Identification of sequence motifs in Lhcx proteins that confer qE-based photoprotection in the diatom Phaeodactylum tricornutum.

Photosynthetic organisms in nature often experience light fluctuations. While low light conditions limit the energy uptake by algae, light absorption exceeding the maximal rate of photosynthesis may go along with enhanced formation of potentially toxic reactive oxygen species. To preempt high light-induced photodamage, photosynthetic organisms evolved numerous photoprotective mechanisms. Among these, energy-dependent fluorescence quenching (qE) provides a rapid mechanism to dissipate thermally the excessively absorbed energy. Diatoms thrive in all aquatic environments and thus belong to the most important primary producers on earth. qE in diatoms is provided by a concerted action of Lhcx proteins and the xanthophyll cycle pigment diatoxanthin. While the exact Lhcx activation mechanism of diatom qE is unknown, two lumen-exposed acidic amino acids within Lhcx proteins were proposed to function as regulatory switches upon light-induced lumenal acidification. By introducing a modified Lhcx1 lacking these amino acids into a Phaeodactylum tricornutum Lhcx1-null qE knockout line, we demonstrate that qE is unaffected by these two amino acids. Based on sequence comparisons with Lhcx4, being incapable of providing qE, we perform domain swap experiments of Lhcx4 with Lhcx1 and identify two peptide motifs involved in conferring qE. Within one of these motifs, we identify a tryptophan residue with a major influence on qE establishment. This tryptophan residue is located in close proximity to the diadinoxanthin/diatoxanthin-binding site based on the recently revealed diatom Lhc crystal structure. Our findings provide a structural explanation for the intimate link of Lhcx and diatoxanthin in providing qE in diatoms.

Pre-purification of diatom pigment protein complexes provides insight into the heterogeneity of FCP complexes.

BACKGROUND: Although our knowledge about diatom photosynthesis has made huge progress over the last years, many aspects about their photosynthetic apparatus are still enigmatic. According to published data, the spatial organization as well as the biochemical composition of diatom thylakoid membranes is significantly different from that of higher plants. RESULTS: In this study the pigment protein complexes of the diatom Thalassiosira pseudonana were isolated by anion exchange chromatography. A step gradient was used for the elution process, yielding five well-separated pigment protein fractions which were characterized in detail. The isolation of photosystem (PS) core complex fractions, which contained fucoxanthin chlorophyll proteins (FCPs), enabled the differentiation between different FCP complexes: FCP complexes which were more closely associated with the PSI and PSII core complexes and FCP complexes which built-up the peripheral antenna. Analysis by mass spectrometry showed that the FCP complexes associated with the PSI and PSII core complexes contained various Lhcf proteins, including Lhcf1, Lhcf2, Lhcf4, Lhcf5, Lhcf6, Lhcf8 and Lhcf9 proteins, while the peripheral FCP complexes were exclusively composed of Lhcf8 and Lhcf9. Lhcr proteins, namely Lhcr1, Lhcr3 and Lhcr14, were identified in fractions containing subunits of the PSI core complex. Lhcx1, Lhcx2 and Lhcx5 proteins co-eluted with PSII protein subunits. The first fraction contained an additional Lhcx protein, Lhcx6_1, and was furthermore characterized by high concentrations of photoprotective xanthophyll cycle pigments. CONCLUSION: The results of the present study corroborate existing data, like the observation of a PSI-specific antenna complex in diatoms composed of Lhcr proteins. They complement other data, like e.g. on the protein composition of the 21 kDa FCP band or the Lhcf composition of FCPa and FCPb complexes. They also provide interesting new information, like the presence of the enzyme diadinoxanthin de-epoxidase in the Lhcx-containing PSII fraction, which might be relevant for the process of non-photochemical quenching. Finally, the high negative charge of the main FCP fraction may play a role in the organization and structure of the native diatom thylakoid membrane. Thus, the results present an important contribution to our understanding of the complex nature of the diatom antenna system.

The evolution of the photoprotective antenna proteins in oxygenic photosynthetic eukaryotes.

Photosynthetic organisms require rapid and reversible down-regulation of light harvesting to avoid photodamage. Response to unpredictable light fluctuations is achieved by inducing energy-dependent quenching, qE, which is the major component of the process known as non-photochemical quenching (NPQ) of chlorophyll fluorescence. qE is controlled by the operation of the xanthophyll cycle and accumulation of specific types of proteins, upon thylakoid lumen acidification. The protein cofactors so far identified to modulate qE in photosynthetic eukaryotes are the photosystem II subunit S (PsbS) and light-harvesting complex stress-related (LHCSR/LHCX) proteins. A transition from LHCSR- to PsbS-dependent qE took place during the evolution of the Viridiplantae (also known as 'green lineage' organisms), such as green algae, mosses and vascular plants. Multiple studies showed that LHCSR and PsbS proteins have distinct functions in the mechanism of qE. LHCX(-like) proteins are closely related to LHCSR proteins and found in 'red lineage' organisms that contain secondary red plastids, such as diatoms. Although LHCX proteins appear to control qE in diatoms, their role in the mechanism remains poorly understood. Here, we present the current knowledge on the functions and evolution of these crucial proteins, which evolved in photosynthetic eukaryotes to optimise light harvesting.

Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes.

Diatoms possess special light-harvesting proteins involved in the photoprotection mechanism called non-photochemical quenching (NPQ). These Lhcx proteins were shown to be subunits of trimeric fucoxanthin-chlorophyll complexes (FCPa) in centric diatoms, but their mode of action is still unclear. Here we investigated the influence of Fcp6, an orthologue to Lhcx1 of Thalassiosira pseudonana in the diatom Cyclotella meneghiniana, by reducing its amount using an antisense approach. Whereas the pigment interactions inside FCPa were not influenced by the presence or absence of Fcp6, as demonstrated by unaltered spectra of circular dichroism, changes could be observed on the level of thylakoids and cells in the mutants compared to WT. This fits to recent models of NPQ in diatoms, where FCP aggregation or supramolecular reorganisation is thought to be a major feature. Thus, Fcp6 (Lhcx1) appears to alter pigment-pigment interactions inside the aggregates, but not inside (un-aggregated) FCPa itself.

The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis.

Diatoms contain a highly flexible capacity to dissipate excessively absorbed light by nonphotochemical fluorescence quenching (NPQ) based on the light-induced conversion of diadinoxanthin (Dd) into diatoxanthin (Dt) and the presence of Lhcx proteins. Their NPQ fine regulation on the molecular level upon a shift to dynamic light conditions is unknown. We investigated the regulation of Dd + Dt amount, Lhcx gene and protein synthesis and NPQ capacity in the diatom Phaeodactylum tricornutum after a change from continuous low light to 3 d of sine (SL) or fluctuating (FL) light conditions. Four P. tricornutum strains with different NPQ capacities due to different expression of Lhcx1 were included. All strains responded to dynamic light comparably, independently of initial NPQ capacity. During SL, NPQ capacity was strongly enhanced due to a gradual increase of Lhcx2 and Dd + Dt amount. During FL, cells enhanced their NPQ capacity on the first day due to increased Dd + Dt, Lhcx2 and Lhcx3; already by the second day light acclimation was accomplished. While quenching efficiency of Dt was strongly lowered during SL conditions, it remained high throughout the whole FL exposure. Our results highlight a more balanced and cost-effective photoacclimation strategy of P. tricornutum under FL than under SL conditions.

Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum.

Diatoms are phytoplanktonic organisms that grow successfully in the ocean where light conditions are highly variable. Studies of the molecular mechanisms of light acclimation in the marine diatom Phaeodactylum tricornutum show that carotenoid de-epoxidation enzymes and LHCX1, a member of the light-harvesting protein family, both contribute to dissipate excess light energy through non-photochemical quenching (NPQ). In this study, we investigate the role of the other members of the LHCX family in diatom stress responses. Our analysis of available genomic data shows that the presence of multiple LHCX genes is a conserved feature of diatom species living in different ecological niches. Moreover, an analysis of the levels of four P. tricornutum LHCX transcripts in relation to protein expression and photosynthetic activity indicates that LHCXs are differentially regulated under different light intensities and nutrient starvation, mostly modulating NPQ capacity. We conclude that multiple abiotic stress signals converge to regulate the LHCX content of cells, providing a way to fine-tune light harvesting and photoprotection. Moreover, our data indicate that the expansion of the LHCX gene family reflects functional diversification of its members which could benefit cells responding to highly variable ocean environments.

Impact of Lhcx2 on Acclimation to Low Iron Conditions in the Diatom Phaeodactylum tricornutum.

Iron is a cofactor of photosystems and electron carriers in the photosynthetic electron transport chain. Low concentrations of dissolved iron are, therefore, the predominant factor that limits the growth of phototrophs in large parts of the open sea like the Southern Ocean and the North Pacific, resulting in "high nutrient-low chlorophyll" (HNLC) areas. Diatoms are among the most abundant microalgae in HNLC zones. Besides efficient iron uptake mechanisms, efficient photoprotection might be one of the key traits enabling them to outcompete other algae in HNLC regions. In diatoms, Lhcx proteins play a crucial role in one of the main photoprotective mechanisms, the energy-dependent fluorescence quenching (qE). The expression of Lhcx proteins is strongly influenced by various environmental triggers. We show that Lhcx2 responds specifically and in a very sensitive manner to iron limitation in the diatom Phaeodactylum tricornutum on the same timescale as the known iron-regulated genes ISIP1 and CCHH11. By comparing Lhcx2 knockout lines with wild type cells, we reveal that a strongly increased qE under iron limitation is based on the upregulation of Lhcx2. Other observed iron acclimation phenotypes in P. tricornutum include a massively reduced chlorophyll a content/cell, a changed ratio of light harvesting and photoprotective pigments per chlorophyll a, a decreased amount of photosystem II and photosystem I cores, an increased functional photosystem II absorption cross section, and decoupled antenna complexes. H2O2 formation at photosystem I induced by high light is lowered in iron-limited cells, while the amount of total reactive oxygen species is rather increased. Our data indicate a possible reduction in singlet oxygen by Lhcx2-based qE, while the other iron acclimation phenotype parameters monitored are not affected by the amount of Lhcx2 and qE.

The Fucoxanthin Chlorophyll a/c-Binding Protein in Tisochrysis lutea: Influence of Nitrogen and Light on Fucoxanthin and Chlorophyll a/c-Binding Protein Gene Expression and Fucoxanthin Synthesis.

We observed differences in lhc classification in Chromista. We proposed a classification of the lhcf family with two groups specific to haptophytes, one specific to diatoms, and one specific to seaweeds. Identification and characterization of the Fucoxanthin and Chlorophyll a/c-binding Protein (FCP) of the haptophyte microalgae Tisochrysis lutea were performed by similarity analysis. The FCP family contains 52 lhc genes in T. lutea. FCP pigment binding site candidates were characterized on Lhcf protein monomers of T. lutea, which possesses at least nine chlorophylls and five fucoxanthin molecules, on average, per monomer. The expression of T. lutea lhc genes was assessed during turbidostat and chemostat experiments, one with constant light (CL) and changing nitrogen phases, the second with a 12 h:12 h sinusoidal photoperiod and changing nitrogen phases. RNA-seq analysis revealed a dynamic decrease in the expression of lhc genes with nitrogen depletion. We observed that T. lutea lhcx2 was only expressed at night, suggesting that its role is to protect \cells from return of light after prolonged darkness exposure.

Flow cytometry-based study of model marine microalgal consortia revealed an ecological advantage of siderophore utilization by the dinoflagellate Amphidinium carterae.

Investigations of phytoplankton responses to iron stress in seawater are complicated by the fact that iron concentrations do not necessarily reflect bioavailability. Most studies to date have been based on single species or field samples and are problematic to interpret. Here, we report results from an experimental cocultivation model system that enabled us to evaluate interspecific competition as a function of iron content and form, and to study the effect of nutritional conditions on the proteomic profiles of individual species. Our study revealed that the dinoflagellate Amphidinium carterae was able to utilize iron from a hydroxamate siderophore, a strategy that could provide an ecological advantage in environments where siderophores present an important source of iron. Additionally, proteomic analysis allowed us to identify a potential candidate protein involved in iron acquisition from hydroxamate siderophores, a strategy that is largely unknown in eukaryotic phytoplankton.

Light harvesting complexes in chlorophyll c-containing algae.

Besides the so-called 'green lineage' of eukaryotic photosynthetic organisms that include vascular plants, a huge variety of different algal groups exist that also harvest light by means of membrane intrinsic light harvesting proteins (Lhc). The main taxa of these algae are the Cryptophytes, Haptophytes, Dinophytes, Chromeridae and the Heterokonts, the latter including diatoms, brown algae, Xanthophyceae and Eustigmatophyceae amongst others. Despite the similarity in Lhc proteins between vascular plants and these algae, pigmentation is significantly different since no Chl b is bound, but often replaced by Chl c, and a large diversity in carotenoids functioning in light harvesting and/or photoprotection is present. Due to the presence of Chl c in most of the taxa the name 'Chl c-containing organisms' has become common, however, Chl b-less is more precise since some harbour Lhc proteins that only bind one type of Chl, Chl a. In recent years huge progress has been made about the occurrence and function of Lhc in diatoms, so-called fucoxanthin chlorophyll proteins (FCP), where also the first molecular structure became available recently. In addition, especially energy transfer amongst the unusual pigments bound was intensively studied in many of these groups. This review summarises the present knowledge about the molecular structure, the arrangement of the different Lhc in complexes, the excitation energy transfer abilities and the involvement in photoprotection of the different Lhc systems in the so-called Chl c-containing organisms. This article is part of a Special Issue entitled Light harvesting, edited by Dr. Roberta Croce.

Light-harvesting protein Lhcx3 is essential for high light acclimation of Phaeodactylum tricornutum.

The light-harvesting protein complexes (Lhc) play key roles in the processes of light absorption and protection in diatoms. However, different Lhc protein carries out distinct function in photosynthesis. For now, roles of many Lhc proteins in light acclimation are largely unknown. Here, function of Lhcx3 in marine diatom Phaeodactylum tricornutum was examined by using reverse genetic technologies. The overexpression of Lhcx3 led to increased diadinoxanthin + diatoxanthin content and elevated non-photochemical fluorescence quenching (NPQ) while knockdown of Lhcx3 reduced NPQ level. In addition, the expression of Lhcx3 could be induced by blue light but not by red light. After addition of the photosynthetic inhibitor, upregulation of Lhcx3 transcript in high light could be inhibited by NH4Cl, but not by DCMU (3-(3,4-dichlorophenyl)-l,l-dim ethylurea). In contrast, DCMU addition increased expression of Lhcx3 in high light. In combination with changes of NPQ after addition of inhibitor, we concluded that the Lhcx3 played key roles in high light acclimation of diatoms. This finding will provide new clues for genetic improvement of P. tricornutum with an aim to cultivate new strains with high growth rate.

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.

Evolution and function of light harvesting proteins.

Photosynthetic eukaryotes exhibit very different light-harvesting proteins, but all contain membrane-intrinsic light-harvesting complexes (Lhcs), either as additional or sole antennae. Lhcs non-covalently bind chlorophyll a and in most cases another Chl, as well as very different carotenoids, depending on the taxon. The proteins fall into two major groups: The well-defined Lhca/b group of proteins binds typically Chl b and lutein, and the group is present in the 'green lineage'. The other group consists of Lhcr/Lhcf, Lhcz and Lhcx/LhcSR proteins. The former are found in the so-called Chromalveolates, where they mostly bind Chl c and carotenoids very efficient in excitation energy transfer, and in their red algae ancestors. Lhcx/LhcSR are present in most Chromalveolates and in some members of the green lineage as well. Lhcs function in light harvesting, but also in photoprotection, and they influence the organisation of the thylakoid membrane. The different functions of the Lhc subfamilies are discussed in the light of their evolution.

A novel cryptochrome in the diatom Phaeodactylum tricornutum influences the regulation of light-harvesting protein levels.

Diatoms possess several genes for proteins of the cryptochrome/photolyase family. A typical sequence for a plant cryptochrome was not found in our analysis of the Phaeodactylum tricornutum genome, but one protein grouped with higher plant and green algal cryptochromes. This protein, CryP, binds FAD and 5,10-methenyltetrahydrofolate, according to our spectroscopic studies on heterologously expressed protein. 5,10-Methenyltetrahydrofolate binding is a feature common to both cyclobutane pyrimidine dimer photolyases and DASH cryptochromes. In recombinant CryP, however, the FAD chromophore was present in its neutral radical state and had a red-shifted absorption maximum at 637 nm, which is more characteristic for a DASH cryptochrome than a cyclobutane pyrimidine dimer photolyase. Upon illumination with blue light, the fully reduced state of FAD was formed in the presence of reductant. Expression of CryP was silenced by antisense approaches, and the resulting cell lines showed increased levels of proteins of light-harvesting complexes, the Lhcf proteins, in vivo. In contrast, the levels of proteins active in light protection, the Lhcx proteins, were reduced. Thus, CryP cannot be directly grouped with known members of the cryptochrome/photolyase family. Of all P. tricornutum proteins, it is the most similar in sequence to a plant cryptochrome, and is involved in the regulation of light-harvesting protein expression, but shows spectroscopic features and a chromophore composition that are most typical of a DASH cryptochrome.