Multiple Biochemical and Morphological Factors Underlie the Production of Methylketones in Tomato Trichomes 1

Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f glabratum , which synthesizes and accumulates insecticidal methylketones (MK), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and Solanum lycopersicum (cultivated tomato), which does not, demonstrated that several genetic loci contribute to MK metabolism in the wild species. A strong correlation was found between the shape of the glandular trichomes and their MK content, and significant associations were seen between allelic states of three genes and the amount of MK produced by the plant. Two genes belong to the fatty acid biosynthetic pathway and the third is the previously identified Methylketone Synthase 1 ( MKS1 ) that mediates divergence to MK from β -ketoacyl intermediates. Comparative transcriptome analysis of the glandular trichomes of F2 progeny grouped into low- and high-MK- containing plants identified several additional genes whose transcripts were either more or less abundant in the high-MK bulk. In particular, a wild-species specific transcript for a gene which we named Methylketone Synthase 2 ( MKS2 ), encoding a protein with some similarity to a well-characterized bacterial thioesterase, was approximately 300-fold more highly expressed in F2 plants with high MK content than in those with low MK content. Genetic analysis in the segregating population showed that MKS2 's significant contribution to MK accumulation is mediated by an epistatic relationship with MKS1 . Furthermore, heterologous expression of MKS2 in Escherichia coli resulted in the production of methylketones in this host. (Bird et al., 2007). Together, these observations suggest a model in which enhanced activity of MK biosynthesis in the wild species may underlie the diversion of the fatty acids to MK at the expense of the synthesis of very-long-chain fatty acids, hence changing the morphology of the trichomes.


INTRODUCTION
Plants exhibit a large range of chemical and morphological variation, reflecting different adaptations to mediating their interactions with the biotic and abiotic environment throughout their life cycle (Ehrlich and Raven, 1964). Some plant chemicals are lipophilic (oily) compounds that have high vapor pressure and therefore volatilize easily when exposed to air. Such volatiles can serve as signal molecules that either attract or repel animals. Many such compounds are also toxic and can damage a predatory organism through external or internal contact, and are therefore synthesized in dedicated cells that also serve to store them (Wagner et al., 2004). In particular, such compounds may be synthesized and accumulated in small epidermal cell extensions on the surface of their leaves, stems and reproductive tissues called glandular trichomes (Schilmiller et al., 2008). Since the initial work on glandular trichomes in mint (Gershenzon et al., 1992) various studies involving transcriptomics, proteomics and metabolomics have indicated that entire metabolic pathways responsible for the production of such compounds operate within the trichomes and that these unique cells require the import of only the basic building blocks to make these chemicals ( various Solanum species. One of these trichome types which has been investigated in some detail, is the Type VI glandular trichome, which is composed of a stalk cell with four cells at the top that form a mushroom-like shape; a cuticular sac wrapped around these cells allows accumulation of secreted compounds similar to an inflating balloon (Snyder and Carter, 1985;Werker, 2000). We  6 species Solanum habrochaites f glabratum, the Type VI glandular trichomes, which are present at high density on both the leaf surfaces and stems, contain two main MK compounds, 2-tridecanone (2TD, containing a 13-C backbone) and 2-undecanone (2UD, containing an 11-C backbone), as well as some 2-pentadecanone (containing a 15-C backbone) and a few other unidentified MK. These MK are synthesized and accumulated to very high levels in these trichomes, up to 5500 µg/g leaf fresh weight (Antonious, 2001;Fridman et al., 2005).
Analysis of a Type VI-specific EST database from a MK-producing S.
habrochaites f glabratum (accession PI126449) showed that transcripts of genes encoding plastidic enzymes of fatty acid biosynthesis are highly represented, in contrast to their relatively low representation in a line that does not make MK (accession LA1777; Fridman et al., 2005). The comparative analysis of the two EST databases also led to the isolation and characterization of a novel gene encoding a protein belonging to the α /β hydrolase family, which was specifically and exclusively expressed in Type VI trichomes of methylketone-producing plants but not in non-producers. Although the protein did not appear to have a transit peptide, the results of plastid import experiments indicated that it could be imported into the plastids.
Since 3-ketoacids are inherently unstable and undergo spontaneous decarboxylation (Kornberg 1948), albeit at low rate at ambient temperature, the evidence of elevated levels of fatty acid biosynthesis in these trichomes suggested that the observed straight-chain methylketones such as 2TD and 2UD could be derived from enzymatic or non-enzymatic decarboxylation of the respective C n+1 3-ketoacids. In plants, 3-ketoacyls of fatty acids mostly occur in plastids (as 3-ketoacyl-ACPs) as intermediates in the fatty acid biosynthesis pathway, and in peroxisomes (as 3-ketoacyl-CoA) as intermediates in the fatty acid degradation pathway Crosses between MK-producing and non-producing lines followed by segregation analysis have indicated that the ability to produce MK requires multiple quantitative trait loci in addition to MKS1 (Zamir et al., 1984). Consequently, it has not been possible to breed cultivated tomato lines that produce high levels of MK in their glands. It is likely that the trait of MK production in S. habrochaites evolved through multiple morphological and biochemical changes that took place gradually during evolution. To uncover the additional factors influencing MK production, we took a quantitative genetic approach to identify QTLs that might affect MK production, including genes encoding biosynthetic enzymes, and tested the possible relationship between trichome characteristics and chemical content. In addition, comparative transcriptomic analysis was used to identify new genes whose differential expression is correlated with MK production in interspecific populations.

Crosses Between the Cultivated Tomato and S. habrochaites f glabratum
The chemical profiles of leaves of the cultivated tomato S. lycopersicum (var. M82) and the wild species S. habrochaites f glabratum (accession PI126449) differ in their shape and chemical content. In particular, leaves of the cultivated tomato contain little or no MK while leaves of the wild species contain high levels of 2UD and 2TD which are synthesized and stored in the Type VI glandular trichomes on the leaf surface (Fridman et al., 2005). A series of crosses were conducted between these accessions to genetically dissect the contribution of candidate genes to MK content. Tomato plants of different genetic backgrounds were then evaluated, including the two parental lines: planted and from each, six young leaflets were removed for chemical characterization and 2TD level determination, since 2TD is the major MK produced in the parental wildspecies (Fridman et al., 2005). Overall, the 2TD levels of most F2 progeny were more similar to the cultivated tomato parent (Fig. 1). This, combined with the observation of very low values in the backcrossed (BC) generations indicated polygenic inheritance of this trait and suggested the recessive characteristic of the wild-species alleles that participate in this pathway.
Digital images of leaflet surfaces were taken to determine trichome density and its association with MK accumulation. While analyzing these images, we noticed that the F2 population segregates not only for trichome number, but also for trichome shape. This observation is in agreement with previously described distinctions in trichome shape between cultivated and wild species of tomato (Snyder and Carter, 1985;Antonious, 2001). While none of the F2 plants showed clear separation of the cells at the tip of the trichomes (as the trichomes of M82), 31% of the population had Type VI trichomes with partial separation of these cells (M82-like; Fig. 2A,B), 18% of the F2 progeny had round Type VI trichomes, basically identical in shape to those of the wild species (PI shape; Fig. 2A, B), and in 51% of the plants, the cells of the Type VI trichomes were not separated similar to the M82 parent, but the trichome appeared more square than round.

Association Between Candidate Genes, Trichome Characteristics, and MK Content
The association between variation in candidate structural genes and 2TD content was examined in genetic mapping experiments employing these genes as simple PCR content. In addition, a significant positive correlation between the density and shape of the trichomes and the amount of MK in the leaves was found (Fig. 4A). This multiple regression reinforced the previous results indicating an association between trichome morphology and 2TD levels (Fig. 2), and overall this model explained approximately one-third of the total 2TD phenotypic variation in the F2 population (R 2 = 0.333; Fig.   4B). To test whether the three candidate genes that are significantly associated with 2TD levels in the segregating population (MKS1, ACC and MaCoA-ACP trans), exhibit differential expression between the wild and cultivated species, quantitative reversetranscription PCR (qRT-PCR) approach was taken. Primer pairs that fully matched both alleles were designed for each gene and qRT-PCR was conducted using RNA from  (Table I).
In particular, one wild-species specific transcript of a gene which we subsequently designated Methylketone synthase 2 (MKS2; see below) was 337-fold more highly expressed in F2 plants with high vs. low MK content while a similar transcript, derived from the cultivated species, was 7.5-fold more highly expressed in the F2 plants with low vs. high MK content (Table I).

MKS2 Shares Sequence Identity with Hotdog-Fold Thioesterases
The MKS2 protein is 52 to 70% identical to several plant proteins with no proven functions, encoded by genes in the Arabidopsis and rice genomes and by many ESTs from various plant species of the angiosperm family, as well as from white spruce (Picea glauca) (Fig. 5

MKS1
Nucleotide differences were used to employ the MKS2 gene as a DNA HRM marker (Fig. 6A) and to investigate the association between the allelic state in this locus and the 2TD-content variation in the segregating population. The allelic variation in MKS2 was significantly associated with 2TD content (P < 0.0001), and ranked as the second-most contributing factor (after MKS1) among the loci thus far identified in this quantitative analysis (Fig. 6B). Moreover, inclusion of this locus in the multiple regression analysis increased the R 2 of the model from 0.333 to 0.485 (Fig. 6C).
Expression analysis by qRT-PCR showed that MKS2 is 980-fold more highly expressed in the trichomes of the high-MK accumulator PI parent than in those of the M82 parent ( Fig. 6D), similarly to MKS1, ACC and MaCoA-ACP trans (Fig. 4C).
In an attempt to define possible epistatic interactions between the different genetic components of the MK network, the genetic factors that significantly contribute to MK variation in the test population were evaluated for possible two-way interactions. This analysis identified a single significant interaction between the MKS2 and MKS1 loci (Fig.   7A). The data showed that to achieve high levels of these compounds, the plant has to carry at least one wild-species allele in each of these two interacting loci. While  7C).

Heterologous Expression of MKS2 in E. coli
To investigate the biochemical activity of MKS2, the full ORFs of the wildspecies allele (ShMKS2) and the cultivated allele (SlMKS2) were amplified and ligated into an E. coli expression vector (see Materials and Methods). These vectors were introduced into E. coli BL21cells and ShMKS2 or SlMKS2 expression was induced by the addition of IPTG (see Materials and Methods). After induction and overnight growth, the culture was analyzed by solid-phase microextraction (SPME) of its headspace followed by gas chromatography-mass spectrometry (GC-MS; see Materials and Methods). The major compound in the headspace of the E. coli cells expressing ShMKS2 was identified as 2TD (Fig. 8A). Lower amounts of 2UD and 2-pentadecanone were also detected, as well as the reduced alcohol forms of 2UD and 2TD (i.e., 2-undecanol and 2-tridecanol).
The headspace of the E. coli cells expressing SlMKS2 contained 2UD as well as 2nonanone as the two main MK, and only trace amounts of 2TD. The headspace also contained 2-nonanol and 2-undecanol (Fig. 8B). However, the major headspace compound produced by SlMKS2-expressing cells eluted slightly later than 2TD (peak labeled "1" in Fig. 8B, and also present at lower levels in the chromatograph in Fig. 8A).
Mass spectrometry (MS) analysis suggested that it is a 2-tridecenone but the position of the double bond has not yet been determined.

Developmental and Biochemical Connection in MK Synthesis
One of the most surprising findings of this study was the tight relationship between the shape of the trichomes and MK content (Fig. 2). The round and globular trichome shape of the wild species and its progeny was significantly associated with higher MK content. While this observation suggests that morphology constitutes a general barrier to accumulation of volatile compounds, analysis of other volatile compounds in the F2 population did not support this. For example, the distribution of one of the other major volatiles in the glandular trichomes of PI126449, ß-caryophellene, was not correlated with trichome shape. Another possible explanation is that since cuticular waxes are complex mixtures of C20-C34 straight-chain aliphatics derived from verylong-chain fatty acids (Jetter and Kunst, 2008), the diversion of fatty acid pool towards MK comes at the expense of cuticle biosynthesis. This is also supported by the three-way relationship between MK content, trichome shape and the genotype of MKS1 in the segregating population ( Fig. 2 and Fig. 3). However, we cannot reject the possibility that the connection between MKS1 variation and trichome shape might be due to genetic linkage with a gene(s) that modulates the development of this specialized organ. The globular shape of the wild-species trichome may be comparable to the "fused" organ morphology seen in mutants with defective cuticle (Sinha and Lynch, 1998). These in the multiple regression analysis were also differentially expressed in the two species ( Fig. 4C, Fig. 6D), and the F2 population genotyped and phenotyped in this study is relatively large, support the conclusion that a major portion of the MK variation observed in this interspecific population can indeed be attributed to diversity in these genes rather than to other genes that may be in LD with these candidates. Overall, it appears that the flux in MK pathway is controlled at the gene-expression level and the alleles from both species encode almost identical proteins that are likely to be equally active.
Interestingly, the wild-type allele of the gene encoding MaCoA-ACP trans, the enzyme that acts immediately after ACC, was inversely associated with MK content. The relative contribution of this locus to the chemical variation was very low (Fig. 6), and our genetic analysis indicated that the genes encoding these two enzymes are tightly linked

The Role of MKS1 and MKS2 in MK Biosynthesis
The protein with the highest sequence similarity to MKS2 that has established enzymatic activity is 4-hydroxybenzoyl-coenzyme A thioesterase from Pseudomonas sp.
strain CBS3. Indeed, the crystallized 1Z54 and 4HBT crystal structures reveal similar homotetrameric assemblies, which our MKS2 model also reflects (Supplemental data Fig.   S3). The conservation of the 4HBT catalytic Asp17 by ShMKS2 and SlMKS2 suggests that they are likely also thioesterases. Moreover, the production of MK in E. coli cells expressing either allele of MKS2 is a strong indication that the heterologous MKS2 enzyme may be capable of hydrolyzing ( Fig. 9; step I) and perhaps also decarboxylating (Fig 9; step II) 3-ketoacyl intermediates, analogous to the reaction catalyzed by MKS1.
However, the production of MK in E. coli expressing MKS2 is not informative in regard to the specific substrates -3-ketoacyl-ACPs or 3-ketoacyl-CoA -because E. coli cells produce both types of substrates.
Proteins with high levels of identity to tomato MKS2 are found throughout the plant kingdom, but interestingly all such sequences outside Solanaceae contain an Nterminal extension predicted to be a plastid or mitochondrial transit sequence (Fig. 5).
The SlMKS2 and ShMKS2 (and also a petunia MKS2 homolog) lack such a transit peptide, raising the possibility that these Solanaceae proteins are not localized in the plastids and their substrates may therefore not be 3-ketoacyl-ACPs but rather a 3ketoacyl-CoAs. The MKS2 proteins, however, do not contain any other obvious subcellular targeting sequences (e.g., no obvious PTS1 or PTS2 sequences that would target the protein to the peroxisomes).
The presence of two distinct enzymes that contribute to the production of the same compound in the same organ, and even in the same cell, is not unprecedented and such functional redundancy was recently reported for eugenol biosynthesis in Clarkia and Alternatively, epistatic interactions may indicate not a physical interaction but that they act sequentially in the pathway from 3-ketoacyl intermediates to methylketones.
A closer analysis of the genetic data reveals that although the two wild-species alleles in MKS1 and MKS2 loci are required for the accumulation of high 2TD levels in tomato, some levels are nevertheless found in plants that carry only the MKS2 wild allele (ShMKS2), but not vice versa (Fig. 7). These results suggest a model for MK biosynthesis in the trichomes in which MKS2 works upstream of MKS1. By this model, MKS2 hydrolyzes the 3-ketoacyl intermediates ( Fig. 9; step I), and a low level of spontaneous decarboxylation ( Fig. 9; step II) can occur to produce MK (Kornberg 1948), a step that is sped-up by MKS1 when present.
A resolution between these competing hypotheses will require a determination of the subcellular localization of the MKS2 protein, whether it physically interacts with MKS1 and the substrate it acts on, either independently or in complex with MKS1. The results of these experiments may in turn require a reassessment of the subcellular localization of MKS1 as well and its substrate specificity.

CONCLUSIONS
The above results present the complex monophyletic evolution of a specialized pathway, and highlight the power of incorporating morphological and chemical data for a detailed understanding of pathways that appear to be isolated in specialized cells. The combined data provide a framework for determining the molecular and biochemical bases for the unexpected relationships between shape and content of the glandular trichomes.
Moreover, the genetic and biochemical relationship between MKS1 and newly identified MKS2 loci highlights the major role of epistasis interactions in determining phenotypic variation among populations, and emphasizes the importance of taking it into account when dissecting the genetic basis of complex phenotypes.

Volatile Analysis
Six young leaflets (the first, second and third from the first or second leaves) were sampled into scintillation vials on ice and volatiles were extracted and analyzed as described in Fridman et al., (2005).

Morphology Indexes
Six young leaflets (opposite those taken for volatile analysis) were sampled into scintillation vials and a digital photo of the central upper surface was taken. Mean trichome number per square millimeter was calculated and trichome shape was classified as follows: wild shape (PI shape), intermediate shape ( . PCR products were analyzed on an agarose gel (3%) and reactions that produced a single product with no primer dimers were selected for HRM analysis on a Rotor-Gene 6000 (Corbett Research, Sydney, Australia). Primers that showed the best allelic discrimination by HRM examination were selected to score the genotype of the F2 population. HRM was performed immediately after the PCR cycles as a single run following the manufacturer's default parameters (see supplemental data).

Transcriptome Analysis
Trichome isolation was performed as described in

Sequence Analysis
Alignment of multiple protein sequences was performed using the ClustalW program (Thompson et al., 1997).

Statistical Analysis
Statistical analyses were conducted with JMP software (SAS Institute, Cary, NC  interaction, was performed by replacing these two singular factors with a new factor representing the haplotype at those loci. Interactions between genes were tested by twoway ANOVA under the "Fit Model" function.

Isolation of Full-Length ShMKS2 and SlMKS2 cDNAs and expression in E. coli
The following primers were used to amplify the full ORF of MKS2 from PI126449               1, homozygous for the cultivated allele; 2, heterozygous; 3, homozygous for the PI allele.
(C) Accumulated variation explained by the model (R 2 ) with each additional factor.