Identification of 1,4-Benzoxazin-3-ones in Maize Extracts by Gas-Liquid Chromatography and Mass Spectrometry

Gas-liquid chromatography-mass spectrometry (GLC-MS) has been used for the separation, detection, and identification of 1,4-benzoxazin-3-ones (hydroxamic acids and lactams) and benzoxazolinones found in maize (Zea mays L.) extracts. Compounds of interest were partitioned into ethyl acetate from aqueous maize seedling extracts. For analysis by GLC-MS, trimethylsilyl derivatives were prepared, chromatographed on a column of 3% OV-1, and detected in the mass spectrometer. Mass spectra were obtained for all peaks present in extracts of four maize lines. A data comparison system was developed for relating unidentified spectra to the spectra of the reference compounds. Based on spectral comparisons, three hydroxamic acids (2,4-dihydroxy-2H-1, 4-benzoxazin-3(4H)-one; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one), three lactams (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one; 2,7-dihydroxy-2H-1,4-benzoxazin-3(4H)-one; and 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one), one benzoxazolinone (6-methoxybenzoxazolinone), and two organic acids (malic and aconitic) were identified in the extracts. In addition, one other hydroxamic acid and one other related compound were tentatively identified based on mass spectral evidence.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, an Inhibitor from Zea mays with Differential Activity against Soft Rotting Erwinia Species

[2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] DIMBOA was extracted with ethyl acetate from acidified water homogenates of corn (Zea mays L.) seedlings. Pure DIMBOA or ethyl acetate extracts of corn tissue were added to bacterial growth medium at five concentrations (measured as hydroxamates). DIMBOA and corn extracts were more inhibitory to soft rot bacteria (Erwinia spp.) that are nonpathogenic to corn than to soft rot bacteria that are corn pathogens. The inhibitory activity of DIMBOA was similar to that of the ethyl acetate extracts. Both corn extracts and DIMBOA prolonged the lag phase of bacterial growth without significantly changing log phase growth rates. At various concentrations of the inhibitor, 50 to 100% of the activity of corn extracts inhibitory to different bacterial isolates was attributable to DIMBOA. Extracts of DIMBOA-deficient plants (genotype bxbx) were not inhibitory to Erwinia spp. It was concluded that DIMBOA is the major active component in those corn extracts which are inhibitory to soft rot Erwinia species.     

<Emphasis Type="Italic">ScBx</Emphasis> gene based association analysis of hydroxamate content in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

Hydroxamates (HX) are major secondary metabolites synthesized by rye and are responsible for some of the unique properties of this cereal, including good tolerance of biotic and abiotic stresses and allelopathy. Recently, five genes encoding enzymes taking part in HX biosynthesis have been sequenced and characterized, which was the starting point to undertake the present study. Association analysis of the content of six HX–HBOA (2-hydroxy-1,4-benzoxazin-3-one), GDIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one glucoside), DIBOA (2,4-dihydroxy-1,4-benzoxazin-3(4H)-one), GDIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one glucoside), DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3(4H)-one) and MBOA (6-methoxy-benzoxazolin-2(3H)-one) in the above-ground parts of plants and roots was performed on a population consisting of 102 and 121 diverse inbred lines, in 2013 and 2014, respectively. Altogether, 48 single nucleotide polymorphisms (SNPs) were found to be associated with the content of at least one HX: 20 SNPs were associated with HX synthesized in the above-ground parts of rye plants (AG-SNP), and 28 were associated with HX synthesized in the roots (R-SNP). The highest number of SNPs was present in genes ScBx1 (9) and ScBx5 (11). The majority of SNPs were affected by environmental factors, except for two: ScBx4_1702 associated with GDIBOA and MBOA contents, and ScBx5_1105 associated with HBOA content in roots.     

Degradation of diazinon by 2,4-dihydroxy-7-methoxy-2h-1, 4-benzoxazin-3(4h)-one in maize

A cyclic hydroxamate, 2,4-dihydroxy-7-methoxy-2H- 1,4-benzoxazin-3(4H)-one (DIMBOA), was isolated and identified from shoots of 6-day-old corn seedlings grown in the dark. From 100 g of plant tissue 100 mg of DIMBOA were isolated. This hydroxamate was very effective in catalysing the hydrolysis of the pyrimidinyl organophosphate insecticide, diazinon (O, O-diethyl- O-[6- methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate) to 6- methyl-2-(1-methylethyl)-4-hydroxypyrimidine and diethyl phosphorothioic acid. The optimum pH for hydrolytic activity was 5 and at pH values equal to or higher than the pK_a of the hydroxamic group (6.95) most of the activity was lost.     

Hydroxamic Acid glucosyltransferases from maize seedlings

Hydroxamic acids occur in several forms in maize (Zea mays L.) with 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) being the predominant form and others including 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) being found at lower concentrations. Two enzymes capable of glucosylating hydroxamic acids were identified in maize protein extracts and partially purified and characterized. The total enzyme activity per seedling increased during the first 4 days of germination and was concurrent with the accumulation of DIMBOA. Purification of the enzymes by ammonium sulfate precipitation followed by Sephadex G-200 and Q-Sepharose gel chromatography resulted in a 13-fold increase in specific activity. The enzymes are initially separated into two peaks (peak 1 and peak 2) of activity by Q-Sepharose gel chromatography. The peak 1 glucosyltransferase had 3.6% of the DIMBOA glucosylating activity when DIBOA was used as substrate, whereas this percentage increased to 57% for the peak 2 enzyme. The enzyme in peak 2 has a K_m of 174 micromolar for DIMBOA and a K_m of 638 micromolar for DIBOA; the enzyme in peak 1 has a K_m of 217 micromolar for DIMBOA and its activity on DIBOA was too low to determine a K_m. The identification of two glucosyltransferases capable of glucosylating hydroxamic acids in vitro serves as an initial step in the characterization of the enzymes involved in production of hydroxamic acids in maize.     

Distribution and variations of three 1,4-benzoxazin-3-ones in maize induced by the Asian corn borer, Ostrinia furnacalis (Guenée)

Contents of three 1,4-benzoxazin-3-ones in tissue samples from different parts (young leaf, second leaf, old leaf, stem and root) of young maize plants of 4-leaves stage, fed by the third instar larvae of the Asian corn borer, Ostrinia furnacalis (Guenée), were analyzed by high-performance liquid chromatography-mass spectroscopy (HPLC-MS). Samples were taken immediately (set A) or 48 h (set B) after larvae had fed on the second leaf for 48 h. The three 1,4-benzoxazin-3-ones investigated in our experiments were 2,4-dihydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (DIMBOA), 2,4-dihydroxy-1,4(2H)-benzoxazin-3-one (DIBOA) and 2-hydroxy-7-methoxy-1,4(2H)-benzoxazin-3-one (HMBOA). In samples of set A, the levels of DIMBOA and HMBOA were significantly lifted in the old leaf (L3) and young leaf (L1), respectively, while amounts of these two chemicals in other plant parts were not significantly different between larvae-fed plants and intact plants. Concentrations of DIBOA in each plant part remained unchanged. In samples of set B, no concentration differences for any of these three 1,4-benzoxazin-3-ones between larvae-fed plants and controls were observed in any plant part. The feeding of the Asian corn borer seems to have limited effects on induction of these three 1,4-benzoxazin-3-ones in young maize plants of the variety investigated.     

Factors That Influence the Activity of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one on Erwinia Species in Growth Assays

Factors affecting the inhibitory activity of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) against Erwinia carotovora, a nonpathogen of Zea mays L., and against a maize pathovar of Erwinia chrysanthemi (ECZ) were examined. Most experiments were performed with DIMBOA dissolved in a bacterial growth medium containing 10 g/liter of sucrose, inorganic salts, and 1 g/liter of casamino acids at pH 6.75. When temperature and pH were held constant, inhibition of E. carotovora varied linearly with the logarithm of the initial cell population. By altering temperatures, assays with constant pH and initial cell populations were performed under conditions of varying DIMBOA stability. When E. carotovora was grown at 24, 28, 32, and 36 C in the presence of 0.1 to 0.5 mm DIMBOA, the inhibition of bacterial growth was maintained long after DIMBOA had decomposed in the medium to levels which, if added initially, would not have been inhibitory. When assays were performed at pH 5.5, the pH of aqueous maize extracts, E. carotovora was more inhibited than at pH 6.75; however, ECZ was substantially less inhibited at the lower pH.     

Enantiomers of 2-methyl- and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid: Preparation by resolution,determination of absolute configuration,and Curtius rearrangement

Both enantiomers of 2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 2 and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 3 were prepared via resolution of the corresponding racemic carboxylic acids with (R)- and (S)-1-phenylethylamine, respectively. Absolute configuration of (−)-(R)-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid was determined by X-ray crystallography. Curtius rearrangement of acyl azides prepared from enantiomers of these heterocyclic carboxylic acids carried out in benzyl alcohol afforded enantiomers of the corresponding benzyl carbamates, which upon hydrogenolysis gave racemic 2-amino-2-methyl-3,4-dihydro-2H-1,4-benzoxazin-3-one 4 and 2-amino-2,4-dimethyl-3,4-dihydro-2H-1,4h-benzoxazin-3-one 5. Chirality 10:791–799, 1998. © 1998 Wiley-Liss, Inc.     

Effects of four benzoxazinoids on gibberellin-induced alpha-amylase activity in barley seeds

Germination of barley seeds was inhibited by 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) at concentrations greater than 0.03mmol/L, and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) and benzoxazolin-2(3H)-one (BOA) at concentrations greater than 0.1mmol/L. These benzoxazinoids also inhibited the induction of alpha-amylase activity in the barley seeds, and inhibited gibberellin-induced alpha-amylase activity in de-embryonated barley seeds. Significant inhibition in the germination and alpha-amylase induction were observed as concentrations of DIMBOA, DIBOA, MBOA and BOA increased. These results suggest that DIMBOA, DIBOA, MBOA and BOA may inhibit the germination of barley seeds by inhibiting the gibberellin-induced process, leading to alpha-amylase production. The inhibitory activities of germination and alpha-amylase induction of DIMBOA and DIBOA were greater than those of their degraded substances MBOA and BOA, respectively, and the inhibitory activities of DIMBOA and MBOA were greater than those of their demethoxylated analogues DIBOA and BOA, respectively.     

Decomposition of 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one in Aqueous Solutions

Cyclic hydroxamic acids present in some species of Gramineae have been reported to be important in resistance of these plants to fungi and insects. Since the nonglucosylated forms of these acids are unstable in aqueous solution, in vitro methods for the measurement of their antibiotic properties have been difficult. Kinetics of the decomposition of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), the major hydroxamate in corn (Zea mays L.) extracts, were studied in buffered aqueous solutions from pH 5 to 7.5 at temperatures from 20 to 80 C. Kinetics were apparently first order under all conditions tested; energies of activation (24 to 28 kcal/mol) were nearly pH-independent. DIMBOA decomposed rapidly (half-life = 5.3 hours at 28 C, pH 6.75) relative to the time required for many procedures which have been used to demonstrate the biological activity of DIMBOA. The rate of disappearance of inhibitory activity of DIMBOA toward Erwinia carotovora was indistinguishable from the rate of decomposition of DIMBOA. Contrary to reports, yields of 6-methoxy-2-benzoxazolinone (MBOA) were not quantitative. Gas-liquid chromatography analytical procedures were developed for quantitation of trimethylsilyl and acetyl derivatives of MBOA. As measured by ultraviolet spectroscopy and/or gas-liquid chromatography, conversion of DIMBOA to MBOA ranged from 40 to 75% of theoretical in aqueous buffers, bacterial growth medium, and ethyl acetate extracts of corn tissue resuspended in buffer. Yields varied with temperature, pH, and constituents in the medium.     

Chemical constituents from the roots of Acanthus ilicifolius var. xiamenensis

Chemical investigation of Acanthus ilicifolius var. xiamenensis led to the isolation of eleven compounds, and their structures were identified to be 2-benzoxazolinone (1), 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one (2), (2R)-2-O-β-d-glucopyranosyl-2H-1,4-benzox azin-3(4H)-one (3), (2R)-2-O-β-d-glucopyranosyl-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one (4), (2R)-2-O-β-d-glucopyranosyl-7-hydroxy-2H-1,4-benzoxazin-3(4H)-one (5), lyoniside (6), 3′-methoxy-luteolin-7-O-β-d-glucopyranoside (7), β-sitosterol-3-O-β-d-glucopyranoside (8), stigmasterol octadecanoate (9), β-sitosterol octadecanoate (10), stigmasterol-3-O-β-d-glucopyranoside (11) on the basis of mass and NMR spectra. This is the first report on the occurrence of compound 6 and 7 in Acanthaceae. This work also represents the first phytochemical work on the roots of A. ilicifolius var. xiamenensis.     

Fluconazole analogues containing 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moieties,a novel class of anti-Candida agents

As a part of our program to develop new antifungal agents, a series of fluconazole analogues was designed and synthesized wherein one of the triazole moieties in fluconazole was replaced with 2H-1,4-benzothiazin-3(4H)-one or 2H-1,4-benzoxazin-3(4H)-one moiety. The new chemical entities thus synthesized were screened against various fungi and it was observed that the compounds 4a and 4i are potent inhibitors of Candida strains. The structure–activity relationship for these compounds is discussed.     

Copper(II) complexes of a hydroxamic acid from maize

The thermodynamics of formation for DIMBOA-Cu(II) complexes (where DIMBOA = 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-4H-one, a hydroxamic acid from maize) has been investigated in aqueous solutions by a potentiometric method. DIMBOA forms 1:1 and 1:2 chelates with Cu(II) at ionic strength 0.05 M (NaCl04). The stability constants measured were about 10⁵ and 10⁴ for the 1:1 and 1:2 complexes respectively, determined at 10, 20 and 30°. The contribution of ΔH and ΔS to the stability of complexes is examined and the pK values are compared with other ligands found in maize. Although DIMBOA has similar or higher constants to form copper complexes than other plant ligands, its possible role as a transport agent in maize remains to be established.     

A thin layer chromatographic technique for detecting inducers of Agrobacterium virulence genes in corn,wheat and rye

Summary A method is described for detecting plant metabolites capable of inducing the virulence genes of Agrobacterium tumefaciens. The method uses A. tumefaciens containing a plasmid with an inducible virulence gene fused to a galactosidase gene (virE::lacZ). Thin layer chromatography plates are overlayed with agar containing the indicator bacterium and a chromogenic galactoside (X-gal). Virulence gene inducing plant metabolites induce galactosidase which releases an aglycone readily oxidized by air to a blue pigmented zone at the Rf of the inducer. The method has been used to demonstrate the presence of virulence gene inducers in corn, wheat and rye. The uninduced background level of galactosidase also permits detection of bacterial growth inhibitors after a longer incubation period.Abbreviations X-gal 5-Bromo-4-chloro-3-indoxyl ß-D-galactopyranoside - TLC Thin Layer Chromatography - AS Acetosyringone - DIMBOA 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one - MBOA 6-Methoxy-2(3H)-benzoxazolone - T-DNA Transfer DNA - Ti Plasmid Tumor-inducing Plasmid     

Identification and quantification of hydroxamic acids in maize seedling root tissue and impact on western corn rootworm (Coleoptera: Chrysomelidae) larval development

Hydroxamic acid content was analyzed in the root tissue of four maize, Zea mays L., lines using high-performance liquid chromatography (HPLC) and related to western corn rootworm, Diabrotica virgifera virgifera LeConte, larval development and survivorship. Maize lines evaluated included Mp710 (PI 596627), MpSWCB-4, (PI 550498), Sc213 (PI 548792), and Dk580 (DeKalb commercial hybrid). Maize plants from each line were grown in test tubes containing a transparent agarose gel medium in a growth chamber. After 8 d of growth, root tissue of each line was harvested and hydroxamic acid content analyzed using HPLC. Three hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), 6-methoxybenzoxazolinone (MBOA), and 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), were identified in the maize roots tested. DIMBOA concentration was quantified and ranged from 246.37 +/- 70.53 micrograms to 91.84 +/- 49.82 micrograms DIMBOA per gram of root tissue. No significant difference was found among lines in D. v. virgifera larval development and survivorship.     

Biotransformation of 2-Benzoxazolinone and 2-Hydroxy-1,4-Benzoxazin-3-one by Endophytic Fungi Isolated from Aphelandra tetragona

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from Aphelandra tetragona was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified. Fusarium sambucinum detoxified BOA and HBOA to N-(2-hydroxyphenyl)malonamic acid. Plectosporium tabacinum, Gliocladium cibotii, and Chaetosphaeria sp. transformed HBOA to 2-hydroxy-N-(2-hydroxyphenyl)acetamide, N-(2-hydroxyphenyl)acetamide, N-(2-hydroxy-5-nitrophenyl)acetamide, N-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3H-phenoxazin-3-one, 2-acetylamino-3H-phenoxazin-3-one, and 2-(N-hydroxy)acetylamino-3H-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-N-(2-hydroxyphenyl)[¹³C₂]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to o-aminophenol as a key intermediate.     

Chromones from the tubers of Eranthis cilicica and their antioxidant activity

Chemical studies on the constituents of Eranthis cilicica led to isolation of ten chromone derivatives, two of which were previously known. Comprehensive spectroscopic analysis, including extensive 1D and 2D NMR data, and the results of enzymatic hydrolysis allowed the chemical structures of the compounds to be assigned as 8,11-dihydro-5-hydroxy-2,9-dihydroxymethyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 5,7-dihydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 5,7-dihydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-2-methyl-4H-1-benzopyran-4-one, 7-[(β-d-glucopyranosyl)oxy]-5-hydroxy-2-hydroxymethyl-8-[(2E)-4-hydroxy-3-methylbut-2-enyl]-4H-1-benzopyran-4-one, 9-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-8,11-dihydro-5,9-dihydroxy-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, 8,11-dihydro-5,9-dihydroxy-9-hydroxymethyl-2-methyl-4H-pyrano[2,3-g][1]benzoxepin-4-one, and 7-[(O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)oxy]methyl-4-hydroxy-5H-furo[3,2-g][1]benzopyran-5-one, respectively. The isolated compounds were evaluated for their antioxidant activity.     

The role of DIMBOA on the feeding of Asian corn borer, Ostrinia furnacalis (Guenée) (Lep., Pyralidae)

The role of 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA), on feeding of Asian corn borer (ACB), Ostrinia furnacalis (Guenée), was observed and quantified. DIMBOA had an antifeedant effect on ACB and a mathematical model was built for the correlation between DIMBOA concentration and antifeedant index. DIMBOA increased the time that the larvae required to reach the pupal stage and the larval weights in treatment were significantly less than those in control. The number of entrances of ACB on the plant sites were positively correlative to the DIMBOA concentrations, suggesting that feeding stimulants in the corn plants could probably override the antifeedant effect of DIMBOA     

Occurrence and Characterization of 2-Hydroxy-1,4-benzoxazin-3-one and Indole Hydroxylases in Juvenile Wheat

Cyclic hydroxamic acids, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy analogue (DIMBOA), occur transiently in high amounts in wheat and maize during the juvenile, non-autotrophic stage of growth. To elucidate the biosynthetic enzymes operating for the transient production of these compounds, we examined the hydroxylating activities for 2-hydroxy-1,4-benzoxazin-3-one (HBOA), the immediate precursor of DIBOA, and indole, the first intermediate of the biosynthetic pathway that branches off from the tryptophan pathway, by using microsomes prepared from wheat seedlings. Both hydroxylases occurred soon after germination, reached a maximum 48 h after germination, and decreased to finally disappear as the plants grew into the autotrophic growth stage. The mode of appearance and disappearance similar to that of hydroxamic acids, suggesting that elevated expression of the whole set of enzymes involved in the biosynthesis after indole was responsible for the transient occurrence of hydroxamic acids. The hydroxylating activity was also observed for 1,4-benzoxazin-3-one, a putative precursor of HBOA, but to significantly less extent than that for HBOA and indole.