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Terpenoids are ubiquitous to all kingdoms of life and are one of the most diverse groups of compounds, both structurally and functionally. Despite being derived from common precursors, isopentenyl diphosphate and dimethylallyl diphosphate, their exceptional diversity is partly driven by the substrate and product promiscuity of terpene synthases that produce a wide array of terpene skeletons. Plant terpene synthases can be subdivided into different subfamilies based on sequence homology and function. However, in many cases, structural architecture of the enzyme is more essential to product specificity than primary sequence alone, and distantly related terpene synthases can often mediate similar reactions. As such, the focus of this brief review is on some of the recent progress in understanding terpene synthase function and diversity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Terpenoids constitute one of the largest and most chemically diverse classes of primary and secondary metabolites in nature with an exceptional breadth of functional roles in plants. Biosynthesis of all terpenoids begins with the universal five‑carbon building blocks, isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), which in plants are derived from two compartmentally separated but metabolically crosstalking routes, the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. Here, we review the current knowledge on the terpenoid precursor pathways and highlight the critical hidden constraints as well as multiple regulatory mechanisms that coordinate and homeostatically govern carbon flux through the terpenoid biosynthetic network in plants., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Recent advances in our understanding of plant metabolism have highlighted the significance of specialized metabolites in the regulation of gene expression associated with biosynthetic networks. This opinion article focuses on the molecular mechanisms of small-molecule-mediated feedback regulation at the transcriptional level and its potential modes of action, including metabolite signal perception, the nature of the sensor, and the signaling transduction mechanisms leading to transcriptional and post-transcriptional regulation, based on evidence available from plants and other kingdoms of life. We also discuss the challenges associated with identifying the occurrences, effects, and localization of small molecule-protein interactions. Further understanding of small-molecule-controlled metabolic fluxes will enable rational design of transcriptional regulation systems in metabolic engineering to produce high-value specialized metabolites., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Plants are constantly exposed to volatile organic compounds (VOCs) that are released during plant-plant communication, within-plant self-signaling, and plant-microbe interactions. Therefore, understanding VOC perception and downstream signaling is vital for unraveling the mechanisms behind information exchange in plants, which remain largely unexplored. Using the hormone-like function of volatile terpenoids in reproductive organ development as a system with a visual marker for communication, we demonstrate that a petunia karrikin-insensitive receptor, PhKAI2ia, stereospecifically perceives the (-)-germacrene D signal, triggering a KAI2-mediated signaling cascade and affecting plant fitness. This study uncovers the role(s) of the intermediate clade of KAI2 receptors, illuminates the involvement of a KAI2ia-dependent signaling pathway in volatile communication, and provides new insights into plant olfaction and the long-standing question about the nature of potential endogenous KAI2 ligand(s).

The biosynthesis of specialized metabolites is strictly regulated by environmental inputs such as the day-night cycle, but the underlying mechanisms remain elusive. In Petunia hybrida cv. Mitchell flowers, the biosynthesis and emission of volatile compounds display a diurnal pattern with a peak in the evening to attract nocturnal pollinators. Using petunia flowers as a model system, we found that chromatin level regulation, especially histone acetylation, plays an essential role in mediating the day-night oscillation of the biosynthetic gene network of specialized metabolites. By performing time-course chromatin immunoprecipitation assays for histone modifications, we uncovered that a specific group of genes involved in the regulation, biosynthesis, and emission of floral volatile compounds, which displays the greatest magnitude in day-night oscillating gene expression, is associated with highly dynamic histone acetylation marks H3K9ac and H3K27ac. Specifically, the strongest oscillating genes featured a drastic removal of histone acetylation marks at night, potentially to shut down the biosynthesis of floral volatile compounds during the morning when they are not needed. Inhibiting daytime histone acetylation led to a compromised evening induction of these genes. Overall, our study suggested an active role of chromatin modification in the diurnal oscillation of specialized metabolic network., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Environmental factors such as light, water, minerals, temperature, and other organisms affect plant growth and development. Unlike animals, plants can't escape from unfavorable biotic and abiotic stresses. Thus, they evolved the ability to biosynthesize specific chemicals referred to as plant specialized metabolites in order to facilitate successful interactions with the surrounding environment, as well as with other organisms including plants, insects, microorganisms, and animals. While the exact number of plant specialized metabolites, historically called secondary metabolites, is currently unknown, it has been estimated to range from 200,000 to 1,000,000 compounds. In contrast to the species-, organ- and tissue-specific nature of plant specialized metabolites, primary metabolites are shared by all living organisms, are vital for growth, development and reproduction, and comprise only about 8,000 compounds. The biosynthesis and storage of plant specialized metabolites are developmentally and temporally regulated and depend on biotic and abiotic factors. Specific cell types, subcellular organelles, microcompartments, and/or anatomical structures are often devoted to producing and storing these compounds. The functions of many specialized metabolites are still not fully understood but are generally considered to be essential for the fitness and survival of plants, partially by interacting with other organisms in both mutualistic (for example, attraction of pollinators) and antagonistic (such as defense against herbivores and pathogens) ways. In this primer, we will focus on specialized-metabolite functions in plant defense interactions and on the genetic, molecular, and biochemical mechanisms leading to the structural diversity of specialized metabolites. Though less understood, we will also touch on the mode of action of specialized metabolites in plant defense., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Terpenoids constitute the largest class of plant primary and secondary metabolites with a broad range of biological and ecological functions. They are synthesized from isopentenyl diphosphate and dimethylallyl diphosphate, which in plastids are condensed by geranylgeranyl diphosphate synthases (GGPPSs) to produce GGPP (C20) for diterpene biosynthesis and by geranyl diphosphate synthases (GPPSs) to form GPP (C10) for monoterpene production. Depending on the plant species, unlike homomeric GGPPSs, GPPSs exist as homo- and heteromers, the latter of which contain catalytically inactive GGPPS-homologous small subunits (SSUs) that can interact with GGPPSs. By combining phylogenetic analysis with functional characterization of GGPPS homologs from a wide range of photosynthetic organisms, we investigated how different GPPS architectures have evolved within the GGPPS protein family. Our results reveal that GGPPS gene family expansion and functional divergence began early in nonvascular plants, and that independent parallel evolutionary processes gave rise to homomeric and heteromeric GPPSs. By site-directed mutagenesis and molecular dynamics simulations, we also discovered that Leu-Val/Val-Ala pairs of amino acid residues were pivotal in the functional divergence of homomeric GPPSs and GGPPSs. Overall, our study elucidated an evolutionary path for the formation of GPPSs with different architectures from GGPPSs and uncovered the molecular mechanisms involved in this differentiation., Competing Interests: Conflict of interest statement. The authors declare no conflict of interest., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Geraniol derived from essential oils of various plant species is widely used in the cosmetic and perfume industries. It is also an essential trait of the pleasant smell of rose flowers. In contrast to other monoterpenes which are produced in plastids via the methyl erythritol phosphate pathway, geraniol biosynthesis in roses relies on cytosolic NUDX1 hydrolase which dephosphorylates geranyl diphosphate (GPP). However, the metabolic origin of cytosolic GPP remains unknown. By feeding Rosa chinensis "Old Blush" flowers with pathway-specific precursors and inhibitors, combined with metabolic profiling and functional characterization of enzymes in vitro and in planta, we show that geraniol is synthesized through the cytosolic mevalonate (MVA) pathway by a bifunctional geranyl/farnesyl diphosphate synthase, RcG/FPPS1, producing both GPP and farnesyl diphosphate (FPP). The downregulation and overexpression of RcG/FPPS1 in rose petals affected not only geraniol and germacrene D emissions but also dihydro-β-ionol, the latter due to metabolic cross talk of RcG/FPPS1-dependent isoprenoid intermediates trafficking from the cytosol to plastids. Phylogenetic analysis together with functional characterization of G/FPPS orthologs revealed that the G/FPPS activity is conserved among Rosaceae species. Site-directed mutagenesis and molecular dynamic simulations enabled to identify two conserved amino acids that evolved from ancestral FPPSs and contribute to GPP/FPP product specificity. Overall, this study elucidates the origin of the cytosolic GPP for NUDX1-dependent geraniol production, provides insights into the emergence of the RcG/FPPS1 GPPS activity from the ancestral FPPSs, and shows that RcG/FPPS1 plays a key role in the biosynthesis of volatile terpenoid compounds in rose flowers.

For volatile organic compounds (VOCs) to be released from the plant cell into the atmosphere, they have to cross the plasma membrane, the cell wall, and the cuticle. However, how these hydrophobic compounds cross the hydrophilic cell wall is largely unknown. Using biochemical and reverse-genetic approaches combined with mathematical simulation, we show that cell-wall localized non-specific lipid transfer proteins (nsLTPs) facilitate VOC emission. Out of three highly expressed nsLTPs in petunia petals, which emit high levels of phenylpropanoid/benzenoid compounds, only PhnsLTP3 contributes to the VOC export across the cell wall to the cuticle. A decrease in PhnsLTP3 expression reduces volatile emission and leads to VOC redistribution with less VOCs reaching the cuticle without affecting their total pools. This intracellular build-up of VOCs lowers their biosynthesis by feedback downregulation of phenylalanine precursor supply to prevent self-intoxication. Overall, these results demonstrate that nsLTPs are intrinsic members of the VOC emission network, which facilitate VOC diffusion across the cell wall., (© 2023. The Author(s).)

Lignin, one of the most abundant polymers in plants, is derived from the phenylpropanoid pathway, which also gives rise to an array of metabolites that are essential for plant fitness. Genetic engineering of lignification can cause drastic changes in transcription and metabolite accumulation with or without an accompanying development phenotype. To understand the impact of lignin perturbation, we analyzed transcriptome and metabolite data from the rapidly lignifying stem tissue in 13 selected phenylpropanoid mutants and wild-type Arabidopsis (Arabidopsis thaliana). Our dataset contains 20,974 expressed genes, of which over 26% had altered transcript levels in at least one mutant, and 18 targeted metabolites, all of which displayed altered accumulation in at least one mutant. We found that lignin biosynthesis and phenylalanine supply via the shikimate pathway are tightly co-regulated at the transcriptional level. The hierarchical clustering analysis of differentially expressed genes (DEGs) grouped the 13 mutants into 5 subgroups with similar profiles of mis-regulated genes. Functional analysis of the DEGs in these mutants and correlation between gene expression and metabolite accumulation revealed system-wide effects on transcripts involved in multiple biological processes., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Terpenoids constitute one of the largest and most diverse classes of plant metabolites. While some terpenoids are involved in essential plant processes such as photosynthesis, respiration, growth, and development, others are specialized metabolites playing roles in the interaction of plants with their biotic and abiotic environment. Due to the distinct functions and properties of specific terpenoid compounds, there is a growing interest to introduce or modify their production in plants by metabolic engineering for agricultural, pharmaceutical, or industrial applications. The MVA and MEP pathways and the prenyltransferases providing the general precursors for terpenoid formation, as well as the enzymes of the various downstream metabolic pathways leading to the formation of different groups of terpenoid compounds have been characterized in detail in plants. In contrast, the molecular mechanisms directing the metabolic flux of precursors specifically toward one of several potentially competing terpenoid biosynthetic pathways are still not well understood. The formation of metabolons, multi-protein complexes composed of enzymes catalyzing sequential reactions of a metabolic pathway, provides a promising concept to explain the metabolic channeling that appears to occur in the complex terpenoid biosynthetic network of plants. Here we provide an overview about examples of potential metabolons involved in plant terpenoid metabolism that have been recently characterized and the first attempts to utilize metabolic channeling in terpenoid metabolic engineering. In addition, we discuss the gaps in our current knowledge and in consequence the need for future basic and applied research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gutensohn, Hartzell and Dudareva.)

Plant cuticles are a mixture of crystalline and amorphous waxes that restrict the exchange of molecules between the plant and the atmosphere. The multicomponent nature of cuticular waxes complicates the study of the relationship between the physical and transport properties. Here, a model cuticle based on the epicuticular waxes of Petunia hybrida flower petals was formulated to test the effect of wax composition on diffusion of water and volatile organic compounds (VOCs). The model cuticle was composed of an n-tetracosane (C 24 H 50 ), 1-docosanol (C 22 H 45 OH), and 3-methylbutyl dodecanoate (C 17 H 34 O 2 ), reflecting the relative chain length, functional groups, molecular arrangements, and crystallinity of the natural waxes. Molecular dynamics simulations were performed to obtain diffusion coefficients for compounds moving through waxes of varying composition. Simulated VOC diffusivities of the model system were found to highly correlate with in vitro measurements in isolated petunia cuticles. VOC diffusivity increased up to 30-fold in completely amorphous waxes, indicating a significant effect of crystallinity on cuticular permeability. The crystallinity of the waxes was highly dependent on the elongation of the lattice length and decrease in gap width between crystalline unit cells. Diffusion of water and higher molecular weight VOCs were significantly affected by alterations in crystalline spacing and lengths, whereas the low molecular weight VOCs were less affected. Comparison of measured diffusion coefficients from atomistic simulations and emissions from petunia flowers indicates that the role of the plant cuticle in the VOC emission network is attributed to the differential control on mass transfer of individual VOCs by controlling the composition, amount, and dynamics of scent emission., (© 2022 Society for Experimental Biology and John Wiley & Sons Ltd.)

Benzaldehyde, the simplest aromatic aldehyde, is one of the most wide-spread volatiles that serves as a pollinator attractant, flavor, and antifungal compound. However, the enzyme responsible for its formation in plants remains unknown. Using a combination of in vivo stable isotope labeling, classical biochemical, proteomics and genetic approaches, we show that in petunia benzaldehyde is synthesized via the β-oxidative pathway in peroxisomes by a heterodimeric enzyme consisting of α and β subunits, which belong to the NAD(P)-binding Rossmann-fold superfamily. Both subunits are alone catalytically inactive but, when mixed in equal amounts, form an active enzyme, which exhibits strict substrate specificity towards benzoyl-CoA and uses NADPH as a cofactor. Alpha subunits can form functional heterodimers with phylogenetically distant β subunits, but not all β subunits partner with α subunits, at least in Arabidopsis. Analysis of spatial, developmental and rhythmic expression of genes encoding α and β subunits revealed that expression of the gene for the α subunit likely plays a key role in regulating benzaldehyde biosynthesis., (© 2022. The Author(s).)

Scent bouquets produced by the flowers of Petunia spp. (petunia) are composed of a complex mixture of floral volatile benzenoid and phenylpropanoid compounds (FVBPs), which are specialized metabolites derived from phenylalanine (Phe) through an interconnected network of enzymes. The biosynthesis and emission of high levels of these volatiles requires coordinated transcriptional activation of both primary and specialized metabolic networks. The petunia R2R3-MYB transcription factor ODORANT 1 (ODO1) was identified as a master regulator of FVBP production and emission; however, our knowledge of the direct regulatory targets of ODO1 has remained limited. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq) in petunia flowers, we identify genome-wide ODO1-bound genes that are enriched not only in genes involved in the biosynthesis of the Phe precursor, as previously reported, but also genes associated with the specialized metabolic pathways involved in generating phenylpropanoid intermediates for FVBPs. ODO1-bound genes are also involved in methionine and S-adenosylmethionine metabolism, which could modulate methyl group supplies for certain FVBPs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA-seq analysis in an ODO1 RNAi knockdown line revealed that ODO1-bound targets are expressed at lower levels when ODO1 is suppressed. A cis-regulatory motif, CACCAACCCC, was identified as a potential binding site for ODO1 in the promoters of genes that are both bound and activated by ODO1, which was validated by in planta promoter reporter assays with wild-type and mutated promoters. Overall, our work presents a mechanistic model for ODO1 controlling an extensive gene regulatory network that contributes to FVBP production to give rise to floral scent., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Nudix hydrolases are conserved enzymes ubiquitously present in all kingdoms of life. Recent research revealed that several Nudix hydrolases are involved in terpenoid metabolism in plants. In modern roses, RhNUDX1 is responsible for formation of geraniol, a major compound of rose scent. Nevertheless, this compound is produced by monoterpene synthases in many geraniol-producing plants. As a consequence, this raised the question about the origin of RhNUDX1 function and the NUDX1 gene evolution in Rosaceae, in wild roses or/and during the domestication process. Here, we showed that three distinct clades of NUDX1 emerged in the Rosoidae subfamily (Nudx1-1 to Nudx1-3 clades), and two subclades evolved in the Rosa genus (Nudx1-1a and Nudx1-1b subclades). We also showed that the Nudx1-1b subclade was more ancient than the Nudx1-1a subclade, and that the NUDX1-1a gene emerged by a trans-duplication of the more ancient NUDX1-1b gene. After the transposition, NUDX1-1a was cis-duplicated, leading to a gene dosage effect on the production of geraniol in different species. Furthermore, the NUDX1-1a appearance was accompanied by the evolution of its promoter, most likely from a Copia retrotransposon origin, leading to its petal-specific expression. Thus, our data strongly suggest that the unique function of NUDX1-1a in geraniol formation was evolved naturally in the genus Rosa before domestication., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Galling insects gain food and shelter by inducing specialized anatomical structures in their plant hosts. Such galls often accumulate plant defensive metabolites protecting the inhabiting insects from predation. We previously found that, despite a marked natural chemopolymorphism in natural populations of Pistacia palaestina, the monoterpene content in Baizongia pistaciae-induced galls is substantially higher than in leaves of their hosts. Here we show a general up-regulation of key structural genes in both the plastidial and cytosolic terpene biosynthetic pathways in galls as compared with non-colonized leaves. Novel prenyltransferases and terpene synthases were functionally expressed in Escherichia coli to reveal their biochemical function. Individual Pistacia trees exhibiting chemopolymorphism in terpene compositions displayed differential up-regulation of selected terpene synthase genes, and the metabolites generated by their gene products in vitro corresponded to the monoterpenes accumulated by each tree. Our results delineate molecular mechanisms responsible for the formation of enhanced monoterpene in galls and the observed intraspecific monoterpene chemodiversity displayed in P. palaestina. We demonstrate that gall-inhabiting aphids transcriptionally reprogram their host terpene pathways by up-regulating tree-specific genes, boosting the accumulation of plant defensive compounds for the protection of colonizing insects., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p -cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto-enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p -cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Carrot psylla is one of the devastating pests of carrot throughout northern Europe and the Mediterranean basin. Here we characterized the behavioral response of psylla females towards different carrot germplasm and identified the chemical cues involved in the host selection of psylla females by oviposition choice experiments and metabolic profiling of leaf volatiles. In choice assays, carrot psylla displayed differential responses to tested 14 germplasm. Among germplasm, wild accessions 21793 and 20465 were highly preferred by carrot psylla, while wild accessions 20465 and the orange cultivar Nairobi were less. In non-choice experiments conducted only with this four-germplasm revealed that the carrot psylla females gave higher preference to the Nairobi and wild accession 20465, indicating the vicinity to other host plants in the same area might affect female preference. Moreover, the nymph development and survival experiments showed the lowest nymphs survival rate on the wild accessions 21793 and 20497. Furthermore, the volatile emissions among different carrot cultivars infested with psylla showed qualitative and quantitative differences versus intact plants. Among these volatiles, apiol, β-asarone, myristicin, and sabinene showed a relationship with psyllas growth and survival. We also showed that myristicin and sabinene exogenous applications caused a dramatic reduction in the number of eggs laid by psylla and subsequent nymph survival. This is an initial study of the volatiles that mediate attraction and oviposition preference of carrot psylla in response to its host plant. The results from this study provide baseline information for the development of new control strategies against carrot psylla., (Copyright © 2021 Elsevier B.V. All rights reserved.)

As sessile organisms, plants evolved elaborate metabolic systems that produce a plethora of specialized metabolites as a means to survive challenging terrestrial environments. Decades of research have revealed the genetic and biochemical basis for a multitude of plant specialized metabolic pathways. Nevertheless, knowledge is still limited concerning the selective advantages provided by individual and collective specialized metabolites to the reproductive success of diverse host plants. Here we review the biological functions conferred by various classes of plant specialized metabolites in the context of the interaction of plants with their surrounding environment. To achieve optimal multifunctionality of diverse specialized metabolic processes, plants use various adaptive mechanisms at subcellular, cellular, tissue, organ and interspecies levels. Understanding these mechanisms and the evolutionary trajectories underlying their occurrence in nature will ultimately enable efficient bioengineering of desirable metabolic traits in chassis organisms., (© 2021. Springer Nature America, Inc.)

Many flowering plants rely on pollinators for their reproductive success. Plant-pollinator interactions usually depend on a complex combination of traits based on a fine-tuned biosynthetic machinery, with many structural and regulatory genes involved. Yet, the physiological mechanisms in plants are the product of evolutionary processes. While evolution has been modifying flowers through millions of years, it is also a rapid process that can change plant traits within few generations. Here we discuss both mechanistic and evolutionary aspects of pollinator attraction. We also propose how latest advances in biotechnology and evolutionary studies, and their combination, will improve the elucidation of molecular mechanisms and evolutionary dynamics of pollinator attraction in changing environments., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gutensohn, Henry, Gentry, Lynch, Nguyen, Pichersky and Dudareva.)

Metabolic engineering is embraced as a method to sustainably enhance production of valuable phytochemicals with beneficial properties. However, successful production of these compounds in plants is not always predictable even when the pathways are fully known, frequently due to the lack of comprehensive understanding of plant metabolism as a whole, and interconnections between different primary, secondary, and hormone metabolic networks. Here, we highlight critical hidden constraints, including substrate availability, silent metabolism, and metabolic crosstalk, that impair engineering strategies. We explore how these constraints have historically been manifested in engineering attempts and propose how modern advancements will enable future strategies to overcome these impediments., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Biosynthesis of secondary metabolites relies on primary metabolic pathways to provide precursors, energy, and cofactors, thus requiring coordinated regulation of primary and secondary metabolic networks. However, to date, it remains largely unknown how this coordination is achieved. Using Petunia hybrida flowers, which emit high levels of phenylpropanoid/benzenoid volatile organic compounds (VOCs), we uncovered genome-wide dynamic deposition of histone H3 lysine 9 acetylation (H3K9ac) during anthesis as an underlying mechanism to coordinate primary and secondary metabolic networks. The observed epigenome reprogramming is accompanied by transcriptional activation at gene loci involved in primary metabolic pathways that provide precursor phenylalanine, as well as secondary metabolic pathways to produce volatile compounds. We also observed transcriptional repression among genes involved in alternative phenylpropanoid branches that compete for metabolic precursors. We show that GNAT family histone acetyltransferase(s) (HATs) are required for the expression of genes involved in VOC biosynthesis and emission, by using chemical inhibitors of HATs, and by knocking down a specific HAT gene, ELP3, through transient RNAi. Together, our study supports that regulatory mechanisms at chromatin level may play an essential role in activating primary and secondary metabolic pathways to regulate VOC synthesis in petunia flowers., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The plant cuticle is the final barrier for volatile organic compounds (VOCs) to cross for release to the atmosphere, yet its role in the emission process is poorly understood. Here, using a combination of reverse-genetic and chemical approaches, we demonstrate that the cuticle imposes substantial resistance to VOC mass transfer, acting as a sink/concentrator for VOCs and hence protecting cells from the potentially toxic internal accumulation of these hydrophobic compounds. Reduction in cuticle thickness has differential effects on individual VOCs depending on their volatility, and leads to their internal cellular redistribution, a shift in mass transfer resistance sources and altered VOC synthesis. These results reveal that the cuticle is not simply a passive diffusion barrier for VOCs to cross, but plays the aforementioned complex roles in the emission process as an integral member of the overall VOC network.

In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.

In plants, high carbon flux is committed to the biosynthesis of phenylalanine, tyrosine, and tryptophan, owing to their roles not only in the production of proteins, but also as precursors to thousands of primary and specialized metabolites. The core plastidial pathways that supply the majority of aromatic amino acids (AAAs) have previously been described in detail. More recently, the discovery of cytosolic enzymes contributing to overall AAA biosynthesis, as well as the identification of intracellular transporters and the continuing elucidation of transcriptional and post-transcriptional regulatory mechanisms, have revealed the complexity of this intercompartmental metabolic network. Here, we review the latest breakthroughs in AAA production and use the newest findings to highlight both longstanding and newly developed questions., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Monoterpenes contribute either favorably or adversely to the flavor of tomato, yet modern tomato varieties generally lack monoterpenes in their fruit. The main immediate biosynthetic precursor of monoterpenes is geranyldiphosphate (GPP), produced by the action of GPP synthases (GPPSs). Plant GPPSs are often heteromeric enzymes consisting of a non-catalytic small subunit (GPPS.SSU) and a large subunit (GPPS.LSU), the latter similar to geranylgeranyldiphosphate synthases (GGPPSs) which generate longer prenylphosphate chains. We show here that LeGGPPS2, an enzyme previously reported to support carotenoid biosynthesis, can synthesize farnesyldiphosphate (FPP) and GPP in vitro, in addition to geranylgeranyldiphosphate, depending on the assay conditions. Moreover, GPP formation is favored in vitro by the interaction of LeGGPPS2 with GPPS.SSU from either Anthirrhinum majus (AmGPPS.SSU) or from a newly discovered GPPS.SSU ortholog present in the genome of M82 tomato. SlGPPS.SSU is not expressed in M82 tomato fruit but its orthologs are expressed in fruit of wild tomato relatives, such as Solanum pimpinelifollium and S. cheesmaniae that accumulate monoterpenes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Species within the mint family, Lamiaceae, are widely used for their culinary, cultural, and medicinal properties due to production of a wide variety of specialized metabolites, especially terpenoids. To further our understanding of genome diversity in the Lamiaceae and to provide a resource for mining biochemical pathways, we generated high-quality genome assemblies of four economically important culinary herbs, namely, sweet basil (Ocimum basilicum L.), sweet marjoram (Origanum majorana L.), oregano (Origanum vulgare L.), and rosemary (Rosmarinus officinalis L.), and characterized their terpenoid diversity through metabolite profiling and genomic analyses. A total 25 monoterpenes and 11 sesquiterpenes were identified in leaf tissue from the 4 species. Genes encoding enzymes responsible for the biosynthesis of precursors for mono- and sesqui-terpene synthases were identified in all four species. Across all 4 species, a total of 235 terpene synthases were identified, ranging from 27 in O. majorana to 137 in the tetraploid O. basilicum. This study provides valuable resources for further investigation of the genetic basis of chemodiversity in these important culinary herbs., (© The Author(s) 2020. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Catnip or catmint ( Nepeta spp.) is a flowering plant in the mint family (Lamiaceae) famed for its ability to attract cats. This phenomenon is caused by the compound nepetalactone, a volatile iridoid that also repels insects. Iridoids are present in many Lamiaceae species but were lost in the ancestor of the Nepetoideae, the subfamily containing Nepeta . Using comparative genomics, ancestral sequence reconstructions, and phylogenetic analyses, we probed the re-emergence of iridoid biosynthesis in Nepeta . The results of these investigations revealed mechanisms for the loss and subsequent re-evolution of iridoid biosynthesis in the Nepeta lineage. We present evidence for a chronology of events that led to the formation of nepetalactone biosynthesis and its metabolic gene cluster. This study provides insights into the interplay between enzyme and genome evolution in the origins, loss, and re-emergence of plant chemical diversity., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Flowers are essential but vulnerable plant organs, exposed to pollinators and florivores; however, flower chemical defenses are rarely investigated. We show here that two clustered terpene synthase and cytochrome P450 encoding genes ( TPS11 and CYP706A3 ) on chromosome 5 of Arabidopsis ( Arabidopsis thaliana ) are tightly coexpressed in floral tissues, upon anthesis and during floral bud development. TPS11 was previously reported to generate a blend of sesquiterpenes. By heterologous coexpression of TPS11 and CYP706A3 in yeast ( Saccharomyces cerevisiae ) and Nicotiana benthamiana , we demonstrate that CYP706A3 is active on TPS11 products and also further oxidizes its own primary oxidation products. Analysis of headspace and soluble metabolites in cyp706a3 and 35S:CYP706A3 mutants indicate that CYP706A3-mediated metabolism largely suppresses sesquiterpene and most monoterpene emissions from opening flowers, and generates terpene oxides that are retained in floral tissues. In flower buds, the combined expression of TPS11 and CYP706A3 also suppresses volatile emissions and generates soluble sesquiterpene oxides. Florivory assays with the Brassicaceae specialist Plutella xylostella demonstrate that insect larvae avoid feeding on buds expressing CYP706A3 and accumulating terpene oxides. Composition of the floral microbiome appears also to be modulated by CYP706A3 expression. TPS11 and CYP706A3 simultaneously evolved within Brassicaceae and form the most versatile functional gene cluster described in higher plants so far.plantcell;31/12/2947/FX1F1fx1., (© 2019 American Society of Plant Biologists. All rights reserved.)

The transition from pollinator-mediated outbreeding to selfing has occurred many times in angiosperms. This is generally accompanied by a reduction in traits attracting pollinators, including reduced emission of floral scent. In Capsella, emission of benzaldehyde as a main component of floral scent has been lost in selfing C. rubella by mutation of cinnamate-CoA ligase CNL1. However, the biochemical basis and evolutionary history of this loss remain unknown, as does the reason for the absence of benzaldehyde emission in the independently derived selfer Capsella orientalis. We used plant transformation, in vitro enzyme assays, population genetics and quantitative genetics to address these questions. CNL1 has been inactivated twice independently by point mutations in C. rubella, causing a loss of enzymatic activity. Both inactive haplotypes are found within and outside of Greece, the centre of origin of C. rubella, indicating that they arose before its geographical spread. By contrast, the loss of benzaldehyde emission in C. orientalis is not due to an inactivating mutation in CNL1. CNL1 represents a hotspot for mutations that eliminate benzaldehyde emission, potentially reflecting the limited pleiotropy and large effect of its inactivation. Nevertheless, even closely related species have followed different evolutionary routes in reducing floral scent., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Plants synthesize volatile organic compounds (VOCs) to attract pollinators and beneficial microorganisms, to defend themselves against herbivores and pathogens, and for plant-plant communication. In general, VOCs accumulate in and are emitted from the tissue of their biosynthesis. However, using biochemical and reverse genetic approaches, we demonstrate a new physiological phenomenon: inter-organ aerial transport of VOCs via natural fumigation. Before petunia flowers open, a tube-specific terpene synthase produces sesquiterpenes, which are released inside the buds and then accumulate in the stigma, potentially defending the developing stigma from pathogens. These VOCs also affect reproductive organ development and seed yield, which are previously unknown functions of terpenoid compounds.

Background: Teak, a member of the Lamiaceae family, produces one of the most expensive hardwoods in the world. High demand coupled with deforestation have caused a decrease in natural teak forests, and future supplies will be reliant on teak plantations. Hence, selection of teak tree varieties for clonal propagation with superior growth performance is of great importance, and access to high-quality genetic and genomic resources can accelerate the selection process by identifying genes underlying desired traits., Findings: To facilitate teak research and variety improvement, we generated a highly contiguous, chromosomal-scale genome assembly using high-coverage Pacific Biosciences long reads coupled with high-throughput chromatin conformation capture. Of the 18 teak chromosomes, we generated 17 near-complete pseudomolecules with one chromosome present as two chromosome arm scaffolds. Genome annotation yielded 31,168 genes encoding 46,826 gene models, of which, 39,930 and 41,155 had Pfam domain and expression evidence, respectively. We identified 14 clusters of tandem-duplicated terpene synthases (TPSs), genes central to the biosynthesis of terpenes, which are involved in plant defense and pollinator attraction. Transcriptome analysis revealed 10 TPSs highly expressed in woody tissues, of which, 8 were in tandem, revealing the importance of resolving tandemly duplicated genes and the quality of the assembly and annotation. We also validated the enzymatic activity of four TPSs to demonstrate the function of key TPSs., Conclusions: In summary, this high-quality chromosomal-scale assembly and functional annotation of the teak genome will facilitate the discovery of candidate genes related to traits critical for sustainable production of teak and for anti-insecticidal natural products., (© The Author(s) 2019. Published by Oxford University Press.)

Carrot (Daucus carota subsp. sativus) is a widely cultivated root vegetable of high economic importance. The aroma of carrot roots and aboveground organs is mainly defined by terpenes. We found that leaves of orange carrot cultivar also produce considerable amounts of the phenylpropenes methyleugenol and methylisoeugenol. Notably, methyleugenol is most abundant in young leaves, while methylisoeugenol is the dominant phenylpropene in mature leaf tissue. The goal of the present study was to shed light on the biochemistry and molecular biology of these compounds' biosynthesis and accumulation. Using the available genomic and transcriptomic data, we isolated a cDNA encoding eugenol/isoeugenol synthase (DcE(I)GS1), an NADPH-dependent enzyme that converts coniferyl acetate to eugenol. This enzyme exhibits dual product specificity and yields propenylphenol isoeugenol alongside allylphenol eugenol. Furthermore, we identified a cDNA encoding S-adenosyl-L-methionine:eugenol/isoeugenol O-methyltransferase 1 (DcE(I)OMT1) that produces methyleugenol and methylisoeugenol via methylation of the para-OH-group of their respective precursors. Both DcE(I)GS1 and DcE(I)OMT1 were expressed in seeds, roots, young and mature leaves, and the DcE(I)OMT1 transcript levels were the highest in leaves. The DcE(I)GS1 protein is 67% identical to anise t-anol/isoeugenol synthase and displays an apparent K m of 247 μM for coniferyl acetate. The catalytic efficiency of DcEOMT1 with eugenol is more than five-fold higher than that with isoeugenol, with K m values of 40 μM for eugenol, and of 115 μM for isoeugenol. This work expands the current knowledge of the enzymes involved in phenylpropene biosynthesis and would enable studies into structural elements defining the regioselectivity of phenylpropene synthases., (Copyright © 2018. Published by Elsevier Ltd.)

In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.

Lignin is a polymer that significantly inhibits saccharification of plant feedstocks. Adjusting the composition or reducing the total lignin content have both been demonstrated to result in an increase in sugar yield from biomass. However, because lignin is essential for plant growth, it cannot be manipulated with impunity. Thus, it is important to understand the control of carbon flux towards lignin biosynthesis such that optimal modifications to it can be made precisely. Phenylalanine (Phe) is the common precursor for all lignin subunits and it is commonly accepted that all biosynthetic steps, spanning multiple subcellular compartments, are known, yet an in vivo model of how flux towards lignin is controlled is lacking. To address this deficiency, we formulated and parameterized a kinetic model based on data from feeding Arabidopsis thaliana basal lignifying stems with ring labeled [ 13 C 6 ]-Phe. Several candidate models were compared by an information theoretic approach to select the one that best matched the experimental observations. Here we present a dynamic model of phenylpropanoid metabolism across several subcellular compartments that describes the allocation of carbon towards lignin biosynthesis in wild-type Arabidopsis stems. Flux control coefficients for the enzymes in the pathway starting from arogenate dehydratase through 4-coumarate: CoA ligase were calculated and show that the plastidial cationic amino-acid transporter has the highest impact on flux., (Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Plant genomes encode isopentenyl phosphate kinases (IPKs) that reactivate isopentenyl phosphate (IP) via ATP-dependent phosphorylation, forming the primary metabolite isopentenyl diphosphate (IPP) used generally for isoprenoid/terpenoid biosynthesis. Therefore, the existence of IPKs in plants raises unanswered questions concerning the origin and regulatory roles of IP in plant terpenoid metabolism. Here, we provide genetic and biochemical evidence showing that IP forms during specific dephosphorylation of IPP catalysed by a subset of Nudix superfamily hydrolases. Increasing metabolically available IP by overexpression of a bacterial phosphomevalonate decarboxylase (MPD) in Nicotiana tabacum resulted in significant enhancement in both monoterpene and sesquiterpene production. These results indicate that perturbing IP metabolism results in measurable changes in terpene products derived from both the methylerythritol phosphate (MEP) and mevalonate (MVA) pathways. Moreover, the unpredicted peroxisomal localization of bacterial MPD led us to discover that the step catalysed by phosphomevalonate kinase (PMK) imposes a hidden constraint on flux through the classical MVA pathway. These complementary findings fundamentally alter conventional views of metabolic regulation of terpenoid metabolism in plants and provide new metabolic engineering targets for the production of high-value terpenes in plants.

Background: Metabolic fluxes represent the functional phenotypes of biochemical pathways and are essential to reveal the distribution of precursors among metabolic networks. Although analysis of metabolic fluxes, facilitated by stable isotope labeling and mass spectrometry detection, has been applied in the studies of plant metabolism, we lack experimental measurements for carbon flux towards lignin, one of the most abundant polymers in nature., Results: We developed a feeding strategy of excised Arabidopsis stems with 13 C labeled phenylalanine (Phe) for the analysis of lignin biosynthetic flux. We optimized the feeding methods and found the stems continued to grow and lignify. Consistent with lignification profiles along the stems, higher levels of phenylpropanoids and activities of lignin biosynthetic enzymes were detected in the base of the stem. In the feeding experiments, 13 C labeled Phe was quickly accumulated and used for the synthesis of phenylpropanoid intermediates and lignin. The intermediates displayed two different patterns of labeling kinetics during the feeding period. Analysis of lignin showed rapid incorporation of label into all three subunits in the polymers., Conclusions: Our feeding results demonstrate the effectiveness of the stem feeding system and suggest a potential application for the investigations of other aspects in plant metabolism. The supply of exogenous Phe leading to a higher lignin deposition rate indicates the availability of Phe is a determining factor for lignification rates.

Peroxisomal β-oxidative degradation of compounds is a common metabolic process in eukaryotes. Reported benzoyl-coenzyme A (BA-CoA) thioesterase activity in peroxisomes from petunia flowers suggests that, like mammals and fungi, plants contain auxiliary enzymes mediating β-oxidation. Here we report the identification of Petunia hybrida thioesterase 1 (PhTE1), which catalyzes the hydrolysis of aromatic acyl-CoAs to their corresponding acids in peroxisomes. PhTE1 expression is spatially, developmentally and temporally regulated and exhibits a similar pattern to known benzenoid metabolic genes. PhTE1 activity is inhibited by free coenzyme A (CoA), indicating that PhTE1 is regulated by the peroxisomal CoA pool. PhTE1 downregulation in petunia flowers led to accumulation of BA-CoA with increased production of benzylbenzoate and phenylethylbenzoate, two compounds which rely on the presence of BA-CoA precursor in the cytoplasm, suggesting that acyl-CoAs can be exported from peroxisomes. Furthermore, PhTE1 downregulation resulted in increased pools of cytoplasmic phenylpropanoid pathway intermediates, volatile phenylpropenes, lignin and anthocyanins. These results indicate that PhTE1 influences (i) intraperoxisomal acyl-CoA/CoA levels needed to carry out β-oxidation, (ii) efflux of β-oxidative products, acyl-CoAs and free acids, from peroxisomes, and (iii) flux distribution within the benzenoid/phenylpropanoid metabolic network. Thus, this demonstrates that plant thioesterases play multiple auxiliary roles in peroxisomal β-oxidative metabolism., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Detrimental effects of hyperaccumulation of the aromatic amino acid phenylalanine (Phe) in animals, known as phenylketonuria, are mitigated by excretion of Phe derivatives; however, how plants endure Phe accumulating conditions in the absence of an excretion system is currently unknown. To achieve Phe hyperaccumulation in a plant system, we simultaneously decreased in petunia flowers expression of all three Phe ammonia lyase (PAL) isoforms that catalyze the non-oxidative deamination of Phe to trans-cinnamic acid, the committed step for the major pathway of Phe metabolism. A total decrease in PAL activity by 81-94% led to an 18-fold expansion of the internal Phe pool. Phe accumulation had multifaceted intercompartmental effects on aromatic amino acid metabolism. It resulted in a decrease in the overall flux through the shikimate pathway, and a redirection of carbon flux toward the shikimate-derived aromatic amino acids tyrosine and tryptophan. Accumulation of Phe did not lead to an increase in flux toward phenylacetaldehyde, for which Phe is a direct precursor. Metabolic flux analysis revealed this to be due to the presence of a distinct metabolically inactive pool of Phe, likely localized in the vacuole. We have identified a vacuolar cationic amino acid transporter (PhCAT2) that contributes to sequestering excess of Phe in the vacuole. In vitro assays confirmed PhCAT2 can transport Phe, and decreased PhCAT2 expression in PAL-RNAi transgenic plants resulted in 1.6-fold increase in phenylacetaldehyde emission. These results demonstrate mechanisms by which plants maintain intercompartmental aromatic amino acid homeostasis, and provide critical insight for future phenylpropanoid metabolic engineering strategies., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Plant glandular trichomes are able to secrete and store large amounts of volatile organic compounds (VOCs). VOCs typically accumulate in dedicated extracellular spaces, which can be either subcuticular, as in the Lamiaceae or Asteraceae, or intercellular, as in the Solanaceae. Volatiles are retained at high concentrations in these storage cavities with limited release into the atmosphere and without re-entering the secretory cells, where they would be toxic. This implies the existence of mechanisms allowing transport of VOCs to the cavity but preventing their diffusion out once they have been delivered. The cuticle and cell wall lining the cavity are likely to have key roles in retaining volatiles, but their exact composition and the potential molecular players involved are largely unknown., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: The phenylpropanoid pathway is a source of a diverse group of compounds derived from phenylalanine, many of which are involved in lignin biosynthesis and serve as precursors for the production of valuable compounds, such as coumarins, flavonoids, and lignans. Consequently, recent efforts have been invested in mechanistically understanding monolignol biosynthesis, making the quantification of these metabolites vital., Objective: To develop an improved and comprehensive analytical method for (i) extensively profiling, and (ii) accurately quantifiying intermediates of the monolignol biosynthetic network, using Arabidopsis thaliana as a model system., Method: A liquid chromatography-tandem mass spectrometry with electrospray ionization was developed to quantify phenylpropanoid metabolites in Arabidopsis wildtype and cinnamoyl CoA reductase1 (CCR1) deficient lines (ccr1)., Results: Vortexing at high temperatures (65°C) enhanced release of phenylpropanoids, specifically the more hydrophobic compounds. A pH of 5.3 and ammonium acetate buffer concentration of 2.5 mM resulted in an optimal analyte response across standards. Ion suppression was estimated using standard spike recovery studies for accurate quantitation. The optimized method was used to profile Arabidopsis wildtype and ccr1 stems. An increase in hydroxycinnamic acid derivatives and a decrease in the hydroxycinnamyl aldehydes and alcohols in ccr1 lines, supports a shift of flux from lignin synthesis to other secondary metabolites and phenylpropanoid derivatives., Conclusions: Compared to existing targeted profiling techniques, our method is capable of quantifying a wider range of intermediates (15 out of 22 in WT Arabidopsis stems) at low in vivo concentrations (~50 pmol/g-FW for certain compounds), while requiring minimal sample preparation. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Plants synthesize a diversity of volatile molecules that are important for reproduction and defense, serve as practical products for humans, and influence atmospheric chemistry and climate. Despite progress in deciphering plant volatile biosynthesis, their release from the cell has been poorly understood. The default assumption has been that volatiles passively diffuse out of cells. By characterization of a Petunia hybrida adenosine triphosphate-binding cassette (ABC) transporter, PhABCG1, we demonstrate that passage of volatiles across the plasma membrane relies on active transport. PhABCG1 down-regulation by RNA interference results in decreased emission of volatiles, which accumulate to toxic levels in the plasma membrane. This study provides direct proof of a biologically mediated mechanism of volatile emission., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In plants, geranylgeranyl diphosphate (GGPP) is produced by plastidic GGPP synthase (GGPPS) and serves as a precursor for vital metabolic branches, including chlorophyll, carotenoid, and gibberellin biosynthesis. However, molecular mechanisms regulating GGPP allocation among these biosynthetic pathways localized in the same subcellular compartment are largely unknown. We found that rice contains only one functionally active GGPPS, OsGGPPS1, in chloroplasts. A functionally active homodimeric enzyme composed of two OsGGPPS1 subunits is located in the stroma. In thylakoid membranes, however, the GGPPS activity resides in a heterodimeric enzyme composed of one OsGGPPS1 subunit and GGPPS recruiting protein (OsGRP). OsGRP is structurally most similar to members of the geranyl diphosphate synthase small subunit type II subfamily. In contrast to members of this subfamily, OsGRP enhances OsGGPPS1 catalytic efficiency and specificity of GGPP production on interaction with OsGGPPS1. Structural biology and protein interaction analyses demonstrate that affinity between OsGRP and OsGGPPS1 is stronger than between two OsGGPPS1 molecules in homodimers. OsGRP determines OsGGPPS1 suborganellar localization and directs it to a large protein complex in thylakoid membranes, consisting of geranylgeranyl reductase (OsGGR), light-harvesting-like protein 3 (OsLIL3), protochlorophyllide oxidoreductase (OsPORB), and chlorophyll synthase (OsCHLG). Taken together, genetic and biochemical analyses suggest OsGRP functions in recruiting OsGGPPS1 from the stroma toward thylakoid membranes, thus providing a mechanism to control GGPP flux toward chlorophyll biosynthesis., Competing Interests: The authors declare no conflict of interest.

Synthetic plant volatile lures attract natural enemies, but may have non-target effects due to the multifunctional nature of volatile signals. For example, methyl salicylate (MeSA) is used to attract predators, yet also serves as a signaling hormone involved in plant pathogen defense. We investigated the consequences of deploying MeSA lures to attract predators for tomato (Solanum lycopersicum) defense against herbivores. To understand the spatial distribution of the lure's effect, we exposed tomatoes in the field to MeSA along a linear distance gradient and induced defenses by simulating feeding by hornworm caterpillars in a fully crossed factorial design (+/- MeSA, +/- herbivory). Subsequently, we analyzed activity of several defensive proteins (protease inhibitors, polyphenol oxidase, peroxidase), development of hornworm larvae (Manduca sexta), growth of fungal pathogens (Cladosporium and Alternaria), and attractiveness to herbivores and predators. Overall, MeSA-exposed plants were more resistant to both insects and pathogens. Secondary pathogen infection was reduced by 25% in MeSA exposed plants, possibly due to elevated polyphenol oxidase activity. Interestingly, we found that lures affected plant pathogen defenses equivalently across all distances (up to 4 m away) indicating that horizontal diffusion of a synthetic volatile may be greater than previously assumed. While thrips avoided colonizing hornworm- damaged tomato plants, this induced resistance was not observed upon pre-exposure to MeSA, suggesting that MeSA suppresses the repellant effect induced by herbivory. Thus, using MeSA lures in biological control may inadvertently protect crops from pathogens, but has mixed effects on plant resistance to insect herbivores.

Certain insect species can induce gall formation on numerous plants species. Although the mechanism of gall development is largely unknown, it is clear that insects manipulate their hosts' anatomy, physiology, and chemistry for their own benefit. It is well known that insect-induced galls often contain vast amounts of plant defensive compounds as compared to non-colonized tissues, but it is not clear if defensive compounds can be produced in situ in the galled tissues. To answer this question, we analyzed terpene accumulation patterns and possible independent biosynthetic potential of galls induced by the aphid Baizongia pistaciae L. on the terminal buds of Pistacia palaestina Boiss. We compared monoterpene levels and monoterpene synthase enzyme activity in galls and healthy leaves from individual trees growing in a natural setting. At all developmental stages, monoterpene content and monoterpene synthase activity were consistently (up to 10 fold on a fresh weight basis) higher in galls than in intact non-colonized leaves. A remarkable tree to tree variation in the products produced in vitro from the substrate geranyl diphosphate by soluble protein extracts derived from individual trees was observed. Furthermore, galls and leaves from the same trees displayed enhanced and often distinct biosynthetic capabilities. Our results clearly indicate that galls possess independent metabolic capacities to produce and accumulate monoterpenes as compared to leaves. Our study indicates that galling aphids manipulate the enzymatic machinery of their host plant, intensifying their own defenses against natural enemies.