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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]

Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the outer pollen wall (exine) are contributed by surrounding sporophytic tapetal cells. Because the biosynthesis and development of the exine occurs in the innermost cell layers of the anther, direct observations of this process are difficult. The objective of this study was to investigate the transport and assembly of exine components from tapetal cells to microspores in the intact anthers of Arabidopsis thaliana. Intrinsically fluorescent components of developing tapetum and microspores were imaged in intact, live anthers using two-photon microscopy. Mutants of ABCG26 , which encodes an ATP binding cassette transporter required for exine formation, accumulated large fluorescent vacuoles in tapetal cells, with corresponding loss of fluorescence on microspores. These vacuolar inclusions were not observed in tapetal cells of double mutants of abcg26 and genes encoding the proposed sporopollenin polyketide biosynthetic metabolon (ACYL COENZYME A SYNTHETASE5 , POLYKETIDE SYNTHASE A [ PKSA ], PKSB , and TETRAKETIDE α-PYRONE REDUCTASE1), providing a genetic link between transport by ABCG26 and polyketide biosynthesis. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides, raising the possibility that they are incorporated into the exine prior to pollen coat deposition. We propose a model where ABCG26-exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with the trafficking of additional constituents, prior to tapetum programmed cell death. [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]

Summary [ABSTRACT FROM AUTHOR]

The effects of reductions in cell wall lignin content, manifested by RNA interference suppression of coumaroyl 3'-hydroxylase, on plant growth, water transport, gas exchange, and photosynthesis were evaluated in hybrid poplar trees (Populus alba X grandidentata). The growth characteristics of the reduced lignin trees were significantly impaired, resulting in smaller stems and reduced root biomass when compared to wild-type trees, as well as altered leaf morphology and architecture. The severe inhibition of cell wall lignification produced trees with a collapsed xylem phenotype, resulting in compromised vascular integrity, and displayed reduced hydraulic conductivity and a greater susceptibility to wall failure and cavitation. In the reduced lignin trees, photosynthetic carbon assimilation and stomatal conductance were also greatly reduced, however, shoot xylem pressure potential and carbon isotope discrimination were higher and water-use efficiency was lower, inconsistent with water stress. Reductions in assimilation rate could not be ascribed to increased stomatal limitation. Starch and soluble sugars analysis of leaves revealed that photosynthate was accumulating to high levels, suggesting that the trees with substantially reduced cell wall lignin were not carbon limited and that reductions in sink strength were, instead, limiting photosynthesis. [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.

Reports on a new model of cell plate formation in higher plants. Cytokinesis in tobacco BY-2 and root tip cells; Stages of cell plate formation; Results of immunolabeling.

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]

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]

Background Secondary cell walls (SCWs) form the architecture of terrestrial plant biomass. They reinforce tracheary elements and strengthen fibres to permit upright growth and the formation of forest canopies. The cells that synthesize a strong, thick SCW around their protoplast must undergo a dramatic commitment to cellulose, hemicellulose and lignin production. Scope This review puts SCW biosynthesis in a cellular context, with the aim of integrating molecular biology and biochemistry with plant cell biology. While SCWs are deposited in diverse tissue and cellular contexts including in sclerenchyma (fibres and sclereids), phloem (fibres) and xylem (tracheids, fibres and vessels), the focus of this review reflects the fact that protoxylem tracheary elements have proven to be the most amenable experimental system in which to study the cell biology of SCWs. Conclusions SCW biosynthesis requires the co-ordination of plasma membrane cellulose synthases, hemicellulose production in the Golgi and lignin polymer deposition in the apoplast. At the plasma membrane where the SCW is deposited under the guidance of cortical microtubules, there is a high density of SCW cellulose synthase complexes producing cellulose microfibrils consisting of 18-24 glucan chains. These microfibrils are extruded into a cell wall matrix rich in SCW-specific hemicelluloses, typically xylan and mannan. The biosynthesis of eudicot SCW glucuronoxylan is taken as an example to illustrate the emerging importance of protein-protein complexes in the Golgi. From the trans-Golgi, trafficking of vesicles carrying hemicelluloses, cellulose synthases and oxidative enzymes is crucial for exocytosis of SCW components at the microtubule-rich cell membrane domains, producing characteristic SCW patterns. The final step of SCW biosynthesis is lignification, with monolignols secreted by the lignifying cell and, in some cases, by neighbouring cells as well. Oxidative enzymes such as laccases and peroxidases, embedded in the polysaccharide cell wall matrix, determine where lignin is deposited. [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]

Background and Aims The Arabidopsis thaliana pollen cell wall is a complex structure consisting of an outer sporopollenin framework and lipid-rich coat, as well as an inner cellulosic wall. Although mutant analysis has been a useful tool to study pollen cell walls, the ultrastructure of the arabidopsis anther has proved to be challenging to preserve for electron microscopy. Methods In this work, high-pressure freezing/freeze substitution and transmission electron microscopy were used to examine the sequence of developmental events in the anther that lead to sporopollenin deposition to form the exine and the dramatic differentiation and death of the tapetum, which produces the pollen coat. Key Results Cryo-fixation revealed a new view of the interplay between sporophytic anther tissues and gametophytic microspores over the course of pollen development, especially with respect to the intact microspore/pollen wall and the continuous tapetum epithelium. These data reveal the ultrastructure of tapetosomes and elaioplasts, highly specialized tapetum organelles that accumulate pollen coat components. The tapetum and middle layer of the anther also remain intact into the tricellular pollen and late uninucleate microspore stages, respectively. Conclusions This high-quality structural information, interpreted in the context of recent functional studies, provides the groundwork for future mutant studies where tapetum and microspore ultrastructure is assessed. [ABSTRACT FROM PUBLISHER]

Lignin, the second most abundant biopolymer, is a promising renewable energy source and chemical feedstock. A key element of lignin biosynthesis is unknown: how do lignin precursors (monolignols) get from inside the cell out to the cell wall where they are polymerized? Modeling indicates that monolignols can passively diffuse through lipid bilayers, but this has not been tested experimentally. We demonstrate significant monolignol diffusion occurs when laccases, which consume monolignols, are present on one side of the membrane. We hypothesize that lignin polymerization could deplete monomers in the wall, creating a concentration gradient driving monolignol diffusion. We developed a two-photon microscopy approach to visualize lignifying Arabidopsis thaliana root cells. Laccase mutants with reduced ability to form lignin polymer in the wall accumulated monolignols inside cells. In contrast, active transport inhibitors did not decrease lignin in the wall and scant intracellular phenolics were observed. Synthetic liposomes were engineered to encapsulate laccases, and monolignols crossed these pure lipid bilayers to form polymer within. A sink-driven diffusion mechanism explains why it has been difficult to identify genes encoding monolignol transporters and why the export of varied phenylpropanoids occurs without specificity. It also highlights an important role for cell wall oxidative enzymes in monolignol export. [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]

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]

The highly resistant biopolymer, sporopollenin, gives the outer wall (exine) of spores and pollen grains their unparalleled strength, shielding these structures from terrestrial stresses. Despite a limited understanding of the composition of sporopollenin, it appears that the synthesis of sporopollenin occurs in the tapetum and requires the transport of one or more sporopollenin constituents to the surface of developing microspores. Here, we describe ABCG26, a member of the ATP-binding cassette (ABC) transporter superfamily, which is required for pollen exine formation in Arabidopsis (Arabidopsis thaliana), abcg26 mutants are severely reduced in fertility, with most siliques failing to produce seeds by self-fertilization and mature anthers failing to release pollen. Transmission electron microscopy analyses revealed an absence of an exine wall on abcg26-1 mutant microspores. Phenotypic abnormalities in pollen wall formation were first apparent in early uninucleate microspores as a lack of exine formation and sporopollenin deposition. Additionally, the highest levels of ABCG26 mRNA were in the tapetum, during early pollen wall formation, sporopollenin biosynthesis, and sporopollenin deposition. Accumulations resembling the trilamellar lipidic coils in the abcg11 and abcg12 mutants defective in cuticular wax export were observed in the anther locules of abcg26 mutants. A yellow fluorescent protein-ABCG26 protein was localized to the endoplasmic reticulum and plasma membrane. Our results show that ABCG26 plays a critical role in exine formation and pollen development and are consistent with a model by which ABCG26 transports sporopollenin precursors across the tapetum plasma membrane into the locule for polymerization on developing microspore walls. [ABSTRACT FROM AUTHOR]

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]

Lignin, a critical phenolic polymer in secondary cell walls of plant cells, enables strength in fibers and water transportation in xylem vessel elements. Secreted enzymes, namely laccases (LACs) and peroxidases (PRXs), facilitate lignin polymerization by oxidizing lignin monomers (monolignols). Previous work in Arabidopsis (Arabidopsis thaliana) demonstrated that AtLAC4 and AtPRX64 localized to discrete lignified cell wall domains in fibers, although the spatial distributions of other enzymes in these large gene families are unknown. Here, we show that characteristic sets of putative lignin-associated LACs and PRXs localize to precise regions during stem development, with LACs and PRXs co-occurring in cell wall domains. AtLAC4, AtLAC17, and AtPRX72 localized to the thick secondary cell wall of xylem vessel elements and fibers, whereas AtLAC4, AtPRX64, and AtPRX71 localized to fiber cell corners. Interestingly, AtLAC4 had a transient cell corner localization early in fiber development that disappeared in the mature stem. In contrast with these secondary cell wall localizations, AtLAC10, AtPRX42, AtPRX52, and AtPRX71 were found in nonlignified tissues. Despite ubiquitous PRX occurrence in cell walls, PRX oxidative activity was restricted to lignifying regions during development, which suggested regulated production of apoplastic hydrogen peroxide. Relative amounts of apoplastic reactive oxygen species differed between lignified cell types, which could modulate PRX activity. Together, these results indicate that precise localization of oxidative enzymes and differential distribution of oxidative substrates, such as hydrogen peroxide, provide mechanisms to control spatiotemporal deposition of lignin during development. [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]

Secondary cell wall (SCW) production during xylem development requires massive up-regulation of hemicellulose (e.g. glucuronoxylan) biosynthesis in the Golgi. Although mutant studies have revealed much of the xylan biosynthetic machinery, the precise arrangement of these proteins and their products in the Golgi apparatus is largely unknown. We used a fluorescently tagged xylan backbone biosynthetic protein (IRREGULAR XYLEM9; IRX9) as a marker of xylan production in the Golgi of developing protoxylem tracheary elements in Arabidopsis (Arabidopsis thaliana). Both live-cell confocal and transmission electron microscopy (TEM) revealed SCW deposition is accompanied by a significant proliferation of Golgi stacks. Furthermore, although Golgi stacks were randomly distributed, the organization of the cytoplasm ensured their close proximity to developing SCWs. Quantitative immuno-TEM revealed IRX9 is present in a specific subdomain of the Golgi stack and was most abundant in the ring of the inner margins of medial cisternae where fenestrations are abundant. Conversely, the xylan product accumulated in swollen trans cisternal margins and the Trans-Golgi network (TGN). The irx9 mutant lacked this expansion for both the cisternal margins and the TGN, whereas Golgi stack proliferation was unaffected. Golgi in irx9 also displayed dramatic changes in their structure, with increases in cisternal fenestration and tubulation. Our data support a new model where xylan biosynthesis and packaging into secretory vesicles are localized in distinct structural and functional domains of the Golgi. Rather than polysaccharide biosynthesis occurring in the center of the cisternae, IRX9 and the xylan product are arranged in successive concentric rings in Golgi cisternae. [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]

Lignin is an important phenolic biopolymer that provides strength and rigidity to the secondary cell walls of tracheary elements, sclereids, and fibers in vascular plants. Lignin precursors, called monolignols, are synthesized in the cell and exported to the cell wall where they are polymerized into lignin by oxidative enzymes such as laccases and peroxidases. In Arabidopsis thaliana, a peroxidase (PRX64) and laccase (LAC4) are shown to localize differently within cell wall domains in interfascicular fibers: PRX64 localizes to the middle lamella whereas LAC4 localizes throughout the secondary cell wall layers. Similarly, laccases localized to, and are responsible for, the helical depositions of lignin in protoxylem tracheary elements. In addition, we tested the mobility of laccases in the cell wall using fluorescence recovery after photobleaching. mCHERRY-tagged LAC4 was immobile in secondary cell wall domains, but mobile in the primary cell wall when ectopically expressed. A small secreted red fluorescent protein (sec-mCHERRY) was engineered as a control and was found to be mobile in both the primary and secondary cell walls. Unlike sec-mCHERRY, the tight anchoring of LAC4 to secondary cell wall domains indicated that it cannot be remobilized once secreted, and this anchoring underlies the spatial control of lignification. [ABSTRACT FROM AUTHOR]

Membrane contact sites are recognized across eukaryotic systems as important nanostructures. Endoplasmic reticulum (ER)-plasma membrane (PM) contact sites (EPCS) are involved in excitation-contraction coupling, signaling, and plant responses to stress. In this report, we perform a multiscale structural analysis of Arabidopsis EPCS that combines live cell imaging, quantitative transmission electron microscopy (TEM) and electron tomography over a developmental gradient. To place EPCS in the context of the entire cortical ER, we examined green fluorescent protein (GFP)-HDEL in living cells over a developmental gradient, then Synaptotagmin1 (SYT1)-GFP was used as a specific marker of EPCS. In all tissues examined, young, rapidly elongating cells showed lamellar cortical ER and higher density of SYT1-GFP puncta, while in mature cells the cortical ER network was tubular, highly dynamic and had fewer SYT1-labeled puncta. The higher density of EPCS in young cells was verified by quantitative TEM of cryo-fixed tissues. For all cell types, the size of each EPCS had a consistent range in length along the PM from 50 to 300 nm, with microtubules and ribosomes excluded from the EPCS. The structural characterization of EPCS in different plant tissues, and the correlation of EPCS densities over developmental gradients illustrate how ER-PM communication evolves in response to cellular expansion. [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]

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]

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]

Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC 17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac41ac17 mutant analysis demonstrating that laceases are necessary for protoxylem TE lignification. Overexpression studies showed that laceases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laceases to secondary cell wall domains directs lignin deposition. [ABSTRACT FROM AUTHOR]

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH- dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. [ABSTRACT FROM AUTHOR]

ATP binding cassette (ABC) transporters play diverse roles, including lipid transport, in all kingdoms. ABCG subfamily transporters that are encoded as half-transporters require dimerization to form a functional ABC transporter. Different dimer combinations that may transport diverse substrates have been predicted from mutant phenotypes. In Arabidopsis thaliana , mutant analyses have shown that ABCG11/WBC11 and ABCG12/CER5 are required for lipid export from the epidermis to the protective cuticle. The objective of this study was to determine whether ABCG11 and ABCG12 interact with themselves or each other using bimolecular fluorescence complementation (BiFC) and protein traffic assays in vivo. With BiFC, ABCG11/ABCG12 heterodimers and ABCG11 homodimers were detected, while ABCG12 homodimers were not. Fluorescently tagged ABCG11 or ABCG12 was localized in the stem epidermal cells of abcg11 abcg12 double mutants. ABCG11 could traffic to the plasma membrane in the absence of ABCG12, suggesting that ABCG11 is capable of forming flexible dimer partnerships. By contrast, ABCG12 was retained in the endoplasmic reticulum in the absence of ABCG11, indicating that ABCG12 is only capable of forming a dimer with ABCG11 in epidermal cells. Emerging themes in ABCG transporter biology are that some ABCG proteins are promiscuous, having multiple partnerships, while other ABCG transporters form obligate heterodimers for specialized functions. [ABSTRACT FROM AUTHOR]

Secondary xylem (wood) formation in gyninosperms requires that the tracheid protoplasts first build an elaborate secondary cell wall from an array of polysaccharides and then reinforce it with lignin, an amorphous, three-dimensional product of the random radical coupling of monolignols. The objective of this study was to track the spatial distribution of monolignols during development as they move from symplasm to apoplasm. This was done by feeding [³H]phenylalanine ([³HIPhe) to dissected cambium/ developing wood from lodgepole pine (Pinus contorta var latifolia) seedlings, allowing uptake and metabolism, then rapidly freezing the cells and performing autoradiography to detect the locations of the monolignols responsible for lignification. Parallel experiments showed that radioactivity was incorporated into polymeric ligrnn and a methanol-soluble pool that was characterized by high-performance liquid chromatography. [³H]Phe was incorporated into expected lignin precursors, such as coniferyl alcohol and p-coumaryl alcohol, as well as pinoresinol. Coniferin, the glucoside of coniferyl alcohol, was detected by high-performance liquid chromatography but was not radioactively labeled. With light microscopy, radiolabeled phenylpropanoids were detected in the rays as well as the tracheids, with the two cell types showing differential sensitivity to inhibitors of protein translation and phenylpropanoid metabolism. Secondary cell walls of developing tracheids were heavily labeled when incubated with [³H]Phe. Inside the cell, cytoplasm was most strongly labeled followed by Golgi and low-vacuole label. Inhibitor studies suggest that the Golgi signal could be attributed to protein, rather than phenylpropanoid, origins. These data, produced with the best microscopy tools that are available today, support a model in which unknown membrane transporters, rather than Golgi vesicles, export monolignols. [ABSTRACT FROM AUTHOR]

Differentiation of the Arabidopsis thaliana seed coat cells includes a secretory phase where large amounts of pectinaceous mucilage are deposited to a specific domain of the cell wall. During this phase, Golgi stacks had cisternae with swollen margins and trans-Golgi networks consisting of interconnected vesicular clusters. The proportion of Golgi stacks producing mucilage was determined by immunogold labeling and transmission electron microscopy using an antimucilage antibody, CCRC-M36. The large percentage of stacks found to contain mucilage supports a model where all Golgi stacks produce mucilage synchronously, rather than having a subset of specialist Golgi producing pectin product. Initiation of mucilage biosynthesis was also correlated with an increase in the number of Golgi stacks per cell. Interestingly, though the morphology of individual Golgi stacks was dependent on the volume of mucilage produced, the number was not, suggesting that proliferation of Golgi stacks is developmentally programmed. Mapping the position of mucilage-producing Golgi stacks within developing seed coat cells and live-cell imaging of cells labeled with a trans-Golgi marker showed that stacks were randomly distributed throughout the cytoplasm rather than clustered at the site of secretion. These data indicate that the destination of cargo has little effect on the location of the Golgi stack within the cell. [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]

The Arabidopsis seed coat epidermis undergoes a complex process of differentiation that includes the biosynthesis and secretion of large quantities of pectinaceous mucilage, cytoplasmic rearrangement, and secondary cell wall biosynthesis. Mutations in MUM4 (MUCILAGE-MODIFIED4) lead to a decrease in seed coat mucilage and incomplete cytoplasmic rearrangement. We show that MUM4 encodes a putative NDP-r-rhamnose synthase, an enzyme required for the synthesis of the pectin rhamnogalacturonan I, the major component of Arabidopsis mucilage. This result suggests that the synthesis of monosaccharide substrates is a limiting factor in the biosynthesis of pectinaceous seed coat mucilage. In addition, the reduced cytoplasmic rearrangement observed in the absence of a key enzyme in pectin biosynthesis in mum4 mutants establishes a causal link between mucilage production and cellular morphogenesis. The cellular phenotype seen in mum4 mutants is similar to that of several transcription factors (AP2 [APETALA2], TTG1 [TRANSPARENT TESTA GLABRA1], TTG2 MYB61, and GL2 [GLABRA2]). Expression studies suggest that MUM4 is developmentally regulated in the seed coat by AP2, TTG1, and GL2, whereas TTG2 and MYB61 appear to be regulating mucilage production through alternate pathway(s). Our results provide a framework for the regulation of mucilage production and secretory cell differentiation. [ABSTRACT FROM AUTHOR]

A waxy protective cuticle coats all primary aerial plant tissues. Its synthesis requires extensive export of lipids from epidermal cells to the plant surface. Arabidopsis cer5 mutants had reduced stem cuticular wax loads and accumulated sheetlike inclusions in the cytoplasm of wax-secreting cells. These inclusions represented abnormal deposits of cuticular wax and resembled inclusions found in a human disorder caused by a defective peroxisomal adenosine triphosphate binding cassette (ABC) transporter. We found that the CER5 gene encodes an ABC transporter localized in the plasma membrane of epidermal cells and conclude that it is required for wax export to the cuticle. [ABSTRACT FROM AUTHOR]