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Cannabis glandular trichomes alter morphology and metabolite content during flower maturation
Abstract
The cannabis leaf is iconic, but it is the flowers of cannabis that are consumed for the psychoactive and medicinal effects of their specialized metabolites. Cannabinoid metabolites, together with terpenes, are produced in glandular trichomes. Superficially, stalked and sessile trichomes in cannabis only differ in size and whether they have a stalk. The objectives of this study were: to define each trichome type using patterns of autofluorescence and secretory cell numbers, to test the hypothesis that stalked trichomes develop from sessile‐like precursors, and to test whether metabolic specialization occurs in cannabis glandular trichomes. A two‐photon microscopy technique of glandular trichome intrinsic autofluorescence was developed which demonstrated that stalked glandular trichomes possessed blue autofluorescence correlated with high cannabinoid levels. These stalked trichomes had 12‐16 secretory disc cells and strongly monoterpene‐dominant terpene profiles. In contrast, sessile trichomes on mature flowers and vegetative leaves possessed red‐shifted autofluorescence, eight secretory disc cells, and less monoterpene‐dominant terpene profiles. Moreover, intrinsic autofluorescence patterns and disc cell numbers supported a developmental model where stalked trichomes develop from apparently sessile trichomes. Transcriptomes of isolated floral trichomes revealed strong expression of cannabinoid and terpene biosynthetic genes, as well as uncharacterized genes highly co‐expressed with CBDA synthase. Identification and characterization of two previously unknown and highly expressed monoterpene synthases highlighted the metabolic specialization of stalked trichomes for monoterpene production. These unique properties and highly‐expressed genes of cannabis trichomes determine the medicinal, psychoactive and sensory properties of cannabis products. This article is protected by copyright. All rights reserved.
... While the role(s) of PCs within C. sativa has not been fully established, the concentration of these compounds surrounding the ovules of female flowers and on the anthers of male flowers suggests they may act as plant defense compounds against (a) biotic stressors (Ries et al., 2000;Sirikantaramas et al., 2005;Liu et al., 2019). Female plants are prolific producers of PCs, with capitate stalked trichomes on the vegetative tissues proximal to pistillate flowers being the major site for PC biosynthesis and accumulation (Livingston et al., 2020). Despite the inherent capacity of C. sativa plants to produce large quantities of PCs, the genetic and molecular components regulating PC content and accumulation within trichomes are only partially resolved (Grassa et al., 2021;Hesami et al., 2023). ...
... Understanding of PC biosynthesis, regulation, and its compartmentalization within trichomes has accelerated in recent years (Livingston et al., 2020), with many of the enzymes having been functionally validated (Hesami et al., 2020;Grassa et al., 2021). ...
... is closely associated with PC biosynthesis based on association mapping in a PC alkyl side chain diversity panel and its co-expression with a cytosolic hexanoyl-CoA synthetase (e.g., AAE) gene Zager et al., 2019;Welling et al., 2020). For scenario 3, an oxylipin biosynthesis origin is predicted, whereby FAs are degraded into smaller carbon chains following oxidation and lyase reactions (Supplementary Figure S1), with genes encoding a linoleate 13S-lipoxygenase (LOX) and hydroperoxide lyase (HPL) highly expressed in glandular trichomes of C. sativa (Marks et al., 2009;Stout et al., 2012;Livingston et al., 2020); the focus of the present study. ...
Cannabis sativa is a multi-use and chemically complex plant which is utilized for food, fiber, and medicine. Plants produce a class of psychoactive and medicinally important specialized metabolites referred to as phytocannabinoids (PCs). The phytohormone methyl jasmonate (MeJA) is a naturally occurring methyl ester of jasmonic acid and a product of oxylipin biosynthesis which initiates and regulates the biosynthesis of a broad range of specialized metabolites across a number of diverse plant lineages. While the effects of exogenous MeJA application on PC production has been reported, treatments have been constrained to a narrow molar range and to the targeted analysis of a small number of compounds. Using high-resolution mass spectrometry with data-dependent acquisition, we examined the global metabolomic effects of MeJA in C. sativa to explore oxylipin-mediated regulation of PC biosynthesis and accumulation. A dose–response relationship was observed, with an almost two-fold increase in PC content found in inflorescences of female clones treated with 15 mM MeJA compared to the control group. Comparison of the inflorescence metabolome across MeJA treatments coupled with targeted transcript analysis was used to elucidate key regulatory components contributing to PC production and metabolism more broadly. Revealing these biological signatures improves our understanding of the role of the oxylipin pathway in C. sativa and provides putative molecular targets for the metabolic engineering and optimization of chemical phenotype for medicinal and industrial end-uses.
... In C. sativa, the phytocannabinoids are synthesized and accumulated in the glandular trichomes distributed on the aerial parts, i.e., leaves, stem and inflorescence (Andre et al., 2016;Chandra et al., 2011;Raman et al., 2017). The earlier detailed studies on dioecious C. sativa reported three types of glandular trichomes, i.e., capitate sessile, capitate stalk and bulbous (Chandra et al., 2019;Dayanandan and Kaufman, 1976;Fairbairn, 1972;Mahlberg, 1977, 1973;Livingston et al., 2020;Mohan Ram and Nath, 1964). Furthermore, the density, distribution and size of the three types of glandular trichomes are well understood in dioecious plants (Dayanandan and Kaufman, Ghosh et al. ...
... Furthermore, the density, distribution and size of the three types of glandular trichomes are well understood in dioecious plants (Dayanandan and Kaufman, Ghosh et al. Journal of Applied Research on Medicinal and Aromatic Plants xxx (xxxx) 100476 1976;Fairbairn, 1972;Hammond and Mahlberg, 1973;Kim and Mahlberg, 2003;Lanyon et al., 1981;Livingston et al., 2020). However, we are first-time demonstrating a detailed trichome study in monoecious C. sativa accessions. ...
... Our study firsttime revealed the organ-specific different glandular trichomes size in monoecious plants. In our observations, the size of glandular trichomes was within the range of earlier reports in dioecious C. sativa plants (Hammond and Mahlberg, 1973;Livingston et al., 2020). Glandular trichomes play an essential role in specialized secondary metabolite production (Azevedo and Otoni, 2010;Choi et al., 2011;Lanyon et al., 1981). ...
Cannabis sativa L., an industrially important plant, is a source of medicinally important cannabidiol (CBD) and Δ9-tetrahydrocannabinol (D9-THC). C. sativa is mainly a dioecious plant. However, rarely monoecious plants with hermaphrodite inflorescence were also reported naturally. The monoecious C. sativa plant research is still inadequate compared to dioecious plants. The present study aims to evaluate four monoecious C. sativa accessions (CH-1, CH-2, CH-3 and CH-4) for organ-specific (leaves, male flowers, and female flowers) distribution of trichomes, histochemical localization phytocannabinoids in trichomes, and phytocannabinoids content and antioxidant potential. Results showed that morphological traits differed in all the accessions (p < 0.05). The phytocannabinoids are produced in capitate sessile and capitate stalked glands. The highest density of phytocannabinoids synthesizing glands was found in female flowers (46.67–57.01 mm-2), followed by male flowers (30.73–33.98 mm-2) and leaves (12.37–23.64 mm-2) in different monoecious accessions. The female flower produces the maximum content of total phytocannabinoids. In the same way, the female flower shows the highest free radical scavenging activity and total antioxidant capacity compared to other studied plant parts. In conclusion, the CH-1 accession is superior to the other in terms of morphological characters, phytocannabinoids synthesizing glands and cannabinoids content. Moreover, the levels of phytocannabinoids are higher in female flowers than the male flowers and leaves. The approach towards monoecious accession with higher cannabinoid content has enormous potential for industrial applications.
... Despite the medicinal and economic significance of trichomes, cannabinoid levels, chemical profile, and its distribution in the trichomes remain uninvestigated and poorly understood [6]. To analyse the trichomes and to extract their chemical composition, a powerful non-destructive Raman spectroscopy technique was adopted, but no systematic information was reported about the spatial distribution of the chemical species in the plant [7,8]. ...
... Here, we aim to differentiate the structure of secretary vesicles in the whole trichome using Raman spectroscopy based on the expected variation in the cannabinoids levels. According to Livingston et al. [6] some regions in the trichomes can be identified as the secretory vesicles that are characterized by the presence of higher level of cannabinoids than others. The bright field image of trichome samples 1 and 2 is depicted in Figure 2a and 2d, respectively. ...
... We notice a large dark spot in the PC2 scoremap. Since the dimensions of the observed dark area is 20 µm, this compares with the size of a trichome structure [6]where the presence of chemical species make it Raman active. The PC3 loading does not show background (fluorescence) contributions. ...
The development of analytical techniques that decode chemical information in complex biochemical samples to discriminate different structural components may open the way for several new findings. In this study, principal component analysis (PCA) is carried out using an ad hoc Matlab coding that provides a transparent access to multivariate analysis of Raman mapping datasets. Here, we illustrated the efficacy of this method to extract meaningful results from Raman images of Cannabis sativa trichomes. A large dataset of Cannabis trichome comprising of 441 Raman spectra was examined for the first time using our OpenPCA. By mapping the chemical distribution in the trichome, we could locate the secretary vesicles in the PC score maps generated from the mapped Raman spectra. Black-box PCA solutions available in commercial software can be limited by rigid input interfaces which may prevent obtaining information by tuning the PCA analysis on selected wavenumber ranges. Hence, the OpenPCA scripts facilitate the task of obtaining key information from widely distributed range of wavenumbers that are characteristic to a specific cannabinoid, namely Δ9-THC and CBD. Overall, the PCA-coding algorithm shows advantages in decoding Raman spectra that could be extended to handle all kinds of datasets with simultaneous spatial and chemical details.
... Female C. sativa inflorescences are a rich source of hundreds of specialized metabolites, including phytocannabinoids Welling et al., 2022). Phytocannabinoid biosynthesis is concentrated within glandular trichomes (Livingston et al., 2020), present on the perigonal bracts as well as other modified floral leaves within pistillate inflorescences. The capitate stalked trichome is the most abundant trichome morphotype in pistillate inflorescences and these and are principally responsible for the high concentration of phytocannabinoids in C. sativa plants (Livingston et al., 2020). ...
... Phytocannabinoid biosynthesis is concentrated within glandular trichomes (Livingston et al., 2020), present on the perigonal bracts as well as other modified floral leaves within pistillate inflorescences. The capitate stalked trichome is the most abundant trichome morphotype in pistillate inflorescences and these and are principally responsible for the high concentration of phytocannabinoids in C. sativa plants (Livingston et al., 2020). ...
Cannabis sativa L. is a phenotypically diverse and multi-use plant used in the production of fiber, seed, oils, and a class of specialized metabolites known as phytocannabinoids. The last decade has seen a rapid increase in the licit cultivation and processing of C. sativa for medical end-use. Medical morphotypes produce highly branched compact inflorescences which support a high density of glandular trichomes, specialized epidermal hair-like structures that are the site of phytocannabinoid biosynthesis and accumulation. While there is a focus on the regulation of phytocannabinoid pathways, the genetic determinants that govern flowering time and inflorescence structure in C. sativa are less well-defined but equally important. Understanding the molecular mechanisms that underly flowering behavior is key to maximizing phytocannabinoid production. The genetic basis of flowering regulation in C. sativa has been examined using genome-wide association studies, quantitative trait loci mapping and selection analysis, although the lack of a consistent reference genome has confounded attempts to directly compare candidate loci. Here we review the existing knowledge of flowering time control in C. sativa , and, using a common reference genome, we generate an integrated map. The co-location of known and putative flowering time loci within this resource will be essential to improve the understanding of C. sativa phenology.
... These tiny hair-like structures are classified into three distinct groups: stalked, sessile, and bulbous. The bulbous trichomes have limited cannabinoid production, whereas the other two types are responsible for producing almost all of the cannabinoids [14]. ...
... Hexanoic acid is an extensively used polyketide starter molecule, and its origin is believed to be from C18 fatty acids that undergo sequential desaturation, peroxygenation, and cleavage. This cascade of reactions produces a C6 compound (hexanoic acid) and a C12 product, achieved through desaturases, lipoxygenases, and hydroperoxide lyases [14]. The formation of C6 alkyl compounds involves a series of reactions where hexanoic acid is transformed into hexanoyl-CoA, a thioester that is activated with the help of acyl-activating enzyme 1 (AAE1). ...
Cannabis is probably the best-known species for the production of alkaloids of medical importance. Recent biotechnological approaches lead to the utilization of biotechnological methods, including metabolic engineering and synthetic biology to increase the productivity of Cannabis secondary metabolites with medical importance and economic value. Hence, this review article presents the biosynthetic pathway of cannabinoid biosynthesis to underline the important genes that could be used for the enhancement of cannabinoid production together with a summary of studies of biotechnological technologies, including in vitro culture, polyploidy induction, gene transformation, gene editing, metabolic engineering, and synthetic biology used in Cannabis.
... However, they do not consider that a tissue can simultaneously present different types of trichomes. In this scenario, isolation by micromanipulation is a more suitable method for studying a specific type of trichome (Livingston et al., 2020). Optimized protocols can produce sufficient quantities of trichomes for analysis of gene expression (Zager & Lange, 2018), profiling of primary and secondary metabolites, and even analysis of 13 C incorporation following labeling experiments (Balcke et al., 2017). ...
... Adaptations of well-established protocols (Lisec et al., 2006) have enabled metabolomics analysis of protoplasts (Robaina-Est evez et al., 2017) or isolated guard cells via GC-MS (Daloso, Antunes, et al., 2015). Similarly, GC-MS and/or LC-MS have also been used to perform metabolomics analysis in guard cell preparations Jin et al., 2013;Misra et al., 2015;Zhu et al., 2020;Zhu & Assmann, 2017), seed embryos (Schwender & Ohlrogge, 2002), epidermal bladder cells (Barkla & Vera-Estrella, 2015), trichomes (Livingston et al., 2020) and other cell types (Misra et al., 2014). Highsensitivity MS and methods for separation and identification of metabolites in complex biological samples have also created the possibility of single-cell metabolomics, though these techniques offer the additional challenge of harvesting sufficient cells to meet the sensitivity threshold of the analytical platform (Guo et al., 2021). ...
Every plant organ contains tens of different cell types, each with a specialized function. These functions are intrinsically associated with specific metabolic flux distributions that permit the synthesis of the ATP, reducing equivalents and biosynthetic precursors demanded by the cell. Investigating such cell-type-specific metabolism is complicated by the mosaic of different cells within each tissue combined with the relative scarcity of certain types. However, techniques for the isolation of specific cells, their analysis in situ by microscopy, or modelling of their function in silico have permitted insight into cell-type-specific metabolism. In this review we present some of the methods used in the analysis of cell-type-specific metabolism before describing what we know about metabolism in several cell types that have been studied in depth; (1) leaf source and sink cells, (2) glandular trichomes which are capable of rapid synthesis of specialised metabolites, (3) guard cells that must accumulate large quantities of the osmolytes needed for stomatal opening, (4) cells of seeds involved in storage of reserves and (5) the mesophyll and bundle sheath cells of C4 plants that participate in a CO2 concentrating cycle. Metabolism is discussed in terms of its principal features, connection to cell function and what factors affect the flux distribution. Demand for precursors and energy, availability of substrates and suppression of deleterious processes are identified as key factors in shaping cell-type specific metabolism.
... However, phytocannabinoid synthesis involves several structures of chemical compounds, such as THC, CBD, CBG, CBC, cannabicyclol (CBL), and cannabidiol type (CBND). Phytocannabinoids are represented by several motifs composed of various moieties: the isoprenyl residue, resorcinol core, and alkyl group [34] (Figure 1). ∆ 9 -tetrahydrocannabinol (∆ 9 -THC) and cannabidiol (CBD) are the most abundant compounds in cannabis plants and are central to their therapeutic application [35,36]. ...
... Phytocannabinoids are stored in glandular trichomes, located all over the aerial part of the plant; the root surface and root tissue do not produce phytocannabinoids [34]. Moreover, plants grown outdoors are exposed to a fair amount of ultraviolet UV-A and UV-B, and exposure to UV rays before harvest causes the plant to produce more THC compounds [35]. ...
Cannabis belongs to the family Cannabaceae, and phytocannabinoids are produced by the Cannabis sativa L. plant. A long-standing debate regarding the plant is whether it contains one or more species. Phytocannabinoids are bioactive natural products found in flowers, seeds, and fruits. They can be beneficial for treating human diseases (such as multiple sclerosis, neurodegenerative diseases, epilepsy, and pain), the cellular metabolic process, and regulating biological function systems. In addition, several phytocannabinoids are used in various therapeutic and pharmaceutical applications. This study provides an overview of the different sources of phytocannabinoids; further, the biosynthesis of bioactive compounds involving various pathways is elucidated. The structural classification of phytocannabinoids is based on their decorated resorcinol core and the bioactivities of naturally occurring cannabinoids. Furthermore, phytocannabinoids have been studied in terms of their role in animal models and antimicrobial activity against bacteria and fungi; further, they show potential for therapeutic applications and are used in treating various human diseases. Overall, this review can help deepen the current understanding of the role of biotechnological approaches and the importance of phytocannabinoids in different industrial applications.
... Cannabis produces over 500 different chemical compounds, with recent reports cataloging as many as 125 cannabinoids and 150 terpenoids, as well as a variety of phenolic, alkaloid, and flavonoid compounds (Booth and Bohlmann 2019;Radwan et al. 2021). Although cannabinoids and terpenes are produced plant-wide, most are produced and stored in glandular structures called trichomes (Booth and Bohlmann 2019;Livingston et al. 2020). There has been considerable interest during the past few years in assembling Cannabis genomes (e.g., Bakel et al. 2011;Laverty et al. 2019;McKernan et al. 2020;Grassa et al. 2021). ...
... However, capitate-stalked glandular trichomes are the richest source of cannabinoids and terpenoids (Turner et al. 1978). The greatest synthetic activity and accumulation of these metabolites coincide with a change in trichome color that is used as a benchmark for sampling at a time most valuable for the cannabis industry (Zager et al. 2019;Booth et al. 2020;Livingston et al. 2020). ...
Cannabis sativa is widely used for fiber, medicinal, and other purposes, and many cultivars exist, yielding varying proportions of cannabinoids and terpenes. There is considerable interest in characterizing genomes and transcriptomes of C. sativa. Alternative splicing (AS) is a fundamental aspect of gene expression that results in multiple types of mRNAs produced by differential splicing. Transcriptome-wide identification of AS events in drug-type cultivars of C. sativa has not been reported. Here we identified AS events using a transcriptome dataset derived from five drug-type cultivars with divergent chemotypes. Intron retention is the most common event type, followed by alternative acceptor, alternative donor, and skipped exons. We also sought to assess conservation of AS events among cultivars. We found 547 events (5%) unique to a single cultivar, 2661 (25%) shared by 2-4 cultivars, and 7569 (70%) common to all five cultivars. Genes with AS events in each set were analyzed for gene ontology enrichment, showing that genes with AS unique to a single cultivar are enriched for molecular functions related to interactions with ATP, and processes involving transport within cells and across membranes. These results provide insights into the conservation and variation of AS events in multiple cultivars of C. sativa.
... They are classified in three types based on their morphologies: bulbous, sessile, and stalked. Trichomes are also different in their metabolic profiles (Livingston et al., 2020) and distribution in leaves and inflorescences (Turner et al., 1980). The stalked glandular trichomes accumulate more total cannabinoids than the other types. ...
... The stalked glandular trichomes accumulate more total cannabinoids than the other types. They are found exclusively on the surface of perigonal bracts and the small companion leaves ("sugar leaves") of the female raceme inflorescences and develop from the sessile trichomes under short day photoperiod (Spitzer-Rimon et al., 2019;Livingston et al., 2020;Tanney et al., 2021). More than 113 different cannabinoids are produced by C. sativa, among which the most abundant are cannabigerolic acid (CBGA), cannabidiolic acid (CBDA), D 9 -tetrahydrocannabinolic acid (THCA), and cannabichromenic acid (CBCA), together with their decarboxylated neutral forms (CBG, CBD, THC, and CBC, respectively) (Gülck and Møller, 2020). ...
Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids.
... Booth et al. analyzed the genome and transciptome of Purple Kush cannabis to identify more than 30 cannabis terpene synthases (CsTPS genes) [35], which has been expanded to over 14 cultivars, representing chemotypes I, II, and III [34,36,37]. The characterized TPS genes of cannabis are documented as being a part of the TPS-a and TPS-b subfamilies [29]. ...
... In addition, non-enzymatic modifications of terpenes, such as cyclization and oxidation, can increase structural diversity independent of enzymatic biochemical reactions. Even post-harvest considerations can Booth et al. analyzed the genome and transciptome of Purple Kush cannabis to identify more than 30 cannabis terpene synthases (CsTPS genes) [35], which has been expanded to over 14 cultivars, representing chemotypes I, II, and III [34,36,37]. The characterized TPS genes of cannabis are documented as being a part of the TPS-a and TPS-b subfamilies [29]. ...
Cannabis is a complex biosynthetic plant, with a long history of medicinal use. While cannabinoids have received the majority of the attention for their psychoactive and pharmacological activities, cannabis produces a diverse array of phytochemicals, such as terpenes. These compounds are known to play a role in the aroma and flavor of cannabis but are potent biologically active molecules that exert effects on infectious as well as chronic diseases. Furthermore, terpenes have the potential to play important roles, such as synergistic and/or entourage compounds that modulate the activity of the cannabinoids. This review highlights the diversity and bioactivities of terpenes in cannabis, especially minor or secondary terpenes that are less concentrated in cannabis on a by-mass basis. We also explore the question of the entourage effect in cannabis, which studies to date have supported or refuted the concept of synergy in cannabis, and where synergy experimentation is headed, to better understand the interplay between phytochemicals within Cannabis sativa L.
... glandular trichomes, which are highly enriched on floral organs, alter morphology and metabolite content during flower maturation (Livingston et al., 2020), and similar changes may occur on leaf surfaces. We concluded that growing C. sativa L. on fertilized soil may have resulted in increased metabolite content and likely changed metabolite profiles in a cultivar-specific manner, thus resulting in the differently enhanced prediction accuracies in our study. ...
Cannabis sativa L. is a versatile crop attracting increasing attention for food, fiber, and medical uses. As a dioecious species, males and females are visually indistinguishable during early growth. For seed or cannabinoid production, a higher number of female plants is economically advantageous. Currently, sex determination is labor‐intensive and costly. Instead, we used rapid and non‐destructive hyperspectral measurement, an emerging means of assessing plant physiological status, to reliably differentiate males and females. One industrial hemp (low tetrahydrocannabinol [THC]) cultivar was pre‐grown in trays before transfer to the field in control soil. Reflectance spectra were acquired from leaves during flowering and machine learning algorithms applied allowed sex classification, which was best using a radial basis function (RBF) network. Eight industrial hemp (low THC) cultivars were field grown on fertilized and control soil. Reflectance spectra were acquired from leaves at early development when the plants of all cultivars had developed between four and six leaf pairs and in three cases only flower buds were visible (start of flowering). Machine learning algorithms were applied, allowing sex classification, differentiation of cultivars and fertilizer regime, again with best results for RBF networks. Differentiating nutrient status and varietal identity is feasible with high prediction accuracy. Sex classification was error‐free at flowering but less accurate (between 60% and 87%) when using spectra from leaves at early growth stages. This was influenced by both cultivar and soil conditions, reflecting developmental differences between cultivars related to nutritional status. Hyperspectral measurement combined with machine learning algorithms is valuable for non‐invasive assessment of C. sativa cultivar and sex. This approach can potentially improve regulatory security and productivity of cannabis farming.
... In Cannabis sativa, there might exist a partially shared biosynthetic pathway in oxylipins and cannabinoids production. In the plant, a specific examination of trichomes' gene expression revealed that the genes responsible for producing oxylipins, namely LIPOXYGENASE (LOX) and HYDROPEROXIDE LYASE (HPL), were found to be co-expressed with genes already known to be involved in the production of cannabinoids (Stout et al., 2012;Livingston et al., 2020). This observation has led to the hypothesis that the oxylipin pathway provides the necessary substrate for the production of cannabinoids (Borrego et al., 2023). ...
Lipids are a principal component of plasma membrane, acting as a protective barrier between the cell and its surroundings. Abiotic stresses such as drought and temperature induce various lipid-dependent signaling responses, and the membrane lipids respond differently to environmental challenges. Recent studies have revealed that lipids serve as signal mediators forreducing stress responses in plant cells and activating defense systems. Signaling lipids, such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, and N-acylethanolamines, are generated in response to stress. Membrane lipids are essential for maintaining the lamellar stack of chloroplasts and stabilizing chloroplast membranes under stress. However, the effects of lipid signaling targets in plants are not fully understood. This review focuses on the synthesis of various signaling lipids and their roles in abiotic stress tolerance responses, providing an essential perspective for further investigation into the interactions between plant lipids and abiotic stress.
... Cannabinoids are also synthesized in vegetative tissues throughout the life cycle in both male and female plants, albeit at lower concentrations (Pacifico et al. 2008;Stack et al. 2021). Trichomes form a secretory cavity bordered by the plant cuticle and secretory disc cells, which effectively isolate the produced cannabinoids from the somatic plant cells (Livingston et al. 2020). Modern cannabis breeding has focused on selecting plants with high trichome densities and cannabinoid content as superior traits for commercial THC/CBD production (Clarke and Merlin 2016); however, it is not known whether selecting for these traits is beneficial to the plant in an ecological or evolutionary context. ...
i>Cannabis sativa is one of the oldest cultivated crops, used for its fiber and medicinal properties. The cannabis plant synthesizes a myriad of secondary metabolites, but the most valuable products from a medical and commercial standpoint are cannabinoids. Despite significant advances in elucidating the biochemistry and genetics that govern cannabinoid accumulation, we still do not have conclusive evidence for the role of these secondary metabolites in the physiology of C. sativa . In line with known functions of other secondary metabolites, the protective functions of cannabinoids against temperature stress, poor micronutrient soil content, drought, UV-B radiation, and as anti-microbial agents have been suggested, but are yet to be conclusively demonstrated. Recent research suggests that the environment has a major effect on cannabis growth and productivity, but the relationship between stress, cannabinoid accumulation, and plant health is complex. Here, we summarize the current insights on how abiotic and biotic stress affect C. sativa biology. We also examine the available evidence to support the hypothesis for the protective function of cannabinoids against environmental stressors. Maintaining optimal growth and high cannabinoid synthesis is a balancing act, one that can only be achieved by better understanding of the effects on the environment on the cannabis plant.
... Tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) are made from the common precursor, cannabigerolic acid (CBGA) whereas CBN is an oxidation product of THC. Acidic forms of cannabinoids are biosynthesized in the trichomes on the female flowers (Sirikantaramas et al., 2005;Livingston et al., 2020). The storage of the toxic cannabinoids in trichomes minimizes the risk of self-intoxication (Sirikantaramas et al., 2005). ...
Cannabis sativa L. is an ancient crop used for fiber and seed production and not least for its content of cannabinoids used for medicine and as an intoxicant drug. Due to the psychedelic effect of one of the compounds, tetrahydrocannabinol (THC), many countries had regulations or bands on Cannabis growing, also as fiber or seed crop. Recently, as many of these regulations are getting less tight, the interest for the many uses of this crop is increasing. Cannabis is dioecious and highly heterogenic, making traditional breeding costly and time consuming. Further, it might be difficult to introduce new traits without changing the cannabinoid profile. Genome editing using new breeding techniques might solve these problems. The successful use of genome editing requires sequence information on suitable target genes, a genome editing tool to be introduced into plant tissue and the ability to regenerate plants from transformed cells. This review summarizes the current status of Cannabis breeding, uncovers potentials and challenges of Cannabis in an era of new breeding techniques and finally suggests future focus areas that may help to improve our overall understanding of Cannabis and realize the potentials of the plant.
... densely produced on female inflorescences of Cannabis sativa (Dayanandan & Kaufman, 1976;Livingston et al., 2020;Turner et al., 1978). The adaptive value of cannabinoid synthesis prior to human cultivation is not known, but theories include protection from herbivores, pathogens, or ultraviolet radiation (Gorelick & Bernstein, 2017;Lydon et al., 1987;Pate, 1983;Tanney et al., 2021). ...
Cannabis sativa is cultivated for multiple uses including the production of cannabinoids. In developing improved production systems for high-cannabinoid cultivars, scientists and cultivators must consider the optimization of complex and interacting sets of morphological, phenological, and biochemical traits, which have historically been shaped by natural and anthropogenic selection. Determining factors that modulate cannabinoid variation within and among genotypes is fundamental to developing efficient production systems and understanding the ecological significance of cannabinoids. Thirty-two high-cannabinoid hemp cultivars were characterized for traits including flowering date and shoot-tip cannabinoid concentration. Additionally, a set of plant architecture traits, as well as wet, dry, and stripped inflorescence biomass were measured at harvest. One plant per plot was partitioned post-harvest to quantify intra-plant variation in inflorescence biomass production and cannabinoid concentration. Some cultivars showed intra-plant variation in cannabinoid concentration, while many had a consistent concentration regardless of canopy position. There was both intra- and inter-cultivar variation in architecture that correlated with intra-plant distribution of inflorescence biomass, and concentration of cannabinoids sampled from various positions within a plant. These relationships among morphological and biochemical traits will inform future decisions by cultivators, regulators, and plant breeders.
... This was consistent with the finding of Aizpurua-Olaizola et al. (2016) for cannabis chemotype I. The reason may be that monoterpenes are biosynthesized in the same glandular trichomes and partly by a similar biochemical pathway as cannabinoids, consisting of alkylresorcinol and monoterpene groups (Booth et al., 2017;Livingston et al., 2020;Sirikantaramas et al., 2007). In contrast, the sesquiterpene, β-caryophyllene, showed a higher constant content in DS in almost all measured weeks (Fig. 7C). ...
Rising temperatures, higher water consumption for irrigation, and the discharge of agricultural wastewater may soon adversely affect water resources across the entire planet and its people. Agriculture consumes large amounts of water, and there is a pressing need to explore possibilities for recirculation and reuse of nutrient solutions to reduce the associated environmental and economic costs of hydroponics. This study compared two different nutrient systems for hydroponic cultivation (recirculation, RS, and drain-to-waste system, DS), and assessed their effect on the growth and concentration of elements and secondary metabolites in medical cannabis (Cannabis sativa L.) plants. The dry decomposition method with flame atomic absorption spectroscopy (FAAS) and inductively coupled plasma optical emission spectroscopy (ICP-OES) were used for the elemental analysis of plant organs. Nitrogen content was measured using the Kjeldahl method. A high-performance liquid chromatograph equipped with a diode array detector (HPLC-DAD) was used to analyze the phytocannabinoid profile in ethanolic flower extracts. Gas chromatography with flame ionization detection (GC-FID) and gas chromatography with mass spectrometry (GC-MS) were employed to determine the content of terpenic compounds in hexane extracts. Cultivation of plants with the RS system, achieved an 87% higher maximum yield of the main cannabinoid of medical cannabis chemotype I, tetrahydrocannabinolic acid (THCA), with lower total water and nutrient consumption, but with a longer cultivation time and a lower concentration of the sesquiterpene, β-caryophyllene. In contrast, the DS system enabled better control over the nutrient solution with stable delivery of the exact concentrations of nutrients. DS accelerated plant maturation, but at the cost of higher water and fertilizer consumption and a significantly lower total yield of THCA and monoterpenes.
... 4,5 The female inflorescence of the cannabis plant forms glandular trichomes, which serve as the primary site for the production and storage of valued cannabinoids and terpenoids. 6,7 Flavonoids represent a third major group of secondary metabolite categories with added value potential; these are found in the highest abundance in leaves, followed by the inflorescence. 8 Cannabis accessions, environmental conditions during cultivation, harvesting techniques, and post-harvest processing are four critical parameters that affect secondary metabolite profiles, in addition to the quality of the cannabis plant biomass or cannabis-derived products; as such, post-harvest activities are considered by some as crucial to the entire economy, requiring technological evaluation and innovation. ...
... Photoperiod, or daylight length, triggers transitions between vegetative and flowering phases in C. sativa, affecting cannabinoid and terpene production [37]- [40]. Indoor cultivators control this transition using an 18/4 to 24/0 (on/off) light cycle during propagation and vegetative stages, ensuring PFR supply [11], [42], [43]. ...
The limited availability of peer-reviewed scientific evidence in the cannabis industry has led many companies to rely on techniques derived from non-peer-reviewed sources for their practices. This study begins by examining the literature on the cultivation of
C. sativa
, investigating optimal conditions and their effects on growth, and characterising the requirements for greenhouse monitoring and control. A systematic review of current technological approaches is then conducted. The review demonstrates that technology-based control of greenhouse environments has the potential to surpass manual or traditional rule-based management techniques by reducing costs and increasing yields. However, the adoption of these technologies is impeded by the lack of publicly available labelled data on growth, pest and disease, environmental, and yield data of multiple indoor cultivation cycles. Currently, much of the research in this field is conducted privately by companies in the cannabis industry. This study recognises substantial gaps in research surrounding
C. sativa
cultivation and emphasises the opportunity for new research to address the absence of available
C. sativa
datasets and peer-reviewed scientific studies outside of private endeavours.
... Female cannabis inflorescences are the primary source of cannabinoids and terpenes (Ohlsson et al., 1971), with stalked glandular trichomes on the surface of floral organs and bracts being the key secretory structures (Mahlberg and Eun, 2004;Livingston et al., 2020). Recent work has used a deep learning pipeline to identify stages of trichome development based on their age-based transition through clear-milky-brown phenotypes, providing a sophisticated tool for cannabis product investigation (Sutton et al., 2023). ...
Cannabis sativa remains under heavy legal restriction around the globe that prevents extensive investigations into agricultural applications for improving its development. This work investigates the potential of specific plant growth-promoting rhizobacteria (PGPR) to improve Cannabis cannabinoid yield through increased trichome densities on floral organs, and to determine if sub-optimal environmental conditions would affect the outcomes of PGPR presence by altering plant development and cannabinoid profiles. Here, Pseudomonas sp. or Bacillus sp. were applied to the root system either separately or in a consortium to determine the effect of this bacterial treatment on the density of stalked glandular trichomes. Further, a low nutrient regime was applied for the first half of plant development to determine if an environmental stressor interacts with the effects of the microbial treatments on stalked trichome densities. Following 8 weeks of flower development, trichome density on calyces and bracts of inflorescences were determined using microscopy. Our findings unexpectedly indicate that recommended nutrient levels were linked to a decreasing trend in trichome densities with PGPR inoculations, but a low nutrient regime coupled with PGPR treatment increased them. Cannabinoid content is partially consistent with these results, in that a low nutrient regime increased the abundance of key cannabinoids compared to recommended regimes, with Bacillus sp. inoculation linked to the greatest number of significant changes between the two nutrient regimes. Overall, this work provides insight into how PGPR presence affects Cannabis stalked trichome development and cannabinoid profiles, and how environmental stressors can affect, and even enhance, trichome densities and influence major cannabinoid production, thereby pointing towards avenues for reducing the reliance on synthetic fertilizers during plant production without compromising yield.
... All TPS-b clade members characterized thus far generated monoterpenes with GPP ( Fig. 1). Some catalyzed the formation of one major product (e.g., b-myrcene by TPS3FN, TPS15CT, and TPS30PK; (E)-b-ocimene by TPS6FN and TPS38FN; (Z)-b-ocimene by TPS13PK; (-)-limonene by TPS1PK, CsTPS1FN, and CsTPS14CT; terpinolene by TPS37FN; and (?)-a-pinene by CsTPS2SK and CsTPS2FN), or two products (a-terpinene and cterpinene by TPS33PK), while others generated mixtures without a major product (TPS5PK, TPS13LS, TPS17BC, TPS31PK, and TPS32PK) (Booth et al. 2017Livingston et al. 2020;Zager et al. 2019;Günnewich et al. 2007) (Table 1 and Fig. 1). Two of these enzymes (TPS14CT and TPS15CT) were found to be inactive with neryl diphosphate (NPP), the cisisomer of GPP, as an alternative substrate (the others ...
Terpene synthases catalyze the first committed step in the biosynthesis of terpenes, a structurally diverse class of natural products that also encompasses volatiles derived from precursors in the C10 to C15 range (termed monoterpenes and sesquiterpenes, respectively). In the review section of this article, we are providing information about all functionally characterized monoterpene synthases (MTSs) and sesquiterpene synthases (STSs) of Cannabis sativa L. We are also exploring the locations of MTSs and STSs in the chromosome-level assembly of the reference chemovar CBDRx. A follow-up computational structure–function analysis focuses on MTSs, as there is already a rich literature available on the topic. More specifically, by employing sequence comparisons and homology structural modeling, we infer which amino acid residues are likely to constrain the available space in the active site of cannabis MTSs. The emphasis of these studies was to investigate why some MTSs accept only a C10 diphosphate as substrate, while mixed MTS/STS enzymes also accommodate a C15 diphosphate. By combining a literature review and computational analyses in a hybrid format, we are laying the foundation for future studies to better understand the determinants of substrate and product specificity in these fascinating enzymes.
... Additionally, as a fast-growing annual plant, C. sativa has the potential to aid carbon sequestration efforts (Finnan and Styles, 2013). However, C. sativa is best known for its medicinal and intoxicating applications, as glandular trichomes on female C. sativa flowers produce high-value, psychoactive compounds called cannabinoids (Livingston et al., 2020;Melzer et al., 2022). One of these cannabinoids, cannabidiol (CBD), is non-intoxicating with anti-cancer and anti-inflammatory effects, currently used to treat conditions like epilepsy and multiple sclerosis . ...
Hemp ( Cannabis sativa L.) is an extraordinarily versatile crop, with applications ranging from medicinal compounds to seed oil and fibre products. Cannabis sativa is a short-day plant, and its flowering is tightly controlled by photoperiod. However, substantial genetic variation exists for photoperiod sensitivity in C. sativa, and photoperiod-insensitive (“autoflower”) cultivars are available.
Using a bi-parental mapping population and bulked segregant analysis, we identified Autoflower2 , a 0.5 Mbp locus significantly associated with photoperiod-insensitive flowering in hemp. Autoflower2 contains an ortholog of the central flowering time regulator FLOWERING LOCUS T ( FT ) from Arabidopsis thaliana which we termed CsFT1 . Extensive sequence divergence between alleles of CsFT1 was identified between photoperiod-sensitive and insensitive cultivars of C. sativa , including a duplication of CsFT1 and sequence differences especially in introns. Genotyping of several mapping populations and a diversity panel confirmed a strong correlation between CsFT1 alleles and photoperiod response as well as affirming that at least two independent loci for this agriculturally important trait, Autoflower1 and Autoflower2 , exist in the C. sativa gene pool.
This study reveals the multiple independent origins of photoperiod insensitivity in C. sativa, supporting the likelihood of a complex domestication history in this species. By integrating the genetic relaxation of photoperiod sensitivity into novel C. sativa cultivars, expansion to higher latitudes will be permitted, thus allowing the full potential of this versatile crop to be reached.
... All TPS-b clade members characterized thus far generated monoterpenes with GPP ( Fig. 1). Some catalyzed the formation of one major product (e.g., b-myrcene by TPS3FN, TPS15CT, and TPS30PK; (E)-b-ocimene by TPS6FN and TPS38FN; (Z)-b-ocimene by TPS13PK; (-)-limonene by TPS1PK, CsTPS1FN, and CsTPS14CT; terpinolene by TPS37FN; and (?)-a-pinene by CsTPS2SK and CsTPS2FN), or two products (a-terpinene and cterpinene by TPS33PK), while others generated mixtures without a major product (TPS5PK, TPS13LS, TPS17BC, TPS31PK, and TPS32PK) (Booth et al. 2017Livingston et al. 2020;Zager et al. 2019;Günnewich et al. 2007) (Table 1 and Fig. 1). Two of these enzymes (TPS14CT and TPS15CT) were found to be inactive with neryl diphosphate (NPP), the cisisomer of GPP, as an alternative substrate (the others Fig. 1 Structures of monoterpenes and C10 prenyl diphosphates mentioned in the narrative. ...
Terpene synthases catalyze the first committed step in the biosynthesis of terpenes, a structurally diverse class of natural products that also encompasses volatiles derived from precursors in the C10 to C15 range (termed monoterpenes and sesquiterpenes, respectively). In the review section of this article, we are providing information about all functionally characterized monoterpene synthases (MTSs) and sesquiterpene synthases (STSs) of Cannabis sativa L. We are also exploring the locations of MTSs and STSs in the chromosome-level assembly of the reference chemovar CBDRx. A follow-up computational structure–function analysis focuses on MTSs, as there is already a rich literature available on the topic. More specifically, by employing sequence comparisons and homology structural modeling, we infer which amino acid residues are likely to constrain the available space in the active site of cannabis MTSs. The emphasis of these studies was to investigate why some MTSs accept only a C10 diphosphate as substrate, while mixed MTS/STS enzymes also accommodate a C15 diphosphate. By combining a literature review and computational analyses in a hybrid format, we are laying the foundation for future studies to better understand the determinants of substrate and product specificity in these fascinating enzymes.
... However, their location in other parts of the plant is possible, although to a lesser extent (Figure 1), e.g., seeds, leaves, roots, and pollen [16,23]. Livingston et al. [24] concluded that revealing the particular and uncommon properties of these economically and biotechnologically important structures provides new opportunities to optimize the harvest time and extraction processing for the obtention of cannabinoids. ...
The Cannabis genus of plants has been widely used in different cultures for various purposes. It is separated into three main species: sativa, indica, and ruderalis. In ancient practices, the plant was used as a multipurpose crop and valued for its fiber, food, and medicinal uses. Since methodologies for the extraction, processing, and identification of components have become available, medical, and food applications have been increasing, allowing potential development in the pharmaceutical and healthy functional food industries. Although the growing legalization and adoption of cannabis for the treatment of diseases are key factors pushing the growth of its market, the biggest challenge is to obtain higher-quality products in a time- and cost-effective fashion, making the process of extraction and separation an essential step. Latin American countries exhibit great knowledge of extraction technologies; nevertheless, it is still necessary to verify whether production costs are economically profitable. In addition, there has been an increase in commercial cannabis products that may or may not be allowed, with or without quality fact sheets, which can pose health risks. Hence, legalization is mandatory and urgent for the rest of Latin American countries. In this article, the phytochemical compounds (cannabinoids, terpenes, and phenolic compounds), the current status of legalization, extraction techniques, and research advances in cannabis in Latin America are reviewed.
... The most abundant secondary metabolites of hemp are terpenes and cannabinoids. Terpenes are derived from 5-carbon precursors, isopentenyl diphosphate, and dimethylallyl diphosphate, which are modified and joined to form mono-, di-, or sesquiterpenes (Chen et al., 2011) and stored in Cannabis glandular trichomes that cover leaves and pistillate bracts (Livingston et al., 2020). Glandular trichomes of Cannabis typically accumulate tetrahydrocannabinolic acid (THCa), cannabidiolic acid (CBDa), or a mixture of both (Toth et al., 2020). ...
Evolved resistance to herbicides necessitates alternative weed control strategies. Allelopathic crops show promise as an alternative to exogenous herbicides and could be used to reduce establishment, growth, and reproduction of weeds. Individual cannabinoids and terpenes found in hemp ( Cannabis sativa L.) show allelopathic potential, but allelopathic effects of plant residue have not been characterized. A laboratory assay found that crude, acetone-soluble hemp extracts diluted to 2.5 mg mL ⁻¹ reduced the germination of a bioindicator species. However, tested concentrations below 2.5 mg mL ⁻¹ were not more effective than the no-extract control treatment at reducing germination. A greenhouse study found that soil-incorporated hemp residue was not more effective than a maize ( Zea mays L.) residue treatment comparison in reducing plant growth. However, a simulated chaff line experiment in the greenhouse showed that the equivalent of 378 g m ⁻² hemp residue on the soil surface can effectively reduce and delay the germination of waterhemp [ Amaranthus tuberculatus (Moq.) Sauer] in comparison to bare-soil, or a maize residue treatment comparison. Together, these results show that incorporated hemp residue will likely not be an effective weed control practice. However, chaff lining hemp residue may be an effective practice for the control of certain weeds and warrants further research in a field setting.
... Cannabinoids are unique phenolic substances found in cannabis, and there are approximately 100 types of cannabis [5]. Female flowers are the main sinks for accumulating large amounts of cannabinoids, such as cannabidiol (CBD), ∆-9tetrahydrocannabinol (THC), and cannabigerol (CBG) [6,7]. Besides cannabinoids, terpenes, which also confer medicinal properties, are produced in floral tissues too [5]. ...
Female Cannabis sativa plants have important therapeutic properties. The sex ratio of the dioecious cannabis is approximately 1:1. Cultivating homozygous female plants by inducing female plants to produce male flowers is of great practical significance. However, the mechanism underlying cannabis male development remains unclear. In this study, single-molecule real-time (SMRT) sequencing was performed using a mixed sample of female and induced male flowers from the ZYZM1 cannabis variety. A total of 15,241 consensus reads were identified, and 13,657 transcripts were annotated across seven public databases. A total of 48 lncRNAs with an average length of 986.54 bp were identified. In total, 8202 transcripts were annotated as transcription factors, the most common of which were bHLH transcription factors. Moreover, tissue-specific expression pattern analysis showed that 13 MADS transcription factors were highly expressed in male flowers. Furthermore, 232 reads of novel genes were predicted and enriched in lipid metabolism, and qRT-PCR results showed that CER1 may be involved in the development of cannabis male flowers. In addition, 1170 AS events were detected, and two AS events were further validated. Taken together, these results may improve our understanding of the complexity of full-length cannabis transcripts and provide a basis for understanding the molecular mechanism of cannabis male development.
... Cannabinoids are structurally isoprenylated resorcinyl polyketides that are biosynthesised in the glandular trichomes of female flowers [8,9] and have been classified into nine structural families: namely, THC, CBD, cannabichromene (CBC), cannabinodiol (CBND), cannabigerol (CBG), cannabitriol (CBT), cannabicyclol (CBL), cannabielsoin (CBE) and cannabinol (CBN) [10]. All cannabinoids share the same initial pathways, namely the polyketide and methylerythritol 4-phosphate (MEP) pathway, which produce olivetolic acid (OLA) and geranyl diphosphate (GPP), respectively. ...
In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous plants and cell cultures are still limited. In this study, we assessed the potential use of N. benthamiana (Nicotiana benthamiana) plants as a heterologous host for producing natural and novel cannabinoids. Transgenic N. benthamiana plants expressing genes encoding cannabis acyl-activating enzyme and olivetol synthase were generated, which were then used for transiently expressing other downstream pathway genes. Production of olivetolic acid and divarinic acid, the universal precursors for major and minor cannabinoids, respectively, was observed in transgenic N. benthamiana plants. To produce novel cannabinoid precursors with different side chains, various fatty acids were infiltrated into the transgenic N. benthamiana plants and the production of novel derivatives was observed. Although we were not able to derive the core intermediate, cannabigerolic acid, from our transgenic plants, possibly due to the low production levels of the precursors, our transgenics plants still serve as a high-potential platform for further development and exploring the N. benthamiana chemical space for generating novel cannabinoids.
... Cannabis exhibits substantial versatility, with various parts of the plant providing fiber, and food, as well as intoxicant and medicinal molecules. These medicinal compounds are secondary metabolites, specifically the cannabinoids, produced in glandular trichomes that protrude from the epidermal layer, predominantly on the floral organs of female plants [3]. The most well-known and comprehensively studied cannabinoids are Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). ...
Cannabis sativa (Cannabis) has recently been legalized in multiple countries globally for either its recreational or medicinal use. This, in turn, has led to a marked increase in the number of Cannabis varieties available for use in either market. However, little information currently exists on the genetic distinction between adopted varieties. Such fundamental knowledge is of considerable value and underpins the accelerated development of both a nascent pharmaceutical industry and the commercial recreational market. Therefore, in this study, we sought to assess genetic diversity across 10 Cannabis varieties by undertaking a reduced representation shotgun sequencing approach on 83 individual plants to identify variations which could be used to resolve the genetic structure of the assessed population. Such an approach also allowed for the identification of the genetic features putatively associated with the production of secondary metabolites in Cannabis. Initial analysis identified 3608 variants across the assessed population with phylogenetic analysis of this data subsequently enabling the confident grouping of each variety into distinct subpopulations. Within our dataset, the most diagnostically informative single nucleotide polymorphisms (SNPs) were determined to be associated with the long-terminal repeat (LTRs) class of retroelements, with 172 such SNPs used to fully resolve the genetic structure of the assessed population. These 172 SNPs could be used to design a targeted resequencing panel, which we propose could be used to rapidly screen different Cannabis plants to determine genetic relationships, as well as to provide a more robust, scientific classification of Cannabis varieties as the field moves into the pharmaceutical sphere.
... C. sativa synthesizes and accumulates more than 500 known secondary metabolites [4]. Considerable efforts are aimed at investigating this plant's secondary metabolites, including cannabinoids and terpenes [5,6]. Major cannabinoids, including ∆9-tetrahydrocannabinol (THC) and cannabidiol (CBD), aid in reducing chronic pain, chemotherapy-induced nausea, vomiting, and improving multiple sclerosis spasticity symptoms [7][8][9]. ...
Light is one of the most crucial parameters for enclosed cannabis (Cannabis sativa) production, as it highly influences growth, secondary metabolite production, and operational costs. The objective of this study was to investigate and evaluate the impact of six light spectra on C. sativa (‘Babbas Erkle Cookies’ accession) growth traits and secondary metabolite (cannabinoid and terpene) profiles. The light spectra evaluated included blue (430 nm), red (630 nm), rose (430 + 630 nm, ratio 1:10), purple (430 + 630 nm, ratio 2:1), and amber (595 nm) LED treatments, in addition to a high-pressure sodium (HPS, amber-rich light) treatment as a control. All the LED light treatments had lower fresh mean inflorescence mass than the control (HPS, 133.59 g plant−1), and monochromatic blue light yielded the least fresh inflorescence mass (76.39 g plant−1). Measurement of Δ9-tetrahydrocannabinol (THC) concentration (%) and total yield (g plant−1) showed how inflorescence mass and THC concentration need to be analyzed conjointly. Blue treatment resulted in the highest THC concentration (10.17% m/m), yet the lowest THC concentration per plant (1.44 g plant−1). The highest THC concentration per plant was achieved with HPS (2.54 g plant−1). As with THC, blue light increased cannabigerol (CBG) and terpene concentration. Conversely, blue light had a lesser impact on cannabidiol (CBD) biosynthesis in this C. sativa chemotype. As the combined effects of the light spectrum on both growth traits and secondary metabolites have important ramifications for the industry, the inappropriate spectral design could cause a reduction in cannabinoid production (20–40%). These findings show promise in helping producers choose spectral designs that meet specific C. sativa production goals.
... Cannabinoids are produced mostly in glandular trichomes, which are located in the bracts of female flowers. However, cannabinoids can also be present in other parts of the plant, such as seeds, but to a lesser extent (Small and Naraine, 2016;Livingston et al., 2020). Cannabinoids are terpenophenols that can be classified in different groups depending on their structure. ...
Nutrition security is a challenge of the XXI century for achieving a sustainable health. Hemp cultivation contributes to the European Green Deal objectives and is a potential solution for producing a more sustainable food chain and contributing to the nutrition security of the global population. Hemp, Cannabis sativa cultivars containing less than 0.2% of Δ9-tetrahydrocannabinol (THC), is a multipurpose crop which can be used to produce feed, food and supplements among other products (biodegradable plastics, paper, paint). Hemp seeds are the hemp component most used in the food context, and the products derived from them (oil, cake, flour and proteins) are gaining popularity in human nutrition. In the European Union (EU), only marketing of hemp seeds and their derivatives, such as hemp seed oil, hemp seed flour, defatted hemp seed, and germinated hemp seed is authorized. Other parts of the plant are considered as novel foods. Nutrition claims “high dietary fiber, high protein, low saturated fat, high omega-3 fatty acids, high polyunsaturated fat, high unsaturated fat” can be attributed to those hemp products. In addition, hemp is a source of bioactive compounds, cannabinoids and others, with great impact in health including that of the brain-gut axis which is essential for achieving optimal physical and emotional conditions. The present chapter represents an updated revision of the state of the art on the potential of hemp in nutrition security.
... Trichome isolation was performed following the protocol developed by Livingston et al. 2020 [81] with several modifications. Trichomes collected were detected using microscope. ...
Wall-associated kinases (WAKs) are receptors that bind pectin or small pectic fragments in the cell wall and play roles in cell elongation and pathogen response. In the Cannabis sativa (Cs) genome, 53 CsWAK/CsWAKL (WAK-like) protein family members were identified and characterized; their amino acid lengths and molecular weights varied from 582 to 983, and from 65.6 to 108.8 kDa, respectively. They were classified into four main groups by a phylogenetic tree. Out of the 53 identified CsWAK/CsWAKL genes, 23 CsWAK/CsWAKL genes were unevenly distributed among six chromosomes. Two pairs of genes on chromosomes 4 and 7 have undergone duplication. The number of introns and exons among CsWAK/CsWAKL genes ranged from 1 to 6 and from 2 to 7, respectively. The promoter regions of 23 CsWAKs/CsWAKLs possessed diverse cis-regulatory elements that are involved in light, development, environmental stress, and hormone responsiveness. The expression profiles indicated that our candidate genes (CsWAK1, CsWAK4, CsWAK7, CsWAKL1, and CsWAKL7) are expressed in leaf tissue. These genes exhibit different expression patterns than their homologs in other plant species. These initial findings are useful resources for further research work on the potential roles of CsWAK/CsWAKL in cellular signalling during development, environmental stress conditions, and hormone treatment.
Cannabinoids are well-known specialised metabolites from the plant Cannabis sativa L. (cannabis). They exhibit various therapeutical to intoxicating psychoactive effects and have potential for medicinal applications. Among the enzymes involved in cannabinoid biosynthesis, cannabinoid oxidocyclases such as the tetrahydrocannabinolic acid (THCA) synthase play a key role in determining cannabis chemotype. To improve our understanding of cannabinoid oxidocyclase structure-function relationship, we proposed a new approach to targeted mutagenesis. By reviewing cannabis natural variation, three cannabinoid oxidocyclase mutations (S355N, CONF, G376R) associated to atypical plant chemotypes were selected. In-vitro characterization of THCA synthase mutants demonstrated these mutations significantly impact enzyme activity, correlating with the associated chemotype: S355N nearly inactivated the THCA synthase, CONF impaired CBGA metabolization and altered product specificity, while G376R drastically reduced enzyme activity and altered product specificity. In-silico docking experiments permitted to model the successive steps of THCA synthase substrate metabolization, revealing that the three mutations hamper substrate binding. Collectively, our results demonstrated how plant diversity can be leveraged to guide enzyme targeted mutagenesis, highlighted a key region of cannabinoid oxidocyclases, and permitted the establishment of a new model of the THCA synthase catalytic mechanism. This provides new insights into enzyme function, which can ultimately help developing medicinal cannabis cultivars and cannabinoid biotechnological production.
Is Cannabis a boon or bane? Cannabis sativa has long been a versatile crop for fiber extraction (industrial hemp), traditional Chinese medicine (hemp seeds), and recreational drugs (marijuana). Cannabis faced global prohibition in the 20th century because of the psychoactive properties of ∆9-tetrahydrocannabinol; however, recently, the perspective has changed with the recognition of additional therapeutic values, particularly the pharmacological potential of cannabidiol. A comprehensive understanding of underlying mechanism of cannabinoid biosynthesis is necessary to cultivate and promote globally the medicinal application of Cannabis resources. Here, we comprehensively review the historical usage of Cannabis, biosynthesis of trichome-specific cannabinoids, regulatory network of trichome development, and synthetic biology of cannabinoids. This review provides valuable insights into the efficient biosynthesis and green production of cannabinoids, and the development and utilization of novel Cannabis varieties.
Lipoxygenase (LOX) enzymes play a pivotal role in the biosynthesis of oxylipins. The phyto-oxilipins have been implicated in diverse aspects of plant biology, from regulating plant growth and development to providing tolerance against biotic and abiotic stresses. C. sativa is renowned for its bioactive secondary metabolites, namely cannabinoids. LOX route is assumed to be involved in the biosynthesis of hexanoic acid, which is one of the precursors of cannabinoids of C. sativa. For obvious reasons, the LOX gene family deserves thorough investigation in the C. sativa. Genome-wide analysis revealed the presence of 21 LOX genes in C. sativa, which can be further grouped into 13-LOX and 9-LOX depending upon their phylogeny as well as the enzyme activity. The promoter regions of the CsLOX genes were predicted to contain cis-acting elements involved in phytohormones responsiveness and stress response. The qRT-PCR-based expression analysis of 21 LOX genes revealed their differential expression in different plant parts (root, stem, young leaf, mature leaf, sugar leaf, and female flower). The majority of CsLOX genes displayed preferential expression in the female flower, which is the primary site for the biosynthesis of cannabinoids. The highest LOX activity and expression level of a jasmonate marker gene were reported in the female flowers among all the plant parts. Several CsLOX genes were found to be upregulated by MeJA treatment. Based on the transient expression in Nicotiana benthamiana and the development of stable Nicotiana tabacum transgenic lines, we demonstrate that CsLOX13 encodes functional lipoxygenase and play an important role in the biosynthesis of oxylipins.
Single-cell metabolomics is a powerful tool that can reveal cellular heterogeneity and can elucidate the mechanisms of biological phenomena in detail. It is a promising approach in studying plants, especially when cellular heterogeneity has an impact on different biological processes. In addition, metabolomics, which can be regarded as a detailed phenotypic analysis, is expected to answer previously unanswered questions which will lead to expansion of crop production, increased understanding of resistance to diseases, and in other applications as well. In this review, we will introduce the flow of sample acquisition, and single-cell metabolomics techniques to facilitate the adoption of single-cell metabolomics. Furthermore, the applications of single cell metabolomics will be summarized and reviewed.
Cannabissativa aromatic prenyltransferase 4 (CsPT4) and 1 (CsPT1) have been shown to catalyze cannabigerolic acid (CBGA) biosynthesis, a step that rate-limits the cannabinoid biosynthetic pathway; both genes are highly expressed in flowers. CsPT4 and CsPT1 promoter driven β-glucuronidase (GUS) activities were detected in leaves of cannabis seedlings, and strong CsPT4 promoter activities were associated with glandular trichomes. Hormonal regulation of cannabinoid biosynthetic genes is poorly understood. An in silico analysis of the promoters identified putative hormone responsive elements. Our work examines hormone-responsive elements in the promoters of CsPT4 and CsPT1 in the context of physiological responses of the pathway to the hormone in planta. Dual luciferase assays confirmed the regulation of promoter activities by the hormones. Further studies with salicylic acid (SA) demonstrated that SA pretreatment increased the expression of genes located downstream of the cannabinoid biosynthetic pathway. The results from all aspects of this study demonstrated an interaction between certain hormones and cannabinoid synthesis. The work provides information relevant to plant biology, as we present evidence demonstrating correlations between molecular mechanisms that regulate gene expression and influence plant chemotypes.
Plant ATP-binding cassette (ABC) transporters contribute the transport of diverse secondary metabolites. However, their roles in cannabinoid trafficking are still unsolved in Cannabis sativa. In this study, 113 ABC transporters were identified and characterized in C. sativa from their physicochemical properties, gene structure, and phylogenic relationship, as well as spatial gene expression patterns. Eventually, seven core transporters were proposed including one member in ABC subfamily B (CsABCB8) and six ABCG members (CsABCG4, CsABCG10, CsABCG11, CsABCG32, CsABCG37, and CsABCG41), harboring potential in participating cannabinoid transport, by combining phylogenetic and co-expression analysis from the gene and metabolite level. The candidate genes exhibited a high correlation with cannabinoid biosynthetic pathway genes and the cannabinoid content, and they were highly expressed where cannabinoids appropriately biosynthesized and accumulated. The findings underpin further research on the function of ABC transporters in C. sativa, especially in unveiling the mechanisms of cannabinoid transport to boost systematic and targeted metabolic engineering.
Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.
Glandular trichomes (GTs) are outgrowths of plant epidermal cells that secrete and store specialized secondary metabolites that protect plants against biotic and abiotic stresses and have economic importance for human use. While extensive work has been done to understand the molecular mechanisms of trichome organogenesis in Arabidopsis (Arabidopsis thaliana), which forms unicellular, non-glandular trichomes (NGTs), little is known about the mechanisms of GT development or regulation of secondary metabolites in plants with multicellular GTs. Here, we identified and functionally characterized genes associated with GT organogenesis and secondary metabolism in GTs of cucumber (Cucumis sativus). We developed a method for effective separation and isolation of cucumber GTs and NGTs. Transcriptomic and metabolomic analyses showed that flavonoid accumulation in cucumber GTs is positively associated with increased expression of related biosynthesis genes. We identified 67 GT development-related genes, the functions of 7 of which were validated by virus-induced gene silencing. We further validated the role of cucumber ECERIFERUM1 (CsCER1) in GT organogenesis by overexpression and RNA interference transgenic approaches. We further show that the transcription factor TINY BRANCHED HAIR (CsTBH) serves as a central regulator of flavonoid biosynthesis in cucumber glandular trichomes. Work from this study provides insight into the development of secondary metabolite biosynthesis in multi-cellular glandular trichomes.
Background:
Glandular capitate trichomes which form on bract tissues of female inflorescences of high THC-containing Cannabis sativa L. plants are important sources of terpenes and cannabinoids. The influence of plant age and cannabis genotype on capitate trichome development, morphology, and maturation has not been extensively studied. Knowledge of the various developmental changes that occur in trichomes over time and the influence of genotype and plant age on distribution, numbers, and morphological features should lead to a better understanding of cannabis quality and consistency.
Methods:
Bract tissues of two genotypes-"Moby Dick" and "Space Queen"-were examined from 3 weeks to 8 weeks of flower development using light and scanning electron microscopy. Numbers of capitate trichomes on upper and lower bract surfaces were recorded at different positions within the inflorescence. Observations on distribution, extent of stalk formation, glandular head diameter, production of resin, and extent of dehiscence and senescence were made at various time points. The effects of post-harvesting handling and drying on trichome morphology were examined in an additional five genotypes.
Results:
Two glandular trichome types-bulbous and capitate (sessile or stalked)-were observed. Capitate trichome numbers and stalk length were significantly (P = 0.05) greater in "Space Queen" compared to "Moby Dick" at 3 and 6 weeks of flower development. Significantly more stalked-capitate trichomes were present on lower compared to upper bract surfaces at 6 weeks in both genotypes, while sessile-capitate trichomes predominated at 3 weeks. Epidermal and hypodermal cells elongated to different extents during stalk formation, producing significant variation in length (from 20 to 1100 μm). Glandular heads ranged from 40 to 110 μm in diameter. Maturation of stalked-capitate glandular heads was accompanied by a brown color development, reduced UV autofluorescence, and head senescence and dehiscence. Secreted resinous material from glandular heads appeared as droplets on the cuticular surface that caused many heads to stick together or collapse. Trichome morphology was affected by the drying process.
Conclusion:
Capitate trichome numbers, development, and degree of maturation were influenced by cannabis genotype and plant age. The observations of trichome development indicate that asynchronous formation leads to different stages of trichome maturity on bracts. Trichome stalk lengths also varied between the two genotypes selected for study as well as over time. The variability in developmental stage and maturation between genotypes can potentially lead to variation in total cannabinoid levels in final product. Post-harvest handling and drying were shown to affect trichome morphology.
Cannabis sativa is a flowering plant belonging to the Cannabinaceae family. There have been numerous ongoing studies globally on C. sativa which include investigating the yield and quality of essential oils. However, this study is the first to assess the chemical composition and biological activities of Jamaican C. sativa essential oils (EOs) extracted via hydrodistillation as the plants mature from week 8 through to week 12. EOs from fresh landrace female plants were analysed by gas chromatography–mass spectrometry (GC–MS) and GC coupled to a flame ionization detector (GC‐FID). Cannabis sativa essential oils were screened for their in vitro antimicrobial activity using the disc diffusion method and further with tube dilution method. The antioxidant properties were investigated using the 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) radical scavenging assay. In total, 36 constituents were identified in the C. sativa EOs by comparison of the Kováts retention indices (RI), the mass spectra data with those in the Natural Institute of Standards and Technology (NIST) library and by co‐elution with authentic samples where available. The major constituents were β‐caryophyllene (25.34%) and α‐humulene (α‐caryophyllene) (10.94%) both at the highest levels in week nine and were the most abundant constituents present at all stages of maturity. All extracted C. sativa essential oils did not exhibit antioxidant properties. However, the C. sativa essential oils exhibit significant to moderate antimicrobial property against the test gram‐positive microorganisms, Enterococcus faecalis, Streptococcus Group A and Streptococcus Group B with MIC values ranging from 16 to 125 μg/mL. Jamaican Cannabis sativa essential oils extracted via hydrodistillation as the plants mature from week 8 through to week 12 were assessed for its chemical composition and biological activities. A total of 36 compounds were identified with the major constituents being β‐caryophyllene (25.34%) and α‐humulene (10.94%) both at highest level in week nine and were the most abundant constituents present at all stages of maturity. The oils showed antimicrobial against three gram‐positive micro‐organisms assayed, however no significant antioxidant activities were observed.
Specialized metabolite (SM) diversification is a core process to plants' adaptation to diverse ecological niches. Here, we implemented a computational mass spectrometry-based metabolomics approach to exploring SM diversification in tissues of 20 species covering Nicotiana phylogenetics sections. To markedly increase metabolite annotation, we created a large in silico fragmentation database, comprising >1 million structures, and scripts for connecting class prediction to consensus substructures. Together, the approach provides an unprecedented cartography of SM diversity and section-specific innovations in this genus. As a case study and in combination with nuclear magnetic resonance and mass spectrometry imaging, we explored the distribution of N-acylnornicotines, alkaloids predicted to be specific to Repandae allopolyploids, and revealed their prevalence in the genus, albeit at much lower magnitude, as well as a greater structural diversity than previously thought. Together, the data integration approaches provided here should act as a resource for future research in plant SM evolution.
Flowering plants have evolved extraordinarily diverse metabolites that underpin the floral visual and olfactory signals enabling plant–pollinator interactions. In some cases, these metabolites also provide unusual rewards that specific pollinators depend on. While some metabolites are shared by most flowering plants, many have evolved in restricted lineages in response to the specific selection pressures encountered within different niches. The latter are designated as specialized metabolites. Recent investigations continue to uncover a growing repertoire of unusual specialized metabolites. Increased accessibility to cutting-edge multi-omics technologies (e.g. genome, transcriptome, proteome, metabolome) is now opening new doors to simultaneously uncover the molecular basis of their synthesis and their evolution across diverse plant lineages. Drawing upon the recent literature, this perspective discusses these aspects and, where known, their ecological and evolutionary relevance. A primer on omics-guided approaches to discover the genetic and biochemical basis of functional specialized metabolites is also provided.
Since time immemorial, humans utilize plants for various needs such as food, shelter, fuelwood, and medicine. Plants exhibit medicinal properties due to the presence of several classes of phytochemicals, especially secondary metabolites, also known as specialized metabolites (SMs). Plants are chemical factories and many important drugs used today are derived from the plants or plant-inspired semi-biosynthetic pathways. Large numbers of plants are being increasingly explored for medicinally important specialized metabolites because there has been an increase in the interest in bioprospecting natural sources of more drugs. However, current bioprospecting approaches are not sustainable. Therefore, modern approaches can be used for the characterization of medicinally important plants for metabolites/phytochemicals present in them and for the validation of their medicinal properties in treating several serious diseases. Recent decades have seen the emergence of new technologies which can be deployed to characterize the phytochemicals present in the plants on a large scale. The genetic biosynthetic pathways of the plants can also be traced using high-throughput technologies such as genomics, proteomics, metabolomics, and transcriptomics. Several studies aim to characterize biosynthetic pathways of phytochemicals, and efforts are on identifying genes, proteins, and metabolomes associated with a particular metabolite. All this has led to an emergence of an entirely new discipline known as phytochemical genomics, which involves the integration of multi-omics approaches, such as genomics, transcriptomics, proteomics, and metabolomics intending to decipher gene-protein-metabolite networks. This chapter introduces the concept of phytochemical genomics, various techniques/technologies, and approaches that are used in phytochemical genomics and some of the examples of medicinal plants, where these technologies have been successfully utilized. This chapter further delves into linking gene editing technologies with omics information for the improvement of medicinal plants.
Cannabis glandular trichomes produce and store an abundance of lipidic specialised metabolites (e.g. cannabinoids and terpenes) that are consumed by humans for medicinal and recreational purposes. Due to a lack of genetic resources and inherent autofluorescence of cannabis glandular trichomes, our knowledge of cannabinoid trafficking and secretion is limited to transmission electron microscopy (TEM). Advances in cryofixation methods has resulted in ultrastructural observations closer to the 'natural state' of the living cell, and recent reports of cryofixed cannabis trichome ultrastructure challenge the long-standing model of cannabinoid trafficking proposed by ultrastructural reports using chemically fixed samples. Here, we compare the ultrastructural morphology of cannabis glandular trichomes preserved using conventional chemical fixation and ultrarapid cryofixation. We show that chemical fixation results in amorphous metabolite inclusions surrounding the organelles of glandular trichomes that were not present in cryofixed samples. Vacuolar morphology in cryofixed samples exhibited homogenous electron density, while chemically fixed samples contained a flocculent electron dense periphery and electron lucent lumen. In contrast to the apparent advantages of cryopreservation, fine details of cell wall fibre orientation could be observed in chemically fixed glandular trichomes that were not seen in cryofixed samples. Our data suggest that chemical fixation results in intracellular artefacts that impact the interpretation of lipid production and trafficking, while enabling greater detail of extracellular polysaccharide organisation.
Mentha haplocalyx Briq (M. haplocalyx) is a herbaceous plant that has long been used as a food, medicinal spice, and flavoring agent in traditional Chinese medicine. Its secondary metabolites, having high commercial values, are mainly produced in tiny specialized structures called glandular trichomes (GTs). The primary purpose of this study was to examine the morphology and metabolites of peltate GTs in M. haplocalyx.Peltate GTs possessed globular dome shapes and intense auto-fluorescence on the surfaces of M. haplocalyx leaves. Structure subsidence and cuticle rupture were found throughout the aging stage of peltate GTs. According to histochemical staining results, the secretion of peltate GTs contained anthraquinone, flavonoids, phenolic acid and terpenoids. In M. haplocalyx peltate GTs and leaf tissues without peltate glandular trichomes, ten and two volatile compounds were identified respectively. Peltate GTs contained 42 non-volatile chemicals with a variety of structural types, including 20 flavonoids, 17 phenolic acids,1 diterpene, 3 anthraquinone and 1 alkane. Meanwhile, 15 non-volatile compounds were discovered in leaf tissues without peltate glandular trichomes, and they were all included in the list of peltate GTs' 41 components. Therefore, Peltate GTs were shown to be the primary site of not just volatile compounds but also non-volatile chemicals in M. haplocalyx. This study provides an important theoretical basis and technical approach for clarifying the bio-active metabolite biosynthesis in M. haplocalyx.
The study of plant secondary metabolites, now designated as specialized metabolites, is motivated by multiple scientific objectives, ranging from bioprospecting for bioactive molecules to answering ecological and evolutionary questions. This paper provides a constructive commentary on chemosystematics, or chemotaxonomy, and chemophenetic analyses to follow these objectives. Discussion of the outcomes of these types of studies and the recent progress in understanding the occurrence and variability of plant metabolites at different spatiotemporal scales is presented. It is also demonstrated that chemosystematics and chemophenetics are not conflicting or mutually exclusive, but they should be used for specific purposes, particularly in the context of understanding ecological and evolutionary relationships between taxa and chemical variability among them. We also demonstrate that inter-specimen studies benefit from chemophenetic and α chemodiversity indices that help to interpret the outcome of ecological and bioprospective studies in the context of space, time, and spatiotemporal scales. Furthermore, at the population level, β and γ chemodiversity indices are useful to address the influence of spatiotemporal scales, in addition to other ecological pressures.
Background: Cannabis has a long history of being credited with centuries of healing powers for millennia. The cannabis plant is a rich source of cannabinoids and terpenes. Each cannabis chemovar exhibits a different flavor and aroma, which are determined by its terpene content.
Methods: In this study, a gas chromatography-flame ionization detector method was developed and validated for the determination of the 10 major terpenes in the main three chemovars of Cannabis sativa L. with n-tridecane used as the internal standard following the standard addition method. The 10 major terpenes (monoterpenes and sesquiterpenes) are α-pinene, β-pinene, β-myrcene, limonene, terpinolene, linalool, α-terpineol, β-caryophyllene, α-humulene, and caryophyllene oxide. The method was validated according to Association of Official Analytical Chemists guidelines. Spike recovery studies for all terpenes were carried out on placebo cannabis material and indoor-growing high THC chemovar with authentic standards.
Results: The method was linear over the calibration range of 1–100 μg/mL with r2>0.99 for all terpenes. The limit of detection and limit of quantification were calculated to be 0.3 and 1.0 μg/mL, respectively, for all terpenes. The accuracy (%recovery) at all levels ranged from 89% to 104% and 90% to 111% for placebo and indoor-growing high THC chemovar, respectively. The repeatability and intermediate precision of the method were evaluated by the quantification of target terpenes in the three different C. sativa chemovars, resulting in acceptable relative standard deviations (less than 10%).
Conclusions: The developed method is simple, sensitive, reproducible, and suitable for the detection and quantification of monoterpenes and sesquiterpenes in C. sativa biomass.
Hamppu (Cannabis sativa L.) on monimuotoinen viljelykasvi, josta on kehitetty lajikkeita eri
käyttötarkoituksiin: öljy-, kuitu-, lääke- ja huumekasviksi. Hyöty- eli teollisuushamppulajikkeisiin luetaan öljy- ja kuituhamppu. Tällä hetkellä Suomessa hampun kannabinoideja sisältävien kasvinosien käyttö
elintarvikkeina ei ole sallittua, vaan edellyttää uuselintarvikelain mukaisen uuselintarvikeluvan hankkimista. Suomessa elintarvikkeet eivät saa sisältää huumausaineeksi luokiteltavaa Δ9-tetrahydrokannabinolia (THC). Koska Δ9-THC:n enimmäismäärälle ole määritelty raja-arvoa, sen tulkitaan tarkoittavan nollatoleranssia. Lainsäädäntö vaihtelee EU-maissa ja on oletettavissa, että Euroopan komissio linjaa kantansa Δ9-THC:n enimmäispitoisuudeksi uuselintarvikkeiden luetteloinnin yhteydessä, jolloin Suomessakin tilanne arvioidaan uudelleen.Hampunsiementuotannon sivuvirtana syntyy kasvimateriaalia, joka sisältää kannabinoideja Tällä hetkellä viljelijät eivät hyödynnä sivuvirtamateriaalia muuten kuin maanparannusaineena.Öljyhamppua tällä hetkellä viljeleville ja aiemmin viljelleille viljelijöille osoitetulla kyselytutkimuksella kartoitettiin heidän näkemyksiään ja kiinnostustaan hampunkorjuusta jäävien
sivuvirtojen hyödyntämiseen. Kyselyyn vastasi 44 viljelijää. Vastaajista noin puolet oli kiinnostuneita hampun sivuvirtamateriaalin hyödyntämisestä ja valmiutta lisäinvestointien tekemiseen löytyi. Vastaavasti noin puolet eivät olisi valmiita tekemään lisäinvestointeja, vaikka sivuvirtamateriaalista tulisi myyntituote. Alustava arvoketjuanalyysi osoittaa, että kustannusten ja riskien hallinta on haastavaa.
Glandular trichomes are specialized anatomical structures that accumulate secretions with important biological roles in plant-environment interactions. These secretions also have commercial uses in the flavor, fragrance, and pharmaceutical industries. The capitate-stalked glandular trichomes of Cannabis sativa (cannabis), situated on the surfaces of the bracts of the female flowers, are the primary site for the biosynthesis and storage of resins rich in cannabinoids and terpenoids. In this study, we profiled nine commercial cannabis strains with purportedly different attributes, such as taste, color, smell and genetic origin. Glandular trichomes were isolated from each of these strains and cell type-specific transcriptome data sets were acquired. Cannabinoids and terpenoids were quantified in flower buds. Statistical analyses indicated that these data sets enable the high-resolution differentiation of strains by providing complementary information. Integrative analyses revealed a coexpression network of genes involved in the biosynthesis of both cannabinoids and terpenoids from imported precursors. Terpene synthase genes involved in the biosynthesis of the major mono- and sesquiterpenes routinely assayed by cannabis testing laboratories were identified and functionally evaluated. In addition to cloning variants of previously characterized genes, specifically CsTPS14CT ((-)-limonene synthase) and CsTPS15CT (β-myrcene synthase) we functionally evaluated genes that encode enzymes with activities not previously described in cannabis, namely CsTPS18VF and CsTPS19BL (nerolidol/linalool synthases); CsTPS16CC (germacrene B synthase); and CsTPS20CT (hedycaryol synthase). This study lays the groundwork for developing a better understanding of the complex chemistry and biochemistry underlying resin accumulation across commercial cannabis strains.
Cannabis sativa (cannabis) produces a resin that is valued for its psychoactive and medicinal properties. Despite being the foundation of a multi-billion dollar global industry, scientific knowledge and research on cannabis is lagging behind compared to other high-value crops. This is largely due to legal restrictions that have prevented many researchers from studying cannabis, its products, and their effects in humans. Cannabis resin contains hundreds of different terpene and cannabinoid metabolites. Many of these metabolites have not been conclusively identified. Our understanding of the genomic and biosynthetic systems of these metabolites in cannabis, and the factors that affect their variability, is rudimentary. As a consequence, there is concern about lack of consistency with regard to the terpene and cannabinoid composition of different cannabis ‘strains’. Likewise, claims of some of the medicinal properties attributed to cannabis metabolites would benefit from thorough scientific validation.
Background
Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type.
Results
Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ⁹-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures.
Conclusion
Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.
Electronic supplementary material
The online version of this article (10.1186/s12870-018-1481-4) contains supplementary material, which is available to authorized users.
The diversity of non-glandular and glandular hairs of Cannabis sativa L. (marihuana) are described by scanning electron microscopy. The non-glandular hairs are of two major types, as distinguished by size differences and locations, and all of them are highly silicified. The presence of silica as well as cystoliths of calcium carbonate help in the identification of marihuana even in its ash residues. X-ray microanalyses of Cannabis hairs are compared with those of Humulus lupulus and Lantana camera, whose hairs have been considered to resemble those of marihuana. Glandular hairs are found to be of two major categories. One group consists of glands whose heads are generally made up of eight cells and the other group whose heads are generally made up of two cells but never more than four cells. All glands of both categories are stalked. Some glands of the first category are massively stalked and these are restricted solely to anthers and bracts of staminate and pistillate plants. The massive stalk is considered to be made up of epidermal and hypodermal cells that have grown in response to some stimulation during anthesis. Fine details of the shoot system of Cannabis, such as cuticular ridges on epidermal cells, warty protuberances on non-glandular hairs, and surface views of glands in developing stages are also reported. Glandular hairs on the bracts of Humulus lupulus resemble those of Cannabis.
The commercially important essential oils of peppermint (Mentha x piperita L.) and its relatives in the mint family (Lamiaceae) are accumulated in specialized anatomical structures called glandular trichomes (GTs). A genome-scale stoichiometric model of secretory phase metabolism in peppermint GTs was constructed based on current biochemical and physiological knowledge. Fluxes through the network were predicted based on metabolomic and transcriptomic data. Using simulated reaction deletions, this model predicted that two processes, the regeneration of ATP and ferredoxin (in its reduced form), exert substantial control over flux toward monoterpenes. Follow-up biochemical assays with isolated GTs indicated that oxidative phosphorylation and ethanolic fermentation were active, and that cooperation to provide ATP depended on the concentration of the carbon source. We also report that GTs with high flux toward monoterpenes express, at very high levels, genes coding for a unique pair of ferredoxin and ferredoxin-NADP+ reductase isoforms. This study provides the first evidence how bioenergetic processes determine flux through monoterpene biosynthesis in GTs.
Multicellular glandular trichomes are epidermal outgrowths characterized by the presence of a head made of cells that have the ability to secrete or store large quantities of specialized metabolites. Our understanding of the transcriptional control of glandular trichome initiation and development is still in its infancy. This review points to some central questions that need to be addressed to better understand how such specialized cell structures arise from the plant protodermis. A key and unique feature of glandular trichomes is their ability to synthesize and secrete large amounts, relative to their size, of a limited number of metabolites. As such, they qualify as true cell factories, making them interesting targets for metabolic engineering. In this review, recent advances regarding terpene metabolic engineering are highlighted, with a special focus on tobacco (Nicotiana tabacum). In particular, the choice of transcriptional promoters to drive transgene expression and the best ways to sink existing pools of terpene precursors are discussed. The bioavailability of existing pools of natural precursor molecules is a key parameter and is controlled by so-called cross talk between different biosynthetic pathways. As highlighted in this review, the exact nature and extent of such cross talk are only partially understood at present. In the future, awareness of, and detailed knowledge on, the biology of plant glandular trichome development and metabolism will generate new leads to tap the largely unexploited potential of glandular trichomes in plant resistance to pests and lead to the improved production of specialized metabolites with high industrial or pharmacological value.
Cannabis (Cannabis sativa) plants produce and accumulate a terpene-rich resin in glandular trichomes, which are abundant on the surface of the female inflorescence. Bouquets of different monoterpenes and sesquiterpenes are important components of cannabis resin as they define some of the unique organoleptic properties and may also influence medicinal qualities of different cannabis strains and varieties. Transcriptome analysis of trichomes of the cannabis hemp variety 'Finola' revealed sequences of all stages of terpene biosynthesis. Nine cannabis terpene synthases (CsTPS) were identified in subfamilies TPS-a and TPS-b. Functional characterization identified mono-and sesqui-TPS, whose products collectively comprise most of the terpenes of 'Finola' resin, including major compounds such as β-myr-cene, (E)-β-ocimene, (-)-limonene, (+)-α-pinene, β-caryophyllene, and α-humulene. Transcripts associated with terpene biosynthesis are highly expressed in trichomes compared to non-resin producing tissues. Knowledge of the CsTPS gene family may offer opportunities for selection and improvement of terpene profiles of interest in different cannabis strains and varieties.
Coordinated transcriptional and metabolic reprogramming ensures a plant's continued growth and survival under adverse environmental conditions. Transcription factors (TFs) act to modulate gene expression through complex cis-regulatory element (CRE) interactions. Genome-wide analysis of known plant CREs was performed for all currently predicted protein-coding gene promoters in grapevine (Vitis vinifera L.). Many CREs such as abscisic acid (ABA)-responsive, drought-responsive, auxin-responsive, and evening elements, exhibit bona fide CRE properties such as strong position bias towards the transcription start site (TSS) and over-representation when compared with random promoters. Genes containing these CREs are enriched in a large repertoire of plant biological pathways. Large-scale transcriptome analyses also show that these CREs are highly implicated in grapevine development and stress response. Numerous CRE-driven modules in condition-specific gene co-expression networks (GCNs) were identified and many of these modules were highly enriched for plant biological functions. Several modules corroborate known roles of CREs in drought response, pathogen defense, cell wall metabolism, and fruit ripening, whereas others reveal novel functions in plants. Comparisons with Arabidopsis suggest a general conservation in promoter architecture, gene expression dynamics, and GCN structure across species. Systems analyses of CREs provide insights into the grapevine cis-regulatory code and establish a foundation for future genomic studies in grapevine.
The industrial relevance of a number of metabolites produced in plant glandular trichomes has spurred research on these specialized organs for a number of years. Most of the research however, has focused on the elucidation of secondary metabolite pathways and comparatively little has been undertaken on the development and differentiation of glandular trichomes. One way to gain insight into these developmental processes is to generate stage-specific transcriptome and metabolome data. The difficulty for this resides in the isolation of early stages of development of the glandular trichomes. Here we describe a method for the separation and isolation of intact young and mature type VI trichomes from the wild tomato species Solanum habrochaites. The final and key step of the method uses cell sorting based on distinct autofluorescence signals of the young and mature trichomes. We demonstrate that sorting by flow cytometry allows recovering pure fractions of young and mature trichomes. Furthermore, we show that the sorted trichomes can be used for transcript and metabolite analyses. Because, many plant tissues or cells have distinct autofluorescence components, the principles of this method can be generally applicable for the isolation of specific cell types without prior labeling.
Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than do other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant’s lifestyle, by transporting various compounds across specific membranes of the plant.
The evolution of major cannabinoids and terpenes during the growth of Cannabis sativa plants was studied. In this work, seven different plants were selected: three each from chemotypes I and III and one from chemotype II. Fifty clones of each mother plant were grown indoors under controlled conditions. Every week, three plants from each variety were cut and dried, and the leaves and flowers were analyzed separately. Eight major cannabinoids were analyzed via HPLC-DAD, and 28 terpenes were quantified using GC-FID and verified via GC-MS. The chemotypes of the plants, as defined by the tetrahydrocannabinolic acid/cannabidiolic acid (THCA/CBDA) ratio, were clear from the beginning and stable during growth. The concentrations of the major cannabinoids and terpenes were determined, and different patterns were found among the chemotypes. In particular, the plants from chemotypes II and III needed more time to reach peak production of THCA, CBDA, and monoterpenes. Differences in the cannabigerolic acid development among the different chemotypes and between monoterpene and sesquiterpene evolution patterns were also observed. Plants of different chemotypes were clearly differentiated by their terpene content, and characteristic terpenes of each chemotype were identified.
Background:
Type VI glandular trichomes represent the most abundant trichome type on leaves and stems of tomato plants and significantly contribute to herbivore resistance, particularly in the wild species. Despite this, their development has been poorly studied so far. The goal of this study is to fill this gap. Using a variety of cell imaging techniques, a detailed record of the anatomy and developmental stages of type VI trichomes in the cultivated tomato (Solanum lycopersicum) and in a related wild species (S. habrochaites) is provided.
Results:
In both species, the development of these structures follows a highly reproducible cell division pattern. The two species differ in the shape of the trichome head which is round in S. habrochaites and like a four-leaf clover in S. lycopersicum, correlating with the presence of a large intercellular cavity in S. habrochaites where the produced metabolites accumulate. In both species, the junction between the intermediate cell and the four glandular cells constitute a breaking point facilitating the decapitation of the trichome and thereby the quick release of the metabolites. A strongly auto-fluorescent compound transiently accumulates in the early stages of development suggesting a potential role in the differentiation process. Finally, immuno-labelling with antibodies recognizing specific cell wall components indicate a key role of pectin and arabinogalactan components in the differentiation of type VI trichomes.
Conclusions:
Our observations explain the adaptive morphologies of type VI trichomes for metabolite storage and release and provide a framework for further studies of these important metabolic cellular factories. This is required to better exploit their potential, in particular for the breeding of pest resistance in tomato.
Despite its cultivation as a source of food, fibre and medicine, and its global status as the most used illicit drug, the genus Cannabis has an inconclusive taxonomic organization and evolutionary history. Drug types of Cannabis (marijuana), which contain high amounts of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC), are used for medical purposes and as a recreational drug. Hemp types are grown for the production of seed and fibre, and contain low amounts of THC. Two species or gene pools (C. sativa and C. indica) are widely used in describing the pedigree or appearance of cultivated Cannabis plants. Using 14,031 single-nucleotide polymorphisms (SNPs) genotyped in 81 marijuana and 43 hemp samples, we show that marijuana and hemp are significantly differentiated at a genome-wide level, demonstrating that the distinction between these populations is not limited to genes underlying THC production. We find a moderate correlation between the genetic structure of marijuana strains and their reported C. sativa and C. indica ancestry and show that marijuana strain names often do not reflect a meaningful genetic identity. We also provide evidence that hemp is genetically more similar to C. indica type marijuana than to C. sativa strains.
In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis thaliana lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then employed to infer flux bottlenecks in the sterol pathway. The extent of metabolic crosstalk - the exchange of isoprenoid intermediates between compartmentalized pathways - was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants.
Copyright © 2015, Plant Physiology.
In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html.
An attempt has been made to define the extent to which metabolic flux in central plant metabolism is reflected by changes in the transcriptome and metabolome, based on an analysis of in vitro cultured immature embryos of two oilseed rape (Brassica napus) accessions which contrast for seed lipid accumulation. Metabolic flux analysis (MFA) was used to constrain a flux balance metabolic model which included 671 biochemical and transport reactions within the central metabolism. This highly confident flux information was eventually used for comparative analysis of flux vs. transcript (metabolite). Metabolite profiling succeeded in identifying 79 intermediates within the central metabolism, some of which differed quantitatively between the two accessions and displayed a significant shift corresponding to flux. An RNA-Seq based transcriptome analysis revealed a large number of genes which were differentially transcribed in the two accessions, including some enzymes/proteins active in major metabolic pathways. With a few exceptions, differential activity in the major pathways (glycolysis, TCA cycle, amino acid, and fatty acid synthesis) was not reflected in contrasting abundances of the relevant transcripts. The conclusion was that transcript abundance on its own cannot be used to infer metabolic activity/fluxes in central plant metabolism. This limitation needs to be borne in mind in evaluating transcriptome data and designing metabolic engineering experiments.
Motivation:
A large choice of tools exists for many standard tasks in the analysis of high-throughput sequencing (HTS) data. However, once a project deviates from standard workflows, custom scripts are needed.
Results:
We present HTSeq, a Python library to facilitate the rapid development of such scripts. HTSeq offers parsers for many common data formats in HTS projects, as well as classes to represent data, such as genomic coordinates, sequences, sequencing reads, alignments, gene model information and variant calls, and provides data structures that allow for querying via genomic coordinates. We also present htseq-count, a tool developed with HTSeq that preprocesses RNA-Seq data for differential expression analysis by counting the overlap of reads with genes.
Availability and implementation:
HTSeq is released as an open-source software under the GNU General Public Licence and available from http://www-huber.embl.de/HTSeq or from the Python Package Index at https://pypi.python.org/pypi/HTSeq.
Although many NGS read pre-processing tools already existed, we could not find any tool or combination of tools which met our requirements in terms of flexibility, correct handling of paired-end data, and high performance. We have developed Trimmomatic as a more flexible and efficient pre-processing tool, which could correctly handle paired-end data.
The value of NGS read pre-processing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output which is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested.
Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available from http://www.usadellab.org/cms/index.php?page=trimmomatic CONTACT: usadel@bio1.rwth-aachen.de SUPPLEMENTARY INFORMATION: Manual and source code are available from http://www.usadellab.org/cms/index.php?page=trimmomatic.
Chromatographic and spectroscopic data was determined for 16 different major cannabinoids from Cannabis sativa plant material as well as 2 human metabolites of Δ‐tetrahydrocannabinol. Spectroscopic analysis included UV absorbance, infrared‐spectral analysis, (GC‐) mass spectrometry, and spectrophotometric analysis. Also, the fluorescent properties of the cannabinoids are presented. Most of this data is available from literature but scattered over a large amount of scientific papers. In this case, analyses were carried out under standardised conditions for each tested cannabinoid so spectroscopic data can be directly compared. Different methods for the analysis of cannabis preparations were used and are discussed for their usefulness in the identification and determination of separate cannabinoids. Data on the retention of the cannabinoids in HPLC, GC, and TLC are presented.
Glandular trichomes are specialized hairs found on the surface of about 30% of all vascular plants and are responsible for a significant portion of a plant's secondary chemistry. Glandular trichomes are an important source of essential oils, i.e., natural fragrances or products that can be used by the pharmaceutical industry, although many of these substances have evolved to provide the plant with protection against herbivores and pathogens. The storage compartment of glandular trichomes usually is located on the tip of the hair and is part of the glandular cell, or cells, which are metabolically active. Trichomes and their exudates can be harvested relatively easily, and this has permitted a detailed study of their metabolites, as well as the genes and proteins responsible for them. This knowledge now assists classical breeding programs, as well as targeted genetic engineering, aimed to optimize trichome density and physiology to facilitate customization of essential oil production or to tune biocide activity to enhance crop protection. We will provide an overview of the metabolic diversity found within plant glandular trichomes, with the emphasis on those of the Solanaceae, and of the tools available to manipulate their activities for enhancing the plant's resistance to pests.
The morphology, histochemistry, and ultrastructural aspects of the secretion of Lavandula pinnata L. (Lamiaceae) glandular trichomes were studied in order to prepare for work on calcium distribution in normal and heat stressed plants. The secretory process was observed in both light and dark-grown plants. The secretion of the capitate glandular trichomes consists of both lipophilic and polysaccharidic substances (mainly the latter), while peltate glandular trichomes only secrete lipophilic substances. Our ultrastructural results are consistent with the finding that plastids are the main organelle that synthesizes the lipophilic substances, especially terpenes, while dictyosomes synthesize polysaccharides. The essential oil is most likely transported directly by the RER, while the polysaccharides reach the plasma membrane via Golgi-derived vesicles. Exocytosis is the main way that secretory products move from the cell to the subcuticular space. There are no differences in gland development or secretion between the light and dark-grown plants.
Glandular secreting trichomes on the surface of tomato plants and many of its relatives in the Solanaceae produce a mixture of O-acyl sugars that contribute to insect resistance. The majority of acyl sucroses produced by the cultivated tomato (Solanum lycopersicum) contain three or four short chain aliphatic acyl esters, and tetra-acyl sucroses have an acetyl group as one of the acyl chains. We previously reported overlapping S. lycopersicum × Solanum pennellii introgression lines (ILs) that fail to accumulate high levels of acetylated tetra-acyl sucroses. A survey of the annotated genes in this region of cultivated tomato chromosome 1 revealed three candidate acyltransferases that were tested for function using virus-induced gene silencing. A member of the BAHD family of acyltransferases (Solyc01g105580, SlAT2) was shown to encode an acetyl-CoA-dependent acyltransferase enzyme capable of acyl sucrose acetylation in vitro. RNAi suppression of SlAT2 in transgenic S. lycopersicum cv. M82 resulted in reduced acyl sugar acetylation, whereas expression of the functional S. lycopersicum allele of SlAT2 in the triacyl sucrose producing IL1-3 restored the ability of the IL to synthesize acetylated tetra-acyl sugars. Transgenic plants with the SlAT2 promoter driving GFP expression showed fluorescence in tips cells of long, slender trichomes that is consistent with acyl sugar acetylation occurring in these cells.
Δ(9)-Tetrahydrocannabinol (THC) and other cannabinoids are responsible for the psychoactive and medicinal properties of Cannabis sativa L. (marijuana). The first intermediate in the cannabinoid biosynthetic pathway is proposed to be olivetolic acid (OA), an alkylresorcinolic acid that forms the polyketide nucleus of the cannabinoids. OA has been postulated to be synthesized by a type III polyketide synthase (PKS) enzyme, but so far type III PKSs from cannabis have been shown to produce catalytic byproducts instead of OA. We analyzed the transcriptome of glandular trichomes from female cannabis flowers, which are the primary site of cannabinoid biosynthesis, and searched for polyketide cyclase-like enzymes that could assist in OA cyclization. Here, we show that a type III PKS (tetraketide synthase) from cannabis trichomes requires the presence of a polyketide cyclase enzyme, olivetolic acid cyclase (OAC), which catalyzes a C2-C7 intramolecular aldol condensation with carboxylate retention to form OA. OAC is a dimeric α+β barrel (DABB) protein that is structurally similar to polyketide cyclases from Streptomyces species. OAC transcript is present at high levels in glandular trichomes, an expression profile that parallels other cannabinoid pathway enzymes. Our identification of OAC both clarifies the cannabinoid pathway and demonstrates unexpected evolutionary parallels between polyketide biosynthesis in plants and bacteria. In addition, the widespread occurrence of DABB proteins in plants suggests that polyketide cyclases may play an overlooked role in generating plant chemical diversity.
For the past 25 years NIH Image and ImageJ software have been pioneers as open tools for the
analysis of scientific images. We discuss the origins, challenges and solutions of these two programs,
and how their history can serve to advise and inform other software projects.
Development of the secretory cavity and formation of the subcuticular wall of glandular trichomes in Cannabis sativa L. was examined by transmission electron microscopy. The secretory cavity originated at the wall‐cuticle interface in the peripheral wall of the discoid secretory cells. During the presecretory phase in development of the glandular trichome, the peripheral wall of the disc cells became laminated into a dense inner zone adjacent to the plasma membrane and a less dense outer zone subjacent to the cuticle. Loosening of wall matrix in the outer zone initiated a secretory cavity among fibrous wall materials. Membrane‐bound hyaline areas, compressed in shape, arose in the wall matrix. They appeared first in the outer and subsequently in the inner zone of the wall. The membrane of the vesicles, and associated dense particles attached to the membrane, arose from the wall matrix. Hyaline areas, often with a conspicuous electron‐dense content, were released into the secretory cavity where they formed rounded secretory vesicles. Fibrous wall material released from the surface of the disc cells became distributed throughout the secretory cavity among the numerous secretory vesicles. This wall material was incorporated into the developing subcuticular wall that increased five‐fold in thickness during enlargement of the secretory cavity. The presence of a subcuticular wall in the cavity of Cannabis trichomes, as contrasted to the absence of this wall in described trichomes of other plants, supports a polyphyletic interpretation of the evolution of the secretory cavity in glandular trichomes among angiosperms.