No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Abstract
Plant cytoskeleton consisting of microtubules (MTs) and actin filaments (AFs) plays diverse irreplaceable roles in plant development and response to environmental stimuli. The cytoskeleton forms a highly dynamic and tightly regulated network which offers microenvironments for intra- and inter-cellular communication and physiological events. Plant endomembrane system is composed of membrane-enclosed compartments including the endoplasmic reticulum (ER), the Golgi apparatus, the endosome, the vacuole, etc. These compartments communicate with each other and are essential for cellular activities such as vesicle trafficking, proliferation, immunity and stress response. The ER is the platform for protein synthesis and the initiation site of secretory pathway. Maintenance of plant ER morphology and dynamics mainly depends on actin cytoskeleton. The ER-Cplasma membrane (PM) contact site (EPCS) is a common feature in eukaryotic organisms which is implicated in endomembrane dynamics, cytoskeleton organization and intercellular communication. EPCSs also function as signaling platforms response to environmental stimuli. Cortical cytoskeleton composed of cortical MTs (cMTs) and cortical AFs (cAFs) cooperates with the PM, cell wall and phytohormone to regulate plant cell division, morphogenesis and defense. The interplay between the cellular structures and signals is strictly controlled by diverse factors and signaling pathways. In brief, the endomembrane system cooperates with cytoskeleton network for regulation of plant development and environmental stimuli response and adaption. Here, we summarize the current knowledge to highlight the synergism of plant cytoskeleton network and endomembrane system in regulation of plant development and defense.
ResearchGate has not been able to resolve any citations for this publication.
The Arabidopsis EH proteins (AtEH1/Pan1 and AtEH2/Pan1) are components of the endo-cytic TPLATE complex (TPC) which is essential for endocytosis. Both proteins are homo-logues of the yeast ARP2/3 complex activator, Pan1p. Here, we show that these proteins are also involved in actin cytoskeleton regulated autophagy. Both AtEH/Pan1 proteins localise to the plasma membrane and autophagosomes. Upon induction of autophagy, AtEH/Pan1 proteins recruit TPC and AP-2 subunits, clathrin, actin and ARP2/3 proteins to autophago-somes. Increased expression of AtEH/Pan1 proteins boosts autophagosome formation, suggesting independent and redundant pathways for actin-mediated autophagy in plants. Moreover, AtEHs/Pan1-regulated autophagosomes associate with ER-PM contact sites (EPCS) where AtEH1/Pan1 interacts with VAP27-1. Knock-down expression of either AtEH1/ Pan1 or VAP27-1 makes plants more susceptible to nutrient depleted conditions, indicating that the autophagy pathway is perturbed. In conclusion, we identify the existence of an autophagy-dependent pathway in plants to degrade endocytic components, starting at the EPCS through the interaction among AtEH/Pan1, actin cytoskeleton and the EPCS resident protein VAP27-1.
In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.
Building a complex structure such as the cell wall, with many individual parts that need to be assembled correctly from distinct sources within the cell, is a well-orchestrated process. Additional complexity is required to mediate dynamic responses to environmental and developmental cues. Enzymes, sugars and other cell wall components are constantly and actively transported to and from the plasma membrane during diffuse growth. Cell wall components are transported in vesicles on cytoskeletal tracks composed of microtubules and actin filaments. Many of these components, and additional proteins, vesicles, and lipids are trafficked to and from the cell plate during cytokinesis. In this review, we first discuss how the cytoskeleton is initially organized to add new cell wall material or to build a new cell wall, focusing on similarities during these processes. Next, we discuss how polysaccharides and enzymes that build the cell wall are trafficked to the correct location by motor proteins and through other interactions with the cytoskeleton. Finally, we discuss some of the special features of newly formed cell walls generated during cytokinesis.
Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.
Myosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely-growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to several exocyst subunits in vitro and functional fluorescently-tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the rate of appearance and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes (CSCs) with high spatiotemporal resolution imaging and pair-wise colocalization of myosin XIK, exocyst subunits and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the efficient localization and normal dynamic behavior of exocyst complex at the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides insights about the dynamic regulation of exocytosis in plants.
Significance
Endocytosis controls the proteome and lipidome of the plasma membrane and therefore the communication between the cell and the outside world. Because of its importance, many attempts have been made to develop tools to interfere with this process. These include genetic approaches and small chemicals. Thus far, however, the tools developed to modulate plant endocytosis either lack specificity, are irreversible, or require several days to produce an effect. Here, we generated a conditional and reversible tool to specifically interfere with endocytosis. Short-term heat treatment destabilizes an engineered adaptor complex subunit, which abolishes the function of the endocytic TPLATE complex. This causes accumulation of endocytic cargoes at the plasma membrane and provides a genetic background for future research.
In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.
Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (β-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.
In living cells, dynamics of the endoplasmic reticulum (ER) are driven by the cytoskeleton motor machinery as well as the action of ER-shaping proteins such as atlastin GTPases including RHD3 in Arabidopsis. It is not known if the two systems interplay, and, if so, how they do. Here we report the identification of ARK1 (Armadillo-Repeat Kinesin1) via a genetic screen for enhancers of the rhd3 mutant phenotype. In addition to defects in microtubule dynamics, ER organization is also defective in mutants lacking a functional ARK1. In growing root hair cells, ARK1 comets predominantly localize on the growing-end of microtubules and partially overlap with RHD3 in the cortex of the subapical region. ARK1 co-moves with RHD3 during tip growth of root hair cells. We show that there is a functional interdependence between ARK1 and RHD3. ARK1 physically interacts with RHD3 via its armadillo domain (ARM). In leaf epidermal cells where a polygonal ER network can be resolved, ARK1, but not ARK1ΔARM, moves together with RHD3 to pull an ER tubule toward another and stays with the newly formed 3-way junction of the ER for a while. We conclude that ARK1 acts together with RHD3 to move the ER on microtubules to generate a fine ER network.
The membrane contact sites (MCSs) enable interorganelle communication by associating organelles at distances of tens of nanometers over extended membrane surfaces and serve to maintain cellular homeostasis through efficient exchange of metabolites, lipid, and calcium between organelles, organelle fission, and movement. Most MCSs and a growing number of tethering proteins especially those involved in mediating the junctions between endoplasmic reticulum (ER) and other organelles have been extensively characterized in mammal and yeast. However, the studies of plant MCSs are still at stages of infancy, at least one reason might be due to the lack of bona fide markers for visualizing these membrane junctions in plant cells. In this study, a series of genetically encoded reporters using split super-folder GFP protein were designed to detect the possible MCSs between ER and three other cellular compartments including chloroplast, mitochondria and plasma membrane (PM) in plant cell. By expressing these genetically encoded reporter in Arabidopsis protoplasts as well as Nicotiana benthamiana leaf, we could intuitively observe the punctate signal surrounding chloroplast upon expression of ER-chloroplast MCS reporter, punctate signal of ER-mitochondria MCS reporter and punctate signal close to the PM upon expression of ER-PM MCS reporter. We also showed that the ER-chloroplast MCSs were dynamic structures that undergo active remodeling with concomitant occurrence of chloroplast dysfunction inside plant cells. This study demonstrates that ER associates with various organelles in close proximity in plant cells and provides tools that might be applicable for visualizing MCSs in plants.
Cellulose is an essential plant cell wall component and represents the most abundant biopolymer on Earth. Supramolecular plant cellulose synthase complexes organize multiple linear glucose polymers into
microfibrils as load-bearing wall components. We determined the structure of a poplar cellulose synthase CesA homotrimer that suggests a molecular basis for cellulose microfibril formation. This complex, stabilized by cytosolic plant-conserved regions and helical exchange within the transmembrane segments, forms three channels occupied by nascent cellulose polymers. Secretion steers the polymers toward a common exit point, which could facilitate protofibril formation. CesA’s N-terminal domains assemble into a cytosolic stalk that interacts with a microtubule-tethering protein and may thus be
involved in CesA localization. Our data suggest how cellulose synthase complexes assemble and provide the molecular basis for plant cell wall engineering.
How the membrane trafficking system spatially organizes intracellular activities and intercellular signaling networks is not well understood in plants. The TRAnsport Protein Particle (TRAPP) complexes play key roles in selective delivery of membrane vesicles to various subcellular compartments in yeast and animals, but are not characterized in plants. Here we interrogate TRAPP complexes in Arabidopsis. Affinity purification of a core subunit AtTRS33 followed by quantitative mass spectrometry identified fourteen interacting proteins; including homologues of all thirteen TRAPP components in yeast and mammals and a novel protein we named TRAPP-interacting plant protein (TRIPP), which is conserved in multi-cellular photosynthetic organisms. Proteomic and molecular analyses showed that TRIPP specifically associates with the TRAPPII complex through binary interactions with two TRAPPII-specific subunits. TRIPP co-localizes with a subset of TRS33 compartments and trans-Golgi network markers in a TRS33-dependent manner. Loss-of-function tripp mutation caused dwarfism, sterility, partial photomorphogenesis in the dark, reduced polarity of the auxin transporter PIN2, incomplete cross wall formation and altered localization of a TRAPPII-specific component. Our study demonstrates that plants possess at least two distinct TRAPP complexes similar to metazoans, and identifies TRIPP as a novel plant-specific component of the TRAPPII complex with important functions in trafficking, plant growth and development.
Autophagy is an intracellular trafficking and degradation system for recycling of damaged organelles, mis-folded proteins and cytoplasmic constituents. Autophagy can be divided into non-selective autophagy and selective autophagy according to the cargo specification. Key to the process is the timely formation of the autophagosome, a double-membrane structure which is responsible for the delivery of damaged organelles and proteins to lysosomes or vacuoles for their turnover. Autophagosomes are formed by the closure of cup-shaped phagophore which depends on the proper communication with membrane contributors. The endoplasmic reticulum (ER) is a major membrane source for autophagosome biogenesis whereby the ER connects with phagophore through membrane contact sites (MCSs). MCSs are closely apposed domains between organelle membranes where lipids and signals are exchanged. Lipid transfer proteins (LTPs) are a large family of proteins including Oxysterol-binding protein related proteins (ORP) which can be found at MCSs and mediate lipid transfer in mammals and yeast. In addition, interaction between autophagosomes and other organelles can also be detected in selective autophagy for selection and degradation of various damaged organelles. Selective autophagy is mediated by the binding of a receptor or an adaptor between a cargo and an autophagosome. Here we summarize what we know about the MCS between autophagosomes and other organelles in eukaryotes. We then discuss progress in our understanding about ORPs at MCSs in plants and the underlying mechanisms of selective autophagy in plants with a focus on receptors/adaptors that are involved in the interaction of the autophagosome with other cytoplasmic constituents, including the Neighbor of BRCA1 gene 1 (NBR1), ATG8-interacting protein 1 (ATI1), Regulatory Particle Non-ATPase 10 (RPN10), and Dominant Suppressor of KAR2 (DSK2).
Eukaryotes transport biomolecules between intracellular organelles and between cells and the environment via vesicle trafficking. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE proteins) play pivotal roles in vesicle and membrane trafficking. These proteins are categorized as Qa, Qb, Qc, and R SNAREs and form a complex that induces vesicle fusion for targeting of vesicle cargos. As the core components of the SNARE complex, the SNAP25 Qbc SNAREs perform various functions related to cellular homeostasis. The Arabidopsis thaliana SNAP25 homolog AtSNAP33 interacts with Qa and R SNAREs and plays a key role in cytokinesis and in triggering innate immune responses. However, other Arabidopsis SNAP25 homologs, such as AtSNAP29 and AtSNAP30, are not well studied; this includes their localization, interactions, structures, and functions. Here, we discuss three biological functions of plant SNAP25 orthologs in the context of AtSNAP33 and highlight recent findings on SNAP25 orthologs in various plants. We propose future directions for determining the roles of the less well-characterized AtSNAP29 and AtSNAP30 proteins.
In plant cells, environmental stressors promote changes in connectivity between the cortical ER and the PM. Although this process is tightly regulated in space and time, the molecular signals and structural components mediating these changes in inter-organelle communication are only starting to be characterized. In this report, we confirm the presence of a putative tethering complex containing the synaptotagmins 1 and 5 (SYT1 and SYT5) and the Ca2+ and lipid binding protein 1 (CLB1/SYT7). This complex is enriched at ER-PM contact sites (EPCS), have slow responses to changes in extracellular Ca2+, and display severe cytoskeleton-dependent rearrangements in response to the trivalent lanthanum (La3+) and gadolinium (Gd3+) rare earth elements (REEs). Although REEs are generally used as non-selective cation channel blockers at the PM, here we show that the slow internalization of REEs into the cytosol underlies the activation of the Ca2+/Calmodulin intracellular signaling, the accumulation of phosphatidylinositol-4-phosphate (PI4P) at the PM, and the cytoskeleton-dependent rearrangement of the SYT1/SYT5 EPCS complexes. We propose that the observed EPCS rearrangements act as a slow adaptive response to sustained stress conditions, and that this process involves the accumulation of stress-specific phosphoinositides species at the PM.
The regulated assembly of multiple filamentous actin (F-actin) networks from an actin monomer pool is important for a variety of cellular processes. Chlamydomonas reinhardtii is a unicellular green alga expressing a conventional and divergent actin that is an emerging system for investigating the complex regulation of actin polymerization. One actin network that contains exclusively conventional F-actin in Chlamydomonas is the fertilization tubule, a mating structure at the apical cell surface in gametes. In addition to two actin genes, Chlamydomonas expresses a profilin (PRF1) and four formin genes (FOR1-4), one of which (FOR1) we have characterized for the first time. We found that unlike typical profilins, PRF1 prevents unwanted actin assembly by strongly inhibiting both F-actin nucleation and barbed end elongation at equimolar concentrations to actin. However, FOR1 stimulates the assembly of rapidly elongating actin filaments from PRF1-bound actin. Furthermore, for1 and prf1-1 mutants, as well as the small molecule formin inhibitor SMIFH2, prevent fertilization tubule formation in gametes, suggesting that polymerization of F-actin for fertilization tubule formation is a primary function of FOR1. Together, these findings indicate that FOR1 and PRF1 cooperate to selectively and rapidly assemble F-actin at the right time and place.
[Media: see text]
Plant trichomes originate from epidermal cell, forming protective structure from abiotic and biotic stresses. Different from the unicellular trichome in Arabidopsis, tomato trichomes are multicellular structure and can be classified into seven different types based on cell number, shape and the presence of glandular cells. Despite the importance of tomato trichomes in insect resistance, our understanding of the tomato trichome morphogenesis remains elusive. In this study, we quantitatively analyzed morphological traits of trichomes in tomato and further performed live imaging of cytoskeletons in stably transformed lines with actin and microtubule markers. At different developmental stages, two types of cytoskeletons exhibited distinct patterns in different trichome cells, ranging from transverse, spiral to longitudinal. This gradual transition of actin filament angle from basal to top cells could correlate with the spatial expansion mode in different cells. Further genetic screen for aberrant trichome morphology led to the discovery of a number of independent mutations in SCAR/WAVE and ARP2/3 complex, which resulted in actin bundling and distorted trichomes. Disruption of microtubules caused isotropic expansion while abolished actin filaments entirely inhibited axial extension of trichomes, indicating that microtubules and actin filaments may control distinct aspects of trichome cell expansion. Our results shed light on the roles of cytoskeletons in the formation of multicellular structure of tomato trichomes.
Plasma membranes are heterogeneous and laterally compartmentalized into distinct microdomains. These membrane microdomains consist of special lipids and proteins and are thought to act as signaling platforms. In plants, membrane microdomains have been detected by super-resolution microscopy, and there is evidence that they play roles in several biological processes. Here, we review current knowledge about the lipid and protein components of membrane microdomains. Furthermore, we summarize the dynamics of membrane microdomains in response to different stimuli. We also explore the biological functions associated with membrane microdomains as signal integration hubs. Finally, we outline challenges and questions for further studies.
Light and gravity are two key determinants in orientating plant stems for proper growth and development. The organization and dynamics of the actin cytoskeleton are essential for cell biology and critically regulated by actin-binding proteins. However, the role of actin cytoskeleton in shoot negative gravitropism remains controversial. In this work, we report that the actin-binding protein Rice Morphology Determinant (RMD) promotes reorganization of the actin cytoskeleton in rice (Oryza sativa) shoots. The changes in actin organization are associated with the ability of the rice shoots to respond to negative gravitropism. Here, light-grown rmd mutant shoots exhibited agravitropic phenotypes. By contrast, etiolated rmd shoots displayed normal negative shoot gravitropism. Furthermore, we show that RMD maintains an actin configuration that promotes statolith mobility in gravisensing endodermal cells, and for proper auxin distribution in light-grown, but not dark-grown, shoots. RMD gene expression is diurnally controlled and directly repressed by the phytochrome-interacting factor-like protein OsPIL16. Consequently, overexpression of OsPIL16 led to gravisensing and actin patterning defects that phenocopied the rmd mutant. Our findings outline a mechanism that links light signaling and gravity perception for straight shoot growth in rice.
Auxin transport inhibitors are essential tools for understanding auxin-dependent plant development. One mode of inhibition affects actin dynamics; however, the underlying mechanisms remain unclear. In this study, we characterized the action of 2,3,5-triiodobenzoic acid (TIBA) on actin dynamics in greater mechanistic detail. By surveying mutants for candidate actin-binding proteins with reduced TIBA sensitivity, we determined that Arabidopsis (Arabidopsis thaliana) villins contribute to TIBA action. By directly interacting with the C-terminal headpiece domain of villins, TIBA causes villin to oligomerize, driving excessive bundling of actin filaments. The resulting changes in actin dynamics impair auxin transport by disrupting the trafficking of PIN auxin efflux carriers and reducing their levels at the plasma membrane (PM). Collectively, our study provides mechanistic insight into the link between the actin cytoskeleton, vesicle trafficking, and auxin transport.
Microtubules are involved in plant development and adaptation to their environment, but the sustaining molecular mechanisms remain elusive. Microtubule-end-binding 1 (EB1) proteins participate in directional root growth in Arabidopsis thaliana. However, a connection to the underlying microtubule array has not been established yet. We show here that EB1 proteins contribute to the organization of cortical microtubules in growing epidermal plant cells, without significant modulation of microtubule dynamics. Using super-resolution stimulated emission depletion (STED) microscopy and an original quantification approach, we also demonstrate a significant reduction of apparent microtubule bundling in cytoplasmic-EB1-deficient plants, suggesting a function for EB1 in the interaction between adjacent microtubules. Furthermore, we observed root growth defects in EB1-deficient plants, which are not related to cell division impairment. Altogether, our results support a role for EB1 proteins in root development, in part by maintaining the organization of cortical microtubules. This article has an associated First Person interview with the first author of the paper.
Background
During sexual reproduction, pollen grains land on the stigma, rehydrate and produce pollen tubes that grow through the female transmitting-tract tissue allowing the delivery of the two sperm cells to the ovule and the production of healthy seeds. Because pollen tubes are single cells that expand by tip-polarized growth, they represent a good model to study the growth dynamics, cell wall deposition and intracellular machineries. Aiming to understand this complex machinery, we used a low throughput chemical screen approach in order to isolate new tip-growth disruptors. The effect of a chemical inhibitor of monogalactosyldiacylglycerol synthases, galvestine-1, was also investigated. The present work further characterizes their effects on the tip-growth and intracellular dynamics of pollen tubes.
Results
Two small compounds among 258 were isolated based on their abilities to perturb pollen tube growth. They were found to disrupt in vitro pollen tube growth of tobacco, tomato and Arabidopsis thaliana. We show that these 3 compounds induced abnormal phenotypes (bulging and/or enlarged pollen tubes) and reduced pollen tube length in a dose dependent manner. Pollen germination was significantly reduced after treatment with the two compounds isolated from the screen. They also affected cell wall material deposition in pollen tubes. The compounds decreased anion superoxide accumulation, disorganized actin filaments and RIC4 dynamics suggesting that they may affect vesicular trafficking at the pollen tube tip.
Conclusion
These molecules may alter directly or indirectly ROP1 activity, a key regulator of pollen tube growth and vesicular trafficking and therefore represent good tools to further study cellular dynamics during polarized-cell growth.
Electronic supplementary material
The online version of this article (10.1186/s12870-019-1743-9) contains supplementary material, which is available to authorized users.
Cellular elongation requires the defined coordination of intra- and extracellular processes, but the underlying mechanisms are largely unknown. The vacuole is the biggest plant organelle, and its dimensions play a role in defining plant cell expansion rates. Here, we show that the increase in vacuolar occupancy enables cellular elongation with relatively little enlargement of the cytosol in Arabidopsis thaliana. We demonstrate that cell wall properties are sensed and impact on the intracellular expansion of the vacuole. Using vacuolar morphology as a quantitative read-out for intracellular growth processes, we reveal that the underlying cell wall sensing mechanism requires interaction of extracellular leucine-rich repeat extensins (LRXs) with the receptor-like kinase FERONIA (FER). Our data suggest that LRXs link plasma membrane-localised FER with the cell wall, allowing this module to jointly sense and convey extracellular signals to the cell. This mechanism coordinates the onset of cell wall acidification and loosening with the increase in vacuolar size. © 2019 The Authors. Published under the terms of the CC BY 4.0 license
Gravitropism is an adaptive response that orients plant growth parallel to the gravity vector. Asymmetric distribution of the phytohormone auxin is a necessary prerequisite to the tropic bending both in roots and shoots. During hypocotyl gravitropic response, the PIN3 auxin transporter polarizes within gravity sensing cells to redirect intercellular auxin fluxes. First gravity‐induced PIN3 polarization to the bottom cell membranes leads to the auxin accumulation at the lower side of the organ, initiating bending and, later, auxin feedback‐mediated repolarization restores symmetric auxin distribution to terminate bending. Here we performed a forward genetic screen to identify regulators of both PIN3 polarization events during gravitropic response. We searched for mutants with defective PIN3 polarizations based on easy‐to‐score morphological outputs of decreased or increased gravity‐induced hypocotyl bending. We identified number of hypocotyl reduced bending (hrb) and hypocotyl hyperbending (hhb) mutants revealing that reduced bending correlated typically with defective gravity‐induced PIN3 relocation whereas all analysed hhb mutants showed defects in the second, auxin‐mediated PIN3 relocation. Next generation sequencing‐aided mutation mapping identified several candidate genes including SCARECROW and ACTIN2 revealing roles of endodermis specification and actin cytoskeleton in the respective gravity‐ and auxin‐induced PIN polarization events. The hypocotyl gravitropism screen thus promises to provide novel insights into mechanisms underlying cell polarity and plant adaptive development.
This article is protected by copyright. All rights reserved.
Defining convergent and divergent mechanisms underlying the biogenesis and function of endomembrane organelles is fundamentally important in cell biology. In all eukaryotes, the Trans-Golgi Network (TGN) is the hub where the exocytic and endocytic pathways converge. To gain knowledge in the mechanisms underlying TGN biogenesis and function, we characterized TGNap1, a protein encoded by a plant gene of unknown function conserved with metazoans. We demonstrate that TGNap1 is a TGN protein required for the homeostasis of biosynthetic and endocytic traffic pathways. We also show that TGNap1 binds Rab6, YIP4 and microtubules. Finally, we establish that TGNap1 contributes to microtubule-dependent biogenesis, tracking and function of a TGN subset, likely through interaction with Rab6 and YIP4. Our results identify an important trafficking determinant at the plant TGN and reveal an unexpected reliance of post-Golgi traffic homeostasis and organelle biogenesis on microtubules in plants.
A population of dynamic apical actin filaments is required for rapid polarized pollen tube growth. However, the cellular mechanisms driving their assembly remain incompletely understood. It was postulated that formin is a major player in nucleating apical actin assembly, but direct genetic and cytological evidence remains to be firmly established. Here we found that both Arabidopsis formin 3 (AtFH3) and formin 5 (AtFH5) are involved in the regulation of apical actin polymerization and actin array construction in pollen tubes, with AtFH3 playing a more dominant role. We found that both formins have plasma membrane (PM) localization signals but exhibit distinct PM localization patterns in the pollen tube, and loss of their function reduces the amount of apical actin filaments. Live-cell imaging revealed that the reduction in filamentous actin is very likely due to the decrease in filament elongation. Furthermore, we found that the rate of tip-directed vesicle transport is reduced and the pattern of apical vesicle accumulation is altered in formin loss-of-function mutant pollen tubes, which explains to some extent the reduction in pollen tube elongation. Thus, we provide direct genetic and cytological evidence showing that formin is an important player in nucleating actin assembly from the PM at pollen tube tips.
Central to the building and reorganizing cytoskeletal arrays is creation of new polymers. Although nucleation has been the major focus of study for microtubule generation, severing has been proposed as an alternative mechanism to create new polymers, a mechanism recently shown to drive the reorientation of cortical arrays of higher plants in response to blue light perception. Severing produces new plus ends behind the stabilizing GTP-cap. An important and unanswered question is how these ends are stabilized in vivo to promote net microtubule generation. Here we identify the conserved protein CLASP as a potent stabilizer of new plus ends created by katanin severing in plant cells. Clasp mutants are defective in cortical array reorientation. In these mutants, both rescue of shrinking plus ends and the stabilization of plus ends immediately after severing are reduced. Computational modeling reveals that it is the specific stabilization of severed ends that best explains CLASP’s function in promoting microtubule amplification by severing and array reorientation.
The plant immune system comprises a complex network of signaling processes, regulated not only by classically defined immune components (e.g., resistance genes) but also by a suite of developmental, environmental, abiotic, and biotic-associated factors. In total, it is the sum of these interactions-the connectivity to a seemingly endless array of environments-that ensures proper activation, and control, of a system that is responsible for cell surveillance and response to threats presented by invading pests and pathogens. Over the past decade, the field of plant pathology has witnessed the discovery of numerous points of convergence between immunity, growth, and development, as well as overlap with seemingly disparate processes such as those that underpin plant response to changes in the environment. Toward defining how immune signaling is regulated, recent studies have focused on dissecting the mechanisms that underpin receptor-ligand interactions, phospho-regulation of signaling cascades, and the modulation of host gene expression during infection. As one of the major regulators of these immune signaling cascades, the plant cytoskeleton is the stage from which immune-associated processes are mobilized and oriented and, in this role, it controls the movement of the organelles, proteins, and chemical signals that support plant defense signaling. In short, the cytoskeleton is the battlefield from which pathogens and plants volley virulence and resistance, transforming resistance to susceptibility. Herein, we discuss the role of the eukaryotic cytoskeleton as a platform for the function of the plant immune system.
During plant reproduction, sperm cells are delivered to ovules through growing pollen tubes. This process involves tip-localized receptor kinases regulating integrity and/or guidance of pollen tubes, whose localizations must be strictly regulated. However, the molecular basis for tip-localization of these molecules remains largely elusive. Here we show that a pair of AP180 N-terminal homology domain-containing proteins, PICALM5a and PICALM5b, is responsible for the tip-localization of ANXUR receptor kinases acting in an autocrine signaling pathway required for pollen tube integrity in Arabidopsis thaliana. The picalm5a picalm5b double mutant exhibits reduced fertility, and the double mutant pollen is defective in pollen tube integrity with premature bursts. The tip localization of ANXUR proteins is severely impaired in picalm5a picalm5b pollen tubes, whereas another receptor kinase PRK6 acting in pollen tube guidance is not affected. Based on these results, we propose that PICALM5 proteins serve as specific loading adaptors to recycle ANXUR proteins.
Cell wall appositions (CWAs) are produced reactively by the plant immune system to arrest microbial invasion through the local inversion of plant cell growth. This process requires the controlled invagination of the plasma membrane (PM) in coordination with the export of barrier material to the volume between the plant PM and cell wall. Plant actin dynamics are essential to this response, but it remains unclear how exocytosis and the cytoskeleton are linked in space and time to form functional CWAs. Here, we show that actin-dependent trafficking to immune response sites of Arabidopsis thaliana delivers membrane-integrated FORMIN4, which in turn contributes to local cytoskeletal dynamics. Total internal reflection fluorescence (TIRF) microscopy combined with controlled induction of FORMIN4-GFP expression reveals a dynamic population of vesicular bodies that accumulate to form clusters at the PM through an actin-dependent process. Deactivation of FORMIN4 and its close homologs partially compromises subsequent defense and alters filamentous actin (F-actin) distribution at mature CWAs. The localization of FORMIN4 is stable and segregated from the dynamic traffic of the endosomal network. Moreover, the tessellation of FORMIN4 at the PM with meso-domains of PEN3 reveals a fine spatial segregation of destinations for actin-dependent immunity cargo. Together, our data suggest a model where FORMIN4 is a spatial feedback element in a multi-layered, temporally defined sequence of cytoskeletal response. This positional feedback makes a significant contribution to the distribution of actin filaments at the dynamic CWA boundary and to the outcomes of pre-invasion defense.
Although cytoskeleton is a driving force for cell division and growth in higher plants, there is little evidence about its components in parasitic angiosperms. Microtubules and actin filaments in cells of shoot apical meristem and root-like structure of stem holoparasites European (C. europaea L.) and Eastern (C. monogyna Vahl.) dodders, as well as in prehaustorium, the specific organ adapted to parasitism, were visualized for the first time by immunolabeling and fluorescence microscopy. The significance of cytoskeletal elements during germination and prehaustorium formation was addressed by treatments with taxol, oryzalin, latrunculin B, cytochalasin B/D, jasplakinolide, and 2,3-butanedione monoxime. In shoot apical meristem many dividing cells were observed, in contrast to root-like structure, devoid of cell divisions. Cortical microtubules were oriented transversely and/or obliquely, while actin filaments were randomly distributed in cells of both organs. Furthermore, longitudinal cortical microtubules were present in digitate cells of prehaustorium, and transverse arrays were found in its file cells. Long and short random actin filaments were also observed in prehaustorium cells. Thus, it was shown that the cytoskeleton in dodder shoot cells is organized in a similar way to non-parasitic dicots, while cytoskeletal organization has some peculiarities in quickly senescing root-like structure and prehaustorium.
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 Gβγ 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 Gβγ 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.
Autophagy, a highly conserved quality control mechanism, is essential for maintaining cellular homeostasis and healthy growth of plants. Compared with extensive research in the cytoplasmic control of autophagy, studies regarding the nuclear events involved in the regulation of plant autophagy are just beginning to emerge. Accumulating evidence reveals a coordinated expression of plant autophagy genes in response to diverse developmental states and growth conditions. Here, we summarize recent progress in the identification of tightly controlled transcription factors and histone marks associated with the autophagic process in plants, and propose several modules, consisting of transcription regulators and epigenetic modifiers, as important nuclear players that could contribute to both short-term and long-term controls of plant autophagy at the transcriptional and post-transcriptional levels.
At the subcellular level, the cytoskeleton regulates cell structure, organelle movement, and cytoplasmic streaming. Autophagy is a process to remove unwanted biomaterials or damaged organelles through double membrane compartments known as autophagosomes. Autophagosome biogenesis requires vesicle trafficking between donor and acceptor compartments, membrane expansion, and fusion, which is very likely to be regulated by the cytoskeleton. Recent studies have demonstrated that by knocking out key actin-regulating proteins, autophagosome biogenesis is inhibited. However, the formation of ATG8 positive structures are not affected when the entire actin network is disrupted. Here, we discuss this paradox and propose the function of the actin cytoskeleton in plant autophagy.
Pollen tubes rapidly elongate, penetrate and navigate through multiple female tissues to reach ovules for sperm delivery by utilizing a specialized form of polar growth known as tip growth. This process requires a battery of cellular activities differentially occurring at the apical growing region of the plasma membrane (PM), such as the differential cellular signaling involving calcium (Ca2+), phospholipids, and ROP-type Rho GTPases, fluctuation of ions and pH, exocytosis and endocytosis, and cell wall construction and remodeling. There is an emerging understanding of how at least some of these activities are coordinated and/or interconnected. The apical active ROP modulates exocytosis to the cell apex for PM and cell wall expansion differentially occurring at the tip. The differentiation of the cell wall involves at least the preferential distribution of deformable pectin polymers to the apex and of non-deformable pectin polymers to the shank of pollen tubes, facilitating the apical cell expansion driven by high internal turgor pressure. Recent studies have generated inroads into how the ROP GTPase-based intracellular signaling spatiotemporally is coordinated with the external wall mechanics to maintain the tubular cell shape and how the apical cell wall mechanics is regulated to allow rapid tip growth while maintaining the cell wall integrity under the turgor pressure. Evidence suggests that exocytosis and endocytosis play crucial but distinct roles in this spatiotemporal coordination. In this review, we summarize some recent advances in the regulation and coordination of the differential pectin distribution and the apical domain of active ROP by exocytosis and endocytosis in pollen tubes.
A complex and dynamic endomembrane system is a hallmark of eukaryotic cells and underpins the evolution of specialised cell types in multicellular organisms. Endomembrane system function critically depends on the ability of the cell to (1) define compartment and pathway identity, and (2) organise compartments and pathways dynamically in space and time. Eukaryotes possess a complex molecular machinery to control these processes, including small GTPases and their regulators, SNAREs, tethering factors, motor proteins, and cytoskeletal elements. Whereas many of the core components of the eukaryotic endomembrane system are broadly conserved, there have been substantial diversifications within different lineages, possibly reflecting lineage-specific requirements of endomembrane trafficking. This Review focusses on the spatio-temporal regulation of post-Golgi exocytic transport in plants. It highlights recent advances in our understanding of the elaborate network of pathways transporting different cargoes to different domains of the cell surface, and the molecular machinery underpinning them (with a focus on Rab GTPases, their interactors and the cytoskeleton). We primarily focus on transport in the context of growth, but also highlight how these pathways are co-opted during plant immunity responses and at the plant-pathogen interface.
Plant growth and development relies on the accurate positioning of the cell plate between dividing cells during cytokinesis. The cell plate is synthetized by a specialized structure called the phragmoplast, which contains bipolar microtubules that polymerize to form a framework with the plus ends at or near the division site. This allows the transport of Golgi-derived vesicles toward the plus ends to form and expand the cell plate. Actin filaments play important roles in cell plate expansion and guidance in plant cytokinesis at the late phase, but whether they are involved at the early phase is unknown. To investigate this further, we disrupted the actin filaments in cell cycle-synchronized tobacco BY-2 cells with latrunculin B (LatB), an actin polymerization inhibitor. We observed the cells under a transmission electron microscope or a spinning-disk confocal laser scanning microscope. We found that disruption of actin filaments by LatB caused the membrane vesicles at the equatorial plane of the cell plate to be dispersed rather than form clusters as they did in the untreated cells. The midzone constriction of phragmoplast microtubules also was perturbed in LatB-treated cells. The live cell imaging and kymograph analysis showed that disruption of actin filaments also changed the accumulation timing of NACK1 kinesin, which plays a crucial role in cell plate expansion. This suggests that there are two functionally different types of microtubules in the phragmoplast. Together, our results show that actin filaments regulate phragmoplast microtubules at the initial phase of plant cytokinesis.
Current models of plasma membrane (PM) postulate its organization in various nano‐ and micro‐domains with distinct protein and lipid composition. While metazoan PM nanodomains usually display high lateral mobility, the dynamics of plant nanodomains is often highly spatially restricted. Here we have focused on the determination of the PM distribution in nanodomains for Arabidopsis thaliana flotillin (AtFLOT) and hypersensitive induced reaction proteins (AtHIR), previously shown to be involved in response to extracellular stimuli. Using in vivo laser scanning and spinning disk (SD) confocal microscopy in Arabidopsis thaliana we present here their nanodomain localization in various epidermal cell types. Fluorescence recovery after photobleaching (FRAP) and kymographic analysis revealed that PM‐associated AtFLOTs contain significantly higher immobile fraction than AtHIRs. In addition, much lower immobile fractions have been found in tonoplast pool of AtHIR3. Although members of both groups of proteins were spatially restricted in their PM distribution by corrals co‐aligning with microtubules (MTs), pharmacological treatments showed no or very low role of actin and microtubular cytoskeleton for clustering of AtFLOT and AtHIR into nanodomains. Finally, pharmacological alteration of cell wall (CW) synthesis and structure resulted in changes in lateral mobility of AtFLOT2 and AtHIR1. Accordingly, partial enzymatic CW removal increased the overall dynamics as well as individual nanodomain mobility of these two proteins. Such structural links to CW could play an important role in their correct positioning during PM communication with extracellular environment.
The plant endomembrane system comprises distinctive membrane-bounded organelles connected with one another by the membrane trafficking system. The RAB GTPase is a key component of the membrane trafficking machinery that regulates the targeting and tethering of trafficking vesicles to target compartments by acting as a molecular switch cycling between active and inactive states. The functions of RAB GTPases are fulfilled through their interactions with several classes of interacting factors, including guanine nucleotide exchange factors (GEFs) and effector proteins. Effector proteins for plant RAB GTPases consist of evolutionarily conserved and plant-unique factors, which are involved in various membrane trafficking events in plant cells in ways unique to plants. In this review, we summarize recent findings on the functions of endosomal RAB GTPases that underwent unique diversification during plant evolution, with a special focus on RAB5/RABF and RAB11/RABA.
The Transport Protein Particle II (TRAPPII) is essential for exocytosis, endocytosis, protein sorting and cytokinesis. In spite of a considerable understanding of its biological role, little is known about Arabidopsis TRAPPII complex topology and molecular function. In this study, independent proteomic approaches initiated with TRAPP components or Rab‐A GTPase variants converge on the TRAPPII complex. We show that the Arabidopsis genome encodes the full complement of 13 TRAPPC subunits, including four previously unidentified components. A dimerization model is proposed to account for binary interactions between TRAPPII subunits. Preferential binding to dominant negative (GDP‐bound) versus wild‐type or constitutively active (GTP‐bound) RAB‐A2a variants discriminates between TRAPPII and TRAPPIII subunits and shows that Arabidopsis complexes differ from yeast but resemble metazoan TRAPP complexes. Analyses of Rab‐A mutant variants in trappii backgrounds provide genetic evidence that TRAPPII functions upstream of RAB‐A2a, allowing us to propose that TRAPPII likely behaves as a Guanine‐nucleotide Exchange Factor (GEF) for the RAB‐A2a GTPase. GEFs catalyze exchange of GDP for GTP; the GTP‐bound, activated, Rab then recruits a diverse local network of Rab effectors to specify membrane identity in subsequent vesicle fusion events. Understanding GEF‐Rab interactions will be crucial to unraveling the co‐ordination of plant membrane traffic. This article is protected by copyright. All rights reserved.
Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as gravitropism. Previously, we have shown that NEGATIVE GRAVITROPIC RESPONSE OF ROOTS (NGR) is essential for the positive gravitropic response of roots. Here we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport (PAT) in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation are reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.
Polarized cell growth in plants is maintained under the strict control and exquisitely choregraphed balance of exocytic and endocytic membrane trafficking. The pollen tube has become a model system for rapid polar growth in which delivery of cell wall material and membrane recycling are controlled by membrane trafficking. Endocytosis plays an important role that is poorly understood. The plant AP180 epsin-N-Homolog (ANTH) proteins are putative homologs of Epsin 1 that recruits clathrin to phosphatidylinositol 4,5-bisphosphate (PIP2) containing membranes to facilitate vesicle budding during endocytosis. Two Arabidopsis ANTH encoded by the genes AtAP180 and AtECA2 are highly expressed in pollen tubes. Pollen tubes from T-DNA inserted knockout mutant lines display significant morphological defects and unique pectin deposition. Fluorescently tagging reveals organization into dynamic foci located at the lateral flanks of the pollen tube. This precisely defined sub-apical domain coincides which clathrin mediated endocytosis and PIP2 localization. Using a liposome-protein binding test, we showed that AtECA2 protein and ANTH domain recombinant proteins have strong affinity to PIP2 and phosphatidic acid (PA) containing liposomes in vitro. Taken together these data suggest that Arabidopsis ANTH proteins may play an important role in clathrin mediated endocytosis, proper cell wall assembly and morphogenesis.
Significance
Interorganelle connectivity and nonvesicular information transfer are hallmarks of biological systems. These processes facilitate communication between organelles, allowing them to adapt to changing cellular environments. In plants, the endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (EPCSs) physically connect the cortical ER and the PM, and act as general platforms for Ca ²⁺ homeostasis regulation and the cellular adaptation to environmental stresses. Our identification of ionic stress and PM phosphoinositides as enhancers of ER–PM connectivity advances our understanding of how stress influences interorganelle communication. Furthermore, our analyses of the spatiotemporal regulation of EPCS expansion highlights unique mechanisms that plants activate to maintain interorganelle communication during long-term exposure to environmental stress, not described in other eukaryotes.
In eukaryotic cells, organelles and the cytoskeleton undergo highly dynamic yet organized interactions capable of orchestrating complex cellular functions. Visualizing these interactions requires noninvasive, long-duration imaging of the intracellular environment at high spatiotemporal resolution and low background. To achieve these normally opposing goals, we developed grazing incidence structured illumination microscopy (GI-SIM) that is capable of imaging dynamic events near the basal cell cortex at 97-nm resolution and 266 frames/s over thousands of time points. We employed multi-color GI-SIM to characterize the fast dynamic interactions of diverse organelles and the cytoskeleton, shedding new light on the complex behaviors of these structures. Precise measurements of microtubule growth or shrinkage events helped distinguish among models of microtubule dynamic instability. Analysis of endoplasmic reticulum (ER) interactions with other organelles or microtubules uncovered new ER remodeling mechanisms, such as hitchhiking of the ER on motile organelles. Finally, ER-mitochondria contact sites were found to promote both mitochondrial fission and fusion. A new approach for visualizing dynamic processes within cells offers insight into membrane-membrane contact interactions and microtubule function.
Cell walls play critical roles in plants, regulating tissue mechanics, defining the extent and orientation of cell expansion, and providing a physical barrier against pathogen attack [1]. Cellulose microfibrils, which are synthesized by plasma membrane-localized cellulose synthase (CESA) complexes, are the primary load-bearing elements of plant cell walls [2]. Cell walls are dynamic structures that are regulated in part by cell wall integrity (CWI)-monitoring systems that feed back to modulate wall properties and the synthesis of new wall components [3]. Several receptor-like kinases have been implicated as sensors of CWI [3-5], including the FEI1/FEI2 receptor-like kinases [4]. Here, we characterize two genes encoding novel plant-specific plasma membrane proteins (SHOU4 and SHOU4L) that were identified in a suppressor screen of the cellulose-deficient fei1 fei2 mutant. shou4 shou4l double mutants display phenotypes consistent with elevated levels of cellulose, and elevated levels of non-crystalline cellulose are present in this mutant. Disruption of SHOU4 and SHOU4L increases the abundance of CESA proteins at the plasma membrane as a result of enhanced exocytosis. The SHOU4/4L N-terminal cytosolic domains directly interact with CESAs. Our results suggest that the SHOU4 proteins regulate cellulose synthesis in plants by influencing the trafficking of CESA complexes to the cell surface.
It's all about your contacts
Membrane contact sites have recently come to the fore of our understanding of interorganelle communication. Wu et al. review how these important structures help to promote a variety of key functions, including organelle division and lipid transfer. Focusing on contacts between the endoplasmic reticulum and a variety of organelles or the plasma membrane reveals the generality and importance of these contacts in cellular homeostasis and organismal health.
Science , this issue p. eaan5835
Microtubules act as "railways" for motor-driven intracellular transport, interact with accessory proteins to assemble into larger structures such as the mitotic spindle, and provide an organizational framework to the rest of the cell. Key to these functions is the fact that microtubules are "dynamic." As with actin, the polymer dynamics are driven by nucleotide hydrolysis and influenced by a host of specialized regulatory proteins, including microtubule-associated proteins. However, microtubule turnover involves a surprising behavior-termed dynamic instability-in which individual polymers switch stochastically between growth and depolymerization. Dynamic instability allows microtubules to explore intracellular space and remodel in response to intracellular and extracellular cues. Here, we review how such instability is central to the assembly of many microtubule-based structures and to the robust functioning of the microtubule cytoskeleton.
Profilin functions with formin in actin assembly, a process that regulates multiple aspects of plant development and immune responses. High-level eukaryotes contain multiple isoforms of profilin, formin, and actin, whose partner-specific interactions in actin assembly are not completely understood in plant development and defense responses. To examine the functionally distinct interactions between profilin and formin, we studied all five Arabidopsis profilins and their interactions with formin by using both in vitro biochemical and in vivo cell biology approaches. Unexpectedly, we found a previously undescribed negative regulatory function of AtPRF3 in AtFH1-mediated actin polymerization. The N-terminal 37 residues of AtPRF3 were identified to play a predominant role in inhibiting formin-mediated actin nucleation via their high affinity for the formin polyproline region and their triggering of the oligomerization of AtPRF3. Both in vivo and in vitro mechanistic studies of AtPRF3 revealed a universal mechanism in which the weak interaction between profilin and formin positively regulates actin assembly by ensuring rapid recycling of profilin, whereas profilin oligomerization negatively regulates actin polymerization. Upon recognition of the pathogen-associated molecular pattern, the gene transcription and protein degradation of AtPRF3 are modulated for actin assembly during plant innate immunity. The prf3 Arabidopsis plants show higher sensitivity to the bacterial flagellum peptide in both the plant growth and ROS responses. These findings demonstrate a profilin-mediated actin assembly mechanism underlying the plant immune responses. Sun et al. demonstrate that AtPRF3 is the unconventional profilin isoform with an N-terminal extension, which causes the protein oligomerization and inhibits the formin-mediated actin assembly in Arabidopsis. Moreover, AtPRF3 regulates the PAMP-triggered immune responses that also modulate the AtPRF3 degradation.