Carroll, Andrew Douglas; (1993) The compartmentation of nitrogenous assimilates in suspension cultured cells of carrot. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Little is yet known of the role of compartmentation of assimilate in the regulation of higher plant nitrogen metabolism. In order to investigate the importance of such compartmentation techniques were developed which enabled intact plastids, mitochondria and vacuoles to be prepared from protoplasts derived from carrot (Daucus carota) suspension cells. When the amino acid content of each organelle was determined by HPLC (high performance liquid chromatography) the results suggested that certain amino acids were unevenly distributed in the protoplast. Glutamine, valine, phenylalanine and asparagine were primarily vacuolar in distribution while other species, glutamate, Gaba and alanine, were chiefly cytoplasmic. By feeding protoplasts with ammonium chloride containing the stable isotope 15N and preparing organelle fractions at regular intervals thereafter it was possible to monitor the intercompartmental fluxes of freshly synthesised amino acids by HPLC and GC-MS (gas chromatography-mass spectrometry). Feeding with ammonium resulted in a considerable increase in the total amino acid content of protoplasts, chiefly due to increased breakdown of proteins in the vacuole, a response characteristically elicited by the onset of cytosolic acidosis. 15N was found to be incorporated chiefly into three different amino acids, Gaba, glutamine and glutamate, which differed in their spatial distribution: freshly synthesised Gaba and glutamate were largely restricted to the cytosol while glutamine was compartmentalised in the vacuole. Of these three Gaba appeared to be the major sink for freshly assimilated nitrogen. The metabolic responses of protoplasts to feeding with ammonium was studied using in vivo 31P-and 15N-NMR (nuclear magnetic resonance). This confirmed that ammonium uptake resulted in a decline in cytosolic pH, which was subsequently restored to its original value. A concomitant increase and decrease were observed in glutamate decarboxylase activity and in the resulting accumulation of Gaba. It is proposed that cytosolic pH is regulated through a biochemical pH stat centred around the decarboxylation of glutamate. 15N-NMR spectra collected from cells made hypoxic prior to the addition of ammonium, to induce cytosolic acidosis, suggested that maintenance of a vacuolar pool of glutamine is an important way in which plant cells conserve nitrogen and presumably also carbon.

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