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

Abstract

Addition of N03-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 N03 assimilation. In contrast, the short-term changes in pyridine nucleotides and metabolites during photosynthetic assimilation of NH4' were not consistent with a stimulation of the oxidative pentose phosphate pathway.electron transport has been shown to be insufficient to supply NO3-assimilation by N-limited S. minutum, and a role for respiration to supply some of this reductive potential has been proposed (24).We have now examined the pyridine nucleotides and two metabolites, G6P and 6PG, to evaluate the potential role of OPP in providing reducing power to N assimilation by S. minutum in the light. Our results suggest that under conditions where the demand for reductive power exceeds that of photosynthetic electron transport, the OPP may be activated in the light to contribute NADPH. MATERIALS AND METHODSUnicellular algae grown under N limitation respire starch during the assimilation of added NH4' and NO3-in both the dark and the light (1,15,17,24). 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).In light, respiratory CO2 release is higher during NO3-assimilation than during NH4' assimilation (24). Photosynthetic electron transport provides reductive energy for N03-assimilation (11, 12, 18) Cultures of the green alga Selenastrum minutum (Naeg.) Collins (UTEX 2459) were maintained under N limitation as previously described (22). Experiments were performed and analyzed as detailed by Vanlerberghe et al. (23) with the following modifications. Cells were concentrated to 5 jig Chl. mL-1 and were incubated in continuous light sufficient to saturate photosynthesis (approximately 400 ,umol quanta m-2 s-at the face of the sampling cuvette) until injection into the appropriate kill solution. After Speed-Vac concentration, samples were brought to 500 AL with distilled H20.Samples for G6P and 6PG were killed and extracted as previously described (21). All samples were stored in liquid N2. RESULTS AND DISCUSSIONThe ...

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). I. INTRACELLULAR DMSP CONCENTRATIONS

“…Iron is used in the assimilation of NO 3 Ϫ , as it is a structural element of nitrate and nitrite reductase-which catalyze the reduction of NO 3 Ϫ into NO 2 Ϫ and NO 2 Ϫ into NH 4 ϩ , respectively-and of ferredoxin-which provides part of the necessary reducing power. In the chloroplasts, photosynthetic CO 2 fixation and nitrite reduction compete for electrons derived from photosynthesis, but N assimilation can also make use of additional reducing power supplied by respiratory carbon metabolism (Turpin 1991, Huppe et al 1992, Huppe and Turpin 1994. Respiration is also essential for the assimilation of NO 3 Ϫ as provider of carbon skeletons for amino acids synthesis via the tricarboxylic acid (TCA) cycle.…”

Subcellular and tissue localization of NAD kinases from Arabidopsis: compartmentalization of de novo NADP biosynthesis

“…Other significant cytosolic NADPH demands in roots include iron and nitrate reduction mediated through ferredoxin NADP ? -oxidoreductase which is fueled by the OPPP (Huppe et al 1992;Connolly et al 2003) and perhaps also by NADP-malic enzymes (Wheeler et al 2005). …”

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)

“…A11 data were calculated from averaging rates determined in three separate time-course experiments, and the SE on any time point was less than 5% in a11 studies. The determination of pyridine nucleotide changes was as described by Huppe et al (1992), except that cells were concentrated to approximately 10 pg Chl mL-' and the sample size for cycling was optimized for each type of nucleotide determined. O2 evolution was determined with an O2 electrode (Hansatech, King's Lynn, UK).…”

“…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.…”

“…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).…”