KOLBER, ZBIGNIEW, KEVIN D. WYMAN. AND PAUL G
Transcription
KOLBER, ZBIGNIEW, KEVIN D. WYMAN. AND PAUL G
hmnol. Oceanogr., 0 by the American 1990, 35(l), 1990, 72-19 Society of’ Limnology and Oceanography, Inc. Natural variability in photosynthetic energy conversion efkiency: A field study in the Gulf of Maine Zbigniew Kolber, Kevin V. Wyman, and Paul G. Falkowski Oceanographic Sciences Division, Brookhavcn National Laboratory, Upton, New York 11973 Abstract The maximal change in the quantum yield of fluorescence (A&,,) is a quantitative measure of photosynthetic conversion efhcicncy of phytoplankton. Using a pump-and-probe fluorometer, we measured A&, along the 100-m isobath in the Gulf of Maine in June 1987. The hydrographic regime was charactcrizcd by a nutrient-rich, vertically mixed region in the northcast and a nutrientdepleted, stratified region to the southwest. The results reveal that A&,, is strongly related to the rate of supply of dissolved inorganic nitrogen and provide strong evidence that photosynthetic energy conversion can be nutrient limited in natural phytoplankton communities. The issue of whether phytoplankton productivity and growth is nutrient limited in the sea is contentious (Thomas 1970; EppIcy 1980; Goldman 1980). Although inorganic nutrient concentrations may be vanishingly low, it has been suggested that the regenerative l’iux of nutrients from hcterotrophic metabolism is adequate to maintain high rates of production and growth (e.g. Kerr 1983; Laws et al. 1987). A variety of physiological indices has provided evidence of nitrogen limitation in natural phytoplankton communrties (Dugdale 1967; Morrisetal. 1971;Yentschctal. 1977;Kanda ct al. 1SSS),but evidence that photosynthetic or relative specific growth rates are nutrient limited in natural populations is scant and contradictory (L,aws et al. 1987; Cleveland et al. 1989). Under controlled laboratory conditions, nitrogen-deficient phytoplankton have low photosynthetic quantum yields (Welschmeyer and L,orenzen 198 1; Chalup 1987; Cleveland and Perry 1987,; Her-zig and Falkowski 1989) and reduced photosynthetic energy conversion efficiencies (Kolber et al. 1988). Therefore, if photosynthesis in natural phytoplankton communities were nitrogen limited, one might expect to measure decreased photosynthetic quantum yields or conversion efficiencies. Here we examine --- variability in photosynthetic energy conversion efficiencies by measuring the change in the maximal quantum yields of fluorescence in natural phytoplankton communitics in the Gulf of Maine in June 1987 with a pump-and-probe fluorometer (Falkowski et al. 1986). Our results suggest that variations in photosynthet-ic energy conversion efficiency in coastal waters are correlated with the supply of dissolved inorganic nitrogen, implying that regenerative fluxes of nitrogen within the euphotic zone are not adequate to maintain maximal photosynthetic rates. Theory of the pump-and-probe technique The pump-and-probe fluorescence technique measures the change in fluorescence yield (A&,,) of a weak “probe” flash preceding (FJ and following (1;1,),at time t, an actinic “pump” flash (Falkowski and Kiefer 1985). We measured Fy with a 70-ps delay following the pump flash. This delay is long enough for quenchers of fluorescence, produced by the pump flash, to have decayed, yet short enough that electrons transferred from photosystem (PS) II reaction centers to the primary electron acceptor (QA) to be stabilized (Mauzerall 1972; Falkowski et al. 1986; Falkowski and Kiefer 1985). If the pump flash is saturating, all functional PS II reaction centers will transfer an electron from the reaction center to QA. ,4ckno wledgments This research was supported by NASA under grant The greater the capacity for the photochemUPN 16l-35-05-08 (2857-OP-46 1), and the Office of ical energy conversion, the larger will be the Health and Environmental Research, U.S. Department difference between the fluorescence yields of Energy, under contract DE-AC02-76CHOOO 16. WC thank John Christensen ofBigelow Laboratory for pro- (PI - F,). The difference between these two fluorescence yields in a dark-adapted samviding shiptime on the RV Cape Ilcnlopen. 72 Photosynthetic ple is the maximal variable Auoresccnce, denoted F,,. Photosynthetic energy convcrsion is calculated by normalizing F,, to either F, or F, (Crofts and Wraight 1983; Genty et al. 1989; Falkowski et al. 1986). For comparing efficiencies between two or more samples, it is more useful to normalize to F,. Thus we use 73 eficiency 8. ( -.-. .__ -_ --_-.- --~- 3 q nu -5 E --I .20 60 -- 2 Ou q n 40-- 000 uno 0 q q __A- 0. q 00 0 no 00~ 0 n [I e----- _______----- W&s,, = 1 - cxP(- (TPS Id9 where es II is the apparent, relative absorption cross-section for PS II and E is relative hash energy (Falkowski et al. 1988). gps rI should not be confused with the optical absorption cross-section normalized to Chl a, denoted kc or a* (e.g. Dubinsky et al. 1986). Measurements of a* include pigment molecules associated with both PS II and PS I and do not distinguish between molecules that transfer excitation energy to the reaction center and those that do not (Dubinsky et al. 1986); cIbs,, is the relative functional cross-section of PS II and includes only molecules that transfer excitation energy to PS II reaction centers (Falkowski in press). The ratio cpS,,/a* is the maximal quantum yield of photosynthesis (Ley and Mauzerall 1982; Dubinsky et al. 1986). 0 -0.80 G 10 % - - Fs 0 A*sat (-jt---.,-+ &sat = K - e7YFo In phytoplankton grown under nutrientreplete conditions, the maximal value of A$,,, is remarkably constant, averaging 1.6, and independent of growth irradiance (Mauzerall 1972; Kolbcr et al. 1988). For cells grown in nitrogen-limited (but not phosphate, R. Herzig pers. comm.) chemostats, however, A&,, varies as an cxponential function of the dilution rate (hence, growth rate) regardless of species (Kolbcr et al. 1988). If, rather than providing a saturating pump flash, A&,, is measured as a function of the change in the intensity of the pump flash, the relative functional absorption cross-section for PS IT (upsrI) can bc calculated from an exponential model (Ley and Mauzerall 1982; Falkowski et al. 1986, 1988). Flash-intensity saturation curves were gencratcd by varying the pump flash intensity, and cps ,1 was calculated from nonlinear regression to a cumulative onehit Poisson function, 1.50 1 -. 0 10 Time of dark adaptation 20 - -r 30 (min) Fig. 1. A typical time-course of changes in in vivo fluorescence measured with a pump-and-probe fluorometer. One liter of a whole seawater sample taken from 10-m depth at 1330 hours with a 5-liter Niskin bottle was removed within 2 min after collection and placed in a darkened (three layers of black lape) reservoir at surface seawater temperature. The sample was pumped through the fluorometer cuvette as described in the text and F,, F,, and A$,,, were measured. The initial solar-induced, nonphotochemical quenching is apparent in both the F,, and F, signals and decays with a halftime of -5 min. Bccausc the concentration of phytoplankton in the sea is low (usually < 10 ,ugliter-‘), and our fluorometer has an optical pathlength of only 0.2 cm, reabsorption of light by the sample itself is negligible. Thus, measurements of A$,,, and cpsII are independent of Chl a concentrations over the range normally encountered in the ocean. Moreover, pheopigmcnts (or other pigments not associated with functional PS II reaction centers) do not contribute to variable fluorescence and therefore have a negligible effect on A&, or cps 11;both A&,, and bpsrI originate only from viable photosynthetic organisms. Dark adaptation When a phytoplankton sample is taken during daylight, the in vivo fluorescence per unit of Chl a is initially low and rises over a period of a few tens of minutes to a steady state value as the cells become dark adapted (Bates 1985). It can be shown from oxygen flash yields that PS IT reaction centers open immediately in the dark (Falkowski et al. 1988), so the light-induced, long-lived fluoresccnce quenching must have other origins. It is presently believed that the longlived quenchers are related to the formation of de-epoxidated xanthophylls which form in the light as part of a xanthophyll cycle 74 Kolber et al. 44 43 42' 41' Fig. Z!. Location of stations occupied along the 100-m isobath in June 1987 in the Gulf of Maine. Arrows indicate general circulation in the upper 75 m (after Brooks 1985). Cold, nutrient-rich water enters Jordan Basin (JB) from the Nova Scotian Shelf. Some of this water is entrained in an anticlockwise gyre, and some is advected southwest into Wilkenson Basin (WB). Along the 100-m isobath, a front between stations 8 and 10 (Fig. 3a) demarks the region of mixing between the two basins. (Demmig et al. 1987). In order to avoid the possible interference of these quenchers with measurements of A&,, and cps rI, we darkadapted phytoplankton samples for 30 min at in situ temperatures. This period is sufficient for all quenchers to decay (Fig. 1). During the measurements of A&,, and pumped flPS II7 sample was continuously through a 2004 flow-through cuvette from the 1-liter, dark-adapted sample reservoir with a diaphragm bellows pump placed between the outlet of the cuvette and the in- flow to the reservoir. The flow rate was - 5 ml min-‘. This system causes little disturbance even to fragile organisms. Measurements of A&,, and clBsII were completed within 10 min and, as actinic pump flashes were provided at a rate of 1 s, samples effectively were exposed to only one “pump” flash. Field measurements We measured total dissolved inorganic nitrogen (DIN) (including NH4+, N02-, and Photosynthetic 75 eficiency STATION STATION 3 4 2 1 5 6 7 14 13 12 11 10 9 8432156 0 . ,. ..” 7 14 <’ . 8 131211109 0 . , r-4 . . 30 30 60 60 90 90 (a) TEMPERATURE (b Tc OC 120 12c -ul * 30 30 60 60 9c 90 G .IE” I E Li CHLOROPHYLL 12a 0 40 80 120 DISTANCE 3c 30 6C 60 90 90 160 200 240 120 280 (kilometers) 120 0 40 80 120 DISTANCE 160 200 240 280 (kilometers) Fig. 3. Sections along the 100-m isobath showing temperature, concentrations of total dissolved inorganic nitrogen (DIN = NO,- + NOJ- + NH$) in pg-atoms N liter-l, Chl a in cLgliter-l, the maximal change in variable fluorescence yield, A&,, (dimensionless), and the relative absorption cross-section of photosystem II, upsI, (dimensionless). The numbers at the top correspond to stations in Fig. 2. Sample depths for each variable are indicated in each panel. The waters northeast of station 10 were cold and well mixed and separated from warmer, stratified water to the southwest by a thermal front between stations 8 and 10. The front was also apparent in sections of DIN and Chl a. Changes in fluorescence yields were measured on whole-water samples after 30 min of dark adaptation with a pump-and-probe fluorometer. 76 Kolber et al. tion of DIN nitrogen in the upper water column is due to upwelling of nutrient-rich 1 2-water from the Jordan Basin and the advection of similarly enriched waters from 0 9 -O s" o" 0 o;o 2 the adjacent Bay of Fundy (Townsend et al. 0 0 ’ 06-a 1987). In the unstratified region, the accumula0 3-tion of phytoplankton was limited by strong 0.0 7 vertical turbulence which mixed cells below 40 60 0 20 Distance from n i tricline (m) the critical depth. We propose that the relFig. 4. Relationship between A$,, and distance from atively low values of A$,,, found at stations the nitricline r = -0.63, n = 29, P < 0.0001). 8 and 9 are due to poorly developed photosynthetic apparatus resulting from low NO,-), Chl a, A$sat,and upsI along the 100-m mean irradiance in this deeply mixed reisobath from the eastern end of the Gulf of gion. As the water column stabilized across Maine to the southwest in June 1987 (Fig. the frontal boundary, however, high Chl a 2). Water samples were collected with 5-liter concentrations immediately downstream of Niskin bottles attached to a CTD ro- the nutrient source were found (Fig. 3~). The sette and immediately analyzed for nu- high Chl a region was dominated by diatrient, pigment, and fluorescent properties. toms, especially Thalassiosria and Melosira Dissolved inorganic N02- + N03- and spp. Chl a decreased toward the southwest NH,- were measured with a Technicon as the waters further stratified and a Chl a AutoAnalyzer as described by Whitledge et maximum formed near the base of the nial. (198 1). For Chl a determinations, 140- tricline. ml samples were filtered on Whatman GF/F Measurements of A&,, in fully darkglass-fiber filters and extracted in 90% ace- adapted samples indicated a strong parabathic gradient, with maxima of 1.5 found tone with a glass mortar and motor-driven Teflon pestle. The extract was clarified by adjacent to the upstream chlorophyll maxfiltration through GF/A glass-fiber filters and imum and minima of 0.5 occurring downthe fluorescence determined before and af- stream of the front (Fig. 3d). The data in ter acidification with a Turner Designs Fig. 3 suggest a relationship between A&,, model 10 fluorometer (Yentsch and Menzel and the hydrographic regime. Upstream of 1963) calibrated with pure Chl a. A$,,, and the upwelling front, where nutrients are cps rI were measured with a specially con- abundant but vertical mixing is deep, A$sat is low. Downstream of the nitrogen source, structed pump-and-probe fluorometer (Kolber et al. 1988) on freshly collected phy- A&,, appears to be related to the nutrient toplankton after a 30-min period of dark gradient. The almost threefold decrease in A&,, in the euphotic zone, downstream of adaptation. the front, suggests that in situ regeneration Results and discussion and diffusion of nutrients across the nitricThe temperature section along the 100-m line affected phytoplankton photosynthetic isobath shows the presence of a front be- energy conversion efficiency. tween stations 9 and 10. Upstream of the To examine the relationship between the front, at stations 8 and 9, water was verti- rate of supply of nitrogen and A$,,, more cally well mixed, while downstream, south- rigorously, we plotted values of A&,, in the west of station 10, a thermocline developed euphotic zone as a function of the distance between 20 and 30 m (Fig. 3a). The tem- from the nitricline (Fig. 4). The nitricline perature section was correlated with a strong was taken asthe center of the inflection depth parabathic gradient in dissolved inorganic of the DIN gradient. The results suggest a nitrogen (DIN) (Fig. 3b). In the upper 20 m statistically inverse correlation between A@,,, of the water column DIN decreased from and distance from the source of DIN (Fig. >4 pg-atoms N liter-l in the northeast to 4). A similar relationship was found in the < 1 in the southwest. The lateral distribuSargasso Sea between the maximal quan1.5 a 0 7 0 0 0 0 00 0 0 0 Photosynthetic turn yield of photosynthetic carbon fixation and distance from the nitricline by Cleveland et al. (1989). Our results and those of Cleveland et al. (1989) strongly suggest that the flux of DIN may limit photosynthetic efficiency in both coastal and oceanic waters. Further evidence of nutrient limitation of photosynthetic energy conversion is implied in the spatial distribution of the absorption cross-section of PS II. In laboratory cultures of phytoplankton, cps rI increases with decreasing growth irradiance (Ley and Mauzerall 1982; Dubinsky et al. 1986; Sukenik et al. 1987)-a phenomenon associated with photoadaptation (Falkowski 1980). If cells were simply light limited in the upper ocean and vertical mixing rates were lower than the rate at which cells photoadapt (Falkowski 1983), we would expect to find an increase in gpsiI with depth. As cells become nitrogen limited (Kolber et al. 1988; Herzig and Falkowski 1989), however, cpsII also increases. The modifying effects of nitrogen availability on photoadaptation have also been described for cultures of dinoflagellates by Prezelin (1982). Consequently, for cells in the upper ocean, ups rI is a complex function of photoadaptive strategies that leads to decreased bpsrI near the surface, vertical mixing rates that tend to distribute cps rI evenly when high, and nitrogen availability that tends to increasegpsIIwhen low. A section of gpsII (Fig. 3e) shows the complex interactions of these concurrent processes.These data suggestthat variations in the absorption cross-section of PS II in natural phytoplankton communities do not simply reflect light history but are also modified by nutrient availability. These measurements are the first reported of the functional absorption cross-sections of PS II in natural phytoplankton communities, and we hope that a clearer understanding of the effects of light, mixing, and nutrient availability on gpsiI will emerge as more observations become available. On a molecular level the relationship bctween A$ps rI and photosynthetic energy conversion efficiency in nitrogen-limited cells appears to arise from a reduction in the synthesis of specific proteins such as CP43 and CP47 (Kolbcr ct al. 1988; Falkowski et al. 1989). These two chlorophyll eficiency 77 protein complexes are ubiquitous in both procaryotic and eucaryotic oxygenic photoautotrophs and mediate the transfer of excitation energy from antenna1 light-harvesting complexes to PS II reaction centers. A reduction in the abundance of these proteins relative to light-harvesting chlorophyll protein complexes leads to a decreased efficiency in the transfer of energy from antennal pigments to the reaction centers of PS II as absorbed light is reradiated (i.e. fluoresces) within the pigment bed. This loss is reflected by a decrease in A@sal(Kolber et al. 1988). On an ecological level, spatial variations in A&,, suggest that the rate of regeneration of nutrients by heterotrophic organisms is not always adequate to meet the rate required to sustain the maximal photosynthetic energy conversion efficiency of phytoplankton. The results shown here are representative of data we have obtained in temperate coastal waters where macrozooplankton are the predominant grazers (Townsend et al. 1987), but may not be representative of the hypothesized tight coupling between nutrient uptake and regeneration in the oligotrophic ocean where microzooplankton are more important (Goldman 1980). Our results and those of Cleveland et al. (1989) suggest, however, that the use of solar-induced fluorescence to estimate the quantum yield of photosynthesis (Kiefer et al. 1989) will be complicated by nutrient limitation because the ratio of the quantum yield of photosynthesis to that of fluorescence is not constant. We suggest that the pump-and-probe fluorescence technique may provide a rapid, nondestructive means of examining the photosynthetic energy conversion of phytoplankton in the ocean. Finally, we note (Kolber et al. 1988) that in laboratory cultures variations in A&, in nitrogen-limited cells are predictable from relative specific growth rates (sensu Goldman 1986). As A&,, is independent of growth irradiance under nutrient-replete conditions, variations in A& with depth reflect changes in relative growth rates independent of irradiance and cannot be explained solely by photoadaptive changes. 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