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Metabolic, Genomic, and Biochemical Analyses of Glandular Trichomes from the Wild Tomato Species Lycopersicon hirsutum Identify a Key Enzyme in the Biosynthesis of Methylketones

^a Department of Molecular, Cellular, and Developmental Biology, University of Michigan Ann Arbor, Michigan 48109-1048
^b Michigan State University, Department of Energy, Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824
^c Department of Plant Biology, University of Arizona, Tuscon, Arizona 85721-0036
^d Department of Plant Biology, Michigan State University, East Lansing, Michigan 48824

² To whom correspondence should be addressed. E-mail lelx{at}umich.edu; fax 734-936-3522.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Medium-length methylketones (C₇-C₁₅) are highly effective in protecting plants from numerous pests. We used a biochemical genomics approach to elucidate the pathway leading to synthesis of methylketones in the glandular trichomes of the wild tomato Lycopersicon hirsutum f glabratum (accession PI126449). A comparison of gland EST databases from accession PI126449 and a second L. hirsutum accession, LA1777, whose glands do not contain methylketones, showed that the expression of genes for fatty acid biosynthesis is elevated in PI126449 glands, suggesting de novo biosynthesis of methylketones. A cDNA abundant in the PI126449 gland EST database but rare in the LA1777 database was similar in sequence to plant esterases. This cDNA, designated Methylketone Synthase 1 (MKS1), was expressed in Escherichia coli and the purified protein used to catalyze in vitro reactions in which C₁₂, C₁₄, and C₁₆ ß-ketoacyl–acyl-carrier-proteins (intermediates in fatty acid biosynthesis) were hydrolyzed and decarboxylated to give C₁₁, C₁₃, and C₁₅ methylketones, respectively. Although MKS1 does not contain a classical transit peptide, in vitro import assays showed that it was targeted to the stroma of plastids, where fatty acid biosynthesis occurs. Levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Plants synthesize a multitude of specialized compounds to help ward off pests. Some of these classes of compounds, like terpenes and phenolics, are widely distributed throughout the plant kingdom (Bennet and Wallsgrove, 1994; McGarvey and Croteau, 1995). Others occur sporadically or are limited to one or a few taxa. Medium-length methylketones (Figure 1), found throughout the plant kingdom, are one class of compounds highly effective in protecting plants against pests (Williams et al., 1980; Kennedy, 2003). Early studies on the composition of the essential oils of many species found medium-length methylketones in lime (Citrus limetta) leaves (Watts, 1886), in clove (Eugenia caryophyllus) and cinnamon (Cinnamomum zeylanicum) oil (Walbaum and Huthig, 1902), in palm kernel (Lodoicea maldivica), peanut (Arachis hypogaea), cottonseed (Gossypium hirsutum), and sunflower (Helianthus annuus) seed oils (Jasperson and Jones, 1947), and in oil of hop (Humulus lupulus) (Sorm et al., 1949). 2-Tridecanone was characterized as a crystalline constituent of the essential oil of matsubasa (Shizandra nigra maxim), a plant in the magnolia family, which is used as a bath perfume (Sengoku, 1933). In some plants, the methylketone and the derived secondary alcohol are found together, for example, 2-heptanone and 2-heptanol in the oil of cloves (Wehmer, 1931).

View larger version (11K): [in this window] [in a new window]   Figure 1. Structures of Common Methylketones Found in Plants.

View larger version (67K): [in this window] [in a new window]   Figure 2. Glandular Trichomes of Tomato. (A) A light microscope picture of the adaxial surface of a young leaf, showing the predominant four-celled glandular trichomes (in focus) and the less abundant long, nonglandular trichomes. (B) Scanning electron micrograph of the abaxial surface of a mature leaf of L. hirsutum. The four-celled glandular trichomes (arrow and in inset) predominate. (C) Light micrograph of the four-celled glandular trichomes isolated from L. hirsutum leaves after final purification.

The prevalence in plants, as well as in other organisms, of methylketones with an odd number of carbons (including the long-chain methylketones [C > 20] found in cuticular waxes) suggests that they are derived from fatty acids, perhaps by decarboxylation of the respective ß-ketoacids, which are intermediates in both the biosynthesis and degradation pathways of fatty acids. Several studies in the 1950s through the 1970s investigating the role of microorganisms in the spoilage of fats attempted to elucidate the biosynthesis of methylketones in fungi (Mukherjee, 1951; Forney and Markovetz, 1971). In these experiments, Penicillium was grown in pure culture on individual fatty acids, and in each case the methylketone formed had one fewer carbon than the progenitor fatty acid. This suggested that a decarboxylation reaction was involved, possibly during an abortive fatty acid degradation process. In fatty acid degradation, the intermediates are CoA esters, and free ß-ketoacids are known to be highly labile and to undergo spontaneous decarboxylation (Ege, 1989). Thus, the enzyme responsible for the production of 2-tridecanone (in the case of myristic acid) may simply be a thioesterase acting on ß-ketomyristoyl-CoA, giving a free ß-ketomyristic acid which then decarboxylates spontaneously. But enzymatic decarboxylation could not be ruled out because a purified enzyme was not obtained and, therefore, the nature of the reaction was not defined further in vitro (Hwang et al., 1976).

Whereas in fungi the methylketones appear to be derived from degradation of fatty acids through a ß-ketoacyl-CoA intermediate (Forney and Markovetz, 1971), their synthesis in plants may be equally likely to be derived from a ß-ketoacyl–acyl-carrier-protein (ACP) intermediate formed during fatty acid biosynthesis. Under this hypothesis, an esterase could cleave ß-ketoacyl-ACP, rather than ß-ketoacyl-CoA, to form the unstable free ß-ketoacid, which will then decarboxylate, either enzymatically or nonenzymatically to form the methylketone. In this study, we used a combination of chemical, biochemical, and genomics approaches to show that methylketones are not only stored but also synthesized through the biosynthetic pathway of fatty acids in tomato glandular trichomes. We were further able to demonstrate the activity of a novel enzyme, Methylketone Synthase 1 (MKS1) that catalyzes the final step of methylketone synthesis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
Localization of Methylketone Storage to the Glandular Trichomes of L. hirsutum f glabratum
Although the presence of methylketones has been detected in several tomato species, including the cultivated tomato L. esculentum, the highest levels have been reported in L. hirsutum f glabratum, including accessions PI126449, PI134417, and LA0407 (Antonious, 2001). We extracted volatiles from intact leaves of several L. hirsutum accessions (see Methods) and confirmed that L. hirsutum f glabratum accession PI126449 had high levels of the methylketones 2-undecanone and 2-tridecanone, as well as 2-pentadecanone (Table 1). We also analyzed the volatile content in the related L. hirsutum accession LA1777 (Table 1), the only L. hirsutum accession for which a gland EST database was available at the beginning of our investigation. LA1777 was found not to make detectable amounts of methylketones (Table 1). Leaves from both lines were shown to contain a variety of terpenes, mostly sesquiterpenes, in agreement with a previous study on the biosynthesis of terpenes in these accessions (van Der Hoeven et al., 2000).

View larger version (22K): [in this window] [in a new window]   Figure 3. Localization of Methylketones in Glandular Trichomes of Tomato. (A) Relative levels of methylketone content in different tissues of the plant, per unit fresh weight. YL, young leaf; ML, mature leaf; ST, stem; RT, root; SE, sepal; PE, petal. Values are shown as percentages of the amount in young leaves. Each value in the graph is an average of three replicates. (B) Comparison of the relative levels of methylketones in leaves and stems with or without a brushing treatment to remove glands, per unit fresh weight. Leaf volatiles were extracted from opposite leaflets of the same leaf. One leaflet was extracted directly with methyl-tert-butyl ether after weighing (Brushed –), and the other was brushed under liquid N2 (Brushed +) before weighing and extraction. Stem samples were cut in half and one half was extracted directly after weighing (Brushed –), and the other was brushed under liquid N2 (Brushed +) before extraction. For each organ, the level of methylketones in unbrushed leaves was set at 100%. Each value in the graph is an average of three replicates. (C) Comparison of the volatile content in the glandular trichomes of L. hirsutum f glabratum (PI126449; top) and L. hirsutum (LA1777; bottom). Glands were isolated from young leaves, and the volatile content was extracted with hexane and injected into a gas chromatograph–mass spectrometer. A portion of the gas chromatograph, from 12.5 to 25 min, is shown. Ter, terpene; MKx, unidentified methylketone derivative; 2UD, 2-undecanone; 2TD, 2-tridecanone; 2PD, 2-pentadecanone. All the major peaks in the LA1777 chromatograph were identified as terpenes, mostly sesquiterpenes. The scale at the y axis is in arbitrary units.

We next isolated glandular trichomes from L. hirsutum accessions PI126449 and LA1777 (Figure 2B) and examined them for the presence of methylketones. After gland isolation (Figure 2C), volatiles were extracted and analyzed by gas chromatography–mass spectrometry (GC-MS). The glands of LA1777 contained mostly sesquiterpenes and one monoterpenene (ocimene), but no methylketones could be detected (Figure 3C). In PI126449, on the other hand, 2-undecanone, 2-tridecanone, and 2-pentadecanone were prominent peaks (Figure 3C) found in similar ratios to those found in extracts from whole leaves. Moreover, additional peaks tentatively identified by mass spectrometry as modified methylketones were also observed. These results confirm that the glandular trichomes are the site of storage of methylketones.

A Gland EST Database of the Methylketone-Producing Accession PI126449 Has High Levels of Sequences Encoding Fatty Acid Biosynthetic Enzymes Compared with an EST Database from LA1777 Glands
To examine whether the methylketones in L. hirsutum f glabratum are derived from fatty acid biosynthesis or degradation, we constructed an EST database from the glands of PI126449. The prevalence of cDNAs encoding enzymes involved in fatty acid biosynthesis and degradation was determined in the analyzed sequences and compared with those found in the previously reported EST database from trichomes of LA1777 (The Institute for Genomic Research, http://www.tigr.org/tdb/Igi/). The PI126449 database contains 5500 ESTs, and the LA1777 database contains 2500. Most of the plant genes encoding both the enzymes of the degradative pathway (which occurs in the peroxisomes) and the enzymes of the biosynthetic pathway (which occurs in the plastids) have been molecularly characterized (Mekhedov et al., 2000; Graham and Eastmond, 2002), so this analysis was relatively straightforward. One of the most striking results of this analysis was the high abundance of the cDNA for the ACP in the PI126449 EST database. ACP is a small (9.5 kD) acidic protein that functions as an essential cofactor in fatty acid synthesis, acyl-ACP desaturation (McKeon and Stumpf, 1982), and plastidic acyl-transferase reactions (Frentzen et al., 1983). Multiple ACP isoforms have been found in evolutionarily diverse species of higher and lower plants, and some are constitutively expressed, while others show developmental and tissue-specific expression (Suh et al., 1999). We identified two distinct contigs in PI126449 that encode two isoforms of the protein (in a 3:1 ratio), which together account for 0.9% of the total transcripts in the glands. The LA1777 EST database, on the other hand, contained only one cDNA (accession number AW616614; 0.04% of total cDNAs) encoding ACP (Figure 4A).

View larger version (18K): [in this window] [in a new window]   Figure 4. In Silico Analysis of the L. hirsutum Leaf Glandular Trichome EST Databases. (A) Analysis of the enzymes of the fatty acid biosynthesis pathway. The frequencies of specific cDNAs encoding each enzyme (labeled A to F) and the ACP are in parentheses (number per 10,000 ESTs; the number to the left of the diagonal is for PI126449; the number to the right is for LA1777). A, acetyl-CoA carboxylase; B, malonyl-CoA:ACP transacylase; CI, ß-ketoacyl-ACP synthase I; CIII, ß-ketoacyl-ACP synthase III; D, ß-ketoacyl-ACP reductase; E, ß-hydroxyacyl-ACP dehydratase; F, enoyl-ACP reductase. The total number of ESTs in accession PI126449 is 5500, and the total number of ESTs in accession LA1777 is 2500. (B) Analysis of the peroxisomal core enzymes of the ß-oxidation of saturated fatty acids. The frequencies of ESTs (number per 10,000 ESTs) encoding each enzyme (labeled A to C) are in parentheses (left number, PI126449; right number, LA1777). A, acyl-CoA oxidase; B, multifunctional protein, with BI-2-trans-enoyl-CoA hydratase and BII-L-ß-hydroxyacyl-CoA dehydrogenase; C, ß-ketoacyl-CoA thiolase.

The prevalence of cDNAs encoding the degradative enzymes in the gland EST databases of the two L. hirsutum accessions was, on average, severalfold lower than the prevalence of the cDNAs for the biosynthetic enzymes, and there was no statistically significant difference in their abundance between the two accessions (Figure 4B). Thus, this analysis suggested that the methylketones in PI126449 are synthesized during de novo fatty acid synthesis rather than via the degradative pathway.

An EST With Homology to Some Esterases Is Highly Expressed in PI126449 Glands and the Encoded Protein Is Correlated with Methylketone Production
The most abundant unknown sequence in the PI126449 database (107 ESTs, 2% of total ESTs, ranked third in abundance after a putative secretory carrier membrane protein and S-adenosyl-L-homocysteinase) encodes a 29-kD protein, with an open reading frame (ORF) containing 265 amino acids (Figure 5). The correct ORF was established by examining several independent full-length cDNAs, two of which contain 182 nucleotides upstream of the initiating ATG (accession number AY701574). There are four stop codons in frame in this 5' nontranslated region, with the 3'-most being located 36 nucleotides upstream of the initiating ATG. Two more cDNAs contain 5' nontranslated sequences that include the stop codon proximal to the initiating ATG. In addition, the genomic sequence of this gene is completely identical to the corresponding sequences of all these cDNAs (E. Fridman and E. Pichersky, unpublished data).

View larger version (32K): [in this window] [in a new window]   Figure 5. Amino Acid Sequence Alignment Comparing the L. hirsutum f glabratum Accession PI126449 MKS1 Protein with Two Functionally Characterized Esterases Belonging to the /ß-Hydrolase Superfamily. The MKS1 accession number is AY701574. LeMJE, methyl jasmonate esterase from L. esculentum (accession number AAS10488); PNAE, polyneuridine aldehyde esterase from R. serpentina (accession number AAF22288). The first amino acid position of the mature PNAE and LeMJE, determined by N-terminal sequencing, is underlined and in bold. When the same amino acid occurs in two or all three proteins in a given position, it is shown in white on a black background.

To investigate the correlation between levels of MKS1 and methylketone content, we first sampled young leaves of 10 different accessions of Lycopersicon. We determined their methylketone content by GC-MS and analyzed the transcript levels of MKS1 and the abundance of its protein product. In RNA gel blots, a signal was observed for mRNA isolated from whole leaves (i.e., leaves containing glands) of PI126449, LA134417, and LA0407 (Figure 6A, lanes 8 to 10, respectively), all L. hirsutum f glabratum accessions that were reported previously to contain high levels of methylketones (Antonious, 2001). Furthermore, high levels of MKS1 transcripts were also detected in L. hirsutum f glabratum accession LA1624 (lane 5), which had not previously been examined for methylketone content, but which our measurements showed to have similar methylketone content to the above three genotypes (Figures 6A and 6C). MKS1 protein content in all these accessions was concordant with MKS1 transcript levels and methylketone content (Figure 6B). On the other hand, MKS1 transcripts or MKS1 protein were not detected in leaves from the cultivated tomato M82 (L. esculentum; lane 1), from two accessions of L. pennellii (lanes 2 and 3), and from L. hirsutum LA1777 (Figures 6A and 6B, lane 6), all lines that did not contain detectable levels of methylketones (Figure 6C). L. hirsutum f glabratum accession PI251305 and L. hirsutum accession PI127826 had low levels of MKS1 transcripts and MKS1 protein (Figures 6A and 6B, lanes 7 and 4, respectively); metabolic profiling of these two accessions showed that the former had barely detectable levels of methylketones, whereas no methylketones could be detected in the latter (Figure 6C).

View larger version (42K): [in this window] [in a new window]   Figure 6. Correlation between MKS1 Transcript and Protein and Methylketone Content in Leaves and Glands of Different Tomato Accessions. (A) RNA gel blots of MKS1 mRNA abundance in young leaves and in glands of various Lycopersicon accessions. Lanes 1 to 10, 5 µg each of total RNA isolated from young leaves; lanes 11 and 12, 2 µg each of total RNA isolated from glands. Lanes 1 to 12, rRNA control. (B) Protein immunoblot of the levels of MKS1 protein in young leaves and glands of various Lycopersicon accessions. Lanes 1 to 10 (15% SDS-PAGE), 2 µg each of total plant protein; lanes 11 and 12, 1 µg each; lane 13, 80 ng of MKS1 purified from E. coli lysate. (C) Total methylketone content in young leaves of various Lycopersicon accessions. The bars and error bars indicate mean and SE, respectively, obtained from four measurements (each from a different plant) for each accession. Le, L. esculentum; Lp, L. pennellii; Lh, L. hirsutum; Lhg, L. hirsutum f glabratum.

To further examine the correlation among MKS1, methylketones, and glands, we measured the concentration of MKS1 in different parts of the plant (Figure 7A). MKS1 protein levels were quantified by SDS-PAGE, followed by immunoblotting of the protein samples together with known amounts of the purified MKS1 protein. MKS1 levels were highest in both young and mature leaves and in sepals and lower in stems and petals, generally correlating with the methylketone content of these organs (Figure 3A). As with methylketone level, MKS1 levels were also correlated with the presence of glands because brushed leaves and stems contained much reduced amounts of MKS1 (75 and 90% reduction in the brushed leaves and stems, respectively) (Figure 7B).

View larger version (13K): [in this window] [in a new window]   Figure 7. Correlation between MKS1 Protein and Glands in the Whole Plant. (A) Relative levels of MKS1 content in different tissues of the plant. YL, young leaf; ML, mature leaf; ST, stem; RT, root; SE, sepal; PE, petal. MKS1 levels (per unit fresh weight) were quantified by immunoblotting. Values are shown as percentages of the amount in young leaves. Each value is an average of three replicates. (B) Comparison of the relative levels of MKS1 in leaves and stems with and without a brushing treatment to remove glands, per unit fresh weight. Leaves and stems treated as in Figure 3B. For each organ, the level of MKS1 in unbrushed leaves was set at 100%. Each bar represents an average of at least three replicates.

The in vitro–translated, radiolabeled tomato MKS1 protein was incubated with isolated pea (Pisum sativum var Little Marvel) chloroplasts and ATP, followed by reisolation of the chloroplasts, incubation with proteases, lysis, fractionation into membrane and soluble components, and SDS-PAGE. The translated protein was found to be imported into the chloroplast (Figure 8B), as deduced from its resistance to proteolytic digestion by both thermolysin and trypsin, compared with the sensitivity of the unprotected MKS1 (treatment of chloroplasts with Triton X-100 before protease treatment makes their membrane permeable to the proteases; Figures 8A and 8B, lanes 7 to 10), and could be recovered from the soluble fraction of the chloroplast.

View larger version (49K): [in this window] [in a new window]   Figure 8. MKS1 Is Targeted to the Stroma of Chloroplasts. (A) Sensitivity of unprotected MKS1 to thermolysin and trypsin. Lane 1, 1 µg MKS1 purified from E. coli, run on SDS-PAGE, and visualized by Coomassie bluee staining; lane 2, in vitro–translated, [35S]-labeled MKS1 product (translated protein [TP]) electrophoresed under identical conditions. Lanes 3 and 4, radioactively labeled MKS1 (same amount as in lane 2) treated with either thermolysin or trypsin for 30 min at 4°C before electrophoresis. The gel containing lanes 2 to 4 was analyzed by fluorography after SDS-PAGE. (B) MKS1 import assay. [35S]-labeled MKS1 was incubated with isolated pea chloroplasts in the presence of 4.0 mM Mg-ATP for 30 min. Lane labeled TP represents 10% of translated product added to a single import assay. After import, chloroplasts were reisolated, Iysed, and fractionated into soluble and insoluble fractions (see Methods). Both fractions were analyzed by SDS-PAGE and fluorography. P, membrane (pellet) component; S, soluble component. (C) Import assay of Arc6, a DnaJ-like protein that contains a cleavable transit peptide and is imported into the plastid envelope (Vitha et al., 2003). Experiments were analyzed in the same way as in (B). The N terminus of the protein precursor (pArc6) is cleaved after import, and the mature protein (mArc6) is partially sensitive to trypsin because it protrudes into the intermembrane space. The asterisk depicts the protected part of the protein, which is embedded in the membrane. (D) Import assay of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase that contains a cleavable transit peptide and is imported into the stroma. The N terminus of the protein precursor small subunit (pSS) is cleaved after import, and the mature small subunit protein (mSS) is therefore not sensitive to trypsin digestion.

The Tomato MKS1 Protein Catalyzes the Formation of 2-Undecanone, 2-Tridecanone, and 2-Pentadecanone from ß-Ketolauroyl-ACP, ß-Ketomyristoyl-ACP, and ß-Ketopalmitoyl-ACP in the Glands
PNAE is a member of the /ß-hydrolase superfamily (Dogru et al., 2000), and it catalyzes the deesterification of a methylester with a ß-keto functionality (Figure 9A). Once the methyl group is removed, the resulting ß-ketoacid decarboxylates, although it is not yet known if this reaction is also catalyzed by PNAE or occurs spontaneously either before or after the ß-ketoacid leaves the active site of the enzyme. The similarity of the PNAE esterase protein sequence to MKS1 suggested that the latter may be the hypothesized MKS enzyme that deesterifies ß-ketoacyl-ACPs to the ß-ketoacids and thus leads to the formation of methylketones after decarboxylation (Figure 9B).

View larger version (7K): [in this window] [in a new window]   Figure 9. The Hypothesized Reaction Leading to Methylketones Is Similar to the Reaction Catalyzed by PNAE. (A) General scheme of the reaction catalyzed by PNAE. (B) Proposed scheme of the reaction catalyzed by MKS.

View larger version (18K): [in this window] [in a new window]   Figure 10. Radio–Thin Layer Chromatography Analysis of the Hexane-Soluble Products from Enzymatic Assays Using [3-14C] ß-Ketomyristoyl-ACP as a Substrate. (A) Enzymatic assays with gland protein and purified MKS1. Arrows indicate the positions of the 2-tridecanone (2TD) standard, the [1-14C] lauric acid standard (C12:0), and the origin. Lane 1, crude protein extract of PI126449 glands; lane 2, crude protein extract of LA1777 glands; lane 3, MKS1 purified from E. coli; lane 4, BSA control. The concentration of the substrate in each reaction was 10 µM. Lanes 1 and 2 contained 5 µg total protein. Lanes 3 and 4 contained 0.5 µg of protein. (B) Comparisons of the relative levels of MKS1 activity in stems and leaves with and without treatment to remove glands. Leaves and stems treated as in Figure 3B. The concentration of the substrate and the amount of protein in each reaction was 3 µM and 3 µg, respectively. A sample thin layer chromatography plate is shown above, and the bars represent averages of three replicates.

ß-Ketolauroyl-ACP and ß-ketopalmitoyl-ACP were synthesized in a similar way, and MKS1 was able to convert ß-ketolauroyl-ACP to 2-undecanone and ß-ketopalmitoyl-ACP to 2-pentadecanone. We next determined the apparent K_m values of purified MKS1 for all three substrates (Figure 11). The enzyme had an apparent K_m value for ß- ketomyristoyl-ACP of 3.1 µM (Figure 11A). The apparent K_m values for ß-ketolauroyl-ACP and ß-ketopalmitoyl-ACP were 1.2 and 10.7 µM, respectively (Figures 11B and 11C). The V_max value for the reaction of MKS1 with [3-¹⁴C] ß- ketomyristoyl-ACP was 10^–8 µmoles·s^–1·µg protein^–1, and similar values were obtained for the reaction with ß-ketolauroyl-ACP and ß-ketopalmitoyl-ACP.

View larger version (12K): [in this window] [in a new window]   Figure 11. Steady State Kinetic Analysis of MKS1 Assayed with [3-14C] ß-Ketoacyl-ACPs. Initial velocities are shown as dots, and the line represents the nonlinear least-squares fit of the initial velocities versus [3-14C] ß-ketoacyl-ACP concentration to the hyperbolic Michaelis-Menten equation. 2UD, 2-undecanone; 2TD, 2-tridecanone; 2PD, 2-pentadecanone. (A) A representative measurement with [3-14C] ß-ketomeristoyl-ACP as the substrate. The apparent Km value shown is an average of three independent assays. (B) A measurement with [3-14C] ß-ketolauroyl-ACP as the substrate. This assay gave a similar result to the one shown in (A) and was done only once because of the low availability of substrate (see Methods). (C) A representative measurement with [3-14C] ß-ketopalmitoyl-ACP as the substrate. The apparent Km value shown is an average of two independent assays.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

 
The Machinery to Accumulate and Produce Methylketones from Fatty Acids Is Located in the Glands of L. hirsutum f glabratum
The four-celled glandular trichomes on the leaves of the wild tomato L. hirsutum f glabratum had previously been recognized to be the site of accumulation of methylketones (Lin et al., 1987). We show that methylketone content correlates well with the presence and density of glands in leaves as well as in other green parts of the plant (Figure 3A). However, the correlation is not perfect; for example, stems have 7% of the methylketone content per unit fresh weight that young leaves do, but young leaves have almost 100-fold more glands per unit fresh weight. This observation suggests that stem glands contain more methylketones because, perhaps, being older, they have had more time to accumulate them. The correlation of methylketone content with glands was further strengthened by examining leaves and stems from which most glands were removed by brushing. Although it was not possible to remove all glands from the surfaces of the plant organs, the great decrease in methylketone content in brushed leaves and stems, as well as the isolation and purification of glands and the demonstration of their methylketone content, support the conclusion that the glands are the main, and perhaps exclusive, site of methylketone storage in the plant (Figures 3B and 3C).

With the demonstration of the glandular trichomes as the major site of methylketone storage, we applied the single-cell EST database approach to determine if these glands might also be the site of synthesis of methylketones and, if so, to identify the genes and enzymes involved in their biosynthesis. Analysis of EST databases constructed from a single type of cell in conjunction with other types of data, such as metabolic profiling, is a powerful method to identify major biochemical pathways operating in the cell. We have previously used this approach to identify genes important in the phenylpropene and terpene pathways by constructing and comparing EST databases from peltate gland cells of several basil varieties (Gang et al., 2001, 2002; Iijima et al., 2004a, 2004b). Gene identification in these studies was based on prevalence of specific sequences, comparisons with known sequences encoding enzymes of similar function (either in terms of substrates or type of reactions), metabolic profiling of the plant material, and enzymatic assays of candidate proteins (Fridman and Pichersky, 2005). This approach has allowed us to determine that methylketones are made via the de novo fatty acid biosynthetic pathway in the chloroplast and to identify MKS1 as the enzyme responsible for the reaction leading from C₁₂, C₁₄, and C₁₆ ß-ketoacyl-ACPs to the C₁₁, C₁₃, and C₁₅ methylketones, respectively. The levels of MKS1 transcripts and protein are closely correlated with the presence of methylketones in the Lycopersicon genus (Figure 6). Furthermore, the levels of MKS1 transcripts, protein, and MKS enzymatic activity were found to correlate well with the presence of glands throughout the plant (Figures 6, 7, and 10). Taken together, these results indicate that these glands are also the major, and perhaps exclusive, site of methylketone biosynthesis.

MKS1 Is a Plastid Protein
The results of the chloroplast import experiments indicate that MKS1 is targeted to the soluble stroma compartment of the chloroplast (Figure 8B), in close proximity to the site of fatty acid biosynthesis (Walker and Harwood, 1985). The routing of a protein to the plastid stroma without the cleavage of a typical transit peptide is rare but not unprecedented; Rathinasabapathi et al. (1994) reported that betaine aldehyde dehydrogenase is imported into the chloroplast and is ultimately localized in the stroma without cleavage of an N-terminal peptide. In addition, several inner and outer membrane proteins are known not to contain a cleavable transit peptide (Froehlich et al., 2001; Miras et al., 2002). Although our results indicate that MKS1 is not substantially processed during import (Figures 6B and 8B), we cannot exclude the possibility that a small peptide is removed from MKS1 during its targeting to the stroma.

The Mechanism of Action of MKS1
Our enzymatic characterization of MKS1 indicated that it can use several ß-ketoacyl-ACPs with high affinity to produce the corresponding methylketones. However, the efficiency of the reaction was too low to account for the amount of methylketones made by the plant. It can be calculated that only 1% of the observed amount of methylketones in the leaves could be made given the amount of MKS1 in the leaves and the V_max value of the enzyme determined in the in vitro assays. There may be several explanations for this discrepancy between the in vitro rate of the MKS1-catalyzed reaction and the actual in vivo synthesis of methylketones in the cell. It is likely that the in vitro assay we developed is not yet optimized. The enzyme may need some cofactors that have not yet been identified. For example, we have found that adding the soluble fraction of an E. coli extract increases the efficiency by 10-fold (E. Fridman and E. Pichersky, unpublished data). In addition, we used spinach (Spinacia oleracea) ACP in our assays, but the enzyme may work faster with L. hirsutum ACPs. Several previous kinetic characterizations of fatty acid biosynthetic enzymes (e.g., acetyl-CoA carboxylase and malonyl-CoA:ACP transacylase) and polyketide synthases, some of which use substrates containing ACP moieties, have reported either low in vitro V_max values or higher K_m values (Alban et al., 1994) that could not explain the observed rate of polyketide or fatty acid synthesis in the organism. In some cases, the ACP moiety was directly tied to the low V_max values (Tang et al., 2003), but in other cases the reasons remained undetermined or were attributed to a lack of components of a multienzyme complex, where the product of one reaction is generated immediately adjacent to the active site of the next enzyme in the reaction sequence (e.g., substrate channeling) (Roughan, 1997; Tang et al., 2003).

There are some indications that the plastidic enzymes involved in fatty acid biosynthesis are part of a complex (Roughan and Ohlrogge, 1996). It is also likely that MKS1 is part of this protein complex in the L. hirsutum glands and that its kinetic parameters are quite different when it acts as part of such a complex. The existence of such a complex may also relate to the intriguing question of how the substrates of MKS1, which are intermediates in the fatty acid biosynthetic pathway, become accessible to MKS1 and why in L. hirsutum f glabratum the main products are 2-tridecanone, 2-undecanone, and 2-pentadecanone (and not smaller or larger methylketones). Furthermore, although MKS1 showed similar specificity to the three different ß-ketoacyl-ACPs that lead to the formation of 2-undecanone, 2-tridecanone, and 2-pentadecanone (Figure 11), the three methylketones exist in different quantities in the glands (Table 1, Figure 3C). It is interesting that in one of the L. hirsutum f glabratum accessions, LA0407, the ratio between 2-tridecanone and 2-undecanone is the opposite of that found in the rest of this subspecies, with 2-undecanone being the major methylketone in the leaf (Antonious, 2001). Yet, the sequence of the LA0407 allele of MKS1 is identical to that of MKS1 from PI126449 (E. Fridman and E. Pichersky, unpublished data). This suggests that the variation in the abundance of the different methylketones in the glands (and the real turnover rate of the enzyme) may be determined by the availability of the substrate rather than the specificity of MKS1 for the different substrates.

Relationship of MKS1 to Other /ß-Hydrolase Enzymes
MKS1 is part of the /ß-hydrolase superfamily that includes several enzymes with a proven esterase function. These include, in addition to PNAE and tomato MJE, potato (Solanum tuberosum) MJE (accession number AY684102) and PIR7, a protein from rice (Oryza sativa) that was shown to have esterase activity with artificial substrates (Waspi et al., 1998). In addition, the tobacco (Nicotiana tabacum) SABP2 protein, which has recently been shown to be methyl salicylate esterase (Forouhar et al., 2005), and an uncharacterized protein encoded by an ethylene-induced citrus EST (Zhong et al., 2001), as well as 20 proteins encoded in the Arabidopsis genome, show similarity to MKS1 (Figure 12). PIR7, PNAE, SABP2, and MJE all appear to have defense-related functions. For example, the defense-related rice Pir7 transcripts were found to accumulate upon infiltration with the resistance-inducing Pseudomonas syringae pv syringae (Waspi et al., 1998), and PNAE is involved in the biosynthesis of defense alkaloids in the Indian medicinal plant R. serpentina. MJE may be involved in the jasmonate signal transduction pathway that turns on many defense genes (Sasaki et al., 2001; Stuhlfelder et al., 2004). The role of MKS1 in the biosynthesis of the highly protective methylketone is therefore not surprising. However, the origin of MKS1 during evolution and the prevalence of MKS1-related sequences in the plant kingdom (and elsewhere) remain to be determined.

View larger version (22K): [in this window] [in a new window]   Figure 12. Unrooted Neighbor-Joining Phylogenetic Tree of Selected Plant Proteins Belonging to the /ß-Hydrolase (Esterase) Family. The source and accession numbers for the non-Arabidopsis proteins that do not appear in the sequence alignment in Figure 5 are as follows: EIE (Ethylene Induced Esterase) from Citrus sinensis, accession AAK58599; SABP2 (Salicylic Acid Binding Protein 2) from Nicotiana tabacum, AAR87711 PIR7 (pathogen-induced) from Oryza sativa, CAA84024 StMJE (methyl jasmonate esterase) from Solanum tuberosum (potato), AY684102. The scale indicates average substitutions per site for each cladogram.

	METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION METHODS REFERENCES

Scanning Electron Microscopy
Young leaves of L. hirsutum f glabratum were prepared for scanning electron microscopy as previously described (Gang et al., 2001).

Leaf Gland Trichome Isolation and Removal for Protein Extraction, Volatile Analysis, and RNA Isolation
Leaf glandular trichomes were isolated from young leaves of different tomato accessions following the procedure described previously (Gang et al., 2001) with a few modifications. The glands were collected on a final 40-µm mesh cloth, and the yield was 1 to 1.5 mL of packed glands per 15 g of leaf sample. Glands were left on ice for 15 min to settle down and were used either for analysis of essential oil constituents or for protein extraction. The crude protein extract of the glands was obtained by resuspending the packed glands in ice-cold gland lysis buffer (10 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM ß-mercaptoethanol, and 10% glycerol) followed by sonication for 2 min and collection of the supernatant after centrifugation at 14,000g at 4°C for 20 min. For RNA isolation, the glands were isolated in a similar way, except that aurintricarboxylic acid (1 mM) was added to all buffers and solutions. After letting the glands settle down on ice for 15 min, the overlaying gland isolation buffer was removed and 450 µL of buffer RLT (Qiagen, Valencia, CA) was added for each 100 mg of glands. The glands were lysed by a 30-s sonication pulse, and the total RNA was purified using the RNeasy plant mini kit (Qiagen).

Leaf and stem samples were collected from five independent PI126449 plants that were randomly planted in pots with other tomato accessions. Partial removal of the glands from leaves and stems was performed in liquid N₂ with a painter's brush before weight measurements and extractions. Compared leaves, with or without treatment, were opposite leaflets on the same leaf, and compared stem samples were from the same stem.

cDNA Library Construction, Sequencing, and EST Analysis
A directional cDNA library was constructed as previously described (Gang et al., 2001), and recombinant plasmids from 3840 colonies (10 x 384-well plates) were randomly and automatically isolated and sequenced from both 5'- and 3'-ends, using the T3 and T7 primers, respectively. After removal of vector and poor quality sequences, the remaining 5496 EST sequences were compared against the SWISS-PRO and nonredundant databases using the tBLASTn search algorithms. The contig assemblies and the singletons were assigned specific functions based on highest similarity and each was BLAST searched against the L. hirsutum (accession LA1777) trichome EST database (The Institute for Genomic Research, http://www.tigr.org/tdb/Igi/) to obtain the number of ESTs for each gene.

GC-MS Analysis of Volatiles
Plant samples were weighted and then placed in 5-mL glass vials containing 1 mL of methyl-tert-butyl ether. The vials were sealed with rubber septa cups and mildly shaken for 2 h. Toluene was added as an internal standard (0.03%), and the resulting extract was used for GC-MS analysis. GC-MS was performed as described previously (Gang et al., 2001). Three microliters of the extract, with no prior concentration, were analyzed by GC-MS for determination of the essential oil constituents. The identities of the main methylketones and terpenes were verified with commercially available standards. For determination of the volatiles in the glands, 100 mg of packed glands (see extraction protocol above) were extracted twice with 200 µL of hexane, followed by concentration and GC-MS analysis.

Chloroplast Import Assays
Pea plants (Pisum sativum var Little Marvel; Olds Seed, Madison, WI) were grown under natural light in the greenhouse at 18 to 20°C. Chloroplasts were isolated from 8- to 12-d-old plants as described previously (Perry and Keegstra, 1994). MKS1, ARC6 (Vitha et al., 2003), and RBCS (Olsen and Keegstra, 1992) genes were transcribed, translated, and labeled with [³⁵S]-Met using the TNT-coupled Reticulocyte Lysate system (Promega, Madison, WI). Import assays were performed in 450 µL of import buffer (50 mM Hepes-KOH, pH 8.0; 330 mM sorbitol) containing 75 µL of chloroplasts, 4 mM Mg-ATP, and 5 x 10⁶ dpm precursor protein for 30 min, at room temperature, in the presence of light. Chloroplasts were recovered by sedimentation through a 40% Percoll cushion and then incubated with (+) or without (–) thermolysin or trypsin for 30 min at 4°C as previously described (Jackson et al., 1998), with or without Triton X-100. Intact chloroplasts not treated with Triton X-100 were again recovered by centrifugation through a 40% Percoll cushion then lysed in a solution containing 25 mM Hepes, pH 8.0, and 5 mM EDTA and fractionated into stroma and total membrane (including outer envelope, inner envelope, and thylakoid). Samples treated with Triton X-100 were centrifuged at 100,00g and separated to pellet and supernatant fractions. All import reactions were analyzed by SDS-PAGE. After electrophoresis, the gels were subjected to fluorography and exposed to x-ray film (Eastman-Kodak, Rochester, NY).

RNA Extraction and Gel Blot Analysis
RNA from glands was isolated using the RNeasy plant mini kit (Qiagen) as described above, and leaf RNA was isolated using TRIzol reagent (Gibco-BRL, Grand Island, NY) according to the manufacturer's protocol. RNA gel blots were performed as described previously (D'Auria et al., 2002) using 2 and 5 µg of RNA in each lane for glands and leaf samples, respectively. An MKS1 probe was synthesized with the rediprime II kit (Amersham Pharmacia Biotech, Piscataway, NJ) using a PCR-amplified fragment of MKS1.

Protein Expression and Purification
The following primers were used to amplify and clone the full ORF of MKS1 from a PI126449 leaf cDNA into the Escherichia coli expression TA cloning vector (pCRT7/CT TOPO-TA; Invitrogen, Carlsbad, CA): forward, 5'-AATGGAGAAAAGCATGTCGC-3'; reverse, 5'-GCTCTTTATTTATACTTGTTAGC-3'. PCR conditions were 30 s at 94°C, followed by 25 cycles of 20 s at 94°C, 20 s at 56°C, 40 s at 68°C, and an additional 10 min at 68°C. The full-length MKS1 construct was transformed into BL21(DE3) Codon+ cells, and a single transformed colony was grown at 37°C overnight in 2 mL of LB media containing 100 and 34 µg/mL of ampicillin and chloramphenicol, respectively. The culture was then used to start a 200-mL culture (with the same antibiotics) that was grown at 37°C until an A₆₀₀ value of 0.4 was obtained. After induction with 0.5 mM isopropyl 1-thio-ß-galactopyranoside, the culture was grown overnight at 18°C. Cells were pelleted, harvested, and resuspended in 20 mL of lysis buffer (10 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 10 mM ß-mercaptoethanol, and 10% [v/v] glycerol). After sonication for 2 min and centrifugation at 14,000g for 20 min at 4°C, the supernatant was collected and kept at –80°C for further applications.

A plasmid containing the spinach (Spinacia oleracea) ACP isoform I gene using codons preferred by E. coli, with a C-terminal His-tag (pSACP-1a; Broadwater and Fox, 1999), was kindly provided to us by Brian Fox (University of Wisconsin, Madison). The plasmid was transformed into BL21(DE3) LysS cells, and a single transformed colony was grown at 37°C overnight in 20 mL of LB medium containing 100 and 34 µg/mL of ampicillin and chloramphenicol, respectively. The culture was then used to start a 500-mL terrific-broth culture (Tartof and Hobbs, 1987) (with the same antibiotics) that was grown at 37°C until an A₆₀₀ value of 1.0 was obtained. After induction with 0.5 mM isopropyl 1-thio-ß-galactopyranoside, the culture was grown overnight at 18°C. Cells were pelleted, harvested, and resuspended in 50 mL of ACP lysis buffer (20 mM Tris-HCl, pH 7.9, 250 mM NaCl, 10 mM imidazole, 1% Tween 20, 10 mM ß-meracptoethanol, and 10% [v/v] glycerol). The lysate was stirred on ice for 1 h with 50 µg mL^–1 of lysozyme and DNase (Sigma-Aldrich, St. Louis, MO). After sonication for 2 min and centrifugation at 30,000g for 20 min at 4°C, the supernatant was collected and kept at –80°C until purification.

MKS1 was purified from E. coli cell lysates by diethylaminoethyl-cellulose anion exchange column chromatography (DE53; Sigma-Aldrich) followed by chromatography on a Hi-Trap Phenyl HP (hydrophopic interactions) column (Amersham Pharmacia Biotech) connected to a fast protein liquid chromatography system. MKS1 did not bind to DE53 and eluted with the flow-through, thus achieving a substantial purification. It bound well to a Phenyl HP column, eluting at 0.3 to 0.35 M (NH₄)₂SO₄. Fractions with highest concentrations of MKS1 eluting from the Phenyl HP column were concentrated by centrifugation as described previously (Iijima et al., 2004a).

Spinach ACP was purified from E. coli cell lysates on a column containing 2 mL of packed Ni²⁺-NTA resin (Qiagen). The column was equilibrated with 40 mL of double distilled water, followed by 40 mL of ACP lysis buffer before loading all 50 mL of lysate. The column was then washed once with 20 mL of ACP lysis buffer followed by a 40 mL wash with the same buffer but with no Tween 20. The His-tagged ACP was eluted with 4 mL of ACP lysis buffer with no Tween 20 and with 250 mM imidazole. The eluted 0.5-mL fractions were analyzed on a 15% SDS-PAGE, and fractions with high levels of ACP-His6 were pooled and dialyzed against 100 to 200 volumes of 10 mM Mes, pH 6.1, with 0.5 mM dithioerythritol overnight at 4°C and stored at –80°C for further applications.

The acyl-acyl carrier protein synthase enzyme (Aas) was overexpressed and purified from E. coli cell line BL21(DE3) LysS, using the plasmid and procedures described previously (Shanklin, 2000).

Preparation of MKS1 Antibodies and Protein Gel Blots
One milligram of the purified protein (see Supplemental Figure 1 online, lane 5) was electrophoresed on a 13% SDS-PAGE gel, stained with Coomassie Brilliant Blue, and the protein excised from the gel. Antibodies were prepared by Cocalico Biologicals (Reamstown, PA) by injecting macerated gel fragments containing the purified MKS1 and following the company's protocol. Specificity of the antibody was tested with the preinjection serum and with different purified proteins (data not shown). Anti-MKS1 antibodies were used at a 1:2000 dilution and incubated with the protein gel blots for 2 to 16 h. All other conditions of the protein gel blots were performed as described previously (Dudareva et al., 1996).

Substrate Preparation, Enzyme Assays, and Product Identification
The coupled enzymatic assay we developed was done in three steps, with the first two steps needed to produce the ß-ketoacyl-ACP substrate for the reaction (third step) catalyzed by the putative MKS (see Supplemental Figure 2 online). The following is a description of the synthesis of [3-¹⁴C] ß-ketomyristoyl-ACP from the starting material [1-¹⁴C] 12:0 lauric acid. The same procedure was used for the synthesis of [3-¹⁴C] ß-ketolauroyl-ACP or [3-¹⁴C] ß-ketopalmitoyl-ACP, starting from [1-¹⁴C] 10:0 capric acid or [1-¹⁴C] 14:0 myristic acid, respectively.

In the first step, [1-¹⁴C] 12:0 lauric acid (ARC 257; American Radiolabeled Chemicals, St. Louis, MO) is converted to an acyl-ACP in a reaction containing spinach ACP and an E. coli acyl-ACP synthetase (Rock and Garwin, 1979). (This E. coli acyl-ACP synthetase couples C12:0 and C14:0 fatty acids efficiently, but it has low coupling efficiency with decanoic acid [C10:0], thereby limiting the amount of [3-¹⁴C] ß-lauroyl-ACP that we were ultimately able to make). Because the ACP that was used in these syntheses carried a His-tag, a Ni²⁺-NTA