J. Phycol. 34, 496–503 (1998) EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). II. PIGMENT COMPOSITION1 Maria A. van Leeuwe 2 Netherlands Institute for Sea Research, P.O. Box 59, 1790 AB Den Burg, The Netherlands and Jacqueline Stefels University of Groningen, Department of Marine Biology, P.O. Box 14, 9750 AA Haren, The Netherlands ABSTRACT nomenon in all seas surrounding the continent (e.g. Palmisano et al. 1986, Wright and Jeffrey 1987, Da- A strain of Phaeocystis sp., isolated in the Southern vidson and Marchant 1992). Considering the im-Ocean, was cultured under iron- and light-limited condi- portance of Phaeocystis sp. in the Antarctic ecosys-tions. The cellular content of chlorophyll a and accessory tem, it is desirable to understand its behavior underlight-harvesting (LH) pigments increased under low light the different conditions experienced in the Antarc-intensities. Iron limitation resulted in a decrease of all tic Ocean. Besides the variable light regimelight-harvesting pigments. However, this decrease was (Laubscher et al. 1993, Kirst and Wiencke 1995),greatly compensated for by a decrease in cell volume. Cel- low iron concentrations may have a strong impactlular concentrations of the LH pigments were similar for on phytoplankton physiology in this region. The is-both iron-replete and iron-deplete cells. Concentrations of sue of iron limitation has been the topic of manychlorophyll a were affected only under low light conditions, studies. Though several ﬁeld experiments—rangingwherein concentrations were suppressed by iron limitation. from bottle experiments (de Baar et al. 1990, MartinRatios of the LH pigments to chlorophyll a were highest for et al. 1990) to large scale in situ experiments (Kol-iron-deplete cells under both light conditions. The photo- ber et al. 1994, Martin et al. 1994)—demonstratedprotective cycle of diato/diadinoxanthin was activated un- the importance of iron for phytoplankton produc-der high light conditions, and enhanced by iron stress. The tivity, few studies examined the physiological aspectsratio of diatoxanthin to diadinoxanthin was highest under of iron limitation (Geider and LaRoche 1994, vanhigh light, low iron conditions. Leeuwe et al. 1997). Effects on the photosynthetic Iron limitation induced synthesis of 19 -hexanoyloxyfu- apparatus have been investigated and described bycoxanthin and 19 -butanoyloxyfucoxanthin at the cost of e.g. Greene et al. (1991, 1992). Insight into the cell’sfucoxanthin. Fucoxanthin formed the main carotenoid in metabolic responses is scarce; however, this may beiron-replete Phaeocystis cells, whereas for iron-deplete cells important considering the vast areas where iron lim-19 -hexanoyloxyfucoxanthin was found to be the main ca- itation may prevail.rotenoid. This shift in carotenoid composition is of impor- Pigment signatures provide a major tool in deter-tance in view of the marker function of both pigments, mining the contribution of distinct taxonomicespecially in areas where Phaeocystis sp. and diatoms oc- groups in the ﬁeld. Studies on the effects of iron oncur simultaneously. A hypothesis is presented to explain algal pigment composition are therefore important.the transformation of fucoxanthin into 19 -hexanoyloxy- A decline in cellular pigment content seems a gen-fucoxanthin and 19 -butanoyloxyfucoxanthin, referring to eral response to iron stress, as observed in extensivetheir roles as a light-harvesting pigment. studies on higher plants (Terry 1980, Abadia et al. 1989, Pascal et al. 1995). As chlorophyll decreasesKey index words: Antarctic Phaeocystis; fucoxanthin; more than carotenoids, the ratio of light-harvestingiron; light; 19 -hexanoyloxyfucoxanthin; pigments pigments to chlorophyll a increases. Furthermore,Abbreviations: BUTA, 19 -butanoyloxyfucoxanthin; iron-limited cells show a high susceptibility to pho-FUCO, fucoxanthin; HEXA, 19 -hexanoyloxyfucoxanthin; tooxidation. To prevent photodamage, synthesis ofLH, light harvesting photoprotective pigments is induced by iron limi- tation, which results in a relative increase of pho- Phaeocystis sp. is a common microalga in the toprotectors compared to light-harvesting pigmentsSouthern Ocean; spring blooms are a yearly phe- (Abadia et al. 1989, Morales et al. 1990, 1994). These effects haven’t been investigated in detail for 1 algae. Besides well-described inhibitory effects on Received 19 April 1996. Accepted 10 March 1998. 2Author for reprint requests and present address: University of chlorophyll synthesis (Guikema and Sherman 1983,Groningen, Department of Marine Biology, P.O. Box 14, 9750 AA Greene et al. 1992), only a few studies have ad-Haren, The Netherlands; e-mail M.A.van.Leeuwe@biol.rug.nl. dressed the total spectrum of algal pigments. Geider 496
IRON, LIGHT, AND PIGMENT COMPOSITION 497et al. (1993) presented a study on the effects of iron MATERIALS AND METHODSlimitation on the pigment composition of Phaeodac- The origin of the Antarctic Phaeocystis strain used is describedtylum tricornutum. In contrast to earlier experiments in Stefels and van Leeuwe (1998). Phaeocystis sp. was grown in 2-(Greene et al. 1991), in which an increased ratio of L polystyrene culture ﬂasks at 4 C and placed on a rolling device ( 2 rpm). Cultures were grown at two irradiances (25 and 110light-harvesting pigments to chlorophyll a was mea- mol photons·m 2·s 1) on a 16-h light:8-h dark regime. Light wassured in iron-limited cultures, Geider et al. (1993) provided by cool white ﬂuorescence tubes (Philips TLD 36W/54,found no signiﬁcant change of this ratio for the measured with a spherical sensor [LI-COR, SPQA III]). Mediumsame species studied. However, the latter authors was prepared as described by Morel et al. (1979). Chelexed, nu- trient-poor seawater originating from northern North Sea was en-did mention an increased ratio for Dunaliella terti- riched with 12.5 M PO43 and 150 M NO32 . FeCl3 and EDTAolecta, as was also described by Greene et al. (1992). were added separately at ﬁnal concentrations of 10 5 M EDTAConﬁrming the studies on higher plants, activation for all cultures, 10 6 M Fe for iron-rich cultures, and 10 9 M forof the photoprotective xanthophyll cycle of diato- iron-poor cultures. Initial iron concentrations (measured by sol- vent extraction followed by atomic absorption spectrophotometry;and diadinoxanthin was indeed observed for the Nolting and de Jong 1994) ranged from 2 to 10 nM Fe.alga P. tricornutum. The ratio of the xanthophyll-cy- The cultures mainly consisted of dense colonies. Before startingcle pigments to chlorophyll a increased markedly; the experiments, cultures were stressed by iron deﬁciency andthe ratio of diatoxanthin to diadinoxanthin in- adapted to light conditions by repeated transfers. Experiments were started by inoculation of several thousands of exponentiallycreased only slightly (Geider et al. 1993). growing cells into a fresh medium. Cultures were sampled at reg- The pigment signature of different strains of the ular intervals. Samples were taken in a Class-100 clean air roomprymnesiophyte Phaeocystis has recently been re- to avoid contamination with both iron and bacteria while sam-viewed by Vaulot et al. (1994). All strains summa- pling. All experiments were performed in duplicate. Cell numbers and volumes were determined by microscopy (asrized contain fucoxanthin as the main carotenoid. described in Stefels and van Leeuwe 1998). To release the cellsAdditional fuco pigments as 19 -hexanoyloxyfuco- from the colonies, cell suspensions were sonicated with a Soni-xanthin and 19 -butanoyloxyfucoxanthin are pres- prep 150 using minimal energy (four cycles of 10 s, one ampli-ent in variable concentrations. None of the fucoxan- tude micron) after ﬁxation with acid Lugol’s solution. Samples taken for pigment analyses were ﬁltered gently (pres-thins is exclusive for Phaeocystis. Whereas fucoxan- sure 0.15 bar) over GF/F Whatman ﬁlters, wrapped in alumi-thin is the major carotenoid in diatoms, 19 -hexan- num foil and stored at 50 C until analysis. Pigments were ex-oyloxyfucoxanthin is important for several members tracted in 90% acetone, placing the ﬁlters in the dark for 6 h atof the Prymnesiophyceae (Arpin et al. 1976, Haxo 4 C, after disruption with a stainless steel spatula. Next, the sam- ples were centrifuged (5 min at 600 g) to remove debris, and1985, Gieskes and Kraay 1986, Bjornland et al. 1988, ¨ then transferred to sample vials, which were placed in an auto-Bjerkeng et al. 1989) but also is characteristic for sampler, and maintained at 2–3 C. The autosampler injected 60some Chrysophyceae (Vesk and Jeffrey 1987, Wright L sample, packed in two times 30 L Milli-Q water. Pigmentsand Jeffrey 1987, Bjornland and Liaanen-Jensen ¨ were analyzed by HPLC on a Pharmacia-LKB liquid chromato- graph, carrying a C18 column (Waters Delta-Pak), protected by a1989) and Dinophyceae (Tangen and Bjornland C18 guard column (Waters Nova-Pak). The elution gradient was1981). 19 -Butanoyloxyfucoxanthin is observed in based upon a binary solvent system (Solvent A: Methanol:Aceto-most of these species in trace concentrations. nitrile:0.5 M Ammonium acetate [6:2:2 v/v]; Solvent B: Methanol: The usefulness of 19 -hexanoyloxyfucoxanthin, Acetonitrile:Ethyl acetate [2:6:2 v/v]). The wavelengths used for19 -butanoyloxyfucoxanthin and fucoxanthin as di- pigment detection were 436 and 410 nM; identiﬁcation was based upon known standards (Villerius et al., pers. commun.).agnostic pigments of Phaeocystis sp. is debatable. The The pigment concentrations described are average values overcontribution of these acyloxyfucoxanthins not only the exponential growth phase (n 5–7) (Stefels and van Leeuwevaries between strains (Wright and Jeffrey 1987, Vau- 1998), excluding the data from the lag and stationary phase. Thelot et al. 1994) but also appears to depend on standard deviation has been calculated using the ‘‘nonbiased’’ (‘‘n-1’’) method.growth phase (Buma et al. 1991). Clear insight intothe conditions that control the concentrations of RESULTSthese pigments is still lacking, but a better under- Antarctic Phaeocystis sp. contained chlorophyll a,standing of these is required in view of their func- chlorophyll c1 2, chlorophyll c3, fucoxanthins, dia-tion as marker pigments in the ﬁeld. As the effects dinoxanthin, and diatoxanthin. Cells grown underof iron limitation on pigment synthesis are still un- high iron concentrations contained fucoxanthinknown, and because of the prominent role of Phaeo- (FUCO) as the major carotenoid, with low levels ofcystis sp. in the Southern Ocean ecosystem, an ex- 19 -hexanoyloxyfucoxanthin (HEXA), whereas 19 -tensive study was carried out. Phaeocystis sp. was sub- butanoyloxyfucoxanthin (BUTA) was virtually ab-jected to two different concentrations of both iron sent (Fig. 1a). For cells grown under low iron con-and light. Metabolic effects are described in a com- centrations, HEXA was the main carotenoid besidespanion paper by Stefels and van Leeuwe (1998). lower amounts of BUTA and FUCO (Fig. 1b).Here, we describe the effects of iron limitation on Though pigment concentrations varied slightly dur-the cell’s pigment composition as determined by ing growth, the overall pigment pattern did notHPLC. Responses in cellular pigment content will change over time.be discussed relative to their function as light-har- The cellular content of chlorophylls and the totalvesting and photoprotective pigments, and as diag- pool of fucoxanthins (FUCO, HEXA, BUTA, allo-nostic pigments. fucoxanthin, and cisfucoxanthin) was highest under
498 MARIA A. VAN LEEUWE AND JACQUELINE STEFELS FIG. 1. Representative chromatograms of the pigment composition of cultures of Phaeocystis sp. cultured under high light conditions. HL : high iron concentrations; HL : low iron concentra- tions. Absorbance at 436 nM. Peak identiﬁcation: 1. chlorophyll c3; 2. chlorophyll c1,2; 3. 19 -butan- oyloxyfucoxanthin; 4. fucoxanthin; 5. 19 -hexan- oyloxyfucoxanthin; 6. diadinoxanthin; 7. diatox- anthin; 8. chlorophyll a. Allo- and cisfucoxanthin form left shoulders of fucoxanthin and 19 -hex- anoyloxyfucoxanthin, respectively.low light and iron-replete conditions. The effects of der low light conditions (Fig. 2a; ANOVA, Piron limitation on cellular pigment content were not 0.005). The interaction between iron and light wasequally strong for all pigments (Table 1). Cellular found signiﬁcant at P 0.05. The cellular contentlevels of both chlorophyll a and c (c1,2 c3) de- of the total group of fucoxanthins also decreasedcreased under iron limitation. The ratio of the total under iron limitation (Table 1), but to a lesser ex-group of chlorophyll c to chlorophyll a was not af- tent than chlorophyll a. Consequently, the ratio offected by iron limitation, and was always highest un- total fucoxanthins to chlorophyll a was highest forTABLE 1. Cellular pigment content in Antarctic Phaeocystis sp. (pg/cell) (SD in parentheses), averaged over the growth phase. Culture conditions:LL : low light, low iron; LL : low light, high iron; HL : high light, low iron; HL : high light, high iron. Experiments performed in duplicate (A,B). Number of observations: LL A, n 5; LL B, n 5; LL A, n 6; LL B, n 6; HL A, n 7; HL B, n 6; HL A, n 5; HL B, n 6. chl c3 chl c1 2 BUTA FUCO HEXA Diadino Diato chl a Total fucosa Total LHbLL A 0.07 (0.03) 0.12 (0.08) 0.06 (0.02) 0.12 (0.05) 0.26 (0.08) 0.10 (0.03) 0.01 (0.01) 0.29 (0.07) 0.44 (0.11) 0.63 (0.21)LL B 0.06 (0.02) 0.11 (0.07) 0.03 (0.02) 0.14 (0.02) 0.15 (0.06) 0.07 (0.03) 0.00 (0.00) 0.22 (0.06) 0.34 (0.08) 0.51 (0.17)LL A 0.15 (0.05) 0.19 (0.11) 0.00 (0.00) 0.32 (0.09) 0.01 (0.00) 0.08 (0.02) 0.00 (0.00) 0.52 (0.12) 0.38 (0.10) 0.72 (0.25)LL B 0.21 (0.11) 0.28 (0.28) 0.00 (0.00) 0.49 (0.25) 0.01 (0.00) 0.11 (0.02) 0.00 (0.00) 0.59 (0.22) 0.58 (0.29) 1.08 (0.68)HL A 0.03 (0.01) 0.05 (0.03) 0.03 (0.01) 0.04 (0.03) 0.11 (0.02) 0.09 (0.03) 0.01 (0.01) 0.17 (0.06) 0.18 (0.04) 0.25 (0.08)HL B 0.01 (0.01) 0.04 (0.03) 0.03 (0.01) 0.02 (0.01) 0.10 (0.03) 0.08 (0.02) 0.01 (0.01) 0.11 (0.03) 0.15 (0.04) 0.20 (0.07)HL A 0.06 (0.02) 0.09 (0.05) 0.01 (0.00) 0.15 (0.03) 0.03 (0.00) 0.08 (0.03) 0.00 (0.00) 0.26 (0.04) 0.20 (0.04) 0.35 (0.10)HL B 0.06 (0.04) 0.10 (0.11) 0.01 (0.00) 0.17 (0.06) 0.03 (0.01) 0.07 (0.03) 0.01 (0.00) 0.29 (0.12) 0.22 (0.08) 0.38 (0.22) a Total fucos BUTA, FUCO, HEXA and allo- and cisfucoxanthin (not listed). b Total LH chlorophyll c’s total fucos.
IRON, LIGHT, AND PIGMENT COMPOSITION 499 FIG. 3. Abundance of the fucoxanthin pigments as a percent- age of the main pool of fucoxanthins (BUTA FUCO HEXA), under the different culture conditions. Abbreviations as described for Figure 2. Cellular content of diadinoxanthin was similar un- der all experimental conditions (Table 1). The dia- toxanthin content increased under iron-deplete conditions, with maximum values measured under high light conditions. Therefore, the ratio of diato- xanthin to diadinoxanthin was highest under low iron, high light conditions (Fig. 4; ANOVA, P 0.005). As cell volumes were substantially smaller in iron- deplete cultures (Stefels and van Leeuwe 1998), cel- lular pigment content showed a different pattern from cellular pigment concentration, when compar- ing different cultures. For cells cultured under low light, iron depletion had a small effect on chloro- phyll a concentrations, whereas under high light conditions, no difference could be observed (Table 2). Cellular concentrations of the total group of fu- coxanthins were elevated for iron-deplete cells, whereas cellular concentrations of the total pool of accessory LH pigments appeared independent of FIG. 2. The ratio of pigment groups to chlorophyll a, under the iron conditions (Table 2). Concentrations ofthe different culture conditions. LL : low light, low iron; LL :low light, high iron; HL : high light, low iron; HL : high light, diadinoxanthin and diatoxanthin were higher forhigh iron. A. total group of chlorophyll cs (c1,2 c3) to chl a. B. iron-deplete cells than for iron-replete cells undertotal pool of fucoxanthins (BUTA FUCO HEXA) to chl a. both high and low light conditions (Table 2).C. total pool of light-harvesting pigments (fucos chl cs) to chl a. The implications of studying cellular concentra- tions rather than cellular content are well illustrated in Figure 5a. Chlorophyll a per cell was affected byiron-deplete cells (Fig. 2b; ANOVA, P 0.005). The iron limitation, whereby effects of iron and light lim-increase appeared most pronounced under low itation induced an inverse change but of similar am-light conditions (P 0.05 for the interaction be-tween iron and light). The cellular content of thetotal pool of accessory light-harvesting (LH) pig-ments (chlorophyll c’s plus fucoxanthins) decreasedunder iron-deplete conditions (Table 1). The ratioof total LH pigments to chlorophyll a was highestfor iron-deplete cells. The increase was most pro-nounced under low light conditions (Fig. 2c; ANO-VA, P 0.005) (P 0.05 for the interaction be-tween iron and light). Important shifts occurred within the group of fu-coxanthins. Synthesis of FUCO was strongly reducedunder iron-deplete conditions, in favor of enhancedsynthesis of HEXA and BUTA (Fig. 3). This wasmost pronounced under high light conditions. Un- FIG. 4. The ratio of diatoxanthin to diadinoxanthin, underder both light conditions, HEXA was the main ca- the different culture conditions. Abbreviations as described forrotenoid for iron-deplete cells. Figure 2.
500 MARIA A. VAN LEEUWE AND JACQUELINE STEFELSTABLE 2. Pigment concentrations in Antarctic Phaeocystis sp. per cell volume (fg/ m3) (SD in parentheses), averaged over the growth phase. Volumestaken from Stefels and van Leeuwe (1998). *Total fucos BUTA, FUCO, HEXA, and allo- and cisfucoxanthin (not listed); total LH chlorophyll c’s total fucos. chl c3 chl c1 2 BUTA FUCO HEXA Diadino Diato chl a Total Fucos* Total LH*LL A 0.74 (0.18) 1.20 (0.34) 0.59 (0.25) 1.34 (0.56) 2.66 (0.97) 1.06 (0.30) 0.09 (0.08) 3.02 (0.76) 4.60 (1.13) 6.53 (1.65)LL B 0.59 (0.14) 0.93 (0.21) 0.30 (0.16) 1.39 (0.34) 1.28 (0.62) 0.59 (0.24) 0.03 (0.02) 2.04 (0.61) 3.06 (0.65) 4.58 (1.01)LL A 0.90 (0.27) 1.05 (0.33) 0.01 (0.01) 1.92 (0.65) 0.05 (0.01) 0.50 (0.12) 0.00 (0.01) 3.16 (0.92) 2.30 (0.71) 4.25 (1.31)LL B 1.30 (0.47) 1.57 (0.59) 0.02 (0.01) 2.91 (1.40) 0.07 (0.02) 0.71 (0.15) 0.01 (0.02) 3.56 (0.66) 3.48 (1.64) 6.36 (2.69)HL A 0.42 (0.14) 0.64 (0.17) 0.44 (0.03) 0.59 (0.51) 1.48 (0.24) 1.21 (0.15) 0.18 (0.12) 2.46 (0.85) 2.51 (0.52) 3.58 (0.83)HL B 0.22 (0.14) 0.52 (0.11) 0.42 (0.07) 0.33 (0.20) 1.42 (0.16) 1.15 (0.17) 0.13 (0.13) 1.77 (0.46) 2.17 (0.36) 2.91 (0.61)HL A 0.41 (0.09) 0.62 (0.14) 0.05 (0.01) 1.10 (0.22) 0.23 (0.02) 0.55 (0.18) 0.03 (0.02) 1.95 (0.37) 1.43 (0.26) 2.46 (0.49)HL B 0.47 (0.13) 0.70 (0.19) 0.05 (0.01) 1.30 (0.17) 0.23 (0.03) 0.52 (0.12) 0.04 (0.03) 2.07 (0.32) 1.63 (0.21) 2.81 (0.53) plitude in chlorophyll a per cell, i.e. reduction un- der iron limitation and induction under light limi- tation. The cellular content of LH pigments was also suppressed by iron limitation, although the induc- ing effects of light limitation were twice as strong as the inhibiting effect of iron limitation. However, the responses per cell volume to iron and light stress show that pigment concentrations were hardly af- fected by iron limitation. Chlorophyll a synthesis was suppressed only under low light conditions; the con- centrations of LH pigments were not at all reduced by iron limitation. Although less pronounced for iron-deplete than for iron-replete cells, the concen- trations of LH pigments were clearly enhanced un- der low light conditions. DISCUSSION Light-harvesting pigments. Light adaptation by algae has been a topic of many studies (reviewed by Rich- ardson et al. 1983, Falkowski and LaRoche 1991). A common response to reduced light availability is an increase in the cellular content of light-harvesting pigments. This response was also observed for Phaeo- cystis sp. The cellular content of chlorophyll a and the main accessory light-harvesting pigments, chlo- rophyll c and the fucoxanthins, increased under re- duced light conditions, irrespective of the iron sta- tus of the medium. A general response of cellular pigment content toward iron deﬁciency is more dif- ﬁcult to describe. A decline is often observed (Gei- der and LaRoche 1994), but changes in pigment ratios are less universal. An increase in the ratio of LH pigments to chlorophyll a, as observed for Phaeo- cystis in iron-deplete cells, was also observed by Greene et al. (1991, 1992) for the diatom Phaeodac- tylum tricornutum. However, the latter observation was not conﬁrmed by Geider et al. (1993), although the same species was studied. Greene et al. (1991, 1992) also found an increase in the ratio of chlo- rophyll c to chlorophyll a; an increase that was ob- served neither by Geider et al. (1993) nor by us. FIG. 5. The relative effects of light and iron limitation on A. As described in an extensive study by Greene etchlorophyll a and LH pigment content vs. concentration. B. the al. (1992), iron limitation leads to a reduction inratio of HEXA and BUTA to FUCO. LL :LL and HL :HL photosynthetic efﬁciency, largely due to a reduceddisplay the effect of iron limitation under respectively low lightand high light conditions. LL :HL and LL :HL display the efﬁciency of electron transfer within the photosys-effect of light limitation under respectively low iron and high iron tems. The accompanying decrease in carbon ﬁxa-conditions. tion can partly be compensated for by a decrease in
IRON, LIGHT, AND PIGMENT COMPOSITION 501cell volume, which is a means of reducing mainte- As FUCO forms the basic structure of BUTA andnance costs. For these cells, a proportionally smaller HEXA (Hertzberg et al. 1977), an active mechanismsized light-harvesting system may be sufﬁcient to must be involved in the generation of BUTA andprovide the photosystems with sufﬁcient electrons, HEXA. The effect of iron depletion on the abun-without imbalancing the process of photosynthesis. dances of the fucoxanthin pigments may be ex-Iron-deplete cells of Phaeocystis sp. indeed showed a plained by their function in the light-harvesting pro-decrease in cellular pigment content in proportion cess. Iron-limited cells suffer from an excess of ex-to a decrease in cell volume, whereby the pigment citation energy. To prevent subsequent photoda-concentrations did not change. Cellular concentra- mage, a reduction in energy supply toward thetions of LH pigments were similar for iron-deplete photosystems may be required. The efﬁciency of en-and iron-replete cells, whereas chlorophyll a con- ergy transfer from light-harvesting pigments to thecentrations were affected only under low light con- core complex depends on the position of the pro-ditions. We therefore conclude that iron limitation tein-pigment complex in the chloroplast mem-had little or no direct effect on pigment synthesis of branes. Slight changes in this conﬁguration may re-Phaeocystis. An indirect effect indeed was observed, duce the transfer efﬁciency (Shimura and Fujitawhereby pigment synthesis paralleled an overall re- 1975). FUCO is a highly efﬁcient light-harvestingduction in photosynthetic capacity, which was re- pigment (Siefermann-Harms 1987), which transfersﬂected in a reduction in growth and cell volume excitation energy with only minor losses to chloro-(also, Stefels and van Leeuwe 1998). phyll a. Hydroxylation of FUCO into HEXA and Photoprotection. Photoprotective responses of cells BUTA may cause a modiﬁcation of the membraneexposed to high light intensities are well document- conﬁguration, which in turn may result in a reduceded (Demmig-Adams and Adams III 1992). An excess efﬁciency of energy supply by the light-harvestingof excitation energy, which results from the absorp- complex. Alternatively, if HEXA and BUTA aretion of more light than can be utilized by the elec- readily transformed into an efﬁciently functioningtron transport system, leads to the formation of su- FUCO, this may be of ecological importance, es-peroxide radicals. These radicals are potentially pecially under the variable light conditions thatdamaging, as they may evolve in the formation of characterize the Southern Ocean. This may result inthe aggressive H2O2. In iron-deﬁcient algae, not only a competitive advantage over those species that dothe production of radicals increases as a result of a not produce acyloxyfucoxanthins, if Phaeocystis sp. isreduced efﬁciency of electron transfer (Greene et able to make use of this fucoxanthin cycle. It mayal. 1991), the concentration of catalase (which con- here be relevant to note the observation that Ant-tains iron; Geider and LaRoche 1994) necessary for arctic strains contain relatively high concentrationsthe breakdown of H2O2 may also be reduced. Thus, of HEXA when compared to strains from temperatethe removal of an energy excess by means of energy latitudes (Vaulot et al. 1994). This could be regard-dissipation is essential for iron-deplete cells under ed as an adaptation to long-term exposure to iron-high light conditions. This involves the activation of limited conditions. However, to conﬁrm these spec-the xanthophyll cycle. Upon exposure to high light, ulations, a more detailed study on the functioningdiadinoxanthin will be deepoxidized, forming dia- of the light-harvesting complex in Phaeocystis sp. istoxanthin, which has the capacity of thermal dissi- required.pation (Demers et al. 1991, Olaizola et al. 1994). In Ecological considerations. Variability in the cellularour experiments, the exposure of Phaeocystis sp. to content of the group of fucoxanthins was describedhigh light induced synthesis and deepoxidation of by Wright and Jeffrey (1987). A more extensive over-diadinoxanthin. This resulted in an increase in the view (Jeffrey and Wright 1994) resulted in a cluster-concentrations of diadino- plus diatoxanthin, and ing of different groups of Phaeocystis strains, butan enhanced ratio of diatoxanthin to diadinoxan- could not account for the variability within groups.thin. Though Buma et al. (1991), in comparing Antarctic FUCO, HEXA, and BUTA. Phaeocystis cells hold Phaeocystis (the same strain as used in our experi-FUCO, HEXA, and BUTA in varying proportions, ments) with a strain isolated from the North Sea,which were inﬂuenced by iron availability. Synthesis hinted at possible effects of nutrient limitation, noof HEXA and BUTA appeared to be induced under causal relationships were established. Our study islow iron conditions at the expense of FUCO synthe- the ﬁrst to describe a clear linkage between nutrientsis, with HEXA as the main carotenoid (Fig. 3). Fig- conditions and the status of the fucoxanthins. Thisure 5b clearly illustrates the strong inducing effects is important in view of the natural distribution ofof iron limitation on HEXA and BUTA relative to Phaeocystis sp. in the Southern Ocean. In open oceanthe much smaller reducing effects resulting from waters iron concentrations below 1 nM have beenlight stress. Although, in iron-deplete cells, BUTA measured, as compared to levels above 5 nM forwas less abundant than HEXA, and virtually absent coastal areas (Martin et al. 1990, Westerlund andin iron-replete cells, the magnitude of induction of ohman 1991, de Baar et al. 1995). Because of its ¨BUTA synthesis following iron limitation appeared distribution in coastal waters as well as in the opento be just as strong as for HEXA. ocean, the pigment composition of this species may
502 MARIA A. VAN LEEUWE AND JACQUELINE STEFELSwell vary with different ambient nutrient conditions. carbon concentration during a Phaeocystis-dominated bloomNear-shore, cellular levels of FUCO may be relatively at an Antarctic coastal site. Polar Biol. 12:387–95. de Baar, H. J. W., Buma, A. G. J., Nolting, R. F., Cadee, G. C., ´high, gradually decreasing moving to more off-shore Jacques, G. & Treguer, P. 1990. On iron limitation of the ´areas. We hypothesize that concurrently with a de- Southern Ocean: experimental observations in the Weddellcrease in iron concentrations, levels of HEXA will and Scotia Seas. Mar. Ecol. Prog. Ser. 65:105–22.increase. In iron-depleted waters, HEXA will thus de Baar, H. J. W., de Jong, J. T. M., Bakker, D. C. E., Loscher, B. M., Veth, C., Bathmann, U. & Smetacek, V. 1995. Impor-constitute the major carotenoid of Phaeocystis sp. tance of iron for plankton blooms and carbon dioxide draw-This indicates that care should be taken when using down in the Southern Ocean. Nature 373:412–5.pigment markers for the description of phytoplank- Demers, S., Roy, S., Gagnon, R. & Vignault, C. 1991. Rapid light-ton distribution patterns. The variability within the induced changes in cell ﬂuorescence and in xanthphyll-cyclegroup of fucoxanthins will not only affect estima- pigments of Alexandrium excavatum (Dinophyceae) ad Thal- assiosira pseudonana (Bacillariophyceae): a photo-protectiontions of Phaeocystis biomass, but may also obscure mechanism. Mar. Ecol. Prog. Ser. 76:185–93.possible contributions of diatoms. In the Southern Demmig-Adams, B. & Adams III, W. W. 1992. PhotoprotectionOcean, both species often occur simultaneously, and other responses of plants to high light stress. Annu. Rev.sharing FUCO as a marker pigment (e.g. Wright Plant Physiol. Plant Mol. Biol. 43:599–626.and Jeffrey 1987). Falkowski, P. G. & LaRoche, J. 1991. Acclimation to spectral ir- radiance in algae. J. Phycol. 27:8–14. Under nitrogen-limited conditions, a similar Geider, R. J. & LaRoche, J. 1994. The role of iron in phytoplank-mechanism may be activated. Like iron-deplete cells, ton photosynthesis, and the potential for iron-limitation ofnitrogen-deﬁcient cells also are more susceptible to primary productivity in the sea. Photosynth. Res. 39:275–301.photodamage (Kolber et al. 1988); a ﬂexible light- Geider, R. J., LaRoche, J., Greene, R. M. & Olaizola, M. 1993.harvesting complex is therefore important. Notably, Response of the photosynthetic apparatus of Phaeodactylum tricornutum (Bacillariophyceae) to nitrate, phosphate, or ironduring bloom development, of Phaeocystis sp., the starvation. J. Phycol. 29:755–66.relative amounts of HEXA increased, whereas the Gieskes, W. W. & Kraay, G. W. 1986. 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