J. Phycol. 41, 1169–1177 (2005)
r 2005 Phycological Society of America
DOI: 10.1111/j.1529-8817.2005.00144.x
EFFECT OF NITRATE CONCENTRATION ON THE RELATIONSHIP BETWEEN
PHOTOSYNTHETIC OXYGEN EVOLUTION AND ELECTRON TRANSPORT RATE
IN ULVA RIGIDA (CHLOROPHYTA)1
Alejandro Cabello-Pasini2
Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, A.P. 453, Ensenada,
Baja California 22800, México
and
Fe´lix L. Figueroa
Dept. Ecologı́a, Facultad de Ciencias, Universidad de Málaga, Campus Universitario Teatinos s/n, E-29071, Spain
The electron transport rate (ETR) versus gross
photosynthesis (GPS) relationship varies as a function of species, temperature, irradiance, and inorganic carbon levels, but less is known about the
effect of nitrogen supply on this relationship. The
objective of this study was to evaluate the effect of
nitrate concentration on the ETR versus GPS relationship in Ulva rigida C. Agardh from the Mediterranean Sea. Chlorophyll content and tissue
absorptance increased 2-fold as nitrate in the media increased from 0 to 50 lM. Whereas internal N
content increases 3-fold at 50 lM, internal C increased slightly. Oxygen evolution and ETR, evaluated as in vivo chl fluorescence using pulse
amplitude modulated fluorometry, in general saturated at irradiances above 100 lmol photons . m 2 .
s 1. Both maximum ETR and GPS values increased
as nitrate concentration increased. In general, the
ETR versus GPS relationship showed a linear response to increasing nitrate with little variance of
the data. This relationship, however, became more
variable at high irradiances and high nitrate concentrations. The ETR/GPS ratio was close to the
theoretical value of 4 at low nitrate concentrations,
and the ratio decreased exponentially when nitrate
concentration in the media increased. The variations of ETR/GPS under different inorganic nitrogen supply are discussed in terms of the effect of
nitrate on the photosynthesis and respiration relationship.
amplitude modulated; UPSII, effective quantum
yield of PSII
Photosynthetic rates of marine macrophytes vary as
a function of light, temperature, nutrients, and water
motion; thus, algae can acclimate to environmental
fluctuations and stress. Although primary production
has been historically evaluated through oxygen evolution or carbon incorporation methods, the use of fluorometric methods for the study of photosynthesis have
become common in the last two decades (Edwards and
Baker 1993). Pulse amplitude modulated (PAM) fluorometry of in vivo chl fluorescence associated with PSII
has been used to evaluate primary reactions and
quenching mechanisms in physiology studies under natural or artificial light conditions (Genty et al. 1989). In
general, electron transport rate (ETR) is positively correlated to oxygen evolution and CO2 fixation, especially
in C4 plants, as long as environmental stresses do not
impose restrictions on photosynthetic CO2 metabolism
(Genty et al. 1989, Edwards and Baker 1993). In aquatic
systems, however, the ETR versus gross photosynthesis
(GPS) relationship is often ambiguous, especially at high
irradiances (Beer et al. 1998, Carr and Bjork 2003).
Electron transport rates in a number of plants have
been shown to increase linearly at low light levels until
ETR saturates at high irradiances (Edwards and Baker
1993). However, the ETR versus GPS relationship in
aquatic plants is species dependent (Beer et al. 1998)
and linear at only low irradiances (Figueroa et al.
2003) or dependent on methodological procedures
(Carr and Bjork 2003). Furthermore, it has been observed that ETR overestimates gross O2 evolution in a
number of seaweeds, especially when carbon availability is low and saturating photon fluxes are provided
(Franklin and Badger 2001, Carr and Bjork 2003). Although the effect of CO2 and temperature on the photosynthetic characteristics has been well established,
little is known about the effect of nitrogen concentration on the relationship between photosynthesis and
Key index words: electron transport rate; fluorescence; nitrate; photosynthesis; Ulva rigida
Abbreviations: ETR, electron transport rate; Fm,
maximum fluorescence; Fo, intrinsic fluorescence;
Fv, variable fluorescence; Fv/Fm, optimum quantum
yield; GPS, gross photosynthesis; PAM, pulse
1
Received 11 March 2005. Accepted 30 August 2005.
Author for correspondence: e-mail [email protected].
2
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ALEJANDRO CABELLO-PASINI AND FÉLIX L. FIGUEROA
ETR. It has been well established that plants growing
under low nitrogen conditions reduce the synthesis of
pigments and proteins, including RUBISCO (Seemann and Sharkey 1986). In addition to changes in
metabolite synthesis, changes in nitrogen levels have
been shown to modify GPS/ETR ratios in Ulva rotundata (Henley et al. 1991). The slopes of the GPS versus
ETR relationship, or effective quantum yield of PSII
(FPSII) versus quantum yield of oxygen evolution, have
been shown to be lower in species with the greatest N
assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents (Figueroa et al.
2003). Although the electron pathways to N assimilation diverts on the level of ferredoxin, this should not
influence the O2 rates but influences CO2 fixation
rates, because the extent of the electron flow depends
on the electron sink (i.e. carbon and nitrogen assimilation). As observed in marine macroalgae, the optimum quantum yield covaries with NO3 , Si (OH)4, and
PO34 concentration in phytoplankton (Kolber et al.
1990, Babin et al. 1996, Young and Beardall 2003).
Consequently, the objective of this study was to evaluate the effect of nitrate concentration on the ETR and
oxygenic photosynthesis relationship of Ulva rigida
under laboratory conditions.
MATERIALS AND METHODS
Plant material. Ulva rigida was collected in the intertidal
zone of Málaga, Spain (36147 0 N, 4119 0 E) and transported in
ice coolers to the laboratory in February 2004. Samples of
approximately 6 cm2 (approximately 2 g total fresh weight)
were incubated from 2 to 3 weeks in six acrylic containers
(2 L, 15 samples per container) with seawater containing
10 mM PO34 and 0, 2, 5, 10, 25, and 50 mM NO3 . Seawater
used to prepare media contained less than 1 mM NO3 and
NH4þ . Nutrients were added, and seawater was changed
every other day. Samples were maintained at 151 C and
100 mmol photons m 2 s 1 using daylight fluorescent
lamps (Osram FL 18 W, Munich, Germany). The tissue was
kept in constant suspension by bubbling air into each container. The tissue-to-volume rate was maintained constant by
trimming growth in each tissue sample every other day.
Pigment and CHN Determination. Chlorophylls a and b
were extracted in N,N-dimethylformamide according to Inskeep and Bloom (1985). Samples (2.5 cm2, n 5 6) were incubated in 3 mL N,N-dimethylformamide for 24 h at 41 C in
darkness. Absorbance was determined using a spectrophotometer, and chl concentration was estimated using the equations proposed by Porra et al. (1989). Total intracellular
carbon and nitrogen content (g/g) was determined (n 5 6)
after the incubation period using an elemental analyzer
(model 2400 CHN, Perkin-Elmer, Wellesley, MA, USA).
Oxygen evolution and chl fluorescence. Photosynthetic rates
in U. rigida were determined using polarographically measured rates of steady-state O2 evolution (Rank Brothers, Inc.,
Cambridge, UK). Tissue (n 5 6) of 2.5 cm2 was incubated in
seawater (2.2 mM DIC) at 151 C in 5-mL jacketed chambers
connected to a water–circulating bath after a 0.5-h preincubation in darkness. Halogen lamps (Quartzline, 150 W, GE,
Fairfield, CT, USA) were used as a light source, and photosynthetic photon flux from 0 to 600 mmol photons m 2 s 1
was varied using neutral–density filters (Lee Filters, Osram,
Hampshire, UK). Maximum oxygenic photosynthesis, the
initial slope of the photosynthesis versus irradiance curve
(aoxy), the threshold for irradiance–saturated photosynthesis,
and respiration were determined by a nonlinear direct fitting
algorithm (Sigma Plot, Jandel Scientific, Chicago, IL, USA) of
the data to the exponential equation described by Webb et al.
(1974). Gross photosynthesis was calculated by adding net
photosynthesis and respiration measured after each irradiance period.
In vivo chl fluorescence of PSII was determined (n 5 6) with
a portable PAM fluorometer (DIVING PAM, Walz, Effeltrich,
Germany). Intrinsic fluorescence (Fo) was determined after
maintaining the tissue in darkness for 1 to 2 h. A saturating
actinic light pulse (9000 mmol photons m 2 s 1, 800 ms) was
applied to obtain maximum fluorescence (Fm) in the darkacclimated samples. Variable fluorescence (Fv) was determined
as the difference between Fm and Fo, and optimum quantum
yield (Fv/Fm) was calculated as the ratio of Fv to Fm (Schreiber
et al. 1994). Effective quantum yield of PSII (FPSII) was determined in light-acclimated tissue according to Schreiber and
Neubauer (1990):
FPSII ¼ ðF0m Ft Þ=F0m
ð1Þ
where F 0 m is the maximal fluorescence of light-acclimated tissue induced by a saturating actinic light pulse (9000 mmol photons m 2 s 1, 800 ms) and Ft is the intrinsic steady-state
fluorescence in light-acclimated tissue. The fiberoptic of the
PAM fluorometer was kept at a 45-degree angle from the
seaweed tissue throughout the experiments.
The ETR was determined according the following formula:
ETRðmmol electrons m2 s1 Þ ¼ AQl FII FPSII
ð2Þ
where AQl is the absorbed photons calculated as the product
of the integration of the spectral absorptance (Al) between 400
and 700 nm and spectral irradiance of the light source (El), FII
is the fraction of AQ directed to PSII including its light-harvesting complexes, and FPSII is the effective quantum yield or
quantum yield of PSII charge separation. Values of FII for different pigment groups can be estimated by determining the
fraction of chl a associated with PSII and its corresponding
light-harvesting complexes (Grzymski et al. 1997). For example, FII values are approximately 0.5 for Chlorophyta (Grzymski et al. 1997, Figueroa et al. 2003). Maximum ETR values
(ETRmax), the initial slope of the ETR versus irradiance curve
(aETR), and the threshold for irradiance–saturated photosynthesis (EkETR) were determined by a nonlinear direct fitting
algorithm (Sigma Plot, Jandel Scientific) of the data to the
exponential equation described by Webb et al. (1974).
Tissue absorptance (Al, n 6) was determined at 1-nm intervals between 400 and 700 nm using an integrating sphere
(LICOR–1802, Lincoln, NE, USA) connected to a spectroradiometer (LICOR–1800 UW) according to the following formula
(Schreiber and Neubauer 1990):
A l ¼ 1 Tl R l
ð3Þ
where Tl is transmittance and Rl is reflectance of the tissue.
Simultaneous measurements of oxygen evolution and FPSII
were conducted by introducing the PAM’s fiberoptic sensor into
the oxygen chamber. The optic fiber was placed at a 45-degree
angle to the algal tissue. Values of FPSII were determined after
light steady oxygen evolution (approximately 5 min) at each experimental irradiance. Irradiance was increased using neutral
density filters after a 10-min period of darkness.
Statistical analysis. Differences in photosynthetic parameters (GPS, ETR) as a function of nitrate treatment were evaluated using a one-way analysis of variance after testing for
normality and homoscedasticity of the data (Sokal and Rohlf
1995). All pair-wise multiple comparisons were conducted
using Tukey’s test. The significance of correlations between
chl levels and absorptance of the tissue as well as between
GPS and ETR were tested using Pearson’s Product-Move-
EFFECT OF NITRATE ON PHOTOSYNTHESIS AND ETR
1171
ment correlations. Minimum significance level was established at Po0.05.
RESULTS
Chlorophyll concentration and absorptance in the
U. rigida tissue varied in relation to the nitrate incubation treatment (Fig. 1). Chlorophyll concentration in
the tissue increased as the nitrate concentration in the
incubation treatment increased (Fig. 1A). Similarly, absorptance of the tissue increased as the nitrate concentration in the incubation treatment increased (Fig. 1B).
There was an increase of absorptance as tissue chl
levels increased in U. rigida (Fig. 1C).
Carbon and nitrogen concentration in the tissue of
U. rigida varied in relation to the nitrate incubation
treatment (Fig. 2). Carbon levels in the tissue increased
from 21.9 1.1% in algae incubated at 0 mM NO3 to
saturating levels (26.2 0.7%) in algae incubated at 25
and 50 mM NO3 (Fig. 2A). Although carbon levels increased 16%, nitrogen levels in the tissue increased
more than 2-fold from 1.1 0.04% in plants incubated
at 0 mM NO3 to 2.6 0.2% in plants incubated at
50 mM NO3 (Fig. 2B). There was a 2-fold decrease in
the carbon to nitrogen ratio (C/N), from 20.6 0.5 in
tissue incubated at 0–2 mM NO3 to 10.4 0.9 in tissue
incubated at 50 mM NO3 (Fig. 2C).
FIG. 2. Carbon concentration (A), nitrogen concentration
(B), and the carbon vs. nitrogen relationship (C) in the tissue
of Ulva rigida incubated at different nitrate levels. Data points
represent the average of six samples SD.
FIG. 1. Chlorophyll a þ b concentration (A), absorptance (B),
and the chl vs. absorptance relationship (C) in the tissue of Ulva
rigida incubated at different nitrate levels. Data points are average of six samples SD.
Maximum rates of oxygen evolution and ETR in
U. rigida increased as a function of nitrate availability
(Fig. 3). There was a 2-fold increase in GPSmax, from
8 mmol O2 m 2 s 1 in tissue incubated from 0 to
5 mM NO3 to 15 mmol O2 m 2 s 1 in tissue incubated from 25 to 50 mM NO3 1 (Fig. 4A). Similar to GPSmax
values, ETRmax values increased from low values in
tissue incubated with 0 to 5 mM NO3 to high values in
tissue incubated with 50 mM NO3 (Fig. 4A). In general,
ETRmax values were approximately 4-fold greater than
GPSmax values. In contrast to GPSmax and ETRmax, the
initial slope of oxygen evolution (aoxy) and the initial
slope of the ETR versus irradiance relationship (aETR)
remained relatively constant at all incubation treatments (Fig. 4B). However, values of aETR were 2-fold
greater than aoxy values at all incubation treatments.
There was a 3-fold increase of EkETR values in U. rigida,
from 100 mmol photons m 2 s 1 in tissue incubated
at 2 mM NO3 to 300 mmol photons m 2 s 1 in tissue
incubated at 50 mmol photons m 2 s 1. In contrast to
EkETR, values of threshold for irradiance-saturated
photosynthesis only increased by 40% from low values
in tissue incubated with 0–2 mM NO3 to high values in
tissue incubated at 50 mM NO3 (Fig. 4C).
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ALEJANDRO CABELLO-PASINI AND FÉLIX L. FIGUEROA
FIG. 3. Oxygen evolution vs. irradiance relationship (A) and
electron transport rate vs. irradiance relationship (B) in Ulva
rigida incubated at different nitrate levels. Data points represent
the average of six samples SD. Error bars not shown are smaller than symbol size.
Gross photosynthesis and ETR followed a linear relationship in tissue incubated from 0 to 50 mM NO3
(Fig. 5). In general, values showed little deviation from
a linear model at irradiances below 300 mmol photons m 2 s 1, and deviation increased when tissues
were incubated at irradiances above 300 mmol photons m 2 s 1. Deviation of the data from the linear
model at high irradiances slightly increased as nitrate
concentration increased in the treatments. The slope
of the ETR versus GPS relationship decreased exponentially from 4.24 0.13 in algae incubated with
0–2 mM NO3 to 3.42 0.11 in plants incubated with
25 to 50 mM NO3 (Fig. 6).
Respiration rates increased from approximately
0.05 mmol O2 m 2 s 1 in tissue incubated with 0–
2 mM NO3 to saturation values of 2.3 0.3 mmol O2 m 2 s 1 in tissue incubated with 25–50 mM NO3
(Fig. 7A). The GPS-to-respiration ratio decreased
from 129 8.9 in plants incubated with 0 mM NO3
to 4.25 0.3 in plants incubated with 5 to 50 mM NO3
(Fig. 7B). The rapid decline in the GPS-to-respiration
ratio was promoted by the rapid increase in respiration
rate as nitrogen increased, relative to the increase in
GPS.
Both Fv/Fm and Fm were significantly correlated
(Po0.001) to nitrate concentration in the media
(Fig. 8A). Optimum quantum yield decreased linearly
from 0.72 0.005 in tissue of U. rigida incubated with
0 mM NO3 to 0.55 0.029 in tissue incubated with
50 mM NO3 . In contrast, Fm in dark-acclimated U.
rigida increased linearly from 849 161 in tissue incu-
FIG. 4. Maximum oxygenic photosynthesis (Pmax) and maximum electron transport rate (ETRmax, A), the initial slope of the
oxygenic photosynthesis vs. irradiance relationship (Alpha) and
the initial slope of the electron transport rate vs. irradiance relationship (AlphaETR, B), and subsaturation coefficient (Ek, C) of
Ulva rigida incubated at different nitrate levels. Data points represent the average of four samples SD. Error bars not shown
are smaller than symbol size.
bated with 0 mM NO3 to 2232 246 in tissue incubated with 50 mM NO3 . Similar to Fm, Fo increased
linearly as nitrate concentration increased in U. rigida
(Fig. 8B).
DISCUSSION
The GPS versus ETR relationship in marine
macrophytes has been shown to depend, among other things, on the species studied, the light history, the
CO2 levels in the water, and the methods used to evaluate oxygen evolution and chl fluorescence (Beer and
Bjork 2000, Franklin and Badger 2001, Gordillo et al.
2001b, Carr and Bjork 2003). Here we demonstrate
that the GPS versus ETR relationship also depends on
the nitrogen status of the tissue studied. Furthermore,
chl concentration in the tissue, absorptance, C/N ratios,
Fv/Fm, Fm, and GPS-to-respiration rates were also
regulated by nitrate availability in the media.
The GPS versus ETR relationship has been examined in the red algae Palmaria palmata (Hanelt and
Nultsch 1995) and Porphyra columbina (Franklin and
Badger 2001); the brown algae Dictyota dichotoma (Hanelt et al. 1995) and Zonaria crenata (Franklin and Badger 2001); the green algae Ulva rotundata (Osmond
EFFECT OF NITRATE ON PHOTOSYNTHESIS AND ETR
1173
FIG. 5. Electron transport rate vs. oxygenic gross photosynthesis relationship of
Ulva rigida incubated at different nitrate
levels.
et al. 1993), U. lactuca, U. fasciata (Beer et al. 2000),
and U. australis (Franklin and Badger 2001); and the
seagrasses Halodule wrightii, Halophila ovalis, Cymodocea
nodosa, and Zostera marina (Beer et al. 1998, Beer and
Bjork 2000), among other species. At moderate irradiance, ETR calculated from FPSII closely matches
gross O2 evolution in U. fasciata and U. lactuca (Beer
et al. 2000). In contrast, Longstaff et al. (2002) found
that in situ measurements of diel photosynthesis of
FIG. 6. Initial slope of the electron transport rate vs. oxygenic
gross photosynthesis relationship of Ulva rigida incubated at different nitrate levels. Data points represent the average of four
samples SD.
U. lactuca revealed a significant correlation between
ETR and O2 evolution at moderate light, but at higher
irradiances oxygen evolution saturated as ETR values
increased. Similarly, Franklin and Badger (2001) reported a significant correlation between GPS and ETR
at limiting irradiances in U. australis and P. columbina,
whereas at saturating photon fluxes, especially when
inorganic carbon availability was low, ETR overestimated gross O2 evolution.
Unlike results of Carr and Bjork (2003), the ETR
versus GPS relationship here in U. rigida showed a linear response, and there was little deviation of the data
from the linear model. Our experiments were conducted by a step-wise increase of irradiance to the same
tissue after a 10-min dark period between irradiance
increments in the same irradiance range as experiments conducted by Carr and Bjork (2003). Clearly,
the relationship between ETR and GPS in this study
only deviated from the linear relationship, and data
become more variable in tissue incubated at high irradiances and high nitrate levels. The lack of linearity
generally results in species where GPS but not ETR
saturate at high irradiances. It is possible then that the
higher variance obtained in other studies is the result
of incubations or preincubations at nonecologically significant nitrate levels. In our experiments, however,
GPS and ETR saturated at irradiances greater than
100 mmol photons m 2 s 1, except in tissue incubat-
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ALEJANDRO CABELLO-PASINI AND FÉLIX L. FIGUEROA
FIG. 7. Maximum photosynthesis-to-respiration ratio of Ulva
rigida incubated at different nitrate concentrations. Data points
represent the average of six samples SD. Error bars not shown
are smaller than symbol size.
ed at 50 mM NO3 , where the greatest variance of the
data was observed at high irradiances. Although the
linearity of the GPS versus ETR relationship observed
FIG. 8. Optimum quantum yield (Fv/Fm) and maximum fluorescence (Fm; A) and (B) intrinsic fluorescence (Fo) in darkacclimated tissue of Ulva rigida incubated at different nitrate
concentrations. Data points represent the average of nine samples SD. Error bars not shown are smaller than symbol size.
in our studies is consistent with results in phytoplankton (Flameling and Kromkamp 1998, Gordillo et al.
2001a), seaweeds (Franklin and Badger 2001), and
seagrasses (Beer and Bjork 2000), it is not consistent
with results observed in other studies, including those
with other species of Ulva (Carr and Bjork 2003, Figueroa et al. 2003). Although a linear relationship was
not observed in the whole range of the GPS versus
ETR relationship in these experiments, there is a clear
linearity when photosynthetic values were below
saturation (Figueroa et al. 2003). The great sampleto-sample variation observed in other studies (Carr
and Bjork 2003) suggests a light history difference or
internal nitrogen differences among samples. The tissues used in our experiments did not show such large
sample-to-sample differences because they came from
a temperature-, irradiance-, and nutrient-homogenous
environment.
The nonlinear relationship between GPS and ETR
can be explained by the action of electron sink processes such as the Mehler reaction and PSII cycling.
The Mehler reaction may contribute to an electron
demand of approximately 20% of the total ETR in
cyanobacteria (Kana 1993) and green algae (Rees et al.
1992). This reaction induces a trans-thylakoid pH gradient that plays a key role in the dissipation of excess
energy at high light intensity (Schreiber et al. 1995).
Thus, in N-deprived samples of U. rigida, both the
Mehler reaction and the electron demand by nitrate
reductase could affect the pH gradient and consequently the energy dissipation of the photosynthetic
apparatus. Franklin and Badger (2001) reported that
the excess electron flow to O2 in U. australis was neither
related to the Mehler-ascorbate peroxidase reaction
nor to the cyclic electron flow around PSII enhanced at
high irradiance or low Ci. However, in our study it is
not possible to exclude the action of these electron sink
processes under nitrate limitation.
The slope of the ETR versus GPS relationship in
U. rigida was close to the theoretical value of 4 mol
electrons per mol oxygen evolved as observed for other Ulva species (Carr and Bjork 2003). However, our
results show that the slope of the ETR versus GPS increases as nitrogen decreases in the thalli. Although
total chl concentration in the tissue of U. rigida increased with nitrate concentration, the chl a/b ratio was
slightly higher in samples incubated at low nitrate concentration (approximately 1.95) than those incubated
at high nitrate concentration (approximately 1.75; data
not shown). This could indicate changes in the antenna
size and/or PSI/PSII ratios. Values of FII for different
pigment groups can be estimated by determining the
fraction of chl a associated with PSII and its corresponding light-harvesting complexes (i.e. light-harvesting complex II) (Grzymski et al. 1997). In the Chlorophyta, values of FII have been shown to be
approximately 0.5 (Grzymski et al. 1997), which indicates a 1:1 distribution of irradiance between PSI and
PSII. It is possible, however, that the contribution of
chl a in PSII may be higher at low nitrate concentra-
EFFECT OF NITRATE ON PHOTOSYNTHESIS AND ETR
tions than that at high nitrate levels. The ETR/GPS
value of less than 4 at low nitrate concentrations may
be the result of an underestimation of ETR because
more than 50% of the absorbed light goes to PSII in
algae grown at low nitrate concentration.
It has been established that electrons are transferred from PSII to an intermembrane plastoquinone
pool, then to a series of carriers, and eventually to ferredoxin and the production of NADPH (Edwards and
Walker 1983). This reductant is spent in pathways such
as nitrogen assimilation. Assimilatory nitrate reductase
catalyzes the first step of nitrate assimilation, the reduction of NO3 to NO2 in plants and fungi, and
spends up to 25% of the photosynthetically generated
NADPH in U. australis and P. columbina (Franklin and
Badger 2001). The enzyme nitrate reductase is activated as nitrate availability increases in the media.
Consequently, it is possible that electrons used to
feed other metabolic pathways such as nitrate reductase are also promoting the variation of the ETR
versus GPS slope. Our result cannot be explained by
the nitrate reductase activity increase because O2 is
formed at PSII, and consequently an increase in the
ETR/GPS ratio would be expected as a result of increasing nitrate concentration. However, nitrate concentration can also decrease the photosynthesis–
respiration relationship in a number of algae. It has
been demonstrated, for example, that the addition of
NO3 and NH4þ resulted in a large stimulation of
dark respiration and dark carbon fixation in the microalga Selenastrum minutum (Elrifi and Turpin 1986).
Consequently, it is possible that there is a transient
suppression of photosynthetic carbon fixation in response to N pulses as result of a competition between
Calvin cycle and nitrogen assimilation. The greater increase of dark respiration than that of net photosynthesis could explain the ETR GPS decrease under high
nitrate availability. The high GPS-to-respiration ratio at
low nitrate concentrations could be explained by a
high release of organic carbon. Gordillo et al. (2001b)
reported a high excretion of organic carbon (approximately 45% of the primary production) in N-deficient
U. rigida compared with samples cultured at N-sufficient conditions (approximately 30% of the primary
production).
External and internal nitrogen contents appear to
have a critical role in the tissue chl levels and absorptance in U. rigida. Changes in the chl content in the
cells of marine algae fluctuate significantly within
hours as a response to changes to nitrate concentration in the media (Young and Beardall 2003). The observed increase of absorptance is consistent with
increasing nitrogen levels in the tissue as observed in
other seaweed and higher plant species (Mercado et al.
1996, Carter and Spiering 2002) and reflects the increasing chl levels within the cells. Furthermore, the
rate of nitrogen incorporation in the tissue was faster
than that of chl synthesis, suggesting that nitrogen is
initially stored or used for the synthesis of nitrogenated
molecules other than chl.
1175
In addition to irradiance, nitrate availability had an
effect on Fv/Fm. Although Fm, Fo, ETR, and GPS increased with increasing nitrate concentration, Fv/Fm
decreased from high values in algae incubated with
0 mM NO3 to low values in thalli incubated with
50 mM NO3 in spite of the increase of chl concentration and absorptance in the tissue. The decrease of
Fv/Fm with increasing nitrate concentration was not
expected because chl concentration and maximal photosynthesis increased with nitrate concentration. Hyperbolic relationships have been observed between the
maximum quantum efficiency and the rate of nitrogen
supply in chemostat phytoplanktonic cultures (Kolber
et al. 1988) and in natural phytoplankton assemblages
(Geider et al. 1993). The decrease of Fv/Fm with increasing nitrate concentrations here may indicate a
change in the antenna composition (i.e. higher accumulation of chl in PSI compared with low nitrate concentration). The possible increase of PSI activity as a
consequence of the antenna increase under high
nitrogen levels can alter the ATP/NADPH ratio (i.e.
increase of PSI cyclic phosphorylation). Thus, the increase of nitrate availability resembles the typical response of macrophytes to high irradiances (high nonphotochemical quenching and a decrease of Fv/Fm).
Although the concentration of total chl increased
above 25 mM NO3 , the maximal quantum yield decreased. This is explained by the anomalous variations
of Fo and Fm. It has been reported that values of Fo can
be influenced by different cell structures and chloroplast morphology in higher plants (Genty et al. 1990)
and algae (Lange et al. 1989). In lichens containing
green algae and cyanobacteria, for example, Lange
et al. (1989) reported large changes in Fo due to cell
shrinkage during dehydratation. In addition, Brugnoli
and Björkman (1992) reported variation in Fo related
to chloroplast movements. Thus, the large variations in
Fo observed in our study may be the result of a morphogenic response to nutrient availability.
The increase of maximal GPS and ETR with nitrate
availability may be the result of a higher demand of
electrons for nitrate assimilation. Although the electron pathways to N assimilation divert on the level of
ferredoxin, this should not influence the O2 rates but
influences CO2 fixation rates, as the extent of the electron flow depends on the electron sink (i.e. carbon and
nitrogen assimilation). Babin et al. (1996) found maximum quantum yield of carbon fixation to covary with
nitrate concentration in phytoplankton. Furthermore,
the slopes of GPS versus ETR or FPSII versus quantum
yield of oxygen evolution were lower in Ulva species
with the greatest N assimilation capacity, estimated as
nitrate reductase activity and internal nitrogen contents (Figueroa et al. 2003). In these experiments, the
increase of GPS was greater than the increase of ETR
as nitrate concentrations was increased.
This study demonstrates that the GPS versus ETR
relationship is dependent on the nitrogen status of the
tissue of U. rigida. Nitrate availability also affected
a number of fluorometry-obtained photosynthetic
1176
ALEJANDRO CABELLO-PASINI AND FÉLIX L. FIGUEROA
parameters such as Fv/Fm, Fm, ETR, and Fo. Consequently, it is possible that the sample-to-sample variation within and among studies may be the result of
tissue nitrogen differences among samples. This study
also suggests that the GPS versus ETR relationship in
natural seaweed populations may vary as a result of
natural fluctuations in nutrient loads (i.e. upwelling
events). Thus, it is necessary to investigate in more detail the effect of nutrient availability (not only nitrate)
on the photosynthesis, respiration, and maximal quantum yield relationships.
Financial support was provided by the Ministry of Education
and Culture and Ministry of Science and Technology of Spain
(AGL 2001-1888-C03-02) and Junta de Andalucı́a (RNM-295).
Alejandro Cabello-Pasini was supported by a grant of the Ministry of Education and Science of Spain (SAB2002-0209). This
project was partially supported by a grant of the Consejo Nacional de Ciencia y Tecnologı́a from México (C01-40144). We
thank Cristobal Lobato for his help in the laboratory.
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