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Acclimation of chlorophyll a and carotenoid levels to different irradiances in four freshwater cyanobacteria
ARTICLE IN PRESS Journal of Plant Physiology 163 (2006) 709—716
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Acclimation of chlorophyll a and carotenoid levels to different irradiances in four freshwater cyanobacteria Michael Schagerl, Brigitte Mu ¨ller Department Marine Biology, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria Received 27 October 2004; accepted 29 September 2005
KEYWORDS Carotenoid; Cyanobacteria; Photoacclimation; Photoprotection; Zeaxanthin
Summary This study investigated carotenoid and chlorophyll a (Chl-a) contents under two different growth irradiances in four freshwater cyanobacterial strains. We found an increased weight ratio of zeaxanthin to Chl-a after exposure to high irradiances over several days. Two out of four strains showed higher zeaxanthin amounts on a biomass basis as well. It appears that cyanobacteria enhance their carotenoid pool in response to high light conditions, as increased production of other carotenoids with photoprotective abilities has also been observed under high irradiance levels. Cyanobacteria do not possess the violaxanthin cycle, which enables a rapid reversible conversion from violaxanthin into zeaxanthin and functioning as a quencher of excessive energy, and elevated zeaxanthin concentrations could therefore be seen as an adaptive strategy against excess light energy. Some differences in the acclimation pattern were revealed between different cyanobacteria. Anabaena torulosa contained higher amounts of every carotenoid, while Nostoc sp. mainly increased zeaxanthin, and myxoxanthophyll. Anabaenopsis elenkinii produced exceptionally high amounts of myxoxanthophyll and b-carotene under higher irradiances. Anabaena cylindrica generally showed less variation of carotenoids under different irradiances. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviations: AFDM, Ash-free dry mass; Chl-a, Chlorophyll a; HI, High growth irradiances; LI, Low growth irradiances; rt, Retention time Corresponding author. Tel.: +43 1 4277 54362; fax: +43 1 4277 9542. E-mail address: [email protected] (M. Schagerl).
Carotenoids, with more than 640 identified substances, comprise the largest class of naturally occurring pigments in organisms. Depending on subtle structural changes, they demonstrate a
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.09.015
ARTICLE IN PRESS 710 remarkable range of spectral characteristics (Hirschberg and Chamovitz, 1994), which result from the association of carotenoids with apoproteins, as well (Grabowski et al., 2001). Carotenoids are involved in photosynthesis in various ways: they act as light-harvesting pigments, contribute to the structure of thylakoid membranes and play an important role in photoprotection (Kohl and Nicklisch, 1988; Richter, 1996; Falkowski and Raven, 1997). Perhaps the most important functions of carotenoids include the dissipation of excess energy of excited chlorophyll and the elimination of reactive oxygen species (Lawlor, 2001). Carotenoid content in cyanobacteria varies considerably with growth conditions, though the predominant pigments remain the same (Hirschberg and Chamovitz, 1994). Cyanobacteria generally contain b-carotene, zeaxanthin, the ketocarotenoids echinenone and canthaxanthin, and the carotenoid-glycoside myxoxanthophyll. Myxoxanthophyll seems to be class-specific, as it has not been detected in any other algal group (Rowan, 1989; Van den Hoek et al., 1993; Schagerl and Donabaum, 2003). However, within cyanobacteria, some differences in pigment content may be observed. For example, marine cyanobacteria often contain a reduced pattern of carotenoids (Schagerl and Donabaum, 2003). Algae that are acclimated to high irradiance levels generally have relatively increased carotenoid:chlorophyll a (Chl-a) ratios (Falkowski and Raven, 1997). In higher plants, the xanthophyll zeaxanthin has been shown to mediate radiationless energy dissipation in the chlorophyll antenna (Demmig-Adams, 1990; Demmig-Adams and Adams III, 1997; Gilmore, 1997). Also, in higher plants such as chlorophyte algae and some chromophyte algae (e.g. phaeophyceae), zeaxanthin is formed rapidly via the xanthophyll cycle if irradiances are increased (Lohr and Wilhelm, 1999). In the remaining chromophyte algae, an alternative cycle is observed, where excessive light causes a reversible conversion of diadinoxanthin into diatoxanthin, which appears to be a quencher of excitation energy (Falkowski and Raven, 1997; Raven and Geider, 2003). Cyanobacteria lack the xanthophyll cycle, and zeaxanthin must be synthesized slowly via b-carotene (Demmig-Adams, 1990). Some authors have found a relatively constant cellular ratio of zeaxanthin in cyanobacteria. As increased zeaxanthin:Chl-a ratios are also found under high irradiances, a lowered cellular Chl-a content must be the cause. On the other hand, b-carotene appears to covary with Chl-a (Kana et al., 1988; Moore et al., 1995; MacIntyre et al., 2002). Some
M. Schagerl, B. Mu ¨ller investigations support a photoprotective function of myxoxanthophyll or canthaxanthin in cyanobacteria (Raps et al., 1983; Leisner et al., 1994; Steiger et al., 1999; Lakatos et al., 2001). This study investigated the Chl-a and carotenoid content of four freshwater cyanobacteria with respect to variations in growth under different irradiances. Some carotenoids, such as zeaxanthin or echinenone, are used to estimate cyanobacterial abundances in water bodies. Therefore, knowledge of the range under which these carotenoids occur in cyanobacteria is of special importance. This investigation aims to contribute further knowledge concerning cyanobacterial carotenoid content.
Materials and methods Plant material The cyanobacteria Anabaena cylindrica Lemm. (strain number ASW 01033), Anabaenopsis elenkinii V. Miller (ASW 01057), Anabaena torulosa (Carm.) Lagerh. (ASW 01023) and Nostoc sp. (ASW 042) were grown as unialgal batch cultures in a nutrient solution according to Ju ¨ttner (1976). The cultures were kept at 20 1C and under continuous light supply (standard fluorescent tubes, Philips, TL M 40W/84 RS) at 120 mmol m2 s1 (HI ¼ high growth irradiance) and at 15 mmol m2 s1 (LI ¼ low growth irradiance), respectively. For each species, two culture vessels (HI, LI) were used.
Harvesting In order to observe the development of the cultures, samples were taken at intervals of 1–3 days depending on the growth rate.
Pigments Algal suspensions were collected on Whatman GF/C filters. After freezing and grounding of the filters for 30 s in 90% acetone by a potterhomogeniser (Elfechiem) under dim light conditions, extraction was carried out in the dark at 4 1C for 12 h. After centrifugation for 10 min at 2500 rpm (Jouan MR 22), the supernatant was transferred to the HPLC system (Merck-Hitachi) with an autosampler (AS-4000), a gradient-pump (L-6200), a RPcolumn (Merck Superspher 100 RP-18 LichroCART 250-4, thermostated at 35 1C) and an UV-VISdetector (L-4250). After dilution of the samples by 30% with distilled water, 50 ml were injected.
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Solvents and analytical gradient protocol were carried out according to Wright and Jeffrey (1997). Peaks were detected at 440 nm and identified by comparison of retention times, peak maxima, band ratios and shoulders of peaks with literature (Foppen, 1971; Mantoura and Llewellyn, 1983; Wright and Shearer, 1984; Wright et al., 1991; Jeffrey et al., 1997), or by comparison with absorption spectra of authentic standards (DHI Bioproducts, Denmark).
Biomass For determination of ash-free dry mass (AFDM), which represents the organic component, samples were harvested on Whatman GF/F filters. AFDM was estimated by the difference between dried material at 95 1C and combustion at 450 1C. Cell counts and biovolume estimations were carried out with the inverted microscope technique according to Utermo ¨hl (1958; Nikon, Diaphot).
Statistics To test for significant differences (po0.001), non-parametric Mann–Whitney U-tests were performed with SPSS 10.0. We took data from the logarithmic growth phase of single batch cultures and pooled the results obtained from different times and different cells (independent treatment, n ¼ 6).
Results A representative chromatogram of A. elenkinii is shown in Fig. 1. Peak 1 represents an unidentified
Anabaenopsis elenkinii
4
mAUFS
5
1
6
2 3
0
14
16
18 20 retention time (min)
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24
Figure 1. Chromatogram of a pigment extract of Anabaenopsis elenkinii (high growth irradiance). Peak 1: unknown (retention time rt: 13.96 min); peak 2: myxoxanthophyll (rt: 15.19 min); peak 3: zeaxanthin (rt: 17.33 min); peak 4: chlorophyll a (rt: 19.27 min); peak 5: echinenone (rt: 19.98 min); peak 6: b-carotene (rt: 22.14 min).
carotenoid, which was detected in A. cylindrica and in the HI cultures of A. torulosa in trace amounts. The remaining peaks were classified as myxoxanthophyll, zeaxanthin, Chl-a, echinenone and b-carotene. In all strains, canthaxanthin was found in trace amounts. Nostoxanthin and caloxanthin were additionally detected in Nostoc sp. subsequently diluting after myxoxanthophyll. All cultures grew more slowly at LI, and did not attain as high concentrations as the HI cultures (Fig. 2). Highest biomass was reached by A. torulosa with approximately 1900 mg Chl-a l1 and 1000 mg total carotenoids l1. With respect to Chl-a based on AFDM, Nostoc sp. showed significantly increased amounts in the LI cultures, whereas differences of the remaining HI and LI cultures were not significant (Fig. 3c). HI and LI cells did not differ significantly in cell weights (Fig. 3d). In all HI samples, the weight ratio of total carotenoids:Chl-a was significantly higher than in the LI cultures (Fig. 3a). In A. elenkinii, total carotenoids frequently exceeded Chl-a (values over 100%), whereas carotenoids of HI A. cylindrica and HI A. torulosa reached about half of the Chl-a content. The LI cultures of A. cylindrica, A. torulosa and A. elenkinii showed less variation relative to the HI cultures and the total carotenoids were between 20% and 50% of Chl-a. The difference between the HI and LI samples of Nostoc sp. was smaller than in the other strains, as total carotenoids were approximately 25% of Chl-a content in HI and 15% in LI. The carotenoid:AFDM ratios showed fewer differences between HI and LI samples than did the Chl-a based ratios (Fig. 3b) and only for A. torulosa and A. elenkinii were significant differences detected. Compared to LI, myxoxanthophyll per Chl-a was significantly higher in all HI cultures, and generally showed the largest differences (Fig. 4c). With the exception of A. cylindrica, ratios of b-carotene, zeaxanthin and echinenone per Chl-a were also higher in HI cultures (Fig. 4). Caloxanthin, nostoxanthin and canthaxanthin were found only in trace amounts. When examining b-carotene to AFDM, a significant increase was detected only in HI of A. torulosa (Fig. 5a). Zeaxanthin and echinenone were higher in HI cultures of A. torulosa and Nostoc sp.; myxoxanthophyll attained significantly increased ratios in A. torulosa and A. elenkinii under high irradiances. Generally, myxoxanthophyll was the most abundant carotenoid in all HI cultures with the exception of Nostoc sp., which showed higher amounts of b-carotene (Figs. 5a and c).
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250 Pigment [µg L-1]
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Figure 2. Time-dependent change of chlorophyll a (circles) and total carotenoids (triangles) of cultures grown in high irradiance (open symbols) and low irradiance (closed symbols).
0.6 b
a Carotenoids AFDM-1 [%]
100 80 60 40 20 0 2.5
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d 100 AFDM cell-1 [pg]
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Figure 3. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of (a) total carotenoids per chlorophyll a; (b) total carotenoids per ash-free dry mass; (c) chlorophyll a per ash-free dry mass; (d) ash-free dry mass per cell.
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Figure 4. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of chlorophyll a: (a) b-carotene; (b) zeaxanthin; (c) myxoxanthophyll; (d) echinenone.
Discussion When comparing HI and LI treatments, low growth irradiances generally caused elevated Chla contents on an AFDM basis with the exception of A. torulosa (Fig. 3c). A significant increase of the carotenoid:Chl-a ratio was observed under higher growth irradiances (Fig. 3a). In A. torulosa and A. elenkinii carotenoids were also increased on an AFDM basis, indicating a cellular accumulation (Fig. 3b). This is in contrast to some authors, who have reported that the relative abundance of carotenoids, but not the total cellular content, vary with irradiance (Raps et al., 1983; Millie et al., 1990). One explanation may be that laboratory cultures usually grow under light conditions free of UV radiation, whereas under natural conditions UV radiation might enhance the synthesis of specific carotenoids (Rao et al., 1995; MacIntyre et al., 2002) favouring dissipation of excess energy (Paerl et al., 1983; Kohl and Nicklisch, 1988; Richter, 1996; Ha ¨der, 1999).
In addition to their photoprotective role, some carotenoids serve as accessory light-harvesting pigments as well. However, with an efficiency of only 30–40%, they absorb photons in the range between 450 and 570 nm and transfer the energy to the reaction centers. Some investigators report that carotenoids in cyanobacteria play only a minor role in light-harvesting (Kana et al., 1988), while others have found that enhanced carotenoid accumulation increases photosynthetic efficiency (Paerl et al., 1983). Therefore, it appears to be necessary to go further into the question of carotenoid patterns. A pigment which has proved to play a central role in photoprotection, especially in cormophytes and some green algae, is zeaxanthin (Gilmore and Yamamoto, 1993; Gilmore et al., 1995, 2000). Cyanobacteria are not capable of rapidly transforming zeaxanthin via the xanthophyll cycle when exposed to excess light because the epoxidized xanthophylls viola- and antheraxanthin are lacking. It can therefore be hypothesized that they will accumulate it slowly during longer periods
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Figure 5. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of ash-free dry mass: (a) bcarotene; (b) zeaxanthin; (c) myxoxanthophyll; (d) echinenone.
of high light exposure for radiationless energy dissipation. The results of our study strengthen this assumption, as all strains examined developed an increased zeaxanthin:Chl-a ratio under higher irradiances (Fig. 4b). With respect to A. torulosa and Nostoc sp., zeaxanthin per AFDM was significantly higher in the HI cultures as well (Fig. 5b). An accumulation of zeaxanthin on a biomass basis most probably caused this increase (the alternative would have been reduced Chl-a amounts). Whether zeaxanthin really plays a central role in radiationless energy dissipation of excess light in cyanobacteria still requires investigation. Some studies indicate this function, e.g. investigations on lichens. Cyanobacterial lichens without detectable amounts of zeaxanthin are highly sensitive to photooxidative damage. In some cases, phycobionts were able to synthesize zeaxanthin after transfer to high irradiances over several days. When compared, those specimens with zeaxanthin formation appeared to be much more tolerant toward excess light than those without zeaxanthin (Demmig-Adams, 1990; Demmig-Adams et al., 1990; Adams III et al., 1993).
However, investigations on Synechococcus have revealed that the cellular content of zeaxanthin remains constant, while b-carotene covaries with Chl-a (Kana et al., 1988; Samson et al., 1994; Moore et al., 1995; MacIntyre et al., 2002). For some cyanobacterial strains, the constant amount of zeaxanthin has been found to be consistent, with a zeaxanthin pool being primarily situated in the cytoplasmic or outer cell wall membrane, and therefore, not being capable of any photosynthetic energy transfer (Kana et al., 1988). In our study, zeaxanthin showed more variation compared to other carotenoids. We hypothesize that in the examined species zeaxanthin might occur in two different fractions, which are located in the cytoplasmic or outer cell wall membrane and in the photosystems as well. Our results show the large HI/LI differences in myxoxanthophyll with higher amounts at HI. These results have been found in other species as well (Raps et al., 1983; Mis´kiewicz et al., 2000). Moreover, in comparison to other pigments in cyanobacteria, myxoxanthophyll was found to be the most effective carotenoid in photoprotection of
ARTICLE IN PRESS Acclimation of pigments in cyanobacteria liposome assays, whereas echinenone has been shown to be the least effective (Steiger et al., 1999). Explanations for the high photoprotective effectiveness of myxoxanthophyll are its high degree of unsaturated bonds and its glycosidic nature (Steiger et al., 1999), but to our knowledge no details are known about the molecular mechanisms in living material. There is no doubt that cyanobacteria are capable of producing photoprotective compounds under high irradiances. Nevertheless, there would be a disadvantage in lacking a self-regulating mechanism like the xanthophyll cycle, for excess energy dissipation because the deactivation of these carotenoids is also slow upon return to low irradiances. Moreover, the threshold for the synthesis of photoprotective carotenoids is relatively low (Ibelings et al., 1994). In addition, the amount of Chl-a and the proportions of light-harvesting to photoprotective carotenoids are reduced under high irradiances. This leads to a decrease of the cellular absorption cross-section. As a consequence, a large amount of excitation energy might be lost under fluctuating irradiances in natural environments. A possible strategy to overcome this problem was found by Subramaniam et al. (1999). These authors discovered a reversible conversion of phycoerythrobilin to phycourobilin in the filamentous marine cyanobacterium Trichodesmium at high growth irradiances. This transformation can be seen as an alternative way to the xanthophyllcycle as phycourobilin dissipates excess energy as fluorescence. However, further studies must verify whether this mechanism is generally found in cyanobacteria or/and if other carotenoids function as photoprotectives as well.
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715 Foppen FH. Tables for the identification of carotenoid pigments. Chromatogr Rev 1971;14:133–298. Gilmore AM. Mechanistic aspects of xanthophyll cycledependent photoprotection in higher plant chloroplasts and leaves. Physiol Plant 1997;99:197–209. Gilmore AM, Yamamoto HY. Linear models relating xanthophylls and lumen acidity to non-photochemical fluorescence quenching. Evidence that antheraxanthin explains zeaxanthin-independent quenching. Photosynth Res 1993;35:67–78. Gilmore AM, Hazlett TL, Govindjee. Xanthophyll cycledependent quenching of photosystem II chlorophyll a fluorescence: Formation of a quenching complex with a short fluorescence lifetime. Proc Natl Acad Sci USA 1995;92:2273–7. Gilmore AM, Itoh S, Govindjee. Global spectral-kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley. Philos Trans R Soc London 2000;335:1371–84. Grabowski B, Cunningham Jr FX, Gantt E. Chlorophyll and carotenoid binding in a simple red algal light-harvesting complex crosses phylogenetic lines. PNAS 2001;98:2911–6. Ha ¨der D-P. Photosynthese. Stuttgart, New York: Georg Thieme Verlag; 1999. Hirschberg J, Chamovitz D. Carotenoids in cyanobacteria. In: Bryant DA, editor. The molecular biology of cyanobacteria. Dordrecht: Kluwer Academic Publishers; 1994. Ibelings BW, Kroon BMA, Mur LR. Acclimation of photosystem II in a cyanobacterium and a eukaryotic green alga to high and fluctuating photosynthetic photon flux densities, simulating light regimes induced by mixing lakes. New Phytol 1994;128:407–24. Jeffrey SW, Mantoura RFC, Wright SW, editors. Phytoplankton pigments in oceanography. France: Unesco Publishing; 1997. Ju ¨ttner F. b-Cyclocitral and alkanes in Microcystis (Cyanophyceae). Z Naturforsch 1976;31c:491–5. Kana TM, Glibert PM, Goericke R, Welschmeyer NA. Zeaxanthin and b-carotene in Synechococcus WH7803 respond differently to irradiance. Limnol Oceanogr 1988;33:1623–7. ¨ kophysiologie der Algen. Berlin: Kohl J-G, Nicklisch A. O Akademie-Verlag; 1988. Lakatos M, Bilger W, Bu ¨del B. Carotenoid composition of terrestrial cyanobacteria: response to natural light conditions in open rock habitats in Venezuela. Eur J Phycol 2001;36:367–75. Lawlor DW. Photosynthesis. Oxford: Bios Scientific Publishers; 2001. Leisner JMR, Bilger W, Czygan F-C, Lange OL. Light exposure and the composition of lipophilous carotenoids in cyanobacterial lichens. J Plant Physiol 1994;143:514–9. Lohr M, Wilhelm C. Algae displaying the diadinoxanthin cycle also possess the violoxanthin cycle. PNAS 1999;96:8784–9. MacIntyre HL, Kana TM, Anning T, Geider RJ. Photoacclimation of photosynthesis irradiance response
ARTICLE IN PRESS 716 curves and pigments in microalgae and cyanobacteria. J Phycol 2002;38:17–38. Mantoura RFC, Llewellyn CA. The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse— phase high—performance liquid chromatography. Anal Chim Acta 1983;151:297–314. Millie DF, Ingram DA, Dionigi CP. Pigment and photosynthetic responses of Oscillatoria agardhii (Cyanophyta) to photon flux density and spectral quality. J Phycol 1990;26:660–6. Mis´kiewicz E, Ivanov AG, Williams JP, Khan MU, Falk S, Huner NPA. Photosynthetic acclimation of the filamentous cyanobacterium, Plectonema boryanum UTEX 485, to temperature and light. Plant Cell Physiol 2000;41:767–75. Moore LR, Goericke R, Chisholm SW. Comparative physiology of Synechococcus and Prochlorococcus: influence of light and temperature on growth, pigments, fluorescence and absorptive properties. Mar Ecol Prog Ser 1995;116:259–75. Paerl HW, Tucker J, Bland PT. Carotenoid enhancement and its role in maintaining blue-green algal (Microcystis aeruginosa) surface blooms. Limnol Oceanogr 1983;28:847–57. Rao MV, Paliyath G, Ormrod DP. Differential responses of photosynthetic pigments, Rubisco activity, and Rubisco protein of Arabidopsis thaliana exposed to UVB and ozone. Photochem Photobiol 1995;62:727–35. Raps S, Wyman K, Siegelman HW, Falkowski PG. Adaption of the cyanobacterium Microcystis aeruginosa to light intensity. Plant Physiol 1983;72:829–32. Richter G. Biochemie der Pflanzen. Stuttgart, New York: Georg Thieme Verlag; 1996. Raven JA, Geider RJ. Adaptation, acclimation and regulation in algal photosynthesis. In: Larkum AW, Douglas SE, Raven JA, editors. Photosynthesis in algae. The Netherlands: Academic Publishers; 2003. p. 385–412.
M. Schagerl, B. Mu ¨ller Rowan KS. Photosynthetic pigments of algae. Cambridge: Cambridge University Press; 1989. Samson G, Herbert SK, Fork DC, Laudenbach DE. Acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus lacking iron superoxide dismutase. Plant Physiol 1994;105:287–94. Schagerl M, Donabaum K. Patterns of major photosynthetic pigments in freshwater algae. 1. Cyanoprokaryota, rhodophyta and cryptophyta. J Limnol 2003;39:35–47. Steiger S, Scha ¨fer L, Sandmann G. High-light-dependent upregulation and their antioxidative properties in the cyanobacterium Synechocystis PCC 6803. J Photochem Photobiol B: Biol 1999;52:14–8. Subramaniam A, Carpenter EJ, Karentz D, Falkowski PG. Bio-optical properties of the marine diazothrophic cyanobacteria Trichodesmuim spp. I. Absorption and photosynthetic action spectra. Limnol Oceanogr 1999;44:608–17. Utermo ¨hl UF. Zur Vervollkommnung der quantitativen Erfassung des Planktons. Verh Int Ver Limnol 1958;5:567–95. Van den Hoek C, Jahns HM, Mann DG. Algen. Stuttgart, New York: Georg Thieme Verlag; 1993. Wright SW, Jeffrey SW. High-resolution HPLC system for chlorophylls and carotenoids of marine phytoplankton. In: Jeffrey SW, Mantoura RFC, Wright SW, editors. Phytoplankton pigments in oceanography. France: UNESCO Publishing; 1997. Wright SW, Shearer JD. Rapid extraction and High performance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton. J Chromatogr 1984;294:281–95. Wright SW, Jeffrey SW, Mantoura RFC, Llewellyn CA, Bjornland T, Repeta D, Welschmeyer NA. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 1991;77:183–96.
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Acclimation of chlorophyll a and carotenoid levels to different irradiances in four freshwater cyanobacteria Michael Schagerl, Brigitte Mu ¨ller Department Marine Biology, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria Received 27 October 2004; accepted 29 September 2005
KEYWORDS Carotenoid; Cyanobacteria; Photoacclimation; Photoprotection; Zeaxanthin
Summary This study investigated carotenoid and chlorophyll a (Chl-a) contents under two different growth irradiances in four freshwater cyanobacterial strains. We found an increased weight ratio of zeaxanthin to Chl-a after exposure to high irradiances over several days. Two out of four strains showed higher zeaxanthin amounts on a biomass basis as well. It appears that cyanobacteria enhance their carotenoid pool in response to high light conditions, as increased production of other carotenoids with photoprotective abilities has also been observed under high irradiance levels. Cyanobacteria do not possess the violaxanthin cycle, which enables a rapid reversible conversion from violaxanthin into zeaxanthin and functioning as a quencher of excessive energy, and elevated zeaxanthin concentrations could therefore be seen as an adaptive strategy against excess light energy. Some differences in the acclimation pattern were revealed between different cyanobacteria. Anabaena torulosa contained higher amounts of every carotenoid, while Nostoc sp. mainly increased zeaxanthin, and myxoxanthophyll. Anabaenopsis elenkinii produced exceptionally high amounts of myxoxanthophyll and b-carotene under higher irradiances. Anabaena cylindrica generally showed less variation of carotenoids under different irradiances. & 2005 Elsevier GmbH. All rights reserved.
Introduction Abbreviations: AFDM, Ash-free dry mass; Chl-a, Chlorophyll a; HI, High growth irradiances; LI, Low growth irradiances; rt, Retention time Corresponding author. Tel.: +43 1 4277 54362; fax: +43 1 4277 9542. E-mail address: [email protected] (M. Schagerl).
Carotenoids, with more than 640 identified substances, comprise the largest class of naturally occurring pigments in organisms. Depending on subtle structural changes, they demonstrate a
0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2005.09.015
ARTICLE IN PRESS 710 remarkable range of spectral characteristics (Hirschberg and Chamovitz, 1994), which result from the association of carotenoids with apoproteins, as well (Grabowski et al., 2001). Carotenoids are involved in photosynthesis in various ways: they act as light-harvesting pigments, contribute to the structure of thylakoid membranes and play an important role in photoprotection (Kohl and Nicklisch, 1988; Richter, 1996; Falkowski and Raven, 1997). Perhaps the most important functions of carotenoids include the dissipation of excess energy of excited chlorophyll and the elimination of reactive oxygen species (Lawlor, 2001). Carotenoid content in cyanobacteria varies considerably with growth conditions, though the predominant pigments remain the same (Hirschberg and Chamovitz, 1994). Cyanobacteria generally contain b-carotene, zeaxanthin, the ketocarotenoids echinenone and canthaxanthin, and the carotenoid-glycoside myxoxanthophyll. Myxoxanthophyll seems to be class-specific, as it has not been detected in any other algal group (Rowan, 1989; Van den Hoek et al., 1993; Schagerl and Donabaum, 2003). However, within cyanobacteria, some differences in pigment content may be observed. For example, marine cyanobacteria often contain a reduced pattern of carotenoids (Schagerl and Donabaum, 2003). Algae that are acclimated to high irradiance levels generally have relatively increased carotenoid:chlorophyll a (Chl-a) ratios (Falkowski and Raven, 1997). In higher plants, the xanthophyll zeaxanthin has been shown to mediate radiationless energy dissipation in the chlorophyll antenna (Demmig-Adams, 1990; Demmig-Adams and Adams III, 1997; Gilmore, 1997). Also, in higher plants such as chlorophyte algae and some chromophyte algae (e.g. phaeophyceae), zeaxanthin is formed rapidly via the xanthophyll cycle if irradiances are increased (Lohr and Wilhelm, 1999). In the remaining chromophyte algae, an alternative cycle is observed, where excessive light causes a reversible conversion of diadinoxanthin into diatoxanthin, which appears to be a quencher of excitation energy (Falkowski and Raven, 1997; Raven and Geider, 2003). Cyanobacteria lack the xanthophyll cycle, and zeaxanthin must be synthesized slowly via b-carotene (Demmig-Adams, 1990). Some authors have found a relatively constant cellular ratio of zeaxanthin in cyanobacteria. As increased zeaxanthin:Chl-a ratios are also found under high irradiances, a lowered cellular Chl-a content must be the cause. On the other hand, b-carotene appears to covary with Chl-a (Kana et al., 1988; Moore et al., 1995; MacIntyre et al., 2002). Some
M. Schagerl, B. Mu ¨ller investigations support a photoprotective function of myxoxanthophyll or canthaxanthin in cyanobacteria (Raps et al., 1983; Leisner et al., 1994; Steiger et al., 1999; Lakatos et al., 2001). This study investigated the Chl-a and carotenoid content of four freshwater cyanobacteria with respect to variations in growth under different irradiances. Some carotenoids, such as zeaxanthin or echinenone, are used to estimate cyanobacterial abundances in water bodies. Therefore, knowledge of the range under which these carotenoids occur in cyanobacteria is of special importance. This investigation aims to contribute further knowledge concerning cyanobacterial carotenoid content.
Materials and methods Plant material The cyanobacteria Anabaena cylindrica Lemm. (strain number ASW 01033), Anabaenopsis elenkinii V. Miller (ASW 01057), Anabaena torulosa (Carm.) Lagerh. (ASW 01023) and Nostoc sp. (ASW 042) were grown as unialgal batch cultures in a nutrient solution according to Ju ¨ttner (1976). The cultures were kept at 20 1C and under continuous light supply (standard fluorescent tubes, Philips, TL M 40W/84 RS) at 120 mmol m2 s1 (HI ¼ high growth irradiance) and at 15 mmol m2 s1 (LI ¼ low growth irradiance), respectively. For each species, two culture vessels (HI, LI) were used.
Harvesting In order to observe the development of the cultures, samples were taken at intervals of 1–3 days depending on the growth rate.
Pigments Algal suspensions were collected on Whatman GF/C filters. After freezing and grounding of the filters for 30 s in 90% acetone by a potterhomogeniser (Elfechiem) under dim light conditions, extraction was carried out in the dark at 4 1C for 12 h. After centrifugation for 10 min at 2500 rpm (Jouan MR 22), the supernatant was transferred to the HPLC system (Merck-Hitachi) with an autosampler (AS-4000), a gradient-pump (L-6200), a RPcolumn (Merck Superspher 100 RP-18 LichroCART 250-4, thermostated at 35 1C) and an UV-VISdetector (L-4250). After dilution of the samples by 30% with distilled water, 50 ml were injected.
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Solvents and analytical gradient protocol were carried out according to Wright and Jeffrey (1997). Peaks were detected at 440 nm and identified by comparison of retention times, peak maxima, band ratios and shoulders of peaks with literature (Foppen, 1971; Mantoura and Llewellyn, 1983; Wright and Shearer, 1984; Wright et al., 1991; Jeffrey et al., 1997), or by comparison with absorption spectra of authentic standards (DHI Bioproducts, Denmark).
Biomass For determination of ash-free dry mass (AFDM), which represents the organic component, samples were harvested on Whatman GF/F filters. AFDM was estimated by the difference between dried material at 95 1C and combustion at 450 1C. Cell counts and biovolume estimations were carried out with the inverted microscope technique according to Utermo ¨hl (1958; Nikon, Diaphot).
Statistics To test for significant differences (po0.001), non-parametric Mann–Whitney U-tests were performed with SPSS 10.0. We took data from the logarithmic growth phase of single batch cultures and pooled the results obtained from different times and different cells (independent treatment, n ¼ 6).
Results A representative chromatogram of A. elenkinii is shown in Fig. 1. Peak 1 represents an unidentified
Anabaenopsis elenkinii
4
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5
1
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2 3
0
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18 20 retention time (min)
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Figure 1. Chromatogram of a pigment extract of Anabaenopsis elenkinii (high growth irradiance). Peak 1: unknown (retention time rt: 13.96 min); peak 2: myxoxanthophyll (rt: 15.19 min); peak 3: zeaxanthin (rt: 17.33 min); peak 4: chlorophyll a (rt: 19.27 min); peak 5: echinenone (rt: 19.98 min); peak 6: b-carotene (rt: 22.14 min).
carotenoid, which was detected in A. cylindrica and in the HI cultures of A. torulosa in trace amounts. The remaining peaks were classified as myxoxanthophyll, zeaxanthin, Chl-a, echinenone and b-carotene. In all strains, canthaxanthin was found in trace amounts. Nostoxanthin and caloxanthin were additionally detected in Nostoc sp. subsequently diluting after myxoxanthophyll. All cultures grew more slowly at LI, and did not attain as high concentrations as the HI cultures (Fig. 2). Highest biomass was reached by A. torulosa with approximately 1900 mg Chl-a l1 and 1000 mg total carotenoids l1. With respect to Chl-a based on AFDM, Nostoc sp. showed significantly increased amounts in the LI cultures, whereas differences of the remaining HI and LI cultures were not significant (Fig. 3c). HI and LI cells did not differ significantly in cell weights (Fig. 3d). In all HI samples, the weight ratio of total carotenoids:Chl-a was significantly higher than in the LI cultures (Fig. 3a). In A. elenkinii, total carotenoids frequently exceeded Chl-a (values over 100%), whereas carotenoids of HI A. cylindrica and HI A. torulosa reached about half of the Chl-a content. The LI cultures of A. cylindrica, A. torulosa and A. elenkinii showed less variation relative to the HI cultures and the total carotenoids were between 20% and 50% of Chl-a. The difference between the HI and LI samples of Nostoc sp. was smaller than in the other strains, as total carotenoids were approximately 25% of Chl-a content in HI and 15% in LI. The carotenoid:AFDM ratios showed fewer differences between HI and LI samples than did the Chl-a based ratios (Fig. 3b) and only for A. torulosa and A. elenkinii were significant differences detected. Compared to LI, myxoxanthophyll per Chl-a was significantly higher in all HI cultures, and generally showed the largest differences (Fig. 4c). With the exception of A. cylindrica, ratios of b-carotene, zeaxanthin and echinenone per Chl-a were also higher in HI cultures (Fig. 4). Caloxanthin, nostoxanthin and canthaxanthin were found only in trace amounts. When examining b-carotene to AFDM, a significant increase was detected only in HI of A. torulosa (Fig. 5a). Zeaxanthin and echinenone were higher in HI cultures of A. torulosa and Nostoc sp.; myxoxanthophyll attained significantly increased ratios in A. torulosa and A. elenkinii under high irradiances. Generally, myxoxanthophyll was the most abundant carotenoid in all HI cultures with the exception of Nostoc sp., which showed higher amounts of b-carotene (Figs. 5a and c).
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Figure 2. Time-dependent change of chlorophyll a (circles) and total carotenoids (triangles) of cultures grown in high irradiance (open symbols) and low irradiance (closed symbols).
0.6 b
a Carotenoids AFDM-1 [%]
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Figure 3. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of (a) total carotenoids per chlorophyll a; (b) total carotenoids per ash-free dry mass; (c) chlorophyll a per ash-free dry mass; (d) ash-free dry mass per cell.
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Figure 4. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of chlorophyll a: (a) b-carotene; (b) zeaxanthin; (c) myxoxanthophyll; (d) echinenone.
Discussion When comparing HI and LI treatments, low growth irradiances generally caused elevated Chla contents on an AFDM basis with the exception of A. torulosa (Fig. 3c). A significant increase of the carotenoid:Chl-a ratio was observed under higher growth irradiances (Fig. 3a). In A. torulosa and A. elenkinii carotenoids were also increased on an AFDM basis, indicating a cellular accumulation (Fig. 3b). This is in contrast to some authors, who have reported that the relative abundance of carotenoids, but not the total cellular content, vary with irradiance (Raps et al., 1983; Millie et al., 1990). One explanation may be that laboratory cultures usually grow under light conditions free of UV radiation, whereas under natural conditions UV radiation might enhance the synthesis of specific carotenoids (Rao et al., 1995; MacIntyre et al., 2002) favouring dissipation of excess energy (Paerl et al., 1983; Kohl and Nicklisch, 1988; Richter, 1996; Ha ¨der, 1999).
In addition to their photoprotective role, some carotenoids serve as accessory light-harvesting pigments as well. However, with an efficiency of only 30–40%, they absorb photons in the range between 450 and 570 nm and transfer the energy to the reaction centers. Some investigators report that carotenoids in cyanobacteria play only a minor role in light-harvesting (Kana et al., 1988), while others have found that enhanced carotenoid accumulation increases photosynthetic efficiency (Paerl et al., 1983). Therefore, it appears to be necessary to go further into the question of carotenoid patterns. A pigment which has proved to play a central role in photoprotection, especially in cormophytes and some green algae, is zeaxanthin (Gilmore and Yamamoto, 1993; Gilmore et al., 1995, 2000). Cyanobacteria are not capable of rapidly transforming zeaxanthin via the xanthophyll cycle when exposed to excess light because the epoxidized xanthophylls viola- and antheraxanthin are lacking. It can therefore be hypothesized that they will accumulate it slowly during longer periods
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Figure 5. Measurements of cultures grown at high irradiance (open plots) or low irradiance (shaded plots). 5th, 25th, 75th and 95th percentiles, and the median are shown. Weight ratios as a percentage of ash-free dry mass: (a) bcarotene; (b) zeaxanthin; (c) myxoxanthophyll; (d) echinenone.
of high light exposure for radiationless energy dissipation. The results of our study strengthen this assumption, as all strains examined developed an increased zeaxanthin:Chl-a ratio under higher irradiances (Fig. 4b). With respect to A. torulosa and Nostoc sp., zeaxanthin per AFDM was significantly higher in the HI cultures as well (Fig. 5b). An accumulation of zeaxanthin on a biomass basis most probably caused this increase (the alternative would have been reduced Chl-a amounts). Whether zeaxanthin really plays a central role in radiationless energy dissipation of excess light in cyanobacteria still requires investigation. Some studies indicate this function, e.g. investigations on lichens. Cyanobacterial lichens without detectable amounts of zeaxanthin are highly sensitive to photooxidative damage. In some cases, phycobionts were able to synthesize zeaxanthin after transfer to high irradiances over several days. When compared, those specimens with zeaxanthin formation appeared to be much more tolerant toward excess light than those without zeaxanthin (Demmig-Adams, 1990; Demmig-Adams et al., 1990; Adams III et al., 1993).
However, investigations on Synechococcus have revealed that the cellular content of zeaxanthin remains constant, while b-carotene covaries with Chl-a (Kana et al., 1988; Samson et al., 1994; Moore et al., 1995; MacIntyre et al., 2002). For some cyanobacterial strains, the constant amount of zeaxanthin has been found to be consistent, with a zeaxanthin pool being primarily situated in the cytoplasmic or outer cell wall membrane, and therefore, not being capable of any photosynthetic energy transfer (Kana et al., 1988). In our study, zeaxanthin showed more variation compared to other carotenoids. We hypothesize that in the examined species zeaxanthin might occur in two different fractions, which are located in the cytoplasmic or outer cell wall membrane and in the photosystems as well. Our results show the large HI/LI differences in myxoxanthophyll with higher amounts at HI. These results have been found in other species as well (Raps et al., 1983; Mis´kiewicz et al., 2000). Moreover, in comparison to other pigments in cyanobacteria, myxoxanthophyll was found to be the most effective carotenoid in photoprotection of
ARTICLE IN PRESS Acclimation of pigments in cyanobacteria liposome assays, whereas echinenone has been shown to be the least effective (Steiger et al., 1999). Explanations for the high photoprotective effectiveness of myxoxanthophyll are its high degree of unsaturated bonds and its glycosidic nature (Steiger et al., 1999), but to our knowledge no details are known about the molecular mechanisms in living material. There is no doubt that cyanobacteria are capable of producing photoprotective compounds under high irradiances. Nevertheless, there would be a disadvantage in lacking a self-regulating mechanism like the xanthophyll cycle, for excess energy dissipation because the deactivation of these carotenoids is also slow upon return to low irradiances. Moreover, the threshold for the synthesis of photoprotective carotenoids is relatively low (Ibelings et al., 1994). In addition, the amount of Chl-a and the proportions of light-harvesting to photoprotective carotenoids are reduced under high irradiances. This leads to a decrease of the cellular absorption cross-section. As a consequence, a large amount of excitation energy might be lost under fluctuating irradiances in natural environments. A possible strategy to overcome this problem was found by Subramaniam et al. (1999). These authors discovered a reversible conversion of phycoerythrobilin to phycourobilin in the filamentous marine cyanobacterium Trichodesmium at high growth irradiances. This transformation can be seen as an alternative way to the xanthophyllcycle as phycourobilin dissipates excess energy as fluorescence. However, further studies must verify whether this mechanism is generally found in cyanobacteria or/and if other carotenoids function as photoprotectives as well.
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M. Schagerl, B. Mu ¨ller Rowan KS. Photosynthetic pigments of algae. Cambridge: Cambridge University Press; 1989. Samson G, Herbert SK, Fork DC, Laudenbach DE. Acclimation of the photosynthetic apparatus to growth irradiance in a mutant strain of Synechococcus lacking iron superoxide dismutase. Plant Physiol 1994;105:287–94. Schagerl M, Donabaum K. Patterns of major photosynthetic pigments in freshwater algae. 1. Cyanoprokaryota, rhodophyta and cryptophyta. J Limnol 2003;39:35–47. Steiger S, Scha ¨fer L, Sandmann G. High-light-dependent upregulation and their antioxidative properties in the cyanobacterium Synechocystis PCC 6803. J Photochem Photobiol B: Biol 1999;52:14–8. Subramaniam A, Carpenter EJ, Karentz D, Falkowski PG. Bio-optical properties of the marine diazothrophic cyanobacteria Trichodesmuim spp. I. Absorption and photosynthetic action spectra. Limnol Oceanogr 1999;44:608–17. Utermo ¨hl UF. Zur Vervollkommnung der quantitativen Erfassung des Planktons. Verh Int Ver Limnol 1958;5:567–95. Van den Hoek C, Jahns HM, Mann DG. Algen. Stuttgart, New York: Georg Thieme Verlag; 1993. Wright SW, Jeffrey SW. High-resolution HPLC system for chlorophylls and carotenoids of marine phytoplankton. In: Jeffrey SW, Mantoura RFC, Wright SW, editors. Phytoplankton pigments in oceanography. France: UNESCO Publishing; 1997. Wright SW, Shearer JD. Rapid extraction and High performance liquid chromatography of chlorophylls and carotenoids from marine phytoplankton. J Chromatogr 1984;294:281–95. Wright SW, Jeffrey SW, Mantoura RFC, Llewellyn CA, Bjornland T, Repeta D, Welschmeyer NA. Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 1991;77:183–96.