Effects of Phosphorus Limitation on Respiratory Metabolism in the Green Alga Selenastrum minutum.

The effects of phosphorus nutrition on several physiological and biochemical parameters of the green alga, Selenastrum minutum, have been examined. Algal cells were cultured in chemostats under conditions of either Pi limitation or nutrient sufficiency. Pi limitation resulted in: (a) a 5-fold lower rate of respiration, (b) a 3-fold decline in rates of photosynthetic carbon dioxide fixation and oxygen evolution, (c) a 3-fold higher rate of dark carbon dioxide fixation, (d) significant increases in activities of phosphoenolpyruvate (PEP) carboxylase and PEP phosphatase (128% and 158% of nutrient sufficient activities, respectively), (e) significant reductions in activities of nonphosphorylating NADP-glyceraldehyde-3-phosphate dehydrogenase and NAD malic enzyme, and (f) no change in levels of ATP:fructose-6-phosphate 1-phosphotransferase, phosphorylating NAD-glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and pyruvate kinase. The intracellular concentrations of Pi, ATP, AMP, soluble protein, and chlorophyll were also significantly reduced in response to Pi limitation. As well, the level of ADP was about 11-fold lower in the Pi-limited cells as compared to the nutrient sufficient controls. It was predicted that because of this low level of ADP, pyruvate kinase catalyzed conversion of PEP to pyruvate may be restricted in Pi-limited cells. During Pi limitation, PEP carboxylase and PEP phosphatase may function to "bypass" the ADP dependent pyruvate kinase, as well as to recycle Pi for its reassimilation into cellular metabolism.

The Relationship between Ribulose Bisphosphate Concentration, Dissolved Inorganic Carbon (DIC) Transport and DIC-Limited Photosynthesis in the Cyanobacterium Synechococcus leopoliensis Grown at Different Concentrations of Inorganic Carbon.

To examine the factors which limit photosynthesis and their role in photosynthetic adaptation to growth at low dissolved inorganic carbon (DIC), Synechococcus leopoliensis was grown at three concentrations (as signified by brackets) of DIC, high (1000-1800 micromolar), intermediate (200-300 micromolar), and low (10-20 micromolar). In all cell types photosynthesis varied from being ribulose bisphosphate (RuBP)-saturated at low external [DIC] to RuBP-limited at high external [DIC]. The maximum rate of photosynthesis (P(max)) was achieved when the internal concentration of RuBP fell below the active site density of RuBP carboxylase/oxygenase (Rubisco). At rates of photosynthesis below P(max), photosynthetic capacity was limited by the ability of the cell to transport inorganic carbon and to supply CO(2) to Rubisco. Adaptation to low DIC was reflected by a decrease in the [DIC] required to half-saturate photosynthesis. Simultaneous mass-spectrometric measurement of rates of photosynthesis and DIC transport showed that the initial slope of the photosynthesis versus [DIC] curve is identical to the initial slope of the DIC transport versus [DIC] curve. This provided evidence that the enhanced capacity for DIC transport which occurs upon adaptation to low [DIC] was responsible for the increase in the initial slope of the photosynthesis versus [DIC] curve and therefore the decrease in the half saturation constant of photosynthesis with respect to DIC. Levels of RuBP and in vitro Rubisco activity varied only slightly between high and intermediate [DIC] grown cells but fell significantly (65-70%) in low [DIC] grown cells. Maximum rates of photosynthesis followed a similar pattern with P(max) only slightly lower in intermediate [DIC] grown cells than in high [DIC] grown cells, but much lower in low [DIC] grown cells. The changing response of photosynthesis to [DIC] during adaptation to low DIC, may be explained by the interaction between DIC-transport limited and [RuBP]-limited photosynthesis.

RuBP Limitation of Photosynthetic Carbon Fixation during NH(3) Assimilation : Interactions between Photosynthesis, Respiration, and Ammonium Assimilation in N-Limited Green Algae.

The effects of ammonium assimilation on photosynthetic carbon fixation and O(2) exchange were examined in two species of N-limited green algae, Chlorella pyrenoidosa and Selenastrum minutum. Under light-saturating conditions, ammonium assimilation resulted in a suppression of photosynthetic carbon fixation by S. minutum but not by C. pyrenoidosa. These different responses are due to different relationships between cellular ribulose bisphosphate (RuBP) concentration and the RuBP binding site density of ribulose bisphosphate carboxylase/oxygenase (Rubisco). In both species, ammonium assimilation resulted in a decrease in RuBP concentration. In S. minutum the concentration fell below the RuBP binding site density of Rubisco, indicating RuBP limitation of carboxylation. In contrast, RuBP concentration remained above the binding site density in C. pyrenoidosa. Compromising RuBP regeneration in C. pyrenoidosa with low light resulted in an ammonium-induced decrease in RuBP concentration below the RuBP binding site density of Rubisco. This resulted in a decrease in photosynthetic carbon fixation. In both species, ammonium assimilation resulted in a larger decrease in net O(2) evolution than in carbon fixation. Mass spectrometric analysis shows this to be a result of an increase in the rate of mitochondrial respiration in the light.

Ammonium Assimilation Requires Mitochondrial Respiration in the Light : A Study with the Green Alga Selenastrum minutum.

Mass spectrometric analysis of O(2) and CO(2) exchange in the green alga Selenastrum minutum (Naeg. Collins) provides evidence for the occurrence of mitochondrial respiration in light. Stimulation of amino acid synthesis by the addition of NH(4)Cl resulted in nearly a 250% increase in the rate of TCA cycle CO(2) efflux in both light and dark. Ammonium addition caused a similar increase in cyanide sensitive O(2) consumption in both light and dark. Anaerobiosis inhibited the CO(2) release caused by NH(4)Cl. These results indicated that the cytochrome pathway of the mitochondrial electron transport chain was operative and responsible for the oxidation of a large portion of the NADH generated during the ammonium induced increase in TCA cycle activity. In the presence of DCMU, ammonium addition also stimulated net O(2) consumption in the light. This implied that the Mehler reaction did not play a significant role in O(2) consumption under our conditions. These results show that both the TCA cycle and the mitochondrial electron transport chain are capable of operation in the light and that an important role of mitochondrial respiration in photosynthesizing cells is the provision of carbon skeletons for biosynthetic reactions.

The Path of Carbon Flow during NO(3)-Induced Photosynthetic Suppression in N-Limited Selenastrum minutum.

Nitrate addition to nitrate-limited cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) resulted in a 70% suppression of photosynthetic carbon fixation. In (14)CO(2) pulse/chase experiments nitrate resupply increased radiolabel incorporation into amino and organic acids and decreased radiolabel incorporation into insoluble material. Nitrate resupply increased the concentration of phosphoenolpyruvate and increased the radiolabeling of phosphoenolpyruvate, pyruvate and tricarboxylic acid cycle intermediates, notably citrate, fumarate, and malate. Furthermore, nitrate also increased the pool sizes and radiolabeling of most amino acids, with alanine, aspartate, glutamate, and glutamine showing the largest changes. Nitrate resupply increased the proportion of radiolabel in the C-4 position of malate and increased the ratios of radiolabel in aspartate to phosphoenolpyruvate and in pyruvate to phosphoenolpyruvate, indicative of increased phosphoenolpyruvate carboxylase and pyruvate kinase activities. Analysis of these data showed that the rate of carbon flow through glutamate (10.6 mumoles glutamate per milligram chlorophyll per hour) and the rate of net glutamate production (7.9 mumoles glutamate per milligram chlorophyll per hour) were both greater than the maximum rate of carbon export from the Calvin cycle which could be maintained during steady state photosynthesis. These results are consistent with the hypothesis that nitrogen resupply to nitrogen-limited microalgae results in a transient suppression of photosynthetic carbon fixation due, in part, to the severity of competition for carbon skeletons between the Calvin cycle and nitrogen assimilation (IR Elrifi, DH Turpin 1986 Plant Physiol 81: 273-279).

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum: II. Effects of NO(3) and NH(4) Addition to CO(2) Efflux in the Light.

The effects of nitrate and ammonium addition on net and gross photosynthesis, CO(2) efflux and the dissolved inorganic carbon compensation point of nitrogen-limited Selenastrum minutum Naeg. Collins (Chlorophyta) were studied. Cultures pulsed with nitrate or ammonium exhibited a marked decrease in both net and gross photosynthetic carbon fixation. During this period of suppression the specific activity of exogenous dissolved inorganic carbon decreased rapidly in comparison to control cells indicating an increase in the rate of CO(2) efflux in the light. The nitrate and ammmonium induced rates of CO(2) efflux were 31.0 and 33.8 micromoles CO(2) per milligram chlorophyll per hour, respectively, and represented 49 and 48% of the rate of gross photosynthesis. Nitrate addition to cells at dissolved inorganic carbon compensation point caused an increase in compensation point while ammonium had no effect. In the presence of the tricarboxylic acid cycle inhibitor fluoroacetate, the nitrate-induced change in compensation point was greatly reduced suggesting the source of this CO(2) was the tricarboxylic acid cycle. These results are consistent with the mechanism of N-induced photosynthetic suppression outlined by Elrifi and Turpin (1986 Plant Physiol 81: 273-279).

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum.

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO(3) (-) or NH(4) (+) induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH(4) (+) addition resulted in O(2) consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO(3) (-) or NH(4) (+) resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O(2) evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.

Effect of N Source on the Steady State Growth and N Assimilation of P-limited Anabaena flos-aquae.

Phosphate-limited chemostat cultures were used to study cell growth and N assimilation in Anabaena flos-aquae under various N sources to determine the relative energetic costs associated with the assimilation of NH(3), NO(3) (-), or N(2). Expressed as a function of relative growth rate, steady state cellular P contents and PO(4) assimilation rates did not vary with N-source. However, N-source did alter the maximal PO(4)-limited growth rate achieved by the cultures: the NO(3) (-) and N(2) cultures attained only 97 and 80%, respectively, of the maximal growth rate of the NH(3) grown cells. Cellular biomass and C contents did not vary with growth rate, but changed with N source. The NO(3) (-)-grown cells were the smallest (627 +/- 34 micromoles C . 10(-9) cells), while NH(3)-grown cells were largest (900 +/- 44 micromoles C . 10(-9) cells) and N(2)-fixing cells were intermediate (726 +/- 48 micromoles C . 10(-9) cells) in size. In the NO(3) (-)-and N(2)-grown cultures, N content per cell was only 57 and 63%, respectively, of that in the NH(3)-grown cells. Heterocysts were absent in NH(3)-grown cultures but were present in both the N(2) and NO(3) (-) cultures. In the NO(3) (-)-grown cultures C(2)H(2) reduction was detected only at high growth rates, where it was estimated to account for a maximum of 6% of the N assimilated. In the N(2)-fixing cultures the acetylene:N(2) ratio varied from 3.4:1 at lower growth rates to 3.0:1 at growth rates approaching maximal.Compared with NH(3), the assimilation of NO(3) (-) and N(2) resulted either in a decrease in cellular C (NO(3) (-) and N(2) cultures) or in a lower maximal growth rate (N(2) culture only). The observed changes in cell C content were used to calculate the net cost (in electron pair equivalents) associated with growth on NO(3) (-) or N(2) compared with NH(3).

Modeling the C Economy of Anabaena flos-aquae: Estimates of Establishment, Maintenance, and Active Costs Associated with Growth on NH(3), NO(3), and N(2).

Steady state cultures of Anabaena flos-aquae were established over a wide range of phosphate-limited growth rates while N was supplied as either NH(3), NO(3) (-), or N(2) gas. At growth rates greater than 0.03 per hour, rates of gross and net carbon fixation were similar on all N sources. However, at lower growth rates (<0.03 per hour) in the NO(3) (-) and N(2) cultures, gross photosynthesis greatly exceeded net photosynthesis. The increase in photosynthetic O(2) evolution with growth rate was greatest when N requirements were met by NO(3) (-) and least when met by NH(3). These results were combined with previously reported measurements of cellular chemical composition, N assimilation, and acetylene reduction (Layzell, Turpin, Elrifi 1985 Plant Physiol 78: 739-745) to construct empirical models of carbon and energy flow for cultures grown at 30, 60, and 100% of their maximal growth rate on all N sources. The models suggested that over this growth range, 89 to 100% of photodriven electrons were allocated to biomass production in the NH(3) cells, whereas only 49 to 74% and 54 to 90% were partitioned to biomass in the NO(3) (-)-and N(2)-grown cells, respectively. The models were used to estimate the relative contribution of active, maintenance, and establishment costs associated with NO(3) (-) and N(2) assimilation over the entire range of growth rates. The models showed that the relative contribution of the component costs of N assimilation were growth rate dependent. At higher growth rates, the major costs for NO(3) (-) assimilation were the active costs, while in N(2)-fixing cultures the major energetic requirements were those associated with heterocyst establishment and maintenance. It was concluded that compared with NO(3) (-) assimilation, N(2) fixation was energetically unfavorable due to the costs of heterocyst establishment and maintenance, rather than the active costs of N(2) assimilation.