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 ...