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

Abstract

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 N03-or NH4' induced a transient suppression of photosyn-thetic 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) NH4W addition resulted in 02 consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of N03-or NH4W 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 photosyn-thetic 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 tricar-boxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regen-eration 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 02 evolution, dark respiration, and dark C-fixation are shown to be consistent with this model. Nitrogen is an important regulator of photosynthetic carbon flow in both higher plants and algae (2, 12, 14, 15, 22, 30). In higher plants, NH4+ enrichment increases the flow of newly fixed carbon into TCA2 cycle intermediates and amino acids while decreasing the flux into starch and sucrose (2, 14). Phosphoen-olpyruvate carboxylase and pyruvate kinase have been identified 'Supported by the Advisory Research Committee and Faculty of Graduate Studies of Queen's University and the Natural Sciences and Engineering Research Council of Canada. 2 Abbreviations: TCA cycle, tricarboxylic acid cycle; RuBP, ribulose bisphosphate; GOGAT, glutamine 2-oxoglutarate aminotransferase (EC. 2.6.1.53); GS, glutamate synthase (EC. 6.3.1.2); DIC, dissolved inorganic carbon; g, growth rate; Rubisco, ribulose bisphosphate carboxylase; PEP, phosphoenolpyruvate; TP, triose phosphate. as key enzymes in regulating this metabolic shift (14, 15, 22). The significance of N in regulating algal carbon flow increases under N-limitation (7, 20, 27). Responses of N-limited microal-gae to N enrichment are light and time dependent. In the dark, N enrichment stimulates both 02 consumption and CO2 uptake (19, 24). In the light, N uptake is very rapid (5, 1 1) and ultimately results in an increase in photosynthesis (13, 27). In many cases, however, N enrichment to N-limited microalgae or natural phy-toplankton assemblages results in a temporary suppression of photosynthetic carbon fixation (4, 7, 9, 10, 13, 16, 17, 20, 25-27). Although there are several reports ofN-induced photosynthetic suppression, the phenomenon has yet to be studied in any detail. In this study chemostat cultures were used to produce steady state NO3-limited cells of Selenastrum minutum (Chlorophyta). The effects of nitrogen resupply on photosynthetic carbon fixa-tion, 02 evolution, dark carbon fixation, dark respiration, and RuBP concentration are reported. The observed changes enable development of a hypothesis which explains the processes of N-induced photosynthetic suppression in N-limited microalgae. MATERIALS AND METHODS Chemostat Culture. Selenastrum minutum Naeg. Collins was isolated from Lake Ontario and grown axenically in chemostat culture under N03-limitation at a growth rate of 0.3 d-'. Complete culture conditions were as previously described (8) with the exception that cultures were buffered at pH 8.0 with 50 mm Hepes and grown at a photon flux density of 165 gE.M2.S-' These conditions resulted in steady state cell densities of 0.90 sg Chl-ml'. This concentration was used in all experiments with the exception of those associated with RuBP measurement. Under steady state conditions, the cellular growth rate in a chemostat depends upon the constant flow of a nutrient limited media (in this study, NO3-limited) into the growth vessel. Upon addition, this media is mixed rapidly and homogeneously throughout the culture by magnetic stirring and continuous aeration. As each drop enters the culture an equal volume is forced out of the reactor. Consequently, the culture volume remains constant and at steady state the growth rate of the cells must equal the dilution rate of the culture (28). Consequently, the lower the growth rate, the greater the degree of N-limitation. In this study cells were extremely N-limited, growing at 18% of the maximum growth rate. Nitrogen sufficient cells were obtained by growing cells at maximum growth rate (1.68 d-') in chemostat culture. Photosynthetic Carbon Fixation. Culture samples (0.90 ug Chl-ml', 10 mm DIC) were incubated in the presence ofH'4CO3 (Atomic Energy Commission of Canada, 50 MCi ml-') with or without nitrogen enrichment. Samples were withdrawn at discrete time intervals and placed in 5.0 ml scintillation vials containing 0.5 ml of stop solution (80% aqueous ethanol, 5% HCOOH), evaporated to dryness and '4C determined as previ-273