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.     

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-β-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-β-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.     

Tryptophan-Independent Indole-3-Acetic Acid Synthesis: Critical Evaluation of the Evidence

Evidence for Trp-independent IAA synthesis is critically reevaluated in the light of tryptophan synthase proteome data, local IAA synthesis and Trp, indole-3-pyruvate, and IAA turnover.Trp-independent synthesis of indole-3-acetic acid (IAA) was proposed back in the early 1990s based on observations from Trp auxotrophs in maize (Zea mays; Wright et al., 1991) and Arabidopsis (Arabidopsis thaliana; Normanly et al., 1993). Recently, Wang et al. (2015) published new data suggesting that a cytosolic indole synthase (INS) may catalyze the first step separating the Trp-dependent and Trp-independent pathways in Arabidopsis. If this is the case, it would be a major breakthrough; however, in this article, I critically evaluate both recent and older evidence for the Trp-independent route and suggest that the INS is more likely to participate in Trp-dependent IAA production.The original work supporting Trp-independent IAA production was carried out prior to the availability of genome/proteome data and before the discovery that the final step of Trp-dependent IAA synthesis is carried out by a large number of YUCCA homologs operating in a highly localized manner (Zhao, 2008). I argue that experimental data supporting the Trp-independent route needs to be reconsidered in light of complete proteome data. Further, the evidence from feeding labeled compounds should be critically evaluated in light of recent data on the highly localized nature of IAA synthesis as well as older quantitative data on Trp, indole-3-pyruvic acid (IPA), and IAA turnover from my own laboratory (Cooney and Nonhebel, 1991). I conclude that evidence for the Trp-independent route is at best equivocal, and that it is not a conserved source of IAA in angiosperms.Figure 1 shows the major Trp-dependent route for IAA production whereby Trp, produced by the concerted action of Trp synthase α- and β-subunits, is converted to IAA in a further two steps catalyzed by Trp aminotransferase and the flavin monooxygenases commonly known as YUCCA (Mashiguchi et al., 2011; Won et al., 2011). This is compared with the Trp-independent route in which IAA may be produced from free indole by an unknown route (Ouyang et al., 2000; Wang et al., 2015).Open in a separate windowFigure 1.Outline of the major pathway for Trp-dependent IAA synthesis and the proposed Trp-independent route. The role proposed for Trp synthase beta (TSB) homologs discussed in the present paper is also shown. For clarity, reactions are simplified to show only the major compounds relevant to IAA synthesis.The Trp-independent route was originally based on data from Trp auxotrophs that have mutations in genes encoding either the α- or β-subunits of Trp synthase. The α-subunit catalyzes the removal of the side chain from indole-3-glycerol phosphate, passing the indole product directly to the β-subunit where the Trp side chain is created from a Ser substrate (Pan et al., 1997). In plants, this is a chloroplast-localized enzyme. Elevated levels of IAA have been reported in Trp auxotrophs of both maize and Arabidopsis. However, the trp3-1 and trp2-1 mutants of Arabidopsis, deficient in the α- and β-subunits, respectively, only showed an increase in total IAA measured following conjugate hydrolysis. No difference in free IAA levels was found (Normanly et al., 1993). The orange pericarp (orp) maize mutant was reported to have 50 times more IAA than the wild type (Wright et al., 1991). However, this was also total IAA; no data on free IAA were published. Work by Müller and Weiler (2000) indicated that IAA measured following conjugate hydrolysis could have originated via the degradation of indole-3-glycerol phosphate that accumulates in trp3-1 mutants. Further doubt regarding the accuracy of IAA measurements following conjugate hydrolysis has recently been published. Yu et al. (2015) have shown that conjugate hydrolysis treatment substantially overestimates the actual conjugated IAA due to degradation of glucobrassicin and proteins. In addition, neither report (Wright et al., 1991; Normanly et al., 1993) described a high auxin phenotype for the Trp auxotrophs. This contrasts with the superroot1 (sur1) and sur2 mutants, where the accumulation of indole intermediates resulted in a high level of free IAA as well as a high auxin phenotype (Boerjan et al., 1995; Delarue et al., 1998). It is therefore doubtful that Trp auxotrophs actually accumulate more IAA than the wild-type plants.In addition, proteome data have revealed new homologs of TSB in both Arabidopsis and maize that may contribute to Trp production in TSB mutants; these have not been considered in arguments supporting Trp-independent IAA synthesis. Maize orp has mutations in two TSB genes, resulting in a seedling lethal phenotype with high levels of accumulated indole. However, proteome sequence information now indicates that maize has three TSB genes. Plants and bacteria have divergent forms of TSB, type 1 and type 2 (Xie et al., 2001); the major TSB genes responsible for Trp synthase activity in maize and Arabidopsis are type 1. The third maize TSB gene, maize locus ID GRMZM2G054465, is a member of the TSB type 2 group. Its product is reported not to interact directly with a Trp synthase alpha (TSA) subunit but has experimentally demonstrated catalytic activity converting indole and Ser to Trp (Yin et al., 2010). This type 2 TSB may allow orp plants to make sufficient Trp for IAA production from the accumulated free indole.When the original work on trp2 mutants of Arabidopsis was carried out, two TSB genes were known (Last et al., 1991). As the trp2 plants were deficient only in TSB1, they were able to make sufficient Trp to survive under low-light conditions. Full proteome data now indicate that Arabidopsis has four TSB-like genes; in addition to TSB1 and TSB2, there is a third type 1 TSB gene, Arabidopsis locus ID AT5G28237. The product of this gene has not been experimentally characterized. The fourth gene, AT5G38530, encodes a type 2 TSB with demonstrated catalytic activity similar to ZmTSB type 2 mentioned above (Yin et al., 2010). Thus, the trp2 plants may also make enough Trp for IAA production. It is even possible that one of the minor forms of TSB has a specific role in IAA production. Type 2 TSBs are conserved throughout the plant kingdom, and the biological role for this protein is not known (Xie et al., 2001).A phylogenetic analysis of type 1 TSBs is shown in Figure 2. This indicates that the product of AT5G28237 belongs to a eudicot-conserved TSB type 1-like clade, divergent from that containing major experimentally characterized TSBs. A multiple sequence alignment (not shown) reveals that members of this divergent clade have a shortened N terminus with respect to the major chloroplast-localized TSB proteins. A localization prediction carried out in CELLO (Yu et al., 2006) suggests a cytosolic location for these proteins. Examination of EST databases indicates that the genes encoding these proteins are expressed. It is possible that the product of AT5G28237 could interact with the cytosolic INS studied by Wang et al. (2015), or separately with its indole product, to produce Trp that is further converted to IAA.Open in a separate windowFigure 2.Phylogeny of TSB type 1 homologs from Oryza sativa (LOC_Os), Sorghum bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), Brassica rapa (Brara), Solanum lycopersicum (Solyc), Populus trichocarpa (Potri), and Physcomitrella patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (http://megasoftware.net; Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with type 1 TSBs from the moss P. patens.The second major line of evidence for Trp-independent IAA synthesis comes from isotopic labeling experiments. Wright et al. (1991) observed greater incorporation of ²H into IAA than Trp in orp seedlings grown on ²H₂O. Normanly et al. (1993) reported higher enrichment of ¹⁵N in IAA than Trp in trp2-1 mutants of Arabidopsis grown on ¹⁵N anthranilate; very poor incorporation of deuterium from ²H-Trp into IAA was reported in the trp2-1 plants. A number of similar reports relating to other plants have been published showing differences in the incorporation of label from Trp into IAA depending on experimental tissue and environmental conditions (e.g. Michalczuk et al., 1992; Rapparini et al., 2002; Sztein et al., 2002). This evidence has been persuasive; however, it assumes a single pool of Trp to which ¹⁵N anthranilate and ²H-Trp contribute and from which IAA is made. If Trp is made at different rates in different parts of the plant, and/or exogenous ²H-Trp does not equilibrate with newly synthesized Trp, then the ratio of ¹⁵N to ²H in Trp will vary in different plant organs/tissues/cells. Trp turnover and thus incorporation of label from ¹⁵N anthranilate are likely to differ substantially throughout the plant, with the highest rates of labeling occurring in cells with high rates of protein synthesis. This would not be a problem for the experiment if IAA is made at equal rates in different parts of the plant, but we know it is not. The Trp aminotransferase/YUCCA pathway of IAA synthesis elegantly shown to be responsible for the bulk of IAA synthesis (Mashiguchi et al., 2011; Won et al., 2011) appears to be locally controlled in Arabidopsis via 11 different YUCCA-encoding genes that have highly localized expression (Zhao, 2008). Adding to the complication is the need for ¹⁵N anthranilate and ²H-Trp to move into and through the plant to regions of Trp and IAA synthesis, respectively. This is likely to occur at different rates due to differing transporter requirements.Data from my own laboratory (Cooney and Nonhebel, 1991) is particularly relevant to this discussion. We monitored incorporation of ²H from deuterated water into IAA and Trp in tomato (S. lycopersicum) shoots. Unlike the other studies, we also measured the incorporation of label into IPA. Our data showed that IPA became labeled at a rate consistent with this compound acting as the major/sole precursor of IAA. Crucially, the proportion of labeled Trp was lower than ²H-IPA. Our interpretation of these data was that IPA and IAA were produced from newly synthesized Trp, and that Trp was not uniformly labeled throughout the shoot. At the time, we suggested different subcellular pools of Trp; this may be the case, but in light of new knowledge of localized IAA synthesis, it is most likely that substantial differences in Trp and IAA turnover in different cells/tissues may be the reason for these observations.The arguments above cast doubt on the existence of the Trp-independent route; however, a recent publication by Wang et al. (2015) claims to provide new evidence for its importance. They present the interesting finding that Arabidopsis plants with a null mutation in INS, a cytosolic TSA homolog previously shown to have indole-3-glycerol phosphate lyase (IGL) activity (Zhang et al., 2008), had reduced levels of IAA. The mutation particularly affected early embryo development. I suggest that the INS may make a contribution to IAA synthesis, but the only specific evidence that it does so via a Trp-independent route is the observation that the ins-1 mutation has an additive effect with the weakly ethylene insensitive8-1 Trp aminotransferase mutation. This evidence is indicative rather than conclusive. The possibility that INS may act in concert with a minor TSB homolog, as suggested in Figure 1, needs to be considered.In addition, Wang et al. (2015) focus on Arabidopsis alone. If INS has a key role in IAA synthesis, then evolutionary theory predicts a conserved protein with wide taxonomic distribution. On the contrary, an exhaustive BLAST search (Altschul et al., 1997) of diverse taxa in Phytozome v10.2 (Goodstein et al., 2012) and GenBank (Benson et al., 2013) revealed that INS orthologs with cytosolic prediction and shortened N terminus occur only in members of the Brassicaceae (Eutrema salsugineum, Arabis alpina, Camelina sativa, Capsella rubella, Brassica napus, Boechera stricta, Arabidopsis lyrata, B. rapa) and in Tarenaya hassleriana from the Brassicaceae sister family, the Cleomaceae. The phylogenetic tree in Figure 3 shows relationships between INS and TSA homologs from several plant species and indicates the separate clade of cytosolic INS homologs in the Brassicaceae. In this diagram, the sequence most closely related to INS from another group is that from tomato. This protein is the only TSA found in tomato and has an unambiguous chloroplast signal peptide. P. trichocarpa and M. truncatula as well as other eudicots outside the Brassicaeae and Cleomaceae also lack cytosolic TSA homologs. Furthermore, INS and its orthologs are phylogenetically distinct from the other experimentally characterized indole-3-glycerol phosphate lyases benzoxazin1 and IGL (Frey et al., 2000) and their orthologs. The latter are restricted to the grasses where they are involved in the production of cyclic hydroxamic acid defense compounds (Frey et al., 2000). The grasses also have an additional separate clade of cytosolic TSA homologs, although work by Kriechbaumer et al. (2008) did not detect any catalytic activity for the product of GRMZM2G046191_T01. The phylogeny of INS and its orthologs would suggest the major role of these proteins may be the production of lineage-specific metabolites such as the indole-derived defense compounds produced in grasses; any role in IAA synthesis may be incidental and restricted to the Brassicaeae and Cleomaceae.Open in a separate windowFigure 3.Phylogeny of TSA homologs from O. sativa (LOC_Os), S. bicolor (Sobic), Z. mays (GRMZM), Arabidopsis (AT), A. lyrata (Alyrata), B. rapa (Brara), S. lycopersicum (Solyc), Medicago truncatula (Medtr), P. trichocarpa (Potri), and P. patens (Phpat). Protein sequences were downloaded from Phytozome v10.2 (Goodstein et al., 2012). The phylogenetic analysis was conducted in MEGA6 (Tamura et al., 2013) with multiple sequence alignment by MUSCLE (Edgar, 2004) and evolutionary history inferred using the neighbor-joining method (Saitou and Nei, 1987). The rooted optimal tree is shown; the percentage of replicate trees in which the associated sequences clustered together in the bootstrap test (500 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale; the scale bar indicates the number of amino acid substitutions per site. It is rooted with the TSA ortholog from the moss P. patens.In conclusion, I contend that experimental data relating to IAA synthesis in Arabidopsis, including that suggesting the involvement of a cytosolic INS, can be explained by the Trp-dependent IAA synthesis pathway. I show that INS and its orthologs are not found outside the Brassicaceae and a closely related sister clade; any alternative IAA synthesis pathway in which they may be involved is likely to have similar limited taxonomic occurrence. Furthermore, Arabidopsis and its relatives contain two additional TSB homologs that could convert free indole into Trp. Curiously, both of these proteins have a wider taxonomic distribution. A priority for further experimental work should be testing the involvement of minor TSB homologs in IAA synthesis, including the highly conserved type 2 TSBs as well as a eudicot-specific clade of possibly cytosolic type 1 TSBs. Work would also have to establish whether free indole exists in plants other than the Brassicaceae and the grasses. Finally, I argue that isotope-labeling experiments do not provide strong support for the Trp-independent route, as IAA production is highly localized. Previously published data from my laboratory clearly show that the main Trp-dependent IAA precursor IPA becomes more highly labeled from ²H₂O than Trp, even though the latter is produced from Trp in a single reaction. Thus, it cannot be argued that differences in isotope enrichment between Trp and IAA demonstrate the existence of a Trp-independent route.     

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.     

Partial purification and characterisation of an aromatic amino acid aminotransferase from mung bean (Vigna radiata L. Wilczek)

An aromatic amino acid aminotransferase (aromAT) was purified over 33 000-fold from the shoots and primary leaves of mung beans (Vigna radiata L. Wilczek). The enzyme was purified by ammonium sulfate precipitation, gel filtration and anion exchange followed by fast protein liquid chromatography using Mono Q and Phenylsuperose. The relative amino transferase activities using the most active amino acid substrates were: tryptophan 100, tyrosine 83 and phenylalanine 75, withK _m values of 0.095, 0.08 and 0.07 mM, respectively. The enzyme was able to use 2-oxoglutarate, oxaloacetate and pyruvate as oxo acid substrates at relative activities of 100, 128 and 116 andK _m values of 0.65, 0.25 and 0.24 mM, respectively. In addition to the aromatic amino acids the enzyme was able to transaminate alanine, arginine, aspartate, leucine and lysine to a lesser extent. The reverse reactions between glutamate and the oxo acids indolepyruvate and hydroxyphenylpyruvate occurred at 30 and 40% of the forward reactions of tryptophan and tyrosine, withK _m, values of 0.1 and 0.8 mM, respectively. The enzyme was not inhibited by indoleacetic acid, although -naphthaleneacetic acid did inhibit slightly. Addition of the cofactor pyridoxal phosphate only slightly increased the activity of the purified enzyme. The aromAT had a molecular weight of 55–59 kDa. The possible role of the aromAT in the biosynthesis of indoleacetic acid is discussed.Abbreviations AAT aspartate aminotransferase - aromAT aromatic amino acid aminotransferase - FPLC fast protein liquid chromatography - IPyA indolepyruvate - OHPhPy hydroxyphenylpyruvate - PLP pyridoxal phosphate - TAT tryptophan aminotransferase     

Impacts of biogas production on nitrogen flows on Dutch dairy system: Multiple level assessment of nitrogen indicators within the biogas production chain

Biogas production on dairy farms is promoted as a climate change measure since it captures methane, a greenhouse gas emitted by manure, and produces renewable energy. Digestate is a by‐product of biogas production and is often used for nutrient recycling in a similar way as traditional manure. Despite having similar functions, manure and digestate have different behaviors related to nitrogen recycling and nitrogen emissions which are significant agricultural and environmental concerns of manure. This paper provides an insight into the impact of biogas production on nitrogen emissions and nitrogen recycling issues of the current dairy farming practice. Using the Substance Flow Analysis (SFA) approach, we analyzed the changes on three levels: manure handling, dairy farm, and the whole chain. Four biogas production options on a Dutch dairy farm related to types and sources of feedstocks were considered. We quantified biogas output, nitrogen fertilizer replacement percentage (%) and consequential nitrogen emissions (kgN/year; kgN/m³ biogas produced) of these productions in comparison with the baseline of current dairy farming without biogas. We conclude that biogas production options with additional feedstocks will cause profound changes in the nitrogen recycling on dairy farms and the nitrogen emissions at the chain level. Besides, the results show that determining the optimal biogas production option can be challenging as the evaluation is highly dependent on the used nitrogen indicator and the included level of analysis. Our findings show how SFA and a multilevel perspective can give a broader understanding of environmental trade‐offs.     

Toxicity of hydrolysis volatile products of Brassica plants to Sclerotinia sclerotiorum,in vitro

Oilseed rape stem rot disease caused by Sclerotinia sclerotiorum causes serious yield losses worldwide. Glucosinolates as specific secondary metabolites of Brassicaceae are produced in various parts of the host plants. Their enzymatic hydrolysis releases chemical components, particularly isothiocyanates, with fungitoxic activity and volatile characteristics. To investigate the effect of volatiles derived from Brassica tissues, the pathogen was exposed to hydrolysis products of Brassica shoot parts as sources of glucosinolates including oilseed rape varieties and two species, black and white mustard. The results showed significant differences in inhibition of S. sclerotiorum growth between varieties and species. All tissues of black mustard inhibited completely the exposed colonies of the pathogen and oilseed rape varieties Dunkeld, Oscar and Rainbow had significant inhibitory effect on the fungus. The genotypes demonstrated significant differences for the production of toxic volatiles, indicating that GSL contents in Brassica species and even cultivars have different potentials for toxic products.     

Redirection of tryptophan metabolism in tobacco by ectopic expression of an Arabidopsis indolic glucosinolate biosynthetic gene

Indole-3-acetaldoxime (IAOx) is a branch point compound of tryptophan (Trp) metabolism in glucosinolate-producing species such as Arabidopsis, serving as a precursor to indole-glucosinolates (IGs), the defense compound camalexin, indole-3-acetonitrile (IAN) and indole-3-acetic acid (IAA). We synthesized [²H₅] and [¹³C₁₀¹⁵N₂]IAOx and [¹³C₆], [²H₅] and [2′,2′-²H₂]IAN in order to quantify endogenous IAOx and IAN in Arabidopsis and tobacco, a non-IG producing species. We found that side chain-labeled [2′,2′-²H₂]IAN overestimated the amount of IAN by 2-fold compared to when [²H₅]IAN was used as internal standard, presumably due to protium-deuterium exchange within the internal standard during extraction of plant tissue. We also determined that [¹³C₁]IAN underestimated the amount of IAN when the ratio of [¹³C₁]IAN standard to endogenous IAN was greater than five to one, whereas either [²H₅]IAN or [¹³C₆]IAN showed a linear relationship with endogenous IAN over a broader range of concentrations. Transgenic tobacco vector control lines did not have detectable levels of IAOx or IAN (limit of detection ∼ 100 pg/g fr. wt), while lines expressing either the IAOx-producing CYP79B2 or CYP79B3 genes from Arabidopsis under CaMV 35S promoter control accumulated IAOx in the range of 1-9 μg/g fr. wt. IAN levels in these lines ranged from 0.6 to 6.7 μg/g fr. wt, and IAA levels were ∼9-14-fold above levels in control lines. An Arabidopsis line expressing the same CYP79B2 overexpression construct accumulated IAOx in two of three lines measured (∼200 and 400 ng/g fr. wt) and accumulated IAN in all three lines. IAN is proposed to be a metabolite of IAOx or an enzymatic breakdown product of IGs induced upon tissue damage. Since tobacco does not produce detectable IGs, the tobacco data are consistent with IAN being a metabolite of IAOx. IAOx and IAN were also examined in the Arabidopsis activation tagged yucca mutant, and no accumulation of IAOx was found above the limits of detection but accumulation of IAN (3-fold above wt) occurred. The latter was surprising in light of recent reports that rule out IAOx and IAN as intermediates in YUCCA-mediated IAA synthesis.     

Metabolism of [14C]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 ¹⁴C-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 [¹⁴C]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 [¹⁴C]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 [¹⁴C]IAA in Zea mays root segments was tentatively identified by high-performance liquid chromatography as oxindole-3-acetic acid.Abbreviations HPLC High-performance liquid chromatography - IAA Indole-3-acetic acid