Reimund Goss

Regulation and function of xanthophyll cycle-dependent photoprotection in algae

The xanthophyll cycle represents one of the important photoprotection mechanisms in plant cells. In the present review, we summarize current knowledge about the violaxanthin cycle of vascular plants, green and brown algae, and the diadinoxanthin cycle of the algal classes Bacillariophyceae, Xanthophyceae, Haptophyceae, and Dinophyceae. We address the biochemistry of the xanthophyll cycle enzymes with a special focus on protein structure, co-substrate requirements and regulation of enzyme activity. We present recent ideas regarding the structural basis of xanthophyll cycle-dependent photoprotection, including different models for the mechanism of non-photochemical quenching of chlorophyll a fluorescence. In a dedicated chapter, we also describe the unique violaxanthin antheraxanthin cycle of the Prasinophyceae, together with its implication for the mechanism of xanthophyll cycle-dependent heat dissipation. The interaction between the diadinoxanthin cycle and alternative electron flow pathways in the chloroplasts of diatoms is an additional topic of this review, and in the last chapter we cover aspects of the importance of xanthophyll cycle-dependent photoprotection for different algal species in their natural environments.

Biodiversity of NPQ

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.

Ancient Recruitment by Chromists of Green Algal Genes Encoding Enzymes for Carotenoid Biosynthesis

Chromist algae (stramenopiles, cryptophytes, and haptophytes) are major contributors to marine primary productivity. These eukaryotes acquired their plastid via secondary endosymbiosis, whereby an early-diverging red alga was engulfed by a protist and the plastid was retained and its associated nuclear-encoded genes were transferred to the host genome. Current data suggest, however, that chromists are paraphyletic; therefore, it remains unclear whether their plastids trace back to a single secondary endosymbiosis or, alternatively, this organelle has resulted from multiple independent events in the different chromist lineages. Both scenarios, however, predict that plastid-targeted, nucleus-encoded chromist proteins should be most closely related to their red algal homologs. Here we analyzed the biosynthetic pathway of carotenoids that are essential components of all photosynthetic eukaryotes and find a mosaic evolutionary origin of these enzymes in chromists. Surprisingly, about one-third (5/16) of the proteins are most closely related to green algal homologs with three branching within or sister to the early-diverging Prasinophyceae. This phylogenetic association is corroborated by shared diagnostic indels and the syntenic arrangement of a specific gene pair involved in the photoprotective xanthophyll cycle. The combined data suggest that the prasinophyte genes may have been acquired before the ancient split of stramenopiles, haptophytes, cryptophytes, and putatively also dinoflagellates. The latter point is supported by the observed monophyly of alveolates and stramenopiles in most molecular trees. One possible explanation for our results is that the green genes are remnants of a cryptic endosymbiosis that occurred early in chromalveolate evolution; that is, prior to the postulated split of stramenopiles, alveolates, haptophytes, and cryptophytes. The subsequent red algal capture would have led to the loss or replacement of most green genes via intracellular gene transfer from the new endosymbiont. We argue that the prasinophyte genes were retained because they enhance photosynthetic performance in chromalveolates, thus extending the niches available to these organisms. The alternate explanation of green gene origin via serial endosymbiotic or horizontal gene transfers is also plausible, but the latter would require the independent origins of the same five genes in some or all the different chromalveolate lineages.

Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of diatoxanthin in the diatom Phaeodactylum tricornutum

Summary Based on our recent findings that in the diatom Phaeodactylum tricornutum, chlororespiration in periods of prolonged darkness leads to the accumulation of diatoxanthin (DT), we have elaborated in detail the interdependence between the chlororespiratory proton gradient and the activation of diadinoxanthin de-epoxidase (DDE). The data clearly demonstrates that activation of DDE in Phaeodactylum occurs at higher pH-values compared to activation of violaxanthin de-epoxidase (VDE) in higher plants. In thylakoid membranes as well as in enzyme assays with isolated DDE, the de-epoxidation of diadinoxanthin (DD) is efficiently catalyzed at pH 7.2. In comparison, de-epoxidation of violaxanthin (Vx) in spinach thylakoids is observed below pH 6.5. Phaeodactylum thylakoids isolated from high light grown cells, that also contain the pigments of the violaxanthin cycle, show violaxanthin de-epoxidation at higher pH-values, thus suggesting that in Phaeodactylum, one de-epoxidase converts both diadinoxanthin and violaxanthin. We conclude that the activation of DDE at higher pH-values can explain how the low rates of chlororespiratory electron flow, that lead to the build-up of a rather small proton gradient, can induce the observed accumulation of diatoxanthin in the dark. Furthermore, we show that dark activation of diadinoxanthin de-epoxidation is not restricted to Phaeodactylum tricornutum but was also found in another diatom, Cyclotella meneghiana

Evidence for the existence of one antenna-associated, lipid-dissolved and two protein-bound pools of diadinoxanthin cycle pigments in diatoms

We studied the localization of diadinoxanthin cycle pigments in the diatoms Cyclotella meneghiniana and Phaeodactylum tricornutum. Isolation of pigment protein complexes revealed that the majority of high-light-synthesized diadinoxanthin and diatoxanthin is associated with the fucoxanthin chlorophyll protein (FCP) complexes. The characterization of intact cells, thylakoid membranes, and pigment protein complexes by absorption and low-temperature fluorescence spectroscopy showed that the FCPs contain certain amounts of protein-bound diadinoxanthin cycle pigments, which are not significantly different in high-light and low-light cultures. The largest part of high-light-formed diadinoxanthin cycle pigments, however, is not bound to antenna apoproteins but located in a lipid shield around the FCPs, which is copurified with the complexes. This lipid shield is primarily composed of the thylakoid membrane lipid monogalactosyldiacylglycerol. We also show that the photosystem I (PSI) fraction contains a tightly connected FCP complex that is enriched in protein-bound diadinoxanthin cycle pigments. The peripheral FCP and the FCP associated with PSI are composed of different apoproteins. Tandem mass spectrometry analysis revealed that the peripheral FCP is composed mainly of the light-harvesting complex protein Lhcf and also significant amounts of Lhcr. The PSI fraction, on the other hand, shows an enrichment of Lhcr proteins, which are thus responsible for the diadinoxanthin cycle pigment binding. The existence of lipid-dissolved and protein-bound diadinoxanthin cycle pigments in the peripheral antenna and in PSI is discussed with respect to different specific functions of the xanthophylls.

Molecular dynamics of the diatom thylakoid membrane under different light conditions

During the last years significant progress was achieved in unraveling molecular characteristics of the thylakoid membrane of different diatoms. With the present review it is intended to summarize the current knowledge about the structural and functional changes within the thylakoid membrane of diatoms acclimated to different light conditions. This aspect is addressed on the level of the organization and regulation of light-harvesting proteins, the dissipation of excessively absorbed light energy by the process of non-photochemical quenching, and the lipid composition of diatom thylakoid membranes. Finally, a working hypothesis of the domain formation of the diatom thylakoid membrane is presented to highlight the most prominent differences of heterokontic thylakoids in comparison to vascular plants and green algae during the acclimation to low and high light conditions.

The xanthophyll cycle of Mantoniella squamata converts violaxanthin into antheraxanthin but not to zeaxanthin: consequences for the mechanism of enhanced non-photochemical energy dissipation

Abstract. The prasinophycean alga Mantoniella squamata uses in vivo an incomplete violaxanthin cycle. Although the violaxanthin cycle in Mantoniella is capable of converting violaxanthin to zeaxanthin, in intact cells only antheraxanthin accumulates during periods of strong illumination. Antheraxanthin enhances non-photochemical quenching of chlorophyll fluorescence. Inhibition of antheraxanthin synthesis by the de-epoxidase inhibitor dithiothreitol abolishes increased thermal energy dissipation. Antheraxanthin-dependent non-photochemical quenching, like zeaxanthin-mediated non-photochemical quenching in higher plants, is uncoupler-sensitive. Mantoniella squamata cells cultivated at high light intensities contain higher amounts of violaxanthin than cells grown at low light. The increased violaxanthin-cycle pool size in high-light-grown Mantoniella cells is accompanied by higher de-epoxidation rates in the light and by a greater capacity to quench chlorophyll fluorescence non-photochemically. Antheraxanthin-dependent amplification of non-photochemical quenching is discussed in the light of recent models developed for zeaxanthin- and diatoxanthin-mediated enhanced heat dissipation.

The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana.

Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.

Investigation of the quenching efficiency of diatoxanthin in cells of Phaeodactylum tricornutum (Bacillariophyceae) with different pool sizes of xanthophyll cycle pigments

A. Schumann, R. Goss, T. Jakob and C. Wilhelm. 2007. Investigation of the quenching efficiency of diatoxanthin in cells of Phaeodactylum tricornutum (Bacillariophyceae) with different pool sizes of xanthophyll cycle pigments. Phycologia 46: 113–117. DOI: 10.2216/06-30.1 In the present study, the content of diadinoxanthin (Dd) + diatoxanthin (Dt) in the diatom Phaeodactylum tricornutum was manipulated by low-light (LL) and high-light (HL) growth conditions. Cells grown in HL almost doubled the pool size of Dd cycle pigments compared to cells from a LL culture. HL-grown cultures exhibited a stronger de-epoxidation and higher amounts of Dt during actinic HL illumination. However, the correlation between Dt and the corresponding nonphotochemical quenching (NPQ) revealed that the quenching efficiency of Dt was drastically decreased in these HL-grown cells compared to cells from the LL cultivation. The correlation between the de-epoxidation state of the Dd cycle and NPQ implied that in HL-grown cells a part of the Dt molecules had lost their ability to contribute to the NPQ. A precise quantification of the amount of free Dd + Dt by anion exchange chromatography and high-performance liquid chromatography showed that only 10% of the additionally synthesized Dd cycle pigments existed as free pigment in the thylakoid membrane and that most of the Dd + Dt was still bound to the light-harvesting proteins. The possible function of the additional free Dt in photo-protection and the antenna-bound Dt in the synthesis of the light-harvesting pigment fucoxanthin is discussed.

A New Multicomponent NPQ Mechanism in the Diatom Cyclotella meneghiniana

In the present study we report that in the diatom Cyclotella meneghiniana the diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence (NPQ) is heterogeneous and consists of three different components. (i) A transient NPQ component that generates immediately upon illumination, depends on the transthylakoid proton gradient as well as on the light intensity, and is modulated by the initial diatoxanthin content of the cells. It is located in the antenna complexes of C. meneghiniana and is comparable with the transient NPQ observed in vascular plants. (ii) A steady-state NPQ component is observed during later stages of the high-light illumination and depends on the diatoxanthin content formed by the light-activated diadinoxanthin cycle. (iii) A fast relaxing NPQ component is seen upon a transition of high-light-illuminated cells to complete darkness. This component relaxes within a time frame of tens of seconds and its extent is correlated with the amount of diatoxanthin formed during the phase of actinic illumination. It cannot be observed in dithiothreitol-treated cells where the de-epoxidation of diadinoxanthin to diatoxanthin is suppressed. The fast relaxing component can be interpreted as a relaxation of part of the steady-state NPQ. The different diatoxanthin-dependent components are characterized by different quenching efficiencies of diatoxanthin. Diatoxanthin involved in the transient NPQ exhibits a 2-fold higher quenching efficiency compared with diatoxanthin participating in the steady-state NPQ. It is proposed that the different quenching efficiencies of diatoxanthin are caused by the existence of different diatoxanthin pools within the antenna system of C. meneghiniana.