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     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Deuterium-labelled indole-3-acetic acid neo-synthesis in plantlets and excised roots of maize

Synthesis of indole-3-acetic acid (IAA), using stable-isotope incorporation, was investigated in Zea mays L. Incorporation of ²H from ²H₂O into IAA molecules was shown to occur in intact plantlets and excised primary roots cultured in vitro. This demonstrates the de-novo formation of IAA, a process which is quantitatively well defined and is initiated early in germination.Abbreviations IAA indole-3-acetic acid     

Effect of Endosperm Removal on 7 Normal NaOH-Labile Indole-3-acetic Acid Conjugates in Shoots and Roots of Zea mays Seedlings

The pool of amide-linked indole-3-acetic acid (amide IAA) in the shoot of growing etiolated seedlings of Zea mays increases between the 3rd and 5th day of germination to equal the amount of free IAA and two-thirds the amount of ester IAA. Deseeding the germinant changes the pool size of free and amide IAA in a manner suggestive of conversion of endogenous free IAA to amide IAA. Deseeding also caused an almost total disappearance of amide IAA from the root, demonstrating that the pool of amide IAA is not inert and can be actively metabolized in young Z. mays seedlings.     

Translocation of Radiolabeled Indole-3-Acetic Acid and Indole-3-Acetyl-myo-Inositol from Kernel to Shoot of Zea mays L.

Either 5-[³H]indole-3-acetic acid (IAA) or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm of kernels of dark-grown Zea mays seedlings. The distribution of total radioactivity, radiolabeled indole-3-acetic acid, and radiolabeled ester conjugated indole-3-acetic acid, in the shoots was then determined. Differences were found in the distribution and chemical form of the radiolabeled indole-3-acetic acid in the shoot depending upon whether 5-[³H]indole-3-acetic acid or 5-[³H]indole-3-acetyl-myo-inositol was applied to the endosperm. We demonstrated that indole-3-acetyl-myo-inositol applied to the endosperm provides both free and ester conjugated indole-3-acetic acid to the mesocotyl and coleoptile. Free indole-3-acetic acid applied to the endosperm supplies some of the indole-3-acetic acid in the mesocotyl but essentially no indole-3-acetic acid to the coleoptile or primary leaves. It is concluded that free IAA from the endosperm is not a source of IAA for the coleoptile. Neither radioactive indole-3-acetyl-myo-inositol nor IAA accumulates in the tip of the coleoptile or the mesocotyl node and thus these studies do not explain how the coleoptile tip controls the amount of IAA in the shoot.     

Indole-3-methylglucosinolate biosynthesis and metabolism in clubroot diseased plants

lndole-3-methylglucosinolate biosynthesis and metabolism in roots of Brassica napus (swede, cv. Danestone II) infected with Plasmodiophora brassicae Wor. were investigated with a pulse feeding technique developed to infiltrate intact tissue segments with labelled substrates. Infected root tissue metabolized [¹⁴C]-L-tryptophan to indole-3-methylglucosinolate, indole-3-acetonitrile, and some other lipophilic indole compounds. The incorporation of radioactivity into these compounds was significantly enhanced in infected tissue compared with control tissue. A time course study showed a high turnover of indole-3-methylglucosinolate and indole-3-acetonitrile in infected tissue. However, thioglucoside glucohydrolase activity was not changed in infected tissue compared with control tissue. Disc electrophoresis revealed the same isoenzyme in both tissues. Control and infected tissues both rapidly hydrolyzed [¹⁴C]-indole-3-acetonitrile in vivo. The possibility of a disease specific biosynthesis of indole-3-acetic acid from indole-3-methylglucosinolate as the result of a changed compartmentation is discussed.     

Auxin-induced changes in the population of translatable messenger RNA in elongating maize coleoptile sections

In-vitro translation products of polyadenylated RNA from untreated and indole-3-acetic acid (IAA)-treated elongating sections of maize (Zea mays L.) coleoptiles were analyzed by twodimensional polyacrylamide gel electrophoresis. Treatment with IAA results in an increased amount of at least four in-vitro translation products. The amounts of two of these translation products are increased within 10 min of IAA treatment.Abbreviation IAA indole-3-acetic acid     

Occurrence and in Vivo Biosynthesis of Indole-3-Butyric Acid in Corn (Zea mays L.)

Indole-3-butyric acid (IBA) was identified as an endogenous compound in leaves and roots of maize (Zea mays L.) var Inrakorn by thin layer chromatography, high-performance liquid chromatography, and gas chromatography-mass spectrometry. Its presence was also confirmed in the variety Hazera 224. Indole-3-acetic acid (IAA) was metabolized to IBA in vivo by seedlings of the two maize varieties. The reaction product was identified by thin layer chromatography, high performance liquid chromatography, and gas chromatography-mass spectrometry after incubating the corn seedlings with [¹⁴C]IAA and [¹³C₆]IAA. The in vivo conversion of IAA to IBA and the characteristics of IBA formation in two different maize varieties of Zea mays L. (Hazera 224 and Inrakorn) were investigated. IBA-forming activity was examined in the roots, leaves, and coleoptiles of both maize varieties. Whereas in the variety Hazera 224, IBA was formed mostly in the leaves, in the variety Inrakorn, IBA synthesis was detected in the roots as well as in the leaves. A time course study of IBA formation showed that maximum activity was reached in Inrakorn after 1 hour and in Hazera after 2 hours. The pH optimum for the uptake of IAA was 6.0, and that for IBA formation was 7.0. The K_m value for IBA formation was 17 micromolar for Inrakorn and 25 micromolar for Hazera 224. The results are discussed with respect to the possible functions of IBA in the plant.     

Analysis of Indole-3-Acetic Acid and Related Indoles in Culture Medium from Azospirillum lipoferum and Azospirillum brasilense

Analysis of neutral and acidic ethyl acetate extracts from culture medium of Azospirillum brasilense 703Ebc by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol, indole-3-methanol, and indole-3-lactic acid. IAA in media of 20 strains of A. brasilense and Azospirillum lipoferum was analyzed quantitatively by both the colorimetric Salkowski assay and HPLC-based isotopic dilution procedures. There was little correlation between the estimates obtained with the two procedures. For instance, the Salkowski assay suggested that the culture medium from A. brasilense 703Ebc contained 26.1 μg of IAA ml⁻¹, whereas HPLC revealed the presence of only 0.5 μg of IAA ml⁻¹. Equivalent estimates with A. brasilense 204Ed were 10.5 and 0.01 μg of IAA ml⁻¹, respectively. The data demonstrate that the Salkowski assay is not a reliable method for measuring the IAA content of Azospirillum culture medium and that estimates in excess of 10 μg of IAA ml⁻¹ should be viewed with particular caution. Metabolism of [2′-¹⁴C]IAA by A. brasilense 703Ebc yielded radiolabeled indole-3-methanol, whereas roots of maize (Zea mays L.) seedlings gave rise to [¹⁴C]oxindole-3-acetic acid and an array of polar metabolites. Metabolism of [2′-¹⁴C]IAA by maize roots inoculated with A. brasilense 703Ebc produced a metabolic profile characteristic of maize rather than Azospirillum species.     

Effects of phenolic substances on metabolism of exogenous indole-3-acetic acid in maize stems

Four-day-old stem segments of Zea mays L. cv. Seneca 60 were treated sequentially with phenolic substances and indole-3-acetic [2-¹⁴C] acid ([2-¹⁴C]IAA). Formation of bound IAA was rapid, but a pretreatment with p-coumaric acid, ferulic acid or 4-methylumbelliferone decreased the level of bound IAA. The decrease is not likely related to the effect of the phenolics on enzymic oxidation of IAA, since the level of free IAA was not limiting and the activity of ferulic acid in enzymic oxidation of IAA is different from that of p-coumaric acid and 4-methylum-belliferone. Apparently these compounds inhibited the formation of bound IAA and consequently caused an accumulation of free IAA. In contrast, caffeic acid, protocatechuic acid and 2,3-dihydro-2, 2-dimethyl-7-benzofuranol had little effect. After the uptake of IAA there was a slow but steady incorporation of the radioactivity into the 80% ethanol-insoluble, 1 M NaOH-soluble fraction. Phenolic substances also affected this process. The compounds which are cofactors of IAA-oxidase increased the incorporation while those which are inhibitors of IAA-oxidase decreased the incorporation. Results suggest that the phenolics also affected the enzymic oxidation of IAA in vivo in the same way as in vitro.     

The relationship between diffusible,extractable and conjugated (base-labile) forms of indole-3-acetic acid in isolated coleoptile tips of Zea mays L.

Isolated, 2.5-mm-long coleoptile tips of Zea mays L. cv. Anjou 210 were analyzed for diffusible and tissue-extractable indole-3-acetic acid (IAA) in comparison with the level of base-labile conjugates at various times after excision. The results indicate that base-labile conjugates of IAA do not serve as major sources of free IAA in maize coleoptile tips.Abbreviations IAA indole-3-acetic acid - TLC thin-layer chromatography     

Identification of indole compounds secreted byFrankia HFPArI3 in defined culture medium

Indole compounds secreted byFrankia sp. HFPArI3 in defined culture medium were identified with gas chromatography-mass spectrometry (GC-MS). WhenFrankia was grown in the presence of¹³C(ring-labelled)-L-tryptophan,¹³C-labelled indole-3-acetic acid (IAA), indole-3-ethanol (IEtOH), indole-3-lactic acid (ILA), and indole-3-methanol (IMeOH) were identified.High performance liquid chromatography (HPLC) and GC-MS with selected ion monitoring were used to quantify levels of IAA and IEtOH inFrankia culture medium. IEtOH was present in greater abundance than IAA in every experiment. When no exogenous trp was supplied, no or only low levels of indole compounds were detected.Seedling roots ofAlnus rubra incubated in axenic conditions in the presence of indole-3-ethanol formed more lateral roots than untreated plants, indicating that IEtOH is utilized by the host plant, with physiological effects that modify patterns of root primordium initiation.     

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.     

Quantification of indol-3-yl acetic acid in pea and maize seedlings by gas chromatography-mass spectrometry

A procedure is described for the identification and quantification of IAA in plant tissues by GC/MS analysis of the N-heptafluorobutyryl ethyl ester of IAA using [²H₅]IAA as an internal standard. The detection limit is ca 3 pmol IAA/tissue sample. By using this method, IAA levels of 30–90 pmol/g fr. wt were obtained for dark-grown Pisum sativum epicotyls and 71–199 pmol/g fr. wt for dark-grown Zea mays seedlings. When either methanol or ethanol was used as extraction solvent, some esterification of IAA during sample preparation was observed. No evidence for the natural occurrence of methyl or ethyl esters of IAA in Pisum sativum seedlings was found.     

Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

The role of lysines in Euglena cytochrome c-552. Chemical modification studies.

2-Hydroxy(p-hydroxyphenyl)-acetaldoxime, the alternative precursor to p-hydroxyphenylacetonitrile in dhurrin biosynthesis, was synthesized and its effectiveness as a substrate was examined in a microsomal enzyme system from sorghum seedlings. The hydroxyaldoxime was slowly converted to p-hydroxymandelonitrile when compared with p-hydroxyphenylacetonitrile and p-hydroxyphenylacetaldoxime. Moreover, radioactivity from [U-¹⁴C]tyrosine was efficiently incorporated by trapping experiments into both the nitrile and aldoxime, but not into the hydroxyaldoxime. The reaction products formed on hydroxylation of the nitrile by the microsomal enzyme were identified as p-hydroxybenzaldehyde, HCN, and H₂O. Under anaerobic conditions, the nitrile was produced from the aldoxime and accumulated without undergoing hydroxylation. These results establish p-hydroxyphenylacetonitrile and not 2-hydroxy(p-hydroxyphenyl)-acetaldoxime as the intermediate in the conversion of p-hydroxyphenylacetaldoxime to p-hydroxymandelonitrile in dhurrin biosynthesis.     

Analysis of Indole-3-acetic Acid Metabolism in Zea mays Using Deuterium Oxide as a Tracer

A method using deuterium oxide (D₂O) as a tracer was used to study indole-3-acetic acid (IAA) metabolism in Zea mays seedlings. Seeds were imbibed and grown for 4 days in 30% D₂O in the dark. IAA was then isolated from roots and shoots and analyzed for deuterium content by mass spectrometry. We found that a significant portion of the IAA isolated from plants had incorporated deuterium at nonexchangeable sites of the indole ring. This indicates that some of the IAA in the germinating seedling is made via de novo indole synthesis. Moreover, we found that the deuterium content of IAA was 2.6 times greater in shoots than in roots. These results indicate that at least some of the IAA in roots and shoots came from different biosynthetic pathways. It appears that the fraction of IAA produced via de novo indole synthesis is greater in shoots than in roots.     

Influence of L-tryptophan and auxins applied to the rhizosphere on the vegetative growth of Zea mays L.

Glasshouse experiments were conducted to evaluate the influence of L-TRP in comparison with indole-3-acetamide (IAM), tryptophol (TOL) and indole-3-acetic acid (IAA) on the growth of Zea mays L. var. Early Sunglow. L-TRP (25 to 2.5×10^–5 mg kg^–1 soil), IAM (22 to 2.2×10^–5 mg kg^–1 soil), TOL (20 to 2.0×10^–5 mg kg^–1 soil), and IAA (22 to 2.2×10^–5 mg kg^–1 soil) were applied as a soil drench to established uniform seedlings. All treatments were applied in a completely randomized design with 10 replicates. IAM had no significant effect on the plant growth parameters. Shoot height, uppermost leaf collar base distance, internodal distance, and shoot dry and fresh weights were significantly improved upon the addition of TOL (2.0×10^–2 mg kg^–1 soil), however, the highest concentration (20 mg kg^–1 soil) caused a 14.6% reduction in leaf width. L-TRP (2.5×10^–3 mg kg^-1 soil) also had a significant influence on shoot height, uppermost leaf collar base distance, internodal distance and fresh weight of shoot compared with the control. The highest concentration of L-TRP (25=mg kg^–1 soil) had a negative effect on leaf width and dry weight of the shoot. The most pronounced response on the corn growth parameters was observed with the application of IAA at lower concentrations (2.2×10^–5 to 2.2×10^–2 mg kg^–1 soil) specifically improving root growth. The highest concentration (22 mg kg^–1 soil) of IAA had a significant negative effect on plant height, leaf width, stem diameter, shoot fresh and dry weight. These findings indicate that L-TRP applied at the appropriate concentrations can have positive effects on corn growth comparable to pure auxins (TOL and IAA).     

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