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Interactions between photosynthesis and ‘light-enhanced dark respiration’ (LEDR) in the flagellate Euglena gracilis after irradiation with ultraviolet radiation
www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 55 (2000) 63–69
Interactions between photosynthesis and ‘light-enhanced dark respiration’ (LEDR) in the flagellate Euglena gracilis after irradiation with ultraviolet radiation Nils G.A. Ekelund * ¨ ¨ Mid Sweden University, Department of Applied Science, S-871 88 Harnosand, Sweden Received 21 April 1999; accepted 14 February 2000
Abstract The effects of ultraviolet radiation (UV-A, 315–400 nm plus UV-B, 280–315 nm) on photosynthesis and ‘light-enhanced dark respiration’ (LEDR) in Euglena gracilis have been investigated by using light pulses (80 s) with increasing photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 and dark periods between the light pulses. LEDR is estimated as the maximum rate of oxygen consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate. Without any exposure to UV radiation, the photosynthetic rate and LEDR increase with increasing photon fluence rate. After 20 and 40 min exposures to UV radiation, the photosynthetic rate and LEDR as functions of photon fluence rate are reduced. After a 20 min UV treatment respiration is greater than photosynthesis after the first light pulse of 59 mmol my2 sy1 radiation, and especially at higher photon fluence rates photosynthesis is lower than the control values. The inhibitory effects of UV radiation on photosynthetic rate and LEDR are greater after a 40 min UV exposure than after a 20 min exposure. Only at 600 mmol my2 sy1 is the rate of oxygen evolution greater than that of oxygen consumption after a 40 min UV treatment. Both photosynthetic rate and LEDR are inhibited by the photosynthetic inhibitor DCMU (10y5 M) in a similar way, which indicates close regulatory interactions between photosynthesis and LEDR. Potassium cyanide (KCN) inhibits dark respiration more than it inhibits LEDR. Dark respiration is not affected to the same degree by UV radiation as are photosynthesis and LEDR. q2000 Elsevier Science S.A. All rights reserved. Keywords: Euglena gracilis; Light-enhanced dark respiration (LEDR); Photoinhibition; Photosynthesis; Respiration; Ultraviolet (UV) radiation
1. Introduction Recent studies have shown that the atmospheric ozone layer is still decreasing above both the Arctic and the Antarctic due to increasing concentrations of greenhouse gases [1]. The result will be that more biologically damaging ultraviolet-B (UV-B, 280–315 nm) radiation will reach the Earth9s surface in the near future. Most of the current knowledge of UV-B effects on phytoplankton and macroalgae is based on the inhibitory effects on photosynthesis [2]. These inhibitory effects are caused either by direct damage to photosynthetic components, which results in a lower rate of photosynthesis, or by damage to DNA, which results in the formation of photoproducts. The degree of inhibition observed when studying phytoplankton depends on the vertical mixing of the water column and the depth of the mixed layer [3]. There are several ways in which phytoplankton can protect them* Corresponding author.
selves against high UV-B irradiances, for example, via pigmentation and different enzyme mechanisms [2]. Photoinhibition is caused by high levels of photosynthetically active radiation (PAR); it can act as a protective mechanism where photosynthesis is downregulated [4]. Photoinhibition is often determined as the decrease in oxygen production and can be viewed as a regulatory mechanism rather than as a simple fault in the photosynthetic machinery [5,6]. This has been shown in macroalgae where the photosynthetic efficiency decreases with an increase in solar radiation at noon [4]. The possibility of repairing damage to photosynthesis is low for phytoplankton in short-term experiments and cold, deeply mixed surface layers [3]. Also, very low rates of respiration and growth in light-limited phytoplankton make the photoinhibitory effects more severe if these phytoplankton are suddenly exposed to high levels of irradiance. The importance of respiration in the repair mechanisms of inhibition has rarely been taken into consideration when studying the effects of UV radiation on phytoplankton, although
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it has been shown that dark respiration has a key role in the inhibition and reactivation of photosynthesis in the cyanobacterium Anacystis nidulans [7]. In Chlamydomonas reinhardtii the susceptibility to inhibition increased when dark respiration was inhibited by potassium cyanide (KCN) [8]. Not only dark respiration but also respiration in light (photorespiration) might prevent inhibition [9]. Earlier investigations indicated that ‘light-enhanced dark respiration’ (LEDR), which in the present investigation is estimated as the maximum rate of oxygen consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate, is a photosynthesis-dependent phenomenon and that it is a function of both the duration of illumination and the photon fluence rate [10]. The aim of this investigation was to study the role of LEDR in relation to photosynthesis in the flagellate Euglena gracilis, after exposure to ultraviolet radiation.
2. Materials and methods 2.1. Organism and culture conditions The flagellate E. gracilis strain Z (obtained from Professor ¨ ¨ Botanik und Pharmazeutische BioD.-P. Hader, Institut fur logie, Erlangen, Germany) was grown in 100 ml Erlenmeyer flasks in a freshwater medium described by Checcuci et al. [11]. The cells were grown in a cultivation cabinet (TERMAKS 6395 F/FL, AB Nino Lab., Sweden) at 208C with a 16 h/8 h light–dark cycle and a photon fluence rate of 90 mmol my2 sy1 (LI-COR quantum sensor, model LI-189) in the visible region (400–700 nm). 2.2. Ultraviolet radiation treatments Quartz glass bottles containing cells of E. gracilis were exposed for 20 and 40 min to UV-A radiation of 1.02 W my2 plus UV-B radiation of 0.73 W my2. UV-A and UV-B radiation were measured with an IL 1400A broad-band radiometer (International Light, Newburyport, MA, USA)
equipped with detectors for UV-A (Wa6259, a12667) and UV-B (W a6272, a12618). Ultraviolet radiation was obtained from two 40 W sunlamps (FS40, Westinghouse Electricity, Lamp Division, Bloomfield, NJ, USA). The ultraviolet radiation was filtered through a cellulose acetate (CA) film (0.13 mm thickness) to remove shorter-wavelength (less than 290 nm) components not encountered in nature. The CA was preburnt for 48 h at a distance of 1 m from four UV lamps in order to minimize changes of the filter properties. 2.3. Measurements of photosynthesis and respiration Photosynthesis and respiration were measured as oxygen evolution and consumption with a Light Pipette (Brammer, Illuminova, Uppsala, Sweden). The instrument consists of a light source that is connected to a cuvette with a micro-oxygen electrode (MI-730, Microelectrodes, 298 Rockingham Rd., Londonderry, Northern Ireland, UK), a quantum sensor, a temperature-controlled water bath and a computer. The light source delivers precise photon fluence rates of 0 to 3500 mmol my2 sy1. The rate of oxygen evolution was calculated assuming an ambient O2 concentration of 0.276 mmol mly1, which in this investigation corresponds to 100%. After centrifugation (30 s, 4000 rpm), the cells of E. gracilis were placed in the cuvette and each measurement of oxygen evolution/consumption lasted 20 min. The experiments were replicated three times. Chlorophyll was determined by extraction in 80% acetone solution and the absorbance was measured with a spectrophotometer (Perkin–Elmer Lambda Bio 20 UV–Vis spectrometer) at 652 nm [12]. In order to measure oxygen evolution and oxygen consumption during the same experiment, the cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses (Fig. 1). Each light pulse and dark period lasted for 80 s, except for the dark periods at the beginning and at the end of each experiment, which lasted for 90 s. The different time periods of light pulses and darkness were controlled by a computer. When dark
Fig. 1. Change in O2 concentration (% (mg Chl mly1)y1 sy1, bold curve) when the cells of E. gracilis were given six different light pulses with photon fluence rates (dashed curve) of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses. The photosynthetic rate was estimated by measuring the average values of O2 evolution during the light pulses, and LEDR (‘light-enhanced dark respiration’) was estimated as the maximum rate of O2 consumption after a period of light minus the rate of O2 consumption 30 s after the maximum rate (dark respiration).
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Fig. 2. Changes in O2 concentration (% (mg Chl mly1)y1 sy1, bold curve) and the amount of oxygen (% oxygen, thin curves) when the cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses. Before the measurements the cells were exposed to no UV radiation (control, A), 20 min of UV radiation (B), 40 min UV radiation (C) and DCMU (10y5 M, D).
3. Results
respiration was measured, no light pulses were given during a 20 min period. The experiments were carried out at 208C. The calculations of the photosynthetic rate were carried out by measuring the average values of oxygen evolution during the light pulses except for the first few seconds of transition (Fig. 1). DCMU (3-(39,49-dichlorophenyl)-1,1,dimethylurea) at a concentration of 10y5 M was used as an inhibitor of photosynthesis. For dark respiration the inhibitor used was KCN (potassium cyanide) at a concentration of 10 mM. LEDR was estimated as the maximum rate of O2 consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate (Fig. 1).
In the control experiments, without ultraviolet radiation, the cells of E. gracilis responded by increasing the rate of oxygen evolution with increased photon fluence rate (Fig. 2(A) and Fig. 3). During the dark periods the rate of oxygen consumption increased with higher photon fluence rates, with a sharp peak in oxygen consumption immediately after the light was turned off (Fig. 2(A)). These sharp peaks at higher photon fluence rates indicate that LEDR is a function of the photon fluence rate (Fig. 4). In the same experiment it was shown that the absolute value of oxygen in the cuvette
Fig. 3. Photosynthetic rates for E. gracilis at six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1. The photosynthetic rate was estimated by measuring the average values of oxygen evolution during the light pulses. Before the measurements the cells were exposed to no UV radiation (control), 20 min of UV radiation (UV20), 40 min of UV radiation (UV40) and DCMU (10y5 M, DCMU). Error bars represent standard deviations (SDs) of three replicated experiments.
Fig. 4. LEDR (‘light-enhanced dark respiration’) of E. gracilis estimated as the maximum rate of O2 consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate. Before the measurements the cells were exposed to no UV radiation (control), 20 min of UV radiation (UV20), 40 min of UV radiation (UV40), DCMU (10y5 M, DCMU) and KCN (10 mM, KCN).
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increased during the 20 min from about 95 to 120% (Fig. 2(A)); distilled water at 208C showed 100% oxygen). After a 20 min UV irradiation, both oxygen evolution and consumption are inhibited (Fig. 2(B) and Fig. 3)). At the first light pulse of 59 mmol my2 sy1 respiration is the dominating process, and at higher photon fluence rates the levels of photosynthesis are lower than the control values. LEDR, and the amount of oxygen, decreased after 20 min of UV radiation (Fig. 2(B) and Fig. 4). With a 40 min UV irradiation the inhibition of photosynthesis was greater than after a 20 min one (Fig. 2(C) and Fig. 3). The rate of oxygen evolution was first greater than the rate of oxygen consumption at 595 mmol my2 sy1 (Fig. 2(C)). The amount of oxygen decreased from about 90 to 30%, and the LEDR was greatly inhibited after 40 min of UV radiation (Fig. 2(C) and Fig.
4). With DCMU (10y5 M) photosynthesis was totally inhibited for all light pulses and the amount of oxygen decreased from about 100 to nearly 0% (Fig. 2(D) and Fig. 4). When dark respiration alone was measured in 20 min darkness, the rate and amount of oxygen consumption slowly decreased without any treatment with UV radiation (Fig. 5(A)). The rate of oxygen consumption after a 20 min UV irradiation showed the same pattern as for the control but after a 40 min UV irradiation the amount of oxygen reached the zero level after about 16 min (Fig. 5(B) and (C)). However, the level of oxygen at the beginning of the experiment with a 40 min UV exposure was lower than the control. The average values of the photosynthetic rates showed that the photosynthetic rate increased with increasing photon fluence rate in the control (Fig. 3). However when the cells
Fig. 5. Dark respiration measured during 20 min darkness after no UV radiation (control, A), 20 min of UV radiation (B) and 40 min of UV radiation (C). Respiration is expressed as oxygen consumption (change in O2 concentration, % (mg Chl mly1)y1 sy1, bold curve), and the amount of oxygen in the cuvette as % oxygen (thin curves).
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Fig. 6. Change in O2 concentration (% (mg Chl mly1)y1 sy1, dashed curve) and the amount of oxygen (thin curve) after treatment with 10 mM KCN. The cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses.
were treated with UV radiation, the photosynthetic rate decreased and differences between ‘control’ and ‘treated’ organisms increased with longer period of UV irradiation (Fig. 3). After both 20 and 40 min exposures to UV radiation, the maximum photosynthetic rate was reached at a photon fluence rate of 600 mmol my2 sy1 (Fig. 3). In contrast to the photosynthetic rates, LEDR increased at higher photon fluence rates in the control and after a 20 min UV irradiation (Fig. 4). However after a 40 min UV irradiation and when treated with DCMU, the levels of LEDR were much lower (Figs. 3 and 4). By using KCN, it was shown that dark respiration was inhibited, but LEDR and photosynthesis were not affected as much as dark respiration (Figs. 4 and 6). The absolute value of oxygen showed only small variations between the light and dark periods, which indicates that dark respiration was inhibited during the dark periods and that oxygen evolution and consumption due to photosynthesis and LEDR were not affected by KCN (Fig. 6).
4. Discussion and conclusions The influence of UV radiation on aquatic organisms has been studied in several investigations [13–15]. Most of the results deal with the inhibitory effects on photosynthesis. The possibility of recovering from UV stress depends not only on the duration and degree of UV irradiation but also on the ability of organisms to protect themselves from UV radiation. Hanelt et al. [4] showed that inhibitory effects of UV radiation on Arctic macroalgae caused a delay in the recovery processes in the afternoon and evening, rather than an inhibitory effect on photosynthesis in the morning. The protection mechanism that has been studied most is the amount of pro-
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tecting pigments and dark and light repair mechanisms, like the turnover rate of the D1 protein in photosynthesis [2]. When the cyanobacterium Anacystis nidulans was exposed to photoinhibitory light, the susceptibility to photoinhibition increased when dark respiration was inhibited. This indicates that dark respiration plays an important role in the process of photoinhibition of photosynthesis [7]. In phytoplankton, marine benthic algae and sea grasses, no effects of UV-B radiation on respiration have been observed [16]. The present study also shows that dark respiration is less affected by UV radiation than photosynthesis (Figs. 2, 5 and 6). In the experiments with 40 min UV exposure, the oxygen level was reduced to zero after about 16 min of darkness, which made it impossible for the cells to continue with dark respiration (Fig. 5). This was due to a lower concentration of oxygen at the beginning of the experiments in comparison with the control experiments. One way to reduce this problem would be to do the experiments with less-dense samples. However, the rate of oxygen consumption was about the same, independent of UV radiation. When studying the level of respiration in connection with light pulses, it was found to be almost constant before and after the first and second light pulses, but after the third light pulse with a photon fluence rate of 600 mmol my2 sy1, the rate of respiration increased (Fig. 2(A)). This increase is due to the additive effects of LEDR, which could be seen as a sharp peak in oxygen consumption immediately after the light was turned off. Depending on the photon fluence rate, the level of respiration will gradually decrease from a higher level to the rate of dark respiration that was documented before the first light pulse (Fig. 2(A)). The rate of LEDR increases as the concentration of oxygen is increased; this is
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shown in the control experiments, where the oxygen concentration at the end of the experiment was found to be about 120% (Fig. 2(A)). The rate of LEDR has earlier been shown to be a good estimate of the rate of respiratory oxygen consumption in the preceeding light period (photorespiration) [9]. Without any photorespiratory activity, the photoinhibition of photosynthesis would probably have been greater at the light pulses with irradiances higher than 600 mmol my2 sy1. In Phaseolus vulgaris linear electron transport decreased when the oxygen concentration was reduced from 21 to 2% in order to inhibit photorespiration, but the photoinhibitory effects on photosystem II did not increase due to drought conditions [17]. In the present study with UV radiation the levels of respiration were, in general, lower than the levels before the light pulses. This indicates that photorespiration could play a significant role in the rate of respiration or that the amount of photosynthate regulates dark respiration (Fig. 2(B) and (C)). In microalgae the rate of respiration often decreases from an initially high rate in the transition from light to darkness to a much lower rate after a period in darkness [18,19]. Beardall et al. [19] have shown that the phenomenon of LEDR is widespread in microalgae and that the respiration rates were enhanced by 10–140% following exposure to a high photon flux. They speculated that the enhanced LEDR could be a consequence of repair of photodamage [19]. However, this enhanced respiration was demonstrated to be inhibited by 92% by 100 mM CNy, which is different from the present results where the inhibitory effect of KCN on LEDR was about 40% at the highest photon fluence rate (Fig. 4) [20]. In leaf protoplasts it has also been shown that LEDR is stimulated by bicarbonate and inhibited by the photosynthetic inhibitor DCMU [21]. Respiration also protects leaf protoplasts against photoinhibition and it is the oxidative electron transport and phosphorylation that are important in these mechanisms of protection against high light intensities [21]. In another investigation dark respiration in Chlorella pyrenoidosa was stimulated by lead (Pb), whereas photosynthesis was inhibited [22]. The increased dark respiration was suggested to compensate the demand for energy to carry out metabolic and growth processes when C. pyrenoidosa was stressed by high levels of Pb [22]. Also photorespiration could act as a protection against photohinibition by an efficient consumption of ATP and reductant [23]. In summary, both the increase in photosynthetic rate and LEDR are negatively affected by UV radiation; this is shown by a lower rate of oxygen evolution/consumption. The effects of UV radiation and DCMU indicate that LEDR and photosynthesis have close regulatory interactions. The present results do not indicate that the effects of UV radiation on LEDR are separated from the effects on photosynthesis. The negative effects on photosynthesis followed by an inhibition of LEDR could be due to a decrease of intracellular pools of photosynthate. Dark respiration is not affected to the same degree as photosynthesis and LEDR. Photorespiration probably acts as a protection against photoinhibition, which makes
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it possible for the cells of E. gracilis to restore a higher photosynthetic capacity.
Acknowledgements ¨ and The author acknowledges Professor Lars Olof Bjorn Dr Ian Max Møller for helpful comments, Ruth Berglund for help with the manuscript and Marie Andersson for skillful technical assistance. This work was supported by Mid Sweden University.
References [1] D.T. Shindell, D. Rind, P. Lonergan, Increased polar stratospheric ozone losses and delayed eventual recovery owing to increasing greenhouse-gas concentrations, Nature 392 (1998) 589–592. [2] L.A. Franklin, R.M. Forster, The changing irradiance environment: consequences for marine macrophyte physiology, productivity and ecology, Eur. J. Phycol. 32 (1997) 207–232. [3] P.J. Neale, R.F. Davis, J.J. Cullen, Interactive effects of ozone depletion and vertical mixing on photosynthesis of Antarctic phytoplankton, Nature 392 (1998) 585–589. [4] D. Hanelt, C. Wiencke, W. Nultsch, Influence of UV radiation on the photosynthesis of Arctic macroalgae in the field, J. Photochem. Photobiol. B: Biol. 38 (1997) 40–47. ´ [5] F.L. Figueroa, J. Mercado, C. Jimenes, S. Salles, J. Aguilera, M.P. ´ ¨ ´ Sanchez-Saavedra, M. Lebert, D-P. Hader, O. Montero, L. Lubian, Relationship between bio-optical characteristics and photoinhibition of phytoplankton, Aqua. Bot. 59 (1997) 237–251. ¨ [6] E. Tyystjarvi, Photoinhibition — struggle between damage and repair of photosystem II. Thesis, University of Turku, ISBN 952-90-4519-0 (1993). [7] R. Shyam, A.S. Raghavendra, P.V. Sane, Role of dark respiration in photoinhibition of photosynthesis and its reactivation in the cyanobacterium Anacystis-nidulans, Physiol. Plant. 88 (1993) 452–466. [8] K.K. Singh, R. Shyam, P.V. Sane, Reactivation of photosynthesis in the photoinhibited green alga Chlamydomonas reinhardtii: role of dark respiration and of light, Photosyn. Res. 49 (1996) 11–20. [9] S. Kromer, Respiration during photosynthesis, Ann. Rev. Plant. Phys. Plant. Mol. Biol. 46 (1995) 45–70. [10] X. Xue, D.A. Gauthier, D.H. Turpin, H.G. Weger, Interactions between photosynthesis and respiration in the green algae Chlamydomonas reinhardtii, Plant. Physiol. 112 (1996) 1005–1014. [11] A. Checcuci, G. Colombetti, R. Ferrara, F. Lenci, Action spectra for photoaccumulation of green and colorless Euglena: evidence for identification of receptor pigments, Photochem. Photobiol. 23 (1976) 51– 54. [12] D.O. Hall, K.K. Rao, Photosynthesis, 5th edn., Cambridge University Press, Cambridge, 1994. [13] O. Holm-Hansen, D. Lubin, E.W. Helbling, Ultraviolet radiation and its effects on organisms in aquatic environments, in: A.R. Young, L.O. ¨ J. Moan, W. Nultsch (Eds.), Environmental UV Photobiology, Bjorn, Plenum, New York, 1993, pp. 379–425. [14] K. Garde, Effects of ultraviolet-B radiation on marine planktonic organisms, and the impact on the structure and function of pelagic ecosystems. Thesis (1998), The International Agency for 14C determination, VKI, Institute for the Water Environments Freshwater Biological Laboratory, University of Copenhagen. ¨ ¨ [15] D.-P. Hader, M. Porst, H. Herrmann, J. Schafer, R. Santas, Photosynthesis of the mediterranean green alga Caulerpa prolifera measured in the field under solar irradiation, J. Photochem. Photobiol. B: Biol. 37 (1997) 66–73.
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[20] P.G. Falkowski, A. Dubinsky, G. Santostefano, Light-enhanced dark respiration in phytoplankton, Verh. Internat. Verein. Limnol. 22 (1985) 2830–2833. [21] A.S. Raghavendra, K. Padmasree, K. Saradevi, Interdependence of photosynthesis and respiration in plant cells: interactions between chloroplasts and mitochondria, Plant Sci. 97 (1994) 1–14. [22] J.W. Poskuta, E. Parys, E. Romanowska, Toxicity of lead to photosynthesis, accumulation of chlorophyll, respiration and growth of Chlorella pyrenoidosa. Protective role of dark respiration, Acta Physiol. Plant. 18 (1996) 165–171. [23] R.C. Leegood, P.J. Lee, M.D. Adcock, R.E. Hausler, ¨ The regulation and control of photorespiration, J. Exp. Bot. 46 (1995) 1397–1414.
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Interactions between photosynthesis and ‘light-enhanced dark respiration’ (LEDR) in the flagellate Euglena gracilis after irradiation with ultraviolet radiation Nils G.A. Ekelund * ¨ ¨ Mid Sweden University, Department of Applied Science, S-871 88 Harnosand, Sweden Received 21 April 1999; accepted 14 February 2000
Abstract The effects of ultraviolet radiation (UV-A, 315–400 nm plus UV-B, 280–315 nm) on photosynthesis and ‘light-enhanced dark respiration’ (LEDR) in Euglena gracilis have been investigated by using light pulses (80 s) with increasing photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 and dark periods between the light pulses. LEDR is estimated as the maximum rate of oxygen consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate. Without any exposure to UV radiation, the photosynthetic rate and LEDR increase with increasing photon fluence rate. After 20 and 40 min exposures to UV radiation, the photosynthetic rate and LEDR as functions of photon fluence rate are reduced. After a 20 min UV treatment respiration is greater than photosynthesis after the first light pulse of 59 mmol my2 sy1 radiation, and especially at higher photon fluence rates photosynthesis is lower than the control values. The inhibitory effects of UV radiation on photosynthetic rate and LEDR are greater after a 40 min UV exposure than after a 20 min exposure. Only at 600 mmol my2 sy1 is the rate of oxygen evolution greater than that of oxygen consumption after a 40 min UV treatment. Both photosynthetic rate and LEDR are inhibited by the photosynthetic inhibitor DCMU (10y5 M) in a similar way, which indicates close regulatory interactions between photosynthesis and LEDR. Potassium cyanide (KCN) inhibits dark respiration more than it inhibits LEDR. Dark respiration is not affected to the same degree by UV radiation as are photosynthesis and LEDR. q2000 Elsevier Science S.A. All rights reserved. Keywords: Euglena gracilis; Light-enhanced dark respiration (LEDR); Photoinhibition; Photosynthesis; Respiration; Ultraviolet (UV) radiation
1. Introduction Recent studies have shown that the atmospheric ozone layer is still decreasing above both the Arctic and the Antarctic due to increasing concentrations of greenhouse gases [1]. The result will be that more biologically damaging ultraviolet-B (UV-B, 280–315 nm) radiation will reach the Earth9s surface in the near future. Most of the current knowledge of UV-B effects on phytoplankton and macroalgae is based on the inhibitory effects on photosynthesis [2]. These inhibitory effects are caused either by direct damage to photosynthetic components, which results in a lower rate of photosynthesis, or by damage to DNA, which results in the formation of photoproducts. The degree of inhibition observed when studying phytoplankton depends on the vertical mixing of the water column and the depth of the mixed layer [3]. There are several ways in which phytoplankton can protect them* Corresponding author.
selves against high UV-B irradiances, for example, via pigmentation and different enzyme mechanisms [2]. Photoinhibition is caused by high levels of photosynthetically active radiation (PAR); it can act as a protective mechanism where photosynthesis is downregulated [4]. Photoinhibition is often determined as the decrease in oxygen production and can be viewed as a regulatory mechanism rather than as a simple fault in the photosynthetic machinery [5,6]. This has been shown in macroalgae where the photosynthetic efficiency decreases with an increase in solar radiation at noon [4]. The possibility of repairing damage to photosynthesis is low for phytoplankton in short-term experiments and cold, deeply mixed surface layers [3]. Also, very low rates of respiration and growth in light-limited phytoplankton make the photoinhibitory effects more severe if these phytoplankton are suddenly exposed to high levels of irradiance. The importance of respiration in the repair mechanisms of inhibition has rarely been taken into consideration when studying the effects of UV radiation on phytoplankton, although
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it has been shown that dark respiration has a key role in the inhibition and reactivation of photosynthesis in the cyanobacterium Anacystis nidulans [7]. In Chlamydomonas reinhardtii the susceptibility to inhibition increased when dark respiration was inhibited by potassium cyanide (KCN) [8]. Not only dark respiration but also respiration in light (photorespiration) might prevent inhibition [9]. Earlier investigations indicated that ‘light-enhanced dark respiration’ (LEDR), which in the present investigation is estimated as the maximum rate of oxygen consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate, is a photosynthesis-dependent phenomenon and that it is a function of both the duration of illumination and the photon fluence rate [10]. The aim of this investigation was to study the role of LEDR in relation to photosynthesis in the flagellate Euglena gracilis, after exposure to ultraviolet radiation.
2. Materials and methods 2.1. Organism and culture conditions The flagellate E. gracilis strain Z (obtained from Professor ¨ ¨ Botanik und Pharmazeutische BioD.-P. Hader, Institut fur logie, Erlangen, Germany) was grown in 100 ml Erlenmeyer flasks in a freshwater medium described by Checcuci et al. [11]. The cells were grown in a cultivation cabinet (TERMAKS 6395 F/FL, AB Nino Lab., Sweden) at 208C with a 16 h/8 h light–dark cycle and a photon fluence rate of 90 mmol my2 sy1 (LI-COR quantum sensor, model LI-189) in the visible region (400–700 nm). 2.2. Ultraviolet radiation treatments Quartz glass bottles containing cells of E. gracilis were exposed for 20 and 40 min to UV-A radiation of 1.02 W my2 plus UV-B radiation of 0.73 W my2. UV-A and UV-B radiation were measured with an IL 1400A broad-band radiometer (International Light, Newburyport, MA, USA)
equipped with detectors for UV-A (Wa6259, a12667) and UV-B (W a6272, a12618). Ultraviolet radiation was obtained from two 40 W sunlamps (FS40, Westinghouse Electricity, Lamp Division, Bloomfield, NJ, USA). The ultraviolet radiation was filtered through a cellulose acetate (CA) film (0.13 mm thickness) to remove shorter-wavelength (less than 290 nm) components not encountered in nature. The CA was preburnt for 48 h at a distance of 1 m from four UV lamps in order to minimize changes of the filter properties. 2.3. Measurements of photosynthesis and respiration Photosynthesis and respiration were measured as oxygen evolution and consumption with a Light Pipette (Brammer, Illuminova, Uppsala, Sweden). The instrument consists of a light source that is connected to a cuvette with a micro-oxygen electrode (MI-730, Microelectrodes, 298 Rockingham Rd., Londonderry, Northern Ireland, UK), a quantum sensor, a temperature-controlled water bath and a computer. The light source delivers precise photon fluence rates of 0 to 3500 mmol my2 sy1. The rate of oxygen evolution was calculated assuming an ambient O2 concentration of 0.276 mmol mly1, which in this investigation corresponds to 100%. After centrifugation (30 s, 4000 rpm), the cells of E. gracilis were placed in the cuvette and each measurement of oxygen evolution/consumption lasted 20 min. The experiments were replicated three times. Chlorophyll was determined by extraction in 80% acetone solution and the absorbance was measured with a spectrophotometer (Perkin–Elmer Lambda Bio 20 UV–Vis spectrometer) at 652 nm [12]. In order to measure oxygen evolution and oxygen consumption during the same experiment, the cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses (Fig. 1). Each light pulse and dark period lasted for 80 s, except for the dark periods at the beginning and at the end of each experiment, which lasted for 90 s. The different time periods of light pulses and darkness were controlled by a computer. When dark
Fig. 1. Change in O2 concentration (% (mg Chl mly1)y1 sy1, bold curve) when the cells of E. gracilis were given six different light pulses with photon fluence rates (dashed curve) of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses. The photosynthetic rate was estimated by measuring the average values of O2 evolution during the light pulses, and LEDR (‘light-enhanced dark respiration’) was estimated as the maximum rate of O2 consumption after a period of light minus the rate of O2 consumption 30 s after the maximum rate (dark respiration).
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Fig. 2. Changes in O2 concentration (% (mg Chl mly1)y1 sy1, bold curve) and the amount of oxygen (% oxygen, thin curves) when the cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses. Before the measurements the cells were exposed to no UV radiation (control, A), 20 min of UV radiation (B), 40 min UV radiation (C) and DCMU (10y5 M, D).
3. Results
respiration was measured, no light pulses were given during a 20 min period. The experiments were carried out at 208C. The calculations of the photosynthetic rate were carried out by measuring the average values of oxygen evolution during the light pulses except for the first few seconds of transition (Fig. 1). DCMU (3-(39,49-dichlorophenyl)-1,1,dimethylurea) at a concentration of 10y5 M was used as an inhibitor of photosynthesis. For dark respiration the inhibitor used was KCN (potassium cyanide) at a concentration of 10 mM. LEDR was estimated as the maximum rate of O2 consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate (Fig. 1).
In the control experiments, without ultraviolet radiation, the cells of E. gracilis responded by increasing the rate of oxygen evolution with increased photon fluence rate (Fig. 2(A) and Fig. 3). During the dark periods the rate of oxygen consumption increased with higher photon fluence rates, with a sharp peak in oxygen consumption immediately after the light was turned off (Fig. 2(A)). These sharp peaks at higher photon fluence rates indicate that LEDR is a function of the photon fluence rate (Fig. 4). In the same experiment it was shown that the absolute value of oxygen in the cuvette
Fig. 3. Photosynthetic rates for E. gracilis at six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1. The photosynthetic rate was estimated by measuring the average values of oxygen evolution during the light pulses. Before the measurements the cells were exposed to no UV radiation (control), 20 min of UV radiation (UV20), 40 min of UV radiation (UV40) and DCMU (10y5 M, DCMU). Error bars represent standard deviations (SDs) of three replicated experiments.
Fig. 4. LEDR (‘light-enhanced dark respiration’) of E. gracilis estimated as the maximum rate of O2 consumption after a period of light minus the rate of oxygen consumption 30 s after the maximum rate. Before the measurements the cells were exposed to no UV radiation (control), 20 min of UV radiation (UV20), 40 min of UV radiation (UV40), DCMU (10y5 M, DCMU) and KCN (10 mM, KCN).
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increased during the 20 min from about 95 to 120% (Fig. 2(A)); distilled water at 208C showed 100% oxygen). After a 20 min UV irradiation, both oxygen evolution and consumption are inhibited (Fig. 2(B) and Fig. 3)). At the first light pulse of 59 mmol my2 sy1 respiration is the dominating process, and at higher photon fluence rates the levels of photosynthesis are lower than the control values. LEDR, and the amount of oxygen, decreased after 20 min of UV radiation (Fig. 2(B) and Fig. 4). With a 40 min UV irradiation the inhibition of photosynthesis was greater than after a 20 min one (Fig. 2(C) and Fig. 3). The rate of oxygen evolution was first greater than the rate of oxygen consumption at 595 mmol my2 sy1 (Fig. 2(C)). The amount of oxygen decreased from about 90 to 30%, and the LEDR was greatly inhibited after 40 min of UV radiation (Fig. 2(C) and Fig.
4). With DCMU (10y5 M) photosynthesis was totally inhibited for all light pulses and the amount of oxygen decreased from about 100 to nearly 0% (Fig. 2(D) and Fig. 4). When dark respiration alone was measured in 20 min darkness, the rate and amount of oxygen consumption slowly decreased without any treatment with UV radiation (Fig. 5(A)). The rate of oxygen consumption after a 20 min UV irradiation showed the same pattern as for the control but after a 40 min UV irradiation the amount of oxygen reached the zero level after about 16 min (Fig. 5(B) and (C)). However, the level of oxygen at the beginning of the experiment with a 40 min UV exposure was lower than the control. The average values of the photosynthetic rates showed that the photosynthetic rate increased with increasing photon fluence rate in the control (Fig. 3). However when the cells
Fig. 5. Dark respiration measured during 20 min darkness after no UV radiation (control, A), 20 min of UV radiation (B) and 40 min of UV radiation (C). Respiration is expressed as oxygen consumption (change in O2 concentration, % (mg Chl mly1)y1 sy1, bold curve), and the amount of oxygen in the cuvette as % oxygen (thin curves).
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Fig. 6. Change in O2 concentration (% (mg Chl mly1)y1 sy1, dashed curve) and the amount of oxygen (thin curve) after treatment with 10 mM KCN. The cells of E. gracilis were given six different light pulses with photon fluence rates of 59, 163, 600, 1180, 2080 and 3340 mmol my2 sy1 with dark periods between the light pulses.
were treated with UV radiation, the photosynthetic rate decreased and differences between ‘control’ and ‘treated’ organisms increased with longer period of UV irradiation (Fig. 3). After both 20 and 40 min exposures to UV radiation, the maximum photosynthetic rate was reached at a photon fluence rate of 600 mmol my2 sy1 (Fig. 3). In contrast to the photosynthetic rates, LEDR increased at higher photon fluence rates in the control and after a 20 min UV irradiation (Fig. 4). However after a 40 min UV irradiation and when treated with DCMU, the levels of LEDR were much lower (Figs. 3 and 4). By using KCN, it was shown that dark respiration was inhibited, but LEDR and photosynthesis were not affected as much as dark respiration (Figs. 4 and 6). The absolute value of oxygen showed only small variations between the light and dark periods, which indicates that dark respiration was inhibited during the dark periods and that oxygen evolution and consumption due to photosynthesis and LEDR were not affected by KCN (Fig. 6).
4. Discussion and conclusions The influence of UV radiation on aquatic organisms has been studied in several investigations [13–15]. Most of the results deal with the inhibitory effects on photosynthesis. The possibility of recovering from UV stress depends not only on the duration and degree of UV irradiation but also on the ability of organisms to protect themselves from UV radiation. Hanelt et al. [4] showed that inhibitory effects of UV radiation on Arctic macroalgae caused a delay in the recovery processes in the afternoon and evening, rather than an inhibitory effect on photosynthesis in the morning. The protection mechanism that has been studied most is the amount of pro-
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tecting pigments and dark and light repair mechanisms, like the turnover rate of the D1 protein in photosynthesis [2]. When the cyanobacterium Anacystis nidulans was exposed to photoinhibitory light, the susceptibility to photoinhibition increased when dark respiration was inhibited. This indicates that dark respiration plays an important role in the process of photoinhibition of photosynthesis [7]. In phytoplankton, marine benthic algae and sea grasses, no effects of UV-B radiation on respiration have been observed [16]. The present study also shows that dark respiration is less affected by UV radiation than photosynthesis (Figs. 2, 5 and 6). In the experiments with 40 min UV exposure, the oxygen level was reduced to zero after about 16 min of darkness, which made it impossible for the cells to continue with dark respiration (Fig. 5). This was due to a lower concentration of oxygen at the beginning of the experiments in comparison with the control experiments. One way to reduce this problem would be to do the experiments with less-dense samples. However, the rate of oxygen consumption was about the same, independent of UV radiation. When studying the level of respiration in connection with light pulses, it was found to be almost constant before and after the first and second light pulses, but after the third light pulse with a photon fluence rate of 600 mmol my2 sy1, the rate of respiration increased (Fig. 2(A)). This increase is due to the additive effects of LEDR, which could be seen as a sharp peak in oxygen consumption immediately after the light was turned off. Depending on the photon fluence rate, the level of respiration will gradually decrease from a higher level to the rate of dark respiration that was documented before the first light pulse (Fig. 2(A)). The rate of LEDR increases as the concentration of oxygen is increased; this is
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shown in the control experiments, where the oxygen concentration at the end of the experiment was found to be about 120% (Fig. 2(A)). The rate of LEDR has earlier been shown to be a good estimate of the rate of respiratory oxygen consumption in the preceeding light period (photorespiration) [9]. Without any photorespiratory activity, the photoinhibition of photosynthesis would probably have been greater at the light pulses with irradiances higher than 600 mmol my2 sy1. In Phaseolus vulgaris linear electron transport decreased when the oxygen concentration was reduced from 21 to 2% in order to inhibit photorespiration, but the photoinhibitory effects on photosystem II did not increase due to drought conditions [17]. In the present study with UV radiation the levels of respiration were, in general, lower than the levels before the light pulses. This indicates that photorespiration could play a significant role in the rate of respiration or that the amount of photosynthate regulates dark respiration (Fig. 2(B) and (C)). In microalgae the rate of respiration often decreases from an initially high rate in the transition from light to darkness to a much lower rate after a period in darkness [18,19]. Beardall et al. [19] have shown that the phenomenon of LEDR is widespread in microalgae and that the respiration rates were enhanced by 10–140% following exposure to a high photon flux. They speculated that the enhanced LEDR could be a consequence of repair of photodamage [19]. However, this enhanced respiration was demonstrated to be inhibited by 92% by 100 mM CNy, which is different from the present results where the inhibitory effect of KCN on LEDR was about 40% at the highest photon fluence rate (Fig. 4) [20]. In leaf protoplasts it has also been shown that LEDR is stimulated by bicarbonate and inhibited by the photosynthetic inhibitor DCMU [21]. Respiration also protects leaf protoplasts against photoinhibition and it is the oxidative electron transport and phosphorylation that are important in these mechanisms of protection against high light intensities [21]. In another investigation dark respiration in Chlorella pyrenoidosa was stimulated by lead (Pb), whereas photosynthesis was inhibited [22]. The increased dark respiration was suggested to compensate the demand for energy to carry out metabolic and growth processes when C. pyrenoidosa was stressed by high levels of Pb [22]. Also photorespiration could act as a protection against photohinibition by an efficient consumption of ATP and reductant [23]. In summary, both the increase in photosynthetic rate and LEDR are negatively affected by UV radiation; this is shown by a lower rate of oxygen evolution/consumption. The effects of UV radiation and DCMU indicate that LEDR and photosynthesis have close regulatory interactions. The present results do not indicate that the effects of UV radiation on LEDR are separated from the effects on photosynthesis. The negative effects on photosynthesis followed by an inhibition of LEDR could be due to a decrease of intracellular pools of photosynthate. Dark respiration is not affected to the same degree as photosynthesis and LEDR. Photorespiration probably acts as a protection against photoinhibition, which makes
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it possible for the cells of E. gracilis to restore a higher photosynthetic capacity.
Acknowledgements ¨ and The author acknowledges Professor Lars Olof Bjorn Dr Ian Max Møller for helpful comments, Ruth Berglund for help with the manuscript and Marie Andersson for skillful technical assistance. This work was supported by Mid Sweden University.
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