Download Relations between electron transport rates determined by pulse

Photosynthesis Research 75: 259–275, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
259
Regular paper
Relations between electron transport rates determined by pulse amplitude
modulated chlorophyll fluorescence and oxygen evolution in macroalgae
under different light conditions
Félix L. Figueroa1,∗ , Rafael Conde-Álvarez1 & Iván Gómez2
1 Departamento
de Ecologı́a, Facultad de Ciencias, Universidad de Málaga, Campus Universitario de Teatinos
s/n, E-29071 Málaga, Spain; 2 Instituto de Biologı́a Marina, Facultad de Ciencias, Universidad Austral de Chile,
Casilla 567, Valdivia, Chile; ∗ Author for correspondence (e-mail: [email protected]; fax: +34-952-132000)
Received 20 September 2002; accepted in revised form 22 January 2003
Key words: chlorophyll fluorescence, light quality, oxygen evolution, photosynthesis, Porphyra, Ulva
Abstract
The relationship between O2 -based gross photosynthesis (GP) and in vivo chlorophyll fluorescence of Photosystem
II-based electron transport rate (ETR) as well as the relationship between effective quantum yield of fluorescence
(PSII ) and quantum yield of oxygen evolution (O2 ) were examined in the green algae Ulva rotundata and Ulva
olivascens and the red alga Porphyra leucosticta collected from the field and incubated for 3 days at 100 µmol
m−2 s−1 in nutrient enriched seawater. Maximal GP was twice as high in Ulva species than that measured in P.
leucosticta. In all species ETR was saturated at much higher irradiance than GP. The initial slope of ETR versus
absorbed irradiance was higher than that of GP versus absorbed irradiance. Only under absorbed irradiances below
saturation or at values of GP <2 µmol O2 m−2 s−1 a linear relationship was observed. In the linear phase, calculated O2 evolved /ETR molar ratios were closed to the theoretical value of 0.25 in Ulva species. In P. leucosticta,
the estimated GP was associated to the estimated ETR only at high irradiances. ETR was determined under white
light, red light emitting by diodes and solar radiation. In Ulva species the maximal ETR was reached under red light
and solar radiation whereas in P. leucosticta the maximal ETR was reached under white light and minimal under
red light. These results are in agreement with the known action spectra for photosynthesis in these species. In the
case of P. leucosticta, GP and ETR were additionally determined under saturating irradiance in algae pre-incubated
for one week under white light at different irradiances and at white light (100 µmol m−2 s−1 ) enriched with far-red
light. GP and growth rate increased at a growth irradiance of 500 µmol m−2 s−1 becoming photoinhibited at higher
irradiances, while ETR increased when algae were exposed to the highest growth irradiance applied (2000 µmol
m−2 s−1 ). The calculated O2 evolved /ETR molar ratios were close to the theoretical value of 0.25 when algae
were pre-incubated under 500–1000 µmol m−2 s−1 . The enrichment by FR light provoked a decrease in both
GP and ETR and an increase of nonphotochemical quenching although the irradiance of PAR was maintained at
a constant level. In addition to C assimilation, other electron sinks, such as nitrogen assimilation, affected the
GP–ETR relationship. The slopes of GP versus ETR or PSII versus O2 were lower in the algae with the highest
N assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents, i.e., Ulva rotundata
and Porphyra leucosticta, than that observed in U. olivascens. The possible mechanisms to explain this discrepancy
between GP and ETR are discussed.
Abbreviations: Aλ – spectral absorptance; Ci – internal carbon; Chl a – chlorophyll a; DMF – N,N, dimethylformamide; DW – dry weight; E – incident irradiance; ETR – electron transport rate; Fm – maximal fluorescence; Fo –
intrinsic fluorescence; FR – far-red light; Ft – current steadystate fluorescence; Fv – variable fluorescence of plant
pre-incubated
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in darkness; Fv /Fm – maximal quantum yield; FW – fresh weight; O2 – quantum yield of oxygen evolution;
PSII – effective quantum yield of fluorescence; GP – gross photosynthesis; GPestimated – estimated gross photosynthesis; LED – red-light emitting diode; Ni – internal nitrogen; NP – net photosynthesis; NRA – nitrate reductase
activity; PAM – pulse amplitude modulated; PAR – photosynthetically active radiation (400–700 nm); PMSF –
phenilmethyl-sulfonylfluorid; PS I – Photosystem I; PS II – Photosystem II; qP – photochemical quenching; qN –
nonphotochemical quenching; R – red light; RGR – relative growth rate; Rλ – spectral reflectance; SP – soluble
proteins; Tλ – spectral transmittance; WL – white light
Introduction
Photosynthetic rates and consequently primary production of marine macrophytes display a wide range
of values (Nielsen and Sand-Jensen 1990; Enríquez
et al. 1995) which depend on different variables such
as light, temperature, nutrients, water motion etc.,
and on the capacity for acclimation to environmental
fluctuations and stress (Franklin and Forster 1997;
Häder and Figueroa 1997). Accurate determinations
of algal photosynthesis present difficulties on both
short and long-term scales due to diverse photoacclimation mechanisms and complex regulation systems
(MacIntyre et al. 2002). The development of nonintrusive methodologies has led to rapid and sensitive measurements of changes in the physiological
status of marine macrophytes subjected to light stress
(Schreiber et al. 1986). Pulse amplitude modulation
(PAM) chlorophyll fluorescence of Photosystem II
(PS II) was primarily developed to assess photosynthetic primary reactions and quenching mechanisms
in plant physiology studies of higher plants (Schreiber
et al. 1995). The application of PAM fluorometry in
macroalgae is relatively recent and has become a useful tool for evaluating photosynthesis under different
natural and artificial light conditions (Henley et al.
1991; Hanelt 1992; Franklin et al. 1996). Taking into
account the differences in the organization of the photosynthetic apparatus between macroalgae and higher
plants, an optimisation of the PAM instrumentation
has been needed to meet accurately the low chlorophyll fluorescence emission of macroalgae (Büchel
and Wilhem 1993; Hanelt 1996). For example, the
presence of phycobilisomes in the light-harvesting
system of red algae results in generally lower maximal
quantum yields (Fv /Fm ) than that measured in green
and brown algae (Büchel and Wilhem 1993).
The usefulness of chlorophyll fluorescence as an
indicator of photosynthesis requires to demonstrate its
relationship with the quantum yield of gas evolution
(O2 or CO2 ). Thus, simultaneous measurements of
both oxygen evolution and chlorophyll fluorescence
are required. Quantum yields of photosynthesis are
usually defined as the quantum yield for oxygen production or C fixation (Genty et al. 1989). Assuming
that excess energy is dissipated as heat through inactivated PS II centers, a linear correlation between ETR
calculated from fluorescence and CO2 exchange can
be demonstrated, as is the case of C4 and C3 plants
at various irradiances under non-photorespiratory conditions (Weis and Berry 1987; Krall and Edwards
1990). This relationship can be curvilinear at low irradiance at normal levels of O2 and CO2 when electrons
flow to O2 via the photosynthetic carbon oxidation
(PCO) cycle and/or Mehler-ascorbate-peroxidase reaction. This reaction in the water cycle competes with
carbon fixation and confounds measurements of O2
evolution (Genty et al. 1989, 1992; Asada 1999).
Extrapolations of PSII to the absolute photosynthetic electron transport rate (ETR) depend on the
specific correlation among quantum yields of oxygen
evolution (O2 ), carbon fixation (CO2 ) and any other
competing sinks for electrons. However, the number
of algal species in which both variables have been
simultaneously analyzed are so far scarce: in some
microalgae, a non-linear relationship between PSII
and O2 and CO2 at both low and high irradiance was
demonstrated (Flameling and Kromkamp 1998; Hartig
et al. 1998).
In our study, simultaneous measurements of oxygen evolution and effective quantum yield were conducted in the red alga Porphyra leucosticta and two
green algae Ulva rotundata and Ulva olivascens under
different irradiances (similar to Franklin and Badger
2001) and also under different light qualities (solar
radiation, white light fluorescent lamps, red lightemitting diodes, white light enriched with far-red
light). Although the studied algae attained similar
morphology, i.e., sheet-like structure, they showed
very different absorption properties due to the different
cell size and cell layers, i.e., one (Porphyra) or two
(Ulva), but mainly due to the pigment composition,
which determines very different spectra quantum yield
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of photosynthesis (Lüning and Dring 1985; Markager
1993; Agustí et al. 1994). In addition, the functionality
of pigments involved in photosynthesis, indicated by
in vivo fluorescence excitation spectra, are very different in Ulva species compared to Porphyra (Grymski
et al. 1997; Figueroa et al. 2003). The wavelength
dependence of the maximum quantum yield of carbon
fixation has been studied in different algae (Kroon et
al. 1993; Schofield et al. 1996). Exposures to enriched
far-red light conditions over several days resulted in
changes in the Photosystem II/Photosystem I ratios
and consequently, maximum quantum yields, dissipation mechanisms and GP–ETR relationships can be
affected (Eskins and Duysen 1984; Chow et al. 1990).
Finally, the GP–ETR relationships were related not
only to carbon assimilation but also to other electron
sinks, e.g., nitrogen assimilation. The capacity for the
nitrate incorporation was estimated as the activity of a
key enzyme nitrate reductase (NR), and as the assimilation of inorganic nitrogen (nitrate and ammonium
added to the medium), i.e., the total internal nitrogen
content.
Materials and methods
Sampling sites and algal material
Porphyra leucosticta Thuret in Le Jolis and Ulva
olivascens P.A. Dangerad were collected at 0–0.2 m
depths from two sites (eulittoral zone) on the coast
of Málaga, southern Spain; Lagos and La Araña
(36◦45 N 4◦ 18 W), respectively. Ulva rotundata
Bliding was collected from the estuarine area of
Palmones River (Cádiz, southern Spain, 36◦ 13 N
5◦ 27 W). The locations are characterized by different environmental conditions: the coast of Málaga
presents salinities of 35–37 SPU and almost no tides.
Palmones River on the other hand, is an estuarine
area with higher contents of particles and nutrients and
consequently, subjected to changing conditions of water transparency and light penetration (Vergara et al.
1997). Thus, U. rotundata is submitted to high solar
radiation when emerged during low tide but submitted
to very low irradiance at 1.5 m depth during high tide
due to water turbidity (Vergara et al. 1997).
Culture conditions
After collection, algae were transported in darkness
in an icebox to the laboratory. Then algae were trans-
ferred into glass cylinders containing Provasoli enriched seawater (PES) (Provasoli 1968) with aeration
at 15 ◦ C and a light/dark regime of 12/12 h. The
illumination of cultures was at 100 µmol m−2 s−1
provided by two fluorescent lamps (True Lite Plus II,
Duro Test, Fairfield, New Jersey) (Figure 1). The algae were incubated during three days in the laboratory
in these conditions for acclimation before the start of
experiments.
For outdoor experiments, the algae were transferred to three trays containing 0.5 l PES medium (35
SPU) supplied with constant aeration. The whole system was placed within a water bath and maintained at
a temperature of 16–20 ◦ C by pumping cooled water.
Light treatments
Effective quantum yield and oxygen evolution were
determined under different irradiances and light qualities provided by fluorescent white light lamps as
Compact True Lite13W (Duro-Test Corp., Fairfield,
New Jersey) denominated WL-1 and True-Lite Plus
II 40W (Duro-Test Corp., Fairfield, New Jersey) denominated WL-1 and red-light provided by red light
emitting diodes (red LED) of the pulse amplitude
modulated fluorometer (PAM-2000, Walz GmbH, Effeltrich, Germany) (Figure 1a). Samples were submitted to increasing irradiances between 0 and 400 µmol
m−2 s−1 at intervals of 30 s each in the case of red
light emitting diodes and between 0 and 1300 µmol
m−2 s−1 in the case of fluorescent white light lamps.
In the outdoor experiments (described above), they
were maintained under natural solar radiation (Figure 1b) and then covered by different neutral filters
(Lee filters, Hampshire, UK) in order to decrease the
solar irradiance, which reached maximal values close
to 2000 µmol photons m−2 s−1 .
In the case of P. leucosticta, effective quantum
yield and oxygen evolution were determined at the
irradiance of 1000 µmol m−2 s−1 (above light saturation for photosynthesis) after 1 week of incubation
in the laboratory in PES medium at different irradiances of PAR provided by a 300 W metal halide lamp
(Optimarc, Duro Test, Fairfield, New Jersey): 50, 100,
500, 1000 and 2000 µmol m−2 s−1 (Figure 1b). In
addition, P. leucostica was incubated for 1 week under
a irradiance of 100 µmol m−2 s−1 provided by a white
light fluorescent lamp (Osram DL 18W) and enriched
with far-red (FR) light (λ > 700 nm), mainly absorbed by Photosystem I (PS I), provided by Linestra
lamps (Osram C428 35W) (Figure 1c). Different red
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(R, λ = 630-680 nm) to far-red (FR, λ = 700-720 m,)
(R:FR: 0.30, 0.56, 1.01, 1.98 and 2.93) ratios were
obtained by increasing the irradiance of far-red light
maintaining red light constant (Figure 1c). Effective
quantum yield and oxygen evolution were determined
at the irradiance of 1000 µmol m−2 s−1 (above light
saturation for photosynthesis).
O2 evolution and chlorophyll fluorescence
determination
Measurements were carried out using computer aided
OXY M-5 equipment (Real Time Computer, Erlangen,
Germany). Thallus pieces (0.15–0.2 g FW) were taken
and put into a measuring chamber (10 ml), fitted with a
Clark-type electrode and a magnetic stirrer. The chamber contained filtered seawater buffered to pH 8.2 with
20 mM Tris and maintained at a temperature close to
15 ◦ C. The content of inorganic C was 2.5 mM. To
simultaneously measure chlorophyll fluorescence, the
optic fiber of the PAM device was integrated in the
measuring chamber. Two fluorescent white light lamps
of small size (Compact white lamp, Duro-Test Corp.,
Fairfield, New Jersey) were used as light sources (Figure 1a). The irradiances reaching the thallus surface
were monitored using a spherical PAR quantum sensor
(Zemoko, Koudekerke, The Netherlands) specially designed for small chambers. Thalli were exposed for
5 to 10 min to an initial dark respiration period and
then to a gradient of increasing irradiances from 3.5 to
1300 µmol m−2 s−1 for 5 to 10 min each.
The photosynthetic parameters were estimated by
fitting a non-linear function (Jassby and Platt 1976) to
each data series:
NP = NPmax ∗ (tanh(α ∗ E/NPmax )
Figure 1. Spectral Irradiance (emission spectra) of the different
light sources used in the experiments (a) white light provided by
fluorescent lamps: (1) Compact True Lite 13 W (WL-1, open triangles) and True Lite PlusII 40 W (WL-2, open squares) and
red-light emitting diodes provided by the PAM instrument (red
LEDs, closed circles), (b) solar radiation (closed circles) and 300
W metal halide lamp (Optimarc) (open circles) and (c) day light
fluorescent lamps (Osram DL 18W) at 100 µmol m−2 s−1 enriched
with different irradiances of far-red light provided by Linestra lamps
(Osram C428 35W).
(1)
where NP is the net photosynthetic rate, NPmax is the
saturated net photosynthesis, tanh is the hyperbolic
tangent function, α is the photosynthetic efficiency at
low irradiance and E is the incident irradiance. Gross
photosynthesis (GP) was calculated as the sum of net
photosynthesis and respiration.
In vivo chlorophyll fluorescence of PSII was determined with a portable pulse modulation fluorometer
(PAM 2000, Waltz GmbH, Effeltrich, Germany). After
5–8 min in darkness to measure Fo a saturating flash
(400 ms) was applied to obtain the maximal fluorescence level (Fm ). Thus, the maximal quantum yield
of fluorescence (Fv /Fm ) was obtained (Schreiber et al.
1986). The variable fluorescence Fv is the difference
between the maximal fluorescence from fully reduced
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PS II reaction center (Fm ) and the intrinsic fluorescence (Fo ) from the antenna of fully oxidized PS II.
The effective quantum yield (PSII ) was calculated
according to Schreiber and Neubaer (1990):
PSII = Fm − Ft /Fm
(2)
F m being the maximal fluorescence which is induced with a saturating white light pulse (400 ms,
approx. 9000 µmol m−2 s−1 . Ft is the current steadystate fluorescence in light adapted algae. The electron
transport rate ETR was determined according to the
following formula:
ETR (µmol electrons m−2 s−1 ) = AQλ∗FII ∗ PSII
(3)
Where AQλ is the absorbed quanta calculated as the
product the integration of the spectral Absorptance
(Aλ ) between 400-700 nm and spectral irradiance of
the light source (Eλ ), FII is the fraction of AQ directed to PS II including its light harvesting complexes
(LHCs) and PSII is the effective quantum yield or
quantum yield of PS II charge separation as it was
defined above. According to Grzymski et al. (1997)
and Johnsen (personal communication), FII for different pigment groups can be estimated by determining
the fraction of Chl a associated with PS II and its
corresponding light-harvesting complexes, i.e., LHC
II. FII for Rhodophyta is about 0.15 and 0.5 for
Chlorophyta (Grzymski et al. 1997; Figueroa et al.
2003).
The absorptance (Aλ ) was determined for each 1
nm by means of an integrating sphere (Licor-1802)
connected to a Licor-1800 UW spectroradiometer according to the formula:
Aλ = 1 − Tλ − Rλ
(4)
with Tλ being the transmittance and Rλ reflectance.
For measurements of GP and ETR at different
irradiances, eight replicates were taken at the end
of the incubation to the highest irradiance applied.
No differences were observed between measurements
conducted at the initial time (at the end of the lowest irradiance applied). In the samples exposed for
one week under different irradiances and light quality
(R:FR ratios) in Porphyra leucosticta, Aλ was determined from eight replicates before and after one week
of exposure.
Gross photosynthesis was estimated (GPe) from
the ETR (3) according to the formula:
GPe(µmol O2 m−2 s−1 ) = AQλ ∗ FII ∗ PSII ∗τ
(5)
with τ being the ratio of oxygen evolved per electron
generated at PS II, i.e., four stable charge separations
are needed at PS II to evolve 1 O2 -molecule, thus τ is
equal to 0.25.
The parameters from the ETR curves were calculated following the model of O2 -based photosynthesis
versus irradiance curves. Here a modification of the
non-linear function from Jassby and Platt (1976) was
made:
ETR = ETRmax ∗ tanh(αETR ∗ E/ETRmax )
(6)
Where ETR is the relative electron transport rate mentioned above, ETRmax is the saturated ETR, tanh is the
hyperbolic tangent function, α ETR is the efficiency of
the electron transport (initial slope of the ETR versus
Irradiance curves) and E is the incident irradiance.
Non-photochemical quenching (qN) was calculated as
qN = 1 − (Fm − Fo )/Fm − Fo
(7)
being Fm the maximal fluorescence of an dark‘adapted’ sample, Fm is the maximal fluorescence
of an light-exposed alga under a given irradiance
and Fo the intrinsic fluorescence of a light-adapted
sample determined after a pulse of far-red light of five
seconds (Schreiber et al. 1986). Five different algal
samples were used for each measurement of oxygen
evolution and chlorophyll fluorescence. Three GP and
ETR versus irradiance curves were determined in each
treatment.
Determinations of pigments, proteins, nitrate
reductase activity and C-N contents
Algal material for chlorophyll determinations was
sampled simultaneously with those used for photosynthesis measurements and was stored in liquid nitrogen
until analysis. Chl a, b and total chlorophylls were
extracted in N,N, dimethylformamide (DMF) following the methodology described by Inskeep and Bloom
(1985). Samples (1 mg FW) from apical thallus regions were thawed at room temperature and incubated
in 2.5 ml DMF for 24 h at 4 ◦ C in darkness. The absorbance was finally measured at 750, 664.5 and 647
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nm in a Beckman DU-7 spectrophotometer (Beckman
Instruments Inc., San Piego, California).
Proteins were extracted in a phosphate buffer 0.1.
M, pH 7.5 at 4 ◦ C containing 10 mM (Na2 -EDTA)
and 4 mM phenilmethyl-sulfonylfluorid (PMSF). The
extracts were centrifuged at 19 000 g for 30 min.
The supernatant was used for soluble protein (SP)
determination according to Bradford (1976).
Six samples (0.25 g FW) were assayed for in
situ nitrate reductase activity after Corzo and Niell
(1991). The concentration of NO2 − was determined
spectrophotometrically according to Snell and Snell
(1949).
Total intracellular carbon and nitrogen content was
determined using a Perkin-Elmer elemental analyser
model 2400 CHN.
Table 1. Photosynthetic parameters measured as oxygen evolution
(GPmeasured ) and estimated (GPestimated ): photosynthetic efficiency
(α) and saturated gross photosynthetic rate (GPmax , µmol O2
m−2 s−1 ). Parameters defined from electron transport rate (ETR)
curves: efficiency of electron transport rate (α) and maximal ETR
(ETRmax , µmol electrons m−2 s−1 ) in the green algae Ulva rotundata
and Ulva olivascens and the red alga Porphyra leucosticta under artificial white light (WL-1, Fluorescent Compact True Lite, Duro Test,
USA). Efficiency of ETR (α) and maximal ETR (ETRmax ) under different light qualities: artificial white light (WL-2, True Lite Plus II,
Duro-Test, Fairfield, New Jersey), solar radiation and red light emitting diodes (red LED) provided by the fluorometer PAM-200 (Walz
GmbH). The light spectra of the different light sources are presented in
Figure 1
Species
U. olivascens
P. leucosticta
GPmeasured (WL-1)
α
0.031 ± 0.001a
Pmax
4.7 ± 0.35a
0.063 ± 0.003a
6.3 ± 0.3b
0.053 ± 0.002b
2.9 ± 0.2c
GPestimated (WL-1)
α
0.069 ± 0.003a
Pmax
46.4 ± 4.1a
0.071 ± 0.006a
23.1 ± 2.6b
0.021 ± 0.001b
3.0 ± 0.23c
ETR (WL-1)
α
0.276 ± 0.02a
ETRmax
186.6 ± 14.3a
0.284 ± 0.02a
92.4 ± 10.5b
0.088 ± 0.006b
10.8 ± 1.9c
(8)
ETR (WL-2)
α
0.293 ± 0.02a
ETRmax
131.0 ± 11.7a
0.283 ± 0.02a
89.0 ± 9.2b
0.145 ± 0.009b
27.4 ± 3.4c
where At is the algal area measured after six days of
incubation, Ai is the algal area at the initial time and t
is the time expressed as days.
ETR (solar radiation)
α
0.353 ± 0.03a
ETRmax
157.4 ± 18.5a
0.214 ± 0.03b
106.2 ± 13.3b
0.127 ± 0.01b
28.1 ± 3.6c
Statistics
ETR (Red LED)
α
0.375 ± 0.03a
ETRmax
78.53 ± 9.3a
0.440 ± 0.05a
13.1 ± 1.5b
0.069 ± 0.007c
5.09 ± 0.6c
Growth rate determination
The thallus area of each disc was determined from disc
diameter, since growth of the circular discs proceeded
isodiametrically. The relative growth rate, expressed
as the percentage increase per day, was computed from
the following expression (Kain 1987).
RGR (% day
−1
) = (ln At − ln Ai )/t
Data treatment included one-way ANOVA and further
mean comparisons by means of LSD Fisher test (Sokal
and Rohlf 1995).
Results
Light response curves of oxygen and chlorophyll
fluorescence
No clear patterns were seen in O2 -based P-E curves.
Measured GPmax was higher in Ulva species than
that in the red alga P. leucosticta. In general, both
measured GP and α were significantly higher (P <
0.05) in U. olivascens than in U. rotundata and P.
leucosticta (Table 1, Figure 2c). However, estimated
gross photosynthesis (GPestimated) according to the
formula (5) showed a clearer pattern: GPmax and α
exhibited the highest values in Ulva species compared
Variable
U. rotundata
Different letters represent significant differences among the species for
each variable at P < 0.05.
to P. lecucosticta. On the other hand, in Ulva species, GPestimated values were higher than GPmeasured .
In P. leucosticta the reverse situation was found for α
values, while, GPmax did not change.
In contrast to GP, electron transport rate (ETR)
presented higher values in U. rotundata than in U.
olivascens, but the ETR-based α was similar in both
species (Figure 3, Table 1). ETRmax was higher in the
Ulva species than in P. leucosticta (Figure 3). Comparatively, ETR was saturated at much higher irradiance
than that required to saturate gross photosynthetic rate.
The ETR-based α was higher than O2 -based α in all
algae, in U. rotundata about nine times, whereas in
U. olivascens and P. leucosticta 4.5 times 1.6 times
higher, respectively (Figure 3, Table 1).
265
Figure 2. Gross photosynthetic rate in µmol O2 m−2 s−1 as a function of the absorbed irradiance in µmol photons m−2 s−1 . Measurements were carried out in 20 mM Tris buffer at pH 8.2 at natural
inorganic carbon concentration (2.5 mM) in (a) Ulva rotundata, (b)
Ulva olivascens and (c) Porphyra leucosticta. The measurements
were conducted under different irradiances of white light provided
by two white light fluorescent lamps (WL-1, Compact True Lite 13
W).
Figure 3. Electron transport rate (ETR) expressed as µmol electrons m−2 s−1 calculated as ETR = AQλ * FII * PSII as function
of absorbed irradiance (µmol photons m−2 s−1 ). Measurements
were performed in 20 mM Tris buffer at pH 8.2 at a natural inorganic carbon (2.5 mM) in (a) Ulva rotundata, (b) Ulva olivascens
and (c) Porphyra leucosticta. The measurements were conducted
under different irradiances of white light provided by two white light
fluorescent (WL-1, Compact True Lite).
The relation between ETR and GP was not linear
for the whole data set (Figure 4). At GP >2 µmol
O2 m−2 s−1 the slope of the function ETR versus
GP drastically increased. The function ETR-GP for
266
the whole data sets can be adjusted to an exponential
function (r 2 = 0.92 P < 0.01). The slopes were
lower in Ulva species than in P. leucosticta, i.e., 0.965
in the case of U. rotundata, 0.645 for U. olivascens
and 1.099 for P. leucosticta. At low irradiances with
values of GP lower than 2 µmol O2 m−2 s−1 , the relation between ETR and GP can be adjusted to a linear
function (r 2 = 0.98 P < 0.01) and the number of
mols of electrons per mol of oxygen was equal to the
theoretical value of four in U. olivascens or close to
that in the case of U. rotundata with a value of 3.4.
However, in the case of P. leucosticta the number of
mols of electrons per mol of oxygen was only two,
half of the theoretical value. In contrast to the exposure to low irradiances, at high irradiances, the ratio
maximal ETR per maximal GP (Table1) was far from
the theoretical value in the Ulva species, i.e., 39.70
in U. rotundata and 14.66 in U. olivascens. However,
at very high irradiance in the case of P. leucosticta, a
ETR / GP ratio value of 3.72 was found, which is very
close to the theoretical value (Table 1).
Values of PSII versus O2 were not linearly correlated for the whole data set (Figure 5). For the three
algae studied, at O2 values < 0.04, a linear relationship in the function PSII versus O2 with slopes of
7.86 (U. rotundata), 6.40 (U. olivascens) and 8.44 (P.
leucosticta) could be determined. In the upper part of
the curve, increases of O2 reflected only slight increases of PSII , with maximal values of about 0.7 for
the green algae and 0.6 for Porphyra (Figure 5).
Pigments, protein and C:N contents
Contents of Chl a and Chl b were about 3 and 1.5
times higher in U. rotundata relative to U. olivascens,
consequently the absorptance in the PAR region of the
spectra was about 45% higher in U. rotundata than in
U. olivascens (Table 2). Thus, the ratio Chl a/Chl b
in U. olivascens was twice as much as found in U.
rotundata. The contents of soluble proteins, total internal N and nitrate reductase activity were increased
in U. rotundata relative to U. olivascens. The Chl concentration, absorptance, soluble protein and internal
N were higher in U. rotundata and P. leucosticta than
in U. olivascens (Table 2). Similarly, nitrate reductase
activity (NRA) was higher in U. rotundata and P. leucosticta compared to U. olivascens. NRA was linearly
related (r 2 = 0.91) to the internal nitrogen content
(data not shown). Internal carbon content was similar in U. olivascens and P. leucosticta and higher
than in U. rotundata. In P. leucosticta, Chl a con-
Figure 4. Electron transport rate (ETR) in µmol electrons m−2
s−1 ) and it calculated as ETR = AQλ * FII * PSII versus gross
photosynthetic rate in µmol O2 m−2 s−1 . Measurements were
made in 20 mM Tris buffer at pH 8.2 at a natural inorganic carbon (2.5 mM) in (a) Ulva rotundata, Ulva olivascens (b) and (c)
Porphyra leucosticta. The measurements were conducted under
different irradiances of white light provided by two white light
fluorescent lamps (WL-1, Compact True Lite).
267
Table 2. Chlorophyll a and chlorophyll b contents expressed a mg m−2 , thallus absorptance
(A), soluble protein (SP) concentration in mg gDW−1 , nitrate reductase activity (NRA) in
µmol NO2 gDW−1 h−1 , internal C and N and C:N ratios in the green algae Ulva rotundata
and Ulva olivascens and in the red alga Porphyra leucosticta
Variable
Chl a (mg m−2 )
Chl b (mg m−2 )
Absorptance (A)
SP (mg gDW−1 )
NRA (µmol NO2 gDW−1 h−1 )
C (mg gDW−1 )
N (mg gDW−1 )
C:N ratio
U. rotundata
Species
U. olivascens
P. leucosticta
79.9 ± 6.4a
24.6 ± 2.8a
0.573 ± 0.04a
59.1 ± 4.3a
7.2 ± 0.6a
344.3 ± 27.5a
43.0 ± 3.44a
8.0 ± 0.8a
24.6 ± 2.8b
16.5 ± 1.8b
0.313 ± 0.02b
33.2 ± 2.8b
5.4 ± 0.4b
363.1 ± 21.77a
26.9 ± 1.61b
13.5 ± 1.1b
63.2 ± 4.5c
–
0.46 ± 0.03c
45.6 ± 4.4c
6.8 ± 0.3a
365.2 ± 18.25a
35.3 ± 2.75c
10.4 ± 1.6b
Different letters represent significant differences among the species for each variable among
the species at P < 0.05.
tent, soluble protein concentration, Aλ and NRA were
higher than in U. olivascens but lower than in U. rotundata (Table 2). The C:N ratio in P. leucosticta was
higher than that in U. rotundata but lower than in U.
olivascens (Table 2).
Species dependent effects of light quality on ETR
In all species, maximal ETR was higher under solar
light and fluorescent white light than that under red
LEDs (Figure 6, Table 1). Both maximal ETR and α
were higher in Ulva species than that in P. leucosticta
in all light qualities (Table 1). In U. rotundata maximal ETR was higher in WL-1 and solar radiation
than under WL-2 (Table 1). In U. olivascens, no significant differences were found in the maximal ETR
under solar radiation compared to both types of artificial white lamps. In P. leucosticta maximal ETR
was higher under solar radiation and WL-2 than that
under WL-1 and red LEDs (Table 1). In U. rotundata,
ETRbased α was similar under solar radiation and red
LEDs and higher than under fluorescent lamps (WL1 and WL-2). However in U. olivascens, the highest
ETR-based α values were reached under red LEDs and
the minimal under solar radiation. Under white light
fluorescent lamps, ETR-based α was higher than that
under solar radiation. In P. leucosticta, the minimal
ETR-based α values were reached under red LEDs
and it was higher under solar radiation and WL-2 than
under WL-1 (Table 1).
Gross photosynthesis and ETR of P. leucosticta grown
for one week at different irradiances and red:far-red
ratios
Maximal GP increased with increasing the growth irradiance up to 500 µmol m−2 s−1 and it decreased
when it was incubated at 1000–2000 µmol m−2 s−1 .
However, ETR increased with increased growth irradiances (Table 3). GP estimated according to Equation
(5) overestimated GP (Table 3) except when it was incubated at 500 µmol m−2 s−1 . The ratio ETR/GP was
close to the theoretical value only at growth irradiance
of 500 µmol m−2 s−1 (3.47) and at 1000 µmol m−2
s−1 (4.79). At growth irradiances of 50 and 100 µmol
m−2 s−1 , the ETR/GP ratio was lower (0.8 and 1.28,
respectively) and at growth irradiances of 2000 µmol
m−2 s−1 it was higher (11.5) than the theoretical
value. Non-photochemical quenching (qN) increased
and Chl a concentration decreased with growth irradiance. The growth rate (% day−1) increased from
growth irradiances of 50 to 500 µmol m−2 s−1 and
it decreased from 1000 to 2000 µmol m−2 s−1 .
GP and ETR in P. leucosticta were affected by
the proportion of red:far-red light under the same irradiance (100 µmol m−2 s−1 ) in the PAR region
of the spectra (Table 4). ETR and GP decreased
with the far-red light enrichment. GP and ETR at
R:FR light ratios of 0.30 and 0.56 were significantly
(P < 0.05) lower than that observed at 1.01, 1.98
and 2.92. GP:ETR ratios were lower than the theoretical value of four. The closest value was reached
when the algae were incubated under the highest
R:FR light ratio (2.85). Non-photochemical quench-
268
Table 3. Gross photosynthesis (GP, µmol O2 m−2 s−1 ) measured and estimated according to the formula (5), electron transport
rate (ETR µmol m−2 s−1 ) and non-photochemical quenching (qN), chlorophyll a concentration (Chl a, mg gDW−1 ) and
growth rates (% day−1 ) in the red alga Porphyra leucostica grown for one week under different growth irradiances (50, 100,
500, 1000 and 2000 µmol O2 m−2 s−1 ) provided by a 300 W metal halide lamp (Optimarc, Duro-Test, Fairfield, New Jersey).
Algae were cultivated for one week at a 12h:12h L:D photoperiod and 16±2 ◦ C. GP and ETR were determined under a fixed
irradiance of 1000 µmol m−2 s−1 white light (WL-1, Compact True Lite 13 W). qN, chlorophyll a concentration and growth
rate are refereed to growth irradiances
Growth
Irradiances
(µmol m−2 s−1 )
Gmeasured
GPestimated
ETR
qN
Chl a
Growth rate
50
100
500
1000
2000
2.8 ± 0.17a
3.2 ± 0.21b
3.8 ± 0.16c
2.9 ± 0.21a,b,d
1.8 ± 0.12e
0.6 ± 0.03a
1.0 ± 0.07b
3.3 ± 0.18c
3.4 ± 0.23c
5.2 ± 0.31d
2.3 ± 0.15a
4.1 ± 0.21b
13.2 ± 1.1c
13.9 ± 1.3c
20.7 ± 2.1d
0.06 ± 0.004a
0.11 ± 0.009b
0.22 ± 0.01c
0.36 ± 0.03d
0.58 ± 0.04e
3.2 ± 0.21a
2.9 ± 0.14a
2.3 ± 0.14b
1.3 ± 0.12c
0.6 ± 0.03d
12.9 ± 1.2a
19.8 ± 1.6b
25.4 ± 2.3c
21.3 ± 2.2b,c,d
18.2 ± 1.6 b,d,e
Different letters represent significant differences among the species for each variable at P < 0.05.
ing (qN) increased with the far-red light enrichment
and it was about three times higher under 0.30 R:FR
light ratio than that at 1.98 or 2.92 (Table 4). Chl
a concentration increased with the far-red light enrichment. Growth rate (% day−1 ) increased with the
R:FR light ratios up to 1.85–2.85 values. Growth rate
(% day−1) was 50% lower at 0.30 and 0.56 R:FR ratios
compared to that at values of 1.98 or 2.92.
Discussion
Gross photosynthetic rates reported in this work for
Ulva rotundata and Porphyra leucosticta were similar
to those previously reported for U. rotundata (Osmond
et al. 1993; Pérez-Llorens et al. 1996; Vergara et al.
1997) and the intertidal P. perforata (Herbert and Waaland 1998) and P. columbina incubated under artificial
white light (Franklin and Badger 2001). The maximal
gross photosynthesis and the efficiency measured in P.
leucosticta was 50% lower than values found in the
two Ulva species.
Effect of irradiance on GP–ETR relationships
The pattern of ETR as a function of the absorbed irradiance was different compared to O2 evolution. Firstly,
ETR is saturated at much higher irradiance than gross
photosynthesis and secondly the initial slope is higher
compared to gross photosynthesis. Consequently, no
linear relation between ETR versus gross photosynthesis was observed for the whole irradiance interval
applied. Only for absorbed irradiance below saturation
of photosynthesis or for values of gross photosynthesis below 2 µmol m−2 s−1 a linear response was
observed. In this case, calculated molar ratios at O2
evolved/ETRs were closed to the theoretical values
of 0.25 in the Ulva species but not in the red alga P.
leucosticta. In the latter species, the theoretical value
is only reached at the highest absorbed irradiances
(300–450 µmol m−2 s−1 ). At low irradiances, the loss
of correlation between ETR and linear photosynthetic
electron flow in Porphyra limits the application of the
PAM technique, whereas the limitation in Ulva species
occurs at high irradiances. In Ulva species at values of
gross photosynthesis > 2–3 µmol O2 m−2 s−1 , the
relation between the mol of electrons (ETR) per mol
of O2 drastically increased because electron sinks are
very active, while O2 reaches the steady state.
The GP-ETR relations have been occasionally examined, e.g., 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), and the green algae Ulva
rotundata (Osmond et al. 1993), U. lactuca, U. fasciata (Beer et al. 2000) and U. australis (Franklin and
Badger 2001). At moderate irradiance, ETR calculated from PSII 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 U. lactuca
revealed a good correlation between ETR and O2 evolution at moderate light but at higher irradiances had a
higher ETR than the expected one. Recently, Franklin and Badger (2001) reported a good correlation
269
Table 4. Gross photosynthesis (GP, µmol O2 m−2 s−1 ), measured and estimated according to the formula (5),
maximal electron transport rate (ETR, µmol m−2 s−1 ), non photochemical quenching (qN), chlorophyll a content
(Chl a, mg gDW−1 ) and growth rates (% day−1 ) in the red alga Porphyra leucostica grown for one week under a
irradiance of 100 µmol m−2 s−1 provided by two white light fluorescent lamps (Day Light, Osram DL 18W) and
enriched with far-red light (λ > 700 nm) provided by Linestra lamps (Osram C428 35W). Different red:far-red
ratios (0.30, 0.56, 1.01, 1.98, 2.92) were obtained by increasing the irradiance of far-red (λ = 700–720 nm) light
and maintaining red light (λ = 660–680 nm). GP and ETR were determined under a fixed irradiance of 1000 µmol
m−2 s−1 white light (WL-1, Compact True Lite 13 W)
R:FR
ratio
GPmeasured
GPestimated
0.30
0.56
1.01
1.98
2.93
1.9 ± 0.3a
2.2 ± 0.2a
3.0 ± 0.2b
3.1 ± 0.2b
3.2 ± 0.2b
1.2 ± 0.09a
1.3 ± 0.10a
1.7 ± 0.12b
1.9 ± 0.09a,c
2.7 ± 0.1a
ETR qN
4.99 ± 0.28a
5.34 ± 0.35a,b
6.71 ± 0.331a,c
7.86 ± 0.32b,c,d
10.82 ± 0.41a,b,c,d
Chl a
Growth rate
0.36 ± 0.03a
0.31 ± 0.02a
0.13 ± 0.01b
0.11 ± 0.008b
0.11 ± 0.009b
4.8 ± 0.4a
4.3 ± 0.3a
3.2 ± 0.2b
3.3 ± 0.2b
3.0 ± 0.3b
11.0 ± 1.2a
11.3 ± 1.8a
15.7 ± 1.3b
20.3 ± 2.2c
20.2 ± 1.6c
Different letters represent significant differences among the species for each variable at P < 0.05.
between GP and ETR at limiting irradiances in Ulva
australis and Porphyra columbina, whereas at saturating photon fluxes, especially when Ci availability was
low, ETR overestimated gross O2 evolution. These authors suggested that excess electron flow was not due
to an increase in gross O2 uptake, neither the Mehlerascorbate-peroxidase reaction nor the photosynthetic
carbon oxidation enhanced at high irradiance or low Ci
(Franklin and Badger 2001). in Ulva australis and Porphyra columbina, Franklin and Badger (2001) found
values close to theoretical ETRs for O2 evolution
(determined by mass spectrometry) at subsaturating
irradiances.
The different response of Ulva species compared to
P. leucosticta could be due to their different photosynthetic acclimation characteristics in response to their
natural environment, i.e., sun type in P. leucosticta and
shade type in Ulva species. P. leucosticta is growing
in the eulittoral system and it has been adapted to very
high solar radiation (Figueroa et al. 1997). The sink
of electrons in the sun type Porphyra is probably less
efficient than that in Ulva species.
The formulation of PSII by the Genty method, as
the product of the reaction centre ‘openness’ and excitation capture efficiency by open centres, assumes that
non-radiative dissipation occurs in the light-harvesting
antenna. Alternatively, non-photochemical quenching can occur in PS II reaction centres (Krause and
Weis 1991), altering the relationship between photochemical and fluorescence yields (Schreiber et al.
1995). The thermal dissipation can be different in
Ulva species compared to P. leucosticta. Dissipation
mechanisms are different among the different groups
of macroalgae: red algae, and specifically Porphyra
species, show more active state transitions than green
or brown algae (Satoh and Fork 1983; Büchel and
Wilhelm 1993). However, the contribution of state
transition to non photochemical quenching compared
to other quenchings such as energy dependent quenching, qE or thermal energy dissipation, qI, is difficult to
evaluate.
At sub-inhibitory fluence rates, thermal energy
dissipation is mainly controlled by the light-induced
formation of the thylakoid pH gradient (energy dependent quenching, qE), which accelerates the rate
of energy supplied to the Calvin cycle. At higher,
photoinhibitory fluence rates, photoinactivation due to
photoinhibition opens an additional path of thermal
energy dissipation (qI), thus optimizing the rate of
photochemical dissipation and diminishing the rate of
photodamage. For Porphyra perforata a new mechanism for adaptation to changes of light intensities and
quality was reported in which light energy reaching
the reaction centres of PS II decreased without any
significant change in PS I activity (Satoh and Fork
1983). The non- photochemical quenching in algae has
usually been related with the xanthophyll cycle (Uhrmacher et al. 1995; Franklin et al. 1996). However,
the xantophyll cycle activity is lacking in Rhodophyta
(Hager 1980) or attenuated in Ulva species (Franklin
et al. 1992), thus, alternative quenching mechanisms
cannot be ruled out.
A further explanation for enhanced ETRs after exposures to high light is the cyclic flow around PS
II from quinone acceptor QB (or pheophytin) via cyt
b559 and chl Z to P689 (Falkowsky et al. 1986).
270
Figure 5. PSII versus O2 . Measurements were made in 20 mM
Tris buffer at pH 8.2 at a natural inorganic carbon (2.5 mM) in (a)
Ulva rotundata, (b) Ulva olivascens and (c) the red alga Porphyra
leucosticta. The measurements were conducted under different irradiances of white light provided by two white light fluorescent lamps
(WL-1, Compact True Lite).
Using a ‘pump and probe’ fluorescence technique.
Falkowsky et al. (1986) and Prasil et al. (1996)
demonstrated an uncoupling between water splitting
activity in PS II and PS I under conditions where the
plastoquinone pool became strongly reduced (e.g., sat-
Figure 6. Electron transport rate (ETR) (µmol electrons m−2 s−1 )
calculated as ETR = AQλ * FII * PSII versus absorbed irradiance
in µmol photons m−2 s−1 of (a), Ulva rotundata, (b) Ulva olivascens and (c) Porphyra leucosticta under different light qualities:
solar radiation (dark circles), white light provided by fluorescent
lamps (WL-2, True Lite PlusII 40 W) (open circles and thick and
pointed line) and red light emitting diodes (red LEDS) provided by
the PAM-2000 fluorometer (dark triangles).
271
urating light), which was accompanied by an enhanced
ETR. In the cases where xantophyll cycle activity is
limited, i.e., Ulva species or non existent, i.e., in Porphyra species, cyclic flow in PS II could be essential
for protecting those species inhabiting the intertidal
system. The indication that cyclic flow around PS II
in macroalgae was supported by changes in the rate
of PSII to O2 relative to Ci (Franklin and Badger
2001). Beer et al. (2000) reported a close linear correlation between ETR and O2 evolution in U. lactuca
at irradiances up to 36% of growth saturating irradiances and at various Ci concentrations at 13% growth
saturating irradiance, which probably are lower than
irradiance levels required to saturate photosynthesis.
The loss of linearity between GP–ETR was also
observed in algae grown for one week at different irradiances. At high irradiances (1000–2000 µmol m−2
s−1 ), GP was saturated but not ETR. A closer relation between GP and growth rate was found compared
to the relationship between ETR and growth rate.
The decrease in O2 with increasing growth irradiance was much higher than that observed for PSII .
This decrease in O2 can be explained, in part, as a
consequence of an increase in cyclic phosphorilation
with respect to non-cyclic phosphorilation under high
light (Dubinsky et al. 1986; Gilmoire and Govindjee
1999). The acclimation to high irradiance is produced
by a decrease in the size of peripheral PS II antenna,
mostly attributed to a decrease in the LHC IIb and subsequent decrease in its component pigment, namely
Chl a + b, lutein and neoxanthin (Gilmore and Govindjee 1999). Under high growth irradiance both Chl a
(Table 3) and biliproteins (data not shown) decreased
in P. leucosticta.
Other explanations for the non-linearity between
PSII and O2 is the PS II heterogeneity (Melis 1991).
Schreiber et al. (1995) observed a linear relationship at
high irradiances but a deviation from non linearity at
high quantum efficiencies, i.e., low irradiances, as has
been observed in this study in P. leucosticta. These authors, using artificial electron acceptors, demonstrated
the presence of two different populations of PS II and
they ascribed the deviations of linearity between PSII
and O2 to this PS II heterogeneity. In P. leucosticta
the similar values of GP and estimated GP only at high
irradiances as in higher plants could also indicate the
existence of PS II heterogeneity in this red alga.
Effects of light quality on the GP–ETR relationship
The calculation of ETR depends on the correct determination of the absorptance, i.e., the fraction of
incident irradiance absorbed by PS II (Beer et al.
2000). In our study, A was determined by using an
integrating sphere taking into account both spectral
transmittance and spectral reflectance. Thus, absorbed
quanta were considered spectrally dependent and according to Grzymski et al. (1997) and Figueroa et al.
(2003), the fraction of Chl a associated with PS II and
its corresponding LHC was about 0.15 in red algae
and 0.5 in green algae. Other authors used the factor
of 0.5 for all algal groups accounting for the presence
of two photosystems, assuming equal involvement in
linear electron flow (Beer et al. 2000; Franklin and
Badger 2001). PS II absorption cross-section also
might change during the course of short experiments,
i.e., during determination of GP versus irradiance,
basically as a response to increased plastoquinone reduction (Fork et al. 1991), rendering our assumption
of a 1:1 distribution of irradiance between PS II and
PS I as erroneous. The optical thickness of the thallus
seems to be a limitation for the application of chlorophyll fluorescence measurements in macroalgae. For
example, absorbed quanta can be determined easily
in species attaining thin thalli, in contrast to thicker
morphs (Lüning and Dring 1985; Markager 1993; Enríquez et al. 1994). For example, the calculated ETR
was equivalent to the theoretical electron requirement
in a thin species such as Ulva or Porphyra, but not
in thicker species such as Zonaria crenata (Franklin and Badger 2001). Changes in the distribution of
excitation between PS II and PS I could affect the absorptance determination. The absorption cross-section
among macroalgae can vary due to differential intracellular self-shading (Grzymski et al. 1997). On the
other hand, the absorption cross-section at high irradiances can be affected by chloroplast movements
(Hanelt and Nultsch 1991). In long term experiments
(one week exposure to different irradiances), absorbed
quanta in P. leucosticta decreased as a consequence of
pigment content variations (photoacclimation). However, during the short time exposure (less than one
hour) necessary for ETR or GP determinations, no
differences in absorbed quanta at the lowest and the
highest irradiance applied were observed (data not
shown).
Light quality affects the ETR relationship in the
analysed macroalgae. In Ulva species, maximal ETRbased α were estimated under solar radiation and red
272
light incubation, whereas P. leucosticta was minimal at
red light. This pattern is according to the action spectra
of O2 -based photosynthesis reported by Lüning and
Dring (1985), i.e., in red light GP at 10 µmol O2 m−2
s−1 was about two times higher in Ulva lactuca than
in Porphyra umbilicalis. Maximal ETR was reached in
all algae under solar radiation and the white light fluorescent lamp True light Plus II (WL-2). Similar effects
of solar radiation and WL-2 (but not WL-1; Fluorescent compact True lite), are explained by the emission
of WL-2 which resembles more closely the solar radiation spectra than WL-1 (see Figure 1). WL-1 showed
narrow peaks in the blue, green and red region of the
spectra, being very different to the sun spectra. Under
red light, ETR decreased because a lesser number of
photons from other wavelengths are being absorbed
through accessory pigments i.e., blue light (in green
algae) and green light (in red algae). Energy imbalances between PS I and PS II as a consequence of differential absorption of different spectral wavelengths
have also been suggested to be responsible for a lack
of correlation between PSII and O2 (Kroon et al.
1993). Light sources with different spectral light proportions, i.e., R:FR light ratios, (red light is mainly
absorbed by PS II and FR light by PS I) can affect
the redox state. In this sense, R:FR light ratios from
solar radiation (1.06) were more closely related to ratios emitted by WL-2 (1.92) than WL-1 ones (0.401).
Kroon et al. (1993) suggested that the rate of cyclic
electron transport around PS I is spectrally regulated
and is less important for cells exposed to broad-band
white light, which encompasses all photosynthetically
active wavelengths. In cells exposed to narrow-band,
spectral illumination disproportionately drives PS II
photochemistry. In P. leucosticta, increases in far-red
light irradiances (changing R:FR light ratios under
constant PAR irradiance) resulted in decreases in both
GP and ETR. Far-red light is absorbed mainly by PS
I and consequently cyclic transport around PS I can
be activated. The increase of cyclic transport around
PS I relative to cyclic photosynthetic transport in both
PS II and PS I can affect ATP/NADPH ratios and
consequently the electron sinks, i.e., C or N assimilation rates (Chow et al. 1990). The excitation pressure
(expressed through the redox state of an intersystem
component of photosynthetic electron transfer chain)
has been suggested as a key signal in the regulation
of photosynthetic proteins (Durnford and Falkowski
1997). Thus, in the studied macroalgae, the change in
the excitation pressure provoked by the different light
qualities could explain the variations in the ETR–GP
relationship throughout the regulation of photosynthetic proteins (Durnford and Falkowski 1997) and
through the changes in the ATP/NADPH ratio, affecting enzyme activities related to electron sinks (Chow
et al. 1990).
The enrichment by FR light also increased the non
photochemical quenching (qN). Non photochemical
quenching can be induced by changes in pH around
Photosystem II (Bruce et al. 1997). Thus a possible
mechanism of increasing qN by FR light is that this
light quality favors the decrease of pH in thylakoid
membranes. In addition, FR light could affect chlorophyll synthesis, carbon assimilation or other electron
sinks such as N assimilation via non-photosynthetic
photoreceptors, i.e., phytochrome (López-Figueroa et
al. 1989; Rüdiger and López-Figueroa 1992) and consequently, the ETR can be affected. Chlorophyll and
biliproteins are regulated by light quality in P. leucosticta, through phytochrome and other red/green
light photoreceptors (López-Figueroa and Niell 1991).
In Porphyra sp., nitrogen assimilation and growth rate
is stimulated by red light whereas they are inhibited by blue and far-red light (Figueroa et al. 1995a,
b; Aguilera et al. 1997). Kroon et al. (1993) found
that enzymatic processes associated with organic C
synthesis appeared to vary depending on the spectral
growth irradiance, which contributes to the observed
variability in quantum yield for C fixation in the
microalga Heterocapsa pygmaea.
In addition to the light conditions (quantity and
quality), it is crucial to investigate if other variables
such as C or N availability affect the GP–ETR relationship. High CO2 levels (1%) in Porphyra leucosticta
(Mercado et al. 1999) can affect the photoinhibitory
rates. Franklin and Badger (2001) demonstrated that
the loss of correlation between ETR and linear photosynthetic flow as irradiance was exacerbated during
low Ci availability. In our study, however, the level of
Ci was maintained at an optimal level (2.5 mM) and no
carbon limitation was expected. In relation to N availability, the algae were incubated in nitrogen enriched
seawater in the laboratory although they grow in the
coastal waters under low levels of nitrate (LópezFigueroa and Niell 1991; Hernández 1993). Changes
in nitrogen levels can affect GP–ETR ratios in U. rotundata (Henley et al. 1991). Plants growing in low
nitrogen environments are limited in their synthesis of
proteins including Rubisco (Logan et al. 1999). The
different GP/ETR ratios in the two species of Ulva
analyzed can be due to the drastic differences not only
in the absorptance and pigment content, but also due
273
to the different N assimilation rates. Nitrogen assimilation is a competing sink for electrons in addition to
C fixation. However, with the exception of a study in
U. rotundata (Henley et al. 1991), a possible relation
of the lost of GP/ETR linear and nitrogen metabolism
in macroalgae has not been intensively examined. The
slopes of ETR versus GP function (Figure 4) or PSII
versus O2 (Figure 5) were higher in the algae with
higher N assimilation (Ulva rotundata and Pophyra
leucosticta). Such findings were supported by higher
internal N contents (total Ni and soluble proteins) and
nitrate reductase activity (Table 2) in these species. Although the electron pathways to N assimilation diverts
on the level of ferredoxin, this should not influence
O2 rates but 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 roughly
to covary with nitrate concentration in phytoplankton.
In our study, algae were incubated in a enriched nitrogen seawater media and in these conditions the sudden
nitrogen assimilation can inhibit Rubisco activity and
electrons are used for nitrate assimilation, and the respiratory C flow increases to provide carbon (Turpin
1991).
Thus, ETR could become higher in U. rotundata
than in U. olivascens because its higher N assimilation
determines a higher sink of electrons. The light and
nutrient status history could also affect the GP/ETR
relationship. Maximal photosynthesis decreased on an
area basis in high-light grown algae but only under
N limitation (Henley et al. 1991b; Pérez-Lloréns et
al. 1996). At limiting light, the maximal photosynthetic rate in U. rotundata decreased not only on an
area basis but also on a N basis (Pérez-Lloréns et al.
1996). This may indicate that the electron transport
chain components (lower catalytic membrane concentration and/or electron transport chain density) limits
light saturated photosynthetic rates (Pérez-Lloréns et
al. 1996).
Acknowledgements
The authors thank the technical assistance of Pilar
Sánchez, Soluna Salles and Luis Escassi. We would
like to thank Kai Bischof and Geir Johnsen for insightful comments and critical reading of the manuscript. Financial support was provided by the Ministry
of Education and Culture and Ministry of Science
and Technology of Spain (CICYT AMB97-1021-C02-
01, AGL 2001-1888-C03) and the European Union
(FEDER, 1FD97-0824).
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