The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

A chromosome-level genome assembly reveals that a bipartite gene cluster formed via an inverted duplication controls monoterpenoid biosynthesis in Schizonepeta tenuifolia.

Biosynthetic gene clusters (BGCs) are regions of a genome where genes involved in a biosynthetic pathway are in proximity. The origin and evolution of plant BGCs as well as their role in specialized metabolism remain largely unclear. In this study, we have assembled a chromosome-scale genome of Japanese catnip (Schizonepeta tenuifolia) and discovered a BGC that contains multiple copies of genes involved in four adjacent steps in the biosynthesis of p-menthane monoterpenoids. This BGC has an unprecedented bipartite structure, with mirrored biosynthetic regions separated by 260 kilobases. This bipartite BGC includes identical copies of a gene encoding an old yellow enzyme, a type of flavin-dependent reductase. In vitro assays and virus-induced gene silencing revealed that this gene encodes the missing isopiperitenone reductase. This enzyme evolved from a completely different enzyme family to isopiperitenone reductase from closely related Mentha spp., indicating convergent evolution of this pathway step. Phylogenomic analysis revealed that this bipartite BGC has emerged uniquely in the S. tenuifolia lineage and through insertion of pathway genes into a region rich in monoterpene synthases. The cluster gained its bipartite structure via an inverted duplication. The discovered bipartite BGC for p-menthane biosynthesis in S. tenuifolia has similarities to the recently described duplicated p-menthane biosynthesis gene pairs in the Mentha longifolia genome, providing an example of the convergent evolution of gene order. This work expands our understanding of plant BGCs with respect to both form and evolution, and highlights the power of BGCs for gene discovery in plant biosynthetic pathways.

Mitogenomics of the Olive Seed Weevil, Anchonocranus oleae Marshall and Implications for Its Phylogenetic Position in Curculionidae.

Anchonocranus oleae Marshall (Coleoptera: Curculionidae) is a seed-feeding weevil native to southern Africa; its larvae are known to develop in the fruits of the African Wild Olive and, more rarely, cultivated olives. The species has been mainly found in the Western Cape province of South Africa, but it has remained in relative obscurity because it does not seem to represent a current threat to commercial olive production. As part of an ongoing effort to produce baseline genetic data for olive-associated entomofauna in South Africa, we generated reference DNA barcodes for A. oleae collected from wild and cultivated olives and sequenced its mitogenome for assessment of the phylogenetic position of the species in the family Curculionidae. The mitochondrial phylogeny estimate indicated that A. oleae shares a common ancestor with Elaidobius (tribe Derelomini), but a definite and close relationship to this tribe and the precise tribal placement of A. oleae in the subfamily Curculioninae could not be inferred due to the lack of representative mitogenomes of other relevant curculionine tribes and genera. This study will assist future work on the DNA-based species identification, genetic diversity, and phylogenetic position of the genus Anchonocranus and related taxa.

Plant biosynthetic gene clusters in the context of metabolic evolution.

Covering: up to 2022Plants produce a wide range of structurally and biosynthetically diverse natural products to interact with their environment. These specialised metabolites typically evolve in limited taxonomic groups presumably in response to specific selective pressures. With the increasing availability of sequencing data, it has become apparent that in many cases the genes encoding biosynthetic enzymes for specialised metabolic pathways are not randomly distributed on the genome. Instead they are physically linked in structures such as arrays, pairs and clusters. The exact function of these clusters is debated. In this review we take a broad view of gene arrangement in plant specialised metabolism, examining types of structures and variation. We discuss the evolution of biosynthetic gene clusters in the wider context of metabolism, populations and epigenetics. Finally, we synthesise our observations to propose a new hypothesis for biosynthetic gene cluster formation in plants.

Seeing the Forest through the (Phylogenetic) Trees: Functional Characterisation of Grapevine Terpene Synthase (VviTPS) Paralogues and Orthologues.

Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.

Surgical practice in a maximum security prison - unique and perplexing problems.

The practice of general surgery in a prison population differs considerably from that in a general surgical practice. We audited surgical consultations at the Mangaung Correctional Centre from December 2003 to April 2009. We found a high incidence of foreign object ingestion and anal pathology. Understanding the medical and social aspects of prison life facilitates the treatment of inmates with surgical problems.