Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii

1 Peculiar properties of chlorophyll thermoluminescence emission of autotrophically or mixotrophically grown Chlamydomonas reinhardtii Jean-Marc Ducruet1,*, Aurelio Serrano2, Mercedes Roncel2, José M. Ortega2 1 Groupe de Biospectroscopie Végétale, Ecophysiologie Végétale, ESE, Bât. 362, Université Paris-Sud-Orsay, 91400 Orsay. France. 2 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Avda. Américo Vespucio 49, 41092 Sevilla, Spain. * Corresponding author. Tel: 33 6 87 70 36 72; fax: 33 1 69 15 73 53, E-mail address: jean- [email protected] ABSTRACT The microalgae Chlamydomonas reinhardtii and Chlorella sp. CCAP 211/84 were grown autotrophically and mixotrophically and their thermoluminescence emissions were recorded after excitation by 1, 2 or 3 xenon flashes or by continuous far-red light. An oscillation of the B band intensity according to the number of flashes was always observed, with a maximum after 2 flashes, accompanied by a downshift of the B band temperature maximum in mixotrophic compared to autotrophic grown cells, indicative of a dark stable pH gradient. Moreover, new flash-induced bands emerged in mixotrophic Chlamydomonas grown cells, at temperatures higher than that of the B band. In contrast to the afterglow band observed in higher plants, in Chlamydomonas these bands were not inducible by far-red light, were fully suppressed by 2 µM antimycin A, and peaked at different temperatures depending on the flash J Photochem Photobiol B. 2011 Jul-Aug;104(1-2):301-7. doi: 10.1016/ j.jphotobiol.2011.02.014. 2 number and growth stage, with higher temperature maxima in cells at a stationary compared to an exponential growth stage. These differences are discussed according to the particular properties of cyclic electron transfer pathways in Chlamydomonas reinhardtii. Keywords Thermoluminescence Microalga Cyclic electron flow Photosystem II Chlororespiration Abbreviations AG band afterglow thermoluminescence band B band thermoluminescence band due to S2/3 QBrecombination CCCP/FCCP chloro/fluoro carbonyl cyanide phenylhydrazone FQR Ferredoxine-plastoQuinone-Reductase cyclic/Chlororespiratory pathway NDH NAD(P)H DeHydrogenase cyclic/Chlororespiratory pathway TL Thermoluminescence Tm Temperature of the maximum of a TL band; Tm(B) for B band.. 3 1. Introduction Photosynthesis relies on the conversion of a quantum of light energy into a charge separation within photosystem I (PSI) and photosystem II (PSII) reaction centers, creating charge pairs stabilized on electron carriers by activation energy barriers that limit charge recombination, i.e. the wasteful back reaction of the forward charge separation [1]. In PSII centers, charge recombination, limited to a slow rate at physiological temperatures, proceeds following different pathways [2], one of these leading to the recreation at a low yield of an exciton in the chlorophyll antenna, with a probability to deactivate as fluorescence. This delayed fluorescence emitted in darkness following an illumination is also called PSII luminescence. Thermoluminescence (TL) is a technique to study luminescence emission that consists in illuminating the sample at a temperature sufficiently low to make negligibly small the recombination rate of the charge pairs under investigation, then to reveal them successively as TL bands by a progressive warming. Due to the strong temperature dependency of charge recombination, TL has a greater resolving power than luminescence multiphasic decays recorded at a constant temperature immediately after an illumination. In unstressed dark-adapted photosynthetic material, one or few flashes give rise essentially to a so-called B band, located between 25 and 40 °C depending on species and conditions, which results from a recombination of an electron stored on the secondary quinonic acceptor QB and a positive charge stored on the S2 or S3 states of the oxygen evolving complex of PSII (OEC). Analysis of luminescence signals becomes more complex for intact photosynthetic systems, in which a delayed luminescence bounce appears superimposed to the exponential decay phases of the different types of charge pairs initially stabilized on PSII electron carriers [3]. This delayed emission called "afterglow" can be related to a dark electron back-transfer from stroma to the acceptor side of PSII [4]. The afterglow (AG) emission, highly dependent on temperature, can be optimally recorded as a sharp TL band peaking at about 45 °C at a 0.5 4 °C/s warming rate in plant leaves [5]. Complex luminescence decays at constant temperatures, sometimes exhibiting one or two successive delayed luminescence bursts, have been reported in some algal species [6,7,8]. The kinetics observed were strongly dependent on the growth conditions, e.g. low or high CO2, phosphate deficiency and ionic composition. Green microalgae are considered by plant biologists as valuable unicellular model systems for photosynthesis studies. In particular, Chlamydomonas reinhardtii (C. reinhardtii), a flagellated photosynthetic protist that belongs to the class Chlorophyceae, has been widely used for biochemical, physiological and genetic studies. This cosmopolite microalga is able to growth in many different environments, normally deriving energy from oxygenic photosynthesis. Chlamydomonas exhibits a remarkable metabolic flexibility and can also thrive in total darkness with acetate as an alternative carbon source. Its adaptability and quick generation time have made it an important model organism for biological research and studies on this microalga have provided major research contributions in diverse areas of cell and molecular biology, like photosynthesis, phototaxis, cell motility, inorganic nutrient assimilation, abiotic stress, etc. [9]. Moreover, its recently sequenced nuclear genome has advanced the understanding of the ancestral eukaryotic cell of animals and plants [10]. Here we show that TL emission properties in C. reinhardtii vary widely depending on growth stage and nutritive medium and are quite different from those observed in Chlorella CCAP 211/84, another well known chlorophycean microalga, or in higher plants. Additional TL bands peaking at temperatures above the B band that appear in mixotrophic grown cells, can be ascribed to an “afterglow” mechanism of electron transfer with, however, noticeable discrepancies compared to what is observed in higher plants: namely, occurrence of several bands peaking at variable temperatures depending on culture growth stage, sensitivity to antimycine A, an inhibitor of the Ferredoxine-plastoQuinone-Reductase (FQR) and failure to induce these bands by a far-red illumination. 5 2. Material and Methods 2.1. Algal strains and growth conditions The unicellular green algae (Chlorophyceae) C. reinhardtii 21gr and Chlorella sp. CCAP 211/84 were grown in Sueoka liquid mineral medium (pH 7.1) under photoautotrophic conditions (continuous white light, 100 moles m-2 s-1) as previously described [11]. When indicated Chlamydomonas and Chlorella cultures were supplemented with 12 mM sodium acetate or 25 mM glucose, respectively, and maintained under continuous illumination (mixotrophic conditions). Growth of the algal batch cultures was monitored by measuring the chlorophyll cell content after methanol extraction. Exponential growth phase and plateau growth phase cells were collected after two-day and five-days culturing, respectively. 2.2. Thermoluminescence TL emission was recorded as previously described [5,12,13,14]. Briefly, temperature regulation, signal recording and flash sequences were driven by a computer through a National Instrument DAQ-Pad1200 interface, using dedicated software. Temperature regulation was performed by means of a Marlow thermoelectric "Peltier" element powered by a variable (0 to 5 A) computer-driven power supply. Luminescence emission was detected by a H5701-50 Hamamatsu photomultiplier module. Illumination was performed through a light guide parallel to the photomultiplier, both of them being attached to the same stand sliding horizontally from the illumination to the measuring position. Single turn-over flashes were provided by a xenon white light (Walz XST-103). Far-red was provided by an Epitex LED 6 emitting at 735 nm with negligibly small intensity below 700 nm. TL signals were analyzed with a dedicated numerical simulation software described in Ducruet [12]. Algal suspensions were maintained in the darkness or under dim green light in Erlenmeyer vessels (about 5 mm layer thickness) that were all shaken horizontally before each TL recording. Samples were then kept at 25 °C 2 minutes in complete darkness in the TL measuring cell, then cooled to 1 °C and submitted to flash or far-red illumination; the TL recording was started immediately. 3. Results 3.1. Variations of TL emission depending on autotrophic or mixotrophic growth conditions (Figure 1) In dark-adapted autotrophically or mixotrophically grown Chlorella cells at stationary phase, single turn-over flash sequences induced a single TL band (Fig. 1) reaching a maximum intensity after 2 flashes and oscillating with a period 4, as classically observed also in plant leaves for the B band [15,16]. The maximal temperature Tm(B) of the B band after 2 or 3 flashes occurred at a lower temperature in mixotrophic compared to autotrophic grown cells. A similar unique TL band was generally observed in Chlamydomonas cells from autotrophic cultures in the exponential growth phase (Fig. 2A). However, when cells from a culture that reached the stationary growth phase were analyzed, 2 or 3 flashes induced a noticeable shoulder at >30 °C that is suppressed by antimycin A, in addition to the B band centered near 25 °C (see Fig.5, Ctrl auto). (Figure 2) 7 A clearly different emission pattern was observed in mixotrophically grown cells at a stationary stage (Fig 2B). The B band peaking at 25 °C after 1 flash was downshifted to lower temperatures and broadened after 2 or 3 flashes. In addition, new bands emerged above 30 °C. A band arised at 50 °C after 1 flash (luminescence emitting centers are 100% S2QB-) and another one at about 40 °C overlapping the smaller 50 °C band after 2 flashes (mixture of S2QB-, S3QB-). Interestingly, 3 flashes (mostly S3QB-) induced a band at 32 °C, not at 40 °C as observed after 2 flashes, with a shoulder towards high temperatures suggesting that the 50 °C band observed after 1 flash is still present after 3 flashes, despite the low amount of S2QB-; this was further confirmed by its sensitivity to antimycin A (Fig. 3C). As a consequence the bands observed at 50 °C, 40 °C and 32 °C cannot be simply ascribed to the proportion of S2 and S3 states in luminescence-emitting centers. A brief 5 s far-red illumination produced a sharp band at about 25 °C both in autotrophic (Fig. 2A) and mixotrophic (Fig. 2B) grown cells, similar to a B band, without any of the >30 °C non-B bands induced by flashes. It is worth noticing that the Tm of this band is about 3 °C lower in mixotrophic compared to autotrophic grown cells. The strongly downshifted B band (Fig. 2B) due to an acidic dark-stable lumen pH is also broadened and flattened increasingly with the S3/S2 ratio, from 1 to 3 flashes. In these conditions, the apparent B band is not a genuine TL band but becomes an envelop of elementary B bands dependent on varied local pHs [17], making a simulation impossible. Nevertheless, discarding the B band, the main band peaking at 42°C after 2 flashes can be tentatively decomposed into 2 components, including a 48°C component similar to that found after 1 flash. (Fig. 2B, dots). This confirms that analyzing the main band after 2 flashes as the sum of 2 elementary TL band is possible, although this does not exclude a higher number of components that also provide a satisfactory fit. 8 In mixotrophically grown Chlamydomonas at an exponential growth stage (Fig. 2C), the same general pattern for flash-induced TL bands was observed with, however, two differences compared to mixotrophic grown cells at a stationary phase (Fig. 2B): - the downshift of the B bands (arrows in Fig. 2) was not so pronounced at exponential stage (Fig. 2C) compared to stationary stage (Fig. 2B), the “relaxed” position of the B band being at 25 °C as observed in autotrophic exponential samples (Fig. 2A). - the 50 °C, 40 °C and 32 °C bands found in mixotrophic grown cells at stationary stage (Fig. 2B) were had their peak temperature Tm dercreased to 44 °C, 33 °C and 25 °C, respectively, in cells at exponential growth stage (Fig. 2C). The TL signal after 3 flashes was tentatively decomposed into 3 elementary components, including the B band (Fig. 2C, dots). Unexpectedly, the intensity of the highest temperature component at 42°C did not decrease with flash number, as it should if it was due to S2 centers. Preillumination during 20 min with blue light (480 nm) did not change significantly the Tm of the 30-35 °C band induced by 2 or 3 flashes (data not shown). 3.2. Effects of inhibitors of photosynthetic cyclic pathways (Figure 3) The origins of the non-B flash-induced bands was further investigated by adding antimycin A, a specific inhibitor of the FQR cyclic pathway at sub-micromolar concentrations [18,19]. It should be noticed that antimycin A had a slight inhibitory effect on the B band in autrophic (Fig. 3A) as well as in mixotrophic conditions (Fig. 3B). In a mixotrophic conditions, at a stationary phase, the band induced by 2 flashes peaking near 40 °C (Fig. 3B, 2F) was sharper in presence of 1% ethanol than that shown in 9 Fig. 2B without ethanol. This 40 °C band was clearly suppressed by antimycin A with a 0.4 µM half-inhibitory concentration (Fig. 3B). In mixotrophic grown cells at an exponential stage (Fig. 3C), the band at 45 °C after 1 flash was also present as a shoulder after 3 flashes, and was in both cases suppressed by 2 µM antimycin A. This suggests an involvement of the FQR pathway in all the non-B bands induced by flashes in mixotrophically grown Chlamydomonas. The main band after 3 flashes appeared unique and was located at about 25 °C (Fig. 2C, 3C), but antimycin A revealed a smaller B band at 18 °C, confirming the 25 °C band was for more than a half of non-B origin and related to FQR pathway. Myxothiazol and HQNO, other inhibitors of the FQR pathway [19], also suppressed the >30 °C TL bands (data not shown). 3.3. Effect of phosphorylation uncouplers (Figure 4) Noticeably, nigericine was active at least in presence 3% ethanol (Fig. 4). Ethanol in the 0.5% to 3% range modified the 32-40 °C band(s) induced by 2 or 3 flashes, with a maximum sharpening and enhancing effect at 1% (Fig. 3B, Fig. 4), followed by a slight downshift at higher ethanol concentrations. This ethanol enhancing effect was less evident when the non-B bands induced by 2 or 3 flashes were peaking at lower temperatures (mixotrophic Chlamydomonas cells at exponential growth stage, control shown in Fig. 2C, Fig. 3C). 6 µM nigericine in 3% ethanol caused a fusion of the two bands of control into one single band at an intermediate position, with a Tm upshift of the B band and a downshift of the 40 °C band, as also observed in leaves of higher plants infiltrated by nigericine [5]. (Figure 5) CCCP, another uncoupler and also an ADRY reagent that impairs the oxygen evolving complex [20], caused a complete suppression of the TL signal in autotrophic cultures, due to 16 [1] E. Tyystjärvi, I. Vass, Light emission as a probe of charge separation and recombination in the photosynthetic apparatus: relation of prompt fluorescence to delayed light emission and thermoluminescence, in: G.C. Papageorgiou, Govindjee, (Eds.), Chlorophyll a Fluorescence: A Signature of Photosynthesis, Advances in Photosynthesis and Respiration, Springer, Dordrecht, 2003, Volume 19, pp. 363-388. [2] F. Rappaport, A. Cuni, L. Xiong, R. Sayre, J. Lavergne, Charge recombination and thermoluminescence in photosystem II, Biophysical J. 88 (2005) 1948-1958. [3] W.F. Bertsch, J. R. Azzi, A relative maximum in the decay of long-term delayed light emission from the photosynthetic apparatus, Biochim. Biophys. Acta 94 (1965)15-26. [4] L.G. Sundblad, W.P. Schröder, H.E. Akerlung, S-state distribution and redox state of QA in barley in relation to luminescence decay kinetics, Biochim. Biophys. Acta 973 (1988) 47-52. [5] T. Miranda, J.M. Ducruet, Characterization of the chlorophyll thermoluminescence afterglow in dark-adapted or far-red-illuminated plant leaves, Plant Physiol. Biochem. 33 (1995) 689-699. [6] S. Mellvig, J. S. Tillberg, Transient peaks in the delayed luminescence from Scenedesmus obtusiusculus induced by phosphorous starvation and carbon dioxide deficiency, Physiol. Plant. 28 (1986) 180-188. [7] K. Palmqvist, L. G. Sundblad, G. Samuelsson, E. Sundbom, A correlation between changes in luminescence decay kinetics and the appearance of a CO2-accumulating mechanism in Scenedesmus obliquus, Photosynt. Res. 10 (1986) 113-123. [8] W. Schmidt, H. Senger, Long-term delayed luminescence in Scenedesmus obliquus, Biochim. Biophys. Acta 891 (1987) 15-27. [9] E.H. Harris, The Chlamydomonas Sourcebook (2nd Edition, Volume 1), Academic Press/Elsevier, Amsterdam (2009). 17 [10] Merchant, S, SE Prochnik, O Vallon, EH Harris, SJ Karpowicz, GB Witman, A Terry, A Salamov, LK Fritz-Laylin, L Maréchal-Drouard, WF Marshall, L-H Qu, DR Nelson, AA Sanderfoot, MH Spalding, et al., DS Rokhsar, AR Grossman, The Chlamydomonas genome reveals evolutionary insights into key animal and plant functions, Science 318 (2007) 245-250. [11] M.R. Gómez-García, M. Losada, A. Serrano, A novel subfamily of monomeric inorganic pyrophosphatases in photosynthetic eukaryotes, Biochem. J. 395 (2006) 211-221. [12] J.M. Ducruet, Chlorophyll thermoluminescence of leaf discs: simple instruments and progress in signal interpretation open the way to new ecophysiological indicators, J. Exp. Bot. 54 (2003) 2419-2430. [13] M. Roncel, J.M. Ortega, Afterglow thermoluminescence as a possible early indicator of changes in the photosynthetic electron transport in leaves, Photosynth. Res 84 (2005) 167-172. [14] J.L. Zurita, M. Roncel, J.M. Ortega, M. Barón, J.M. Ducruet, Changes in photosynthetic metabolism induced by tobamovirus infection in Nicotiana benthamiana studied in vivo by chlorophyll thermoluminescence, New Phytol. 175 (2007) 120-130. [15] A.W. Rutherford, A.R. Crofts, Y. Inoue, Thermoluminescence as a probe of Photosystem II photochemistry: the origin of the flash-induced glow peaks, Biochim. Biophys. Acta 689 (1983) 457-465. [16] A.W. Rutherford, Govindjee, Y. Inoue, Charge accumulation and photochemistry in leaves studied by thermoluminescence and delayed light emission, Proc. Natl. Acad. Sci. USA 81 (1984) 1107-1111. [17] T. Miranda, J. M. Ducruet, Effects of darkand light-induced proton gradients in thylakoids on the Q and B thermoluminescence bands, Photosynth. Res. 43 (1995) 251262. 18 [18] K. Tagawa, H.Y. Tsujimoto, D.I. Arnon, Role of chloroplast ferredoxin in the energy conversion process of photosynthesis, Proc. Natl. Acad. Sci. USA 49 (1963) 567-572. [19] J. Ravenel, G. Peltier, M. Havaux, The cyclic electron pathway around photosystem-I in Chlamydomonas reinhardtii as determined in vivo by photoacoustic measurements of energy storage, Planta (1994) 193, 251-259. [20] G. Renger, Reaction of CCCP in photosynthesis on an intermediate between chlorophylla and water, Naturwissenschaften 56 (1969), 370. [21] P. Joliot, A. Joliot, Dependence of delayed luminescence upon adenosine triphosphatase activity in Chlorella, Plant Physiol. 65 (1980) 691-696. [22] J. Lavergne, F. Rappaport, Redox and ATP control of photosynthetic cyclic electron flow in Chlamydomonas reinhardtii (I) aerobic conditions, Biochim. Biophys. Acta 1797 (2010) 44-51. [23] J. Lavergne, W. Jünge, Proton release during the redox cycle of the water oxidase, Photosynth. Res 38 (1992) 279-296. [24] P. Bennoun, Evidence for a respiratory chain in the chloroplast. Proc. Natl. Acad. Sci. USA 79 (1982) 4352-4356. [25] G. Peltier, L. Cournac, Chlororespiration, Annual Review Plant Biol. 53 (2002) 523-550. [26] P. Tapie, Y. Choquet, J. Breton, P. Delepelaire, F.A. Wollman, Orientation of photosystem I pigments. Investigation by low-temperature linear dichroism and polarized fluorescence emission, Biochim. Biophys. Acta 767 (1984) 57-69. [27] D.S. Bendall, R.S. Manasse, Cyclic photophosphorylation and electron transport, Biochim. Biophys. Acta 1229 (1995) 23-38. [28] M. Havaux, D. Rumeau, J.M. Ducruet, Probing the FQR and NDH activities involved in cyclic electron transport around Photosystem I by the ‘afterglow’ luminescence, Biochim. Biophys. Acta 1709 (2005) 203-213. 19 [29] T. Shikanai, Cyclic electron transport around Photosystem I: Genetic approaches, Ann. Rev. Plant Biol. 58 (2007) 199-217. [30] M. Martin, B. Sabater, Plastid ndh genes in plant evolution, Plant Physiol. Biochem. 48 (2010) 636-645. [31] G. Peltier, D. Tolleter, E. Billon, L. Cournac, Auxiliary electron transport pathways in chloroplasts in microalgae, Photosynth. Res. 106 (2010) 19-31. [32] N. Bukhov, R. Carpentier, Alternative Photosystem-I driven electron transport routes: mechanisms and functions, Photosynth. Res. 82 (2004) 17-33. [33] J. Alric, Cyclic electron flow around photosystem I in unicellular green algae, Photosynth. Res. 106 (2010) 47-56. [34] J.M. Ducruet, L. Kovacs, G. Garab, Thermoluminescence bands in unfrozen cells of Chlamydomonas reinhardtii, in: R. Carpentier (Ed.), Proceedings of the 13th International Congress of Photosynthesis, Montréal, 2004. [35] G. Peltier, G.W. Schmidt, Chlororespiration: an adaptation to nitrogen deficiency in Chlamydomonas reinhardtii, Proc. Nat. Acad. Sci. USA 88 (1991) 4791-4795. [36] D. Petroutsos, A.M. Teraushi, A. Busch, I. Hirschmann, S.S. Merchant, G. Finazzi, M. Hippler, PGRL1 participate in iron-induced remodelling of the photosynthesis apparatus and in energy metabolism in Chlamydomonas reinhardtii, J. Biol. Chem. 284 (2009) 32770-32781. [37] J.M. Ducruet, I. Vass,. Thermoluminescence: experimental. Photosynth. Res. 101 (2009) 195 – 204. 20 Legends Fig. 1 - Thermoluminescence emission curves of Chlorella sp. CCAP 211/84 cells grown autotrophically (line) or mixotrophically (dots) at the stationary phase. Cells were excited by 1, 2, or 3 flashes at 1 °C. Fig. 2 - Thermoluminescence emission curves of C. reinhardtii cells. A: autotrophic growth, exponential phase. B: mixotrophic growth, stationary phase. C: mixotrophic growth, exponential phase. Excitation by 1, 2, 3 flashes (lines) or 5 s far red-light (dashes). The arrows point at the maximum of the B band. Symbol ? indicates the estimated position of B band. Simulated signal for 2 flashes in thicks dots, components in thin dots (B: 2 components, excluding B band. C: 3 components, including B band). Fig. 3 - Effect of antimycin A on thermoluminescence of C. reinhardtii cells. A: autotrophic growth, after 3 flashes in samples supplemented with 1% ethanol (line, Ctrl); after 3 flashes without ethanol (dots) and with 2 µM antimycin A and 1% ethanol (dashed). B: mixotrophic growth, stationary phase, after 2 flashes in samples with 1% ethanol (2F), 0.4 µM, 1 µM or 2 µM antimycin A and 1% ethanol. C: mixotrophic growth, exponential phase, after 1 or 3 flashes in samples with 1% ethanol (lines) or 2 µM antimycin A and 1% ethanol (dashed and dot lines, respectively). Fig. 4 - Effect of nigericine on thermoluminescence of C. reinhardtii. Cells grown mixotrophically at stationary phase. After 2 flashes without addition (thin line), with 1% ethanol (dots), 3% ethanol (thick line, Ctrl) or 3% ethanol and 6 µM nigericine (dashed line, Nig.). 21 Fig. 5 - Effect of CCCP on the thermoluminescence of C. reinhardtii. Autotrophically (thin and dashed lines, auto) and mixotrophically (thick and dashed lines, mixo) cells grown, at stationary stage. Where indicated 1 µM CCCP was added. Fig. 6 - Effect of phosphate deficiency on thermoluminescence of C. reinhardtii. Cells growth mixotrophically, at exponential phase. Previous to TL experiments cells were incubated three days with (A) and without phosphate (B). For the experiment without phosphate, cells were grown in Sueoka liquid mineral medium in which phosphate salts were substituted by potassium nitrate (see Materials and Methods). TL emission curves were recorded after 3 flashes. Control without addition (dots); with 1% ethanol (lines); with 1% ethanol and 2 µM antimycin A (dashed lines).