Reinventing metabolic pathways: Independent evolution of benzoxazinoids in flowering plants.

Benzoxazinoids (BXDs) form a class of indole-derived specialized plant metabolites with broad antimicrobial and antifeedant properties. Unlike most specialized metabolites, which are typically lineage-specific, BXDs occur sporadically in a number of distantly related plant orders. This observation suggests that BXD biosynthesis arose independently numerous times in the plant kingdom. However, although decades of research in the grasses have led to the elucidation of the BXD pathway in the monocots, the biosynthesis of BXDs in eudicots is unknown. Here, we used a metabolomic and transcriptomic-guided approach, in combination with pathway reconstitution in Nicotiana benthamiana, to identify and characterize the BXD biosynthetic pathways from both Aphelandra squarrosa and Lamium galeobdolon, two phylogenetically distant eudicot species. We show that BXD biosynthesis in A. squarrosa and L. galeobdolon utilize a dual-function flavin-containing monooxygenase in place of two distinct cytochrome P450s, as is the case in the grasses. In addition, we identified evolutionarily unrelated cytochrome P450s, a 2-oxoglutarate-dependent dioxygenase, a UDP-glucosyltransferase, and a methyltransferase that were also recruited into these BXD biosynthetic pathways. Our findings constitute the discovery of BXD pathways in eudicots. Moreover, the biosynthetic enzymes of these pathways clearly demonstrate that BXDs independently arose in the plant kingdom at least three times. The heterogeneous pool of identified BXD enzymes represents a remarkable example of metabolic plasticity, in which BXDs are synthesized according to a similar chemical logic, but with an entirely different set of metabolic enzymes.

Origin and early evolution of the plant terpene synthase family.

As a midsized gene family conserved more by lineage than function, the typical plant terpene synthases (TPSs) could be a valuable tool to examine plant evolution. TPSs are pivotal in biosynthesis of gibberellins and related phytohormones as well as in formation of the extensive arsenal of specialized plant metabolites mediating ecological interactions whose production is often lineage specific. Yet the origin and early evolution of the TPS family is not well understood. Systematic analysis of an array of transcriptomes and sequenced genomes indicated that the TPS family originated after the divergence of land plants from charophytic algae. Phylogenetic and biochemical analyses support the hypothesis that the ancestral TPS gene encoded a bifunctional class I and II diterpene synthase producing the ent-kaurene required for phytohormone production in all extant lineages of land plants. Moreover, the ancestral TPS gene likely underwent duplication at least twice early in land plant evolution. Together these two gave rise to three TPS lineages leading to the extant TPS-c, TPS-e/f, and the remaining TPS (h/d/a/b/g) subfamilies, with the latter dedicated to secondary rather than primary metabolism while the former two contain those genes involved in ent-kaurene production. Nevertheless, parallel evolution from the ent-kaurene­producing class I and class II diterpene synthases has led to roles for TPS-e/f and -c subfamily members in secondary metabolism as well. These results clarify TPS evolutionary history and provide context for the role of these genes in producing the vast diversity of terpenoid natural products observed today in various land plant lineages.

Biosynthesis of iridoid sex pheromones in aphids.

Iridoid monoterpenes, widely distributed in plants and insects, have many ecological functions. While the biosynthesis of iridoids has been extensively studied in plants, little is known about how insects synthesize these natural products. Here, we elucidated the biosynthesis of the iridoids cis-trans-nepetalactol and cis-trans-nepetalactone in the pea aphid Acyrthosiphon pisum (Harris), where they act as sex pheromones. The exclusive production of iridoids in hind legs of sexual female aphids allowed us to identify iridoid genes by searching for genes specifically expressed in this tissue. Biochemical characterization of candidate enzymes revealed that the iridoid pathway in aphids proceeds through the same sequence of intermediates as described for plants. The six identified aphid enzymes are unrelated to their counterparts in plants, conclusively demonstrating an independent evolution of the entire iridoid pathway in plants and insects. In contrast to the plant pathway, at least three of the aphid iridoid enzymes are likely membrane bound. We demonstrated that a lipid environment facilitates the cyclization of a reactive enol intermediate to the iridoid cyclopentanoid-pyran scaffold in vitro, suggesting that membranes are an essential component of the aphid iridoid pathway. Altogether, our discovery of this complex insect metabolic pathway establishes the genetic and biochemical basis for the formation of iridoid sex pheromones in aphids, and this discovery also serves as a foundation for understanding the convergent evolution of complex metabolic pathways between kingdoms.

Functional and Mechanistic Characterization of the 4,5-diepi-Isoishwarane Synthase from the Liverwort Radula lindenbergiana.

The microbial type sesquiterpene synthase RlMTPSL4 from the liverwort Radula lindenbergiana was investigated for its products, showing the formation of several sesquiterpene hydrocarbons. The main product was structurally characterized as the new compound 4,5-diepi-isoishwarane, while the side products included the known hydrocarbons germacrene A, α-selinene, eremophilene and 4,5-diepi-aristolochene. The cyclization mechanism towards 4,5-diepi-isoishwarane catalyzed by RlMTPSL4 was investigated through isotopic labeling experiments, revealing the stereochemical course for the deprotonation step to the neutral intermediate germacrene A, a reprotonation for its further cyclization, and a 1,2-hydride shift along the cascade. The absolute configuration of 4,5-diepi-isoishwarane was determined using a stereoselective deuteration approach, revealing an absolute configuration typically observed for a microbial type sesquiterpene.

HDR, the last enzyme in the MEP pathway, differently regulates isoprenoid biosynthesis in two woody plants.

Dimethylallyl diphosphate (DMADP) and isopentenyl diphosphate (IDP) serves as the universal C5 precursors of isoprenoid biosynthesis in plants. These compounds are formed by the last step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, catalyzed by (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR). In this study, we investigated the major HDR isoforms of two woody plant species, Norway spruce (Picea abies) and gray poplar (Populus × canescens), to determine how they regulate isoprenoid formation. Since each of these species has a distinct profile of isoprenoid compounds, they may require different proportions of DMADP and IDP with proportionally more IDP being needed to make larger isoprenoids. Norway spruce contained two major HDR isoforms differing in their occurrence and biochemical characteristics. PaHDR1 produced relatively more IDP than PaHDR2 and it encoding gene was expressed constitutively in leaves, likely serving to form substrate for production of carotenoids, chlorophylls, and other primary isoprenoids derived from a C20 precursor. On the other hand, Norway spruce PaHDR2 produced relatively more DMADP than PaHDR1 and its encoding gene was expressed in leaves, stems, and roots, both constitutively and after induction with the defense hormone methyl jasmonate. This second HDR enzyme likely forms a substrate for the specialized monoterpene (C10), sesquiterpene (C15), and diterpene (C20) metabolites of spruce oleoresin. Gray poplar contained only one dominant isoform (named PcHDR2) that produced relatively more DMADP and the gene of which was expressed in all organs. In leaves, where the requirement for IDP is high to make the major carotenoid and chlorophyll isoprenoids derived from C20 precursors, excess DMADP may accumulate, which could explain the high rate of isoprene (C5) emission. Our results provide new insights into the biosynthesis of isoprenoids in woody plants under conditions of differentially regulated biosynthesis of the precursors IDP and DMADP.

Biosynthesis, herbivore induction, and defensive role of phenylacetaldoxime glucoside.

Aldoximes are well-known metabolic precursors for plant defense compounds such as cyanogenic glycosides, glucosinolates, and volatile nitriles. They are also defenses themselves produced in response to herbivory; however, it is unclear whether aldoximes can be stored over a longer term as defense compounds and how plants protect themselves against the potential autotoxic effects of aldoximes. Here, we show that the Neotropical myrmecophyte tococa (Tococa quadrialata, recently renamed Miconia microphysca) accumulates phenylacetaldoxime glucoside (PAOx-Glc) in response to leaf herbivory. Sequence comparison, transcriptomic analysis, and heterologous expression revealed that 2 cytochrome P450 enzymes, CYP79A206 and CYP79A207, and the UDP-glucosyltransferase UGT85A123 are involved in the formation of PAOx-Glc in tococa. Another P450, CYP71E76, was shown to convert PAOx to the volatile defense compound benzyl cyanide. The formation of PAOx-Glc and PAOx in leaves is a very local response to herbivory but does not appear to be regulated by jasmonic acid signaling. In contrast to PAOx, which was only detectable during herbivory, PAOx-Glc levels remained high for at least 3 d after insect feeding. This, together with the fact that gut protein extracts of 3 insect herbivore species exhibited hydrolytic activity toward PAOx-Glc, suggests that the glucoside is a stable storage form of a defense compound that may provide rapid protection against future herbivory. Moreover, the finding that herbivory or pathogen elicitor treatment also led to the accumulation of PAOx-Glc in 3 other phylogenetically distant plant species suggests that the formation and storage of aldoxime glucosides may represent a widespread plant defense response.

Mechanistic characterisation of a sesquiterpene synthase for asterisca-1,6-diene from the liverwort Radula lindenbergiana and implications for pentalenene biosynthesis.

A sesquiterpene synthase from the liverwort Radula lindenbergiana was characterised and shown to produce the new sesquiterpene hydrocarbon (3R,9R)-asterisca-1,6-diene, besides small amounts of pentalenene. The biosynthesis of asterisca-1,6-diene was studied through isotopic labelling experiments, giving additional insights into the long discussed biosynthesis of pentalenene.

Predicting functions of putative fungal sesquiterpene synthase genes based on multiomics data analysis.

Sesquiterpenes (STs) are secondary metabolites, which mediate biotic interactions between different organisms. Predicting the species-specific ST repertoires can contribute to deciphering the language of communication between organisms of the same or different species. High biochemical plasticity and catalytic promiscuity of sesquiterpene synthases (STSs), however, challenge the homology-based prediction of the STS functions. Using integrated analyses of genomic, transcriptomic, volatilomic, and metabolomic data, we predict product profiles for 116 out of 146 putative STS genes identified in the genomes of 30 fungal species from different trophic groups. Our prediction method is based on the observation that STSs encoded by genes closely related phylogenetically are likely to share the initial enzymatic reactions of the ST biosynthesis pathways and, therefore, produce STs via the same reaction route. The classification by reaction routes allows to assign STs known to be emitted by a particular species to the putative STS genes from this species. Gene expression information helps to further specify these ST-to-STS assignments. Validation of the computational predictions of the STS functions using both in silico and experimental approaches shows that integrated multiomic analyses are able to correctly link cyclic STs of non-cadalane type to genes. In the process of the experimental validation, we characterized catalytic properties of several putative STS genes from the mycorrhizal fungus Laccaria bicolor. We show that the STSs encoded by the L.bicolor mycorrhiza-induced genes emit either nerolidol or α-cuprenene and α-cuparene, and discuss the possible roles of these STs in the mycorrhiza formation.

Quantifying chemodiversity considering biochemical and structural properties of compounds with the R package chemodiv.

Plants produce large numbers of phytochemical compounds affecting plant physiology and interactions with their biotic and abiotic environment. Recently, chemodiversity has attracted considerable attention as an ecologically and evolutionary meaningful way to characterize the phenotype of a mixture of phytochemical compounds. Currently used measures of phytochemical diversity, and related measures of phytochemical dissimilarity, generally do not take structural or biosynthetic properties of compounds into account. Such properties can be indicative of the compounds' function and inform about their biosynthetic (in)dependence, and should therefore be included in calculations of these measures. We introduce the R package chemodiv, which retrieves biochemical and structural properties of compounds from databases and provides functions for calculating and visualizing chemical diversity and dissimilarity for phytochemicals and other types of compounds. Our package enables calculations of diversity that takes the richness, relative abundance and - most importantly - structural and/or biosynthetic dissimilarity of compounds into account. We illustrate the use of the package with examples on simulated and real datasets. By providing the R package chemodiv for quantifying multiple aspects of chemodiversity, we hope to facilitate investigations of how chemodiversity varies across levels of biological organization, and its importance for the ecology and evolution of plants and other organisms.

Biosynthesis and antifungal activity of fungus-induced O-methylated flavonoids in maize.

Fungal infection of grasses, including rice (Oryza sativa), sorghum (Sorghum bicolor), and barley (Hordeum vulgare), induces the formation and accumulation of flavonoid phytoalexins. In maize (Zea mays), however, investigators have emphasized benzoxazinoid and terpenoid phytoalexins, and comparatively little is known about flavonoid induction in response to pathogens. Here, we examined fungus-elicited flavonoid metabolism in maize and identified key biosynthetic enzymes involved in the formation of O-methylflavonoids. The predominant end products were identified as two tautomers of a 2-hydroxynaringenin-derived compound termed xilonenin, which significantly inhibited the growth of two maize pathogens, Fusarium graminearum and Fusarium verticillioides. Among the biosynthetic enzymes identified were two O-methyltransferases (OMTs), flavonoid OMT 2 (FOMT2), and FOMT4, which demonstrated distinct regiospecificity on a broad spectrum of flavonoid classes. In addition, a cytochrome P450 monooxygenase (CYP) in the CYP93G subfamily was found to serve as a flavanone 2-hydroxylase providing the substrate for FOMT2-catalyzed formation of xilonenin. In summary, maize produces a diverse blend of O-methylflavonoids with antifungal activity upon attack by a broad range of fungi.

A peroxisomal ß-oxidative pathway contributes to the formation of C6-C1 aromatic volatiles in poplar.

Benzenoids (C6-C1 aromatic compounds) play important roles in plant defense and are often produced upon herbivory. Black cottonwood (Populus trichocarpa) produces a variety of volatile and nonvolatile benzenoids involved in various defense responses. However, their biosynthesis in poplar is mainly unresolved. We showed feeding of the poplar leaf beetle (Chrysomela populi) on P. trichocarpa leaves led to increased emission of the benzenoid volatiles benzaldehyde, benzylalcohol, and benzyl benzoate. The accumulation of salicinoids, a group of nonvolatile phenolic defense glycosides composed in part of benzenoid units, was hardly affected by beetle herbivory. In planta labeling experiments revealed that volatile and nonvolatile poplar benzenoids are produced from cinnamic acid (C6-C3). The biosynthesis of C6-C1 aromatic compounds from cinnamic acid has been described in petunia (Petunia hybrida) flowers where the pathway includes a peroxisomal-localized chain shortening sequence, involving cinnamate-CoA ligase (CNL), cinnamoyl-CoA hydratase/dehydrogenase (CHD), and 3-ketoacyl-CoA thiolase (KAT). Sequence and phylogenetic analysis enabled the identification of small CNL, CHD, and KAT gene families in P. trichocarpa. Heterologous expression of the candidate genes in Escherichia coli and characterization of purified proteins in vitro revealed enzymatic activities similar to those described in petunia flowers. RNA interference-mediated knockdown of the CNL subfamily in gray poplar (Populus x canescens) resulted in decreased emission of C6-C1 aromatic volatiles upon herbivory, while constitutively accumulating salicinoids were not affected. This indicates the peroxisomal ß-oxidative pathway participates in the formation of volatile benzenoids. The chain shortening steps for salicinoids, however, likely employ an alternative pathway.

An unbiased approach elucidates variation in (S)-(+)-linalool, a context-specific mediator of a tri-trophic interaction in wild tobacco.

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.

Evolution of DIMBOA-Glc O-Methyltransferases from Flavonoid O-Methyltransferases in the Grasses.

O-Methylated benzoxazinoids (BXs) and flavonoids are widespread defenses against herbivores and pathogens in the grasses (Poaceae). Recently, two flavonoid O-methyltransferases (FOMTs), ZmFOMT2 and ZmFOMT3, have been reported to produce phytoalexins in maize (Zea mays). ZmFOMT2 and ZmFOMT3 are closely related to the BX O-methyltransferases (OMTs) ZmBX10-12 and ZmBX14, suggesting a common evolutionary origin in the Poaceae. Here, we studied the evolution and enzymatic requirements of flavonoid and BX O-methylation activities in more detail. Using BLAST searches and phylogenetic analyses, we identified enzymes homologous to ZmFOMT2 and ZmFOMT3, ZmBX10-12, and ZmBX14 in several grasses, with the most closely related candidates found almost exclusively in species of the Panicoideae subfamily. Biochemical characterization of candidate enzymes from sorghum (Sorghum bicolor), sugar cane (Saccharum spp.), and teosinte (Zea nicaraguensis) revealed either flavonoid 5-O-methylation activity or DIMBOA-Glc 4-O-methylation activity. However, DIMBOA-Glc 4-OMTs from maize and teosinte also accepted flavonols as substrates and converted them to 3-O-methylated derivatives, suggesting an evolutionary relationship between these two activities. Homology modeling, sequence comparisons, and site-directed mutagenesis led to the identification of active site residues crucial for FOMT and BX OMT activity. However, the full conversion of ZmFOMT2 activity into BX OMT activity by switching these residues was not successful. Only trace O-methylation of BXs was observed, indicating that amino acids outside the active site cavity are also involved in determining the different substrate specificities. Altogether, the results of our study suggest that BX OMTs have evolved from the ubiquitous FOMTs in the PACMAD clade of the grasses through a complex series of amino acid changes.

Evolution of a Novel and Adaptive Floral Scent in Wild Tobacco.

Many plants emit diverse floral scents that mediate plant-environment interactions and attain reproductive success. However, how plants evolve novel and adaptive biosynthetic pathways for floral volatiles remains unclear. Here, we show that in the wild tobacco, Nicotiana attenuata, a dominant species-specific floral volatile (benzyl acetone, BA) that attracts pollinators and deters florivore is synthesized by phenylalanine ammonia-lyase 4 (NaPAL4), isoflavone reductase 3 (NaIFR3), and chalcone synthase 3 (NaCHAL3). Transient expression of NaFIR3 alone in N. attenuata leaves is sufficient and necessary for ectopic foliar BA emissions, and coexpressing NaIFR3 with NaPAL4 and NaCHAL3 increased the BA emission levels. Independent changes in transcription of NaPAL4 and NaCHAL3 contributed to intraspecific variations of floral BA emission. However, among species, the gain of expression of NaIFR3 resulted in the biosynthesis of BA, which was only found in N. attenuata. This study suggests that novel metabolic pathways associated with adaptation can arise via reconfigurations of gene expression.

Poplar protease inhibitor expression differs in an herbivore specific manner.

BACKGROUND: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar (P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles. RESULTS: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes. CONCLUSIONS: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Combinatorial Evolution of a Terpene Synthase Gene Cluster Explains Terpene Variations in Oryza.

Terpenes are specialized metabolites ubiquitously produced by plants via the action of terpene synthases (TPSs). There are enormous variations in the types and amounts of terpenes produced by individual species. To understand the mechanisms responsible for such vast diversity, here we investigated the origin and evolution of a cluster of tandemly arrayed TPS genes in Oryza In the Oryza species analyzed, TPS genes occur as a three-TPS cluster, a two-TPS cluster, and a single TPS gene in five, one, and one species, respectively. Phylogenetic analysis revealed the origins of the two-TPS and three-TPS clusters and the role of species-specific losses of TPS genes. Within the three-TPS clusters, one orthologous group exhibited conserved catalytic activities. The other two groups, both of which contained pseudogenes and/or nonfunctional genes, exhibited distinct profiles of terpene products. Sequence and structural analyses combined with functional validation identified several amino acids in the active site that are critical for catalytic activity divergence of the three orthologous groups. In the five Oryza species containing the three-TPS cluster, their functional TPS genes showed both conserved and species-specific expression patterns in insect-damaged and untreated plants. Emission patterns of volatile terpenes from each species were largely consistent with the expression of their respective TPS genes and the catalytic activities of the encoded enzymes. This study indicates the importance of combinatorial evolution of TPS genes in determining terpene variations among individual species, which includes gene duplication, retention/loss/degradation of duplicated genes, varying selection pressure, retention/divergence in catalytic activities, and divergence in expression regulation.

The Occurrence of Sulfated Salicinoids in Poplar and Their Formation by Sulfotransferase1.

Salicinoids form a specific class of phenolic glycosides characteristic of the Salicaceae. Although salicinoids accumulate in large amounts and have been shown to be involved in plant defense, their biosynthesis is unclear. We identified two sulfated salicinoids, salicin-7-sulfate and salirepin-7-sulfate, in black cottonwood (Populus trichocarpa). Both compounds accumulated in high amounts in above-ground tissues including leaves, petioles, and stems, but were also found at lower concentrations in roots. A survey of salicin-7-sulfate and salirepin-7-sulfate in a subset of poplar (Populus sp.) and willow (Salix sp.) species revealed a broader distribution within the Salicaceae. To elucidate the formation of these compounds, we studied the sulfotransferase (SOT) gene family in P trichocarpa (PtSOT). One of the identified genes, PtSOT1, was shown to encode an enzyme able to convert salicin and salirepin into salicin-7-sulfate and salirepin-7-sulfate, respectively. The expression of PtSOT1 in different organs of P trichocarpa matched the accumulation of sulfated salicinoids in planta. Moreover, RNA interference-mediated knockdown of SOT1 in gray poplar (Populus × canescens) resulted in decreased levels of sulfated salicinoids in comparison to wild-type plants, indicating that SOT1 is responsible for their formation in planta. The presence of a nonfunctional SOT1 allele in black poplar (Populus nigra) was shown to correlate with the absence of salicin-7-sulfate and salirepin-7-sulfate in this species. Food choice experiments with leaves from wild-type and SOT1 knockdown trees suggest that sulfated salicinoids do not affect the feeding preference of the generalist caterpillar Lymantria dispar A potential role of the sulfated salicinoids in sulfur storage and homeostasis is discussed.

Phylogeny and abiotic conditions shape the diel floral emission patterns of desert Brassicaceae species.

A key facet of floral scent is diel fluctuations in emission, often studied in the context of plant-pollinator interactions, while contributions of environment and phylogeny remain overlooked. Here, we ask if these factors are involved in shaping temporal variations in scent emission. To that end, we coupled light/dark floral emission measurements of 17 desert Brassicaceae species with environmental and phylogenetic data to explore the individual/combined impacts of these predictors on diel emission patterns. We further investigated these patterns by conducting high-resolution emission measurements in a subset of genetically distant species with contrasting temporal dynamics. While diel shifts in magnitude and richness of emission were strongly affected by genetic relatedness, they also reflect the environmental conditions under which the species grow. Specifically, light/dark emission ratios were negatively affected by an increase in winter temperatures, known to impact both plant physiology and insect locomotion, and sandy soil fractions, previously shown to exert stress that tempers with diel metabolic rhythms. Additionally, the biosynthetic origins of the compounds were associated with their corresponding production patterns, possibly to maximize emission efficacy. Using a multidisciplinary chemical/ecological approach, we uncover and differentiate the main factors shaping floral scent diel fluctuations, highlighting their consequences under changing global climate.

Growing up aspen: ontogeny and trade-offs shape growth, defence and reproduction in a foundation species.

BACKGROUND AND AIMS: Intraspecific variation in foundation species of forest ecosystems can shape community and ecosystem properties, particularly when that variation has a genetic basis. Traits mediating interactions with other species are predicted by simple allocation models to follow ontogenetic patterns that are rarely studied in trees. The aim of this research was to identify the roles of genotype, ontogeny and genotypic trade-offs shaping growth, defence and reproduction in aspen. METHODS: We established a common garden replicating >500 aspen genets in Wisconsin, USA. Trees were measured through the juvenile period into the onset of reproduction, for growth, defence chemistry (phenolic glycosides and condensed tannins), nitrogen, extrafloral nectaries, leaf morphology (specific leaf area), flower production and foliar herbivory and disease. We also assayed the TOZ19 sex marker and heterozygosity at ten microsatellite loci. KEY RESULTS: We found high levels of genotypic variation for all traits, and high heritabilities for both the traits and their ontogenetic trajectories. Ontogeny strongly shaped intraspecific variation, and trade-offs among growth, defence and reproduction supported some predictions while contradicting others. Both direct resistance (chemical defence) and indirect defence (extrafloral nectaries) declined during the juvenile stage, prior to the onset of reproduction. Reproduction was higher in trees that were larger, male and had higher individual heterozygosity. Growth was diminished by genotypic allocation to both direct and indirect defence as well as to reproduction, but we found no evidence of trade-offs between defence and reproduction. CONCLUSIONS: Key traits affecting the ecological communities of aspen have high levels of genotypic variation and heritability, strong patterns of ontogeny and clear trade-offs among growth, defence and reproduction. The architecture of aspen's community genetics - its ontogeny, trade-offs and especially its great variability - is shaped by both its broad range and the diverse community of associates, and in turn further fosters that diversity.

Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthases from Dictyostelium purpureum and Streptomyces coelicolor.

The main product of DpTPS9 from the social amoeba Dictyostelium purpureum was identified as (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol that is also known as an intermediate of bacterial geosmin synthase, but the experimentally verified cyclisation mechanisms differ. Together with the low sequence identity this points to convergent evolution. The functionality of selected residues in DpTPS9 was investigated via site-directed mutagenesis experiments.