Research - Institute of Plant Biology - Laboratory of Plant Lipid Function and Structure

Zoltán GOMBOS
scientific adviser

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Mihály KIS senior research associate
Ildikó RACSKÓNÉ DOMONKOS research associate
Hajnalka HEVÉRNÉ LACZKÓ-DOBOS research associate
Tünde TÓTH research associate
Tomás ZAKAR junior research associate
Sindhujaa VAJRAVEL Ph.D. student
Istvánné KIRI laboratory assistant

FUNCTIONAL AND STRUCTURAL IMPORTANCE OF LIPIDS IN PHOTOSYNTHETIC MEMBRANES

The lipid composition of the photosynthetic membranes is essential in the construction of the photosynthetic machinery (Fig. 1) and in developing resistance to various stress conditions. We use a combination of physiological, biophysical and molecular biological approaches to investigate the structural and functional importance of phosphatidylglycerol in the functions and the formation of the photosynthetic apparatus, the role of lipid-protein, lipid-carotenoid and carotenoid-protein interaction in stress responses.




Figure 1. A scheme of the electron transport chain in cyanobacterial cells.


The role of phosphatidylglycerol in photosynthetic electron transport processes

Besides glycoglycerolipids, dominant lipids of photosynthetic membranes, there is only one phospholipid species, phosphatidylglycerol (PG). PG is an indispensable lipid in cyanobacteria and higher plants, which are able to carry out oxygenic photosynthesis. Crystallographic studies of protein complexes involved in photosynthesis have revealed the presence of PG molecules in the reaction centre (RC) of PSII (Fig. 2) and PSI (Fig. 3) complexes.




Figure 2. Locations of negatively charged lipids, PG and SQDG (SQ), at the reducing-side surface of PS II RC monomer structure at 1.9 Å-resolution. (A) Locations of cofactors, PG, SQDG and plastoquinone (QA and QB) are shown. Green arrows indicate the positions of head groups of PG. (B) Locations of cofactors and protein subunits of the RC. (C) A view of PSII surface on the reducing side.




Figure 3. Localization of PG molecules in a PS I RC as shown by X-ray crystallography


We generated several PG deficient mutants of various cyanobacterial strains. These PG mutants needed an exogenous supply of PG for their growth. The growth rate of the mutant cells in the presence of PG was hardly distinguishable from that of the wild-type, however, that of the mutant cells gradually suppressed during PG depletion. Electron micrographs showed bigger size of PG-depleted cells compared to PG-supplemented ones and also morphological aberrations were occurred among PG-deleted cells (Fig.4). Re-addition of PG molecules restored cell division. We observed and proved by MS measurements the remodeling of PG molecules in the PG-deficient mutant Synechocystis PCC6803. The exogenously added artificial dioleoyl-PG was transformed into photosynthetically more essential natural PG derivatives. Our experiments demonstrated remodeling of lipids in a prokaryotic photosynthetic bacterium.




Figure 4. I. Electron micrographs of PG-deficient Synechocystis pgsA cells, grown in the presence (A) and in the absence (B) of PG; dividing cells and morphological aberrations in PG-depleted cultures (C). Bar, 1 μm. II. Electron micrographs of PG-deficient Synechococcus cdsA cells, grown in the presence (A) and in the absence (B) of PG. Morphological aberration of a PG-depleted cell (C). Bar, 0.6 μm (Courtesy of Á. Párducz).



In the PG-deficient cells short-term depletion of PG hardly influenced the activity of PSI, however, that of PSII was severely affected. PG depletion suppressed the electron transport in PSII around primary and secondary quinones, inhibited the assembly of CP43 subunit to PSII RC and disturbed the formation of the functionally active dimers of PSII. Long-term PG depletion suppressed the oligomerization of PSI RCs (Fig. 5) and decreased the PSI activity of the PG-deficient cells.



Figure 5. Top view of a PSI trimer, and disintegration of a trimer to monomers occured after the PG depletion.



We concluded that PG depletion affects the cyanobacterial cell division and PG is necessary for the oligomerization and function of photosynthetic reaction centres.


Carotenoids, indispensable components for assembly and functions of photosystems

A gradual elimination of PG from the photosynthetic membranes of the mutant cells resulted in increased sensitivity to light which resulted in photobleaching of photosynthetic pigments and ultimately to cell death. The induction of photobleaching can be explained by the formation of a triplet state of chlorophyll, which initiates the formation of reactive oxygen species that induce the degradation of photosynthetic pigments in the PG-depleted cells. We demonstrated that in a PG-deficient mutant Synechocystis PCC6803 cells, PG depletion induces an accumulation of myxoxanthophyll and echinenone, and that in both thylakoid and cytoplasmic membranes. An increase in the content of myxoxanthophyll and echinenone upon PG depletion suggests that PG depletion regulates the biosynthetic pathway of specific carotenoids.

In order to get better understanding of carotenoid involvement in photosynthetic functions we constructed the first oxygenic photosynthetic prokaryotic mutant which is completely deficient in carotenoid (Car) synthesis (Fig. 6).




Figure 6. HPLC chromatograms of the pigments of wild-type and carotenoidless mutant Synechocystis PCC6803 cells.


Complete elimination of Cars did not significantly affect the assembly of PSI and cytochrome b6f complexes. The carotenoid deficient mutant strain of Synechocystis PCC6803 did not have any oxygen-evolving activity. The cells became light sensitive. The lack of Cars blocked the construction of PSII RCs and the functions related to PSII protein complexes. Elimination of Cars does not affect severely the transcription of the studied photosynthetic RC genes, although the translation of protein subunits of PSII was remarkably suppressed.

We concluded that Cars are indispensable constituents of the photosynthetic apparatus and essential for protection of chlorophylls to photooxidation. Furthermore they are also required for the synthesis and assembly of PSII subunits.


Roles of carotenoids in supramolecular organization of phosynthetic complexes

The thylakoid membranes of photosynthetic organisms that perform the photosynthetic energy transduction are very complex and dynamic systems. The pigment-protein complexes are organised to form multiprotein complexes, which also form large domains with long-range order in a hierarchic way. This hierarchic order can vary according to the taxonomic position of the given species reflecting the evolutionary adaptation to a particular ecological niche or to the environmental factors which the individual organisms exposed to. The self assembly of multiprotein complexes and formation of long range ordered structures are not well understood. It is mainly determined of the inherent traits of pigment-protein complexes. Evolution produced many similar but distinct pigment binding proteins that encoded by a few multi gene superfamilies. Slight variations of the structure and pigment content of these proteins can modulate the interactions of protein subunits resulting altered supramolecular or domain structures that may serve the fine tuning of photosynthetic processes. The carotenoid constituents of the pigment-protein complexes have an essential role in the proper protein folding. Some proteins are not folded at all in the absent of proper carotenoids. In other cases the removal or replacement of certain carotenoid provides a structural flexibility to the protein. The effects of carotenoids on the subunit interaction of multiprotein complexes or in long range interaction within macrodomains are less studied, although some recently published data and our preliminary results indicate the role of carotenoids in the supramolecular organization of thylakoid membrane proteins.


Selected publications

Domonkos I, Kis M, Gombos Z, Ughy B (2013) Carotenoids, versatile components of oxygenic photosynthesis. Prog Lipid Res. 52:539-561

Dobrikova AG, Domonkos I, Sözer Ö, Laczkó-Dobos H, Kis M, Párducz Á, Gombos Z, Apostolova EL. (2013) Effect of partial or complete elimination of light-harvesting complexes on the surface electric properties and the functions of cyanobacterial photosynthetic membranes. Physiol Plant. 147:248-60.

Itoh S, Kozuki T, Nishida K, Fukushima Y, Yamakawa H, Domonkos I, Laczkó-Dobos H, Kis M, Ughy B, Gombos Z. (2012) Two functional sites of phosphatidylglycerol for regulation of reaction of plastoquinone Q(B) in photosystem II. Biochim Biophys Acta. 1817:287-97 Ozge Sozer, Josef Komenda, Bettina Ughy, Ildikó Domonkos, Hajnalka Laczkó-Dobos, Przemyslaw Malec, Zoltán Gombos and Mihály Kis (2010) Involvement of Carotenoids in the Synthesis and Assembly of Protein Subunits of Photosynthetic Reaction Centers of Synechocystis sp. PCC 6803. Plant Cell Physiol. 51: 823–835.

Domonkos, I., Malec, P., Laczkó-Dobos, H., Sözer, O., Klodawska, K., Wada, H., Strzalka, K. and Gombos, Z. (2009). Phosphatidylglycerol depletion induces an increase in myxoxanthophyll biosynthetic activity in Synechocystis PCC6803 cells. Plant Cell Physiol. 50: 374-382.

Domonkos, I., Laczkó-Dobos, H. and Gombos, Z. (2008). Lipid-assisted protein-protein interactions that support photosynthetic and other cellular activities. Prog. Lipid Res. 47: 422-435.

Laczkó-Dobos, H., Ughy, B., Tóth, S.Z., Komenda, J., Zsiros, O., Domonkos, I., Párducz, Á., Bogos, B., Komura, M., Itoh, S. and Gombos, Z. (2008). Role of phosphatidylglycerol in the function and assembly of Photosystem II reaction center, studied in a cdsA-inactivated PAL mutant strain of Synechocystis sp. PCC6803 that lacks phycobilisomes. Biochim. Biophys. Acta 1777: 1184-1194.

Domonkos, I., Malec, P., Sallai, A., Kovács, L., Itoh, K., Shen, G., Ughy, B., Bogos, B., Sakurai, I., Kis, M., Strzalka, K., Wada, H., Itoh, S., Farkas, T. and Gombos, Z. (2004). Phosphatidylglycerol is essential for oligomerization of Photosystem I reaction center. Plant Physiol. 134: 1471-1478.

Várkonyi, Zs., Masamoto, K., Debreceny, M., Zsiros, O., Ughy, B., Gombos, Z., Domonkos, I., Farkas, T., Wada, H. and Szalontai, B. (2002). Low-temperature-induced accumulation of xanthophylls and its structural consequences in the photosynthetic membranes of the cyanobacterium Cylindrospermopsis raciborskii. An FTIR spectroscopic study. Proc. Natl. Acad. Sci. USA 99: 2410-2415.

Gombos, Z., Várkonyi, Zs., Hagio, M., Iwaki, M., Kovács, L., Masamoto, K., Itoh, S. and Wada, H. (2002). Phosphatidylglycerol requirement for the function of elektron acceptor plastoquinone QB in the photosystem II reaction center. Biochemistry 41: 3796-3802

Hagio, M., Gombos, Z., Vákonyi, Zs., Masamoto, K., Sato, N., Tsuzuki, M. and Wada, H. (2000). Direct evidence for requirement of phosphatidylglycerol in photosystem II of photosynthesis. Plant Physiol. 124: 795-804.

Kis, M., Zsiros, O., Farkas, T., Wada, H., Nagy, F. and Gombos, Z. (1998). Light-induced expression of fatty acid desaturase genes. Proc. Natl. Acad. Sci. USA 95: 4209-4214.

Gombos, Z., Kanervo, E., Tsvetkova, N., Sakamoto, T., Aro, E.M. and Murata, N. (1997). Genetic enhancement of the ability to tolerate photoinhibition by introduction of unsaturated bonds into membrane glycerolipids. Plant Physiol. 115: 551-559.

Gombos, Z., Wada, H. and Murata, N. (1994). The recovery of photosynthesis from low-temperature photoinhibition. Proc. Natl. Acad. Sci. USA 91: 8787-8791.

Gombos, Z., Wada, H. and Murata, N. (1992). Unsaturation of fatty acids in membrane lipids enhances the tolerance of the cyanobacterium Synechocystis PCC6803 to low-temperature photoinhibition. Proc. Natl. Acad. Sci. USA 89: 9959-9963.

Wada, H., Gombos, Z. and Murata, N. (1990). Enhancement of chilling tolerance of a cyanobacterium by genetic manipulation of fatty acid desaturation. Nature (London) 347: 200-203.

Gombos, Z., Kis, M., Páli, T. and Vígh, L. (1987). Nitrate starvation induces homeoviscous regulation of lipids in the cell envelope of the blue-green alga, Anacystis nidulans. Eur. J. Biochem. 165: 461-465.