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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...

This study investigates how the ILR1-like indole acetic acid (IAA) amidohydrolase family of genes has functionally evolved in the monocotyledonous species wheat (Triticum aestivum). An ortholog for the Arabidopsis IAR3 auxin amidohydrolase gene has been isolated from wheat (TaIAR3). The TaIAR3 protein hydrolyzes negligible levels of IAA-Ala and no other IAA amino acid conjugates tested, unlike its ortholog IAR3. Instead, TaIAR3 has low specificity for the ester conjugates IAA-Glc and IAAmyoinositol and high specificity for the conjugates of indole-3-butyric acid (IBA-Ala and IBA-Gly) and indole-3-propionic-acid (IPA-Ala) so far tested. TaIAR3 did not convert the methyl esters of the IBA conjugates with Ala and Gly. IBA and IBA conjugates were detected in wheat seedlings by gas chromatography-mass spectrometry, where the conjugate of IBA with Ala may serve as a natural substrate for this enzyme. Endogenous IPA and IPA conjugates were not detected in the seedlings. Additionally, crude protein extracts of wheat seedlings possess auxin amidohydrolase activity. Temporal expression studies of TaIAR3 indicate that the transcript is initially expressed at day 1 after germination. Expression decreases through days 2, 5, 10, 15, and 20. Spatial expression studies found similar levels of expression throughout all wheat tissues examined.In vascular plants, auxins, primarily indole-3-acetic acid (IAA), regulate gene expression, cell division, and cell elongation and differentiation in plant tissue. Auxins also affect vascularization, phototropism, geotropism, fruit development, flower development, and apical dominance (Davies, 1995). While IAA in low concentrations stimulates growth and development, higher concentrations can be toxic to the plant (Bandurski et al., 1995). Therefore, tight control of IAA concentration is necessary for proper plant development.IAA is stored in conjugated forms that are mostly considered to be inactive. Two main types of conjugated molecules have been studied: the amide-linked IAA forms bound to one or more amino acids and the ester-linked forms primarily bound to a sugar(s). These two types of conjugates appear to be found at varying concentrations in the diverse tissues of angiosperms (Domagalski et al., 1987). On average, 95% of all IAA in a plant is conjugated into these storage forms (Cohen and Bandurski, 1982;Bandurski et al., 1995;Campanella et al., 1996;Walz et al., 2002).There have been a variety of amide conjugates found in the plants studied to date. IAA-Asp has been identified as a natural conjugate in Scots pine (Pinus sylvestris; Andersson and Sandberg, 1982) and, together with IAA-Glu, in cucumber (Sonner and Purves, 1985) and soybean (Cohen, 1982). IAA-Ala has been detected in Picea abies Karst (Ö stin et al., 1992). Additionally, IAA-Ala, IAA-Asp, IAA-Leu, and IAA-Glu have been detected in Arabidopsis L. Heynh (Tam et al., 2000;Kowalczyk and Sandberg, 2001), although recent data suggest that IAA peptides may account for the majority of amide conjugates in this and other plant ...