Comparison by PAM Fluorometry of Photosynthetic Activity of Nine Marine
Phytoplankton Grown Under Identical Conditions
Author(s): P. Juneau and P. J. Harrison
Source: Photochemistry and Photobiology, 81(3):649-653. 2005.
Published By: American Society for Photobiology
DOI: 10.1562/2005-01-13-RA-414.1
URL: http://www.bioone.org/doi/full/10.1562/2005-01-13-RA-414.1
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Photochemistry and Photobiology, 2005, 81:
649–653
Comparison by PAM Fluorometry of Photosynthetic Activity of
Nine Marine Phytoplankton Grown Under Identical Conditions{
P. Juneau* and P. J. Harrisony
Department of Earth and Ocean Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada
Received 7 January 2005; accepted 26 January 2005
structure in the natural environment where light energy is
highly variable. Our results suggest that for an accurate
evaluation of primary productivity from fluorescence measurements, it is essential to know the species composition of the
algal community and the individual photosynthetic capacity
related to the major phytoplankton species present in the
natural phytoplankton assemblage.
ABSTRACT
The photosynthetic activity of marine phytoplankton from five
algal classes (Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira oceanica, Thalassiosira weissflogii, Dunaliella tertiolecta, Mantoniella squamata, Emiliania huxleyi,
Pavlova lutheri and Heterosigma akashiwo) was investigated
under identical growth conditions to determine interspecies
differences. Primary photochemistry and electron transport
capacity of individual species were examined by pulse
amplitude–modulated (PAM) fluorescence. Although few
differences were found in maximal photosystem II (PSII)
photochemical efficiency between various species, large differences were noticed in their PSII–photosystem I (PSI) electron
transport activity. We found that species such as T. oceanica
and M. squamata have much lower photochemical activity than
H. akashiwo. It appeared that processes involved in electron
transport activity were more susceptible to change during algal
evolution compared with the primary photochemical act close
to PSII. Large variations in the nonphotochemical energy
dissipation event among species were also observed. Light
energy required to saturate photosynthesis was very different
between species. We have shown that M. squamata and H.
akashiwo required higher light energy (>1300 lmol m22 s21) to
saturate photosynthesis compared with S. costatum and E.
huxleyi (ca 280 lmol m22 s21). These differences were
interpreted to be the result of variations in the size of lightharvesting complexes associated with PSII. These disparities in
photosynthetic activity might modulate algal community
INTRODUCTION
Marine phytoplankton biomass represents only 0.2% of the
photosynthetically active carbon biomass on Earth but accounts
for about 45–50% of the global net primary production (1). The
large variability in the accessory light-harvesting pigments of
phytoplankton participating in primary production may induce
heterogeneity concerning the efficiency of light absorption and
energy dissipation processes in photosynthesis (2). Furthermore,
when phytoplankton are grown under different environmental
conditions, such as light, temperature and nutritional status,
photosynthetic activity can change through adaptation or acclimation processes (3). On the other hand, although phytoplankton
species are phylogenetically diverse (see Fig. 1 for a diagram
representing the evolution of photosynthetic eukaryote groups used
in this study), the molecular structure of the photosystem II and I
(PSII and PSI) core complexes responsible for primary photochemistry has been remarkably conserved (4,5). Such specificity in
photosynthetic activity could lead to a problem if one compares
energy storage and dissipation processes through photosynthesis
between different algal species and attempts to evaluate global net
primary productivity in aquatic ecosystems. Therefore, study of the
differences in photosynthetic activity between algal species could
facilitate the understanding of how different algal species can
contribute to primary productivity in aquatic ecosystems.
Evaluation of primary productivity or photosynthetic capacity by
either classical (14C-uptake or O2 evolution) or recently developed
methods on the basis of chlorophyll a fluorescence has shown wide
variations among algal species. For example, the maximal PSII
photochemical yield, M, which is proportional to maximal
photosynthetic efficiency, can change from 0.4 to 0.8 for different
phytoplankton classes (6). Green algae such as Dunaliella
tertiolecta and Dunaliella salina have M values around 0.8,
whereas the M values for diatoms Phaeodactylum tricornutum and
Thalassiosira weisfflogii were shown to be between 0.6 and 0.7 (7–
10). On the other hand, very low maximal PSII photochemical yield
(M 5 0.37) was reported for the prasinophyte Mantoniella
squamata (11). The operational PSII photochemical yield, 9M ,
{Posted on the website on 1 February 2005
*To whom correspondence should be addressed: Department of Biological
Sciences Laboratory of Environmental Toxicology (TOXEN), University
of Québec in Montréal, C.P. 8888, succursale Centre-Ville, Montreal,
Quebec H3C 3P8, Canada. Fax: 514-987-4647; e-mail: [email protected]
Current address: AMCE Program, Hong Kong University of Science and
Technology. Clear Water Bay, Kowloon, Hong Kong.
Abbreviations: M and 9M , maximal and operational PSII photochemical
yields; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; ESAT, energy
to saturate photosynthesis; ETR, electron transport rate; FM and F9M ,
maximal fluorescence yield of a dark-adapted and light-adapted sample;
FO and F9O , dark- and light-adapted state constant fluorescence yields; FS,
steady state fluorescence yield; FV, variable fluorescence; PAM, pulse
amplitude–modulated; P/E, photosynthesis/irradiance; PQ, plastoquinone; PSI and PSII, photosystems I and II; qP(rel) and qN(rel), relative
photochemical and nonphotochemical quenchings; UQF(rel), relative
unquenched fluorescence.
Ó 2005 American Society for Photobiology 0031-8655/05
649
650 P. Juneau and P. J. Harrison
Table 1. Strains and growth rates of the different species used in this
study. The strain numbers are from the Northeast Pacific Culture Collection
(NEPCC) and Provasoli–Guillard National Center for Culture of Marine
Phytoplankton (CCMP). Values are means 6 1 SD (n 5 3)
Figure 1. Diagram representing the evolution of photosynthetic eukaryotes. The primary and the secondary endosymbioses are represented by the
numbers 1 and 2 respectively.
which characterizes steady state photosynthesis, appeared also to be
widely variable among algal species, as seen for Emiliania huxleyi,
Phaeocystis globosa and P. tricornutum (9M : 0.35, 0.5 and 0.6
respectively) (9,12). These variations in the maximal and
operational PSII photochemical yields were explained to be the
result of functional and structural properties specific for the
photosynthetic apparatus of different species (2,6). One could
consider also that these differences in photosynthetic activity might
be influenced by various algal growth conditions (13), but so far
little information is known on how the differences in the maximal
and operational PSII photochemical yields are attributed to species
properties when growth and measurement conditions are the same.
Furthermore, besides the maximal and the operational PSII
photochemical yields, it is also worthwhile to investigate how
other photosynthetic fluorescence parameters reflecting energy
storage and dissipation processes are variable among algal species.
In this study we investigated various aspects of photosynthetic
activity and energy dissipation mechanisms of several ecologically
important phytoplankton belonging to different taxonomic groups.
Different photosynthetic parameters were examined by pulse amplitude–modulated (PAM) fluorometry for algal species grown under
the same light, temperature and nutrient conditions. This investigation
of the photosynthetic capacity of individual algal species will contribute to better estimates of primary productivity in aquatic ecosystems.
MATERIALS AND METHODS
The different marine phytoplankton strains used in this study are listed in
Table 1. Cultures (except E. huxleyi) were grown in filter-sterilized modified
ESAW medium (14) in which the concentrations of nitrate, silicate and
phosphate were 0.2, 0.5 and 0.1 mM respectively. For E. huxleyi the medium
was modified to minimize coccolith detachment (15); the trace metal and
selenium concentrations were respectively 1/50 and 1/100 of the original
ESAW concentrations, whereas nitrate and phosphate concentrations were
reduced to 0.03 and 0.02 mM respectively, and silicate was omitted (16).
Algae were grown in 40 mL culture tubes under continuous illumination
provided by white fluorescent lamps (Vita-Lite 40 W, DURO-TEST,
Philadelphia, PA). The light intensity was 130 lmol m2 s1, and the
temperature was 178C. Algae were grown at or near their maximum growth
rates (Table 1) and were sampled in their midexponential growth phase.
Similar results were obtained when the algae were grown in 1 L flasks (data
Growth rate (day1)
Species
Strain number
Bacillariophyceae
Phaeodactylum tricornutum
Skeletonema costatum
Thalassiosira oceanica
Thalassiosira weissflogii
NEPCC 640
NEPCC 676
CCMP 1003
NEPCC 636
1.11
1.18
0.93
0.90
Chlorophyceae
Dunaliella tertiolecta
NEPCC 001
1.11 6 0.03
Prasinophyceae
Mantoniella squamata
NEPCC 524
0.76 6 0.03
Prymnesiophyceae
Emiliania huxleyi
Pavlova lutheri
NEPCC 732*
NEPCC 634
0.52 6 0.01
0.72 6 0.01
Raphidophyceae
Heterosigma akashiwo
NEPCC 522
0.59 6 0.05
6
6
6
6
0.03
0.04
0.05
0.01
*This isolate was kept in low macro- and micronutrient North Pacific
(Station Papa; 508N, 1458W) water to retain the organism’s ability to
form coccoliths.
not shown). Growth rates were calculated from plots of the log of fluorescence
against incubation time with a Turner Designs fluorometer (Sunnyvale, CA).
All measurements were carried out at the same time of day to minimize the
error associated with any endogenous rhythms within the cultures.
Fluorescence induction measurements were made with a PAM fluorometer 101 (Heinz Walz GmbH, Effeltrich, Germany) after dark adaptation of
the algae for 25 min, according to Schreiber et al. (17). Following dark
adaptation, the dark-adapted state constant fluorescence yield, FO, was
recorded under modulated light (2 lmol m2 s1). The maximal fluorescence
yield for a dark-adapted sample, FM, was obtained with a saturating flash
(700 ms, 3000 lmol m2 s1) permitting all the plastoquinone (PQ) pool to be
in maximum reduced state. The flash intensity was saturating because the
addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (20 lM) did not permit
further increases of the fluorescence level (data not shown). Fluorescence
induction dependent on PSII–PSI electron transport was measured under
continuous actinic light (130 lmol m2 s1). Simultaneously, saturating
flashes (700 ms, 3000 lmol m2 s1) given periodically (every 30 s) provided
the maximal fluorescence yield for a light-adapted sample, F9M . At steady
state fluorescence yield (FS), actinic light was turned off and, following
a short dark period, the light-adapted state constant fluorescence yield, F9O ,
was evaluated. Fluorescence parameters were calculated according to the
following equations after subtraction of the blank fluorescence value
obtained by measuring the fluorescence of a 0.22 lm filtered sample.
M ¼ ðFM FO Þ=FM ¼ FV =FM ð18Þ
9M ¼ ðF9M FS Þ=F9M ð19Þ
qPðrelÞ ¼ ðF9M FS Þ=ðFM F9O Þð20Þ
qNðrelÞ ¼ ðFM F9M Þ=ðFM F9O Þð20Þ
UQFðrelÞ ¼ ðFS F9O Þ=ðFM F9O Þð21Þ
FV is variable fluorescence, qP(rel) and qN(rel) are the relative photochemical
and nonphotochemical quenching values and UQF(rel) is the relative
unquenched fluorescence. Triplicate cultures were grown and data were
analyzed by one-way analysis of variance.
The relative photosynthetic electron transport rate (ETR) was estimated
from the operational PSII photochemical yield measured at different
photosynthetic photon flux densities (PPFD).
ETR ¼ 9M 3 PPFD
The photosynthesis/irradiance (P/E) curve measurements were taken on
the same algal sample by increasing the actinic light intensity stepwise
between 0 and 1600 lmol m2 s1 with saturating flashes (700 ms) every
30 s according to White and Critchley (22). After each increase in actinic
Photochemistry and Photobiology, 2005, 81
651
Table 2. Fluorescence parameters for nine algal species. Parameters are maximal PSII photochemical yield (M), operational PSII photochemical yield
(9M ), relative photochemical and nonphotochemical quenching coefficients (qP(rel) and qN(rel)) and relative unquenched fluorescence (UQF(rel)) . Values are
means 6 1 SD (n 5 3). For each parameter, values with the same letters are not significantly different at P . 0.05
M
Species
P. tricornutum
S. costatum
T. oceanica
T. weissflogii
D. tertiolecta
M. squamata
E. huxleyi
P. lutheri
H. akashiwo
0.687
0.684
0.628
0.690
0.690
0.692
0.628
0.619
0.717
6
6
6
6
6
6
6
6
6
0.034a
0.018a
0.021b
0.014a
0.017a
0.016a
0.018b
0.042b
0.031a
9M
0.296
0.317
0.178
0.347
0.397
0.233
0.296
0.335
0.473
6
6
6
6
6
6
6
6
6
0.008c
0.054c
0.032d
0.022c
0.015c
0.009d
0.010c
0.017c
0.010e
light intensity, the sample was adapted for 2 min to the new light intensity,
and then three flashes were triggered. The use of this method to obtain the
P/E curve gave the same result as the usual method of using a new sample
for each light intensity (22). The fitting procedure was performed in Origin
6.0 (Microcal Software, Northampton, MA) according to the photosynthesis
model described in Eilers and Peeters (23).
RESULTS AND DISCUSSION
It has been reported for different algal species that M can vary by
up to 100% depending on the taxonomic group (6). This wide
variation was proposed to be due to different organization and
functional properties of the photosynthetic apparatus (6,24). As seen
from our study, when algae were grown under the same conditions,
much smaller variations in M values were noticed among different
algal species (;15%) compared with earlier studies (Table 2).
Therefore, we can assume that the previously reported large
variations were probably caused not only by specificity of different
photosynthetic apparatus but by various culture conditions.
However, fluorescence induction of E. huxleyi, Thalassiosira
weissflogii and Pavlova lutheri revealed important variations in the
maximal fluorescence yields measured after dark and light
adaptation (FM and F9M respectively; Fig. 2). Important variations
were also noticed in the fluorescence yield at steady state electron
transport (FS). Smaller differences in the fluorescence kinetics were
also observed for the other species (data not shown). These variations might indicate real differences among species concerning
the balance of the energy dissipation associated with PSII–PSI
electron transport. The high value of FS seen for E. huxleyi (Fig. 2A)
could reflect that an important fraction of PSII reaction centers was
closed, indicating that few PSII participate in electron transport
toward PSI. On the other hand, the decrease in FS close to F9O
seen in P. lutheri (Fig. 2C) could indicate high PQ pool oxidation
state and an active electron transport activity toward PSI.
One might expect that fluorescence indicators obtained at steady
state electron transport vary importantly between algal species
because the variations in the fluorescence kinetics were evident.
Indeed, variations were noticed for 9M , qP(rel), qN(rel) and UQF(rel).
For example, unlike M, which tends to be similar between species
(Table 2), 9M , qP(rel), qN(rel) and UQF(rel) showed variation of 55,
68, 75 and 100% respectively. Therefore, low variation of M
values reflect functional properties of the conservative part of PSII
electron transport (PSII core complex), whereas other parameters
are influenced by various factors related to organization and
function of photosynthetic electron transport and related processes.
The quenching components evaluated by qP and qN (17) showed
larger variations compared with the relative quenching coefficients
qP(rel)
0.354
0.362
0.282
0.383
0.373
0.129
0.409
0.365
0.414
6
6
6
6
6
6
6
6
6
0.058f
0.065f
0.032g
0.025f
0.038f
0.004h
0.015f
0.029f
0.030f
qN(rel)
0.427
0.329
0.217
0.504
0.330
0.869
0.222
0.512
0.325
6
6
6
6
6
6
6
6
6
0.017i
0.056j
0.005k
0.028l
0.021j
0.011m
0.001k
0.042l
0.002j
UQF(rel)
0.219
0.309
0.501
0.113
0.297
0.002
0.369
0.123
0.261
6
6
6
6
6
6
6
6
6
0.013n
0.034o
0.014p
0.021q
0.025o
0.009r
0.008s
0.031q
0.021o
(qP(rel) and qN(rel); data not shown). This might be explained by the
fact that qP and qN are not normalized to the same reference signal
and because the calculation of qP is based on a photosynthetic unit
model (puddle model) known not to be representative of the real
situation (20,21,25).
We found that all algal species except T. oceanica and
M. squamata have similar 9M and photochemical quenching
(qP(rel)). This might be explained by PSII and PSI core complexes
that are highly conserved among all classes of eukaryotic algae
(5,26). However, reasons for lower photochemical energy dissipation in T. oceanica and M. squamata compared with the other
species are not clear yet. It has been found that M. squamata has
high chlororespiratory activity (27), which might explain the high
nonphotochemical energy dissipation that was noticed in our study.
Furthermore, M. squamata appears to lack some of the lightharvesting complexes associated with PSI (28), which might reduce
the light energy funneled to the PSI reaction centers and consequently diminish PSII–PSI electron transport. On the other hand,
the low PSII electron transport for T. oceanica (see 9M and qP(rel) in
Table 2) could be interpreted by the higher PSII:PSI ratio
characteristic for this species compared with the other algal species
(29–31). Decreased PSII–PSI electron transport will induce a high
reduced state of PSII and therefore the high FS yield and high
UQF(rel) value seen for T. oceanica (Fig. 2A, Table 2).
Although the photochemical energy dissipation (qP(rel)) of
P. tricornutum, S. costatum, T. weissflogii, D. tertiolecta, E.
huxleyi, P. lutheri and Heterosigma akashiwo was similar, significant differences were found in nonphotochemical energy
dissipation (qN(rel)), which varied from 0.222 to 0.512. It is known
that nonphotochemical energy dissipation processes are affected by
mechanisms and structures that are not linked only to the PSII and
PSI core complexes (32,33). Therefore we may suppose that these
processes have passed through important modifications during the
evolution of algal species. The relative unquenched fluorescence
parameter also showed wide variation among species, suggesting
that the processes involved in the control of the proportion of closed
PSII reaction centers at steady state electron transport were not
conserved during the evolution of phytoplankton.
Significant differences in the photosynthetic capacity and
structural properties among the examined species are also shown
by the change in the P/E response (Fig. 3). The initial slope of the
P/E curve was reported to be proportional to the PSII antenna size
(34). Therefore, the smaller initial slopes of the P/E curves for
T. oceanica, H. akashiwo and M. squamata compared with
P. tricornutum, S. costatum and E. huxleyi can be interpreted as
smaller PSII antenna sizes. This might explain the high energy
652 P. Juneau and P. J. Harrison
Figure 2. Fluorescence induction kinetics of E. huxleyi (A), T. weissflogii
(B) and P. lutheri (C). The different fluorescence yields (FO, FM, F9M , FS
and F9O ) used for the PAM fluorescence parameter calculation are indicated
in panel A.
radiation required to reach photosynthetic saturation for these
species (Fig. 4). Indeed, a good correlation between the energy to
saturate photosynthesis (ESAT) and the initial slope of the P/E curve
(a) was obtained for the investigated species. However, the fact that
T. oceanica, which has a small antenna size, shows saturation of
photosynthesis at relatively low energy (280 lmol m2 s1; see u in
Figure 4), might be explained by its high PSII:PSI ratio noted earlier
(30). It appeared that photosynthesis of the majority of investigated
algae was saturated at intensities ,1300 lmol m2 s1. However for
some species such as H. akashiwo and M. squamata, the electron
transport rate was saturated above 1300 lmol m2 s1. The saturation
at relatively low light intensity for E. huxleyi and S. costatum might
suggest that modest requirements in ATP and NADPH are necessary
for CO2 fixation. Variations in light intensity in the natural habitat
could influence the contribution of individual algae to global
Figure 3. Relative electron transport rate (ETR) of M. squamata (D),
H. akashiwo (s), T. oceanica (h), D. tertiolecta (m), P. lutheri ( ),
T. weissflogii (n), E. huxleyi (*), P. tricornutum ()) and S. costatum (3)
as a function of the photosynthetic photon flux density (PPFD). Lines
represent fit according to the photosynthesis model of Eilers and Peeters
(23). Error bars 5 61 SD, n 5 3.
photosynthesis in aquatic ecosystems. Indeed, as indicated by the
decline in photosynthesis (see Fig. 3), some species could be
photoinhibited at relatively low light intensity, making them less
likely to be productive near the surface of the ocean during sunny
days. However, light intensity is only one of the possible ecological
factors that can influence the contribution of individual algae to the
entire algal community. Therefore additional effects on the
photosynthetic capacity of other environmental factors, such as
temperature and nutrient status, have also to be considered.
We have shown that the photosynthetic activity of different algal
species can differ widely even if they were grown under the same
conditions. Consequently, for an accurate evaluation of primary
productivity on the basis of fluorescence measurements, it is useful
Photochemistry and Photobiology, 2005, 81
12.
13.
14.
15.
16.
Figure 4. Relationship between the initial slope (a) of the P/E curve and
the energy required to saturate photosynthesis (ESAT). Symbols representing
different algae are as in Fig. 3. T. oceanica (h) was not included in the
curve fitting and correlation factor determination (see text for details).
17.
18.
to know the structure of the algal community and individual
photosynthetic capacity related to the major phytoplankton species.
Indeed, the knowledge of algal species composition is essential if
one wants to obtain an accurate evaluation of primary productivity
because, as we have shown here, an estimate of the photosynthetic
capacity of an algal assemblage is closely linked to specific
properties of each algal species.
19.
20.
Acknowledgements—This research was supported by a Natural Sciences
and Engineering Research Council of Canada (NSERC) Research Grant.
P.J. received support from a NSERC Postdoctoral Fellowship.
21.
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