Centrum Algatech

Mikrobiologický ústav AV ČR, v.v.i. - vědecké pracoviště Třeboň

GROUP MEMBERS

Group picture (Bára Šedivá, Myriam Canonico, Eliška Trsková, Radek Kaňa, Aurelie Crepin, Greg Konert), year 2020

Research topics: We study organization of thylakoid membrane proteins into MICRODOMAINS and mechanisms of photoprotection in photothrophs (NPQ - Non-photochemical quenching).

(1)  Heterogenerous mosaic of proteins in thylakoids of cyanobacteria - MICRODOMAINS

Biological membranes were originally described as uniform fluid-mosaic of lipids and proteins. Later, heterogeneous membrane areas have been found in many membrane systems including cyanobacterial thylakoids. There, the heterogeneous areas are formed by pigment-protein complexes (photosystems, light-harvesting antennas) responsible for bioenergetics of light-harvesting in photosynthesis. These complexes are naturally self-organized into homo-/hetero- oligomers (nanodomains) that create sub-micrometer-sized in vivo microdomains (e.g. granal/stromal thylakoids in higher plants or cyanobacterial microdomains). In the project, we study imporrtance of thylakoid membranes heterogeneity, especially for light-harvesting in cyanobacteria. We use up-to-date methods of superresolution microscopy (Zeiss LSM 880 with Airyscan detector) and time/spatial cross-correlation microcopy (FRAP, FCS etc. ) address microdomains heterogeneity caused by nanodomains. Thanks to the molecular biology methods (specific mutants) we want to establish a link between membrane heterogeneity and the ligh-harvesting tfunction of photosystems and phycobilisoms in vivo.

 

Localisation of main thylakoid membrane protein complexes in cyanobacteria Anabaena sp. PCC 7120 (see Steinbach et al. 2015)

 

Proposed organization of thylakoid membrane complexes (Photosystem I, Photosystem II and Phycobilisomes) in cyanobacterial thylakoids (see Strašková et al. 2019)

 

We have identified and described heterogeneous organisation of cyanobacterial thylakoid membrane proteins into MICRODOMAINS [1]. We study importance of the mosaic-like organization of Photosystems (PSI and PSII) and Phycobilisomes for overall function of photosynthesis especially for light-harvesting. The microdomains organization of thylakoids is based on PSI/PSII/PBS co-localization (visible as Red-Green-Blue color coding pictures), its is stable in minutes/hours, and it can ponly slowly adapt to different growing conditions [3]. Microdomains seems to be typical for cyanobacterial strains [4] and and they resembles grana/stromal heterogenity in PSI/PSII distribution typical for higher plants thylakoids.

[1] Strašková A et al. (2019) Pigment-protein complexes are organized into stable microdomains in cyanobacterial thylakoids Biochimica et Biophysica Acta (BBA) - Bioenergetics 1860
[2] Konert G, Steinbach G, Canonico M, Kaňa R (2019) Protein arrangement factor: a new photosynthetic parameter characterizing the organization of thylakoid membrane proteins Physiol Plantarum 166:264-277
[3] Canonico M, Konert G, Kaňa R (2020) Plasticity of Cyanobacterial Thylakoid Microdomains Under Variable Light Conditions Frontiers in Plant Science 11
[4] Steinbach G, Schubert F, Kana R (2015) Cryo-imaging of photosystems and phycobilisomes in Anabaena sp PCC 7120 cells J Photochem Photobiol B-Biol 152:395-399

Proteins mobility: Thylakoid is a higly crowded membrane formed from more then 70% by proteins. It results in restricted diffusion of proteins in thylakoid membrane  (see Kaňa et al. 2013). We try to explore factors affecting photosynthetic proteins immobility and how it is reflected in effeciency of light-harvesting (Kaňa et al. 2014).

The protein mobility is explored in situ single cell level by confocal microscopy methods including FRAP (Fluorescence Recovery After Photobleaching) and FCS (Fluorescence Correlation Spectroscopy). We want to address interconnection between thylakoid membrane structure (in a sence of proteins organisation), protein dynamic and their function in photosynthesis.

 

Typical setup of FRAP method (for details, see e.g. Kaňa et a. 2013).

Typical FRAP image series for PBilisome fluorescence emissiotn (for details, see e.g. Kaňa et a. 2013, 2014).


(2) Mechanisms of photoprotection - NON-PHOTOCHEMICAL QUENCHING

Photosynthetic organisms are often exposed to highly fluctuating light-conditions with short periods of excessive irradiation. Therefore, they had to developed a several photoprotective mechanisms that can cope with with conditions when excessive light can produced .ROS and destroy photosynthetic apparatus. We have studied several model organisms including green plants (spinach, arabidopsis), algae (chromerids, cryptophytes alga), cyanobacteria to resolve molecular mechanism mechanisms of photoprotection. It includes non-photochemical quenching, photoinhibition, phycobilisome decoupling or state transitions. It is still an open question how much are those processes are connected with a short or a long-term TM re-organization on nano- and micro-scale levels.  Non-photochemical quenching NPQ - represents a photoprotective mechanisms dissipating excessive irradiation into heat (see Kaňa and Vass 2008). It is a preciselly controlled process affecting excesive energy dissipation in reaction centre (e.g. in extremophilic red algae, see Krupnik et al. 2013) or more often in light-harvesting antennae (see e.g. Kaňa et al. 2012).

 

 

Slow temperature increase induced by irradiation measured by theromocamera (for details, see Kaňa and Vass 2008).

Typical quenching analysis of Rhodomonas salina cells. (for more details, see Kaňa et al. 2012)

 

Isolated antennae complexes of Rhodomonas salina cells. (for more details, see Kaňa et al. 2012)

We focuse on mechanisms of NPQ in red-clade of photosynthetic organisms that are typical in huge variability in antennae proteins and pigment compositions. Our main model organisms are represented by Chromera velia (photosynthetic colpedelid algae) and cryptophytes (Rhodomonas salina, Guillardia Theta). These algae species represents a good models to study NPQ mechanisms dependent on xanthophyle cycle (C. velia - see Kotabová et al. 2011) and independent to any xanthophyll cycle (see Kaňa et al. 2012. Cheregi et al. 2015).

(3) NEW BIOPHYSICAL METHODS IN PHOTOSYNTHESIS

We have adapted several new biophysical methods for photosynthesis research. It includes spectrally resolved fluorescence induction (SRFI) allowing spectral detection of fluorescence induction and fluorescence parameters (e.g. NPQ, see .....) and and detection fluorescence induction (PAM-like curves) for various chromophores simultaneously.

We have also developed new method of cryoimaging (see Steinbach et al. 2015) allowing separate depetection of PSII fluorescence and red shifted PSI fluorescence at 77K.

 

Typical time-course of Spectrally Resolved Fluorescnece Induction Method (SRFI) for cyanobacteria.