PINOID positively regulates auxin efflux in Arabidopsis root hair cells and tobacco cells

Intercellular transport of auxin is mediated by influx and efflux carriers in the plasma membrane and subjected to developmental and environmental regulation. Here, using the auxin-sensitive Arabidopsis thaliana root hair cell system and the tobacco (Nicotiana tabacum) suspension cell system, we demonstrate that the protein kinase PINOID (PID) positively regulates auxin efflux. Overexpression of PID (PIDox) or the auxin efflux carrier component PINFORMED3 (PIN3, PIN3ox), specifically in the root hair cell, greatly suppressed root hair growth. In both PIDox and PIN3ox transformants, root hair growth was nearly restored to wild-type levels by the addition of auxin, protein kinase inhibitors, or auxin efflux inhibitors. Localization of PID or PIN3 at the cell boundary was disrupted by brefeldin A and staurosporine. A mutation in the kinase domain abrogated the ability of PID to localize at the cell boundary and to inhibit root hair growth. These results suggest that PIDox- or PIN3ox-enhanced auxin efflux results in a shortage of intracellular auxin and a subsequent inhibition of root hair growth. In an auxin efflux assay using transgenic tobacco suspension cells, PIDox or PIN3ox also enhanced auxin efflux. Collectively, these results suggest that PID positively regulates cellular auxin efflux, most likely by modulating the trafficking of PIN and/or some other molecular partners involved in auxin efflux.     

PGP4, an ATP binding cassette P-glycoprotein, catalyzes auxin transport in Arabidopsis thaliana roots

Members of the ABC (for ATP binding cassette) superfamily of integral membrane transporters function in cellular detoxification, cell-to-cell signaling, and channel regulation. More recently, members of the multidrug resistance P-glycoprotein (MDR/PGP) subfamily of ABC transporters have been shown to function in the transport of the phytohormone auxin in both monocots and dicots. Here, we report that the Arabidopsis thaliana MDR/PGP PGP4 functions in the basipetal redirection of auxin from the root tip. Reporter gene studies showed that PGP4 was strongly expressed in root cap and epidermal cells. PGP4 exhibits apolar plasma membrane localization in the root cap and polar localization in tissues above. Root gravitropic bending and elongation as well as lateral root formation were reduced in pgp4 mutants compared with the wild type. pgp4 exhibited reduced basipetal auxin transport in roots and a small decrease in shoot-to-root transport consistent with a partial loss of the redirective auxin sink in the root. Seedlings overexpressing PGP4 exhibited increased shoot-to-root auxin transport. Heterologous expression of PGP4 in mammalian cells resulted in 1-N-naphthylthalamic acid-reversible net uptake of [3H]indole-3-acetic acid. These results indicate that PGP4 functions primarily in the uptake of redirected or newly synthesized auxin in epidermal root cells.     

Cytoplasm localization of aminopeptidase M1 and its functional activity in root hair cells and BY-2 cells

Aminopeptidase M1 (APM1) was the first M1 metallopeptidase family member identified in Arabidopsis, isolated by its affinity for the auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). A loss-of-function mutation showed various developmental defects in cell division and auxin transport. APM1 was shown to be localized in endomembrane structures, the cytoplasm, and the plasma membrane. These previous results suggested that APM1 has diverse functional roles in different cell and tissue types. Here we report that APM1 localized to the cytoplasm, and its over-expression in the root hair cell caused longer root hair phenotypes. Treatment of aminopeptidase inhibitors caused internalization of auxin efflux PIN-FORMED proteins in root hair cells and suppressed short root hair phenotype of PIN3 overexpression line (PIN3ox). APM1 also localized to the cytoplasm in tobacco BY-2 cells, its over-expression had little effect on auxin transport in these cells.     

Molecular players of auxin transport systems: advances in genomic and molecular events*

Phytohormone auxin plays an indispensable role in the plethora of plant developmental process starting from the cell division, and cell elongation to morphogenesis. Auxins are transported to different parts of the plant by different sophisticated transporter molecules known as ‘auxin transporters’.There are four auxin transporter families that have been reported so far in the plant kingdom which includes AUX/LAX (AUXIN-RESISTANT1–LIKES), PIN (PIN-FORMED, auxin efflux carriers), ABCB ((ATP-binding cassette-B (ABCB)/P-glycoprotein (PGP)) and PILS (PIN-Likes). Auxin influx and efflux carriers are distributed in a polar fashion in the plasma membrane whereas ABCB and PILS are present in a non-polar fashion. Other than AUX/LAX, other auxin transporters harbor N-and C-terminal conserved domains along with a variable hydrophilic loop in the transmembrane domain. The AUX/LAX, ABCB and PIN transporters mediate long distance auxin transport whereas PILS and PIN5 protein involved in intracellular auxin homeostasis.     

ATP-binding cassette B4, an auxin-efflux transporter, stably associates with the plasma membrane and shows distinctive intracellular trafficking from that of PIN-FORMED proteins

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.     

The ER-localized TWD1 immunophilin is necessary for localization of multidrug resistance-like proteins required for polar auxin transport in Arabidopsis roots

Multidrug resistance ABC transporters in plants are required for polar transport of the hormone auxin (indole-3-acetic acid). They are studied in animals primarily because their overexpression confers resistance to anticancer agents. Immunophilins are studied in both plants and animals for their roles in folding and trafficking of proteins, particularly those with signal transducing functions and susceptibility to immunosuppressant drugs. Previous genetic and molecular studies in Arabidopsis thaliana established a physical and functional interaction between some ABCB transporters and the TWISTED DWARF1 (TWD1) immunophilin. In this work, confocal microscopy of fluorescently tagged TWD1 shows it to reside at the endoplasmic reticulum (ER). Mutations in TWD1 caused mislocalization of ABCB1, ABCB4, and ABCB19 to the ER instead of the plasma membrane as shown by confocal microscopy of fluorescently tagged fusion proteins and transmission electron microscopy of immunogold-labeled samples in the case of ABCB19. Localization of the unrelated PIN-FORMED2 auxin transporter or plasma membrane marker proteins was not affected by loss of TWD1. Abnormal spread of auxin signaling into the elongation zone of twd1 roots, attributable to mislocalized ABCB transporters and suppressed by an auxin transport inhibitor, appeared to cause the twisted cell files characteristic of twd1 roots.     

Role of the Arabidopsis PIN6 Auxin Transporter in Auxin Homeostasis and Auxin-Mediated Development

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin–regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.     

The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

Arabidopsis ATP-binding cassette B4 (ABCB4) is a root-localised auxin efflux transporter with reported auxin uptake activity in low auxin concentrations. Results reported here demonstrate that ABCB4 is a substrate-activated regulator of cellular auxin levels. The contribution of ABCB4 to shootward auxin movement at the root apex increases with auxin concentration, but in root hair elongation assays ABCB4-mediated uptake is evident at low concentrations as well. Uptake kinetics of ABCB4 heterologously expressed in Schizosaccharomyces pombe differed from the saturation kinetics of AUX1 as uptake converted to efflux at threshold indole-3-acetic acid (IAA) concentrations. The concentration dependence of ABCB4 appears to be a direct effect on transporter activity, as ABCB4 expression and ABCB4 plasma membrane (PM) localisation at the root apex are relatively insensitive to changes in auxin concentration. However, PM localization of ABCB4 decreases with 1-naphthylphthalamic acid (NPA) treatment. Unlike other plant ABCBs studied to date, and consistent with decreased detergent solubility, ABCB4(pro) :ABCB4-GFP is partially internalised in all cell types by 0.05% DMSO, but not 0.1% ethanol. In trichoblasts, ABCB4(pro) :ABCB4-GFP PM signals are reduced by >200 nm IAA and 2,4-dichlorophenoxyacetic acid (2,4-D). In heterologous systems and in planta, ABCB4 transports benzoic acid with weak affinity, but not the oxidative catabolism products 2-oxindole-3-acetic-acid and 2-oxindole-3-acetyl-β-D-glucose. ABCB4 mediates uptake, but not efflux, of the synthetic auxin 2,4-D in cells lacking AUX1 activity. Results presented here suggest that 2,4-D is a non-competitive inhibitor of IAA transport by ABCB4 and indicate that ABCB4 is a target of 2,4-D herbicidal activity.     

The ABC of auxin transport: the role of p-glycoproteins in plant development

A surprising outcome of the Arabidopsis genome project was the annotation of a large number of sequences encoding members of the ABC transporter superfamily, including 22 genes encoding the p-glycoprotein (PGP) subfamily. As mammalian PGP orthologs are associated with multiple drug resistance, plant PGPs were initially presumed to function in detoxification, but were soon seen to have a developmental role. Here, we summarise recent studies of plant PGPs indicating that PGPs mediate the cellular and long-distance transport of the plant hormone auxin. One class of PGPs, represented by AtPGP1, catalyze auxin export, while another class with at least one member, AtPGP4, appears to function in auxin import. Current models on the physiological role of PGPs, their functional interaction and their involvement in cell-to cell (polar) auxin transport are discussed.     

Over-expression of OsAGAP, an ARF-GAP, interferes with auxin influx, vesicle trafficking and root development

Development and organogenesis in both dicot and monocot plants are highly dependent on polar auxin transport (PAT), which requires the proper asymmetric localization of both auxin influx and efflux carriers. In the model dicot plant Arabidopsis thaliana, the trafficking and localization of auxin efflux facilitators such as PIN-FORMED1 (PIN1) are mediated by GNOM, a guanine-nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, but molecular regulators of the auxin influx facilitators remain unknown. Here, we show that over-expression of OsAGAP, an ARF-GTPase-activating protein (ARF-GAP) in rice, impaired PAT and interfered with both primary and lateral root development. The lateral root phenotype could be rescued by the membrane-permeable auxin 1-naphthyl acetic acid, but not by indole 3-acetic acid (IAA) or by 2,4-dichloro-phenoxyacetic acid, which require influx facilitators to enter the cells. OsAGAP-over-expressing plants had alterations in vesicle trafficking and localization of the presumptive A. thaliana auxin-influx carrier AUX1, but not in the localization of the auxin efflux facilitators. Together, our data suggest that OsAGAP has a specific role in regulating vesicle trafficking pathways such as the auxin influx pathway, which in turn controls auxin-dependent root growth in plants.     

Mutations in Arabidopsis multidrug resistance-like ABC transporters separate the roles of acropetal and basipetal auxin transport in lateral root development

Auxin affects the shape of root systems by influencing elongation and branching. Because multidrug resistance (MDR)-like ABC transporters participate in auxin transport, they may be expected to contribute to root system development. This reverse genetic study of Arabidopsis thaliana roots shows that MDR4-mediated basipetal auxin transport did not affect root elongation or branching. However, impaired acropetal auxin transport due to mutation of the MDR1 gene caused 21% of nascent lateral roots to arrest their growth and the remainder to elongate 50% more slowly than the wild type. Reporter gene analyses indicated a severe auxin deficit in the apex of mdr1 but not mdr4 lateral roots. The mdr1 deficit was explained by 40% less acropetal auxin transport within the mdr1 lateral roots. The slow elongation of mdr1 lateral roots was rescued by auxin and phenocopied in the wild type by an inhibitor of polar auxin transport. Confocal microscopy analysis of a functional green fluorescent protein-MDR1 translational fusion showed the protein to be auxin inducible and present in the tissues responsible for acropetal transport in the primary root. The protein also accumulated in lateral root primordia and later in the tissues responsible for acropetal transport within the lateral root, fully supporting the conclusion that auxin levels established by MDR1-dependent acropetal transport control lateral root growth rate to influence root system architecture.     

Cellular efflux of auxin catalyzed by the Arabidopsis MDR/PGP transporter AtPGP1

Directional transport of the phytohormone auxin is required for the establishment and maintenance of plant polarity, but the underlying molecular mechanisms have not been fully elucidated. Plant homologs of human multiple drug resistance/P-glycoproteins (MDR/PGPs) have been implicated in auxin transport, as defects in MDR1 (AtPGP19) and AtPGP1 result in reductions of growth and auxin transport in Arabidopsis (atpgp1, atpgp19), maize (brachytic2) and sorghum (dwarf3). Here we examine the localization, activity, substrate specificity and inhibitor sensitivity of AtPGP1. AtPGP1 exhibits non-polar plasma membrane localization at the shoot and root apices, as well as polar localization above the root apex. Protoplasts from Arabidopsis pgp1 leaf mesophyll cells exhibit reduced efflux of natural and synthetic auxins with reduced sensitivity to auxin efflux inhibitors. Expression of AtPGP1 in yeast and in the standard mammalian expression system used to analyze human MDR-type proteins results in enhanced efflux of indole-3-acetic acid (IAA) and the synthetic auxin 1-naphthalene acetic acid (1-NAA), but not the inactive auxin 2-NAA. AtPGP1-mediated efflux is sensitive to auxin efflux and ABC transporter inhibitors. As is seen in planta, AtPGP1 also appears to mediate some efflux of IAA oxidative breakdown products associated with apical sites of high auxin accumulation. However, unlike what is seen in planta, some additional transport of the benzoic acid is observed in yeast and mammalian cells expressing AtPGP1, suggesting that other factors present in plant tissues confer enhanced auxin specificity to PGP-mediated transport.     

Arabidopsis TRAPPII is functionally linked to Rab-A,but not Rab-D in polar protein trafficking in trans-Golgi network

The trans-Golgi network (TGN) in plant cells is an independent organelle, displaying rapid association and dissociation with Golgi bodies. In plant cells, the TGN is the site where secretory and endocytic membrane trafficking meet. Cell wall components, signaling molecules and auxin transporters have been found to undergo intracellular trafficking around the TGN. However, how different trafficking pathways are regulated and how different cargoes are sorted in the TGN is poorly defined in plant cells. Using a combined approach of genetic and in vivo imaging, we recently demonstrated that Arabidopsis TRAPPII acts in the TGN and is required for polar targeting of PIN2, but not PIN1, auxin efflux carrier in root tip cells. Here, we report that, TRAPPII in Arabidopsis is required for polar distribution of AUX1, an auxin influx carrier in protophloem cells and epidermal cells of Arabidopsis root tips. In yeast cells, TRAPPII serves as a guanine-nucleotide exchange factor (GEF) for Ypt1 and Ypt31/32 in late Golgi trafficking, while in mammalian cells, TRAPPII acts as a GEF for Rab1 (homolog of yeast Ypt1) in early Golgi trafficking. We show here that TRAPPII in Arabidopsis is functionally linked to Rab-A proteins, homologs of yeast Ypt31/32, but not Rab-D proteins, homologs of yeast Ypt1 and animal Rab1 proteins.     

Phospholipase A2 Is Required for PIN-FORMED Protein Trafficking to the Plasma Membrane in the Arabidopsis Root

Phospholipase A₂ (PLA₂), which hydrolyzes a fatty acyl chain of membrane phospholipids, has been implicated in several biological processes in plants. However, its role in intracellular trafficking in plants has yet to be studied. Here, using pharmacological and genetic approaches, the root hair bioassay system, and PIN-FORMED (PIN) auxin efflux transporters as molecular markers, we demonstrate that plant PLA₂s are required for PIN protein trafficking to the plasma membrane (PM) in the Arabidopsis thaliana root. PLA₂α, a PLA₂ isoform, colocalized with the Golgi marker. Impairments of PLA₂ function by PLA₂α mutation, PLA₂-RNA interference (RNAi), or PLA₂ inhibitor treatments significantly disrupted the PM localization of PINs, causing internal PIN compartments to form. Conversely, supplementation with lysophosphatidylethanolamine (the PLA₂ hydrolytic product) restored the PM localization of PINs in the pla₂α mutant and the ONO-RS-082–treated seedling. Suppression of PLA₂ activity by the inhibitor promoted accumulation of trans-Golgi network vesicles. Root hair–specific PIN overexpression (PINox) lines grew very short root hairs, most likely due to reduced auxin levels in root hair cells, but PLA₂ inhibitor treatments, PLA₂α mutation, or PLA₂-RNAi restored the root hair growth of PINox lines by disrupting the PM localization of PINs, thus reducing auxin efflux. These results suggest that PLA₂, likely acting in Golgi-related compartments, modulates the trafficking of PIN proteins.     

Cell polarity signaling in Arabidopsis involves a BFA-sensitive auxin influx pathway

Coordination of cell and tissue polarity commonly involves directional signaling. In the Arabidopsis root epidermis, cell polarity is revealed by basal, root tip-oriented, hair outgrowth from hair-forming cells (trichoblasts). The plant hormone auxin displays polar movements and accumulates at maximum concentration in the root tip. The application of polar auxin transport inhibitors evokes changes in trichoblast polarity only at high concentrations and after long-term application. Thus, it remains open whether components of the auxin transport machinery mediate establishment of trichoblast polarity. Here we report that the presumptive auxin influx carrier AUX1 contributes to apical-basal hair cell polarity. AUX1 function is required for polarity changes induced by exogenous application of the auxin 2,4-D, a preferential influx carrier substrate. Similar to aux1 mutants, the vesicle trafficking inhibitor brefeldin A (BFA) interferes with polar hair initiation, and AUX1 function is required for BFA-mediated polarity changes. Consistently, BFA inhibits membrane trafficking of AUX1, trichoblast hyperpolarization induced by 2,4-D, and alters the distal auxin maximum. Our results identify AUX1 as one component of a novel BFA-sensitive auxin transport pathway polarizing cells toward a hormone maximum.     

Analysis of subcellular localization of auxin carriers PIN,AUX/LAX and PGP in Sorghum bicolor

Auxin transport at least correlates to the three gene families: efflux carriers PIN-formed (PIN), p-glycoprotein (PGP), and influx carrier auxin resistant 1/like aux1(AUX/LAX) in Arabidopsis thaliana. In monocotyledon Sorghum bicolor, the biological function of these genes retains unclear. Our previous study reported that the member analysis, organ-specific expression and expression profiles of the auxin transporter PIN, PGP and AUX/LAX gene families in Sorghum bicolor under IAA, brassinosteroid, polar auxin transport inhibitors and abiotic stresses. Here we further supply the prediction of subcellular localization of SbPIN, SbLAX and SbPGP proteins and discuss the potential relationship between the subcellular localization and stress response. The predicted results showed that the most of SbPIN, SbLAX and SbPGP proteins are localized to the plasma membrane, except few localized to vacuolar membrane and endoplasmic reticulum. This data set provides novel information for investigation of auxin transporters in Sorghum bicolor.     

MDR-like ABC transporter AtPGP4 is involved in auxin-mediated lateral root and root hair development

Previous data have suggested an involvement of MDR/PGP-like ABC transporters in transport of the plant hormone auxin and, recently, AtPGP1 has been demonstrated to catalyze the primary active export of auxin. Here we show that related isoform AtPGP4 is expressed predominantly during early root development. AtPGP4 loss-of-function plants reveal enhanced lateral root initiation and root hair lengths both known to be under the control of auxin. Further, atpgp4 plants show altered sensitivities toward auxin and the auxin transport inhibitor, NPA. Finally, mutant roots reveal elevated free auxin levels and reduced auxin transport capacities. These results together with yeast growth assays suggest a direct involvement of AtPGP4 in auxin transport processes controlling lateral root and root hair development.     

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.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.