Directionality in the intercellular transport of the plant hormone auxin is determined by polar plasma membrane localization of PIN-FORMED (PIN) auxin transport proteins. However, apart from PIN phosphorylation at conserved motifs, no further determinants explicitly controlling polar PIN sorting decisions have been identified. Here we present Arabidopsis WAVY GROWTH 3 (WAV3) and closely related RING-finger E3 ubiquitin ligases, whose loss-of-function mutants show a striking apical-to-basal polarity switch in PIN2 localization in root meristem cells. WAV3 E3 ligases function as essential determinants for PIN polarity, acting independently from PINOID/WAG-dependent PIN phosphorylation. They antagonize ectopic deposition of de novo synthesized PIN proteins already immediately following completion of cell division, presumably via preventing PIN sorting into basal, ARF GEF-mediated trafficking. Our findings reveal an involvement of E3 ligases in the selective targeting of apically localized PINs in higher plants.

• MeSH
• Arabidopsis * metabolism   MeSH
• Plant Roots metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins * genetics   metabolism   MeSH
• Protein Transport MeSH
• Ubiquitin-Protein Ligases genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Arabidopsis Proteins * MeSH
• Ubiquitin-Protein Ligases MeSH

The analysis of dynamic cellular processes such as plant cytokinesis stands and falls with live-cell time-lapse confocal imaging. Conventional approaches to time-lapse imaging of cell division in Arabidopsis root tips are tedious and have low throughput. Here, we describe a protocol for long-term time-lapse simultaneous imaging of multiple root tips on a vertical-stage confocal microscope with automated root tracking. We also provide modifications of the basic protocol to implement this imaging method in the analysis of genetic, pharmacological or laser ablation wounding-mediated experimental manipulations. Our method dramatically improves the efficiency of cell division time-lapse imaging by increasing the throughput, while reducing the person-hour requirements of such experiments.

• Keywords
• Arabidopsis, Automation, Confocal microscopy, Cytokinesis, Laser ablation, Root meristem, Time-lapse imaging,
• MeSH
• Arabidopsis * MeSH
• Cell Division MeSH
• Time-Lapse Imaging MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• Meristem MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

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

Spontaneously arising channels that transport 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 feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. 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 feedback that coordinates polarization of individual cells during auxin canalization.

• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Biological Transport MeSH
• Indoleacetic Acids metabolism   MeSH
• Protein Interaction Mapping MeSH
• Membrane Transport Proteins metabolism   MeSH
• Protein Kinases genetics   metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Transcription Factors 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
• PIN1 protein, Arabidopsis MeSH Browser
• Protein Kinases MeSH
• Arabidopsis Proteins MeSH
• Transcription Factors MeSH
• WRKY23 protein, Arabidopsis MeSH Browser

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

Cell polarity is crucial for the coordinated development of all multicellular organisms. In plants, this is exemplified by the PIN-FORMED (PIN) efflux carriers of the phytohormone auxin: The polar subcellular localization of the PINs is instructive to the directional intercellular auxin transport, and thus to a plethora of auxin-regulated growth and developmental processes. Despite its importance, the regulation of PIN polar subcellular localization remains poorly understood. Here, we have employed advanced live-cell imaging techniques to study the roles of microtubules and actin microfilaments in the establishment of apical polar localization of PIN2 in the epidermis of the Arabidopsis root meristem. We report that apical PIN2 polarity requires neither intact actin microfilaments nor microtubules, suggesting that the primary spatial cue for polar PIN distribution is likely independent of cytoskeleton-guided endomembrane trafficking.

• Keywords
• PIN auxin efflux carriers, actin, cell polarity, cytoskeleton, live-cell imaging, microtubules, polarity establishment,
• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Cytoskeleton metabolism   MeSH
• Intracellular Space metabolism   MeSH
• Arabidopsis Proteins metabolism   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