Molecular cloning and sequence analysis of an Azospirilium brasilense indole-3-pyruvate decarboxylase gene

Azospirillum brasilense isolated from the rhizosphere of different plants has the ability to excrete indole-3-acetic acid (IAA) into the culture media. Cosmid p0.2, isolated from an A. brasilense Sp245 genome library in pLAFR1, complements the Tn5-induced mutant SpM7918 of A. brasilense Sp6 which excretes reduced amounts of IAA. Restriction mapping and gene expression studies identified a BglII-EcoRI 4.3 kb fragment of p0.2 sufficient for the restoration of high levels of IAA production in mutant SpM7918. Tn5 mutagenesis localized the gene responsible on a 1.8 kb SmaI fragment. Nucleotide sequence analysis revealed that this fragment contains one complete open reading grame. The predicted protein sequence shows extensive homology with the indole-3-pyruvate decarboxylase of Enterobacter cloacae and the pyruvate decarboxylases of Saccharomyces cerevisiae and Zymomonas mobilis. The A. brasilense mutant Sp245a, constructed by homogenotization of a Tn5 insertion derivative of the 1.8 kb SmaI fragment, also displayed reduced IAA production. Introduction of the cloned wild-type gene into Rhizobium meliloti 1021 resulted in increased IAA production. Cell-free extracts prepared from R. meliloti and A. brasilense transconjugants harboring this gene could convert indole-3-pyruvic acid to indole-3-acetaldehyde and tryptophol. These results clearly demonstrate that IAA production in A. brasilense is mediated by indole-3-pyruvate decarboxylase.     

Identification of an aldehyde oxidase involved in indole-3-acetic acid synthesis in Bombyx mori silk gland

Auxin is thought to be an important factor in the induction of galls by galling insects. We have previously shown that both galling and nongalling insects synthesize indole-3-acetic acid (IAA) from tryptophan (Trp) via two intermediates, indole-3-acetaldoxime (IAOx) and indole-3-acetaldehyde (IAAld). In this study, we isolated an enzyme that catalyzes the last step “IAAld → IAA” from a silk-gland extract of Bombyx mori. The enzyme, designated “BmIAO1”, contains two 2Fe–2S iron–sulfur-cluster-binding domains, an FAD-binding domain, and a molybdopterin-binding domain, which are conserved in aldehyde oxidases. BmIAO1 causes the nonenzymatic conversion of Trp to IAAld and the enzymatic conversion of IAOx to IAA, suggesting that BmIAO1 alone is responsible for IAA production in B. mori. However, a detailed comparison of pure BmIAO1 and the crude silk-gland extract suggested the presence of other enzymes involved in IAA production from Trp.

Abbreviations: BA: benzoic acid; CE: collision energy; CXP: collision cell exit potential; DP: declustering potential; IAA: indole-3-acetic acid; IBI1: IAA biosynthetic inhibitor-1; IAAld: indole-3-acetaldehyde; ICA: indole-3-carboxylic acid; IAOx: indole-3-acetaldoxime; IEtOH: indole-3-ethanol; LC–MS/MS: liquid chromatography–tandem mass spectrometry; Trp: tryptophan     

An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

Biosynthetic pathway of the phytohormone auxin in insects and screening of its inhibitors

Insect galls are abnormal plant tissues induced by galling insects. The galls are used for food and habitation, and the phytohormone auxin, produced by the insects, may be involved in their formation. We found that the silkworm, a non-galling insect, also produces an active form of auxin, indole-3-acetic acid (IAA), by de novo synthesis from tryptophan (Trp). A detailed metabolic analysis of IAA using IAA synthetic enzymes from silkworms indicated an IAA biosynthetic pathway composed of a three-step conversion: Trp → indole-3-acetaldoxime → indole-3-acetaldehyde (IAAld) → IAA, of which the first step is limiting IAA production. This pathway was shown to also operate in gall-inducing sawfly. Screening of a chemical library identified two compounds that showed strong inhibitory activities on the conversion step IAAld → IAA. The inhibitors can be efficiently used to demonstrate the importance of insect-synthesized auxin in gall formation in the future.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Biosynthesis of indole-3-acetic acid in tomato shoots: Measurement,mass-spectral identification and incorporation of −2H from −2H2O into indole-3-acetic acid,d- and l-tryptophan,indole-3-pyruvate and tryptamine

Indole-3-acetic acid (IAA) and its putative precursors, l- and d-tryptophan, indole-3-pyruvate, and tryptamine were isolated from tomato (Lycopersicon esculentum (L.) Mill.) shoots, identified by mass spectrometry, and measured using capillary gas chromatography with an electron capture detector and radioactive internal standards. Average amounts present were 7.9ng · (g FW)^–-1 IAA, 5.7ng · (g FW)^–-1 indole-3-pyruvate, 132 ng · (g FW)^–-1 tryptamine, 103 ng · (g FW)^–-1 d-tryptophan, and 2250 ng · (g FW)^–-1 l-tryptophan. Indole-3-acetaldoxime was not found; detection limits were less than 1ng · (g FW)^–-1. When tomato shoots were incubated for 6, 10 and 21 h in 30% ^–2H₂O, up to four positions in IAA, l- and d-tryptophan, tryptamine and indole-3-pyruvate became labelled with ^–2H. Compounds became labelled rapidly with 10% of IAA molecules containing ^–2H after 6 h. The percentage of labelled molecules of IAA and l-tryptophan increased up to 10 h but then decreased again, correlating with an increase in the total shoot tryptophan and presumably a result of protein hydrolysis in the excised, slowly senescing tissue. The amount of ^–2H in d-tryptophan also showed an increase followed by a decrease, but the proportion of labelled molecules was much less than in l-tryptophan and IAA. Tryptamine became labelled initially at a similar rate to IAA but continued to accumulate ^–2H up to 21 h. We conclude that tryptamine is synthesized from a different pool of tryptophan from that used in IAA synthesis, and is not a major endogenous precursor of IAA in tomato shoots. Indole-3-pyruvate was the most heavily labelled compound after 6 and 10 h incubation (21-h data not available). Furthermore, the proportion of ^–2H-labelled indole-3-pyruvate molecules was quantitatively consistent with the amount of label in IAA. On the other hand, a quantitative comparison of the IAA turnover rate and the rate of ^–2H incorporation into both l- and d-tryptophan indicates that IAA is not made from the total shoot pool of either l- or d-tryptophan. Instead IAA appears to be synthesized from a restricted pool which is turning over rapidly and which has access to both newly synthesized tryptophan and that from protein hydrolysis.Abbreviations GC-ecd gas chromatography with electroncapture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - IAOX indole-3-acetaldoxime - IPyA indole-3-pyruvate - PFB pentafluorobenzyl - R_T retention time - TNH₂ tryptamine - Trp tryptophan - SIM selected ion monitoring We wish to thank Ms. Sue Alford for running the mass spectra and Dr Harry Young for advice with the mass spectrometry. The work was supported by grants from the University of Auckland Research Committee and the C. Alma Baker Trust fund. The mass spectrometer was supported jointly by the University Grants Commitee (NZ) and the DSIR Division of Horticulture and Processing.     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

Production of indole-3-acetic acid, aromatic amino acid aminotransferase activities and plant growth promotion by Pantoea agglomerans rhizosphere isolates

The production of auxins, such as indole-3-acetic acid (IAA), by rhizobacteria has been associated with plant growth promotion, especially root initiation and elongation. Six indole-producing bacteria isolated from the rhizosphere of legumes grown in Saskatchewan soils and identified as Pantoea agglomerans spp. were examined for their ability to promote the growth of canola, lentil and pea under gnotobiotic conditions and for tryptophan (Trp)-dependent IAA production. Five of the isolates enhanced root length, root weight or shoot weight by 15–37% in at least one of the plant species, but isolates 3–117 and 5–51 were most consistent in enhancing plant growth across the three species. Indole concentrations in the rhizosphere of plants grown under gnotobiotic conditions increased in the presence of the rhizosphere isolates and when Trp was added 3 days prior to plant harvest. Isolates 3–117, 5–51 and 5–105 were most effective in increasing rhizosphere indole concentrations. Colony hybridization confirmed that all of the isolates possessed the ipdC gene which codes for a key enzyme in the Trp-dependent IAA synthetic pathway. The activity of amino acid aminotransferase (AAT), catalyzing the first step in the Trp-dependent synthetic pathway, was examined in the presence of Trp and other aromatic amino acids. All of the isolates accumulated Trp internally and released different amounts of IAA. The production of IAA from the isolates was greatest in the presence of Trp, ranging from 2.78 to 16.34 μg mg protein⁻¹ in the presence of 250 μg of Trp ml⁻¹. The specific activity of AAT was correlated with the concentration of IAA produced in the presence of Trp but not when tyrosine (Tyr), phenylalanine (Phe) or aspartate (Asp) was used as a sole nitrogen source. Isolate 3–117, which produced significant concentrations of IAA in the presence and absence of Trp, was able to use aromatic amino acids as sole sources of nitrogen and was most consistent in enhancing the growth of canola, lentil and pea may have potential for development as a plant growth-promoting inoculant. Responsible Editor: Peter A. H. Bakker.     

Current aspects of auxin biosynthesis in plants

Auxin is an important plant hormone essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the most studied auxin in plants, and its biosynthesis pathway has been investigated for over 70 years. Although the complete picture of auxin biosynthesis remains to be elucidated, remarkable progress has been made recently in understanding the mechanism of IAA biosynthesis. Genetic and biochemical studies demonstrate that IAA is mainly synthesized from l-tryptophan (Trp) via indole-3-pyruvate by two-step reactions in Arabidopsis. While IAA is also produced from Trp via indole-3-acetaldoxime in Arabidopsis, this pathway likely plays an auxiliary role in plants of the family Brassicaceae. Recent studies suggest that the Trp-independent pathway is not a major route for IAA biosynthesis, but they reveal an important role for a cytosolic indole synthase in this pathway. In this review, I summarize current views and future prospects of IAA biosynthesis research in plants.     

Metabolism of auxin in pine tissues: Indole-3-acetic acid conjugation

Incubation of sections of various tissues of Pinus pinea L. with a relatively low concentration (3.6 μM) of indole-3-acetic acid-2-¹⁴C (IAA) resulted in the formation of two major metabolites. The first, which has not been identified, seemed to be a polar acidic compound and the second was identified as indole-3-acetylaspartic acid (IAAsp). The polar acidic metabolite has been found to be the major metabolite in needles, shoot wood and roots, while IAAsp has been found to be the major metabolite in shoot bark. Increasing the concentration of IAA in the incubation medium resulted in an increase in the formation of a third metabolite which proved to be l-O-(indole-3-acetyl)-β-d -glucose (IAGlu) and a concomitant decrease in the amount of the polar acidic metabolite. This phenomenon was prominent particularly in needles. IAGlu was isolated from needles and IAAsp was isolated from shoot bark by means of polyvinylpolypyrrolidone column chromatography and preparative thin-layer chromatography. IAGlu was identified by comparison with authentic material by co-chromatography in three different solvent systems and by ¹H-nuclear magnetic resonance analysis. IAAsp was identified by comparison with authentic material by gas-liquid chromatography and ¹H-nuclear magnetic resonance analysis. Several aspects of formation, separation and isolation of IAA metabolites are discussed.     

Lateral root formation in rice (Oryza Sativa L.): differential effects of indole-3-acetic acid and indole-3-butyric acid

Comparative effects of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) on lateral root (LR) formation were studied using 2-day-old seedlings of IR8 rice (Oryza sativa L.). Results showed that IBA at all concentrations (0.8–500 nmol/L) increased the number of LRs in the seminal root. However exogenous IAA, failed to increase the number of LRs. On the other hand, both IBA and IAA caused inhibition of seminal root elongation and promotion of LR elongation, but IAA can only reach to the same degree of that of IBA at a more than 20-fold concentration. Exogenous IBA had no effect on endogenous IAA content. We conclude from the results that IBA could act directly as a distinct auxin, promoting LR formation in rice, and that the signal transduction pathway for IBA is at least partially different from that for IAA.     

Relationship between tryptophan biosynthesis and indole-3-acetic acid production in Azospirillum: identification and sequencing of a trpGDC cluster

Summary Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb Sall — SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI — SalI fragment including the kb SalI — SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.     

Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin

Germination of orchid seeds is a complex process. In this paper we focus on interactions between the host-plant and its bacterial partners via indole-3-acetic acid (IAA). Originally isolated from the roots of the epiphytic orchid Dendrobium moschatum, the strains of Rhizobium, Microbacterium, Sphingomonas, and Mycobacterium genera were among the most active IAA producers. Addition of exogenous tryptophan significantly enhanced auxin formation both in mineral and complex media. The presence of IAA and indole-3-acetaldehyde was confirmed by HPLC. Indole-3-pyruvic and indole-3-lactic acids were also detected in supernatants of culture filtrates of Sphingomonas sp., Rhizobium sp., and Microbacterium sp., while indole-3-acetamide was identified only in Mycobacterium sp. Some concentration- and strain-dependent effects of exogenous IAA on bacterial development were also established. Treatment of the cultures with 10 and 100 μg/ml of auxin resulted in an increase in microbial yield. None of the investigated strains was able to utilize IAA as a source of carbon and energy. Furthermore, inoculation of D. moschatum seeds with Sphingomonas sp. and Mycobacterium sp. resulted in considerable enhancement of orchid seeds germination. This growth-promoting activity was observed in the absence of any plant growth stimulators or mycorrhizal fungi, usually required for orchid germination.     

DDT降解菌株Chryseobacterium sp. PYR2对小麦的促生作用及其机理

【背景】前期结果表明,DDT降解菌株Chryseobacterium sp. PYR2可高效去除土壤中的DDT等污染物,具有潜在的应用价值,但该菌对植物的影响尚不清楚。【目的】探讨菌株Chryseobacterium sp. PYR2对植物的促生作用及其机理,为后续开发DDT降解及植物促生双效功能菌剂提供理论依据。【方法】配制该菌株的不同梯度稀释菌悬液,用纸卷发芽法和盆栽法研究菌悬液对小麦种子萌发和植株生长的影响;Salkowski法测定PYR2合成吲哚-3-乙酸(Indole-3-acetic acid,IAA)量;单因素实验研究不同培养条件对菌株生长及IAA合成的影响;液相色谱-串联质谱-多反应监测(LC-MS/MS-MRM)方法分析IAA在PYR2菌体内的生物合成途径。【结果】PYR2菌悬液可明显提高小麦种子萌发率并促进小麦植株的生长,小麦的侧根数、株高、鲜重、干重等指标均明显提高。该作用是由于菌株PYR2可以合成植物生长激素IAA。最适IAA合成条件:温度30°C,pH 7.0-8.0,盐浓度0.5%,L-色氨酸50mg/L。代谢液中检测到色醇、色胺和吲哚-3-乙酰胺3种中间代谢产物,推测PYR2体内存在3条IAA合成途径,分别为吲哚-3-丙酮酸(IPy A)、TAM和IAM途径。【结论】菌株PYR2对小麦具有明显的促生效果,是由于其具有多条高效合成IAA的代谢途径,表明其在农药污染土壤的生物修复及作物种植中具有潜在的应用前景。     

Characterization of tryptophan-overproducing potato transgenic for a mutant rice anthranilate synthase α-subunit gene (<Emphasis Type="Italic">OASA1D</Emphasis>)

Potato plants (Solanum tuberosum cv. May Queen) transgenic for OASA1D, which encodes a point mutant of an -subunit of rice (Oryza sativa) anthranilate synthase (AS, EC 4.1.3.27), were generated in order to determine the effects of the mutant gene on levels of free tryptophan (Trp) and AS activity in this important crop. Expression of OASA1D in potato induced a 2- to 20-fold increase in the amount of free Trp. This increase was likely due to a reduction in the sensitivity of AS containing the mutant -subunit to feedback inhibition by Trp. Nontargeted metabolite profiling by high-performance liquid chromatography coupled with ultraviolet photodiode array detection as well as targeted profiling by liquid chromatography coupled with mass spectrometry revealed no marked changes in the levels of other metabolites, with the exception of indole-3-acetic acid (IAA), in the transgenic plants. The level of IAA in the upper part of the shoot was increased by a factor of 8.3–39, depending on the transgenic lines, with no detectable effect on plant growth or development. The effects of transformation thus appeared limited to the biosynthesis of Trp and IAA, with the overall metabolic network in potato being virtually unaffected. These results suggest that transformation with OASA1D may prove effective for the breeding of crops with an increased level of free Trp.     

Different Behaviour of Indole-3-Acetic Acid and Indole-3-Butyric Acid in Stimulating Lateral Root Development in Rice (Oryza sativa L.)

The plant hormone auxin has been shown to be involved in lateral root development and application of auxins, indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), increases the number of lateral roots in several plants. We found that the effects of two auxins on lateral root development in the indica rice (Oryza sativa L. cv. IR8) were totally different from each other depending on the application method. When the roots were incubated with an auxin solution, IAA inhibited lateral root development, while IBA was stimulatory. In contrast, when auxin was applied to the shoot, IAA promoted lateral root formation, while IBA did not. The transport of [³H]IAA from shoot to root occurred efficiently (% transported compared to supplied) but that of [³H]IBA did not, which is consistent with the stimulatory effect of IAA on lateral root production when applied to the shoot. The auxin action of IBA has been suggested to be due to its conversion to IAA. However, in rice IAA competitively inhibited the stimulatory effect of IBA on lateral root formation when they were applied to the incubation solution, suggesting that the stimulatory effect of IBA on lateral root development is not through its conversion to IAA.     

Auxin Biosynthesis in Pea: Characterization of the Tryptamine Pathway

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.Open in a separate windowFigure 1.Left: The putative tryptamine (red), Trp-independent (light blue), IPyA (green), indoleacetamide (yellow), and IAOx (dark blue) biosynthetic pathways to IAA in Arabidopsis. The steps shown in gray appear not to occur in peas. Right: The simplified pathway scheme suggested to occur in pea based on the present results and Sugawara et al. (2009). N-hydroxytryptamine was not detected as a metabolite in this study, suggesting that tryptamine might be converted directly to IAAld in pea roots. The Trp-independent, indoleacetamide, and IPyA pathways were not studied.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), despite being present in tomato (Solanum lycopersicum; Cooney and Nonhebel, 1991), rice (Oryza sativa; Ishihara et al., 2008), Arabidopsis (Sugawara et al., 2009), and barley (Hordeum vulgare; Schneider et al., 1972). In tomato, although tryptamine is relatively abundant, and early metabolism studies indicated the conversion of tryptamine to IAA via IAAld (Schneider et al., 1972), Cooney and Nonhebel (1991) cast doubt on the role of tryptamine after studying patterns of labeling after incubation of plants with deuterated water. Again, in tobacco (Nicotiana tabacum), Songstad et al. (1990) showed that while tobacco plants overexpressing a Trp decarboxylase accumulated very high levels of tryptamine, IAA levels were unaffected. Although this result has been interpreted as evidence against the involvement of tryptamine (Bartel et al., 2001), another explanation is that excess tryptamine is converted to compounds via a side branch or side branches, although these are not well studied. Finally, the compound N-hydroxytryptamine is relatively unknown, with no reports of its presence in plants to date.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.     

Growth and indole-3-acetic acid biosynthesis of Azospirillum brasilense Sp245 is environmentally controlled

Batch and fed batch cultures of Azospirillum brasilense Sp245 were conducted in a bioreactor. Growth response, IAA biosynthesis and the expression of the ipdC gene were monitored in relation to the environmental conditions (temperature, availability of a carbon source and aeration). A. brasilense can grow and produce IAA in batch cultures between 20 and 38 degrees C in a standard minimal medium (MMAB) containing 2.5 gl(-1)l-malate and 50 microgml(-1) tryptophan. IAA synthesis requires depletion of the carbon source from the growth medium in batch culture, causing growth arrest. No significant amount of IAA can be detected in a fed batch culture. Varying the concentration of tryptophan in batch experiments has an effect on both growth and IAA synthesis. Finally we confirmed that aerobic growth inhibits IAA synthesis. The obtained profile for IAA synthesis coincides with the expression of the indole-3-pyruvate decarboxylase gene (ipdC), encoding a key enzyme in the IAA biosynthesis of A. brasilense.