Subcellular trafficking of PIN auxin efflux carriers in auxin transport

The directional transport of the plant hormone auxin is a unique process mediating a wide variety of developmental processes. Auxin movement between cells depends on AUX1/LAX, PGP and PIN protein families that mediate auxin transport across the plasma membrane. The directionality of auxin flow within tissues is largely determined by polar, subcellular localization of PIN auxin efflux carriers. PIN proteins undergo rapid subcellular dynamics that is important for the process of auxin transport and its directionality. Furthermore, various environmental and endogenous signals can modulate trafficking and polarity of PIN proteins and by this mechanism change auxin distribution. Thus, the subcellular dynamics of auxin transport proteins represents an important interface between cellular processes and development of the whole plant. This review summarizes our recent contributions to the field of PIN trafficking and auxin transport regulation.     

Maintenance of Asymmetric Cellular Localization of an Auxin Transport Protein through Interaction with the Actin Cytoskeleton

In shoots, polar auxin transport is basipetal (that is, from the shoot apex toward the base) and is driven by the basal localization of the auxin efflux carrier complex. The focus of this article is to summarize the experiments that have examined how the asymmetric distribution of this protein complex is controlled and the significance of this polar distribution. Experimental evidence suggests that asymmetries in the auxin efflux carrier may be established through localized secretion of Golgi vesicles, whereas an attachment of a subunit of the efflux carrier to the actin cytoskeleton may maintain this localization. In addition, the idea that this localization of the efflux carrier may control both the polarity of auxin movement and more globally regulate developmental polarity is explored. Finally, evidence indicating that the gravity vector controls auxin transport polarity is summarized and possible mechanisms for the environmentally induced changes in auxin transport polarity are discussed.     

Mechanisms of auxin-dependent cell and tissue polarity

The establishment of cellular asymmetries and their coordination within the tissue layer are fundamental to the development of multicellular organisms. In plants, the induction and coordination of cell polarity have classically been attributed to involve the hormone auxin and its flow. However, the underlying mechanisms have only recently been addressed at the molecular level. We review progress on the characterisation of the auxin influx and efflux carrier properties of specific plasma membrane proteins, mechanisms underlying their delivery to and internalisation from the plasma membrane, their endocytic transport and degradation. We discuss mechanisms of auxin gradient, transport and response action during the coordination of polarity, along with the downstream involvement of Rho-of-plant small GTPases during the execution of cell polarity.     

Regulation of the polarity of protein trafficking by phosphorylation

The asymmetry of environmental stimuli and the execution of developmental programs at the organism level require a corresponding polarity at the cellular level, in both unicellular and multicellular organisms. In plants, cell polarity is important in major developmental processes such as cell division, cell enlargement, cell morphogenesis, embryogenesis, axis formation, organ development, and defense. One of the most important factors controlling cell polarity is the asymmetric distribution of polarity determinants. In particular, phosphorylation is implicated in the polar distribution of the determinant protein factors, a mechanism conserved in both prokaryotes and eukaryotes. In plants, formation of local gradients of auxin, the morphogenic hormone, is critical for plant developmental processes exhibiting polarity. The auxin efflux carriers PIN-FORMEDs (PINs) localize asymmetrically in the plasma membrane and cause the formation of local auxin gradients throughout the plant. The asymmetry of PIN distribution in the plasma membrane is determined by phosphorylationmediated polar trafficking of PIN proteins. This review discusses recent studies on the role of phosphorylation in polar PIN trafficking.     

Regulation of auxin transport polarity by AGC kinases

The plant hormone auxin controls plant development through gradients and maxima that are generated by PIN efflux carrier driven polar auxin transport. PIN proteins direct this cell-to-cell auxin transport, and thus orient plant development through their asymmetric subcellular distribution. PIN polarity is regulated by PINOID and the phototropins, members of the AGC protein serine/threonine kinase family. Here we review the signaling pathways of these kinases and the role of calcium and BTB proteins in translating both internal and external signals into developmental responses via PIN relocalization, to adapt plant development to changing environmental conditions.     

Auxin transport

Polar transport of auxin is essential for normal plant growth and development. On a cellular level, directional auxin transport is primarily controlled by an efflux carrier complex that is characterized by the PIN-FORMED (PIN) family of proteins. Detailed developmental studies of PIN distribution and subcellular localization have been combined with the analysis of changes in localized auxin levels to map PIN-mediated auxin movement throughout Arabidopsis tissues. Plant orthologs of mammalian multidrug-resistance/P-glycoproteins (MDR/PGPs) also function in auxin efflux. MDR/PGPs appear to stabilize efflux complexes on the plasma membrane and to function as ATP-dependent auxin transporters, with the specificity and directionality of transport being provided by interacting PIN proteins.     

Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development

The signalling molecule auxin controls plant morphogenesis via its activity gradients, which are produced by intercellular auxin transport. Cellular auxin efflux is the rate-limiting step in this process and depends on PIN and phosphoglycoprotein (PGP) auxin transporters. Mutual roles for these proteins in auxin transport are unclear, as is the significance of their interactions for plant development. Here, we have analysed the importance of the functional interaction between PIN- and PGP-dependent auxin transport in development. We show by analysis of inducible overexpression lines that PINs and PGPs define distinct auxin transport mechanisms: both mediate auxin efflux but they play diverse developmental roles. Components of both systems are expressed during embryogenesis, organogenesis and tropisms, and they interact genetically in both synergistic and antagonistic fashions. A concerted action of PIN- and PGP-dependent efflux systems is required for asymmetric auxin distribution during these processes. We propose a model in which PGP-mediated efflux controls auxin levels in auxin channel-forming cells and, thus, auxin availability for PIN-dependent vectorial auxin movement.     

Polar auxin transport: controlling where and how much.

Auxin is transported through plant tissues, moving from cell to cell in a unique polar manner. Polar auxin transport controls important growth and developmental processes in higher plants. Recent studies have identified several proteins that mediate polar auxin transport and have shown that some of these proteins are asymmetrically localized, paving the way for studies of the mechanisms that regulate auxin transport. New data indicate that reversible protein phosphorylation can control the amount of auxin transport, whereas protein secretion through Golgi-derived vesicles and interactions with the actin cytoskeleton might regulate the localization of auxin efflux complexes.     

Polar-localized NPH3-like proteins regulate polarity and endocytosis of PIN-FORMED auxin efflux carriers

PIN-FORMED (PIN)-dependent auxin transport is essential for plant development and its modulation in response to the environment or endogenous signals. A NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3)-like protein, MACCHI-BOU 4 (MAB4), has been shown to control PIN1 localization during organ formation, but its contribution is limited. The Arabidopsis genome contains four genes, MAB4/ENP/NPY1-LIKE1 (MEL1), MEL2, MEL3 and MEL4, highly homologous to MAB4. Genetic analysis disclosed functional redundancy between MAB4 and MEL genes in regulation of not only organ formation but also of root gravitropism, revealing that NPH3 family proteins have a wider range of functions than previously suspected. Multiple mutants showed severe reduction in PIN abundance and PIN polar localization, leading to defective expression of an auxin responsive marker DR5rev::GFP. Pharmacological analyses and fluorescence recovery after photo-bleaching experiments showed that mel mutations increase PIN2 internalization from the plasma membrane, but affect neither intracellular PIN2 trafficking nor PIN2 lateral diffusion at the plasma membrane. Notably, all MAB4 subfamily proteins show polar localization at the cell periphery in plants. The MAB4 polarity was almost identical to PIN polarity. Our results suggest that the MAB4 subfamily proteins specifically retain PIN proteins in a polarized manner at the plasma membrane, thus controlling directional auxin transport and plant development.     

Cell polarity, auxin transport, and cytoskeleton-mediated division planes: who comes first?

In plants, cell polarity is an issue more recurring than in other systems, because plants, due to their adaptive and flexible development, often change cell polarity postembryonically according to intrinsic cues and demands of the environment. Recent findings on the directional movement of the plant signalling molecule auxin provide a unique connection between individual cell polarity and the establishment of polarity at the tissue, organ, and whole-plant levels. Decisions about the subcellular polar targeting of PIN auxin transport components determine the direction of auxin flow between cells and consequently mediate multiple developmental events. In addition, mutations or chemical interference with PIN-based auxin transport result in abnormal cell divisions. Thus, the complicated links between cell polarity establishment, auxin transport, cytoskeleton, and oriented cell divisions now begin to emerge. Here we review the available literature on the issues of cell polarity in both plants and animals to extend our understanding on the generation, maintenance, and transmission of cell polarity in plants.     

Sterol-dependent endocytosis mediates post-cytokinetic acquisition of PIN2 auxin efflux carrier polarity

The polarization of yeast and animal cells relies on membrane sterols for polar targeting of proteins to the plasma membrane, their polar endocytic recycling and restricted lateral diffusion. However, little is known about sterol function in plant-cell polarity. Directional root growth along the gravity vector requires polar transport of the plant hormone auxin. In Arabidopsis, asymmetric plasma membrane localization of the PIN-FORMED2 (PIN2) auxin transporter directs root gravitropism. Although the composition of membrane sterols influences gravitropism and localization of two other PIN proteins, it remains unknown how sterols contribute mechanistically to PIN polarity. Here, we show that correct membrane sterol composition is essential for the acquisition of PIN2 polarity. Polar PIN2 localization is defective in the sterol-biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) which displays altered sterol composition, PIN2 endocytosis, and root gravitropism. At the end of cytokinesis, PIN2 localizes initially to both newly formed membranes but subsequently disappears from one. By contrast, PIN2 frequently remains at both daughter membranes in endocytosis-defective cpi1-1 cells. Hence, sterol composition affects post-cytokinetic acquisition of PIN2 polarity by endocytosis, suggesting a mechanism for sterol action on establishment of asymmetric protein localization.     

PIN polarity maintenance by the cell wall in Arabidopsis

A central question in developmental biology concerns the mechanism of generation and maintenance of cell polarity, because these processes are essential for many cellular functions and multicellular development. In plants, cell polarity has an additional role in mediating directional transport of the plant hormone auxin that is crucial for multiple developmental processes. In addition, plant cells have a complex extracellular matrix, the cell wall, whose role in regulating cellular processes, including cell polarity, is unexplored. We have found that polar distribution of PIN auxin transporters in plant cells is maintained by connections between polar domains at the plasma membrane and the cell wall. Genetic and pharmacological interference with cellulose, the major component of the cell wall, or mechanical interference with the cell wall disrupts these connections and leads to increased lateral diffusion and loss of polar distribution of PIN transporters for the phytohormone auxin. Our results reveal a plant-specific mechanism for cell polarity maintenance and provide a conceptual framework for modulating cell polarity and plant development via endogenous and environmental manipulations of the cellulose-based extracellular matrix.     

Complex regulation of Arabidopsis AGR1/PIN2-mediated root gravitropic response and basipetal auxin transport by cantharidin-sensitive protein phosphatases

Polar auxin transport, mediated by two distinct plasma membrane-localized auxin influx and efflux carrier proteins/complexes, plays an important role in many plant growth and developmental processes including tropic responses to gravity and light, development of lateral roots and patterning in embryogenesis. We have previously shown that the Arabidopsis AGRAVITROPIC 1/PIN2 gene encodes an auxin efflux component regulating root gravitropism and basipetal auxin transport. However, the regulatory mechanism underlying the function of AGR1/PIN2 is largely unknown. Recently, protein phosphorylation and dephosphorylation mediated by protein kinases and phosphatases, respectively, have been implicated in regulating polar auxin transport and root gravitropism. Here, we examined the effects of chemical inhibitors of protein phosphatases on root gravitropism and basipetal auxin transport, as well as the expression pattern of AGR1/PIN2 gene and the localization of AGR1/PIN2 protein. We also examined the effects of inhibitors of vesicle trafficking and protein kinases. Our data suggest that protein phosphatases, sensitive to cantharidin and okadaic acid, are likely involved in regulating AGR1/PIN2-mediated root basipetal auxin transport and gravitropism, as well as auxin response in the root central elongation zone (CEZ). BFA-sensitive vesicle trafficking may be required for the cycling of AGR1/PIN2 between plasma membrane and the BFA compartment, but not for the AGR1/PIN2-mediated root basipetal auxin transport and auxin response in CEZ cells.     

PIN Auxin Efflux Carrier Polarity Is Regulated by PINOID Kinase-Mediated Recruitment into GNOM-Independent Trafficking in Arabidopsis

The phytohormone auxin plays a major role in embryonic and postembryonic plant development. The temporal and spatial distribution of auxin largely depends on the subcellular polar localization of members of the PIN-FORMED (PIN) auxin efflux carrier family. The Ser/Thr protein kinase PINOID (PID) catalyzes PIN phosphorylation and crucially contributes to the regulation of apical-basal PIN polarity. The GTP exchange factor on ADP-ribosylation factors (ARF-GEF), GNOM preferentially mediates PIN recycling at the basal side of the cell. Interference with GNOM activity leads to dynamic PIN transcytosis between different sides of the cell. Our genetic, pharmacological, and cell biological approaches illustrate that PID and GNOM influence PIN polarity and plant development in an antagonistic manner and that the PID-dependent PIN phosphorylation results in GNOM-independent polar PIN targeting. The data suggest that PID and the protein phosphatase 2A not only regulate the static PIN polarity, but also act antagonistically on the rate of GNOM-dependent polar PIN transcytosis. We propose a model that includes PID-dependent PIN phosphorylation at the plasma membrane and the subsequent sorting of PIN proteins to a GNOM-independent pathway for polarity alterations during developmental processes, such as lateral root formation and leaf vasculature development.     

Auxin regulation of axial growth in bryophyte sporophytes: its potential significance for the evolution of early land plants

To identify developmental mechanisms that might have been involved in the evolution of axial sporophytes in early land plants, we examined the effects of auxin-regulatory compounds in the sporophytes of the hornwort Phaeoceros personii, the liverwort Pellia epiphylla, and the moss Polytrichum ohioense, members of the three divisions of extant bryophytes. The altered growth of isolated young sporophytes exposed to applied auxin (indole-3-acetic acid) or an auxin antagonist (p-chlorophenoxyisobutyric acid) suggests that endogenous auxin acts to regulate the rates of axial growth in all bryophyte divisions. Auxin in young hornwort sporophytes moved at very low fluxes, was insensitive to an auxin-transport inhibitor (N-[1-naphthyl]phthalamic acid), and exhibited a polarity ratio close to 1.0, implying that auxin moves by simple diffusion in these structures. Emerging liverwort sporophytes had somewhat higher auxin fluxes, which were sensitive to transport inhibitors but lacked any measurable polarity. Thus, auxin movement in liverwort sporophytes appears to result from a unique type of apolar facilitated diffusion. In young Polytrichum sporophytes, auxin movement was predominantly basipetal and occurred at high fluxes exceeding those measured in maize coleoptiles. In older Polytrichum sporophytes, acropetal auxin flux had increased beyond the level measured for basipetal flux. Insofar as acropetal and basipetal fluxes had different inhibitor sensitivities, these results suggested that moss sporophytes carry out bidirectional polar transport in different cellular pathways, which resembles the transport in certain angiosperm structures. Therefore, the three lineages of extant bryophytes appear to have evolved independent innovations for auxin regulation of axial growth, with similar mechanisms operating in moss sporophytes and vascular plants.     

Cell polarity in plants: when two do the same, it is not the same...

In unicellular and multicellular organisms, cell polarity is essential for a wide range of biological processes. An important feature of cell polarity is the asymmetric distribution of proteins in or at the plasma membrane. In plants such polar localized proteins play various specific roles ranging from organizing cell morphogenesis, asymmetric cell division, pathogen defense, nutrient transport and establishment of hormone gradients for developmental patterning. Moreover, flexible respecification of cell polarities enables plants to adjust their physiology and development to environmental changes. Having evolved multicellularity independently and lacking major cell polarity mechanisms of animal cells, plants came up with alternative solutions to generate and respecify cell polarity as well as to regulate polar domains at the plasma membrane.     

Auxins and tropisms

Differential growth of plants in response to the changes in the light and gravity vectors requires a complex signal transduction cascade. Although many of the details of the mechanisms by which these differential growth responses are induced are as yet unknown, auxin has been implicated in both gravitropism and phototropism. Specifically, the redistribution of auxin across gravity or light-stimulated tissues has been detected and shown to be required for this process. The approaches by which auxin has been implicated in tropisms include isolation of mutants altered in auxin transport or response with altered gravitropic or phototropic response, identification of auxin gradients with radiolabeled auxin and auxin-inducible gene reporter systems, and by use of inhibitors of auxin transport that block gravitropism and phototropism. Proteins that transport auxin have been identified and the mechanisms which determine auxin transport polarity have been explored. In addition, recent evidence that reversible protein phosphorylation controls this process is summarized. Finally, the data in support of several hypotheses for mechanisms by which auxin transport could be differentially regulated during gravitropism are examined. Although many details of the mechanisms by which plants respond to gravity and light are not yet clear, numerous recent studies demonstrate the role of auxin in these processes.     

The gene MACCHI-BOU 4/ENHANCER OF PINOID encodes a NPH3-like protein and reveals similarities between organogenesis and phototropism at the molecular level

Intercellular transport of the phytohormone auxin is a significant factor for plant organogenesis. To investigate molecular mechanisms by which auxin controls organogenesis, we analyzed the macchi-bou 4 (mab4) mutant identified as an enhancer of pinoid (pid). Although mab4 and pid single mutants displayed relatively mild cotyledon phenotypes, pid mab4 double mutants completely lacked cotyledons. We found that MAB4 was identical to ENHANCER OF PINOID (ENP), which has been suggested to control PIN1 polarity in cotyledon primordia. MAB4/ENP encodes a novel protein, which belongs to the NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) family thought to function as a signal transducer in phototropism and control lateral translocation of auxin. MAB4/ENP mRNA was detected in the protodermal cell layer of the embryo and the meristem L1 layer at the site of organ initiation. In the mab4 embryo, the abundance of PIN1:GFP was severely decreased at the plasma membrane in the protodermal cell layer. In addition, subcellular localization analyses indicated that MAB4/ENP resides on a subpopulation of endosomes as well as on unidentified intracellular compartments. These results indicate that MAB4/ENP is involved in polar auxin transport in organogenesis.     

Elementary auxin response chains at the plasma membrane involve external abp1 and multiple electrogenic ion transport proteins

Studies of membrane electrical responses of isolated protoplasts to auxin have demonstrated the existence of elementary response chains to auxin at the plasma membrane, presently defined only by their uttermost ends. At one side, as demonstrated by several lines of evidence, the auxin perception unit involves proteins homologous to ZmER-abp1 (abp1), the most abundant auxin-binding protein from maize coleoptiles. At the other side, multiple ion transport proteins appear as targets of the auxin signal; the proton pump ATPase, an anion channel and potassium channels. We investigated early electrical responses to auxin at the plasma membrane of tobacco protoplasts. The work presented here will initially focus on abp1 and its functional role at the membrane. The C-terminus abp1 peptide (Pz151–163) was recently reported to modulate K⁺ currents at the plasma membrane of intact guard cells from broad bean [23] and induce plasma membrane hyperpolarisation of tobacco mesophyll protoplasts. These results further demonstrate that proteins involved in plasma membrane responses to auxin are related to maize abp1, and provide clues as to the region of the protein possibly involved in the interaction of abp1 with the plasma membrane. Secondly, this report concentrates on one of the targets of auxin, a voltage-dependent and ATP-regulated anion channel that we characterised on protoplasts from tobacco cell suspensions. This anion channel was specifically modulated by auxin, as already observed for the anion channel of guard cells [14]. Further work will be needed to assess if this auxin modulation involves a direct interaction between the hormone and the anion channel protein(s), or follows from the activation of a perception chain including abp1 homologues.