Compartmentation of amino acid biosynthesis in Lemna minor L.

Dennis Gene Brunk, Purdue University

Abstract

Metabolic inhibitors and stable isotopes of carbon and nitrogen were used to investigate the fluxes and compartmentation of amino acid metabolism in Lemna minor L. The transaminase inhibitor, aminooxyacetate, causes metabolic perturbations which appear consistent with the operation of the photorespiratory nitrogen cycle, and release of individual amino acids from protein at rates of 20 to 40 nmol/h.gfw. Nitrogen-15 tracer studies indicate that the chloroplast-localized glutamine synthetase/glutamate synthase cycle is the primary pathway of ammonia assimilation. The labeling of glutamine suggests a secondary site of glutamine synthesis in the cytosol. Asparagine appears to be derived from heavily labeled chloroplastic pools of glutamine-amide and aspartate. Determinations of the in vivo rates of serine and glycine synthesis suggest that glycine is not the sole precursor of serine. When Lemna minor is supplied with high concentrations (2000 ppm) of $\sp{13}$CO$\sb2$ under nonphotorespiratory conditions, the $\sp{13}$C labeling kinetics of glycine and serine are consistent with a pathway of serine synthesis from 3-phosphoglycerate, not glycine. Respiration rates in Lemna minor were determined to be 7 to 8 $\mu$mol CO$\sb2$ released/h.gfw in the light (one seventh of the CO$\sb2$ uptake rate in normal air; 340 ppm CO$\sb2$, 21% O$\sb2$), and 15 to 16 $\mu$mol CO$\sb2$ released/h.gfw in the dark. Ammonium uptake rates in the light range from 4 to 6 $\mu$mol/h.gfw. Changes in ammonium uptake rate associated with light/dark transitions are shown to be independent of modulation of glutamine synthetase by reversible deactivation. In a pulse-chase labeling experiment with $\sp{15}$NH$\sb4\sp+$ and $\sp{13}$CO$\sb2$ conducted under near steady-state conditions which should favor photorespiration (340 ppm CO$\sb2$; 21% O$\sb2$), the rate of glycine synthesis was determined to be 178 nmol/h.gfw, whereas that of serine was 3671 nmol/h.gfw. This suggests that even under photorespiratory conditions, serine must be largely synthesized from 3-phosphoglycerate rather than from glycine. Quantitative flux analysis of the labeling of glutamine and glutamine confirms the importance of the glutamine synthetase/glutamate synthase cycle (and associated recycling of 2-oxoglutarate) in ammonia assimilation, but provides little evidence for a massive flux of NH$\sb3$ via this pathway due to photorespiratory nitrogen cycling. This flux can not exceed 2.9 $\mu$mol/h.gfw.

Degree

Ph.D.

Advisors

Rhodes, Purdue University.

Subject Area

Botany

COinS