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Originally published In Press as doi:10.1074/jbc.M706587200 on November 12, 2007

J. Biol. Chem., Vol. 283, Issue 6, 3231-3247, February 8, 2008
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A Functional Genomics Investigation of Allelochemical Biosynthesis in Sorghum bicolor Root Hairs*Formula

Scott R. Baerson{ddagger}1, Franck E. Dayan{ddagger}, Agnes M. Rimando{ddagger}, N. P. Dhammika Nanayakkara§, Chang-Jun Liu, Joachim Schröder||, Mark Fishbein**, Zhiqiang Pan{ddagger}, Isabelle A. Kagan{ddagger}2, Lee H. Pratt{ddagger}{ddagger}, Marie-Michèle Cordonnier-Pratt{ddagger}{ddagger}, and Stephen O. Duke{ddagger}

From the {ddagger}United States Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, Mississippi 38677, the §National Center for Natural Products Research, School of Pharmacy, University of Mississippi, University, Mississippi 38677, the Biology Department, Brookhaven National Laboratory, Upton, New York 11973, the ||Universität Freiburg, Institut für Biologie II, Schänzlestrasse 1, D-79104 Freiburg, Germany, the **Department of Biology, Portland State University, Portland, Oregon 97207, and the {ddagger}{ddagger}Department of Plant Biology, University of Georgia, Athens, Georgia 30602

Received for publication, August 8, 2007 , and in revised form, October 9, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sorghum is considered to be one of the more allelopathic crop species, producing phytotoxins such as the potent benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone) and its analogs. Sorgoleone likely accounts for much of the allelopathy of Sorghum spp., typically representing the predominant constituent of Sorghum bicolor root exudates. Previous and ongoing studies suggest that the biosynthetic pathway for this plant growth inhibitor occurs in root hair cells, involving a polyketide synthase activity that utilizes an atypical 16:3 fatty acyl-CoA starter unit, resulting in the formation of a pentadecatrienyl resorcinol intermediate. Subsequent modifications of this resorcinolic intermediate are likely to be mediated by S-adenosylmethionine-dependent O-methyltransferases and dihydroxylation by cytochrome P450 monooxygenases, although the precise sequence of reactions has not been determined previously. Analyses performed by gas chromatography-mass spectrometry with sorghum root extracts identified a 3-methyl ether derivative of the likely pentadecatrienyl resorcinol intermediate, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. An expressed sequence tag data set consisting of 5,468 sequences selected at random from an S. bicolor root hair-specific cDNA library was generated to identify candidate sequences potentially encoding enzymes involved in the sorgoleone biosynthetic pathway. Quantitative real time reverse transcription-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from the expressed sequence tag data set have led to the identification of a novel O-methyltransferase highly and predominantly expressed in root hairs (designated SbOMT3), which preferentially utilizes alk(en)ylresorcinols among a panel of benzene-derivative substrates tested. SbOMT3 is therefore proposed to be involved in the biosynthesis of the allelochemical sorgoleone.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Allelopathy, the chemical inhibition of one plant species by another, represents a form of chemical warfare between neighboring plants competing for limited light, water, and nutrient resources (1, 2). Allelopathic interactions have been proposed to have profound effects on the evolution of plant communities through the loss of susceptible species via chemical interference, and by imposing selective pressure favoring individuals resistant to inhibition from a given allelochemical (2, 3). In addition, allelochemicals released by grain crop species such as barley, rye, and sorghum are thought to play a significant role in their efficacy as weed suppressants when used as cover crops or within intercropping systems (4, 5).

Sorghum bicolor (L.) Moench is one of the most important cereal crops worldwide (6), surpassed only by wheat, rice, corn, and barley in total acreage, with the United States currently accounting for a major portion of total world production and exports (FAOSTAT data). The allelopathic properties of sorghum were first suggested from observations of reduced growth of other crop species when grown in rotation; moreover, certain sorghum species such as Sudan grass (Sorghum sudanense) can produce largely weed-free monocultures without the use of synthetic herbicides (reviewed in Ref. 7). Current evidence suggests that a family of allelochemicals active at micromolar concentrations, referred to as sorgoleones, may account for much of the allelopathic properties of Sorghum spp. (8–10). The term sorgoleone is most frequently used to describe the compound corresponding to the predominant congener identified in sorghum root exudates (11), 2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone (Fig. 1), which has been estimated to account for as much as 85% of the exudate material (w/w) in some varieties (10). The remaining exudate consists largely of sorgoleone congeners differing in the length or degree of saturation of the aliphatic side chain and in the substitution pattern of the quinone ring (11, 12). The fact that sorgoleone acts as a potent broad-spectrum inhibitor active against many agronomically important monocot and dicot weed species, exhibits a long half-life in soil, and appears to affect multiple cellular targets (e.g. 8–10, 13–17) may make it promising for development as a natural product alternative to synthetic herbicides (18, 19).


Figure 1
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  		FIGURE 1.
Proposed biosynthetic pathway for sorgoleone.

 
Sorgoleone biosynthesis likely occurs exclusively in root hairs, which appear as cytoplasmically dense cells in sorghum, containing large osmiophilic globules deposited between the plasmalemma and cell wall, presumably associated with sorgoleone rhizosecretion (10, 20). Labeling studies have demonstrated that the biosynthesis of sorgoleone involves the convergence of the fatty acid and polyketide pathways (21, 22) through the action of a polyketide synthase activity utilizing fatty acyl-CoA starter units, resulting in the addition of a quinone head via iterative condensations of acetate extender units (Fig. 1).3 Subsequent modifications of the alkylresorcinol intermediate are likely to be mediated by AdoMet4-dependent O-methyltransferases and by hydroxylases (possibly P450 monooxygenases), yielding the reduced form of sorgoleone (dihydrosorgoleone). Upon exudation, the less stable hydroquinone rapidly oxidizes to the highly phytotoxic benzoquinone form, which can persist in soil for extended periods (9, 10, 23). In addition, recent studies have shown that sorgoleone biosynthesis occurs constitutively during early seedling establishment, and its accumulation is apparently distinct from that of phytoalexins as levels were not observed to increase following various elicitor treatments (24).

The specific sequence of biosynthetic reactions leading to the formation of dihydrosorgoleone, starting from the proposed 5-pentadecatrienyl resorcinol intermediate (Fig. 1), has not been determined previously. Moreover, despite the ecological and agronomic importance of this family of allelochemicals, a paucity of information exists concerning the genes and corresponding enzymes participating in their biosynthesis. In this study, we have identified a 3-methyl ether derivative of the previously characterized 5-pentadecatrienyl resorcinol intermediate by GC-MS analysis of sorghum root extracts, indicating that dihydroxylation of the resorcinol ring is preceded by O-methylation at the 3'-position by a novel 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase activity. To identify candidate O-methyltransferase sequences, as well as candidates representing other steps in the biosynthetic pathway, an annotated EST data set consisting of 5,468 quality 5'-sequences was generated from an S. bicolor root hair-specific cDNA library. Follow-up real time RT-PCR and recombinant enzyme studies with putative O-methyltransferase sequences obtained from this library have led to the identification of a root hair-specific O-methyltransferase (designated SbOMT3) utilizing alkylresorcinolic substrates, proposed to be involved in the biosynthesis of sorgoleone. Furthermore, the annotated root hair-specific data set we have generated directly complements the existing public sorghum EST sequences, and expands our understanding of the transcriptome of a highly specialized and unique cell type.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Plant Material and Growth Conditions—Seeds of S. bicolor genotype BTx623 were purchased from Crosbyton Seed Co. (Crosbyton, TX), and SX-17 sorghum-Sudangrass hybrid seeds (S. bicolor x sudanense) were purchased from Dekalb Genetics (Dekalb, IL). SX-17 was used for sorgoleone content comparisons with BTx623; all other experiments described in this work involved only BTx623. Root tissues used for sorgoleone content determinations, analysis of C15:3 resorcinols, root hair preparations, and whole root systems used for real time RT-PCR experiments were obtained from 5- or 8-day-old dark-grown seedlings grown under soil-free conditions using a capillary mat system devised by Czarnota and co-workers (10). Immature leaves and shoot apices used for real time RT-PCR experiments were isolated from seedlings maintained in a growth chamber at 28 °C for 8 days in standard (~20 x 40 cm) nursery flats using Premier Pro Mix PGX potting media (Hummert International, Earth City, MO) under a combination of cool-white fluorescent and incandescent lighting at an intensity of ~400 µmol m-2 s-1 and a 16-h photoperiod. Developing panicles, mature leaves, and culm (stem) tissues used for real time RT-PCR experiments were isolated from 10-week-old greenhouse-grown plants. At the time of harvest, panicles were partially exerted from flag leaf sheaths, just prior to anthesis. All harvested plant material was directly flash-frozen in liquid nitrogen and stored at -80 °C prior to analysis, with the exception of material used for sorgoleone content determinations, which involved fresh tissue extractions.

Sorgoleone Content Determinations—Root systems from 5-day-old seedlings were weighed, immersed in chloroform, and agitated for 30 s. Extracts were filtered through Whatman 110-mm number 1 filter disks (Whatman Inc., Florham Park, NJ) to remove debris, concentrated in vacuo at 30 °C using a rotary evaporator (Büchi Rotovapor, Brinkmann Instruments), and dried to completion under nitrogen gas, then weighed on an analytical balance. Dried extracts were then re-dissolved in acetonitrile (1.0 mg sample per ml of acetonitrile) and analyzed by high performance liquid chromatography (HPLC) using a Hewlett-Packard 1050 HPLC System (Agilent Technologies, Palo Alto, CA) equipped with an Alltech EPS C18 column (100 Å, 3 µm, 150-mm length, 4.5-mm internal diameter; Alltech Associates Inc., Deerfield, IL). The sample was eluted as follows (solvent A is 2.5% acetic acid in water; solvent B is acetonitrile): 0–15-min 45% A, 55% B isocratic; 15–22-min linear gradient from 55 to 100% B; 22–25-min 100% B; 25–26-min 100 to 55% B; 26–30-min 45% A/55% B isocratic. A flow rate of 2 ml/min was used, and the sample injection volume was 20 µl. The peak corresponding to sorgoleone was monitored at 280 nm. Quantitation was based on a calibration curve using purified sorgoleone as an external standard.

GC-MS Analysis of C15:3 Resorcinols—Root systems from 8-day-old seedlings were first immersed in chloroform with agitation for 30 s to remove sorgoleone and then lyophilized. Lyophilized material was pulverized using a mortar and pestle, followed by homogenization in methanol (~10 g per 50 ml) for 1 min at 25,000 rpm. Homogenates were then filtered through Whatman number 1 filter disks, then evaporated using a rotary evaporator (Büchi Rotovapor, Brinkmann Instruments) at 30 °C. Residues were then re-dissolved in methanol and transferred to GC vials. GC-MS analysis was performed with a JEOL GCMate II System (JEOL USA Inc., Peabody, MA) using a J & W DB-5 capillary column (0.25-mm internal diameter, 0.25-µm film thickness, 30-m length; Agilent Technologies, Foster City, CA). The GC temperature program was initially set to 110 °C, raised to 300 °C at a rate of 6 °C/min, and then held at this temperature for 2.3 min. Ultra-high purity helium was used as carrier at a flow rate of 1.0 ml/min. The inlet (splitless), GC interface, and ion chamber temperatures were 250, 250, and 230 °C, respectively. The sample injection volume used was 2.0 µl.

The mass spectrum of the peak at 21.8 min (Fig. 2, B and C) showed fragment ions m/z 314 [M+], 313 [M+ - H], 269 [313+ - CH=CH2, -OH], 255 [269+ - CH2], 241 [255+ - CH2], 227 [241+ - CH2], 213 [227+ - CH2], 199 [213+ - CH2], 187 [313+ - CH(CH2CH=CH)2H, - 2O], 171 [185+ - CH2], 159 [314+ - (CH2CH=CH)3H, -2OH], 143 [314+ - (CH2)2(CH2CH = CH)3H, -2H2O], 131 [159+ - 2CH2], 129 [143+ - CH2], and 117 [131+ - CH2] supporting the identification of the 5-(8',11',14')-pentadecatrienyl resorcinol intermediate. Similarly, the mass spectrum of the peak at 18.3 min (Fig. 2C) showed fragment ions m/z 328 [M+], 313 [M+ - CH3], 285 [M+ - H - CH=CH2, -OH], 269 [M+ - CH2CH=CH2, -H2O], 243 [269+ - CH=CH], 229 [243+ - CH2], 201 [M+ - 2H - (CH2CH=CH)2H, -OH, -OCH3], 187 [201+ - CH2], 171 [M+ - H, -(CH=CHCH2)2CH=CH2, -H2O, -OCH3], 159 [M+ - (CH2CH=CH)3H, -OH, -OCH3], 145 [159+ - CH2], 132 [145+ - CH], 129 [171+ - (CH2)3], and 117 [159+ - (CH2)3], supporting the identification of the 5-(8',11',14')-pentadecatrienyl resorcinol-3-methyl ether intermediate.

cDNA Library Construction—Root hairs were isolated from dark-grown 8-day-old BTX623 seedling root systems using the method devised by Bucher et al. (25), involving immersion in liquid nitrogen with gentle stirring, followed by filtration through a 250-µm aluminum mesh to remove root system debris. Purity of the root hair preparations was assessed by bright field microscopy, and only highly enriched preparations were retained for subsequent cDNA library construction. Root hair preparations were stored at -80 °C prior to RNA extraction. Total RNAs were isolated from root hairs using the TRIzol reagent (Invitrogen) per the manufacturer's instructions, with an additional homogenization step of 30 s at 25,000 rpm using a hand-held homogenizer. RNA purity was determined spectrophotometrically, and integrity was assessed by agarose gel electrophoresis. Poly(A)+ mRNA was prepared from root hair total RNA using an Oligotex mRNA Midi Kit (Qiagen, Valencia, CA), and ~1.5 µg was used for construction of a directional cDNA library with the Uni-Zap XR cDNA library construction kit (Stratagene, La Jolla, CA), per the manufacturer's instructions. A primary library of ~3 x 106 plaque-forming units was obtained. To obtain an estimate of average insert size, 36 randomly selected plaques from a primary library plating were sampled from NZY plates using a sterile 1.0-ml pipette tip, then transferred into culture tubes containing 1.0 ml of SM buffer, and allowed to elute overnight at 4 °C with shaking. Phage eluates (2.5 µl) were then used as templates in 50-µl PCRs containing T3- and T7-specific PCR primers (Stratagene) using an Expand High Fidelity PCR kit (Roche Diagnostics) per the manufacturer's instructions. After an initial denaturation step of 94 °C for 5 min, a thermal profile of 94 °C for 30 s, then 48 °C for 1 min 30 s, followed by 72 °C for 2 min for 35 cycles was used, and aliquots of the reactions were subsequently analyzed by agarose gel electrophoresis. By this analysis, the average insert size was estimated to be ~0.93 kb, ranging between 2.4 and 0.2 kb.

EST Sequencing and Data Analysis—Recombinant plasmid-bearing colonies were obtained from the nonamplified S. bicolor root hair phagemid library by mass excision, and then plasmid mini-preparations were performed for 6,624 randomly selected isolates arrayed into 69 96-well plates. 5' DNA sequencing reactions were performed using ABI BigDye Terminator Cycle Sequence Ready Reaction kits (versions 2 and 3; Applied Biosystems, Foster City, CA) as described previously (26). Base calling on raw sequence trace data were performed using PHRED software (27), and vector, adapter, and low quality sequence ends were identified using an in-house processing script (28), resulting in 5,468 high quality sequences, or an 82.6% success rate. The average trimmed EST length, determined using a moving window with a PHRED quality score of 16, was 451 bp, of which on average 432 bp were called with a quality score equal to or greater than 20. The resulting 5,468 root hair ESTs were assembled using TGICL (29). Provisional annotation of all EST and contig consensus sequences was performed by BLASTX analysis against all full-coding length entries from the PIR-NREF data base (30, 31). EST data mining was performed using the MAGIC Gene Discovery software (32), and by BLASTN and TBLASTN analysis (30). Provisional gene ontology (GO) categorization of the assembled data set was performed by BLASTX analysis against all UniProt peptide sequences downloaded from the European Bioinformatics Institute web site (EBI UniProt release 9.0). An E value cutoff of E < 10-10 was applied to the BLASTX returns, and the corresponding full GO terms from significant matches were retrieved using the association table provided by the Gene Ontology Consortium web site. Full GO terms retrieved were finally mapped to their Plant GOSlim counterparts (33) using the "map2slim.pl" tool also available on line.

Quantitative Real Time RT-PCR Analysis—Quantitative real time PCRs were performed in triplicate using the GenAmp® 5700 sequence detection system (Applied Biosystems, Foster City, CA) as described previously (34). First strand cDNAs were synthesized from 2 µg of total RNA in a 100-µl reaction volume using the TaqMan reverse transcription reagents kit (Applied Biosystems) per the manufacturer's instructions. Independent PCRs were performed using the same cDNA for both the gene of interest and 18 S rRNA, using the SYBR® Green PCR Master Mix (Applied Biosystems) with the following gene-specific primer pairs: SbOMT1 forward, 5'-GCATCTTCGTTCATGTACTTGTTACAC-3', and reverse, 5'-CGACGAAGCACATCCTTACTATGAG-3'; SbOMT2 forward, 5'-GCGCCTCGTTTTCGTATGC-3', and reverse, 5'-GAACATACAGCTCACCTTCTCTGC-3'; SbOMT3 forward, 5'-CAATTTCCCTTTTATGTTTAGCCTGATAG-3', and reverse, 5'-TGCCAGGGTGTGATATGTGC-3'; polyubiquitin forward, 5'-CTTCCTCTGTCCCTCTGATGGAG-3', and reverse, 5'-AAGACACGACCACGACATGC-3'; chlorophyll a/b-binding protein forward, 5'-TGGATTGATTGATGCTGCAAG-3', and reverse, 5'-CGTGAAACAAGAGACACACATGC-3'; 18 S rRNA forward, 5'-GGCTCGAAGACGATCAGATACC-3', and reverse, 5'-TCGGCATCGTTTATGGTT-3'. Primers were designed using Primer Express® software (Applied Biosystems) and the Amplify program (35). A dissociation curve was generated at the end of each PCR cycle to verify that a single product was amplified using software provided with the GenAmp® 5700 sequence detection system. A negative control reaction in the absence of template (no template control) was also routinely performed in triplicate for each primer pair. The change in fluorescence of SYBR® Green I dye in every cycle was monitored by the GenAmp® 5700 system software, and the threshold cycle (CT) above background for each reaction was calculated. The CT value of 18 S rRNA was subtracted from that of the gene of interest to obtain a {Delta}CT value. The CT value of an arbitrary calibrator (e.g. the tissue sample from which the largest {Delta}CT values were obtained) was subtracted from the {Delta}CT value to obtain a {Delta}{Delta}CT value. The fold changes in expression level relative to the calibrator were expressed as 2-{Delta}{Delta}CT.

Southern Blot Analysis—Genomic DNA from S. bicolor genotype BTx623 was prepared from young leaf tissue using the Plant DNAzol Reagent (Invitrogen). Approximately 1 g of powdered tissue was mixed with 3.0 ml of plant DNAzol reagent supplemented with RNase A at a final concentration of 1.0 mg/ml and then incubated at room temperature for 10 min with gentle shaking. The remainder of the extraction procedure was carried out per the manufacturer's instructions, with an additional chloroform:isoamyl alcohol (24:1, v/v) extraction step performed prior to ethanol precipitation. Restriction endonuclease digestions and Southern blotting procedures were performed according to standard protocols (36). Probe sequences containing partial 3'-UTR and 3' coding sequences were generated by PCR amplification from cloned SbOMT1, SbOMT2, and SbOMT3 cDNA templates with an Expand High Fidelity PCR kit (Roche Diagnostics) using a thermal profile of 94 °C for 30 s, then 55 °C for 1 min 30 s, followed by 72 °C for 1 min for 25 cycles. The following PCR primer pairs were used to generate probe sequences for the three S. bicolor OMTs: SbOMT1 forward, 5'-CACCAGAAGAAACACTAGCATCG-3', and reverse, 5'-TTAAGGACCAATAAGCAAGCTAGTACA-3'; SbOMT2 forward, 5'-GAAGCACAGCTGCTGATGG-3', and reverse, 5'-CAGCAAGCAACACACATCAAGTATG-3'; and SbOMT3 forward, 5'-ATGACTGGAGCAATGATGAGTG-3', and reverse 5'-CAGGGTGCCAGGGTGTG-3'. For SbOMT1, the amplified regions correspond to nucleotides 966–1410 (445 bp in length; GenBankTM accession number EF189707), containing 191 bp of 3'-coding sequence and 254 bp of contiguous 3'-UTR sequence; for SbOMT2, amplified regions correspond to nucleotides 961–1424 (464 bp in length; GenBankTM accession number EF189706), containing 159 bp of 3'-coding sequence and 305 bp of contiguous 3'-UTR sequence; and for SbOMT3, amplified regions correspond to nucleotides 893–1376 (484 bp in length; GenBankTM accession number EF189708), containing 290 bp of 3'-coding sequence and 194 bp of contiguous 3'-UTR sequence. The resulting PCR products were cloned using a Zero Blunt TOPO PCR cloning kit (Invitrogen) and confirmed by DNA sequence analysis. Prior to use in labeling reactions, probe sequences were excised from the cloning vectors by restriction endonuclease digestion and then gel-purified.

Heterologous Expression and Purification of Recombinant OMTs—DNA manipulations and Escherichia coli transformation protocols used during the preparation of plasmid vectors for recombinant OMT experiments were performed according to standard procedures (36). Full-length open reading frames for SbOMT1, SbOMT2, and SbOMT3 were determined from assembled EST sequence data obtained from the root hair ESTs. E. coli overexpression vectors were constructed by PCR amplification of SbOMT1, SbOMT2, and SbOMT3 coding regions using PCR primers designed with flanking NdeI (forward primer) and BamHI (reverse primer) restriction sites to facilitate direct cloning into pET15b (EMD Biosciences, La Jolla, CA), using the following PCR primer pairs: SbOMT1 forward, 5'-GCAATTCCATATGGCCAGCTATACTAGTACTAGTGG-3', and reverse, 5'-GACTAGGGATCCTCACTTTGTGAATTCATGGG-3'; SbOMT2 forward, 5'-GCAATTCCATATGGCCGCGTCTTCTCATGC-3', and reverse, 5'-GACTAGGGATCCTTATGGGTAGACTTCGATGACACCAC-3'; and SbOMT3 forward, 5'-GCAATTCCATATGGTACTCATCAGCGAGGAC-3', and reverse, 5'-GACTAGGGATCCTCATGGATATAGCTCAATGATCG-3'. Primers were added at a final concentration of 0.4 mM to 50-µl PCRs, using 2 µl of first strand cDNA as template, prepared from root hair total RNA with a SuperScript first strand synthesis system (Invitrogen) per the manufacturer's instructions. PCR amplifications were performed with an Expand High Fidelity PCR kit (Roche Diagnostics), using a thermal profile of 94 °C for 30 s, then 55 °C for 2 min, followed by 75 °C for 3 min, for a total of 30 cycles. The resulting PCR products were then gel-purified, digested with NdeI and BamHI, then ligated with NdeI- and BamHI-digested pET15b, resulting in the final overexpression vectors containing the three S. bicolor OMT predicted open reading frames, as confirmed by DNA sequence analysis. The expression vectors were then transformed into E. coli strain BL21/DE3 (EMD Biosciences) for recombinant enzyme studies.

For recombinant protein production, E. coli cultures were grown at 37 °C to an absorbance of 0.6 at 600 nm, then induced with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside, and allowed to grow an additional 5 h at 25 °C. Cells were harvested by centrifugation at 3000 x g for 20 min at 4 °C, washed with cold 0.9% NaCl, and then collected by re-centrifugation at 3000 x g. Pellets were resuspended in cold lysis buffer (50 mM Tris-HCl, pH 7.5, 1 M NaCl, 5 mM imidazole, 10% glycerol, 1 µg/ml leupeptin) and extracted using a French press (Thermo IEC, Needham Heights, MA) at a pressure of 10,000 kPa. Benzonase (25 units/ml) and 1 mM phenylmethylsulfonyl fluoride were added immediately to the lysate. After 15 min of incubation at room temperature, lysates were centrifuged at 15,000 x g for 20 min, and the supernatant was loaded onto His Gravi-Trap columns (Amersham Biosciences) activated with 2 ml of 0.1 M NiSO4 and washed with 10 ml of distilled water. The column was previously equilibrated with 10 ml of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM imidazole). The column was washed with 4 ml of buffer A following each 2 ml of supernatant added. Once sample loadings were complete, the columns were washed with 8 ml of buffer A, followed by 8 ml of buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 100 mM imidazole) to remove nonspecifically bound proteins. Recombinant proteins were then eluted with 2.5 ml of elution buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 250 mM imidazole). Columns were washed with 10 ml of wash buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 M imidazole), followed by 10 ml of distilled water after each use to remove contaminating proteins. Recombinant protein-containing fractions (250 mM imidazole) were desalted on a PD-10 column equilibrated with cold desalting buffer (20 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol, 10% glycerol). Protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad). All recombinant proteins were at least 95% pure, as estimated by SDS-PAGE. Enzyme preparations were stored at -80 °C prior to use.

OMT Enzymatic Assays—Substrate specificities and kinetic parameters for recombinant OMTs were determined using a modified protocol based on Wang and Pichersky (37). All enzymatic assays consisted of 90 µl of assay buffer (250 mM Tris-HCl, pH 7.5, 10 mM dithiothreitol), 200 µl of purified enzyme preparation (200 µg protein/ml), 5 µl of a 10 mM substrate stock solution (dissolved in 100% ethanol), and 5 µl of S-[methyl-14C]adenosyl-L-methionine (40–60 mCi/mmol, 0.1 mCi/ml; ICN Biomedicals, Irvine, CA). Reactions were incubated for 30 min at 30 °C using a Thermomixer (Brinkmann Instruments) and then quenched by addition of 25 µl of 6 N HCl. Radiolabeled products were subsequently extracted by the addition of 1 ml of hexane:ethyl acetate (1:1 v/v), and 300 µl of the (upper) organic phase were transferred to scintillation vials containing 5 ml of Ultima Gold scintillation fluid (Packard BioScience, Meriden, CT). Scintillation counts were performed using a Tri-Carb 1600TR liquid scintillation analyzer (Packard BioScience). Protein concentrations and time points used for activity measurements were controlled to ensure linearity of the assays. Kinetic parameters were determined from assays performed in triplicate as described above but with substrate concentrations ranging from 10 µM to 10 mM. Data from enzyme kinetics experiments were fit to the Michaelis-Menten equation using the SigmaPlot version 9.01 enzyme kinetics module (Systat Software, Inc., Point Richmond, CA).

Homology Modeling and Automated Substrate Docking—The SbOMT3 amino acid sequence was aligned to Medicago sativa I-7-OMT using ClustalW (version 1.82). The model of SbOMT3 was built as described (38), using the Modeler software package (39, 40), with S-adenosyl-L-homocysteine and formononetin complexed to I-7-OMT (Protein Data Bank code 1FP2) as the structural template. For docking analysis, the GOLD (Genetic Optimization for Ligand Docking) program (CCDC Software Ltd., Cambridge UK) was employed. The parameters controlling the precise operation of the genetic algorithm were set as described previously (38). The validity of the settings was first confirmed as follows: the isoformononetin bound in the active site of the I-7-OMT model was removed, and the isoflavone daidzein was then docked to the structure to compare the in silico docking solutions to the actual structural complex. The confirmed settings were then used for the automated docking of 5-(8',11',14')-pentadecatrienyl resorcinol to the SbOMT3 model. The size of the SbOMT3 active site was defined within 15 Å around the N-{epsilon}2 atom of His-322, which projects into the center of the putative substrate-binding site of the SbOMT3 model. Ten docking calculations were run, and the GOLD score was used to identify the lowest energy docking results.

Substrates—Resorcinol and orcinol were purchased from Sigma. 5-n-Pentyl-resorcinol was purchased from Fisher, and 5-n-pentadecyl-resorcinol was purchased from Chem Service, Inc. (West Chester, PA). For synthesis of 5-n-propyl-, 5-n-butyl-, 5-n-hexyl-, 5-n-heptyl-, and 5-n-nonyl-resorcinol, 3,5-bis-(benzyloxy)benzaldehyde was first prepared from methyl 3,5-dihydroxybenzoate (Lancaster Synthesis, Inc., Pelham, NH) using the method developed by Franke and Binder (41). A mixture of the appropriate alkyltriphenylphosphonium bromide (5 mmol) (Lancaster Synthesis, Inc.) with sodium hydride (5 mmol) was stirred for 15 min at room temperature in dry methylene chloride, and then 3,5-bis(benzyloxy)benzaldehyde (5 mmol) was added. The reaction mixture was refluxed for 4 h, filtered, and evaporated. The residue obtained was chromatographed over silica gel, and eluted with hexane:ethyl acetate (49:1 v/v), to afford a mixture of cis- and trans-1-(2-alkenylinyl)-3, 5-dihydroxybenzene isomers in 70–80% yield. The mixtures were hydrogenated at 275 kPa for 10 h in the presence of 10% palladium-carbon catalyst, filtered through celite, and then evaporated. Residues were then chromatographed over silica gel and eluted with hexane:ethyl acetate (17:3 v/v), quantitatively yielding the final n-alkylresorcinol product. The identities of all compounds were confirmed using both physical and spectroscopic methods, including 1H NMR, 13C NMR, and high resolution mass spectroscopy. 1H NMR and 13C NMR spectra were recorded using an Avance DPX-300 spectrometer (300 MHz for 1H NMR, 75.45 MHz for 13C NMR; Bruker Biospin Corp., Billerica, MA) and an Avance DRX-500 spectrometer (500 MHz for 1H NMR, 125 MHz for 13C NMR; Bruker Biospin Corp.) in CDCl3 and MeOH-D4, using tetramethylsilane as an internal standard. High resolution mass spectroscopy data were obtained by direct probe, using a Bioapex-FTMS spectrometer with electrospray ionization (Bruker Biospin Corp.). 5-n-[8',11',14']-Pentadecatrienyl-resorcinol was purified from Anacardium occidentale (cashew) nutshell liquid using the method developed by Paramashivappa et al. (42). All other substrates described were purchased from Sigma.

Phylogenetic Analysis—Amino acid sequences of putative O-methyltransferases were retrieved from the NCBI nonredundant peptide sequence data base (ncbi.nlm.nih.gov) by BLASTP analysis and from the TIGR plant gene indices data base by TBLASTN analysis, using default parameters. A candidate list was screened for redundancy and errors, resulting in a data set consisted of 134 sequences, including the three S. bicolor OMT sequences described in this work (SbOMT1, SbOMT2, and SbOMT3). Multiple sequence alignments were constructed with ClustalX version 1.81 (43). Three parameter sets were investigated to assess sensitivity of the alignment to gap costs: default (gap opening = 10.0, gap extension = 0.2); gap opening = 10.0, gap extension = 1.0; gap opening = 1.0, gap extension = 1.0. All other parameters were set at default values (in particular, the Gonnet weight matrix was employed). The alignments differed substantially in length (853, 698, and 817 residues, respectively).

Phylogenetic estimates of the relationships among sequences were conducted separately for each alignment. The neighbor-joining method (44) as implemented in PAUP* version 4.0b10 (45) was used to estimate trees. Default parameters were used except that ties were broken randomly. Trees were midpoint-rooted and nodal support was estimated by the bootstrap approach (46), employing 5000 pseudoreplicate data sets. Phylogenetic trees estimated from the three alignments were extremely similar with differences restricted to minor rearrangements within clades and the relationships among several moderately sized clades. The third alignment, with equivalent gap opening and extension penalties, was selected for discussion and further analysis. However, all interpretations made here would be identical on trees estimated from the other two alignments. To clarify relationships in the presentation of the results, the 134 sequences in the third alignment were reduced to 76 sequences by removing highly similar sequences and reducing representation in clades distantly related to SbOMT1, SbOMT2 and SbOMT3. The resulting alignment was reanalyzed using the same methods, and the resulting phylogeny (Fig. 6) was highly congruent with the estimate from the 134-sequence data set.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Identification of Pentadecatrienyl Resorcinol and Pentadecatrienyl Resorcinol-3-methyl Ether Intermediates in S. bicolor Genotype BTx623—Given the likelihood that sorgoleone is synthesized predominantly in root hair cells (10, 20), it follows that the biosynthetic enzymes are exclusively or predominantly localized in this cell type. Furthermore, the significant quantity of sorgoleone-containing exudate produced by these cells suggests that the corresponding mRNAs encoding these enzymes could be among the most abundant. Expressed sequence tag (EST) analysis was therefore chosen as a gene isolation strategy to identify genes encoding enzymes involved in the biosynthesis of the allelochemical sorgoleone, as this approach is ideally suited for profiling the more abundant transcripts in a specific cell or tissue type (47–50). The majority of the existing sorghum genomics infrastructure is based on the S. bicolor genotype BTx623 (e.g. 26, 51), a parental line commonly used in commercial breeding programs and also used for the development of detailed genetic maps (52, 53). BTx623 was therefore initially selected as a model for the present work to utilize, as well as expand, the existing sorghum genomics infrastructure.

To first assess the suitability of genotype BTx623 as a model, sorgoleone levels were determined by HPLC analysis of root exudates collected from 5-day-old, etiolated seedlings. For comparison, an identical analysis was also performed with SX-17 seedlings, a previously characterized S. bicolor x S. sudanense hybrid (10, 54, 55). The results of this comparison are shown in Fig. 2A. The total sorgoleone content of ~1500 µg/g fresh weight measured in SX-17 seedlings is in agreement with previously reported levels for this genotype (54, 55), and was ~30% higher than those observed for BTx623 seedlings (Fig. 2A). Even though significant variation in sorgoleone content among different sorghum accessions is typically observed (14, 54), the results obtained in this study nevertheless clearly indicate that the sorgoleone content of genotype BTx623 is comparable with levels observed in other accessions. Also of interest is the observation that the predominant sorgoleone congener, 2-hydroxy-5-methoxy-3-[(Z,Z)-8',11',14'-pentadecatriene]-p-benzoquinone (Fig. 1), represents a major constituent (41%, w/w) of the exudate material produced by BTx623 seedlings (Fig. 2A). Taken together, the data clearly demonstrate the suitability of the genotype BTx623 as a model system for identifying genes associated with the biosynthesis of sorgoleone.

The likely pathway intermediate directly resulting from the polyketide synthase activity involved in sorgoleone biosynthesis, 5-pentadecatrienyl resorcinol (5-[(8'Z,11'Z)-8',11',14'-pentadecatrienyl]resorcinol; Fig. 1), was previously identified by GC-MS analysis (22). This finding, along with labeling studies demonstrating the polyketide origin of the quinone ring (21, 22), lends support for the initial steps in the proposed pathway shown (Fig. 1). Given the ultimate goal of identifying all of the key enzymes required for sorgoleone biosynthesis, which would include the biochemical characterization of fatty acid desaturases (DESs), polyketide synthases (PKSs), O-methyltransferases (OMTs), and possibly P450 monooxygenases (Fig. 1), the identification of additional pathway intermediates is crucial for the understanding of the in vivo substrates used by the O-methyltransferase and hydroxylase activities likely to be involved. Toward this end, methanol extracts were prepared from roots of 8-day-old BTx623 and SX-17 seedlings and analyzed by GC-MS, as shown in Fig. 2, B and C (representative chromatograms for genotype BTx623 shown). Significantly, in addition to the 5-pentadecatrienyl resorcinol previously identified, the 3-methyl ether derivative (5-[(8'Z,11'Z)-8',11',14'-pentadecatrienyl resorcinol-3-methyl ether) was also clearly observed (Fig. 2, B and C). The 5-pentadecatrienyl resorcinol and its 3-methyl ether derivative were identified from the total ion chromatograms of the root methanol extracts (retention time 21.8 and 18.3 min, respectively; Fig. 2, B and C) by extracted ion monitoring at m/z 314 for 5-pentadecatrienyl resorcinol and m/z 328 for the 3-methyl ether derivative (Fig. 2C), and the corresponding mass spectra for the two peaks revealed characteristic fragment ions supporting their identity (Fig. 2C; see also "Experimental Procedures"). Collectively, these data support the pathway model proposed in Fig. 1, where dihydroxylation of the resorcinol ring is preceded by 3'-O-methylation, likely catalyzed by a novel S-adenosyl-L-methionine-dependent OMT utilizing 5-[(8'Z,11'Z)-8',11',14'-pentadecatriene]resorcinol as a substrate in vivo. Consistent with this hypothesis, subsequent feeding studies performed using purified sorghum root hair preparations have detected an OMT activity capable of methylating the structurally related compound 5-n-pentadecyl resorcinol (56).


Figure 2
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  		FIGURE 2.
Comparison of sorgoleone production in S. bicolor genotype BTx623 versus SX-17 and identification of sorgoleone biosynthetic pathway intermediates. A, sorgoleone levels were determined by HPLC analysis of root exudates prepared from 5-day-old etiolated seedlings of S. bicolor genotypes BTx623 and SX-17. Data are shown as mean ± S.D. Open bars represent total sorgoleone content (µg/g fresh weight), and closed bars represent sorgoleone content as a percent of total exudate (w/w). B, GC-MS analysis was performed on methanol extracts prepared from roots of 8-day-old seedlings of S. bicolor genotype BTx623. The graph represents the total ion chromatogram showing the peaks of 5-(8',11',14')-pentadecatrienyl resorcinol-3-methyl ether at 18.3 min and 5-(8',11',14')-pentadecatrienyl resorcinol at 21.8 min. C, extracted ion chromatogram defined at m/z 314 showing the peaks for 5-(8',11',14')-pentadecatrienyl resorcinol-3-methyl and 5-(8',11',14')-pentadecatrienyl resorcinol, with the corresponding mass spectra shown as insets.

 
Survey of S. bicolor Root Hair ESTs—To create an EST data set useful for the identification of candidate OMT, DES, PKS, and P450 sequences potentially encoding enzymes involved in sorgoleone biosynthesis, highly enriched root hair preparations were obtained using the two-step liquid nitrogen immersion-microfiltration procedure devised by Bucher et al. (25). An aliquot of the nonamplified primary library obtained was used directly as the source of randomly selected clones for high throughput 5'-sequence analysis, resulting in 5,468 high quality ESTs, which assembled into 3,204 unique clusters, suggesting a broad estimation of ~9,000 for the transcriptome size of root hair cells (supplemental Fig. 1, A–C). Additionally, the 3,204 unique sequences identified in the root hair data set were assigned to available GO terms (Fig. 3), based on the Plant GOSlim classification scheme (33). Of particular interest, "other metabolic processes" (GO:0008152) were the second largest assignment within the "biological process" category (10.1% of all terms assigned), consistent with previous ultrastructural studies suggesting a high level of metabolic activity for this cell type associated with exudate production (10, 20). Not unexpectedly, "transport" (GO:0006810) was also identified as one of the most frequent assignments within the biological process (8.3% of all terms assigned), given the pivotal role played by root hair cells in soil mineral and organic nutrient uptake (57, 58).

Importantly, OMT, DES, P450, and PKS-like sequences were identified within the EST data set, and thus all of the major enzyme classes predicted to be required for the biosynthesis of sorgoleone appear to be highly represented (Table 1). Moreover, several OMT and DES-like sequences were included among the most highly expressed sequences in root hairs (Table 2), and we have recently demonstrated that contigs 2_32 and 2_162 (accounting for 0.4572% and 0.0914% of all ESTs, respectively) represent desaturases likely involved in generating the unusual 16:3{Delta}9,12,15 fatty acyl-CoA precursor used for sorgoleone biosynthesis in planta (designated SbDES2 and SbDES3; 59). A root hair-specific desaturase-like sequence nearly identical to contig 2_32 (designated SOR1), was also isolated from a sorghum-Sudangrass hybrid (60), and although the biochemical function of SOR1 was not determined, it is likely identical to that of SbDES3 (59).


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  		TABLE 1
Fatty acid desaturase-, polyketide synthase-, O-methyltransferase-, and P450 monooxygenase-like sequences identified in S. bicolor root hair EST data set

 


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  		TABLE 2
Most abundant sequences in S. bicolor root hair EST data set

 


Figure 3
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  		FIGURE 3.
Distribution of unique sequences identified in the S. bicolor root hair EST data set into GO-based categories. Sequences were assigned various GO terms based on the "Plant GOSlim" classification scheme (33). Percentages within each GO category (biological process, molecular function, cellular component) are indicated on the x axis and include all putative functions assigned to a given sequence using a cutoff threshold of E > 10-10.

 
To identify potentially novel gene sequences within the root hair ESTs, all contig consensus sequences were compared by BLASTN analysis against extensive nucleotide sequence data sets available for sorghum (Sorghum Milestone version 3.0; 22), corn (PlantGDB-assembled unique transcripts for Zea mays; and rice (Rice Annotation Project Data base; 61), using thresholds of E < 10-10 as well as E < 10-25 for the identification of potentially homologous sequences (supplemental Table 1, A–F). In addition, BLASTX comparisons against all Rice Annotation Project Data base (RAP-DB) proteins were performed using the same thresholds (supplemental Table 1G–H). At the more stringent E < 10-10 BLASTN criterion, 457 of the 3,204 root hair contigs (~14%) had no match with corn, 939 (~29%) had no match with rice, and 416 (~12%) had no match with publicly available sorghum sequences. Similar results for rice were obtained from BLASTX comparisons, with 827 sequences (~26%) finding no match. Thus, in addition to serving as a source of candidate sequences for the present work (Table 1), the root hair EST data significantly augment the existing EST data for S. bicolor and provide a useful resource for investigating novel genes specifically expressed in this cell type.

Identification of OMTs Preferentially Expressed in Root Hairs—As mentioned, 58 ESTs potentially encoding O-methyltransferases were identified in the root hair data set, which assembled into 12 contigs, and included 6 singletons (Table 1). Given the likelihood that sorgoleone biosynthesis occurs primarily or exclusively in root hairs (discussed above), a secondary screen using quantitative real time PCR was employed to identify OMT sequences expressed specifically or predominantly in this cell type. Gene-specific primer pairs were designed for monitoring OMT expression patterns in SYBR Green I-based real time PCR assays, using cDNAs prepared from total RNAs isolated from root hairs, root systems, developing panicles, stems, immature and fully expanded leaves, and shoot apices. As controls, gene-specific primer pairs were also designed for an S. bicolor chlorophyll a/b-binding protein (CAB; uniscript ID 2_9279) and polyubiquitin-like sequence (uniscript ID 2_9314), selected by in silico expression analysis using the MAGIC Gene Discovery software (32). Based on these analyses, three OMT-like contig sequences (2_81, 2_47, and 2_53; Tables 1 and 2) preferentially expressed in root hairs were selected for more detailed characterization and designated SbOMT1, SbOMT2, and SbOMT3, respectively. As can be seen in Fig. 4A, SbOMT1, SbOMT2, and SbOMT3 transcripts were predominantly expressed in root hairs, as well as total root systems harvested from 8-day-old seedlings grown under conditions identical to those used for root hair preparations, and thus contained extensive amounts of root hairs. In all cases, however, expression in root hairs was much higher than that observed in roots, suggesting that the root hairs contributed a significant percentage of the transcripts detected in the total root samples. Specifically, SbOMT1 transcript levels were ~88-fold higher, SbOMT2 transcript levels were ~7-fold higher, and SbOMT3 transcript levels were ~32-fold higher in root hairs as compared with that observed for total root systems (Fig. 4A). As expected, expression of the CAB control sequence was detected predominantly in chloroplast-containing tissues, particularly in immature leaves, using the identical cDNA samples prepared for the OMT expression studies described above (Fig. 4B). For the polyubiquitin control, transcript levels were remarkably consistent among all of the samples analyzed, varying ~4-fold between the highest and lowest samples (Fig. 4B).

The deduced full-length open reading frames for SbOMT1, SbOMT2, and SbOMT3 exhibited extensive sequence similarity at the amino acid level to previously characterized plant type I S-adenosyl-L-methionine-dependent O-methyltransferases (62), and contained conserved residues and motifs putatively associated with catalysis and substrate binding, based on the crystal structures determined for several M. sativa type I enzymes (63, 64; Fig. 5A). All three sequences encode ~41-kDa proteins with predicted isoelectric points of 4.86 (SbOMT1), 5.43 (SbOMT2), and 5.11 (SbOMT3). Southern analyses performed using ~450-bp probe sequences derived from contiguous 3'-UTR and coding regions for all three genes (see "Experimental Procedures") indicated that SbOMT1 and SbOMT2 likely do not share extensive nucleotide identity with other OMT genes within the S. bicolor genotype BTx623 genome (Fig. 5B). For SbOMT3, the hybridization patterns obtained suggest the existence of one or more closely related OMT sequences in BTx623, based on the high stringency wash conditions employed.


Figure 4
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  		FIGURE 4.
Identification of root hair-specific OMT sequences by quantitative real time RT-PCR analysis. Relative expression levels were determined by quantitative real time RT-PCR using gene-specific primers. Data were normalized to an internal control (18 S rRNA), and the {Delta}{Delta}CT method was used to obtain the relative expression levels for each sequence, expressed as mean ± S.D. A, relative expression levels of SbOMT1, SbOMT2, and SbOMT3 in different S. bicolor tissues. For clarity, a different y axis scale was used for displaying SbOMT2 values (right side of graph). B, relative expression levels of two S. bicolor control genes: chlorophyll a/b-binding protein (CAB 2_9279) and a polyubiquitin-like sequence (UBI 2_9314). For clarity, different y axis scales were used for displaying CAB 2_9279 and UBI 2_9314 values.

 
OMT Functional Characterization—Follow-up recombinant enzyme studies with SbOMT1, SbOMT2, and SbOMT3 were next performed to determine their preferred substrates among a panel of benzene derivatives containing different functional groups and substitution patterns (Table 3), including a series of 5-n-alkylresorcinols of increasing chain length, as well as 5-n-pentadecatrienyl resorcinol, the proposed sorgoleone pathway intermediate and physiological substrate for the participating OMT enzyme (Fig. 1). For these analyses, full-length open reading frames for all three OMTs were overexpressed in E. coli as N-terminal polyhistidine fusions, purified using an activated nickel column, and then radiometrically assayed in the presence of different substrates as described by Wang and Pichersky (37), with slight modifications (see "Experimental Procedures"). The results of these experiments are shown in Table 3. For SbOMT1, eugenol was clearly the preferred substrate among the various benzene derivatives analyzed, but significant activity was also obtained with the monomethyl ethers of orcinol and resorcinol. Aside from all three compounds containing a methoxy group and a simple benzene ring, it is difficult to discern a clear pattern governing the substrate specificity for SbOMT1. For example, guaiacol, ferulic acid, and 4-methoxyphenol all possess a single benzene ring containing a methoxy group, yet were much less efficiently utilized by recombinant SbOMT1. By definition, SbOMT1 could be classified as a eugenol OMT (EOMT), functionally related to the previously characterized EOMT isolated from sweet basil (65; GenBankTM accession number AAL30424 [GenBank] ; Fig. 5A), and the (iso)EOMT isolated from Clarkia breweri (66; GenBankTM accession number AAC01533 [GenBank] ). The substrate specificity of SbOMT1 is particularly reminiscent of that observed for EOMT from sweet basil, which also efficiently utilized guaiacol but exhibited little to no detectable activity with resorcinol, caffeic acid, ferulic acid, or catechol. The sweet basil enzyme did efficiently use chavicol as a substrate, which lacks the methoxy ring substituent; however, chavicol is otherwise structurally identical to eugenol (65). Recombinant SbOMT2, in contrast to SbOMT1 and SbOMT3, showed no detectable activity against any of the substrates indicated in Table 3, although the protein did associate with the soluble fraction of the E. coli protein extracts, and migrated at the expected position by SDS-PAGE analysis. Presumably, either SbOMT2 encodes a nonfunctional protein or the enzyme does not act on any of the substrates tested in the present work.


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  		TABLE 3
Relative activity (%) of recombinant SbOMT1 and SbOMT3 with various substrates

 
In contrast to SbOMT1, the substrate specificity for SbOMT3 indicated a clear preference for the alkylresorcinolic substrates listed in Table 3. SbOMT3 enzyme activity with alkylresorcinols increased dramatically as the alkyl side chain was increased up to five carbons in length, and near-maximal activity was observed using hexyl resorcinol, after which enzyme activity decreased precipitously with increasing side chain length (Table 3). Evaluation of steady-state kinetic parameters for reaction of SbOMT3 with C3–C7 alkylresorcinols (Table 4) suggested an increase in both the affinity of the enzyme for these substrates (Km) with increasing chain length, as well as an increase in catalytic efficiency (kcat/Km). Because of the limited activity with alkylresorcinols larger than C7 (for reasons discussed below), kinetic parameters were not determined for these substrates. Interestingly, activity of SbOMT3 with the proposed sorgoleone pathway intermediate, 5-pentadecatrienyl resorcinol (Figs. 1 and 2B), was ~3-fold higher than that for the 5-pentadecyl resorcinol, which contains a saturated (alkyl) side chain of identical length (Table 3). Overall, SbOMT3 activity appeared somewhat promiscuous, as a low level of residual activity occurred with almost all of the substrates analyzed.


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  		TABLE 4
Kinetic parameters for recombinant SbOMT3 with alkylresorcinols and co-substrate (AdoMet)

 


Figure 5
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  		FIGURE 5.
Sequence alignment and genomic organization of SbOMT1, SbOMT2, and SbOMT3. A, deduced amino acid sequences of SbOMT1, SbOMT2, and SbOMT3 were aligned with three related plant type I OMTs as follows: M. sativa I-7-OMT, GenBankTM accession number AAC49927 (76); Rosa hybrid cultivar "Fragrant Cloud" OOMT1, GenBankTM accession number AAM23004 (67); Ocimum basilicum EOMT1, GenBankTM accession number AAL30424 (65) using ClustalW. Residues important for catalysis, substrate binding, and AdoMet binding are indicated based on OMT crystallography studies performed by Zubieta et al. (63, 64), and by computational homology modeling of SbOMT3 (see text). Identical or similar amino acids shared by at least two sequences within the alignment are shaded with black and gray, respectively. B, 10 µg of genomic DNA isolated from S. bicolor genotype BTx623 were digested with either DraI (lane 1), EcoRV (lane 2), ScaI (lane 3), or SphI (lane 4) and then size-fractionated on 0.8% (w/v) agarose gels and transferred to nylon membranes. Blots were then hybridized using 32P-labeled 3' probe sequences for SbOMT1, SbOMT2, and SbOMT3, washed at high stringency, and then subjected to autoradiography.

 
The alk(en)ylresorcinols used for the present study have not been previously tested as substrates in other OMT studies performed to date, and thus it is somewhat difficult to draw direct parallels between SbOMT3 and other characterized OMT enzymes. For example, OOMT1 isolated from rose (Fig. 5A) has been shown to preferentially utilize orcinol (5-methylresorcinol) as a substrate; however, its activity against 5-n-resorcinolic derivatives with longer alkyl side chains has not been determined (67). Moreover, OOMT1 efficiently utilizes other simple phenolic substrates such as guaiacol and orcinol monomethyl ether, which SbOMT3 showed only marginal activity against (Table 3). Thus, SbOMT3 likely represents a novel subclass of type I plant-specific OMT enzymes, exhibiting a substrate profile that has not been demonstrated previously for any plant OMT to our knowledge. Moreover, phylogenetic analyses of putative and functionally characterized plant-specific type I OMT enzymes indicate that SbOMT3 falls within a distinct clade of OMTs, which include predicted sequences from rice and corn whose function at the present time remains obscure (Fig. 6). Within this group, SbOMT3 was most closely related (52% identity) to a putative O-methyltransferase from Z. mays expressed predominantly in root endodermal cells (accession number P47917 [GenBank] ; 68). The observed preference for alkylresorcinolic substrates is particularly intriguing with respect to the consideration of a possible role for SbOMT3 in the sorgoleone biosynthetic pathway. It is highly tempting to speculate that this enzyme could correspond to the OMT involved in sorgoleone biosynthesis, given the strong preference for compounds within the same closely related structural family as the likely physiological substrate (Figs. 1 and 2). Interestingly, OMT enzymatic activity(s) recently detected in ex vivo assays with purified sorghum root hair preparations also utilized C3–C15 5-alkylresorcinolic substrates (56), and likely corresponds to the activity directed by SbOMT3 and/or closely related enzymes expressed at high levels in these cells (Tables 1 and 2).


Figure 6
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  		FIGURE 6.
Phylogenetic analysis of SbOMT1, SbOMT2, and SbOMT3 relatives. The phylogram was generated using the neighbor-joining method as implemented in PAUP version 4.0b10 (45). To assess clade support, the bootstrap method was used with 5000 pseudoreplicates. Numbers at nodes indicate the percentage of bootstrap pseudoreplicates in which the clade was recovered.

 
To examine the potential structural basis for the observed substrate specificity of SbOMT3, computational homology modeling and automated substrate dockings were also performed (Fig. 7). Several previously determined plant type I OMT crystal structures (63, 64, 69) provide a useful basis for an in silico homology-based structure-function analysis of OMT homologs. Among the crystallized proteins, the alfalfa (M. sativa) I-7-OMT (see also Fig. 5) revealed the highest relationship score with SbOMT3 (37% identity), and therefore its crystal structure (Protein Data Bank code IFP2) was used to model SbOMT3.

Similar to the M. sativa I-7-OMT (63), the overall structure of the SbOMT3 model (Fig. 7A) indicates a small N-terminal domain that, as in the other type I OMTs, likely plays a major role facilitating the dimerization of the two subunits within the homodimer. The larger C-terminal domain contains both the AdoMet and the substrate-binding sites. The SbOMT3 substrate docking model with 5-(8',11',14')-pentadecatrienyl resorcinol (Fig. 7B) shows the best fit with the 3-hydroxyl group of the resorcinol head pointing to the methyl donor AdoMet and His-279, consistent with the histidine functioning as general base in the deprotonation of the hydroxyl nucleophile in type I OMTs (63, 64, 69). The binding of the resorcinolic substrates appears to be primarily by van der Waals hydrophobic interactions, with the resorcinol ring positioned by a series of hydrophobic residues (Met-126, Phe-183, Met-187, Ile-336, and the side chains of Trp-170 and Trp-276; Fig. 7B). Val-130 and Phe-161 form a hydrophobic sandwich-like clamp that constrains the center portion of the aliphatic side chain, whereas the tip of the very hydrophobic alkyl portion protrudes into a hydrophobic cave formed at the protein dimer interface by the side chains of Lys-335, Met-334, Leu-139 (from protein subunit A), and Phe-25 and Met-26 (from subunit B).


Figure 7
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  		FIGURE 7.
Molecular modeling of SbOMT3 based on the M. sativa I-7-OMT crystal structure. A, ribbon diagram of the SbOMT3 monomer three-dimensional structure. {alpha}-Helices are indicated in red, connecting loops in green, and β-strands in yellow. N and C termini are also shown. B, close-up view of the three-dimensional model of the SbOMT3 active site containing AdoMet and a docked molecule of 5-(8',11',14')-pentadecatrienyl resorcinol. C, close-up molecular surface views of the I-7-OMT and SbOMT3 active sites, with I-7-OMT shown complexed with the isoflavone daidzein. Numbering for amino acid residues shown were based on the deduced amino acid sequence of SbOMT3 (for B and right panel in C, GenBankTM accession number EF189708), and of M. sativa I-7-OMT (left panel in C, GenBankTM accession number AAC49927; 76).

 
The positioning and binding of the methyl donor AdoMet and the location of the putative catalytic histidine (His-279; Fig. 7B) are very similar to that in the crystallized proteins, indicating that SbOMT3 uses the same Sn2 catalytic mechanism for methyl transfer as proposed for the other enzymes. However, the active site architecture of SbOMT3 (Fig. 7B) reveals significant differences with that of the M. sativa I-7-OMT, most readily visible by comparison of the active site surface views of the two enzymes (Fig. 7C). Most notable is the position of the side chain of His-332 in SbOMT3 (corresponding to Asn-310 in I-7-OMT), which projects into the center of the binding site, producing a curved cavity appearing particularly well suited to accommodate the stereochemical conformation of 5-pentadecatrienyl resorcinol, consistent with the proposal that 5-pentadecatrienyl resorcinol represents the physiological substrate for this enzyme. The observation that the otherwise very similar substrate 5-pentadecyl resorcinol possessing a saturated alkyl side chain is a poorer substrate (Table 3) suggests that the shape of the cavity plays a significant role in determining the substrate preferences. Furthermore, given the size of the active site cavity, it is not surprising that nonphysiological substrates possessing a resorcinol head but shorter alkyl side chains are also excellent substrates of SbOMT3 (Table 3).

When considering the alk(en)yl-resorcinolic substrates used in this work (Tables 3 and 4), it is important to take into account the amphiphilicity of these compounds, which contain separate hydrophilic (resorcinolic head group) and hydrophobic (aliphatic side chain) substituents (reviewed in Ref. 70), as well as the relatively low aqueous solubilities for those having longer aliphatic side chains (supplemental Table 2). Because of their amphiphilicity, resorcinolic lipids can form monolayers in aqueous solution, and this propensity increases as a function of aliphatic chain length and reduced saturation (71, 72). Thus, the in vitro studies shown in Table 3 for alkylresorcinolic substrate utilization by SbOMT3 likely reflect a combination of substrate preference, solubility, and the extent to which a given substrate exists in monomeric form in solution.

It is reasonable to speculate that the in vivo situation involves biochemical mechanisms that circumvent the inherent difficulties associated with the physical properties of these molecules, such as "substrate channeling" through the active sites of multienzyme biosynthetic complexes (73, 74), or the involvement of carrier protein co-enzymes such as acyl-CoA-binding proteins (75) (see also Table 2). These additional considerations, in combination with the SbOMT3 functional and in silico results described in the present work, argue strongly in favor of a potential role for SbOMT3 in the sorgoleone biosynthetic pathway. Experiments are currently being developed to directly address this question, including overexpression and RNA interference studies with transgenic sorghum plants.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) EF189706, EF189707, EF189708, and EH406574 to EH412041.

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and Tables 1 and 2. Back

2 Present address: U. S. Department of Agriculture, Agricultural Research Service, FAPRU, N220 Agricultural Sciences North, Lexington, KY 40546. Back

1 To whom correspondence should be addressed: U. S. Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, P. O. Box 8048, University, MS 38677. Tel.: 662-915-7965; Fax: 662-915-1035; E-mail: sbaerson{at}ars.usda.gov.

3 D. Cook, A. M. Rimando, T. E. Clemente, J. Schröder, N. P. Nanayakkara, M. Fishbein, Z. Pan, I. Abe, F. E. Dayan, and S. R. Baerson, unpublished results. Back

4 The abbreviations used are: AdoMet, S-adenosylmethionine; GC-MS, gas chromatography-mass spectrometry; RT, reverse transcription; EST, expressed sequence tag; HPLC, high pressure liquid chromatography; DES, desaturase; OMT, O-methyltransferase; PKS, polyketide synthase; UTR, untranslated region; GO, gene ontology; I-7-OMT, isoflavone 7-O-methyltransferase; EOMT, eugenol OMT; CAB, chlorophyll a/b-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Joel Parker of Expression Analysis, Inc. (Durham, NC), for assistance with bioinformatic analyses and Melanie Mask, Susan Watson, Gloria Hervey, Haiming Wang, Zheng Xia, Virgil Ed Johnson, Krishna Reddy, and Albert Tidwell for providing materials and excellent technical support. We are also grateful to Dr. Joseph Noel for numerous helpful discussions and suggestions pertaining to this work.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Inderjit, and Duke, S. O. (2003) Planta 217, 529-539[CrossRef][Medline] [Order article via Infotrieve]
  2. Bais, H. P., Park, S. W., Weir, T. L., Callaway, R. M., and Vivanco, J. M. (2004) Trends Plant Sci. 9, 26-32[CrossRef][Medline] [Order article via Infotrieve]
  3. Schulz, M., and Wieland, I. (1999) Chemoecology 9, 133-141[CrossRef]
  4. Duke, S. O., Rimando, A. M., Baerson, S. R., Scheffler, B. E., Ota, E., and Belz, R. G. (2002) J. Pestic. Sci. 27, 298-306
  5. Weston, L. A., and Duke, S. O. (2003) Crit. Rev. Plant Sci. 22, 367-389[CrossRef]
  6. Dogget, H. (1988) Sorghum, 2nd Ed., pp. 1-13, John Wiley & Sons, Inc., New York
  7. Duke, S. O., Belz, R. G., Baerson, S. R., Pan, Z., Cook, D. D., and Dayan, F. E. (2005) Outlooks on Pest Management 16, 64-68
  8. Netzly, D. M., and Butler, L. G. (1986) Crop Sci. 26, 775-778[Abstract/Free Full Text]
  9. Einhellig, F. A., and Souza, I. F. (1992) J. Chem. Ecol. 18, 1-11[CrossRef]
  10. Czarnota, M. A., Paul, R. N., Dayan, F. E., Nimbal, C. I., and Weston, L. A. (2001) Weed Technol. 15, 813-825[CrossRef]
  11. Kagan, I. A., Rimando, A. M., and Dayan, F. E. (2003) J. Agric. Food Chem. 51, 7589-7595[CrossRef][Medline] [Order article via Infotrieve]
  12. Rimando, A. M., Dayan, F. E., and Streibig, J. C. (2003) J. Nat. Prod. (Lloydia) 66, 42-45[CrossRef]
  13. Rasmussen, J. A., Hejl, A. M., Einhellig, F. A., and Thomas, J. A. (1992) J. Chem. Ecol. 18, 197-207[CrossRef]
  14. Nimbal, C. I., Pedersen, J. F., Yerkes, C. N., Weston, L. A., and Weller, S. C. (1996) J. Agric. Food Chem. 44, 1343-1347[CrossRef]
  15. Rimando, A. M., Dayan, F. E., Czarnota, M. A., Weston, L. A., and Duke, S. O. (1998) J. Nat. Prod. (Lloydia) 61, 927-930[CrossRef]
  16. Meazza, G., Scheffler, B. E., Tellez, M. R., Rimando, A. M., Nanayakkara, N. P. D., Khan, I. A., Abourashed, E. A., Romagni, J. G., Duke, S. O., and Dayan, F. E. (2002) Phytochemistry 59, 281-288
  17. Hejl, A. M., and Koster, K. L. (2004) J. Chem. Ecol. 30, 2181-2191[CrossRef][Medline] [Order article via Infotrieve]
  18. Bertin, C., Yang, X., and Weston, L. A. (2003) Plant Soil. 256, 67-83[CrossRef]
  19. Duke, S. O. (2003) Trends Biotechnol. 21, 192-195[CrossRef][Medline] [Order article via Infotrieve]
  20. Czarnota, M. A., Paul, R. N., Weston, L. A., and Duke, S. O. (2003) Int. J. Plant Sci. 164, 861-866[CrossRef]
  21. Fate, G. D., and Lynn, D. G. (1996) J. Am. Chem. Soc. 118, 11369-11376[CrossRef]
  22. Dayan, F. E., Kagan, I. A., and Rimando, A. M. (2003) J. Biol. Chem. 278, 28607-28611[Abstract/Free Full Text]
  23. Netzly, D. M., Riopel, J. L., Ejeta, G., and Butler, L. G. (1988) Weed Sci. 36, 441-446
  24. Dayan, F. E. (2006) Planta 224, 339-346[CrossRef][Medline] [Order article via Infotrieve]
  25. Bucher, M., Schroeer, B., Willmitzer, L., and Riesmeier, J. W. (1997) Plant Mol. Biol. 35, 497-508[CrossRef][Medline] [Order article via Infotrieve]
  26. Pratt, L. H., Liang, C., Shah, M., Sun, F., Wang, H., Reid, S. P., Gingle, A. R., Paterson, A. H., Wing, R., Dean, R., Klein, R., Nguyen, H. T., Ma, H. M., Zhao, X., Morishige, D. T., Mullet, J. E., and Cordonnier-Pratt, M.-M. (2005) Plant Physiol. 139, 869-884[Abstract/Free Full Text]
  27. Ewing, B., Hillier, L., Wendl, M. C., and Green, P. (1998) Genome Res. 8, 175-185[Abstract/Free Full Text]
  28. Liang, C., Sun, F., Wang, H., Qu, J., Freeman, R. M., Jr., Pratt, L. H., and Cordonnier-Pratt, M.-M. (2006) BMC Bioinformatics 7, 115[CrossRef][Medline] [Order article via Infotrieve]
  29. Pertea, G., Huang, X., Liang, F., Antonescu, V., Sultana, R., Karamycheva, S., Lee, Y., White, J., Cheung, F., Parvizi, B., Tsai, J., and Quackenbush, J. (2003) Bioinformatics (Oxf.) 19, 651-652[Abstract/Free Full Text]
  30. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res. 25, 3389-3402[Abstract/Free Full Text]
  31. Wu, C. H., Huang, H., Arminski, L., Castro-Alvear, J., Chen, Y., Hu, Z. Z., Ledley, R. S., Lewis, K. C., Mewes, H. W., Orcutt, B. C., Suzek, B. E., Tsugita, A., Vinayaka, C. R., Yeh, L. S., Zhang, J., and Barker, W. C. (2002) Nucleic Acids Res. 30, 35-37[Abstract/Free Full Text]
  32. Cordonnier-Pratt, M.-M., Liang, C., Wang, H., Kolychev, D. S., Sun, F., Freeman, R., Sullivan, R., and Pratt, L. H. (2004) Comp. Funct. Genom. 5, 268-275[CrossRef]
  33. Berardini, T. Z., Mundodi, S., Reiser, L., Huala, E., Garcia-Hernandez, M., Zhang, P., Mueller, L. A., Yoon, J., Doyle, A., Lander, G., Moseyko, N., Yoo, D., Xu, I., Zoeckler, B., Montoya, M., Miller, N., Weems, D., and Rhee, S. Y. (2004) Plant Physiol. 135, 1-11[Free Full Text]
  34. Baerson, S. R., Sánchez-Moreiras, A., Pedrol-Bonjoch, N., Schulz, M., Kagan, I. A., Agarwal, A. K., Reigosa, M. J., and Duke, S. O. (2005) J. Biol. Chem. 280, 21867-21881[Abstract/Free Full Text]
  35. Engels, W. R. (1993) Trends Biochem. Sci. 18, 448-450[CrossRef][Medline] [Order article via Infotrieve]
  36. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, pp. 9.31-9.57, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  37. Wang, J., and Pichersky, E. (1999) Arch. Biochem. Biophys. 368, 172-180[CrossRef][Medline] [Order article via Infotrieve]
  38. Coiner, H., Schröder, G., Wehinger, E., Liu, C. J., Noel, J. P., Schwab, W., and Schröder, J. (2006) Plant J. 46, 193-205[CrossRef][Medline] [Order article via Infotrieve]
  39. Marti-Renom, M. A., Stuart, A. C., Fiser, A., Sánchez, R., and Sali, A. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 291-325[CrossRef][Medline] [Order article via Infotrieve]
  40. Fiser, A., Do, R. K., and Sali, A. (2000) Protein Sci. 9, 1753-1773[Medline] [Order article via Infotrieve]
  41. Franke, I., and Binder, M. (1980) Helv. Chim. Acta 63, 2508-2514[CrossRef]
  42. Paramashivappa, R., Kumar, P. P., Vithayathil, P. J., and Rao, A. S. (2001) J. Agric. Food Chem. 49, 2548-2551[CrossRef][Medline] [Order article via Infotrieve]
  43. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins, D. G. (1997) Nucleic Acids Res. 25, 4876-4882[Abstract/Free Full Text]
  44. Saitou, N., and Nei, M. (1987) Mol. Biol. Evol. 4, 406-425[Abstract]
  45. Swofford, D. (2001) PAUP* Phylogenetic Analysis Using Parsimony (* and Other Methods), Version 4, Sinauer Associates, Sunderland, MA
  46. Felsenstein, J. (1985) Evolution 39, 783-791[CrossRef]
  47. Ohlrogge, J., and Benning, C. (2000) Curr. Opin. Plant Biol. 3, 224-228[Medline] [Order article via Infotrieve]
  48. Lange, B. M., Wildung, M. R., Stauber, E. J., Sánchez, C., Pouchnik, D., and Croteau, R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2934-2939[Abstract/Free Full Text]
  49. Guterman, I., Shalit, M., Menda, N., Piestun, D., Dafny-Yelin, M., Shalev, G., Bar, E., Davydov, O., Ovadis, M., Emanuel, M., Wang, J., Adam, Z., Pichersky, E., Lewinsohn, E., Zamir, D., Vainstein, A., and Weiss, D. (2002) Plant Cell 10, 2325-2338
  50. Fridman, E., Wang, J., Iijima, Y., Froehlich, J. E., Gang, D. R., Ohlrogge, J., and Pichersky, E. (2005) Plant Cell 17, 1252-1267[Abstract/Free Full Text]
  51. Paterson, A. H. (2006) Genome Biol. 7, 311[CrossRef][Medline] [Order article via Infotrieve]
  52. Menz, M. A., Klein, R. R., Mullet, J. E., Obert, J. A., Unruh, N. C., and Klein, P. E. (2002) Plant Mol. Biol. 48, 483-499[CrossRef][Medline] [Order article via Infotrieve]
  53. Bowers, J. E., Abbey, C., Anderson, S., Chang, C., Draye, X., Hoppe, A. H., Jessup, R., Lemke, C., Lennington, J., Li, Z., Lin, Y. R., Liu, S. C., Luo, L., Marler, B. S., Ming, R., Mitchell, S. E., Qiang, D., Reischmann, K., Schulze, S. R., Skinner, D. N., Wang, Y. W., Kresovich, S., Schertz, K. F., and Paterson, A. H. (2003) Genetics 165, 367-386[Abstract/Free Full Text]
  54. Czarnota, M. A., Rimando, A. M., and Weston, L. A. (2003) J. Chem. Ecol. 29, 2073-2083[CrossRef][Medline] [Order article via Infotrieve]
  55. Rimando, A. M., Kagan, I. A., Dayan, F. E., Czarnota, M. A., and Weston, L. A. (2005) in Semiochemicals in Pest and Weed Control: ACS Symposium Series (Petroski, R. J., Tellez, M. R., and Behle, R. W., eds) Vol. 906, pp. 59-70, American Chemical Society, Washington, D. C.
  56. Dayan, F. E., Watson, S. B., and Nanayakkara, D. N. P. (2007) J. Exp. Bot. 58, 3263-3272[Abstract/Free Full Text]
  57. Cutter, E. G. (1978) Plant Anatomy, pp. 94-106, Addison-Wesley Publishing Co., Boston
  58. Grierson, C., and Schiefelbein, J. (2002) in The Arabidopsis Book (Somerville, C. R., and Meyerowitz, E. M., eds) pp. 1-22, American Society of Plant Biologists, Rockville, MD
  59. Pan, Z., Rimando, A. M., Baerson, S. R., Fishbein, M., and Duke, S. O. (2007) J. Biol. Chem. 282, 4326-4335[Abstract/Free Full Text]
  60. Yang, X., Scheffler, B. E., and Weston, L. A. (2004) J. Exp. Bot. 55, 2251-2259[Abstract/Free Full Text]
  61. Ohyanagi, H., Tanaka, T., Sakai, H., Shigemoto, Y., Yamaguchi, K., Habara, T., Fujii, Y., Antonio, B. A., Nagamura, Y., Imanishi, T., Ikeo, K., Itoh, T., Gojobori, T., and Sasaki, T. (2006) Nucleic Acids Res. 1, D741-D744
  62. Noel, J. P., Dixon, R. A., Pichersky, E., Zubieta, C., and Ferrer, J.-L. (2003) Recent Adv. Phytochem. 37, 37-58
  63. Zubieta, C., He, X.-Z., Dixon, R. A., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 271-279[CrossRef][Medline] [Order article via Infotrieve]
  64. Zubieta, C., Kota, P., Ferrer, J.-L., Dixon, R. A., and Noel, J. P. (2002) Plant Cell 14, 1265-1277[Abstract/Free Full Text]
  65. Gang, D. R., Lavid, N., Zubieta, C., Chen, F., Beuerle, T., Lewinsohn, E., Noel, J. P., and Pichersky, E. (2002) Plant Cell 14, 505-519[Abstract/Free Full Text]
  66. Wang, J., Dudareva, N., Bhakta, S., Raguso, R. A., and Pichersky, E. (1997) Plant Physiol. 114, 213-221[Abstract]
  67. Lavid, N., Wang, J., Shalit, M., Guterman, I., Bar, E., Beuerle, T., Menda, N., Shafir, S., Zamir, D., Adam, Z., Vainstein, A., Weiss, D., Pichersky, E., and Lewinsohn, E. (2002) Plant Physiol. 129, 1899-1907[Abstract/Free Full Text]
  68. Held, B. M., Wang, H., John, I., Wurtele, E. S., and Colbert, J. T. (1993) Plant Physiol. 102, 1001-1008[Abstract]
  69. Liu, C. J., Deavours, B. E., Richard, S. B., Ferrer, J. L., Blount, J. W., Huhman, D., Dixon, R. A., and Noel, J. P. (2006) Plant Cell 18, 3656-3669[Abstract/Free Full Text]
  70. Kozubek, A., and Tyman, J. H. P. (1999) Chem. Rev. 99, 1-25[CrossRef][Medline] [Order article via Infotrieve]
  71. Kozubek, A. (1989) Acta Univ. Wratislav. 868, 27-32
  72. Kozubek, A. (1995) Acta Biochim. Pol. 42, 241-246[Medline] [Order article via Infotrieve]
  73. Achnine, L., Blancaflor, E. B., Rasmussen, S., and Dixon, R. A. (2004) Plant Cell 16, 3098-3109[Abstract/Free Full Text]
  74. Facchini, P. J., and St-Pierre, B. (2005) Curr. Opin. Plant Biol. 8, 657-666[CrossRef][Medline] [Order article via Infotrieve]
  75. Gossett, R. E., Frolov, A. A., Roths, J. B., Behnke, W. D., Kier, A. B., and Schroeder, F. (1996) Lipids 31, 895-918[Medline] [Order article via Infotrieve]
  76. He, X.-Z., Reddy, J. T., and Dixon, R. A. (1998) Plant Mol. Biol. 36, 43-54[CrossRef][Medline] [Order article via Infotrieve]

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