Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid

Abstract

We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of L-['5Njtryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled L-tryptophan was extract-able from plant tissue after feeding and showed no measurable isomerization into D-tryptophan. D-['5N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous D-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of L-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that L-tryptophan is a more direct precursor to IAA than the D isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that L-tryptophan also may not be a primary precursor to IAA in plants. Many studies of IAA biosynthesis have shown that TRP4 is the primary precursor of IAA by one of several different pathways (for reviews, see refs. 1, 24). However, studies of in vivo biosynthesis ofIAA in maize seedlings have demonstrated that conversion of TRP to IAA does not occur at significant rates (9, 17, 21). The simple model for TRP involvement in IAA biosynthesis is further complicated by the natural occurrence of the 12 carbon indole, IBA, in some plant tissues (8, ' 3Present address: USDA/ARS Plant Hormone Laboratory, Belts-ville, MD 20705. 4Abbreviations: TRP, tryptophan; IBA, indole-3-butyric acid; PL, parental line; GC-MS-SIM, selected ion monitoring GC-MS; DIP-MS, direct insertion probe-MS; El, electron impact. 1203 23). The metabolic conversion of an indolic precursor to IBA has not been demonstrated; however, the side chain reduction of IBA to IAA has been shown to occur in plants (10, 12). These results present an interesting problem with respect to the hypothesis that TRP is the primary precursor to indoleal-kanoic acids in plants. The in vivo role of TRP and other potential intermediates in pathways that could lead to indole derivatives clearly needs to be reexamined. Recently, D-TRP, rather than the much more abundant L isomer, has been proposed to be a direct biosynthetic precursor of IAA based on evidence of gibberellin enhanced race-mization of L-TRP to D-TRP, which could then be transa-minated to indolepyruvic acid (19, 20). The evidence for the in vivo operation of this pathway is difficult to interpret because correction of the specific activity of the applied labeled compounds, necessitated by the differences in uptake and size of internal pools, was not done. Our studies of IAA biosynthesis in vivo have been facilitated by the use of an IAA overp...

Indole-3-acetic acid metabolism in normal and dwarf micropropagated banana plants (Musa spp. AAA)

“…These results corroborate the suggestion given by Baldi et al (1991) that indole-3-acetic acid is synthesized from Ltryptophan and not from D-tryptophan, as proposed by Law G.R. ZAFFARI et al (1987).…”

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

“…Trp has long been considered to be the immediate precursor to IAA (15), but it also was suggested (2,22) Black and Hamilton (12), who observed that excised bean shoots convert radioactive indole to IAA with Trp as the apparent intermediate.…”

Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover

“…The reason for the higher levels of indole found in the other plant species is unknown. In Lemna, the non-Trp pathway for IAA biosynthesis is probably the predominant pathway during vegetative growth based on the low level of Trp conversion to IAA (Baldi et al, 1991), and, where the non-Trp pathway is active, anthranilate synthase may have a more pronounced effect on IAA biosynthesis than would be seen in plants where the Trp degradation pathway predominated.…”

“…Unlike most other plants, however, Lemna cultures allow rapid isotope labeling and pulse/chase experiments under sterile conditions (Slovin and Tobin, 1982;Baldi et al, 1991), making them well suited for turnover studies. In addition, problems related to wounding encountered with tissue sections can be avoided with the Lemna whole plant system.…”