Biogenesis and light regulation of the major light harvesting chlorophyll-protein of diatoms

The apoprotein of the major light harvesting pigment-protein complex from the diatom Phaeodactylum tricornutum (UTEX 646) is composed of two similar polypeptides of 17.5 and 18.0 kilodaltons (kD). The in vivo synthesis of these polypeptides is inhibited by the 80s protein synthesis inhibitor cycloheximide, but not by the 70s ribosome inhibitor chloramphenicol. When total poly(A)⁺ RNA was used in in vitro protein synthesis, a number of polypeptides were synthesized with a dominant product at 22 kD. When the polypeptides were immunoprecipitated with monospecific antibodies to the 17.5 and 18.0 polypeptides, a single protein zone of 22 kD was detected. Immunoprecipitation with preimmune serum failed to precipitate detectable levels of protein at any relative molecular weight (M_r). These findings indicate that the two apoprotein polypeptides of the diatom light harvesting pigment-protein are translated from polyadenylated message on cytoplasmic ribosomes as either a single or two (or more) similar M_r precursor proteins. These findings also suggest that this protein is encoded in the nucleus.

Photosynthetic light adaptation features of P. tricornutum UTEX 646 indicate that it responds to low light by increasing cell size and numbers of photosystem I and II reaction centers per cell, but does not change photosynthetic rate per cell or photosynthetic unit sizes significantly. When low light cells are exposed to higher photon flux densities, the in vivo incorporation of label into the apoprotein of the light harvesting complex decreases. In contrast, high light grown cells show rapid (<3 hour) increases in apoprotein synthesis when exposed to low light levels. This is the first demonstration of a specific role of photon flux density in regulating the synthesis of a major light harvesting pigment-protein during photosynthetic light adaptation.

     

Interaction between saline stress and photoinhibition of photosynthesis in the freshwater green algae Chlamydomonas reinhardtii. Implications for glycerol photoproduction

Light intensity is the main limiting factor for the photosynthetic bioconversion of CO₂ into glycerol which takes place when Chlamydomonas reinhardtii cells are exposed to saline stress conditions. Although productivity increases with light intensity for low irradiances, a strong inhibition is observed for high light intensity values. Saline stress enhances the damage caused by excess of light on the photosynthetic apparatus. The aim of this work is to evaluate the effect of high light intensity and saline stress on photosynthetic activity, cell growth and glycerol photoproduction by C. reinhardtii. The effect of light intensity on C. reinhardtii cells was studied immediately after transfer to a saline medium and after 24 h of adaptation to saline stress conditions. The influence of light intensity on the glycerol production rate was also evaluated for C. reinhardtii cultured in bioreactors of different radius. The factors that significantly affected photoinhibition were light intensity, cell density, radius of the bioreactor and time of exposure to the high light intensity. Our results suggest that bioreactors with a high surface/volume ratio will enable the achievement of high productivities with relatively low light intensities on the surface and will miminise the photoinhibition effect.     

Acclimation of Nannochloropsis gaditana to different illumination regimes: effects on lipids accumulation

Algae are interesting potential sources of biodiesel, although research is still needed to develop efficient large scale productions. One major factor affecting productivity is light use efficiency. The effect of different light regimes on the seawater alga Nannochloropsis gaditana was accessed monitoring growth rate and photosynthetic performances. N. gaditana showed the capacity of acclimating to different light intensities, optimizing its photosynthetic apparatus to illumination. Thanks to this response, N. gaditana maintained similar growth rates under a wide range of irradiances, suggesting that this organism is a valuable candidate for outdoor productions in variable conditions. In the conditions tested here, without external CO₂ supply, light intensity alone was not found to be a major signal affecting lipids accumulation showing the absence of a direct regulatory link between the light stress and lipids accumulation. Strong illumination can nevertheless indirectly influences lipid accumulation if combined with other stresses or in the presence of excess CO₂.     

Interactions between Glucose and Inorganic Carbon Metabolism in Chlorella vulgaris Strain UAM 101

Chlorella vulgaris strain UAM 101 has been isolated from the effluent of a sugar refinery. This alga requires glucose to achieve maximal growth rate even under light saturating conditions. The growth rate of cultures grown on light + CO₂ + glucose (3.16 per day) reaches the sum of those grown on light + CO₂ (1.95 per day) and on dark + glucose (1.20 per day). Unlike other Chlorella strains, uptake of glucose (about 2 micromoles per milligram dry weight per hour) was induced to the same extent in the light and dark and was not photosensitive. The rate of dark respiration was not affected by light and was strongly stimulated by the presence of glucose (up to about 40% in 4 hours). The rate of photosynthetic O₂ evolution was measured as a function of the CO₂ concentration. These experiments were conducted with cells which experienced different concentrations of CO₂ or glucose during growth. The maximal photosynthetic rate was inhibited severely by growing the cells in the presence of glucose. A rather small difference in the apparent photosynthetic affinity for extracellular inorganic carbon (from 10-30 micromolar) was found between cells grown under low and high CO₂. Growth with glucose induced a reduction in the apparent affinity (45 micromolar) even though cells had not been provided with CO₂. Experiments performed at different pH values indicate CO₂ as the major carbon species taken from the medium by Chlorella vulgaris UAM 101.     

Cell cycle regulation of the mixotrophic dinoflagellate Dinophysis acuminata: Growth,photosynthetic efficiency and toxin production

The mixotrophic dinoflagellate Dinophysis acuminata is a widely distributed diarrhetic shellfish poisoning (DSP) producer. Toxin variability of Dinophysis spp. has been well studied, but little is known of the manner in which toxin production is regulated throughout the cell cycle in these species, in part due to their mixotrophic characteristics. Therefore, an experiment was conducted to investigate cell cycle regulation of growth, photosynthetic efficiency, and toxin production in D. acuminata. First, a three-step synchronization approach, termed “starvation-feeding-dark”, was used to achieve a high degree of synchrony of Dinophysis cells by starving the cells for 2 weeks, feeding them once, and then placing them in darkness for 58 h. The synchronized cells started DNA synthesis (S phase) 10 h after being released into the light, initiated G2 growth stage eight hours later, and completed mitosis (M phase) 2 h before lights were turned on. The toxin content of three dominant toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1) and pectenotoxin-2 (PTX2), followed a common pattern of increasing in G1 phase, decreasing on entry into the S phase, then increasing again in S phase and decreasing in M phase during the diel cell cycle. Specific toxin production rates were positive throughout the G1 and S phases, but negative during the transition from G1 to S phase and late in M phase, the latter reflecting cell division. All toxins were initially induced by the light and positively correlated with the percentage of cells in S phase, indicating that biosynthesis of Dinophysis toxins might be under circadian regulation and be most active during DNA synthesis.     

Culture conditions and the development of the photosynthetic mechanism; influence of light intensity on photosynthetic characteristics of Chlorella

1. Chlorella pyrenoidosa has been grown in a continuous-culture apparatus under various light intensities provided by incandescent lamps, other conditions of culture being maintained constant. Light intensity curves for cells immersed in the No. 11 Warburg buffer and in Knop''s solution + 4.4 per cent CO₂ at a saturating light intensity were determined as characteristics of the photosynthetic mechanism. These characteristics were referred to the centrifuged cell volume as an index of quantity of cellular material. 2. Cells grown at intensities in the range of about 35 f.-c. develop a capacity for a high rate of photosynthesis (c.mm. O₂/hour/c.mm. cells). At culture intensities above or below this range the cells produced have a lower capacity for photosynthesis. A similar effect is observed for rate of photosynthesis per unit dry weight or rate per unit cell nitrogen. 3. The rate of photosynthesis per cell or rate per unit chlorophyll shows no maximum at any light intensity of culture but increases continuously throughout the range of light intensities studied. 4. Maximum rate of growth is attained at a light intensity of about 100 f.-c. The hypothesis is advanced that at culture intensities above that needed to give maximum rate of growth (100 f.-c.) a mechanism is developed which opposes the photosynthetic process and removes the photosynthetic products. 5. The low capacity for photosynthesis shown by cells grown at culture intensities below 35 f.-c. finds no immediate explanation. 6. The shape of the light intensity curve is markedly affected by the light intensity at which the cells have been cultured. Cells grown at lower intensities give light intensity curves approaching the Blackman type with a short transitional region between light limitation and light saturation.     

Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO₂ concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO₂ concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.     

The Thylakoid Membrane Protein CGL160 Supports CF1CF0 ATP Synthase Accumulation in Arabidopsis thaliana

The biogenesis of the major thylakoid protein complexes of the photosynthetic apparatus requires auxiliary proteins supporting individual assembly steps. Here, we identify a plant lineage specific gene, CGL160, whose homolog, atp1, co-occurs with ATP synthase subunits in an operon-like arrangement in many cyanobacteria. Arabidopsis thaliana T-DNA insertion mutants, which no longer accumulate the nucleus-encoded CGL160 protein, accumulate less than 25% of wild-type levels of the chloroplast ATP synthase. Severe cosmetic or growth phenotypes result under either short day or fluctuating light growth conditions, respectively, but this is ameliorated under long day constant light growth conditions where the growth, ATP synthase activity and photosynthetic electron transport of the mutants are less affected. Accumulation of other photosynthetic complexes is largely unaffected in cgl160 mutants, suggesting that CGL160 is a specific assembly or stability factor for the CF₁CF₀ complex. CGL160 is not found in the mature assembled complex but it does interact specifically with subunits of ATP synthase, predominantly those in the extrinsic CF₁ sub-complex. We suggest therefore that it may facilitate the assembly of CF₁ into the holocomplex.     

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.     

Photosynthetic response of Chlamydomonas reinhardtii to short-term storage on ice in darkness. Dependence of the response on growth conditions

The effect of storage of the unicellular green alga Chlamydomonas reinhardtii (strain 137+) in the pelleted state in darkness on ice (0.2–0.5°C) (further simply “SPDI-treatment”) on its photosynthetic and respiratory activities was studied. To this end, the steady-state rates of O₂ exchange in darkness (dark respiration) and under saturating light (apparent photosynthesis) as well as the induction periods (IP) of apparent photosynthesis were measured at 25°C in the SPDI-untreated and SPDI-treated for the period from ～0.5 to ～30 h algal cells. In contrast to expectations, the SPDI-treatment consistently affected the rate and IP of photosynthesis depending on the physiological state of C. reinhardtii. Dark respiration was affected by the SPDI-treatment as well. However, in absolute values the respiratory changes were much less than the photosynthetic ones, and they were insufficiently reproducible. The SPDI-treatment affected photosynthesis most significantly in high-CO₂-grown cells (cells grown at 5% CO₂ in white light). The rate of photosynthesis in these cells declined quasi-exponentially as a function of time during the SPDI-treatment with a t _1/2 ～1.5 h and finally became by about 60% lower than that before the SPDI-treatment. This decline of photosynthesis was accompanied by continuous and essential increase in the photosynthetic IP. The SPDI-induced photosynthetic changes in high-CO₂-grown cells resulted from the firm disfunction of the photosynthetic apparatus. After switch from growth at 5% CO₂ in white light to growth at ～0.03% CO₂ (air) in white, blue, or red light, the alga gradually transited to a physiological state, in which the negative effects of the SPDI-treatment on the rate and IP of photosynthesis became weak and absent, respectively. Remarkably, this transition was faster in blue (≤5 h) than in white and red light (>10 h). Similar changes in the response of the alga to the SPDI-treatment occurred when high-CO₂-grown cells (5% CO₂, white light, 26°C) were incubated in darkness (air, 24–26°C) for 20–25 h. The results of study were analyzed in the light of literature data relating to the effects of CO₂ concentration, darkness, and light quality on carbohydrates in plant organisms. The analysis led to suggestion that there is connection between the negative effect of the SPDI-treatment on C. reinhardtii and nonstructural carbohydrates presented in the alga: the more carbohydrates contain the alga, the more extensive inactivation of the photosynthetic apparatus occurs in it during its storage in the dense (pelleted) state in darkness on ice.     

Regulating photoprotection improves photosynthetic growth and biomass production in Q<Subscript>C</Subscript>-site mutant cells of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

We characterized the photosynthetic growth of wild-type (WT) and QC-site mutant cells of the cyanobacterium Synechocystis sp. PCC 6803 grown in a photobioreactor under medium-intensity [~70 μmol(photon) m^–2 s^–1] and high-intensity [~200 μmol(photon) m^–2 s^–1] light conditions. Photosynthetic growth rate (the exponential phase) increased about 1.1–1.2 fold for the A16FJ, S28Aβ, and V32Fβ mutant compared with WT cells under medium-intensity light and about 1.2–1.3 fold under high-intensity light. Biomass production increased about 17–20% for A16FJ and S28Aβ mutant cells as compared with WT cells under medium-intensity light and about 14–17% for A16FJ and V32Fβ mutant cells under high-intensity light. The greater photosynthetic growth rate and biomass production of these Q_C-site mutant cells could be attributed to the increased photosynthesis efficiency and decreased dissipation of wasteful energy from phycobilisomes in mutants vs. WT cells. Our results support that manipulation of photoprotection may improve photosynthesis and biomass production of photosynthetic organisms.     

Assembly of photosynthetic apparatus in Rhodobacter sphaeroides as revealed by functional assessments at different growth phases and in synchronized and greening cells

The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0–4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10⁴ s^?1) and yield (F _v/F _max ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10⁴ s^?1). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.     

High performance analysis of the cyanobacterial metabolism via liquid chromatography coupled to a LTQ-Orbitrap mass spectrometer: evidence that glucose reprograms the whole carbon metabolism and triggers oxidative stress

Cyanobacteria are environmentally important photosynthetic microorganisms attracting a growing attention in various areas of basic and applied researches. To better understand their metabolism, we presently report on the development of a robust and simple protocol for facile extraction and high throughput analysis of the metabolites of the widely-used strain Synechocystis PCC6803 through liquid chromatography coupled to high resolution mass spectrometry (LC/MS). Our analytical method was developed and tested with 102 reference compounds representative of the chemical diversity of polar cell metabolites, and Synechocystis cell extracts spiked with 37 reference compounds. These samples were analyzed with two chromatographic systems, each coupled to a LTQ-Orbitrap mass spectrometer: a liquid chromatographic system equipped with a pentafluorophenylpropyl column (the PFPP-LC/MS system), and an ultra-high performance liquid chromatographic system with a C₁₈-reversed phase column (the C₁₈-UHPLC/MS system). We showed that the PFPP-LC/MS method performs better than the C₁₈-UHPLC/MS method in terms of retention, separation and detection of metabolites. Consequently, we applied the PFPP-LC/MS method to analyze the metabolome of Synechocystis growing under various conditions of light and glucose, which strongly influence cell growth. We found that glucose increases glucose storage (synthesis of glycogen-like polysaccharide) and catabolism (oxidative pentose phosphate pathway and glycolysis), while it decreases the Calvin–Benson cycle that consumes photosynthetic electrons for CO₂ assimilation. Depending on light and glucose availabilities, this global metabolic reprogramming can generate an oxidative stress, likely through the recombination of the glucose-spared electrons with the photosynthetic oxygen thereby producing toxic reactive oxygen species.     

Proteomic analysis of Synechocystis sp. PCC6803 responses to low-temperature and high light conditions

The global changes in protein expression of Synechocystis sp. PCC6803, a photosynthetic bacterium for the production of secondary metabolites as a green cell factory, were investigated by proteome separation and a subsequent tandem mass spectrometry. Two different proteome separation techniques, strong cation exchange chromatography and off-gel electrophoresis, were applied. The combination of the two proteome separation techniques enabled the comparative analysis of the differential regulation of the Synechocystis proteome in response to two different environmental factors, temperature and light. A total of 1,483 proteins were identified, which represent over 40% of the genes in Synechocystis. Our data showed that fatty acid metabolism was inhibited by (3R)-hydroxymyristol acyl carrier protein dehydrase (Sll1605) under low temperature conditions. The expression of UDP-N-acetylglucosamine acyltransferase (Sll0379) and 3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase (Slr0776), which is involved in lipopolysaccharide metabolism, was not observed under high light conditions. Under high light exposure, proteins related to iron-sulfur metabolism were detected, which may be responsible for maintaining the redox potential of the photosystem. High light under low temperature caused severe damage to the photosystem. Some of the responses to these stresses were similar to those previously reported for other photosynthetic organisms. Notably, this study revealed the followings: (i) low temperature inhibits fatty acid synthesis; (ii) high light inhibits lipopolysaccharides synthesis and stimulates the expression of iron-sulfur related proteins; and (iii) high light under low temperature induces the photorespiratory cycle. The global proteomic analysis clearly showed that stress conditions such as low temperature and/or high light induce cellular metabolisms related with the protection of their photosystems in the model microalga Synechocystis sp. PCC6803.     

Denitrifying and photoheterotrophic growth of Rhodobacter sphaeroides S under anaerobic-dark and -light conditions

Denitrifying growth characteristics of Rhodobacter shaeroides S were examined in batch and continuous cultures under anaerobic-dark and -light conditions. Growth yield based on the electron equivalent, Y_eq., in anaerobic-dark conditions was 2–3 times lower than that in aerobic-dark conditions (aerobic-heterotropic growth). Under anaerobic-light conditions, denitrifying growth was observed with photosynthetic growth, but decreased with the increase of light intensity. The value of Y_eq. in anaerobic-light conditions increased with the increase of light intensity. This phenomenon could be explained as the change of the fraction of cell mass grown by denitrification and photosynthesis in the total cell mass from the energetic analysis.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

Growth and photosynthetic response of the cyanobacterium Microcystis aeruginosa in relation to photoperiodicity and irradiance

Growth and photosynthetic characteristics, P _max (maximum light-saturated oxygen production rate) and (photosynthetic affinity), of Microcystis aeruginosa were studied in continuous cultures under a range of photoperiod lengths and growth irradiances. Microcystis showed a low specific maintenance rate constant and a high growth affinity for light (typical cyanobacterial features), but required a dark period to obtain maximum growth rate. P _max and per unit dry weight increased, as did pigment content, when less light became available. By regulation in and P _max (crucial in light-limiting and high-light conditions, respectively) this buoyant species can flourish in low light, but also in high-light environments which may arise when buoyancy is lost.The two different types of light conditions affected growth, and photosynthesis, in different ways. One needs thus to discriminate between photoperiod- and irradiance-limitation, which restricts the utility of simple algal growth models. It was emphasized that photosynthetic adaptation patterns of light-limited species may resemble short-term nutrient uptake kinetics of nutrient-limited organisms.With prior knowledge of the growth limitation, we were able to assess the growth rate of a natural population of Microcystis from its photosynthetic response and from data of laboratory cultures of a known physiological state.