Biodiversity of NPQ.

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Physiology

Biodiversity of NPQ Reimund Goss a,∗ , Bernard Lepetit b a b

Institut für Biologie, Universität Leipzig, Johannisallee 21-23, D-04103 Leipzig, Germany Institut für Biologie, Universität Konstanz, Universitätsstrasse 10, D-78457 Konstanz, Germany

a r t i c l e

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Article history: Received 16 January 2014 Received in revised form 10 March 2014 Accepted 11 March 2014 Available online xxx This paper is dedicated to Prof. Dr. Christian Wilhelm on the occasion of his 60th birthday. Keywords: Lhcsr Lhcx NPQ PsbS Xanthophyll cycle

s u m m a r y In their natural environment plants and algae are exposed to rapidly changing light conditions and light intensities. Illumination with high light intensities has the potential to overexcite the photosynthetic pigments and the electron transport chain and thus induce the production of toxic reactive oxygen species (ROS). To prevent damage by the action of ROS, plants and algae have developed a multitude of photoprotection mechanisms. One of the most important protection mechanisms is the dissipation of excessive excitation energy as heat in the light-harvesting complexes of the photosystems. This process requires a structural change of the photosynthetic antenna complexes that are normally optimized with regard to efficient light-harvesting. Enhanced heat dissipation in the antenna systems is accompanied by a strong quenching of the chlorophyll a fluorescence and has thus been termed non-photochemical quenching of chlorophyll a fluorescence, NPQ. The general importance of NPQ for the photoprotection of plants and algae is documented by its wide distribution in the plant kingdom. In the present review we will summarize the present day knowledge about NPQ in higher plants and different algal groups with a special focus on the molecular mechanisms that lead to the structural rearrangements of the antenna complexes and enhanced heat dissipation. We will present the newest models for NPQ in higher plants and diatoms and will compare the features of NPQ in different algae with those of NPQ in higher plants. In addition, we will briefly address evolutionary aspects of NPQ, i.e. how the requirements of NPQ have changed during the transition of plants from the aquatic habitat to the land environment. We will conclude with a presentation of open questions regarding the mechanistic basis of NPQ and suggestions for future experiments that may serve to obtain this missing information. © 2014 Elsevier GmbH. All rights reserved.

Introduction Higher plants and algae have to cope with rapidly changing light conditions in their natural habitat. Land plants can experience sudden changes of the light intensity caused by clouds or changes in shading by other leaves or plants, a situation that is also valid for the macrophytic marine algae. Planktonic algae are passively transported within the water body and, especially in estuarine habitats, even moderate water mixing can bring algae from darkness to full sunlight within minutes (Schubert and Forster, 1997; MacIntyre et al., 2000). Since high light intensities can lead to an overexcitation of the photosynthetic apparatus, which results in the production of harmful reactive oxygen species (ROS, for a review see Triantaphylidès and Havaux, 2009), terrestrial plants and aquatic algae have developed a variety of photoprotective mechanisms. One of the important mechanisms, which helps plants and algae

∗ Corresponding author. Universität Leipzig Institut für Biologie I Johannisallee 21-23 04103 Leipzig Germany. Tel.: +49 341 9736873; fax: +49 341 9736899. E-mail address: [email protected] (R. Goss).

to survive in environments characterized by rapidly changing light conditions, is the so-called non-photochemical quenching of chlorophyll (Chl) a fluorescence (NPQ, for recent reviews see Horton and Ruban, 2005; Jahns and Holzwarth, 2012; Niyogi and Truong, 2013). In the mechanism of NPQ the excessive excitation energy which cannot be used for photosynthesis is dissipated as heat. Since the thermal dissipation of excessive excitation energy becomes visible as a pronounced quenching of the Chl a fluorescence in fluorescence measurements (for a review see Krause and Jahns, 2004), the process has been termed NPQ. NPQ is induced rapidly on a time-scale of seconds to a few minutes and is thus perfectly suited to cope with sudden fluctuations in the light intensity. NPQ consists of different components, i.e. is the sum of different mechanisms which contribute to the overall heat dissipation. These components are the high-energy-state quenching qE, the photoinhibitory quenching qI and fluorescence quenching caused by state transitions qT. Note, however, that state transitions in higher plants are not necessarily a reaction to illumination with excessive light and are not associated with the thermal dissipation of excitation energy. Since qE represents the main component of NPQ, most of the results presented in this review are actually results on the

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Haptophytes (Dd-Dt/Lhcsr)

Diatoms (Dd-Dt/Lhcsr) Eusgmatophytes (VAZ/Lhcsr) Chrysophytes (VAZ)

Cryptophytes Green algae/plants (VAZ/Lhcsr+PsbS)

Stramenopiles

Archaeplasds Hacrobionts

Chromera (VAZ) Dinophytes (Dd-Dt)*

Phaeophyceae (VAZ/Lhcsr) Raphidophytes (VAZ)

Red algae

Fig. 1. Simplified tree of eukaryotic phototrophs whose NPQ characteristics are described in the present article, modified after Keeling (2013). The colour of the chloroplasts indicates their origin as green or red chloroplasts, respectively, while the number of chloroplast membranes points towards the respective chloroplast evolution by primary endosymbiosis (two membranes) or secondary endosymbiosis (four membranes). In parentheses, the prevailing type of XC, as well as the existence of PsbS or Lhcsr (=LI818/Lhcx), according to the present knowledge, is indicated. Note that the absence of Lhcx identification in some groups does not exclude future findings of these proteins in more and better annotated genome databases of the respective organisms. The asterisk highlights the fact that dinophytes also contain chloroplasts which have evolved by serial secondary or even tertiary endosymbiosis, which also has consequences for the existence of Lhcsr proteins. For further details, see text.

mechanism of high-energy-state quenching qE. If other components, like photoinhibitory quenching qI or transient quenching, contribute to NPQ, an explanation is given in the respective parts of the text. The importance of NPQ for the protection of the photosynthetic apparatus also becomes clear by its wide distribution in the plant kingdom (Fig. 1, for reviews see Goss and Jakob, 2010; Niyogi and Truong, 2013). All major algal taxa and terrestrial plants exhibit NPQ, although the underlying mechanisms of the enhanced heat dissipation of excessive excitation energy are different. In the present review we will, on the one hand, give an overview of the molecular mechanism of NPQ in higher plants, as this process is well understood due to a wealth of information which has been gathered during recent years. On the other hand, we will try to present the biodiversity of NPQ by explaining the features, and if known, the molecular basis of NPQ in different algal groups including the diatoms, the green and brown algae and the cyanobacteria. With regard to the comparison of NPQ in algae and higher plants, we will also focus on evolutionary aspects of NPQ. We will discuss the appearance and loss of specific antenna proteins and modifications of the xanthophyll cycle (XC) reactions which represent two important components of NPQ during the evolution from the aquatic algae to the terrestrial plants.

Properties of VAZ cycle enzymes. The VDE is a protein which belongs to the lipocalin family of proteins (Hieber et al., 2000; Grzyb et al., 2006). It has a pH-optimum of pH 5.2 (Hager, 1969; Pfündel et al., 1994) and requires the reduced form of ascorbate as cosubstrate (Hager, 1969). The VDE in its inactive form is a water-soluble enzyme and is localized in the thylakoid lumen (Fig. 2, Hager and Holocher, 1994). After the drop of the lumenal pH to a value of around 5, which is caused by the light-driven electron transport and the build-up of the transmembrane proton gradient, the VDE is activated and binds to the thylakoid membrane (Hager and Holocher, 1994; Schaller et al., 2010, see also “Role of MGDG in V de-epoxidation”). According to recent results, which were obtained with an artificial VDE, which included the catalytic site formed by the barrel structure typical for the lipocalins (Hieber et al., 2000) but lacked other important protein domains, the inactive VDE is a monomer (Fig. 2, Arnoux et al., 2009; Saga et al., 2010). Upon the decrease of the pH-value to 5 the monomeric VDE forms a dimer which represents the active form of the enzyme. According to these results the VDE dimer contains a catalytic site which is able to incorporate the complete V molecule and two molecules of the VDE cosubstrate ascorbate. The VDE dimer realizes the simultaneous de-epoxidation of the two epoxy groups of V and releases the final product of the forward reaction, Z. With regard to this attractive model of VDE action it should, however, be noted, that under natural conditions, de-epoxidation of V often results in an accumulation of the intermediate A (Adams and Demmig-Adams, 1992) which is not in line with the simultaneous de-epoxidation as predicted by the dimer model. Like the VDE the ZEP belongs to the lipocalin group of proteins (Hieber et al., 2000; Grzyb et al., 2006). It is characterized by a broad pH-optimum and is usually active at neutral or slightly basic pHvalues that are typical for the chloroplast stroma (Hager, 1975; Siefermann and Yamamoto, 1975). The ZEP requires NADPH and O2 as cosubstrates to reintroduce the epoxy group into the Z and A molecules (Büch et al., 1995). In contrast to the VDE, the ZEP of higher plants is active both during darkness and illumination (Siefermann and Yamamoto, 1975; Gilmore et al., 1994; Goss et al., 1998). Due to the extremely low conversion rates of Z to A and V, which are a factor of 10 lower than the respective de-epoxidation rates (Siefermann and Yamamoto, 1975; Goss et al., 2006b), the conversion of Z to V does not inhibit the fast accumulation of Z during periods of high light illumination. According to recent results (Schaller et al., 2012a,b), the ZEP is a peripheral membrane protein located at the stromal side of the thylakoid membrane. The ZEP seems to be rather weakly bound to the membrane, possibly by specialized hydrophobic domains which realize the partial integration of the protein into the lipid phase of thylakoid membrane.

NPQ in higher plants Requirements of NPQ in higher plants The VAZ cycle One of the important factors for the induction of NPQ in higher plants is the operation of the violaxanthin, antheraxanthin, zeaxanthin (VAZ) cycle. The VAZ cycle consists of a forward reaction in which the di-epoxy xanthophyll V is de-epoxidized to the epoxy-free xanthophyll Z (Yamamoto et al., 1962; Hager, 1967a). The intermediate of this reaction sequence is the xanthophyll A which contains one epoxy group. The forward reaction takes place under illumination with high light intensities and is catalyzed by the enzyme V de-epoxidase (VDE, see “Properties of VAZ cycle enzymes”). The back reaction of the VAZ cycle, which regains the V molecule by a stepwise re-introduction of the two epoxy groups into the Z molecule, is normally observed during periods of low light illumination or in darkness (Hager, 1967b). It is catalyzed by the enzyme Z epoxidase (ZEP, see “Properties of VAZ cycle enzymes”).


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Role of MGDG in V de-epoxidation. The presence of the main thylakoid membrane lipid monogalactosyldiacylglycerole (MGDG) is essential for the efficient de-epoxidation of V to A and Z (Fig. 2, Latowski et al., 2002; Goss et al., 2005, 2007). The role of MGDG is twofold: first, it serves as solubilizing agent for the hydrophobic xanthophyll cycle pigment V, thus making the substrate accessible for the enzyme VDE (Goss et al., 2005). In the absence of MGDG V forms aggregates in aqueous media which cannot be converted to A and Z. Due to the special molecule structure, i.e. the small headgroup area represented by the single galactose residue and the rather spacious area covered by the desaturated fatty acid moieties, the MGDG molecule forms the so-called inverted hexagonal phases in aqueous surroundings (van den Brink-van der Laan et al., 2004, Fig. 2). It has been shown that these HII phases, which can also be formed by other lipids, e.g. the phospholipid PE, strongly increase the efficiency of V de-epoxidation (Latowski et al., 2004; Goss et al., 2007). With respect to the possible occurrence of inverted hexagonal phases in the native thylakoid membrane (Fig. 2), the results are

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Fig. 2. The most important requirements of the VAZ cycle and NPQ in higher plants are summarized. It shows that upon the decrease of the pH of the thylakoid lumen the monomeric, inactive VDE forms a dimer and becomes activated. The dimeric VDE then binds to lipid inverted hexagonal phases in the thylakoid membrane. These HII phases are realized by MGDG molecules that surround the light-harvesting complexes and are possibly segregated from the bilayer upon the aggregation of LHCII. The HII phases target the VDE to the LHCII. Thus, de-epoxidation of V to Z takes place in close vicinity of the antenna complexes and allows for a fast rebinding of Z to the LHCII and the minor, monomeric antenna proteins, i.e. CP29, CP26 and CP24. Enhanced heat dissipation of excessive excitation energy (NPQ) takes place at two different quenching sites termed the Q1 and Q2 sites, respectively. Q1 consists of LHCII complexes which, due to the interaction with the protonated PsbS protein, have detached from the PSII core complex and have aggregated. NPQ at the Q1 site does not depend on the presence of Z, although rebinding of Z to the LHCII certainly takes place. The Q2 site is realized by minor PSII antenna proteins which stay attached to the PSII core. NPQ at Q2 depends on the presence of Z which is found in higher concentration in the minor antenna complexes compared to the LHCII. For further details and the respective references refer to the text.

still ambiguous, but it has been suggested that these lipid phases can exist either within the thylakoid membrane (Jahns et al., 2009) or as attachment to the lumenal side of the membrane, while retaining an association with the membrane bilayer (Garab et al., 2000). A recent study has shown that MGDG forms a shield around the main light-harvesting complex of PSII, the LHCII (Schaller et al., 2010). This lipid shield incorporates a part of the VAZ cycle pigment pool which is not bound to specific protein binding sites of the LHCII apoproteins. It has been proposed (Schaller et al., 2010) that the MGDG shield forms an attraction site for the VDE which targets the enzyme to the places of the thylakoid membrane where the majority of the VAZ cycle pigments are located and where, after the synthesis of Z and its rebinding to the LHCII, the mechanism of NPQ is induced (see “Recent models for NPQ”). Role of the VAZ cycle in NPQ. The first evidence for an involvement of the VAZ cycle in NPQ emerged during the late 1980s and early 1990s when Barbara Demmig-Adams (Demmig et al., 1987, 1988) observed a link between the kinetics of Z synthesis and the thermal dissipation of excitation energy, measured as NPQ, in several plant species. Later, inhibition of Z synthesis with the VDE inhibitor dithiothreitol (DTT, Yamamoto and Kamite, 1972), which caused a major decrease of NPQ, confirmed the first results and showed the importance of Z for the mechanism of enhanced heat dissipation (Demmig-Adams, 1990; Demmig-Adams et al., 1990). Additional experiments by Demmig-Adams and co-workers concentrated on the correlation between Z and different NPQ components and it became clear that Z is important for the fast developing and relaxing qE component of NPQ, the so-called high-energy-state quenching, but that it also contributes to the slowly relaxing qI component, the photoinhibitory quenching (Demmig-Adams et al., 2006; DemmigAdams and Adams, 2006). Since qI consists of more than one component, the Z dependent, slowly relaxing component which is observed in low light adapted plants that are illuminated with high light intensities, has been termed qZ (Demmig-Adams and Adams, Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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2006, Nilkens et al., 2010). Interestingly, more than 20 years ago Demmig-Adams (1990) proposed that Z induces the thermal dissipation of excitation energy by taking over the excitation energy of singlet excited Chl a molecules. This concept was later supported by the experimental determination of the levels of the excited singlet states of Chl a and the VAZ pigments (Frank et al., 1994). It was also challenged by other groups (for a review see Polivka and Sundström, 2004) but, surprisingly, the concept is still valid for some mechanistic models of NPQ which have been put forward only recently (Bode et al., 2009, see also “Recent models for NPQ”). Role of the proton gradient Role of the proton gradient for NPQ. The proton gradient across the thylakoid membrane is the most important component for the mechanism of NPQ in higher plants since it is needed as a trigger for various mechanisms contributing to NPQ (see the respective chapters). It can be seen as a sensor which detects the state of the photosynthetic apparatus. Under low light illumination the magnitude of the proton gradient is low and the antenna system of PSII remains in a light-harvesting state. Illumination with high light intensities increases the magnitude of the proton gradient until, under illumination with excessive light intensities, a threshold value is surpassed and different mechanisms, which induce NPQ, are triggered. The first mechanism, which depends on the magnitude of the proton gradient, is the de-epoxidation of V to Z (see “The VAZ cycle”). Besides the activation of the VDE the proton gradient exerts a more direct control on NPQ. Uncoupler experiments in the beginning of the 1990s, which were mainly conducted by the group of Peter Horton (Noctor et al., 1991, 1993), showed that the breakdown of the light-driven proton gradient led to a complete collapse of NPQ. Later, with the use of dicyclohexylcarbodiimide (DCCD, Walters et al., 1994), it was shown that the LHCII contains special amino acid residues which become protonated after the establishment of the light-driven proton gradient and it was proposed that this protonation leads to the structural rearrangement, i.e.

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aggregation of the LHCII, which provides the basis for NPQ (Horton and Ruban, 1992; Horton et al., 2005, see also “Role of LHCII aggregation”). With the finding that the PsbS protein plays an important role in NPQ in higher plants (Li et al., 2000, see “The PsbS protein”), the role of the proton gradient for the structural change of the PSII antenna was modified. It became clear that the PsbS protein acts as the sensor for the proton gradient and has the ability to bind protons (Li et al., 2004). The protonation of the PsbS protein during high light illumination was shown with DCCD experiments and by side-directed mutagenesis of putative PsbS proton binding sites. Role of the proton gradient in Z synthesis. The proton gradient also plays an important role in the synthesis of NPQ-effective Z. The first evidence that the efficiency of the NPQ enhancement by Z depends on the mode of the synthesis of the xanthophyll was presented by Goss et al. (1995). These authors could show that Z, which was formed by a V de-epoxidation driven by a light-induced proton gradient, was able to enhance NPQ significantly. In contrast, Z which was converted from V in the dark, by adjusting isolated thylakoid membranes to a low pH which corresponds to the pH-optimum of the VDE, was unable to increase NPQ beyond the value that was found in Z-free membranes. Later, in a more detailed study on the effect of the mode of Z synthesis on NPQ (Goss et al., 2008), it was demonstrated that the missing NPQ induction by Z formed in darkness at pH 5 is not caused by a lack of Z rebinding to the LHCII apoproteins. LHCII, that was prepared from illuminated thylakoid membranes, which thus experienced a real light-driven proton gradient, contained a comparable amount of Z as LHCII that was isolated from thylakoids incubated at pH 5 in the dark. Based on these results it was argued that NPQ-effective Z can only be synthesized if the lumenal side of the thylakoid membrane experiences a high proton concentration, i.e. a low pH, whereas the stromal side is exposed to a slightly basic pH, as it is typical for the native situation during illumination and photosynthetic electron transport. Hence, a pH difference between lumen and stroma, and not only acidic conditions in the thylakoid lumen, are indispensable for the induction of NPQ. It was concluded that the rebinding of Z to the LHCII under these conditions leads to a special configuration of the Z binding site or the complete LHCII that enables high NPQ. This conformation cannot be established when Z rebinds to the LHCII in a thylakoid membrane where both sides of the membrane are exposed to high proton concentrations, i.e. pH 5. Importance of grana and LHCII structure for NPQ Importance of grana structures. Since the antenna system of PSII, which represents the site where the majority of the VAZ cycle pigments is located and where NPQ is taking place, is situated in the grana membranes of higher plants (for a review see Dekker and Boekema, 2005), the importance of the grana structure for NPQ has been investigated (Goss et al., 2007). Varying the grana stacking by incubation of isolated thylakoid membranes in buffers with different contents of salt, i.e. Mg2+ , and osmotically active substances like sorbitol, did not affect the de-epoxidation of V to Z. In contrast, the NPQ values differed drastically. High NPQ could only be detected in thylakoid membranes where grana stacking was conserved by addition of Mg2+ and sorbitol. Membranes with partially unstacked grana regions were characterized by a significantly decreased NPQ and in membranes, where a complete unstacking of the grana had been induced by the absence of Mg2+ and sorbitol, only very low values of NPQ could be detected. Since the different membranes contained comparable amounts of Z, this resulted in extremely different quenching capacities of the de-epoxidized VAZ cycle pigment, thus indicating that Z-dependent NPQ strongly depends on the amount of grana stacking in the native membrane. The stimulating effect of Mg2+ for NPQ is in line with earlier data that also showed a strong enhancement of NPQ in the presence of the divalent Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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cation (Noctor et al., 1993). With respect to the importance of grana stacking for NPQ, it has been proposed that the macrodomain organization of LHCII, which is realized in the stacked grana regions, is essential for efficient NPQ (Goss et al., 2007). The long-range order of the photosynthetic pigments in the macrodomains, in conjunction with the delocalization of the excitation energy (Barzda et al., 1994, 1996), could provide the basis for efficient Z-dependent NPQ. The introduction of quenching sites into a system, whose excitonic coupling may span several adjacent grana membranes, would strongly enhance the efficiency of the quencher. The importance of grana stacking for NPQ has also been discussed by Horton et al. (2008), who stress that the assembly of PSII-LHCII supercomplexes into organized arrays in the grana membranes represents the prerequisite for the formation of the quenching site. According to their opinion, the quenching locus consists not only of LHCII, but is formed by the interplay of the major LHCII and the minor PSII antenna proteins CP29, CP26 and CP24. They suggest that NPQ should be regarded as a property of the supercomplex rather than of any specific component of the complex and that the characteristics of the supercomplex determine the regulation of the structural rearrangement by the proton gradient, the xanthophyll composition and the PsbS protein. Role of LHCII aggregation. The first evidence for an aggregation of the light-harvesting complex of PSII as the basis for NPQ emerged during the beginning of 1990s, when Horton and co-workers studied aggregation of isolated LHCII by 77K fluorescence emission spectroscopy and observed a long-wavelength emission band associated with the aggregated state of the complex (Ruban et al., 1992). Later, the influence of low pH-values and the conversion of V to Z on the aggregation state of LHCII were investigated and it was observed that both high proton concentrations and the presence of Z are beneficial for LHCII aggregation (Phillip et al., 1996; Ruban et al., 1997). Additional measurements with isolated LHCII showed that Z- and pH-dependent aggregation induced a strong reduction of the fluorescence yield of the antenna complex, i.e. quenching of the Chl a fluorescence (Ruban et al., 1997, see also Schaller et al., 2013). Aggregation dependent quenching is not restricted to the LHCII but can also be found in the isolated minor PSII antenna proteins CP29 and CP26 (Wentworth et al., 2001), which are enriched in VAZ cycle pigments compared to the major LHCII (Bassi et al., 1993; Ruban et al., 1994; Goss et al., 1997). Recent investigations have shown that for the structural rearrangement of the PSII antenna system in the native thylakoid membrane the PsbS protein plays an important role (Li et al., 2000, for more details see “The PsbS protein”). In addition, it has become clear that for full activation of NPQ the presence of the minor antenna proteins is required and mutants of Arabidopsis thaliana, which are devoid of one or more of the minor proteins, show a significantly reduced NPQ (Andersson et al., 2001; Kovacs et al., 2006). This has been interpreted in such a way that for the establishment of an efficient quenching site the complete PSII antenna system, consisting of the major LHCII and the minor antenna proteins CP29, CP26 and CP24, is needed (Horton et al., 2008). However, in the frame of the recent model for NPQ by Holzwarth et al. (2009, see “Recent models for NPQ”) the LHCII and the minor PSII antenna proteins form two different quenching sites, the Q1 and Q2 sites, respectively. The PsbS protein The analysis of A. thaliana mutants, which are unable to develop fast and efficient NPQ, has revealed that the PsbS protein is important for NPQ in higher plants (Li et al., 2000). The PsbS protein belongs to the family of light-harvesting proteins (Bonente et al., 2008). The pigmentation of the PsbS protein is still unclear and there have been contradicting results concerning the ability of PsbS to bind xanthophyll, i.e. Z, and Chl molecules (Holt et al., 2005;

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Bonente et al., 2008). For the role of the PsbS protein in the process of NPQ different mechanisms have been proposed. It has been suggested that the PsbS protein represents the actual quenching site where heat is dissipated from PSII (Holt et al., 2005, see also “Recent models for NPQ”). According to this proposal, energy transfer from Chl a molecules to a Chl a-Z heterodimer, which then undergoes charge separation, represents the mechanism of energy dissipation during NPQ. However, later results have indicated that this process is located in the minor PSII antenna proteins where Z receives excitation energy from Chl a, followed by an energy transfer to a Chl a dimer which finally leads to the charge separated state (Ahn et al., 2008). In addition, it has been suggested that PsbS plays a more indirect role in the establishment of NPQ and facilitates the structural change that is needed for the transformation of LHCII from the lightharvesting to the heat dissipating state (Kiss et al., 2008; Kereiche et al., 2010). The role of PsbS as antenna organizer is supported by the observations that the protein is mobile in the thylakoid membrane (Teardo et al., 2007), that it can associate with both the PSII core complex and the LHCII (Bergantino et al., 2003) and that it plays a role in the Mg2+ -dependent macro-organization of the PSII antenna (Kiss et al., 2008). However, both proposed mechanisms have in common that PsbS is activated by protonation, caused by the increase of the proton concentration of the thylakoid lumen driven by the photosynthetic electron transport. With respect to the importance of the lumenal pH for the establishment of NPQ, it has recently been shown that high NPQ is possible in the absence of the PsbS protein but requires very low pH values of the thylakoid lumen (Johnson and Ruban, 2011). This observation is in line with the role of the PsbS protein as antenna organizer and indicates that the protonation of PsbS allows for a fast and efficient response and rearrangement of the PSII antenna, which in the absence of PsbS would still be possible but occur on a significantly longer time scale. Recent models for NPQ The LHCII conformation model for NPQ has been first introduced by Horton and Ruban (1992) at the beginning of 1990s. Since then it has been refined in many ways (for an updated version of the model see Horton et al., 2005, 2008) but the basics of that first model are still valid. The model depicts the LHCII in four different quenching states and explains how V de-epoxidation and the generation of a light-driven proton gradient control the LHCII conformation and thus NPQ. State 1 represents the LHCII as it is present in dark-adapted plants or plants that are illuminated with low light intensities. The LHCII is in a non-aggregated state due to the absence of amino acid protonation and the presence of V in the VAZ binding pocket. The LHCII in State 1 is in an unquenched, light-harvesting state and the largest part of the excitation energy is used to drive the photosynthetic electron transport. State 4 describes the LHCII as it is found in plants that are illuminated with high light intensities where a part of the excitation energy is in excess and thus cannot be used for photosynthesis. The LHCII in State 4 exists in a strongly aggregated state (see “Role of LHCII aggregation” and references therein) which is characterized by high NPQ. Aggregation of the LHCII is triggered by the protonation of special amino acid residues and the transformation of V to Z and the rebinding of Z to the VAZ binding pocket. Since the PsbS protein has been shown to be essential for NPQ (see “The PsbS protein” and references therein), it has been incorporated into the LHCII conformation model. However, the role of the PsbS protein in NPQ is still not completely clear and thus two possible modes of PsbS action have been proposed. In both cases the PsbS protein acts as a sensor of the change of the proton concentration of the thylakoid lumen during high light illumination. PsbS becomes protonated and either catalyses the conformational change of the LHCII, which leads to aggregation and NPQ, or binds Z and forms the quenching Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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site where the excited states of Chl a molecules are deactivated. Recent spectroscopic measurements have, however, suggested that PsbS does not represent the actual quenching site. According to this data, the heat dissipating site in the aggregated LHCII consists of either a Chl a-lutein heterodimer or a Chl a-Chl a homodimer (Pascal et al., 2005; Ruban et al., 2007). The LHCII conformation model describes two further quenching states: State 3 is characterized by LHCII complexes which are protonated but still contain V in the VAZ binding pocket. These LHCII complexes are aggregated to a lesser extent than those in State 4 and exhibit lower NPQ. LHCII in State 3 can be present in the thylakoid membranes immediately after the onset of illumination, since the buildup of the proton gradient exhibits faster kinetics than the de-epoxidation of V to Z. Due to its transient nature, NPQ found in complexes in State 3 has also been termed transient NPQ (Finazzi et al., 2004; Kalituho et al., 2007). LHCII in State 3 may also be found in membranes where V de-epoxidation is inhibited or perturbed. LHCII in State 2 consists of unprotonated complexes with Z present in the VAZ binding site. This situation is found in thylakoid membranes after a transition from high light illumination to darkness or low light. LHCII in State 2 is characterized by a lower NPQ than LHCII in State 4, but quenching may persist for longer times due to the slow removal of Z from the VAZ binding site by the inefficient Z epoxidation. NPQ that relaxes with slow kinetics has been termed photoinhibitory quenching qI or, when it is related to the presence of Z, qZ (Demmig-Adams et al., 2006; Demmig-Adams and Adams, 2006; Nilkens et al., 2010). A second, attractive NPQ model has been put forward by Holzwarth and coworkers during the last years (Fig. 2, Holzwarth et al., 2009; Jahns and Holzwarth, 2012). This NPQ model is based on time-resolved fluorescence measurements that showed that two distinct and independent quenching sites are responsible for the total NPQ in the thylakoid membranes of higher plants (Holzwarth et al., 2009). According to this model, the first quenching site, which is termed Q1 site, consists of the major LHCII that, during high light illumination, detaches from the PSII core complex. These detached LHCIIs form aggregates (see “Role of LHCII aggregation”) which are characterized by a high dissipation of excitation energy as heat. The formation of the Q1 site requires the interaction with the protonated PsbS protein (see “The PsbS protein”) which regulates the detachment, migration and aggregation/disaggregation of LHCII. It has been proposed that quenching at the Q1 site, which does not require the presence of Z but the protonation of PsbS, reflects the fast part of NPQ (qE) which forms and relaxes on a time scale of 1 to 5 min (Li et al., 2002). The second quenching site, Q2, consists of minor LHCII proteins (CP29, CP26 and CP24) which stay attached to the PSII core complex. Formation of NPQ at the Q2 site exhibits slower kinetics than NPQ at the Q1 site and is established on a time scale of 10 to 15 min, which corresponds to the kinetics of the conversion of V to Z (see “The VAZ cycle”). Together with former observations that the minor LHCII antenna proteins are enriched in the VAZ cycle pigments (Bassi et al., 1993; Ruban et al., 1994; Goss et al., 1997), this led to the conclusion that NPQ at the Q2 site strongly depends on the VAZ cycle. Interestingly, recent experiments on diatoms (Miloslavina et al., 2009) have implicated that the total NPQ in these algae also depends on the formation of two quenching sites consisting of detached, aggregated FCP complexes and FCP proteins which stay associated with the PSII core complex (see “The mechanism of NPQ in diatoms”). NPQ in mosses With regard to the biodiversity of NPQ, mosses represent an interesting group of organisms, since they bridge the gap between land plants and the aquatic algae. Recently, it has been shown (Alboresi et al., 2010) that the moss Physcomitrella patens contains

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both the PsbS protein, that is typical for land plants and plays an important role in the establishment of NPQ (see “The PsbS protein” and references therein), and the Lhcsr proteins, which are found within the green algae and replace the PsbS protein with respect to its function in NPQ (Peers et al., 2009, see Requirements of NPQ in green algae). Knock-out mutants of P. patens where either the PsbS or one of the two Lhcsr proteins was missing, showed a significantly reduced NPQ, which demonstrates that in the moss both proteins are needed for efficient and high NPQ. Double and triple mutants, in which PsbS and the Lhcsr1 and Lhcsr2 proteins were absent, were characterized by an almost complete absence of NPQ. These mutants exhibited an increased susceptibility to high light stress compared to the wild type, which proved that the PsbS and Lhcsr proteins indeed provide efficient photoprotection for the moss. Interestingly, the strongest effect on NPQ stimulation was found for the Lhcsr1 protein. The data presented by Alboresi et al. (2010) suggest that upon land colonization, the higher plants evolved a unique mechanism for the dissipation of excessive excitation energy which relies on the single presence of the PsbS protein, before losing the ancestral Lhcsr protein found in the green algae. Newest results with VDE, PsbS and Lhcsr mutants (Pinnola et al., 2013) show that Z is important for NPQ in P. patens. It is of further importance that the Lhcsr-dependent NPQ exhibited a strong dependence on the presence of Z, while the PsbS dependent quenching was less influenced by the VAZ cycle pigment. In accordance with this observation, the authors were able to show that the Lhcsr protein contained a binding site for Z. Interestingly, the Lhcsr protein of Chlamydomonas reinhardtii (see chapter 4.1.3) can also bind Z, but Z has no or only little effect on the Lhcsr-dependent NPQ in the green alga (Bonente et al., 2011). From the results on P. patens Pinnola et al. (2013) conclude that Z plays an important role in the adaptation of higher plants to the land environment. Taking into account that the higher plants have lost the Lhcsr-dependent NPQ, Z might have helped to switch from the PsbS/Lhcsr mechanism to a mechanism which relies on a single protein, namely the PsbS protein (Alboresi et al., 2010). NPQ in green algae Requirements of NPQ in green algae While in general the requirements of NPQ in green algae, i.e. the VAZ cycle, the proton gradient and LHCII aggregation, are comparable to those of higher plants (see chapters above), there are also important differences. For the model green alga C. reinhardtii it has been shown that the requirements for NPQ activation are different and that significant NPQ is only induced after a high light acclimation of the alga, whereas it is constitutive in higher plants (Peers et al., 2009; Bonente et al., 2011). It also has to be mentioned that most green algae do not show a differentiation of the thylakoid membranes into grana and stroma regions (Gunning and Schwartz, 1999) and that two minor PSII antenna proteins, namely Lhcb3 and Lhcb6 (CP24), are missing (Koziol et al., 2007; Alboresi et al., 2010). This implies that the LHCII supercomplexes, which exist in the grana regions of higher plants and have been proposed to represent the quenching locus for efficient NPQ (Horton et al., 2008), are not present in most of the green algae and that the antenna structure required for high NPQ in green algae is of a different nature. With respect to the VAZ cycle it has to be mentioned that for C. reinhardtii no de-epoxidase gene has been found in the genome to the present day (Grossman et al., 2010). However, the most prominent difference in the requirements of NPQ between higher plants and green algae is that in several green algae the PsbS protein is not present and is replaced by the so-called Lhcsr proteins, ancient members of the LHC protein superfamily (Peers et al., 2009; Bonente et al., 2011; Gerotto and Morosinotto, Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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2013). The importance of the Lhcsr proteins for NPQ in green algae was found when NPQ-mutants of C. reinhardtii were analyzed with respect to their antenna protein composition and the absence of the Lhcsr protein was detected (Peers et al., 2009). In this study it was also demonstrated that the strongly reduced NPQ in the mutants resulted in a significantly decreased fitness upon a shift from moderate to high light intensities compared to the C. reinhardtii wild-type. Interestingly, the Lhcsr proteins were also found in other groups of the green algae, including the Prasinophyceae (Peers et al., 2009, see “NPQ in prasinophyceae”). In addition, it has been shown recently that Lhcsr proteins are also required for NPQ in diatoms and brown algae (see “NPQ in diatoms” and references therein). Although significant differences exist between Lhcsr proteins and PsbS, i.e. the former are three helix proteins and bind Chl and xanthophyll molecules while the latter contains four helices and most probably is pigment free (Li et al., 2002; Bonente et al., 2011), a recent study has demonstrated that the Lhcsr3 protein of C. reinhardtii also shows features which are comparable to those of PsbS (Bonente et al., 2011). Lhcsr3 is a strong quencher of Chl a fluorescence and is highly active in the formation of a carotenoid radical cation which has been proposed as the quenching site in higher plants (Holt et al., 2005). Like the PsbS protein, Lhcsr3 is also sensitive to an increase in the proton concentration during the establishment of the photosynthetic proton gradient. NPQ in prasinophyceae The VA cycle The VAZ cycle of higher plants (see “The VAZ cycle”), which is found in the majority of the algae belonging to the chlorophyta (“Requirements of NPQ in green algae”), exists in a modified form in the Prasinophyceae. These green algae have been shown to exhibit an incomplete XC (Goss et al., 1998). This VA cycle has been extensively studied in the alga Mantoniella squamata which reacts with a strong accumulation of the intermediate reaction product of the XC, A, to a high light illumination. Z, that is normally found in high concentrations of high light exposed higher plants and green algae (see chapters “The VAZ cycle” and “Requirements of NPQ in green algae”), does not amount in significant concentration. Further studies have shown that the Z-depleted VA cycle of M. squamata is in part the result of an extremely slow second de-epoxidation step from A to Z, while the first de-epoxidation step from V to A proceeds with fast kinetics (Frommolt et al., 2001). In addition, the simultaneous epoxidation reaction from Z to A is rather fast, thus efficiently converting the few Z molecules, which are formed by the slow second de-epoxidation step, back to A. Thus it is the interplay of the modified de-epoxidation reaction and the epoxidation reaction that leads to the observed strong accumulation of A during periods of high light illumination. The strongly reduced rate of A de-epoxidation is an intrinsic feature of the VDE of M. squamata (Goss, 2003) and is described in more detail in “Properties of the VDE of Prasinophyceae”. The VA cycle has also been observed in other members of the Prasinophyceae like Micromonas pusilla (Goss et al., unpublished) and Ostreococcus tauri (Cardol et al., 2008). Recently, evidence has occurred that other green algae, which do not belong to the Prasinophyceae, can also exhibit the reduced VA cycle (Stamenkovic et al., 2013). Properties of the VDE of Prasinophyceae The VDE of the Prasinophyceae M. squamata, although in general performing the same enzymatic reaction, shows pronounced differences to the VDE of higher plants (see “Properties of VAZ cycle enzymes”). It is characterized by a significantly reduced substrate affinity for the substrate A, while the KM value for V is comparable to that of the higher plant VDE (Frommolt et al., 2001). A more

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detailed examination of the substrate specifity of the M. squamata VDE (Goss, 2003) revealed that the low substrate affinity of the enzyme is not restricted to the mono-epoxide A. Other xanthophyll mono-epoxides, like diadinoxanthin, lutein epoxide, cryptoxanthin epoxide and neoxanthin, proved to be similarly poor substrates for the M. squamata VDE. Xanthophylls with a second epoxy group, like V and cryptoxanthin di-epoxide, could, on the other hand, be de-epoxidized with a higher efficiency. Such a preference for xanthophyll di-epoxides was not observed for the higher plant VDE, where no marked differences in the pigment de-epoxidation rates exist between xanthophyll mono- and di-epoxides. The VDE of the higher plant spinach, on the contrary, shows the highest pigment conversion rates in the presence of the mono-epoxides antheraxanthin and lutein epoxide. Despite this substantial difference between the VDEs of M. squamata and Spinacia oleracea, both enzymes share common features. In in vitro experiments both VDEs were unable to convert xanthophylls with a 9-cis configuration in the acyclic polyene chain and both enzymes relied on substrates in the all-trans configuration (see also Yamamoto and Higashi, 1978; Grotz et al., 1999). This indicates that the catalytic site of the M. squamata VDE, in general, has a comparable three-dimensional structure as the VDE of higher plants (see also “Properties of VAZ cycle enzymes”). Based upon recent experiments with the isolated LHC and VDE of M. squamata and a comparison of the VDE sequences of higher plants and the Prasinophyceae O. tauri (Schaller et al., 2012a,b), the differences in the substrate affinity of the VDEs have been discussed within the frame of the VDE dimerization model (Arnoux et al., 2009). It has been observed that, despite the high homology of the VDE sequences between higher plants and the Prasinophyceae, certain important amino acids located in the vicinity of the V binding site have been exchanged in the algal sequence. These amino acids have also been shown, by mutational analysis, to play an important role in the activity of the VDE of higher plants (Saga et al., 2010). It has also been suggested that the amino acid exchanges in O. tauri have an impact on the binding sites for the cosubstrate of the de-epoxidation reaction, ascorbate (Schaller et al., 2012a,b). A limitation in ascorbate binding to the VDE would increase the possibility that only one epoxy group of V is converted and thus lead to the observed accumulation of A in M. squamata and O. tauri. After its formation, A would need to detach from the VDE, perform a thermodynamically unfavourable flip-flop mechanism in the thylakoid membrane and rebind to the enzyme to become completely de-epoxidized to Z. This series of events is in line with the characteristics of the de-epoxidation reaction observed in the Prasinophyceae, namely a fast first de-epoxidation step from V to A and a slow second de-epoxidation step from A to Z (see “The VAZ cycle”). A as quencher For the VA cycle containing alga M. squamata, it was observed that A is able to strongly enhance NPQ. This observation is of high importance, since it shows that A can completely replace Z in the mechanism of enhanced thermal dissipation of excitation energy. The suitability of A as replacement for Z in the mechanism of NPQ was also shown for higher plants (Gilmore and Yamamoto, 1993) and the green alga Chlorella vulgaris (Goss et al., 2006b). While Gilmore et al. (1994) differentiated between A formed at the stromal side of the thylakoid membrane during the epoxidation reaction, which, according to their results, did not exert the same quenching capacity as A synthesized at the lumenal side during Vx deepoxidation, Goss et al. (2006b) did not observe a difference in the quenching capacity between A formed during the de-epoxidation and epoxidation, respectively in C. vulgaris. With respect to the recent NPQ models (see “Recent models for NPQ” and references therein), this indicates that A can replace Z as a direct Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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quencher of singlet excited Chl a states or that it is able to form a radical pair with a special Chl molecule (Holt et al., 2005) of the LHC or the Lhcsr protein, which is used by the Prasinophyceae as a replacement for the PsbS protein (Peers et al., 2009, see chapter 4.1.3). Taking into consideration that the LHCII aggregation model (Horton et al., 2005) might also provide the mechanistic basis of NPQ in the Prasinophyceae, A would act as an aggregation enhancer of the algal LHC, as it has been proposed for Z in higher plants. With respect to the LHC aggregation model, experimental evidence has been provided that the LHC of M. squamata is able to aggregate under conditions of a low pH, which leads to a strong quenching of the Chl a fluorescence (Goss and Garab, 2001). NPQ in diatoms Requirements of NPQ in diatoms Diatoms are unicellular algae, which are ubiquitous in aquatic environments and contribute significantly to the global carbon fixation (Geider et al., 2001). They belong to the stramenopiles, whose photosynthetic members acquired their chloroplast by secondary endosymbiosis from a red algal ancestor (Keeling, 2013). From the three NPQ components qE, qT and qI, qT was argued to be absent in diatoms due to experimental results which were based on the assumption of a specific far red light absorbance of PSI like in land plants (Owens, 1986). However recent results of a long wavelength absorbing component of PSII in diatoms (Fujita and Ohki, 2004) suggest a re-evaluation of the possibility of state transitions in diatoms using other experimental approaches. The distinction between qE and qI in higher plants is mainly based on the different relaxation kinetics, which occur on the timescale of minutes or hours, respectively. A differentiation between qE and qI in diatoms is more difficult since an extremely rapid relaxation of NPQ caused by the breakdown of the proton gradient is missing (see below). However, the processes we will describe in the following concern the relatively fast reversible part of NPQ, which is not related to photoinhibition of PSII due to D1 impairment and hence belongs to the category of qE. For the sake of simplicity, we will designate this process NPQ instead of qE. It relies on three interacting components: (1) The proton gradient generated between the thylakoid lumen and the chloroplast stroma during light exposure, (2) The xanthophyll diatoxanthin (Dt), which is synthesized from diadinoxanthin (Dd) by the enzyme diadinoxanthin de-epoxidase (DDE) in the presence of a proton gradient and (3) The chloroplast located, but nuclear encoded, antenna proteins called Lhcx. In the last years a huge progress has been made regarding the localization of Dt in the membrane, its binding to antenna proteins and lipids, the distribution of lipids in the chloroplast, the organization of the antenna, the involvement of Lhcx in NPQ, and the ecophysiological significance of NPQ for diatoms. In this chapter we will discuss only those findings which are directly related to NPQ. For the other aspects we recommend the reader to the reviews by Goss and Wilhelm (2009), Lepetit et al. (2012) and Lavaud and Goss (2014, in press). Role of the proton gradient The involvement of the proton gradient in the formation of NPQ in diatoms was first demonstrated by Caron et al. (1987) and Ting and Owens (1993). The acidic conditions in the thylakoid lumen are needed to activate the de-epoxidation from Dd to Dt by the DDE (see “The Dd–Dt cycle”). In higher plants and green algae, besides the activation of the VDE, the proton gradient alone can induce significant NPQ (see “Role of the proton gradient” and “Requirements of NPQ in green algae”, Horton et al., 1991; Niyogi, 1999; Ruban et al., 2012) and Z-dependent NPQ immediately relaxes once the proton gradient is abolished (Gilmore and Yamamoto, 1991).

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In contrast, in diatoms the pH is unable to induce NPQ in the absence of Dt (Lavaud et al., 2002a). Moreover, once diatoms have established NPQ, the breakdown of the proton gradient does not lead to a direct relaxation of NPQ which in turn depends on the epoxidation of Dt to Dd (Goss et al., 2006a). However, some alterations of the inter-dependency of the proton gradient and Dt in the establishment of NPQ can be achieved. In cells of the centric (radial symmetric) diatom Cyclotella meneghiniana that contain a significant concentration of Dt due to the cultivation conditions, a very fast and large NPQ is induced by the establishment of the proton gradient which is independent of the formation of additional Dt. The magnitude of this so-called transient NPQ depends, on the one hand, on the amount of initially available Dt but, on the other hand, can also be amplified by an increased proton gradient (Grouneva et al., 2008). This transient NPQ is not present in the pennate (bilateral symmetric) Phaeodactylum tricornutum. However, the relationship between NPQ and Dt can be modified employing special experimental conditions which avoid long term acclimation processes. This was interpreted in terms of a need for the protonation of proton binding sites of antenna proteins for the development of NPQ (Lavaud and Kroth, 2006). Both results indicate that the role of the proton gradient might not be restricted to the mere activation of the DDE but that the pH itself is important for the NPQ process under certain experimental conditions, possibly by switching Dt into an activated, quenching state (Lavaud and Lepetit, 2013; Ruban et al., 2004). It has to be kept in mind that most of the results concerning the role of the proton gradient were obtained by the application of several inhibitors of the pH, which always have significant side effects, like, e.g. influences on the electric potential of the chloroplast or the redox state of the electron transport chain components. Hence, these results need to be interpreted with care, leaving up the possibility of other or additional triggers of NPQ besides the proton gradient. To clarify the role of the proton gradient, the exact determination of the magnitude of the pH will be helpful. Very recently, it was shown in T. pseudonana that the use of the electrochromic absorbance shift provides a tool to measure the extent of the proton gradient which can be used in future measurements to answer these important questions (Thamatrakoln et al., 2013).

The Dd–Dt cycle The conversion of the xanthophyll Dd to Dt during illumination and the reverse reaction in darkness was already described in 1970 (Hager and Stransky, 1970; Stransky and Hager, 1970). During the Dd to Dt conversion, which is triggered by the presence of the proton gradient, the epoxy group of Dd is removed by the enzyme DDE (see “Properties of Dd–Dt cycle enzymes”). It is reintroduced during the epoxidation reaction, which usually takes place in the absence of a proton gradient, i.e. in darkness or low light, by the enzyme Dt epoxidase (DEP, see also “Properties of Dd–Dt cycle enzymes”). To distinguish the XC of diatoms from the VAZ cycle of higher plants and green algae (see “The VAZ cycle” and “Requirements of NPQ in green algae”), it is normally termed the Dd–Dt cycle. Interestingly, the actual involvement of Dt in the formation of NPQ in diatoms was unravelled more than 20 years after the discovery of the Dd–Dt cycle (Demers et al., 1991; Arsalane et al., 1994; Olaizola et al., 1994). Since then, numerous studies have been performed on the function of the Dd–Dt cycle (reviewed, e.g. in Wilhelm et al., 2006; Lavaud, 2007; Jahns et al., 2009; Bertrand, 2010; Brunet and Lavaud, 2010; Goss and Jakob, 2010; Lepetit et al., 2012), and the overwhelming majority of the experimental results points towards a direct involvement of Dt in the NPQ mechanism and will be discussed in the following chapters. Moreover, a role of Dt as an antioxidant against the damaging effects of ROS under prolonged high light illumination was proposed (Lepetit et al., 2010).


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Properties of Dd–Dt cycle enzymes. The DDE and the DEP have a comparable structure to the VDE and ZEP of green plants (for details see “Properties of VAZ cycle enzymes”), which is also demonstrated by their ability to convert V to Z, via the intermediate A (VDE), and vice versa (ZEP, Lohr and Wilhelm, 1999). Furthermore, both enzymes show similar cosubstrate requirements (see also “Properties of VAZ cycle enzymes”) However, recent investigations have shown that both enzymes exhibit several major differences in their properties compared to the VDE and ZEP of higher plants and green algae. First, unlike green plants, the DEP is strongly regulated by the proton gradient, making it virtually inactive once the transmembrane pH has been established (Goss et al., 2006a; Mewes and Richter, 2002). In the absence of the proton gradient, the epoxidation in diatoms reaches extremely high rates, which can be more than 20 times higher than the epoxidation rates observed in higher plants or the green alga Chlorella vulgaris (Goss et al., 2006a). Taking into account that in diatoms NPQ does not relax by the simple breakdown of the proton gradient, there is a strong need to convert Dt back to Dd as fast as possible, thereby converting the antenna system from the state of thermal dissipation back to the light-harvesting state. It is of further interest that the pennate diatom P. tricornutum contains three and the centric diatom T. pseudonana two DEP genes in their genome (annotated as ZEP), while only one copy of the gene is present in green plants (Coesel et al., 2008; Frommolt et al., 2008; Montsant et al., 2007). It needs to be clarified if these additional enzymes are also involved in xanthophyll cycling or if they fulfil different, yet unknown functions. The mode of DEP inactivation by the proton gradient is yet unknown. However, different mechanisms are possible: (1) In contrast to the ZEP, the DEP might be extremely sensitive to the minor pH changes of the chloroplast stroma that occur during illumination (a value of around 0.5 has been reported for plants by Antal et al., 2013). (2) The proton gradient might lead to the activation or generation of a special DEP inhibitor (putatively localized in the lumen to sense minor pH changes) whose nature is unclear. (3) Unlike the ZEP, the DEP might not be localized at the stromal side of the membrane but in the thylakoid lumen or at the lumenal membrane side. In this respect, it is interesting that all three DEP sequences found in P. tricornutum contain remarkably different structural parts compared to the ZEP of higher plants (Coesel et al., 2008). (4) The DEP might be localized at the stromal membrane side but possesses a membrane spanning domain with a proton sensing residue exposed to the lumen. With regard to this aspect it is noteworthy, that one of the three DEPs present in P. tricornutum possesses one membrane spanning domain (Coesel et al., 2008). For points 2 and 4, a permanent binding of the DEP to the thylakoid membrane would be required which in turn has been proposed recently for the ZEP of higher plants (see “Properties of VAZ cycle enzymes”, Schaller et al., 2012a,b). Like the DEP, the lumenal DDE shows remarkable differences compared to the VDE of higher plants. Most importantly, it is already active at higher pH values and exhibits a broader pHoptimum than the VDE (Jakob et al., 2001). At the slightly acidic pH of 6.5 the DDE shows more than 50% of its maximum activity, which is reached in a range between pH 5 to 6 (Grouneva et al., 2006), and even at a pH value of 7 the DDE is still able to convert some Dd to Dt (Jakob et al., 2001). The low concentration of protons needed to activate the DDE might be related to the lower amount of charged glutamic acid residues (Coesel et al., 2008), which are thought to be responsible for the binding of the protein to the thylakoid membrane (Bugos and Yamamoto, 1996). In addition, the DDE has a lower KM -value for the cosubstrate ascorbate than the VDE, which means that full DDE activity can be obtained at significantly lower ascorbate concentrations in the thylakoid lumen (Grouneva et al., 2006). Interestingly, the pH-dependency of the DDE is linked to the availability of ascorbate, i.e. at low ascorbate concentrations

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the pH-optimum is shifted to lower pH values whereas at high ascorbate concentrations a broad pH-optimum is observed. In conclusion, due to the specific properties of DDE and DEP, slight alterations in the magnitude of the proton gradient can cause large changes in the ratio of de-epoxidized to epoxidized xanthophylls thus allowing diatoms to rapidly acclimate to changes in the environmental light conditions. However, due to the influence of reduced ascorbate on the pH dependence of the DDE, the same proton gradient under different physiological conditions of the chloroplast may result in different de-epoxidation states, which adds another level of complexity to the XC of diatoms compared to that of higher plants. Similarly to the DEP, several DDE sequences are found in the diatom genome. E.g., P. tricornutum contains, besides the DDE (annotated as VDE), two VDE like enzymes (VDL) and one VDE related enzyme (VDR) (Coesel et al., 2008). Also in the genome of T. pseudonana, one VDL and one VDR are encoded besides the classical DDE (Montsant et al., 2007; Coesel et al., 2008). However, the VDL and VDR genes do not contain the charged domains that have been proposed to realize the binding of the enzyme to the thylakoid membrane. Their gene expression differs from that of the classical DDE genes (Coesel et al., 2008, Nymark et al., 2009), and to date their function is unclear. As some of the VDL and VDR genes are up-regulated during light stress (Nymark et al., 2009), it was hypothesized that they might be involved in the somewhat different de-epoxidation characteristics of high light synthesized Dd (Lepetit et al., 2013). Moreover, it was assumed that in P. tricornutum cells where the DDE was silenced, these enzymes might perform the Dd to Dt conversion (Lavaud et al., 2012). In addition, it is not unlikely that the alternative DDEs and DEPs are involved in the biosynthesis of the XC pigments and the main light-harvesting pigment of diatoms, fucoxanthin (Lohr, 2011).

Role of MGDG in Dd de-epoxidation. As it has been described in “Role of MGDG in V de-epoxidation” for higher plants, the inverted hexagonal structure forming lipid MGDG is also essential for efficient Dd de-epoxidation in diatoms (Goss et al., 2005, 2007). By comparing green plants and diatoms it was found that the concentration of MGDG needed for the complete solubilization of Dd is much lower than that needed for the solubilization of V (Goss et al., 2005, 2007). In line with this finding it was demonstrated that efficient de-epoxidation of Dd to Dt can be realized at significantly lower MGDG concentrations than the de-epoxidation of V to Z. This basically means that the MGDG phases in the diatom thylakoid membrane can harbour a higher number of Dd/Dt molecules compared to the V/Z containing MGDG parts of the higher plant membrane. Indeed, it has been shown that in HL acclimated diatoms the Dd/Dt content can reach values of up to 0.7 mol per mol Chl a (Lepetit et al., 2010). These values are by far higher than those that are typically observed in sun-acclimated plants (DemmigAdams and Adams, 1992; Demmig et al., 1987). The majority of these Dd/Dt molecules are dissolved in MGDG molecules which are tightly connected to the diatom antenna complex (FCP) (Lepetit et al., 2010). This lipid shield forms an attraction site for the DDE and represents that part of the membrane where the efficient Dd de-epoxidation is taking place. Furthermore, diatom thylakoids are characterized by a very high content of the negatively charged lipid SQDG (Vieler et al., 2007; Goss et al., 2009). Interestingly, this lipid inhibits Dd de-epoxidation (Goss et al., 2009). The DDE inhibition by SQDG, and the findings regarding the function of MGDG, allowed us to develop a model of the diatom thylakoid membrane where most of the Dd–Dt cycle pigments synthesized during high light acclimation are localized in MGDG phases located in the inner membranes of the stacks of three membranes that are typical for the diatom chloroplast (Lepetit et al., 2012).


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The Lhcx proteins The Lhcx proteins belong to the large family of light-harvesting proteins (LHC, Koziol et al., 2007; Engelken et al., 2012,). In diatoms, mainly three LHC protein classes can be differentiated: (1) The Lhcf proteins which constitute the peripheral fucoxanthin chlorophyll binding protein (FCP) complex, the main peripheral diatom antenna system (Lepetit et al., 2010; Grouneva et al., 2011; Gundermann et al., 2013; Nagao et al., 2013a); (2) The Lhcr proteins which comprise the specific antenna of photosystem I (Veith et al., 2009; Lepetit et al., 2010; Grouneva et al., 2011; Ikeda et al., 2013); (3) The Lhcx proteins which were identified as components of the peripheral FCP antenna complex (Beer et al., 2006; Lepetit et al., 2010; Grouneva et al., 2011; Nagao et al., 2013a) and the PSI specific light-harvesting system (Grouneva et al., 2011). Recently, another member of the LHC family was identified in diatoms, the so called RedCAPs, whose function is so far not resolved (Sturm et al., 2013). The Lhcx proteins are grouped with the LI818/Lhcsr proteins, which are also present in green algae and mosses (see “NPQ in mosses” and “Requirements of NPQ in green algae”, Koziol et al., 2007). Recently, Bailleul et al. (2010) and Zhu and Green (2010) unambiguously showed the pivotal role of these proteins in the NPQ of diatoms and demonstrated that, besides the proton gradient and the Dd–Dt conversion, special antenna proteins are needed for efficient NPQ. The Lhcx proteins act as a modulator of NPQ, i.e. in the presence of comparable Dt concentrations the reduced or increased amount of Lhcx1 protein strongly diminishes or amplifies the NPQ capacity, respectively (Bailleul et al., 2010). Lhcx1 is already highly abundant in low light acclimated P. tricornutum cells (Bailleul et al., 2010; Lepetit et al., 2010, 2013; Grouneva et al., 2011; Nymark et al., 2013). The same holds true for the Lhcx1 protein of T. pseudonana (Zhu and Green, 2010; Grouneva et al., 2011; Wu et al., 2012) and the FCP6 of Cyclotella cryptica/meneghiniana (Westermann and Rhiel, 2005; Beer et al., 2006, 2011), both of them probably exerting a similar function like the Lhcx1 protein in P. tricornutum. In the genome of P. tricornutum 4 Lhcx genes are present (Bowler et al., 2008), while in T. pseudonana 6 Lhcx genes were identified (Armbrust et al., 2004). The pennate diatom Fragilariopsis cylindrus contains even 11 Lhcx sequences (http://genome.jgi-psf.org/Fracy1/Fracy1.home.html). In P. tricornutum, Lhcx1, but especially Lhcx2 and Lhcx3, have been shown to strongly react to changes in the light intensity, both on the genetic and protein level (Nymark et al., 2009; Bailleul et al., 2010; Lepetit et al., 2013; Nymark et al., 2013). Moreover, not only the light intensity but also the characteristics of the illumination, i.e. illumination with continuous or fluctuating light, have a pronounced impact on specific light induced Lhcx proteins (Lepetit et al., unpublished results). In darkness, Lhcx4 seems to be up-regulated (Nymark et al., 2013) and it also exhibits a circadian expression pattern (Lepetit et al., 2013). Lastly, iron starvation induces gene expression of Lhcx2 (Allen et al., 2008). In T. pseudonana the Lhcx1, Lhcx4 and Lhcx6 proteins react to changes in the light intensity (Zhu and Green, 2010), while nutrient availability (Zhu et al., 2010) and temperature (Wu et al., 2012) influence the expression of Lhcx1 and Lhcx6. The expression of FCP6 in C. cryptica/meneghiniana is, besides the regulation by the light intensity (Oeltjen et al., 2004; Beer et al., 2006), also triggered by the iron content of the culture medium (Beer et al., 2011). In the psychrophilic diatom Chaetoceros neogracile several high light induced Lhcx genes were identified (Park et al., 2010) and one of them responded also to elevated temperatures (Hwang et al., 2008). Overall, it seems that some Lhcx proteins are always present to provide a basal capacity for NPQ, while others are expressed upon changes in the environmental conditions, i.e. to realize enhanced photoprotection during periods of increased stress. It is reasonable to believe that the enhanced, Lhcx-dependent NPQ serves to prevent the formation of ROS during the stress period, but the function of Lhcx proteins might go beyond photosynthesis related processes,

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as the example of a dark induced Lhcx4 demonstrates. Most likely, the application of several other stress conditions will reveal further expression patterns of Lhcx genes and proteins, thus enlightening the complex regulation of these proteins and their role in diatom photoprotection. The mechanism of NPQ in diatoms Dt as quencher As mentioned above, NPQ in diatoms is more closely linked to the amount of Dt than the NPQ in higher plants and green algae to the Z concentration (Lavaud et al., 2002a, 2004; Goss et al., 2006a). The importance of Dt for NPQ can be observed in intact cells but also in isolated FCP complexes where the fluorescence emission can be reduced significantly upon the replacement of Dd by Dt (Gundermann and Büchel, 2008). Strikingly, diatoms can even synthesize Dt during periods of prolonged darkness by a chlororespiratory electron transport and proton gradient (Jakob et al., 1999). Although Dt is unable to enhance NPQ significantly during the dark phase, it becomes activated once the cells are transferred to illumination (Cruz et al., 2011). Due to the different enzyme properties of the diatom DDE and DEP (see “Properties of Dd–Dt cycle enzymes”), only a weak chlororespiratory proton gradient can activate the DDE and inhibit the DEP. This chlororespiratory pH is most probably induced by the oxidation of stromal reducing equivalents via the concomitant action of a plastoquinol oxidase and a proton pumping NADPH dehydrogenase (Jakob et al., 2001; Grouneva et al., 2009). Also a shortage of NADPH, and hence the inhibition of the epoxidation under dark conditions (Goss et al., 2006a), might allow the de-epoxidation due to the activity of the DDE even at almost neutral pH. As Dt plays such a prominent role in the diatom NPQ, a direct quenching function of this xanthophyll molecule was discussed (Frank et al., 1996). According to the gearshift model the energy level of the S1 state of the excited Dd molecule should be higher than that of excited Chl a, while the S1 energy level of Dt should be lower. This would allow for a direct transfer of excitation energy from excited Chl a molecules to Dt molecules, which eventually would dissipate the energy as heat (Frank et al., 1996). A similar mechanism was discussed for V and Z in plants (see “Role of the VAZ cycle in NPQ”, Frank et al., 1994), but the determination of the energy levels of the XC pigments was later challenged (Polivka and Sundström, 2004). By applying more sophisticated spectroscopic methods, both the energy levels of the S1 state of Dd and Dt were recently demonstrated to be lower than the energy level of the Chl a S1 state (Enriquez et al., 2010). Hence, the conversion from Dd to Dt would only allow for a direct quenching of the Chl a excitation, if the energy levels of the protein bound molecules were different or if, in addition, the steric configuration of the protein-associated molecules would prevent (Dd) or enable (Dt) the energy transfer from the excited Chl a molecule. In analogy to the proposal of the formation of a Chl a-Z radical pair as the basis of NPQ in higher plants (Ahn et al., 2008; Holt et al., 2005), Dt might also be able to form such a radical pair with Chl a in diatoms. In that case, a Dt molecule would receive energy from an excited Chl a molecule, followed by electron transfer to a closely coupled • • chlorophyll dimer, thereby producing a Chl− Dt+ charge transfer state. In the following recombination event the energy would then be dissipated as heat. In order to participate in NPQ, Dt needs to be bound to proteins (Lepetit et al., 2012). This is illustrated by the fact that high amounts of unbound Dt are synthesized during long term high light illumination (timescale of days) which are unable to enhance NPQ (Schumann et al., 2007; Lepetit et al., 2010). On the other hand, during medium term high light stress (starting after 30 min of illumination) the magnitude of NPQ is strongly increased, because the Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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increase of the Dd/Dt pool size is coupled to the synthesis of Lhcx proteins. Once nuclear gene expression, and hence Lhcx synthesis, is blocked, the newly synthesized Dt does not lead to higher NPQ (Lepetit et al., 2013). Based on these experimental data three Dd/Dt pools can be differentiated: 1. Dd and Dt molecules which are bound to the proteins of the FCP antenna. During high light acclimation this pool increases, visualized by a complex 77K resonance Raman spectral fingerprint (Alexandre et al., 2013); 2. Dd/Dt dissolved in the thylakoid lipid MGDG which is in close association with the FCP antenna proteins. The size of this Dd/Dt pool is much higher in high light cultivated diatoms compared to cultures acclimated to low light conditions and is detected by a specific absorption fingerprint. Lipid dissolved Dt seems to be involved in the antioxidative response of the chloroplast (Lepetit et al., 2010); 3. Dd and Dt bound to the Lhcr proteins of the PSI antenna. Dd in this pool acts as lightharvesting pigment and transfers the absorbed excitation energy to Chl a, as revealed by 77K fluorescence excitation spectra. The size of the PSI Dd/Dt pool is comparable in low or high light acclimated cells (Lepetit et al., 2010). Hence, an involvement of pool 3 in NPQ, at least under high light conditions, is unlikely, even though the presence of Lhcx proteins in the PSI of centric diatoms has been reported (Grouneva et al., 2011). The largest part of Dt-dependent NPQ is related to the Dt molecules found in pool 1. This Dt can be traced by the absorption difference at 522 nm (522) that exists between cells that are illuminated with low or high light intensities (Ruban et al., 2004). Interestingly, the correlation of 522 and NPQ is the same in different diatom species (Lavaud and Lepetit, 2013), pointing towards similar quenching mechanisms in all diatoms despite the obvious differences in antenna structures (see “Aggregation of antenna complexes”). The LHC proteins that bind the majority of Dd/Dt molecules of pool 1 are most likely the Lhcx proteins, based on several evidences: the overexpression of Lhcx1 leads to a much higher NPQ with the same amount of Dt (Bailleul et al., 2010). Lhcx genes are, besides a few members of the Lhcr genes, the only Lhc genes overexpressed during high light stress, and hence only their protein products are able to bind the newly synthesized Dt molecules, which confer the higher NPQ (Nymark et al., 2009). Indeed, by blocking their translation, no additional NPQ is formed by newly synthesized Dt molecules (Lepetit et al., 2013). Lhcr proteins build up the antenna of PSI (Veith et al., 2009; Lepetit et al., 2010) and therefore most probably do not contribute to the binding of Dt molecules from pool 1. In contrast, Lhcx proteins have been identified in the FCP antenna (Beer et al., 2006; Lepetit et al., 2010). This putative binding of Dd/Dt by Lhcx is also corroborated by studies with the Lhcx related Lhcsr protein from C. reinhardtii (Bonente et al., 2011) and the moss Physcomitrella patens (Pinnola et al., 2013), which demonstrated a high xanthophyll binding capacity of these proteins (see “NPQ in mosses” and “Requirements of NPQ in green algae”). Finally, it has to be mentioned that diatoms show Dtindependent NPQ: 1. A putative reaction centre quenching in PSII, which can be seen in the presence of the protonophore DCP (Eisenstadt et al., 2008); 2. NPQ during a dark recovery period after prolonged high light stress, which is independent of Dt or the proton gradient (Lepetit et al., 2013). 3. NPQ induced by a cyclic electron transfer around PSII (Lavaud et al., 2002b; Onno Feikema et al., 2006; Lavaud, 2007). Aggregation of antenna complexes In 2009 a time resolved fluorescence study with the pennate P. tricornutum and the centric C. meneghiniana revealed the formation of two different quenching sites under high light illumination (Miloslavina et al., 2009). The first site is located in an oligomeric antenna complex which becomes detached from the photosystems (quenching site 1, Q1). Its spectral feature is a long wavelength (red shifted) Chl a fluorescence component. The second quenching site

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is located in an antenna attached to the PSII (Q2) and might directly depend on the formation of Dt, comparable to Z-dependent NPQ in the minor LHCII antenna complexes of higher plants (Holzwarth et al., 2009). So far, it is difficult to bring the presence of two quenching sites in agreement with the information obtained with isolated antenna complexes. First of all, despite the comparable fluorescence decays with two independent emission wavelengths obtained for both centric and pennate diatoms (Miloslavina et al., 2009), these two major groups of diatoms contain different antenna structures. The centricdiatoms C. meneghiniana, T. pseudonana and C. neogracile are characterized by the presence of a trimeric antenna, hereafter called FCP-A, and an oligomeric, probably hexameric FCPB complex (Büchel, 2003; Beer et al., 2006; Lepetit et al., 2010; Nagao et al., 2013a). In contrast, the pennate diatom P. tricornutum contains only a trimeric FCP as basic unit (Lepetit et al., 2007; Joshi-Deo et al., 2010; Nagao et al., 2013a), although both, the FCP of P. tricornutum and the FCP-A and FCP-B of C. meneghiniana can be isolated as higher oligomeric complexes (Lepetit et al., 2007, 2010). Furthermore, time resolved spectroscopic measurements on isolated FCP-A and FCP-B complexes could not identify similar fluorescence spectroscopic fingerprints like those observed in intact cells (Nagao et al., 2013b). Artificial oligomerization of trimeric FCP-A, but not hexameric FCP-B, gave rise to a red shifted stark fluorescence component (Wahadoszamen et al., 2014), indicating that rather FCP-A than FCP-B represents the red shifted Q1 site. This would imply that FCP-B contributes to the Q2 site, which is in contradiction with the Dt requirement of this quenching site (Miloslavina et al., 2009), since it has been shown that FCP-B contains much lower amounts of Dd/Dt than FCP-A (Gildenhoff et al., 2010; Lepetit et al., 2010; Beer et al., 2011; Nagao et al., 2013b). Although the results on isolated antenna complexes have to be interpreted with care, further studies with FCP preparations underline the importance of aggregation for the diatom NPQ. By removing detergent and thus aggregating FCP complexes, a strong fluorescence quenching was induced (Gundermann and Büchel, 2008; Miloslavina et al., 2009). Moreover, a lower pH decreased the fluorescence emission of detergent deprived FCP-A, but not FCP-B (Gundermann and Büchel, 2008). Similarly, when the FCP-A complex of C. meneghiniana was incorporated into liposomes, a higher aggregation and quenching was observed at low lipid concentrations (Gundermann and Büchel, 2012). This quenching could be increased further at low and high pH values, while at pH 6.5 it reached its minimum. Also, the conversion of Dd to Dt increased the fluorescence quenching of FCP-A in liposomes. In a recent paper, Schaller et al. (2013) demonstrated that Mg2+ and low pH values lead to an aggregation and fluorescence quenching of the isolated P. tricornutum FCP. However, this aggregation is significantly less pronounced than that of the LHCII of higher plants. This is in line with the observation that Mg2+ is able to modulate the macro-aggregation of FCP complexes in the native thylakoid membrane of P. tricornutum, indicating that Mg2+ plays a comparable role in the macro-organization of the thylakoid membrane as in higher plants (Szábo et al., 2008). According to Gundermann and Büchel (2012), the actual aggregation of the FCP could be triggered by the proton gradient via the protonation of one glutamate residue in the lumenal loop of both the Lhcf and Lhcx proteins. However, in the native membrane these residues might be masked by the negatively charged lipids SQDG and PG (Schaller et al., 2013). Taking into account the disaggregating effect of the anionic lipids, it is noteworthy that the FCP seems to preferentially bind the neutral lipid MGDG (Lepetit et al., 2010). Besides these evidences for a correlation of FCP aggregation and NPQ in vitro, recent results on intact cells have also pointed towards a dependence of NPQ on structural changes of the FCP. It has been observed that in P. tricornutum a long wavelength fluorescence component, peaking at around 710 nm, is present (Fujita and Ohki, 2004). Its extent Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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correlates with the NPQ capacity, i.e. at higher ratios of the 710 to 685 nm 77K fluorescence an increased NPQ value can be obtained upon high light illumination (Lavaud and Lepetit, 2013). This long wavelength fluorescence component was much more pronounced in the pennate P. tricornutum compared to the centrics C. meneghiniana and Skeletonema costatum. Interestingly, the spectral signature of the 710 nm component resembles the characteristics of the Q1 site proposed by Miloslavina et al. (2009) and might thus represent some disconnected oligomeric FCP complexes. As the Q1 site was proposed to be independent of Dt (Miloslavina et al., 2009), the question arises which pigments are actually involved in the quenching mechanism. Miloslavina et al. (2009) suggested that fluorescence quenching at Q1 is based on Chl a-Chl a interactions. This could be induced by conformational changes within the antenna subunits, i.e. the trimers, or by newly established chlorophyll interactions between adjacent trimers, which get into close contact due to the oligomerization (Miloslavina et al., 2008). A quenching site composed of a Chl a homodimer might exist in the LHCII of higher plants (see “Recent models for NPQ”). This site is characterized by a long wavelength fluorescence component that is also visible in diatoms (see above). Alternatively, a conformational change caused by FCP aggregation might induce the formation of a Chl-fucoxanthin charge transfer state in the oligomeric antenna complexes (Wahadoszamen et al., 2014). In this case, besides its role in light-harvesting, fucoxanthin would also exert a photoprotective function. A model for NPQ in diatoms Recently, several models have been proposed to summarize the growing knowledge about NPQ in diatoms (e.g. Gundermann and Büchel, 2012; Lepetit et al., 2012; Lavaud and Goss, 2014, in press). All of them are based on the initial study by Miloslavina et al. (2009). Fig. 3 is another update of the current knowledge and resembles the model of Lavaud and Goss (2014, in press). Despite the obvious existing difference in antenna structure and quenching characteristics between pennates and centrics (see “Role of the proton gradient” and “Aggregation of antenna complexes”), there are also remarkable similarities, and the model by Miloslavina et al. (2009) highlights major characteristics in NPQ which are valid for the two main diatom groups. From the present state of knowledge it is clear that, despite the obvious in vitro effects of aggregation/oligomerization, pH and Mg2+ on the chlorophyll fluorescence, the XC pigment Dt represents the most important component in the overall NPQ process in vivo (possibly assisted by the protonation of specific antenna sites which helps shifting Dt in an NPQ effective state). For the Q2 quenching site a direct involvement of Dt, either via energy transfer from the S1 state of Chl a (see “Dt as quencher”) or the formation of a Chl-Dt charge transfer state (see also “‘Dt as quencher”), is likely (Miloslavina et al., 2009). Taking into account that in intact cells high NPQ is only visible in the presence of Dt and that the proton gradient alone is not sufficient to induce NPQ (Lavaud et al., 2002a), it has to be assumed that Dt also plays a role in the formation of the Q1 site, which consists of detached, aggregated FCP complexes. Moreover, relaxation of NPQ directly depends on the epoxidation of Dt to Dd, while the relaxation of the pH without conversion of Dt to Dd does not decrease the light-induced NPQ (Goss et al., 2006a). This makes it likely that the conversion of aggregated FCP complexes into smaller unquenched subunits more strongly depends on the interconversion of the XC pigments than on the protonation/deprotonation of FCP proteins. The question then arises how Dt, if it is not bound to the FCP complexes that form the Q1 site, could promote NPQ at this site. One can speculate that the conversion of Dd to Dt in the FCP complexes at the Q2 site induces a conformational change in the Dt binding proteins, most likely the Lhcx proteins, or the trimers themselves. This structural change would then lead to the dissociation of adjacent

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Fig. 3. Model of the diatom NPQ mechanism in PSII of low and high NPQ cells. After transfer from low light intensities to excessive light conditions, several structural rearrangements are taking place in the PSII antenna, which are reversed by transferring the cells back to low light. The conversion of Dd to Dt at the Lhcx protein within the FCP trimer leads to the formation of the Q2 site. The concomitant conformational change of the trimer promotes an expulsion of adjacent trimers, which form oligomeric complexes, which then can build up Q1. The extent of the structural changes upon high light acclimation depends on the pre-acclimation conditions of the cells, which directly influence the initial amount of Dd/Dt and Lhcx proteins as well as the presence of oligomeric antenna complexes due to an altered thylakoid membrane lipid composition. For a detailed explanation, see “A model for NPQ in diatoms”.

trimers from the PSII FCP supercomplex, followed by an aggregation of these complexes and the formation of Q1. This disconnection might also render the FCPs more prone to changes of the proton gradient, which would further facilitate their aggregation. Such a sequence of events, i.e. first the establishment of the Dt-dependent quenching at the Q2 site, which is directly followed by the disconnection and aggregation of FCP complexes for the establishment of the Q1 site, is also more in line with the results obtained with isolated FCPs: higher pH values around 6.5 already induce a significant de-epoxidation of Dd to Dt (Jakob et al., 2001), while lower pH values are necessary to induce fluorescence quenching due to the aggregation of FCP complexes (Gundermann and Büchel, 2012; Schaller et al., 2013). Under certain conditions oligomerization of FCP trimers might be triggered by other factors than the conversion of Dd to Dt, as seen by the 710 nm fluorescence emission maximum in cells in the stationary growth phase (see “Aggregation of antenna complexes”, Lavaud and Lepetit, 2013). These factors include an altered FCP to lipid ratio as well as changes in the composition of lipids associated with the FCP trimers. Still, these complexes would need to be activated to form the Q1 site. This activation most likely also depends on the increase of the proton concentration and the conversion of Dd to Dt, which in this case might also take place within the oligomeric complexes. The current model is based on the assumption that higher concentrations of protein bound Dt induce a higher amount of both NPQ at the Q2 site, which directly depends on Dt, and NPQ at the Q1 site, which requires the indirect action of Dt during detachment and aggregation of FCPs. Hence, a higher Lhcx protein expression, which may provide additional Dt binding sites, correlates with a higher NPQ (Bailleul et al., 2010), while higher concentrations of Dt alone only increase the antioxidant activity (Lepetit et al., 2010). As the Q1 site is only indirectly coupled to the


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presence of Dt, the partial uncoupling of Dt and NPQ under special experimental conditions (Lavaud and Kroth, 2006; Eisenstadt et al., 2008; Lepetit et al., 2013) might be explained by a stabilization of the Q1 site for a certain time under non-physiological conditions, i.e. in the presence of inhibitors or during a sudden change from high light illumination to darkness. As outlined above (see “Aggregation of antenna complexes”), isolated FCP-B does not fulfil the prerequisites for the formation of either the Q1 or Q2 site. Consequently, FCP-A, which binds Dt and has a high capacity for aggregation, might provide both the Q1 and Q2 site (Miloslavina et al., 2009; Gundermann and Büchel, 2012; Wahadoszamen et al., 2014). This poses the question how one type of antenna complex is able to realize several quenching mechanisms. In this respect, it has to be noted that the terms FCPA and FCP-B do not really stand for completely different protein complexes but merely describe different organization states of the antenna system. In P. tricornutum, where only trimers like FCP-A exist as basic antenna units, it has been shown that the trimers consist of different polypeptide combinations (Gundermann et al., 2013). In T. pseudonana, which contains a very similar antenna organization to C. meneghiniana (Nagao et al., 2013a), 12 proteins of the Lhcf and Lhcx family were identified in the FCP-A complex (Grouneva et al., 2011). Even if not all possible combinations of proteins might be used in vivo to form the FCP trimers, the least amount of different trimers would be four. This example illustrates that, although the organization state of the antenna system is comparable, individual complexes might be different and hence serve as building blocks for the formation of different quenching sites. If the connectivity of the individual antenna units is high, excess excitation energy might even be trapped from adjacent FCP complexes, either of the Q1 or Q2 type. Evidently, an increase in the amount

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of quenching sites increases the probability of trapping absorbed photons within the light-harvesting system, before the excitation energy reaches the photosynthetic reaction centres. NPQ in brown algae Brown algae (Phaeophyceae) are multicellular macroalgae mainly localized in benthic marine communities where they are often keystone members (Amsler and Fairhead, 2005). Like the diatoms, they belong to the stramenopiles and thus obtained their chloroplast by secondary endosymbiosis from a red algal ancestor (Fig. 1, Keeling, 2013). Compared to diatoms the knowledge about NPQ in brown algae is less profound, although some progress has been achieved during the last years. A major difference to diatoms is that the brown algae use the VAZ cycle that is typical for higher plants and green algae (Hager and Stransky, 1970). Several studies have shown that the conversion of V to Z is related to the formation of NPQ in the brown algae (e.g. Benet et al., 1994; Uhrmacher et al., 1995; Harker et al., 1999). Comparable to diatoms, the transthylakoid proton gradient alone does not induce NPQ, which is only estabslished when the light-driven pH is accompanied by the accumulation of Z (García-Mendoza and Colombo-Pallotta, 2007; García-Mendoza et al., 2011; Li et al., 2014). The extent of NPQ that can be achieved by brown algae is highly variable (Harker et al., 1999; Rodrigues et al., 2002) and pronounced differences in NPQ can be found even within one organism. Especially the macrophytic brown algae are characterized by a very high NPQ in the blades that are located near the water surface and are exposed to full sunlight, whereas the blades in deeper water regions exhibit a much lower capacity for NPQ (García-Mendoza and Colombo-Pallotta, 2007; Ocampo-Alvarez et al., 2013). Hereby, the extent of NPQ correlates with the size of the XC pigment pool (Harker et al., 1999; ColomboPallotta et al., 2006; García-Mendoza and Colombo-Pallotta, 2007; Ocampo-Alvarez et al., 2013). So far, information about the kinetics of the de-epoxidation and epoxidation reaction in brown algae is scarce. However, recent experiments have shown that, while in the higher plant Hordeum vulgare the de-epoxidation reaction is approximately 10 times faster than the respective epoxidation (Hartel et al., 1996), in the brown alga Macrocystis pyrifera the ratio between de-epoxidation and epoxidation is significantly lower, with an only twofold faster de-epoxidation compared to the epoxidation (García-Mendoza and Colombo-Pallotta, 2007). Interestingly, this relatively fast epoxidation is found in the surface blades while it is strongly reduced in blades located in deeper water layers. Although Z epoxidation is faster than that observed in higher plants, the epoxidation kinetics do not reach similar values than those reported by Goss et al. (2006a) for diatoms. Also in contrast to diatoms (Goss et al., 2006a), no difference in the epoxidation kinetics during illumination (in the presence of an uncoupler of the pH) and the kinetics during darkness could be observed (GarcíaMendoza and Colombo-Pallotta, 2007). As brown algae are mostly anchored to a solid substrate, they do not experience such huge fluctuations of the light intensities on a time scale of several minutes as it is typical for the diatoms in their natural environment (Brunet and Lavaud, 2010). Thus, a comparably fast relaxation of NPQ, as it is observed for diatoms, is not needed. From the available data it cannot be conclusively decided if the brown algal Z epoxidase is constitutively active or regulated by the proton gradient as it is typical for diatoms. However, the observation that a strong de-epoxidation can be induced in Pelvetia canaliculata in darkness under several stress conditions like dehydration, anoxia or heat treatment or even in the absence of a proton gradient (FernandezMarin et al., 2011) led to the hypothesis that the epoxidase is strongly regulated while the de-epoxidase exhibits some sort of basal activity (Fernandez-Marin et al., 2011; Ocampo-Alvarez et al., 2013). Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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To be able to quench non-photochemically, Z (and probably also A) need to be bound to proteins and, in analogy to diatoms and green algae, the Lhcx/Lhcsr proteins were suggested to confer the respective binding sites (Ocampo-Alvarez et al., 2013). Indeed, in the genome of the brown alga Ectocarpus siliculosus 13 Lhcsr protein sequences were identified (Dittami et al., 2010). In line with the possible importance of the Lhcsr proteins in NPQ, in Macrocystis pyrifera some Lhcsr genes showed a higher expression in surface blade tissues than in the blades in deeper water layers (Konotchick et al., 2013). The expression profile is comparable to the differences in the XC pigment pool size and the NPQ capacity in the different blade types (Colombo-Pallotta et al., 2006). However, to obtain a direct prove of the involvement of the Lhcsr proteins in the NPQ mechanism of brown algae, reverse genetic approaches are necessary. Finally, it should not go unnoticed that, as described above for Dt in diatoms (see “Dt as quencher”), Z in brown algae seems to be able to quench the fluorescence after the complete relaxation of the pH (García-Mendoza and Colombo-Pallotta, 2007). This is in contrast to higher plants and green algae (see “Role of the VAZ cycle in NPQ” and “Requirements of NPQ in green algae”), where the largest part of Z-dependent NPQ relaxes with the breakdown of the proton gradient. The stable Z- or Dt-dependent NPQ implies that the nature of the light-harvesting complex and not of the XC determines the mode of xanthophyll-dependent NPQ.

NPQ in other organisms with secondary plastids derived from red algae Other stramenopiles In chrysophytes the VAZ cycle (Hager and Stransky, 1970) is functional in NPQ (Lichtlé et al., 1995; Dimier et al., 2009). In some chrysophyte snow algae, besides the VAZ cycle pigments, also Dd was detected. However, while V was converted via A to Z, no conversion from Dd to Dt was found (Tanabe et al., 2011). This is in contrast to diatoms, where both XCs are present under specific light treatments, and where it was demonstrated that the DDE can convert both Dd and V (Lohr and Wilhelm, 1999). Moreover, also the isolated VDE of higher plants has been shown to be able to convert Dd into Dt (see also “Properties of the VDE of Prasinophyceae”, Yamamoto and Higashi, 1978; Goss, 2003). This implies that in chrysophytes the low amount of Dd present in the cells is probably bound to light-harvesting proteins in such a way that it is not accessible by the VDE, which in turn asks for the functional role of Dd in these organisms. Eustigmatophyceae also exhibit an NPQ which relies on the operation of the VAZ cycle and shows similar characteristics to the NPQ of brown algae and diatoms, i.e. the NPQ primarily depends on the concentration of A and Z and the role of the proton gradient is mainly restricted to the activation of the VDE (Gentile and Blanch, 2001; Cao et al., 2013). Two proteins belonging to the Lhcsr family were detected in the genome of Nannochloropsis, while no PsbS homologue was identified, which further argues for a comparability of the NPQ mechanisms in Eustigmatophyceae and diatoms and brown algae (Vieler et al., 2012). In isolates of the raphidophyte Chatonella subsalsa NPQ is almost negligible, while it is pronounced in Prorocentrum minimum (Warner and Madden, 2007). In Heterosigma akashiwo only a relatively low NPQ was observed (Hennige et al., 2013), but the existence of the VAZ cycle was demonstrated. However, the A and Z concentrations did not correlate well with NPQ, different to what was observed for the other stramenopiles. Hence, other still unresolved mechanisms have been proposed to be responsible for fast NPQ and are assumed to be of higher importance than the VAZ cycle dependent NPQ (Hennige et al., 2013).

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Alveolata In Chromeria velia, a recently discovered photosynthetic alveolata which, together with the rhizaria and stramenopiles form the SAR group (Fig. 1, Keeling, 2013), the VAZ cycle is active in NPQ (Kotabová et al., 2011). Interestingly, the de-epoxidation and epoxidation kinetics are so fast, that the intermediate of both reaction sequences, A, cannot be detected. As V is the main lightharvesting carotenoid in these algae, its conversion to Z does not only increase NPQ, but simultaneously decreases the effective absorption cross section of the antenna system. Like in diatoms and brown algae, the proton gradient is unable to induce NPQ alone, but is merely needed to activate V de-epoxidation (Kotabová et al., 2011). Dinophytes contain the Dd–Dt cycle, which is functional in NPQ (Brown et al., 1999). Two different main antenna complexes are identified in dinophytes: an intrinsic LHC related antenna (acpCP) (Hiller et al., 1993) and a water soluble, peridinin chlorophyll protein complex (PCP), which is probably located in the thylakoid lumen (Hofmann et al., 1996). The XC-dependent NPQ is thought to be located in the membrane bound acpCP (Iglesias-Prieto and Trench, 1997). In some symbiotic dinophytes of corals an NPQ mechanism was identified which decouples the PCP from PSII (Reynolds et al., 2008). Recently, it was suggested that both the acpCP and the PCP might disconnect from PSII once the maximum capacity of the xanthophyll cycle to dissipate the excessive excitation energy as heat is exhausted and still more photons are absorbed than can be quenched photochemically and nonphotochemically by the XC dependent NPQ (Hill et al., 2012). The evolution of dinophytes is very complex: all members of the dinophytes originally obtained their red chloroplast in a secondary endosymbiosis event. Later, some lineages acquired their final chloroplast from a green alga in a serial secondary event, while others eventually received their chloroplast from a haptophyte or a diatom by tertiary endosymbiosis (Keeling, 2013). Interestingly, putatively functional Lhcsr related proteins were so far only detected in the dinophyte Karlodinium, which obtained its chloroplasts in a tertiary endosymbiosis from a haptophyte ancestor (Patron et al., 2006), but not in other dinophytes (Boldt et al., 2012). This suggests that Lhcsr related proteins are not involved in Dt-dependent NPQ within the acpCP, which is found in all photosynthetic dinophytes (Boldt et al., 2012).

Hacrobionts Haptophytes also contain secondary plastids derived from a red algal ancestor, but are phylogenetically more distantly related compared to the SAR group (Fig. 1, Keeling, 2013). However, they possess a Dd–Dt cycle and are able to build-up NPQ (Harris et al., 2005). In Prymnesium parvum, NPQ was shown to depend directly on the amount of Dt, while the role of the proton gradient is restricted to the activation of the xanthophyll cycle, comparable to the situation in diatoms (Goss et al., 2006a, Dimier et al., 2009). Nevertheless, for Emiliana huxleyi it was discussed that the amount of Dd rather than Dt correlates with NPQ, at least during longer illumination periods (Harris et al., 2009). Such significant species-dependent differences in the NPQ mechanisms need to be verified in further experiments. Like in diatoms, haptophytes can also synthesize the VAZ cycle pigments under prolonged high light exposure (Lohr and Wilhelm, 1999). Interestingly, the enormous increase of XC pigments under high light acclimation, compared to low light conditions, by a factor of seven was paralleled by an increase of Lhcsr related proteins, suggesting a photoprotective role of the latter in the haptophytes, too (McKew et al., 2013). Please cite this article in press as: http://dx.doi.org/10.1016/j.jplph.2014.03.004

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Cryptophytes, which group together with the haptophytes (Keeling, 2013), exhibit a completely different NPQ. They lack a XC and instead possess a rapidly inducible and reversible proton gradient-dependent NPQ, which is, however, located in the LHC antenna and not in the phycobiliproteins which are present in ˇ et al., 2012). Consequently, NPQ in R. salina these organisms (Kana shows the same fast induction and relaxation kinetics as the proton gradient-dependent qE component of NPQ in higher plants (see “Role of the proton gradient”). Apparently, neither a PsbS-like nor an Lhcsr-like protein is involved in this type of quenching (Funk et al., 2011). NPQ in cyanobacteria and red algae Research during recent years has shown that cyanobacteria are capable of variable NPQ. The thermal dissipation of excessive excitation energy takes place on the level of the cyanobacterial light-harvesting system, the phycobilisome. Induction of NPQ is triggered by the orange carotenoid protein (OCP), a soluble protein which binds the keto-carotenoid 3 -hydroxyechinenone (for a review see Kirilovsky, 2010). OCP is activated by the absorption of blue-green light and converted into a metastable form (Wilson et al., 2008). By a reconstitution of cyanobacterial NPQ in vitro, Gwizdala et al. (2011) were able to determine mechanistic details of the process. They showed that the activated form of OCP binds to the phycobilisome, which in return stabilizes the active OCP. Binding of OCP to the phycobilisome takes place in the core domain, not in the outer rod parts. With respect to the quenching of excitation energy, it has been observed that the OCP interacts with the APC660 trimers and quenches their fluorescence and that one OCP is able to quench the Chl a fluorescence of one phycobilisome. In contrast to higher plants, green algae, diatoms and brown algae with their membrane intrinsic light-harvesting systems (see “NPQ in higher plants”, “NPQ in mosses”, “NPQ in green algae”, “NPQ in diatoms” and “NPQ in brown algae”), NPQ relaxation in the cyanobacteria is not triggered by the relaxation of the proton gradient or the removal of de-epoxidized XC pigments, but requires the action of another, specific protein, the fluorescence recovery protein (FRP, Boulay et al., 2010). The FRP is needed to detach the active and stable OCP from the phycobilisome (Gwizdala et al., 2011), thereby relaxing the quenching and restoring the light-harvesting function of the antenna. Interestingly, the OCP has been shown to bind Z (Punginelli et al., 2009) which, despite the lack of a functional VAZ cycle, can be present in high concentrations in cyanobacteria. However, Z binding to the OCP does not activate the OCP and seems to have no specific function in the variable NPQ of cyanobacteria. The eukaryotic red algae are another algal group which contains phycobilisomes as light-harvesting systems. With respect to the mechanism of NPQ in red algae, knowledge is quite scarce and also the presence of an active VAZ cycle is still under debate (see Goss and Jakob, 2010 and references therein). First experiments with the red alga Rhodella violacea showed that the quenching of PSII fluorescence in this alga was not induced by state transitions but by the establishment of a pH (Delphin et al., 1996, 1998). Interestingly, this quenching could also be observed during low light illumination or in darkness after illumination. It was caused by the inactivity of the ATPase which thus was unable to dissipate the proton gradient across the thylakoid membrane (Delphin et al., 1996, 1998). Later, it became clear that Rhodella violacea, besides the pH-dependent NPQ, reacts to high light illumination with a decrease of the phycobilisome antenna and changes in the structure and number of the thylakoid membranes (Ritz et al., 2000). Recent experiments with Porphyridium cruentum have corroborated these findings and also show that NPQ in red algae is a process which depends on the establishment of the proton gradient and results in a physical decoupling of neighbouring phycobilisomes (Liu et al., 2008).

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Conclusion/outlook

Acknowledgements

Our present review shows that all algal taxa and higher plants use NPQ as an important photoprotection mechanism. A common theme in all organisms is the structural reorganization of antenna complexes as the basis for NPQ. The molecular mechanisms that induce these structural changes are, however, diverse and depend on the type of antenna system. Organisms with a membrane intrinsic LHC, i.e. most of the algal taxa, mosses and higher plants, show structural rearrangements of the antenna complexes without a degradation of antenna proteins. These structural changes include an aggregation of antenna complexes and/or a possible detachment from the PS core complexes. Aggregation of the antenna system relies on special proteins which belong to the LHC family of proteins. These proteins differ in higher plants, mosses and the different algal taxa. Another important factor that controls the antenna aggregation is the cycling of xanthophylls. Again higher plants and the different algal groups use different xanthophyll cycles to induce and enhance NPQ. Algae with a membrane extrinsic antenna, i.e. cyanobacteria and red algae, are characterized by a degradation of phycobilisomes followed by a detachment of the antenna complexes from the membrane intrinsic core complexes. In cyanobacteria special proteins are needed for the structural changes of the antenna and thus for the establishment and relaxation of NPQ. In red algae NPQ seems to be triggered by the proton gradient across the thylakoid membrane. It has to be analyzed if the different molecular mechanisms of NPQ are related to the switch from membrane extrinsic to membrane intrinsic light-harvesting systems. Despite the wealth of information concerning NPQ and its molecular basis that has been gathered during the last years, many questions, especially with regard to the mechanism of NPQ in the different algal groups, remain open and have to be clarified in the following years. In higher plants the molecular mechanism of the PsbS function is still not completely clear and more data on the interaction of PsbS with the other PSII antenna proteins is needed. Future measurements on the nature of the actual quenching site within the FCP complexes of diatoms and brown algae have to be performed to answer the question if thermal dissipation in these organisms also relies on the specific interaction of xanthophyll molecules, i.e. Dt or Z, and Chl a molecules, or if specific Chl a dimers represent the quenching locus. The topic of a functional XC in red algae is also still under debate and no measurements exist until today, that can either unequivocally exclude or support the presence of an active XC in these algae. With regard to the LHC proteins that are essential for NPQ, more information about the Lhcx proteins of diatoms and the Lhcsr proteins is needed. Specifically, further data about their in vivo pigment binding, their localization in the thylakoid membrane and their exact mechanistic role in NPQ are required. With respect to differences between algae and higher plants, it would also be interesting to address the question why the Lhcsr protein was lost during the transition to land. Another important topic with regard to evolutionary aspects is the observation that the NPQ mechanism in most algae with secondary plastids derived from a red algal ancestor is remarkably different to the NPQ mechanism in red algae. Except for the cryptophytes, all of these algae possess a functional XC and most of them, when investigated, also contain Lhcsr related proteins. However, both are most likely not present in red algae. First analyses have indicated that both the XC enzymes and the Lhsr proteins are derived from green algae (Frommolt et al., 2008; Moustafa et al., 2009), but more studies are clearly needed to elaborate the evolutionary relationships in closer detail. Finally, a quantitative analysis of the beneficial effects of NPQ for the survival of plants and algae under excessive illumination would be needed to compare the importance of the NPQ mechanism to other photoprotective processes in the plant.

RG thanks the Deutsche Forschungsgemeinschaft (DFG) for financial support (grant Go818/7-1). BL acknowledges the EU FP7 Marie Curie Zukunftskolleg Incoming Fellowship Programme, University of Konstanz (grant no. 291784) for financial support. Both authors thank Dr. Susann Schaller for developing and drawing the model depicted in Fig. 2 and Dr. Johann Lavaud for his help in developing the model shown in Fig. 3. Both authors also thank Dr. Benjamin Bailleul for fruitful discussions.


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Lepetit

B.

Biodiversity

of

NPQ.

J

Plant

Physiol

(2014),

Comparison of the protective effectiveness of NPQ in Arabidopsis plants deficient in PsbS protein and zeaxanthin.

Biodiversity processing.

Physiological functions of PsbS-dependent and PsbS-independent NPQ under naturally fluctuating light conditions.

Secondary extinctions of biodiversity.

Biodiversity supported.

Origins of Biodiversity.

Generalized model of island biodiversity.

The biodiversity of carbon assimilation.

Biodiversity: The ecological deficit.

Overlooked local biodiversity loss.

Maintenance of biodiversity on islands.

Continental-level biodiversity collapse.

Brazil's drought: protect biodiversity.

Biodiversity: Land use matters.

Biodiversity. Preserving the present.

Biodiversity: Supply and demand.

Brazil's government attacks biodiversity

Biodiversity: broaden the search.

Biodiversity Areas under Threat: Overlap of Climate Change and Population Pressures on the World's Biodiversity Priorities.

Biodiversity of Jinggangshan Mountain: the importance of topography and geographical location in supporting higher biodiversity.

Evolution and Biodiversity: the evolutionary basis of biodiversity and its potential for adaptation to global change.

Biodiversity in cities needs space: a meta-analysis of factors determining intra-urban biodiversity variation.

Belowground biodiversity and ecosystem functioning.

Biodiversity: Life--a status report.