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First published online August 26, 2009; 10.1104/pp.109.141507 Plant Physiology 151:1130-1138 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Auxin Biosynthesis in Pea: Characterization of the Tryptamine Pathway1,[W],[OA]School of Plant Science (L.J.Q., N.D.T., J.J.R.), School of Chemistry (J.A.S., P.P.M.), and Central Science Laboratory (N.W.D.), University of Tasmania, Hobart, Tasmania 7001, Australia
One pathway leading to the bioactive auxin, indole-3-acetic acid (IAA), is known as the tryptamine pathway, which is suggested to proceed in the sequence: tryptophan (Trp), tryptamine, N-hydroxytryptamine, indole-3-acetaldoxime, indole-3-acetaldehyde (IAAld), IAA. Recently, this pathway has been characterized by the YUCCA genes in Arabidopsis (Arabidopsis thaliana) and their homologs in other species. YUCCA is thought to be responsible for the conversion of tryptamine to N-hydroxytryptamine. Here we complement the genetic findings with a compound-based approach in pea (Pisum sativum), detecting potential precursors by gas chromatography/tandem-mass spectrometry. In addition, we have synthesized deuterated forms of many of the intermediates involved, and have used them to quantify the endogenous compounds, and to investigate their metabolic fates. Trp, tryptamine, IAAld, indole-3-ethanol, and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence: Trp, tryptamine, IAAld, IAA, with indole-3-ethanol as a side-branch product of IAAld. N-hydroxytryptamine was not detected, but we cannot exclude that it is an intermediate between tryptamine and IAAld, nor can we rule out the possibility of a Trp-independent pathway operating in pea roots.
Auxin is a key plant growth hormone, involved in processes as diverse as branching, gravitropism, phototropism, and seed development (Davies, 2004  ). However, the biosynthetic pathways leading to the main auxin in plants, indole-3-acetic acid (IAA), are not well understood. Although there is good evidence that the amino acid Trp is an early precursor (Gibson et al., 1972; Wright et al., 1991; Tsurusaki et al., 1997), several routes from Trp to IAA have been proposed, and for any given species it is not clear which route or routes occur. The possible Trp-dependent pathways in higher plants are the indole-3-pyruvic acid (IPyA) pathway (Stepanova et al., 2008; Tao et al., 2008), the tryptamine (YUCCA) pathway (Zhao et al., 2001), the indole-3-acetaldoxime (IAOx) pathway (Bartel et al., 2001), and the indoleacetamide pathway (Pollmann et al., 2002), on the basis of the first metabolite of Trp (Fig. 1
). In addition, a possible Trp-independent pathway has been proposed (Normanly et al., 1993), bypassing Trp completely, further complicating the process of IAA biosynthesis.
Since 2001, there has been renewed interest in the tryptamine route to IAA, after the discovery and functional analysis of the Arabidopsis (Arabidopsis thaliana) YUCCA gene, reported to encode the enzyme for converting tryptamine to N-hydroxytryptamine (Zhao et al., 2001 , 2002). On the basis of the Zhao et al. (2001, 2002) reports, tryptamine pathways have generally been proposed in the sequence: Trp, tryptamine, N-hydroxytryptamine, IAOx, indole-3-acetaldehyde (IAAld), IAA (Fig. 1; Woodward and Bartel, 2005). Recently, however, Sugawara et al. (2009) suggested that IAOx be removed from the tryptamine pathway, and that IAOx-dependent IAA biosynthesis operates only in the Brassicaceae. In Arabidopsis, this pathway can be important, at least in some circumstances, because when a side branch is impaired, as in the sur1 mutant, IAA levels increase dramatically (Sugawara et al., 2009). On the other hand, the IAOx pathway is not the only pathway operating in Arabidopsis, because genetically blocking the step Trp to IAOx does not always reduce IAA content, compared with wild-type plants (Sugawara et al., 2009). This means that in Arabidopsis, the tryptamine and/or IPyA and/or indoleacetamide pathways compensate for the loss of the IAOx pathway. Interestingly, Sugawara et al. (2009) do not include IAAld in their tryptamine pathway, and their model implies instead that N-hydroxytryptamine is directly converted to IAA.
Turning to other species, it has been reported that tryptamine is not present in pea (Pisum sativum; Schneider et al., 1972 It is clear, therefore, that the tryptamine pathway to IAA remains poorly understood. In this article, we further characterize the pathway, using the garden pea as a model species. We report on the presence/absence and levels of the putative endogenous intermediates, as determined by gas chromatography/tandem mass spectrometry (GC/MS/MS), and investigate their metabolic fates using [14C] and deuterated versions of the compounds. Our evidence indicates that key elements of the tryptamine pathway are operative in pea roots.
Chemical Synthesis of IAA Precursors
We synthesized labeled forms of L-Trp, tryptamine, IAOx, and indole-3-acetonitrile (IAN), as confirmed by GC/MS/MS, all with up to five deuteriums on the indole ring, as well as a dideuterated form of indole-3-ethanol (IEt). For the [2H5]-labeled compounds, over 50% of the material was [2H5] labeled, as shown in Supplemental Table S1. However, IAAld was too unstable to allow the production of a labeled form, and in fact, spontaneously converted to IAA when left at room temperature (as previously suggested by Ernstsen et al., 1986
The identification and quantification of putative IAA precursors provides insight into both their involvement within the pathway and their distribution in the plant. The putative IAA precursors Trp, tryptamine, IAAld, and IEt were identified and quantified in pea shoot and root extracts. The identifications were based on GC/MS/MS analyses, using synthesized standards for reference (as shown for tryptamine in Fig. 2
). The putative precursor IAN was not detected in pea tissue (the [2H5]IAN internal standard added to the extract was not diluted by endogenous IAN), nor was IAOx, despite both being present in Arabidopsis seedling extracts (IAN: 9,720 ng g fresh weight [FW]–1; IAOx: 1.7 ng g [FW]–1; Sugawara et al., 2009
In pea, Trp levels ranged from 6,000 to 30,000 ng g (FW)–1, with a pattern of distribution very similar to that of IAA itself (10–75 ng g [FW]–1). Levels of both these compounds were higher in internode and apical bud tissue than in root and leaf tissue (P < 0.05; Table I ). After IAAld was converted to IEt, and total IEt quantified, IAAld levels were calculated by subtracting from that total the IEt level found in extracts not treated with sodium borohydride. The level of IAAld was between 2 and 21 ng g (FW)–1, higher in the roots compared with the apical bud, leaf, and internode tissue (P < 0.001; Table I). The levels of IEt, and particularly tryptamine (as shown in Fig. 2, in root tissue), were very low in all tissues.
[2H5]L-Trp Metabolism Studies The labeled forms synthesized in this study were essential also for metabolism studies. Roots of sterile whole pea seedlings were incubated with [2H5]L-Trp for 24 h, and both the products and the substrates were quantified using appropriately labeled internal standards. A time-course study found that [2H5]Trp moved into the roots by 6 h, and the compound was detected in roots at all subsequent time points (every 6 h for 24 h). In the experiment represented in Table II , endogenous IAA was diluted with [2H5]IAA, showing that [2H5]L-Trp was taken up and converted to IAA; the deuterium incorporation was approximately 8.3% of the total root IAA pool (Fig. 3 ). The percentage of labeling with [2H5] material for each putative IAA precursor is shown in Table II. The percentage of [2H5]tryptamine was 160% of the endogenous tryptamine pool and that of IAAld, 61.5%. Interestingly, IEt had a relatively high (190%) [2H5] incorporation (Table II). In no case was deuterium found when extracts were tested for IAN, nor was any endogenous IAN found within the plant material. Similarly, no conversion of [2H5]Trp to [2H5]IAOx was found after 24 h. Furthermore, in none of the experiments was any deuterium-labeled compound found in the control cases (i.e. tubes with plants but no substrate), and in selected experiments no label was found in shoot material. Moreover, [2H5]L-Trp left in tubes without plant material showed no conversion to any of the compounds analyzed.
[2H5] and [14C]Tryptamine Metabolism Studies For this study, it was important to determine whether tryptamine can be converted to subsequent intermediates within the pathway. After conducting a dose-response study, it was found that a [2H5]tryptamine concentration of 7 to 10 µM was sufficient to allow the roots to take up and metabolize tryptamine to intermediates in the pathway. Quantitation of [2H5]tryptamine in roots using a [14C]-labeled internal standard indicated that the substrate was taken up rapidly (within 6 h), and this compound was detected in roots at all subsequent time points (every 6 h for 24 h). The extent of uptake was also monitored by using [14C]tryptamine as the substrate (7 µM). For example, in one experiment, 88% of the label was taken up by the roots after 24 h, as determined by scintillation counting. Relative to endogenous levels, this value was very high: Endogenous levels of tryptamine were approximately 0.4 ng g (FW)–1.
After incubation of sterile roots with [2H5]tryptamine, GC/MS/MS analysis showed that approximately 5% of the total IAA pool (Fig. 4
), and 32% of the IAAld pool was [2H5] labeled (Table III
). A high degree of deuterium labeling was also detected in IEt, with levels of [2H5]IEt 30.2 times higher than endogenous IEt, at 4.98 ng g (FW)–1 (Table III). No evidence for the conversion from [2H5]tryptamine to [2H5]IAOx was found. However, a HPLC profile of [14C]tryptamine-incubated sterile roots (Fig. 5
) showed strong conversion of [14C]tryptamine to an initially unidentified product (fraction 20; Fig. 5). On the basis of first principles MS evidence—the loss of 59 mass units (acetamide) from [M + H]+ 205 to mass-to-charge ratio (m/z) 146—this was postulated to be [14C]N
Analyses by LC/MS and GC/MS showed that the conversion of [14C]tryptamine to [14C]N  -acetyltryptamine far exceeded the conversion of the substrate to [14C]IAA or [14C]IEt. N-acetyltryptamine is not thought to be involved in IAA biosynthesis. Again, tubes containing [2H5]tryptamine, but no plant material, showed no evidence of metabolism of the substrate; the compound appeared stable when left in distilled water. After feeds of [14C]tryptamine, there was no evidence from LC/MS of [14C]N-hydroxytryptamine as a metabolite (this compound elutes approximately 1 min later than tryptamine with the LC/MS method used, as shown with a standard synthesized in our laboratory) and it was therefore concluded that [14C]tryptamine was not converted to [14C]N-hydroxytryptamine in pea roots, or if it was, that the levels were too low to detect. Endogenous N-hydroxytryptamine was likewise not detected (detection limit approximately 0.5 ng g [FW]–1), although the absence of a labeled internal standard and the instability of N-hydroxytryptamine made the detection of this compound difficult.
Despite the lack of labeling found in the IAN pool after incubation with L-Trp, and failure to detect IAN in pea root extracts, it was important to determine whether the machinery needed for the production of IAA from IAN was present within pea. It was formally possible that IAN was being created (albeit at an undetectable level) via another pathway, and subsequently converted to IAA. However, after incubation with [2H5]IAN, no detectable amount of [2H5]IAA was found in any case, indicating that the compound is not, in fact, involved. This finding was repeated and the plants analyzed for the presence of [2H5]IAN itself; the compound was, indeed, taken up by the roots.
As for IAN, although no metabolism of [2H5]Trp to [2H5]IAOx nor evidence of an endogenous pool of the compound was found in pea (Supplemental Fig. S1), we assessed the ability of the plants to metabolize [2H5]IAOx. After incubation of pea roots with IAOx, a dilution of the IAA and IEt pools by their [2H5] versions was found (Supplemental Figs. S2 and S3). The IAA pool was diluted with 10.46% [2H5], and the IEt pool more than 500%. No evidence of conversion of [2H5]IAOx to [2H5]IAN was observed.
Recently, there has been renewed focus on the tryptamine pathway as a route to IAA biosynthesis, based on genetic studies with Arabidopsis (for review, see Zhao, 2008 ). The present study provides clear physicochemical evidence that key elements of the tryptamine pathway (tryptamine, IAAld, IAA) can operate in the garden pea.
First, Trp, tryptamine, IAAld, and IEt were identified and quantified in pea tissues. It appears that in this species the tryptamine pathway does not involve high endogenous levels of intermediates: Although Trp was present at µg g (FW)–1 levels, and IAA itself is reasonably abundant, the intermediates were present at lower levels, comparable to those of other hormone pathways, e.g. gibberellins (Ross, 1998
Second, metabolism studies with labeled compounds indicated that tryptamine is an intermediate in peas, with [2H5]Trp converted to [2H5]tryptamine, and [2H5]tryptamine to [2H5]IAA. It has been reported previously that labeled Trp can be converted to IAA in pea roots (Mitchell and Davies, 1972
It appears that a pathway including IAN and/or IAOx is not operative in pea, consistent with the findings of Sugawara et al. (2009)
Our results clearly indicate that IAAld can be formed from tryptamine in pea. This is significant because a recent model includes N-hydroxytryptamine as the intermediate between tryptamine and IAA in Arabidopsis (Sugawara et al., 2009
The conversion of [14C]tryptamine to IAA has been previously reported in tobacco apices (Phelps and Sequeira, 1967
Our results are consistent with previous evidence that IAA is produced in the roots. Deuterated IAA precursors fed to pea roots in metabolism studies were not transported to the shoots, but were metabolized within the roots themselves; that is, no deuterated compound was found in shoot extracts after the metabolism studies carried out herein. This is consistent with the findings of Ljung et al. (2001)
It cannot be ruled out that IPyA is a pathway constituent in peas. Because IAAld is an intermediate to IAA on both the tryptamine and IPyA pathways, it could be said that some accumulation of labeled IAAld after Trp incubation occurred via the IPyA route. However, our studies show that at least a portion of the IAAld pool can be formed from tryptamine, indicating that this pathway may be active in vivo. Recently, Tao et al. (2008)
The renewed focus on the tryptamine pathway stemmed from the discovery of the Arabidopsis YUCCA gene, which encodes a monooxygenase-like enzyme that appears to oxidize tryptamine to N-hydroxytryptamine (Zhao et al., 2001
Chemicals
Labeled forms used for metabolism studies and internal standards were synthesized in our laboratories (see Supplemental Data), apart from [2H4]tryptamine ( All deuterated compounds were checked for both the level of [2H5] incorporation as determined by GC/MS/MS (Supplemental Table S1), as well as for target products of metabolism studies to eliminate the possibility that any observed positive results were due to contamination from the deuterium-fed treatments. After purification, no such contamination was found in the compounds. The conditions of metabolism were also tested—deuterated compounds (tryptamine, L-Trp, and IAN) were left in the same conditions without plant material and tested for spontaneous degradation to the target compounds; none was found. Nor was any loss of deuterium observed, and the labeled compounds were as such considered stable.
Pea (Pisum sativum) plants of line 107 were used throughout. Line 107 (Torsdag) is wild type with respect to internode length genes. For quantification purposes, nicked seeds were grown, two per pot, in experimental and glasshouse conditions as described previously (Jager et al., 2008
For metabolism purposes, pea seeds were sterilized in 70% ethanol for 1 min, followed by a sodium hypochlorite solution containing 0.2% available chlorine for 5 min, before being rinsed several times in sterile distilled water. Seeds were planted in 10 mL of autoclaved revised Murashige and Skoog (1962) Selected experiments included a harvest of the whole shoot tissue above the seed, separately, to examine possible uptake of labeled compounds from the root to the shoot tissue. In two studies, involving the assessment of uptake of [2H5]tryptamine and [2H5]Trp, both sterile root material and the distilled water incubation medium, were sampled every 6 h for 24 h, and the subsequent extracts tested for the substrates themselves.
Apart from those experiments involving Trp quantification, once harvested and weighed the solutions were homogenized, and filtered as described previously for IAA (Jager et al., 2005
Unless otherwise stated, extracts were reduced under vacuum at 30°C to a small volume (1–4 mL) using a rotary evaporator. Extracts used for IAA and IAN analysis were purified with Sep-Pak C18 cartridges (VAC RC 500 mg; Waters) and prepared as described for IAA previously (Jager et al., 2005
Before drying, extracts for the quantification of IAAld, including the [2H2]IEt internal standard, were stirred on a block heater, with 1 g of sodium borohydride per 50 mL extract (Sigma) added twice over a 9 h period to reduce IAAld to IEt (as previously noted by Moore and Shaner, 1968
For analysis of tryptamine, extracts in 80% methanol were dried and then taken up in 2 mL of 2% acetic acid in distilled water, and partitioned three times with 1 mL chloroform. The organic phase was discarded, and the aqueous phase taken to pH 11, using 1 N NaOH, and partitioned with chloroform again. In some tryptamine quantification or metabolism experiments (Figs. 2 and 5), extracts were subjected to HPLC as previously described (Ross et al., 1993
For LC/MS analyses of N For the analysis of IAOx, extracts were loaded on to Sep Pak C18 cartridges in 0.4% acetic acid and eluted with 50% methanol (0.4% acetic acid). IAOx samples were then subjected to HPLC as described for tryptamine, but with solvent A being 0.4% acetic acid in distilled water and solvent B methanol, on a gradient of 20% to 75% B over 25 min; flow rate 2 mL min–1. IAOx eluted in fractions 14 to 15. Samples were then trimethylsilylated as described for IEt.
Aqueous Trp-containing extracts were acidified to pH 2 using 1 N HCl, 400 µL penta-fluoro-propanol (Fluka) and 100 µL pyridine were added, and the samples were vortexed for 5 s. After this, 100 µL of ethyl chloroformate (Merck) was added and the sample was vortexed for 10 s and left to stand for 10 min (as described previously by Perrine et al., 2004 All samples were then dried under N2 and taken up in 50 µL of chloroform to transfer to an autosampler vial for subsequent GC/MS/MS analysis.
Identification and quantification of the compounds were performed using GC/MS/MS. A Varian 3800 GC coupled to a Varian 1200 triple quadrupole MS was employed, with quantitation via Varian Star software. The column used was a Varian Factor Four VF-5 ms (30 m x 0.25 mm internal diameter and 0.25 micron film), using a Varian 1177 split/splitless injector in splitless mode. The ion source was held at 220°C, the injector 250°C, and the transfer line 290°C, with the typical injection volume being 1 µL. The helium carrier gas was supplied at a constant flow of 1.4 mL per min. For tryptamine and IEt the column oven temperature program was 50°C for 2 min, then to 190°C at 30 degrees per min, then to 220°C at 10 degrees per min, and finally to 270°C at 30 degrees per min. For Trp the column oven temperature program was 50°C for 2 min, then to 270°C at 10 degrees per min. The cycle time was 0.3 s. The GC/MS/MS conditions and specifications for IAA were as described (Jones et al., 2005 For tryptamine, IEt, IAN, and Trp full-scan MS/MS spectra of standards were acquired to determine suitable MS/MS transitions for quantitation. The molecular ion (or in some cases a prominent fragment ion) were selected as the precursor ions. Once relevant product ions were observed, the conditions for quantitation were optimized, including the collision energy for each transition. For tryptamine, IEt (including IAAld converted to IEt using sodium borohydride), Trp, IAOx, and IAN, the GC/MS/MS conditions were as shown in Supplemental Table S2. Compounds were all separable by GC/MS/MS, and eluted in the order tryptamine, followed 71 s later by IAN, then (5 s) IEt, (5 s) IAA, (52.6 s) IAOx, and finally (213.4 s) Trp. Compounds were identified on the basis of retention times and by monitoring a selected transition (or two transitions when possible) characteristic of the compound. Calculations of endogenous levels were performed by comparing peak areas of a transition derived from the endogenous hormone and the corresponding stable-isotope-labeled internal standard. To calculate the endogenous hormone level, the corrected endogenous product ion intensity was divided by the internal standard product ion intensity, and this figure was multiplied by the amount of internal standard added, divided by the FW of the tissue, to give results in ng g (FW)–1. Corrections were made for the small amount of unlabeled material in the deuterated standards, and for contributions from natural isotopes in unlabeled material to peaks corresponding to internal standards. For measurement of endogenous IAAld after its conversion to IEt, the endogenous actual IEt was subtracted from the total IEt (including IAAld), after the conversion was complete.
The following materials are available in the online version of this article.
We thank Ian Cummings and Tracey Winterbottom for technical assistance, and the Australian Research Council for financial assistance. We also thank Hiroyuki Kasahara for providing a sample of N-hydroxytryptamine. Received May 18, 2009; accepted August 24, 2009; published August 26, 2009.
1 This work was supported by the Australian Research Council.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: John J. Ross (john.ross{at}utas.edu.au).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.141507 * Corresponding author; e-mail john.ross{at}utas.edu.au.
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-[2H2],β-[2H2]; Medical Isotopes Inc.), [13C6]IAA (Cambridge Isotope Laboratories), [14C]Trp (sidechain 3-[14C]; American Radiolabelled Chemicals, Inc.), and [14C]tryptamine (
al K, Schlereth A, Jürgens G, Alonso JM (2008) TAA1-mediated auxin biosynthesis is essential for hormone crosstalk and plant development. Cell 133: 177–191