Intracellular Concentrations and Metabolism of Carbon Compounds in Tobacco Callus Cultures: Effects of Light and Auxin

Abstract

Callus cultures derived from pith tissue of Nicotiana tabacum were grown on two media either under continuous illumination or in complete darkness. The first medium limited greening ability of callus grown in the light (3 milligrams per liter naphthalene acetic acid, 0.3 milligram per liter 2-isopentenylaminopurine, Murashige and Skoog salts, and 2% sucrose). The second medium encouraged chlorophyll synthesis (greening) though not shoot formation (0.3 milligram per liter naphthalene acetic acid; 0.3 milligrans per liter 2-isopentylaminopurine). To measure intracellular concentrations, calli were grown for 15 days on these standard media containing [U-(14)C]sucrose. The dry weight proportions of the calli (as a fraction of fresh weight) and many metabolite concentrations nearly doubled in light-grown cells compared to dark-grown cells and increased 30 to 40% on low-auxin media relative to high-auxin media. Glutamine concentrations (from 4 to 26 millimolar) were very high, probably due to the NH(3) content of the media. Proline concentrations were 20-fold higher in calli grown on low-auxin media in the light (green cells), possibly a stress response to high osmotic potentials in these cells. To analyze sucrose metabolism, callus cells were allowed to take up 0.2% (weight per volume) [U-(14)C]sucrose for up to 90 minutes. In callus tissues and in pith sections from stems of tobacco plants, sucrose was primarily metabolized through invertase activity, producing equal amounts of labeled glucose and fructose. Respiration of (14)CO(2) followed the labeling patterns of tricarboxylic acid cycle intermediates. Photorespiration activity was low.