Eyal Fridman

Characterization of Geraniol Synthase from the Peltate Glands of Sweet Basil

The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn2+ as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 μm were obtained for geranyl diphosphate and Mn2+, respectively. In the presence of 18O-labeled water, GES catalyzed the formation of 18O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.

The Biochemical and Molecular Basis for the Divergent Patterns in the Biosynthesis of Terpenes and Phenylpropenes in the Peltate Glands of Three Cultivars of Basil

Surface glandular trichomes distributed throughout the aerial parts of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles. Three distinct basil chemotypes were used to examine the molecular mechanisms underlying the divergence in their monoterpene and sesquiterpene content. The relative levels of specific terpenes in the glandular trichomes of each cultivar were correlated with the levels of transcripts for eight genes encoding distinct terpene synthases. In a cultivar that produces mostly (R)-linalool, transcripts of (R)-linalool synthase (LIS) were the most abundant of these eight. In a cultivar that synthesizes mostly geraniol, transcripts of geraniol synthase were the most abundant, but the glands of this cultivar also contained a transcript of an (R)-LIS gene with a 1-base insertion that caused a frameshift mutation. A geraniol synthase-LIS hybrid gene was constructed and expressed in Escherichia coli, and the protein catalyzed the formation of both geraniol and (R)-linalool from geranyl diphosphate. The total amounts of terpenes were correlated with total levels of terpene synthase activities, and negatively correlated with levels of phenylpropanoids and phenylalanine ammonia lyase activity. The relative levels of geranyl diphosphate synthase and farnesyl diphosphate synthase activities did not correlate with the total amount of terpenes produced, but showed some correlation with the ratio of monoterpenes to sesquiterpenes.

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

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 β-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.

Reduction of Benzenoid Synthesis in Petunia Flowers Reveals Multiple Pathways to Benzoic Acid and Enhancement in Auxin Transport

In plants, benzoic acid (BA) is believed to be synthesized from Phe through shortening of the propyl side chain by two carbons. It is hypothesized that this chain shortening occurs via either a β-oxidative or non-β-oxidative pathway. Previous in vivo isotope labeling and metabolic flux analysis of the benzenoid network in petunia (Petunia hybrida) flowers revealed that both pathways yield benzenoid compounds and that benzylbenzoate is an intermediate between l-Phe and BA. To test this hypothesis, we generated transgenic petunia plants in which the expression of BPBT, the gene encoding the enzyme that uses benzoyl-CoA and benzyl alcohol to make benzylbenzoate, was reduced or eliminated. Elimination of benzylbenzoate formation decreased the endogenous pool of BA and methylbenzoate emission but increased emission of benzyl alcohol and benzylaldehyde, confirming the contribution of benzylbenzoate to BA formation. Labeling experiments with 2H5-Phe revealed a dilution of isotopic abundance in most measured compounds in the dark, suggesting an alternative pathway from a precursor other than Phe, possibly phenylpyruvate. Suppression of BPBT activity also affected the overall morphology of petunia plants, resulting in larger flowers and leaves, thicker stems, and longer internodes, which was consistent with the increased auxin transport in transgenic plants. This suggests that BPBT is involved in metabolic processes in vegetative tissues as well.

Functional Divergence of a Syntenic Invertase Gene Family in Tomato, Potato, and Arabidopsis

Comparative analysis of complex developmental pathways depends on our ability to resolve the function of members of gene families across taxonomic groups. LIN5, which belongs to a small gene family of apoplastic invertases in tomato (Lycopersicon esculentum), is a quantitative trait locus that modifies fruit sugar composition. We have compared the genomic organization and expression of this gene family in the two distantly related species: tomato and Arabidopsis. Invertase family members reside on segmental duplications in the near-colinear genomes of tomato and potato (Solanum tuberosum). These chromosomal segments are syntenically duplicated in the model plant Arabidopsis. On the basis of phylogenetic analysis of genes in the microsyntenic region, we conclude that these segmental duplications arose independently after the separation of the tomato/potato clade from Arabidopsis. Rapid regulatory divergence is characteristic of the invertase family. Interestingly, although the processes of gene duplication and specialization of expression occurred separately in the two species, synteny-based orthologs from both clades acquired similar organ-specific expression. This similar expression pattern of the genes is evidence of comparable evolutionary constraints (parallel evolution) rather than of functional orthology. The observation that functional orthology cannot be identified through analysis of expression similarity highlights the caution that needs to be exercised in extrapolating developmental networks from a model organism.

Enzymatic Functions of Wild Tomato Methylketone Synthases 1 and 2

The trichomes of the wild tomato species Solanum habrochaites subsp. glabratum synthesize and store high levels of methylketones, primarily 2-tridecanone and 2-undecanone, that protect the plants against various herbivorous insects. Previously, we identified cDNAs encoding two proteins necessary for methylketone biosynthesis, designated methylketone synthase 1 (ShMKS1) and ShMKS2. Here, we report the isolation of genomic sequences encoding ShMKS1 and ShMKS2 as well as the homologous genes from the cultivated tomato, Solanum lycopersicum. We show that a full-length transcript of ShMKS2 encodes a protein that is localized in the plastids. By expressing ShMKS1 and ShMKS2 in Escherichia coli and analyzing the products formed, as well as by performing in vitro assays with both ShMKS1and ShMKS2, we conclude that ShMKS2 acts as a thioesterase hydrolyzing 3-ketoacyl-acyl carrier proteins (plastid-localized intermediates of fatty acid biosynthesis) to release 3-ketoacids and that ShMKS1 subsequently catalyzes the decarboxylation of these liberated 3-ketoacids, forming the methylketone products. Genes encoding proteins with high similarity to ShMKS2, a member of the “hot-dog fold” protein family that is known to include other thioesterases in nonplant organisms, are present in plant species outside the genus Solanum. We show that a related enzyme from Arabidopsis (Arabidopsis thaliana) also produces 3-ketoacids when recombinantly expressed in E. coli. Thus, the thioesterase activity of proteins in this family appears to be ancient. In contrast, the 3-ketoacid decarboxylase activity of ShMKS1, which belongs to the α/β-hydrolase fold superfamily, appears to have emerged more recently, possibly within the genus Solanum.

Strong correlation of wild barley (Hordeum spontaneum) population structure with temperature and precipitation variation.

In this study, we present the genetic analysis of a new collection of wild barley (Hordeum spontaneum) using 42 simple sequence repeat (SSR) markers that represent the seven chromosomes. The Barley1K (B1K) infrastructure consists of 1020 accessions collected in a hierarchical sampling mode (HSM) from 51 sites across Israel and represents the wide adaptive niche of the modern barleys ancestor. According to the genetic structure analysis, the sampled sites can be divided into seven groups, and sampled microsites located on opposing slopes or in different soil types did not show significant genetic differentiation. Although the genetic analysis indicates a simple isolation‐by‐distance model among the populations, examination of the genetic populations’ structure with abiotic parameters in an ordination analysis revealed that the combination of elevation, mid‐day temperature and rainfall explains a high proportion of the variance in the principal components analysis. Our findings demonstrate that the current populations have therefore been shaped and distinguished by non‐selective forces such as migration; however, we suggest that aridity and temperature gradients played major roles as selective forces in the adaptation of wild barley in this part of the Fertile Crescent. This unique collection is a prelude for the investigation of the molecular basis underlying plant adaptation and responsiveness to harsh environments.

The Small Subunit of Snapdragon Geranyl Diphosphate Synthase Modifies the Chain Length Specificity of Tobacco Geranylgeranyl Diphosphate Synthase in Planta

Geranyl diphosphate (GPP), the precursor of many monoterpene end products, is synthesized in plastids by a condensation of dimethylallyl diphosphate and isopentenyl diphosphate (IPP) in a reaction catalyzed by homodimeric or heterodimeric GPP synthase (GPPS). In the heterodimeric enzymes, a noncatalytic small subunit (GPPS.SSU) determines the product specificity of the catalytic large subunit, which may be either an active geranylgeranyl diphosphate synthase (GGPPS) or an inactive GGPPS-like protein. Here, we show that expression of snapdragon (Antirrhinum majus) GPPS.SSU in tobacco (Nicotiana tabacum) plants increased the total GPPS activity and monoterpene emission from leaves and flowers, indicating that the introduced catalytically inactive GPPS.SSU found endogenous large subunit partner(s) and formed an active snapdragon/tobacco GPPS in planta. Bimolecular fluorescence complementation and in vitro enzyme analysis of individual and hybrid proteins revealed that two of four GGPPS-like candidates from tobacco EST databases encode bona fide GGPPS that can interact with snapdragon GPPS.SSU and form a functional GPPS enzyme in plastids. The formation of chimeric GPPS in transgenic plants also resulted in leaf chlorosis, increased light sensitivity, and dwarfism due to decreased levels of chlorophylls, carotenoids, and gibberellins. In addition, these transgenic plants had reduced levels of sesquiterpene emission, suggesting that the export of isoprenoid intermediates from the plastids into the cytosol was decreased. These results provide genetic evidence that GPPS.SSU modifies the chain length specificity of phylogenetically distant GGPPS and can modulate IPP flux distribution between GPP and GGPP synthesis in planta.

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

Genetic analysis of interspecific populations derived from crosses between the wild tomato species Solanum habrochaites f. sp. glabratum, which synthesizes and accumulates insecticidal methylketones (MK), mostly 2-undecanone and 2-tridecanone, in glandular trichomes, and cultivated tomato (Solanum lycopersicum), 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 Synthase1 (MKS1) that mediates conversion to MK of 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 that we named 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 MKS2s 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.

Evolution of Cinnamate/p-Coumarate Carboxyl Methyltransferases and Their Role in the Biosynthesis of Methylcinnamate

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase–like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.