Polymethylated myricetin in trichomes of the wild tomato species Solanum habrochaites and characterization of trichome-specific 3'/5'- and 7/4'-myricetin O-methyltransferases.

Flavonoids are a class of metabolites found in many plant species. They have been reported to serve several physiological roles, such as in defense against herbivores and pathogens and in protection against harmful ultraviolet radiation. They also serve as precursors of pigment compounds found in flowers, leaves, and seeds. Highly methylated, nonglycosylated derivatives of the flavonoid myricetin flavonoid, have been previously reported from a variety of plants, but O-methyltransferases responsible for their synthesis have not yet been identified. Here, we show that secreting glandular trichomes (designated types 1 and 4) and storage glandular trichomes (type 6) on the leaf surface of wild tomato (Solanum habrochaites accession LA1777) plants contain 3,7,3'-trimethyl myricetin, 3,7,3',5'-tetramethyl myricetin, and 3,7,3',4',5'-pentamethyl myricetin, with gland types 1 and 4 containing severalfold more of these compounds than type 6 glands and with the tetramethylated compound predominating in all three gland types. We have also identified transcripts of two genes expressed in the glandular trichomes and showed that they encode enzymes capable of methylating myricetin at the 3' and 5' and the 7 and 4' positions, respectively. Both genes are preferentially expressed in secreting glandular trichome types 1 and 4 and to a lesser degree in storage trichome type 6, and the levels of the proteins they encode are correspondingly higher in types 1 and 4 glands compared with type 6 glands.


INTRODUCTION
Flavonoids constitute a large and structurally diverse family of metabolites synthesized in plants. The core structure of flavonoids is either a 2-phenylchromen-4-one (flavonoids), 3-phenylchromen-4-one (isoflavonoids), or 4-phenylcoumarin (neoflavonoids). The great structural diversity of flavonoids stems from the possible substitution on up to 10 carbons of the core structure. Some common functional group substitutions include hydroxylation, methylation, sulfonation, methylation, and (iso)prenylation ( Ibrahim and Anzellotti, 2003). In addition to these core substitutions, hydroxyl functional groups can be further modified by the addition of a wide range of different sugar moieties, which can be further modified themselves. Current estimates of the number of structurally distinct, plant-derived, flavonoids probably exceed 9,000 (Williams and Grayer, 2004). This rich structural diversity extends well into the functional diversity of flavonoids. They play crucial roles in plants in pathogen and herbivore defense, protection from harmful UV radiation, pigmentation of flowers, fruits and seeds. They also act as plant-microbe signaling molecules, inhibitors in biochemical pathways, and developmental regulators (reviewed in Buer et al., 2010;Taylor and Grotewold, 2005;Treutter, 2005).
The flavonoid pathway in flowering plants can be traced back to the first plants to colonize land. The most primitive form of the pathway probably terminated at the production of flavonols (Rausher, 2008). Dihydroflavonols, the reduced forms of flavonols, represent an important step in the evolution of the structural and functional diversity of flavonoids seen in extant flowering plants. All anthocyanins, flavonols, and derivatives of these come from one of the three dihydroflavonols -dihydrokaempferol, dihydroquercetin, and dihydromyricetin -the latter being the most highly substituted, with hydroxyl groups on the 3, 5, 7, 3′, 4′ and 5′ carbons. The enzyme flavonol synthase (FLS) converts the dihydroflavonoids to their corresponding flavonols; kaempferol, quercetin, and myricetin, by oxidation of the C2-C3 bond of the C ring (Figure 1).
In plants that synthesize highly methylated flavonols, the process occurs in a stepwise manner with O-methylation at position 3 being the first step in the process (Huang et al., 2004;Ibrahim et al., 1987;Thresh and Ibrahim, 1985;Macheix and Ibrahim, 1984). In Chrysosplenium americanum, methylation of quercetin (Q) proceeds from 3-methylquercetin (3-MeQ) to 3,7-MeQ to 3,7,4′-MeQ. Several species of the genus Aeonium accumulate highly methylated quercetin and myricetin. In these species the methylation pattern appears to follow the same stepwise addition of methyl groups beginning with position 3. The myricetin methyl ethers that accumulate in the leaves include 3,7, 3′-trimethylmyricetin, 3,7,3′,4′-tetramethylmyricetin, and 3,7,3′,4′,5′pentamethylmyricetin (Stevens et al., 1995). To date an enzyme responsible for the synthesis of polymethylated myricetin in these species has not been identified. An enzyme isolated from Catharanthus roseus was shown to methylate free myricetin in vitro, and this reaction was hypothesized to occur in vivo prior to the further modifications of myricetin into the anthocyanins observed in the plant, but analysis of the kinetic parameters of the enzyme was not reported (Cacace et al. 2003).
Glandular trichomes are specialized storage and secreting organs that develop on the surface of areal parts of a wide variety of different plant species (Wagner, 1991;Schilmiller et al., 2008). They synthesize, store, and secrete specialized metabolites important to plant defense, and serve as the major source of essential oils (Ambrsio et al., elucidating specialized biochemical pathways because they are biochemically highly active in select pathways, metabolite accumulation is species-specific, and their metabolites and gene transcripts can be easily extracted and analyzed (Schilmiller et al., 2008 and2010). In the Solanum genus, glandular trichomes can be divided into two main groups, secreting glands and storage glands (Luckwill, 1943;Schilmiller et al., 2010).
The secreting glands (of which there are two types, 1 and 4, with type 4 being shorter) are supported atop a relatively long multicellular stalk that varies in length, and the gland itself appears to be unicellular. Droplets rich in specialized metabolites are often observed on the surface of these glands or on the stalk near the gland. The storage glands, defined as type 6 glands, are multicellular and sit atop a relatively short multicellular stalk. The storage glands consist of four cells arranged such that each makes up one quarter of the round structure.
Here we report the identification of polymethylated myricetin from isolated types 1, 4 and 6 glandular trichomes from the wild tomato Solanum habrochaites. We also report the identification and the biochemical characterization of two myricetin Omethyltransferases encoded by transcripts found in the Solanum habrochaites glandular trichomes, and show that one of them, ShMOMT1, is likely responsible for Omethylation of the 3′ and 5′ hydroxyl groups and the second, ShMOMT2, is likely responsible for O-methylation of the 7 and 4′ hydroxyl groups.
To examine the relative distribution of the non-glycosylated myricetins in specific types of trichomes, secreting glands and storage glands were collected individually from leaves of Solanum habrochaites for metabolic profiling (Figure 3 and Supplemental Figure 1). Secreting glands (types 1 and 4) contained higher levels per gland of all three methylated myricetins compared to storage glands (type 6). Levels of myricetin tetramethyl ether (3,7,3′,5′-MeM) were greatest in both secreting and storage gland types compared to the myricetin trimethyl ether (3,7,3′-MeM) and myricetin pentamethyl ether (3,7,3′,4′,5′-MeM). However, the levels of all myricetin methyl ethers were 5 to 6 fold greater in secreting type 1 and 4 glands compared to the corresponding levels in storage glands. Also, in secreting glands the levels of myricetin pentamethyl ether were slightly higher than levels of myricetin trimethyl ether, whereas in storage glands levels of the triand penta-methylated myricetins were not significantly different.

Characterization of substrate specificity of ShMOMT1 and ShMOMT2
We have recently constructed EST libraries from the secreting and storage glands (types 1, 4, and type 6, respectively) of Solanum habrochaites leaves (http://www.trichome.msu.edu/; McDowell et al., 2011). A bioinformatics search of these libraries using BLAST sequence comparisons with known O-methyltransferase (OMT) sequences identified three OMT sequences in S. habrochaites trichomes. All three cDNAs were expressed in E. coli and the crude extracts were tested for OMT activity with a battery of substrates (Table I) Table I for list of tested compounds). Consequently, it was not investigated further. A second cDNA encoded a protein, subsequently named ShMOMT1, with methylating activity toward myricetin and quercetin but not kaempferol, suggesting that this protein has 3′/5′ O-methyltransferase activity. A third cDNA encoded a protein, subsequently named ShMOMT2, with methylating activity against all these three flavonols.
ShMOMT1 and ShMOMT2 were further tested with a range of substrates related to myricetin that could be obtained in sufficient concentrations for these assays.  Table I). When the 3′ hydroxyl of the substrate was already methylated, as in laricitin, ShMOMT1 transferred a methyl group to the 5′ hydroxyl, as determined by co-migration with an authentic standard of 3′,5′-methylmyricetin in RTLC and by LC/MS (Table I and Supplemental Figures 2 and 3). When both 3′ and 5′ hydroxyls were already methylated, for example in the substrate 3′,5′-dimethyl myrecitin (i.e. syringetin), ShMOMT1 could not transfer a methyl group to any other hydroxyl (Table I).
ShMOMT2 transferred a methyl group to the 4′ hydroxyl of kaempferol, but to the 7 position of quercetin and myricetin (Table I). When the hydroxyl at the 7 position was already methylated, it transferred a methyl to the 4′ hydroxyl (e.g. with substrate 7methyl quercetin), and when the 4′ hydroxyl was already methylated, ShMOMT2 transferred the methyl group to the hydroxyl at the 7 position (e.g. with the substrate 3′,4′,5′-trimethyl myrecitin). It did not transfer a methyl group to any hydroxyl other than at the 7 or 4′ position (Table I). Radioactive thin-layer chromatography of the reaction with myricetin revealed a single product that migrated between myricetin and 3′-methyl myricetin (Supplemental Figure 2). This product was identified by LC/MS as 7-methyl myricetin. When myricetin was incubated with ShMOMT2 overnight, the product obtained was 7, 4′methyl myricetin (Supplemental Figure 4).
The protein encoded by the ShMOMT1 cDNA is 362 amino acids long, with a calculated molecular mass of 40.7 kD, and it contains all of the recognized plant OMT domains known or hypothesized to be involved in binding to SAM and metal cofactors (Ibrahim, 1997) (analysis not shown). ShMOMT1 is most similar (40-49% identity) to a number of mostly 3′ and 3′/5′ O-methyltransferases (Figure 4), consistent with its regiospecificity for the 3′ and 5′ positions.
The protein encoded by the ShMOMT2 cDNA is 355 amino acids long, with a calculated molecular mass of 39.4 kD, and it also contains all of the recognized plant OMT domains known or hypothesized to be involved in binding to SAM and metal cofactors (analysis not shown). ShMOMT2 is most similar (29-47% identity) to several O-methyltransferases identified (with one exception) as specific for the 7 and/or 4′ position (Figure 4), consistent with its regiospecificity for these positions. ShMOMT2 is only 27% identical to ShMOMT1.

Distribution of ShMOMT1 and ShMOMT2 transcripts and protein in trichome glands
We used quantitative RT-PCR (qRT-PCR) and Western blot analyses to localize ShMOMT1 and ShMOMT2 transcript, and ShMOMT1 and ShMOMT2 proteins, respectively, in the different types of trichome glands. Extracts of collections of individual types of glands were compared to whole leaf extracts in both types of experiments. ShMOMT1 transcript levels were 3.5 to 12.5 fold higher in secreting glands from types 4 and 1 trichomes, respectively, compared to storage glands of type 6 trichomes ( Figure 5). ShMOMT2 transcript levels were 2 to 4 fold higher in secreting glands from types 4 and 1 trichomes, respectively, compared to storage glands of type 6 trichomes ( Figure 5). Comparison of transcript levels from leaf tissue with trichomes vs. leaf tissue from which the trichomes had been mechanically removed indicated that transcripts of both ShMOMT1 and ShMOMT2 are present exclusively in trichomes ( Figure 5). Protein blot analysis indicated that levels of ShMOMT1 protein were 7 to 8.6 fold higher in secreting glands compared to storage glands, and 1.9 to 2.4 fold higher compared to whole leaf extracts ( Figure 6). Levels of ShMOMT2 protein were 5 to 6.6 fold higher in secreting glands compared to storage glands, while ShMOMT2 was not detectable in whole leaf extracts ( Figure 6).

Characterization of the kinetic parameters of ShMOMT1 and ShMOMT2
ShMOMT1 and ShMOMT2 were expressed in E. coli BL21 (DE3) cells and the recombinant proteins were purified to near homogeneity by two successive anion exchange chromatography steps (Figure 7). The purified ShMOMT1 protein catalyzed the formation of laricitrin (3′-methyl myricetin) from myricetin with an apparent K m value of 0.46 µM and an apparent K cat value of 1.59 s -1 . An apparent K m value of 0.21 µM was measured for ShMOMT1 with laricitrin (giving the product syringetin, 3′,5′dimethyl myricetin) as the substrate, with an apparent K cat value of 0.45 s -1 . The apparent K m value for SAM, with myricetin as co-substrate, was 16.64 µM with an apparent K cat value of 0.47 s -1 (Table II and Supplemental Figure 5).
Purified ShMOMT2 catalyzed methylation of the 7 hydroxyl group of myricetin, the 7 hydroxyl group of kaempferide (4′-methyl kaempferol), and the 4′ hydroxyl group of rhamnetin (7-methyl quercetin). An apparent K m of 1.68 µM was determined for myricetin with an apparent K cat value of 7.4x10 -3 s -1 . An apparent K m of 2.27 µM was determined for kaempferide with an apparent K cat value of 5.76x10 -3 s -1 . And, an apparent K m of 2.30 µM was determined for rhamnetin with an apparent K cat value of 6.40x10 -3 s -1 .
The apparent K m value for SAM with kaempferide as co-substrate was 18.71 µM with an apparent K cat value of 1.64x10 -2 s -1 (Table III and Supplemental Figure 6).

Characterization of optimal conditions for catalysis revealed that both ShMOMT1
and ShMOMT2 do not require the addition of Mg 2+ or Mn 2+ for activity. At levels below 2.5 mM, Mg 2+ had little negative effect on activity (≤10%); however, concentrations above 2.5 mM had increasing inhibitory effects on activity with myricetin. Similarly, addition of Mn 2+ to enzyme assays, using myricetin as substrate, had little negative effect (≤10%) on activity until levels exceeded 2.5 mM. ShMOMT1 activity with myricetin was observed in the pH range of 6.0-8.5 with optimal activity observed at pH 7.5. And, ShMOMT2 activity with myricetin was observed in the pH range of 6.0-9.0 with optimal activity observed at pH 8.0.
By isolating individual types of glands, we were able to show that these compounds are found in three types of glandular trichomes -1, 4, and 6 ( Figure 3), although they are most abundant in the secreting glands (types 1 and 4).
All of the myricetin methyl ethers that we detected in the glands of glandular trichomes were methylated at the 3 position (in the C ring). This position is often glycosylated, and the glycosylated form is then transported to the vacuole (Vogt and Jones, 2000). We did not find any glycosylated myricetin in the trichomes nor myricetin species that are not modified at the 3 position, suggesting that the 3-OMT responsible for this methylation reaction is quite efficient. However, our attempts to detect OMT activity in crude extracts of glands or whole leaves capable of adding a methylgroup to the 3hydroxyl position of myreicetin was unsuccessful, nor could we identify a cDNA encoding such an enzyme in our EST databases. To our knowledge, no 3-OMTs capable of methylating myricetin or any other flavonols have been identified from any plant, although a cellular activity capable of methylating quercetin at the 3 position has been reported (De Luca and Ibrahim, 1985;Huang et al., 2004).
Our analyses of S. habrochaites leaves with trichomes and leaves with trichomes removed revealed that 3,7,3′-trimethyl myricetin and 3,7,3′,5′-tetramethyl myricetin were found in trichome gland cells only, and 3,7,3′,4′,5′-pentamethyl myricetin was found in both trichomes and the rest of the leaf organ ( Figure 2). All other flavonol compounds were apparently confined mostly to non-trichome leaf cells since their levels did not decrease significantly when trichomes were removed (Figure 2). The presence of our analysis indicates that ShMOMT1 and ShMOMT2 are not expressed in non-trichome leaf cells (Figure 5).

ShMOMT1 is a 3′/5′ myricetin methyltransferase and ShMOMT2 is a 7 and 4′ myricetin methyltransferase
The characterization of the enzymatic properties of ShMOMT1 in vitro showed that it has high affinity for both myricetin and 3′-methyl myricetin, and its products are 3′-methyl myricetin (laricitrin) and 3′,5′-dimethyl myricetin (syringetin) (Supplemental Figure 3). In previous studies, OMTs have been identified that can methylate myricetin at these positions, but in all such cases myricetin was not the best substrate for the enzyme and the tissue source of the enzyme did not actually contain methylated myricetin but only related compounds, such as quercetin, kaempferol, tricin, tricetin and luteolin (Muzac et al., 2000;Lee et al., 2008;Zhou et al., 2006). The catalytic efficiency of ShMOMT1 with both myricetin and laricitrin are significantly higher than for such 3′,5′-OMTs (Table IV), and these enzymes had higher affinity to the substrates whose methylation led to the compounds actually observed in the plant.
ShMOMT2 is most similar to some enzymes characterized as 4′ methyltransferases and some characterized as 7 methyltransferases, with one exception (Figure 4). This exception is Catharanthus roseus flavonol 3′/5′ O-methyltransferase (Cacace et. al., 2003), which is very similar to Cantharanthus roseus flavonol 4′ Omethyltransferase (Schröder et. al., 2004) (Figure 4), and may represent a recent case of gene duplication and divergence. When ShMOMT2 was incubated with kaempferol, a substrate missing both a 3′ and a 5′-hydroxyl, it added a methyl group to the 4′-hydroxyl (Table I). However, with either quercetin or myricetin, ShMOMT2 initially added a methyl group to the 7-hydroxyl, suggesting that 3′ and/or 5′-hydroxyls might inhibit its activity with the 4′-hydroxyl. This is consistent with the observation that 3,7,3′-trimethyl myricetin is found in the glands, but no 3,3′,4′-trimethyl myricetin is observed ( Figure   3). Thus, it appears that after the 3-hydroxyl is methylated, the next hydroxyls to be methylated are at the 7 and 3′ position, although which of these two is methylated first cannot yet be resolved. This is also consistent with what has been shown in Chrysosplenium americanum, where methylation of quercetin proceeds first to 3www.plantphysiol.org on July 9, 2020 -Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. methylquercetin, then to 3,7-dimethyl quercetin (De Luca and Ibrahim, 1985). It can be deduced that the next hydroxyl to be methylated is at the 5′ position, since we see accumulation of 3,7,3′,5′-tetra methyl myricetin but no 3,7,3′,4′-tetra methyl myricetin, and also because it appears that ShMOMT1 is not active with a substrate that has a methyl group at both the 3′,4′ positions (Table I). ShMOMT2 clearly is capable of methylating the 4′-hydroxyl after it methylated the 7-hydroxyl (tested with 7-methyl quercetin for lack of 7-methyl myricetin, see Table I, and also by incubating myricetin with ShMOMT2 for an extended period (>10 h), after which the major product is 7,4′myricetin (Supplemental Figure 4)). However, it seems to be less efficient at methylating the 4′ hydroxyl once the 3′ and/or the 5′ hydroxyls have been methylated (Table I), consistent with the lower levels of 3,7,3′,4′,5′-pentamethyl myricetin observed in the trichomes.
A caveat for the kinetic analysis of ShMOMT1 and ShMOMT2 presented here is that, for lack of availability, we were not able to test them with 3-methylmyricetin or other combinations of polymethylated myricetin with one methyl group at the 3-position (for example, 3,7-dimethylmyricetin). However, we did obtain and test both enzymes with 3-methyl quercetin. The results with ShMOMT1 indicated that it had higher activity with the 3-methyl quercetin than with quercetin, although ShMOMT2 had lower activity (Table I). It has been shown for many OMTs that they are regiospecific but not substratespecific, meaning that their specificity is determined by the part of the molecule which is modified by their catalytic activity (Vogt, 2004). However, we note that the turnover rate of ShMOMT2 with the in vitro substrates tested were substantially lower than the turnover rates observed for ShMOMT1 (Tables II, III Our data indicate that ShMOMT1 and ShMOMT2 transcripts and proteins are found in all three types of glandular trichomes in S. habrochaites that are metabolically active. The levels of the transcripts and proteins in these gland types -1, 4, and 6correlate well with the amount of methylated myricetin found in them, with type 6 glands containing an order of magnitude less of each compared with type 1 and 4 glands, with the exception that type 4 glands have somewhat reduced amounts of both transcripts compared to type 1 glands. However, the level of ShMOMT1 and ShMOMT2 transcripts in type 4 glands are still 4-fold and 2-fold higher, respectively, than that found in type 6 glands. In addition to localizing ShMOMT1 and ShMOMT2 in glandular trichomes, we have detected transcripts of putative genes involved in flavonoid and flavonol biosynthesis in our EST databases created from isolated trichome glands (types 1, 4, and 6) (Supplemental Table I). Transcripts of both flavonol 3′ hydroxylase and flavonol 3′,5′ hydroxylase, required for the synthesis of myricetin, were detected in these databases, with highest representation in type 1 glands.

Possible roles of methylated myricetins in tomato glandular trichomes
Flavonoids in general have been hypothesized to as act as UV protectants, chemical defense compounds, and in plant-insect, plant-microbe, plant-pathogen, and plant-plant interactions (reviewed by Treutter, 2005). While evidence for some of these roles (e.g., in plant-microbe interactions) is strong, other roles are still tentative (Treutter, 2005). Furthermore, since flavonoids often occur as a mixture, assigning roles to specific compounds is difficult. Currently, no physiological function has been postulated specifically for laricitrin and syringetin in plants, nor for the more highly methylated myricetins found in the tomato trichomes. Laricitrin and syringetin, but not the more highly methylated myricetin ethers, are found in red grape and are probably responsible, along with several other flavonols and methylated derivatives, for the antioxidant potency of red grapes and wine (Mattivi et al., 2006). However, there is no evidence to support specific roles for these compounds in grape. Myricetin has also been linked to radical scavenging activity, xanthine oxidase inhibitory activity, and antioxidant activity in Thus, we can only hypothesize that in tomato trichomes the methylated myricetins contribute to some of the general roles postulated for flavonoids. Their synthesis and accumulation in glandular trichomes along with their relatively lipophilic nature suggest that they are likely targeted to the cuticlular space surrounding the secretory cells. In this location they are well placed to serve roles in chemical defense against herbivores, as UV protectants, or as radical scavengers to aid in preventing peroxidation of lipids.

Plant Material and Growth Conditions
Solanum habrochaites (accession LA1777) seeds were obtained from the C.M.
Rick Tomato Genetics Resource Center (TGRC, University of California at Davis). The seeds were germinated on sterile filter paper in germination boxes and kept for approximately 5-7 days before transfer of seedlings to soil. Plants were grown in a mixture of regular soil:fine sand (3:1, v/v) in a growth chamber under a 14-h light/ 10-h dark photoperiod. Temperature was maintained at 22°C throughout the light period and 18°C during the dark period.
Gland cells were collected from glandular trichomes by hand with micropipettes under a dissecting microscope (Leica MZ6). Micropipettes were hand pulled and shaped from either 9" disposable pasteur pipettes or 1.8 mm X 100 mm capillary tubes. The micropipettes were approximately 6 cm in length and shaped to taper from one end, approximately 1.5-2.0 mm, down to approximately 0.25 mm diameter at the opposite end. Both ends of the pipette were flame sealed to prevent capillary action. Gland cells were picked from the top of glandular trichome structures using the thin tip of the micropipette. The cells adhered to the tip until being put into an appropriate buffer for downstream analyses. Trichomes were removed from leaf material using the same type of micropipettes, except, they were lightly scraped across the leaf surface in order to remove the bulk of trichomes without disturbing the leaf surface.

Metabolic profiling of leaf and trichome gland cells and metabolite identification
Approximately 50 mg fresh weight of leaf material were extracted in 100 µL of ice-cold acetonitrile:isopropanol:water (3:3:2 v/v/v) at room temperature overnight.
Samples were evaporated to near dryness and resuspended in 50% methanol (v/v) for LC/MS analysis. For leaf material with trichomes removed, a glass probe (described previously) was used to gently scrape trichomes from the surface of the leaf prior to extraction with the 3:3:2 solvent mixture.
A total of 50 gland cells from each type of glandular trichome (types 1, 4, and 6) were collected with micropipettes and extracted in 50 µL of ice-cold acetonitrile:isopropanol:water (3:3:2 v/v/v). Samples were stored overnight at -20°C, evaporated to near dryness and resuspended in 50% methanol (v/v) for LC/MS analysis.
Samples were analyzed on a QTRAP™ 3200 mass spectrometer from Applied Biosystems/MDS Sciex (Concord, Ontario, Canada) coupled to a Shimadzu UFLC LC-20AD system and SIL-HTc autosampler. Separation was achieved with a Thermo Betabasic C18 column (150 mm × 1.0 mm, 5 μ m) at 30 C. The mobile phases were, (A) 0.5% formic acid, (B) 0.5% formic acid in 60% methanol+ 40% acetonitrile. A 15 min reverse phase gradient at a flow rate of 0.100 mL/min was used for separation. The linear gradient elution program was as follows: 10% B for 0.3 min, 40% B and linear increase to 100% from 0.31 to 8.5 min, followed by an isocratic hold at 100% B for 2.5 min. At 11 min. B was returned to 10% and the column was equilibrated for 4 min before the next injection.
The mass spectrometer was operated in the positive ion mode with a TurboIonSpray source. Enhanced product ion (MS/MS) scanning was accomplished with www.plantphysiol.org on July 9, 2020 -Published by Downloaded from Copyright © 2011 American Society of Plant Biologists. All rights reserved. none of the metabolites gave MS/MS fragments suggestive of two methyl groups on the A-ring. One additional feature, the loss of 16 Da from the [M+H] + precursor, was shown using deuterium labeling to specifically occur when at least two methyl ether groups were present on the B-ring (3', 4', or 5' positions). The combinations of these features in the MS/MS spectra allow us to use a process of elimination to generate unambiguous evidence for the assignments of methyl group positions in methylated myricetin metabolites.

RNA isolation
Total RNA was extracted from 100 mg fresh weight of young leaf material or young leaf material from which trichomes had been removed. Tri Reagent (Molecular Research Center, Inc.) was used in accordance with the manufacturer's instructions to extract total RNA from leaf and from leaf with trichomes removed. First-strand cDNA was synthesized with SuperScript II reverse transcriptase (Invitrogen) using an anchored poly-T primer supplied by the manufacturer.

Quantitative RT-PCR
Total RNA from young leaf material and young leaf material with trichome removed were extracted as described above then treated with DNase using the DNA-free kit (Ambion). Superscript II Reverse Transcriptase (Invitrogen) and an anchored poly-T primer were used for first-strand cDNA synthesis. A negative control sample was run in parallel without reverse transcriptase added to the reaction mixture. All samples were normalized to the amplification of a Solanum lycopersicum actin gene (accession: BT013707). Quantitative expression analysis was performed using the StepOnePlus Real-Time PCR System (Applied Biosystems). The Fast Sybr Green Master Mix (Applied Biosystems) reagent was used according to the manufacturers' instructions in preparation of the qPCR reactions. The cycling conditions were: 40X 15 sec/95°C, 30 sec/60°C, 30 sec/72°C. Cycling was followed by a melting stage that ramped up from 55 to 95°C with an increasing gradient of 0.5°C, and a 10-s pause at each temperature. The entire experiment was performed in triplicate starting with total RNA isolation from gland cells, leaves, or leaves with trichomes removed. The threshold cycle (Ct) values from each experiment were averaged and the relative expression level of ShMOMT1 in each tissue was calculated using the comparative Ct method (Schmittgen and Livak, 2008). The results were expressed relative to expression levels of ShMOMT1 or ShMOMT2 in leaf material with trichomes.

Isolation, Expression, and purification of recombinant ShMOMT1
The full-length ShMOMT1 and ShMOMT2 ORF's were cloned from cDNA made from S. habrochaites leaf RNA. Tri Reagent (Molecular Research center, Inc.) was used to extract total RNA from approximately 100 mg of material and SuperScript II Reverse Transcriptase (Invitrogen) was used to synthesize first-strand cDNA. ShMOMT1 Induced cultures were pelleted by centrifugation, resuspended in 1/10 volume lysis buffer (50 mM Tris, 10 mM NaCl, 1 mM EDTA, 10% glycerol, 14 mM β mercaptoethanol, pH 8.0), and lysed at 4°C by sonication. The cell lysate was cleared by centrifugation, and the supernatant was partially purified with DE53 anion exchanger (Whatman International, Ltd.). ShMOMT1 and ShMOMT2 were each purified by anionexchange chromatography on an HiTrap Q HP column (GE Healthcare). A linear gradient of (10 -1000 mM) NaCl in lysis buffer was used for the initial purification on the DE53 anion exchanger, and a linear gradient of (250 -500 mM) NaCl in lysis buffer was used for the second round of purification on the HiTrap Q HP anion exchanger. ShMOMT1 eluted in the 400 -500 mM and ShMOMT2 eluted in the 300-400 mM fractions from the DE53 anion exchanger and in the 350 -400 mM and the 300 -350 mM fractions from the HiTrap Q HP anion exchanger, respectively. The active fractions were identified by radiochemical enzyme assays as described above, using myricetin as substrate. SDS-PAGE was used to visualize the degree of homogeneity of the active fractions.

Protein Blot Analysis
Total protein was extracted from collections of 500 gland cells from each of the different types of glandular trichomes (type 1, 4, and 6) in 50 µL of SDS-PAGE sample buffer (100 mM Tris, 2% SDS, 5% ß-mercaptoethanol, 15% glycerol, 0.1% bromophenol blue). Total protein extraction from leaves followed the protocol given in Dudareva et al. 1996. Polyclonal antibodies to ShMOMT1 or ShMOMT2 were generated at Cocalico Biologicals (Reamstown, PA) in rabbit from recombinant ShMOMT1 or ShMOMT2 protein (Supplemental Figure 7). Anti-α-tubulin was from Sigma-Aldrich and served as an internal control to standardize samples from gland cells and leaves. All antibodies (anti-ShMOMT1, anti-ShMOMT2, and antiα -tubulin) were used at a 1:3,000 dilution and incubated with gel blots for 1 h. All other conditions of the protein gel blots were performed as described previously (Dudareva et al., 1996)

ACKNOWLEGMENTS
We would like to express our thanks to Drs. Robert Last and Anthony Schillmiller, and to Ms. Jeongwoon Kim (Michigan State University) for sharing their results with us prior to publication, and for helpful advice.