The investigation of spatial heterogeneity within the thylakoid membrane (TM) proteins has gained increasing attention in photosynthetic research. The recent advances in live-cell imaging have allowed the identification of heterogeneous organisation of photosystems in small cyanobacterial cells. These sub-micrometre TM regions, termed microdomains in cyanobacteria, exhibit functional similarities with granal (Photosystem II dominant) and stromal (Photosystem I dominant) regions observed in TM of higher plants. This study delves into microdomain heterogeneity using super-resolution Airyscan-based microscopy enhancing resolution to approximately ~125 nm in x-y dimension. The new data reveal membrane areas rich in Photosystem I within the inner TM rings. Moreover, we identified analogous dynamics in the mobility of Photosystem II and phycobilisomes; countering earlier models that postulated differing mobility of these complexes. These novel findings thus hold significance for our understanding of photosynthesis regulation, particularly during state transitions.

• Klíčová slova
• Airyscan, FRAP, cyanobacteria, microdomain, photosystem, protein mobility, super-resolution microscopy, thylakoid membrane heterogeneity,
• Publikační typ
• časopisecké články 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

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment-protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound 'free' proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 - 2.95 µm2s-1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50-500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII-light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein-protein interactions in the mobility restrictions for large thylakoid protein complexes.

• Klíčová slova
• FCS, cyanobacteria, photosynthesis, proteins mobility, thylakoids,
• Publikační typ
• časopisecké články MeSH

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.

• Klíčová slova
• cyanobacteria, membrane organization, microdomains and rafts, phenotypic heterogeneity, photosynthesis, photosystems, phycobilisomes decoupling, thylakoid membrane,
• Publikační typ
• časopisecké články MeSH