Photosynthesis Research 75: 259–275, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Regular paper

Relations between electron transport rates determined by pulse amplitudemodulated chlorophyll fluorescence and oxygen evolution in macroalgaeunder different light conditions

Felix L. Figueroa1,∗, Rafael Conde-Alvarez1 & Ivan Gomez2

1Departamento de Ecologıa, Facultad de Ciencias, Universidad de Malaga, Campus Universitario de Teatinoss/n, E-29071 Malaga, Spain; 2Instituto de Biologıa Marina, Facultad de Ciencias, Universidad Austral de Chile,Casilla 567, Valdivia, Chile; ∗Author for correspondence (e-mail: Felix_lopez@uma.es; 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 PhotosystemII-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 Ulvaolivascens and the red alga Porphyra leucosticta collected from the field and incubated for 3 days at 100 µmolm−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 versusabsorbed irradiance was higher than that of GP versus absorbed irradiance. Only under absorbed irradiances belowsaturation or at values of GP <2 µmol O2 m−2 s−1 a linear relationship was observed. In the linear phase, calcu-lated 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 whitelight, red light emitting by diodes and solar radiation. In Ulva species the maximal ETR was reached under red lightand solar radiation whereas in P. leucosticta the maximal ETR was reached under white light and minimal underred light. These results are in agreement with the known action spectra for photosynthesis in these species. In thecase of P. leucosticta, GP and ETR were additionally determined under saturating irradiance in algae pre-incubatedfor one week under white light at different irradiances and at white light (100 µmol m−2 s−1) enriched with far-redlight. GP and growth rate increased at a growth irradiance of 500 µmol m−2 s−1 becoming photoinhibited at higherirradiances, while ETR increased when algae were exposed to the highest growth irradiance applied (2000 µmolm−2 s−1). The calculated O2 evolved /ETR molar ratios were close to the theoretical value of 0.25 when algaewere pre-incubated under 500–1000 µmol m−2 s−1. The enrichment by FR light provoked a decrease in bothGP and ETR and an increase of nonphotochemical quenching although the irradiance of PAR was maintained ata constant level. In addition to C assimilation, other electron sinks, such as nitrogen assimilation, affected theGP–ETR relationship. The slopes of GP versus ETR or �PSII versus �O2 were lower in the algae with the highestN assimilation capacity, estimated as nitrate reductase activity and internal nitrogen contents, i.e., Ulva rotundataand Porphyra leucosticta, than that observed in U. olivascens. The possible mechanisms to explain this discrepancybetween GP and ETR are discussed.

Abbreviations: Aλ – spectral absorptance; Ci – internal carbon; Chl a – chlorophyll a; DMF – N,N, dimethylform-amide; 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 plantpre-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 photo-synthesis; LED – red-light emitting diode; Ni – internal nitrogen; NP – net photosynthesis; NRA – nitrate reductaseactivity; 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 – solubleproteins; Tλ – spectral transmittance; WL – white light

Introduction

Photosynthetic rates and consequently primary pro-duction of marine macrophytes display a wide rangeof values (Nielsen and Sand-Jensen 1990; Enríquezet al. 1995) which depend on different variables suchas light, temperature, nutrients, water motion etc.,and on the capacity for acclimation to environmentalfluctuations and stress (Franklin and Forster 1997;Häder and Figueroa 1997). Accurate determinationsof algal photosynthesis present difficulties on bothshort and long-term scales due to diverse photoaccli-mation mechanisms and complex regulation systems(MacIntyre et al. 2002). The development of non-intrusive methodologies has led to rapid and sens-itive measurements of changes in the physiologicalstatus 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 photosyn-thetic primary reactions and quenching mechanismsin plant physiology studies of higher plants (Schreiberet al. 1995). The application of PAM fluorometry inmacroalgae is relatively recent and has become a use-ful tool for evaluating photosynthesis under differentnatural and artificial light conditions (Henley et al.1991; Hanelt 1992; Franklin et al. 1996). Taking intoaccount the differences in the organization of the pho-tosynthetic apparatus between macroalgae and higherplants, an optimisation of the PAM instrumentationhas been needed to meet accurately the low chloro-phyll fluorescence emission of macroalgae (Bücheland Wilhem 1993; Hanelt 1996). For example, thepresence of phycobilisomes in the light-harvestingsystem of red algae results in generally lower maximalquantum yields (Fv/Fm) than that measured in greenand brown algae (Büchel and Wilhem 1993).

The usefulness of chlorophyll fluorescence as anindicator of photosynthesis requires to demonstrate itsrelationship with the quantum yield of gas evolution(O2 or CO2). Thus, simultaneous measurements of

both oxygen evolution and chlorophyll fluorescenceare required. Quantum yields of photosynthesis areusually defined as the quantum yield for oxygen pro-duction or C fixation (Genty et al. 1989). Assumingthat excess energy is dissipated as heat through inac-tivated PS II centers, a linear correlation between ETRcalculated from fluorescence and CO2 exchange canbe demonstrated, as is the case of C4 and C3 plantsat various irradiances under non-photorespiratory con-ditions (Weis and Berry 1987; Krall and Edwards1990). This relationship can be curvilinear at low irra-diance at normal levels of O2 and CO2 when electronsflow to O2 via the photosynthetic carbon oxidation(PCO) cycle and/or Mehler-ascorbate-peroxidase re-action. This reaction in the water cycle competes withcarbon fixation and confounds measurements of O2evolution (Genty et al. 1989, 1992; Asada 1999).

Extrapolations of �PSII to the absolute photosyn-thetic electron transport rate (ETR) depend on thespecific correlation among quantum yields of oxygenevolution (�O2), carbon fixation (�CO2) and any othercompeting sinks for electrons. However, the numberof algal species in which both variables have beensimultaneously analyzed are so far scarce: in somemicroalgae, a non-linear relationship between �PSIIand �O2 and �CO2 at both low and high irradiance wasdemonstrated (Flameling and Kromkamp 1998; Hartiget al. 1998).

In our study, simultaneous measurements of oxy-gen evolution and effective quantum yield were con-ducted in the red alga Porphyra leucosticta and twogreen algae Ulva rotundata and Ulva olivascens underdifferent irradiances (similar to Franklin and Badger2001) and also under different light qualities (solarradiation, white light fluorescent lamps, red light-emitting diodes, white light enriched with far-redlight). Although the studied algae attained similarmorphology, i.e., sheet-like structure, they showedvery different absorption properties due to the differentcell 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; Markager1993; Agustí et al. 1994). In addition, the functionalityof pigments involved in photosynthesis, indicated byin vivo fluorescence excitation spectra, are very differ-ent in Ulva species compared to Porphyra (Grymskiet al. 1997; Figueroa et al. 2003). The wavelengthdependence of the maximum quantum yield of carbonfixation has been studied in different algae (Kroon etal. 1993; Schofield et al. 1996). Exposures to enrichedfar-red light conditions over several days resulted inchanges in the Photosystem II/Photosystem I ratiosand consequently, maximum quantum yields, dissip-ation mechanisms and GP–ETR relationships can beaffected (Eskins and Duysen 1984; Chow et al. 1990).

Finally, the GP–ETR relationships were related notonly to carbon assimilation but also to other electronsinks, e.g., nitrogen assimilation. The capacity for thenitrate incorporation was estimated as the activity of akey enzyme nitrate reductase (NR), and as the assim-ilation of inorganic nitrogen (nitrate and ammoniumadded to the medium), i.e., the total internal nitrogencontent.

Materials and methods

Sampling sites and algal material

Porphyra leucosticta Thuret in Le Jolis and Ulvaolivascens P.A. Dangerad were collected at 0–0.2 mdepths from two sites (eulittoral zone) on the coastof Málaga, southern Spain; Lagos and La Araña(36◦45′ N 4◦18′ W), respectively. Ulva rotundataBliding was collected from the estuarine area ofPalmones River (Cádiz, southern Spain, 36◦13′ N5◦27′ W). The locations are characterized by differ-ent environmental conditions: the coast of Málagapresents salinities of 35–37 SPU and almost no tides.Palmones River on the other hand, is an estuarinearea with higher contents of particles and nutrients andconsequently, subjected to changing conditions of wa-ter transparency and light penetration (Vergara et al.1997). Thus, U. rotundata is submitted to high solarradiation when emerged during low tide but submittedto very low irradiance at 1.5 m depth during high tidedue to water turbidity (Vergara et al. 1997).

Culture conditions

After collection, algae were transported in darknessin an icebox to the laboratory. Then algae were trans-

ferred into glass cylinders containing Provasoli en-riched seawater (PES) (Provasoli 1968) with aerationat 15 ◦C and a light/dark regime of 12/12 h. Theillumination 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 al-gae were incubated during three days in the laboratoryin these conditions for acclimation before the start ofexperiments.

For outdoor experiments, the algae were trans-ferred to three trays containing 0.5 l PES medium (35SPU) supplied with constant aeration. The whole sys-tem was placed within a water bath and maintained ata temperature of 16–20 ◦C by pumping cooled water.

Light treatments

Effective quantum yield and oxygen evolution weredetermined under different irradiances and light qual-ities provided by fluorescent white light lamps asCompact True Lite13W (Duro-Test Corp., Fairfield,New Jersey) denominated WL-1 and True-Lite PlusII 40W (Duro-Test Corp., Fairfield, New Jersey) de-nominated WL-1 and red-light provided by red lightemitting diodes (red LED) of the pulse amplitudemodulated fluorometer (PAM-2000, Walz GmbH, Ef-feltrich, Germany) (Figure 1a). Samples were submit-ted to increasing irradiances between 0 and 400 µmolm−2 s−1 at intervals of 30 s each in the case of redlight emitting diodes and between 0 and 1300 µmolm−2 s−1 in the case of fluorescent white light lamps.In the outdoor experiments (described above), theywere maintained under natural solar radiation (Fig-ure 1b) and then covered by different neutral filters(Lee filters, Hampshire, UK) in order to decrease thesolar irradiance, which reached maximal values closeto 2000 µmol photons m−2 s−1.

In the case of P. leucosticta, effective quantumyield and oxygen evolution were determined at theirradiance of 1000 µmol m−2 s−1 (above light satur-ation for photosynthesis) after 1 week of incubationin the laboratory in PES medium at different irradi-ances 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). Inaddition, P. leucostica was incubated for 1 week undera irradiance of 100 µmol m−2 s−1 provided by a whitelight fluorescent lamp (Osram DL 18W) and enrichedwith far-red (FR) light (λ > 700 nm), mainly ab-sorbed by Photosystem I (PS I), provided by Linestralamps (Osram C428 35W) (Figure 1c). Different red

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Figure 1. Spectral Irradiance (emission spectra) of the differentlight sources used in the experiments (a) white light provided byfluorescent lamps: (1) Compact True Lite 13 W (WL-1, open tri-angles) and True Lite PlusII 40 W (WL-2, open squares) andred-light emitting diodes provided by the PAM instrument (redLEDs, closed circles), (b) solar radiation (closed circles) and 300W metal halide lamp (Optimarc) (open circles) and (c) day lightfluorescent lamps (Osram DL 18W) at 100 µmol m−2 s−1 enrichedwith different irradiances of far-red light provided by Linestra lamps(Osram C428 35W).

(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 wereobtained by increasing the irradiance of far-red lightmaintaining red light constant (Figure 1c). Effectivequantum yield and oxygen evolution were determinedat the irradiance of 1000 µmol m−2 s−1 (above lightsaturation for photosynthesis).

O2 evolution and chlorophyll fluorescencedetermination

Measurements were carried out using computer aidedOXY M-5 equipment (Real Time Computer, Erlangen,Germany). Thallus pieces (0.15–0.2 g FW) were takenand put into a measuring chamber (10 ml), fitted with aClark-type electrode and a magnetic stirrer. The cham-ber contained filtered seawater buffered to pH 8.2 with20 mM Tris and maintained at a temperature close to15 ◦C. The content of inorganic C was 2.5 mM. Tosimultaneously measure chlorophyll fluorescence, theoptic fiber of the PAM device was integrated in themeasuring chamber. Two fluorescent white light lampsof small size (Compact white lamp, Duro-Test Corp.,Fairfield, New Jersey) were used as light sources (Fig-ure 1a). The irradiances reaching the thallus surfacewere monitored using a spherical PAR quantum sensor(Zemoko, Koudekerke, The Netherlands) specially de-signed for small chambers. Thalli were exposed for5 to 10 min to an initial dark respiration period andthen to a gradient of increasing irradiances from 3.5 to1300 µmol m−2 s−1 for 5 to 10 min each.

The photosynthetic parameters were estimated byfitting a non-linear function (Jassby and Platt 1976) toeach data series:

NP = NPmax ∗ (tanh(α ∗ E/NPmax) (1)

where NP is the net photosynthetic rate, NPmax is thesaturated net photosynthesis, tanh is the hyperbolictangent function, α is the photosynthetic efficiency atlow irradiance and E is the incident irradiance. Grossphotosynthesis (GP) was calculated as the sum of netphotosynthesis and respiration.

In vivo chlorophyll fluorescence of PSII was de-termined with a portable pulse modulation fluorometer(PAM 2000, Waltz GmbH, Effeltrich, Germany). After5–8 min in darkness to measure Fo a saturating flash(400 ms) was applied to obtain the maximal fluores-cence level (Fm). Thus, the maximal quantum yieldof fluorescence (Fv/Fm) was obtained (Schreiber et al.1986). The variable fluorescence Fv is the differencebetween the maximal fluorescence from fully reduced

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PS II reaction center (Fm) and the intrinsic fluores-cence (Fo) from the antenna of fully oxidized PS II.The effective quantum yield (�PSII) was calculatedaccording to Schreiber and Neubaer (1990):

�PSII = F′m − Ft/F′

m (2)

F′m being the maximal fluorescence which is in-

duced with a saturating white light pulse (400 ms,approx. 9000 µmol m−2 s−1. Ft is the current steady-state fluorescence in light adapted algae. The electrontransport rate ETR was determined according to thefollowing formula:

ETR (µmol electrons m−2s−1) = AQλ∗FII ∗ �PSII(3)

Where AQλ is the absorbed quanta calculated as theproduct the integration of the spectral Absorptance(Aλ) between 400-700 nm and spectral irradiance ofthe light source (Eλ), FII is the fraction of AQ direc-ted to PS II including its light harvesting complexes(LHCs) and �PSII is the effective quantum yield orquantum yield of PS II charge separation as it wasdefined above. According to Grzymski et al. (1997)and Johnsen (personal communication), FII for differ-ent pigment groups can be estimated by determiningthe fraction of Chl a associated with PS II and itscorresponding light-harvesting complexes, i.e., LHCII. FII for Rhodophyta is about 0.15 and 0.5 forChlorophyta (Grzymski et al. 1997; Figueroa et al.2003).

The absorptance (Aλ) was determined for each 1nm by means of an integrating sphere (Licor-1802)connected to a Licor-1800 UW spectroradiometer ac-cording 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 endof the incubation to the highest irradiance applied.No differences were observed between measurementsconducted at the initial time (at the end of the low-est irradiance applied). In the samples exposed forone week under different irradiances and light quality(R:FR ratios) in Porphyra leucosticta, Aλ was determ-ined from eight replicates before and after one weekof exposure.

Gross photosynthesis was estimated (GPe) fromthe 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 electrongenerated at PS II, i.e., four stable charge separationsare needed at PS II to evolve 1 O2-molecule, thus τ isequal to 0.25.

The parameters from the ETR curves were calcu-lated following the model of O2-based photosynthesisversus irradiance curves. Here a modification of thenon-linear function from Jassby and Platt (1976) wasmade:

ETR = ETRmax ∗ tanh(αETR ∗ E/ETRmax) (6)

Where ETR is the relative electron transport rate men-tioned above, ETRmax is the saturated ETR, tanh is thehyperbolic tangent function, αETR is the efficiency ofthe electron transport (initial slope of the ETR versusIrradiance curves) and E is the incident irradiance.

Non-photochemical quenching (qN) was calcu-lated as

qN = 1 − (F′m − F′

o)/Fm − Fo (7)

being Fm the maximal fluorescence of an dark-‘adapted’ sample, Fm

′ is the maximal fluorescenceof an light-exposed alga under a given irradianceand Fo

′ the intrinsic fluorescence of a light-adaptedsample determined after a pulse of far-red light of fiveseconds (Schreiber et al. 1986). Five different algalsamples were used for each measurement of oxygenevolution and chlorophyll fluorescence. Three GP andETR versus irradiance curves were determined in eachtreatment.

Determinations of pigments, proteins, nitratereductase activity and C-N contents

Algal material for chlorophyll determinations wassampled simultaneously with those used for photosyn-thesis measurements and was stored in liquid nitrogenuntil analysis. Chl a, b and total chlorophylls wereextracted in N,N, dimethylformamide (DMF) follow-ing the methodology described by Inskeep and Bloom(1985). Samples (1 mg FW) from apical thallus re-gions were thawed at room temperature and incubatedin 2.5 ml DMF for 24 h at 4 ◦C in darkness. The ab-sorbance was finally measured at 750, 664.5 and 647

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nm in a Beckman DU-7 spectrophotometer (BeckmanInstruments 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). Theextracts 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 insitu nitrate reductase activity after Corzo and Niell(1991). The concentration of NO2

− was determinedspectrophotometrically according to Snell and Snell(1949).

Total intracellular carbon and nitrogen content wasdetermined using a Perkin-Elmer elemental analysermodel 2400 CHN.

Growth rate determination

The thallus area of each disc was determined from discdiameter, since growth of the circular discs proceededisodiametrically. The relative growth rate, expressedas the percentage increase per day, was computed fromthe following expression (Kain 1987).

RGR (% day−1) = (ln At − ln Ai)/t (8)

where At is the algal area measured after six days ofincubation, Ai is the algal area at the initial time and tis the time expressed as days.

Statistics

Data treatment included one-way ANOVA and furthermean comparisons by means of LSD Fisher test (Sokaland Rohlf 1995).

Results

Light response curves of oxygen and chlorophyllfluorescence

No clear patterns were seen in O2-based P-E curves.Measured GPmax was higher in Ulva species thanthat in the red alga P. leucosticta. In general, bothmeasured GP and α were significantly higher (P <

0.05) in U. olivascens than in U. rotundata and P.leucosticta (Table 1, Figure 2c). However, estimatedgross photosynthesis (GPestimated) according to theformula (5) showed a clearer pattern: GPmax and α

exhibited the highest values in Ulva species compared

Table 1. Photosynthetic parameters measured as oxygen evolution(GPmeasured) and estimated (GPestimated): photosynthetic efficiency(α) and saturated gross photosynthetic rate (GPmax, µmol O2m−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 rotundataand Ulva olivascens and the red alga Porphyra leucosticta under arti-ficial white light (WL-1, Fluorescent Compact True Lite, Duro Test,USA). Efficiency of ETR (α) and maximal ETR (ETRmax) under dif-ferent light qualities: artificial white light (WL-2, True Lite Plus II,Duro-Test, Fairfield, New Jersey), solar radiation and red light emit-ting diodes (red LED) provided by the fluorometer PAM-200 (WalzGmbH). The light spectra of the different light sources are presented inFigure 1

Variable Species

U. rotundata U. olivascens P. leucosticta

GPmeasured (WL-1)

α 0.031 ± 0.001a 0.063 ± 0.003a 0.053 ± 0.002b

Pmax 4.7 ± 0.35a 6.3 ± 0.3b 2.9 ± 0.2c

GPestimated (WL-1)

α 0.069 ± 0.003a 0.071 ± 0.006a 0.021 ± 0.001b

Pmax 46.4 ± 4.1a 23.1 ± 2.6b 3.0 ± 0.23c

ETR (WL-1)

α 0.276 ± 0.02a 0.284 ± 0.02a 0.088 ± 0.006b

ETRmax 186.6 ± 14.3a 92.4 ± 10.5b 10.8 ± 1.9c

ETR (WL-2)

α 0.293 ± 0.02a 0.283 ± 0.02a 0.145 ± 0.009b

ETRmax 131.0 ± 11.7a 89.0 ± 9.2b 27.4 ± 3.4c

ETR (solar radiation)

α 0.353 ± 0.03a 0.214 ± 0.03b 0.127 ± 0.01b

ETRmax 157.4 ± 18.5a 106.2 ± 13.3b 28.1 ± 3.6c

ETR (Red LED)

α 0.375 ± 0.03a 0.440 ± 0.05a 0.069 ± 0.007c

ETRmax 78.53 ± 9.3a 13.1 ± 1.5b 5.09 ± 0.6c

Different letters represent significant differences among the species foreach variable at P < 0.05.

to P. lecucosticta. On the other hand, in Ulva spe-cies, 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 bothspecies (Figure 3, Table 1). ETRmax was higher in theUlva species than in P. leucosticta (Figure 3). Compar-atively, ETR was saturated at much higher irradiancethan that required to saturate gross photosynthetic rate.The ETR-based α was higher than O2-based α in allalgae, in U. rotundata about nine times, whereas inU. olivascens and P. leucosticta 4.5 times 1.6 timeshigher, respectively (Figure 3, Table 1).

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Figure 2. Gross photosynthetic rate in µmol O2 m−2 s−1 as a func-tion of the absorbed irradiance in µmol photons m−2 s−1. Meas-urements were carried out in 20 mM Tris buffer at pH 8.2 at naturalinorganic carbon concentration (2.5 mM) in (a) Ulva rotundata, (b)Ulva olivascens and (c) Porphyra leucosticta. The measurementswere conducted under different irradiances of white light providedby two white light fluorescent lamps (WL-1, Compact True Lite 13W).

The relation between ETR and GP was not linearfor the whole data set (Figure 4). At GP >2 µmol

Figure 3. Electron transport rate (ETR) expressed as µmol elec-trons m−2 s−1 calculated as ETR = AQλ * FII * �PSII as functionof absorbed irradiance (µmol photons m−2 s−1). Measurementswere performed in 20 mM Tris buffer at pH 8.2 at a natural inor-ganic carbon (2.5 mM) in (a) Ulva rotundata, (b) Ulva olivascensand (c) Porphyra leucosticta. The measurements were conductedunder different irradiances of white light provided by two white lightfluorescent (WL-1, Compact True Lite).

O2 m−2 s−1 the slope of the function ETR versusGP drastically increased. The function ETR-GP for

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the whole data sets can be adjusted to an exponentialfunction (r2 = 0.92 P < 0.01). The slopes werelower in Ulva species than in P. leucosticta, i.e., 0.965in the case of U. rotundata, 0.645 for U. olivascensand 1.099 for P. leucosticta. At low irradiances withvalues of GP lower than 2 µmol O2 m−2 s−1, the rela-tion between ETR and GP can be adjusted to a linearfunction (r2 = 0.98 P < 0.01) and the number ofmols of electrons per mol of oxygen was equal to thetheoretical value of four in U. olivascens or close tothat in the case of U. rotundata with a value of 3.4.However, in the case of P. leucosticta the number ofmols of electrons per mol of oxygen was only two,half of the theoretical value. In contrast to the expos-ure to low irradiances, at high irradiances, the ratiomaximal ETR per maximal GP (Table1) was far fromthe theoretical value in the Ulva species, i.e., 39.70in U. rotundata and 14.66 in U. olivascens. However,at very high irradiance in the case of P. leucosticta, aETR / GP ratio value of 3.72 was found, which is veryclose to the theoretical value (Table 1).

Values of �PSII versus �O2 were not linearly cor-related for the whole data set (Figure 5). For the threealgae studied, at �O2 values < 0.04, a linear relation-ship in the function �PSII versus �O2 with slopes of7.86 (U. rotundata), 6.40 (U. olivascens) and 8.44 (P.leucosticta) could be determined. In the upper part ofthe curve, increases of �O2 reflected only slight in-creases of �PSII, with maximal values of about 0.7 forthe 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 1.5times higher in U. rotundata relative to U. olivascens,consequently the absorptance in the PAR region of thespectra was about 45% higher in U. rotundata than inU. olivascens (Table 2). Thus, the ratio Chl a/Chl bin U. olivascens was twice as much as found in U.rotundata. The contents of soluble proteins, total in-ternal N and nitrate reductase activity were increasedin U. rotundata relative to U. olivascens. The Chl con-centration, absorptance, soluble protein and internalN were higher in U. rotundata and P. leucosticta thanin U. olivascens (Table 2). Similarly, nitrate reductaseactivity (NRA) was higher in U. rotundata and P. leu-costicta compared to U. olivascens. NRA was linearlyrelated (r2 = 0.91) to the internal nitrogen content(data not shown). Internal carbon content was sim-ilar in U. olivascens and P. leucosticta and higherthan 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 grossphotosynthetic rate in µmol O2 m−2 s−1. Measurements weremade in 20 mM Tris buffer at pH 8.2 at a natural inorganic car-bon (2.5 mM) in (a) Ulva rotundata, Ulva olivascens (b) and (c)Porphyra leucosticta. The measurements were conducted underdifferent irradiances of white light provided by two white lightfluorescent lamps (WL-1, Compact True Lite).

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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) inµmol NO2 gDW−1 h−1, internal C and N and C:N ratios in the green algae Ulva rotundataand Ulva olivascens and in the red alga Porphyra leucosticta

Variable Species

U. rotundata U. olivascens P. leucosticta

Chl a (mg m−2) 79.9 ± 6.4a 24.6 ± 2.8b 63.2 ± 4.5c

Chl b (mg m−2) 24.6 ± 2.8a 16.5 ± 1.8b –

Absorptance (A) 0.573 ± 0.04a 0.313 ± 0.02b 0.46 ± 0.03c

SP (mg gDW−1) 59.1 ± 4.3a 33.2 ± 2.8b 45.6 ± 4.4c

NRA (µmol NO2 gDW−1 h−1) 7.2 ± 0.6a 5.4 ± 0.4b 6.8 ± 0.3a

C (mg gDW−1) 344.3 ± 27.5a 363.1 ± 21.77a 365.2 ± 18.25a

N (mg gDW−1) 43.0 ± 3.44a 26.9 ± 1.61b 35.3 ± 2.75c

C:N ratio 8.0 ± 0.8a 13.5 ± 1.1b 10.4 ± 1.6b

Different letters represent significant differences among the species for each variable amongthe species at P < 0.05.

tent, soluble protein concentration, Aλ and NRA werehigher than in U. olivascens but lower than in U. ro-tundata (Table 2). The C:N ratio in P. leucosticta washigher 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 solarlight and fluorescent white light than that under redLEDs (Figure 6, Table 1). Both maximal ETR and α

were higher in Ulva species than that in P. leucostictain all light qualities (Table 1). In U. rotundata max-imal ETR was higher in WL-1 and solar radiationthan under WL-2 (Table 1). In U. olivascens, no sig-nificant differences were found in the maximal ETRunder solar radiation compared to both types of ar-tificial white lamps. In P. leucosticta maximal ETRwas higher under solar radiation and WL-2 than thatunder WL-1 and red LEDs (Table 1). In U. rotundata,ETRbased α was similar under solar radiation and redLEDs and higher than under fluorescent lamps (WL-1 and WL-2). However in U. olivascens, the highestETR-based α values were reached under red LEDs andthe minimal under solar radiation. Under white lightfluorescent lamps, ETR-based α was higher than thatunder solar radiation. In P. leucosticta, the minimalETR-based α values were reached under red LEDsand it was higher under solar radiation and WL-2 thanunder WL-1 (Table 1).

Gross photosynthesis and ETR of P. leucosticta grownfor one week at different irradiances and red:far-redratios

Maximal GP increased with increasing the growth ir-radiance up to 500 µmol m−2 s−1 and it decreasedwhen it was incubated at 1000–2000 µmol m−2 s−1.However, ETR increased with increased growth irra-diances (Table 3). GP estimated according to Equation(5) overestimated GP (Table 3) except when it was in-cubated at 500 µmol m−2 s−1. The ratio ETR/GP wasclose to the theoretical value only at growth irradianceof 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 µmolm−2 s−1, the ETR/GP ratio was lower (0.8 and 1.28,respectively) and at growth irradiances of 2000 µmolm−2 s−1 it was higher (11.5) than the theoreticalvalue. Non-photochemical quenching (qN) increasedand Chl a concentration decreased with growth irra-diance. The growth rate (% day−1) increased fromgrowth irradiances of 50 to 500 µmol m−2 s−1 andit decreased from 1000 to 2000 µmol m−2 s−1.

GP and ETR in P. leucosticta were affected bythe proportion of red:far-red light under the same ir-radiance (100 µmol m−2 s−1) in the PAR regionof the spectra (Table 4). ETR and GP decreasedwith the far-red light enrichment. GP and ETR atR:FR light ratios of 0.30 and 0.56 were significantly(P < 0.05) lower than that observed at 1.01, 1.98and 2.92. GP:ETR ratios were lower than the theor-etical value of four. The closest value was reachedwhen the algae were incubated under the highestR:FR light ratio (2.85). Non-photochemical quench-

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Table 3. Gross photosynthesis (GP, µmol O2 m−2 s−1) measured and estimated according to the formula (5), electron transportrate (ETR µmol m−2 s−1) and non-photochemical quenching (qN), chlorophyll a concentration (Chl a, mg gDW−1) andgrowth 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 fixedirradiance of 1000 µmol m−2 s−1 white light (WL-1, Compact True Lite 13 W). qN, chlorophyll a concentration and growthrate are refereed to growth irradiances

Growth Gmeasured GPestimated ETR qN Chl a Growth rate

Irradiances

(µmol m−2 s−1)

50 2.8 ± 0.17a 0.6 ± 0.03a 2.3 ± 0.15a 0.06 ± 0.004a 3.2 ± 0.21a 12.9 ± 1.2a

100 3.2 ± 0.21b 1.0 ± 0.07b 4.1 ± 0.21b 0.11 ± 0.009b 2.9 ± 0.14a 19.8 ± 1.6b

500 3.8 ± 0.16c 3.3 ± 0.18c 13.2 ± 1.1c 0.22 ± 0.01c 2.3 ± 0.14b 25.4 ± 2.3c

1000 2.9 ± 0.21a,b,d 3.4 ± 0.23c 13.9 ± 1.3c 0.36 ± 0.03d 1.3 ± 0.12c 21.3 ± 2.2b,c,d

2000 1.8 ± 0.12e 5.2 ± 0.31d 20.7 ± 2.1d 0.58 ± 0.04e 0.6 ± 0.03d 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 enrichmentand it was about three times higher under 0.30 R:FRlight ratio than that at 1.98 or 2.92 (Table 4). Chla concentration increased with the far-red light en-richment. Growth rate (% day−1) increased with theR: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 ratioscompared to that at values of 1.98 or 2.92.

Discussion

Gross photosynthetic rates reported in this work forUlva rotundata and Porphyra leucosticta were similarto those previously reported for U. rotundata (Osmondet al. 1993; Pérez-Llorens et al. 1996; Vergara et al.1997) and the intertidal P. perforata (Herbert and Waa-land 1998) and P. columbina incubated under artificialwhite light (Franklin and Badger 2001). The maximalgross photosynthesis and the efficiency measured in P.leucosticta was 50% lower than values found in thetwo Ulva species.

Effect of irradiance on GP–ETR relationships

The pattern of ETR as a function of the absorbed irra-diance was different compared to O2 evolution. Firstly,ETR is saturated at much higher irradiance than grossphotosynthesis and secondly the initial slope is highercompared to gross photosynthesis. Consequently, nolinear relation between ETR versus gross photosyn-thesis was observed for the whole irradiance intervalapplied. Only for absorbed irradiance below saturation

of photosynthesis or for values of gross photosyn-thesis below 2 µmol m−2 s−1 a linear response wasobserved. In this case, calculated molar ratios at O2evolved/ETRs were closed to the theoretical valuesof 0.25 in the Ulva species but not in the red alga P.leucosticta. In the latter species, the theoretical valueis only reached at the highest absorbed irradiances(300–450 µmol m−2 s−1). At low irradiances, the lossof correlation between ETR and linear photosyntheticelectron flow in Porphyra limits the application of thePAM technique, whereas the limitation in Ulva speciesoccurs at high irradiances. In Ulva species at values ofgross photosynthesis > 2–3 µmol O2 m−2 s−1, therelation between the mol of electrons (ETR) per molof O2 drastically increased because electron sinks arevery active, while O2 reaches the steady state.

The GP-ETR relations have been occasionally ex-amined, e.g., the red algae Palmaria palmata (Haneltand Nultsch 1995) and Porphyra columbina (Frank-lin and Badger 2001), the brown algae Dictyota di-chotoma (Hanelt et al. 1995) and Zonaria crenata(Franklin and Badger 2001), and the green algae Ulvarotundata (Osmond et al. 1993), U. lactuca, U. fasci-ata (Beer et al. 2000) and U. australis (Franklin andBadger 2001). At moderate irradiance, ETR calcu-lated from �PSII closely matches gross O2 evolutionin U. fasciata and U. lactuca (Beer et al. 2000). Incontrast, Longstaff et al. (2002) found that in situmeasurements of diel photosynthesis of U. lactucarevealed a good correlation between ETR and O2 evol-ution at moderate light but at higher irradiances had ahigher ETR than the expected one. Recently, Frank-lin and Badger (2001) reported a good correlation

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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 airradiance of 100 µmol m−2 s−1 provided by two white light fluorescent lamps (Day Light, Osram DL 18W) andenriched with far-red light (λ > 700 nm) provided by Linestra lamps (Osram C428 35W). Different red:far-redratios (0.30, 0.56, 1.01, 1.98, 2.92) were obtained by increasing the irradiance of far-red (λ = 700–720 nm) lightand maintaining red light (λ = 660–680 nm). GP and ETR were determined under a fixed irradiance of 1000 µmolm−2 s−1 white light (WL-1, Compact True Lite 13 W)

R:FR GPmeasured GPestimated ETR qN Chl a Growth rate

ratio

0.30 1.9 ± 0.3a 1.2 ± 0.09a 4.99 ± 0.28a 0.36 ± 0.03a 4.8 ± 0.4a 11.0 ± 1.2a

0.56 2.2 ± 0.2a 1.3 ± 0.10a 5.34 ± 0.35a,b 0.31 ± 0.02a 4.3 ± 0.3a 11.3 ± 1.8a

1.01 3.0 ± 0.2b 1.7 ± 0.12b 6.71 ± 0.331a,c 0.13 ± 0.01b 3.2 ± 0.2b 15.7 ± 1.3b

1.98 3.1 ± 0.2b 1.9 ± 0.09a,c 7.86 ± 0.32b,c,d 0.11 ± 0.008b 3.3 ± 0.2b 20.3 ± 2.2c

2.93 3.2 ± 0.2b 2.7 ± 0.1a 10.82 ± 0.41a,b,c,d 0.11 ± 0.009b 3.0 ± 0.3b 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 Ulvaaustralis and Porphyra columbina, whereas at saturat-ing photon fluxes, especially when Ci availability waslow, ETR overestimated gross O2 evolution. These au-thors suggested that excess electron flow was not dueto an increase in gross O2 uptake, neither the Mehler-ascorbate-peroxidase reaction nor the photosyntheticcarbon oxidation enhanced at high irradiance or low Ci(Franklin and Badger 2001). in Ulva australis and Por-phyra columbina, Franklin and Badger (2001) foundvalues close to theoretical ETRs for O2 evolution(determined by mass spectrometry) at subsaturatingirradiances.

The different response of Ulva species compared toP. leucosticta could be due to their different photosyn-thetic acclimation characteristics in response to theirnatural environment, i.e., sun type in P. leucosticta andshade type in Ulva species. P. leucosticta is growingin the eulittoral system and it has been adapted to veryhigh solar radiation (Figueroa et al. 1997). The sinkof electrons in the sun type Porphyra is probably lessefficient than that in Ulva species.

The formulation of �PSII by the Genty method, asthe product of the reaction centre ‘openness’ and excit-ation capture efficiency by open centres, assumes thatnon-radiative dissipation occurs in the light-harvestingantenna. Alternatively, non-photochemical quench-ing can occur in PS II reaction centres (Krause andWeis 1991), altering the relationship between pho-tochemical and fluorescence yields (Schreiber et al.1995). The thermal dissipation can be different inUlva species compared to P. leucosticta. Dissipationmechanisms are different among the different groups

of macroalgae: red algae, and specifically Porphyraspecies, show more active state transitions than greenor brown algae (Satoh and Fork 1983; Büchel andWilhelm 1993). However, the contribution of statetransition to non photochemical quenching comparedto other quenchings such as energy dependent quench-ing, qE or thermal energy dissipation, qI, is difficult toevaluate.

At sub-inhibitory fluence rates, thermal energydissipation is mainly controlled by the light-inducedformation of the thylakoid pH gradient (energy de-pendent quenching, qE), which accelerates the rateof energy supplied to the Calvin cycle. At higher,photoinhibitory fluence rates, photoinactivation due tophotoinhibition opens an additional path of thermalenergy dissipation (qI), thus optimizing the rate ofphotochemical dissipation and diminishing the rate ofphotodamage. For Porphyra perforata a new mechan-ism for adaptation to changes of light intensities andquality was reported in which light energy reachingthe reaction centres of PS II decreased without anysignificant change in PS I activity (Satoh and Fork1983). The non- photochemical quenching in algae hasusually been related with the xanthophyll cycle (Uhr-macher et al. 1995; Franklin et al. 1996). However,the xantophyll cycle activity is lacking in Rhodophyta(Hager 1980) or attenuated in Ulva species (Franklinet al. 1992), thus, alternative quenching mechanismscannot be ruled out.

A further explanation for enhanced ETRs after ex-posures to high light is the cyclic flow around PSII from quinone acceptor QB (or pheophytin) via cytb559 and chl Z to P689 (Falkowsky et al. 1986).

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Figure 5. �PSII versus �O2. Measurements were made in 20 mMTris 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 Porphyraleucosticta. The measurements were conducted under different irra-diances 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 splittingactivity in PS II and PS I under conditions where theplastoquinone 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 irradiancein µmol photons m−2 s−1 of (a), Ulva rotundata, (b) Ulva olivas-cens and (c) Porphyra leucosticta under different light qualities:solar radiation (dark circles), white light provided by fluorescentlamps (WL-2, True Lite PlusII 40 W) (open circles and thick andpointed line) and red light emitting diodes (red LEDS) provided bythe PAM-2000 fluorometer (dark triangles).

271

urating light), which was accompanied by an enhancedETR. In the cases where xantophyll cycle activity islimited, i.e., Ulva species or non existent, i.e., in Por-phyra species, cyclic flow in PS II could be essentialfor protecting those species inhabiting the intertidalsystem. The indication that cyclic flow around PS IIin macroalgae was supported by changes in the rateof �PSII to �O2 relative to Ci (Franklin and Badger2001). Beer et al. (2000) reported a close linear cor-relation between ETR and O2 evolution in U. lactucaat irradiances up to 36% of growth saturating irradi-ances and at various Ci concentrations at 13% growthsaturating irradiance, which probably are lower thanirradiance levels required to saturate photosynthesis.

The loss of linearity between GP–ETR was alsoobserved in algae grown for one week at different ir-radiances. At high irradiances (1000–2000 µmol m−2

s−1), GP was saturated but not ETR. A closer rela-tion between GP and growth rate was found comparedto the relationship between ETR and growth rate.The decrease in �O2 with increasing growth irradi-ance was much higher than that observed for �PSII.This decrease in �O2 can be explained, in part, as aconsequence of an increase in cyclic phosphorilationwith respect to non-cyclic phosphorilation under highlight (Dubinsky et al. 1986; Gilmoire and Govindjee1999). The acclimation to high irradiance is producedby a decrease in the size of peripheral PS II antenna,mostly attributed to a decrease in the LHC IIb and sub-sequent decrease in its component pigment, namelyChl a + b, lutein and neoxanthin (Gilmore and Govind-jee 1999). Under high growth irradiance both Chl a(Table 3) and biliproteins (data not shown) decreasedin 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 athigh irradiances but a deviation from non linearity athigh quantum efficiencies, i.e., low irradiances, as hasbeen observed in this study in P. leucosticta. These au-thors, using artificial electron acceptors, demonstratedthe presence of two different populations of PS II andthey ascribed the deviations of linearity between �PSIIand �O2 to this PS II heterogeneity. In P. leucostictathe similar values of GP and estimated GP only at highirradiances as in higher plants could also indicate theexistence 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 de-termination of the absorptance, i.e., the fraction ofincident irradiance absorbed by PS II (Beer et al.2000). In our study, A was determined by using anintegrating sphere taking into account both spectraltransmittance and spectral reflectance. Thus, absorbedquanta were considered spectrally dependent and ac-cording to Grzymski et al. (1997) and Figueroa et al.(2003), the fraction of Chl a associated with PS II andits corresponding LHC was about 0.15 in red algaeand 0.5 in green algae. Other authors used the factorof 0.5 for all algal groups accounting for the presenceof two photosystems, assuming equal involvement inlinear electron flow (Beer et al. 2000; Franklin andBadger 2001). PS II absorption cross-section alsomight change during the course of short experiments,i.e., during determination of GP versus irradiance,basically as a response to increased plastoquinone re-duction (Fork et al. 1991), rendering our assumptionof a 1:1 distribution of irradiance between PS II andPS I as erroneous. The optical thickness of the thallusseems to be a limitation for the application of chloro-phyll fluorescence measurements in macroalgae. Forexample, absorbed quanta can be determined easilyin species attaining thin thalli, in contrast to thickermorphs (Lüning and Dring 1985; Markager 1993; En-ríquez et al. 1994). For example, the calculated ETRwas equivalent to the theoretical electron requirementin a thin species such as Ulva or Porphyra, but notin thicker species such as Zonaria crenata (Frank-lin and Badger 2001). Changes in the distribution ofexcitation between PS II and PS I could affect the ab-sorptance determination. The absorption cross-sectionamong macroalgae can vary due to differential intra-cellular self-shading (Grzymski et al. 1997). On theother hand, the absorption cross-section at high ir-radiances can be affected by chloroplast movements(Hanelt and Nultsch 1991). In long term experiments(one week exposure to different irradiances), absorbedquanta in P. leucosticta decreased as a consequence ofpigment content variations (photoacclimation). How-ever, during the short time exposure (less than onehour) necessary for ETR or GP determinations, nodifferences in absorbed quanta at the lowest and thehighest irradiance applied were observed (data notshown).

Light quality affects the ETR relationship in theanalysed macroalgae. In Ulva species, maximal ETR-based α were estimated under solar radiation and red

272

light incubation, whereas P. leucosticta was minimal atred light. This pattern is according to the action spectraof O2-based photosynthesis reported by Lüning andDring (1985), i.e., in red light GP at 10 µmol O2 m−2

s−1 was about two times higher in Ulva lactuca thanin Porphyra umbilicalis. Maximal ETR was reached inall algae under solar radiation and the white light fluor-escent lamp True light Plus II (WL-2). Similar effectsof solar radiation and WL-2 (but not WL-1; Fluores-cent compact True lite), are explained by the emissionof WL-2 which resembles more closely the solar radi-ation spectra than WL-1 (see Figure 1). WL-1 showednarrow peaks in the blue, green and red region of thespectra, being very different to the sun spectra. Underred light, ETR decreased because a lesser number ofphotons from other wavelengths are being absorbedthrough accessory pigments i.e., blue light (in greenalgae) and green light (in red algae). Energy imbal-ances between PS I and PS II as a consequence of dif-ferential absorption of different spectral wavelengthshave also been suggested to be responsible for a lackof correlation between �PSII and �O2 (Kroon et al.1993). Light sources with different spectral light pro-portions, i.e., R:FR light ratios, (red light is mainlyabsorbed by PS II and FR light by PS I) can affectthe redox state. In this sense, R:FR light ratios fromsolar radiation (1.06) were more closely related to ra-tios emitted by WL-2 (1.92) than WL-1 ones (0.401).Kroon et al. (1993) suggested that the rate of cyclicelectron transport around PS I is spectrally regulatedand is less important for cells exposed to broad-bandwhite light, which encompasses all photosyntheticallyactive wavelengths. In cells exposed to narrow-band,spectral illumination disproportionately drives PS IIphotochemistry. In P. leucosticta, increases in far-redlight irradiances (changing R:FR light ratios underconstant PAR irradiance) resulted in decreases in bothGP and ETR. Far-red light is absorbed mainly by PSI and consequently cyclic transport around PS I canbe activated. The increase of cyclic transport aroundPS I relative to cyclic photosynthetic transport in bothPS II and PS I can affect ATP/NADPH ratios andconsequently the electron sinks, i.e., C or N assimil-ation rates (Chow et al. 1990). The excitation pressure(expressed through the redox state of an intersystemcomponent of photosynthetic electron transfer chain)has been suggested as a key signal in the regulationof photosynthetic proteins (Durnford and Falkowski1997). Thus, in the studied macroalgae, the change inthe excitation pressure provoked by the different lightqualities could explain the variations in the ETR–GP

relationship throughout the regulation of photosyn-thetic proteins (Durnford and Falkowski 1997) andthrough the changes in the ATP/NADPH ratio, affect-ing enzyme activities related to electron sinks (Chowet al. 1990).

The enrichment by FR light also increased the nonphotochemical quenching (qN). Non photochemicalquenching can be induced by changes in pH aroundPhotosystem II (Bruce et al. 1997). Thus a possiblemechanism of increasing qN by FR light is that thislight quality favors the decrease of pH in thylakoidmembranes. In addition, FR light could affect chloro-phyll synthesis, carbon assimilation or other electronsinks such as N assimilation via non-photosyntheticphotoreceptors, i.e., phytochrome (López-Figueroa etal. 1989; Rüdiger and López-Figueroa 1992) and con-sequently, the ETR can be affected. Chlorophyll andbiliproteins are regulated by light quality in P. leu-costicta, through phytochrome and other red/greenlight photoreceptors (López-Figueroa and Niell 1991).In Porphyra sp., nitrogen assimilation and growth rateis stimulated by red light whereas they are inhib-ited by blue and far-red light (Figueroa et al. 1995a,b; Aguilera et al. 1997). Kroon et al. (1993) foundthat enzymatic processes associated with organic Csynthesis appeared to vary depending on the spectralgrowth irradiance, which contributes to the observedvariability in quantum yield for C fixation in themicroalga Heterocapsa pygmaea.

In addition to the light conditions (quantity andquality), it is crucial to investigate if other variablessuch as C or N availability affect the GP–ETR relation-ship. High CO2 levels (1%) in Porphyra leucosticta(Mercado et al. 1999) can affect the photoinhibitoryrates. Franklin and Badger (2001) demonstrated thatthe loss of correlation between ETR and linear pho-tosynthetic flow as irradiance was exacerbated duringlow Ci availability. In our study, however, the level ofCi was maintained at an optimal level (2.5 mM) and nocarbon limitation was expected. In relation to N avail-ability, the algae were incubated in nitrogen enrichedseawater in the laboratory although they grow in thecoastal waters under low levels of nitrate (López-Figueroa and Niell 1991; Hernández 1993). Changesin nitrogen levels can affect GP–ETR ratios in U. ro-tundata (Henley et al. 1991). Plants growing in lownitrogen environments are limited in their synthesis ofproteins including Rubisco (Logan et al. 1999). Thedifferent GP/ETR ratios in the two species of Ulvaanalyzed can be due to the drastic differences not onlyin the absorptance and pigment content, but also due

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to the different N assimilation rates. Nitrogen assim-ilation is a competing sink for electrons in addition toC fixation. However, with the exception of a study inU. rotundata (Henley et al. 1991), a possible relationof the lost of GP/ETR linear and nitrogen metabolismin macroalgae has not been intensively examined. Theslopes of ETR versus GP function (Figure 4) or �PSIIversus �O2 (Figure 5) were higher in the algae withhigher N assimilation (Ulva rotundata and Pophyraleucosticta). Such findings were supported by higherinternal N contents (total Ni and soluble proteins) andnitrate reductase activity (Table 2) in these species. Al-though the electron pathways to N assimilation divertson the level of ferredoxin, this should not influenceO2 rates but CO2 fixation rates, as the extent of theelectron flow depends on the electron sink, i.e., carbonand nitrogen assimilation. Babin et al. (1996) foundmaximum quantum yield of carbon fixation roughlyto covary with nitrate concentration in phytoplankton.In our study, algae were incubated in a enriched nitro-gen seawater media and in these conditions the suddennitrogen assimilation can inhibit Rubisco activity andelectrons are used for nitrate assimilation, and the res-piratory C flow increases to provide carbon (Turpin1991).

Thus, ETR could become higher in U. rotundatathan in U. olivascens because its higher N assimilationdetermines a higher sink of electrons. The light andnutrient status history could also affect the GP/ETRrelationship. Maximal photosynthesis decreased on anarea basis in high-light grown algae but only underN limitation (Henley et al. 1991b; Pérez-Lloréns etal. 1996). At limiting light, the maximal photosyn-thetic rate in U. rotundata decreased not only on anarea basis but also on a N basis (Pérez-Lloréns et al.1996). This may indicate that the electron transportchain components (lower catalytic membrane concen-tration and/or electron transport chain density) limitslight saturated photosynthetic rates (Pérez-Lloréns etal. 1996).

Acknowledgements

The authors thank the technical assistance of PilarSánchez, Soluna Salles and Luis Escassi. We wouldlike to thank Kai Bischof and Geir Johnsen for in-sightful comments and critical reading of the manu-script. Financial support was provided by the Ministryof Education and Culture and Ministry of Scienceand Technology of Spain (CICYT AMB97-1021-C02-

01, AGL 2001-1888-C03) and the European Union(FEDER, 1FD97-0824).

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