Short-Term Metabolite Changes during Transient Ammonium Assimilation by the N-Limited Green Alga Selenastrum minutum

In this study, we measured the total pool sizes of key cellular metabolites from nitrogen-limited cells of Selenastrum minutum before and during ammonium assimilation in the light. This was carried out to identify the sites at which N assimilation is acting to regulate carbon metabolism. Over 120 seconds following NH(4) (+) addition we found that: (a) N accumulated in glutamine while glutamate and alpha-ketoglutarate levels fell; (b) ATP levels declined within 5 seconds and recovered within 30 seconds of NH(4) (+) addition; (c) ratios of pyruvate/phosphoenolpyruvate, malate/phosphoenolpyruvate, Glc-1-P/Glc-6-P and Fru-1,6-bisphosphate/Fru-6-P increased; and (d) as previously seen, photosynthetic carbon fixation was inhibited. Further, we monitored starch degradation during N assimilation over a longer time course and found that starch breakdown occurred at a rate of about 110 micromoles glucose per milligram chlorophyll per hour. The results are consistent with N assimilation occurring through glutamine synthetase/glutamate synthase at the expense of carbon previously stored as starch. They also indicate that regulation of several enzymes is involved in the shift in metabolism from photosynthetic carbon assimilation to carbohydrate oxidation during N assimilation. It seems likely that pyruvate kinase, phosphoenolpyruvate carboxylase, and starch degradation are all activated, whereas key Calvin cycle enzyme(s) are inactivated within seconds of NH(4) (+) addition to N-limited S. minutum cells. The rapid changes in glutamate and triose phosphate, recently shown to be regulators of cytosolic pyruvate kinase, are consistent with them contributing to the short-term activation of this enzyme.