. 1987. Organic solute accumulation in osmotically-stressed Enteromorpha intestinalis. Marine Biology, 95, 583-592. Gibson, J.A.E., R.C. Garrick, H.R. Burton, and A.R. McTaggart. 1990. Dimethylsulfide and the alga Phaeocystis pouchetii in Antarctic coastal water. Marine Biology, 104, 339-346. Grone, T., and G.O. Kirst. 1992. The effect of nitrogen deficiency, methionine and inhibitors of methionine metabolism on the DMSP contents of Tetraselmis subcordiformis (Stein). Marine Biology, 112, 497-503. Kadota, J., and Y. Ishida. 1968. Effects of salts on enzymatic produc-
A
I11
10
tion of dimethylsulfide from Gyrodinium cohnii. Bulletin ese Society of Science Fish, 34, 512-518.
20
Karl, D. 1993. Microbial processes in the southern oceans. In E.I. Freedmann (Ed.), Antarctic microbiology. New York: Wiley-Liss. Karsten, U., K. Kuck, C. Daniel, C. Wiencke, and others. 1994. A method for complete determination of dimethylsulphoniopropionate (DMSP) in marine macroalgae from different geographical regions. Phycologia, 33, 171-176. Karsten, U., C. Wiencke, and G.O. Kirst. 1990. The effect of light intensity and daylength on the B-dimethylsulfoniopropionate (DMSP) content of marine green macroalgae from Antarctica. P1. Cell. and Environ., 13, 989-993. Karsten, U., C. Wiencke, and G.O. Kirst. 1991. Growth pattern and Bdimethylsulfoniopropionate (DMSP) content of green macroalgae at different irradiances. Marine Biology, 108, 151-155. Keller, M.D. 1989. Dimethyl sulfide production and marine phytoplankton: The importance of species composition and cell size. Limnology and Oceanography, 6,375-382. Keller, M.D., W.K. Bellows, and R.R.L. Guillard. 1989a. Dimethyl sulfide production in marine phytoplankton. In E.S. Saltzmann and W.J. Cooper (Eds.) Biogenic sulfur in the environment (ACS Symposium Series, 383). Keller, M.D., W.K. Bellows, and R.R.L. Guillard. 1989b. Dimethylsulfide production in marine phytoplankton: An additional impact of unusual blooms. In E.M. Cosper, V.M. Bricelj, and E.J. Carpenter (Eds.) Novel phytoplankton blooms. Berlin: Springer-Verlag. Kottmeier, S.T. Personal communication. McTaggarert, A.R., and H. Burton. 1992. Dimethyl sulfide concentrations in the surface waters of the australasian antarctic and subantarctic oceans during and austral summer. Journal of Geophysical Research, 97(C9), 14407-14412. Nishiguchi, M.K., and L.J. Goff. In preparation. Isolation, purification, and characterization of dimethylpropiothetin dethiomethylase (4.4.1.3) from the red alga, Polysiphonia paniculata Montagne.
L1
Figure 3. A. Sephacryl S300 separation of DMSP lyase from P. pouchetii. Filled diamonds represent the activity of DMSP lyase from each fraction measured with FPD-GC. Open squares represent the presence of Carbonyl Sulfides (COS) or dimethyldisulfide (DMDS) present in the sample. Activity of enzyme is measured in tiM of DMSP converted into DMS. Exclusion volume of the column was 6 milliliters at fraction 3 and nonexclusion volume of the column was 38 milliliters, from fractions 13-30. (uM denotes micromolar.) B. Lineweaver- Burke plot for the enzyme DMSP Lyase in P. pouchetii. v= tM of substrate converted per minute, s=DMSP substrate concentration (tM). Sample points represent the mean of 3 samples. Total volume of enzyme added was 50 microliters. Incubations were in 0.05 M potassium phosphate buffer, pH 6.8. All incubations were completed at 12°C for the time measured. (uM/min denotes micromolar per minute.) !
Journal
of Phycology.
Palmisano, A.C., J.B. SooHoo, S.L. SooHoo, S.T. Kottmeier, L.L. Craft, and C.W. Sullivan. 1986. Photoadaptation in Phaeocystis pouchetii advected beneath annual sea ice in McMurdo Sound, Antarctica. Journal of Planktonic Research, 8, 891-906. Reed, R.H. 1983. The osmotic responses of Polysiphonia lanosa (L.) Tandy from marine and estuarine sites: Evidence for incomplete
References Andreae, M.O. 1991. Ocean-atmosphere interactions in the global biogeochemical sulfur cycle. Marine Chemistry, 30,1-29. Andreae, M.O., and H. Raemdonck. 1983. Dimethyl sulfide in the surface ocean and the marine atmosphere: A global view. Science, 221,744-747. Cantoni, G.L., and D.G. Anderson. 1956. Enzymatic cleavage of
dimethylpropiothetin by Polysiphonia lanosa. Journal cal Chemistry, 222,171-177.
ofJa pan-
recovery of turgor. Journal of Experimental Marine Biology and Ecology, 68,169-193. Sieburth, J. McN. 1979. Sea Microbes. New York: Oxford University Press. Stefels, I., and W.H.M. van Boekel. 1993. Production of DMS from dissolved DMSP in axenic cultures of the marine phytoplankton species of Phaeocystis sp. Marine Ecology Progress Series, 97, 11-18. Turner, S.M., G. Malin, P.S. Liss, D.S. Harbour, and P.M. Holligan. 1988. The seasonal variation of dimethyl sulfide and dimethylsulfoniopropionate concentrations in nearshore waters. Limnology and Oceanography, 33, 364-375. Vairavamurthy, A., M.O. Andreae, and R.L. Iverson. 1985. Biosynthesis of dimethylsulfide and dimethylpropiothetin by Hymenomonas carterae in relation to sulfur source and salinity variations. Limnology and Oceangraphy, 30, 59-70.
of Biologi-
Dickson, D.M.J., and G.O. Kirst. 1986. The role of B-dimethylsulfoniopropionate, glycine betaine and homarine in the osmoacclimation of Platymonas subcordiformis. Planta, 167, 536-543. Dickson, D.M,J., R.G. Wyn Jones, and J. Davenport. 1982. Steady state osmotic adaptation in Ulva lactuca. Planta, 155, 409-415. Dieckmann, G.S., K. Arrigo, and C.W. Sullivan. 1992. A high resolution sampler for nutrient and chlorophyll a profiles of the sea ice platelet layer and underlying water column below fast ice in polar oceans: Preliminary results. Marine Ecology Progress Series, 80, 291-300.
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chemicalmodels that incorporate depth-dependent light processes. We thank our graduate students (B.M. Brown, J.G. Qian, and B.H. Yocis) and postdoctoral investigator (S.R. Konduru). We also thank the chief scientist, Patrick Neale, and the captain and crew of the R/V Nathaniel B. Palmer for their support throughout the cruise. Thanks are also extended to the Antarctic Research Associates and the Chilean agents (Agunsa) for their logistical support. We gratefully acknowledge the financial support from National Science Foundation grants OPP 93-12767 to David J. Kieber and OPP 92-21598 to Kenneth Mopper.
30 25 20 15 10 >..
a
0 C
5 0
C C.,
30
-D
25 20
0
CL
References
B OH-
0
.4-
Cooper, W.J., R.G. Zika, R.G. Petasne, and A.M. Fischer. 1989. Sunlight-induced photochemistry of humic substances in natural waters: Major reactive species. In I.H. Suffet and P. MacCarthy (Eds), Aquatic humic substances. Washington, D.C.: American Chemical Society. Kieber, R.J., X. Zhou, and K. Mopper. 1990. Formation of carbonyl compounds from UV-induced photodegradation of humic substances in natural waters: Fate of riverine carbon in the sea. Limnology and Oceanography, 35(7), 1503-1515. Kirchman, D.L. 1990. Limitation of bacterial growth by dissolved organic matter in the subarctic pacific. Marine Ecology Progress Series, 62, 47-54. Miller, W.L, and D.R. Kester. 1988. Hydrogen peroxide measurement in seawater by (p-hydroxyphenyl) acetic acid dimerization. Analytical Chemistry, 60(24), 2711-2715. Mopper, K., and X. Zhou. 1990. Hydroxyl radical photoproduction in the sea and its potential impact on marine processes. Science, 250, 661-664. Vastano, S.E., P.J. Mime, W.L. Stahovec, and K. Mopper. 1987. Determination of picomolar levels of flavins in natural waters by solid phase ion-pair extraction and liquid chromatography. Analytica ChimicaActa, 201,127-133. Zepp, R.G., M.M. Gumz, D.J. Bertino, and W.L. Miller. 1992. Use of valerophenone as an ultraviolet-B actinometer for environmental
15 10 5 0
0
2
4
6 10 15 20
Depth (m) Figure 3. In situ photochemical production rates of (A) hydrogen peroxide and (B) the OH radical as a function of depth in the water column. Error bars denote the 95 percent Cl.
Perhaps the most exciting aspect of this study was that we were able to determine in situ photochemical production rates for hydrogen peroxide and the OH radical. The buoy study also provided evidence for photochemical destruction and/or decreased production of hydrogen peroxide in surface waters. In situ measurements will form the basis for photo-
studies. American Chemical Society Environmental Chemistry Symposium Volume (203rd National Meeting of the American
Chemical Society, San Francisco, 1992). Washington, D.C.: Ameri can Chemical Society.
Analysis of dimethylsulfoniopropionate from Phaeocystis pouchetii in the waters of McMurdo Sound, Antarctica MICHELE K. NISHIGucHI, Department of Biology, University of California, Santa Cruz, Santa Cruz, California 95064 BRIAN DUVAL, Department of Microbiology, University of Massachusetts, Amherst, Amherst, Massachusetts 01003 DuANE P. MOSER, Center for Great Lakes Studies, Milwaukee, Wisconsin 53204
haeocystis pouchetii is a marine microalga that forms P globular, gelatinous colonies up to several millimeters in diameter. Phaeocystis also produces dimethyl sulfide (DMS), a sulfur-based compound that is released into the atmosphere and is transformed into sulfate aerosols (Stefels and van Boekel 1993). From these aerosols, cloud condensation nuclei
(CCN) are formed and are the basis of cloud cover and possibly global climate change (Sieburth 1979; Andreae 1991). The importance of this solute has global implications; DMS contributes approximately 30 percent of the total sulfur found on the Earth today (Andreae 1991; Andreae and Raemdonck 1983). Certain species of phytoplankton are responsible for
ANTARCTIC JOURNAL - REVIEW 1994 102
1 percent of the measured irradiation at the surface (3,100 microeinsteins) was recorded in the water column below 1.0-1.5m. Chlorophyll-a concentrations were determined from 1-, 3-, 6-, 9-, 12-, and 15-m depths. Water samples were collected with a Wildco Alpha Bottle and transferred into light impermeable Nalgene containers. Samples were kept on ice until filtration, usually within 2 hours of collection. Filters were treated in 90 percent acetone, vortexed, and kept cold for a period of 15 hours. Samples were shaken 1 hour prior to taking fluorometric measurements using a Turner Designs model 10-Au Fluorometer. All manipulations and measurements were performed in the dark. Readings were timed and calibrated against a standard curve to determine chlorophylla concentrations in micrograms per liter (tgIL), R2=0.997. Vertical plankton tows were made to a depth of 18-19 m, seining through approximately 65,000 L of water column and concentrating the samples in 1 -L bottles. DMS concentrations in the community fraction were determined via DMSP hydrolysis using 10-molar (Al) sodium hydroxide (NaOH) (Karsten et al. 1994). Three replicate samples along with filtered sea-water controls were analyzed by gas chromatography with a Hewlitt Packard Gas Chromatograph fitted with a Flame Photometric Detector (FPD). Known- concentration DMSP samples were measured during each run to calculate a standard curve. Most of the isolation techniques for DMPT dethiomethylase were taken from Nishiguchi and Goff (in preparation). Because of the nature of most Chrysophyte algae, a french press was used to break open cell walls. Purification of the enzyme involved differential centrifugation, two different column chromatography methods (gel sieve and affinity), and activity assays monitored via FPD GC. Western blot analysis was performed from rabbit polyclonal antibodies made from DMPT dethiomethylase from Polysiphonia paniculata (Nishiguchi and Goff in preparation). Western blot analysis and Km studies were completed by Nishiguchi following many runs of the isolation protocol. From the CTD profiles, average water temperature at Tulip Hut increased from -1.5°C in early January to -0.4°C by the end of the month, perhaps due to the southerly current from the open-water regions. The temperature profile of the deeper waters bordering the ice shelf ranged from -1.4°C near the surface to -1.9°C at 470 m (figure 1). Salinity was between 34.2 and 34.3 parts per thousand (ppt) at Tulip Hut and from 34.3 to 34.7 ppt at the ice barrier. Secchi disk readings were recorded daily to note any changes in water-column productivity due to turbidity. Water clarity remained relatively constant throughout the study period. Light transmittance was 0.84 percent at 9 m and 0.40 percent at 18 m. Chlorophyll-a and phytoplankton decreased with depth but at a lesser rate than light extinction. Chlorophyll-a concentrations at Tulip Hut ranged from 0.060 to 0.172 .tg/L. These chlorophyll-a values are an order of magnitude lower than most of the Research on Antarctic Coastal Ecosystem Rates (RACER) field data from the peninsular coastal region (Karl 1993, pp. 1-63) and probably represent an
the large amounts of DMS produced in the ocean (Keller 1989; Keller et al. 1989a, pp. 167-182; Keller et al. 1989b, pp. 101-115), but clearly no mechanism for explaining this efflux of DMS in a homeostatic environment has been proven in vivo.
The process of conversion is accelerated by an enzyme called dimethyipropiothetin dethiomethylase (DMPT dethiomethylase), or better known as dimethylsulfoniopropionate lyase (DMSP lyase) (Nishiguchi and Goff in preparation). The substrate DMSP is found in some species of macroalgae and phytoplankton (Cantoni and Anderson 1956; Reed 1983; Dickson, Wyn Jones, and Davenport 1982; Edwards et al. 1987) and has been extensively studied for its properties as an osmoregulatory solute (Vairavamurthy, Andreae, and Iverson 1985; Kadota and Ishida 1968). DMSP production in macro- and microalgae has been associated with numerous variables in marine habitats including fluctuations in salinity (Vairavamurthy et al. 1985; Dickson et al. 1982; Dickson and Kirst 1986), light intensity (Karsten, Wiencke, and Kirst 1990, 1991), and nitrate concentration (Turner et al. 1988; Grone and Kirst 1992). These previous studies indicate that abiotic factors have a substantial influence on the production of DMSP, but little evidence about the mechanisms which control the accumulation and subsequent conversion of DMSP into DMS is available. Although the rates of production and accumulation of DMSP in algae have been studied extensively, little research on the identification, characterization, and isolation of DMSP lyase has been done. Cantoni and Anderson (1956) were the first to isolate and partially characterize this enzyme. They isolated proteins from the red alga Polysiphonia lanosa (Ceramiales, Rhodomelaceae), and obtained a crude extract containing DMSP lyase activity. The enzyme occurred only in an insoluble protein fraction and contained sulffiydryl groups bound to a membrane-intact system. The sulfydryl compounds are necessary for enzyme activity. Nishiguchi and Goff (in preparation) have recently punfled this enzyme from the northeast Pacific taxon, Polysiphonia paniculata, and have characterized the molecular weight, p1, Km, and the active site that binds with a number of protease inhibitors and is affected by calcium ions in solution. By comparison, we wanted to measure DMSP and DMS concentrations around McMurdo Sound, Antarctica, and correlate them with the amount of Phaeocystis in the water column. We also isolated the DMSP lyase enzyme from P. pouchetii from our sampling stations and compared the enzymatic affinities and activity of this protein. Although the preliminary study was not completed on a purified culture, further studies on pure cultures in vitro have been planned. Conductivity-temperature-depth (CTD) profiles were taken from a depth of 1 meter (m) to a maximum depth of 19 mat Tulip Hut (20 m to bottom). Percentage of light penetration was determined at Tulip Hut using a LI-COR spherical SPQA bulb and recorded on a LI- 1000 data logger. Solar irradiation and percentage of light penetration was measured at the surface and through the water column at 1-m intervals to 18 m. Due to light attenuation by snow-covered ice, less than
ANTARCTIC JOURNAL - REVIEW 1994 103
oligotrophic condition (Kottmeier personal communication; Dieckmann, Arrigo, and Sullivan 1992). Sea water at the 1-m depth was 35-45 percent more productive (per chlorophyll-a) than water at the 15-rn depth at Tulip Hut (figure 2). Palmisano et al. (1986) report higher amounts of chlorophylla, 12 tg/mL, during period of Phaeocystis bloom. From the vertical plankton tows, DMS concentrations ranged between 1.3 and 64.0 nanomoles per liter (nmol/L) sea water. These values are lower than normal DMS levels. In previous blooms, DMS concentrations resulting from Phaeocystis blooms averaged approximately 290 nmol/L (Gibson et al. 1990). Andreae and Raemdonck (1983) report a global weighted-average of 102 nMIL DMS in surface sea water. The observation that coastal sea-water samples (Tulip Hut) had higher DMSP concentrations than water collected from either the marginal ice or the ice-shelf zones may be due to a richer phytoplankton community found in these coastal waters (McTaggert and Burton 1992). Results of the DMSP lyase isolation are shown in figure 3. Apparently another type of sulfur product is being produced by the organism, and that other type is also a sulfur-related compound (figure 3A). Although earlier elution times indicate a less organic-type sulfur product, we had no standards with which to compare this for estimating and evaluating the product. The likelihood of this product being either carbonyl sulfide, dimethyldisulfide, or dimethylsulfoxide is highly possible. From the SDS-PAGE analysis, three proteins that had activity on the FPD GC were isolated from the glutathione column. The first has a molecular weight greater than 100 kD. This weight may be due to some of the membrane-bound protein not being solubilized and running only partially into the gel matrix. Although solubiization of the enzyme was performed overnight, the polymeric structure of this protein may be composed of many subunits that allow the enzyme to function due to osmotic changes or solute control inside the cell. This band is visualized by Western analysis from the Polysiphonia antibody. The second band isolated runs approximately 80 kD and is also positive with the Polysiphonia antibody as well. A third band runs just below band 2 (this has an approximate molecular weight of 72 1W) and does not have a positive reaction in the Western Blot analysis. Because this protein was eluted on the glutathione affinity column, it may be one that is responsible for the production of the other sulfur compound on the GC. The Michaelis-Menton constant (K m) of DMPT dethiomethylase was also evaluated using the enzyme from the Phaeocystis consortia preparation (figure 3B). The assay displayed biphasic kinetics, a characteristic that is different from the Km produced from the Polysiphonia enzyme. Although this study was on the mixed consortia of Phaeocystis, the possibility of other dinofiagellates in the water column producing this enzyme could show another type of binding affinity for DMSP. We report low Phaeocystis numbers in the water off McMurdo station and vicinity for January 1994. Few Phaeocystis blooms were observed in other parts of the Ross Sea (Dunbar personal communication). Because this organism is
IBZ3JN.C141): Ice barrier, hole 4. 23 Ju, 1994, 7752 0125s,166 513452
aI IIn
588.888
Figure 1. CTD profile at the ice shelf barrier site. Salinity (fainter line) increases with depth as temperature decreases 0.5°C through a thermocline of 100 m. (PSU denotes practical salinity units.)
Chlorophyll a in Water column, Tulip Hut
01/25/94 depth (m)
0 03
01 /24/94
• 12 • 15
01/22/94
0.0
0.2
Figure 2. Chlorophyll-a concentrations in the water column at Tulip Hut, McMurdo Station. A 35-45 percent decrease was noted from 1to 15-rn depths. (mg/m3 denotes milligrams per cubic meter.) a major component of the antarctic marine ecosystem, its population cycles (i.e., blooms and population declines) may affect the marine life around McMurdo Sound and could have major control of the amount of sulfate aerosols being produced from antarctic coastal waters. This work was undertaken as part of the McMurdo Biology Course, Biological Adaptations in Extreme Environments, supported by National Science Foundation grant OPP 9317696 to the University of Southern California. We thank Rich Bartlett and Rob Edwards of the McMurdo Dry Valley Long-Term Ecological Research (LTER) project and Lynda J. Goff, who was the algal project group leader, for their assistance.
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0.1
CHL a (mg/m3)
Dunbar, R. Personal communication. Edwards, D.M., R.H. Reed, J.A. Chudek, R. Foster, and W.D.P. Stewart. 1987. Organic solute accumulation in osmotically-stressed Enteromorpha intestinalis. Marine Biology, 95, 583-592. Gibson, J.A.E., R.C. Garrick, H.R. Burton, and A.R. McTaggart. 1990. Dimethylsulfide and the alga Phaeocystis pouchetii in Antarctic coastal water. Marine Biology, 104, 339-346. Grone, T., and G.O. Kirst. 1992. The effect of nitrogen deficiency, methionine and inhibitors of methionine metabolism on the DMSP contents of Tetraselmis subcordiformis (Stein). Marine Biology, 112, 497-503. Kadota, J., and Y. Ishida. 1968. Effects of salts on enzymatic produc-
A
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tion of dimethylsulfide from Gyrodinium cohnii. Bulletin ese Society of Science Fish, 34, 512-518.
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Karl, D. 1993. Microbial processes in the southern oceans. In E.I. Freedmann (Ed.), Antarctic microbiology. New York: Wiley-Liss. Karsten, U., K. Kuck, C. Daniel, C. Wiencke, and others. 1994. A method for complete determination of dimethylsulphoniopropionate (DMSP) in marine macroalgae from different geographical regions. Phycologia, 33, 171-176. Karsten, U., C. Wiencke, and G.O. Kirst. 1990. The effect of light intensity and daylength on the B-dimethylsulfoniopropionate (DMSP) content of marine green macroalgae from Antarctica. P1. Cell. and Environ., 13, 989-993. Karsten, U., C. Wiencke, and G.O. Kirst. 1991. Growth pattern and Bdimethylsulfoniopropionate (DMSP) content of green macroalgae at different irradiances. Marine Biology, 108, 151-155. Keller, M.D. 1989. Dimethyl sulfide production and marine phytoplankton: The importance of species composition and cell size. Limnology and Oceanography, 6,375-382. Keller, M.D., W.K. Bellows, and R.R.L. Guillard. 1989a. Dimethyl sulfide production in marine phytoplankton. In E.S. Saltzmann and W.J. Cooper (Eds.) Biogenic sulfur in the environment (ACS Symposium Series, 383). Keller, M.D., W.K. Bellows, and R.R.L. Guillard. 1989b. Dimethylsulfide production in marine phytoplankton: An additional impact of unusual blooms. In E.M. Cosper, V.M. Bricelj, and E.J. Carpenter (Eds.) Novel phytoplankton blooms. Berlin: Springer-Verlag. Kottmeier, S.T. Personal communication. McTaggarert, A.R., and H. Burton. 1992. Dimethyl sulfide concentrations in the surface waters of the australasian antarctic and subantarctic oceans during and austral summer. Journal of Geophysical Research, 97(C9), 14407-14412. Nishiguchi, M.K., and L.J. Goff. In preparation. Isolation, purification, and characterization of dimethylpropiothetin dethiomethylase (4.4.1.3) from the red alga, Polysiphonia paniculata Montagne.
L1
Figure 3. A. Sephacryl S300 separation of DMSP lyase from P. pouchetii. Filled diamonds represent the activity of DMSP lyase from each fraction measured with FPD-GC. Open squares represent the presence of Carbonyl Sulfides (COS) or dimethyldisulfide (DMDS) present in the sample. Activity of enzyme is measured in tiM of DMSP converted into DMS. Exclusion volume of the column was 6 milliliters at fraction 3 and nonexclusion volume of the column was 38 milliliters, from fractions 13-30. (uM denotes micromolar.) B. Lineweaver- Burke plot for the enzyme DMSP Lyase in P. pouchetii. v= tM of substrate converted per minute, s=DMSP substrate concentration (tM). Sample points represent the mean of 3 samples. Total volume of enzyme added was 50 microliters. Incubations were in 0.05 M potassium phosphate buffer, pH 6.8. All incubations were completed at 12°C for the time measured. (uM/min denotes micromolar per minute.) !
Journal
of Phycology.
Palmisano, A.C., J.B. SooHoo, S.L. SooHoo, S.T. Kottmeier, L.L. Craft, and C.W. Sullivan. 1986. Photoadaptation in Phaeocystis pouchetii advected beneath annual sea ice in McMurdo Sound, Antarctica. Journal of Planktonic Research, 8, 891-906. Reed, R.H. 1983. The osmotic responses of Polysiphonia lanosa (L.) Tandy from marine and estuarine sites: Evidence for incomplete
References Andreae, M.O. 1991. Ocean-atmosphere interactions in the global biogeochemical sulfur cycle. Marine Chemistry, 30,1-29. Andreae, M.O., and H. Raemdonck. 1983. Dimethyl sulfide in the surface ocean and the marine atmosphere: A global view. Science, 221,744-747. Cantoni, G.L., and D.G. Anderson. 1956. Enzymatic cleavage of
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