Role of an ancient light-harvesting protein of PSI in light absorption and photoprotection.

Diverse algae of the red lineage possess chlorophyll a-binding proteins termed LHCR, comprising the PSI light-harvesting system, which represent an ancient antenna form that evolved in red algae and was acquired through secondary endosymbiosis. However, the function and regulation of LHCR complexes remain obscure. Here we describe isolation of a Nannochloropsis oceanica LHCR mutant, named hlr1, which exhibits a greater tolerance to high-light (HL) stress compared to the wild type. We show that increased tolerance to HL of the mutant can be attributed to alterations in PSI, making it less prone to ROS production, thereby limiting oxidative damage and favoring growth in HL. HLR1 deficiency attenuates PSI light-harvesting capacity and growth of the mutant under light-limiting conditions. We conclude that HLR1, a member of a conserved and broadly distributed clade of LHCR proteins, plays a pivotal role in a dynamic balancing act between photoprotection and efficient light harvesting for photosynthesis.

Basic pH-induced modification of excitation-energy dynamics in fucoxanthin chlorophyll a/c-binding proteins isolated from a pinguiophyte, Glossomastix chrysoplasta.

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs.

Enhancement of excitation-energy quenching in fucoxanthin chlorophyll a/c-binding proteins isolated from a diatom Phaeodactylum tricornutum upon excess-light illumination.

Photosynthetic organisms regulate pigment composition and molecular oligomerization of light-harvesting complexes in response to solar light intensities, in order to improve light-harvesting efficiency. Here we report excitation-energy dynamics and relaxation of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a diatom Phaeodactylum tricornutum grown under high-light (HL) illumination. Two types of FCP complexes were prepared from this diatom under the HL condition, whereas one FCP complex was isolated from the cells grown under a low-light (LL) condition. The subunit composition and oligomeric states of FCP complexes under the HL condition are different from those under the LL condition. Absorption and fluorescence spectra at 77 K of the FCP complexes also vary between the two conditions, indicating modifications of the pigment composition and arrangement upon the HL illumination. Time-resolved fluorescence curves at 77 K of the FCP complexes under the HL condition showed shorter lifetime components compared with the LL condition. Fluorescence decay-associated spectra at 77 K showed distinct excitation-energy-quenching components and alterations of energy-transfer pathways in the FCP complexes under the HL condition. These findings provide insights into molecular and functional mechanisms of the dynamic regulation of FCPs in this diatom under excess-light conditions.

Spectral Properties and Excitation Relaxation of Novel Fucoxanthin Chlorophyll a/c-Binding Protein Complexes.

Fucoxanthin chlorophyll a/c-binding proteins (FCPs) are unique light harvesters for some photosynthetic organisms. There were several reports for the alterations of FCPs in response to light conditions. Here, we present the spectral profiles and excitation dynamics of novel FCP complexes isolated from the diatom Chaetoceros gracilis. Under a red-light condition, C. gracilis cells expressed three types of FCP complexes, two of which are very similar to FCP complexes found in the white-light grown cells, and one of which is the novel FCP complex. The combination of steady-state absorption and fluorescence spectra and time-resolved fluorescence spectra revealed that, compared to other types of FCP complexes, the novel FCP complexes exhibited red-shifted absorption and fluorescence spectra and fast decay of excitation. This finding will provide new insights into not only the light-harvesting strategies of diatoms but also the diversity of light adaptation machinery for photosynthetic organisms.

Influence of pH, Mg²âº, and lipid composition on the aggregation state of the diatom FCP in comparison to the LHCII of vascular plants.

In the present study, the influence of Mg²âº ions and low pH values on the aggregation state of the diatom FCP and the LHCII of vascular plants was studied. In addition, the concentration of thylakoid membrane lipids associated with the complexes was determined. The results demonstrate that the FCP, which contained a significantly higher concentration of the negatively charged lipids SQDG and PG, was less sensitive to Mg²âº and low pH values than the LHCII which was characterized by lower amounts of SQDG and a higher concentration of MGDG. High MgCl2 concentrations and pH values below pH 6 induced significant changes of the absorption and 77K fluorescence emission spectra of the LHCII, indicating a strong aggregation of the light-harvesting complex. This aggregation was also visible as a pellet after centrifugation on a sucrose cushion. Although the FCP responded with changes of the absorption and fluorescence spectra to low pH and Mg²âº incubation, these spectral changes were less pronounced than those observed for the LHCII. In addition, the FCP complexes did not show a visible pellet after incubation with either low pH values or high Mg²âº concentrations. Only the combined action of Mg²âº and pH 5 led to FCP aggregates of a size that could be pelleted by centrifugation. The decreased sensitivity of FCP aggregation to Mg²âº and low pH is discussed with respect to the differences in the concentration of the lipids surrounding the FCP and LHCII and the different thylakoid membrane organizations of diatoms and vascular plants.

Molecular cloning and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild radish.

Hydrophilic chlorophyll (Chl)-binding proteins have been isolated from various Brassicaceae plants and are categorized into Class II water-soluble Chl-binding proteins (WSCPs). Although the molecular properties of class II WSCPs including Brassica-type (e.g., cauliflower WSCP, Brussels sprouts WSCP and BnD22, a drought- and salinity-stress-induced 22 kDa protein of rapeseed), a Lepidium-type, and an Arabidopsis-type WSCPs have been well characterized, those of Raphanus-type WSCPs are poorly understood. To gain insight into the molecular diversity of Class II WSCPs, we cloned a novel cDNA encoding a Raphanus sativus var. raphanistroides (Japanese wild radish called 'Hamadaikon') WSCP (RshWSCP). Sequence analysis revealed that the open reading frame of the RshWSCP gene consisted of 666 bp encoding 222 aa residues, including 23 residues of a deduced signal peptide. Functional recombinant RshWSCP was expressed in Escherichia coli as a hexa-histidine fusion protein (RshWSCP-His). Although the RshWSCP-His was expressed as a soluble protein in E. coli, the apo-protein was highly unstable and tended to aggregate during a series of purification steps. When the soluble fraction of RshWSCP-His-expressing E. coli was mixed immediately with homogenate of spinach leaves containing thylakoid, RshWSCP-His was able to remove Chl molecules from the thylakoid and formed a stable Chl-WSCP complex with high hydrophilicity. UV-visible absorption spectra of the reconstituted RshWSCP-His revealed that RshWSCP-His is one of the Class IIA WSCP with the highest Chl a/b ratio analyzed thus far. A semi-quantitative reverse transcription-polymerase chain reaction analysis revealed that RshWSCP was transcribed in buds and flowers but not in roots, stems and various leaves.

Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms.

Fucoxanthin-chlorophyll complexes (FCP) from the centric diatom Cyclotella meneghiniana were isolated and the trimeric FCPa complex was reconstituted into liposomes at different lipid to Chl a ratios. The fluorescence yield of the complexes in different environments was calculated from room temperature fluorescence emission spectra and compared to the aggregated state of FCPa. FCPa surrounded by high amounts of lipids resembled detergent solubilised complexes and with decreasing lipid levels, i.e. in a situation where protein contacts were increasingly favoured, the fluorescence yield of FCPa gradually decreased. In addition, the yield displayed a strong pH-dependency in case of lower lipid contents. The further reduction in fluorescence yield brought about by the conversion of diadinoxanthin to diatoxanthin was pH independent and only depended on the amount of diatoxanthin synthesised. The implications of these data for non-photochemical quenching in centric diatoms are discussed.

Isolation and purification of the major photosynthetic antenna, fucoxanthin-Chl a/c protein, from cultured discoid germilings of the brown Alga, Cladosiphon okamuranus TOKIDA (Okinawa Mozuku).

A chlorophyll c binding membrane intrinsic light-harvesting complex, the fucoxanthin-chlorophyll a/c protein (FCP), was isolated from cultured discoid germilings of an edible Japanese brown alga, Cladosiphon (C.) okamuranus TOKIDA (Okinawa Mozuku in Japanese). The discoid germiling is an ideal source of brown algal photosynthetic pigment-protein complexes in terms of its size and easiness of cultivation on a large scale. Ion-exchange chromatography was crucial for the purification of FCP from solubilized thylakoid proteins. The molecular weight of the purified FCP assembly was estimated to be ~56 kDa using blue native-PAGE. Further subunit analyses using 2D-PAGE revealed that the FCP assembled as a trimer consisting of two distinguishable subunits having molecular weights of 18.2 (H) and 17.5 (L) kDa. Fluorescence and fluorescence-excitation spectra confirmed that the purified FCP assembly was functionally intact.

The pigment stoichiometry in a chlorophyll a/c type photosynthetic antenna.

The trimeric fucoxanthin-chlorophyll a/c protein (FCP) was purified from a Japanese brown alga, Cladosiphon okamuranus TOKIDA. Its pigment stoichiometry was determined to be chlorophyll (Chl) a:Chl c (1):Chl c (2):fucoxanthin = 4.6:1.1:1.0:5.5 by a combination of binary HPLC and (1)H NMR spectroscopy. No violaxanthin found bound to the FCP. The ratio of Chl c/Chl a in this FCP is amongst the highest so far reported.