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.
- 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
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.
- 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
Photosynthetic organisms had to evolve diverse mechanisms of light-harvesting to supply photosynthetic apparatus with enough energy. Cryptophytes represent one of the groups of photosynthetic organisms combining external and internal antenna systems. They contain one type of immobile phycobiliprotein located at the lumenal side of the thylakoid membrane, together with membrane-bound chlorophyll a/c antenna (CAC). Here we employ femtosecond transient absorption spectroscopy to study energy transfer pathways in the CAC proteins of cryptophyte Rhodomonas salina. The major CAC carotenoid, alloxanthin, is a cryptophyte-specific carotenoid, and it is the only naturally-occurring carotenoid with two triple bonds in its structure. In order to explore the energy transfer pathways within the CAC complex, three excitation wavelengths (505, 590, and 640 nm) were chosen to excite pigments in the CAC antenna. The excitation of Chl c at either 590 or 640 nm proves efficient energy transfer between Chl c and Chl a. The excitation of alloxanthin at 505 nm shows an active pathway from the S2 state with efficiency around 50%, feeding both Chl a and Chl c with approximately 1:1 branching ratio, yet, the S1-route is rather inefficient. The 57 ps energy transfer time to Chl a gives ~25% efficiency of the S1 channel. The low efficiency of the S1 route renders the overall carotenoid-Chl energy transfer efficiency low, pointing to the regulatory role of alloxanthin in the CAC antenna.
A proper spatial distribution of photosynthetic pigment-protein complexes - PPCs (photosystems, light-harvesting antennas) is crucial for photosynthesis. In plants, photosystems I and II (PSI and PSII) are heterogeneously distributed between granal and stromal thylakoids. Here we have described similar heterogeneity in the PSI, PSII and phycobilisomes (PBSs) distribution in cyanobacteria thylakoids into microdomains by applying a new image processing method suitable for the Synechocystis sp. PCC6803 strain with yellow fluorescent protein-tagged PSI. The new image processing method is able to analyze the fluorescence ratios of PPCs on a single-cell level, pixel per pixel. Each cell pixel is plotted in CIE1931 color space by forming a pixel-color distribution of the cell. The most common position in CIE1931 is then defined as protein arrangement (PA) factor with xy coordinates. The PA-factor represents the most abundant fluorescence ratio of PSI/PSII/PBS, the 'mode color' of studied cell. We proved that a shift of the PA-factor from the center of the cell-pixel distribution (the 'median' cell color) is an indicator of the presence of special subcellular microdomain(s) with a unique PSI/PSII/PBS fluorescence ratio in comparison to other parts of the cell. Furthermore, during a 6-h high-light (HL) treatment, 'median' and 'mode' color (PA-factor) of the cell changed similarly on the population level, indicating that such microdomains with unique PSI/PSII/PBS fluorescence were not formed during HL (i.e. fluorescence changed equally in the whole cell). However, the PA-factor was very sensitive in characterizing the fluorescence ratios of PSI/PSII/PBS in cyanobacterial cells during HL by depicting a 4-phase acclimation to HL, and their physiological interpretation has been discussed.
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.
In the present paper, we report an improved method combining sucrose density gradient with ion-exchange chromatography for the isolation of pure chlorophyll a/c antenna proteins from the model cryptophytic alga Rhodomonas salina. Antennas were used for in vitro quenching experiments in the absence of xanthophylls, showing that protein aggregation is a plausible mechanism behind non-photochemical quenching in R. salina. From sucrose gradient, it was also possible to purify a functional photosystem I supercomplex, which was in turn characterized by steady-state and time-resolved fluorescence spectroscopy. R. salina photosystem I showed a remarkably fast photochemical trapping rate, similar to what recently reported for other red clade algae such as Chromera velia and Phaeodactylum tricornutum. The method reported therefore may also be suitable for other still partially unexplored algae, such as cryptophytes.
The slow kinetic phases of the chlorophyll a fluorescence transient (induction) are valuable tools in studying dynamic regulation of light harvesting, light energy distribution between photosystems, and heat dissipation in photosynthetic organisms. However, the origin of these phases are not yet fully understood. This is especially true in the case of prokaryotic oxygenic photoautotrophs, the cyanobacteria. To understand the origin of the slowest (tens of minutes) kinetic phase, the M-T fluorescence decline, in the context of light acclimation of these globally important microorganisms, we have compared spectrally resolved fluorescence induction data from the wild type Synechocystis sp. PCC 6803 cells, using orange (λ = 593 nm) actinic light, with those of mutants, ΔapcD and ΔOCP, that are unable to perform either state transition or fluorescence quenching by orange carotenoid protein (OCP), respectively. Our results suggest a multiple origin of the M-T decline and reveal a complex interplay of various known regulatory processes in maintaining the redox homeostasis of a cyanobacterial cell. In addition, they lead us to suggest that a new type of regulatory process, operating on the timescale of minutes to hours, is involved in dissipating excess light energy in cyanobacteria.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chlorofyl chemie genetika metabolismus MeSH
- diuron chemie MeSH
- fluorescence MeSH
- fluorescenční spektrometrie MeSH
- fykobilizomy genetika metabolismus MeSH
- kyanid draselný chemie MeSH
- luminiscenční měření MeSH
- světlo MeSH
- Synechocystis chemie genetika metabolismus MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The pKa for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are 'natural quenchers' ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching.
- MeSH
- Alveolata fyziologie MeSH
- bílkoviny řas metabolismus MeSH
- fotosyntéza * MeSH
- protony * MeSH
- protozoální proteiny metabolismus MeSH
- rostlinné proteiny metabolismus MeSH
- Spinacia oleracea fyziologie MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
It has previously been shown that the long-term treatment of Arabidopsis thaliana with the chloroplast inhibitor lincomycin leads to photosynthetic membranes enriched in antennas, strongly reduced in photosystem II reaction centers (PSII) and with enhanced nonphotochemical quenching (NPQ) (Belgio et al. Biophys J 102:2761-2771, 2012). Here, a similar physiological response was found in the microalga Chromera velia grown under high light (HL). In comparison to cells acclimated to low light, HL cells displayed a severe re-organization of the photosynthetic membrane characterized by (1) a reduction of PSII but similar antenna content; (2) partial uncoupling of antennas from PSII; (3) enhanced NPQ. The decrease in the number of PSII represents a rather unusual acclimation response compared to other phototrophs, where a smaller PSII antenna size is more commonly found under high light. Despite the diminished PSII content, no net damage could be detected on the basis of the Photosynthesis versus irradiance curve and electron transport rates pointing at the excess capacity of PSII. We therefore concluded that the photoinhibition is minimized under high light by a lower PSII content and that cells are protected by NPQ in the antennas.
- MeSH
- aklimatizace účinky záření MeSH
- Alveolata cytologie fyziologie účinky záření MeSH
- chlorofyl metabolismus MeSH
- fluorescence MeSH
- fotochemické procesy účinky záření MeSH
- fotosyntéza účinky záření MeSH
- fotosystém II - proteinový komplex metabolismus MeSH
- rozpustnost MeSH
- světlo * MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
In the present work, we report the first comparative spectroscopic investigation between Photosystem I (PSI) complexes isolated from two red clade algae. Excitation energy transfer was measured in PSI from Chromera velia, an alga possessing a split PsaA protein, and from the model diatom Phaeodactylum tricornutum. In both cases, the estimated effective photochemical trapping time was in the 15-25ps range, i.e. twice as fast as higher plants. In contrast to green phototrophs, the trapping time was rather constant across the whole emission spectrum. The weak wavelength dependence was attributed to the limited presence of long-wavelength emitting chlorophylls, as verified by low temperature spectroscopy. As the trapping kinetics of C. velia PSI were barely distinguishable from those of P. tricornutum PSI, it was concluded that the scission of PsaA protein had no significant impact on the overall PSI functionality. In conclusion, the two red clade algae analysed here, carried amongst the most efficient charge separation so far reported for isolated Photosystems.
- MeSH
- Alveolata metabolismus MeSH
- chlorofyl metabolismus MeSH
- fluorescenční spektrometrie MeSH
- fotosystém I - proteinový komplex metabolismus MeSH
- kinetika MeSH
- přenos energie fyziologie MeSH
- Rhodophyta metabolismus MeSH
- rozsivky metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
