Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms.

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

The trichome-specific acetolactate synthase NtALS1 gene, is involved in acylsugar biosynthesis in tobacco (Nicotiana tabacum L.).

MAIN CONCLUSION: NtALS1 is specifically expressed in glandular trichomes, and can improve the content of acylsugars in tobacco. ABTRACT: The glandular trichomes of many species in the Solanaceae family play an important role in plant defense. These epidermal outgrowths exhibit specialized secondary metabolism, including the production of structurally diverse acylsugars that function in defense against insects and have substantial developmental potential for commercial uses. However, our current understanding of genes involved in acyl chain biosynthesis of acylsugars remains poor in tobacco. In this study, we identified three acetolactate synthase (ALS) genes in tobacco through homology-based gene prediction using Arabidopsis ALS. Quantitative real-time PCR (qRT-PCR) and tissue distribution analyses suggested that NtALS1 was highly expressed in the tips of glandular trichomes. Subcellular localization analysis showed that the NtALS1 localized to the chloroplast. Moreover, in the wild-type K326 variety background, we generated two ntals1 loss-of-function mutants using the CRISPR-Cas9 system. Acylsugars contents in the two ntals1 mutants were significantly lower than those in the wild type. Through phylogenetic tree analysis, we also identified NtALS1 orthologs that may be involved in acylsugar biosynthesis in other Solanaceae species. Taken together, these findings indicate a functional role for NtALS1 in acylsugar biosynthesis in tobacco.

Enzymology of monoterpene functionalization in glandular trichomes.

The plant kingdom supports an extraordinary chemical diversity, with terpenoids representing a particularly diversified class of secondary (or specialized) metabolites. Volatile and semi-volatile terpenoids in the C10-C20 range are often formed in specialized cell types and secretory structures. In the angiosperm lineage, glandular trichomes play an important role in enabling the biosynthesis and storage (or in some cases secretion) of functionalized terpenoids. The 'decoration' of a terpenoid scaffold with functional groups changes its physical and chemical properties, and can therefore affect the perception of a specific metabolite by other organisms. Because of the ecological implications (e.g. plant-herbivore interactions) and commercial relevance (e.g. volatiles used in the flavor and fragrance industries), terpenoid functionalization has been researched extensively. Recent successes in the cloning and functional evaluation of genes as well as the structural and biochemical characterization of enzyme catalysts have laid the foundation for an improved understanding of how pathways toward functionalized monoterpenes may have evolved. In this review, we will focus on an up-to-date account of functionalization reactions present in glandular trichomes.

A Geranylfarnesyl Diphosphate Synthase Provides the Precursor for Sesterterpenoid (C25) Formation in the Glandular Trichomes of the Mint Species Leucosceptrum canum.

Plant sesterterpenoids, an important class of terpenoids, are widely distributed in various plants, including food crops. However, little is known about their biosynthesis. Here, we cloned and functionally characterized a plant geranylfarnesyl diphosphate synthase (Lc-GFDPS), the enzyme producing the C25 prenyl diphosphate precursor to all sesterterpenoids, from the glandular trichomes of the woody plant Leucosceptrum canum. GFDPS catalyzed the formation of GFDP after expression in Escherichia coli. Overexpressing GFDPS in Arabidopsis thaliana also gave an extract catalyzing GFDP formation. GFDPS was strongly expressed in glandular trichomes, and its transcript profile was completely in accordance with the sesterterpenoid accumulation pattern. GFDPS is localized to the plastids, and inhibitor studies indicated its use of isoprenyl diphosphate substrates supplied by the 2-C-methyl-D-erythritol 4-phosphate pathway. Application of a jasmonate defense hormone induced GFDPS transcript and sesterterpenoid accumulation, while reducing feeding and growth of the generalist insect Spodoptera exigua, suggesting that these C25 terpenoids play a defensive role. Phylogenetic analysis suggested that GFDPS probably evolved from plant geranylgeranyl diphosphate synthase under the influence of positive selection. The isolation of GFDPS provides a model for investigating sesterterpenoid formation in other species and a tool for manipulating the formation of this group in plants and other organisms.

In vitro reconstruction and analysis of evolutionary variation of the tomato acylsucrose metabolic network.

Plant glandular secreting trichomes are epidermal protuberances that produce structurally diverse specialized metabolites, including medically important compounds. Trichomes of many plants in the nightshade family (Solanaceae) produce O-acylsugars, and in cultivated and wild tomatoes these are mixtures of aliphatic esters of sucrose and glucose of varying structures and quantities documented to contribute to insect defense. We characterized the first two enzymes of acylsucrose biosynthesis in the cultivated tomato Solanum lycopersicum. These are type I/IV trichome-expressed BAHD acyltransferases encoded by Solyc12g006330--or S. lycopersicum acylsucrose acyltransferase 1 (Sl-ASAT1)--and Solyc04g012020 (Sl-ASAT2). These enzymes were used--in concert with two previously identified BAHD acyltransferases--to reconstruct the entire cultivated tomato acylsucrose biosynthetic pathway in vitro using sucrose and acyl-CoA substrates. Comparative genomics and biochemical analysis of ASAT enzymes were combined with in vitro mutagenesis to identify amino acids that influence CoA ester substrate specificity and contribute to differences in types of acylsucroses that accumulate in cultivated and wild tomato species. This work demonstrates the feasibility of the metabolic engineering of these insecticidal metabolites in plants and microbes.

The subfamily II catalytic subunits of protein phosphatase 2A (PP2A) are involved in cortical microtubule organization.

MAIN CONCLUSION: The subfamily II catalytic subunits of protein phosphatase 2A (PP2A) regulate the cortical microtubule dynamics in Arabidopsis, through interaction with TONNEAU2 (TON2)/FASS and modulation of α-tubulin dephosphorylation. Protein phosphatase 2A is a major protein phosphatase in eukaryotes that dephosphorylates many different substrates to regulate their function. PP2A is assembled into a heterotrimeric complex of scaffolding A subunit, regulatory B subunit, and catalytic C subunit. Plant PP2A catalytic C subunit (PP2AC) isoforms are classified into two subfamilies. In this study, we investigated the cellular functions of the Arabidopsis PP2AC subfamily II genes PP2AC-3 and PP2AC-4, particularly regarding the cortical microtubule (MT) organization. PP2AC-3 and PP2AC-4 strongly interacted with the B'' regulatory subunit TON2. Simultaneous silencing of PP2AC-3 and PP2AC-4 by virus-induced gene silencing (PP2AC-3,4 VIGS) significantly altered plant morphology in Arabidopsis, increasing cell numbers in leaves and stems. The leaf epidermis of PP2AC-3,4 VIGS plants largely lost its jigsaw-puzzle shape and exhibited reduced trichome branch numbers. VIGS of PP2AC-3,4 in Arabidopsis transgenic plants that expressed GFP-fused ß-tubulin 6 isoform (GFP-TUB6) for the visualization of MTs caused a reduction in the cortical MT array density in the pavement cells. VIGS of TON2 also led to similar cellular phenotypes and cortical MT patterns compared with those after VIGS of PP2AC-3,4, suggesting that PP2AC-3,4 and their interaction partner TON2 play a role in cortical MT organization in leaf epidermal cells. Furthermore, silencing of PP2AC-3,4 did not affect salt-induced phosphorylation of α-tubulin but delayed its dephosphorylation after salt removal. The reappearance of cortical MT arrays after salt removal was impaired in PP2AC-3,4 VIGS plants. These results suggest an involvement of PP2AC subfamily II in the regulation of cortical MT dynamics under normal and salt-stress conditions in Arabidopsis.

Photosynthetic Trichomes Contain a Specific Rubisco with a Modified pH-Dependent Activity.

Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme in plants and is responsible for CO2 fixation during photosynthesis. This enzyme is assembled from eight large subunits (RbcL) encoded by a single chloroplast gene and eight small subunits (RbcS) encoded by a nuclear gene family. Rubisco is primarily found in the chloroplasts of mesophyll (C3 plants), bundle-sheath (C4 plants), and guard cells. In certain species, photosynthesis also takes place in the secretory cells of glandular trichomes, which are epidermal outgrowths (hairs) involved in the secretion of specialized metabolites. However, photosynthesis and, in particular, Rubisco have not been characterized in trichomes. Here, we show that tobacco (Nicotiana tabacum) trichomes contain a specific Rubisco small subunit, NtRbcS-T, which belongs to an uncharacterized phylogenetic cluster (T). This cluster contains RbcS from at least 33 species, including monocots, many of which are known to possess glandular trichomes. Cluster T is distinct from the cluster M, which includes the abundant, functionally characterized RbcS isoforms expressed in mesophyll or bundle-sheath cells. Expression of NtRbcS-T in Chlamydomonas reinhardtii and purification of the full Rubisco complex showed that this isoform conferred higher Vmax and Km values as well as higher acidic pH-dependent activity than NtRbcS-M, an isoform expressed in the mesophyll. This observation was confirmed with trichome extracts. These data show that an ancient divergence allowed for the emergence of a so-far-uncharacterized RbcS cluster. We propose that secretory trichomes have a particular Rubisco uniquely adapted to secretory cells where CO2 is released by the active specialized metabolism.

Comparative glandular trichome transcriptome-based gene characterization reveals reasons for differential (-)-menthol biosynthesis in Mentha species.

The genes involved in menthol biosynthesis are reported earlier in Mentha × piperita. But the information on these genes is not available in Mentha arvensis. To bridge the gap in knowledge on differential biosynthesis of monoterpenes leading to compositional variation in the essential oil of these species, a comparative transcriptome analysis of the glandular trichome (GT) was carried out. In addition to the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathway genes, about 210 and 196 different terpene synthases (TPSs) transcripts were identified from annotation in M. arvensis and M. × piperita, respectively, and correlated to several monoterpenes present in the essential oil. Six isoforms of (-)-menthol dehydrogenases (MD), the last enzyme of the menthol biosynthetic pathway, were identified, cloned and characterized from the transcriptome data (three from each species). Varied expression levels and differential enzyme kinetics of these isoforms indicated the nature and composition of the product, as these isoforms generate both (-)-menthol and (+)-neomenthol from (-)-menthone and converts (-)-menthol to (-)-menthone in the reverse reaction, and hence together determine the quantity of (-)-menthol in the essential oil in these two species. Several genes for high value minor monoterpenes could also be identified from the transcriptome data.

A Feedback-Insensitive Isopropylmalate Synthase Affects Acylsugar Composition in Cultivated and Wild Tomato.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

Unexpected roles for ancient proteins: flavone 8-hydroxylase in sweet basil trichomes is a Rieske-type, PAO-family oxygenase.

Most elucidated hydroxylations in plant secondary metabolism are catalyzed by oxoglutarate- or cytochrome P450-dependent oxygenases. Numerous hydroxylations still evade clarification, suggesting that they might be performed by alternative enzyme types. Here, we report the identification of the flavone 8-hydroxylase (F8H) in sweet basil (Ocimum basilicum L.) trichomes as a Rieske-type oxygenase. Several features of the F8H activity in trichome protein extracts helped to differentiate it from a cytochrome P450-catalyzed reaction and identify candidate genes in the basil trichome EST database. The encoded ObF8H proteins share approximately 50% identity with Rieske-type protochlorophyllide a oxygenases (PTC52) from higher plants. Homology cloning and DNA blotting revealed the presence of several PTC52-like genes in the basil genome. The transcripts of the candidate gene designated ObF8H-1 are strongly enriched in trichomes compared to whole young leaves, indicating trichome-specific expression. The full-length ObF8H-1 protein possesses a predicted N-terminal transit peptide, which directs green fluorescent protein at least in part to chloroplasts. The F8H activity in crude trichome protein extracts correlates well with the abundance of ObF8H peptides. The purified recombinant ObF8H-1 displays high affinity for salvigenin and is inactive with other tested flavones except cirsimaritin, which is 8-hydroxylated with less than 0.2% relative activity. The efficiency of in vivo 8-hydroxylation by engineered yeast was improved by manipulation of protein subcellular targeting. blast searches showed that occurrence of several PTC52-like genes is rather common in sequenced plant genomes. The discovery of ObF8H suggests that Rieske-type oxygenases may represent overlooked candidate catalysts for oxygenations in specialized plant metabolism.

Identification of a unique 2-oxoglutarate-dependent flavone 7-O-demethylase completes the elucidation of the lipophilic flavone network in basil.

Small molecule demethylation is considered unusual in plants. Of the studied instances, the N-demethylation of nicotine is catalyzed by a Cyt P450 monooxygenase, while the O-dealkylation of alkaloids in Papaver somniferum is mediated by 2-oxoglutarate-dependent dioxygenases (2-ODDs). This report describes a 2-ODD regiospecifically catalyzing the 7-O-demethylation of methoxylated flavones in peltate trichomes of sweet basil (Ocimum basilicum L.). Three candidate 2-ODDs were identified in the basil trichome transcriptome database. Only the candidate designated ObF7ODM1 was found to be active with and highly specific for the proposed natural substrates, gardenin B and 8-hydroxysalvigenin. Of the characterized 2-ODDs, ObF7ODM1 is most closely related to O-demethylases from Papaver. The demethylase activity in trichomes from four basil chemotypes matches well with the abundance of ObF7ODM1 peptides and transcripts in the same trichome preparations. Treatment of basil plants with a 2-ODD inhibitor prohexadione-calcium significantly reduced the accumulation of 7-O-demethylated flavone nevadensin, confirming the involvement of a 2-ODD in its formation. Notably, the full-length open reading frame of ObF7ODM1 contains a second in-frame AUG codon 57 nucleotides downstream of the first translation initiation codon. Both AUG codons are recognized by bacterial translation machinery during heterologous gene expression. The N-truncated ObF7ODM1 is nearly inactive. The N-terminus essential for activity is unique to ObF7ODM1 and does not align with the sequences of other 2-ODDs. Further studies will reveal whether alternative translation initiation plays a role in regulating the O-demethylase activity in planta. Molecular identification of the flavone 7-O-demethylase completes the biochemical elucidation of the lipophilic flavone network in basil.

Cloning and functional characterization of two monoterpene acetyltransferases from glandular trichomes of L. x intermedia.

MAIN CONCLUSION: Two alcohol acetyltransferases, LiAAT-3 and LiAAT-4, from L. x intermedia were cloned, expressed in bacteria, and functionally characterized. Two monoterpene acetyltransferase cDNA clones (LiAAT-3 and LiAAT-4) were isolated from L. x intermedia glandular trichomes, expressed in bacteria to produce, and functionally characterize the encoded proteins in vitro. The recombinant LiAAT-3 and LiAAT-4 proteins had molecular weights of ca. 47 and 49 kDa, respectively, as evidenced by SDS-PAGE. The K m (mM) values for the recombinant LiAAT-3 and LiAAT-4 were 1.046 and 0.354 for lavandulol, 1.31 and 0.279 for geraniol, and 0.87 and 0.113 for nerol, respectively. The V max (pkat/mg) values for LiAAT-3 and LiAAT-4 were 92.13 and 105.1 for lavandulol, 81.07 and 52.17 for geraniol, and 15.02 and 15.8 for nerol, correspondingly. Catalytic efficiencies (mM(-1) min(-1)) for LiAAT-3 and LiAAT-4 were 0.27 and 0.85 for lavandulol, 0.19 and 0.54 for geraniol, and 0.052 and 0.4 for nerol, respectively. These kinetic properties are in the range of those reported for other plant acetyltransferases, and indicate that LiAAT-4 has a better catalytic efficiency than LiAAT-3, with lavandulol serving as the preferred substrate for both enzymes. Transcripts for both genes were abundant in L. angustifolia and L. x intermedia flowers, where monoterpene acetates are produced, and were undetectable (or present in trace quantities) in L. latifolia flowers, which do not accumulate significant amounts of these metabolites.

Determination of residues responsible for substrate and product specificity of Solanum habrochaites short-chain cis-prenyltransferases.

Isoprenoids are diverse compounds that have their biosynthetic origin in the initial condensation of isopentenyl diphosphate and dimethylallyl diphosphate to form C10 prenyl diphosphates that can be elongated by the addition of subsequent isopentenyl diphosphate units. These reactions are catalyzed by either cis-prenyltransferases (CPTs) or trans-prenyltransferases. The synthesis of volatile terpenes in plants typically proceeds through either geranyl diphosphate (C10) or trans-farnesyl diphosphate (C15), to yield monoterpenes and sesquiterpenes, respectively. However, terpene biosynthesis in glandular trichomes of tomato (Solanum lycopersicum) and related wild relatives also occurs via the cis-substrates neryl diphosphate (NPP) and 2Z,6Z-farnesyl diphosphate (Z,Z-FPP). NPP and Z,Z-FPP are synthesized by neryl diphosphate synthase1 (NDPS1) and Z,Z-farnesyl diphosphate synthase (zFPS), which are encoded by the orthologous CPT1 locus in tomato and Solanum habrochaites, respectively. In this study, comparative sequence analysis of NDPS1 and zFPS enzymes from S. habrochaites accessions that synthesize either monoterpenes or sesquiterpenes was performed to identify amino acid residues that correlate with the ability to synthesize NPP or Z,Z-FPP. Subsequent structural modeling, coupled with site-directed mutagenesis, highlighted the importance of four amino acids located within conserved domain II of CPT enzymes that form part of the second α-helix, for determining substrate and product specificity of these enzymes. In particular, the relative positioning of aromatic amino acid residues at positions 100 and 107 determines the ability of these enzymes to synthesize NPP or Z,Z-FPP. This study provides insight into the biochemical evolution of terpene biosynthesis in the glandular trichomes of Solanum species.

Cloning of a sesquiterpene synthase from Lavandula x intermedia glandular trichomes.

The essential oil (EO) of Lavandula is dominated by monoterpenes, but can also contain small amounts of sesquiterpenes, depending on species and environmental conditions. For example, the sesquiterpene 9-epi-caryophyllene can make up to 8 % of the EO in a few species, including those commercially propagated for EO production. Here, we report the cloning and functional characterization of 9-epi-caryophyllene synthase (LiCPS) from the glandular trichomes of Lavandula x intermedia, cv. Grosso. The 1,617 bp open reading frame of LiCPS, which did not encode a transit peptide, was expressed in Escherichia coli and the recombinant protein purified by Ni-NTA agarose affinity chromatography. The ca. 60 kDa recombinant protein specifically converted farnesyl diphosphate to 9-epi-caryophyllene. LiCPS also produced a few monoterpenes when assayed with the monoterpene precursor geranyl diphosphate (GPP), but--unlike most monoterpene synthases--was not able to derive detectable amounts of any products from the cis isomer of GPP, neryl diphosphate. The LiCPS transcripts accumulated in developing L. x intermedia flowers and were highly enriched in glandular trichomes, but were not detected in leaves suggesting that the transcriptional expression of this gene is spatially and developmentally regulated.

Characterization of a sesquiterpene cyclase from the glandular trichomes of Leucosceptrum canum for sole production of cedrol in Escherichia coli and Nicotiana benthamiana.

Cedrol is an extremely versatile sesquiterpene alcohol that was approved by the Food and Drug Administration of the United States as a flavoring agent or adjuvant and has been commonly used as a flavoring ingredient in cosmetics, foods and medicine. Furthermore, cedrol possesses a wide range of pharmacological properties including sedative, anti-inflammatory and cytotoxic activities. Commercial production of cedrol relies on fractional distillation of cedar wood oils, followed by recrystallization, and little has been reported about its biosynthesis and aspects of synthetic biology. Here, we report the cloning and functional characterization of a cedrol synthase gene (Lc-CedS) from the transcriptome of the glandular trichomes of a woody Lamiaceae plant Leucosceptrum canum. The recombinant Lc-CedS protein catalyzed the in vitro conversion of farnesyl diphosphate into the single product cedrol, suggesting that Lc-CedS is a high-fidelity terpene synthase. Co-expression of Lc-CedS, a farnesyl diphosphate synthase gene and seven genes of the mevalonate (MVA) pathway responsible for converting acetyl-CoA into farnesyl diphosphate in Escherichia coli afforded 363 µg/L cedrol as the sole product under shaking flask conditions. Transient expression of Lc-CedS in Nicotiana benthamiana also resulted in a single product cedrol with a production level of 3.6 µg/g fresh weight. The sole production of cedrol by introducing of Lc-CedS in engineered E. coli and N. benthamiana suggests now alternative production systems using synthetic biology approaches that would better address sufficient supply of cedrol.

Analyzing trichomes and spatio-temporal expression of a cysteine protease gene Mucunain in Mucuna pruriens L. (DC).

Mucuna pruriens is a well-known legume for the itching attributes of the trichome and a valuable medicinal herb that is used for the treatment of Parkinson's disease, sexual debilities, etc. Its cultivation was deprived due to its itching behavior. The wild genotype of M. pruriens have the largest trichome length (2015 ± 29 µm) compared to other genotype and mutants. The white-seeded variety of M. pruriens was found to be the most suitable for large-scale cultivation due to the small trichome size and less trichome density on the pod. The external surface trichomes have protuberance with unknown function. The unicellular trichomes of Mucuna show the flowing fluid or cytoplasm inside the trichome. The unigenes regulating the differentiation and development of the trichome such as GLABRA-1, GLABRA-2, and cpr-5 have been identified in M. pruriens transcriptome of the leaf. The Mucunain shows a higher transcript abundance in the flower and pod cover compared to the seeds. The Mucunain was found in every stage of plant growth, but it was highly expressed during maturity (about 170 days) with a high fragment per kilobase per million value.

Evolution of a flipped pathway creates metabolic innovation in tomato trichomes through BAHD enzyme promiscuity.

Plants produce hundreds of thousands of structurally diverse specialized metabolites via multistep biosynthetic networks, including compounds of ecological and therapeutic importance. These pathways are restricted to specific plant groups, and are excellent systems for understanding metabolic evolution. Tomato and other plants in the nightshade family synthesize protective acylated sugars in the tip cells of glandular trichomes on stems and leaves. We describe a metabolic innovation in wild tomato species that contributes to acylsucrose structural diversity. A small number of amino acid changes in two acylsucrose acyltransferases alter their acyl acceptor preferences, resulting in reversal of their order of reaction and increased product diversity. This study demonstrates how small numbers of amino acid changes in multiple pathway enzymes can lead to diversification of specialized metabolites in plants. It also highlights the power of a combined genetic, genomic and in vitro biochemical approach to identify the evolutionary mechanisms leading to metabolic novelty.

Evolutionary routes to biochemical innovation revealed by integrative analysis of a plant-defense related specialized metabolic pathway.

The diversity of life on Earth is a result of continual innovations in molecular networks influencing morphology and physiology. Plant specialized metabolism produces hundreds of thousands of compounds, offering striking examples of these innovations. To understand how this novelty is generated, we investigated the evolution of the Solanaceae family-specific, trichome-localized acylsugar biosynthetic pathway using a combination of mass spectrometry, RNA-seq, enzyme assays, RNAi and phylogenomics in different non-model species. Our results reveal hundreds of acylsugars produced across the Solanaceae family and even within a single plant, built on simple sugar cores. The relatively short biosynthetic pathway experienced repeated cycles of innovation over the last 100 million years that include gene duplication and divergence, gene loss, evolution of substrate preference and promiscuity. This study provides mechanistic insights into the emergence of plant chemical novelty, and offers a template for investigating the ~300,000 non-model plant species that remain underexplored.

Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.

A trichome-specific linoleate lipoxygenase expressed during pyrethrin biosynthesis in pyrethrum.

The lipid precursor alcohols of pyrethrins-jasmolone, pyrethrolone and cinerolone-have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases, TcLOX1 and TcLOX2, that have a similar pattern to TcCDS. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as TcCDS, TcLOX1, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.