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Metabolism and Enzymology

Activation of Respiration to Support Dark NO₃^– and NH₄⁺ Assimilation in the Green Alga Selenastrum minutum ¹

Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4

Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO₃^– or NH₄⁺ assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH₄⁺ 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, NO₃^– 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 NO₃^– assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO₃^– assimilation correlates with a slow activation of pyruvate kinase and phosphoenolpyruvate carboxylase. Together, these results indicate that during NH₄⁺ assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO₃^– 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.

¹ Supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). H.C.H. acknowledges support from a NSERC International Fellowship.

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