Chemical inhibition of Arabidopsis PIN-FORMED auxin transporters by the anti-inflammatory drug naproxen

The phytohormone auxin plays central roles in many growth and developmental processes in plants.Development of chemical tools targeting the auxin pathway is useful for both plant biology and agriculture.Here we reveal that naproxen,a synthetic compound with anti-inflammatory activity in humans,acts as an auxin transport inhibitor targeting PIN-FORMED(PIN)transporters in plants.Physiological experiments indicate that exogenous naproxen treatment affects pleiotropic auxin-regulated developmental processes.Additional cellular and biochemical evidence indicates that naproxen suppresses auxin transport,specifically PIN-mediated auxin efflux.Moreover,biochemical and structural analyses confirm that naproxen binds directly to PIN1 protein via the same binding cavity as the indole-3-acetic acid substrate.Thus,by combining cellular,biochemical,and structural approaches,this study clearly establishes that naproxen is a PIN inhibitor and elucidates the underlying mechanisms.Further use of this compound may advance our understanding of the molecular mechanisms of PIN-mediated auxin transport and expand our toolkit in auxin biology and agriculture.

Distinct functions of TiR1 and AFB1 receptors in auxin signaling

Dear Editor,Auxin is the major plant hormone regulating growth and development(Friml,2022).Forward genetic approaches have identified major components of auxin signaling and established the canonical mechanism mediating transcriptional and thus developmental reprogramming in Arabidopsis thaliana.In this textbook view,TRANSPORT INHIBITOR RESPONSE 1(TIR1)/AUXIN-SIGNALING F-BOX(AFB)proteins are auxin receptors,which act as F-box subunits determining the substrate specificity of the Skp1-Cullin1-F box protein(SCF)type E3 ubiquitin ligase complex.Auxin acts as a"molecular glue,"increasing the affinity between TIR1/AFBs and the Auxin/lndole-3-Acetic Acid(Aux/IAA)repressors.Subsequently,Aux/IAAs are ubiquitinated and degraded,thus releasing auxin transcription factors from their repression and making them free to mediate transcription of auxin response genes(Yu et al.,2022).

Pho-view of Auxin:Reversible Protein Phosphorylation in Auxin Biosynthesis,Transport and Signaling

The phytohormone auxin plays a central role in shaping plant growth and development.With decades of genetic and biochemical studies,numerous core molecular components and their networks,underlying auxin biosynthesis,transport,and signaling,have been identified.Notably,protein phosphorylation,catalyzed by kinases and oppositely hydrolyzed by phosphatases,has been emerging to be a crucial type of post-translational modification,regulating physiological and developmental auxin output at all levels.In this review,we comprehensively discuss earlier and recent advances in our understanding of genetics,biochemistry,and cell biology of the kinases and phosphatases participating in auxin action.We provide insights into the mechanisms by which reversible protein phosphorylation defines developmental auxin responses,discuss current challenges,and provide our perspectives on future directions involving the integration of the control of protein phosphorylation into the molecular auxin network.

Osmotic Stress Modulates the Balance between Exocytosis and Clathrin-Mediated Endocytosis in Arabidopsis thaliana

The sessile life style of plants creates the need to deal with an often adverse environment, in which water availability can change on a daily basis, challenging the cellular physiology and integrity. Changes in os- motic conditions disrupt the equilibrium of the plasma membrane： hypoosmotic conditions increase and hyperosmotic environment decrease the cell volume. Here, we show that short-term extracellular osmotic treatments are closely followed by a shift in the balance between endocytosis and exocytosis in root mer- istem cells. Acute hyperosmotic treatments （ionic and nonionic） enhance clathrin-mediated endocytosis simultaneously attenuating exocytosis, whereas hypoosmotic treatments have the opposite effects. In addition to clathrin recruitment to the plasma membrane, components of early endocytic trafficking are essential during hyperosmotic stress responses. Consequently, growth of seedlings defective in elements of clathrin or early endocytic machinery is more sensitive to hyperosmotic treatments. We also found that the endocytotic response to a change of osmotic status in the environment is dominant over the presum- ably evolutionary more recent regulatory effect of plant hormones, such as auxin. These results imply that osmotic perturbation influences the balance between endocytosis and exocytosis acting through clathrin- mediated endocytosis. We propose that tension on the plasma membrane determines the addition or removal of membranes at the cell surface, thus preserving cell integrity.

Cellular and Molecular Requirements for Polar PIN Targeting and Transcytosis in Plants

The polar, sub-cellular localization of PIN auxin efflux carriers determines the direction of intercellular auxin flow, thus defining the spatial aspect of auxin signalling. Dynamic, transcytosis-like relocalizations of PIN proteins occur in response to external and internal signals, integrating these signals into changes in auxin distribution. Here, we examine the cellular and molecular mechanisms of polar PIN delivery and transcytosis. The mechanisms of the ARF-GEF-dependent polar targeting and transcytosis are well conserved and show little variations among diverse Arabidopsis ecotypes consistent with their fundamental importance in regulating plant development. At the cellular level, we refine previous findings on the role of the actin cytoskeleton in apical and basal PIN targeting, and identify a previously unknown role for microtubules, specifically in basal targeting. PIN protein delivery to different sides of the cell is mediated by ARFdependent trafficking with a previously unknown complex level of distinct ARF-GEF vesicle trafficking regulators. Our data suggest that alternative recruitment of PIN proteins by these distinct pathways can account for cell type- and cargo-specific aspects of polar targeting, as well as for polarity changes in response to different signals. The resulting dynamic PIN positioning to different sides of cells defines a three-dimensional pattern of auxin fluxes within plant tissues.

Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment （PVC） Function in Arabidopsis

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 （pat3） mutant that we identified by an epifluorescence-based for- ward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFR While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compart- ments （PVC） with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A （VPS35A）, a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lyric vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.

Three-dimensional visualization of planta clathrin-coated vesicles at ultrastructural resolution

Biological systems are the sum of their dynamic three-dimensional(3D)parts.Therefore,it is critical to study biological structures in 3D and at high resolution to gain insights into their physiological functions.Electron microscopy of metal replicas of unroofed cells and isolated organelles has been a key technique to visualize intracellular structures at nanometer resolution.However,many of these methods require specialized equipment and personnel to complete them.Here,we present novel accessible methods to analyze biological structures in unroofed cells and biochemically isolated organelles in 3D and at nanometer resolution,focusing on Arabidopsis clathrin-coated vesicles(CCVs).While CCVs are essential trafficking organelles,their detailed structural information is lacking due to their poor preservation when observed via classical electron microscopy protocols experiments.First,we establish a method to visualize CCVs in unroofed cells using scanning transmission electron microscopy tomography,providing sufficient resolution to define the clathrin coat arrangements.Critically,the samples are prepared directly on electron microscopy grids,removing the requirement to use extremely corrosive acids,thereby enabling the use of this method in any electron microscopy lab.Secondly,we demonstrate that this standardized sample preparation allows the direct comparison of isolated CCV samples with those visualized in cells.Finally,to facilitate the high-throughput and robust screening of metal replicated samples,we provide a deep learning analysis method to screen the“pseudo 3D”morphologies of CCVs imaged with 2D modalities.Collectively,our work establishes accessible ways to examine the 3D structure of biological samples and provide novel insights into the structure of plant CCVs.

Enquiry into the Topology of Plasma Membrane- Localized PIN Auxin Transport Components

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regard- less of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immuno- localization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane （TM） bundles of five m-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.