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Spontaneously arising channels transporting the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feed-back between auxin signaling and transport as underlying mechanism but molecular players await discovery. Here we identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related, Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feed-back that coordinates polarization of individual cells during auxin canalization.

Summary To adapt to the diverse array of biotic and abiotic cues, plants have evolved sophisticated mechanisms to sense changes in environmental conditions and modulate their growth. Growth‐promoting hormones and defence signalling fine tune plant development antagonistically. During host–pathogen interactions, this defence–growth trade‐off is mediated by the counteractive effects of the defence hormone salicylic acid (SA) and the growth hormone auxin.Here we revealed an underlying mechanism of SA regulating auxin signalling by constraining the plasma membrane dynamics of PIN2 auxin efflux transporter in Arabidopsis thaliana roots.The lateral diffusion of PIN2 proteins is constrained by SA signalling, during which PIN2 proteins are condensed into hyperclusters depending on REM1.2‐mediated nanodomain compartmentalisation. Furthermore, membrane nanodomain compartmentalisation by SA or Remorin (REM) assembly significantly suppressed clathrin‐mediated endocytosis. Consequently, SA‐induced heterogeneous surface condensation disrupted asymmetric auxin distribution and the resultant gravitropic response.Our results demonstrated a defence–growth trade‐off mechanism by which SA signalling crosstalked with auxin transport by concentrating membrane‐resident PIN2 into heterogeneous compartments.

Significance Plasmodesmata (PD) create cytoplasmic and membrane continuities between adjacent cells to facilitate cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity against plant pathogens, including the accumulation of the defense hormone, salicylic acid (SA). However, the mechanism of how this occurs is not well understood. Here, we uncover a mechanism by which SA triggers Remorin-dependent membrane lipid nanodomain assembly, leading to enhancement of the liquid-ordered phase. The higher-ordered lipids, which are particularly enriched at PD membrane, decreased PD membrane plasticity, and thus restricted PD opening and impeded virus spreading. Our findings address a knowledge gap in plant defense mechanisms at the membrane level that rely on SA-controlled lipid order and PD closure., Plasmodesmata (PD) are plant-specific membrane-lined channels that create cytoplasmic and membrane continuities between adjacent cells, thereby facilitating cell–cell communication and virus movement. Plant cells have evolved diverse mechanisms to regulate PD plasticity in response to numerous environmental stimuli. In particular, during defense against plant pathogens, the defense hormone, salicylic acid (SA), plays a crucial role in the regulation of PD permeability in a callose-dependent manner. Here, we uncover a mechanism by which plants restrict the spreading of virus and PD cargoes using SA signaling by increasing lipid order and closure of PD. We showed that exogenous SA application triggered the compartmentalization of lipid raft nanodomains through a modulation of the lipid raft-regulatory protein, Remorin (REM). Genetic studies, superresolution imaging, and transmission electron microscopy observation together demonstrated that Arabidopsis REM1.2 and REM1.3 are crucial for plasma membrane nanodomain assembly to control PD aperture and functionality. In addition, we also found that a 14-3-3 epsilon protein modulates REM clustering and membrane nanodomain compartmentalization through its direct interaction with REM proteins. This study unveils a molecular mechanism by which the key plant defense hormone, SA, triggers membrane lipid nanodomain reorganization, thereby regulating PD closure to impede virus spreading.

Polarsubcellular localization of the PINexporters of the phytohormoneauxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGCkinase-mediated PINphosphorylation andMAB4/MEL recruitment.Wethusuncover themolecularmechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

The quality control system for messenger RNA (mRNA) is fundamental for cellular activities in eukaryotes. To elucidate the molecular mechanism of 3′-phosphoinositide-dependent protein kinase1 (PDK1), a master regulator that is essential throughout eukaryotic growth and development, we employed a forward genetic approach to screen for suppressors of the loss-of-function T-DNA insertion double mutant pdk1.1 pdk1.2 in Arabidopsis thaliana. Notably, the severe growth attenuation of pdk1.1 pdk1.2 was rescued by sop21 (suppressor of pdk1.1 pdk1.2), which harbors a loss-of-function mutation in PELOTA1 (PEL1). PEL1 is a homolog of mammalian PELOTA and yeast (Saccharomyces cerevisiae) DOM34p, which each form a heterodimeric complex with the GTPase HBS1 (HSP70 SUBFAMILY B SUPPRESSOR1, also called SUPERKILLER PROTEIN7, SKI7), a protein that is responsible for ribosomal rescue and thereby assures the quality and fidelity of mRNA molecules during translation. Genetic analysis further revealed that a dysfunctional PEL1–HBS1 complex failed to degrade the T-DNA-disrupted PDK1 transcripts, which were truncated but functional, and thus rescued the growth and developmental defects of pdk1.1 pdk1.2. Our studies demonstrated the functionality of a homologous PELOTA–HBS1 complex and identified its essential regulatory role in plants, providing insights into the mechanism of mRNA quality control.

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate‐dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments., Arabidopsis roots adjust their growth to the type of nitrogen source via phosphorylation of auxin carrier PIN2 and adjustment of auxin distribution between neighboring tissues.

Summary Cell and tissue polarization is fundamental for plant growth and morphogenesis. The polar, cellular localization of Arabidopsis PIN‐FORMED (PIN) proteins is crucial for their function in directional auxin transport. The clustering of PIN polar cargoes within the plasma membrane has been proposed to be important for the maintenance of their polar distribution. However, the more detailed features of PIN clusters and the cellular requirements of cargo clustering remain unclear.Here, we characterized PIN clusters in detail by means of multiple advanced microscopy and quantification methods, such as 3D quantitative imaging or freeze‐fracture replica labeling. The size and aggregation types of PIN clusters were determined by electron microscopy at the nanometer level at different polar domains and at different developmental stages, revealing a strong preference for clustering at the polar domains.Pharmacological and genetic studies revealed that PIN clusters depend on phosphoinositol pathways, cytoskeletal structures and specific cell‐wall components as well as connections between the cell wall and the plasma membrane.This study identifies the role of different cellular processes and structures in polar cargo clustering and provides initial mechanistic insight into the maintenance of polarity in plants and other systems.

Summary: The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are derivatives of the phytohormone salicylic acid (SA). SA is well known to regulate plant immunity and development, whereas there have been few reports focusing on the effects of NSAIDs in plants. Our studies here reveal that NSAIDs exhibit largely overlapping physiological activities to SA in the model plant Arabidopsis. NSAID treatments lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the protein phosphatase 2A (PP2A), NSAIDs also exhibit PP2A-independent effects. Cell biological and biochemical analyses reveal that many NSAIDs bind directly to and inhibit the chaperone activity of TWISTED DWARF1, thereby regulating actin cytoskeleton dynamics and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants and provide insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds.

Nitrogen is an essential macronutrient and its availability in soil plays a critical role in plant growth, development and impacts agricultural productivity. Plants have evolved different strategies to sense and respond to heterogeneous nitrogen distribution. Modulating root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to varying nitrogen sources are poorly understood. Here, using a combination of physiological, live in vivo high- and super resolution imaging, we describe a novel adaptation strategy of root growth on available nitrogen source. We show that growth, i.e. tissue-specific cell division and elongation rates are fine-tuned by modulating auxin flux within and between tissues. Changes in auxin redistribution are achieved by nitrogen source dependent post-translational modification of PIN2, a major auxin efflux carrier, at an uncharacterized, evolutionary conserved phosphosite. Further, we generate a computer model based on our results which successfully recapitulate our experimental observations and creates new predictions that could broaden our understanding of root growth mechanisms in the dynamic environment.

The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.

In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.

Summary Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense., Graphical Abstract, Highlights • SA modulates root development independently of NPR1-mediated canonical signaling • SA attenuates growth through crosstalk with the auxin transport network • SA upregulates the phosphorylation status of PIN auxin efflux carriers through PP2A • SA directly targets A subunits of PP2A, inhibiting the activity of the complex, Besides the canonical role in immunity, the defense hormone salicylic acid also shapes plant growth and development. Tan et al. show that salicylic acid regulates Arabidopsis growth through directly binding to A subunits of the PP2A enzyme and thereby modulating its activity.

Directional intercellular transport of the phytohormone auxin mediated by PIN FORMED (PIN) efflux carriers plays essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. Multilevel regulations of PIN activity under internal and external cues are complicated; however, the underlying molecular mechanism remains elusive. Here we demonstrate that 3’-Phosphoinositide-Dependent Protein Kinase1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub integrating the upstream lipid signalling and the downstream substrate activity through phosphorylation. Genetic analysis uncovers that loss-of-function Arabidopsis mutant pdk1.1 pdk1.2 exhibits a plethora of abnormalities in organogenesis and growth, due to the defective PIN-dependent auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 Protein Kinase to facilitate its activity towards PIN proteins. Our studies establish a lipid-dependent phosphorylation cascade connecting membrane composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.

Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3′-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes. The PDK1 lipid-dependent kinase controls PIN1 and auxin transport through a phosphorylation cascade that includes AGC-type kinase D6PK. The double pdk1 mutant reveals auxin-related phenotypes such as reduced gravitropism and lateral roots.

Abiotic stress poses constant challenges for plant survival and is a serious problem for global agricultural productivity. On a molecular level, stress conditions result in elevation of reactive oxygen species (ROS) production causing oxidative stress associated with oxidation of proteins and nucleic acids as well as impairment of membrane functions. Adaptation of root growth to ROS accumulation is facilitated through modification of auxin and cytokinin hormone homeostasis. Here, we report that in Arabidopsis root meristem, ROS-induced changes of auxin levels correspond to decreased abundance of PIN auxin efflux carriers at the plasma membrane (PM). Specifically, increase in H2O2 levels affects PIN2 endocytic recycling. We show that the PIN2 intracellular trafficking during adaptation to oxidative stress requires the function of the ADP-ribosylation factor (ARF)-guanine-nucleotide exchange factor (GEF) BEN1, an actin-associated regulator of the trafficking from the PM to early endosomes and, presumably, indirectly, trafficking to the vacuoles. We propose that H2O2 levels affect the actin dynamics thus modulating ARF-GEF-dependent trafficking of PIN2. This mechanism provides a way how root growth acclimates to stress and adapts to a changing environment.

Normal leaf margin development is important for leaf morphogenesis and contributes to diverse leaf shapes in higher plants. We here show the crucial roles of an atypical type II phosphatidylinositol 4-kinase, PI4Kγ5, in Arabidopsis leaf margin development. PI4Kγ5 presents a dynamics expression pattern along with leaf development and a T-DNA mutant lacking PI4Kγ5, pi4kγ5–1, presents serrated leaves, which is resulted from the accelerated cell division and increased auxin concentration at serration tips. Studies revealed that PI4Kγ5 interacts with and phosphorylates a membrane-bound NAC transcription factor, ANAC078. Previous studies demonstrated that membrane-bound transcription factors regulate gene transcription by undergoing proteolytic process to translocate into nucleus, and ANAC078 undergoes proteolysis by cleaving off the transmembrane region and carboxyl terminal. Western blot analysis indeed showed that ANAC078 deleting of carboxyl terminal is significantly reduced in pi4kγ5–1, indicating that PI4Kγ5 is important for the cleavage of ANAC078. This is consistent with the subcellular localization observation showing that fluorescence by GFP-ANAC078 is detected at plasma membrane but not nucleus in pi4kγ5–1 mutant and that expression of ANAC078 deleting of carboxyl terminal, driven by PI4Kγ5 promoter, could rescue the leaf serration defects of pi4kγ5–1. Further analysis showed that ANAC078 suppresses the auxin synthesis by directly binding and regulating the expression of auxin synthesis-related genes. These results indicate that PI4Kγ5 interacts with ANAC078 to negatively regulate auxin synthesis and hence influences cell proliferation and leaf development, providing informative clues for the regulation of in situ auxin synthesis and cell division, as well as the cleavage and functional mechanism of membrane-bound transcription factors., Author Summary Transcription factors (TFs) regulate the expression of target genes at nucleus, while interestingly, a few of TFs locate at plasma membrane or ER membrane, stored as resting precursors to secure a rapid response post endogenous or environmental stimuli. Membrane-bound TFs undergo post-translational processing to be liberated and translocate into the nucleus. There are two kinds of liberating mechanisms: regulated intramembrane proteolysis (RIP) by certain membrane-associated proteases and regulated ubiquitin/26S proteasome-dependent processing (RUP). However, the detailed regulatory mechanism is largely unclear. By studying a T-DNA mutant lacking an atypical type II phosphatidylinositol 4-kinase (PI4Kγ5), pi4kγ5–1, which presents serrated leaves that resulted from the accelerated cell division and increased auxin concentration at serration tips, we here showed the crucial roles of PI4Kγ5 on the function of membrane-bound TFs, possibly through a phosphorylation-dependent process. Yeast two-hybrid screening identifies ANAC078, a plasma membrane-bound NAC family TF that contains a one-pass transmembrane and undergoes RIP for release, an interacting TF of PI4Kγ5. Biochemical analysis reveal that PI4Kγ5 phosphorylates ANAC078 and further genetic analysis confirmed that PI4Kγ5 is crucial for the cleavage and normal function of ANAC078, indicating that PI4Kγ5-ANAC078 module downregulates auxin level during leaf development (serration). These findings provide new insights into the regulatory and functional mechanism of membrane-bound TFs and in situ auxin synthesis and effects during leaf morphogenesis.

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.

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.