Seeds of oat, coconut, soybean, sunflower, rice, millet, kidney bean, buckwheat, wheat, and corn and vegetative tissue of oit, pea, and corn were assayed for free indole-3-acetic acid (IAA), esterifled IAA, and peptidyl IAA. Three condusions were drwn: (a) al plat tissues examined contained most of their IAA as derivatives, either esterified or as a peptide; (b) the cereal grins examined contained mainly ester IAA; (c) the legume seeds examined contained mainly peptidyl IAA. Errors in analysis of free and bound IAA are dicsed.Previous studies from this laboratory have described the isolation and characterization of esters of IAA and myo-inositol, and of IAA and myo-inositol glycosides (cf. 19, 28) from Zea mays. The isolation of the 2-0, 4-0, and 6-0 esters of IAA and glucose (7) and esters having 2 or 3 mol of IAA/mol of inositol (9) have also been described. In (3,21) and an abstract of some of these data has appeared (25). This is the first attempt to assay simultaneously for free, ester and peptidyl IAA in a variety of plants.The validity of the isotope dilution method was established for IAA from Zea and Avena by demonstrating identity of the IAA by mass spectrometry and agreement in quantity when estimated by spectral, colorimetric, and gas chromatographic methods (3). Isotope dilution analysis of IAA has been accomplished earlier by Hamilton et al. (14) and by Belli et al. (4 Sunflower seed (Helianthus annuus), millet (Panicum miliaceum), buckwheat (Fagopyrum esculentum), and wheat (Triticum aestivum) were from a local health food store and gave 84, 23, 99, and 98% germination, respectively. Avena and Zea seed were as previously described (3, 21) and peas were from Vaughan's Seed Co., Ovid, Mich. Yeast (Saccharomyces cerevisiae) was a bakers' yeast from Anheuser Busch.Extraction. Vegetative tissue was extracted by homogenization in 70% acetone as previously described (3). The "4C-IAA and unlabeled, protective, indole-3-butyric acid were added immediately after homogenization. Yeast was treated as vegetative tissue except that 1,000 g were suspended in 1,700 ml of acetone to yield a final acetone concentration of 63%. Seeds were ground to a coarse powder in a hammer mill and samples of 300 g extracted for 10 hr in 2 liters of 70% acetone to which the "4C-IAA and indole-3-butyric acid had been added (3). The acetone extract was collected by filtration and the residue resuspended in 2 liters of 70% acetone and reextracted for 12 additional hr. Following filtration, the extracts were combined and reduced in volume (3).Hydrolysis and Extraction of IAA. Free IAA was extracted into ether from the acidified concentrated extract as described (3). Ester IAA was defined as IAA which was liberated by hydrolysis for 1 hr in 1 N NaOH at 22 to 25 C. Following hydrolysis the mixture was acidified and IAA plus hydrolyzed ester IAA were extracted into ether as described (3). Peptidic IAA was defined as IAA liberated during 3 hr of hydrolysis in 7 N NaOH at 100 C. For hydrolysis of peptidic IAA, the 70% acetone ...

An isotope-dilution method has been developed for the assay of free indole-3-acetic acid and ester indole-3-acetic acid as measured by indole-3-acetic acid liberated by mild alkaline hydrolysis. Application of this method to seedlings of Avena sativa and Zea mays indicates the upper limit of free indole-3-acetic acid in Avena to be about 16,ug To our knowledge, the present work is the first quantitative estimation of IAA and IAA liberated by alkaline hydrolysis in A vena and Zea seedlings in which the IAA has been chemically characterized and in which the extraction methods were designed to minimize hydrolysis of IAA complexes. Previous studies provide mass-spectral characterization of the IAA in exudates of Zea coleoptiles (2), but no prior mass spectral characterization of the IAA of Avena has been published. MATERIAIS AND METHODSUV spectra were recorded with a Cary 15 Spectrophotometer. GLC was with an F and M Model 402 equipped with flame ionization detectors, using nitrogen as carrier gas at a flow rate of 60 ml/min. MS3 was with an LKB-9000 analyzer unit coupled to a 1.8 m, 3% SP-2401 column at 210 C, using He as carrier gas at 25 ml/min. The separator was at 250 C, and the ion source was at 290 C. weight, 6.5% dry matter with the dry weight ratios of coleoptile-primary leaf-mesocotyl being 1:0.45:2.3, respectively. For A vena the average shoot weighed 0.05 g fresh weight, 5.7 % dry matter with the above ratios being 1.00: 1.04:0.97.Extraction. Except as indicated, manipulations were at room temperature and with minimum light. Four lots, each of 1 kg of freshly harvested tissue, were extracted by grinding for 2 min with 1.5 liters of acetone in a 1-gallon size Waring Blendor. Ten ml of acetone, containing 1 mg of indole-3-butyric acid as carrier, and about 2 ug of 14C IAA (5 X 10' cpm), were added to one of the homogenates, and all four lots were mixed and extracted in the dark at 22 C for 12 hr. The resultant aqueous acetone extract (about 60% acetone) was collected by filtration, and the residue was re-extracted with an additional 4 liters of 70% acetone for 12 hr. After collection by filtration, the extracts were combined, and the volume was reduced to 500 ml in a flash evaporator (12 mm Hg, bath temperature of 50 C) using 1-octanol to control foaming.Extraction of Free LIA. The 500 ml of concentrate, pH 5.3, was adjusted to pH 2.5 with 12 N H,SO, and extracted five times with 400 ml of ether. The combined ether phases were washed three times with 100 ml of water and the ether was reduced in volume to 400 ml. The IAA was extracted from the ether with 3 X 50 ml of 1 N NaHCO, at 4 C. After washing of the NaHCO, phase with 2 X 50 ml of ether, the NaHCO3 solution was adjusted from pH 8.4 to pH 2.5 with HSO4, and the IAA was extracted back into ether using 3 X 100 ml. The ether phase was washed with 2 X 100 ml of water, dried over anhydrous, granular Na,SO4, and filtered and evaporated in vacuo. The residue was dissolved in 4 ml of CHClI plus 1 ml of CHSOH and stored at -20 C. Average isotope rec...

Syntheses are described for tetra and pentadeutero indole-3-acetic acid (IAA) labeled in positions 4, 5, 6, 7 or 2, 4, 5, 6, 7 of the indole moiety. Polydeuterated IAA is proposed as an internal standard for gas chromatographic-mass spectrometric analysis of IAA by selected ion monitoring. Nanogram amounts of IAA may be assayed by monitoring the base peak of IAA at m/z = 130 (134 for d4-IAA) and the molecular ion of the methyl ester of IAA at 189 (193 for d4-IAA). Deuterium in positions 4,5,6,and 7 and, to only a slightly lesser extent, that in position 2 of IAA is retained during alkali treatment, thus permitting use of these compounds as internal standards for assay of IAA released by alkaline hydrolysis of ester and amide conjugates. The use of polydeutero internal standards separates the standards from the "isotope cluster" caused by the normal abundance of heavy isotopes and also permits use of reduced mass resolution, thus leading to a 10-fold increase in sensitivity.Tetradeutero IAA was used as an internal standard for determining free plus ester IAA in alkaline hydrolysates of Zea mays, and showed exact agreement between estimates based on the molecular ion of the methyl ester and those based upon base peak. Application of the method to measuring free IAA in the upper and lower halves of geotropically stimulated Zea shoots showed 61 ± 4% of the free IAA to be on the lower side.Indolylic compounds, other than tryptophan, occur in plant (cf. 3, 4) and in animal tissue (cf. 23) in ylM amounts. Low tissue concentrations of the indoles and their lability (16,22,24) make assay by GC-SIM-MS4, with deuterated IAA as an internal standard, an attractive procedure. A number of procedures have been described using side chain d2-indoles as standards for assay of IAA and 5-hydroxy-IAA in plants and animals (1, 6, 10, 26). However, plants contain most of their IAA as ester or amide conjugates, conveniently assayed as free IAA, following hydrolysis of the esters by 1 N NaOH and the amide conjugates in 7 N NaOH (4). Alkaline hydrolysis precludes use of d2-IAA with deuterium in the side chain, since these deuterium are lost during base treatment. Deuterium in positions 4, 5, 6, and 7 is not exchanged with hydrogen during alkaline hydrolysis. Even deuterium in the 2 position is only slightly exchanged. ' To whom reprint requests should be directed.4Abbreviations: GC-SIM-MS, gas chromatography-selected ion monitoring-mass spectrometry; mp, melting point; NMR, nuclear magnetic resonance. synthesis for 4,5,6,7-d4-IAA and 2,4,5,6,7-d5-IAA and use of these compounds as internal standards.In addition to alkali stability, polydeuterated compounds provide additional advantages as internal standards for GC-SIM-MS. The ions generated by d4-and d5-IAA during MS are remote from the ions of IAA and, thus, the background caused by the normal abundances of heavy ions at I and 2 mass units above the IAA is eliminated. Further, providing sample purity is adequate, mass resolution may be broadened to ±+ amu (fat peak monitoring) y...

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Gravistimulation induces an asymmetric distribution of free indole-3-acetic acid (IAA) Induction of an asymmetric distribution of the plant growth hormone IAA, by a gravitational stimulus has been well documented. Using a bioassay for IAA, Dolk (4) early showed that stimulus times as short as 5 min induced the asymmetry with