From cyanobacteria and cyanophages to chloroplasts: the fate of the genomes of oxyphototrophs and the genes encoding photosystem II proteins.

Chloroplasts are the result of endosymbiosis of cyanobacterial organisms with proto-eukaryotes. The psbA, psbD and psbO genes are present in all oxyphototrophs and encode the D1/D2 proteins of photosystem II (PSII) and PsbO, respectively. PsbO is a peripheral protein that stabilizes the O2-evolving complex in PSII. Of these genes, psbA and psbD remained in the chloroplastic genome, while psbO was transferred to the nucleus. The genomes of selected cyanobacteria, chloroplasts and cyanophages carrying psbA and psbD, respectively, were analysed. The highest density of genes and coding sequences (CDSs) was estimated for the genomes of cyanophages, cyanobacteria and chloroplasts. The synonymous mutation rate (rS) of psbA and psbD in chloroplasts remained almost unchanged and is lower than that of psbO. The results indicate that the decreasing genome size in chloroplasts is more similar to the genome reduction observed in contemporary endosymbiotic organisms than in streamlined genomes of free-living cyanobacteria. The rS of atpA, which encodes the α-subunit of ATP synthase in chloroplasts, suggests that psbA and psbD, and to a lesser extent psbO, are ancient and conservative and arose early in the evolution of oxygenic photosynthesis. The role of cyanophages in the evolution of oxyphototrophs and chloroplastic genomes is discussed.

Application of Spectroscopic Methods for the Identification of Superoxide Dismutases in Cyanobacteria.

Superoxide dismutases (SODs) belong to the group of metalloenzymes that remove superoxide anion radicals and they have been identified in three domains of life: Bacteria, Archaea and Eucarya. SODs in Synechocystis sp. PCC 6803, Gloeobacter violaceus CCALA 979, and Geitlerinema sp. ZHR1A were investigated. We hypothesized that iron (FeSOD) and/or manganese (MnSOD) dominate as active forms in these cyanobacteria. Activity staining and three different spectroscopic methods of SOD activity bands excised from the gels were used to identify a suitable metal in the separated samples. FeSODs or enzymes belonging to the Fe-MnSOD superfamily were detected. The spectroscopic analyses showed that only Fe is present in the SOD activity bands. We found FeSOD in Synechocystis sp. PCC 6803 while two forms in G. violaceus and Geitlerinema sp. ZHR1A: FeSOD1 and FeSOD2 were present. However, no active Cu/ZnSODs were identified in G. violaceus and Geitlerinema sp. ZHR1A. We have shown that selected spectroscopic techniques can be complementary to the commonly used method of staining for SOD activity in a gel. Furthermore, the occurrence of active SODs in the cyanobacteria studied is also discussed in the context of SOD evolution in oxyphotrophs.

Cyanophages as an important factor in the early evolution of oxygenic photosynthesis.

Cyanophages are viruses that infect cyanobacteria. An interesting feature of many of them is the presence of psbA and psbD, genes that encode D1 and D2 proteins, respectively. The D1 and D2 are core proteins of the photosystem II (PSII) in cyanobacteria, algae and plants and influence the proper function of oxygenic photosynthesis (OP) in all oxyphototrophs on Earth. The frequent occurrence of psbA and psbD in cyanophages raises the question whether these genes coevolved with hosts during the early stages of cyanophage and cyanobacterial evolution, or whether they are direct descendants of genes adopted from the genomes of cyanobacterial hosts. The phylogeny of D1/D2 proteins encoded in the genomes of selected cyanophages and oxyphototrophs was reconstructed. In addition, common ancestral sequences of D1 and D2 proteins were predicted for cyanophages and oxyphototrophs. Based on this, the reconstruction of the 3D structures of D1 and D2 proteins was performed. In addition, the ratio of non-synonymous to synonymous (dN/dS) nucleotide substitutions in the coding sequences (CDSs) of psbA and psbD was determined. The results of the predicted spatial structures of the D1 and D2 proteins and purifying selection for the CDSs of psbA and psbD suggest that they belong to the ancient proteins, which may have formed the primordial PSII. It cannot be ruled out that they involved in water oxidation in cyanobacteria-like organisms at early stages of the evolution of life on Earth and coevolved with ancient cyanophages. The data are also discussed in the context of the origin of viruses.

Genes encoding the photosystem II proteins are under purifying selection: an insight into the early evolution of oxygenic photosynthesis.

The molecular evolution concerns coding sequences (CDSs) of genes and may affect the structure and function of proteins. Non-uniform use of synonymous codons during translation, known as codon usage bias (CUB), depends on the balance between mutations bias and natural selection. We estimated different CUB indices, i.e. the effective number of codons (ENC), G + C content in the 3rd codon positions (GC3), and codon adaptation index for CDSs of intrinsic proteins of photosystem II (PSII), such as psbA (D1), psbD (D2), psbB (CP47), psbC (CP43), and CDSs of the extrinsic protein psbO (PsbO). These genes occur in all organisms that perform oxygenic photosynthesis (OP) on Earth: cyanobacteria, algae and plants. Comparatively, a similar analysis of codon bias for CDSs of L and M subunits that constitute the core proteins of the type II reaction centre (RCII) in anoxygenic bacteria was performed. Analysis of CUB indices and determination of the number of synonymous (dS) and nonsynonymous substitutions (dN) in all analysed CDSs indicated that the crucial PSII and RCII proteins were under strong purifying (negative) selection in course of evolution. Purifying selection was also estimated for CDSs of atpA, the α subunit of ATP synthase, an enzyme that was most likely already present in the last universal common ancestor (LUCA). The data obtained point to an ancient origin of OP, even in the earliest stages of the evolution of life on Earth.

The Role of Selected Wavelengths of Light in the Activity of Photosystem II in Gloeobacter violaceus.

Gloeobacter violaceus is a cyanobacteria species with a lack of thylakoids, while photosynthetic antennas, i.e., phycobilisomes (PBSs), photosystem II (PSII), and I (PSI), are located in the cytoplasmic membrane. We verified the hypothesis that blue-red (BR) light supplemented with a far-red (FR), ultraviolet A (UVA), and green (G) light can affect the photosynthetic electron transport chain in PSII and explain the differences in the growth of the G. violaceus culture. The cyanobacteria were cultured under different light conditions. The largest increase in G. violaceus biomass was observed only under BR + FR and BR + G light. Moreover, the shape of the G. violaceus cells was modified by the spectrum with the addition of G light. Furthermore, it was found that both the spectral composition of light and age of the cyanobacterial culture affect the different content of phycobiliproteins in the photosynthetic antennas (PBS). Most likely, in cells grown under light conditions with the addition of FR and G light, the average antenna size increased due to the inactivation of some reaction centers in PSII. Moreover, the role of PSI and gloeorhodopsin as supplementary sources of metabolic energy in the G. violaceus growth is discussed.

The activity of RubisCO and energy demands for its biosynthesis. Comparative studies with CO2-reductases.

Most CO2 on Earth is fixed into organic matter via reactions catalysed by enzymes called carboxylases. CO2-fixation via carboxylases occurs in the Calvin-Benson-Bassham (CBB) cycle, and the crucial role in this cycle is played by RubisCO (D-ribulose 1,5-bisphosphate carboxylase/oxygenase). CO2 can also be fixed by pathways, where a reduction of CO2 to formate or carbon monoxide (CO) occurs. The latter reactions are performed by so-called CO2-reductases e.g. formate dehydrogenase (FDH), carbon-monooxide (CO) dehydrogenase (CODH), and crotonyl-CoA reductase/carboxylase (CCR). In general, a simple model of enzymatic activity based only on a turnover rate of an enzyme for an appropriate substrate (kcat) is insufficient. Based on estimated metabolic costs of each amino acid, the average energetic costs of amino acid biosynthesis (Eaa), and the total costs (ET) for selected CO2-fixing enzymes were analyzed concerning 1) kcat for CO2 (kC), and 2) specificity factor (Srel) for RubisCO. A comparison of Eaa and ET to their kC showed that CODH and FDHs do not need to be more efficient enzymes in CO2 capturing pathways than some forms of RubisCO. CCR was the only both low-cost and highly active CO2-fixing enzyme. The obtained results showed also that there exists an evolutionarily conserved trade-off between Srel of RubisCOs and the energetic demands needed for their biosynthesis. Phylogenetic analysis demonstrated that RubisCO, CODH, FDH, and CCR are enzymes formed as a result of parallel evolution. Moreover, the kinetic parameters (kC) of CO2-fixing enzymes were plausibly optimized already at the early stages of life evolution on Earth.

How to define obligatory anaerobiosis? An evolutionary view on the antioxidant response system and the early stages of the evolution of life on Earth.

One of the former definitions of "obligate anaerobiosis" was based on three main criteria: 1) it occurs in organisms, so-called obligate anaerobes, which live in environments without oxygen (O2), 2) O2-dependent (aerobic) respiration, and 3) antioxidant enzymes are absent in obligate anaerobes. In contrast, aerobes need O2 in order to grow and develop properly. Obligate (or strict) anaerobes belong to prokaryotic microorganisms from two domains, Bacteria and Archaea. A closer look at anaerobiosis covers a wide range of microorganisms that permanently or in a time-dependent manner tolerate different concentrations of O2 in their habitats. On this basis they can be classified as obligate/facultative anaerobes, microaerophiles and nanaerobes. Paradoxically, O2 tolerance in strict anaerobes is usually, as in aerobes, associated with the activity of the antioxidant response system, which involves different antioxidant enzymes responsible for removing excess reactive oxygen species (ROS). In our opinion, the traditional definition of "obligate anaerobiosis" loses its original sense. Strict anaerobiosis should only be restricted to the occurrence of O2-independent pathways involved in energy generation. For that reason, a term better than "obligate anaerobes" would be O2/ROS tolerant anaerobes, where the role of the O2/ROS detoxification system is separated from O2-independent metabolic pathways that supply energy. Ubiquitous key antioxidant enzymes like superoxide dismutase (SOD) and superoxide reductase (SOR) in contemporary obligate anaerobes might suggest that their origin is ancient, maybe even the beginning of the evolution of life on Earth. It cannot be ruled out that c. 3.5 Gyr ago, local microquantities of O2/ROS played a role in the evolution of the last universal common ancestor (LUCA) of all modern organisms. On the basis of data in the literature, the hypothesis that LUCA could be an O2/ROS tolerant anaerobe is discussed together with the question of the abiotic sources of O2/ROS and/or the early evolution of cyanobacteria that perform oxygenic photosynthesis.

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) affects development, photosynthesis, and hormonal homeostasis in hybrid aspen (Populus tremula L. × P. tremuloides).

ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) was first described as a protein involved in salicylic acid (SA)-, ethylene-, and reactive oxygen species (ROS)-dependent defense and acclimation responses. It is a molecular regulator of biotic and abiotic stress-induced programmed cell death. Its role is relatively well known in annual plants, such as Arabidopsis thaliana or Nicotiana benthamiana. However, little is known about its functions in woody plants. Therefore, in this study, we aimed to characterize the function of EDS1 in the Populus tremula L. × P. tremuloides hybrid grown for several seasons in the natural environment. We used two transgenic lines, eds1-7 and eds1-12, with decreased EDS1 expression levels in this study. The observed changes in physiological and biochemical parameters corresponded with the EDS1 silencing level. Both transgenic lines produced more lateral shoots in comparison to the wild-type (WT) plants, which resulted in the modification of tree morphology. Photosynthetic parameters, such as quantum yield of photosystem II (ϕPSII), photochemical and non-photochemical quenching (qP and NPQ, respectively), as well as chlorophyll content were found to be increased in both transgenic lines, which resulted in changes in photosynthetic efficiency. Our data also revealed lower foliar concentrations of SA and ROS, the latter resulting most probably from more efficient antioxidant system in both transgenic lines. In addition, our data indicated significantly decreased rate of leaf senescence during several autumn seasons. Transcriptomic analysis revealed deregulation of 2215 and 376 genes in eds1-12 and eds1-7, respectively, and also revealed 207 genes that were commonly deregulated in both transgenic lines. The deregulation was primarily observed in the genes involved in photosynthesis, signaling, hormonal metabolism, and development, which was found to agree with the results of biochemical and physiological tests. In general, our data proved that poplar EDS1 affects tree morphology, photosynthetic efficiency, ROS and SA metabolism, as well as leaf senescence.

The activity of superoxide dismutases (SODs) at the early stages of wheat deetiolation.

Unbound tetrapyrroles, i.e. protochlorophyllide (Pchlide), chlorophyllide and chlorophylls, bring the risk of reactive oxygen species (ROS) being generated in the initial stages of angiosperm deetiolation due to inefficient usage of the excitation energy for photosynthetic photochemistry. We analyzed the activity of superoxide dismutases (SODs) in etiolated wheat (Triticum aestivum) leaves and at the beginning of their deetiolation. Mn-SOD and three isoforms of Cu/Zn-SODs were identified both in etiolated and greening leaves of T. aestivum. Two Cu/Zn-SODs, denoted as II and III, were found in plastids. The activity of plastidic Cu/Zn-SOD isoforms as well as that of Mn-SOD correlated with cell aging along a monocot leaf, being the highest at leaf tips. Moreover, a high Pchlide content at leaf tips was observed. No correlation between SOD activity and the accumulation of photoactive Pchlide, i.e. Pchlide bound into ternary Pchlide:Pchlide oxidoreductase:NADPH complexes was found. Cu/Zn-SOD I showed the highest activity at the leaf base. A flash of light induced photoreduction of the photoactive Pchlide to chlorophyllide as well as an increase in all the SODs activity which occurred in a minute time-scale. In the case of seedlings that were deetiolated under continuous light of moderate intensity (100 µmol photons m-2 s-1), only some fluctuations in plastidic Cu/Zn-SODs and Mn-SOD within the first four hours of greening were noticed. The activity of SODs is discussed with respect to the assembly of tetrapyrroles within pigment-protein complexes, monitored by fluorescence spectroscopy at 77 K.

RubisCO Early Oxygenase Activity: A Kinetic and Evolutionary Perspective.

RubisCO (D-ribulose 1,5-bisphosphate carboxylase/oxygenase) is Earth's main enzyme responsible for CO2 fixation via carboxylation of ribulose-1,5-bisphosphate (RuBP) into organic matter. Besides the carboxylation reaction, RubisCO also catalyzes the oxygenation of RuBP by O2 , which is probably as old as its carboxylation properties. Based on molecular phylogeny, the occurrence of the reactive oxygen species (ROS)-removing system and kinetic properties of different RubisCO forms, we postulated that RubisCO oxygenase activity appeared in local microoxic areas, yet before the appearance of oxygenic photosynthesis. Here, in reviewing the literature, we present a novel hypothesis: the RubisCO early oxygenase activity hypothesis. This hypothesis may be compared with the exaptation hypothesis, according to which latent RubisCO oxygenase properties emerged later during the oxygenation of the Earth's atmosphere. The reconstruction of ancestral RubisCO forms using ancestral sequence reconstruction (ASR) techniques, as a promising way for testing of RubisCO early oxygenase activity hypothesis, is presented.

Enzymatic Antioxidant Systems in Early Anaerobes: Theoretical Considerations.

UNLABELLED: It is widely accepted that cyanobacteria-dependent oxygen that was released into Earth's atmosphere ca. 2.5 billion years ago sparked the evolution of the aerobic metabolism and the antioxidant system. In modern aerobes, enzymes such as superoxide dismutases (SODs), peroxiredoxins (PXs), and catalases (CATs) constitute the core of the enzymatic antioxidant system (EAS) directed against reactive oxygen species (ROS). In many anaerobic prokaryotes, the superoxide reductases (SORs) have been identified as the main force in counteracting ROS toxicity. We found that 93% of the analyzed strict anaerobes possess at least one antioxidant enzyme, and 50% have a functional EAS, that is, consisting of at least two antioxidant enzymes: one for superoxide anion radical detoxification and another for hydrogen peroxide decomposition. The results presented here suggest that the last universal common ancestor (LUCA) was not a strict anaerobe. O2 could have been available for the first microorganisms before oxygenic photosynthesis evolved, however, from the intrinsic activity of EAS, not solely from abiotic sources. KEY WORDS: Archaea-Atmospheric gases-Evolution-H2O2 resistance-Oxygenic photosynthesis. Astrobiology 16, 348-358.

Titanium dioxide nanoparticles (100-1000 mg/l) can affect vitamin E response in Arabidopsis thaliana.

In the present study we analyze the effect of seed treatment by a range of nano-TiO2 concentrations on the growth of Arabidopsis thaliana plants, on the vitamin E content and the expression of its biosynthetic genes, as well as activity of antioxidant enzymes and lipid peroxidation. To conduct the mechanistic analysis of nano-TiO2 on plants growth and antioxidant status we applied nanoparticles concentrations that are much higher than those reported in the environment. We find that as the concentration of nano-TiO2 increases, the biomass, and chlorophyll content in 5-week-old Arabidopsis thaliana plants decrease in a concentration dependent manner. In opposite, higher nano-TiO2 concentration enhanced root growth. Our results indicate that a high concentration of nano-TiO2 induces symptoms of toxicity and elevates the antioxidant level. We also find that the expression levels of tocopherol biosynthetic genes were either down- or upregulated in response to nano-TiO2. Thermoluminescence analysis shows that higher nano-TiO2 concentrations cause lipid peroxidation. To the best of our knowledge, this is the first report concerning the effect of nano-TiO2 on vitamin E status in plants. We conclude that nano-TiO2 affects the antioxidant response in Arabidopsis thaliana plants. This could be an effect of a changes in vitamin E gene expression that is diminished under lower tested nano-TiO2 concentrations and elevated under 1000 µg/ml.

Role of phytochromes A and B in the regulation of cell death and acclimatory responses to UV stress in Arabidopsis thaliana.

Plants coordinate their responses to various biotic and abiotic stresses in order to optimize their developmental and acclimatory programmes. The ultimate response to an excessive amount of stress is local induction of cell death mechanisms. The death of certain cells can help to maintain tissue homeostasis and enable nutrient remobilization, thus increasing the survival chances of the whole organism in unfavourable environmental conditions. UV radiation is one of the environmental factors that negatively affects the photosynthetic process and triggers cell death. The aim of this work was to evaluate a possible role of the red/far-red light photoreceptors phytochrome A (phyA) and phytochrome B (phyB) and their interrelations during acclimatory responses to UV stress. We showed that UV-C treatment caused a disturbance in photosystem II and a deregulation of photosynthetic pigment content and antioxidant enzymes activities, followed by increased cell mortality rate in phyB and phyAB null mutants. We also propose a regulatory role of phyA and phyB in CO2 assimilation, non-photochemical quenching, reactive oxygen species accumulation and salicylic acid content. Taken together, our results suggest a novel role of phytochromes as putative regulators of cell death and acclimatory responses to UV.

PHYTOALEXIN DEFICIENT 4 affects reactive oxygen species metabolism, cell wall and wood properties in hybrid aspen (Populus tremula L. × tremuloides).

The phytoalexin deficient 4 (PAD4) gene in Arabidopsis thaliana (AtPAD4) is involved in the regulation of plant--pathogen interactions. The role of PAD4 in woody plants is not known; therefore, we characterized its function in hybrid aspen and its role in reactive oxygen species (ROS)-dependent signalling and wood development. Three independent transgenic lines with different suppression levels of poplar PAD expression were generated. All these lines displayed deregulated ROS metabolism, which was manifested by an increased H2O2 level in the leaves and shoots, and higher activities of manganese superoxide dismutase (MnSOD) and catalase (CAT) in the leaves in comparison to the wild-type plants. However, no changes in non-photochemical quenching (NPQ) between the transgenic lines and wild type were observed in the leaves. Moreover, changes in the ROS metabolism in the pad4 transgenic lines positively correlated with wood formation. A higher rate of cell division, decreased tracheid average size and numbers, and increased cell wall thickness were observed. The results presented here suggest that the Populus tremula × tremuloides PAD gene might be involved in the regulation of cellular ROS homeostasis and in the cell division--cell death balance that is associated with wood development.

Isochorismate synthase 1 is required for thylakoid organization, optimal plastoquinone redox status, and state transitions in Arabidopsis thaliana.

Isochorismate synthase 1 (ICS1) is a crucial enzyme in the salicylic acid (SA) synthesis pathway, and thus it is important for immune defences. The ics1 mutant is used in experiments on plant-pathogen interactions, and ICS1 is required for the appropriate hypersensitive disease defence response. However, ICS1 also takes part in the synthesis of phylloquinone, which is incorporated into photosystem I and is an important component of photosynthetic electron transport in plants. Therefore, photosynthetic and molecular analysis of the ics1 mutant in comparison with wild-type and SA-degrading transgenic NahG Arabidopsis thaliana plants was performed. Photosynthetic parameters in the ics1 mutant, when compared with the wild type, were changed in a manner observed previously for state transition-impaired plants (STN7 kinase recessive mutant, stn7). In contrast to stn7, deregulation of the redox status of the plastoquinone pool (measured as 1-q p) in ics1 showed significant variation depending on the leaf age. SA-degrading transgenic NahG plants targeted to the cytoplasm or chloroplasts displayed normal (wild-type-like) state transition. However, ics1 plants treated with a phylloquinone precursor displayed symptoms of phenotypic reversion towards the wild type. ics1 also showed altered thylakoid structure with an increased number of stacked thylakoids per granum which indicates the role of ICS1 in regulation of state transition. The results presented here suggest the role of ICS1 in integration of the chloroplast ultrastructure, the redox status of the plastoquinone pool, and organization of the photosystems, which all are important for optimal immune defence and light acclimatory responses.

Ozone fumigation results in accelerated growth and persistent changes in the antioxidant system of Brassica oleracea L. var. capitata f. alba.

The growth response and antioxidant capacity of Brassica oleracea var. capitata f. alba plants treated with 70ppb of ozone was examined. Four week old cabbage seedlings were fumigated with O3 for 3 days before being transplanted into the growing field. The effect of O3 treatment was determined directly after fumigation and over the course of field cultivation. Plants subjected to O3 treatment had an increased diameter of rosettes and number of leaves after 3 and 7 weeks in agriculture, respectively. In addition, the vast majority of fumigated plants reached marketable quality faster than control plants, indicating a positive role of episodes of increased O3 concentrations during vegetation on growth and yielding. Our analysis revealed that by fumigating juvenile white cabbage plants with moderate doses of O3 the activity of catalases (CAT) and peroxidases was elevated. The activity of the examined enzymes was not affected directly after fumigation, but it increased after several weeks in the experimental field. Increased CAT activity was accompanied by changes in 2 out of the 3 CAT genes CAT1 and CAT2, where CAT2 seemed to be responsible for the induced CAT activity. The biosynthesis of low-molecular stress protectants - tocopherols and the glucosinolate (GLS) sinigrin was transiently affected by ozone. γ-Tocopherol (γ-toc) content significantly increased directly after fumigation, but after 3 weeks of vegetation in the field its concentration reached values similar to control. The biosynthesis of α-tocopherol (α-toc) and sinigrin seemed to be upregulated in fumigated plants. However, the response was delayed; no differences were registered directly after treatment, but 3 weeks after transplanting the concentration of sinigrin and α-toc was elevated.

Lesion simulating disease1, enhanced disease susceptibility1, and phytoalexin deficient4 conditionally regulate cellular signaling homeostasis, photosynthesis, water use efficiency, and seed yield in Arabidopsis.

There is growing evidence that for a comprehensive insight into the function of plant genes, it is crucial to assess their functionalities under a wide range of conditions. In this study, we examined the role of lesion simulating disease1 (LSD1), enhanced disease susceptibility1 (EDS1), and phytoalexin deficient4 (PAD4) in the regulation of photosynthesis, water use efficiency, reactive oxygen species/hormonal homeostasis, and seed yield in Arabidopsis (Arabidopsis thaliana) grown in the laboratory and in the field. We demonstrate that the LSD1 null mutant (lsd1), which is known to exhibit a runaway cell death in nonpermissive conditions, proves to be more tolerant to combined drought and high-light stress than the wild type. Moreover, depending on growing conditions, it shows variations in water use efficiency, salicylic acid and hydrogen peroxide concentrations, photosystem II maximum efficiency, and transcription profiles. However, despite these changes, lsd1 demonstrates similar seed yield under all tested conditions. All of these traits depend on EDS1 and PAD4. The differences in the pathways prevailing in the lsd1 in various growing environments are manifested by the significantly smaller number of transcripts deregulated in the field compared with the laboratory, with only 43 commonly regulated genes. Our data indicate that LSD1, EDS1, and PAD4 participate in the regulation of various molecular and physiological processes that influence Arabidopsis fitness. On the basis of these results, we emphasize that the function of such important regulators as LSD1, EDS1, and PAD4 should be studied not only under stable laboratory conditions, but also in the environment abounding in multiple stresses.

Oxygen and hydrogen peroxide in the early evolution of life on earth: in silico comparative analysis of biochemical pathways.

In the Universe, oxygen is the third most widespread element, while on Earth it is the most abundant one. Moreover, oxygen is a major constituent of all biopolymers fundamental to living organisms. Besides O(2), reactive oxygen species (ROS), among them hydrogen peroxide (H(2)O(2)), are also important reactants in the present aerobic metabolism. According to a widely accepted hypothesis, aerobic metabolism and many other reactions/pathways involving O(2) appeared after the evolution of oxygenic photosynthesis. In this study, the hypothesis was formulated that the Last Universal Common Ancestor (LUCA) was at least able to tolerate O(2) and detoxify ROS in a primordial environment. A comparative analysis was carried out of a number of the O(2)-and H(2)O(2)-involving metabolic reactions that occur in strict anaerobes, facultative anaerobes, and aerobes. The results indicate that the most likely LUCA possessed O(2)-and H(2)O(2)-involving pathways, mainly reactions to remove ROS, and had, at least in part, the components of aerobic respiration. Based on this, the presence of a low, but significant, quantity of H(2)O(2) and O(2) should be taken into account in theoretical models of the early Archean atmosphere and oceans and the evolution of life. It is suggested that the early metabolism involving O(2)/H(2)O(2) was a key adaptation of LUCA to already existing weakly oxic zones in Earth's primordial environment.

Ferredoxin:NADP+ oxidoreductase bound to cytochrome b6f complex is active in plastoquinone reduction: implications for cyclic electron transport.

In this study, we have compared three isolation methods of cytochrome b6f complex, obtained from spinach (Spinacia oleracea), differing in the preservation of the cytochrome b6f-associated ferredoxin:NADP+ oxidoreductase (FNR). Although the complexes isolated by all the methods showed the presence of the FNR peptide(s), when incorporated into liposome membranes, the NADPH-PQ (plastoquinone) oxidoreductase activity was not detected for the cytochrome b6f complex isolated with the original method including a NaBr wash. Some activity was found for the complex isolated with the omission of the wash, but the highest activity was detected for the complex isolated with the use of digitonin. The reaction rate of PQ reduction of the investigated complexes in liposomes was not significantly influenced by the addition of free FNR or ferredoxin. The reaction was inhibited by about 60% in the presence of 2 µM 2-n-nonyl-4-hydroxyquinoline N-oxide, an inhibitor of the cytochrome b6 f complex at the Q(i) site, while it was not affected by triphenyltin or isobutyl cyanide that interacts with the recently identified heme c(i) . The obtained data indicate that FNR associated with the cytochrome b6 f complex can participate in the cyclic electron transport as PSI-PQ or NADPH-PQ oxidoreductase. Moreover, we have shown that PQ can be non-enzymatically reduced by ascorbate in liposomes and this reaction might be involved in non-photochemical reduction pathways of the PQ-pool in chloroplasts.

The key role of the redox status in regulation of metabolism in photosynthesizing organisms.

The redox status of the cell is described by the ratio of reduced to non-reduced compounds. Redox reactions which determine the redox state are an essential feature of all living beings on Earth. However, the first life forms evolved under strongly anoxic conditions of the young Earth, and the redox status probably was based on iron and sulphur compounds. Nowadays, redox reactions in cells have developed in strict connection to molecular oxygen and its derivatives i.e. reactive oxygen species (ROS). Oxygen has started to accumulate on the Earth due to oxygenic photosynthesis. All aspects of aerobic life involve ROS, reactive nitrogen species (RNS), antioxidants and redox regulation. Many different redox-active compounds are involved in the complex of redox processes, including pyridine nucleotides, thioredoxins, glutaredoxins and other thiol/disulphide-containing proteins. Redox regulation is integrated with the redox-reactions in photosynthesis and respiration to achieve an overall energy balance and to maintain a reduced state necessary for the biosynthetic pathways that are reductive in nature. It underlies the physiological and developmental flexibility in plant response to environmental signals.