Polar subcellular localization of the PIN exporters of the phytohormone auxin 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 AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

• Keywords
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetics   metabolism   MeSH
• Biological Transport MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids MeSH
• Membrane Transport Proteins genetics   MeSH
• Cell Polarity MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Cells metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• Arabidopsis Proteins * MeSH

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.

• MeSH
• ADP-Ribosylation Factors metabolism   MeSH
• Arabidopsis drug effects   metabolism   MeSH
• Biological Transport drug effects   MeSH
• Brefeldin A pharmacology   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Epistasis, Genetic drug effects   MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Mutation genetics   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Phospholipid Transfer Proteins metabolism   MeSH
• Nicotiana metabolism   MeSH
• trans-Golgi Network drug effects   metabolism   MeSH
• Protein Binding drug effects   MeSH
• Guanine Nucleotide Exchange Factors metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• ADP-Ribosylation Factors MeSH
• Brefeldin A MeSH
• GTP Phosphohydrolases MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Phospholipid Transfer Proteins MeSH
• Guanine Nucleotide Exchange Factors MeSH

The Arp2/3 complex is an actin nucleator shown to be required throughout plant morphogenesis, contributing to processes such as cell expansion, tissue differentiation or cell wall assembly. A recent publication demonstrated that plants lacking functional Arp2/3 complex also present defects in auxin distribution and transport. This work shows that Arp2/3 complex subunits are predominantly expressed in the provasculature, although other plant tissues also show promoter activity (e.g., cotyledons, apical meristems, or root tip). Moreover, auxin can trigger subunit expression, indicating a role of this phytohormone in mediating the complex activity. Further investigation of the functional interaction between Arp2/3 complex and auxin signaling also reveals their cooperation in determining pavement cell shape, presumably through the role of Arp2/3 complex in the correct auxin carrier trafficking. Young seedlings of arpc5 mutants show increased auxin-triggered proteasomal degradation of DII-VENUS and altered PIN3 distribution, with higher levels of the protein in the vacuole. Closer observation of vacuolar morphology revealed the presence of a more fragmented vacuolar compartment when Arp2/3 function is abolished, hinting a generalized role of Arp2/3 complex in endomembrane function and protein trafficking.

• Keywords
• Arp2/3 complex, actin, auxin, cell expansion, cytoskeleton,
• Publication type
• Journal Article MeSH

The fine tuning of hormone (e.g., auxin and gibberellin) levels and hormone signaling is required for maintaining normal embryogenesis. Embryo polarity, for example, is ensured by the directional movement of auxin that is controlled by various types of auxin transporters. Here, we present pieces of evidence for the auxin-gibberellic acid (GA) hormonal crosstalk during embryo development and the regulatory role of the Arabidopsis thaliana Calcium-Dependent Protein Kinase-Related Kinase 5 (AtCRK5) in this regard. It is pointed out that the embryogenesis of the Atcrk5-1 mutant is delayed in comparison to the wild type. This delay is accompanied with a decrease in the levels of GA and auxin, as well as the abundance of the polar auxin transport (PAT) proteins PIN1, PIN4, and PIN7 in the mutant embryos. We have previously showed that AtCRK5 can regulate the PIN2 and PIN3 proteins either directly by phosphorylation or indirectly affecting the GA level during the root gravitropic and hypocotyl hook bending responses. In this manuscript, we provide evidence that the AtCRK5 protein kinase can in vitro phosphorylate the hydrophilic loops of additional PIN proteins that are important for embryogenesis. We propose that AtCRK5 can govern embryo development in Arabidopsis through the fine tuning of auxin-GA level and the accumulation of certain polar auxin transport proteins.

• Keywords
• Arabidopsis thaliana, Calcium-Dependent Protein Kinase-Related Kinase (CRK), GA3, auxin gradient, embryogenesis, polar auxin transport (PAT) proteins,
• MeSH
• Arabidopsis growth & development   metabolism   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Gibberellins analysis   metabolism   MeSH
• Germination * MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Proteins genetics   metabolism   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Receptors, Cell Surface metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Seeds anatomy & histology   growth & development   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• CRK5 protein, Arabidopsis MeSH Browser
• gibberellic acid MeSH Browser
• Gibberellins MeSH
• Indoleacetic Acids MeSH
• Membrane Transport Proteins MeSH
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface MeSH

Seedling establishment following germination requires the fine tuning of plant hormone levels including that of auxin. Directional movement of auxin has a central role in the associated processes, among others, in hypocotyl hook development. Regulated auxin transport is ensured by several transporters (PINs, AUX1, ABCB) and their tight cooperation. Here we describe the regulatory role of the Arabidopsis thaliana CRK5 protein kinase during hypocotyl hook formation/opening influencing auxin transport and the auxin-ethylene-GA hormonal crosstalk. It was found that the Atcrk5-1 mutant exhibits an impaired hypocotyl hook establishment phenotype resulting only in limited bending in the dark. The Atcrk5-1 mutant proved to be deficient in the maintenance of local auxin accumulation at the concave side of the hypocotyl hook as demonstrated by decreased fluorescence of the auxin sensor DR5::GFP. Abundance of the polar auxin transport (PAT) proteins PIN3, PIN7, and AUX1 were also decreased in the Atcrk5-1 hypocotyl hook. The AtCRK5 protein kinase was reported to regulate PIN2 protein activity by phosphorylation during the root gravitropic response. Here it is shown that AtCRK5 can also phosphorylate in vitro the hydrophilic loops of PIN3. We propose that AtCRK5 may regulate hypocotyl hook formation in Arabidopsis thaliana through the phosphorylation of polar auxin transport (PAT) proteins, the fine tuning of auxin transport, and consequently the coordination of auxin-ethylene-GA levels.

• Keywords
• Arabidopsis thaliana, Ca2+/calmodulin-dependent kinase-related kinases (CRKs), polar auxin transport (PAT) proteins, GA3, auxin gradient, ethylene, skotomorphogenesis,
• MeSH
• Arabidopsis drug effects   physiology   MeSH
• Biomarkers MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Hypocotyl physiology   MeSH
• Germination MeSH
• Morphogenesis * drug effects   genetics   MeSH
• Protein Serine-Threonine Kinases metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Receptors, Cell Surface metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Genes, Reporter MeSH
• Signal Transduction MeSH
• Plant Development * drug effects   genetics   MeSH
• Xanthones pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• CRK5 protein, Arabidopsis MeSH Browser
• GA3 compound MeSH Browser
• Protein Serine-Threonine Kinases MeSH
• Arabidopsis Proteins MeSH
• Receptors, Cell Surface MeSH
• Xanthones MeSH

Polar auxin transport plays a pivotal role in plant growth and development. PIN-FORMED (PIN) auxin efflux carriers regulate directional auxin movement by establishing local auxin maxima, minima, and gradients that drive multiple developmental processes and responses to environmental signals. Auxin has been proposed to modulate its own transport by regulating subcellular PIN trafficking via processes such as clathrin-mediated PIN endocytosis and constitutive recycling. Here, we further investigated the mechanisms by which auxin affects PIN trafficking by screening auxin analogs and identified pinstatic acid (PISA) as a positive modulator of polar auxin transport in Arabidopsis (Arabidopsis thaliana). PISA had an auxin-like effect on hypocotyl elongation and adventitious root formation via positive regulation of auxin transport. PISA did not activate SCFTIR1/AFB signaling and yet induced PIN accumulation at the cell surface by inhibiting PIN internalization from the plasma membrane. This work demonstrates PISA to be a promising chemical tool to dissect the regulatory mechanisms behind subcellular PIN trafficking and auxin transport.

• MeSH
• Arabidopsis drug effects   metabolism   MeSH
• Biological Transport drug effects   MeSH
• Cell Membrane drug effects   metabolism   MeSH
• Endocytosis * drug effects   MeSH
• Phenotype MeSH
• Phenylacetates pharmacology   MeSH
• Gravitropism drug effects   MeSH
• Hypocotyl drug effects   growth & development   MeSH
• Plant Roots drug effects   growth & development   MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Signal Transduction MeSH
• Plant Shoots metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Phenylacetates MeSH
• Indoleacetic Acids MeSH
• phenylacetic acid MeSH Browser
• pinstatic acid MeSH Browser
• Arabidopsis Proteins MeSH

Cell polarity, manifested by the localization of proteins to distinct polar plasma membrane domains, is a key prerequisite of multicellular life. In plants, PIN auxin transporters are prominent polarity markers crucial for a plethora of developmental processes. Cell polarity mechanisms in plants are distinct from other eukaryotes and still largely elusive. In particular, how the cell polarities are propagated and maintained following cell division remains unknown. Plant cytokinesis is orchestrated by the cell plate-a transient centrifugally growing endomembrane compartment ultimately forming the cross wall1. Trafficking of polar membrane proteins is typically redirected to the cell plate, and these will consequently have opposite polarity in at least one of the daughter cells2-5. Here, we provide mechanistic insights into post-cytokinetic re-establishment of cell polarity as manifested by the apical, polar localization of PIN2. We show that the apical domain is defined in a cell-intrinsic manner and that re-establishment of PIN2 localization to this domain requires de novo protein secretion and endocytosis, but not basal-to-apical transcytosis. Furthermore, we identify a PINOID-related kinase WAG1, which phosphorylates PIN2 in vitro6 and is transcriptionally upregulated specifically in dividing cells, as a crucial regulator of post-cytokinetic PIN2 polarity re-establishment.

• MeSH
• Arabidopsis cytology   genetics   physiology   MeSH
• Cell Membrane metabolism   MeSH
• Cell Division * MeSH
• Cytokinesis MeSH
• Endocytosis MeSH
• Phenotype MeSH
• Phosphorylation MeSH
• Plant Roots cytology   genetics   physiology   MeSH
• Cell Polarity * MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Recombinant Fusion Proteins MeSH
• Genes, Reporter MeSH
• Protein Transport MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• PIN2 protein, Arabidopsis MeSH Browser
• Arabidopsis Proteins MeSH
• Recombinant Fusion Proteins MeSH

Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterized compared with that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing tandem affinity purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologs of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in Arabidopsis caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like1/2 loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the ongoing characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in Arabidopsis.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Endocytosis genetics   physiology   MeSH
• Clathrin genetics   metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Seedlings genetics   metabolism   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Clathrin MeSH
• Arabidopsis Proteins MeSH