Synergistic Effects of Metabolically Related Amino Acids

The synergistic inhibition of the growth of Marchantia polymorpha gemmalings by lysine and threonine and its prevention by methionine has been investigated utilizing '4C-labeled amino acids. Experiments involving the uptake of '4C-lysine or '4C-threonine in the presence or absence of methionine indicated that the synergistic growth effects were not a result of altered amino acid uptake. These data, as well as direct chemical analysis, indicated that growth inhibition was correlated with an inhibition of protein synthesis. Experiments utilizing '4C-aspartic acid revealed that the presence of lysine and threonine resulted in increased 14CO2 production and an accumulation of soluble 14C-aspartic acid and labeled ninhydrin-positive compounds. These metabolic alterations were prevented when methionine was also included in the growth media. A model depicting a sequence of events which involve the interaction of regulatory mechianisnms is suggested to account for the effects of specific amino acids on plant growth. biosynthetic pathway) which is subject to concerted (multivalent) feedback inhibition by these amino acids (8, 26). Thus, inhibition of bacterial growth has been postulated to result from inhibition of the activity of this enzyme which could, in turn, prevent the synthesis of methionine (3, 6, 26). The over-all characteristics of the effects of lysine, threonine, and methionine on the growth of Marchantia gemmalings suggested that a similar mechanism might be applicable (11). Preliminary attempts to assay aspartokinase activity in extracts of Marchantia have so far been unsuccessful. Therefore, '4C-labeled amino acids were utilized to study the effects of lysine and threonine on gemmalings. The results of these experiments suggest that a number of metabolic changes, including a decrease in protein synthesis, accompany inhibition of growth. These changes were not observed in plants grown on media containing lysine, threonine, and methionine. Mechanisms are suggested whereby specific exogenous amino acids could influence the over-all metabolic pattern of a cell and, thus, inhibit growth. MATERIALS AND METHODS It has recently been shown that two natural amino acids, lysine and threonine, synergistically inhibit the growth of gemmalings of the thalloid liverwort Marchantia polymorpha (11). Inhibition of growth is accompanied by loss of normal pigmentation at amino acid concentrations as low as 1 ,UM. These effects are only observed in the presence of both L-lysine and L-threonine, are reversible upon removal of these effectors, and can be specifically prevented by low concentrations of L-methionine or L-homoserine. The inhibitory amino acids and those that prevent synergistic inhibition of growth are metabolically related, in that aspartic acid is considered to serve as a general biosynthetic precursor of lysine, homoserine, methionine, threonine, and isoleucine in bacteria (7), and higher plants (9, 18). The growth of certain bacterial species has also been shown to be inhibited by combinations of lysine and threonine. In these cases, the bacteria possess an aspartokinase (the first enzyme in the highly branched Supported by National Science Foundation Grants GB 6862 and GB 13427. 2Taken in part from a dissertation submitted by Valgene L. Dunham to the Graduate School of Syracuse University in partial fulfillment of the requirements for the Ph.D. degree. I Present address: Department of Horticulture, Purdue University, Lafayette, Indiana 47907. 91 Gemmae were collected from stock plants of Marclantia polymorpha L., transferred to nylon mesh, surface sterilized, and grown as previously described (10). When "4C-labeled amino acids were employed, the nylon mesh containing 200 to 300 gemmae was placed on the surface of 1 (w/v) purified agar (Difco) containing a standard mixture of inorganic nutrients (10) and the appropriate amino acids. After incubation, gemmalings were washed repeatedly, homogenized in 75% ethanol, and fractionated by a slight modification of the procedure described by Roberts et al. (29). This procedure yielded ethanol-, ethanolether-, trichloroacetic acid-, acidified ethanol-, and ether-soluble fractions plus an insoluble residue. Since most of the radioactivity was present in the ethanol-soluble and residue fractions, only these fractions were collected in the experiments involving 4Caspartic acid. The residue fractions were further fractionated by hydrolysis in 6 N HCI for 12 hr at 100 C. The hydrolysate was washed and concentrated by repeated addition of H20 and reduction in volume at 35 C under a partial vacuum. Two dimensional chromatography was employed to identify individual amino acids. Two separate solvent systems were employed in all experiments. System 1 was composed of: 1-butanolacetic acid-water (40:10:50, v/v) in the first dimension, and pyridine-water (65:35, v/v) in the second dimension. System 2 contained 2-butanol-formic acid-water (70:10:20, v/v) in the first dimension, and phenol-ammonium hydroxide-water (80: 0.3:20, v/v) in the second. Control experiments, using mixtures of commercially prepared amino acids, were conducted to identify the positions of specific compounds. A mixture of unlabeled www.plantphysiol.org on October 30, 2017 Published by Downloaded from Copyright © 1971 American Society of Plant Biologists. All rights reserved.