The diffusible factor synthase XanB2 is a bifunctional chorismatase that links the shikimate pathway to ubiquinone and xanthomonadins biosynthetic pathways

The diffusible factor synthase XanB2, originally identified in Xanthomonas campestris pv. campestris (Xcc), is highly conserved across a wide range of bacterial species, but its substrate and catalytic mechanism have not yet been investigated. Here, we show that XanB2 is a unique bifunctional chorismatase that hydrolyses chorismate, the end‐product of the shikimate pathway, to produce 3‐hydroxybenzoic acid (3‐HBA) and 4‐HBA. 3‐HBA and 4‐HBA are respectively associated with the yellow pigment xanthomonadin biosynthesis and antioxidant activity in Xcc. We further demonstrate that XanB2 is a structurally novel enzyme with three putative domains. It catalyses 3‐HBA and 4‐HBA biosynthesis via a unique mechanism with the C‐terminal YjgF‐like domain conferring activity for 3‐HBA biosynthesis and the N‐terminal FGFG motif‐containing domain responsible for 4‐HBA biosynthesis. Furthermore, we show that Xcc produces coenzyme Q8 (CoQ8) via a new biosynthetic pathway independent of the key chorismate‐pyruvate lyase UbiC. XanB2 is the alternative source of 4‐HBA for CoQ8 biosynthesis. The similar CoQ8 biosynthetic pathway, xanthomonadin biosynthetic gene cluster and XanB2 homologues are well conserved in the bacterial species within Xanthomonas, Xylella, Xylophilus, Pseudoxanthomonas, Rhodanobacter, Frateuria, Herminiimonas and Variovorax, suggesting that XanB2 may be a conserved metabolic link between the shikimate pathway, ubiquinone and xanthomonadin biosynthetic pathways in diverse bacteria.     

Fungal siderophore biosynthesis is partially localized in peroxisomes

Siderophores play a central role in iron metabolism and virulence of most fungi. Both Aspergillus fumigatus and Aspergillus nidulans excrete the siderophore triacetylfusarinine C (TAFC) for iron acquisition. In A. fumigatus, green fluorescence protein‐tagging revealed peroxisomal localization of the TAFC biosynthetic enzymes SidI (mevalonyl‐CoA ligase), SidH (mevalonyl‐CoA hydratase) and SidF (anhydromevalonyl‐CoA transferase), while elimination of the peroxisomal targeting signal (PTS) impaired both, peroxisomal SidH‐targeting and TAFC biosynthesis. The analysis of A. nidulans mutants deficient in peroxisomal biogenesis, ATP import or protein import revealed that cytosolic mislocalization of one or two but, interestingly, not all three enzymes impairs TAFC production during iron starvation. The PTS motifs are conserved in fungal orthologues of SidF, SidH and SidI. In agreement with the evolutionary conservation of the partial peroxisomal compartmentalization of fungal siderophore biosynthesis, the SidI orthologue of coprogen‐type siderophore‐producing Neurospora crassa was confirmed to be peroxisomal. Taken together, this study identified and characterized a novel, evolutionary conserved metabolic function of peroxisomes.     

Functional analyses of differentially expressed isoforms of the Arabidopsis inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic plasma membranes that are involved in many functions, including the formation signal transduction complexes. In addition, these lipid species and their catabolites function as secondary signalling molecules in, amongst other processes, apoptosis. The biosynthetic pathway for the formation of sphingolipid is largely conserved. However, unlike mammalian cells, fungi, protozoa and plants synthesize inositol phosphorylceramide (IPC) as their primary phosphosphingolipid. This key step involves the transfer of the phosphorylinositol group from phosphatidylinositol (PI) to phytoceramide, a process catalysed by IPC synthase in plants and fungi. This enzyme activity is at least partly encoded by the AUR1 gene in the fungi, and recently the distantly related functional orthologue of this gene has been identified in the model plant Arabidopsis. Here we functionally analysed all three predicted Arabidopsis IPC synthases, confirming them as aureobasidin A resistant AUR1p orthologues. Expression profiling revealed that the genes encoding these orthologues are differentially expressed in various tissue types isolated from Arabidopsis.     

Phylogeny-Aware Chemoinformatic Analysis of Chemical Diversity in Lamiaceae Enables Iridoid Pathway Assembly and Discovery of Aucubin Synthase

Countless reports describe the isolation and structural characterization of natural products, yet this information remains disconnected and underutilized. Using a cheminformatics approach, we leverage the reported observations of iridoid glucosides with the known phylogeny of a large iridoid producing plant family (Lamiaceae) to generate a set of biosynthetic pathways that best explain the extant iridoid chemical diversity. We developed a pathway reconstruction algorithm that connects iridoid reports via reactions and prunes this solution space by considering phylogenetic relationships between genera. We formulate a model that emulates the evolution of iridoid glucosides to create a synthetic data set, used to select the parameters that would best reconstruct the pathways, and apply them to the iridoid data set to generate pathway hypotheses. These computationally generated pathways were then used as the basis by which to select and screen biosynthetic enzyme candidates. Our model was successfully applied to discover a cytochrome P450 enzyme from Callicarpa americana that catalyzes the oxidation of bartsioside to aucubin, predicted by our model despite neither molecule having been observed in the genus. We also demonstrate aucubin synthase activity in orthologues of Vitex agnus-castus, and the outgroup Paulownia tomentosa, further strengthening the hypothesis, enabled by our model, that the reaction was present in the ancestral biosynthetic pathway. This is the first systematic hypothesis on the epi-iridoid glucosides biosynthesis in 25 years and sets the stage for streamlined work on the iridoid pathway. This work highlights how curation and computational analysis of widely available structural data can facilitate hypothesis-based gene discovery.     

Evolutionary trajectory of phytoalexin biosynthetic gene clusters in rice

Plants frequently possess operon‐like gene clusters for specialized metabolism. Cultivated rice, Oryza sativa, produces antimicrobial diterpene phytoalexins represented by phytocassanes and momilactones, and the majority of their biosynthetic genes are clustered on chromosomes 2 and 4, respectively. These labdane‐related diterpene phytoalexins are biosynthesized from geranylgeranyl diphosphate via ent‐copalyl diphosphate or syn‐copalyl diphosphate. The two gene clusters consist of genes encoding diterpene synthases and chemical‐modification enzymes including P450s. In contrast, genes for the biosynthesis of gibberellins, which are labdane‐related phytohormones, are scattered throughout the rice genome similar to other plant genomes. The mechanism of operon‐like gene cluster formation remains undefined despite previous studies in other plant species. Here we show an evolutionary insight into the rice gene clusters by a comparison with wild Oryza species. Comparative genomics and biochemical studies using wild rice species from the AA genome lineage, including Oryza barthii, Oryza glumaepatula, Oryza meridionalis and the progenitor of Asian cultivated rice Oryza rufipogon indicate that gene clustering for biosynthesis of momilactones and phytocassanes had already been accomplished before the domestication of rice. Similar studies using the species Oryza punctata from the BB genome lineage, the distant FF genome lineage species Oryza brachyantha and an outgroup species Leersia perrieri suggest that the phytocassane biosynthetic gene cluster was present in the common ancestor of the Oryza species despite the different locations, directions and numbers of their member genes. However, the momilactone biosynthetic gene cluster evolved within Oryza before the divergence of the BB genome via assembly of ancestral genes.     

Haplotype hitchhiking promotes trait coselection in Brassica napus

Local haplotype patterns surrounding densely spaced DNA markers with significant trait associations can reveal information on selective sweeps and genome diversity associated with important crop traits. Relationships between haplotype and phenotype diversity, coupled with analysis of gene content in conserved haplotype blocks, can provide insight into coselection for nonrelated traits. We performed genome‐wide analysis of haplotypes associated with the important physiological and agronomic traits leaf chlorophyll and seed glucosinolate content, respectively, in the major oilseed crop species Brassica napus. A locus on chromosome A01 showed opposite effects on leaf chlorophyll content and seed glucosinolate content, attributed to strong linkage disequilibrium (LD) between orthologues of the chlorophyll biosynthesis genes EARLY LIGHT‐INDUCED PROTEIN and CHLOROPHYLL SYNTHASE, and the glucosinolate synthesis gene ATP SULFURYLASE 1. Another conserved haplotype block, on chromosome A02, contained a number of chlorophyll‐related genes in LD with orthologues of the key glucosinolate biosynthesis genes METHYLTHIOALKYMALATE SYNTHASE‐LIKE 1 and 3. Multigene haplogroups were found to have a significantly greater contribution to variation for chlorophyll content than haplotypes for any single gene, suggesting positive effects of additive locus accumulation. Detailed reanalysis of population substructure revealed a clade of ten related accessions exhibiting high leaf chlorophyll and low seed glucosinolate content. These accessions each carried one of the above‐mentioned haplotypes from A01 or A02, generally in combination with further chlorophyll‐associated haplotypes from chromosomes A05 and/or C05. The phenotypic rather than pleiotropic correlations between leaf chlorophyll content index and seed GSL suggest that LD may have led to inadvertent coselection for these two traits.     

Highly conserved progesterone 5β-reductase genes (P5βR) from 5β-cardenolide-free and 5β-cardenolide-producing angiosperms

Most cardenolides used in the therapy of cardiac insufficiency are 5β-configured and thus the stereo-specific reduction of the Δ^4,5-double bond of a steroid precursor is a crucial step in their biosynthesis. This step is thought to be catalysed by progesterone 5β-reductases. We report here on the isolation of 11 progesterone 5β-reductase (P5βR) orthologues from 5β-cardenolide-free and 5β-cardenolide-producing plant species belonging to five different angiosperm orders (Brassicales, Gentianales, Lamiales, Malvales and Solanales). Amino acid sequences of the P5βR described here were highly conserved. They all contain certain motifs qualifying them as members of a class of stereo-selective enone reductases capable of reducing activated CC double bonds by a 1,4-addition mechanism. Protein modeling revealed seven conserved amino acids in the substrate-binding/catalytic site of these enzymes which are all supposed to exhibit low substrate specificity. Eight P5βR genes isolated were expressed in Escherichia coli. Recombinant enzymes reduced progesterone stereo-specifically to 5β-pregane-3,20-dione. The progesterone 5β-reductases from Digitalis canariensis and Arabidopsis thaliana reduced activated CC double bonds of molecules much smaller than progesterone. The specific role of progesterone 5β-reductases of P5βRs in cardenolide metabolism is challenged because this class of enone reductases is widespread in higher plants, and they accept a wide range of enone substrates.     

The evolutionary appearance of non‐cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous β–glucosidases resulting from a crucial amino acid substitution

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α–hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific β–glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non‐cyanogenic γ‐ and β–hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only β–glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site‐directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non‐flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic β–glucosidase, resembling BGD2, and required no more than a single amino acid substitution.     

Phenylalanine derived cyanogenic diglucosides from Eucalyptus camphora and their abundances in relation to ontogeny and tissue type

The cyanogenic glucoside profile of Eucalyptus camphora was investigated in the course of plant ontogeny. In addition to amygdalin, three phenylalanine-derived cyanogenic diglucosides characterized by unique linkage positions between the two glucose moieties were identified in E. camphora tissues. This is the first time that multiple cyanogenic diglucosides have been shown to co-occur in any plant species. Two of these cyanogenic glucosides have not previously been reported and are named eucalyptosin B and eucalyptosin C. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora produce only the cyanogenic monoglucoside prunasin, and genetically based variation was observed in the age at which seedlings initiate prunasin biosynthesis. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. The role of multiple cyanogenic glucosides in E. camphora is unknown, but may include enhanced plant defense and/or a primary role in nitrogen storage and transport.     

Molecular phylogeny and evolution of host-plant use in conifer seed chalcids in the genus Megastigmus (Hymenoptera: Torymidae)

Abstract. Phylogenetic relationships amongst Megastigmus species (Chalcidoidea: Torymidae) associated with conifer seeds were inferred from DNA sequence data. Twenty‐nine species of seed chalcids were analysed using two different genes, cytochrome b (mitochondrial DNA) and the D2 domain of the 28S ribosomal DNA. Maximum‐parsimony and maximum‐likelihood analyses showed that taxa formed two monophyletic groups, one clade comprising all species associated with Cupressaceae and Taxodiaceae hosts with the exception of Chamaecyparis, and the other clade composed of species associated with Pinaceae. Species infesting Cupressaceae and Taxodiaceae seemed to be specialized to particular host genera or even to be species specific, which was consistent with a taxonomic radiation following initial host adaptation. By contrast, Megastigmus species associated with Pinaceae appeared capable of shifting onto different congeneric species or even onto a new host genus, with their evolution apparently less constrained by plant association. We hypothesized that the Megastigmus group associated with Pinaceae may have a much higher invasive potential than that related to Cupressaceae. The study also confirmed the presence of invasive Nearctic species in the Palaearctic, and demonstrated the existence of a cryptic species complex.     

Chemical Traits of Hemiparasitism in Odontites luteus

The study of the monoterpene glycosides content of Odontites luteus has shown the presence of a total of fifteen iridoid glucosides. The presence of compounds 1  –  5 and 7  –  10 is perfectly on‐line with both the biogenetic pathway for iridoids biosynthesis in Lamiales and the current botanical classification of the species. On the other side, the presence of compounds like agnuside ( 6 ), adoxosidic acid ( 11 ), monotropein ( 12 ), 6,7‐dihydromonotropein ( 13 ), methyl oleoside ( 14 ) and methyl glucooleoside ( 15 ) is of high interest because, first of all, they have never been reported before in Lamiales. In second instance, the majority of the last compounds are formally derived from a different biogenetic pathway which involves deoxyloganic acid/loganin and led to the formation of decarboxylated iridoid showing the 8β‐configuration. Furthermore, a second abnormality was found during our study and this regards compounds 14 and 15 which are seco‐iriodids and thus not typical for this family. The presence of these unusual compounds, biogenetically not related to species belonging to Lamiales, is a clear evidence of the metabolites transfer from the hosts. In fact, the collection area is also populated by species belonging to Oleaceae and Ericaceae which could be the possible hosts since the biosynthesis of seco‐iridoids and or iridoids related to deoxyloganic acid/loganin pathway, with the 8β‐configuration, is well documented in these species.     

Severity of impacts of an introduced species corresponds with regional eco‐evolutionary experience

Invasive plant impacts vary widely across introduced ranges. We tested the hypothesis that differences in the eco‐evolutionary experience of native communities with the invader correspond with the impacts of invasive species on native vegetation, with impacts increasing with ecological novelty. We compared plant species richness and composition beneath Pinus contorta to that in adjacent vegetation and other P. contorta stands across a network of sites in its native (Canada and USA) and non‐native (Argentina, Chile, Finland, New Zealand, Scotland, Sweden) ranges. At sites in North America and Europe, within the natural distribution of the genus Pinus, P. contorta was not associated with decreases in diversity. In the Southern Hemisphere, where there are no native Pinaceae, plant communities beneath P. contorta were less diverse than in other regions and compared to uninvaded native vegetation. Effects on native vegetation were particularly pronounced where P. contorta was a more novel life form and exhibited higher growth rates. Our results support the hypothesis that the eco‐evolutionary experience of the native vegetation, and thus the novelty of the invader, determines the magnitude of invader impacts on native communities. Understanding the eco‐evolutionary context of invasions will help to better understand and predict where invasion impacts will be greatest and to prioritize invasive species management.     

Species mixture effects on flammability across plant phylogeny: the importance of litter particle size and the special role for non‐Pinus Pinaceae

Fire affects and is affected by plants. Vegetation varies in flammability, that is, its general ability to burn, at different levels of ecological organization. To scale from individual plant traits to community flammability states, understanding trait effects on species flammability variation and their interaction is important. Plant traits are the cumulative result of evolution and they show, to differing extents, phylogenetic conservatism. We asked whether phylogenetic distance between species predicts species mixture effects on litterbed flammability. We conducted controlled laboratory burns for 34 phylogenetically wide‐ranging species and 34 random two‐species mixtures from them. Generally, phylogenetic distance did not predict species mixture effects on flammability. Across the plant phylogeny, most species were flammable except those in the non‐Pinus Pinaceae, which shed small needles producing dense, poorly ventilated litterbeds above the packing threshold and therefore nonflammable. Consistently, either positive or negative dominance effects on flammability of certain flammable or those non‐flammable species were found in mixtures involving the non‐Pinus Pinaceae. We demonstrate litter particle size is key to explaining species nonadditivity in fuelbed flammability. The potential of certain species to influence fire disproportionately to their abundance might increase the positive feedback effects of plant flammability on community flammability state if flammable species are favored by fire.     

Kdo2‐lipid A: structural diversity and impact on immunopharmacology

3‐deoxy‐d ‐manno‐octulosonic acid‐lipid A (Kdo₂‐lipid A) is the essential component of lipopolysaccharide in most Gram‐negative bacteria and the minimal structural component to sustain bacterial viability. It serves as the active component of lipopolysaccharide to stimulate potent host immune responses through the complex of Toll‐like‐receptor 4 (TLR4) and myeloid differentiation protein 2. The entire biosynthetic pathway of Escherichia coli Kdo₂‐lipid A has been elucidated and the nine enzymes of the pathway are shared by most Gram‐negative bacteria, indicating conserved Kdo₂‐lipid A structure across different species. Yet many bacteria can modify the structure of their Kdo₂‐lipid A which serves as a strategy to modulate bacterial virulence and adapt to different growth environments as well as to avoid recognition by the mammalian innate immune systems. Key enzymes and receptors involved in Kdo₂‐lipid A biosynthesis, structural modification and its interaction with the TLR4 pathway represent a clear opportunity for immunopharmacological exploitation. These include the development of novel antibiotics targeting key biosynthetic enzymes and utilization of structurally modified Kdo₂‐lipid A or correspondingly engineered live bacteria as vaccines and adjuvants. Kdo₂‐lipid A/TLR4 antagonists can also be applied in anti‐inflammatory interventions. This review summarizes recent knowledge on both the fundamental processes of Kdo₂‐lipid A biosynthesis, structural modification and immune stimulation, and applied research on pharmacological exploitations of these processes for therapeutic development.     

Evolution and diversity of the 2–oxoglutarate‐dependent dioxygenase superfamily in plants

The 2–oxoglutarate‐dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes – DOXA, DOXB and DOXC – based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4–hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC‐class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm‐specific clades were found to be involved in various lineage‐specific specialized metabolisms, but 31 of the 57 DOXC‐class clades were only found in a single species. Therefore, the evolution and diversification of DOXC‐class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.