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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 260 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 261 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 262 (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 263 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 647 264 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). 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