Protein arrangement factor: a new photosynthetic parameter characterizing the organization of thylakoid membrane proteins.

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.

Gradual Response of Cyanobacterial Thylakoids to Acute High-Light Stress-Importance of Carotenoid Accumulation.

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.

Plasticity of Cyanobacterial Thylakoid Microdomains Under Variable Light Conditions.

Photosynthetic light reactions proceed in thylakoid membranes (TMs) due to the activity of pigment-protein complexes. These complexes are heterogeneously organized into granal/stromal thylakoids (in plants) or into recently identified cyanobacterial microdomains (MDs). MDs are characterized by specific ratios of photosystem I (PSI), photosystem II (PSII), and phycobilisomes (PBS) and they are visible as sub-micrometer sized areas with different fluorescence ratios. In this report, the process of long-term plasticity in cyanobacterial thylakoid MDs has been explored under variable growth light conditions using Synechocystis sp. PCC6803 expressing YFP tagged PSI. TM organization into MDs has been observed for all categorized shapes of cells independently of their stage in cell cycle. The heterogeneous PSI, PSII, and PBS thylakoid areas were also identified under two types of growth conditions: at continuous light (CL) and at light-dark (L-D) cycle. The acclimation from CL to L-D cycle changed spatial distribution of photosystems, in particular PSI became more evenly distributed in thylakoids under L-D cycle. The process of the spatial PSI (and partially also PSII) redistribution required 1 week and was accompanied by temporal appearance of PBS decoupling probably caused by the re-organization of photosystems. The overall acclimation we observed was defined as TM plasticity as it resembles higher plants grana/stroma reorganization at variable growth light conditions. In addition, we observed large cell to cell variability in the actual MDs organization. It leads us to suggest that the plasticity, and cell to cell variability in MDs could be a manifestation of phenotypic heterogeneity, a recently broadly discussed phenomenon for prokaryotes.

Ecotoxicology impact of silica-coated CdSe/ZnS quantum dots internalized in Chlamydomonas reinhardtii algal cells.

The use of quantum dots (QD) in various medical and industrial applications may cause these nanoparticles to leak into waterways and subsequently enter the food chain. Therefore, if we intend to use QD, we must first know their potential environmental implications. In this work, cadmium selenide/zinc sulfide core/shell QD were synthesized, and then, biocompatible, water-dispersed QD were coated with silica (Si-QD). The QD were characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM) combined with energy-dispersive X-ray spectroscopy (EDX), and UV-Vis absorption analysis, which revealed that these surface-engineered QD have a highly crystalline, homogeneous spherical shape measuring approximately 25 nm. The cytotoxicity of the nanoparticles in the green algae Chlamydomonas reinhardtii was studied by incubating the algae cells with Si-QD and determining the optical density of algal cell culture, cell counts, and cells sizes by microflow cytometry. These measurements indicated that Si-QD are biocompatible up to a concentration of 25 ng/ml. Finally, the cellular uptake of Si-QD into C. reinhardtii was monitored by confocal laser scanning microscopy (CLSM). In conclusion, our results reveal that surface-engineered Cd-QD can penetrate the cells of aquatic organisms, which ensures a serious impact on the food chain and consequently the environment. On the other hand, the results also highlight a new potential method for bioremediation of Cd-QD by green algae, especially C. reinhardtii.