The style and substance of plant flavonoid decoration; towards defining both structure and function

Conifers have evolved complex oleoresin terpene defenses against herbivores and pathogens. In co-evolved bark beetles, conifer terpenes also serve chemo-ecological functions as pheromone precursors, chemical barcodes for host identification, or nutrients for insect-associated microbiomes. We highlight the genomic, molecular and biochemical underpinnings of the large chemical space of conifer oleoresin terpenes and volatiles. Conifer terpenes are predominantly the products of the conifer terpene synthase (TPS) gene family. Terpene diversity is increased by cytochromes P450 of the CYP720B class. Many conifer TPS are multiproduct enzymes. Multisubstrate CYP720B enzymes catalyse multistep oxidations. We summarise known terpenoid gene functions in various different conifer species with reference to the annotated terpenoid gene space in a spruce genome. Overall, biosynthesis of terpene diversity in conifers is achieved through a system of biochemical radiation and metabolic grids. Expression of TPS and CYP720B genes can be specific to individual cell types of constitutive or traumatic resin duct systems. Induced terpenoid transcriptomes in resin duct cells lead to dynamic changes of terpene composition and quantity to fend off herbivores and pathogens. While terpenoid defenses have contributed much to the evolutionary success of conifers, under new conditions of climate change, these defences may become inconsequential against range-expanding forest pests.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/nph.15984

Oleoresin defenses in conifers: chemical diversity, terpene synthases and limitations of oleoresin defense under climate change.

Plantation-grown trees have to cope with an increasing pressure of pest and disease in the context of climate change, and breeding approaches using genomics may offer efficient and flexible tools to face this pressure. In the present study, we targeted genetic improvement of resistance of an introduced conifer species in Canada, Norway spruce (Picea abies (L.) Karst.), to the native white pine weevil (Pissodes strobi Peck). We developed single- and multi-trait genomic selection (GS) models and selection indices considering the relationships between weevil resistance, intrinsic wood quality, and growth traits. Weevil resistance, acoustic velocity as a proxy for mechanical wood stiffness, and average wood density showed moderate-to-high heritability and low genotype-by-environment interactions. Weevil resistance was genetically positively correlated with tree height, height-to-diameter at breast height (DBH) ratio, and acoustic velocity. The accuracy of the different GS models tested (GBLUP, threshold GBLUP, Bayesian ridge regression, BayesCπ) was high and did not differ among each other. Multi-trait models performed similarly as single-trait models when all trees were phenotyped. However, when weevil attack data were not available for all trees, weevil resistance was more accurately predicted by integrating genetically correlated growth traits into multi-trait GS models. A GS index that corresponded to the breeders' priorities achieved near maximum gains for weevil resistance, acoustic velocity, and height growth, but a small decrease for DBH. The results of this study indicate that it is possible to breed for high-quality, weevil-resistant Norway spruce reforestation stock with high accuracy achieved from single-trait or multi-trait GS.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/eva.12823

Multi-trait genomic selection for weevil resistance, growth, and wood quality in Norway spruce.

Cannabis (Cannabis sativa L.) is one of the oldest cultivated plants purported to have unique medicinal properties. However, scientific research of cannabis has been restricted by the Single Convention on Narcotic Drugs of 1961, an international treaty that prohibits the production and supply of narcotic drugs except under license. Legislation governing cannabis cultivation for research, medicinal and even recreational purposes has been relaxed recently in certain jurisdictions. As a result, there is now potential to accelerate cultivar development of this multi-use and potentially medically useful plant species by application of modern genomics technologies. Whilst genomics has been pivotal to our understanding of the basic biology and molecular mechanisms controlling key traits in several crop species, much work is needed for cannabis. In this review we provide a comprehensive summary of key cannabis genomics resources and their applications. We also discuss prospective applications of existing and emerging genomics technologies for accelerating the genetic improvement of cannabis.

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Recent advances in Cannabis sativa genomics research.

Specialized phenolic compounds in seeds: structures, functions, and regulations

Cannabis (Cannabis sativa) resin is the foundation of a multibillion dollar medicinal and recreational plant bioproducts industry Major components of the cannabis resin are the cannabinoids and terpenes Variations of cannabis terpene profiles contribute much to the different flavor and fragrance phenotypes that affect consumer preferences A major problem in the cannabis industry is the lack of proper metabolic characterization of many of the existing cultivars, combined with sometimes incorrect cultivar labeling We characterized foliar terpene profiles of plants grown from 32 seed sources and found large variation both within and between sets of plants labeled as the same cultivar We selected five plants representing different cultivars with contrasting terpene profiles for clonal propagation, floral metabolite profiling, and trichome-specific transcriptome sequencing Sequence analysis of these five cultivars and the reference genome of cv Purple Kush revealed a total of 33 different cannabis terpene synthase (CsTPS) genes, as well as variations of the CsTPS gene family and differential expression of terpenoid and cannabinoid pathway genes between cultivars Our annotation of the cv Purple Kush reference genome identified 19 complete CsTPS gene models, and tandem arrays of isoprenoid and cannabinoid biosynthetic genes An updated phylogeny of the CsTPS gene family showed three cannabis-specific clades, including a clade of sesquiterpene synthases within the TPS-b subfamily that typically contains mostly monoterpene synthases The CsTPSs described and functionally characterized here include 13 that had not been previously characterized and that collectively explain a diverse range of cannabis terpenes

/pdf/terpene-synthases-and-terpene-variation-in-cannabis-sativa-1nun9rv34c.pdf

Terpene Synthases and Terpene Variation in Cannabis sativa

Plant specialized metabolism serves as a rich resource of biologically active molecules for drug discovery. The acylated flavonol glycoside montbretin A (MbA) and its precursor myricetin 3-O-(6’-O-caffeoyl)-glucosyl rhamnoside (mini-MbA) are potent inhibitors of human pancreatic α-amylase and are being developed as drug candidates to treat type-2 diabetes. MbA occurs in corms of the ornamental plant montbretia (Crocosmia x crocosmiiflora), but a system for large-scale MbA production is currently unavailable. Biosynthesis of MbA from the flavonol myricetin and MbA accumulation occur during early stages of corm development. We established myricetin 3-O-rhamnoside (MR), myricetin 3-O-glucosyl rhamnoside (MRG), and mini-MbA as the first three intermediates of MbA biosynthesis. Contrasting the transcriptomes of young and old corms revealed differentially expressed UDP-sugar-dependent glycosyltransferases (UGTs) and BAHD-acyltransferases (BAHD-ATs). UGT77B2 and UGT709G2 catalyze the consecutive glycosylation of myricetin to produce MR and of MR to give MRG, respectively. In addition, two BAHD-ATs, CcAT1 and CcAT2, catalyze the acylation of MRG to complete the formation of mini-MbA. Transcript profiles of UGT77B2, UGT709G2, CcAT1, and CcAT2 during corm development matched the metabolite profile of MbA accumulation. Expression of these enzymes in wild tobacco (Nicotiana benthamiana) resulted in the formation of a surrogate mini-MbA, validating the potential for metabolic engineering of mini-MbA in a heterologous plant system.

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana. We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3′-hydroxylase (F3′H), and a flavonoid 3′5′-hydroxylase (F3′5′H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3′5′H shared higher sequence relatedness with F3′Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3′5′H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g−1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.

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Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A

Conifers depend on complex defense systems against herbivores Stone cells (SC) and oleoresin are physical and chemical defenses of Sitka spruce that have been separately studied in previous work Weevil oviposit at the tip of the previous year's apical shoot (PYAS) We investigated interactions between weevil larvae and trees in controlled oviposition experiments with resistant (R) and susceptible (S) Sitka spruce R trees have an abundance of SC in the PYAS cortex SC are mostly absent in S trees R trees and S trees also differ in the composition of oleoresin terpenes Transcriptomes of R and S trees revealed differences in long-term weevil-induced responses Performance of larvae was significantly reduced on R trees compared with S trees under experimental conditions that mimicked natural oviposition behavior at apical shoot tips and may be attributed to the effects of SC In oviposition experiments designed for larvae to feed below the area of highest SC abundance, larvae showed an unusual feeding behavior and oleoresin appeared to function as the major defense The results support a role for both SC and oleoresin terpenes and possible synergies between these traits in the defense syndrome of weevil-resistant Sitka spruce

https://nph.onlinelibrary.wiley.com/doi/pdf/10.1111/nph.15477

Functions of stone cells and oleoresin terpenes in the conifer defense syndrome.

Diabetes and obesity are affecting human health worldwide. Their occurrence is increasing with lifestyle choices, globalization of food systems, and economic development. The specialized plant metabolite montbretin A (MbA) is being developed as an antidiabetes and antiobesity treatment due to its potent and specific inhibition of the human pancreatic α-amylase. MbA is a complex acylated flavonol glycoside formed in small amounts in montbretia (Crocosmia × crocosmiiflora) corms during the early summer. The spatial and temporal patterns of MbA accumulation limit its supply for drug development and application. We are exploring MbA biosynthesis to enable metabolic engineering of this rare and valuable compound. Genes and enzymes for the first four steps of MbA biosynthesis, starting from the flavonol precursor myricetin, have recently been identified. Here, we describe the gene discovery and functional characterization of the final two enzymes of MbA biosynthesis. The UDP-glycosyltransferases, CcUGT4 and CcUGT5, catalyze consecutive reactions in the formation of the disaccharide moiety at the 4'-hydroxy position of the MbA flavonol core. CcUGT4 is a flavonol glycoside 4'-O-xylosyltransferase that acts on the second to last intermediate (MbA-XR2) in the pathway. CcUGT5 is a flavonol glycoside 1,4-rhamnosyltransferase that converts the final intermediate (MbA-R2) to complete the MbA molecule. Both enzymes belong to the UGT family d-clade and are specific for flavonol glycosides and their respective sugar donors. This study concludes the discovery of the MbA biosynthetic pathway and provides the complete set of genes to engineer MbA biosynthesis. We demonstrate successful reconstruction of MbA biosynthesis in Nicotiana benthamiana.

Macaire Man Saint Yuen

Papers

Terpene Synthases and Terpene Variation in Cannabis sativa

Discovery of UDP-Glycosyltransferases and BAHD-Acyltransferases Involved in the Biosynthesis of the Antidiabetic Plant Metabolite Montbretin A.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A

Functions of stone cells and oleoresin terpenes in the conifer defense syndrome.

Complete Biosynthesis of the Anti-Diabetic Plant Metabolite Montbretin A.