Improving the photosynthetic H2-productivity of the green alga Chlorella fusca by physiologically directed O2 avoidance and ammonium stimulation

Low production rates and sensitivity to O₂ are two major obstacles which prevent the technical exploitation of the ability of green algae to produce H₂ from water. Both problems were addressed in the present work. The inhibitory effect of O₂ on the hydrogen photoproduction of the green alga Chlorella fusca could be minimized by using algal cells which had not yet fully restored their oxygen evolving capacities after an artificially induced chloroplast de/regeneration cycle (de-/regreening). The H₂ photoproductivity peaked after 30 h of greening light while the O₂ evolution at this time reached only 59% of its normal capacity. The H₂PP yields could be further increased if NH₄Cl was added to the reaction medium at the beginning of the anaerobic preincubation period. No stimulatory effect was observed when NH₄Cl was added just before illumination, i.e. at the end of the 5-h-preincubation period. It is assumed that NH₄Cl inhibited the photosynthetic reduction of nitrite, which competed with hydrogen photoproduction indirectly by feedback repression of the NO ₂ ^- /NO ₃ ^- -reductive system. The impacts of the given results on an optimized H₂-production in green algae based on photosynthesis are discussed.Abbreviations H₂PP H₂ photoproduction - H₂ase hydrogenase - DA dark adaptation - LRG light regreening - DCMU 3-(3,4-dichlorophenyl)-l, 1-dimethylurea - Dit sodium dithionite - HEPES N-2-hydroxyethylpiperazin-N-2-ethan-sulfonic acid - PS I/II photosystem I/II     

HYDROGEN SULFIDE PRODUCTION BY SYNECHOCOCCUS LIVIDUS Y52-s1

Under anaerobic conditions and in the absence of CO₂, the thermophilic blue-green alga Synechococcus lividus Y52-s, evolved hydrogen sulfide in both darkness and light. The mechanism of this process was investigated and compared with photo- and dark reductions in organisms representing several phyla. The photoproduction of H₂S from either sulfate or thiosulfate was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethyl urea (DCMU) and carbonyl m-chlorophenyl-hydrazone (m-Cl-CCP). The inhibitory effect of DCMU showed the requirement for photosystem II as electron donor. Inhibition by m-Cl-CCP also implicated ATP as an energy source. Monofluoroacetate partially inhibited photoproduction of H₂S. This indicated that oxidative metabolism may act us a source of electrons to reduce the photooxidant under certain conditions. Thiosulfate acts only as electron acceptor and is reductively cleaved to S⁼ and SO₃⁼. Thiosulfate and sulfate appeared to replace CO₂ in the light and O₂ in darkness as electron acceptors. The phosphorylation uncouplers dinitrophenol and m-Cl-CCP stimulated dark H₂S production.     

Down‐regulation of catalase activity allows transient accumulation of a hydrogen peroxide signal in Chlamydomonas reinhardtii

In photosynthetic organisms, excess light is a stress that induces production of reactive oxygen species inside the chloroplasts. As a response, the capacity of antioxidative defence mechanisms increases. However, when cells of Chlamydomonas reinhardtii were shifted from dark to high light, a reversible partial inactivation of catalase activity was observed, which correlated with a transient increase in the level of H₂O₂ in the 10 μm range. This concentration range seems to be necessary to activate H₂O₂‐dependent signalling pathways stimulating the expression of H₂O₂ responsive genes, such as the heat shock protein HSP22C. Catalase knock‐down mutants had lost the transient accumulation of H₂O₂, suggesting that a decrease in catalase activity was the key element for establishing a transient H₂O₂ burst. Catalase was inactivated by a one‐electron event consistent with the reduction of a single cysteine. We propose that under high light intensity, the redox state of the photosynthetic electron transport chain is sensed and transmitted to the cytosol to regulate the catalase activity. This allows a transient accumulation of H₂O₂, inducing a signalling event that is transmitted to the nucleus to modulate the expression of chloroplast‐directed protection enzymes.     

Action of Heavy Metals on Hill Activity and O(2) Evolution in Anacystis nidulans

Addition of 5 micromolar Cu²⁺, Cd²⁺, and Zn²⁺ was inhibitory to 10 micromolar H₂O₂-supported Hill activity (dichlorophenolindophenol reduction) and O₂ evolution in membrane preparation from Anacystis nidulans. The reversal of Cd²⁺ and Zn²⁺ inhibition, in contrast to Cu²⁺, by exogenously added catalase (EC 1.11.1.6) suggested that the former cations were inhibitory to H₂O₂ degradation. Ascorbic acid (20 micromolar) supported 27% of the Hill activity which was insensitive to DCMU (10 micromolar) and the remaining activity, attributable to the DCMU sensitive process, was sensitive to inhibition by Cu²⁺ only. It is suggestive that the action site of Cd²⁺ and Zn²⁺ is located between the electron donation sites of H₂O₂ and ascorbic acid, while that of Cu²⁺ is located beyond it. Electron donation by reduced glutathione was insensitive to DCMU and Cu²⁺, indicating that the action site of Cu²⁺ is prior to its electron donation site. Further, the phenanthroline (10 micromolar) reversal of Cu²⁺ inhibition of Hill activity suggested a tentative action site of Cu²⁺ at the level of cytochrome.     

Heterogeneous photosystem 2 activity in isolated spinach chloroplasts

A study was made of the fluorescence induction curves from gently-broken spinach chloroplasts inhibited with DCMU. It was found that there were four kinetically different phases associated with such curves of which only the fastest did not appear to follow exponential kinetics. A comparison of the effects of various concentrations of DCMU on the rate of oxygen evolution and on the fluorescence induction curve did not support the hypothesis that any of the kinetic phases was simply an artefact caused by incomplete inhibition of electron transport. It was also found that 5 min of dark incubation did not maximally oxidize the electron acceptors to photosystem 2 since some acceptors were only oxidized following far-red illumination, suggesting a heterogeneity among these acceptors with respect to their re-oxidation properties. Investigation of the effect of the Q₄₀₀ oxidation state on the fluorescence induction curve revealed that it only influenced the slowest kinetic phase and that Q₄₀₀ did not seem to be associated with the other phases.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1 - 1 dimethylurea - PS 1 photosystem 1 - PS2 photosystem 2 - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - EDTA ethylene-diaminetetraacetic acid - F_max maximum yield of fluorescence emission - F₀ initial yield of fluorescence emission - F_v variable yield of fluorescence emission - N.E. non-exponential kinetics     

Exogenous H2O2 increased catalase and peroxidase activities and proline content in Nitraria tangutorum callus

Antioxidative responses and proline accumulation induced by exogenous H₂O₂ were investigated in the callus from halophyte Nitraria tangutorum Bobr. H₂O₂-treated callus exhibited higher H₂O₂ content than untreated callus. The activities of catalase (CAT) and peroxidase (POD) significantly increased in the callus treated with H₂O₂, while ascorbate peroxidase (APX) activity decreased. In addition, significantly enhanced proline content was observed in the callus treated by H₂O₂, which could be alleviated by H₂O₂ scavenger dimethylthiourea and calcium (Ca) chelator ethylene glycol bis-(β-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (EGTA). Moreover, γ-glutamyl kinase (GK) activity increased in H₂O₂-treated callus, but proline dehydrogenase (PDH) activity decreased significantly, and the reduction was partly abolished by EGTA or Ca channel blocker verapamil. Assays using a scanning electron microscope showed significantly enhanced Ca content in H₂O₂-treated callus.     

H2-Photoproduction by green algae: The significance of anaerobic pre-incubation periods and of high light intensities for H2-photoproductivity ofChlorella fusca

Using sodium-dithionite as an oxygen scavenger, the influences of different light intensities and periods of anaerobic pre-incubation in the dark on H₂-photoproductivity were studied with the green algaChlorella fusca. By measuring hydrogen production in the light using manometric and gas chromatographic methods the effectiveness of sodium dithionite in stabilizing photoproduction was established. For high rates of H₂-photoproduction high light intensities up to 30,000 lux (580 W m^-2) were necessary; these are comparable to those required for light saturation of oxygen photoproduction by this alga. AlthoughChlorella fusca produces H₂ immediately after transition to anaerobic conditions, the optimum rate of H₂ production was reached after a 5 h dark adaptation period only. The results obtained are discussed with respect to characteristics of H₂-photoproduction by green algae: the initial burst kinetics, the light saturation, and the obligate period of anaerobic adaptation. It is concluded that H₂-photoproduction byChlorella is an anaerobic photosynthetic process which occurs in the absence of CO₂ and can be experimentally stabilized by exogenous oxygen scavengers.Abbreviations DCMU (3-(3,4-Dichlorophenyl)-1,1-dimethylurea) - HEPES (2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid)     

Mass spectrometric determination of O2 gas exchange during a dark-to-light transition in higher-plant cells

The exchange of O₂ and CO₂ by photoautotrophic cells of Euphorbia characias L. was measured using a mass-spectrometry technique. During a dark-tolight transition the O₂ uptake rate was little affected whereas CO₂ efflux was decreased by 40%. In order to differentiate eventual superimposed O₂-uptake processes, the kinetics of O₂ exchange resulting from brief illuminations were measured with a highly sensitive device. When the cells were exposed to a saturating light for short periods, the rate of O₂ uptake passed through a series of transients: there was first a stimulation occurring 2–3 s after the appearance of O₂ from water-splitting, followed 30 s later by an inhibition. These two transients were reduced 80% by 3-(3,4-dichlorophenyl)1, 1-dimethylurea (DCMU), indicating that they relied on the linear transport of electrons in the chloroplasts. The first transient (stimulation of an O₂ uptake) was little affected by mitochondrial inhibitors such as antimycin A and oligomycin or the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but was increased in presence of KCN. When spaced flashes (2 us duration; 100-ms intervals) were used instead of continuous light, this transient was almost suppressed indicating that it was dependent on the saturation of some component of the chloroplastic chain. The second transient (inhibition of O₂ uptake) was present when spaced flashes were used instead of continuous light. It was markedly decreased by addition of CCCP and mitochondrial inhibitors (antimycin A, oligomycin, KCN) which strongly indicates that it relied on mitochondrial respiration. It is concluded from these experiments that illumination of the cells resulted in an inhibition of mitochondrial respiration, but the resulting inhibition of O₂ uptake was hidden by the appearance of an O₂-uptake process of extramitochondrial origin, presumably located in the chloroplast.Abbreviations CCCP carbonylcyanide mchlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Rubisco ri-bulose-1,5-bisphosphate carboxylase/oxygenase The authors thank Drs A. Vermeglio, P. Thibault and P. Gans for helpful discussions.     

16O2/18O2 analysis of oxygen exchange in Dunaliella tertiolecta. Evidence for the inhibition of mitochondrial respiration in the light

A mass spectrometric ¹⁶O₂/¹⁸O₂-isotope technique was used to analyse the rates of gross O₂ evolution, net O₂ evolution and gross O₂ uptake in relation to photon fluence rate by Dunaliella tertiolecta adapted to 0.5, 1.0, 1.5, 2.0 and 2.5 M NaCl at 25°C and pH 7.0.At concentrations of dissolved inorganic carbon saturating for photosynthesis (200 M) gross O₂ evolution and net O₂ evolution increased with increasing salinity as well as with photon fluence rate. Light compensation was also enhanced with increased salinities. Light saturation of net O₂ evolution was reached at about 1000 mol m^-2s^-1 for all salt concentrations tested. Gross O₂ uptake in the light was increased in relation to the NaCl concentration but it was decreased with increasing photon fluence rate for almost all salinities, although an enhanced flow of light generated electrons was simultaneously observed. In addition, a comparison between gross O₂ uptake at 1000 mol photons m^-2s^-1, dark respiration before illumination and immediately after darkening of each experiment showed that gross O₂ uptake in the light paralleled but was lower than mitochondrial O₂ consumption in the dark.From these results it is suggested that O₂ uptake by Dunaliella tertiolecta in the light is mainly influenced by mitochondrial O₂ uptake. Therefore, it appears that the light dependent inhibition of gross O₂ uptake is caused by a reduction in mitochondrial O₂ consumption by light.Abbreviations DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea - DHAP dihydroxy-acetonephosphate - DIC dissolved inorganic carbon - DR_a rate of dark respiration immediately after illumination - DR_b rate of dark respiration before illumination - E₀ rate of gross oxygen evolution in the light - NET rate of net oxygen evolution in the light - PFR photon fluence rate - RubP rubulose-1,5-bisphosphate - SHAM salicyl hydroxamic acid - U₀ rate of gross oxygen uptake in the light     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts

Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H₂O₂. The in vitro activation by H₂O₂ was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H₂O₂ of SH groups on the enzyme which were reduced in the light by the light effect mediator system.     

Scavenging of Hydrogen Peroxide in Prokaryotic and Eukaryotic Algae: Acquisition of Ascorbate Peroxidase during the Evolution of Cyanobacteria

Ascorbate (AsA) peroxidase was found in six species of cyanobacteriaamong ten species tested. Upon the addition of H₂¹⁸O₂ to thecells of AsA peroxidase-containing cyanobacteria, ¹⁶O₂ derivedfrom water and ¹⁸O₂ derived from H₂^I8O₂ were evolved in thelight. The evolution of ¹⁶O₂ was inhibited by DCMU and did notoccur in the dark, but ^I8O₂ was evolved even in the dark orin the presence of DCMU. Similar light-dependent evolution of¹⁶O₂ was observed in the cells of AsA peroxidase-containingEuglena and Chlamydomonas. However, the cells of AsA perox-idase-lackingcyanobacteria evolved only ¹⁸O₂ in either the light or dark.Furthermore, the quenching of chlorophyll fluorescence inducedby hydrogen peroxide was observed only in the cells of the AsAperoxidase-containing Synechocystis 6803, and not in the cellsof Anacystis nidulans which lacks AsA peroxidase. Thus, cyanobacteriacan be divided into two groups, those that has and those thatlacks AsA peroxidase. The first group scavenges hydrogen peroxidewith the peroxidase using a photoreductant as the electron donor,and the second group only scavenges hydrogen peroxide with catalase. (Received July 23, 1990; Accepted October 18, 1990)     

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts

Intact spinach chloroplasts, washed four times in buffered sorbitol to decrease catalase contamination, supported O₂ evolution in the dark at very low rates (less than 2 μmol/mg Chl per h) in the presence of low concentrations of H₂O₂ (0.25 mM); H₂O₂ was not significantly metabolished under these conditions. In the light, washed chloroplasts supported H₂O₂-dependent O₂ evolution at rates of 28–46 μmol/mg Chl per h in the presence of 0.1–0.25 mM H₂O₂; the concentration of H₂O₂ supporting 0.5V_max was estimated to be 25 μM. O₂ evolution in the light was associated with H₂O₂ consumption and ceased after the production of 0.45 mol per mol H₂O₂ consumed. Both O₂ evolution and H₂O₂ consumption were abolished by 5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Washed intact chloroplasts contained endogenous pools of GSH and ascorbate estimated at 10 and 33 mM, respectively. H₂O₂-dependent O₂ evolution in the light was associated with a decrease in these levels which increased as O₂ evolution gradually ceased. The results are consistent with the hypothesis that H₂O serves as eventual electron donor for the reduction of H₂O₂ in illuminated chloroplasts and that GSH/GSSG and ascorbate/dehydroascorbate serve as intermediate electron carriers. Preincubation of chloroplasts in the dark with 0.1 mM H₂O₂ abolished O₂ evolution in the light.     

Inhibition of O2 evolution by NADP in isolated potato tuber chloroplast and its identity with 3-(3,4-dichlorophenyl)-1,1-dimethyl urea inhibition site.

Chloroplast from greening potato tuber showed good photosynthetic capacity. The evolution of O₂ was dependent upon the intensity of light. A light intensity of 30 lux gave maximum O₂ evolution. At higher intensities inhibition was observed. The presence of bicarbonate in the reaction mixture was essential for O₂ evolution. NADP was found to be a potent inhibitor of O₂ evolution in this system. NADP and 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) inhibited the O₂ evolution completely at a 3 μm concentration level, which was reversed by oxidized 2,6-dichlorophenol-indophenol (DCIP). Cyanide (CN)-treated chloroplasts showed full O₂ evolution capacity, when a lipophilic electron acceptor like N-tetramethyl-p-phenylenediamine (TMPD) or DCIP was used along with ferricyanide. Ferricyanide alone showed only 20% reduction. NADP or DCMU could inhibit O₂ evolution only when TMPD was the acceptor but not with DCIP. Photosystem II (PS II) isolated from these chloroplasts also showed inhibition by NADP or DCMU and its reversal by DCIP. Here also the evolution of O₂ with only TMPD as acceptor was sensitive to NADP or DCMU. In the presence of added silicotungstate in PS II NADP or DCMU did not affect ferricyanide reduction or oxygen evolution. The chloroplasts were able to bind exogenously added NADP to the extent of 120 nmol/mg chlorophyll. It is concluded that the site of inhibition of NADP is the same as in DCMU, and it is between the DCIP and TMPD acceptor site in the electron transport from the quencher (Q) to plastoquinone (PQ).     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

Activation and Deactivation of H-ATPase in Intact Chloroplasts

The light activation mechanism of the latent H⁺-ATPase was investigated in intact spinach (Spinacia oleracea, Hybrid 424) chloroplasts. The following observations were made. (a) Photosystem I electron acceptors such as methyl viologen, nitrite, oxaloacetate, etc., inhibit the light activation of the enzyme. (b) The electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) fully inhibits the process. (c) Ascorbate plus diaminodurene or dithionite can restore light activation in DCMU-poisoned chloroplasts. (d) The activated state of the enzyme decays rather slowly (within a few minutes) after illumination of the intact chloroplasts. (e) The rate of dark decay is accelerated by oxidants (H₂O₂ or ferricyanide) and slowed down by dithiothreitol.

It is suggested that the physiological mechanism for regulation of the H⁺-ATPase involves oxidation and reduction reactions in a manner which resembles the regulation of the light-activated carbon cycle enzymes.

     

Inhibition of photosynthetic reactions by light

Illumination of isolated intact chloroplasts of Spinacia oleracea L. for 10 min with 850 W m^-2 red light in the absence of substrate levels of bicarbonate caused severe inhibition of subsequently measured photosynthetic activities. The capacity of CO₂-dependent O₂ evolution and of non-cyclic electron transport were impaired to similar degrees. This photoinactivation was prevented by addition of bicarbonate which allowed normal carbon metabolism to proceed during preillumination. Photoinhibition of electron transport was observed likewise upon illumination of intact or broken chloroplasts when efficient electron acceptors were absent. Addition of uncouplers did not influence the extent of inhibition. Studies of partial electron-transport reactions indicated that the activity of both photosystems was affected by light. In addition, the water-oxidation system or its connection to photosystem II seemed to be impaired. Preillumination did not cause uncoupling of photophosphorylation. Chlorophyll-fluorescence data obtained at room temperature and at 77 K are consistent with the view that photosystem-II reaction centers were altered. Addition of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6) or 1,4-diazabicyclo(2,2,2)octane to isolated thylakoids prior to preillumination substantially diminished photoinhibition. This result shows that reactive oxygen species were involved in the damage. It is concluded that bright light, which normally does not damage the photosynthetic apparatus, may exert the described destructive effects under conditions that restrict metabolic turnover of photosynthetic energy.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI photosystem I - PSII photosystem II     

Salicylic acid and H2O2 function by independent pathways in the induction of freezing tolerance in potato

The effects of salicylic acid (SA) and hydrogen peroxide (H₂O₂) on freezing tolerance were studied in two potato (Solanum tuberosum) cultivars (Alpha and Atlantic) that differ in cold sensitivity, Alpha being more tolerant to freezing than Atlantic. Lowest freezing survival rates were observed in 4-week-old plants. Freezing treatments consisting of exposure to 6° C for 4 h in the dark were applied 24 h after plants had been transferred from in vitro culture to soil. Catalase activity and H₂O₂ were estimated at the following harvest points: stage (a) 4-week-old in vitro plants treated with either 0.1 mM SA or 5 mM H₂O₂; stage (b) as in (a) but 24 h following transfer to soil prior to freezing treatment; stage (c) as in (b) but measured 15 days after a 4-h freezing treatment. The results show that (1) SA induced freezing tolerance in both cultivars; (2) SA inhibited ascorbate peroxidase activities in both cultivars at all harvest points but inhibited catalase activities in only at stage (a); (3) SA induced H₂O₂ accumulation only in Atlantic at stage (a); (4) H₂O₂ enhanced shoot catalase activities in Atlantic at stages (a) and (b) whereas this treatment had no effect on shoot catalase activities in Alpha; (5) H₂O₂ treatment induced freezing tolerance in Atlantic, even though shoot catalase activities were lower than those of the controls following exposure to freezing temperatures. We conclude that SA does not always lead to H₂O₂ accumulation even though catalase and ascorbate peroxidase activities are decreased as a result of the treatment. Moreover, H₂O₂ accumulation is not always associated with the induction of freezing tolerance, for example at stage (a) where SA-induced tolerance in Alpha was not accompanied by H₂O₂ accumulation. H₂O₂ was able to induce freezing tolerance only in Atlantic, even though H₂O₂ accumulated in both cultivars following this treatment.     

Simultaneous measurement of oxygen and hydrogen exchange from the blue-green alga anabaena

Two Clark-type polarographic electrodes were used to measure simultaneous H₂ and O₂ exchange from three species of the blue-green alga Anabaena. Maximum H₂ photoevolution from N₂-fixing cultures of Anabaena required only the removal of dissolved O₂ and N₂; no adaptation period was necessary. No correlation of H₂ photoproduction with photosynthetic O₂ evolution, beyond their mutual light requirement, was found. Hydrogen photoevolution has the following characteristics in common with N₂ fixation in these organisms: DCMU insensitivity; similar white light dependency with very low dark production rates; maximum efficiency in photosystem I light; inhibition by N₂, O₂ and acetylene; and an apparent requirement for the presence of heterocysts. Growth on nitrate medium reduces, and on ammonium medium obliterates, both reactions. Cultures grown under limiting CO₂ conditions have H₂ photoproduction rates proportional to their growth rates. Hydrogenase activity is inferred from H₂ uptake in the dark, but this activity apparently is independent of the photoevolution of H₂ which is ascribed strictly to the nitrogenase system.     

Stimulation of nitrogenase activity and photosynthesis in some cyanobacteria by glyoxylate

Abstract The effect og glyoxylate on nitrogenase activity (C₂H₂ reduction) and photosynthesis (H¹⁴CO₃ fixation and O₂ evolution) was in vestigated in the three heterocystous cyanobacteria Anabaena cylindrica, A. variabiltis and N. muscorum. Glyoxylate had virtually no effect on the rate of dark respiration and was unable to sustain photoheterotrophic growth, though some slight stimulation (= 30%) of photorophic growth was noted. A considerable stimulation of both nitrogenase and photosynthetic activities was observed in presence of glyoxylate. In the light the stimulation increased with time up to about 15-25 h after adding optimal concentrations of 4–6 mM glyoxylate. Placing glyoxylate treated samples in the dark or adding DCMU (30 μM) in the light, showed that glyoxylate initially supported significantly higher nitrogenase activity than did samples in absence of glyoxylate. However, after a prolonged incubation in the dark or in presence of DCMU glyoxylate is unable to relieve the adverse effects of such conditions. The stimulation of the nitrogenase activity was even more pronounced when the glyoxylate was added to cells preincubated in the dark (“carbon starved”) than for cells kept constantly in light. The results suggest that glyoxylate, or a metabolite, may act as an inhibitor of cyanobacterial photorespiration and this hypothesis is discussed.