One of the mechanisms by which signalling molecules regulate cellular behaviour is modulating subcellular protein translocation. This mode of regulation is often based on specialized vesicle trafficking, termed constitutive cycling, which consists of repeated internalization and recycling of proteins to and from the plasma membrane. No such mechanism of hormone action has been shown in plants although several proteins, including the PIN auxin efflux facilitators, exhibit constitutive cycling. Here we show that a major regulator of plant development, auxin, inhibits endocytosis. This effect is specific to biologically active auxins and requires activity of the Calossin-like protein BIG. By inhibiting the internalization step of PIN constitutive cycling, auxin increases levels of PINs at the plasma membrane. Concomitantly, auxin promotes its own efflux from cells by a vesicle-trafficking-dependent mechanism. Furthermore, asymmetric auxin translocation during gravitropism is correlated with decreased PIN internalization. Our data imply a previously undescribed mode of plant hormone action: by modulating PIN protein trafficking, auxin regulates PIN abundance and activity at the cell surface, providing a mechanism for the feedback regulation of auxin transport.

Summary Spatial distribution of the plant hormone auxin regulates multiple aspects of plant development. These self-regulating auxin gradients are established by the action of PIN auxin transporters, whose activity is regulated by their constitutive cycling between the plasma membrane and endosomes. Here, we show that auxin signaling by the auxin receptor AUXIN-BINDING PROTEIN 1 (ABP1) inhibits the clathrin-mediated internalization of PIN proteins. ABP1 acts as a positive factor in clathrin recruitment to the plasma membrane, thereby promoting endocytosis. Auxin binding to ABP1 interferes with this action and leads tothe inhibition of clathrin-mediated endocytosis. Our study demonstrates that ABP1 mediates a nontranscriptional auxin signaling that regulates the evolutionarily conserved process of clathrin-mediated endocytosis and suggests that this signaling may be essential for the developmentally important feedback of auxin on its own transport.

All eukaryotic cells present at the cell surface a specific set of plasma membrane proteins that modulate responses to internal and external cues and whose activity is also regulated by protein degradation. We characterized the lytic vacuole-dependent degradation of membrane proteins in Arabidopsis thaliana by means of in vivo visualization of vacuolar targeting combined with quantitative protein analysis. We show that the vacuolar targeting pathway is used by multiple cargos including PIN-FORMED (PIN) efflux carriers for the phytohormone auxin. In vivo visualization of PIN2 vacuolar targeting revealed its differential degradation in response to environmental signals, such as gravity. In contrast to polar PIN delivery to the basal plasma membrane, which depends on the vesicle trafficking regulator ARF-GEF GNOM, PIN sorting to the lytic vacuolar pathway requires additional brefeldin Asensitive ARF-GEF activity. Furthermore, we identified putative retromer components SORTING NEXIN1 (SNX1) and VACUOLAR PROTEIN SORTING29 (VPS29) as important factors in this pathway and propose that the retromer complex acts to retrieve PIN proteins from a late/pre-vacuolar compartment back to the recycling pathways. Our data suggest that ARF GEF-and retromer-dependent processes regulate PIN sorting to the vacuole in an antagonistic manner and illustrate instrumentalization of this mechanism for fine-tuning the auxin fluxes during gravitropic response.gravitropism ͉ polar auxin transport ͉ posttranslational regulation ͉ vesicle trafficking

Phototropism is an adaptation response, through which plants grow towards the light. It involves light perception and asymmetric distribution of the plant hormone auxin. Here we identify a crucial part of the mechanism for phototropism, revealing how light perception initiates auxin redistribution that leads to directional growth. We show that light polarizes the cellular localization of the auxin efflux carrier PIN3 in hypocotyl endodermis cells, resulting in changes in auxin distribution and differential growth. In the dark, high expression and activity of the PINOID (PID) kinase correlates with apolar targeting of PIN3 to all cell sides. Following illumination, light represses PINOID transcription and PIN3 is polarized specifically to the inner cell sides by GNOM ARF GTPase GEF (guanine nucleotide exchange factor)-dependent trafficking. Thus, differential trafficking at the shaded and illuminated hypocotyl side aligns PIN3 polarity with the light direction, and presumably redirects auxin flow towards the shaded side, where auxin promotes growth, causing hypocotyls to bend towards the light. Our results imply that PID phosphorylation-dependent recruitment of PIN proteins into distinct trafficking pathways is a mechanism to polarize auxin fluxes in response to different environmental and endogenous cues.

The phytohormone auxin acts as a prominent signal, providing, by its local accumulation or depletion in selected cells, a spatial and temporal reference for changes in the developmental program [1][2][3][4][5][6][7] . The distribution of auxin depends on both auxin metabolism (biosynthesis, conjugation and degradation) [8][9][10] and cellular auxin transport [11][12][13][14][15] . We identified in silico a novel putative auxin transport facilitator family, called PIN-LIKES (PILS). Here we illustrate that PILS proteins are required for auxin-dependent regulation of plant growth by determining the cellular sensitivity to auxin. PILS proteins regulate intracellular auxin accumulation at the endoplasmic reticulum and thus auxin availability for nuclear auxin signalling. PILS activity affects the level of endogenous auxin indole-3-acetic acid (IAA), presumably via intracellular accumulation and metabolism. Our findings reveal that the transport machinery to compartmentalize auxin within the cell is of an unexpected molecular complexity and demonstrate this compartmentalization to be functionally important for a number of developmental processes.Prominent auxin carriers with fundamental importance during plant development are PIN-FORMED (PIN) proteins [1][2][3]6,9,15 . PIN1-type auxin carriers regulate the directional intercellular auxin transport at the plasma membrane. In contrast, atypical family member PIN5 regulates intracellular auxin compartmentalization into the lumen of the endoplasmic reticulum and its role in auxin homeostasis was recently identified 15,16 . PIN proteins have a predicted central hydrophilic loop, flanked at each side by five transmembrane domains. We screened in silico for novel PIN-like putative carrier proteins with a predicted topology similar to PIN proteins ( Fig. 1a and Supplementary Fig. 2) and identified a protein family of seven members (Fig. 1b) in Arabidopsis thaliana, which we designated as the PILS proteins. In contrast to the similarities in the predicted protein topology, PIN and PILS proteins do not show pronounced protein sequence identity (10-18%), which limits the identification of PILS proteins by conventional, reciprocal basic local alignment search tool (BLAST) approaches. However, the distinct PIN and PILS protein families contain both the InterPro auxin carrier domain which is an insilico-defined domain, aiming to predict auxin transport function (http://www.ebi.ac.uk/panda/InterPro.html). The PILS putative carrier family is conserved throughout the whole plant lineage, including unicellular algae (such as Ostreococcus tauri and Chlamydomonas reinhardtii) (Supplementary Fig. 3) where PIN proteins are absent 16 , indicating that PILS proteins are evolutionarily older.PILS genes are broadly expressed in various tissues (Fig. 1c) and PILS2-PILS7 were transcriptionally upregulated by auxin application in wild-type seedlings (Fig. 1d-f and Supplementary Fig. 4), indicating a role in auxin-dependent processes. To investigate the potential function of the putative PILS auxi...

Auxin is an essential plant-specific regulator of patterning processes that also controls directional growth of roots and shoots. In response to gravity stimulation, the PIN3 auxin transporter polarizes to the bottom side of gravity-sensing root cells, presumably redirecting the auxin flux toward the lower side of the root and triggering gravitropic bending. By combining live-cell imaging techniques with pharmacological and genetic approaches, we demonstrate that PIN3 polarization does not require secretion of de novo synthesized proteins or protein degradation, but instead involves rapid, transient stimulation of PIN endocytosis, presumably via a clathrindependent pathway. Moreover, gravity-induced PIN3 polarization requires the activity of the guanine nucleotide exchange factors for ARF GTPases (ARF-GEF) GNOM-dependent polar-targeting pathways and might involve endosome-based PIN3 translocation from one cell side to another. Our data suggest that gravity perception acts at several instances of PIN3 trafficking, ultimately leading to the polarization of PIN3, which presumably aligns auxin fluxes with gravity vector and mediates downstream root gravitropic response.polarity | exocytosis | gravitropism | plant cell biology

Growth and development are coordinated by an array of intercellular communications. Known plant signaling molecules include phytohormones and hormone peptides. Although both classes can be implicated in the same developmental processes, little is known about the interplay between phytohormone action and peptide signaling within the cellular microenvironment. We show that genes coding for small secretory peptides, designated GOLVEN (GLV), modulate the distribution of the phytohormone auxin. The deregulation of the GLV function impairs the formation of auxin gradients and alters the reorientation of shoots and roots after a gravity stimulus. Specifically, the GLV signal modulates the trafficking dynamics of the auxin efflux carrier PIN-FORMED2 involved in root tropic responses and meristem organization. Our work links the local action of secretory peptides with phytohormone transport.

Cell polarity manifested by the polar cargo delivery to different plasma-membrane domains is a fundamental feature of multicellular organisms. Pathways for polar delivery have been identified in animals; prominent among them is transcytosis, which involves cargo movement between different sides of the cell [1]. PIN transporters are prominent polar cargoes in plants, whose polar subcellular localization determines the directional flow of the signaling molecule auxin [2, 3]. In this study, we address the cellular mechanisms of PIN polar targeting and dynamic polarity changes. We show that apical and basal PIN targeting pathways are interconnected but molecularly distinct by means of ARF GEF vesicle-trafficking regulators. Pharmacological or genetic interference with the Arabidopsis ARF GEF GNOM leads specifically to apicalization of basal cargoes such as PIN1. We visualize the translocation of PIN proteins between the opposite sides of polarized cells in vivo and show that this PIN transcytosis occurs by endocytic recycling and alternative recruitment of the same cargo molecules by apical and basal targeting machineries. Our data suggest that an ARF GEF-dependent transcytosis-like mechanism is operational in plants and provides a plausible mechanism to trigger changes in PIN polarity and hence auxin fluxes during embryogenesis and organogenesis.