1. 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 under
light-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 regime
light-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 impact
lular 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 many
chlorophyll a were affected only under low light conditions, studies. Though several field experiments—ranging
wherein concentrations were suppressed by iron limitation. from bottle experiments (de Baar et al. 1990, Martin
Ratios 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)—demonstrated
protective 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 aspects
ratio of diatoxanthin to diadinoxanthin was highest under of iron limitation (Geider and LaRoche 1994, van
high 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 by
coxanthin and 19 -butanoyloxyfucoxanthin at the cost of e.g. Greene et al. (1991, 1992). Insight into the cell’s
fucoxanthin. Fucoxanthin formed the main carotenoid in metabolic responses is scarce; however, this may be
iron-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 taxonomic
especially in areas where Phaeocystis sp. and diatoms oc- groups in the field. Studies on the effects of iron on
cur 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 extensive
their roles as a light-harvesting pigment. studies on higher plants (Terry 1980, Abadia et al.
1989, Pascal et al. 1995). As chlorophyll decreases
Key index words: Antarctic Phaeocystis; fucoxanthin; more than carotenoids, the ratio of light-harvesting
iron; 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 of
LH, 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 pigments
Southern 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

2. IRON, LIGHT, AND PIGMENT COMPOSITION 497
et al. (1993) presented a study on the effects of iron MATERIALS AND METHODS
limitation on the pigment composition of Phaeodac- The origin of the Antarctic Phaeocystis strain used is described
tylum 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 110
light-harvesting pigments to chlorophyll a was mea- mol photons·m 2·s 1) on a 16-h light:8-h dark regime. Light was
sured 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]). Medium
same 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 EDTA
olecta, as was also described by Greene et al. (1992). were added separately at final concentrations of 10 5 M EDTA
Confirming the studies on higher plants, activation for all cultures, 10 6 M Fe for iron-rich cultures, and 10 9 M for
of 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 starting
cle pigments to chlorophyll a increased markedly; the experiments, cultures were stressed by iron deficiency and
the ratio of diatoxanthin to diadinoxanthin in- adapted to light conditions by repeated transfers. Experiments
were started by inoculation of several thousands of exponentially
creased 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 room
prymnesiophyte 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 (as
rized contain fucoxanthin as the main carotenoid. described in Stefels and van Leeuwe 1998). To release the cells
Additional 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 at
of 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, and
1985, 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 60
some Chrysophyceae (Vesk and Jeffrey 1987, Wright L sample, packed in two times 30 L Milli-Q water. Pigments
and 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 a
1989) and Dinophyceae (Tangen and Bjornland C18 guard column (Waters Nova-Pak). The elution gradient was
1981). 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 for
19 -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 over
contribution of these acyloxyfucoxanthins not only the exponential growth phase (n 5–7) (Stefels and van Leeuwe
varies between strains (Wright and Jeffrey 1987, Vau- 1998), excluding the data from the lag and stationary phase. The
lot 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 into
the conditions that control the concentrations of RESULTS
these 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 under
of iron limitation on pigment synthesis are still un- high iron concentrations contained fucoxanthin
known, and because of the prominent role of Phaeo- (FUCO) as the major carotenoid, with low levels of
cystis 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 besides
panion 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 not
HPLC. 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 total
vesting and photoprotective pigments, and as diag- pool of fucoxanthins (FUCO, HEXA, BUTA, allo-
nostic pigments. fucoxanthin, and cisfucoxanthin) was highest under

3. 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, P
iron limitation on cellular pigment content were not 0.005). The interaction between iron and light was
equally strong for all pigments (Table 1). Cellular found significant at P 0.05. The cellular content
levels of both chlorophyll a and c (c1,2 c3) de- of the total group of fucoxanthins also decreased
creased 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 of
fected by iron limitation, and was always highest un- total fucoxanthins to chlorophyll a was highest for
TABLE 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 LHb
LL 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.

4. 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 of
the 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 for
high iron. A. total group of chlorophyll cs (c1,2 c3) to chl a. B. iron-deplete cells than for iron-replete cells under
total 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 by
iron-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 the
total pool of accessory light-harvesting (LH) pig-
ments (chlorophyll c’s plus fucoxanthins) decreased
under iron-deplete conditions (Table 1). The ratio
of total LH pigments to chlorophyll a was highest
for 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 reduced
under iron-deplete conditions, in favor of enhanced
synthesis of HEXA and BUTA (Fig. 3). This was
most pronounced under high light conditions. Un- FIG. 4. The ratio of diatoxanthin to diadinoxanthin, under
der both light conditions, HEXA was the main ca- the different culture conditions. Abbreviations as described for
rotenoid for iron-deplete cells. Figure 2.

5. 500 MARIA A. VAN LEEUWE AND JACQUELINE STEFELS
TABLE 2. Pigment concentrations in Antarctic Phaeocystis sp. per cell volume (fg/ m3) (SD in parentheses), averaged over the growth phase. Volumes
taken 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 et
chlorophyll a and LH pigment content vs. concentration. B. the al. (1992), iron limitation leads to a reduction in
ratio of HEXA and BUTA to FUCO. LL :LL and HL :HL photosynthetic efficiency, largely due to a reduced
display the effect of iron limitation under respectively low light
and 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

6. IRON, LIGHT, AND PIGMENT COMPOSITION 501
cell volume, which is a means of reducing mainte- As FUCO forms the basic structure of BUTA and
nance costs. For these cells, a proportionally smaller HEXA (Hertzberg et al. 1977), an active mechanism
sized light-harvesting system may be sufficient to must be involved in the generation of BUTA and
provide 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 the
tions 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 the
centrations 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 Fujita
whereby pigment synthesis paralleled an overall re- 1975). FUCO is a highly efficient light-harvesting
duction in photosynthetic capacity, which was re- pigment (Siefermann-Harms 1987), which transfers
flected 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 membrane
exposed to high light intensities are well document- configuration, which in turn may result in a reduced
ed (Demmig-Adams and Adams III 1992). An excess efficiency of energy supply by the light-harvesting
of excitation energy, which results from the absorp- complex. Alternatively, if HEXA and BUTA are
tion of more light than can be utilized by the elec- readily transformed into an efficiently functioning
tron 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 that
damaging, as they may evolve in the formation of characterize the Southern Ocean. This may result in
the aggressive H2O2. In iron-deficient algae, not only a competitive advantage over those species that do
the production of radicals increases as a result of a not produce acyloxyfucoxanthins, if Phaeocystis sp. is
reduced efficiency of electron transfer (Greene et able to make use of this fucoxanthin cycle. It may
al. 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 concentrations
the breakdown of H2O2 may also be reduced. Thus, of HEXA when compared to strains from temperate
the 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 functioning
diadinoxanthin will be deepoxidized, forming dia- of the light-harvesting complex in Phaeocystis sp. is
toxanthin, which has the capacity of thermal dissi- required.
pation (Demers et al. 1991, Olaizola et al. 1994). In Ecological considerations. Variability in the cellular
our experiments, the exposure of Phaeocystis sp. to content of the group of fucoxanthins was described
high 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, but
an 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, no
of HEXA and BUTA appeared to be induced under causal relationships were established. Our study is
low iron conditions at the expense of FUCO synthe- the first to describe a clear linkage between nutrient
sis, with HEXA as the main carotenoid (Fig. 3). Fig- conditions and the status of the fucoxanthins. This
ure 5b clearly illustrates the strong inducing effects is important in view of the natural distribution of
of iron limitation on HEXA and BUTA relative to Phaeocystis sp. in the Southern Ocean. In open ocean
the much smaller reducing effects resulting from waters iron concentrations below 1 nM have been
light stress. Although, in iron-deplete cells, BUTA measured, as compared to levels above 5 nM for
was less abundant than HEXA, and virtually absent coastal areas (Martin et al. 1990, Westerlund and
in 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 open
to be just as strong as for HEXA. ocean, the pigment composition of this species may

7. 502 MARIA A. VAN LEEUWE AND JACQUELINE STEFELS
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