[Show abstract][Hide abstract] ABSTRACT: Conifers possess an array of physical and chemical defenses against stem boring insects. Stone cells provide a physical defense associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical, and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan, and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared to S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process and fortification of shoot tips in R trees an effective strategy against insect feeding.
Plant Cell and Environment 10/2015; DOI:10.1111/pce.12654 · 6.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Grindelia robusta or gumweed, is a medicinal herb of the sunflower family that forms a diverse suite of diterpenoid natural products. Its major constituents, grindelic acid and related grindelane diterpenoids accumulate in a resinous exudate covering the plants' surface, most prominently the unopened composite flower. Recent studies demonstrated potential pharmaceutical applications for grindelic acid and its synthetic derivatives. Mining of the previously published transcriptome of G. robusta flower tissue identified two additional diterpene synthases (diTPSs). We report the in vitro and in vivo functional characterization of an ent-kaurene synthase of general metabolism (GrTPS4) and a class II diTPS (GrTPS2) of specialized metabolism that converts geranylgeranyl diphosphate (GGPP) into labda-7,13E-dienyl diphosphate as verified by nuclear magnetic resonance (NMR) analysis. Tissue-specific transcript abundance of GrTPS2 in leaves and flowers accompanied by the presence of an endocyclic 7,13 double bond in labda-7,13E-dienyl diphosphate suggest that GrTPS2 catalyzes the first committed reaction in the biosynthesis of grindelic acid and related grindelane metabolites. With the formation of labda-7,13E-dienyl diphosphate, GrTPS2 adds an additional function to the portfolio of monofunctional class II diTPSs, which catalytically most closely resembles the bifunctional labda-7,13E-dien-15-ol synthase of the lycopod Selaginella moellendorffii. Together with a recently identified functional diTPS pair of G. robusta producing manoyl oxide, GrTPS2 lays the biosynthetic foundation of the diverse array of labdane-related diterpenoids in the genus Grindelia. Knowledge of these natural diterpenoid metabolic pathways paves the way for developing biotechnology approaches toward producing grindelic acid and related bioproducts. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The Plant Journal 06/2015; 83(5). DOI:10.1111/tpj.12925 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: White spruce (Picea glauca), a gymnosperm tree, has been established as one of the models for conifer genomics. We describe the draft genome assemblies of two white spruce genotypes, PG29 and WS77111, innovative tools for the assembly of very large genomes, and the conifer genomics resources developed in this process. The two white spruce genotypes originate from distant geographic regions of western (PG29) and eastern (WS77111) North America, and represent elite trees in two Canadian tree breeding programs. We present an update (V3 and V4) for a previously reported PG29 V2 draft genome assembly and introduce a second white spruce genome assembly for genotype WS77111. Assemblies of the PG29 and WS77111 genomes confirm the reconstructed white spruce genome size in the 20 Gbp range, and show broad synteny. Using the PG29 V3 assembly and additional white spruce genomics and transcriptomics resources, we performed MAKER-P annotation and meticulous expert annotation of very large gene families of conifer defense metabolism, the terpene synthases and cytochrome P450s. We also comprehensively annotated the white spruce mevalonate, methylerythritol phosphate and phenylpropanoid pathways. These analyses highlighted the large extent of gene and pseudogene duplications in a conifer genome, in particular for genes of secondary (i.e. specialized) metabolism, and the potential for gain and loss of function for defense and adaptation. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The Plant Journal 05/2015; 83(2). DOI:10.1111/tpj.12886 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: When working on an ongoing genome sequencing and assembly project, it is rather inconvenient when gene identifiers change from one build of the assembly to the next. The gene labelling system described here, UniqTag, addresses this common challenge. UniqTag assigns a unique identifier to each gene that is a representative k-mer, a string of length k, selected from the sequence of that gene. Unlike serial numbers, these identifiers are stable between different assemblies and annotations of the same data without requiring that previous annotations be lifted over by sequence alignment. We assign UniqTag identifiers to ten builds of the Ensembl human genome spanning eight years to demonstrate this stability. The implementation of UniqTag in Ruby and an R package are available at https://github.com/sjackman/uniqtag sjackman/uniqtag. The R package is also available from CRAN: install.packages ("uniqtag"). Supplementary material and code to reproduce it is available at https://github.com/sjackman/uniqtag-paper.
PLoS ONE 05/2015; 10(5):e0128026. DOI:10.1371/journal.pone.0128026 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance
against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures
in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays
with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that transsabin-
3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length
complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the
enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC
genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for
substrate specificity supported a biological role of CYP750B1 in a- and b-thujone biosynthesis. This P450 belongs to the apparently
gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In
contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon,
and its transcript profiles are not well correlated with thujone accumulation.
[Show abstract][Hide abstract] ABSTRACT: : Ambrox and related ambroxides are highly priced in the fragrance industry, and valued for their delicate odor and fixative properties. Historically, ambrox was obtained from ambergris, a waxy excretion produced by sperm whales, now an endangered species. Synthetic ambroxides have replaced ambergris in perfume manufacture. Plant labdane diterpenoids can serve as starting material for ambroxide synthesis. Among these, the diterpene alcohol sclareol is the major industrial precursor obtained from cultivated clary sage (Salvia sclarea). In plants, a large family of diterpene synthase (diTPS) enzymes controls key reactions in diterpenoid biosynthesis. Advanced metabolite profiling and high-throughput sequencing of fragrant and medicinal plants have accelerated discovery of novel diTPS functions, providing a resource for combinatorial synthetic biology and metabolic engineering approaches. This chapter highlights recent progress on the discovery, characterization, and engineering of plant diTPSs with potential uses in ambroxide production. It features biosynthesis of sclareol, cis-abienol, and diterpene resin acids, as sources of genes and enzymes for diterpenoid bioproducts.
[Show abstract][Hide abstract] ABSTRACT: The bark beetle-associated fungus Grosmannia clavigera participates in the large-scale destruction
of pine forests. In the tree, it must tolerate saturating levels of toxic conifer defense
chemicals (e.g. monoterpenes). The fungus can metabolize some of these compounds
through the ß-oxidation pathway and use them as a source of carbon. It also uses carbon
from pine triglycerides, where oleic acid is the most common fatty acid. High levels of free
fatty acids, however, are toxic and can cause additional stress during host colonization.
Fatty acids induce expression of neighboring genes encoding a cytochrome P450
(CYP630B18) and its redox partner, cytochrome P450 reductase (CPR2). The aim of this
work was to study the function of this novel P450 system. Using LC/MS, we biochemically
characterized CYP630 as a highly specific oleic acid ω-hydroxylase. We explain oleic acid
specificity using protein interaction modeling. Our results underscore the importance of ω-
oxidation when the main ß-oxidation pathway may be overwhelmed by other substrates
such as host terpenoid compounds. Because this CYP-CPR gene cluster is evolutionarily
conserved, our work has implications for metabolism studies in other fungi.
PLoS ONE 03/2015; 10(3). DOI:10.1371/journal.pone.0120119 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We tested the hypothesis that Arabidopsis can recognize and respond differentially to insect species at the transcriptional level using a genome wide microarray. Transcriptional reprogramming was characterized using co-expression analysis in damaged and undamaged leaves at two times in response to mechanical wounding and four insect species. In all, 2778 (10.6%) of annotated genes on the array were differentially expressed in at least one treatment. Responses differed mainly between aphid and caterpillar and sampling times. Responses to aphids and caterpillars shared only 10% of up-regulated and 8% of down-regulated genes. Responses to two caterpillars shared 21 and 12% of up- and down-regulated genes, whereas responses to the two aphids shared only 7 and 4% of up-regulated and down-regulated genes. Overlap in genes expressed between 6 and 24 h was 3-15%, and depended on the insect species. Responses in attacked and unattacked leaves differed at 6 h but converged by 24 h. Genes responding to the insects are also responsive to many stressors and included primary metabolism. Aphids down-regulated amino acid catabolism; caterpillars stimulated production of amino acids involved in glucosinolate synthesis. Co-expression analysis revealed 17 response networks. Transcription factors were a major portion of differentially expressed genes throughout and responsive genes shared most of the known or postulated binding sites. However, cis-element composition of genes down regulated by the aphid M. persicae was unique, as were those of genes down-regulated by caterpillars. As many as 20 cis-elements were over-represented in one or more treatments, including some from well-characterized classes and others as yet uncharacterized. We suggest that transcriptional changes elicited by wounding and insects are heavily influenced by transcription factors and involve both enrichment of a common set of cis-elements and a unique enrichment of a few cis-elements in responding genes.
[Show abstract][Hide abstract] ABSTRACT: We developed proteome profiles for host colonizing mountain pine beetle adults, Dendroctonus ponderosae Hopkins (Coleoptera: Curculionidae). Adult insects were fed in pairs on fresh host lodgepole pine, Pinus contorta Dougl. ex Loud, phloem tissue. The proteomes of fed individuals were monitored using iTRAQ and compared to those of starved beetles, revealing 757 and 739 expressed proteins in females and males, respectively, for which quantitative information was obtained. Overall functional category distributions were similar for males and females, with the majority of proteins falling under carbohydrate metabolism (glycolysis, gluconeogenesis, citric acid cycle), structure (cuticle, muscle, cytoskeleton), and protein and amino acid metabolism. Females had 23 proteins with levels that changed significantly with feeding (p<0.05, FDR<0.20), including chaperones and enzymes required for vitellogenesis. In males, levels of 29 proteins changed significantly with feeding (p<0.05, FDR<0.20), including chaperones as well as motor proteins. Only two proteins, both chaperones, exhibited a significant change in both females and males with feeding. Proteins with differential accumulation patterns in females exhibited higher fold changes with feeding than did those in males. This difference may be due to major and rapid physiological changes occurring in females upon finding a host tree during the physiological shift from dispersal to reproduction. The significant accumulation of chaperone proteins, a cytochrome P450, and a glutathione S-transferase, indicate secondary metabolite-induced stress physiology related to chemical detoxification during early host colonization. The females' activation of vitellogenin only after encountering a host indicates deliberate partitioning of resources and a balancing of the needs of dispersal and reproduction.
PLoS ONE 10/2014; 9(10):e110673. DOI:10.1371/journal.pone.0110673 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Insights from sequenced genomes of major land plant lineages have advanced research in almost every aspect of plant biology. Until recently, however, assembled genome sequences of gymnosperms have been missing from this picture. Conifers of the pine family (Pinaceae) are a group of gymnosperms that dominate large parts of the world's forests. Despite their ecological and economic importance, conifers seemed long out of reach for complete genome sequencing, due in part to their enormous genome size (20-30 Gbp) and highly repetitive nature of their genomes. Technological advances in genome sequencing and assembly enabled the recent publication of three conifer genomes: White spruce (Picea glauca), Norway spruce (P. abies), and loblolly pine (Pinus taeda). These genome sequences revealed distinctive features compared to other plant genomes, and may represent a window into the past of seed plant genomes. This update highlights recent advances, remaining challenges, and opportunities in light of the publication of the first conifer and gymnosperm genomes.
[Show abstract][Hide abstract] ABSTRACT: The sesquiterpenoid rich essential oils of sandalwood (Santalum album L.) stems and roots are a highly sought commodity in the fragrance industry. Plantations of sandalwood are being established in northern Australia, however the valuable heartwood essential oils do not accumulate in substantial amounts before 10 years, while commercially viable harvests do not normally take place for at least 15 years. Inducing essential oil accumulation at an earlier stage, or increasing oil yield in mature trees, may have the potential to enhance the oil productivity of plantations. In this study, we investigated the effects of foliar application of methyl jasmonate on less than one-year-old sandalwood seedlings. Essential oil accumulation was unaffected in both stems and roots. However, at the gene transcript level, several key genes early in the biosynthesis of sandalwood oil components were induced in both leaves and stems. These results suggest that terpenoid biosynthesis in S. album does indeed respond to foliar application of methyl jasmonate, however the effects are small and the full biosynthesis of santalols is likely to be developmentally regulated.
[Show abstract][Hide abstract] ABSTRACT: Periodic outbreaks of spruce budworm (SBW) affect large areas of ecologically and economically important conifer forests in North America causing tree mortality and reduced forest productivity. Host resistance against SBW has been linked to growth phenology and chemical composition of foliage, but the underlying molecular mechanisms and population variation are largely unknown. Using a genomics approach, we discovered a β-glucosidase gene, Pgβglu-1, whose expression levels and function underpin natural resistance to SBW in mature white spruce (Picea glauca) trees. In phenotypically resistant trees, Pgβglu-1 transcripts were up to 1000 times more abundant compared to non-resistant trees and highly enriched in foliage. The encoded PgβGLU-1 enzyme catalyzed the cleavage of acetophenone sugar conjugates to release the aglycons piceol and pungenol. These aglycons were previously shown to be active against SBW. Levels of Pgβglu-1 transcripts and biologically active acetophenone aglycons were substantially different between resistant and non-resistant trees over time, positively correlated to each other, and highly variable in a natural white spruce population. These results suggest that expression of Pgβglu-1 and accumulation of acetophenone aglycons is a constitutive defense mechanism in white spruce. Progeny of resistant trees had higher Pgβglu-1 gene expression than non-resistant progeny indicating that the trait is heritable. With reported increases of the intensity of SBW outbreaks, influenced by climate, variation of Pgβglu-1 transcript expression, PgβGLU-1 enzyme activity, and acetophenone accumulation may serve as resistance markers to better predict impacts of SBW both in managed and wild spruce populations.This article is protected by copyright. All rights reserved.
The Plant Journal 10/2014; 81(1). DOI:10.1111/tpj.12699 · 5.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The monoterpene (+)-3-carene is associated with resistance of Sitka spruce against white pine weevil, a major North American forest insect pest of pine and spruce. High and low levels of (+)-3-carene in, respectively, resistant and susceptible Sitka spruce genotypes are due to variation of (+)-3-carene synthase gene copy number, transcript and protein expression levels, enzyme product profiles, and enzyme catalytic efficiency. A family of multi-product (+)-3-carene synthase-like genes of Sitka spruce include the three (+)-3-carene synthases, PsTPS-3car1, PsTPS-3car2, PsTPS-3car3, and the (-)-sabinene synthase PsTPS-sab. Of these, PsTPS-3car2 is responsible for the relatively higher levels of (+)-3-carene in weevil-resistant trees. Here, we identified features of the PsTPS-3car1, PsTPS-3car2, PsTPS-3car3 and PsTPS-sab proteins that determine different product profiles. A series of domain swap and site-directed mutations, supported by structural comparisons, identified the amino acid in position 596 as critical for product profiles dominated by either (+)-3-carene in PsTPS-3car1, PsTPS-3car2 and PsTPS-3car3 or (-)-sabinene in PsTPS-sab. A leucine in this position promotes formation of (+)-3-carene while phenylalanine promotes (-)-sabinene. Homology modeling predicts position 596 directs product profiles through differential stabilization of the reaction intermediate. Kinetic analysis revealed position 596 also plays a role in catalytic efficiency. Mutations of position 596 with different side chain properties resulted in a series of enzymes with different product profiles, further highlighting the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defence against insects.
[Show abstract][Hide abstract] ABSTRACT: Marrubium vulgare (Lamiaceae) is a medicinal plant whose major bioactive compounds, marrubiin and other labdane–related furanoid diterpenoids, have potential applications as anti–diabetics, analgesics or vasorelaxants. Metabolite and transcriptome profiling of M. vulgare leaves identified five different candidate diterpene synthases (diTPSs) of the TPS–c and TPS–e/f clades. We describe the in vitro and in vivo functional characterization of the M. vulgare diTPS family. In addition to MvEKS ent–kaurene synthase of general metabolism, we identified three diTPSs of specialized metabolism: MvCPS3 (+)-copalyl diphosphate synthase, and the functional diTPS pair MvCPS1 and MvELS. In a sequential reaction, MvCPS1 and MvELS produce a unique oxygenated diterpene scaffold 9,13-epoxy-labd-14-ene en route to marrubiin and an array of related compounds. In contrast to previously known diTPSs that introduce a hydroxyl group at carbon C–8 of the labdane backbone, the MvCPS1-catalyzed reaction proceeds via oxygenation of an intermediate carbocation at C–9, yielding the bicyclic peregrinol diphosphate. MvELS belongs to a subgroup of the diTPS TPS–e/f clade with unusual βα–domain architecture. MvELS is active in vitro and in vivo with three different prenyl diphosphate substrates forming the marrubiin precursor 9,13-epoxy-labd-14-ene, as identified by NMR, manoyl oxide and miltiradiene. MvELS fills a central position in the biosynthetic system that forms the foundation for the diverse repertoire of Marrubium diterpenoids. Co-expression of MvCPS1 and MvELS in engineered E. coli and Nicotiana benthamiana offers opportunities for producing precursors for an array of biologically active diterpenoids.This article is protected by copyright. All rights reserved.
The Plant Journal 07/2014; 79(6). DOI:10.1111/tpj.12589 · 5.97 Impact Factor