Multilevel analysis between Physcomitrium patens and Mortierellaceae endophytes explores potential long-standing interaction among land plants and fungi.

The model moss species Physcomitrium patens has long been used for studying divergence of land plants spanning from bryophytes to angiosperms. In addition to its phylogenetic relationships, the limited number of differential tissues, and comparable morphology to the earliest embryophytes provide a system to represent basic plant architecture. Based on plant-fungal interactions today, it is hypothesized these kingdoms have a long-standing relationship, predating plant terrestrialization. Mortierellaceae have origins diverging from other land fungi paralleling bryophyte divergence, are related to arbuscular mycorrhizal fungi but are free-living, observed to interact with plants, and can be found in moss microbiomes globally. Due to their parallel origins, we assess here how two Mortierellaceae species, Linnemannia elongata and Benniella erionia, interact with P. patens in coculture. We also assess how Mollicute-related or Burkholderia-related endobacterial symbionts (MRE or BRE) of these fungi impact plant response. Coculture interactions are investigated through high-throughput phenomics, microscopy, RNA-sequencing, differential expression profiling, gene ontology enrichment, and comparisons among 99 other P. patens transcriptomic studies. Here we present new high-throughput approaches for measuring P. patens growth, identify novel expression of over 800 genes that are not expressed on traditional agar media, identify subtle interactions between P. patens and Mortierellaceae, and observe changes to plant-fungal interactions dependent on whether MRE or BRE are present. Our study provides insights into how plants and fungal partners may have interacted based on their communications observed today as well as identifying L. elongata and B. erionia as modern fungal endophytes with P. patens.

The Mitragyna speciosa (Kratom) Genome: a resource for data-mining potent pharmaceuticals that impact human health.

Mitragyna speciosa (kratom) produces numerous compounds with pharmaceutical properties including the production of bioactive monoterpene indole and oxindole alkaloids. Using a linked-read approach, a 1,122,519,462 bp draft assembly of M. speciosa "Rifat" was generated with an N50 scaffold size of 1,020,971 bp and an N50 contig size of 70,448 bp that encodes 55,746 genes. Chromosome counting revealed that "Rifat" is a tetraploid with a base chromosome number of 11, which was further corroborated by orthology and syntenic analysis of the genome. Analysis of genes and clusters involved in specialized metabolism revealed genes putatively involved in alkaloid biosynthesis. Access to the genome of M. speciosa will facilitate an improved understanding of alkaloid biosynthesis and accelerate the production of bioactive alkaloids in heterologous hosts.

Genome assembly of Chiococca alba uncovers key enzymes involved in the biosynthesis of unusual terpenoids.

Chiococca alba (L.) Hitchc. (snowberry), a member of the Rubiaceae, has been used as a folk remedy for a range of health issues including inflammation and rheumatism and produces a wealth of specialized metabolites including terpenes, alkaloids, and flavonoids. We generated a 558 Mb draft genome assembly for snowberry which encodes 28,707 high-confidence genes. Comparative analyses with other angiosperm genomes revealed enrichment in snowberry of lineage-specific genes involved in specialized metabolism. Synteny between snowberry and Coffea canephora Pierre ex A. Froehner (coffee) was evident, including the chromosomal region encoding caffeine biosynthesis in coffee, albeit syntelogs of N-methyltransferase were absent in snowberry. A total of 27 putative terpene synthase genes were identified, including 10 that encode diterpene synthases. Functional validation of a subset of putative terpene synthases revealed that combinations of diterpene synthases yielded access to products of both general and specialized metabolism. Specifically, we identified plausible intermediates in the biosynthesis of merilactone and ribenone, structurally unique antimicrobial diterpene natural products. Access to the C. alba genome will enable additional characterization of biosynthetic pathways responsible for health-promoting compounds in this medicinal species.

A database-driven approach identifies additional diterpene synthase activities in the mint family (Lamiaceae).

Members of the mint family (Lamiaceae) accumulate a wide variety of industrially and medicinally relevant diterpenes. We recently sequenced leaf transcriptomes from 48 phylogenetically diverse Lamiaceae species. Here, we summarize the available chemotaxonomic and enzyme activity data for diterpene synthases (diTPSs) in the Lamiaceae and leverage the new transcriptomes to explore the diTPS sequence and functional space. Candidate genes were selected with an intent to evenly sample the sequence homology space and to focus on species in which diTPS transcripts were found, yet from which no diterpene structures have been previously reported. We functionally characterized nine class II diTPSs and 10 class I diTPSs from 11 distinct plant species and found five class II activities, including two novel activities, as well as a spectrum of class I activities. Among the class II diTPSs, we identified a neo-cleroda-4(18),13E-dienyl diphosphate synthase from Ajuga reptans, catalyzing the likely first step in the biosynthesis of a variety of insect-antifeedant compounds. Among the class I diTPSs was a palustradiene synthase from Origanum majorana, leading to the discovery of specialized diterpenes in that species. Our results provide insights into the diversification of diterpene biosynthesis in the mint family and establish a comprehensive foundation for continued investigation of diterpene biosynthesis in the Lamiaceae.

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

The terpene synthase gene family in Tripterygium wilfordii harbors a labdane-type diterpene synthase among the monoterpene synthase TPS-b subfamily.

Tripterygium wilfordii (Celastraceae) is a medicinal plant with anti-inflammatory and immunosuppressive properties. Identification of a vast array of unusual sesquiterpenoids, diterpenoids and triterpenoids in T. wilfordii has spurred investigations of their pharmacological properties. The tri-epoxide lactone triptolide was the first of many diterpenoids identified, attracting interest due to the spectrum of bioactivities. To probe the genetic underpinning of diterpenoid diversity, an expansion of the class II diterpene synthase (diTPS) family was recently identified in a leaf transcriptome. Following detection of triptolide and simple diterpene scaffolds in the root, we sequenced and mined the root transcriptome. This allowed identification of the root-specific complement of TPSs and an expansion in the class I diTPS family. Functional characterization of the class II diTPSs established their activities in the formation of four C-20 diphosphate intermediates, precursors of both generalized and specialized metabolism and a novel scaffold for Celastraceae. Functional pairs of the class I and II enzymes resulted in formation of three scaffolds, accounting for some of the terpenoid diversity found in T. wilfordii. The absence of activity-forming abietane-type diterpenes encouraged further testing of TPSs outside the canonical class I diTPS family. TwTPS27, close relative of mono-TPSs, was found to couple with TwTPS9, converting normal-copalyl diphosphate to miltiradiene. The phylogenetic distance to established diTPSs indicates neo-functionalization of TwTPS27 into a diTPS, a function not previously observed in the TPS-b subfamily. This example of evolutionary convergence expands the functionality of TPSs in the TPS-b family and may contribute miltiradiene to the diterpenoids of T. wilfordii.

Oxidation and cyclization of casbene in the biosynthesis of Euphorbia factors from mature seeds of Euphorbia lathyris L.

The seed oil of Euphorbia lathyris L. contains a series of macrocyclic diterpenoids known as Euphorbia factors. They are the current industrial source of ingenol mebutate, which is approved for the treatment of actinic keratosis, a precancerous skin condition. Here, we report an alcohol dehydrogenase-mediated cyclization step in the biosynthetic pathway of Euphorbia factors, illustrating the origin of the intramolecular carbon-carbon bonds present in lathyrane and ingenane diterpenoids. This unconventional cyclization describes the ring closure of the macrocyclic diterpene casbene. Through transcriptomic analysis of E. lathyris L. mature seeds and in planta functional characterization, we identified three enzymes involved in the cyclization route from casbene to jolkinol C, a lathyrane diterpene. These enzymes include two cytochromes P450 from the CYP71 clan and an alcohol dehydrogenase (ADH). CYP71D445 and CYP726A27 catalyze regio-specific 9-oxidation and 5-oxidation of casbene, respectively. When coupled with these P450-catalyzed monooxygenations, E. lathyris ADH1 catalyzes dehydrogenation of the hydroxyl groups, leading to the subsequent rearrangement and cyclization. The discovery of this nonconventional cyclization may provide the key link to complete elucidation of the biosynthetic pathways of ingenol mebutate and other bioactive macrocyclic diterpenoids.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis.

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Manoyl oxide (13R), the biosynthetic precursor of forskolin, is synthesized in specialized root cork cells in Coleus forskohlii.

Forskolin, a complex labdane diterpenoid found in the root of Coleus forskohlii (Lamiaceae), has received attention for its broad range of pharmacological activities, yet the biosynthesis has not been elucidated. We detected forskolin in the root cork of C. forskohlii in a specialized cell type containing characteristic structures with histochemical properties consistent with oil bodies. Organelle purification and chemical analysis confirmed the localization of forskolin and of its simplest diterpene precursor backbone, (13R) manoyl oxide, to the oil bodies. The labdane diterpene backbone is typically synthesized by two successive reactions catalyzed by two distinct classes of diterpene synthases. We have recently described the identification of a small gene family of diterpene synthase candidates (CfTPSs) in C. forskohlii. Here, we report the functional characterization of four CfTPSs using in vitro and in planta assays. CfTPS2, which synthesizes the intermediate copal-8-ol diphosphate, in combination with CfTPS3 resulted in the stereospecific formation of (13R) manoyl oxide, while the combination of CfTPS1 and CfTPS3 or CfTPS4 led to formation of miltiradiene, precursor of abietane diterpenoids in C. forskohlii. Expression profiling and phylogenetic analysis of the CfTPS family further support the functional diversification and distinct roles of the individual diterpene synthases and the involvement of CfTPS1 to CfTPS4 in specialized metabolism and of CfTPS14 and CfTPS15 in general metabolism. Our findings pave the way toward the discovery of the remaining components of the pathway to forskolin, likely localized in this specialized cell type, and support a role of oil bodies as storage organelles for lipophilic bioactive metabolites.

Gene discovery of modular diterpene metabolism in nonmodel systems.

Plants produce over 10,000 different diterpenes of specialized (secondary) metabolism, and fewer diterpenes of general (primary) metabolism. Specialized diterpenes may have functions in ecological interactions of plants with other organisms and also benefit humanity as pharmaceuticals, fragrances, resins, and other industrial bioproducts. Examples of high-value diterpenes are taxol and forskolin pharmaceuticals or ambroxide fragrances. Yields and purity of diterpenes obtained from natural sources or by chemical synthesis are often insufficient for large-volume or high-end applications. Improvement of agricultural or biotechnological diterpene production requires knowledge of biosynthetic genes and enzymes. However, specialized diterpene pathways are extremely diverse across the plant kingdom, and most specialized diterpenes are taxonomically restricted to a few plant species, genera, or families. Consequently, there is no single reference system to guide gene discovery and rapid annotation of specialized diterpene pathways. Functional diversification of genes and plasticity of enzyme functions of these pathways further complicate correct annotation. To address this challenge, we used a set of 10 different plant species to develop a general strategy for diterpene gene discovery in nonmodel systems. The approach combines metabolite-guided transcriptome resources, custom diterpene synthase (diTPS) and cytochrome P450 reference gene databases, phylogenies, and, as shown for select diTPSs, single and coupled enzyme assays using microbial and plant expression systems. In the 10 species, we identified 46 new diTPS candidates and over 400 putatively terpenoid-related P450s in a resource of nearly 1 million predicted transcripts of diterpene-accumulating tissues. Phylogenetic patterns of lineage-specific blooms of genes guided functional characterization.

Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production.

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm-specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production.

Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects.

Diterpene resin acids (DRAs) are specialized (secondary) metabolites of the oleoresin defense of conifers produced by diterpene synthases and cytochrome P450s of the CYP720B family. The evolution of DRA metabolism shares common origins with the biosynthesis of ent-kaurenoic acid, which is highly conserved in general (primary) metabolism of gibberellin biosynthesis. Transcriptome mining in species of spruce (Picea) and pine (Pinus) revealed CYP720Bs of four distinct clades. We cloned a comprehensive set of 12 different Sitka spruce (Picea sitchensis) CYP720Bs as full-length cDNAs. Spatial expression profiles, methyl jasmonate induction, and transcript enrichment in terpenoid-producing resin ducts suggested a role of CYP720B4 in DRA biosynthesis. CYP720B4 was characterized as a multisubstrate, multifunctional enzyme by the formation of oxygenated diterpenoids in metabolically engineered yeast, yeast in vivo transformation of diterpene substrates, in vitro assays with CYP720B4 protein produced in Escherichia coli, and alteration of DRA profiles in RNA interference-suppressed spruce seedlings. CYP720B4 was active with 24 different diterpenoid substrates, catalyzing consecutive C-18 oxidations in the biosynthesis of an array of diterpene alcohols, aldehydes, and acids. CYP720B4 was most active in the formation of dehydroabietic acid, a compound associated with insect resistance of Sitka spruce. We identified patterns of convergent evolution of CYP720B4 in DRA metabolism and ent-kaurene oxidase CYP701 in gibberellin metabolism and revealed differences in the evolution of specialized and general diterpene metabolism in a gymnosperm. The genomic and functional characterization of the gymnosperm CYP720B family highlights that the evolution of specialized metabolism involves substantial diversification relative to conserved, general metabolism.

Transcriptome mining, functional characterization, and phylogeny of a large terpene synthase gene family in spruce (Picea spp.).

BACKGROUND: In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. RESULTS: The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) and full-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca × P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases. CONCLUSIONS: The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling.

An integrated genomic, proteomic and biochemical analysis of (+)-3-carene biosynthesis in Sitka spruce (Picea sitchensis) genotypes that are resistant or susceptible to white pine weevil.

Conifers are extremely long-lived plants that have evolved complex chemical defenses in the form of oleoresin terpenoids to resist attack from pathogens and herbivores. In these species, terpenoid diversity is determined by the size and composition of the terpene synthase (TPS) gene family and the single- and multi-product profiles of these enzymes. The monoterpene (+)-3-carene is associated with resistance of Sitka spruce (Picea sitchensis) to white pine weevil (Pissodes strobi). We used a combined genomic, proteomic and biochemical approach to analyze the (+)-3-carene phenotype in two contrasting Sitka spruce genotypes. Resistant trees produced significantly higher levels of (+)-3-carene than susceptible trees, in which only trace amounts were detected. Biosynthesis of (+)-3-carene is controlled, at the genome level, by a small family of closely related (+)-3-carene synthase (PsTPS-3car) genes (82-95% amino acid sequence identity). Transcript profiling identified one PsTPS-3car gene (PsTPS-3car1) that is expressed in both genotypes, one gene (PsTPS-3car2) that is expressed only in resistant trees, and one gene (PsTPS-3car3) that is expressed only in susceptible trees. The PsTPS-3car2 gene was not detected in genomic DNA of susceptible trees. Target-specific selected reaction monitoring confirmed this pattern of differential expression of members of the PsTPS-3car family at the proteome level. Kinetic characterization of the recombinant PsTPS-3car enzymes identified differences in the activities of PsTPS-3car2 and PsTPS-3car3 as a factor contributing to the different (+)-3-carene profiles of resistant and susceptible trees. In conclusion, variation of the (+)-3-carene phenotype is controlled by copy number variation of PsTPS-3car genes, variation of gene and protein expression, and variation in catalytic efficiencies.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome.

BACKGROUND: Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. RESULTS: We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. CONCLUSION: We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes.

Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation.

Different stages of vascular and interfascicular fiber differentiation can be identified along the axis of bolting stems in Arabidopsis. To gain insights into the metabolic, developmental, and regulatory events that control this pattern, we applied global transcript profiling employing an Arabidopsis full-genome longmer microarray. More than 5000 genes were differentially expressed, among which more than 3000 changed more than twofold, and were placed into eight expression clusters based on polynomial regression models. Within these, 182 upregulated transcription factors represent candidate regulators of fiber development. A subset of these candidates has been associated with fiber development and/or secondary wall formation and lignification in the literature, making them targets for functional studies and comparative genomic analyses with woody plants. Analysis of differentially expressed phenylpropanoid genes identified a set known to be involved in lignin biosynthesis. These were used to anchor co-expression analyses that allowed us to identify candidate genes encoding proteins involved in monolignol transport and monolignol dehydrogenation and polymerization. Similar analyses revealed candidate genes encoding enzymes that catalyze missing links in the shikimate pathway, namely arogenate dehydrogenase and prephenate aminotransferase.