Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Glyoxylate and glutamate effects on photosynthetic carbon metabolism in isolated chloroplasts and mesophyll cells of spinach

Addition of millimolar sodium glyoxylate to spinach (Spinacia oleracea) chloroplasts was inhibitory to photosynthetic incorporation of ¹⁴CO₂ under conditions of both low (0.2 millimolar or air levels) and high (9 millimolar) CO₂ concentrations. Incorporation of ¹⁴C into most metabolites decreased. Labeling of 6-P-gluconate and fructose-1,6-bis-P increased. This suggested that glyoxylate inhibited photosynthetic carbon metabolism indirectly by decreasing the reducing potential of chloroplasts through reduction of glyoxylate to glycolate. This hypothesis was supported by measuring the reduction of [¹⁴C]glyoxylate by chloroplasts. Incubation of isolated mesophyll cells with glyoxylate had no effect on net photosynthetic CO₂ uptake, but increased labeling was observed in 6-P-gluconate, a key indicator of decreased reducing potential. The possibility that glyoxylate was affecting photosynthetic metabolism by decreasing chloroplast pH cannot be excluded. Increased ¹⁴C-labeling of ribulose-1,5-bis-P and decreased 3-P-glyceric acid and glycolate labeling upon addition of glyoxylate to chloroplasts suggested that ribulose-bis-P carboxylase and oxygenase might be inhibited either indirectly or directly by glyoxylate. Glyoxylate addition decreased ¹⁴CO₂ labeling into glycolate and glycine by isolated mesophyll cells but had no effect on net ¹⁴CO₂ fixation. Glutamate had little effect on net photosynthetic metabolism in chloroplast preparations but did increase ¹⁴CO₂ incorporation by 15% in isolated mesophyll cells under air levels of CO₂.     

Glyoxylate Induced Changes in the Carbon and Nitrogen Metabolism of the Cyanobacterium Anabaena cylindrica

Addition of millimolar concentrations of glyoxylate to nitrogen-fixing cultures of Anabaena cylindrica, grown aerobically in the light, caused the following effects: an increase in the number of glycogen granules and in the excretion of carbohydrates; a decreased phycocyanin concentration, but an increase in the chlorophyll a to phycocyanin ratio. Also, an enhancement in the carbon to nitrogen ratio was noted, but this was restored if NH₄⁺ was added simultaneously. The most pronounced effect of glyoxylate addition was a 20-fold increase in the glycine pool. The effect of glyoxylate on N₂ fixation (acetylene reduction) was enhanced at high light intensities, but it did not affect the in vitro ribulose-1,5-bisphosphate carboxylase activity. However, addition of millimolar concentrations of glycolate did not cause changes in nitrogenase activity, CO₂ fixation, and NH₃ release comparable to those caused by glyoxylate. The primary mechanism of action of glyoxylate appears to be within the glycolate pathway of the vegetative cells and metabolically downstream from glycolate.     

Effect of Glyoxylate on the Sensitivity of Net Photosynthesis to Oxygen (the Warburg Effect) in Tobacco

The addition of glyoxylate to tobacco (Nicotiana tabacum) leaf discs inhibited glycolate synthesis and photorespiration and increased net photosynthetic ¹⁴CO₂ fixation. This inhibition of photorespiration was investigated further by studying the effect of glyoxylate on the stimulation of photosynthesis that occurs when the atmospheric O₂ level was decreased from 21 to 3% (the Warburg effect). The Warburg effect is usually ascribed to the increased glycolate synthesis and metabolism that occurs at higher O₂ concentrations. Photosynthesis in control discs increased from 59.1 to 94.7 micromoles of CO₂ per gram fresh weight per hour (a 60% increase) when the O₂ level was lowered from 21 to 3%, while the rate for discs floated on 15 millimolar glyoxylate increased only from 82.0 to 99.7 micromoles of CO₂ per gram fresh weight per hour (a 22% increase). The decrease in the O₂ sensitivity of photosynthesis in the presence of glyoxylate was explained by changes in the rate of glycolate synthesis under the same conditions.

The rate of metabolism of the added glyoxylate by tobacco leaf discs was about 1.35 micromoles per gram fresh weight per hour and was not dependent on the O₂ concentration in the atmosphere. This rate of metabolism is about 10% the amount of stimulation in the rate of CO₂ fixation caused by the glyoxylate treatment on a molar carbon basis. Glyoxylate (10 millimolar) had no effect on the carboxylase/oxygenase activity of isolated ribulose diphosphate carboxylase. Although the biochemical mechanism by which glyoxylate inhibits glycolate synthesis and photorespiration and thereby decreases the Warburg effect is still uncertain, these results show that cellular metabolites can regulate the extent of the Warburg effect.

     

Metabolism of Glycolate in Isolated Spinach Leaf Peroxisomes : KINETICS OF GLYOXYLATE, OXALATE, CARBON DIOXIDE, AND GLYCINE FORMATION

The flow of glyoxylate derived from glycolate into various metabolic routes in the peroxisomes during photorespiration was assessed. Isolated spinach leaf peroxisomes were fed [¹⁴C] glycolate in the absence or presence of exogenous glutamate, and the formation of radioactive glyoxylate, CO₂, glycine, oxalate, and formate was monitored at time intervals. In the absence of glutamate, 80% of the glycolate was consumed within 2 hours and concomitantly glyoxylate accumulated; CO₂, oxalate, and formate each accounted for less than 5% of the consumed glycolate. In the presence of equal concentration of glutamate, glycolate was metabolized at a similar rate, and glycine together with some glyoxylate accumulated; CO₂, oxalate, and formate each accounted for an even lesser percentage of the consumed glycolate. CO₂ and oxalate were not produced in significant amounts even in the absence of glutamate, unless glycolate had been consumed completely and glyoxylate had accumulated for a prolonged period. These in vitro findings are discussed in relation to the extent of CO₂ and oxalate generated in leaf peroxisomes during photorespiration.     

Photorespiratory ammonia does not inhibit photosynthesis in glutamate synthase mutants of Arabidopsis

Exposure of ferredoxin-dependent glutamate synthase (EC 1.4.7.1) mutants of Arabidopsis thaliana to photorespiratory conditions resulted in the accumulation of NH₄⁺ and the inhibition of photosynthesis. However, upon transfer from 2% O₂, 350 microliters per liter CO₂, to 21% O₂, 350 microliters per liter CO₂, net photosynthesis declined at a slower rate in methionine sulfoximine treated leaf discs relative to controls. The recovery of photosynthesis was also more rapid in MSO-treated leaf discs although ammonia levels were more than threefold higher. Photosynthesis in leaf discs treated with azaserine was inhibited more than controls when transferred to 21% O₂ and recovered less than controls when returned to 2% O₂ although NH₄⁺ levels were not significantly different. The results obtained are consistent with the view that the rapid inhibition of photosynthesis in the glutamate synthase mutants in photorespiratory conditions is not due to the accumulation of NH₄⁺ but rather to the depletion of amino donors for glyoxylate and the consequent effects of glyoxylate on the lack of return of carbon to the chloroplast.     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

Formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis

The formation and metabolism of glycolate in the cyanobacterium Coccochloris peniocystis was investigated and the activities of enzymes of glycolate metabolism assayed. Photosynthetic ¹⁴CO₂ incorporation was O₂ insensitive and no labelled glycolate could be detected in cells incubated at 2 and 21% O₂. Under conditions of 100% O₂ glycolate comprised less than 1% of the acid-stable products indicating ribulose 1,5 bisphosphate (RuBP) oxidation only occurs under conditions of extreme O₂ stress. Metabolism of [1-¹⁴C] glycolate indicated that as much as 62% of ¹⁴C metabolized was released as ¹⁴CO₂ in the dark. Metabolism of labelled glycolate, particularly incorporation of ¹⁴C into glycine, was inhibited by the amino-transferase inhibitor amino-oxyacetate. Metabolism of [2-¹⁴C] glycine was not inhibited by the serine hydroxymethyltransferase inhibitor isonicotinic acid hydrazide and little or no labelled serine was detected as a result of ¹⁴C-glycolate metabolism. These findings indicate that a significant amount of metabolized glycolate is totally oxidized to CO₂ via formate. The remainder is converted to glycine or metabolized via a glyoxylate cycle. The conversion of glycine to serine contributes little to glycolate metabolism and the absence of hydroxypyruvate reductase confirms that the glycolate pathway is incomplete in this cyanobacterium.Abbreviations AAN aminoacetonitrile - AOA aminooxyacetate - DIC dissolved inorganic carbon - INH isonicotinic acid hydrazide - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - PG phosphoglycolate - PGA phosphoglyceric acid - PGPase phosphoglycolate phosphatase - PR photorespiration - Rubisco ribulose-1,5-bisphosphate carboxylase oxygenase - TCA trichloroacetic acid - RuBP ribulose-1,5-bisphosphate     

Effect of phosphinothricin (glufosinate) on photosynthesis and photorespiration of C3 and C4 plants

Phosphinothricin (glufosinate), an irreversible inhibitor of glutamine synthetase, causes an inhibition of photosynthesis in C₃ (Sinapis alba) and C₄ (Zea mays) plants under atmospheric conditions (400 ppm CO₂, 21% O₂). This photosynthesis inhibition is proceeding slower in C₄ leaves. Under non-photorespiratory conditions (1000 ppm CO₂, 2% O₂) there is no inhibition of photosynthesis. The inhibition of glutamine synthetase by phosphinothricin results in an accumulation of NH₄ ⁺. The NH₄ ⁺-accumulation is lower in C₄ plants than in C₃ plants. The inhibition of glutamine synthetase through phosphinothricin in mustard leaves results in a decrease in glutamine, glutamate, aspartate, asparagine, serine, and glycine. In contrast to this, a considerable increase in leucine and valine following phosphinothricin treatment is measured. With the addition of either glutamine, glutamate, aspartate, glycine or serine, photosynthesis inhibition by phosphinothricin can be reduced, although the NH₄ ⁺-accumulation is greatly increased. This indicates that NH₄ ⁺-accumulation cannot be the primary cause for photosynthesis inhibition by phosphinothricin. The investigations demonstrate the inhibition of transmination of glyoxylate to glycine in photorespiration through the total lack of amino donors. This could result in a glyoxylate accumulation inhibiting ribulose-1,5-bisphosphate-carboxylase and consequently CO₂-fixation.Abbreviations GOGAT glutamine-2-oxoglutarate-amidotransferase - GS glutamine synthetase - PPT phosphinothricin - MSO methionine sulfoximine - RuBP ribulose-1,5-bisphosphate     

Anaerobic Carbon Metabolism by the Tricarboxylic Acid Cycle : Evidence for Partial Oxidative and Reductive Pathways during Dark Ammonium Assimilation

Nitrogen-limited cells of Selenastrum minutum (Naeg.) Collins are able to assimilate NH₄⁺ in the dark under anaerobic conditions. Addition of NH₄⁺ to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO₂ efflux and an eightfold increase in the rate of anaplerotic carbon fixation via phosphoenolpyruvate carboxylase. Both of these observations are consistent with increased TCAC carbon flow to supply intermediates for amino acid biosynthesis. Addition of H¹⁴CO₃⁻ to anaerobic cells assimilating NH₄⁺ results in the incorporation of radiolabel into the α-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH₄⁺ addition as the specific activity of glutamic acid increases over time. This indicates that this alga is able to maintain partial oxidative TCAC carbon flow while under anoxia to supply α-ketoglutarate for glutamate production. During dark aerobic NH₄⁺ assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH₄⁺ assimilation, these metabolites contain a large proportion of the total radiolabel and radiolabel accumulates in succinate over time. Also, the ratio of dark carbon fixation to NH₄⁺ assimilation is much higher under anaerobic than aerobic conditions. These observations suggest the operation of a partial reductive TCAC from oxaloacetic acid to malate, fumarate, and succinate. Such a pathway might contribute to redox balance in an anaerobic cell maintaining partial oxidative TCAC activity.     

Role of Glycine and Glyoxylate Decarboxylation in Photorespiratory CO(2) Release

Mechanically isolated soybean leaf cells metabolized added glycolate by two mechanisms, the direct oxidation of glyoxylate and the decarboxylation of glycine. The rate of glyoxylate oxidation was dependent on the cellular glyoxylate concentration and was linear between 0.58 and 2.66 micromoles glyoxylate per milligram chlorophyll. The rate extrapolated to zero at a concentration of zero. The concentration and, therefore, the rate of oxidation of glyoxylate could be decreased by adding glutamate or serine to the cells. These substrates were amino donors for the transamination of glyoxylate to glycine. In the presence of these amino acids more CO₂ was released from added glycolate via the glycine decarboxylation reaction and less by the direct oxidation of glyoxylate.     

Effects of glyoxylate on photosynthesis by intact chloroplasts

Because glyoxylate inhibits CO₂ fixation by intact chloroplasts and purified ribulose bisphosphate carboxylase/oxygenase, glyoxylate might be expected to exert some regulatory effect on photosynthesis. However, ribulose bisphosphate carboxylase activity and activation in intact chloroplasts from Spinacia oleracea L. leaves were not substantially inhibited by 10 millimolar glyoxylate. In the light, the ribulose bisphosphate pool decreased to half when 10 millimolar glyoxylate was present, whereas this pool doubled in the control. When 10 millimolar glyoxylate or formate was present during photosynthesis, the fructose bisphosphate pool in the chloroplasts doubled. Thus, glyoxylate appeared to inhibit the regeneration of ribulose bisphosphate, but not its utilization.

The fixation of CO₂ by intact chloroplasts was inhibited by salts of several weak acids, and the inhibition was more severe at pH 6.0 than at pH 8.0. At pH 6.0, glyoxylate inhibited CO₂ fixation by 50% at 50 micromolar, and glycolate caused 50% inhibition at 150 micromolar. This inhibition of CO₂ fixation seems to be a general effect of salts of weak acids.

Radioactive glyoxylate was reduced to glycolate by chloroplasts more rapidly in the light than in the dark. Glyoxylate reductase (NADP⁺) from intact chloroplast preparations had an apparent K_m (glyoxylate) of 140 micromolar and a V_max of 3 micromoles per minute per milligram chlorophyll.

     

Mechanism of decarboxylation of glycine and glycolate by isolated soybean cells

Isolated soybean leaf mesophyll cells decarboxylated exogenously added [1-¹⁴C]glycolate and [1-¹⁴C]glycine in the dark. The rate of CO₂ release from glycine was inhibited over 90% by isonicotinic acid hydrazide and about 80% by KCN, two inhibitors of the glycine to serine plus CO₂ reaction. The release of CO₂ from glycolate was inhibited by less than 50% under the same conditions. This indicates that about 50% of the CO₂ released from glycolate occurred at a site other than the glycine to serine reaction. The sensitivity of this alternative site of CO₂ release to an inhibitor of glycolate oxidase (methyl-2-hydroxy-3-butynoate) but not an inhibitor of the glutamate:glyoxylate aminotransferase (2,3-epoxypropionate) indicates that this alternative (isonicotinic acid hydrazide insensitive) site of CO₂ release involved glyoxylate. Catalase inhibited this CO₂ release. Under the conditions used it is suggested that about half of the CO₂ released from glycolate occurred at the conversion of glycine to serine plus CO₂ while the remaining half of the CO₂ loss resulted from the direct oxidation of glyoxylate by H₂O₂.     

Regulation of non-autotrophic carbon dioxide assimilation by ammonia in cultured cells of Acer pseudoplatanus L

The assimilation of ¹⁴CO₂ by Acer pseudoplatanus cells in the dark was stimulated by the addition of either NH₄Cl or methylamine. Results were obtained demonstrating that methylamine was not metabolised to any appreciable extent by Acer cells. This suggests that the mechanism of stimulation of dark fixation by methylamine does not involve metabolism via the glutamine synthetase reaction. NH₄⁺ stimulation of CO₂ fixation also occurred in cells pretreated with the glutamine-synthetase inhibitor methionine sulfoximine. This further supports the conclusion that neither NH₄⁺ nor methylamine exerts its effect on CO₂-assimilation via a mechanism that depends upon the assimilation of NH₃ by glutamine synthetase.     

Heterotrophic Carbon Dioxide Fixation Products of Euglena: EFFECTS OF AMMONIUM

The metabolic products of heterotrophic (dark) CO₂ fixation by Euglena gracilis Klebs strain Z Pringsheim were separated and identified. They consisted of amino acids, phosphorylated compounds, tricarboxylic acid cycle intermediates, and nucleotides. Exposure of the cells to NH₄⁺ after a period of NH₄⁺ deprivation stimulated heterotrophic CO₂ fixation almost 4-fold, modifying the spectrum of the fixation products. In particular, the NH₄⁺ treatment stimulated fixation of CO₂ into glutamine, glycine, alanine, and serine.     

Aminooxyacetate stimulation of glycolate formation and excretion by chlamydomonas

Aminooxyacetate (1 millimolar) did not inhibit photosynthetic ¹⁴CO₂ fixation by Chlamydomonas reinhardtii Dangeard, (−) strain (N.90) but greatly stimulated the biosynthesis and excretion of glycolate. Similar results were obtained from cells grown with 5% CO₂ or low CO₂ (air). After 2 minutes with air-grown cells, [¹⁴C]glycolate increased from 0.3% of the total ¹⁴C fixed by the control to 11.7% in the presence of aminooxyacetate and after 10 minutes from 3.8% to 41.1%. Ammonium nitrate (0.2 millimolar) in the media blocked the aminooxyacetate stimulation of glycolate excretion. Chromatographic analyses of the labeled products in the cells and supernatant media indicated that aminooxyacetate also completely inhibited the labeling of alanine while some pyruvate accumulated and was excreted. A high percentage (35%) of initial ¹⁴CO₂ fixation was into C₄ acids. Initial products of ¹⁴CO₂ fixation included phosphate esters as well as malate, aspartate, and glutamate in treated or untreated cells. Lactate was also a major early product of photosynthesis, and its labeling was reduced by aminooxyacetate. Inasmuch as lactate was not excreted, glycolate excretion seemed to be specific. When photosynthesis was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, labeled organic and amino acids but not phosphate esters were lost from the cells. Aminooxyacetate did not inhibit the enzymes associated with glycolate synthesis from ribulose bisphosphate.     

Steady-state photosynthesis in alfalfa leaflets: effects of carbon dioxide concentration

When the CO₂ concentration to which Medicago sativa L. var. El Unico leaflets were exposed was increased from half-saturation to saturation (doubled rate of photosynthesis), glycolate and glycine production apparently decreased due to inhibition of a portion of the glycolate pathway. Serine and glycerate production was not inhibited. We conclude that serine and glycerate were made from 3-phosphoglycerate and not from glycolate and that the conversion of glycine to serine may not be the major source of photorespiratory CO₂ in alfalfa. In investigations of glycolate and photorespiratory metabolism, separate labeling data should be obtained for glycine and serine as those two amino acids may be produced from different precursors and respond differently to environmental perturbations. The increased photosynthetic rate (at saturating CO₂) resulted in greater labeling of both soluble and insoluble products. Sucrose labeling increased sharply, but there was no major shift of tracer carbon flow into sucrose relative to other metabolites. The flow of carbon from the reductive pentose phosphate cycle into the production of tricarboxylic acid cycle intermediates and amino acids increased. Only small absolute increases occurred in steady-state pool sizes of metabolites of the reductive pentose phosphate cycle at elevated CO₂, providing further evidence that the cycle is well regulated.     

Glutamic Acid metabolism and the photorespiratory nitrogen cycle in wheat leaves: metabolic consequences of elevated ammonia concentrations and of blocking ammonia assimilation

The effects of methionine sulfoximine and ammonium chloride on [¹⁴C] glutamate metabolism in excised leaves of Triticum aestivum were investigated. Glutamine was the principal product derived from [U¹⁴C]glutamate in the light and in the absence of inhibitor or NH₄Cl. Other amino acids, organic acids, sugars, sugar phosphates, and CO₂ became slightly radioactive. Ammonium chloride (10 mm) increased formation of [¹⁴C] glutamine, aspartate, citrate, and malate but decreased incorporation into 2-oxoglutarate, alanine, and ¹⁴CO₂. Methionine sulfoximine (1 mm) suppressed glutamine synthesis, caused NH₃ to accumulate, increased metabolism of the added radioactive glutamate, decreased tissue levels of glutamate, and decreased incorporation of radioactivity into other amino acids. Methionine sulfoximine also caused most of the ¹⁴C from [U-¹⁴C]glutamate to be incorporated into malate and succinate, whereas most of the ¹⁴C from [1-¹⁴C]glutamate was metabolized to CO₂ and sugar phosphates. Thus, formation of radioactive organic acids in the presence of methionine sulfoximine does not take place indirectly through “dark” fixation of CO₂ released by degradation of glutamate when ammonia assimilation is blocked. When illuminated leaves supplied with [U-¹⁴C] glutamate without inhibitor or NH₄Cl were transferred to darkness, there was increased metabolism of the glutamate to glutamine, aspartate, succinate, malate, and ¹⁴CO₂. Darkening had little effect on the labeling pattern in leaves treated with methionine sulfoximine.     

Nitrogen source and pCO2 synergistically affect carbon allocation,growth and morphology of the coccolithophore Emiliania huxleyi: potential implications of ocean acidification for the carbon cycle

Coccolithophores are unicellular phytoplankton that produce calcium carbonate coccoliths as an exoskeleton. Emiliania huxleyi, the most abundant coccolithophore in the world's ocean, plays a major role in the global carbon cycle by regulating the exchange of CO₂ across the ocean‐atmosphere interface through photosynthesis and calcium carbonate precipitation. As CO₂ concentration is rising in the atmosphere, the ocean is acidifying and ammonium (NH₄⁺) concentration of future ocean water is expected to rise. The latter is attributed to increasing anthropogenic nitrogen (N) deposition, increasing rates of cyanobacterial N₂ fixation due to warmer and more stratified oceans, and decreased rates of nitrification due to ocean acidification. Thus, future global climate change will cause oceanic phytoplankton to experience changes in multiple environmental parameters including CO₂, pH, temperature and nitrogen source. This study reports on the combined effect of elevated pCO₂ and increased NH₄⁺ to nitrate (NO₃^?) ratio (NH₄⁺/NO₃^?) on E. huxleyi, maintained in continuous cultures for more than 200 generations under two pCO₂ levels and two different N sources. Herein, we show that NH₄⁺ assimilation under N‐replete conditions depresses calcification at both low and high pCO₂, alters coccolith morphology, and increases primary production. We observed that N source and pCO₂ synergistically drive growth rates, cell size, and the ratio of inorganic to organic carbon. These responses to N source suggest that, compared to increasing CO₂ alone, a greater disruption of the organic carbon pump could be expected in response to the combined effect of increased NH₄⁺/NO₃^? ratio and CO₂ level in the future acidified ocean. Additional experiments conducted under lower nutrient conditions are needed prior to extrapolating our findings to the global oceans. Nonetheless, our results emphasize the need to assess combined effects of multiple environmental parameters on phytoplankton biology to develop accurate predictions of phytoplankton responses to ocean acidification.     

AMMONIUM STIMULATION OF DARK CARBON FIXATION AS AN INDICATOR OF NITROGEN DEFICIENCY IN PHYTOPLANKTON: POTENTIAL ERRORS CAUSED BY AMMONIUM-OXIDIZING BACTERIA1

Stimulation of dark fixation of carbon by NH₄⁺ is often used as an indicator of phytoplankton N deficiency. This assay is based on the influence of available NH₄⁺ on anaplerotic CO₂ fixation by algae. However, carbon fixation by chemoautotrophic NH₄⁺-oxidizing bacteria may also be stimulated by NH₄⁺ enrichment, a process that can mask the algal response in natural communities. NH₄⁺ addition enhanced dark carbon fixation up to 300%, relative to unamended controls, in organisms collected on a 0.7-μm retention filter in oligotrophic Flathead Lake, Montana, but the effect was not detectable in the presence of nitrapyrin, an inhibitor of NH₄⁺-oxidizing bacteria. Dark carbon fixation was enhanced with addition of NH₄⁺ in organisms retained on 2-μm filters (which should allow passage of most bacteria). NH₄⁺ stimulated dark carbon fixation in N-deficient axenic cultures of Chlamydomonas reinhardtii Dang but not in N-replete cultures in both the presence and absence of nitrapyrin. Application of nitrapyrin or size fractionation treatments, to separate the processes of dark carbon fixation by nitrifiers and phytoplankton, may improve the efficacy of assays using NH₄⁺ stimulation of dark carbon fixation to specifically indicate N deficiency in natural algal communities.