Benzoxazinoids-cyclic hydroxamic acids, lactams and their corresponding glucosides in the genus Aphelandra (Acanthaceae)

An improved method of sample preparation and simultaneous HPLC separation was developed that allowed the separation of 2,4-dihydroxy-1,4-benzoxazine-3(4H)-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazine-3(4H)-one (DIMBOA), 2-hydroxy-1,4-benzoxazine-3(2H)-one (HBOA), 2-hydroxy-7-methoxy-1,4-benzoxazine-3(2H)-one (HMBOA) and their corresponding glucosides as well as the benzoxazolinones BOA and MBOA. The amount and distribution of these compounds was determined in the roots of Aphelandra squarrosa and A. fuscopunctata plants. There is a significant difference in the amount and distribution of this substance class in the two species analyzed. The results are discussed in relation to their function as defence compounds and allelochemicals.     

In vitro analysis of DIMBOA catabolism in the Asian corn borer <Emphasis Type="Italic">Ostrinia furnacalis</Emphasis> (Lepidoptera: Crambidae)

Larvae of the Asian corn borer Ostrinia furnacalis (Guenée) must cope with 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), a major toxic allelochemical present in its host plant, maize Zea mays L. UDP-glucosyltransferase (UGT), which conjugates glucose to various lipophilic toxic compounds and thereby makes them more hydrophilic for easier excretion, has been suggested to be involved in the detoxification of DIMBOA in several insects. Our previous in vitro assays using O. furnacalis midgut homogenates demonstrated that DIMBOA was catabolized only when UDP-glucose, a glucose donor for UGT activity, was included in the reaction mixture; however, DIMBOA glucoside, the expected product of UGT activity, was only detected in trace amounts in assay products. The present study revealed that DIMBOA glucoside was produced, but was immediately degraded by unidentified enzymes in the midgut homogenate that do not require UDP-glucose for their activities, suggesting the presence of a novel route for DIMBOA catabolism in O. furnacalis.     

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.     

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.     

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.     

Quantitation of 1,4-Benzoxazin-3-ones in Maize by Gas-Liquid Chromatography

A gas-liquid chromatographic (GLC) procedure is reported for the quantitation of the trimethylsilyl (TMS) derivatives of substituted 2-hydroxy-2H-1,4-benzoxazin-3(4H)-ones (2-hydroxy-2H-1,4-benzoxazin-3(4H)-one[HBOA]; 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[HMBOA];2,4- dihydroxy-2H-1,4-benzoxazin-3(4H)-one[DIBOA]; 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one[DIMBOA]; and 2,4-dihydroxy-7,8-dimethoxy-2H-1,4-benzoxazin-3(4H)-one[DIM ₂BOA]) found in maize (Zea mays L.) extracts. Derivatized samples were chromatographed on columns with liquid phases of 2% DC-11 and 3% OV-17 and detected by flame ionization. Internal standards were methyl palmitate and methyl stearate on DC-11 and methyl behenate on OV-17. Detector response was linear to at least 5 nanomoles for TMS₂-HBOA and TMS₂-DIBOA and to 19 nanomoles for TMS₂-DIMBOA. Standard errors of 2% or less were obtained when four replicate samples were analyzed. For each of the 15 maize lines examined, the amount of DIMBOA determined by GLC was directly proportional to the amount of ferric chloride-reactive material determined colorimetrically.     

Hydroxamic acids in Secale cereale L. and the relationship with their antifeedant and allelopathic properties

Contents of the hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in leaves and roots of 14 cultivars of rye, Secale cereale L., were determined. Dynamics of accumulation in three cultivars were evaluated. DIBOA was the main cyclic hydroxamic acid in leaves but the contents differed significantly between the cultivars. Both DIBOA and DIMBOA were present in the roots. Maximum concentration of DIBOA in leaves and DIMBOA in roots was reached between 48-54 h and 54-72 h after germination, respectively. Antifeedant activity of DIBOA towards the aphid Rhopalosiphum padi and the feeding behavior were studied by electronic recording in barley leaves treated with different contents of DIBOA. The deleterious activity of DIBOA could arise by starvation and/or a toxic effect. Additionally, allelopathic potential of pure DIBOA and aqueous extracts of leaves and roots of rye (Tetra-Baer) on the germination of lettuce (Lactuca sativa) and rye (Tetra-Baer) seeds was evaluated. A high percentage of germination inhibition of pure DIBOA and the extracts of leaves and roots was observed. The activity is in agreement with the contents of hydroxamic acids in the plants. The substrates had no allelopathic effect on rye seeds.     

Detoxification of corn antimicrobial compounds as the basis for isolating Fusarium verticillioides and some other Fusarium species from corn.

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.     

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.     

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.     

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.     

Induction of beta-glucosidase activity in maize coleoptiles by blue light illumination

The role of beta-glucosidase during the phototropic response in maize (Zea mays) coleoptiles was investigated. Unilateral blue light illumination abruptly up-regulated the activity of beta-glucosidase in the illuminated halves, 10 min after the onset of illumination, peaking after 30 min and decreasing thereafter. The level of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), which is released from DIMBOA glucoside (DIMBOA-Glc) by beta-glucosidase, and its degradation compound 6-methoxy-benzoxazolinone (MBOA) were elevated within 30 min in the illuminated halves as compare to the shaded halves, prior to the phototropic curvature. Furthermore, beta-glucosidase inhibitor treatment significantly decreased the phototropic curvature and decreased growth suppression in the illuminated sides. These results suggest that blue light induces the activity of beta-glucosidase in the illuminated halves of coleoptiles causing an increase in DIMBOA biosynthesis and the growth inhibition that leads to a phototropic curvature.     

Detoxification of Corn Antimicrobial Compounds as the Basis for Isolating Fusarium verticillioides and Some Other Fusarium Species from Corn

The preformed antimicrobial compounds produced by maize, 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3-one and its desmethoxy derivative 2,4-dihydroxy-2H-1,4-benzoxazin-3-one, are highly reactive benzoxazinoids that quickly degrade to the antimicrobials 6-methoxy-2-benzoxazolinone (MBOA) and 2-benzoxazolinone (BOA), respectively. Fusarium verticillioides (= F. moniliforme) is highly tolerant to MBOA and BOA and can actively transform these compounds to nontoxic metabolites. Eleven of 29 Fusarium species had some level of tolerance to MBOA and BOA; the most tolerant, in decreasing order, were F. verticillioides, F. subglutinans, F. cerealis (= F. crookwellense), and F. graminearum. The difference in tolerance among species was due to their ability to detoxify the antimicrobials. The limited number of species having tolerance suggested the potential utility of these compounds as biologically active agents for inclusion within a semiselective isolation medium. By replacing the pentachloronitrobenzene in Nash-Snyder medium with 1.0 mg of BOA per ml, we developed a medium that resulted in superior frequencies of isolation of F. verticillioides from corn while effectively suppressing competing fungi. Since the BOA medium provided consistent, quantitative results with reduced in vitro and taxonomic efforts, it should prove useful for surveys of F. verticillioides infection in field samples.     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

Elucidation of the final reactions of DIMBOA-glucoside biosynthesis in maize: characterization of Bx6 and Bx7

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate K(m) values (below 0.4 mm) and k(cat) values of 2.10 s(-1) and 0.25 s(-1), respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.     

Variation of DIMBOA and related compounds content in relation to the age and plant organ in maize

We report the variation of all 1,4-benzoxazin-3-one derivatives content detectable in maize with plant age in roots and aerial parts. Our results show that the concentration of hydroxamic acids, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc) and its 8-methoxylated analogue (DIM2BOA-Glc) is high after seed germination and then decreases with plant age. Nevertheless, these compounds continue to be biosynthesised during 6-10 days after germination. Variation in concentration of N-O-methylated DIMBOA-Glc (HDMBOA-Glc) is similar to the one of hydroxamic acids in aerial parts. On the contrary, in roots, its concentration remains relatively stable with plant age. After 10 days, HDMBOA-Glc becomes the main compound in roots. This compound is also present in higher concentration than hydroxamic acids in the oldest leaf of 20-day-old maize. The presence of four other DIMBOA related compounds in maize plants depends on variety, age and tissue. The role of these compounds in plant resistance to aphids is discussed.     

Leaf-rust resistance in rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.). 2. Genetic analysis and mapping of resistance genes <Emphasis Type="Italic">Pr3</Emphasis>, <Emphasis Type="Italic">Pr4</Emphasis>, and <Emphasis Type="Italic">Pr5</Emphasis>

Three dominant resistance genes, Pr3, Pr4, and Pr5, were identified by genetic analysis of resistance to leaf rust in rye (Puccinia recondita f. sp. secalis). Each of the three genes confers resistance to a broad scale of single-pustule isolates (SPIs), but differences could be observed for specific Pr gene/SPI combinations. Resistance conferred by the three genes was effective in both detached-leaf tests carried out on seedlings and in field tests of adult plants. Molecular marker analysis mapped Pr3 to the centromeric region of rye chromosome arm 1RS, whereas Pr4 and Pr5 were assigned to the centromeric region of 1RL. Chromosomal localization and reaction patterns to specific SPIs provide evidence that the three Pr genes represent distinct and novel leaf-rust resistance genes in rye. The contributions of these genes to resistance breeding in rye and wheat are discussed.The authors dedicate this paper to Prof. Dr. H.H. Geiger, University of Hohenheim, on the occasion of his 65th birthday.An erratum to this article can be found at     

Inhibition of ATPase from chloroplasts by a hydroxamic acid from the gramineae

-DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one), a hydroxamic acid from the Gramineae involved in the resistance of cereals to aphids, inhibits     

Genotypes,haplotypes and diplotypes of <Emphasis Type="Italic">IGF</Emphasis>-<Emphasis Type="Italic">II</Emphasis> SNPs and their association with growth traits in largemouth bass (<Emphasis Type="Italic">Micropterus salmoides</Emphasis>)

Insulin-like growth factor II (IGF-II) is involved in the regulation of somatic growth and metabolism in many fishes. IGF-II is an important candidate gene for growth traits in fishes and its polymorphisms were associated with the growth traits. The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the largemouth bass (Micropterus salmoides) IGF-II gene and to analyze potential association between IGF-II gene polymorphisms and growth traits in largemouth bass. Four SNPs (C127T, T1012G, C1836T and C1861T) were detected and verified by DNA sequencing in the largemouth bass IGF-II gene. These SNPs were found to organize into seven haplotypes, which formed 13 diplotypes (haplotype pairs). Association analysis showed that four individual SNPs were not significantly associated with growth traits. Significant associations were, however, noted between diplotypes and growth traits (P < 0.05). The fish with H1H3 (CTCC/CGCC) and H1H5 (CTCC/TTTT) had greater body weight than those with H1H1 (CTCC/CTCC), H1H2 (CTCC/TGTT) and H4H4 (TGCT/TGCT/) did. Our data suggest a significant association between genetic variations in the largemouth bass IGF-II gene and growth traits. IGF-II SNPs could be used as potential genetic markers in future breeding programs of largemouth bass.     

The possible role of hydroxylation in the detoxification of atrazine in mature vetiver (Chrysopogon zizanioides Nash) grown in hydroponics

The resistance mechanism of vetiver (Chrysopogon zizanioides) to atrazine was investigated to evaluate its potential for phytoremediation of environment contaminated with the herbicide. Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones, conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated by cytochromes P450. All three possibilities were explored in mature vetiver grown in hydroponics during this research project. Here we report on the chemical role of benzoxazinones in the transformation of atrazine. Fresh vetiver roots and leaves were cut to extract and study their content in benzoxazinones known to hydroxylate atrazine, such as 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA), 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and their mono- and di-glucosylated forms. Identification of benzoxazinones was performed by thin layer chromatography (TLC) and comparison of retention factors (Rf) and UV spectra with standards: although some products exhibited the same Rf as standards, UV spectra were different. Furthermore, in vitro hydroxylation of atrazine could not be detected in the presence of vetiver extracts. Finally, vetiver organs exposed to [14C]-atrazine did not produce any significant amount of hydroxylated products, such as hydroxyatrazine (HATR), hydroxy-deethylatrazine (HDEA), and hydroxy-deisopropylatrazine (HDIA). Altogether, these metabolic features suggest that hydroxylation was not a major metabolic pathway of atrazine in vetiver.