Increased respiratory restriction during phosphate-limited growth in transgenic tobacco cells lacking alternative oxidase.

We found that mitochondrial alternative oxidase (AOX) protein and the capacity for CN-resistant respiration are dramatically increased in wild-type tobacco (Nicotiana tabacum) suspension-cultured cells in response to growth under P limitation, and antisense (AS8) tobacco cells unable to induce AOX under these conditions have altered growth and metabolism. Specifically, we found that the respiration of AS8 cells was restricted during P-limited growth, when the potential for severe adenylate control of respiration (at the level of C supply to the mitochondrion and/or at the level of oxidative phosphorylation) is high due to the low cellular levels of ADP and/or inorganic P. As a result of this respiratory restriction, AS8 cells had altered growth, morphology, cellular composition, and patterns of respiratory C flow to amino acid synthesis compared with wild-type cells with abundant AOX protein. Also, AS8 cells under P limitation displayed high in vivo rates of generation of active oxygen species compared with wild-type cells. This difference could be abolished by an uncoupler of mitochondrial oxidative phosphorylation. Our results suggest that induction of non-phosphorylating AOX respiration (like induction of adenylate and inorganic P-independent pathways in glycolysis) is an important plant metabolic adaptation to P limitation. By preventing severe respiratory restriction, AOX acts to prevent both redirections in C metabolism and the excessive generation of harmful active oxygen species in the mitochondrion.

Molecular Genetic Evidence of the Ability of Alternative Oxidase to Support Respiratory Carbon Metabolism.

With the cytochrome pathway inhibited, AOX was able to support considerable growth of cultured tobacco (Nicotiana tabacum cv Petit Havana SR1) cells but the efficiency of carbon utilization decreased dramatically. Antisense cells with decreased AOX protein did not grow, whereas sense cells with elevated AOX protein had higher growth and respiration rates than the wild type. In antisense cells a large accumulation of pyruvate resulted in aerobic ethanolic fermentation.

Signals Regulating the Expression of the Nuclear Gene Encoding Alternative Oxidase of Plant Mitochondria.

Suspension cells of tobacco (Nicotiana tabacum L. cv Bright Yellow) were used to investigate signals regulating the expression of the nuclear gene Aox1 encoding the mitochondrial alternative oxidase (AOX) protein responsible for cyanide-resistant respiration in plants. We found that an increase in the tricarboxylic acid cycle intermediate citrate (either after its exogenous supply to cells or after inhibition of aconitase by monofluoroacetate) caused a rapid and dramatic increase in the steady-state level of Aox1 mRNA and AOX protein. This led to a large increase in the capacity for AOX respiration, defined as the amount of salicylhydroxamic acid-sensitive O2 uptake by cells in the presence of potassium cyanide. The results indicate that citrate may be an important signal metabolite regulating Aox1 gene expression. A number of other treatments were also identified that rapidly induced the level of Aox1 mRNA and AOX capacity. These included short-term incubation of cells with 10 mM acetate, 2 [mu]M antimycin A, 5 mM H2O2, or 1 mM cysteine. For some of these treatments, induction of AOX occurred without an increase in cellular citrate level, indicating that other signals (possibly related to oxidative stress conditions) are also important in regulating Aox1 gene expression. The signals influencing Aox1 gene expression are discussed with regard to the potential function(s) of AOX to modulate tricarboxylic acid cycle metabolism and/or to prevent the generation of active oxygen species by the mitochondrial electron transport chain.

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation).

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

Molecular Genetic Alteration of Plant Respiration (Silencing and Overexpression of Alternative Oxidase in Transgenic Tobacco).

The alternative oxidase (AOX) of plant mitochondria is encoded by the nuclear gene Aox1. Sense and antisense DNA constructs of Nicotiana tabacum Aox1 were introduced into tobacco, and transgenic plants with both increased and decreased levels of mitochondrial AOX protein were identified. Suspension cells derived from wild-type and transgenic plants were grown in heterotrophic batch culture. Transgenic cells with increased AOX protein had an increased capacity for cyanide-resistant, salicylhydroxamic acid-sensitive respiration compared to wild-type cells, whereas transgenic cells with decreased AOX protein had a decreased capacity for such respiration. Thus, genetic alteration of the level of AOX protein was sufficient to alter the capacity for electron transport through the alternative pathway. Under our standard growth conditions, "antisense" cells with dramatically reduced levels of AOX protein had growth and respiration rates similar to the wild type. However, whereas wild-type cells were able to grow under conditions that severely suppressed cytochrome pathway activity, antisense cells could not survive this treatment. This suggests that a critical function of AOX may be to support respiration when the cytochrome pathway is impaired. The much higher level of AOX protein in "sense" cells compared to the wild type did not appreciably alter the steady-state partitioning of electrons between the cytochrome path and the alternative pathway in vivo, suggesting that this partitioning may be subject to additional regulatory factors.

Mitochondrial electron transport regulation of nuclear gene expression. Studies with the alternative oxidase gene of tobacco.

We have isolated a cDNA representing the tobacco (Nicotiana tabacum L. cv Bright Yellow) nuclear gene Aox1, which encodes the alternative oxidase of plant mitochondria. The clone contains the complete coding region (1059 base pairs) of a precursor protein of 353 amino acids with a calculated molecular mass of 39.8 kD. A putative transit peptide contains common signals believed to be important for import and processing of mitochondrially localized proteins. We have studied changes in Aox1 gene expression in tobacco in response to changes in cytochrome pathway activity. Inhibition of the cytochrome pathway by antimycin A resulted in a rapid and dramatic accumulation of Aox1 mRNA, whereas the level of mRNAs encoding two proteins of the cytochrome pathway did not change appreciably. This was accompanied by a dramatic increase in alternative pathway capacity and engagement in whole cells. Respiration under these conditions was unaffected by the uncoupler p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, levels of Aox1 mRNA returned to control levels, alternative pathway capacity and engagement declined, and respiration could once again be stimulated by FCCP. The results show that a mechanism involving changes in Aox1 gene expression exists whereby the capacity of the alternative pathway can be adjusted in response to changes in the activity of the cytochrome pathway.

Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO(3) but Not NH(4) by a Green Alga.

Addition of NO(3) (-) to N-limited Selenastrum minutum during photosynthesis resulted in an immediate drop in the NADPH/NADP ratio and a slower increase of the NADH/NAD ratio. These changes were accompanied by a rapid decrease in glucose-6-phosphate and increase in 6-phosphogluconate, indicating activation of glucose-6-phosphate dehydrogenase and a role for the oxidation pentose phosphate pathway during photosynthetic NO(3) (-) assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH(4) (+) were not consistent with a stimulation of the oxidative pentose phosphate pathway.

Normal growth of transgenic tobacco plants in the absence of cytosolic pyruvate kinase.

The coding sequence of the cytosolic isozyme of potato tuber pyruvate kinase (PK) was attached to the transit peptide of the small subunit of pea ribulose-1,5-bisphosphate carboxylase oxygenase and placed under the control of the cauliflower mosaic virus 35S promoter. This construct was transformed into Nicotiana tabacum. Unexpectedly, two primary transformants were recovered in which PK activity in leaves was greatly reduced. The reduction in PK activity appeared to result from the complete absence of the cytosolic form of the enzyme (PK(c)). In addition, no PK(c) could be detected on western blots of leaf extracts. Metabolite analyses indicated that the levels of phosphoenolpyruvate are substantially higher in PK(c)-deficient leaves than in wild-type leaves, consistent with a block in glycolysis at the step catalyzed by PK. PK(c) deficiency in the leaves does not appear to adversely affect plant growth. Analysis of progeny indicates that PK(c) deficiency is a heritable trait. The leaves of PK(c)-deficient transformants have normal rates of photosynthetic O(2) evolution and respiratory O(2) consumption, indicating that these plants are using alternative pathways to bypass PK.

Lower growth temperature increases alternative pathway capacity and alternative oxidase protein in tobacco.

Suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow) have been used to study the effect of growth temperature on the CN-resistant, salicylhydroxamic acid-sensitive alternative pathway of respiration. Mitochondria isolated from cells maintained at 30 degrees C had a low capacity to oxidize succinate via the alternative pathway, whereas mitochondria isolated from cells 24 h after transfer to 18 degrees C displayed, on average, a 5-fold increase in this capacity (from 7 to 32 nanoatoms oxygen per milligram protein per minute). This represented an increase in alternative pathway capacity from 18 to 45% of the total capacity of electron transport. This increased capacity was lost upon transfer of cells back to 30 degrees C. A monoclonal antibody to the terminal oxidase of the alternative pathway (the alternative oxidase) from Sauromatum guttatum (T.E. Elthon, R.L. Nickels, L. McIntosh [1989] Plant Physiology 89: 1311-1317) recognized a 35-kilodalton mitochondrial protein in tobacco. There was an excellent correlation between the capacity of the alternative path in isolated tobacco mitochondria and the levels of this 35-kilodalton alternative oxidase protein. Cycloheximide could inhibit both the increased level of the 35-kilodalton alternative oxidase protein and the increased alternative pathway capacity normally seen upon transfer to 18 degrees C. We conclude that transfer of tobacco cells to the lower temperature increases the capacity of the alternative pathway due, at least in part, to de novo synthesis of the 35-kilodalton alternative oxidase protein.

Activation of Respiration to Support Dark NO(3) and NH(4) Assimilation in the Green Alga Selenastrum minutum.

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO(3) (-) or NH(4) (+) assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH(4) (+) was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phosphoenolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO(3) (-) addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO(3) (-) assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO(3) (-) assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH(4) (+) assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO(3) (-) assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: III. Alanine Is the Product of Anaerobic Ammonium Assimilation.

We have determined the flow of (15)N into free amino acids of the N-limited green alga Selenastrum minutum (Naeg.) Collins after addition of (15)NH(4) (+) to aerobic or anaerobic cells. Under aerobic conditions, only a small proportion of the N assimilated was retained in the free amino acid pool. However, under anaerobic conditions almost all assimilated NH(4) (+) accumulates in alanine. This is a unique feature of anaerobic NH(4) (+) assimilation. The pathway of carbon flow to alanine results in the production of ATP and reductant which matches exactly the requirements of NH(4) (+) assimilation. Alanine synthesis is therefore an excellent strategy to maintain energy and redox balance during anaerobic NH(4) (+) assimilation.

Demonstration of Both a Photosynthetic and a Nonphotosynthetic CO(2) Requirement for NH(4) Assimilation in the Green Alga Selenastrum minutum.

Nitrogen-limited and nitrogen-sufficient cell cultures of Selenastrum minutum (Naeg.) Collins (Chlorophyta) were used to investigate the dependence of NH(4) (+) assimilation on exogenous CO(2). N-sufficient cells were only able to assimilate NH(4) (+) maximally in the presence of CO(2) and light. Inhibition of photosynthesis with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron also inhibited NH(4) (+) assimilation. These results indicate that NH(4) (+) assimilation by N-sufficient cells exhibited a strict requirement for photosynthetic CO(2) fixation. N-limited cells assimilated NH(4) (+) both in the dark and in the light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, indicating that photosynthetic CO(2) fixation was not required for NH(4) (+) assimilation. Using CO(2) removal techniques reported previously in the literature, we were unable to demonstrate CO(2)-dependent NH(4) (+) assimilation in N-limited cells. However, employing more stringent CO(2) removal techniques we were able to show a CO(2) dependence of NH(4) (+) assimilation in both the light and dark, which was independent of photosynthesis. The results indicate two independent CO(2) requirements for NH(4) (+) assimilation. The first is as a substrate for photosynthetic CO(2) fixation, whereas the second is a nonphoto-synthetic requirement, presumably as a substrate for the anaplerotic reaction catalyzed by phosphoenolpyruvate carboxylase.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: I. Regulation of Carbon Metabolism and Succinate as a Fermentation Product.

The onset of anaerobiosis in darkened, N-limited cells of the green alga Selenastrum minutum (Naeg.) Collins elicited the following metabolic responses. There was a rapid decrease in energy charge from 0.85 to a stable lower value of 0.6 accompanied by rapid increases in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also a large increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase, can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase activity. These changes resulted in an approximately twofold increase in the rate of starch breakdown indicating a Pasteur effect. The Pasteur effect was accompanied by accumulation of d-lactate, ethanol and succinate as fermentation end-products, but not malate. Accumulation of succinate was facilitated by reductive carbon metabolism by a partial TCA cycle (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). An initial stoichiometric decline in aspartate and increases in succinate and alanine suggests that aspartate catabolism provides an initial source of carbon for reduction to succinate under anoxic conditions. These observations allow us to develop a model for the regulation of anaerobic carbon metabolism and a model for short-term and long-term strategies for succinate accumulation in a green alga.

Anaerobic Metabolism in the N-Limited Green Alga Selenastrum minutum: II. Assimilation of Ammonium by Anaerobic Cells.

The green alga Selenastrum minutum (Naeg.) Collins is able to assimilate NH(4) (+) in the dark under anaerobic conditions (GC Vanlerberghe, AK Horsey, HG Weger, DH Turpin [1989] Plant Physiol 91: 1551-1557). In the present study, analysis of metabolites following addition of NH(4) (+) to cells acclimated to anaerobic conditions has shown the following. There was a transient decline in adenylate energy charge from 0.6 to 0.4 followed by a recovery back to ~0.6. This was accompanied by a rapid increase in pyruvate/phosphoenolpyruvate and fructose-1,6-bisphosphate/fructose-6-phosphate ratios indicating activation of pyruvate kinase and 6-phosphofructokinase, respectively. There was also an increase in fructose-2,6-bisphosphate, which, since this alga lacks pyrophosphate dependent 6-phosphofructokinase can be inferred to inhibit gluconeogenic fructose-1,6-bisphosphatase. These changes resulted in an increase in the rate of anaerobic starch breakdown. Anaerobic NH(4) (+) assimilation also resulted in a two-fold increase in the rate of production of the major fermentative end-products in this alga, d-lactate and ethanol. There was no change in the rate of accumulation of the fermentative end product succinate but malate accumulated under anoxia during NH(4) (+) assimilation. A rapid increase in Gln and decline in Glu indicates that primary NH(4) (+) assimilation under anoxia was via glutamine synthetase-glutamate synthase. Almost all N assimilated under these conditions was sequestered in alanine. These results allow us to propose a model for the regulation of carbon metabolism during anaerobic NH(4) (+) assimilation.

Relationship between NH(4) Assimilation Rate and in Vivo Phosphoenolpyruvate Carboxylase Activity : Regulation of Anaplerotic Carbon Flow in the Green Alga Selenastrum minutum.

The rate of NH(4) (+) assimilation by N-limited Selenastrum minutum (Naeg.) Collins cells in the dark was set as an independent variable and the relationship between NH(4) (+) assimilation rate and in vivo activity of phosphoenolpyruvate carboxylase (PEPC) was determined. In vivo activity of PEPC was measured by following the incorporation of H(14)CO(-) (3) into acid stable products. A linear relationship of 0.3 moles C fixed via PEPC per mole N assimilated was observed. This value agrees extremely well with the PEPC requirement for the synthesis of the amino acids found in total cellular protein. Determinations of metabolite levels in vivo at different rates of N assimilation indicated that the known metabolite effectors of S. minutum PEPC in vitro (KA Schuller, WC Plaxton, DH Turpin, [1990] Plant Physiol 93: 1303-1311) are important regulators of this enzyme during N assimilation. As PEPC activity increased in response to increasing rates of N assimilation, there was a corresponding decline in the level of PEPC inhibitors (2-oxoglutarate, malate), an increase in the level of PEPC activators (glutamine, dihydroxyacetone phosphate), and an increase in the Gln/Glu ratio. Treatment of N-limited cells with azaserine caused an increase in the Gln/Glu ratio resulting in increased PEPC activity in the absence of N assimilation. We suggest glutamate and glutamine play a key role in regulating the anaplerotic function of PEPC in this C(3) organism.

Regulation of Carbon Partitioning to Respiration during Dark Ammonium Assimilation by the Green Alga Selenastrum minutum.

The assimilation of NH(4) (+) causes a rapid increase in respiration to provided carbon skeletons for amino acid synthesis. In this study we propose a model for the regulation of carbon partitioning from starch to respiration and N assimilation in the green alga Selenastrum minutum. We provide evidence for both a cytosolic and plastidic fructose-1,6-bisphosphatase. The cytosolic form is inhibited by AMP and fructose-1,6-bisphosphate and the plastidic form is inhibited by phosphate. There is only one ATP dependent phosphofructokinase which, based on immunological cross reactivity, has been identified as being localized in the plastid. It is inhibited by phosphoenolpyruvate and activated by phosphate. No pyrophosphate dependent phosphofructokinase was found. The initiation of dark ammonium assimilation resulted in a transient increase in ADP which releases pyruvate kinase from adenylate control. This activation of pyruvate kinase causes a rapid 80% drop in phosphoenolpyruvate and a 2.7-fold increase in pyruvate. The pyruvate kinase mediated decrease in phosphoenolpyruvate correlates with the activation of the ATP dependent phosphofructokinase increasing carbon flow through the upper half of glycolysis. This increased the concentration of triosephosphate and provided substrate for pyruvate kinase. It is suggested that this increase in triosephosphate coupled with the glutamine synthetase mediated decline in glutamate, serves to maintain pyruvate kinase activation once ADP levels recover. The initiation of NH(4) (+) assimilation causes a transient 60% increase in fructose-2,6-bisphosphate. Given the sensitivity of the cytosolic fructose-1,6-bisphosphatase to this regulator, its increase would serve to inhibit cytosolic gluconeogenesis and direct the triosephosphate exported from the plastid down glycolysis to amino acid biosynthesis.

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 NH(4) (+) in the dark under anaerobic conditions. Addition of NH(4) (+) to anaerobic cells results in a threefold increase in tricarboxylic acid cycle (TCAC) CO(2) 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 H(14)CO(3) (-) to anaerobic cells assimilating NH(4) (+) results in the incorporation of radiolabel into the alpha-carboxyl carbon of glutamic acid. Incorporation of radiolabel into glutamic acid is not simply a short-term phenomenon following NH(4) (+) 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 alpha-ketoglutarate for glutamate production. During dark aerobic NH(4) (+) assimilation, no radiolabel appears in fumarate or succinate and only a small amount occurs in malate. During anaerobic NH(4) (+) 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 NH(4) (+) 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.

Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum.

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH(4) (+) addition we found that: (a) N accumulated in glutamine while glutamate and alpha-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH(4) (+) addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH(4) (+) addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.

Significance of Phosphoenolpyruvate Carboxylase during Ammonium Assimilation: Carbon Isotope Discrimination in Photosynthesis and Respiration by the N-Limited Green Alga Selenastrum minutum.

The effect of N-assimilation on the partitioning of carbon fixation between phosphoenolpyruvate carboxylase (PEPcase) and ribulose bisphosphate carboxylase/oxygenase (Rubisco) was determined by measuring stable carbon isotope discrimination during photosynthesis by an N-limited green alga, Selenastrum minutum (Naeg.) Collins. This was facilitated by a two process model accounting for simultaneous CO(2) fixation and respiratory CO(2) release. Discrimination by control cells was consistent with the majority of carbon being fixed by Rubisco. During nitrogen assimilation however, discrimination was greatly reduced indicating an enhanced flux through PEPcase which accounted for upward of 70% of total carbon fixation. This shift toward anaplerotic metabolism supports a large increase in tricarboxylic acid cycle activity primarily between oxaloacetate and alpha-ketoglutarate thereby facilitating the provision of carbon skeletons for amino acid synthesis. This provides an example of a unique set of conditions under which anaplerotic carbon fixation by PEPcase exceeds photosynthetic carbon fixation by Rubisco in a C(3) organism.