Activation of respiration to support dark NO3- and NH4+ assimilation in the green alga Selenastrum minutum

Abstract

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/NAOP 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/NAO ratio during NO3- assimilation correlates with a slow activation of pyruvate kinase and phosphoeno/pyruvate 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.