Parallel determination of enzyme activities and in vivo fluxes in Brassica napus embryos grown on organic or inorganic nitrogen source

• Published in: Phytochemistry (Oxford) 2007-08, Vol.68 (16), p.2232-2242
• Main Authors: Junker, Björn H., Lonien, Joachim, Heady, Lindsey E., Rogers, Alistair, Schwender, Jörg
• Format: Article
• Language: English
• Subjects: alanine; Alanine - metabolism; Amino Acids - analysis; Amino Acids - metabolism; ammonium nitrate; biochemical pathways; Brassica napus; Brassica napus - embryology; Brassica napus - enzymology; Central metabolism; Citric Acid Cycle; Culture Techniques; embryo (plant); enzyme activity; Enzyme activity profiling; Fatty Acids - analysis; Fatty Acids - metabolism; Glucose - analysis; Glucose - metabolism; glutamine; Glutamine - metabolism; inorganic compounds; Metabolic flux analysis; Models, Biological; Nitrates - metabolism; nitrogen; Nitrogen - metabolism; organic compounds; Seeds - enzymology; Seeds - growth & development; tricarboxylic acid cycle
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2007.03.032

Brassica napus embryos were grown in culture with either glutamine and alanine or ammonium nitrate as the sole nitrogen source. Dependent on the nitrogen source, in vivo metabolic fluxes around the TCA cycle changed distinctly. The changes observed in enzyme activity were not consistent with the changes in metabolic flux. It is suggested that the observed flux adjustments are driven by mass balances rather than transcriptional regulation. After the completion of the genomic sequencing of model organisms, numerous post-genomic studies, integrating transcriptome and metabolome data, are aimed at developing a more complete understanding of cell physiology. Here, we measure in vivo metabolic fluxes by steady state labeling, and in parallel, the activity of enzymes in central metabolism in cultured developing embryos of Brassica napus. Embryos were grown on either the amino acids glutamine and alanine as an organic nitrogen source, or on ammonium nitrate as an inorganic nitrogen source. The type of nitrogen made available to developing embryos caused substantial differences in fluxes associated with the tricarboxylic acid cycle, including flux reversion. The changes observed in enzyme activity were not consistent with our estimates of metabolic flux. Furthermore, most extractable enzyme activities are in large surplus relative to the requirements for the observed in vivo fluxes. The results demonstrate that in this model system the metabolic response of central metabolism to changes in environmental conditions can be achieved largely without regulatory reprogramming of the enzyme machinery.