Mixed linkage (1,3;1,4)-β-glucan (MLG) is a major non-cellulosic polysaccharide of commelinid monocot cell walls and an important soluble dietary fibre component found in cereal grains, including those of barley. Despite its importance, relatively little is known about the post-translational regulation of cellulose synthase-like F6 (CSLF6), the major MLG synthase (MLGS) isoform involved in MLG biosynthesis in developing grain and young vegetative tissues. Following the detection of higher-order CSLF6-containing protein complexes under non-denaturing conditions, CSLF6 co-immunoprecipitation (co-IP) using detergent-solubilised barley microsomal membranes (MM) identified 16 high confidence (class I) potential candidate HvCSLF6-interacting proteins and 37 medium-confidence (class II) candidate interactors. Biochemical analyses of in vitro MLGS activity in 4-day-old barley seedling MMs, as a proxy for CSLF6 activity, indicated that alkaline pH in the presence of Mg2+ and Ca2+ enhanced MLGS activity. The effect of a buffer-soluble cytoplasmic protein fraction (>100,000 g supernatant) and a mild detergent-treated soluble fraction previously shown to impact MLGS activity in maize coleoptiles was investigated. A higher stimulatory effect on MLGS activity was observed when the cytoplasmic-soluble protein fraction, lacking CSLF6, was incubated with MM compared to MM assayed alone. Based on our co-IP results and MLGS activity reconstitution assays in barley seedlings (and other grass species), we conclude that ancillary protein factor(s) are likely involved in CSLF6 regulation.

Glandular trichomes (GTs) are epidermal outgrowths in which diverse specialised (secondary) metabolites are synthesised and stored. Cannabis (Cannabis sativa L.) and its close relative hop (Humulus lupulus L.) have pharmaceutical and industrial significance due to the presence of these metabolites in their GTs. We examined the conservation or divergence of the specific transcriptional programmes underlying GT biology. To achieve this, we generated transcriptome atlases of trichomes, flower, leaf, stem and root for cannabis, hop and tomato. We found that 12.9, 10.1 and 16.8% of cannabis, hop and tomato genes, respectively, were expressed organ/tissue specifically across all organs/tissues. Transcription factors (TFs) on average accounted for 7.5% of the organ-specific transcriptome and likely regulate organ-specific functions. We also conducted weighted gene co-expression network analysis and gene regulatory network (GRN) analysis to identify key regulators of GT function across the species and validated our predictions by DNA affinity purification sequencing for a subset of the cannabis and tomato GT TFs. The GRNs specific to cannabis or hop GTs were enriched for TFs and target genes associated with specialised metabolism, reflecting their species-specific nature. Conversely, the shared GRN components (identified via orthology analysis) were involved in highly conserved processes, such as flavonoid biosynthesis, solute transport and metabolite storage. Together, these GRNs and the associated transcriptome atlases are valuable resources to improve our knowledge of GT function and organ-specific genome regulation.

Cannabis sativa L. is a recognised medicinal plant containing a diverse array of phytocannabinoids, which are largely responsible for cannabis' therapeutic efficacy in treating psychiatric syndromes, chronic pain and epilepsy. The global market for cannabinoid products is rapidly expanding, necessitating high throughput and sustainable cannabinoid extraction processes to meet this growing demand. Liquid-liquid extraction (LLE) is a superior candidate for industrial cannabinoid isolation but there are still many knowledge gaps regarding its application in drug-type cannabis chemotype and minor cannabinoids recovery. This study filled these gaps by developing an efficient and scalable leaching method to recover cannabinoids from drug-type cannabis flowers using green modifier/water leaching solutions. The impacts of different cannabis forms (i.e., fresh (primarily acidic forms) and heat-treated (primarily decarboxylated forms)), modifier/water systems (i.e., ethanol/water and methanol/water), modifier contents (i.e., 0-100 % v/v) and solid-to-liquid ratios on the leaching efficiency of 15 of the most common cannabinoids were investigated. Results showed that leaching conditions of either >= 50 % v/v ethanol/water or >= 70 % v/v methanol/water were required for the efficient recovery of triangle(9)-tetrahydrocannabinolic acid A (triangle(9)-THCAA), the primary cannabinoid in fresh cannabis, while either >= 70 % v/v ethanol/water or >= 80 % v/v methanol/water leaching solutions were required for complete recovery of triangle(9)-tetrahydrocannabinol (triangle(9)-THC), the predominant cannabinoid in decarboxylated cannabis. The stability of cannabinoids in alcohol/water leachates for up to 40 d was also evaluated, the results of which suggests that alcohol/water leachates containing triangle(9)-THCAA required rapid processing to the next liquid-liquid extraction stage or cool-storing to enhance its stability in solution. Furthermore, this study successfully utilised Hansen Solubility Parameters (HSPs) to predict the experimental leaching trends of cannabinoids, indicating that HSPs is an effective tool for screening alternative solvent systems for cannabis processing.

Cannabis sativa L. is one of the oldest domesticated crops. Hemp-type cultivars, which predominantly produce non-intoxicating cannabidiol (CBD), have been selected for their fast growth, seed, and fibre production, while drug-type chemovars were bred for high accumulation of tetrahydrocannabinol (THC). We investigated how the generation of CBD-dominant chemovars by introgression of hemp- into drug-type Cannabis impacted plant performance. The THC-dominant chemovar showed superior sink strength, higher flower biomass, and demand-driven control of nutrient uptake. By contrast, the CBD-dominant chemovar hyperaccumulated phosphate in sink organs leading to reduced carbon and nitrogen assimilation in leaves, which limited flower biomass and cannabinoid yield. RNA-seq analyses determined organ- and chemovar-specific differences in expression of genes associated with nitrate and phosphate homeostasis as well as growth-regulating transcription factors that were correlated with measured traits. Among these were genes positively selected for during Cannabis domestication encoding an inhibitor of the phosphate starvation response, SPX DOMAIN GENE3, nitrate reductase, and two nitrate transporters. Altered nutrient sensing, acquisition, or distribution are likely a consequence of adaption to growth on marginal, low-nutrient-input lands in hemp. Our data provide evidence that such ancestral traits may become detrimental for female flower development and consequently overall CBD yield in protected cropping environments.

Medicinal plants are integral to traditional medicine systems worldwide, being pivotal for human health. Harvesting plant material from natural environments, however, has led to species scarcity, prompting action to develop cultivation solutions that also aid conservation efforts. Biotechnological tools, specifically plant tissue culture and genetic transformation, offer solutions for sustainable, large-scale production and enhanced yield of valuable biomolecules. While these techniques are instrumental to the development of the medicinal plant industry, the challenge of inherent regeneration recalcitrance in some species to in vitro cultivation hampers these efforts. This review examines the strategies for overcoming recalcitrance in medicinal plants using a holistic approach, emphasizing the meticulous choice of explants (e.g. embryonic/meristematic tissues), plant growth regulators (e.g. synthetic cytokinins), and use of novel regeneration-enabling methods to deliver morphogenic genes (e.g. GRF/GIF chimeras and nanoparticles), which have been shown to contribute to overcoming recalcitrance barriers in agriculture crops. Furthermore, it highlights the benefit of cost-effective genomic technologies that enable precise genome editing and the value of integrating data-driven models to address genotype-specific challenges in medicinal plant research. These advances mark a progressive step towards a future where medicinal plant cultivation is not only more efficient and predictable but also inherently sustainable, ensuring the continued availability and exploitation of these important plants for current and future generations.

The major phytocannabinoid bioactives produced by Cannabis sativa L. (cannabis) are Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and cannabidiol (CBD), with many minor phytocannabinoids (PCs) also thought to contribute to the pharmacological efficacy. Cannabis typically segregates into three main chemical phenotypes (chemotypes) based on their Delta(9)-THC/CBD ratio, a highly heritable trait determined by the segregation of two closely related berberine bridge-like enzymes that perform stereoselective oxidative cyclisation on the geranyl moiety of cannabigeroid intermediates. Apart from a small number of metabolome-wide association studies, few attempts have been made to either understand metabolite coupling among Delta(9)-THC/CBD chemotypes or to examine the inheritance of alternative biomarkers that could be used to discriminate chemotype. Here, we examined the metabolomes of 108 F-2 segregants derived from a cross between a Delta(9)-THC-predominant chemotype I and a CBD-predominant chemotype III plant. Although segregation of the Delta(9)-THC/CBD ratio followed Mendelian genetics expectations, covariation in the inheritance of minor PCs, including cannabichromene (CBC)-types, indicated changes in cannabinoid synthase product specificity among chemotypes. In addition, several non-PC related metabolites were identified that may serve as potential biomarkers for chemotype prediction. These data have important implications for the pre-breeding and selection of cannabis chemovars and highlight the need to adopt metabolic engineering strategies to optimise PC production.

This study investigates the leaching of cannabinoids, primarily in their acidic forms, from Cannabis sativa using a pH-controlled aqueous solvent. Key factors including the initial form of cannabis biomass (fresh vs. dry, ground vs. unground), solvent pH and ionic concentration (NaCl content), solid-to-liquid (S/L) ratio, leaching kinetics and temperature were assessed. The results indicated that the highest cannabinoid yield is obtained from fresh flowers, eliminating the need for drying and grinding, pre-treatment steps that are both time-consuming and energy-intensive. Peleg's model was applied to describe the leaching kinetics, demonstrating a high correlation (R2 >= 0.98) with experimental data. The model's parameters indicated rapid leaching, requiring less than 10 min for whole fresh and dried flowers and less than 30 sec for ground dried samples. Temperature analysis showed that heating was not preferred as elevated temperature converted acidic cannabinoids to neutral forms, lowering leaching yields. Thermodynamic analysis indicated an exothermic reaction. Additionally, salt addition of 0.5 % (w/v) NaCl to the pH 13.0 aqueous solvent enhanced the yield, while higher concentrations led to a sharp decrease in leaching yield due to cannabinoid precipitation. These findings demonstrate the efficiency of this method as a viable alternative for industrial cannabinoid extraction, reducing both energy use and process complexity.

The mechanical response and structure of cellulose hydrogel composites (CHCs) are modelled as cellulose microfibrils using a bead-spring coarse-graining approach. Our results demonstrate that varying the adhesive contact energy between the flexible chains, as well as the number of contacts and structural anisotropy, significantly impacts the mechanical response of the network structure under tensile forces. Specifically, decreasing the contact energy and increasing its range is sufficient to increase the network's extensibility while decreasing its overall modulus. This key finding aligns qualitatively with experiments where the inclusion of polysaccharides in bacterial cellulose-based CHCs had an analogous effect. We hypothesise that polysaccharides, including hemicelluloses, facilitate alignment under strain by increasing the range ("softening") of the contact forces between microfibrils. Conversely, their absence results in "hard" contacts between microfibrils that are more energetic and can only act over short distances. In contrast to finite element models for CHCs, this coarse-grained approach incorporates non-permanent contacts between flexible microfibrils that permits structural rearrangement of the network in response to deformation, whereby controlling the density and proportion of long- and short- range contact forces suffice to qualitatively describe experiments. Controlling interactions between microfibrils thus provides a lever for designing CHCs with specific mechanical properties for various applications. Additionally, we suggest that plants naturally tune these variables in plant cell development to balance wall rigidity and extensibility.

The liverwort Marchantia polymorpha has emerged as an important plant model for developmental studies and may become central to elucidate the complex process of cell wall polysaccharide biosynthesis. This study comprehensively analyses the composition and structure of cell wall glycans across eight different M. polymorpha tissue types. We show that while the cell walls largely mirror known land plant cell wall composition, they also exhibit some unique characteristics. For example, M. polymorpha cell walls displayed a remarkably low overall pectin content, yet the relative abundance of pectic α-(1,5)-arabinan in sporophytes hints at its putative role in the evolution and complexity of spermatophyte cell walls. Furthermore, through comparative analyses of glycosyltransferase (GT) families across plant species, we found that while M. polymorpha generally has low genetic redundancy in most cell wall-related GT families, it also exhibits a diversified GT repertoire in four families, indicating uniqueness in certain cell wall biosynthesis pathways. To support research underpinning cell wall biosynthesis, we developed a Gateway compatible compendium of 87 M. polymorpha GTs, providing a valuable resource for genetic and functional studies. Our study thus works as a foundation to drive new insights into cell wall evolution, structure and function across the plant kingdom.

Efficient regeneration protocols are essential for large-scale propagation and genetic manipulation of recalcitrant medicinal species such as Cannabis sativa. Existing direct and indirect regeneration methods are highly genotype and explant-dependent, limiting broader applicability. Here, we report a five-stage (S0–S4) optimised protocol that is reproducible and achieves high-efficiency direct de novo regeneration using cotyledonary node explants from both hemp and medicinal cannabis genotypes. A 1

Linear, unbranched (1,3;1,4)-β-glucans (mixed-linkage glucans or MLGs) are commonly found in the cell walls of grasses, but have also been detected in basal land plants, algae, fungi and bacteria. Here we show that two family GT2 glycosyltransferases from the Gram-positive bacterium Sarcina ventriculi are capable of synthesizing MLGs. Immunotransmission electron microscopy demonstrates that MLG is secreted as an exopolysaccharide, where it may play a role in organizing individual cells into packets that are characteristic of Sarcina species. Heterologous expression of these two genes shows that they are capable of producing MLGs in planta, including an MLG that is chemically identical to the MLG secreted from S. ventriculi cells but which has regularly spaced (1,3)-β-linkages in a structure not reported previously for MLGs. The tandemly arranged, paralogous pair of genes are designated SvBmlgs1 and SvBmlgs2. The data indicate that MLG synthases have evolved different enzymic mechanisms for the incorporation of (1,3)-β- and (1,4)-β-glucosyl residues into a single polysaccharide chain. Amino acid variants associated with the evolutionary switch from (1,4)-β-glucan (cellulose) to MLG synthesis have been identified in the active site regions of the enzymes. The presence of MLG synthesis in bacteria could prove valuable for large-scale production of MLG for medical, food and beverage applications.

Plant adaptation from aquatic to terrestrial environments required modifications to cell wall structure and function to provide tolerance to new abiotic and biotic stressors. Here, we investigate the nature and function of red auronidin pigment accumulation in the cell wall of the liverwort Marchantia polymorpha. Transgenic plants with auronidin production either constitutive or absent were analysed for their cell wall properties, including fractionation of polysaccharide and phenolic components. While small amounts of auronidin and other flavonoids were loosely associated with the cell wall, the majority of the pigments were tightly associated, similar to what is observed in angiosperms for polyphenolics such as lignin. No evidence of covalent binding to a polysaccharide component was found: we propose auronidin is present in the wall as a physically entrapped large molecular weight polymer. The results suggested auronidin is a dual function molecule that can both screen excess light and increase wall strength, hydrophobicity and resistance to enzymatic degradation by pathogens. Thus, liverworts have expanded the core phenylpropanoid toolkit that was present in the ancestor of all land plants, to deliver a lineage-specific solution to some of the environmental stresses faced from a terrestrial lifestyle.

Adhesion and consequent adoption of a sessile habit is a common feature of many green algae and was likely a key mechanism in terrestrialization by an ancient zygnematophyte (i.e., the Zygnematophyceae, the group of algae ancestral to land plants). Penium margaritaceum is a unicellular zygnematophyte that exhibits a multistep adhesion mechanism, which leads to the establishment of the sessile habit. Based on microscopic and immunological data, a dense aggregate of fibrils containing arabinogalactan-protein (AGP)-like components covers the cell surface and is responsible for initial adhesion. The AGP-like fibrils are 20 mu m in diameter and possess chemical profiles similar to land plant AGPs. The fibrils attach to the inner cell wall layers and are very likely connected to the plasma membrane as glycophosphatidylinositol (GPI) lipid-anchored proteins, as they are susceptible to phospholipase C treatment. The presence of GPI-anchored AGPs in Penium is further supported by the identification of putative Penium homologs of land plant AGP genes responsible for GPI-anchor synthesis. After adhesion, cells secrete a complex heteropolysaccharide-containing extracellular polymeric substance (EPS) that facilitates gliding motility and the formation of cell aggregates. Fucoidan-like polymers, major components of brown algal CWs, are a major constituent of both the EPS and the adhesive layer of the CW and their role in the adhesion process is still to be examined.

Cotton provides the most abundant natural fiber for the textile industry. The mature cotton fiber largely consists of secondary cell walls with the highest proportion of cellulose and a small amount of hemicellulose and lignin. To dissect the roles of hemicellulosic polysaccharides during fiber development, four IRREGULAR XYLEM 15 (IRX15) genes, GhIRX15-1/-2/-3/-4, were functionally characterized in cotton. These genes encode DUF579 domain-containing proteins, which are homologs of AtIRX15 involved in xylan biosynthesis. The four GhIRX15 genes were predominantly expressed during fiber secondary wall thickening, and the encoded proteins were localized to the Golgi apparatus. Each GhIRX15 gene could restore the xylan deficient phenotype in the Arabidopsis irx15irx15l double mutant. Silencing of GhIRX15s in cotton resulted in shorter mature fibers with a thinner cell wall and reduced cellulose content as compared to the wild type. Intriguingly, GhIRX15-2 and GhIRX15-4 formed homodimers and heterodimers. In addition, the GhIRX15s showed physical interaction with glycosyltransferases GhGT43C, GhGT47A and GhGT47B, which are responsible for synthesis of the xylan backbone and reducing end sequence. Moreover, the GhIRX15s can form heterocomplexes with enzymes involved in xylan modification and side chain synthesis, such as GhGUX1/2, GhGXM1/2 and GhTBL1. These findings suggest that GhIRX15s participate in fiber xylan biosynthesis and modulate fiber development via forming large multiprotein complexes.

The cell wall is a defining feature of plants that has contributed to their successful colonisation of land. Its complexity, diversity and recalcitrance have challenged practitioners of plant biology and biotechnology and impeded progress in understanding the roles of walls in directing and controlling processes during plant growth and development, and in unlocking their potential for biotechnological innovation. Historically viewed as a rigid, inert structural support, we now recognise a dynamic metabolically active organelle that acts as cell surface mechanosensor. In this chapter, we provide a potted history of some of the major bottlenecks that advances in techniques and technology have decongested to reveal the rich and alluring tapestry that is the plant wall. This lays the foundation for some insights into future challenges for research on the plant wall to further define its function and utility.

Plant proteins that are secreted without a classical signal peptide leader sequence are termed leaderless secretory proteins (LSPs) and are implicated in both plant development and (a)biotic stress responses. In plant proteomics experimental workflows, identification of LSPs is hindered by the possibility of contamination from other subcellar compartments upon purification of the secretome. Applying machine learning algorithms to predict LSPs in plants is also challenging due to the rarity of experimentally validated examples for training purposes. This work attempts to address this issue by establishing criteria for identifying potential plant LSPs based on experimental observations and training random forest classifiers on the putative datasets. The resultant plant protein database LSPDB and bioinformatic prediction tools LSPpred and SPLpred are available at lsppred.lspdb.org. The LSPpred and SPLpred modules are internally validated on the training dataset, with false positives controlled at 5%, and are also able to classify the limited number of established plant LSPs (SPLpred (3/4, LSPpred 4/4). Until such time as a larger set of bona fide (independently experimentally validated) LSPs is established using imaging technologies (light/fluorescence/electron microscopy) to confirm sub-cellular location, these tools represent a bridging method for predicting and identifying plant putative LSPs for subsequent experimental validation.

Cannabis sativa is a remarkable plant containing a wide variety of valuable secondary metabolites, especially cannabinoids, which are valued for their clinical relevance. In this investigation, an efficient and environmentally friendly method to recover cannabinoids from cannabis plant tissues using organic solvent-free pH-controlled solution has been developed. The impact of leaching pH and solid/liquid ratios on the recovery of thirteen acidic and neutral cannabinoids from a wide range of cannabis plant tissues, which are either high tetrahydrocannabinol (THC) or high cannabidiol (CBD) cultivars in fresh, dried and decarboxylated forms, was investigated. The results revealed acidic cannabinoids, the predominant cannabinoids in fresh and cannabis dried at low temperature, were recovered efficiently at pH & GE; 12, demonstrating the potential of alkaline leaching to replace the conventional alcohol leaching widely utilized in the cannabis industry. Alkaline leaching also enables a highly efficient means of obtaining a cannabinoid concentrate directly from fresh material and avoids the long (4-6 d) and energy-intensive drying process. Alkaline leaching was also viable for neutral cannabinoid recovery from decarboxylated cannabis at low solid/liquid ratio. Finally, the stability of cannabinoids in leached solutions at room temperature and in the dark was tracked and analyzed for up to 15 d. These data offer the cannabis industry a range of options for cannabinoid storage and extraction during the enrichment processes.

Oat (Avena sativa) is a cereal crop whose grains are rich in (1,3;1,4)-β-D-glucan (mixed-linkage glucan or MLG), a soluble dietary fiber. In our study, we analyzed oat endosperm development in 2 Canadian varieties with differing MLG content and nutritional value. We confirmed that oat undergoes a nuclear type of endosperm development but with a shorter cellularization phase than barley (Hordeum vulgare). Callose and cellulose were the first polysaccharides to be detected in the early anticlinal cell walls at 11 days postemergence (DPE) of the panicle. Other polysaccharides such as heteromannan and homogalacturonan were deposited early in cellularization around 12 DPE after the first periclinal walls are laid down. In contrast to barley, heteroxylan deposition coincided with completion of cellularization and was detected from 14 DPE but was only detectable after demasking. Notably, MLG was the last polysaccharide to be laid down at 18 DPE within the differentiation phase, rather than during cellularization. In addition, differences in the spatiotemporal patterning of MLG were also observed between the 2 varieties. The lower MLG-containing cultivar AC Morgan (3.5% w/w groats) was marked by the presence of a discontinuous pattern of MLG labeling, while labeling in the same walls in CDC Morrison (5.6% w/w groats) was mostly even and continuous. RNA-sequencing analysis revealed higher transcript levels of multiple MLG biosynthetic cellulose synthase-like F (CSLF) and CSLH genes during grain development in CDC Morrison compared with AC Morgan that likely contributes to the increased abundance of MLG at maturity in CDC Morrison. CDC Morrison was also observed to have smaller endosperm cells with thicker walls than AC Morgan from cellularization onwards, suggesting the processes controlling cell size and shape are established early in development. This study has highlighted that the molecular processes influencing MLG content and deposition are more complex than previously imagined.

Cannabinoids attract worldwide attention due to their well-known medicinal and psychoactive properties. Their efficient recovery from plant material is a significant challenge due to the presence of a complex mixture of secondary metabolites. In this work, 4-tert-amylphenol (4-TAP) was selected as a cannabinoid mimic from five mimic candidates based on an evaluation matrix that included several criteria - toxicity, market price, physi-ochemical properties, and extraction performance. The COnductor-like Screening MOdel (COSMO) was used to predict the physiochemical properties and extraction performance of the mimic candidates and cannabinoids, and assist in the mimic selection and mimic liquid-liquid extraction (LLE) process development. Three aqueous systems, three volatile organic compound (VOC) solvents, three biodegradable green solvents and one extractant were used to investigate the dissolution, extraction, and stripping of 4-TAP. A three stage LLE recovery process was developed for the selected cannabinoid mimic including leaching, extraction, and stripping. The COSMO model predictions show comparable trends to the experimental data and is shown to be useful for integrating fundamental modelling work into cannabinoid extraction process design.

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.