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First published online October 19, 2007; 10.1105/tpc.107.054155 The Plant Cell 19:3212-3229 (2007) © 2007 American Society of Plant Biologists Evolution of Cinnamate/p-Coumarate Carboxyl Methyltransferases and Their Role in the Biosynthesis of Methylcinnamate[W]
a Department of Plant Sciences and BIO5 Institute for Collaborative Bioresearch, University of Arizona, Tucson, Arizona 85721 2 Address correspondence to gang{at}ag.arizona.edu.
Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase–like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.
Methylcinnamate and methyl-p-coumarate play important roles in plant–insect interactions and are widely distributed throughout the plant kingdom. They have been identified in both aerial and underground tissues from monocots and dicots as well as from ferns (Konda and Kawazu, 1979  ; Schaefers and Herrmann, 1982; Williams and Whitten, 1983; Hooper et al., 1984; Bandara et al., 1988; Cambie et al., 1990; Garcia et al., 1990; Seifert and Unger, 1994; Hiraga et al., 1996; Daayf et al., 1997a; Odell et al., 1999; Wu et al., 1999; Dias et al., 2003; Bruni et al., 2004; Azah et al., 2005; bin Jantan et al., 2005). Methylcinnamate, a known component of the floral scent from various orchid species, acts as a preferred attractant for euglossine bees and is electrophysiologically active toward excised bee antennae (Dodson et al., 1969; Ackerman, 1989; Schiestl and Roubik, 2003; Eltz and Lunau, 2005). Methylcinnamate production, however, is not strictly associated with flowers and has been found to accumulate to high levels in other tissues, including the leaves of sweet basil (Ocimum basilicum). In contrast with methylcinnamate, its p-hydroxylated derivative methyl-p-coumarate has been shown to have insecticidal or insect-deterrent (Hattori et al., 1992; Seifert and Unger, 1994) as well as antifungal (Bandara et al., 1988; Seifert and Unger, 1994) properties. Moreover, in cucumber (Cucumis sativus), methyl-p-coumarate can act as both an anticipin and an elicitor-inducible phytoalexin (Daayf et al., 1997a, 1997b, 2000).
The rapid growth, the presence of secretory glandular trichomes (glands) that produce a diversity of compounds from several different major biosynthetic pathways, and the availability of lines producing copious amounts of volatile compounds make sweet basil an excellent system to investigate the production of methylcinnamate and methyl-p-coumarate in planta. In sweet basil, the glands represent highly active metabolic factories that produce large amounts of terpenoids and phenylpropanoids as well as fatty acid–derived metabolites. Several different basil lines have been developed that produce different compounds from these various pathways as their major volatile and aroma components. Sweet Dani lemon basil (line SD) produces large amounts of citral, a mixture of the monoterpenoid compounds geranial and neral, which gives this variety its strong lemon scent. By contrast, basil lines SW and EMX-1 contain the phenylpropanoid pathway–derived phenylpropenes eugenol and methylchavicol, respectively, as well as mixtures of monoterpenoids and sesquiterpenoids. A fourth basil line, cinnamon basil (line MC), produces large amounts of methylcinnamate. The availability of these basil lines with distinct metabolite profiles and the development of methods for isolating the cells responsible for natural product biosynthesis have enabled us to utilize a comparative systems biology approach to identify genes involved in the production of the various volatile components unique to the different basil lines (Gang et al., 2001
The development of a large glandular trichome EST database made from these four distinct basil lines (J. Kapteyn, C. Soderlund, and D. Gang, unpublished data) allowed us to identify the genes responsible for the ultimate step in methylcinnamate biosynthesis (Figure 1
). As described in this report, methylcinnamate is produced in sweet basil by the activity of a newly identified member of the SABATH family of carboxyl methyltransferases (D'Auria et al., 2003
Gas Chromatography–Mass Spectrometry Analysis of Methylcinnamate in Basil Tissues Methylcinnamate is the key volatile component of several basil varieties and chemotypes, one of which, line MC, was selectively bred to produce high levels of this compound. To determine the biosynthetic commitment of line MC toward methylcinnamate, we used gas chromatography–mass spectrometry (GC-MS) analysis to evaluate the quantity of methylcinnamate relative to other volatile components produced in the leaves across the four referenced basil lines. Methylcinnamate constitutes 63% (based on total ion chromatogram peak area) of the total extracted volatiles from line MC leaves (Figure 2A ). Line SW contains much smaller levels of methylcinnamate (<10% of that produced by line MC, based on relative peak area); instead, it accumulates large amounts of eugenol. Line SD produces large amounts of citral but only trace amounts of methylcinnamate (Figure 2B); in line EMX-1, no methylcinnamate could be detected in ethyl acetate extracts from leaves, whereas large amounts of the phenylpropene methylchavicol were present instead. These differences in methylcinnamate production between the basil lines were used in a functional genomics approach to identify the enzyme(s) responsible for the formation of this methyl ester in basil.
Identification of CCMT Activity in Basil Glandular Trichomes Possible routes to methylcinnamate biosynthesis could include either a methyltransferase that uses AdoMet as a methyl donor and the carboxyl group of cinnamate as the acceptor or an acyltransferase that couples the cinnamoyl moiety from cinnamoyl-CoA to methanol, similar to what was recently shown for the production of the flavor compound methyl anthranilate in Concord grape (Vitis vinifera) (Wang and Luca, 2005 ). In order to distinguish which enzyme type is in fact responsible for the formation of methylcinnamate in sweet basil, cell-free protein extracts were produced from both young leaf glandular trichomes and whole young leaves of all four basil lines and analyzed for potential enzyme activities. To evaluate whether methylcinnamate in MC basil trichomes might be formed by an acyltransferase activity, we incubated MC gland protein extracts with cinnamoyl-CoA in the presence of D3-methanol (CD3OH) and analyzed the products by GC-MS. No labeled methylcinnamate was detected in these assays, suggesting that methylcinnamate production in sweet basil is not due to the activity of an acyltransferase.
By contrast, the formation of methylcinnamate from cinnamic acid and AdoMet was readily detectable in several basil lines. The highest level of this methyltransferase activity was found in protein extracts from glandular trichomes of line MC (35 pkat/mg; Figure 3A
), whereas protein extracts from young leaves of this line possessed only 6% of this activity. This dilution of activity in whole leaves relative to glandular trichomes suggested that CCMT is active specifically in the latter, a pattern that we observed previously for other characterized enzymes of specialized metabolism in sweet basil (Gang et al., 2001
To unequivocally identify the product formed in initial radiolabel-based activity assays, we incubated [U-13C]cinnamate with unlabeled AdoMet and total protein isolated from MC glands. The use of [U-13C]cinnamate permitted us to discriminate between the endogenous and de novo–synthesized methylcinnamate in the GC-MS analysis of the products formed in these assays due to a shift of +9 m/z units for the molecular ion and diagnostic fragment ions in the mass spectrum of the product formed. Results of these assays clearly show de novo methylcinnamate formation (Figure 4 ).
We observed that CCMT activity from protein extracts of MC glands exhibited substrate inhibition, as AdoMet concentrations were increased from 10 µM to 1 mM, with significant inhibition observed at concentrations >200 µM (see Supplemental Figure 1 online), which matches a classical substrate inhibition profile (Cornish-Bowden, 1979 ). Assays with both nondesalted and desalted crude protein extracts from MC whole leaves or isolated trichomes displayed the same substrate inhibition effect, ruling out the possibility of a small endogenous metabolite present in the protein extracts as the cause of the inhibition. In addition, we used LC-MS to analyze the purity of the AdoMet used in these assays and found no detectible S-adenosylhomocysteine (AdoHcy), although this compound is readily detected by this analytical method (data not shown). This suggests that potential product inhibition by AdoHcy contamination in the AdoMet substrate was not the cause. Interestingly, this substrate inhibition effect was not observed in similar assays with recombinant CCMT enzymes (see below), even at high (2 mM) AdoMet concentrations. At present, the reason for the susceptibility of native CCMT activity but not recombinant CCMT enzymes to inhibition by AdoMet is unknown. This inhibition is likely to be caused by some form of posttranslational modification of the polypeptide chain, such as by proteolytic cleavage, phosphorylation of specific amino acid residues, etc. Future research will seek to answer this interesting question.
Identification of Basil Genes Encoding CCMT Twelve of the 22 contigs contained full-length open reading frames, whereas 6 contigs encoded partial open reading frames of 200 amino acids or more. Alignment of the translated amino acids sequences and subsequent cluster analysis revealed that the 22 contigs represent allelic versions of three distinct although very similar (96 to 97% amino acids sequence identity) proteins identified as new members of the SABATH family. These three isoforms were designated CCMT1, CCMT2, and CCMT3 (see below for activity determination) in order of decreasing number of total cDNA clones (allelic variants) for each isoform. The basil CCMT proteins are 373 amino acids in length and 57% identical (75% similar) to Arabidopsis thaliana IAMT1, the closest biochemically characterized relative to basil CCMTs. To estimate the number of CCMTs present in basil lines MC, SW, and SD and to determine why line EMX-1 lacked CCMT transcripts, we performed DNA gel blot analysis using genomic DNA from all four basil lines (see Supplemental Figure 2 online). Line MC appeared to possess several CCMTs, while lines SW, SD, and EMX-1 all appeared to possess fewer. Another interesting finding was that the banding pattern was nearly identical for lines SD and EMX-1, with the pattern for SW showing some similarities to these two, while the pattern for MC showed little resemblance to the other lines. The increased number of copies and the distinct pattern suggest that gene duplication(s) may have occurred in line MC relative to the other lines. It is possible that changes in genome structure and the distribution of CCMT copies in line MC are linked to the increased pattern of CCMT expression in this line. This approach shows the relative differences between basil lines, while sequencing of the genomes of these basil lines will provide in the future the exact copy number of CCMT genes.
qRT-PCR Analysis of CCMT Expression
Proteomics Analysis Supports the Presence of All CCMT Isoforms in MC Trichomes
Biochemical Characterization of CCMT Isoforms
The molecular masses of the three recombinant CCMTs determined by gel filtration chromatography were  88 kD, whereas on SDS-PAGE the denatured proteins exhibited a single band corresponding to a molecular mass of 44 kD, suggesting that the active enzymes exist as homodimers in solution. CCMT proteins were active from pH 6.0 to 9.0, with maximum activity at pH 7.5, 90% of maximum at pH 7.0 and 8.0, and <50% of maximum at pH 6.0 and 9.0. CCMTs did not require cations for their activities; however, strong inhibition (>65%) was observed when Cu2+, Ni2+, or Zn2+ was added at 5 mM to the reaction. Fe2+, Mg2+, Mn2+, and NH4+ led to mild inhibition (<35%), and the addition of Ca2+, K+, or Na+ had no effect on enzyme activities. Since recombinant proteins were purified using Ni2+ agarose columns, the potential inhibitory effect of Ni2+ on enzyme activity was evaluated at six different concentrations: 0.01, 0.05, 0.1, 0.5, 1.0, and 5.0 mM. These experiments showed that Ni2+ concentrations of <1 mM had no effect on enzyme activity.
Steady state kinetics of each CCMT enzyme revealed that the recombinant proteins had a high affinity toward AdoMet, with apparent Km values ranging from 1 to 13 µM depending on the second substrate and CCMT isoform tested (Table 2
). All three enzymes displayed similar apparent Km values for CA, ranging from 124 µM (CCMT1) to 196 µM (CCMT3). CCMT1 and CCMT2 had lower apparent Km values for p-coumarate and BA (from 1.8- to 2.7-fold lower) compared with that for CA, in contrast with CCMT3, which had an 11-fold lower apparent Km value for BA and an almost 2-fold higher apparent Km value toward p-coumarate. CCMT1 and CCMT2 had nearly identical catalytic efficiencies (kcat/Km ratio) with CA, which were almost fivefold higher than that for CCMT3, suggesting that the contribution of CCMT3 to the formation of methylcinnamate in the plant could be small. Interestingly, CCMT2 can use p-coumarate and CA with nearly equal efficiency, while for CCMT1 the difference between catalytic efficiencies with these two substrates was 1.3-fold, favoring CA. The similar catalytic efficiencies of CCMT1 and CCMT2 toward p-coumarate and CA in addition to the higher Vmax with CA suggest that these enzymes can use both substrates efficiently in vivo. Thus, we designated the identified enzymes CCMT, for S-adenosyl-L-Met:cinnamate/p-coumarate carboxyl methyltransferases. These parameters were similar to apparent kinetic parameters determined for CCMT activity in crude protein extracts from line MC glandular trichomes (e.g., apparent Km of
Structural Modeling of CCMT Proteins Alignment of the basil CCMT genes with IAMT, SAMT, and several other biochemically characterized members of the SABATH family (Figure 7 ) revealed that all of the residues implicated in AdoMet binding, as well as other active site residues previously identified in the crystal structure of SAMT from C. breweri (Cb SAMT) (Zubieta et al., 2003 ), are conserved in basil CCMTs. More specifically, this includes residues equivalent to Trp-151 and Gln-25 of Cb SAMT, which have been implicated (Zubieta et al., 2003) as hydrogen bond donors for salicylate to ensure proper orientation and proximity to the donated methyl group of AdoMet. The most striking difference between the CCMT proteins and their closest relative, IAMT, is a gap of 11 amino acids in CCMT spanning residues 126 to 136 of IAMT1. These residues were not predicted to be in the active site based on the initial model of IAMT (Zubieta et al., 2003), and the impact of the gap on CCMT function could not be determined based on the alignment alone.
To further investigate the role of specific residues in substrate binding and CCMT catalysis, we used the solved crystal structure of Cb SAMT to generate structural models of the three basil CCMTs and snapdragon (Antirrhinum majus) Am SAMT. We then compared these models against the structure of Cb SAMT as well as a modeled structure of Arabidopsis IAMT. The first models generated were based only on alignments between Cb SAMT and Ob CCMT amino acid sequences. These initial models suggested that the residues corresponding to Cb SAMT Met-150 and Met-308 are altered in CCMTs to His-160 and a null residue (deleted), respectively. These two Met residues have been proposed to provide a molecular clamp in Cb SAMT for the aromatic ring of SA (Zubieta et al., 2003 ). Based on these initial results, we replaced the His-160 codon in CCMT with a Met codon (CCMT1-H160M mutant) and generated the reciprocal mutation in Am SAMT (SAMT-M150H mutant) to determine whether these changes might alter the substrate preference of the enzymes as the models predicted. In an effort to generate the molecular clamp mentioned above, we also created a mutant CCMT1 protein with an introduced Met codon at amino acid position 323 (CCMT1-M323 insertional mutant) as well as a mutant CCMT1 protein containing both modifications (CCMT1-H160M/M323 double mutant). Insertion of the Met residue at position 323 of CCMT1 completely abolished the activity of the enzyme in both the single mutant and the double mutant. The lack of any catalytic activity in the insertional mutant suggested that the initial alignment was likely incorrect at this position and that insertion of the Met residue led to a shift in structure affecting the active site or some other critical structural component of the enzyme, causing the protein to be nonfunctional. Later multiple sequence alignments with additional SABATH family member sequences, some newly available, led us to modify our initial alignments and structural models in which we had created a gap opposite Cb SAMT Met-308 and instead align CCMT Cys-322 at this position. Future research will pursue a C322M substitution to determine the effects of this mutation on the activity of the enzyme. CCMT1 with the single H160M change, however, retained activity with CA while demonstrating a dramatically increased activity toward BA as a substrate (Tables 1 and 2). This CCMT1-H160M mutant protein also acquired the ability to accommodate SA as a substrate, although with lower activity than that toward CA. Although Arabidopsis IAMT1 was the closest relative to CCMTs, none of the CCMT1 mutants were able to utilize indole-3-acetic acid as a substrate, suggesting that some other feature of the protein or active site conferred the selectivity of CCMT against indole-3-acetic acid, such as the substitution of a larger, more polar Ser-243 in CCMT versus the comparably positioned Gly-256 in IAMT. Once the crystal structure of IAMT is solved, this question may be able to be answered. New CCMT and Am SAMT models based on the revised alignments (Figure 7) predicted several nonconserved residues to be in the active site cavity and suggested that they may contribute to or be responsible for the differential substrate preference of these enzymes.
Docking Experiments Explain the Differences in Substrate Preferences of CCMTs and SAMTs
One of the most interesting of the docking experiment results was that of SA to the CCMT isoforms, which suggested that SA could be a good inhibitor of CCMT enzymes because it appeared to bind well in the active site in an orientation that prevented methyl group transfer (Figure 8E). In vitro inhibition studies validated this prediction: SA was found to be a good inhibitor of CA methylation by both CCMT1 and CCMT3 enzymes (CCMT2 was not tested due to its high similarity to CCMT1). However, the inhibition of CCMT1 and CCMT3 by SA did not fit classical competitive, noncompetitive, uncompetitive, or mixed linear inhibition models (Figure 9 ; see Supplemental Figure 4 online). Instead, it appeared to fit parabolic inhibition models most closely, with the inhibition approximating competitive inhibition at low (<250 µM) SA concentrations, leading to apparent Ki values of 85 and 90 µM for SA with CCMT3 and CCMT1, respectively. However, at higher concentrations (500 to 2000 µM), SA appeared to function more like a noncompetitive inhibitor. These results have significant implications regarding the catalytic mechanism and function of the CCMT enzymes. First, they suggest that SA binds with differing affinities to at least two sites on the functional CCMT proteins. Since these proteins exist as homodimers in solution, it is possible that these sites are the active sites of each of the monomers. These results also suggest that nonproductive binding of substrate to one site affects the affinity of the other (active) site for substrate. Such reaction kinetics have not yet been reported for members of the SABATH family of carboxyl methyltransferases. More detailed kinetics analysis will be required to determine the binding order of substrates and to identify where SA binds to CCMT enzymes.
Phylogenetic Relationships of CCMTs within the SABATH Family We conducted an exhaustive search of currently available protein and genomic sequence databases to identify all sequences available (from all organisms) having homology with the SABATH gene family. In total, >400 sequences were identified from dozens of plant taxa (including Cb SAMT, all known and characterized SABATH members, and our basil CCMT sequences) as well as many sequences representing nonplant genes and sequences derived from marine environmental sampling. A maximum likelihood tree produced from SABATH family sequences representing as much taxonomic diversity as sequence and alignment quality would permit (Figure 10) suggests that the SABATH family is split into three primary clusters. The first cluster comprises microbial sequences that are distinct from terrestrial plant sequences. The second cluster consists of two subclades that are not distinct based on bootstrap support and includes the basil CCMT genes, At IAMT, and Arabidopsis farnesoic and gibberellic acid carboxyl methyltransferases (At FAMT and At GAMT), along with several groups of genes of unknown function from Arabidopsis, Brassica napus, poplar (Populus sp), rice (Oryza sativa), and other monocots as well as all genes identified from conifers and lower plants. The third major cluster comprises sequences from higher plants and includes the characterized SAMTs, BSMTs, BAMT, JMT, and the N-methyltransferases as well as several distinct clusters of uncharacterized monocot and eudicot SAMT-like sequences. It was surprising that basil CCMT does not cluster with benzenoid carboxyl methyltransferases (SAMTs, BSMTs, BAMT, etc.), whereas jasmonic acid carboxyl methyltransferase and the N-methyltransferases do.
Conservation of Primary Structure in SABATH Family Proteins The newly available basil CCMT protein sequences along with those from recently cloned and characterized SABATH genes allowed us to obtain an updated perspective on important primary sequence features of SABATH family proteins and to identify regions of conservation among the protein sequences of the expanded gene family. While the residues surrounding the presumed active site cavities can be quite variable, those that were previously indicated by the solved crystal structure of C. breweri SAMT to be involved in AdoMet/AdoHcy binding (Zubieta et al., 2003 ) are well conserved in basil CCMTs, as they are in most plant-derived members of this gene family that have been characterized to date (indicated by stars in Figure 7). The strict conservation of many residues across the SABATH proteins characterized to date suggests an essential role in this enzyme family, even if this role is not yet known. There are 24 amino acid residues distributed throughout the primary amino acid sequences that are absolutely conserved in the 14 biochemically characterized SABATH family members from plants but that are not identified as active site residues or implicated as having direct interaction with the substrates or products based on solved crystal structures and structural models. Thus, individual solved crystal structures or models, while essential to help us understand the function of a protein and powerful for predictive purposes, may not by themselves explain the contribution of each building block of an enzyme structure to its function.
Substrate Preference and Active Site Structure
Evolution of Methylcinnamate Production in Plants
The distribution of methylcinnamate production throughout several plant families, presumably via biosynthesis by the CCMT homologs identified in this phylogenetic analysis, suggests the possibility that this function may have arisen early in plant evolution, possibly prior to the divergence of monocots and dicots. Several sequence homologs of basil CCMT and Arabidopsis IAMT were found for rice, but it remains to be determined whether these genes encode true CCMTs or IAMTs. The biochemical functions of the related genes from lower plants and of the microbial enzymes are also unknown. Future functional characterization of these genes will be essential to identify whether CCMT activity represents an ancient biochemical process that has been conserved throughout the history of plant evolution or whether it arose via convergent (repeated) evolution in multiple instances throughout evolution of the plant kingdom (Pichersky and Gang, 2000
Origin and Evolution of the SABATH Family Phylogenetic analysis based on the maximum likelihood tree (Figure 10) suggests that the progenitor of the SABATH gene family evolved long before the divergence of vascular plants, since several taxa of eubacteria representing at least three different major groups, the cyanobacteria Synechococcus, Prochlorococcus, and Gloeobacter, all possess SABATH family members. The presence of these genes among a group of taxa thought to be involved in endosymbiosis with early eukaryotes suggests that this family of genes may have been inherited from prokaryotes. BLAST searches, however, did not result in the identification of SABATH homologs among any sequence data available from green or red algae, including Chlamydomonas reinhardtii and Cyanidioschyzon merolae, for which the entire or extensive genome sequences are available, indicating potential gene loss in these taxa. The identification of a homolog in the genome sequence of the diatom Thalassiosira, however, suggests that the loss of this gene in red algae may have occurred after the secondary endosymbiotic event that gave rise to the modern brown algae. This raises interesting questions about the biological role of these SABATH homologs in the taxa in which they have been maintained versus those for which the sequence has been lost. Nevertheless, the tree topology suggests that plant SABATH genes most likely originated from an ancient ancestor shared with the microbial taxa and that the ancestral form of this gene may have undergone a duplication early in plant evolutionary history prior to the divergence of monocots and eudicots, with subsequent duplication events and additional diversification taking place later to generate the contemporary groups and diverse functions within the gene family seen here.
CCMT Activity Assays CCMT activity was determined by measuring the formation of radiolabeled methylcinnamate by total protein extracted from isolated glandular trichomes and intact leaves of four basil (Ocimum basilicum) lines. Assays were performed in 50-µL reactions consisting of 50 mM potassium phosphate buffer, pH 8.0, 9.33 µM S-[methyl-14C]adenosyl-L-Met, 1 mM cinnamate, and 200 ng to 10 µg of protein. Reactions were incubated at 30°C for 30 to 60 min and stopped by the addition of 6 µL of 6 N HCl. Labeled methylcinnamate was extracted with ethyl acetate, and a portion of the separated organic phase was used for scintillation counting. Assays were performed in quadruplicate for each protein sample. Controls included assays with all reagents to which no protein was added and assays including protein with cinnamate omitted. Assays to detect the activity of an acyltransferase capable of producing methylcinnamate were performed using the same assay conditions (similar to those used previously for acyltransferase assays [Gang et al., 2002a ]), with the exceptions that 50 µM cinnamoyl-CoA (synthesized according to a method reported previously [Beuerle and Pichersky, 2002] and purified using a Chromabond C18 column [Macherey-Nagel] and 25 µM D3-methanol [343854; Sigma-Aldrich]) was substituted for AdoMet and cinnamate, the reactions were incubated for 2 h, and detection was performed via GC-MS rather than radiometrically. The standard assay mixture for the recombinant proteins consisted of 20 µL of 250 mM potassium phosphate buffer at pH 7.5 with 10 mM ß-mercaptoethanol, 1 µL of 50 mM disodium EDTA, 2 µL of acid substrate dissolved in DMSO, 0.2 to 2 µL of enzyme solution (48 to 480 ng), 5 µL of radiolabeled AdoMet (389 µM; specific activity of 10.28 mCi/mmol), and water to a final volume of 100 µL. After incubation for 15 to 30 min, the reactions were stopped by the addition of 150 µL of organic solvent (hexane or ethyl acetate) and vortexing for 5 s After centrifugation (1 min at 16,600g) to separate the phases, 75 µL of the organic phase was used for liquid scintillation counting. Verification of the assay product was determined using nonradiolabeled AdoMet in a 100x scaled reaction and analyzing the concentrated organic phase by GC-MS.
GC-MS Analysis of Basil Tissues and CCMT Product The identity of the product formed in the radiometric CCMT assays was confirmed by GC-MS analysis of ethyl acetate extracts from assays performed using stably labeled cinnamate and unlabeled AdoMet. Briefly, [U-13C]L-Phe (Cambridge Isotope Laboratories) was enzymatically converted into cinnamic acid using Phe ammonia-lyase from Rhodotorula glutinis (P1016; Sigma-Aldrich) and subsequently purified. Purified [U-13C]cinnamate was resuspended in ethanol, and the purity and concentration were checked using thin layer chromatography. CCMT enzyme assays were performed as described above with the exception of a higher AdoMet concentration and longer assay period (200 µM, 3 h). The product was extracted with ethyl acetate and analyzed by GC-MS. Controls were performed as described above, with the addition of assays from which AdoMet was omitted. Methylcinnamate was identified via library matching of mass spectra, and the mass spectrum and fragment pattern of labeled methylcinnamate were compared with those of an unlabeled, authentic methylcinnamate standard. Verification of the product formed by recombinant CCMT enzyme was performed by adding nonradiolabeled AdoMet to a 100x scaled reaction and analyzing the concentrated organic phase by GC-MS.
GC-MS analysis was performed using a Thermo Electron Trace GC Ultra apparatus linked to a DSQ mass spectrometer. Volatile compounds were separated on an Alltech ECONO-CAP-EC-5 capillary column (30 m x 0.25 i.d. x 0.25 mm film thickness) and identified using a previously reported method (Ma and Gang, 2005
cDNA Library Construction for Line MC
DNA Gel Blot Analysis
qRT-PCR To further assess the relative gene expression levels for the three major CCMT isoforms identified from the EST analysis, we attempted to design gene-specific primers for qRT-PCR. However, the number of adjacent nucleotides that were polymorphic between the gene sequences and that could be confidently used for primer design was very low. Primer pairs were designed that were 3' anchored at one to two polymorphic bases, but the amplification efficiency of these primers under PCR conditions sufficiently stringent to give discriminatory amplification was too low to permit confidence in any resulting relative quantification. Thus, the qRT-PCR results could not yield any further data regarding differential transcriptional regulation of the three putative CCMT isoforms identified in the basil EST database.
Proteomics Analysis
Recombinant Protein Expression and Purification
Preparation of Crude Cell-Free Extracts from E. coli
Partial Enzyme Purification
Determination of Native Molecular Mass
Enzyme Characterization
Kinetic Properties
Modeling of CCMT Structures
Phylogenetic Analysis of the SABATH Family
Accession Numbers
Supplemental Data
We thank Joseph Noel, Florence Pojer, and Jeannine Ross for valuable discussion and advice on the protein modeling and structural analysis; Joseph Bischoff for valuable discussion regarding the SABATH matrix and phylogenetic analysis; James Simon for valuable discussion regarding methylcinnamate formation in sweet basil; Brenda Jackson for assistance with GC-MS maintenance and operation; Paul Haynes for advice on the shotgun proteomics experimental design; and Linda Breci, George Tsaprailis, and Fatimah Hickman at the Arizona Proteomics Consortium for assistance with proteomics sample analysis and data mining. We thank the National Science Foundation (Grant MCB-0210170 to D.R.G. and Grant MCB-0615700 to N.D.) and the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service (Grant 2005-35318-16207 to N.D.) for financial support.
1 These authors contributed equally to this work.  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: David R. Gang (gang{at}ag.arizona.edu).
[W] Online version contains Web-only data. www.plantcell.org/cgi/doi/10.1105/tpc.107.054155 Received July 16, 2007; Revision received September 18, 2007. accepted September 24, 2007.
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-terpineol; 10.31, linalool; 11.9, borneol; 12.49, 
-terpineol; 18.48, Z-methylcinnamate; 18.94, E-methylcinnamate; 20.35, 
-cadinene; 24.7, Z-nerolidol; 26.33, cubenol; 27.4, 
