Non-photochemical fluorescence quenching in Chromera velia is enabled by fast violaxanthin de-epoxidation

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Non-photochemical fluorescence quenching in Chromera velia is enabled by fast violaxanthin de-epoxidation Article in FEBS letters · June 2011 DOI: 10.1016/j.febslet.2011.05.015 · Source: PubMed

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FEBS Letters 585 (2011) 1941–1945

journal homepage: www.FEBSLetters.org

Non-photochemical fluorescence quenching in Chromera velia is enabled by fast violaxanthin de-epoxidation Eva Kotabová a,b,⇑, Radek Kanˇa a,b, Jana Jarešová a, Ondrˇej Prášil a,b a b

´ mly ´n, 379 81 Trˇebonˇ, Czech Republic Institute of Microbiology, Academy of Sciences of the Czech Republic, Opatovicky Institute of Physical Biology, Nové Hrady, University of South Bohemia in Cˇeské Budeˇjovice, Czech Republic

a r t i c l e

i n f o

Article history: Received 22 February 2011 Revised 21 April 2011 Accepted 4 May 2011 Available online 10 May 2011 Edited by Richard Cogdell Keywords: Non-photochemical fluorescence quenching Light-harvesting Violaxanthin Xanthophyll cycle Chromerida (Chromera velia) Eustigmatophyceae (Nannochloropsis limnetica)

a b s t r a c t Non-photochemical quenching (NPQ) is a mechanism protecting photosynthetic organisms against excessive irradiation. Here, we analyze a unique NPQ mechanism in the alga Chromera velia, a recently discovered close relative of apicomplexan parasites. NPQ in C. velia is enabled by an operative and fast violaxanthin de-epoxidation to zeaxanthin without accumulation of antheraxanthin. In C. velia violaxanthin also serves as a main light-harvesting pigment. Therefore, in C. velia violaxanthin acts as a key factor in both light harvesting and photoprotection. This is in contrast to a similar alga, Nannochloropsis limnetica, where violaxanthin has only light-harvesting function. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction The mechanism of NPQ is a subject of intensive research in vascular plants [1,2], but was shown to have an important role also in algae [3–8]. In vascular plants NPQ operates in light-harvesting system of Photosystem II (PS II), is triggered by DpH and modulated by the presence of de-epoxidated xanthophylls [1,2,9]. This is facilitated by the PsbS protein acting as pH sensor [10]. In green algae [4] and in diatoms [11] another protein – LI818 has been found to be involved in NPQ. Despite the amount of work already done, the precise molecular mechanism and site of quenching are still under extensive discussion. The kinetics of NPQ correlates with the formation of zeaxanthin in vascular plants [12], brown [7] and partly in green algae [3] or with diatoxanthin in diatoms [5]. These de-epoxidised xanthophylls are formed in the so-called xanthophyll cycle that is driven by light and represents the important mechanism directly con-

Abbreviations: DEP, diatoxanthin epoxidase; DDE, diadinoxanthin de-epoxidase; FM, maximal fluorescence; FO, minimal fluorescence; NPQ, non-photochemical fluorescence quenching; PS II, photosystem II; VDE, violaxanthin de-epoxidase; ZE, zeaxanthin epoxidase ⇑ Corresponding author at: Fax: +420 384340415. E-mail address: [email protected] (E. Kotabová).

nected with NPQ. Two main forms of xanthophyll cycle exist; the violaxanthin cycle (reviewed in [9]) and the diadinoxanthin cycle (reviewed in [13]). These two xanthophyll cycles differ not only in the structure of the carotenoids involved, but also in mechanisms of their regulation and kinetics. The standard violaxanthin cycle involves three xanthophylls (violaxanthin, antheraxanthin and zeaxanthin); their interconversion is catalysed by enzymes violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZE). The diadinoxanthin cycle involves only single-step reaction: the monoepoxide diadinoxanthin is de-epoxidised to diatoxanthin by diadinoxanthin de-epoxidase (DDE) and the opposite reaction is catalysed by diatoxanthin epoxidase (DEP). Both de-epoxidase enzymes (VDE and DDE) are activated by acidification of the thylakoid lumen, use acidic form of ascorbate as a co-substrate and require the presence of MGDG (for recent review, see [13]). However, the kinetics of VDE and DDE is different, as the pH dependency of enzyme activation and ascorbate requirement substantially differ for both enzymes (see [13]). This makes the diadinoxanthin cycle considerably faster compared to the violaxanthin cycle (compare reaction rates in [6,7,14]). Recently, a new photosynthetic alga Chromera velia was discovered [15]. This unicellular alga belongs to the supergroup Chromalveolata where it forms a distinct phyllum Chromerida among Alveolata [15]. C. velia is considered to be an important

0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.05.015

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evolutionary link between red algae and apicomplexan parasites [15,16]. Its single big chloroplast originated from the secondary endosymbiosis and is bounded by four membranes [17]. Latest phylogenetic analyses support its close relationship to plastids of heterokont algae [16]. Long thylakoids do not form grana and are regularly arranged in stacks of three [17]. C. velia lacks any chlorophylls other than chlorophyll a [15]. Violaxanthin and a novel isofucoxanthin-like pigment are the major carotenoids in C. velia [15]. In this work, we describe photoprotective NPQ and its regulation in C. velia. NPQ in this alga is connected with the unusually fast (in comparison to vascular plants) violaxanthin to zeaxanthin exchange that is driven by the buildup of the transthylakoid proton gradient. We discuss the possible reasons for such fast violaxanthin de-epoxidation in C. velia. 2. Materials and methods 2.1. Cell growth Chromera velia (strain RM 12) was grown in artificial seawater medium with f/2 nutrient addition. Nannochloropsis limnetica (KR1998/3) was grown in freshwater 1=4 Šetlík and Simmer medium with 8 mM NaHCO3 addition. Cells were cultivated in batch mode in glass tubes at 28 °C (C. velia) or 23 °C (N. limnetica), bubbled with air and continuously illuminated by 80 lmol quanta m2 s1. 2.2. Chlorophyll fluorescence measurements Chlorophyll fluorescence kinetics were measured at room temperature (24 °C) by a chlorophyll fluorometer FL-3000 (PSI, Czech Republic) using standard protocol for quenching analysis. Samples were adjusted to the OD750 = 0.5. Dark adapted cells (20 min) were subjected to a saturating light pulse and then illuminated with an orange actinic light (625 nm) of adjustable intensity. The non-photochemical chlorophyll fluorescence quenching parameter (NPQ) was calculated as NPQ = (FM  FM0 )/FM0 . 2.3. Pigments extraction and HPLC analysis

3. Results 3.1. Non-photochemical quenching The ability to dissipate excessive light energy by NPQ was tested in C. velia and N. limnetica grown under the same light conditions. Fig. 1 shows a light dependency of NPQ. While the maximal NPQ in C. velia was almost 3.5, at the same conditions NPQ in N. limnetica reached value only around 2. Moreover, NPQ in N. limnetica was almost linearly dependent on the actinic light dosage that is considered to be rather a typical sign of photoinhibition [18] than of the protective energetic quenching. Fig. 2 shows characteristic courses of chlorophyll a fluorescence quenching analysis at moderate and high actinic light intensities in C. velia (Fig. 2A and C) and N. limnetica (Fig. 2B and D). C. velia showed a strong NPQ reflecting quenching of maximal fluorescence (FM) and also minimal fluorescence (FO), since the fluorescence after irradiation decreased well below the FO level. Such a strong quenching of minimal fluorescence is an indirect sign of its presence in antennae. The NPQ was not accompanied by rapid reversal of fluorescence signal in the dark and both the FM and the FO recovered slowly. The intensive NPQ in C. velia was observed even at moderate light intensities (Fig. 2A). Moreover, the strong actinic light did not induce photodamage of PS II reaction centers as maximal fluorescence slowly recovered in the dark (Fig. 2C). In many respects, NPQ in C. velia resembles functionally the kinetics observed in diatoms (Heterokontophyta/Bacillariophyceae): the fast fluorescence quenching when exposed to actinic light followed by slow recovery at dark. On the contrary, N. limnetica did not show any quenching of fluorescence at moderate light intensities (Fig. 2B). The reduction in FM0 occurred solely at stronger actinic light, but in that case was irreversible in the dark (Fig. 2D). Suppression of the fluorescence recovery in the dark after high actinic light exposure again indicates photoinhibition of PS II in N. limnetica. These differences suggest different strategies of how C. velia and N. limnetica cope with excessive irradiation. Only C. velia can dissipate excessive irradiance by very efficient NPQ. The NPQ capacity of N. limnetica is limited and in consequence excessive irradiation causes photoinhibition of PS II.

Cells were collected on GF/F filters (Whatman, England) which were then soaked in 100% methanol (overnight at 20 °C) and disrupted using mechanical tissue grinder. Filter and cell debris were removed by centrifugation (12 000g, 15 min) before HPLC analysis on Agilent 1200 chromatography system equipped with the DAD detector. Pigments were separated on Phenomenex column (Luna 3l C8, size 100  4.60 mm) by applying the 0.028 M ammonium acetate/MeOH gradient (20/80). Eluted pigments were quantified at 440 nm with consideration of different extinction coefficients. The de-epoxidation state of xanthophyl cycle pigments (DEPS) was calculated as DEPS = [zeaxanthin]/([violaxanthin] + [zeaxanthin]) using molar concentrations. 2.4. Fluorescence excitation and absorption spectroscopy Room temperature fluorescence excitation spectra were measured in 2 mm quartz cuvette using the Aminco Bowman spectrofluorimeter (Spectronic Unicam, USA) at 735 nm emission (8 nm bandwidth) with cut-off filter before emission monochromator. Quantum correction for the excitation lamp intensity was performed with basic blue (Sigma Aldrich). Absorptance spectra (1T) were detected by Unicam UV/vis 500 spectrometer (Thermo Spectronic, UK) on membrane filters (pore diameter 0.6 lm; Pragochema, Czech Republic) placed in an integrating sphere (4 nm detection bandwidth).

Fig. 1. Light dependency of NPQ in C. velia and N. limnetica. NPQ values were calculated from FM0 measured after 5 min on the light. The maximal efficiency of PS II was 0.49 and 0.66 for C. velia and N. limnetica, respectively.

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Fig. 2. Time courses of chlorophyll a fluorescence quenching analysis and NPQ for C. velia and N. limnetica exposed to 250 (panels A and B) and 1660 (panels C and D) lmol quanta m2 s1. Exposure to actinic irradiance is indicated at the bottom of the figures.

3.2. Light induced xanthophyll cycle in C. velia The typical pigment composition of C. velia and N. limnetica is illustrated in Table 1. In both algae, chlorophyll c is absent and violaxanthin represents the major carotenoid. They differ in the second major carotenoid: isofucoxanthin in C. velia, and vaucheriaxanthin ester in N. limnetica. It is already known from literature that the violaxanthin role in eustigmatophytes consists mainly in light-harvesting [19] and that the activity of the photoprotective violaxanthin cycle is restricted to prolonged high irradiance or temperature stress [20]. Therefore, in order to check if violaxanthin cycle operates in C. velia, we have measured the deepoxidation kinetics during the first minutes of actinic irradiation (250 and 1000 lmol quanta m2 s1) (Fig. 3). We have found that violaxanthin in C. velia undergoes light induced de-epoxidation and furthermore that this reaction is unusually fast (see Fig. 3). The pronounced amount of zeaxanthin was detectable already 1 min after the exposure to light. The conversion was so fast, that we did not observe any significant amount of the intermediate product antheraxanthin. This reaction was reversible in dark as zeaxanthin was partly converted back to violaxanthin during the following 20 min of dark relaxation (see Fig. 3). Again, no antheraxanthin was detected. We conclude that NPQ in C. velia (see Figs. 1 and 2) is accompanied by zeaxanthin formation (shown as DEPS in Fig. 3), and moreover these two processes show similar kinetics. As shown in Fig. 4 both NPQ and DEPS in C. velia are clearly pH dependent as addition of 1 mM of the uncoupler ammonium chloride (NH4Cl) completely eliminated formation of NPQ as well as

Fig. 3. De-epoxidation kinetics in C. velia cells during an illumination (white panel) of 250 lmol quanta m2 s1 (open symbols) and 1000 lmol quanta m2 s1 (full symbols). The 20 min light period was followed by 20 min dark relaxation (grey panel). DEPS (de-epoxidation state of xanthophyl cycle pigments) was calculated as DEPS = [zeaxanthin]/([violaxanthin] + [zeaxanthin]).

DEPS with no effect on the maximal efficiency of PS II (FV/FM = 0.49 ± 0.01). Moreover, NPQ was proportional to DEPS as the relation between NPQ and DEPS was linearly correlated (see Inset of Fig. 4). Furthermore, the inhibition of VDE enzyme with

Table 1 The pigment composition of dark adapted C. velia and N. limnetica. Values are depicted as a % of total pigment content. [% of total]

Chlorophyll a

Chlorophyll c

Violaxanthin

Isofucoxanthin

Vaucheriaxanthin

b-carotene

C. velia N. limnetica

52 ± 4 59.0 ± 0.1

0 0

17 ± 3 25.8 ± 0.1

25 ± 2 0

0 11.6 ± 0.4

2.0 ± 0.2 3.3 ± 0.3

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Fig. 4. Effect of increasing concentration of NH4Cl on NPQ (circles) and DEPS (triangles) in C. velia. Cells were incubated in the dark for 20 min with different concentrations of NH4Cl prior to illumination (5 min at 500 lmol quanta m2 s1, k = 625 nm). Inset: Corresponding correlation of NPQ and DEPS (R2 = 0.95).

40 mM dithiothreitol decreased the NPQ and de-epoxidation equally: NPQ to 40% ± 1 and DEPS to 46% ± 5 of the level of untreated cells. These results indicate that violaxanthin de-epoxidation is driven by the trans-thylakoid DpH that further facilitates the NPQ. In summary, we have observed relatively fast stimulation of photoprotective NPQ (Fig. 2A and C) and a very rapid violaxanthin to zeaxanthin exchange (Fig. 3) in C. velia. As these two processes were correlated (see Fig. 4) we assume a photoprotective role of violaxanthin in C. velia. The violaxanthin cycle in C. velia is unusually fast as the same process in higher plants [14] or in brown algae [7] is generally much slower. 4. Discussion We have described the presence of non-photochemical quenching in the newly discovered chlorophyll c lacking chromalveolate, Chromera velia, the closest photosynthetic relative of the Apicomplexan parasites [15]. We have compared the NPQ in C. velia with other chlorophyll c lacking chromalveolate, Nannochloropsis limnetica (Heterokontophyta/Eustigmatophyceae) (Figs. 1 and 2). We have found that these two chlorophyll c lacking algae cope with the excess irradiation in different ways. While fluorescence quenching in C. velia is pronounced and flexible (Figs. 1 and 2A and C), there is no photoprotective energetic type of quenching in N. limnetica (Figs. 1 and 2B and D) where the observed decline of fluorescence at high light intensities reflects rather photoinhibitory damage of PS II. It seems that NPQ in C. velia serves as effective photoprotective mechanism. We have found that in C. velia the buildup of a transthylakoid proton gradient is crucial for NPQ as well as for the xanthophyll de-epoxidation (Fig. 4). This fact is already well known for both vascular plants [2] and for fucoxanthin containing heterokonts (brown algae and diatoms) [5,7]; however, the mechanism of pH control considerably differs between them. In vascular plants, immediately upon the onset of illumination, the pH dependent NPQ phase is observed that is then subsequently enhanced by the formation of zeaxanthin [2]. This is facilitated by the presence of PsbS protein acting as pH sensor [10]. However, in diatoms, where no PsbS is present [21], the DpH alone is not sufficient to induce NPQ [5,22]. NPQ is linearly related to the concentration of diatoxanthin (zeaxanthin analogue), and if diatoxanthin is not present, NPQ cannot be formed [5,6]. Thus, NPQ mechanism in

diatoms [5] and also in brown algae [7] appears to be more tightly related to the xanthophylls than in vascular plants. Our results with C. velia show similar relation between DpH formation, xanthophylls cycling and NPQ as is known for heterokonts. The NPQ in C. velia is accompanied by unusually fast zeaxanthin formation due to violaxanthin de-epoxidation (Fig. 3). In vascular plants [14] and in brown algae [7] the kinetics of violaxanthin de-epoxidation is much slower than we have observed for C. velia. Moreover, unlike in C. velia, the intermediate, single-epoxidised form, antheraxantin is often found in considerable amounts. The rapid rate of the xanthophyll conversion observed in C. velia is comparable only to the rate of xanthophyll conversion observed in diatoms where diadinoxanthin is de-epoxidised to diatoxanthin in single step reaction [13]. There are several possibilities how to explain the unusually fast violaxanthin de-epoxidation observed in C. velia. One of them could be the absence of grana stacks in C. velia [17], because the presence of grana stacks in vascular plants limits the violaxanthin de-epoxidation [23]. However, this hypothesis could be ruled out as the brown algae lack grana as well [24], but show the ordinary kinetics of violaxanthin de-epoxidation [7]. We exclude also the role of possibly different DpH control characteristics of the enzymes of the xanthophyll cycle, VDE and ZE, like it was found for diadinoxanthin cycle in diatoms [22,25] as it was already shown that DpH is not the limiting factor for the activity of violaxanthin de-epoxidase enzyme [26,27]. The more probable reason for the fast operation of VDE could be the better availability of the pigment substrate, violaxanthin. In C. velia violaxanthin is a major carotenoid accounting for about 40% of total carotenoids (see Table 1). This is several times more than in vascular plants. The relative amount of violaxanthin per chlorophyll a in C. velia in dark adapted state is 0.36 (mol/mol), about eight times higher than in wheat leaves (Triticum aktivum, Kotabova, unpublished results). As the availability of violaxanthin is considered to be the main limiting step of violaxanthin de-epoxidation in vascular plants [28], its abundance in C. velia might possibly result in faster zeaxanthin formation (Fig. 3). It is logical that even with the same DEPS, there is more zeaxanthin accumulated in C. velia in comparison to vascular plants. However, we have found that beyond its involvement in xanthophyll cycle, violaxanthin in C. velia does also notably contribute to light-harvesting like in eustigmatophytes [19], including N. limnetica. The comparison of absorption and fluorescence excitation spectra indicates very similar energy transfer efficiency from violaxanthin (see 460–490 nm region in Fig. 5) to chlorophyll a in both C. velia and N. limnetica. This result indicates the importance of violaxanthin abundance in C. velia. In addition to chlorophyll a and isofucoxanthin, violaxanthin serves in light-harvesting and under excessive light it is easily de-epoxided to zeaxanthin for very efficient and fast photoprotection. The other critical factor influencing the rate of de-epoxidase reaction is the accessibility of the reaction co-substrate, ascorbate [25,29]. It is one of the factors making DDE enzyme in diatoms faster in comparison to VDE in vascular plants [13]. The recent dimeric model [30] shows that VDE is a di-deepoxidase with two active ascorbate binding sites providing the reducing power for de-epoxidation of two epoxy groups at once. This could explain why we haven’t observed the intermediate antheraxanthin production in C. velia. If ascorbate supply is not limiting, then zeaxanthin can be formed directly from violaxanthin, without the intermediate antheraxanthin, as suggested by the dimeric model. This would in consequence result in the faster operational rate of xanthophyll cycle in C. velia. In conclusion, we have found that C. velia is able to effectively quench the excessive light energy by NPQ. This process is enabled by fast violaxanthin to zeaxanthin exchange that is driven by the buildup of the transthylakoid proton gradient and possibly enabled by the availability of violaxanthin and/or ascorbate. Beyond its role

E. Kotabová et al. / FEBS Letters 585 (2011) 1941–1945

Fig. 5. Absorption (1-T) and fluorescence excitation spectra in vivo of C. velia (A) and N. limnetica (B). Fluorescence excitation was detected at 735 nm. Spectra were normalized at the 680 nm.

in photoprotection, violaxanthin is also active in light harvesting. On the other hand, in eustigmatophytes violaxanthin serves mainly in light harvesting [19], its de-epoxidation is negligible [20] and NPQ is not observed. Acknowledgments This work was supported by grants GAAV IAA601410907 of the Grant Agency of Academy of Sciences, GACR 206/09/094 of the Grant Agency of the Czech Republic, by the institutional research concepts AV0Z50200510, MSM6007665808 and by the project Algatech (CZ.1.05/2.1.00/03.0110). References [1] Horton, P., Wentworth, M. and Ruban, A. (2005) Control of the light harvesting function of chloroplast membranes: the LHCII-aggregation model for nonphotochemical quenching. FEBS Lett. 579, 4201–4206. [2] Horton, P., Johnson, M.P., Perez-Bueno, M.L., Kiss, A.Z. and Ruban, A.V. (2008) Photosynthetic acclimation: does the dynamic structure and macroorganisation of photosystem II in higher plant grana membranes regulate light harvesting states? FEBS J. 275, 1069–1079. [3] Niyogi, K.K., Bjorkman, O. and Grossman, A.R. (1997) The roles of specific xanthophylls in photoprotection. Proc. Natl. Acad. Sci. USA 94, 14162–14167. [4] Peers, G., Truong, T.B., Ostendorf, E., Busch, A., Elrad, D., Grossman, A.R., Hippler, M. and Niyogi, K.K. (2009) An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature 462, 518–522.

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