Porphyra laciniata and Chondrus crispus and its relation to the diurnal changes of underwater light quality and quantity. Pubbl. Staz zool Napoli (I: Mar Ecol) ...
Marine Biology (1996) 127:61-66
© Springer-Verlag 1996
D . - P . Hiider • M . Lebert • J. M e r c a d o • J. A g u i l e r a S. Salles • A. F l o r e s - M o y a • C. J i m 6 n e z F. L. F i g u e r o a
Photosynthetic oxygen production and PAM fluorescence in the brown alga Padinapavonicameasured in the field under solar radiation Received: 29 March 1996/Accepted 7 June 1996
Abstract The effects of solar radiation on photosyn-
thetic oxygen production, pulse amplitude-modulated (PAM) fluorescence and pigmentation were measured in the Mediterranean brown macroalga Padina pavonica (Linnaeus) Lamouroux under field conditions and natural sunlight. Exposure of thalli to solar radiation for 1 h caused a dramatic decrease of their photosynthetic quantum yield, which recovered to initial levels after they had been placed in the shade for 3 h. Photoinhibition also occurred at the natural growth site of P. pavonica during the hours of maximal solar irradiance. Photosynthetic oxygen production was also affected by high levels of solar radiation both in algae harvested from the surface and from 6 m depth; oxygen production started to decrease after a few minutes of exposure, and negative values were found after 1 h of solar exposure. Chlorophyll a content in P. pavonica also decreased during the hours of maximal solar irradiation. These results suggest that photoinhibition of P. pavonica occurs during part of a typical summer day on Mediterranean coasts.
Communicated by A. Rodriguez, Puerto Real D.-P. H~ider. M. Lebert Institut fiir Botanik und Pharmazeutische Biologic, Friedrich-Alexander-Universitiit,Staudtstrage 5, D-91058 Erlangen, FRG J. Mercado • J. Aguilera. S. Salles. C. Jim6nez ( ~ ) . F. L. Figueroa Departamento de Ecologia, Universidad de M/tlaga, Campus Universitario de Teatinos, E-29071 Mfilaga, Spain A. Flores-Moya Departamento de Biologia Vegetal, Universidad de Mfilaga, Campus Universitario de Teatinos, E-29071 Malaga, Spain
Introduction
In contrast to phytoplankton, which are free to move vertically in the water column, macroalgae are restricted to their site of growth, and are thus exposed to the ambient conditions in their habitat (L~ining 1985). One important factor controlling the abundance and species distribution of algae is exposure to solar radiation. Some algae are adapted to the bright sunlight at the surface and tolerate being exposed in rock pools, while other algae thrive only in crevices or under overhanging rocks where light exposure is limited. In recent years there has been increasing interest in the photoecology of macroalgae that are exposed to rapidly changing light conditions in their environment due to tides and changes of turbidity in addition to varying solar irradiation. Recent studies have been shown that in many macroalgae net photosynthesis is limited to a specific range of irradiances (Hanelt 1992; Hanelt et al. 1992, 1993). At low irradiances, photosynthetic activity may fall below the light-compensation point; on the other hand, excessive radiation may damage the photosynthetic apparatus of the cells (Coutinho and Zingmark 1987; Franklin et al. 1992). In the past, photosynthetic measurements in macroalgae have been restricted to the laboratory. This involved removing the plants from their habitat and subjecting them to highly artificial conditions including changes in temperature, irradiance and salinity which may impose severe stress factors on the specimens. Only after the development of portable and robust instrumentation did it become possible to determine essential photosynthetic parameters in the field. Recently, a portable instrument has been developed which is suitable for measuring pulse amplitude-modulated (PAM) fluorescence of algal thalli at their site of growth (Schreiber and Bilger 1993; Schreiber et al. 1994). PAM measurements are based on chlorophyll a fluorescence. In the preparation of the quenching analysis,
62
the initial fluorescence in the dark-adapted state (Fo) is measured. Fluorescence is induced by a constant, weak, red-light source, and is measured in dark-adapted thalli. Under these conditions all Photosystem II (PS II) reaction centers are believed to be in the open (oxidized) state. Subsequently, a saturating white-light pulse is applied, which closes (reduces) all PS II reaction centers and results in maximal fluorescence (Fro). The difference between F0 and Fm is calculated as Fv, i.e., variable fluorescence. When the thalli are adapted to moderate or high irradiances, maximal fluorescence in the light-adapted state (Fff) usually decreases, and initial fluorescence in the light-adapted state (Fo') either increases or decreases. The following fluorescence-quenching analysis is based on the measured signals characterized above. The underlying hypothesis assumes that two different processes contribute to the reduction of the maximal fluorescence yield, Fro. The faster of the two is photochemical quenching (qP = (Fff - Ft)/(Fm' - Fo'), where Ft is the current steady-state fluorescence), quantifying the utilization of light energy by the photosynthetic apparatus. Photochemical quenching quickly declines after the application of saturating pulses which close PSII reaction centers. The second process, non-photochemical quenching ( q N = 1 - ( F m ' - Fo')/(Fm - Fo)), occurs on a much slower time scale and is believed to be correlated with the energization of the thylakoid membrane (Krause and Weis 1991; Schreiber et al. 1995). Empirical expressions for the quantum yield have been developed based on the fluorescence parameters measured during quenching analysis (Weis and Berry 1987; Genty et al. 1989). This approach does not require the knowledge of the previously measured fluorescence parameters Fo and F~. Concomitant gasexchange measurements have verified this approach (Schreiber and Bilger 1993). Evaluation of oxygen exchange is another important tool for the ecophysiological properties of algal photosynthesis. As for PAM measurements, only recently did portable instrumentation become available. Besides land-based equipment, a portable and submersible device has been developed which allows on-line, computer-controlled measurements in the water column under solar irradiation (H~ider and Sch~ifer 1994 a, b). The aim of this paper is to describe photosynthetic oxygen production, photoinhibition, and its recovery under field conditions in the brown alga P a d i n a pavonica. A specific question is whether ambient irradiation values impair photosynthesis in this organism in its natural habitat. Materials and methods Plant material The common Mediterranean brown alga P a d i n a pavonica (Linnaeus) Lamouroux was used for all experiments. Thalli of 5 cm
diam were collected fi'om rocky shores on the coast of Saronikos Gulf, near Korinth (Greece), and Punta Carnero (Algeciras, Southern Spain), and were immediately measured on site. The experiments were carried out during the summers of 1994 and 1995.
Measurements of PAM fluorescence In vivo chlorophyl1 fluorescence was determined on site with a portable pulse amplitude-modulated fluorometer (PAM 2000, Waltz, Effeltrich, FRG) (Schreiber et al. 1986). Thalli were harvested immediately before use, and were transferred to custom-made, open, UV-B translucent Plexiglas frames (GS 2458, R6hm and Haas, Darmstadt, FRG). The thalli were kept submersed in shallow water in the shade for dark adaptation, after which PAM fluorescence was measured and optimal photosynthetic quantum yield was determined. After these initial measurements, the specimens were exposed to solar radiation during local noon in shallow water. Any resulting photoinhibition was indicated by a decrease in the effective photosynthetic quantum yield. Subsequently, the samples were transferred back into the shade, and recovery of the quantum yield was measured at predefined time intervals during the following 6 h. The natural daily course of photoinhibition was determined by collecting thalli every hour from sunrise to sunset. The fluorescence parameters were estimated in each specimen immediately after harvest. The PAM instrument permits to run programmed experimental sequences, for example to evaluate the dependence of the fluorescence parameters on the irradiance of the actinic light produced from a red-light-emitting diode (LED). Light was increased in 11 steps, from 1 to 79 W m -2, each lasting 6.5 rain.
Oxygen-exchange measurements Photosynthetic and respiratory oxygen exchange were measured at discrete time intervals under solar irradiance. 500 to 700 mg fresh weight of algae were incubated in cylindrical glass bottles of 250 ml volume for 15 to 20 rain. Five mmol of sodium bicarbonate was added to the seawater at the beginning of the measurements to prevent carbon limitation. Temperature was controlled by placing the bottles in aquaria filled with seawater. Respiration was estimated as oxygen depletion in dark bottles. Oxygen concentration in the seawater was estimated at the beginning and at the end of the experiments by a Crison OXI-92 oxygen electrode. Other measurements were performed at the surface or in the water column with a submersible device (Hiider and Sch~ifer 1994 a,b) using solar radiation as the light source. This apparatus allowed the simultaneous determination of oxygen concentration, photosynthetically active radiation (PAR), temperature and depth. The time course of photoinhibition was determined in thalli exposed to solar radiation after previous dark adaptation (30 rain). Recovery was investigated by storing the samples at 5 m depth for a predefined period of time in a translucent container. In another type of experiment, photosynthetic oxygen production was determined at various depths in the water column. The exposed area of the thalli was measured as well as their dry weight for each experimental run.
Pigmentation Chlorophyll a (chl a) concentration in the experimental thalli of P a d i n a p a v o n i c a was determined every 2 h. Chl a was extracted from 60 to 70 mg fresh wt with 90% pH-neutralized (with sodium carbonate) acetone over a period of 24 h at 4°C in the dark and after grinding the thalli. Its concentration was determined spectrophotometrically with the equation of Jeffrey and Humphrey (1975).
63 Statistics
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At least eight independent P A M fluorescence measurements were performed on different parts of the thallus or from different specimens for each data point from which mean values and standard deviation were calculated. Photosynthetic oxygen-exchange was measured at least three times in independent samples for each treatment. All experimental runs were repeated several times. Pigment concentrations were determined in triplicate samples.
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Measurement of solar radiation Solar irradiance was measured on a permanent basis using a newly developed filter instrument (ELDONET, Real Time Computer, M/Shrendorf, FRG). The dosimeter takes reading in three wavelength bands (UV-B, 280 to 315 nm; UV-A, 315 to 400 nm; PAR, 400 to 700 nm) at 1 s intervals, averages them over 1 min intervals, and stores them on a computer. From these values, doses are calculated on an hourly and daily basis for each channel. Typical irradiances under cloudless skies were 4 2 5 W m -2 for PAR, 49 W m -2 for UV-A and 1.2 W m 2 for UV-B at local noon. Daily doses were 9 8 6 5 k J m 2 for PAR, 1 0 7 0 k J m -2 for UV-A and 22.1 k J m -2 for UV-B.
Results
0 07:00
. . . . . . . . . 09:00
11:00
0
13:00
15:00
17:00
19:00
21:00
Local time (hrs) Fig. 1 Solar irradiances ofUV-B ( x 100), UV-A ( x 5) and photosynthetic active radiation (PAR) from dawn to dusk on 20 September 1995 in P u n t a Carnero, Algeciras, Southern Spain, measured with E L D O N E T filter radiometer
IlIL.__.
PAM fluorescence Fig. 1 shows the irradiances in the UV-B, UV-A and PAR range for one of the experimental days. The measuring instrument was placed at a site where it was not shaded by any obstacles during the day, and measurements were performed throughout the daylight hours. During preparation of the inhibition experiments, the fluorescence parameters of Padina pavonica were determined as a function of actinic irradiation (Fig. 2). Samples were harvested, and Fo and Fm were measured after dark adaptation. The thalli were then adapted to an irradiance of 23.3 W m -2 using an array of redlight-emitting diodes built into the P A M instrument. Subsequently, actinic light irradiance was increased in 11 steps from 1 to 79 W m -2 and the fluorescence parameters were measured. The steady-state fluorescence, Ft, showed no significant variation in this light range. Fo' followed a similar pattern. Fro' showed an initial increase at low irradiances, with a peak at 6.5 W m -2, and then dropped to values of < 0.4. The photosynthetic yield remained about constant up to 10 W m - 2 and then declined steadily. The photochemical quenching declined steadily from its initial value close to 1 to about 0.6 at the highest irradiance. The non-photochemical quenching showed an inverse behaviour to that of F,,', with a dramatic drop at low irradiances, a permanent rise from 6 W m 2, and with final values slightly > 0.9. Specimens of Padina pavonica were harvested from their site of growth and mounted in a UV-transmitting Plexiglas container to keep the algae in place so that
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,
,
,
,
10 Irradiance (W m-2)
.
.
.
.
.
.
.
100
Fig. 2 Padina pavonica. Fluorescence parameters measured in algae harvested from rock pool as a function of the fluence rate of actinic red light. Before experiments, thalli were adapted to intermediate fluence rate of 23 W m 2 for 10 min and then exposed to increasing irradiances for periods of 6.5 min each. At end of each period, fluorescence parameters were determined [ 0 current steady-state fluorescence (Ft); • photosynthetic quantum yield; A photochemical quenching (qP): C) nonphotochemical quenching (qN); -k maximal fluorescence in light-adapted state (F,,'); • initial fluorescence in light-adapted state (Fo'). Experimental runs were repeated several times; error bars have been omitted for clarity
exposure and measurement area could be controlled. Seawater circulated through the container which was kept in shallow water. First, the samples were darkadapted for 1 h to determine the optimal quantum yield (Fig. 3); subsequently, they were exposed to solar radiation for 1 h. The photosynthetic yield decreased dramatically. After exposure to solar radiation, the thalli were transferred back into the shade and recovery was measured at predetermined intervals. After 3 h of recovery, the yield had already reached its initial value. After 6 h, the yield was also measured in specimens
64 0,77
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30 min rec
1 h expos
2 h rec 1 h rec
4 h rec 3 h rec
08:30 09:30 10:30 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30
Local time (hrs)
unexposed 6 h rec
Fig. 3 Padina pavonica. Photosynthetic quantum yield of algae harvested from rock pool and measured after 1 h adaptation in shade (unexposed), after 1 h exposure to solar radiation in shallow water (1 h expos), and during recovery in the shade (30 rain rec to 6 h rec) (Bar on extreme right yield for thalli subjected to same experimental procedure except for solar radiation) For each data point, at least eight measurements were averaged and standard deviation was calculated
Fig. 4 Padina pavonica. Photosynthetic quantum yield from dawn to dusk of algae harvested from rock pool. Thalli were retrieved from their site of growth and measured immediately after harvest. For each data point, at least eight measurements were averaged and standard deviation was calculated
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subjected to the same experimental procedure except for exposure to solar radiation. This control was designed to determine whether there were any other stress factors, in addition to high solar irradiance, affecting the yield. The high yield value indicated that the experimental handling had no effect on the photosynthetic capacity of the algae. In the foregoing experiment, the thalli were exposed to direct solar radiation whilst confined to a container stored in shallow water. In order to determine whether photoinhibition also occurs at their natural site of growth, photosynthetic yield was measured in specimens immediately after harvesting from their original site. In order to follow the natural daily course of photosynthetic yield, this experiment was carried out from dawn to dusk at 1 h intervals. The yield values were close to 0.7 in the early morning hours, indicating no sign of photoinhibition, but decreased substantially later in the day (Fig. 4); only late in the afternoon did the yield values return to uninhibited levels.
Photosynthetic oxygen production Thalli of Padina pavonica were harvested from close to the surface in a rock pool and immediately transferred to the submersible instrument to determine oxygen exchange. After measuring dark respiration, they were exposed at different depths between 4 and 0 m, starting at 4 m (Fig. 5). The highest net photosynthetic oxygen production was found at 0m. This experiment was repeated with algae harvested from 6 m depth, with the same response (data not shown). When thalli harvested from the surface were exposed to solar radiation
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0.14 0.12
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Fig. 5 Padina pavonica. Photosynthetic oxygen exchange of algae harvested from rock pool, and estimated in darkness and at decreasing depths from 4 to 0 m, and solar illuminance at measurement sites. All values integrated over 5 min periods each
close to the surface, oxygen production started to decline after a few minutes of exposure, and negative values were recorded after ~ 1 h of exposure (Fig. 6). Negative oxygen production on P. pavonica was also found by non-continuous oxygen measurements (Table 1). Net photosynthesis increased from 11:00 hrs local time (09:00 hrs solar time) to 13:45 hrs local time; from thereon, net photosynthesis dramatically decreased to negative values at 16:00 hrs local time. High values of positive net photosynthesis were found again at 17:30hrs local time, when effective photosynthetic quantum yield had mostly recovered.
Pigmentation Chlorophyll a content in Padina pavonica also varied during the day; it was maximal during the morning and
65 0.30
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Fig. 6 Padina pavonica. Photosynthetic oxygen exchange of algae harvested from rock pool, and measured under solar radiation at surface, and solar illuminance during measurements. Before exposure, dark respiration was determined, and oxygen exchange then measured; all values integrated over 5 min periods each
Table 1 Padina pavonica. Photosynthetic oxygen exchange (pmol 02 g - 1 fresh wt h 1) and chlorophyll a content (mg g - 1 fresh wt) of algae harvested from rock pool at various times of day. Photosynthesis was measured immediately after harvesting under solar radiation at surface. Values are means of at least 3 replicates _+ SD Local time (hrs) 11:00 13:45 16:00 17:30
02 evolution
17.5 32.0 - 13.2 39.0
_+ 2.5 ± 4.5 ± 2.5 + 7.0
Chl a content
0.40 0.34 0.23 0.29
_+ 0.10 _+ 0.05 _+ 0.02 + 0.07
then started to decrease, reaching a minimum of 0.23 mg g-1 fresh wt at 16.00 hrs. Thereafter, chl a content increased slightly until the end of the day (Table 1).
Discussion
It has been previously reported that solar irradiation of high fluence rate may induce photoinhibition in higher plants (Bj6rkman and Demmig 1987; Schreiber et al. 1994), macroalgae (Franklin et al. 1992; Hanelt et al. 1992, 1993; Larkum and Wood 1993; Herrmann et al. 1995) and phytoplankton (Leverenz et al. 1990; Helbling et al. 1992). The mechanism of photoinhibition is still controversial (Crofts and Yerkes I994); nevertheless, there is a general agreement that during photoinhibition the photosynthetic quantum yield and photochemical quenching decrease, and often nonphotochemciM quenching increases, both in algae (red macroalgae and unicellular green algae; Leverenz et al. 1990; Hanelt et al. 1992) and leaves of higher plants (Ogren and Sj6str6m 1990). In the present work, the Mediterranean brown alga Padina pavonica has been shown not to be an exception to this general behaviour found in other groups of
seaweeds; photosynthetic yield steadily decreases at irradiances > 10 W m - 2, both in algae harvested at the surface and at 6 m depth. Photochemcial quenching (qP) also decreases from values of 1 at very low irradiances, indicating no presence of photoinhibition processes, to values of 0.6 at 79 W m - 2 ; non-photochemical quenching (qN) increases with rising photonftuence rates, with a minimum at ~ 6 W 121-2, showing a perfect inverse response to that of maximal fluorescence in light-adapted thalli (F,,'), indicating that as irradiance is being increased energy dissipation by means of fluorescence is reduced, and non-photochemical processes (e.g. heat dissipation) begin to minimize possible deleterious effects produced by excess energy and by the accumulation of active oxygen species. Photoinhibitory processes are present in Padina pavonica even in its natural habitat: exposure of thalli from both the surface and from 6 m depth to solar radiation for 1 h resulted in a drop in photosynthetic yield to values of < 30% of initial values; however, recovery is also a fast process, and after only 30 min in the shade yield had recovered to levels 77% of the original. Complete recovery took place within 3 h of removal from direct solar radiation. This is a natural process that occurs under clear skies at the site of growth of P. pavonica, as demonstrated by the decrease in its effective quantum yield at noon and during the afternoon, and its almost total recovery close to dusk. Strong photoinhibition of Padina pavonica thalli is also reflected in its oxygen evolution and decreases in pigment content. Short-term acclimation of the photosynthetic apparatus of macroalgae to increasing irradiances by reduction of their pigment content (chl a and phycobiliproteins) has previously been shown in some red algae both in laboratory experiments (Algarra and Niell 1990) and in the field (Ldpez-Figueroa 1992; Riidiger and L6pez-Figueroa 1992). In the case of P. pavonica, a significant decrease in the chl a content was recorded, indicating its ability to acclimate quickly to increasing irradiances. Nevertheless, both continuous and discrete oxygen measurements indicated that net photosynthesis of P. pavonica is highly affected by solar radiation of high fluence rates. A drastic reduction in oxygen production takes place after a few minutes exposure to maximum solar radiation, leading to negative net oxygen production after 1 h exposure. Thus, P. pavonica shows no net oxygen production during part of the day during the summer on the Mediterranean coasts, due to photoinhibition. Acknowledgements This work was financially supported by the European Community (EV5V-CT91-0026) to D.-P.H., by the Acci6n Integrada Hispano-Alemana No. 133-B, DAAD (322- M-e-dr), and by the CICYT Projects Nos. AMB93-1211 and AMB94- 0684CO2-02 to F . L . F . J . M . and J.A. were supported by doctoral fellowships of the Spanish Ministry of Education and Science and of the Junta de Andalucia, respectively. The authors gratefully acknowledge the skilful technical assistance of J. Sch~ifer and H. Wagner.
66
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