The occurrence of the cis/trans geometric isomerism of myxoxanthophyll and 4-ketomyxoxanthophyll in the cyanobacterium Anabaena sp. PCC7120.

The cyanobacteria of the genus Anabaena have recently been recognized as a potential source of secondary metabolites of pharmacological and biotechnological importance. In particular, myxoxanthophylls - specific carotenoid glycosides that accumulate in cyanobacterial cells, are attracting increasing interest. Anabaena (Nostoc) sp. PCC7120, a filamentous, mesophilic, nitrogen-fixing cyanobacterium, is a model organism used in biochemical and genetic studies. The carotenoid pool of Anabaena sp. PCC7120 consists of five main species of pigments, namely ß-carotene, echinenone, canthaxanthin and two derivatives of myxoxanthophyll: myxoxanthophyll ((3R,2'S)-myxol 2'-fucoside) and 4-ketomyxoxanthophyll ((3S,2'S)-4-ketomyxol 2'-fucoside). Recent findings show that the carotenoid biosynthesis pathway functions in Anabaena sp. PCC7120 cells are affected by environmental factors. Specifically, the balance between ß-carotene and ketocarotenoids alters according to the temperature conditions. In this study, a new method, based on single-step liquid adsorption chromatography was developed and applied to separate a fraction containing myxoxanthophyll and 4-ketomyxoxanthophyll from Anabaena sp. PCC7120 cells. It was found that this method allowed a high purity fraction of carotenoid glycosides to be obtained from pigment pools as extracted from cyanobacterial cells. The subsequent analysis using the methods HPLC and LC/MS demonstrated that this fraction consists of a mixture of compounds with different retention times. On the basis of their fragmentation spectra and optical properties, these compounds were identified as geometrical isomers of myxoxanthophyll and 4-ketomyxoxanthophyll, including the dominant all-trans forms and less abundant cis forms. Proposals regarding the structures of myxoxanthophyll isomers are made.

Cyanophage infections reduce photosynthetic activity and expression of CO2 fixation genes in the freshwater bloom-forming cyanobacterium Aphanizomenon flos-aquae.

Cyanobacteria play a significant role in ecosystem functioning as photosynthetic and CO2 fixing microorganisms. Whether and to what extent cyanophages alter these carbon and energy cycles in their cyanobacterial hosts is still poorly understood. In this study, we investigated changes in photosynthetic activity (PSII), expression of genes associated with the light phase of photosynthesis (psbA, petA, ndhK) and carbon metabolism (rbcL, zwf) as well as intracellular ATP and NADHP concentrations in freshwater bloom-forming filamentous cyanobacterium Aphanizomenon flos-aquae infected by cyanophage vB_AphaS-CL131. We found that PSII activity and expression level of rbcL genes, indicating potential for CO2 fixation, had decreased in response to cyanophage adsorption and DNA injection. During the period of viral DNA replication and assembly, PSII performance and gene expression remained at this decreased level and did not change significantly, indicating lack of transcriptional shutdown by the cyanophage. Combined, these observations suggest that although there is little to no interference between cyanophage DNA replication, host transcription and cellular metabolism, A. flos-aquae underwent a physiological state-shift toward lower efficiency of carbon and energy cycling. This further suggest potential cascading effect for co-occurring non-infected members of the microbial community.

Acclimation and Characterization of Marine Cyanobacterial Strains Euryhalinema and Desertifilum for C-Phycocyanin Production.

This study involves evaluation of two native cyanobacterial strains Euryhalinema and Desertifilum isolated from a mangrove pond in Haikou (China) for their possible phycocyanin (C-PC) production. Maximal growth rate with highest chlorophyll and C-PC accumulation were observed at 28°C and 60 µmol photons m-2 s-1 photon flux density for Euryhalinema sp., while for Desertifilum sp. at 32°C and 80 µmol photons m-2 s-1. Nitrogen and iron concentration trails revealed that double strength concentration of sodium nitrate and ferric ammonium citrate in original BG11 media increased growth rate and accumulation of C-PC for both strains. Three different C-PC extraction methods were tested. The combined extraction protocol of freeze-thaw and ultrasonication markedly increased the C-PC extraction efficiency and attained the food grade purity (A 620/A 280 ratio >0.7), whereas a higher C-PC yield was found with Na-phosphate buffer. Furthermore, the clarified crude extract was used to purify C-PC by fractional ammonium sulfate [(NH4)2SO4] precipitation, Sephadex G-25 gel filtration chromatography, and DEAE-sephadex ion exchange chromatography and attained analytical grade purity (A 620/A 280 ratio >3.9). Taken together, both strains showed their potential to be domesticated for valuable phycocyanin production.

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.

Effect of the exogenous anthocyanin extract on key metabolic pathways and antioxidant status of Brazilian elodea (Egeria densa (Planch.) Casp.) exposed to cadmium and manganese.

Present study deals with the effect of 24 h pre-incubation with exogenous anthocyanins (ANTH), extracted from red cabbage leaves, on key metabolic processes (photosynthesis and respiration) and pro-/antioxidant balance in the aquatic macrophyte Egeria densa (Planch.) Casp., Hydrocharitaceae family, treated with Cd and Mn (in sulfate form) at a concentration of 100 µmol. After five days of metal treatments, Cd was accumulated and the damage caused to metabolic processes was stronger than Mn. In Cd-treated leaves, the protein level, chlorophyll concentration and maximal photochemical efficiency of PS II decreased twofold, and net-photosynthesis was significantly inhibited, whereas lipid peroxidation and H2O2 production increased. In turn, protective responses developed, including an increase in the total soluble thiols, alternative respiratory pathway capacity and the activity of superoxide dismutase and peroxidases. Pre-incubation in the ANTH-enriched extract caused an increase in foliar ANTH content, enhanced Cd and reduced Mn uptake into the tissue. A decrease in the level of oxidative reactions, an increase in the protein and chlorophyll concentration compared to the control values and a partial improvement of the photosynthetic parameters confirmed the ability of ANTH to reduce Cd-induced damage effects and to mitigate ROS-driven stress reactions. Stimulation of catalase and ascorbate peroxidase activity, an alternative respiration capacity and non-enzymatic antioxidant (carotenoids, ascorbate and proline) synthesis by ANTH were also revealed. These data suggest that ANTH-enriched extract from red cabbage leaves has a protective action against metal toxicity in Egeria plants.

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.

Lead accumulation and distribution in maize seedlings: Relevance to biomass production and metal phytoextraction.

Among trace metals, lead is a highly toxic contaminant, being hazardous to humans and animals. Application of maize plants for phytoremediation of polluted soils and waters has recently been of particular interest. The aim of this work is to investigate the Pb-phytoextraction potential of the maize cv. Tzariza used widely in Eastern European agriculture. Maize seedlings were exposed in a nutrient solution to 1-10000 µM of Pb2+ for 21 days. Lead accumulated mostly in conductive tissues and shoots at 0.1 mM and higher concentrations of Pb in growth medium. Pb at concentrations of 1 and 10 mM caused an increase in the superoxide anion level and the catalase activity in maize leaves. Lead ions were tolerable to maize seedlings within a concentration range up to 1000 µM of Pb2+. The levels of lead in the nutrient solution above 1 mM resulted in inhibition of the growth of axial organs, decrease in leaf area, inhibition of water absorption, and reduction in accumulation of biomass. Theoretical considerations indicate that in the temperate climates of the phytoremediation with maize may allow annual removal up to 90 kg of Pb per km2, depending on the initial level of soil contamination.

Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

MAIN CONCLUSION: The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

Kinetics of nickel bioaccumulation and its relevance to selected cellular processes in leaves of Elodea canadensis during short-term exposure.

Elodea canadensis is an aquatic macrophyte used widely as a bioindicator for the monitoring of water quality and in the phytoremediation of metal-contaminated waters. This study considers the kinetics of nickel bioaccumulation and changes in accompanying metabolic and stress-related physiological parameters. These include photosynthetic activity, pigment content, the accumulation of thiol-containing compounds, thiobarbituric acid-reactive substance (TBARS) products, and the activity of selected antioxidant enzymes (catalase, glutathione reductase, superoxide dismutase). Elodea leaves accumulated nickel according to pseudo-second-order kinetics, and the protective responses followed a time sequence which was related to the apparent rates of nickel accumulation. The applicability of second-order kinetics to the Ni uptake by Elodea leaves during the first 8 h of exposure to the metal suggested that the passive binding of metal ions (chemisorption) was a rate-limiting step at the initial phase of Ni accumulation. This phase was accompanied by an increase in photosynthetic activity together with elevated photosynthetic pigments and protein synthesis, the enhanced activity of antioxidant enzymes, and increased thiol concentration. In contrast, there was a decrease in metabolic activity upon the accumulation of TBARS, and the decline in enzyme activity was observed in the saturation phase of Ni accumulation (8-24 h). These results show that a correlation exists between the protective response and the apparent kinetic rate of Ni uptake. Thus, the time of exposure to the toxicant is a crucial factor in the activation of specific mechanisms of Ni detoxification and stress alleviation.

High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N(6)-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).

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.

The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds.

Effect of copper on pro- and antioxidative reactions in radish (Raphanus sativus L.) in vitro and in vivo.

The generation of superoxide radicals, lipid peroxidation (as measured by malone dialdehyde formation) and the activity of selected antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase) were assessed in radish (Raphanus sativus L.), in response to elevated concentrations of copper ions in the culture medium in vitro and in vivo. Experiments were performed on 7-day-old seedlings and 5-week-old calluses grown on media supplemented with CuSO4 in concentrations of 10, 100 and 1000µÐœ. The exposure to elevated Cu concentrations in the medium significantly reduced both callogenesis and the proliferation of radish calluses in vitro. Cu treatment resulted in the increased generation of the superoxide radical (O2(-)) in radish seedlings and calluses indicating the occurrence of oxidative stress in radish cells, whereas the level of lipid peroxidation (LPO) remained unchanged. Both in calluses and in radish seedlings in vivo, the relative level of oxidative stress was maximal at micromolar Cu concentrations and became attenuated with increasing Cu concentrations. Stronger oxidative stress occurred in the radish seedlings in vivo, compared with radish calluses in vitro. The observed lower sensitivity of calluses to Cu-induced oxidative stress and their ability to proliferate upon exposure to Cu concentrations of up to 1000µÐœ demonstrate the potential of in vitro cell-selection to obtain metal-tolerant radish plant lines.

Arabidopsis BPG2: a phytochrome-regulated gene whose protein product binds to plastid ribosomal RNAs.

BPG2 (Brz-insensitive pale green 2) is a dark-repressible and light-inducible gene that is required for the greening process in Arabidopsis. Light pulse experiments suggested that light-regulated gene expression of BPG2 is mediated by phytochrome. The T-DNA insertion mutant bpg2-2 exhibited a reduced level of chlorophyll and carotenoid pigmentation in the plastids. Measurements of time resolved chlorophyll fluorescence and of fluorescence emission at 77 K indicated defective photosystem II and altered photosystem I functions in bpg2 mutants. Kinetic analysis of chlorophyll fluorescence induction suggested that the reduction of the primary acceptor (QA) is impaired in bpg2. The observed alterations resulted in reduced photosynthetic efficiency as measured by the electron transfer rate. BPG2 protein is localized in the plastid stroma fraction. Co-immunoprecipitation of a formaldehyde cross-linked RNA-protein complex indicated that BPG2 protein binds with specificity to chloroplast 16S and 23S ribosomal RNAs. The direct physical interaction with the plastid rRNAs supports an emerging model whereby BPG2 provides light-regulated ribosomal RNA processing functions, which are rate limiting for development of the plastid and its photosynthetic apparatus.

Variations in xanthophyll composition in etiolated seedlings of Arabidopsis thaliana correlate with protochlorophyllide accumulation.

Protochlorophyllide (Pchlide) accumulation and xantophyll composition were studied in 5-day old etiolated seedlings of three ecotypes of Arabidopsis thaliana: Columbia (Col-0), Landsberg erecta (Ler) and Wassiliewska (Ws). The total Pchlide level as measured by fluorescence spectroscopy varied significantly between ecotypes. A rapid HPLC method revealed quantitative differences in carotenoid composition. It was found that in the Ler ecotype any enhanced accumulation of Pchlide correlates with an increased level of lutein, suggesting the role of enzymes involved in lutein synthesis in cross-regulation between chlorophyll and carotenoid biosynthetic pathways. The function of the dark-accumulated carotenoid pool in seedling de-etiolation is discussed.

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.

Involvement of carotenoids in the synthesis and assembly of protein subunits of photosynthetic reaction centers of Synechocystis sp. PCC 6803.

The crtB gene of Synechocystis sp. PCC 6803, encoding phytoene synthase, was inactivated in the Delta crtH mutant to generate a carotenoidless Delta crtH/B double mutant. Delta crtH mutant cells were used because they had better transformability than wild-type cells, most probably due to their adaptation to partial carotenoid deficiency. Cells of the Delta crtH/B mutant were light sensitive and could grow only under light-activated heterotrophic growth conditions in the presence of glucose. Carotenoid deficiency did not significantly affect the cellular content of phycobiliproteins while the chlorophyll content of the mutant cells decreased. The mutant cells exhibited no oxygen-evolving activity, suggesting the absence of photochemically active PSII complexes. This was confirmed by 2D electrophoresis of photosynthetic membrane complexes. Analyses identified only a small amount of a non-functional PSII core complex lacking CP43, while the monomeric and dimeric PSII core complexes were absent. On the other hand, carotenoid deficiency did not prevent formation of the cytochrome b(6)f complex and PSI, which predominantly accumulated in the monomeric form. Radioactive labeling revealed very limited synthesis of inner PSII antennae, CP47 and especially CP43. Thus, carotenoids are indispensable constituents of the photosynthetic apparatus, being essential not only for antioxidative protection but also for the efficient synthesis and accumulation of photosynthetic proteins and especially that of PSII antenna subunits.

Responses of Lemna trisulca L. (Duckweed) exposed to low doses of cadmium: thiols, metal binding complexes, and photosynthetic pigments as sensitive biomarkers of ecotoxicity.

Lemna species are reported to accumulate a variety of metals from contaminated/polluted sites. Cadmium is a nonessential element for plant metabolism. In this work, we aimed to investigate physiological responses to low doses of cadmium (up to 100 microM). From exposure to the lowest Cd concentration (1 microM) to the highest (100 microM), photosynthetic pigments (Chl a, b, carotenoids) and the ratios of Chl a/b, Chl (a + b)/carotenoids decreased as a function of the Cd dose. The content of soluble proteins decreased in a dose-dependent manner, while total soluble thiols drastically increased. In Cd-treated fronds, the dose-dependent accumulation of a polypeptide with an apparent molecular weight of 24 kDa, as well as the appearance of two smaller polypeptides with molecular weights <6.5 kDa, was observed in sodium dodecyl sulfate polyacrylamide gel electrophoresis. Our results show that in Lemna trisulca, different adaptative mechanisms may be involved in counterbalancing low and high doses of a particular toxicant (cadmium). This feature makes this plant potentially useful material in biomonitoring and phytotoxicity testing.