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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. LAY DESCRIPTION: For millennia humans have grown and consumed cannabis flowers for use in traditional medicine and intoxication, due to the plant's active ingredients called cannabinoids. The most well‐known cannabinoid is tetrahydrocannabidiol (THC). Despite this long history, and recent relaxation of cannabis uses for medicinal and recreational consumption in many parts of the world, little is known about how the plant makes the cannabinoids in its cells. Small mushroom‐shaped structures on the flower surface, called glandular trichomes, produce and store the cannabinoids. To image inside the cells of THC‐rich cannabis flowers, it is necessary to use electron microscopy, and this has revealed complex internal membrane networks and specialised cell structures in these tiny biofactories. In this study, we explored different ways to preserve the cannabis glandular trichomes for electron microscopy, using either cryogenic preservation or chemical crosslinking, in order to determine the benefits and drawbacks of each method. The chemical crosslinking method produced dramatic re‐arrangement of cell contents that were not seen in the cryofixation, where the near‐instantaneous immobilisation of the cell structure led to exceptional preservation of the THC‐producing cells. Despite the benefits of using cryofixation for preserving the complex cell structures, it was not as useful for seeing details of the cell walls surrounding the glandular trichome. Together, our data show stark differences in cannabis glandular trichome cell structure that result from cryofixation and chemical fixation. This is important because the chemical fixation protocol was historically used and informed previous models of cannabinoid production and trafficking. Cryofixation produces exciting new models of THC production in cannabis, due to its high‐fidelity preservation of cell structures. In contrast, studies of cell walls may still benefit from the traditional chemical cross‐linking technique of sample preservation for electron microscopy. https://www.cell.com/current‐biology/fulltext/S0960‐9822(22)01115‐0 [ABSTRACT FROM AUTHOR]

The hemicelluloses comprise a group of matrix glycans that interact with cellulose microfibrils in plant cell walls and play important roles in establishing wall architecture. The structures of hemicelluloses are determined by carbohydrate‐active enzymes (CAZymes) that synthesize, integrate, and break down these polymers. Specifically, endo‐glucanase 16 (EG16) enzymes, which are related to the well‐known xyloglucan endotransglycosylase/hydrolase (XTH) gene products in Glycoside Hydrolase Family 16 (GH16), have been implicated in the degradation of the β(1,4)‐linked backbone of mixed‐linkage β(1,3);β(1,4)‐glucans (MLG) and xyloglucans. EG16 members are single‐copy genes found in most plant clades but are absent from many eudicots, including the model plant Arabidopsis thaliana. Until recently, EG16 members had only been characterized in vitro, establishing their substrate specificity, protein structure, and phylogenetic history, but their biological function was unknown. Here we used a hybrid polar, Populus alba × Populus grandidentata (P39), as a model to examine EG16 expression, subcellular localization, and pheno‐ and chemotypes of EG16‐downregulated P39 plants. PopulusEG16 expression is strong in young tissues, but RNAi‐mediated downregulation did not impact plant growth nor the fine structure of the hemicellulose xyloglucan, suggesting a restricted or currently unknown role in angiosperm physiology. [ABSTRACT FROM AUTHOR]

Plants maintain large repertoires of carbohydrate‐active enzymes (CAZymes)—comprising between 3% and 10% of their genomes—to synthesize, modify, and degrade the polysaccharide components of the cell wall. We recently identified a unique group of plant endo‐glucanases from Glycoside Hydrolase Family 16, viz. EG16 orthologs, which constitute a sister clade to the well‐known XYLOGLUCAN ENDO‐TRANSGLYCOSYLASE/HYDROLASE (XTH) gene products. Biochemical analysis of EG16 orthologs from poplar (Populus trichocarpa), grapevine (Vitis vinifera), and spreading earthmoss (Physcomitrium patens) has demonstrated that these endo‐glucanases are distinctly active on cell wall matrix glycans, mixed‐linkage β(1,3);β(1,4)‐glucan and xyloglucan (XyG), and that enzyme structure and specificity is highly conserved across diverse plant lineages. However, the physiological role of EG16 orthologs in any species is presently unknown. To shed light on EG16 function in vivo, here we performed reverse genetics and protein localization analyses of the single EG16 ortholog in the model moss P. patens, where this gene is highly expressed in young, expanding tissues, particularly in protonema. Surprisingly, deletion of the PpEG16 gene by homologous recombination led to an increase in growth, as well as accelerated senescence. Notably, the PpEG16 protein was shown to co‐localize with XyG in the cell wall of protonema tissue, specifically at cell tips, despite lacking a secretion signal peptide. Although the precise biological role of EG16 orthologs remains elusive, our results implicate these highly conserved glycoside hydrolases in cell wall polysaccharide remodeling and recycling. We anticipate that these foundational results will inform future studies on EG16 function across plant lineages. [ABSTRACT FROM AUTHOR]

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1 , was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs. [ABSTRACT FROM AUTHOR]

Results demonstrate that in donor bacteria containing conjugative plasmids, distinctive junctions are found between outer membranes of the mating cells. Data also indicate that the junctions are not composed of pilin.

The valuable cannabinoid and terpenoid metabolites of Cannabis sativa L. are produced by floral glandular trichomes. The trichomes consist of secretory disk cells, which produce the abundant lipidic metabolites, and an extracellular storage cavity. The mechanisms of apoplastic cavity formation to accumulate and store metabolites in cannabis glandular trichomes remain wholly unexplored. Here, we identify key wall components and how they change during cannabis trichome development. While glycome and monosaccharide analyses revealed that glandular trichomes have loosely bound xyloglucans and pectic polysaccharides, quantitative immunolabeling with wall-directed antibodies revealed precise spatiotemporal distributions of cell wall epitopes. An epidermal-like identity of early trichome walls matured into specialized wall domains over development. Cavity biogenesis was marked by separation of the subcuticular wall from the underlying surface wall in a homogalacturonan and α-1,5 arabinan epitope-rich zone and was associated with a reduction in fucosylated xyloglucan epitopes. As the cavity filled, a matrix with arabinogalactan and α-1,5 arabinan epitopes enclosed the metabolite droplets. At maturity, the disk cells' apical wall facing the storage cavity accumulated rhamnogalacturonan-I epitopes near the plasma membrane. Together, these data indicate that cannabis glandular trichomes undergo spatiotemporal remodeling at specific wall subdomains to facilitate storage cavity formation and metabolite storage. [ABSTRACT FROM AUTHOR]

Two isolates of Turnip mosaic virus (UK 1 and JPN 1), representative of two different viral strains, induced differential alterations on secondary cell wall (SCW) development in Arabidopsis thaliana , suggesting cell-type specific effects of these viral infections. These potential effects were analyzed in inflorescence stems and flowers of infected plants, together with other possible cellular effects of the infections. Results obtained from macroscopic and histochemical analyses showed that infection with either virus significantly narrowed stem area, but defects in SCW were only found in JPN 1 infections. In flowers, reduced endothecium lignification was also found for JPN 1, while UK 1 infections induced severe floral cell and organ development alterations. A transcriptomic analysis focused on genes controlling and regulating SCW formation also showed notable differences between both viral isolates. UK 1 infections induced a general transcriptional decrease of most regulatory genes, whereas a more complex pattern of alterations was found in JPN 1 infections. The role of the previously identified viral determinant of most developmental alterations, the P3 protein, was also studied through the use of viral chimeras. No SCW alterations or creeping habit growth were found in infections by the chimeras, indicating that if the P3 viral protein is involved in the determination of these symptoms, it is not the only determinant. Finally, considerations as to the possibility of a taxonomical reappraisal of these TuMV viral strains are provided. [ABSTRACT FROM AUTHOR]

Summary: The transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is a Class II KNOTTED1‐like homeobox (KNOX2) gene that, in interfascicular fibres, acts as a negative regulator of secondary cell wall biosynthesis. In addition, knat7 loss‐of‐function mutants display an irregular xylem (irx) phenotype, suggesting a potential positive regulatory role in xylem vessel secondary cell wall deposition. Although our understanding of the role of KNAT7 is evolving, the function(s) of the closely related KNOX2 genes, KNAT3, KNAT4, and KNAT5, in secondary wall formation still remain unclear. We found that all four Arabidopsis KNOX2 genes were expressed in the inflorescence stems. However, only the knat3 knat7 double mutants showed a phenotype, displaying an enhanced irx phenotypes relative to the single mutants, as well as decreased interfascicular fibre cell wall thickness. Moreover, knat3 knat7 double mutants had reduced stem tensile and flexural strength compared with wild‐type and single mutants. In contrast, KNAT3 overexpression resulted in thicker interfascicular fibre secondary cell walls in inflorescence stems, suggesting a potential positive regulation in interfascicular fibre secondary wall development. This work identifies KNAT3 as a potential transcriptional activator working together with KNAT7 to promote secondary cell wall biosynthesis in xylem vessels, while concurrently acting antagonistically with KNAT7 to influence secondary wall formation in interfascicular fibres. Significance Statement: This work identifies KNAT3 as a potential transcriptional activator working together with KNAT7 to promote secondary cell wall biosynthesis in xylem vessels, while concurrently acting antagonistically with KNAT7 to influence secondary wall formation in interfascicular fibres. [ABSTRACT FROM AUTHOR]

Summary: 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 using 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. Significance Statement: The unique monoterpene‐rich cannabis stalked trichomes that determine the medicinal, psychoactive and sensory properties of cannabis flowers develop from sessile‐like intermediates. [ABSTRACT FROM AUTHOR]

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs. [ABSTRACT FROM AUTHOR]

Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce ( Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil ( Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. [ABSTRACT FROM AUTHOR]

Developing plant embryos depend on nutrition from maternal tissues via the seed coat and endosperm, but the mechanisms that supply nutrients to plant embryos have remained elusive. Sucrose, the major transport form of carbohydrate in plants, is delivered via the phloem to the maternal seed coat and then secreted from the seed coat to feed the embryo. Here, we show that seed filling in Arabidopsis thaliana requires the three sucrose transporters SWEET11, 12, and 15. SWEET11, 12, and 15 exhibit specific spatiotemporal expression patterns in developing seeds, but only a sweet11;12;15 triple mutant showed severe seed defects, which include retarded embryo development, reduced seed weight, and reduced starch and lipid content, causing a "wrinkled" seed phenotype. In sweet11;12;15 triple mutants, starch accumulated in the seed coat but not the embryo, implicating SWEET-mediated sucrose efflux in the transfer of sugars from seed coat to embryo. This cascade of sequentially expressed SWEETs provides the feeding pathway for the plant embryo, an important feature for yield potential. [ABSTRACT FROM AUTHOR]

Summary: •The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions.•A membrane‐bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken.•Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1‐1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure.•Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants. [ABSTRACT FROM AUTHOR]

As an initial step to develop plants as systems to produce enzymes for the treatment of lysosomal storage disorders, Arabidopsis thaliana wild-type (Col-0) plants were transformed with a construct to express human α-l-iduronidase (IDUA; EC 3.2.1.76) in seeds using the promoter and other regulatory sequences of the Phaseolus vulgaris arcelin 5-I gene. IDUA protein was easily detected on Western blots of extracts from the T2 seeds, and extracts contained IDUA activity as high as 2.9 nmol 4-methylumbelliferone (4 MU)/min/mg total soluble protein (TSP), corresponding to approximately 0.06 µg IDUA/mg TSP. The purified protein reacted with an antibody specific for xylose-containing plant complex glycans, indicating its transit through the Golgi complex. In an attempt to avoid maturation of the N-linked glycans of IDUA, the same IDUA transgene was introduced into the Arabidopsis cgl background, which is deficient in the activity of N-acetylglucosaminyl transferase I (EC 2.4.1.101), the first enzyme in the pathway of complex glycan biosynthesis. IDUA activity and protein levels were significantly higher in transgenic cgl vs. wild-type seeds (e.g. maximum levels were 820 nmol 4 MU/min/mg TSP, or 18 µg IDUA/mg TSP). Affinity-purified IDUA derived from cgl mutant seeds showed a markedly reduced reaction with the antibody specific for plant complex glycans, despite transit of the protein to the apoplast. Furthermore, gel mobility changes indicated that a greater proportion of its N-linked glycans were susceptible to digestion by Streptomyces endoglycosidase H, as compared to IDUA derived from seeds of wild-type Arabidopsis plants. The combined results indicate that IDUA produced in cgl mutant seeds contains glycans primarily in the high-mannose form. This work clearly supports the viability of using plants for the production of human therapeutics with high-mannose glycans. [ABSTRACT FROM AUTHOR]

All vascular plants are protected from the environment by a cuticle, a lipophilic layer synthesized by epidermal cells and composed of a cutin polymer matrix and waxes. The mechanism by which epidermal cells accumulate and assemble cuticle components in rapidly expanding organs is largely unknown. We have begun to address this question by analyzing the lipid compositional variance, the surface micromorphology, and the transcriptome of epidermal cells in elongating Arabidopsis (Arabidopsis thaliana) stems. The rate of cell elongation is maximal near the apical meristem and decreases steeply toward the middle of the stem, where it is 10 times slower. During and after this elongation, the cuticular wax load and composition remain remarkably constant (32 μg/cm2), indicating that the biosynthetic flux into waxes is closely matched to surface area expansion. By contrast, the load of polyester monomers per unit surface area decreases more than 2-fold from the upper (8 μg/cm2) to the lower (3 μg/cm2) portion of the stem, although the compositional variance is minor. To aid identification of proteins involved in the biosynthesis of waxes and cutin, we have isolated epidermal peels from Arabidopsis stems and determined transcript profiles in both rapidly expanding and nonexpanding cells. This transcriptome analysis was; validated by the correct classification of known epidermis-specific genes. The 15% transcripts preferentially expressed in the epidermis were enriched in genes encoding proteins predicted to be membrane associated and involved in lipid metabolism. An analysis of lipid-related subset is presented. [ABSTRACT FROM AUTHOR]

Investigates conjugative junctions in RP4-mediated mating of Escherichia coli. Preparatory procedures done on E. coli mating aggregates; Significance of conjugative plasmids in donor bacteria; Composition of electron-dense layer between outer membranes of conjugating cells; Role of Tra operon in the transfer of RP4.

Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible polysaccharide-based extracellular matrix: the cell wall. Cell wall composition varies among plant species, across cell types within a plant, within different regions of a single cell wall, and in response to intrinsic or extrinsic signals. This diversity in cell wall makeup is underpinned by common cellular mechanisms for cell wall production. Cellulose synthase complexes function at the plasma membrane and deposit their product into the cell wall. Matrix polysaccharides are synthesized by a multitude of glycosyltransferases in hundreds of mobile Golgi stacks, and an extensive set of vesicle trafficking proteins govern secretion to the cell wall. In this review, we discuss the different subcellular locations at which cell wall synthesis occurs, review the molecular mechanisms that control cell wall biosynthesis, and examine how these are regulated in response to different perturbations to maintain cell wall homeostasis. Organelles of the plant cell cooperate to synthesize and secrete a strong yet flexible cell wall. Hoffmann et al. describe the subcellular locations at which cell wall synthesis occurs and review the mechanisms underlying biosynthesis. They also examine how synthesis is regulated in response to different perturbations to maintain cell wall homeostasis. [ABSTRACT FROM AUTHOR]

The final stage of lignin polymerization in plant cell walls requires oxidation of lignin monomers by secreted enzymes, laccases and peroxidases. Identifying the role of oxidative enzymes during lignin deposition has been hindered by large gene families, functional redundancy, and involvement in diverse biological processes. In this chapter, we discuss how the location and composition of lignin in the secondary cell wall could be fine-tuned by localization and substrate specificity of specific oxidative enzymes. We use Populus trichocarpa (poplar) as a case study to demonstrate the initial steps for identifying putative lignin-associated oxidative enzymes that could be used for further experimental validation. [ABSTRACT FROM AUTHOR]

The coordination of multiple metabolic activities in plants relies on an interorganelle communication network established through membrane contact sites (MCS). The MCS are maintained in transient or durable configurations by tethering structures which keep the two membranes in close proximity, and create chemical microdomains that allow localized and targeted exchange of small molecules and possibly proteins. The past few years have witnessed a dramatic increase in our understanding of the structural and molecular organization of plant interorganelle MCS, and their crucial roles in plant specialized functions including stress responses, cell to cell communication, and lipid transport. In this review we summarize recent advances in understanding the molecular components, structural organization, and functions of different plant-specific MCS architectures. [ABSTRACT FROM AUTHOR]

Summary: In the leaf epidermis, intricately lobed pavement cells use Rho of plants (ROP) small GTPases to integrate actin and microtubule organization with trafficking through the secretory pathway []. Cell signaling occurs because guanine nucleotide exchange factors (GEFs) promote ROP activation and their interactions with effector proteins that direct the cell growth machineries []. In Arabidopsis, SPIKE1 (SPK1) is the lone DOCK family GEF []. SPK1 promotes polarized growth and cell-cell adhesion in the leaf epidermis; however, its mode of action in cells is not known. Vertebrate DOCK proteins are deployed at the plasma membrane []. Likewise, current models place SPK1 activity and/or active ROP at the plant plasma membrane and invoke the localized patterning of the cortical cytoskeleton as the mechanism for shape control []. In this paper, we find that SPK1 is a peripheral membrane protein that accumulates at, and promotes the formation of, a specialized domain of the endoplasmic reticulum (ER) termed the ER exit site (ERES). SPK1 signals are generated from a distributed network of ERES point sources and maintain the homeostasis of the early secretory pathway. The ERES is the location for cargo export from the ER []. Our findings open up unexpected areas of plant G protein biology and redefine the ERES as a subcellular location for signal integration during morphogenesis. [ABSTRACT FROM AUTHOR]

For centuries, humans have cultivated cannabis for the pharmacological properties that result from consuming its specialized metabolites, primarily cannabinoids and terpenoids. Today, cannabis is a multi-billion-dollar industry whose existence rests on the biological activity of tiny cell clusters, called glandular trichomes, found mainly on flowers. Cannabinoids are toxic to cannabis cells,1 and how the trichome cells can produce and secrete massive quantities of lipophilic metabolites is not known. 1 To address this gap in knowledge, we investigated cannabis glandular trichomes using ultra-rapid cryofixation, quantitative electron microscopy, and immuno-gold labeling of cannabinoid pathway enzymes. We demonstrate that the metabolically active cells in cannabis form a "supercell," with extensive cytoplasmic bridges across the cell walls and a polar distribution of organelles adjacent to the apical surface where metabolites are secreted. The predicted metabolic role of the non-photosynthetic plastids is supported by unusual membrane arrays in the plastids and the localization of the start of the cannabinoid/terpene pathway in the stroma of the plastids. Abundant membrane contact sites connected plastid paracrystalline cores with the plastid envelope, plastid with endoplasmic reticulum (ER), and ER with plasma membrane. The final step of cannabinoid biosynthesis, catalyzed by tetrahydrocannabinolic acid synthase (THCAS), was localized in the cell-surface wall facing the extracellular storage cavity. We propose a new model of how the cannabis cells can support abundant metabolite production, with emphasis on the key role of membrane contact sites and extracellular THCA biosynthesis. This new model can inform synthetic biology approaches for cannabinoid production in yeast or cell cultures. • Glandular cells form a polarized syncytium during THCA production and secretion • GPPS is located in plastids that contain conspicuous membrane fusions • THCAS is located exclusively on the extracellular surface of trichomes • Membrane contacts among plastids, ER, and PM inform a new trafficking model Cannabinoids and terpenes are biosynthesized, secreted, and stored in cannabis glandular trichomes. Livingston et al. reveal the nanoscale distribution of the enzymes involved in cannabinoid biosynthesis and the unique cellular environment where these enzymes operate. A new model of metabolite trafficking from the plastid to the cell wall is proposed. [ABSTRACT FROM AUTHOR]