Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement, mass-spectral identification and incorporation of (-2)H from (-2)H 2O into indole-3-acetic acid, D- and L-tryptophan, indole-3-pyruvate and tryptamine.

Indole-3-acetic acid (IAA) and its putative precursors, L- and D-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)(--1) IAA, 5.7ng · (g FW)(--1) indole-3-pyruvate, 132 ng · (g FW)(--1) tryptamine, 103 ng · (g FW)(--1) D-tryptophan, and 2250 ng · (g FW)(--1) L-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)(--1). When tomato shoots were incubated for 6, 10 and 21 h in 30% (-2)H2O, up to four positions in IAA, L- and D-tryptophan, tryptamine and indole-3-pyruvate became labelled with (-2)H. Compounds became labelled rapidly with 10% of IAA molecules containing (-2)H after 6 h. The percentage of labelled molecules of IAA and L-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of (-2)H in D-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in L-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate (-2)H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of (-2)H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of (-2)H incorporation into both L- and D-tryptophan indicates that IAA is not made from the total shoot pool of either L- or D-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.

The measurement and mass spectral identification of indole-3-pyruvate from tomato shoots.

Endogenous indole-3-pyruvate has been identified by full-scan combined gas chromatography-mass spectrometry and measured using gas chromatography with an electron capture detector. High specific-activity [5-3H]indole-3-pyruvate was synthesized from [5-3H]tryptophan and used as an internal standard. In order to allow purification of the labile indole-3-pyruvate it was stabilised by the formation in the crude extract of its pentafluorobenzyl oxime derivative. This derivative also allowed sensitive detection and measurement of indole-3-pyruvate in the picogram range using a gas chromatograph with an electron capture detector. Endogenous levels were found to be between 8-10 ng/g f.wt. of tomato shoots which is comparable to that of the indole-3-acetic acid pool size, 11 ng/g f.wt., in this tissue.

Measurement of the rates of oxindole-3-acetic acid turnover, and indole-3-acetic acid oxidation in Zea mays seedlings.

Oxindole-3-acetic acid is the principal catabolite of indole-3-acetic acid in Zea mays seedlings. In this paper measurements of the turnover of oxindole-3-acetic acid are presented and used to calculate the rate of indole-3-acetic acid oxidation. [3H]Oxindole-3-acetic acid was applied to the endosperm of Zea mays seedlings and allowed to equilibrate for 24 h before the start of the experiment. The subsequent decrease in its specific activity was used to calculate the turnover rate. The average half-life of oxindole-3-acetic acid in the shoots was found to be 30 h while that in the kernels had an average half-life of 35h. Using previously published values of the pool sizes of oxindole-3-acetic acid in shoots and kernels from seedlings of the same age and variety, and grown under the same conditions, the rate of indole-3-acetic acid oxidation was calculated to be 1.1 pmol plant-1 h-1 in the shoots and 7.1 pmol plant-1 h-1 in the kernels.

Indole-3-acetic acid catabolism in Zea mays seedlings. Metabolic conversion of oxindole-3-acetic acid to 7-hydroxy-2-oxindole-3-acetic acid 7'-O-beta-D-glucopyranoside.

A new metabolite of the plant growth substance indole-3-acetic acid has been extracted from Zea mays seedlings and characterized as the 7'-O-beta-D-glucopyranoside of 7-hydroxy-2-oxindole-3-acetic acid. This compound was the major product formed from [5-3H] 2-oxindole-3-acetic acid, incubated with intact plants or root and coleoptile sections. Identification was by gas chromatography-mass spectrometry of the trimethylsilyl derivative and by analysis of the hydrolysis products. A synthesis is reported for 7-hydroxy-2-oxindole-3-acetic acid. These results and prior work demonstrate the following catabolic route for indole-3-acetic acid in Zea: indole-3-acetic acid----2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid----7-hydroxy-2-oxindole-3-acetic acid glucoside.

Metabolism of [(14)C]indole-3-acetic acid by the cortical and stelar tissues of Zea mays L. roots.

Reverse-phase high-performance liquid chromatography was used to analyse (14)C-labelled metabolites of indole-3-acetic acid (IAA) formed in the cortical and stelar tissues of Zea mays roots. After a 2-h incubation in [(14)C]IAA, stelar segments had metabolised between 1-6% of the methanol-extractable radioactivity compared with 91-92% by the cortical segments. The pattern of metabolites produced by cortical segments was similar to that produced by intact segments bathed in aqueous solutions of [(14)C]IAA. In contrast, when IAA was supplied in agar blocks to stelar tissue protruding from the basal ends of segments, negligible metabolism was evident. On the basis of its retention characteristics both before and after methylation, the major metabolite of [(14)C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.

Oxidation of indole-3-acetic acid and oxindole-3-acetic acid to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7'-O-beta-D-glucopyranoside in Zea mays seedlings.

Radiolabeled oxindole-3-acetic acid was metabolized by roots, shoots, and caryopses of dark grown Zea mays seedlings to 2,3-dihydro-7-hydroxy-2-oxo-1H indole-3-acetic acid-7'-O-beta-D-glycopyranoside with the simpler name of 7-hydroxyoxindole-3-acetic acid-glucoside. This compound was also formed from labeled indole-3-acetic acid supplied to intact seedlings and root segments. The glucoside of 7-hydroxyoxindole-3-acetic acid was also isolated as an endogenous compound in the caryopses and shoots of 4-day-old seedlings. It accumulates to a level of 4.8 nanomoles per plant in the kernel, more than 10 times the amount of oxindole-3-acetic acid. In the shoot it is present at levels comparable to that of oxindole-3-acetic acid and indole-3-acetic acid (62 picomoles per shoot). We conclude that 7-hydroxyoxindole-3-acetic acid-glucoside is a natural metabolite of indole-3-acetic acid in Z. mays seedlings. From the data presented in this paper and in previous work, we propose the following route as the principal catabolic pathway for indole-3-acetic acid in Zea seedlings: Indole-3-acetic acid --> Oxindole-3-acetic acid --> 7-Hydroxyoxindole-3-acetic acid --> 7-Hydroxyoxindole-3-acetic acid-glucoside.