Selection and characterization of α-methyltryptophan-resistant lines of Lemna gibba showing a rapid rate of indole-3-acetic acid turnover

Abstract

Turnover rate is an important aspect of the regulation of plant processes by plant growth substances. To study turnover of indole-3-acetic acid (IAA), two α-methyltryptophan-resistant lines (MTR1 and MTR2) of lemna gibba were generated by nitrosomethyl urea treatment of an inbred line derived from L. gibba G-3. In this report we describe: (a) the development of a selection system using this near isogenic line of L. gibba; (b) techniques for chemical mutation of the lines and selection for α-methyltryptophan resistance; and (c) the partial characterization of the selected lines. MTR lines contained 3-fold higher levels of anthranilate synthase activity. The enzyme in the MTR lines required higher levels of tryptophan for feedback inhibition. MTR lines also contained 8-fold higher levels of tryptophan, 3-fold higher levels of free IAA, and similar levels of total IAA compared to the inbred line. Turnover rates in the inbred and selected lines were calculated, using the first-order rate equation, based on the decrease over time in isotopic enrichment of 13C6-IAA introduced into L. gibba during a 1-h pulse period. Isotope enrichment in IAA was determined by using gas chromatography-mass spectrometry. Both MTR lines had an approximately 10-fold higher rate of IAA turnover than the parent inbred line.