Antenna proteins play a major role in the regulation of light-harvesting in photosynthesis. However, less is known about a possible link between their sizes (oligomerization state) and fluorescence intensity (number of photons emitted). Here, we used a microscopy-based method, Fluorescence Correlation Spectroscopy (FCS), to analyze different antenna proteins at the particle level. The direct comparison indicated that Chromera Light Harvesting (CLH) antenna particles (isolated from Chromera velia) behaved as the monomeric Light Harvesting Complex II (LHCII) (from higher plants), in terms of their radius (based on the diffusion time) and fluorescence yields. FCS data thus indicated a monomeric oligomerization state of algal CLH antenna (at our experimental conditions) that was later confirmed also by biochemical experiments. Additionally, our data provide a proof of concept that the FCS method is well suited to measure proteins sizes (oligomerization state) and fluorescence intensities (photon counts) of antenna proteins per single particle (monomers and oligomers). We proved that antenna monomers (CLH and LHCIIm) are more "quenched" than the corresponding trimers. The FCS measurement thus represents a useful experimental approach that allows studying the role of antenna oligomerization in the mechanism of photoprotection.

• Klíčová slova
• Chromera velia, antenna proteins, fluorescence correlation spectroscopy, light-harvesting, microscopy, photosynthesis, protein diffusion, protein oligomerization,
• MeSH
• bílkoviny řas chemie   metabolismus   MeSH
• fluorescence * MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza * MeSH
• kinetika MeSH
• multimerizace proteinu MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bílkoviny řas MeSH

Light plays an essential role in photosynthesis; however, its excess can cause damage to cellular components. Photosynthetic organisms thus developed a set of photoprotective mechanisms (e.g., non-photochemical quenching, photoinhibition) that can be studied by a classic biochemical and biophysical methods in cell suspension. Here, we combined these bulk methods with single-cell identification of microdomains in thylakoid membrane during high-light (HL) stress. We used Synechocystis sp. PCC 6803 cells with YFP tagged photosystem I. The single-cell data pointed to a three-phase response of cells to acute HL stress. We defined: (1) fast response phase (0-30 min), (2) intermediate phase (30-120 min), and (3) slow acclimation phase (120-360 min). During the first phase, cyanobacterial cells activated photoprotective mechanisms such as photoinhibition and non-photochemical quenching. Later on (during the second phase), we temporarily observed functional decoupling of phycobilisomes and sustained monomerization of photosystem II dimer. Simultaneously, cells also initiated accumulation of carotenoids, especially ɣ-carotene, the main precursor of all carotenoids. In the last phase, in addition to ɣ-carotene, we also observed accumulation of myxoxanthophyll and more even spatial distribution of photosystems and phycobilisomes between microdomains. We suggest that the overall carotenoid increase during HL stress could be involved either in the direct photoprotection (e.g., in ROS scavenging) and/or could play an additional role in maintaining optimal distribution of photosystems in thylakoid membrane to attain efficient photoprotection.

• Klíčová slova
• Synechocystis, carotenoids, high light, microdomains, non-photochemical quenching, photoinhibition, photoprotection, photosystems, thylakoid membrane,
• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• fotosystém I (proteinový komplex) genetika   metabolismus   MeSH
• fotosystém II (proteinový komplex) genetika   metabolismus   MeSH
• karotenoidy metabolismus   MeSH
• světlo * MeSH
• Synechocystis metabolismus   účinky záření   MeSH
• tylakoidy metabolismus   účinky záření   MeSH
• velikost buňky účinky záření   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• fotosystém I (proteinový komplex) MeSH
• fotosystém II (proteinový komplex) MeSH
• karotenoidy MeSH

Antenna protein aggregation is one of the principal mechanisms considered effective in protecting phototrophs against high light damage. Commonly, it is induced, in vitro, by decreasing detergent concentration and pH of a solution of purified antennas; the resulting reduction in fluorescence emission is considered to be representative of non-photochemical quenching in vivo. However, little is known about the actual size and organization of antenna particles formed by this means, and hence the physiological relevance of this experimental approach is questionable. Here, a quasi-single molecule method, fluorescence correlation spectroscopy (FCS), was applied during in vitro quenching of LHCII trimers from higher plants for a parallel estimation of particle size, fluorescence, and antenna cluster homogeneity in a single measurement. FCS revealed that, below detergent critical micelle concentration, low pH promoted the formation of large protein oligomers of sizes up to micrometers, and therefore is apparently incompatible with thylakoid membranes. In contrast, LHCII clusters formed at high pH were smaller and homogenous, and yet still capable of efficient quenching. The results altogether set the physiological validity limits of in vitro quenching experiments. Our data also support the idea that the small, moderately quenching LHCII oligomers found at high pH could be relevant with respect to non-photochemical quenching in vivo.

• Klíčová slova
• antenna proteins, detergent critical micelle concentration, fluorescence correlation spectroscopy, non-photochemical quenching, photoprotection, photosynthesis, protein oligomerization,
• MeSH
• chlorofyl chemie   genetika   účinky záření   MeSH
• fluorescence MeSH
• fluorescenční spektrometrie MeSH
• fotosyntéza genetika   MeSH
• fotosystém II (proteinový komplex) genetika   účinky záření   MeSH
• fototrofní procesy genetika   MeSH
• homeodoménový protein Antennapedia chemie   genetika   MeSH
• koncentrace vodíkových iontů MeSH
• proteinové agregáty genetika   MeSH
• shluková analýza MeSH
• světlo škodlivé účinky   MeSH
• světlosběrné proteinové komplexy chemie   genetika   MeSH
• tylakoidy chemie   genetika   účinky záření   MeSH
• zeaxanthiny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl MeSH
• fotosystém II (proteinový komplex) MeSH
• homeodoménový protein Antennapedia MeSH
• proteinové agregáty MeSH
• světlosběrné proteinové komplexy MeSH
• zeaxanthiny MeSH

We explored photoprotective strategies in a cryptophyte alga Rhodomonas salina. This cryptophytic alga represents phototrophs where chlorophyll a/c antennas in thylakoids are combined with additional light-harvesting system formed by phycobiliproteins in the chloroplast lumen. The fastest response to excessive irradiation is induction of non-photochemical quenching (NPQ). The maximal NPQ appears already after 20 s of excessive irradiation. This initial phase of NPQ is sensitive to Ca2+ channel inhibitor (diltiazem) and disappears, also, in the presence of non-actin, an ionophore for monovalent cations. The prolonged exposure to high light of R. salina cells causes photoinhibition of photosystem II (PSII) that can be further enhanced when Ca2+ fluxes are inhibited by diltiazem. The light-induced reduction in PSII photochemical activity is smaller when compared with immotile diatom Phaeodactylum tricornutum. We explain this as a result of their different photoprotective strategies. Besides the protective role of NPQ, the motile R. salina also minimizes high light exposure by increased cell velocity by almost 25% percent (25% from 82 to 104 μm/s). We suggest that motility of algal cells might have a photoprotective role at high light because algal cell rotation around longitudinal axes changes continual irradiation to periodically fluctuating light.

• MeSH
• chlorofyl a metabolismus   MeSH
• chlorofyl metabolismus   MeSH
• Cryptophyta cytologie   metabolismus   účinky záření   MeSH
• fotosystém II (proteinový komplex) metabolismus   MeSH
• pohyb buněk účinky záření   MeSH
• světlo MeSH
• vápník metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chlorofyl a MeSH
• chlorofyl MeSH
• chlorophyll c MeSH Prohlížeč
• fotosystém II (proteinový komplex) MeSH
• vápník MeSH