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

Benzoxazinoids have been 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, the main aglucone moieties are DIBOA (2,4-dihydroxy-2 H -1,4-benzoxazin-3(4 H )-one) and DIMBOA (2,4-dihydroxy-7-methoxy-2 H -1,4-benzoxazin-3(4 H )-one). The genes of DIBOA-glucoside 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 BX6, the produced TRIBOA-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 cytoplasmatic 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. responsible for methylation of plant hormones (salicylic acid, jasmonic acid). The third cluster includes loosely related O -methyltransferases involved in methylation of intermediates of different secondary metabolite pathways. BX7 is linked to this group.


Introduction
obtained. However, signal peptides and the requirements for protein targeting to the vacuole are poorly defined (Carter et al., 2004). These targeting signals can be located either at the amino or the carboxy terminus of the protein. We therefore sandwiched the GFP gene between the amino terminal half of Bx6 and the complete Bx6 sequence (see Materials and Methods). Bx6 is highly expressed in young plants, transcript levels exceed the levels of the housekeeping gene GAP C ( Figure 6). Since the native promoter of Bx6 is not yet defined the 35S-promotor, conferring moderate to high transcript levels in monocot plants (Schledzewski and Mendel, 1994), was employed to drive the gene. The chimeric gene Bx6part-GFP-Bx6 was expressed transiently in maize protoplasts and transformed into maize plants. In transient and stably transformed cells GFP was consistently detected in the cytoplasm and there is no indication for a vacuolar localization of BX6 ( Figure 4). Therefore, hydroxylation of DIBOA-glc most likely takes place in the cytosol.

Isolation and Characterization of Bx7
TRIBOA-glc was used as substrate to purify the putative OMT that catalyzes the last step in benzoxazinoid biosynthesis of maize. Benzoxazinoid-glucosides are not stable in raw protein extracts from maize plantlets under standard OMT conditions. Therefore, affinity chromatography on adenosine agarose was adopted as a first purification step to get rid of unfavorable enzyme activities. Protein fractions eluting during application of a gradient of S-adenosyl methionine were tested for the conversion of TRIBOA-glc to DIMBOA-glc.
Active fractions were pooled and applied to anion exchange chromatography. Analysis of individual fractions on SDS-PAGE gels revealed a band with a molecular weight of about 40 kD that correlated with TRIBOA-O-methyltransferase activity in the enzyme assay (Supplemental data, Figure S1). This band was excised and digested following the protocol of Schäfer et al., 2001. The sequence of several fragments of the trypsin digest was determined by mass spectroscopy (Supplemental data Figure S2). The deduced peptide sequences were compared with a set of predicted maize OMT sequences. The genes were selected from the Pioneer Hi-Bred Int. Inc. expressed sequence tag collection by the criterion of high expression in seedling tissue. One gene demonstrated significant similarity to the deduced peptides (Supplemental data Figure S2 A), especially in a unique sequence stretch of 14 amino acids at the amino terminus. The corresponding peptide is not present in other OMT sequences, the other peptides are located in more conserved regions of the protein and are less indicative. cDNA was isolated for the candidate gene and expressed in E. coli as a His-tagged protein.
Subsequently, trypsin-digested purified protein was subjected to MALDI-MS analysis. The generated peptide pattern was congruent with pattern of the protein isolated from maize (Supplemental data Figure S2 B). The recombinant enzyme was tested in vitro for the methylation of different substrates. The only substance that served as a substrate was TRIBOA-glc, neither the aglucone TRIBOA nor well-known substrates of plant OMTs (Table 1) were efficiently converted by the enzyme. In case of quercetin, a minor activity was detected (just above detection level). The reaction was independent of Mg 2+ . The steady-state kinetic constants of BX7 for the substrate TRIBOA-glc are in a reasonable range (Table 1), v max -values of 0.45 µkat * g -1 (Christensen et al., 1998) (Li et al., 2006, Scalliet et al., 2002. The pH-optimum for the reaction is at 7.0, pH-optima of 7 to 8 are often displayed by OMTs (Lavid et al., 2002, Gang et al., 2002.

Phylogenetic Relationship of Bx7 in the O-Methyltransferase Gene Family
Plant OMTs have been categorized into two classes, based primarily on protein sequences (Joshi and Chiang, 1998). According to the occurrence and spacing of conserved amino acid sequence motifs, BX7 belongs to Class II OMT. The most comprehensively studied representatives of this class are caffeic acid O-methyltransferases (COMTs), which are involved in the biosynthesis of lignin, the preferred substrates being caffeoyl aldehyde and 5-hydroxyconiferaldehyde (Osakabe et al., 1999, Parvathi et al., 2001. Other Class II OMTs catalyze the methylation of flavonoids and phenolics. In the phylogenetic network Bx7 is joined to a web that connects OMTs of berberine alkaloid, isoflavone and phenylpropene biosynthesis ( Figure 5). BX7 has no catalytic activity towards phenylpropanoids and flavonoids (Table 1). Especially, apigenin, the substrate of a flavonoid-7-O-methyltransferase from Hordeum vulgare is no substrate although this gene is the closest relative in phylogenetic analysis ( Figure 5). BX7 shares the origin in the net with this OMT from Hordeum vulgare and the maize OMT ZRP4. It has been proposed that ZRP4 is involved in suberin biosynthesis (Held et al., 1993); however, no experimental evidence is available to our knowledge. The common root may simply reflect the fact that three enzymes of grasses are compared and does not indicate related functions.
A comprehensive survey of TUSC, the Pioneer reverse genetic resource (Benson et al., 1995), and public collections of maize mutants did not result in the identification of a Bx7 mutant. Hence, the genetic proof that Bx7 is not only capable to perform the last step in DIMBOA-glc biosynthesis but is the only enzyme in maize that catalyzes this step is not yet given.

Bx6 and Bx7 Are Predominantly Expressed in the Maize Seedling
The benzoxazinoid content is highest in the young maize plant and correlating with this distribution, major amounts of Bx1 to Bx5 transcripts are present in seedling tissue (von Rad et al., 2001;Frey et al., 2003). The same expression pattern is displayed for the 2ODD It was previously shown that the Bx genes are clustered at the short arm of chromosome 4 (Frey et al., 1997, von Rad et al., 2001, Frey et al., 2003. The set of recombinant inbred lines based on the cross CM37xT232 (Burr and Burr, 1991) was used for mapping of the

All Genes of Benzoxazinone Biosynthesis in Maize Are Isolated
The isolation of Bx6 and Bx7 completes the characterization of benzoxazinoid biosynthesis. The pathway displays the typical features of plant secondary metabolic biosynthesis: the branch point from the primary metabolism is created by gene duplication, in this case of the TSA (tryptophan synthase alpha) gene, and subsequent modification of the duplicated gene to Bx1 (Frey et al., 1997). The same sequence of events is reported for the branch point reaction of pyrrolizidine alkaloids and saponins (Ober and Hartmann,1999;Qi et al., 2004). A set of enzymatic functions commonly found in secondary metabolic pathways, namely cytochrome P450s (BX2 to BX5), a 2ODD (BX6), and an OMT (BX7) are recruited for functionalization of the primary product. The enzymes employed in the pathway are quite specific: It has been shown that the P450 enzymes are substrate-specific and produce only one oxygenation product (Glawischnig et al., 1999). Substrate specificity was also observed for the last enzymes in the pathway, the µkat * g -1 that is in the higher range determined for 2ODDs (e.g. Petunia FHT, substrate naringenin, 31,8 µkat * g -1 , Wellmann et al., 2004) and a relatively high K m of 373 µM for DIBOA-glc. Since benzoxazinoids in maize seedlings reach concentrations of 40 mM (data not shown), the natural milieu contains probably sufficient substrate concentrations for BX6.
OMTs have been originally categorized as promiscuous enzymes that methylate phenylpropanoid and alkaloid compounds (Frick and Kutchan, 1999). Progress in cloning of OMTs and the advent of first crystal structures (Zubieta et al.,, 2001, 2002, Gang et al., 2002 revealed that O-methyltransferases may have subtle structure function relationships and are substrate specific. Recently, the evolution of defined substrate requirements was demonstrated for compounds in the scent of Vanilla planifolia (Li et al., 2006). BX7 may represent an OMT with a narrow substrate spectrum. TRIBOA-glc is the only known substrate that is metabolized, the aglucone is not accepted. Several common substrates of OMTs were assayed but no methylation was observed. Especially, apigenin, the substrate of the flavonoid-7-methyltransferase isolated from Hordeum vulgare (Christensen et al., 1998) is not metabolized; flavonoid-7-methyltransferase is the closest known relative to BX7 with a defined substrate requirement.
In conclusion, BX7 and BX6 represent enzymes that perform each precisely one catalytic step each in the benzoxazinoid biosynthetic pathway. Both enzymes have K m values at the upper level found for the enzyme class combined with a sound v max .
Secondary metabolites are often stored in the vacuole. Glycosylation is discussed as an essential feature for transport across the tonoplast. In maize and petunia, cyanidine 3glucoside is transported to the tonoplast by a carrier protein (BZ2 and AN9, respectively) and delivered to a multidrug resistance-like protein in the vacuolar membrane (Goodman et al.,2004). However, glycosylation is not necessarily the final step of the biosynthetic pathway. Examples for glucosylated intermediates are given in the glucosinolate biosynthesis of Arabidopsis thaliana (Grubb et al., 2004), in loganin biosynthesis of Lonicera japonica (Katano et al., 2001) and in aurone biosynthesis of Antirrhinum majus (Nakayama et al., 2000). These pathways include a sulfotransferase, a cytochrome P450 enzyme and a polyphenol oxidase, respectively, that modifies the glucosylated substrate.
To our knowledge there are no reports of plant 2-oxoglutarate dependent dioxygenases and class II O-methyltransferases that are specific for a glucosylated substrate. At first glance, one would expect the biosynthetic pathway of benzoxazinoid biosynthesis to proceed through the final modification step to DIMBOA and to conclude with glucosylation.
However, DIBOA is the first toxic intermediate of the pathway (see below). Reduction of its reactivity by glucosylation might be required in order to reduce autotoxicity and to provide a stable metabolite for further modifications.
Modification of glucosides may be catalyzed by enzymes with vacuolar location, e.g. the aureusidin synthase AmAS1 of Antirrhinum majus is located in the vacuole (Ono et al., 2006). However, cytoplasmic localization of BX6 was substantiated by the BX6-GFP fusion analysis. According to our findings both enzymes, BX6 and BX7, display no characteristics of vacuolar proteins: no signal peptides are detectable and the pH-optima are not shifted to the acidic range of vacuoles. Hence, it is inferred that DIBOA-glc is converted to DIMBOA-glc prior to translocation to the vacuole.

Evolution of DIMBOA-Glucoside Biosynthesis
Upon cell damage, the benzoxazinone glucoside is released from the vacuole that is used as a storage compartment. The toxic aglucone is produced by a specific glucosidase (Esen, 1992). The toxicity depends largely on the reactivity of both the N-OH function and the presence of a cyclic hemiacetal unit. The hemiacetal undergoes an oxo-cyclo tautomerization. It has been shown that the aldehyde group of the oxo-form reacts with the ε-NH2 group of N-α-acetyl lysine, a model substrate for lysine residues in proteins (Perez and Niemeyer, 1989). DIBOA and DIMBOA function as enzyme inhibitor of, e.g. αchymotrypsin (Cuevas et al., 1990), aphid cholinesterase (Cuevas and Niemeyer, 1993) and plasma membrane H+-ATPase (Friebe et al., 1997). DIBOA-biosynthesis appears as the core pathway for benzoxazinoids. In some species (e.g. maize and wheat), DIBOA is further modified to yield DIMBOA. DIMBOA provides the plant with additional functions. It is a distinctively more reactive compound than DIBOA. As a donor, the 7-MeO-group facilitates N-O bond heterolysis (Hashimoto and Shudo, 1996) and the dehydration of DIMBOA (Hofmann and Sicker, 1999). The first process leads to the formation of a reactive, multi-centered cationic electrophile, the latter results in the generation of a reactive formyl donor towards -NH2, -OH, and -SH groups. Hence, biosynthesis of DIMBOA instead of DIBOA seems to be of evolutionary advantage for the plant by producing a more reactive chemical defense.
glucosyltransferase and the ODD Bx6 are linked within 6 cM on the short arm of chromosome 4 (Figure 7). The OMT Bx7 is more loosely associated but also located on the short arm of chromosome 4. In wheat, the genes of the core biosynthesis are found on two chromosomes and it was proposed that clustering was similar to maize in the original state of a putative a wheat progenitor (Nomura et al., 2003). One other example for genetic linkage of defense pathway related genes is reported in oats. Five genes of saponin biosynthesis, including a gene for a glycosylating enzyme, map within 3.6 cM in Avena (Qi et al., 2004). It has been suggested that clustering has the potential to facilitate coordinate regulation of expression at the chromatin level (Qi et al., 2004). Additionally, we suggest that clustering of the genes is of selective advantage in a population once beneficial allelic combinations are established in coupling phase.
There is ample evidence that the core benzoxazinoid biosynthesis is of monophyletic origin in grasses. It has been shown for wheat that a (yet not cloned) 2ODD is responsible for C-7 oxygenation (Frey et al., 2003), hence, the same enzyme class is used for modification of DIBOA in wheat and maize. However, it remains to be shown if the C-7-hydroxylation and methylation are carried out by orthologous functions in the poaceae. and to NMR analysis) and showed the correct mp 172-173 °C (off-white microcrystalline powder).
TRIBOA-glucoside was generated by two alternative approaches. For the generation of the reference substance chemically synthesized TRIBOA was glucosylated in vitro using the heterologously expressed UDP-glucosyltransferase BX8 (von Rad et al., 2001). Routine large scale production of TRIBOA-glucoside was carried out by incubation of DIBOAglucoside with raw extracts of IPTG-induced E. coli BL21(DE3) expressing Bx6 from the plasmid pET3a (Studier and Moffatt, 1986). Three hours after induction with 1 mM IPTG the cells were resuspended in 100 mM Tris-HCl (pH 7.5), 20 mM NaCl, 5 mM DTT, 1 mM PMSF, 20% glycerol, 1 mg/ml lysosyme (1g of bacteria pellet per 3 ml buffer). Five cycles of freeze (liquid N 2 ) and thaw (ice) were applied, RNase and DNase (10 µg/ml each) were added and solution was cleared by centrifugation. Aliquots were stored at -70°C. For TRIBOA-glucoside synthesis, 100 µl of the protein solution were used for 1ml assay volume. The reaction buffer contained 100 mM MES-NaOH (pH 6.0), 5 mM DTT, 10 mM ascorbate, 10 mM 2-oxoglutarate, 4 mM FeSO 4 and 3 mM DIBOA-glucoside. The reaction was incubated at 30°C with gentle agitation and after 45 min stopped by the addition of 1 volume of methanol. Precipitated protein was removed by centrifugation and the supernatant was applied to HPLC. TRIBOA-glucoside was eluted with 9% acetonitrile and 91% of 0.3% formic acid using Merck LiChroCART® RP-18e. 100% B. Under these conditions, the following retention times were obtained for each compound: DIBOA-glucoside 12 min; TRIBOA-glucoside 6.4 min; DIMBOA-glucoside 16.1 min. The detection was carried out at: 254 nm for DIBOA-glucoside and 266 nm for DIMBOA-glucoside and TRIBOA-glucoside.

Mass spectrometry
The HPLC -MS-investigations were performed with a HP 1100 liquid chromatograph equipped with a diode array detector (280 nm) and a triple quadrupole mass spectrometer (API 2000, Applied Biosystems). A Nucleosil C 18 (Jasco) column was used, 3 mm x 250 mm x 5 µm. The solvent system consisted of pure water (solvent A) and methanol/ isopropanol (95/5), containing 0.025% acetic acid (solvent B), respectively. The mobile phase was used as three step gradient: first part was isocratic (8% B for 2 minutes), subsequently followed by a linear gradient (from 8% to 50% B in 9 minutes) and followed by a third, again isocratic part (50% B). The flow was 150 µl/min, mass spectra were recorded in the negative mode.

Purification of BX7
Maize seedlings were grown on wet paper for 4 days in the dark at 28°C. All purification procedures were carried out at 4°C. Protein was extracted from 120 g seedling shoots with 4 volumes (w/v) extraction buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA and 10% glycerol) and 0.3 volumes (w/w) Polyclar (Serva, Heidelberg). After filtration through miracloth and centrifugation for 1 h at 10 000g the supernatant was sterile filtrated
BX6 initial rate data were obtained using the assay as described above, using 100 mM potassium phosphate buffer (pH 7.0) buffer and 20 µg/ml His-tag purified protein. Except for DIBOA-glucoside, the assay components were mixed and incubated at 30°C for 10 min. The reaction was then initiated by the addition of DIBOA-glucoside. When 2oxoglutarate was the varied substrate, 2-oxoglutarate instead of DIBOA-glucoside was used to initiate the assay, DIBOA-glc was fixed at 2.5 mM concentration. Initial rates were calculated from progress curves of TRIBOA-glucoside formation using the "exact" numerical method described by Cornish-Bowden (1975). Lineweaver-Burk plots were constructed to determine apparent K m and v max values. Calculations were performed using GraphPad Prism v.4.0 software. BX7 initial rate data were obtained using an assay as described previously (Schröder et al., For the determination of subcellular localization of BX6, the complete Bx6-coding sequence was translationally fused to the C-terminus of green fluorescence protein variant mGFP6 (Curtis and Grossniklaus, 2003) via a 10-mer Ala-linker. Ligation of the respective restriction fragments yields the cassette mGFP-(Ala) 10 -Bx6. The first 440 base pairs of Bx6 were amplified by PCR, and a 10-mer Ala-linker was introduced at the C-terminus of the encoded polypeptide (Bx6N-part-(Ala) 10 ). The sequenced PCR fragment was joint with mGFP-(Ala) 10 -Bx6 to yield the translational fusion Bx6(N-part)-(Ala) 10 -mGFP6-(Ala) 10 -Bx6 (Bx6part-GFP-Bx6). The fusion construct was joined with the 2x35S-promoter and nos-terminator of pMDC201 (Curtis and Grossniklaus, 2003) in pUC19. The cassette 2x35S promoter-mGFP6-nos-terminator in pUC19 was used as a control construct in the expression analysis. For stable transformation, the respective promoter-gene-terminator cassettes were isolated as HindIII-EcoRI-restriction fragments and ligated into the Ti-Plasmid pTF101Ubi, a derivative of pTF101.1 that drives the bar-gene with the ubiquitin promoter. Transgenic lines were generated as described by Frame et al. (2002).
For transient expression of the GFP-reporter-constructs, protoplasts were isolated from aseptically grown plants, 12 days after imbibition. Preparation and electroporation was as described by J. Sheen (http://genetics.mgh.harvard.edu/sheenweb). 30 µg of the pUC19based plasmid were used for each electroporation of 1 to 2x10 5 protoplasts. Analysis was 24 hours after electroporation with Zeiss Axiphot equipped with HQ-Filterset for Enhanced-GFP (AHF Analysentechnik AG, Tübingen, Germany). Photographs were taken with DCS 670 Digital Nikon F5 SLR camera and analyzed with the software Kodak DCS Photodesk. Protoplasts from transgenic plants were isolated and analyzed accordingly.
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Supplemental Data
Supplemental Figure        and shoot (leaves and coleoptile); after 6 days mesocotyl, leaves and coleoptile were pooled (mshoot). Tissues of older plants were taken after 3 weeks (root) and 10 weeks.
Steady-state mRNA levels were measured by real-time RT-PCR using gene-specific primer pairs. GAP C mRNA quantities were determined in parallel and used for normalization.