Activation of Respiration to Support Dark NO₃⁻ and NH₄⁺ Assimilation in the Green Alga Selenastrum minutum

Abstract

Glu, Gln), thus activating respiration to provide carbon for biosynthesis.Less is understood concerning the response of respiration during NO3-assimilation. NO3-must first be reduced to NH4+ via NR and nitrite reductase; in the dark this reduction requires that respiration generate eight electron pairs. Only then is there a demand for ATP and carbon skeletons to assimilate the NH4+ produced from NO3-into amino acids. This paper examines the activation of respiratory carbon flow to support either NO3-or NH4' assimilation. Changes in pyridine nucleotides and the time frame of PK and PEPC activation indicate that the initial demand of NO3-assimilation is reductant. Only subsequently is TCA cycle carbon flow activated to provide carbon skeletons for amino acid synthesis. MATERIALS AND METHODS Organism, Culture, and Experimental ConditionsThe green alga Selenastrum minutum (Naeg.) Collins (UTEX 2459) was grown autotrophically in N-limited chemostats as previously described (23). Experiments were done with cells in the dark as previously described (25), except cells were kept aerobic by continuous bubbling with air. Cells were concentrated to approximately 20 ,ug Chl mL-' for measurements of pyridine nucleotides and to approximately 40 yg Chl-mL-' for other metabolites. NH4Cl or NaNO3 was added to a final concentration of 2 to 5 mm as indicated.The assimilation of inorganic N into amino acids, protein, and nucleic acids requires ATP, reductant power, and carbon skeletons. In the dark, N-limited cells of the green alga Selenastrum minutum must respire endogenous starch reserves to meet these demands (20). Recently, we developed a model for the activation of carbon flow for respiration during the assimilation of NH4+ in S. minutum (21). When N is supplied as NH4', consumption of ATP and carbon skeletons by GS/ GOGAT3 causes changes in several key metabolites (ADP,

Evidence for Activation of the Oxidative Pentose Phosphate Pathway during Photosynthetic Assimilation of NO3− but Not NH4+ by a Green Alga

“…The physiological and biochemical responses to N resupply have been extensively characterized using N-limited Selenastrum minutum (19,20, and references within). Recently, we reported the changes in the pyridine nucleotides and key metabolites in N-limited S. minutum upon N resupply in the dark, which indicated that during NH4' assimilation, starch is initially respired to provide carbon skeletons and energy for synthesis of amino acids, whereas the initial requirement of NO3-assimilation is electrons to reduce NO3-to NH4' (23). Provision of electrons in the dark was facilitated by the production of NADPH via activation of carbon flow through the OPP3 pathway (23), a pathway that exists in both a chloroplastic and cytosolic form (10, 16).…”

Nitrogen assimilation following NH4+ pulses in the red alga Gracilariopsis lemaneiformis:effect on C metabolism

“…Transient internal NH,' or amino acid accumulation, if any, will occur within a period of less than 2 h. The accumulation of these metabolites has been observed during N assimilation (Dortch 1982, Fujita et al 1988. In microalgae, the transient increase of the amino acid pool, particularly glutanline (Flynn et al 1989), occurs within a matter of minutes , Vanlerberghe et al 1992. The high capacity of microalgae to incorporate and assimilate an intermittent pulse of NH,' allows them to maintain balanced growth in a patchy environment, because the importance of an enhanced N uptake depends on the coupling between the uptake and the incorporation of N into new cell material (Collos 1986).…”

Coordination of Chloroplastic Metabolism in N-Limited Chlamydomonas reinhardtii by Redox Modulation (II. Redox Modulation Activates the Oxidative Pentose Phosphate Pathway during Photosynthetic Nitrate Assimilation)

“…Therefore, we can use this cell wall-less alga to investigate the response of enzyme activity during the activation of N assimilation. Recent work in our laboratory on the metabolite changes in S. minutum at the onset of N assimilation in the light and dark has provided strong evidence for an increased carbon flux through G6PDH (Vanlerberghe et al, 1991;Huppe et al, 1992), the key regulatory enzyme of the OPP pathway. The increased activity of this enzyme should increase the rate at which carbon is respired via the OPP pathway and implicates the OPP pathway in providing some reductive energy during both photosynthetic and heterotrophic NO3-assimilation.…”

“…Cells were incubated in darkness or saturating light (400 pE m-' s-') for a11 experiments in sampling cuvettes as detailed by Vanlerberghe et al (1991). Enzyme activation analysis was as described by Farr et al (1994).…”

“…In S. minutum, the demand for carbon skeletons to synthesize amino acids at the onset of the assimilation of either NH4+ or NOs-stimulates respiration in the light and the dark (Weger and Turpin, 1989). The additional redox potential required to assimilate NOs-appears to be supplied in part by carbon respiration via the OPP pathway (Vanlerberghe et al, 1991;Huppe et al, 1992).…”