Biochemistry and genetics of floral scent: a historical perspective.

Floral scent plays a crucial role in the reproductive process of many plants. Humans have been fascinated by floral scents throughout history, and have transported and traded floral scent products for which they have found multiple uses, such as in food additives, hygiene and perfume products, and medicines. Yet the scientific study of how plants synthesize floral scent compounds began later than studies on most other major plant metabolites, and the first report of the characterization of an enzyme responsible for the synthesis of a floral scent compound, namely linalool in Clarkia breweri, a California annual, appeared in 1994. In the almost 30 years since, enzymes and genes involved in the synthesis of hundreds of scent compounds from multiple plant species have been described. This review recapitulates this history and describes the major findings relating to the various aspects of floral scent biosynthesis and emission, including genes and enzymes and their evolution, storage and emission of scent volatiles, and the regulation of the biochemical processes.

Many different flowers make a bouquet: Lessons from specialized metabolite diversity in plant-pollinator interactions.

Flowering plants have evolved extraordinarily diverse metabolites that underpin the floral visual and olfactory signals enabling plant-pollinator interactions. In some cases, these metabolites also provide unusual rewards that specific pollinators depend on. While some metabolites are shared by most flowering plants, many have evolved in restricted lineages in response to the specific selection pressures encountered within different niches. The latter are designated as specialized metabolites. Recent investigations continue to uncover a growing repertoire of unusual specialized metabolites. Increased accessibility to cutting-edge multi-omics technologies (e.g. genome, transcriptome, proteome, metabolome) is now opening new doors to simultaneously uncover the molecular basis of their synthesis and their evolution across diverse plant lineages. Drawing upon the recent literature, this perspective discusses these aspects and, where known, their ecological and evolutionary relevance. A primer on omics-guided approaches to discover the genetic and biochemical basis of functional specialized metabolites is also provided.

Synthesis of 4-methylvaleric acid, a precursor of pogostone, involves a 2-isobutylmalate synthase related to 2-isopropylmalate synthase of leucine biosynthesis.

We show here that the side chain of pogostone, one of the major components of patchouli oil obtained from Pogostemon cablin and possessing a variety of pharmacological activities, is derived from 4-methylvaleric acid. We also show that 4-methylvaleric acid is produced through the one-carbon α-ketoacid elongation pathway with the involvement of the key enzyme 2-isobutylmalate synthase (IBMS), a newly identified enzyme related to isopropylmalate synthase (IPMS) of leucine (Leu) biosynthesis. Site-directed mutagenesis identified Met132 in the N-terminal catalytic region as affecting the substrate specificity of PcIBMS1. Even though PcIBMS1 possesses the C-terminal domain that in IPMS serves to mediate Leu inhibition, it is insensitive to Leu. The observation of the evolution of IBMS from IPMS, as well as previously reported examples of IPMS-related genes involved in making glucosinolates in Brassicaceae, acylsugars in Solanaceae, and flavour compounds in apple, indicate that IPMS genes represent an important pool for the independent evolution of genes for specialised metabolism.

Engineering of tomato type VI glandular trichomes for trans-chrysanthemic acid biosynthesis, the acid moiety of natural pyrethrin insecticides.

Glandular trichomes, known as metabolic cell factories, have been proposed as highly suitable for metabolically engineering the production of plant high-value specialized metabolites. Natural pyrethrins, found only in Dalmatian pyrethrum (Tanacetum cinerariifolium), are insecticides with low mammalian toxicity and short environmental persistence. Type I pyrethrins are esters of the monoterpenoid trans-chrysanthemic acid with one of the three rethrolone-type alcohols. To test if glandular trichomes can be made to synthesize trans-chrysanthemic acid, we reconstructed its biosynthetic pathway in tomato type VI glandular trichomes, which produce large amounts of terpenoids that share the precursor dimethylallyl diphosphate (DMAPP) with this acid. This was achieved by coexpressing the trans-chrysanthemic acid pathway related genes including TcCDS encoding chrysanthemyl diphosphate synthase and the fusion gene of TcADH2 encoding the alcohol dehydrogenase 2 linked with TcALDH1 encoding the aldehyde dehydrogenase 1 under the control of a newly identified type VI glandular trichome-specific metallocarboxypeptidase inhibitor promoter. Whole tomato leaves harboring type VI glandular trichomes expressing all three aformentioned genes had a concentration of total trans-chrysanthemic acid that was about 1.5-fold higher (by mole number) than the levels of ß-phellandrene, the dominant monoterpene present in non-transgenic leaves, while the levels of ß-phellandrene and the representative sesquiterpene ß-caryophyllene in transgenic leaves were reduced by 96% and 81%, respectively. These results suggest that the tomato type VI glandular trichome is an alternative platform for the biosynthesis of trans-chrysanthemic acid by metabolic engineering.

Deep roots and many branches: Origins of plant-specialized metabolic enzymes in general metabolism.

Collectively, plants produce hundreds of thousands of specialized metabolites from simple building blocks such as amino acids, fatty acids, and isoprenoids. As additional specialized metabolic enzymes are described, there is increasing recognition of the importance of cooption of general metabolic enzymes to specialized metabolism by gene duplication, narrowing of expression, and alteration of enzymatic activities. Here, we examine how several classes of enzymes were each coopted multiple times. We demonstrate the simplicity of achieving the synthesis of analogous chemicals by coopting existing enzymes and summarize emerging insights that could inform rational metabolic engineering of both general and specialized metabolic enzymes.

Overcoming Bottlenecks for Metabolic Engineering of Sesquiterpene Production in Tomato Fruits.

Terpenoids are a large and diverse class of plant metabolites that also includes volatile mono- and sesquiterpenes which are involved in biotic interactions of plants. Due to the limited natural availability of these terpenes and the tight regulation of their biosynthesis, there is strong interest to introduce or enhance their production in crop plants by metabolic engineering for agricultural, pharmaceutical and industrial applications. While engineering of monoterpenes has been quite successful, expression of sesquiterpene synthases in engineered plants frequently resulted in production of only minor amounts of sesquiterpenes. To identify bottlenecks for sesquiterpene engineering in plants, we have used two nearly identical terpene synthases, snapdragon (Antirrhinum majus) nerolidol/linalool synthase-1 and -2 (AmNES/LIS-1/-2), that are localized in the cytosol and plastids, respectively. Since these two bifunctional terpene synthases have very similar catalytic properties with geranyl diphosphate (GPP) and farnesyl diphosphate (FPP), their expression in target tissues allows indirect determination of the availability of these substrates in both subcellular compartments. Both terpene synthases were expressed under control of the ripening specific PG promoter in tomato fruits, which are characterized by a highly active terpenoid metabolism providing precursors for carotenoid biosynthesis. As AmNES/LIS-2 fruits produced the monoterpene linalool, AmNES/LIS-1 fruits were found to exclusively produce the sesquiterpene nerolidol. While nerolidol emission in AmNES/LIS-1 fruits was 60- to 584-fold lower compared to linalool emission in AmNES/LIS-2 fruits, accumulation of nerolidol-glucosides in AmNES/LIS-1 fruits was 4- to 14-fold lower than that of linalool-glucosides in AmNES/LIS-2 fruits. These results suggest that only a relatively small pool of FPP is available for sesquiterpene formation in the cytosol. To potentially overcome limitations in sesquiterpene production, we transiently co-expressed the key pathway-enzymes hydroxymethylglutaryl-CoA reductase (HMGR) and 1-deoxy-D-xylulose 5-phosphate synthase (DXS), as well as the regulator isopentenyl phosphate kinase (IPK). While HMGR and IPK expression increased metabolic flux toward nerolidol formation 5.7- and 2.9-fold, respectively, DXS expression only resulted in a 2.5-fold increase.

Characterization of a Cytosolic Acyl-Activating Enzyme Catalyzing the Formation of 4-Methylvaleryl-CoA for Pogostone Biosynthesis in Pogostemon Cablin.

Pogostone, a compound with various pharmaceutical activities, is a major constituent of the essential oil preparation called Pogostemonis Herba, which is obtained from the plant Pogostemon cablin. The biosynthesis of pogostone has not been elucidated, but 4-methylvaleryl-CoA (4MVCoA) is a likely precursor. We analyzed the distribution of pogostone in P. cablin using gas chromatography-mass spectrometry (GC-MS) and found that pogostone accumulates at high levels in the main stems and leaves of young plants. A search for the acyl-activating enzyme (AAE) that catalyzes the formation of 4MVCoA from 4-methylvaleric acid was launched, using an RNAseq-based approach to identify 31 unigenes encoding putative AAEs including the PcAAE2, the transcript profile of which shows a strong positive correlation with the distribution pattern of pogostone. The protein encoded by PcAAE2 was biochemically characterized in vitro and shown to catalyze the formation of 4MVCoA from 4-methylvaleric acid. Phylogenetic analysis showed that PcAAE2 is closely related to other AAE proteins in P. cablin and other species that are localized to the peroxisomes. However, PcAAE2 lacks a peroxisome targeting sequence 1 (PTS1) and is localized in the cytosol.

Degradation of salicylic acid to catechol in Solanaceae by SA 1-hydroxylase.

The hormone salicylic acid (SA) plays crucial roles in plant defense, stress responses, and in the regulation of plant growth and development. Whereas the biosynthetic pathways and biological functions of SA have been extensively studied, SA catabolism is less well understood. In this study, we report the identification and functional characterization of an FAD/NADH-dependent SA 1-hydroxylase from tomato (Solanum lycopersicum; SlSA1H), which catalyzes the oxidative decarboxylation of SA to catechol. Transcript levels of SlSA1H were highest in stems and its expression was correlated with the formation of the methylated catechol derivatives guaiacol and veratrole. Consistent with a role in SA catabolism, SlSA1H RNAi plants accumulated lower amounts of guaiacol and failed to produce any veratrole. Two O-methyltransferases involved in the conversion of catechol to guaiacol and guaiacol to veratrole were also functionally characterized. Subcellular localization analyses revealed the cytosolic localization of this degradation pathway. Phylogenetic analysis and functional characterization of SA1H homologs from other species indicated that this type of FAD/NADH-dependent SA 1-hydroxylases evolved recently within the Solanaceae family.

How Plants Synthesize Pyrethrins: Safe and Biodegradable Insecticides.

Natural pyrethrin insecticides produced by Dalmatian pyrethrum (Tanacetum cinerariifolium) have low mammalian toxicity and short environmental persistence, providing an alternative to widely used synthetic agricultural insecticides that pose a threat to human health and the environment. A recent surge of interest in the use of pyrethrins as agricultural insecticides coincides with the discovery of several new genes in the pyrethrin biosynthetic pathway. Elucidation of this pathway facilitates efforts to breed improved pyrethrum varieties and to engineer plants with improved endogenous defenses or hosts for heterologous pyrethrin production. We describe the current state of knowledge related to global pyrethrum production, the pyrethrin biosynthetic pathway and its regulation, and recent efforts to engineer the pyrethrin pathway in diverse plant hosts.

A Trichome-Specific, Plastid-Localized Tanacetum cinerariifolium Nudix Protein Hydrolyzes the Natural Pyrethrin Pesticide Biosynthetic Intermediate trans-Chrysanthemyl Diphosphate.

Tanacetum cinerariifolium flowers synthesize six pyrethrins that function as effective insecticides. trans-Chrysanthemol is an early intermediate in the synthesis of the monoterpene moiety of pyrethrins. Previously, the pyrethrum enzyme chrysanthemyl diphosphate synthase (TcCDS) was shown to catalyze the formation of the prenyl diphosphate compound chrysanthemyl diphosphate (CPP) by condensing two molecules of dimethylallyl diphosphate (DMAPP). Later work also showed that with a low concentration of DMAPP, TcCDS can also remove the diphosphate group to give chrysanthemol. The removal of the phosphate groups from other prenyl diphosphates, such as DMAPP, isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP), was previously shown to occur in two steps. In those cases, the first phosphate group is removed by a member of the Nudix hydrolase protein family, and the second by other unidentified phosphatases. These previously characterized Nudix proteins involved in the hydrolysis of prenyl diphosphates were shown to be cytosolic. Here we report that a plastidic Nudix protein from pyrethrum, designated TcNudix1, has high specificity for CPP and can hydrolyze it to chrysanthemol monophosphate (CMP). TcNudix1 is expressed specifically in the trichomes of the ovaries, where chrysanthemol is produced. TcNudix1 expression patterns and pathway reconstitution experiments presented here implicate the TcNudix1 protein in the biosynthesis of chrysanthemol.

More is better: the diversity of terpene metabolism in plants.

All plants synthesize a diverse array of terpenoid metabolites. Some are common to all, but many are synthesized only in specific taxa and presumably evolved as adaptations to specific ecological conditions. While the basic terpenoid biosynthetic pathways are common in all plants, recent discoveries have revealed many variations in the way plants synthesized specific terpenes. A major theme is the much greater number of substrates that can be used by enzymes belonging to the terpene synthase (TPS) family. Other recent discoveries include non-TPS enzymes that catalyze the formation of terpenes, and novel transport mechanisms.

The complete functional characterisation of the terpene synthase family in tomato.

Analysis of the updated reference tomato genome found 34 full-length TPS genes and 18 TPS pseudogenes. Biochemical analysis has now identified the catalytic activities of all enzymes encoded by the 34 TPS genes: one isoprene synthase, 10 exclusively or predominantly monoterpene synthases, 17 sesquiterpene synthases and six diterpene synthases. Among the monoterpene and sesquiterpene and diterpene synthases, some use trans-prenyl diphosphates, some use cis-prenyl diphosphates and some use both. The isoprene synthase is cytosolic; six monoterpene synthases are plastidic, and four are cytosolic; the sesquiterpene synthases are almost all cytosolic, with the exception of one found in the mitochondria; and three diterpene synthases are found in the plastids, one in the cytosol and two in the mitochondria. New trans-prenyltransferases (TPTs) were characterised; together with previously characterised TPTs and cis-prenyltransferases (CPTs), tomato plants can make all cis and trans C10 , C15 and C20 prenyl diphosphates. Every type of plant tissue examined expresses some TPS genes and some TPTs and CPTs. Phylogenetic comparison of the TPS genes from tomato and Arabidopsis shows expansions in each clade of the TPS gene family in each lineage (and inferred losses), accompanied by changes in subcellular localisations and substrate specificities.

Pyrethrin Biosynthesis: The Cytochrome P450 Oxidoreductase CYP82Q3 Converts Jasmolone To Pyrethrolone.

The plant pyrethrum (Tanacetum cinerariifolium) synthesizes highly effective natural pesticides known as pyrethrins. Pyrethrins are esters consisting of an irregular monoterpenoid acid and an alcohol derived from jasmonic acid (JA). These alcohols, referred to as rethrolones, can be jasmolone, pyrethrolone, or cinerolone. We recently showed that jasmolone is synthesized from jasmone, a degradation product of JA, in a single hydroxylation step catalyzed by jasmone hydroxylase (TcJMH). TcJMH belongs to the CYP71 clade of the cytochrome P450 oxidoreductase family. Here, we used coexpression analysis, heterologous gene expression, and in vitro biochemical assays to identify the enzyme responsible for conversion of jasmolone to pyrethrolone. A further T cinerariifolium cytochrome P450 family member, CYP82Q3 (designated Pyrethrolone Synthase; TcPYS), appeared to catalyze the direct desaturation of the C1-C2 bond in the pentyl side chain of jasmolone to produce pyrethrolone. TcPYS is highly expressed in the trichomes of the ovaries in pyrethrum flowers, similar to TcJMH and other T cinerariifolium genes involved in JA biosynthesis. Thus, as previously shown for biosynthesis of the monoterpenoid acid moiety of pyrethrins, rethrolones are synthesized in the trichomes. However, the final assembly of pyrethrins occurs in the developing achenes. Our data provide further insight into pyrethrin biosynthesis, which could ultimately be harnessed to produce this natural pesticide in a heterologous system.

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.

In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N. benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

Duplication and selection in ß-ketoacyl-ACP synthase gene lineages in the sexually deceptive Chiloglottis (Orchidaceace).

BACKGROUND AND AIMS: The processes of gene duplication, followed by divergence and selection, probably underpin the evolution of floral volatiles crucial to plant-insect interactions. The Australian sexually deceptive Chiloglottis orchids use a class of 2,5-dialkylcyclohexan-1,3-dione volatiles or 'chiloglottones' to attract specific male wasp pollinators. Here, we explore the expression and evolution of fatty acid pathway genes implicated in chiloglottone biosynthesis. METHODS: Both Chiloglottis seminuda and C. trapeziformis produce chiloglottone 1, but only the phylogenetically distinct C. seminuda produces this volatile from both the labellum callus and glandular sepal tips. Transcriptome sequencing and tissue-specific contrasts of the active and non-active floral tissues was performed. The effects of the fatty acid synthase inhibitor cerulenin on chiloglottone production were tested. Patterns of selection and gene evolution were investigated for fatty acid pathway genes. KEY RESULTS: Tissue-specific differential expression of fatty acid pathway transcripts was evident between active and non-active floral tissues. Cerulenin significantly inhibits chiloglottone 1 production in the active tissues of C. seminuda. Phylogenetic analysis of plant ß-ketoacyl synthase I (KASI), a protein involved in fatty acid biosynthesis, revealed two distinct clades, one of which is unique to the Orchidaceae (KASI-2B). Selection analysis indicated a strong signal of positive selection at the split of KASI-2B followed by relaxed purifying selection in the Chiloglottis clade. CONCLUSIONS: By capitalizing on a phylogenetically distinct Chiloglottis from earlier studies, we show that the transcriptional and biochemical dynamics linked to chiloglottone biosynthesis in active tissues are conserved across Chiloglottis. A combination of tissue-specific expression and relaxed purifying selection operating at specific fatty acid pathway genes may hold the key to the evolution of chiloglottones.

Robust predictions of specialized metabolism genes through machine learning.

Plant specialized metabolism (SM) enzymes produce lineage-specific metabolites with important ecological, evolutionary, and biotechnological implications. Using Arabidopsis thaliana as a model, we identified distinguishing characteristics of SM and GM (general metabolism, traditionally referred to as primary metabolism) genes through a detailed study of features including duplication pattern, sequence conservation, transcription, protein domain content, and gene network properties. Analysis of multiple sets of benchmark genes revealed that SM genes tend to be tandemly duplicated, coexpressed with their paralogs, narrowly expressed at lower levels, less conserved, and less well connected in gene networks relative to GM genes. Although the values of each of these features significantly differed between SM and GM genes, any single feature was ineffective at predicting SM from GM genes. Using machine learning methods to integrate all features, a prediction model was established with a true positive rate of 87% and a true negative rate of 71%. In addition, 86% of known SM genes not used to create the machine learning model were predicted. We also demonstrated that the model could be further improved when we distinguished between SM, GM, and junction genes responsible for reactions shared by SM and GM pathways, indicating that topological considerations may further improve the SM prediction model. Application of the prediction model led to the identification of 1,220 A. thaliana genes with previously unknown functions, each assigned a confidence measure called an SM score, providing a global estimate of SM gene content in a plant genome.

Jasmone Hydroxylase, a Key Enzyme in the Synthesis of the Alcohol Moiety of Pyrethrin Insecticides.

Pyrethrins are synthesized by the plant pyrethrum (Tanacetum cinerariifolium), a chrysanthemum relative. These compounds possess efficient insecticidal properties and are not toxic to humans and most vertebrates. Pyrethrum flowers, and to a smaller extent leaves, synthesize six main types of pyrethrins, which are all esters of a monoterpenoid acid moiety and an alcohol moiety derived from jasmonic acid. Here, we identified and characterized the enzyme responsible for the conversion of jasmone, a derivative of jasmonic acid, to jasmolone. Feeding pyrethrum flowers with jasmone resulted in a 4-fold increase in the concentration of free jasmolone as well as smaller but significant proportional increases in free pyrethrolone and all three type I pyrethrins. We used floral transcriptomic data to identify cytochrome P450 genes whose expression patterns were most highly correlated with that of a key gene in pyrethrin biosynthesis, T. cinerariifolium chrysanthemyl diphosphate synthase The candidate genes were screened for jasmone hydroxylase activity through transient expression in Nicotiana benthamiana leaves fed with jasmone. The expression of only one of these candidate genes produced jasmolone; therefore, this gene was named T. cinerariifolium jasmolone hydroxylase (TcJMH) and given the CYP designation CYP71AT148. The protein encoded by TcJMH localized to the endoplasmic reticulum, and microsomal preparations from N. benthamiana leaves expressing TcJMH were capable of catalyzing the hydroxylation of jasmone to jasmolone in vitro, with a Km value of 53.9 µm TcJMH was expressed almost exclusively in trichomes of floral ovaries and was induced in leaves by jasmonate.

MeNA, Controlled by Reversible Methylation of Nicotinate, Is an NAD Precursor that Undergoes Long-Distance Transport in Arabidopsis.

Nicotinamide adenine dinucleotide (NAD) biosynthesis, including synthesis from aspartate via the de novo pathway and from nicotinate (NA) via the Preiss-Handler pathway, is conserved in land plants. Diverse species of NA conjugates, which are mainly involved in NA detoxification, were also found in all tested land plants. Among these conjugates, MeNA (NA methyl ester) has been widely detected in angiosperm plants, although its physiological function and the underlying mechanism for its production in planta remain largely unknown. Here, we show that MeNA is an NAD precursor undergoing more efficient long-distance transport between organs than NA and nicotinamide in Arabidopsis. We found that Arabidopsis has one methyltransferase (designated AtNaMT1) capable of catalyzing carboxyl methylation of NA to yield MeNA and one methyl esterase (MES2) predominantly hydrolyzing MeNA back to NA. We further uncovered that the transfer of [14C]MeNA from the root to leaf was significantly increased in both MES2 knockdown and NaMT1-overexpressing lines, suggesting that both NaMT1 and MES2 fine-tune the long-distance transport of MeNA, which is ultimately utilized for NAD production. Abiotic stress (salt, abscisic acid, and mannitol) treatments, which are known to exacerbate NAD degradation, induce the expression of NaMT1 but suppress MES2 expression, suggesting that MeNA may play a role in stress adaption. Collectively, our study indicates that reversible methylation of NA controls the biosynthesis of MeNA in Arabidopsis, which presumably functions as a detoxification form of free NA for efficient long-distance transport and eventually NAD production especially under abiotic stress, providing new insights into the relationship between NAD biosynthesis and NA conjugation in plants.

Evidence for the Involvement of Fatty Acid Biosynthesis and Degradation in the Formation of Insect Sex Pheromone-Mimicking Chiloglottones in Sexually Deceptive Chiloglottis Orchids.

Hundreds of orchid species secure pollination by sexually luring specific male insects as pollinators by chemical and morphological mimicry. Yet, the biochemical pathways involved in the synthesis of the insect sex pheromone-mimicking volatiles in these sexually deceptive plants remain poorly understood. Here, we explore the biochemical pathways linked to the chemical mimicry of female sex pheromones (chiloglottones) employed by the Australian sexually deceptive Chiloglottis orchids to lure their male pollinator. By strategically exploiting the transcriptomes of chiloglottone 1-producing Chiloglottis trapeziformis at distinct floral tissues and at key floral developmental stages, we identified two key transcriptional trends linked to the stage- and tissue-dependent distribution profiles of chiloglottone in the flower: (i) developmental upregulation of fatty acid biosynthesis and ß-oxidation genes such as KETOACYL-ACP SYNTHASE, FATTY ACYL-ACP THIOESTERASE, and ACYL-COA OXIDASE during the transition from young to mature buds and flowers and (ii) the tissue-specific induction of fatty acid pathway genes in the callus (the insectiform odor-producing structure on the labellum of the flower) compared to the labellum remains (non-odor-producing) regardless of development stage of the flower. Enzyme inhibition experiments targeting KETOACYL-ACP SYNTHASE activity alone in three chiloglottone-producing species (C. trapeziformis, C. valida, and C. aff. valida) significantly inhibited chiloglottone biosynthesis up to 88.4% compared to the controls. These findings highlight the role of coordinated (developmental stage- and tissue-dependent) fatty acid gene expression and enzyme activities for chiloglottone production in Chiloglottis orchids.

Metabolic reconstructions identify plant 3-methylglutaconyl-CoA hydratase that is crucial for branched-chain amino acid catabolism in mitochondria.

The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant-prokaryote comparative genomics detected candidates for 3-methylglutaconyl-CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non-homologous N-terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein-fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3-hydroxymethylglutaryl-CoA into 3-methylglutaconyl-CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark-induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3-methylglutaconyl-CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.