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...

Ester conjugates of indole-3-acetic 16,18,22,23) following various protocols suggested that a more systematic evaluation of this process was needed. In this report we present data on the kinetics of hydrolysis of reagent IAA-GLU2 at selected pH values and compare the lability of IAA-GLU to that of IAA-MI and several other ester conjugates of IAA. In addition, we report the behavior of a relatively polar conjugate, IAA-GLU, and a relatively apolar conjugate, IAA-PNP, in the 'three phase' system described by Liu and Tillberg (16). These data indicate that problems with inadvertent hydrolysis are apparent even at moderate pH values and in multiphase extraction systems.IAA is present in plant tissues as the free acid and also in the form of covalently linked conjugates (5,7,14,20). Two classes of conjugates, based on their alkaline lability, have been recognized. Ester conjugates (including ester, acyl anhydride, and S-acyl anhydrides) are hydrolyzed easily in basic solutions. Initial studies of auxin extraction techniques (1-3, 12-15, 21) established that IAA conjugates could be hydrolyzed during extraction, either by apparently enzymatic mechanisms or by exposure to alkaline environments. For example, Berger and Avery (2, 3) used treatment at pH 9.6 for 5 min to liberate free IAA from its 'complex' in maize kernels. At the time many of these classic studies were done the identity of the bound forms of IAA were unknown. However, since that time many endogenous auxin conjugates have been isolated and structurally characterized (7,20).The presence ofthese bound forms and their lability dictates that in analysis of the free hormone content of plant tissues

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