Plants derive a number of important secondary metabolites from the amino acid tryptophan (Trp), including the growth regulator indole-3-acetic acid (IAA) and defense compounds against pathogens and herbivores. In previous work, we found that a dominant overexpression allele of the Arabidopsis (Arabidopsis thaliana) Myb transcription factor ATR1, atr1D, activates expression of a Trp synthesis gene as well as the Trp-metabolizing genes CYP79B2, CYP79B3, and CYP83B1, which encode enzymes implicated in production of IAA and indolic glucosinolate (IG) antiherbivore compounds. Here, we show that ATR1 overexpression confers elevated levels of IAA and IGs. In addition, we show that an atr1 loss-of-function mutation impairs expression of IG synthesis genes and confers reduced IG levels. Furthermore, the atr1-defective mutation suppresses Trp gene dysregulation in a cyp83B1 mutant background. Together, this work implicates ATR1 as a key homeostatic regulator of Trp metabolism and suggests that ATR1 can be manipulated to coordinately control the suite of enzymes that synthesize IGs.In plants, the Trp pathway provides precursors for a variety of important secondary metabolites. For example, indole-3-acetic acid (IAA), a central regulator of cell division and elongation, is derived via several metabolic routes from Trp pathway compounds (Bartel et al., 2001;Ljung et al., 2002). One route of IAA synthesis elucidated in Arabidopsis (Arabidopsis thaliana) involves the conversion of Trp to an indole-3-acetaldoxime (IAOx) intermediate by a pair of functionally redundant cytochrome P450 enzymes, CYP79B2 and CYP79B3 (Zhao et al., 2002; Fig. 1). IAOx is then converted to IAA, likely via indole-3-acetonitrile and/or indole-3-acetaldehyde.In Brassicas, including Arabidopsis, an important class of Trp secondary metabolites is indolic glucosinolate (IG) defense compounds. Upon tissue damage, such as during insect or herbivore attack, IGs and other glucosinolates are metabolized by myrosinases into biologically active nitrile, isothiocyanate, or thiocyanate forms that give Brassicas their distinctive mustard flavor (Wittstock and Halkier, 2002). In the human diet, glucosinolate-derived isothiocyanates act as anticancer compounds by inducing carcinogendetoxifying enzymes (Talalay and Fahey, 2001).In Arabidopsis, IG synthesis involves the CYP79B2/ CYP79B3-catalyzed conversion of Trp to IAOx (Hull et al., 2000;Mikkelsen et al., 2000;Zhao et al., 2002). IAOx is then converted by the cytochrome P450 enzyme CYP83B1 to the next intermediate in the IG pathway, which is proposed to be 1-aci-nitro-2-indolylethane Hansen et al., 2001a). Thus, IAOx lies at a metabolic branch point between the synthesis of IAA and IGs (Fig. 1). Plants that overexpress CYP79B2 from the cauliflower mosaic virus (CaMV) 35S promoter display elevated levels of both IAA and IGs (Zhao et al., 2002). Conversely, Arabidopsis cyp79B2 cyp79B3 double mutants are strongly deficient in IGs and partially deficient in IAA, suggesting that IAOx produced by CYP79B2 and CYP79B3 is the ...

Plant mutants with defects in intermediate enzymes of the tryptophan biosynthetic pathway often display a blue fluorescent phenotype. This phenotype results from the accumulation of the fluorescent tryptophan precursor anthranilate, the bulk of which is found in a glucose-conjugated form. To elucidate factors that control fluorescent tryptophan metabolites, we conducted a genetic screen for suppressors of blue fluorescence in the Arabidopsis trp1-100 mutant, which has a defect in the second enzymatic step of the tryptophan pathway. This screen yielded loss-of-function mutations in the UDP-glucosyltransferase gene UGT74F2. The bacterially expressed UGT74F2 enzyme catalyzed a conjugation reaction, with free anthranilate and UDP-glucose as substrates, that yielded the same fluorescent glucose ester compound as extracted from the trp1-100 mutant. These results indicate that sugar conjugation of anthranilate by UGT74F2 allows its stable accumulation in plant tissues. A highly related Arabidopsis enzyme UGT74F1 could also catalyze this reaction in vitro and could complement the ugt74F2 mutation when overexpressed in vivo. However, the UGT74F1 gene is expressed at a lower level than the UGT74F2 gene. Therefore, even though UGT74F1 and UGT74F2 have redundant conjugating activities toward anthranilate, UGT74F2 is the major source of this activity in the plant. The Arabidopsis genome encodes over 100 predicted UDPglucosyltransferase (UGT) 1 genes (1). These genes are identified by a conserved amino acid motif in the carboxyl-terminal region of the protein sequence that binds the common UDPglucose substrate molecule. UGT enzymes can form either glucose esters or glucosides with a wide range of substrate molecules. These enzymes serve a variety of important biological functions, including converting metabolically active molecules into inactive storage/transport forms or in some cases generating intermediates for the subsequent conversion into other metabolites. To understand the substrate specificity of the Arabidopsis UGTs, ninety of these enzymes have been expressed in bacteria and are being systematically tested for activity against a battery of potential substrate compounds. This strategy has identified specific Arabidopsis UGTs that can use the plant growth regulator indole-3-acetic acid (IAA) (2), various phenylpropanoid compounds (3), and various hydroxybenzoic acid compounds (4) as substrates in vitro. However, understanding the roles of specific UGTs in the plant is only in its early stages.The previous in vitro screening strategy identified the UGT74F1 and UGT74F2 enzymes as being able to use benzoic acid, 2-hydroxybenzoic acid (salicylic acid), and 3-hydroxybenzoic acid as substrates for glucose conjugation (4). Here we report that UGT74F1 and UGT74F2 can also use the tryptophan precursor compound 2-aminobenzoic acid (anthranilate) as a substrate for glucose ester conjugation both in vitro and in vivo. This finding has important implications for understanding the flux of metabolites in the tryptophan pathway.The ...