Role of natural benzoxazinones in the survival strategy of plants

Dieter Sicker*,Monika Freyt, Margot Schulz$,and Alfons Gierlt *Institute of Organic Chemistry, University of Leipzig, D-04103 Leipzig, Germany; bstitut fiir Genetik, Technische UniversitS Miinchen, D-85747 Garching, Germany; and SInstitute of Agricultural Botany, University of Bonn, D-53115 Bonn, Germany

Benzoxazinoidacetal glucosidesare a unique class of natural products abundant in Gramineae, includingthe major agriculturalcrops maize, wheat, and rye. These secondary metabolites are also found in several dicotyledonousspecies. Benzoxazinoids serve as important factors of host plant resistanceagainst microbialdiseasesand insects and as allelochemicalsand endogenous ligands. Interdisciplinaryinvestigationsby biologists,biochemists,and chemists are stimulated by the intention to make agricultural use of the benzoxazinonesas natural pesticides.These natural products are not only constituents of a plant defense system but also part of an active allelochemicalsystem used In the competition with other plants. This review covers biologicaland chemical aspects of benzoxazinone researchover the last decade with special emphasis on recent advances in the elucidationof the biosyntheticpathway. KEY WORDS: Benzoxazinones,Cyclichydroxamicacids, Acetal Glucosides, Biosynthesis,Defence, Allelopathy. o 2000Academicpress.

I. Introduction

Natural benzoxazinones were discovered 40 years ago in rye when resistance against pathogenic fungi was investigated. The first benzoxazinone aghicone and the first glucoside were reported in two successive papers (Virtanen and Hietala, 1960; Hietala and Virtanen, 1960). The discovery that natural benzoxazinones function as pesticides resulted in interdisciplinary research for understanding all aspects of this unique class of natural products. The natural occurrence of beuzoxazinoids and their role in host plant resistance International Review 0074.7696100 $35.00

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Copyright 0 2000 by Academic Press All rights OF reproduction in my form reserved.

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to pests and diseases have been reviewed by several authors (Meyer, 1988; Niemeyer, 1988; Gross, 1989). Structural requirements for the biological activity of benzoxazinoids and possible molecular mechanisms of action have been discussed (Hashimoto and Shudo, 1996). Recently, synthetic approaches to acetal glucosides and aglucones as well as to analogues have been documented (Sicker et al, 1997). A substantial part of benzoxazinoid biosynthesis has been elucidated (Frey et aZ., 1997). It is the aim of this review to summarize the genetic, biochemical, and cellular information on biosynthesis and to relate this information to plant host defense and allelopathic aspects of benzoxazinone research of the last decade.

II. General A. Structure

Characteristics

of Benzoxazinones

and Distribution

Benzoxazinones are stored in the vacuole of plant cells as D-glucosides. Because of active endogenous glucosidases, aglucones are produced when aqueous extracts are made from plant material. The 2-hydroxy-2H-1,4benzoxazin3(4H)-one skeleton and the D-glucose unit show (2R)-2-Plinkage without exception, although four stereochemical possibilities exist (Fig. 1). Acronyms are used in the literature that are derived from the chemical designation, e.g. DIMBOA, one of the best-studied benzoxazinones, is derived from 2,4-dihydroxy-7-methoxy-2H-l,Cbenzoxazin3(4H)-one. The term cyclic hydvoxamic acids or hydroxamic acids is often used for these natural products, although some of them do not contain a cyclic hydroxamic acid but the related lactam. However, it is only with the combination of hydroxamic acid with the cyclic hemiacetal unit that these secondary metabolites receive their unique bioactive properties. Furthermore, the bioactivity can be clearly enhanced by a 7-methoxy or 7-hydroxy group as a donor substituent. To avoid misinterpretation of the chemical structures responsible for biological effects, a neutral term like benzoxazinones or benzoxazinoids is used here. A unique feature of benzoxazinones is the presence of a nitrogen atom in the cyclic hemiacetal ring of the aglucone. From the chemist’s point of view, this is the structural source of a certain instability essential to obtain the chemical reactivity required for the defence reaction. An overview on benzoxazinones is given in Fig. 1. Note that TRIBOA has only been detected as an aglucone, not as a corresponding ~-P-Dglucoside, whereas HMzBOA was only described as glucoside. Further-

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acetal glucosides I ,

IOH

OH

H

I3H

OMe

H

I3H ,OMe IH

OMe OMe H

OMe H H

IH IH

OH OMe

H H

I

aglucones (racemic) plant (family) reference Secale cereale (Gramineae) ‘, Triticum aestivum (Gramineae) b, Zea mays (Gramineae) ‘, Hordeum vulgare (Gramineae) ‘, Agropyron repens (Gramineae) “, Acanthus mollis (Acanfhaceae) ‘, Aphelandra tetragona (Acanfhaceae) g, Consolida orientalis [i?anunculaceae) h Zea mays ‘, Crqssandra pungens (Acanfhaceae) ’ Triticum aestivum “, Zea mays K,‘, Agropyron repens m, Aphelandra auriantica, A. sauarrosa (Abanfhaceae) i Zea mays ’ Zea mays * Blepharis edulis Pers. (Acanfhaceae) ‘, Zea mays 4 Scoparia dulois (Scrophulariaceae) 4 Aphelandra tetragona g Zea mays c Coix lachryma jobi r, Triticum aestivum “, Zea mays u Zea mays ’

FIG. 1 Bezoxazinods from plants. # All aglucones are released from corresponding (2R)-2P-D-glucosides by enzymatic cleavage. References for isolation of the glucosides are omitted, except for HMzBOA. References:” Virtanen and Hietala (1959),b Niemeyer (1988),): Woodward et al. (1979),d Barria et al. (1992).” Schulz et al. (1994)f Wolf et al. (1985),g Todorova et al. (1994),h &den et al. (1992): Pratt et al. (1995),k Wahlroos and Virtanen (1959): Hartenstein et al. (1992),” Friebe et al. (1995),n Hedin et al. (1993)p Chatterjee et al. (1990): Bailey and Larson (1991),4 Kamperdick et al. (1997); Nagao et al (1985); Grambow et al. (1986), Hofman and Masojidkova (1973).

more, the hydroxamic acid ester HDIBOA could only be proved by some spectroscopic methods (Hedin et al., 1993). In the aglucone a racemic mixture of the (2R)- and (2s) -enantiomers is always found with respect to the C(2)-0 bond. Benzoxazinones have been predominantly found in genera of the Gramineae. Outside the Gramineae, they have been isolated from various species

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of the Acanthaceae, Ranunculaceae, and Scrophulariaceae (Fig. 1). A systematic search for benzoxazinones has yet to be performed and might identify more species containing these natural products. There is a single report that bezoxazinones are synthezised in bacteria. The antibiotic C-1027, an antitumor chromoprotein has been isolated from Streptomyces globisporus (Otani et al., 1988). The absolute configuration of the C-1027 chromophore has been determined (Fig. 2; Iida et al., 1996). C-1027 belongs to the enediyne antibiotics and contains a 1,4-benzoxazin3(4H)-one structural moiety which resembles the benzoxazinoids from plant origin except for the 2-methylene group in place of the hemiacetalic 2-OH group. Evidence has been presented that the benzoxazinone moiety confers intercalative DNA binding of the C-1027 chromophore (Yu et al., 1995).

6. Degradation of Acetal Glucosides Benzoxazolin-2-[3H]-ones

to

Benzoxazinoid acetal glucosides are compounds of low toxicity that can undergo enzymatic and chemical degradation. In the intact plant cell, the acetal glucosides and the P-glucosidases are stored in two different cell compartments, the vacuole, and the plastid, respectively (Esen, 1992; Babcock and Esen, 1994). The benzoxazinone glucoside content in seedlings can reach concentrations between 1 and 10 mmoles per kilogram of fresh weight (Long et al., 1974; Tang et al., 1975). After cell damage, the glycoside

Me2N

0

NHz FIG.2 C-1027 Chromphor

from Streptomyces

globispoms.

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BENZOXAZINONESIN SURVIVAL STRATEGYOF PLANTS

and the enzyme are released and the hemiacetalic aglucone is produced (Fig 3). In maize the enzymatic release of DIMBOA after wounding is complete within half an hour. The aglucone has been found to be the toxic principle against microbial and insect pests (see Section IV). Interestingly, all aglucones containing the 2,4-dihydroxy-2H-l,4-benzoxazin-3(4H)-one skeleton (i.e., the direct combination of cyclic hydroxamic acid and cyclic hemiacetal unit) have been found to be chemically instable. As a result of the chemical degradation, benzoxazolin2(3H)ones are formed in a ring contraction reaction accompanied by the loss of formic acid. The half-life of DIMBOA in the exudate of injured maize cells is about 24 hr (Woodward et al., 1378). In fact, benzoxazinone acetal glucosides were detected from this end of the cascade, starting with the discovery of benzoxazolin-2(3N)-one (BOA) from rye (Virtanen and Hietala, 1955). Meanwhile, several substituted benzoxazolin-2(3H)-ones have been isolated from plants (Fig. 4). The glucosidic origin has only been established in the case of BOA and its 6-methoxy derivative MBOA. Very likely, DMBOA, 4-ABOA, 4-ClDMBOA and 5-Cl-MBOA are also derived from glucosides, although this has yet to be proven.

,,,,> ldZH RTc-)$L 0

&H

R=H,

OMe

Jtbglucosidase I

I

- HCOOH

FIG.3 Enzymatic and chemical degradation of a benzoxazinoid

acetal glucoside.

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SICKER ET AL. R4 R3

/ I

R”’ 0

R’

O)=o ‘;’ H

acronym BOA MBOA DMBOA 4-ABOA 4-Cl-DMBOA 5Cl-MBOA

R’ H H H AC Cl H

R2 H H H H H Cl

R3

R4

:Me OMe H OMe OMe

; OMe H OMe H

FIG.4 Structure and occurrence of natural benzoxazolin-2(3H)-ones. BOA was isolated from rye (Virtanen and Hietala, 1955; Barnes et al., 1987), from maize (Kosemura et aZ., 1995), from Blepharisedulis (Chatterjee et al., 1990), and from Aphelandra tetragona (Werner et al., 1993). MBOA was isolated from maize and wheat (Virtanen et al., 19.57), from Aphelundru tetrugona (Werner eta& 1993), from Coix-lachrymajobi (Nagao et al., 1985), and from Scopariu d&is (Chen and Chen, 1976; Hayashi et al., 1994). DMBOA, (Klun et al, 1970; Kosemura er al., 1995), 4-ABOA, (Fielder et al, 1994), and 4-Cl-DMBOA, (Kosemura et aZ., 1995), and 5-Cl-MBOA (Kato-Noguchi et aZ., 1998) were each isolated from maize.

III. Biosynthesis

of Benzoxazinoids

The benzoxazinoid pathway has been elucidated in maize. First, the genes encoding enzymes integrated in biosynthesis were isolated. The expression of these genes in microbial systems has enabled the determination of the enzymatic reactions in vitro. Gene isolation was facilitated by the genetic tools that are available for maize and by the substantial biochemical data that had accumulated. The pathway is genetically defined by the Bxl gene. The maize mutation bxl (benzoxazineless) abolishes DIMBOA synthesis (Hamilton, 1964). The DIMBOA and tryptophan biosynthetic pathways share certain intermediates because labeled tryptophan precursors such as anthranilic acid (Tipton et al., 1973) and indole (Desai et al., 1996) are incorporated into DIMBOA, although incorporation of labeled tryptophan was not detected. The conversion of HBOA to DIBOA was identified as a cytochrom P450 monooxygenase catalysed reaction (Bailey and Larson, 1991). Later it was determined that all five oxygen atoms of DIMBOA are derived from molecular oxygen (Glawischnig et al, 1997). Therefore, key features of the pathway were expected to be a mechanism that produces indole and the incorporation of oxygen atoms by P450 enzymes in order to convert indole to a benzoxazinone.

A. Isolation

and Function

of 5x1

The Bxl gene was molecularly identified by directed transposon tagging using the Mutator (MU) transposon system of maize (Chomet, 1994). A

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A4u-induced recessive bxl allele was identified. Subsequently, a genomic DNA fragment flanking the MU insert was isolated by the so-called AIMS method (a PCR-based method, Frey et al., 1998). This fragment was used to isolate the wild-type Bxl and the recessive bx1 alleles from genomic lambda-libraries as well as the full length cDNA clones (Frey et al., 1997). Another allele of Bxl was fortuitously isolated (Kramer and Koziel, 1995) by searching for genes that were not expressed in the maize seed. It was found that Bxl is similar to tryptophan synthase alpha subunits (TSA) and when expressed in E. coli, Bxl complemented a bacterial TSA mutation Therefore, originally this gene was falsely classified as TSA from maize. However, BXl is not involved in trypthophan biosynthesis but represents the mechanism that produces indole for secondary metabolism (see discussion that follows) and manifests the branch point from primary metabolism for benzoxazinoid biosynthesis (Fig. 5). The bxl mutation can be complemented with indole (Frey et aZ., 1997). The immersion of shoots of bxl seedings into a 1 miM solution of indole restored the formation of DIMBOA. This confirmed that indole is a specific intermediate for DIMBOA biosynthesis. BXl catalyzes the conversion of indole3-glycerol phosphate to indole (Frey et aZ., 1997). BXl protein was expressed in E. coEi and purified to homogeneity. The steady-state kinetic constants (K, 0.013 m&f, K,,t 2.8 s-‘) indicate that BXl acts efficiently as an indole3-glycerol phophate lyase (Frey et aZ., 1997). In a separate study, BXl enzyme was given the name indole synthase (Melanson et aZ., 1997). The sequence similarity to TSA suggests that BXl function originated from the penultimate reaction in tryptophan biosynthesis and was modified during evolution to open the secondary metabolic pathway. These modifications essentially permitted BXl to act as a free enzyme, whereas tryptophan synthase is typically a tetrameric heterosubunit complex that is formed by two TSA-TSB (tryptophan synthase beta) complexes that are linked via TSB (Creighton and Yanofsky, 1966). These complexes were originally described in bacteria; however, an analogous heterosubunit tryptophan synthase exists in plants (Radwanski et aZ., 1995). Indole is usually not released from the tryptophan synthase complex but is directly converted to tryptophan. TSA activity is almost completely dependent on complex formation. Tryptophan levels in the bxl mutants are normal; the natural maize TSA subunit must, therefore, be encoded by gene(s) different from Bad. The product of these genes would presumably form heterosubunit complexes with TSB for which two genes have been characterized in maize (Wright et cd., 1992). The BXl protein, like tryptophan synthase, is localized in the stroma of the chloroplast. In vitro import experiments with spinach plastids revealed the cleavage of a signal peptid. When the mature BXl protein is isolated

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SICKER ET AL. Indole-3-glycerol

phosphate

lndole

TSB

BX2

Indolin-2(1H)-one 0 Tryptophan BX3

3-Hydroxyindolin-2(1H)+me H

HBOA

0 H 2H-1,4-Benzoxazin-3(4H)-one

DIBOA

HyJyy DIMBOA

~ bH

“““a;$ HMBOA

FIG. 5 DIMBOA biosynthesis. The branchpoint of DIMBOA and tryptophan biosynthesis is indicated on top. Abbreviations for enzymes are indicated for each reaction (see text). The enzymes for conversion of DIBOA to DIMBOA have not yet been identified.

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from maize, the amino terminus is characteristically shorter than that predicted from the Bxl cDNA sequence (C. Stettner, unpublished). The synthesis of several other secondary metabolites in plants, such as the indole glucosinates, anthranilate-derived alkaloids, and tryptamine derivatives (Radwanski and Last 199.5, Kutchan 19951, could depend on indole as an intermediate. Indole-3-glycerol phosphate was proposed as a branchpoint from the tryptophan pathway for the synthesis of the indolic phytoalexin camalexin (3-thiazol-2’yl-indole) in Arubidopsis thaliana (Tsuji et aZ., 1993, Zook, 1998). In maize and cotton, indole is produced as part of the volatile mixture or “cocktail” that is released following attack by army beet worm catapillars and other insect larvae (Turlings et al., 1990). The indole-3-glycerol phosphate lyase function of RX1 exemplifies how indole can be generated in plants to serve either as intermediate for secondary metabolism or as an end product.

6. Isolation and Function

of /3x2-Bx5

The following reactions in DIBOA biosynthesis are catalyzed by four cytochrome P450-dependent monooxygenases. These enzymes are membranebound heme-containing mixed function oxidases. They use NADPH or NADH to reductively cleave molecular oxygen to produce functionalized organic products and a molecule of water. In this generalized reaction, reducing equivalents from NADPH are transferred to the P45U enzyme via a flavin-containing NADPH-P450 reductase. In plants, P450 enzymes are involved mainly in hydroxylation or oxidative demethylation reactions of a large variety of primary and secondary metabolites including hormones, phytoalexins, xenobiotics, and pharmaceutically relevant compounds. Four maize P450 genes, one of which was isolated by substractive cDNA cloning from high versus low DIMBOA ,accumulating lines (Frey et al., 1995), are in the CYP71 C subfamily of plant cytochrome P450 genes. These genes are strongly expressed in young maize seedlings and share an overall amino acid identity of 45-65%. The observation that all oxygen atoms of DIMBOA are incorporated from molecular oxygen (Glawischnig et a& 1997) led to the speculation that these cytochrome P450 enzymes might be involved in this pathway. The genes encoding these enzymes are designated Bx2, Bx3, Bx4 and Bx5. Direct evidence for the involvement of Bx3 in DIMBOA biosynthesis is provided by a mutant allele (Bx3::Mu) isolated by a reverse genetic approach to screen for 1Mu insertions in the P450 genes (Frey et #al., 1997). In maize seedlings homozygous for the recessive mutant allele,, no DIMBOA could be detected by HPLC analysis. In contrast, DIMBOA

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was detected in seedlings that were either heterozygous or homozygous wild type. Thus, a functional Bx3 gene is required for DIMBOA biosynthesis. The cDNAs of the four I’450 enzymes were expressed in a yeast system (Truan et aZ., 1993; Urban et al, 1994). Microsomes were isolated from the transgenic yeast strains in order to demonstrate a function in benzoxazinoid biosynthesis. BX2-BX5 convert indole to DIBOA by catalyzing a series of four specific hydroxylations (Frey et aZ., 1997, Fig. 5). First, BX2 catalyzes the formation of indolin-2(1H)-one, which is converted to 3-hydroxyindolin-2(1H)-one by BX3. Then, BX4 catalyzes the conversion of 3hydroxy-indolin-2(W)-one to 2-hydroxy-2H-l,Cbenzoxazin3(4H)-one (HBOA). The mechanism for the unusual ring expansion associated with this reaction is presently being analyzed. Finally, HBOA is hydroxylated by BX5 to DIBOA. Although the four cytochrome P450 enzymes are homologous proteins, they are substrate specific. Only one intermediate in the pathway was converted by each respective P450 enzyme to a specific product. The question of enzyme specificity was addressed with the yeast expression system. The intermediate metabolites of the DIBOA pathway indole, indolin2(W)-one, 3-hydroxy-indolin-2(1N)-one, and HBOA (Frey et al., 1997) were incubated with microsomal preparations each containing one of the P450 enzymes. No detectable conversions occurred in other enzyme/substrate combinations. Each enzyme is therefore specific for the introduction of only one of the oxygen atoms in the DIBOA molecule. Enzymatic reactions, identical to the ones with the different yeast microsomal preparations, could be performed with maize microsomes, indicating that identical reactions occur naturally in maize. In addition, feeding bxl mutant seedlings with all the intermediates from indole to DIBOA results in the formation of DIMBOA in the plantlets. These findings suggest that the reaction sequence in maize from indole to DIMBOA is as depicted in Fig. 5. In addition to the reactions already described, 2H-l,Cbenzoxazin3(4H)one and 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA) were tested as possible substrates for BX2-BX5. The hydroxylation of 2H1,4-benzoxazin3(4H)-one to HBOA is catalyzed by BX3 (Glawischnig et aZ., 1999). This reaction is approximately half as efficient as the hydroxylation of indolin-a-one. In both cases, a C atom at an equivalent position is hydroxylated (Fig. 5). In contrast, for HMBOA, a derivative present in maize seedlings, no enzymatic conversions are detectable with BX2-BX5 expressed in yeast or with maize microsomes. In particular, HMBOA is not N-hydroxylated by BX5. This indicates that the 7-methoxy group of HMBOA would interfere with BX5 action. The relatively high specificity of the enzymes seems to support the idea that plant P45Os generally have a much greater substrate specificity than their animal homologues. However, there is emerging evidence that

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plant P45Os in addition to their normal physiological function, can also convert certain xenobiotics with varying efficiencies. The artificial substrate p-chloro-N-methylaniline (pCMA) is for example efficiently demethylated by BX2 and by several other plant P450 enzymes (Glawischnig et al., 1999).

The cellular compartments that comprise the benzoxazinoid biosynthetic enzymes and the glucosidases required for producing the active aglucones have also been identified (Fig. 6). Formation of indole by BXl takes place in the plastid (C. Stettner, unpublished). The conversion of indole to DIBOA by consecutive oxidation is catalized by BXZ-3X5. These P45O enzymes are localized in the endoplasmatic reticulum (Frey et al., 1997). Very likely, further hydroxylation and methylation reactions take also place in the cytoplasm. Biosynthesis commences by glycoylzation followed by transport and storage of the glucosides in the vacuole. The glycosidases required for activation of the glucosides are stored in the plastid (Cicek and Esen, 1998). In the case of cell wounding, the two cellular organelles are damaged, and the toxic aglucones are produced.

C. Genomic

Organization

of the Bx Gene Cluster

have been grouped into the CYWlC subfamily (Frey et clL, 1995) of cytochrome P450-dependent monooxygenases. These genes have probably evolved by duplication events as indicated by the clustering of the genes on the short arm of chromosome four, their similar exoniintron organization, and their sequence homology (Fig. 7). Such a case illustrates that a substantial part of a pathway can evolve by gene duplication followed by sequence modification. The cluster of P450 genes is tightly linked to the Bxl gene encoding the indoleglycerol-3-phosphate lyase that produces indole which is hydroxylated by the P450 enzyme BX2. The genes of Bxl and Bx2 are separated by only 2.5 kb. At present, there is no other example of two nonhomologous genes that are comparably closely linked in maize. All genes of the & gene cluster are located within 6 CM. These genes will therefore frequently be transferred to the next generation as one functional unit, encoding all. enzymes required for the biosynthesis of DIBOA. Bx2-Bx5

D. Evolution of the DIBOA Pathway

The benzoxazinoids are widely distributed in grasses and are also found in several dicotyledoneous species (Fig. l), suggesting that the acquisition of this pathway occurred relatively early in the evolution of the Gramineae

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FIG. 6 Cellular compartimentation of DIMBOA biosynthesis. Indol, synthesized in the chloroplast, is the substrate for P450 enzymes localized in the endoplasmic reticulum. The benzoxazinones are readily glucosylated by cytosolic UDP-glucosyl-transferases and the glucoside is stored in the vacuole. The specific glucosidase is found in the chloroplast. When the structural integrity of the cell is destroyed, the glucosidase and the glucosides encounter each other, and the toxic aglucon is produced.

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OF PLANTS

Chromosome 4

TAG

ATG

ATG

Bxl

-

TAG

ATG ;._

I’/’

)1

I j .,1>1 !I “,


TAG

5x2

2.5 kb FIG. 7 Structure and chromosomal location of the Bx genes in maize. A schematic presentation of the BX gene cluster at the short arm of chromosome 4 is given; genetic distances are indicated in centimorgans. The exon-intron structure of Bxl through Bx5 is outlined. Exons are represented by boxes, and the position of translation start and stop codons are indicated. Introns of Bx2-Bx5 are present at equivalent positions but diier in sequence and size.

and probably even before the monocots and dicots diverged. The activity of the DIBOA-specific P450 enzymes has been assayed in two other cereals: rye (containing benzoxazinoids) and barley (without benzoxazinoids). These two species are much more closely related to each other than either is to maize (Devos and Gale, 1997). The predominant benzoxazinoid of rye is DIBOA. As in maize, there are relatively high concentrations (up to 1 mgig fresh weight) present in the rye seedling. The cytochrome P450-dependent reactions are very similar to those detected in maize. Indole, indolin2(lli)-one, 3-hydroxy-indolin2(M)-one, and HBOA were converted to the same products that were obtained with maize microsomes (Glawischmg et aZ., 1999). No additional products were detected. The reactions were strictly dependent on NADPH, indicating true cytochrome P450 enzyme reactions. The similarity of the reactions in maize and in rye suggests identical DIBOA biosynthetic pathways for both species. In microsomes prepared from barley seedlings, no activities of the P450 enzymes of the DIBOA pathway were detectable, although the total P450 content in microsomes and the NADPH-P450 reductase activity were similar to maize (Glawischnig et al, 1999).

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Although maize and rye are distantly related, the DIBOA biosynthetic pathway seems to be identical in both species. Therefore, a set of proteins homologous to BXl-BX5 could be expected to exist in other grasses. If this were the case, the gene duplications responsible for the evolution of the &Z-&5 gene cluster must have occurred early in the development of the Gramineae, possibly even before the devergence of the monocots and the dicots when the presence of benzoxazinones in the Acanthaceae, Ranunculaceae, and Scrophulariaceae is taken into account. The isolation of genes homologs to Bx2-Bx5 from other species will give an insight into the evolution of the Bx gene cluster. Barley microsomes show no significant DIBOA-specific P450 activities. It remains to be shown whether the loss of enzyme activity is due to gene inactivation, or whether the whole Bx2-Bx5 cluster has been lost. This might have occurred during agricultural breeding from wild barley varieties in which DIBOA was initially present (Niemeyer, 1988; Barria et al., 1992). A similar loss of enzyme activity has also been observed for the UDPglucose : DIBOA glycosyltransferase. Glycosyltransferase activity is present in wild varieties and was susequently lost during barley cultivation (Leighton et al., 1994).

IV. Mode

of Action

A. Interaction

Benzoxazinoid

of Benzoxazinoid

Aglucones

Aglucones

with Pests

Benzoxazinones are part of the so-called nonhost or general defense of plants (Health, 1985). In contrast to specific or cultivar resistance, a wider range of pathogens and pests are controlled by the general toxicity of such ecochemicals. A decrease of pathogen or pest injury has been positively correlated with increased benzoxazinone content by several authors for maize and wheat (Givovich and Niemeyer 1996; references in Niemeyer 1988) and Triticum (Thackray et al., 1990; Niemeyer et al., 1992). High benzoxazinone levels reduce the growth rate and enhance the mortality of bacteria, fungi, and insects; the spread of plant viruses is indirectly affected by controlling their insect hosts (Bravo and Lazo, 1993; Freymark et aZ,, 1993; Givovich and Niemeyer, 1996). The antialgal, antifungal, and antimicrobial potential of benzoxazinones can be demonstrated by including benzoxazinones in the growth media for these organisms (Bravo and Lazo, 1996; Bravo et aZ., 1997). Interestingly, DIMBOA blocks both virulence and growth of Agrobacteria (Sahi et aZ., 1990). This protection against infection by tumor-

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inducing bacteria might be one reason why cereals have been recalcitrant to Agrobacteriaum tumefaciens-mediated transformation. The correlation between benzoxazinone content and insect defense in relation to loss of yield is perhaps the most important aspect of study. Two main approaches are used: (i) larval development and insect performance are analyzed on benzoxazinone diets (Argandona et al., 1983, Campos et al., 1989) and (ii) insect damage is monitored in the greenhouse or by field trials on high and low benzoxazinone cultivars (references in Niemeyer, 1988). These investigations have shown that benzoxazinones can act as feed deterrents and reduce the viability of larvae. In maize, the control of the main insect pest, the European corn borer (ECB, Ostrinia nubilalis), can be correlated with high DIMBOA content. The genetic basis of benzoxazinone-mediated resistance against ECB was demonstrated very early on, and the DIMBOA content was successfully elevated in breeding programs (Klun et al., 1970; Grombacher et aZ., 1989). The proposed major gene on short arm of chromosome 4 and the major quantitative trait locus (QTL) at this position turned out to coincide with the Bx gene cluster. Benzoxazinone-mediated resistance is restricted to the first brood of the European corn borer (Barry et al, 1994). The secon brood of ECB can circumvent benzoxazinone control since DIMBOA synthesis and concentration is highest in young plants and is much reduced when the second generation larvae develop. Also in wheat, rye, and perennial triticeae, benzoxazinone levels are highest in the seedling (Copaja et al., 1991a, 1991b). A detailed accumulation pattern has been elucidated in wheat (Iwamura et al., 1996), and the observed benzoxazinone distribution fits exactly with the expression pattern of the genes Bx2-Bx.5 (Frey et al., 1995). As would be expected for a defense-related compound, the organs facing the environment like the coleoptile or the root tip have the highest Bx gene activity and benzoxazinone content. In hexaploid and tetraploid Triticum, an elevated benzoxazinone content is not restricted to the seedling, but the concentration is also high in young leaves (Thackray et al., 1990). This finding is paralleled by in situ hybridization work that shows Bx2-Bx5 gene expression not only in the seedling root but also in young side roots and young emerging adventitious roots (M. Frey unpublished). Clearly, an intrinsic “seedling” and “young tissue” expression program exists for benzoxazinone synthesis in the grasses, and protection of these tissues might be especially important in the defense strategy of the plant. In the seedling, the benzoxazinone content and Bx2-Bx.5 gene expression is not detectably influenced by wounding. However, a different expression program might exist in older plants. A localized, nonsystemic increase of benzoxazinone was detected in reaction to aphid damage in wheat and wild wheat (Niemeyer et al., 1989; Gianoli and Niemeyer, 1998) and mechanical

334

SICKER ET AL.

damage in maize (Morse et al., 1991). The induction is variable with respect to the cultivar and requires high levels of aphid infestation.

6. Molecular

Mode of Action of Aglucones

Several proposals have been made to explain the defense mechanism of benzoxazinoids based on model experiments with DIMBOA and its structural analogs. DIMBOA is known as an enzyme inhibitor of the chloroplast ATPase coupling factor (Queirolo et aZ., 1983), papain (Perez and Niemeyer, 1989a), a-chymotrypsin (Cuevas et al., 1990), aphid cholinesterases (Cuevas and Niemeyer, 1993), and plasma membrane H+-ATPase (Friebe et al, 1997). DIMBOA, but not its glucoside, has a range of different activities: l

l

l

Reduction of the electron transport in both mitochondria and chloroplasts of maize (Massardo et al., 1994). Stimulation of the rate of NADH oxidation catalyzed by horseradish peroxidase isoenzyme C that catalyzes Hz02 formation, required for oxidative cross-linking of polysaccharides and proteins of the cell wall (Rojas G. et aZ., 1997). Reduction of the activity of glutathione S-transferase of the grain aphid, Sitobion avenae (Leszczynski and Dixon, 1992).

It was suggested that these effects may result from reaction with NH2 and SH nucleophilic groups of biomolecules. The oxo-cycle tautomerism of DIMBOA offers two possibilities for a structural understanding. (i) The aldehyde group of the 0x0 form should be a strong electrophile. It has been shown that it reacts with the E-NH, group of N-Lu-acetyl lysine used as a model for lysine residues of proteins (Perez and Niemeyer, 1989b). (ii) Toward mercapto groups, the hemioxidized cyclic hydroxamic acid may behave as an oxidant, with the corresponding lactam as a reduction product. Hence, reactions of DIMBOA with mercaptanes have been studied in detail (Perez and Niemeyer, 198.5); 2,4-dihydroxy-2H-l,4-benzoxazin-3(4H)-ones substituted at the aromatic ring with 2-mercaptoethanol gave evidence that an electron-donating substituted at position 7 (para to the hydroxamic acid N atom) is required to obtain reduction (Atkinson et al., 1991). Siderophores of microorganisms of the hydroxamate type are known to be efficient carriers of Fe3+ ions in the form of iron complexes from the soil into the microorganisms. Benzoxazinoids secreted by maize, wheat, and rye seedlings have a phytosiderophore-like function (Petho et al., 1997). The importance of the substitution pattern of DIMBOA was studied by removing either the 2-OH group or the 7-Me0 group with concomittant activation of the analogs by acetylation at N&Bl;OH. Both model

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compounds, 4-acetoxy-7-methoxy-2N-l,Cbenzoxazin3(4H)-one and 4acetoxy-2-hydroxy-2Hl,4-benzoxazin-3(4H)-one, were shown to react with various C-, N-, or S-nucleophiles, regioselectively (Hashimoto et al., 1991). Also DNA reacted covalently with the hydroxamic acid N atom (Ishizaki et al., 1982). The results suggest that DIMBOA, after metabolic 0-acylation, may act as an alkylating agent toward biomolecules, like proteins or nucleic acids, due to the hemiacetal and the 7-donor-activated cyclic hydroxamic moiety (Hashimoto and Shudo, 1996). Recently, it was reported that a DIMBOA dehydration product is a reactive formyl donor (Hofmann and Sicker, 1999). Therefore formylation could also contribute to the biological effects of benzoxazinoids. In summary, the high reactivity of DIMBOA is the basis for the wide antimicrobial and mutagenic activities of benzoxazinoids.

V. Molecular Allelopathy and kmzoxazolnones

of Benroxazinones

Allelopathic interactions between individuals of different plant species or those of the same species are caused by plant-produced allelochemicals. Once released into the environment, passively or actively, they can influence germination, growth, and development of neighboring plants either negatively or positively (Harborne, 1977; Rice 1984). Most alleIochemicaIs are characterized by multifunctional phytotoxicity and are often also important for general defense. The allelopathic potential of cereals has been extensively studied in rye. A dramatic reduction (more than 90%) of the biomass of dicotyledonous weeds, such as Chenopodium album by rye mulch (Putnam and DeFrank, 1983) and living rye plants was reported by Perez and Ormeno-Nunez (1993). In 1987, Barnes et al. identified DIBOA and BOA as first-order reaction allelochemicals in rye. According to the calculations of Barnes et al. (1986) 35day-old rye can release 14.3 kg/ha of DIBOA. Fresh rye mulch contains 20-50 mmol benzoxazinoids. After 12 days, this concentration is reduced to half, and after about four months benzoxazinoids are no longer detectable (Yenish et al., 1995). Rye actively releases the DIBOA into the environment via root exudation. The exudate should influence the species composition of the neighboring vegetation and the composition of rhizosphere-colonizing mieroorganisms. DIBOA exudation was also observed with Agropyron repens (common couch grass), another member of the grass family. Here, phytotoxic effects were highest in exudates of young rhizome-borne roots, that

SICKER ET

336

AL.

appear during spring time. The effects decreased with increasing age of the roots (Schulz et al., 1994). The position and the nature of substitutions of benzoxazolinone molecules may influence effects on monocots and dicots. Barnes et al. (1987) observed that DIBOA had a significant effect on monocots. Perez and Ormeno-Nunez (1991) demonstrated a 100% suppession of Avena fatia radicle growth in the presense of 1 m&f MBOA. In a recent study, KatoNoguchi et al. (1998) reported on 5chloro-6-methoxy-benzoxazolin-2(3H)one as a potent growth inhibitor of several Gramineae. The relatively high capacity of certain Gramineae to detoxify benzoxazinoids could reflect the abundance of these secondary products in this family. The biosynthesis of endogenous benzoxazinoids commences with glycosylation and storage of the compounds in the vacuole. It seems that the same mechanism is used for detoxification of exogeneous benzoxazinoids (Fig. 8). Glycosylation of BOA was observed when BOA uptake experiments with oats, wheat, rye, and corn were analyzed (Wieland et al, 1998, 1999). BOA-6-0-glucoside was the major metabolite in oat, wheat, and rye. In maize, however, BOA-N-glucoside was the dominant

1. Plants

6-Hydroxybenzoxazolin2(3H)-one (BOA-6-OH)

benzoxazolin-2(3H)-one (BOA-N-glucoside)

2. Soil bacteria

2-aminophenol

2-amino-3%phenoxazin-3-one

2-acetylamino-3H-phenoxazin-3-one

FIG. 8 Metabolization products of BOA. References for plants (Wieland et aZ., 1998, 1999) and for soil bacteria (Chase et aZ.,1991, 1991b; Friebe et al, 1996; Gerber and Le Chevalier, 1964; Kumar et aZ., 1993; Nair et al., 1990).

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metabolite. Whereas BOA-N-glucoside shows no phytotoxicity, BOA-Oglucoside is still inhibitory but drastically lower than BOA. Thus, BOAN-glucoside presents a true detoxification product, particularly as it is not hydrolysable by common /3-glucosidases. In contrast, BOAd-0-glucoside can be easily hydrolyzed, resulting in highly toxic BOA-&OH. However, in the plant cell, the 0-glucoside is certainly transported into and stored within the vacuole. Generally, the phytotoxic effects of benzoxazinoids depend on the dose and the target species, with monocots showing a lower sensitivity compared to dicots. Avena sativa, Avena fatua, and Trilicum aestivum were compared with the dicot Vicia faba. Roots or whole plants were incubated in the presence of BOA (Wieland et al., 1999). The four species were able to absorb the substance when 100 ph4 were applied, however with different kinetics (Table I). With 500~@4 BOA, the root tips of Vi&a faba were damaged during the course of incubation, showing extensive blackening. The cereals were still able to absorb BOA, Triticum aestivum without a lag phase, and Avena sativa after lo-15 hr. Microorganisms that live within the rhizosphere or colonize roots seem to be also an important factor in overcoming the phytotoxic effects of BOA. The benzoxazolinone was converted to %amino-H-phenoxazin-3one and 2-acetylamino-3EiT-phenoxazin-3-one (Friebe et al., 1996) by bacteria present on the root surfaces of oats and rye (Table I). Both substances are known as the natural antibiotics questiomycin and Nacetylquestiomycin (Gerber and LeChevalier, 1964). BOA-mediated growth inhibition was reduced in the absence of antibiotics, when phenoxazinone production was allowed. Phenoxazinones can also be produced by bacteria associated with the roots of several dicotyledonous species (Schulz and Wieland, 1999). The different detoxification capacities seem to explain the variation in sensitivity to BOA observed in the plant species. Constitutive and inducible enzymes involved in detoxification reactions must be regarded as causative factors. The ability to detoxify may not always be essential, but it is an essential function during germination or seedling development, which are very sensitive stages in the life cycle of the plant. In a balanced plant community, individuals must endure allelochemicals released by neighboring plants of the same or of different species. There is a certain selection pressure for taxa of the community to detoxify characteristic allelochemicals highly abundant in that plant association. This can be regarded as a co-evolutionary process that comprises a whole plant community. In order to test this hypothesis, 15 species were chosen and evaluated (Schulz and Wieland, 1999): seven species of field weed communities (vegetation class Secalieta); three species of hoed vegetable communities (vegetation class Chenopodietea); one species belonging to hoed vege-

Capacity

of

Fast 0.1 mM in < 3 days, 0.5 mMin < 5 days (with antibiotica) BOAd-OH BOAd-OH, BOA6-0-glucoside, BOA-N-glucoside

Avena saliva Fast 0.1 mM in < 4 days, 0.5 mM in <5 days (without antibiotica) BOA-N-glucoside BOA-6-OH, BOA-60-glucoside, BOAN-glucoside

Triticum aestivum

Avena sativa, Trihm aestivum,Avena f&a, and Vicia faba

(Wieland et al. 1999).

Exudation Products in root extracts

BOA uptake

Detoxification

TABLE I

BOA-6-OH, BOA-N-glucoside BOAd-OH, BOA-6-0-glucoside, BOA-N-glucoside

Fast 0.5 mM < 4 days phenoxazinone production in absence of antibiotica

Avena futua

0.1 mM < 5 days root tip blackening in presence of 0.5 mM Not detectable BOAd-OH traces of BOA-6-0glucoside after 10 days of incubation with 0.1 tnM BOA

Slow

Viciu faba

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339

table communities but occurring in grain field weed communities as well; two species of the vegetation subclass Artemisietea vulgaris; Flantago major, a member of Agrostietea stolonifera communities; and Galinsoga ciliata, native to the Andes and a neophyte in Germany since 1850. Data obtained resulted in four groups of species with high to low detoxification capacity (Table II): Group I consists of Plantago major and Coriandrum sativum. Both species exhibited a high detoxification capacity but are in some respect exceptional: Plantago major is a perennial species in contrast to the others, and Coriandrum sativum is a cultivated plant that occasionally behaves as a weed in wheat fields. Group II represents species of grain field weeds and Artemisietea vulgaris communities with a rather good detoxification capacity. Carduus nutans and Daucus carota often grow closely associated with Agropyron repens, which exudes DIBOA, at least temporarily. The other species are weeds occurring in wheat and rye fields. Group III, with Consolida regalis, Agrostemma githago, Capsella bursapastoris, and Legousia speculum veneris, is a group with moderate to low detoxification capacity. Capsella bursa-pastoris and Chenopodium album (group IV), can be found only occasionally in wheat fields, although they belong to Chenopodietea communities. Both species are able to perfor some N-glucosylation. Chenopodium album was integrated into group IV, because N-glucosylation broke down at higher BOA concentrations. Polygonum aviculare, Urtica urens, and Galinsoga ciliata in group IV are characterized by a low detoxification potential and cannot perform N-glucosylation, but 0-glucosylation is detectable. Polygonurn aviculare represents an exception in group IV because it can appear in both Secalietea and Chenopodieta vegetation classes but lacks the ability to perform N-glucosylation. Polygonum aviculare and Chenopodium album have, however, a high allelopathic potential and may inhibit hydroxamic acid containing species in their close neighborhood. In summary, there seems to be indeed a correlation between the occurrence of a species in certain plant communities and the existence of effective metabolization pathways that result in detoxification products. Evidently, the ecobiochemical potential of a species to detoxify benzoxazolinone can be regarded as an essential secondary function in rye and wheat fields, which is manifested in those European plant associations. Exact explanations at the molecular level for the differences in species reactions to allelochemicals are not common. Benzoxazinoids are, however, an example where biochemical and physiological mechanisms that improve our knowledge about allelochemical interactions have been identified. The understanding of molecular aspects of allelopathic interactions is a prerequisite if the application of allelochemicals in modern agrotechnology is to be considered.

IV

III

II

I

Metabolization

TABLE II

class

Agrostietea stolonifera Secalietea Secalietea Artemisietea vulgaris Secalietea Secalietea Artemisietea vulgaris Secalietea Secalietea Chenopodietea Secalietea Chenopodietea Chenopodietea Chenopodietea Neophyte

Vegetation

Classes Species

(European

Plant Communities)

Plantago major Coriandrum sativum Centaurea cyanus Carduus nutans Papaver rhoeas Matricaria chamomilla Daucus carota Consolida regalis Agrostemma githago Capsella bursa-pastoris Legousia speculum-veneris Chenopodium album Polygonum aviculare Urtica urens Galinsoga ciliata

Capacity of Species Belonging to Different Vegetation

Plantaginaceae Apiaceae Asteraceae Asteraceae Papaveraceae Asteraceae Apiaceae Ranunculaceae Caryophyllaceae Brassicaceae Campanulaceae Chenopodiaceae Polygonaceae Urticaceae Asteraceae

Family

High High High to moderate High to moderate Moderate Moderate Moderate Moderate to low Moderate to low Moderate to low Moderate to low Low No BOA-N-glucoside No BOA-N-glucoside No BOA-N-glucoside

Metabolization

capacity

BENZOXAZtN~NES

Vi: Concluding

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341

OF PLANTS

Remarks

Benzoxazinones are an important component of general defense in certain plants. Substantial data concerning the biosynthesis, its realization in cellular compartments, the temporal and spatial expression pattern, the molecular mode of action, and the allelopathic interactions have accumulated for these secondary metabolites. There are also indications as to how the pathway could have evolved. Some enzymes still remain to be isolated and characterized. Completely absent from this relatively complex picture are data about the factors that govern the concerted expression of the biosynthetic genes during plant development. This information could be relevant for elevating benzoxazinone levels and extending the expression of the biosynthetic genes into older developmental stages. The biosynthetic pathway to DIBOA is relatively short and begins with indale-3-glycerol phosphate, a metabolite ubiquitous to plants. Therefore, DIBOA biosynthesis could be. transgenically introduced into other plant species. If successful, such species would gain a powerful component of general defense that should improve their disease resistance against a wide range of pathogens.

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