NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Disrupting cell wall integrity impacts endomembrane trafficking to promote secretion over endocytic trafficking.

The plant cell wall provides a strong yet flexible barrier to protect cells from the external environment. Modifications of the cell wall, either during development or under stress conditions, can induce cell wall integrity responses and ultimately lead to alterations in gene expression, hormone production, and cell wall composition. These changes in cell wall composition presumably require remodelling of the secretory pathway to facilitate synthesis and secretion of cell wall components and cell wall synthesis enzymes from the Golgi apparatus. Here, we used a combination of live-cell confocal imaging and transmission electron microscopy to examine the short-term and constitutive impact of isoxaben, which reduces cellulose biosynthesis, and Driselase, a cocktail of cell-wall-degrading fungal enzymes, on cellular processes during cell wall integrity responses. We show that both treatments altered organelle morphology and triggered rebalancing of the secretory pathway to promote secretion while reducing endocytic trafficking. The actin cytoskeleton was less dynamic following cell wall modification, and organelle movement was reduced. These results demonstrate active remodelling of the endomembrane system and actin cytoskeleton following changes to the cell wall.

Two roads diverge for cellulose synthase complex trafficking.

Cellulose, an abundant and essential component of plant cell walls, is made by cellulose synthase complexes at the plasma membrane (PM). Recently, Liu et al. uncovered molecular mechanisms that suggest the existence of two distinct pathways for cellulose synthase trafficking from the Golgi apparatus to the PM.

Open questions in plant cell wall synthesis.

Plant cells are surrounded by strong yet flexible polysaccharide-based cell walls that support cells while also allowing growth by cell expansion. Plant cell wall research has advanced tremendously in recent years. Sequenced genomes of model and crop plants have facilitated cataloguing and characterization of many enzymes involved in cell wall synthesis. Structural information has been generated for several important cell wall-synthesizing enzymes. Important tools have been developed including antibodies raised against a variety of cell wall polysaccharides and glycoproteins, collections of enzyme clones and synthetic glycan arrays for characterizing enzymes, herbicides that specifically affect cell wall synthesis, live-cell imaging probes to track cell wall synthesis, and an inducible secondary cell wall synthesis system. Despite these advances, and often because of the new information they provide, many open questions about plant cell wall polysaccharide synthesis persist. This article highlights some of the key questions that remain open, reviews the data supporting different hypotheses that address these questions, and discusses technological developments that may answer these questions in the future.

Running a research group in the next generation: combining sustainable and reproducible research with values-driven leadership.

In the summer of 2021, we held a community workshop at the International Congress of Arabidopsis Research (ICAR) aimed at early career researchers and focused on values-based lab leadership. Here, we elaborate on ideas emerging from the workshop that we hope will allow current and future group leaders to reflect on and adjust to the rapidly evolving nature of the academic scientific enterprise.

The TOR complex controls ATP levels to regulate actin cytoskeleton dynamics in Arabidopsis.

Energy is essential for all cellular functions in a living organism. How cells coordinate their physiological processes with energy status and availability is thus an important question. The turnover of actin cytoskeleton between its monomeric and filamentous forms is a major energy drain in eukaryotic cells. However, how actin dynamics are regulated by ATP levels remain largely unknown in plant cells. Here, we observed that seedlings with impaired functions of target of rapamycin complex 1 (TORC1), either by mutation of the key component, RAPTOR1B, or inhibition of TOR activity by specific inhibitors, displayed reduced sensitivity to actin cytoskeleton disruptors compared to their controls. Consistently, actin filament dynamics, but not organization, were suppressed in TORC1-impaired cells. Subcellular localization analysis and quantification of ATP concentration demonstrated that RAPTOR1B localized at cytoplasm and mitochondria and that ATP levels were significantly reduced in TORC1-impaired plants. Further pharmacologic experiments showed that the inhibition of mitochondrial functions led to phenotypes mimicking those observed in raptor1b mutants at the level of both plant growth and actin dynamics. Exogenous feeding of adenine could partially restore ATP levels and actin dynamics in TORC1-deficient plants. Thus, these data support an important role for TORC1 in coordinating ATP homeostasis and actin dynamics in plant cells.

Regulation of cellulose synthesis via exocytosis and endocytosis.

Cellulose is a critical component of plant cell walls. Cellulose is made at the plasma membrane by cellulose synthase (CESA) enzymes organized into large, multi-subunit cellulose synthase complexes (CSCs). Although CESAs are only active at the plasma membrane, fluorescently-tagged CESAs also substantially label the Golgi apparatus and other intracellular compartments, even when cellulose synthesis rates are high. These data imply that CESA activity is regulated by trafficking to the plasma membrane (exocytosis) and removal from the plasma membrane (endocytosis), as well as recycling of endocytosed CESAs back to the plasma membrane. Key molecular components and events of CESA exocytosis and endocytosis have recently been defined, primarily using mutant analysis and live-cell imaging in Arabidopsis thaliana. Here, we integrate these data into a working model of CESA regulation by exocytosis and endocytosis and highlight key outstanding questions. We present the hypothesis that cycling of CESAs between the plasma membrane and the endomembrane system is important for regulating cellulose synthesis and for maintaining a robust population of active CSCs in the plasma membrane.

Plant Trans-Golgi Network/Early Endosome pH regulation requires Cation Chloride Cotransporter (CCC1).

Plant cells maintain a low luminal pH in the trans-Golgi-network/early endosome (TGN/EE), the organelle in which the secretory and endocytic pathways intersect. Impaired TGN/EE pH regulation translates into severe plant growth defects. The identity of the proton pump and proton/ion antiporters that regulate TGN/EE pH have been determined, but an essential component required to complete the TGN/EE membrane transport circuit remains unidentified - a pathway for cation and anion efflux. Here, we have used complementation, genetically encoded fluorescent sensors, and pharmacological treatments to demonstrate that Arabidopsis cation chloride cotransporter (CCC1) is this missing component necessary for regulating TGN/EE pH and function. Loss of CCC1 function leads to alterations in TGN/EE-mediated processes including endocytic trafficking, exocytosis, and response to abiotic stress, consistent with the multitude of phenotypic defects observed in ccc1 knockout plants. This discovery places CCC1 as a central component of plant cellular function.

Analysis of cellulose synthase activity in Arabidopsis using spinning disk microscopy.

We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021.

Small but Mighty: An Update on Small Molecule Plant Cellulose Biosynthesis Inhibitors.

Cellulose is one of the most abundant biopolymers on Earth. It provides mechanical support to growing plant cells and important raw materials for paper, textiles and biofuel feedstocks. Cellulose biosynthesis inhibitors (CBIs) are invaluable tools for studying cellulose biosynthesis and can be important herbicides for controlling weed growth. Here, we review CBIs with particular focus on the most widely used CBIs and recently discovered CBIs. We discuss the effects of these CBIs on plant growth and development and plant cell biology and summarize what is known about the mode of action of these different CBIs.

A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis.

Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gßγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gßγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.

Subcellular coordination of plant cell wall synthesis.

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.

Insights from the Structure of a Plant Cellulose Synthase Trimer.

Cellulose is an essential component of plant cell walls and the most abundant biopolymer on Earth. Despite its chemical simplicity, questions remain regarding the mechanisms of cellulose synthesis. A cryo-electron microscopy structure of a simplified plant cellulose synthase enzyme complex provides new insights into assembly, localization, and regulation of this complex.

EPSIN1 and MTV1 define functionally overlapping but molecularly distinct trans-Golgi network subdomains in Arabidopsis.

The plant trans-Golgi network (TGN) is a central trafficking hub where secretory, vacuolar, recycling, and endocytic pathways merge. Among currently known molecular players involved in TGN transport, three different adaptor protein (AP) complexes promote vesicle generation at the TGN with different cargo specificity and destination. Yet, it remains unresolved how sorting into diverging vesicular routes is spatially organized. Here, we study the family of Arabidopsis thaliana Epsin-like proteins, which are accessory proteins to APs facilitating vesicle biogenesis. By comprehensive molecular, cellular, and genetic analysis of the EPSIN gene family, we identify EPSIN1 and MODIFIED TRANSPORT TO THE VACUOLE1 (MTV1) as its only TGN-associated members. Despite their large phylogenetic distance, they perform overlapping functions in vacuolar and secretory transport. By probing their relationship with AP complexes, we find that they define two molecularly independent pathways: While EPSIN1 associates with AP-1, MTV1 interacts with AP-4, whose function is required for MTV1 recruitment. Although both EPSIN1/AP-1 and MTV1/AP-4 pairs reside at the TGN, high-resolution microscopy reveals them as spatially separate entities. Our results strongly support the hypothesis of molecularly, functionally, and spatially distinct subdomains of the plant TGN and suggest that functional redundancy can be achieved through parallelization of molecularly distinct but functionally overlapping pathways.

The Cytoskeleton and Its Role in Determining Cellulose Microfibril Angle in Secondary Cell Walls of Woody Tree Species.

Recent advances in our understanding of the molecular control of secondary cell wall (SCW) formation have shed light on molecular mechanisms that underpin domestication traits related to wood formation. One such trait is the cellulose microfibril angle (MFA), an important wood quality determinant that varies along tree developmental phases and in response to gravitational stimulus. The cytoskeleton, mainly composed of microtubules and actin filaments, collectively contribute to plant growth and development by participating in several cellular processes, including cellulose deposition. Studies in Arabidopsis have significantly aided our understanding of the roles of microtubules in xylem cell development during which correct SCW deposition and patterning are essential to provide structural support and allow for water transport. In contrast, studies relating to SCW formation in xylary elements performed in woody trees remain elusive. In combination, the data reviewed here suggest that the cytoskeleton plays important roles in determining the exact sites of cellulose deposition, overall SCW patterning and more specifically, the alignment and orientation of cellulose microfibrils. By relating the reviewed evidence to the process of wood formation, we present a model of microtubule participation in determining MFA in woody trees forming reaction wood (RW).

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization.

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant that the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform separation of early to late Golgi subcompartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in vivo using high-resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance, and Phe bilayer asymmetry that change across the Golgi. Overall, our results suggest that a continuum of transmembrane features, rather than discrete rules, guide proteins to earlier or later locations within the Golgi stack.

The companion of cellulose synthase 1 confers salt tolerance through a Tau-like mechanism in plants.

Microtubules are filamentous structures necessary for cell division, motility and morphology, with dynamics critically regulated by microtubule-associated proteins (MAPs). Here we outline the molecular mechanism by which the MAP, COMPANION OF CELLULOSE SYNTHASE1 (CC1), controls microtubule bundling and dynamics to sustain plant growth under salt stress. CC1 contains an intrinsically disordered N-terminus that links microtubules at evenly distributed points through four conserved hydrophobic regions. By NMR and live cell analyses we reveal that two neighboring residues in the first hydrophobic binding motif are crucial for the microtubule interaction. The microtubule-binding mechanism of CC1 is reminiscent to that of the prominent neuropathology-related protein Tau, indicating evolutionary convergence of MAP functions across animal and plant cells.

A Golgi UDP-GlcNAc transporter delivers substrates for N-linked glycans and sphingolipids.

Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cytosolic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphorylceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids.

Inhibition of TOR Represses Nutrient Consumption, Which Improves Greening after Extended Periods of Etiolation.

Upon illumination, etiolated seedlings experience a transition from heterotrophic to photoautotrophic growth. During this process, the tetrapyrrole biosynthesis pathway provides chlorophyll for photosynthesis. This pathway has to be tightly controlled to prevent the accumulation of photoreactive metabolites and to provide stoichiometric amounts of chlorophyll for its incorporation into photosynthetic protein complexes. Therefore, plants have evolved regulatory mechanisms to synchronize the biosynthesis of chlorophyll and chlorophyll-binding proteins. Two phytochrome-interacting factors (PIF1 and PIF3) and the DELLA proteins, which are controlled by the gibberellin pathway, are key regulators of this process. Here, we show that impairment of TARGET OF RAPAMYCIN (TOR) activity in Arabidopsis (Arabidopsis thaliana), either by mutation of the TOR complex component RAPTOR1B or by treatment with TOR inhibitors, leads to a significantly reduced accumulation of the photoreactive chlorophyll precursor protochlorophyllide in darkness but an increased greening rate of etiolated seedlings after exposure to light. Detailed profiling of metabolic, transcriptomic, and physiological parameters revealed that the TOR-repressed lines not only grow slower, they grow in a nutrient-saving mode, which allows them to resist longer periods of low nutrient availability. Our results also indicated that RAPTOR1B acts upstream of the gibberellin-DELLA pathway and its mutation complements the repressed greening phenotype of pif1 and pif3 after etiolation.