The photoreactivation of 6 - 4 photoproducts in chloroplast and nuclear DNA depends on the amount of the Arabidopsis UV repair defective 3 protein.

BACKGROUND: 6 - 4 photoproducts are the second most common UV-induced DNA lesions after cyclobutane pyrimidine dimers. In plants, they are mainly repaired by photolyases in a process called photoreactivation. While pyrimidine dimers can be deleterious, leading to mutagenesis or even cell death, 6 - 4 photoproducts can activate specific signaling pathways. Therefore, their removal is particularly important, especially for plants exposed to high UV intensities due to their sessile nature. Although photoreactivation in nuclear DNA is well-known, its role in plant organelles remains unclear. In this paper we analyzed the activity and localization of GFP-tagged AtUVR3, the 6 - 4 photoproduct specific photolyase. RESULTS: Using transgenic Arabidopsis with different expression levels of AtUVR3, we confirmed a positive trend between these levels and the rate of 6 - 4 photoproduct removal under blue light. Measurements of 6 - 4 photoproduct levels in chloroplast and nuclear DNA of wild type, photolyase mutants, and transgenic plants overexpressing AtUVR3 showed that the photoreactivation is the main repair pathway responsible for the removal of these lesions in both organelles. The GFP-tagged AtUVR3 was predominantly located in nuclei with a small fraction present in chloroplasts and mitochondria of transgenic Arabidopsis thaliana and Nicotiana tabacum lines. In chloroplasts, this photolyase co-localized with the nucleoid marked by plastid envelope DNA binding protein. CONCLUSIONS: Photolyases are mainly localized in plant nuclei, with only a small fraction present in chloroplasts and mitochondria. Despite this unbalanced distribution, photoreactivation is the primary mechanism responsible for the removal of 6 - 4 photoproducts from nuclear and chloroplast DNA in adult leaves. The amount of the AtUVR3 photolyase is the limiting factor influencing the photoreactivation rate of 6 - 4 photoproducts. The efficient photoreactivation of 6 - 4 photoproducts in 35S: AtUVR3-GFP Arabidopsis and Nicotiana tabacum is a promising starting point to evaluate whether transgenic crops overproducing this photolyase are more tolerant to high UV irradiation and how they respond to other abiotic and biotic stresses under field conditions.

Trimeric organization of photosystem I is required to maintain the balanced photosynthetic electron flow in cyanobacterium Synechocystis sp. PCC 6803.

In Synechocystis sp. PCC 6803 and some other cyanobacteria photosystem I reaction centres exist predominantly as trimers, with minor contribution of monomeric form, when cultivated at standard optimized conditions. In contrast, in plant chloroplasts photosystem I complex is exclusively monomeric. The functional significance of trimeric organization of cyanobacterial photosystem I remains not fully understood. In this study, we compared the photosynthetic characteristics of PSI in wild type and psaL knockout mutant. The results show that relative to photosystem I trimer in wild-type cells, photosystem I monomer in psaL- mutant has a smaller P700+ pool size under low and moderate light, slower P700 oxidation upon dark-to-light transition, and slower P700+ reduction upon light-to-dark transition. The mutant also shows strongly diminished photosystem I donor side limitations [quantum yield Y(ND)] at low, moderate and high light, but enhanced photosystem I acceptor side limitations [quantum yield Y(NA)], especially at low light (22 µmol photons m-2 s-1). In line with these functional characteristics are the determined differences in the relative expression genes encoding of selected electron transporters. The psaL- mutant showed significant (ca fivefold) upregulation of the photosystem I donor cytochrome c6, and downregulation of photosystem I acceptors (ferredoxin, flavodoxin) and proteins of alternative electron flows originating in photosystem I acceptor side. Taken together, our results suggest that photosystem I trimerization in wild-type Synechocystis cells plays a role in the protection of photosystem I from photoinhibition via maintaining enhanced donor side electron transport limitations and minimal acceptor side electron transport limitations at various light intensities.

Effect of growth temperature on biosynthesis and accumulation of carotenoids in cyanobacterium Anabaena sp. PCC 7120 under diazotrophic conditions.

Carotenoid composition has been studied in mesophilic, nitrogen-fixing cyanobacterium Anabaena sp. PCC7120 grown photoautotrophically, under diazotrophic conditions at four different temperatures (15 °C, 23 °C, 30 °C and 37 °C). The relative accumulation of chlorophyll, carotenoids and proteins was the highest at temperature of 23 °C. At a suboptimal temperature (15 °C) ß-carotene was the dominant carotenoid compound, whereas the increase in temperature caused ketocarotenoids (echinenone, canthaxanthin, keto-myxoxanthophyll) to accumulate. A significant increase in the accumulation of phytoene synthase (CrtB) transcript was observed at both extreme growth temperatures (15 °C and 37 °C). The relative amount of ß-carotene ketolase (CrtW) transcript directly corresponded to the accumulation of its product (keto-myxoxanthophyll) with a maximum at 30 °C and a profound decrease at 37 °C, whereas the transcription level of ß-carotene ketolase (CrtO) was significantly decreased only at a suboptimal temperature (15 °C). These results show that temperature affects the functioning of the carotenoid biosynthesis pathway in Anabaena cells under photoautotrophic growth. Specifically, the balance between ß-carotene and ketocarotenoids is altered according to temperature conditions. The transcriptional regulation of genes encoding enzymes active both at the early (CrtB) and the final steps (CrtO, CrtW) of the carotenoid biosynthetic pathway may participate in the acclimation mechanism of cyanobacteria to low and high temperatures.

Photosystem I oligomerization affects lipid composition in Synechocystis sp. PCC 6803.

In cyanobacteria, increasing growth temperature decreases lipid unsaturation and the ratio of monomer/trimer photosystem I (PSI) complexes. In the present study we applied Fourier-transform infrared (FTIR) spectroscopy and lipidomic analysis to study the effects of PSI monomer/oligomer ratio on the physical properties and lipid composition of thylakoids. To enhance the presence of monomeric PSI, a Synechocystis sp. PCC6803/ΔpsaL mutant strain (PsaL) was used which, unlike both trimeric and monomeric PSI-containing wild type (WT) cells, contain only the monomeric form. The protein-to-lipid ratio remained unchanged in the mutant but, due to an increase in the lipid disorder in its thylakoids, the gel to liquid-crystalline phase transition temperature (Tm) is lower than in the WT. In thylakoid membranes of the mutant, digalactosyldiacylglycerol (DGDG), the most abundant bilayer-forming lipid is accumulated, whereas those in the WT contain more monogalactosyldiacylglycerol (MGDG), the only non-bilayer-forming lipid in cyanobacteria. In PsaL cells, the unsaturation level of sulphoquinovosyldiacylglycerol (SQDG), a regulatory anionic lipid, has increased. It seems that merely a change in the oligomerization level of a membrane protein complex (PSI), and thus the altered protein-lipid interface, can affect the lipid composition and, in addition, the whole dynamics of the membrane. Singular value decomposition (SVD) analysis has shown that in PsaL thylakoidal protein-lipid interactions are less stable than in the WT, and proteins start losing their native secondary structure at much milder lipid packing perturbations. Conclusions drawn from this system should be generally applicable for protein-lipid interactions in biological membranes.

Zeaxanthin and echinenone modify the structure of photosystem I trimer in Synechocystis sp. PCC 6803.

The function of xanthophylls in the organisation and structure of the photosynthetic complexes is not completely clarified yet. Recently, we observed a reduced level of the photosystem oligomers upon xanthophyll deficiency, although xanthophylls are not considered to be part of the photosynthetic complexes of cyanobacteria. The present study aimed at further investigating the relationship between xanthophylls and photosytem I (PSI) complex in the cyanobacterium Synechocystis sp. PCC 6803. Interestingly, we recorded the presence of echinenone and zeaxanthin in the isolated PSI trimers. These two xanthophyll species are among the most abundant xanthophylls in this cyanobacterial species. Various xanthophyll biosynthesis mutants were used to investigate the specific role of these xanthophylls. Our spectroscopic results revealed specific structural changes manifested in altered pigment-pigment or pigment-protein interactions within PSI complex in the absence of zeaxanthin and echinenone. These structural modifications of the complexes seem to destabilize the PSI trimeric complexes and eventually result in an increased propensity for monomerization. Our results clearly demonstrate that xanthophylls are important for the fine-tuning of the PSI trimer structure. These xanthophylls could be part of the complex or be embedded in the membrane in the vicinity of PSI.

Elevated growth temperature can enhance photosystem I trimer formation and affects xanthophyll biosynthesis in Cyanobacterium Synechocystis sp. PCC6803 cells.

In the thylakoid membranes of the mesophilic cyanobacterium Synechocystis PCC6803, PSI reaction centers (RCs) are organized as monomers and trimers. PsaL, a 16 kDa hydrophobic protein, a subunit of the PSI RC, was previously identified as crucial for the formation of PSI trimers. In this work, the physiological effects accompanied by PSI oligomerization were studied using a PsaL-deficient mutant (ΔpsaL), not able to form PSI trimers, grown at various temperatures. We demonstrate that in wild-type Synechocystis, the monomer to trimer ratio depends on the growth temperature. The inactivation of the psaL gene in Synechocystis grown phototropically at 30°C induces profound morphological changes, including the accumulation of glycogen granules localized in the cytoplasm, resulting in the separation of particular thylakoid layers. The carotenoid composition in ΔpsaL shows that PSI monomerization leads to an increased accumulation of myxoxantophyll, zeaxanthin and echinenone irrespective of the temperature conditions. These xanthophylls are formed at the expense of ß-carotene. The measured H2O→CO2 oxygen evolution rates in the ΔpsaL mutant are higher than those observed in the wild type, irrespective of the growth temperature. Moreover, circular dichroism spectroscopy in the visible range reveals that a peak attributable to long-wavelength-absorbing carotenoids is apparently enhanced in the trimer-accumulating wild-type cells. These results suggest that specific carotenoids are accompanied by the accumulation of PSI oligomers and play a role in the formation of PSI oligomer structure.

EPR study of thylakoid membrane dynamics in mutants of the carotenoid biosynthesis pathway of Synechocystis sp. PCC6803.

EPR spectroscopy using 5-doxylstearic acid (5-SASL) and 16-doxylstearic acid (16-SASL) spin probes was used to study the fluidity of thylakoid membranes. These were isolated from wild type Synechocystis and from several mutants in genes encoding selected enzymes of the carotenoid biosynthesis pathway and/or acyl-lipid desaturases. Cyanobacteria were cultivated at 25°C and 35°C under different light regimes: photoautotrophically (PAG) and/or in light-activated heterotrophic conditions (LAHG). The relative fluidity of membranes was estimated from EPR spectra based on the empirical outermost splitting parameter in a temperature range from 15°C to 40°C. Our findings demonstrate that in native thylakoid membranes the elimination of xanthophylls decreased fluidity in the inner membrane region under optimal growth conditions (25°C) and increased it under sublethal heat stress (35°C). This indicated that the overall fluidity of native photosynthetic membranes in cyanobacteria may be influenced by the ratio of polar to non-polar carotenoid pools under different environmental conditions.

Phosphatidylglycerol depletion induces an increase in myxoxanthophyll biosynthetic activity in Synechocystis PCC6803 cells.

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolving activity of Synechocystis PCC6803 pgsA mutant cells. Shortage of PG led to decreased photosynthetic activity, which, similar to the effect of high light exposure, is likely to generate the production of reactive oxygen species (ROS) or free radicals. Protection of the PG-depleted cells against light-induced damage increased the echinenone and myxoxanthophyll content of the cells. The increased carotenoid content was localized in a soluble fraction of the cells as well as in isolated thylakoid and cytoplasmic membranes. The soluble carotenoid fraction contained carotene derivatives, which may bind to proteins. These carotene-protein complexes are similar to orange carotenoid protein that is involved in yielding protection against free radicals and ROS. An increase in the content of myxoxanthophyll and echinenone upon PG depletion suggests that PG depletion regulates the biosynthetic pathway of specific carotenoids.