Leonora Reinhold

Genes encoding A-type flavoproteins are essential for photoreduction of O2 in cyanobacteria.

O(2) photoreduction by photosynthetic electron transfer, the Mehler reaction, was observed in all groups of oxygenic photosynthetic organisms, but the electron transport chain mediating this reaction remains unidentified. We provide the first evidence for the involvement of A-type flavoproteins that reduce O(2) directly to water in vitro. Synechocystis sp. strain PCC 6803 mutants defective in flv1 and flv3, encoding A-type flavoproteins, failed to exhibit O(2) photoreduction but performed normal photosynthesis and respiration. We show that the light-enhanced O(2) uptake was not due to respiration or photorespiration. After dark acclimation, photooxidation of P(700) was severely depressed in mutants Deltaflv1 and Deltaflv3 but recovered after light activation of CO(2) fixation, which gives P(700) an additional electron acceptor. Inhibition of CO(2) fixation prevented recovery but scarcely affected P(700) oxidation in the wild-type, where the Mehler reaction provides an alternative route for electrons. We conclude that the source of electrons for O(2) photoreduction is PSI and that the highly conserved A-type flavoproteins Flv1 and Flv3 are essential for this process in vivo. We propose that in cyanobacteria, contrary to eukaryotes, the Mehler reaction produces no reactive oxygen species and may be evolutionarily related to the response of anaerobic bacteria to O(2).

Sustained net CO2 evolution during photosynthesis by marine microorganism

BACKGROUND Many aquatic photosynthetic microorganisms possess an inorganic-carbon-concentrating mechanism that raises the CO2 concentration at the intracellular carboxylation sites, thus compensating for the relatively low affinity of the carboxylating enzyme for its substrate. In cyanobacteria, the concentrating mechanism involves the energy-dependent influx of inorganic carbon, the accumulation of this carbon--largely in the form of HCO3(-)-in the cytoplasm, and the generation of CO2 at carbonic anhydrase sites in close proximity to the carboxylation sites. RESULTS During measurements of inorganic carbon fluxes associated with the inorganic-carbon-concentrating mechanism, we observed the surprising fact that several marine photosynthetic microorganisms, including significant contributors to oceanic primary productivity, can serve as a source of CO2 rather than a sink during CO2 fixation. The phycoerythrin-possessing cyanobacterium Synechococcus sp. WH7803 evolved CO2 at a rate that increased with light intensity and attained a value approximately five-fold that for photosynthesis. The external CO2 concentration reached was significantly higher than that predicted for chemical equilibrium between HCO3- and CO2, as confirmed by the rapid decline in the CO2 concentration upon the addition of carbonic anhydrase. Measurements of oxygen exchange between water and CO2, by means of stable isotopes, demonstrated that the evolved CO2 originated from HCO3- taken up and converted intracellularly to CO2 in a light-dependent process. CONCLUSIONS We report net, sustained CO2 evolution during photosynthesis. The results have implications for energy balance and pH regulation of the cells, for carbon cycling between the cells and the marine environment, and for the observed fractionation of stable carbon isotopes.

Physiological and Molecular Studies on the Response of Cyanobacteria to Changes in the Ambient Inorganic Carbon Concentration

The ability of cyanobacteria to adapt to a wide range of ambient CO2 concentrations involves modulation of the activity of an inorganic carbon-concentrating mechanism (CCM), as well as other changes at various cellular levels including the biosynthetic pathway of purines. Studies of high-CO2-requiring mutants have identified several of the genes involved in the operation of the CCM and in the ability to grow under changing ambient CO2 concentration. In the case of Synechococcus sp. strain PCC 7942 most of these genes have been mapped in the genomic region of the rbcLS operon. Higher levels of detectable transcripts originating from some of these genes have been observed after exposure of the cells to low CO2 concentration. Studies of mutants have confirmed quantitative models postulating crucial roles for carboxysomes and carboxysome-located carbonic anhydrase (CA) in cyanobacterial photosynthesis. A central role is also proposed for cytoplasmic-membrane-associated CA activity: CA may function to scavenge escaping CO2 by intracellular conversion to bicarbonate against the chemical potential.

Passive Entry of CO2 and Its Energy-dependent Intracellular Conversion to HCO in Cyanobacteria Are Driven by a Photosystem I-generated ΔμH+

CO2 entry intoSynechococcus sp. PCC7942 cells was drastically inhibited by the water channel blocker p-chloromercuriphenylsulfonic acid suggesting that CO2 uptake is, for the most part, passive via aquaporins with subsequent energy-dependent conversion to HCO 3 − . Dependence of CO2uptake on photosynthetic electron transport via photosystem I (PSI) was confirmed by experiments with electron transport inhibitors, electron donors and acceptors, and a mutant lacking PSI activity. CO2 uptake was drastically inhibited by the uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and ammonia but substantially less so by the inhibitors of ATP formation arsenate and N, N,-dicyclohexylcarbodiimide (DCCD). Thus a ΔμH+ generated by photosynthetic PSI electron transport apparently serves as the direct source of energy for CO2 uptake. Under low light intensity, the rate of CO2 uptake by a high-CO2-requiring mutant ofSynechococcus sp. PCC7942, at a CO2concentration below its threshold for CO2 fixation, was higher than that of the wild type. At saturating light intensity, net CO2 uptake was similar in the wild type and in the mutant IL-3 suggesting common limitation by the rate of conversion of CO2 to HCO 3 − . These findings are consistent with a model postulating that electron transport-dependent formation of alkaline domains on the thylakoid membrane energizes intracellular conversion of CO2 to HCO 3 − .

An essential role for sodium in the bicarbonate transporting system of the cyanobacterium Anabaena variabilis

The apparent photosynthetic affinity of Anabaena variabilis for extracellular inorganic carbon (Ci) was strikingly increased by Na+. The effect was highly specific for Na+ and was maximal at 40 mM Na+. Na+ supply decreased the apparent K m (Ci) of the Ci transporting system and to a lesser extent increased V max. It did not affect photosynthetic rate expressed as a function of intracellular Ci. We infer an effect of Na+ on the Ci transporting system rather than on the photosynthetic machinery itself. We propose several possible models, including Na+‐H+ antiport for maintenance of intracellular pH during HCO3 uptake, and Na+‐HCO− 3 symport.

Inorganic Carbon Fluxes and Photosynthesis in Cyanobacteria — A Quantitative Model

The late 1960’s saw the important discovery of the C4-dicarboxylic pho-tosynthetic pathway, which was followed by the revelation that a carbon dioxide concentrating mechanism operates in certain terrestrial higher plants to raise the CO2: O2 concentration ratio at the site of the carboxylating enzyme, Rubisco. A decade or so later a group of researchers at the Carnegie Laboratory at Stanford led by Joseph Berry demonstrated that a CO2-concentrating mechanism also operates in unicellular aquatic plants. Unlike its counterpart in terrestrial plants, this mechanism does not depend on a metabolic cycle. The lower plant CO2 concentrating mechanism has not in the past received the general attention given to the higher plant counterpart, but the fact that the Organizing Committee of this Congress has judged it worthy of being the topic of our symposium here today is a clear sign of growing interest in the phenomenon.

Low Activation State of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Carboxysome-Defective Synechococcus Mutants

The high-CO2-requiring mutant of Synechococcus sp. PCC 7942, EK6, was obtained after extension of the C terminus of the small subunit of ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). The carboxysomes in EK6 were much larger than in the wild type, but the cellular distribution of the large and small sub-units of Rubisco was not affected. The kinetic parameters of in vitro-activated Rubisco were similar in EK6 and in the wild type. On the other hand, Rubisco appeared to be in a low state of activation in situ in EK6 cells pretreated with an air level of CO2. This was deduced from the appearance of a lag phase when carboxylation was followed with time in cells permeabilized by detergent and subsequently supplied with saturating CO2 and RuBP. Pretreatment of the cells with high CO2 virtually abolished the lag. After low-CO2 treatment, the internal RuBP pool was much higher in mutant cells than in the wild-type cells; pretreatment with high CO2 reduced the pool in mutant cells. We suggest that the high-CO2-requiring phenotype in mutants that possess aberrant carboxysomes arises from the inactivated state of Rubisco when the cells are exposed to low CO2.

Acclimation of photosynthetic microorganisms to changing ambient CO2 concentration

Photosynthetic microorganisms can acclimate to a wide range of CO2 concentration, from as low as 0.001% to ≈10% CO2 (vol/vol in the air in equilibrium with their environment). Some can even grow in the presence of 40% CO2 (1). Acclimation to a limiting CO2 level, well below the Km(CO2) of their carboxylating enzyme, Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase), is achieved by substantial physiological and structural changes at various cell levels (2–4). The most prominent of these is the induction of a CO2 concentrating mechanism (CCM), which raises the [CO2] in close proximity to Rubisco. The latter is for the most part located in pyrenoids or carboxysomes in eukaryotes and prokaryotes photosynthetic microorganisms, respectively (5). This CCM involves light energy-dependent inorganic carbon uptake and accumulation of HCO within the cell. In cyanobacteria, the accumulated HCO penetrates the carboxysomes where carbonic anhydrase (CA) catalyzes the formation of CO2 in the close vicinity of Rubisco. The elevated CO2 concentration is thus confined to the carboxysomes (3). This model is most likely also applicable to eukaryotic algae where pyrenoids, densely packed with Rubisco and also containing CA, may have the same function as carboxysomes (2–7). The elevated [CO2] in these bodies compensates for the relatively low affinity of Rubisco for CO2 and consequently also decreases photorespiration.

ACCLIMATION OF SYNECHOCOCCUS STRAIN WH7803 TO AMBIENT CO2 CONCENTRATION AND TO ELEVATED LIGHT INTENSITY1

A CO2 concentrating mechanism has been identified in the phycoerythrin‐possessing Synechococcus sp. WH7803 and has been observed to be severely inhibited by short exposure to elevated light intensities. A light treatment of 300–2000 μmol quanta·m−2·s−1 resulted in a considerable decay in the variable fluorescence of PSII with time, suggesting decreased efficiency of energy transfer from the phycobilisomes, direct damage to the reaction center II, or both. Measurements of the activity of PSII and changes in fluorescence emission spectra during a light treatment of 1000 μmol quanta·m−2·s−1 indicated considerable reduction in the energy flow from the phycocyanin to the phycobilisome terminal acceptor and chlorophyll a. Consequently, whereas the maximal photosynthetic rate, at saturating light and Co2 concentration, was hardly affected by a light treatment of 1000 μmol quanta·m−2·s−1 for 2 h, the light intensity required to reach that maximum increased with the duration of the light treatment.

The "acid growth effect" and geotropism.

SummaryThe curvature developed by segments of sunflower hypocotyl exposed to gravitational stimulus was enhanced in buffer solutions between pH 3.4 and 4.0 in the absence of added auxin. This effect was observed both when the segments were submerged during the stimulus and when they floated near the surface of the solution. 5–10 min in a horizontal position was sufficient to induce subsequent curvature.Straight growth of the segments was also promoted in buffers of this pH range.The acid effect on curvature was insensitive to KAsO2, HgCl2 and cycloheximide, inhibitors which drastically reduced auxin-induced curvature. Furthermore, acid buffer, but not auxin, restored the ability of segments taken from etiolated and “starved” plants to respond to gravity. These results suggest that the polarisation following gravistimulus may not be resticted to the asymmetric distribution of auxin and auxin co-factors but may involve a general physiological asymmetry.