Variation of terpene alkaloids in Daphniphyllum macropodum across plants and tissues.

Daphniphyllum macropodum produces alkaloids that are structurally complex with polycyclic, stereochemically rich carbon skeletons. Understanding how these compounds are formed by the plant may enable exploration of their biological function and bioactivities. We employed multiple metabolomics techniques, including a workflow to annotate compounds in the absence of standards, to compare alkaloid content across plants and tissues. Different alkaloid structural types were found to have distinct distributions between genotypes, between tissues and within tissues. Alkaloid structural types also showed different isotope labelling enrichments that matched their biosynthetic relationships. The work suggests that mevalonate derived 30-carbon alkaloids are formed in the phloem region before their conversion to 22-carbon alkaloids which accumulate in the epidermis. This sets the stage for further investigation into the biosynthetic pathway.

Plant glucosinolate biosynthesis and breakdown pathways shape the rhizosphere bacterial/archaeal community.

Rhizosphere microbial community assembly results from microbe-microbe-plant interactions mediated by small molecules of plant and microbial origin. Studies with Arabidopsis thaliana have indicated a critical role of glucosinolates in shaping the root and/or rhizosphere microbial community, likely through breakdown products produced by plant or microbial myrosinases inside or outside of the root. Plant nitrile-specifier proteins (NSPs) promote the formation of nitriles at the expense of isothiocyanates upon glucosinolate hydrolysis with unknown consequences for microbial colonisation of roots and rhizosphere. Here, we generated the A. thaliana triple mutant nsp134 devoid of nitrile formation in root homogenates. Using this line and mutants lacking aliphatic or indole glucosinolate biosynthesis pathways or both, we found bacterial/archaeal alpha-diversity of the rhizosphere to be affected only by the ability to produce aliphatic glucosinolates. In contrast, bacterial/archaeal community composition depended on functional root NSPs as well as on pathways of aliphatic and indole glucosinolate biosynthesis. Effects of NSP deficiency were strikingly distinct from those of impaired glucosinolate biosynthesis. Our results demonstrate that rhizosphere microbial community assembly depends on functional pathways of both glucosinolate biosynthesis and breakdown in support of the hypothesis that glucosinolate hydrolysis by myrosinases and NSPs happens before secretion of products to the rhizosphere.

Are seven amino acid substitutions sufficient to explain the evolution of high l-DOPA 4,5-dioxygenase activity leading to betalain pigmentation? Revisiting the gain-of-function mutants of Bean et al. (2018).

This work revisits a publication by Bean et al. (2018) that reports seven amino acid substitutions are essential for the evolution of l-DOPA 4,5-dioxygenase (DODA) activity in Caryophyllales. In this study, we explore several concerns which led us to replicate the analyses of Bean et al. (2018). Our comparative analyses, with structural modelling, implicate numerous residues additional to those identified by Bean et al. (2018), with many of these additional residues occurring around the active site of BvDODAα1. We therefore replicated the analyses of Bean et al. (2018) to re-observe the effect of their original seven residue substitutions in a BvDODAα2 background, that is the BvDODAα2-mut3 variant. Multiple in vivo assays, in both Saccharomyces cerevisiae and Nicotiana benthamiana, did not result in visible DODA activity in BvDODAα2-mut3, with betalain production always 10-fold below BvDODAα1. In vitro assays also revealed substantial differences in both catalytic activity and pH optima between BvDODAα1, BvDODAα2 and BvDODAα2-mut3 proteins, explaining their differing performance in vivo. In summary, we were unable to replicate the in vivo analyses of Bean et al. (2018), and our quantitative in vivo and in vitro analyses suggest a minimal effect of these seven residues in altering catalytic activity of BvDODAα2. We conclude that the evolutionary pathway to high DODA activity is substantially more complex than implied by Bean et al. (2018).

Ecological generalism drives hyperdiversity of secondary metabolite gene clusters in xylarialean endophytes.

Although secondary metabolites are typically associated with competitive or pathogenic interactions, the high bioactivity of endophytic fungi in the Xylariales, coupled with their abundance and broad host ranges spanning all lineages of land plants and lichens, suggests that enhanced secondary metabolism might facilitate symbioses with phylogenetically diverse hosts. Here, we examined secondary metabolite gene clusters (SMGCs) across 96 Xylariales genomes in two clades (Xylariaceae s.l. and Hypoxylaceae), including 88 newly sequenced genomes of endophytes and closely related saprotrophs and pathogens. We paired genomic data with extensive metadata on endophyte hosts and substrates, enabling us to examine genomic factors related to the breadth of symbiotic interactions and ecological roles. All genomes contain hyperabundant SMGCs; however, Xylariaceae have increased numbers of gene duplications, horizontal gene transfers (HGTs) and SMGCs. Enhanced metabolic diversity of endophytes is associated with a greater diversity of hosts and increased capacity for lignocellulose decomposition. Our results suggest that, as host and substrate generalists, Xylariaceae endophytes experience greater selection to diversify SMGCs compared with more ecologically specialised Hypoxylaceae species. Overall, our results provide new evidence that SMGCs may facilitate symbiosis with phylogenetically diverse hosts, highlighting the importance of microbial symbioses to drive fungal metabolic diversity.

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

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

Evolution of l-DOPA 4,5-dioxygenase activity allows for recurrent specialisation to betalain pigmentation in Caryophyllales.

The evolution of l-DOPA 4,5-dioxygenase activity, encoded by the gene DODA, was a key step in the origin of betalain biosynthesis in Caryophyllales. We previously proposed that l-DOPA 4,5-dioxygenase activity evolved via a single Caryophyllales-specific neofunctionalisation event within the DODA gene lineage. However, this neofunctionalisation event has not been confirmed and the DODA gene lineage exhibits numerous gene duplication events, whose evolutionary significance is unclear. To address this, we functionally characterised 23 distinct DODA proteins for l-DOPA 4,5-dioxygenase activity, from four betalain-pigmented and five anthocyanin-pigmented species, representing key evolutionary transitions across Caryophyllales. By mapping these functional data to an updated DODA phylogeny, we then explored the evolution of l-DOPA 4,5-dioxygenase activity. We find that low l-DOPA 4,5-dioxygenase activity is distributed across the DODA gene lineage. In this context, repeated gene duplication events within the DODA gene lineage give rise to polyphyletic occurrences of elevated l-DOPA 4,5-dioxygenase activity, accompanied by convergent shifts in key functional residues and distinct genomic patterns of micro-synteny. In the context of an updated organismal phylogeny and newly inferred pigment reconstructions, we argue that repeated convergent acquisition of elevated l-DOPA 4,5-dioxygenase activity is consistent with recurrent specialisation to betalain synthesis in Caryophyllales.

Detailed Profiling of 17-Hydroxygeranyllinalool Diterpene Glycosides from Nicotiana Species Reveals Complex Reaction Networks of Conjugation Isomers.

BACKGROUND: Specialised anti-herbivory metabolites are abundant in the solanaceous genus Nicotiana. These metabolites include the large family of 17-hydroxygeranyllinalool diterpene glycosides (HGL-DTGs). Many HGL-DTGs occur exclusively within the Nicotiana genus, but information from the molecular model species N. tabacum, N. benthamiana, and the tree tobacco N. glauca is limited. OBJECTIVES: We studied HGL-DTG occurrence and complexity in these species with the aim of providing in-depth reference annotations and comprehensive HGL-DTG inventories. METHODS: We analysed polar metabolite extracts in comparison to the previously investigated wild reference species N. attenuata using positive ESI(+) and negative ESI(-) mode electrospray ionisation LC-MS and MS/MS. RESULTS: We provide annotations of 66 HGL-DTGs with in-source and MS/MS fragmentation spectra for selected HGL-DTGs with exemplary fragment interpretations of ESI(+) as well as less studied ESI(-) spectra. We assemble a potential biosynthesis pathway comparing the presence of HGL-DTGs in N. tabacum, N. glauca, and N. benthamiana to N. attenuata. Approximately one-third of HGL-DTGs are chromatographically resolved isomers of hexose, deoxyhexose, or malonate conjugates. The number of isomers is especially high for conjugates with low numbers of deoxyhexose moieties. CONCLUSIONS: We extend the number of known HGL-DTGs with a focus on Nicotiana model species and demonstrate that the HGL-DTG family of N. tabacum plants can be surprisingly complex. Our study provides an improved basis with detailed references to previous studies of wild Nicotiana species and enables inference of HGL-DTG pathways with required enzymes for the biosynthesis of this important family of specialised defence metabolites.

Design and validation of a PCR screen for γ-butyrolactone-like regulatory systems in Streptomyces.

γ-butyrolactone and related signalling systems are found in Streptomyces and other actinobacteria where they control the production of secondary or specialized metabolites such as antibiotics. Genetic manipulation of these regulatory systems therefore leads to changes in the secondary metabolite profile of a strain and has been used to activate previously silent secondary metabolite gene clusters. However, there is no easy way to assess the presence of γ-butyrolactone-like systems in Streptomyces strains without whole-genome sequencing. We have therefore developed and tested a PCR screen that is able to detect homologues of the commonly co-located butenolide synthase and γ-butyrolactone receptor genes. This PCR screen could be employed for the screening of strain libraries to detect signalling systems without the necessity for whole-genome sequencing.

Genetic and physiological requirements for high-level sesquiterpene-production in tomato glandular trichomes.

Type-VI glandular trichomes of wild tomato Solanum habrochaites PI127826 produce high levels of the sesquiterpene 7-epizingiberene and its derivatives, making the plant repellent and toxic to several pest insects and pathogens. How wild tomato trichomes achieve such high terpene production is still largely unknown. Here we show that a cross (F1) with a cultivated tomato produced only minute levels of 7-epizingiberene. In the F2-progeny, selected for the presence of the 7-epizingiberene biosynthesis genes, only three percent produced comparable amounts the wild parent, indicating this trait is recessive and multigenic. Moreover, trichome density alone did not explain the total levels of terpene levels found on the leaves. We selected F2 plants with the "high-production active-trichome phenotype" of PI127826, having trichomes producing about 150 times higher levels of terpenes than F2 individuals that displayed a "low-production lazy-trichome phenotype". Terpene quantities in trichomes of these F2 plants correlated with the volume of the storage cavity and shape of the gland. We found that trichome morphology is not a predetermined characteristic, but cavity volume rather depended on gland-cell metabolic activity. Inhibitor assays showed that the plastidial-precursor pathway (MEP) is fundamental for high-level production of both cytosolic as well as plastid-derived terpenes in tomato trichomes. Additionally, gene expression profiles of isolated secretory cells showed that key enzymes in the MEP pathway were higher expressed in active trichomes. We conclude that the MEP pathway is the primary precursor-supply route in wild tomato type-VI trichomes and that the high-production phenotype of the wild tomato trichome is indeed a multigenic trait.

Glycine max (L.) Merr. (Soybean) metabolome responses to potassium availability.

Potassium (K+) has vital physiological and metabolic functions in plants and its availability can impact tolerance to biotic and abiotic stress conditions. Limited studies have investigated the effect of K+ fertilization on soybean metabolism. Using integrated omics, ionomics and metabolomics, we investigated the field-grown Glycine max (soybean) response, after four K+ soil fertilization rates. Soybean leaf and pod tissue (valves and immature seeds) extracts were analysed by ultra-performance liquid chromatography coupled to high-resolution mass spectrometry (UPLC-HRMS) and inductively coupled plasma optical emission spectroscopy (ICP-OES). Multivariate analyses (PCA-X&Y e O2PLS-DA) showed that 51 compounds of 19 metabolic pathways were regulated in response to K+ availability. Under very low potassium availability, soybean plants accumulated of Ca2+, Mg2+, Fe2+, Cu2+, and B in young and old leaves. Potassium fertilization upregulated carbohydrate, galactolipid, and flavonol glycoside biosynthesis in leaves and pod valves, while K+ deficient pod tissues showed increasing amino acids, oligosaccharides, benzoic acid derivatives, and isoflavones contents. Severely K+ deficient soils elicited isoflavones, coumestans, pterocarpans, and soyasaponins in trifoliate leaves, likely associated to oxidative and photodynamic stress status. Additionally, results demonstrate that L-asparagine content is higher in potassium deficient tissues, suggesting this compound as a biomarker of K+ deficiency in soybean plants. These results demonstrate that potassium soil fertilization did not linearly contribute to changes in specialised constitutive metabolites of soybean. Altogether, this work provides a reference for improving the understanding of soybean metabolism as dependent on K+ availability.

Identification of early quassinoid biosynthesis in the invasive tree of heaven (Ailanthus altissima) confirms evolutionary origin from protolimonoids.

The tree of heaven, Ailanthus altissima (MILL.) SWINGLE, is a globally invasive plant known to secrete allelopathic metabolites called quassinoids. Quassinoids are highly modified triterpenoids. So far, nothing has been known about the biochemical basis of quassinoid biosynthesis. Here, based on transcriptome and metabolome data of Ailanthus altissima, we present the first three steps of quassinoid biosynthesis, which are catalysed by an oxidosqualene cyclase and two cytochrome P450 monooxygenases, resulting in the formation of the protolimonoid melianol. Strikingly, these steps are identical to the first steps of the biosynthesis of limonoids, structurally different triterpenoids from sister plant families within the same order Sapindales. Our results are therefore not only important to fully understand the biosynthesis of complex triterpenoids in plants, but also confirm the long-standing hypothesis that quassinoids and limonoids share an evolutionary origin. In addition, our transcriptome data for Ailanthus altissima will be beneficial to other researchers investigating the physiology and ecology of this invasive tree.

Automatic Identification of Players in the Flavonoid Biosynthesis with Application on the Biomedicinal Plant Croton tiglium.

The flavonoid biosynthesis is a well-characterised model system for specialised metabolism and transcriptional regulation in plants. Flavonoids have numerous biological functions such as UV protection and pollinator attraction, but also biotechnological potential. Here, we present Knowledge-based Identification of Pathway Enzymes (KIPEs) as an automatic approach for the identification of players in the flavonoid biosynthesis. KIPEs combines comprehensive sequence similarity analyses with the inspection of functionally relevant amino acid residues and domains in subjected peptide sequences. Comprehensive sequence sets of flavonoid biosynthesis enzymes and knowledge about functionally relevant amino acids were collected. As a proof of concept, KIPEs was applied to investigate the flavonoid biosynthesis of the medicinal plant Croton tiglium on the basis of a transcriptome assembly. Enzyme candidates for all steps in the biosynthesis network were identified and matched to previous reports of corresponding metabolites in Croton species.