Overexpression of the Auxin Binding PROTEIN1 Modulates PIN-Dependent Auxin Transport in Tobacco Cells

Background

Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive.

Methodology/Principal Findings

Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations.

Conclusions/Significance

The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.     

Clathrin Light Chains Regulate Clathrin-Mediated Trafficking,Auxin Signaling,and Development in Arabidopsis

Plant clathrin-mediated membrane trafficking is involved in many developmental processes as well as in responses to environmental cues. Previous studies have shown that clathrin-mediated endocytosis of the plasma membrane (PM) auxin transporter PIN-FORMED1 is regulated by the extracellular auxin receptor AUXIN BINDING PROTEIN1 (ABP1). However, the mechanisms by which ABP1 and other factors regulate clathrin-mediated trafficking are poorly understood. Here, we applied a genetic strategy and time-resolved imaging to dissect the role of clathrin light chains (CLCs) and ABP1 in auxin regulation of clathrin-mediated trafficking in Arabidopsis thaliana. Auxin was found to differentially regulate the PM and trans-Golgi network/early endosome (TGN/EE) association of CLCs and heavy chains (CHCs) in an ABP1-dependent but TRANSPORT INHIBITOR RESPONSE1/AUXIN-BINDING F-BOX PROTEIN (TIR1/AFB)-independent manner. Loss of CLC2 and CLC3 affected CHC membrane association, decreased both internalization and intracellular trafficking of PM proteins, and impaired auxin-regulated endocytosis. Consistent with these results, basipetal auxin transport, auxin sensitivity and distribution, and root gravitropism were also found to be dramatically altered in clc2 clc3 double mutants, resulting in pleiotropic defects in plant development. These results suggest that CLCs are key regulators in clathrin-mediated trafficking downstream of ABP1-mediated signaling and thus play a critical role in membrane trafficking from the TGN/EE and PM during plant development.     

Altered Expression of Auxin-binding Protein 1 Affects Cell Expansion and Auxin Pool Size in Tobacco Cells

Auxin-binding protein 1 (ABP1) has an essential role in auxin-dependent cell expansion, but its mechanisms of action remain unknown. Our previous study showed that ABP1-mediated cell expansion is auxin concentration dependent. However, auxin distribution in plant tissue is heterogeneous, complicating the interpretation of ABP1 function. In this study, we used cells in culture that have altered expression of ABP1 to address the mechanism of ABP1 action at the cellular level, because cells in culture have homogeneous cell types and could potentially circumvent the heterogeneous auxin-distributions inherent in plant tissues. We found that cells overexpressing ABP1 had altered sensitivity to auxin and were larger, with nuclei that have undergone endoreduplication, a finding consistent with other data that support an auxin extracellular receptor role for ABP1. These cells also had a higher free auxin pool size, which cannot be explained by altered auxin transport. In cells lacking detectable ABP1, a higher rate of auxin metabolism was observed. The results suggest that ABP1 has, beyond its proposed role as an auxin extracellular receptor, a role in mediating auxin availability.     

The AUXIN BINDING PROTEIN 1 Is Required for Differential Auxin Responses Mediating Root Growth

Background

In plants, the phytohormone auxin is a crucial regulator sustaining growth and development. At the cellular level, auxin is interpreted differentially in a tissue- and dose-dependent manner. Mechanisms of auxin signalling are partially unknown and the contribution of the AUXIN BINDING PROTEIN 1 (ABP1) as an auxin receptor is still a matter of debate.

Methodology/Principal Findings

Here we took advantage of the present knowledge of the root biological system to demonstrate that ABP1 is required for auxin response. The use of conditional ABP1 defective plants reveals that the protein is essential for maintenance of the root meristem and acts at least on the D-type CYCLIN/RETINOBLASTOMA pathway to control entry into the cell cycle. ABP1 affects PLETHORA gradients and confers auxin sensitivity to root cells thus defining the competence of the cells to be maintained within the meristem or to elongate. ABP1 is also implicated in the regulation of gene expression in response to auxin.

Conclusions/Significance

Our data support that ABP1 is a key regulator for root growth and is required for auxin-mediated responses. Differential effects of ABP1 on various auxin responses support a model in which ABP1 is the major regulator for auxin action on the cell cycle and regulates auxin-mediated gene expression and cell elongation in addition to the already well known TIR1-mediated ubiquitination pathway.     

The auxin-binding pocket of auxin-binding protein 1 comprises the highly conserved boxes a and c

The auxin-binding protein 1 (ABP1) has already been proved to be an extracellular receptor of auxin in single cell systems. Protoplasts of maize coleoptiles respond to auxin with an increase in volume. The 2-naphthaleneacetic acid (2-NAA), an inactive auxin analog, acts as an anti-auxin in protoplast swelling, as it suppresses the effect of indole-3-acetic acid (IAA). Antibodies raised against box a of ABP1 induce protoplast swelling in the absence of auxin. This response is inhibited by pre-incubation with 2-NAA. The effect of 2-NAA on swelling induced by agonistic antibodies appears to depend on the binding characteristics of the antibody. ScFv12, an antibody directed against box a, box c and the C-terminal domain of ABP1 also exhibits auxin-agonist activity which is, however, not abolished by 2-NAA. Neither does 2-NAA affect the activity of the C-terminal peptide of ABP1, which is predicted to interact with putative binding proteins of ABP1. These results support the view that box a and box c of ABP1 are auxin-binding domains.     

Comparison of Site I auxin binding and a 22-kilodalton auxin-binding protein in maize

Several properties of a 43-kilodalton (kDa) auxin-binding protein (ABP) having 22-kDa subunits are shared by a class of auxin binding designated Site I. The spatial distribution of the ABP in the maize (Zea mays L.) mesocotyl corresponds with the distribution of growth induced by naphthalene-1-acetic acid and with the distribution of Site I binding as previously shown by J.D. Walton and P.M. Ray (1981, Plant Physiol. 68, 1334–1338). The greatest abundance of both ABP and Site I activity is at the apical region of the mesocotyl. The ABP and Site I activity co-migrate in isopycnic centrifugation with the endoplasmic-reticulum marker, cytochrome-c reductase. Red light, at low and high fluence, far-red and white light were used to alter the elongation rate of apical 1-cm sections of etiolated maize mesocotyls, the amount of auxin binding, and the abundance of the ABP. Relative changes in auxin binding and the ABP were correlated, but the growth rate was not always correlated with the abundance of the ABP.Abbreviations ABP auxin-binding protein - ER endoplasmic reticulum - FR far-red light - kDa kilodalton - NAA naphthalene-1-acetic acid - PM plasma membrane - R red light - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Auxin binding protein: curiouser and curiouser

Auxin is implicated in a variety of plant developmental processes, yet the molecular mechanism of auxin response remains largely unknown. Auxin binding protein 1 (ABP1) mediates cell expansion and might be involved in cell cycle control. Structural modeling shows that it is a β-barrel dimer, with the C terminus free to interact with other proteins. We do not know where ABP1 performs its receptor function. Most ABP1 is detected within the endoplasmic reticulum but the evidence indicates that it functions at the plasma membrane. ABP1 is established as a crucial component of auxin signaling, but its precise mechanism remains unclear.     

Plasmalemma hyperpolarity and culture of sense and antisenseabp transgenic tobacco protoplasts

The relationship among transfer and expression of auxin binding protein gene (abp), auxin (NAA)-induced plasmalemma hyperpolarity and sensibility to auxin during protoplast culture was studied by measuring transmembrane potential difference (Em) and culturing the protoplasts of sense and antisenseabp transgenic tobacco. The concentration of NAA inducing the highest degree of hyperpolarity of senseabp transgenic tobacco protoplasts was lower than the control, and in protoplast culture, their sensibility to auxin increased. The concentration of antisenseabp transgenic tobacco protoplasts was higher than the control, and in protoplast culture, their sensibility to auxin decreased. These results demonstrated that ABP synthesized in endoplasmic reticulum needed to transport to cell membrane and functioned there.     

Arabidopsis FAB1A/B is possibly involved in the recycling of auxin transporters

Fab1/PIKfyve produces Phosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) from Phosphatidylinositol-3-phosphate (PtdIns 3-P), and is involved not only in vacuole/lysosome homeostasis, but also in transporting various proteins to the vacuole or recycling proteins on the plasma membrane (PM) through the use of endosomes in a variety of eukaryotic cells. We previously demonstrated that Arabidopsis FAB1A/B functions as PtdIns-3,5-kinase in both Arabidopsis and fission yeast and plays a key role in vacuolar acidification and endocytosis. Although the conditional FAB1A/B knockdown mutant revealed an auxin-resistant phenotype to a membrane-impermeable auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), the mutant did not exhibit this phenotype to a membrane-permeable artificial auxin, naphthalene 1-acetic acid (NAA). The difference in the sensitivities to 2,4-D and NAA is similar to those of the auxin-resistant mutants such as aux1. Taken together, these results suggest that impairment of the function of Arabidopsis FAB1A/B might cause a defect in the membrane recycling capabilities of the auxin transporters and inhibit proper auxin transport into the cells in Arabidopsis.Key words: auxin signaling, auxin transporter, recycling of plasma membrane proteinsPhosphatidylinositol-3,5-bisphosphate (PtdIns (3,5) P₂) exists on the external membrane of multi-vesicular bodies (MVBs) at very low levels in eukaryotic cells,^1,2 and plays key roles in endomembrane homeostasis including endocytosis, vacuole/lysosome formation and vacuolar acidification.^1,3 PtdIns (3,5) P₂ deficiency causes an enlarged vacuolar structure in yeast and mammalian cells.^4,5 FAB1 forms a protein complex with its regulatory molecules, and synthesizes PtdIns (3,5) P₂ from PtdIns 3P.^6–9 In Arabidopsis, there are four Fab1/PIKfyve orthologs (FAB1A, FAB1B, FAB1C and FAB1D) in the genome, and the double homozygous mutant of FAB1A and FAB1B exhibited the male gametophyte lethal phenotype.¹⁰ Previously, we reported that conditional loss-of-function and gain-of-function mutants of FAB1A/B impair endomembrane homeostasis and reveal various developmental phenotypes.¹¹ Interestingly, lateral root formation by exogenous auxin, which is known as a typical auxin-responsive phenotype, was largely impaired when FAB1A/B expression was conditionally downregulated or upregulated. From these results, we speculated that the defect in the endocytosis process in fab1a/b mutants might inhibit the precise recycling process of auxin transporters on the PM, thereby inhibiting proper auxin transport into the plant cells.¹¹ In this report, we tested this hypothesis to assess the sensitivity on auxin-dependent lateral root formation to a membrane permeable auxin, NAA, in the fab1a/b knockdown mutant.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

ARF-GTPase as a Molecular Switch for Polar Auxin Transport Mediated by Vesicle Trafficking in Root Development

Polar auxin transport (PAT), which is controlled precisely by both auxin efflux and influx facilitators and mediated by the cell trafficking system, modulates organogenesis, development and root gravitropism. ADP-ribosylation factor (ARF)-GTPase protein is catalyzed to switch to the GTP-bound type by a guanine nucleotide exchange factor (GEF) and promoted for hybridization to the GDP-bound type by a GTPase-activating protein (GAP). Previous studies showed that auxin efflux facilitators such as PIN1 are regulated by GNOM, an ARF-GEF, in Arabidopsis. In the November issue of The Plant Journal, we reported that the auxin influx facilitator AUX1 was regulated by ARF-GAP via the vesicle trafficking system.¹ In this addendum, we report that overexpression of OsAGAP leads to enhanced root gravitropism and propose a new model of PAT regulation: a loop mechanism between ARF-GAP and GEF mediated by vesicle trafficking to regulate PAT at influx and efflux facilitators, thus controlling root development in plants.Key Words: ADP-ribosylation factor (ARF), ARF-GAP, ARF-GEF, auxin, GNOM, polar transport of auxinPolar auxin transport (PAT) is a unique process in plants. It results in alteration of auxin level, which controls organogenesis and development and a series of physiological processes, such as vascular differentiation, apical dominance, and tropic growth.² Genetic and physiological studies identified that PAT depends on efflux facilitators such as PIN family proteins and influx facilitators such as AUX1 in Arabidopsis.Eight PIN family proteins, AtPIN1 to AtPIN8, exist in Arabidopsis. AtPIN1 is located at the basal side of the plasma membrane in vascular tissues but is weak in cortical tissues, which supports the hypothesis of chemical pervasion.³ AtPIN2 is localized at the apical side of epidermal cells and basally in cortical cells.^1,4 GNOM, an ARF GEF, modulates the localization of PIN1 and vesicle trafficking and affects root development.^5,6 The PIN auxin-efflux facilitator network controls root growth and patterning in Arabidopsis.⁴ As well, asymmetric localization of AUX1 occurs in the root cells of Arabidopsis plants,⁷ and overexpression of OsAGAP interferes with localization of AUX1.¹ Our data support that ARF-GAP mediates auxin influx and auxin-dependent root growth and patterning, which involves vesicle trafficking.¹ Here we show that OsAGAP overexpression leads to enhanced gravitropic response in transgenic rice plants. We propose a model whereby ARF GTPase is a molecular switch to control PAT and root growth and development.Overexpression of OsAGAP led to reduced growth in primary or adventitious roots of rice as compared with wild-type rice.¹ Gravitropism assay revealed transgenic rice overxpressing OsAGAP with a faster response to gravity than the wild type during 24-h treatment. However, 1-naphthyl acetic acid (NAA) treatment promoted the gravitropic response of the wild type, with no difference in response between the OsAGAP transgenic plants and the wild type plants (Fig. 1). The phenotype of enhanced gravitropic response in the transgenic plants was similar to that in the mutants atmdr1-100 and atmdr1-100/atpgp1-100 related to Arabidopsis ABC (ATP-binding cassette) transporter and defective in PAT.⁸ The physiological data, as well as data on localization of auxin transport facilitators, support ARF-GAP modulating PAT via regulating the location of the auxin influx facilitator AUX1.¹ So the alteration in gravitropic response in the OsAGAP transgenic plants was explained by a defect in PAT.Open in a separate windowFigure 1Gravitropism of OsAGAP overexpressing transgenic rice roots and response to 1-naphthyl acetic acid (NAA). (A) Gravitropism phenotype of wild type (WT) and OsAGAP overexpressing roots at 6 hr gravi-stimulation (top panel) and 0 hr as a treatment control (bottom panel). (B) Time course of gravitropic response in transgenic roots. (C and D) results correspond to those in (A and B), except for treatment with NAA (5 × 10⁻⁷ M).The polarity of auxin transport is controlled by the asymmetric distribution of auxin transport proteins, efflux facilitators and influx carriers. ARF GTPase is a key member in vesicle trafficking system and modulates cell polarity and PAT in plants. Thus, ARF-GDP or GTP bound with GEF or GAP determines the ARF function on auxin efflux facilitators (such as PIN1) or influx ones (such as AUX1).ARF1, targeting ROP2 and PIN2, affects epidermal cell polarity.⁹ GNOM is involved in the regulation of PIN1 asymmetric localization in cells and its related function in organogenesis and development.⁶ Although VAN3, an ARF-GAP in Arabidopsis, is located in a subpopulation of the trans-Golgi transport network (TGN), which is involved in leaf vascular network formation, it does not affect PAT.¹⁰ OsAGAP possesses an ARF GTPase-activating function in rice.¹¹ Specifically, our evidence supports that ARF-GAP bound with ARF-GTP modulates PAT and gravitropism via AUX1, mediated by vesicle trafficking, including the Golgi stack.¹Therefore, we propose a loop mechanism between ARF-GAP and GEF mediated by the vascular trafficking system in regulating PAT at influx and efflux facilitators, which controls root development and gravitropism in plants (Fig. 2). Here we emphasize that ARF-GEF catalyzes a conversion of ARF-bound GDP to GTP, which is necessary for the efficient delivery of the vesicle to the target membrane.¹² An opposite process of ARF-bound GDP to GTP is promoted by ARF-GTPase-activating protein via binding. A loop status of ARF-GTP and ARF-GDP bound with their appurtenances controls different auxin facilitators and regulates root development and gravitropism.Open in a separate windowFigure 2Model for ARF GTPase as a molecular switch for the polar auxin transport mediated by the vesicle traffic system.     

Molecular analysis of auxin-specific signal transduction

The auxin-binding protein (ABP1) of maize has been purified, cloned and sequenced. Homologues have been found in a wide range of plants and at least seven ABP sequences from four different species are now known. We have developed a range of anti-ABP antibodies and these have been applied to analysis of the structure, localization and receptor function of ABP. ABP1 is a glycoprotein with two identical subunits of apparent M _r =22 kDa. The regions recognised by our five monoclonal antibodies (MAC 256–260) and by polyclonal antisera from our own and other laboratories have been specified by epitope mapping and fragmentation studies. All polyclonal anti-ABP sera recognise two or three dominant epitopes around the single glycosylation site. Two monoclonals (MAC 256, 259) are directed at the endoplasmic reticulum (ER) retention sequence KDEL at the C-terminus. Early biochemical data pointed to six amino acids likely to be involved in the auxin binding site. Inspection of the deduced sequence of ABP1 showed a hexapeptide (HRHSCE) containing five of these residues. Antibodies were raised against a polypeptide embracing this region and recognised ABP homologs in many species, suggesting that the region is highly conserved. This is confirmed by more recent information showing that the selected polypeptide contains the longest stretch of wholly conserved sequence in ABP1. Most strikingly, the antibodies show auxin agonist activity against protoplasts in three different electrophysiological systems-hyperpolarization of tobacco transmembrane potential; stimulation of outward ATP-dependent H⁺ current in maize; modulation of anion channels in tobacco. The biological activity of these antibodies indicates that the selected peptide does form a functionally important part of the auxin binding site and strongly supports a role for ABP1 as an auxin receptor. Although ABP contains a KDEL sequence and is located mainly in the ER lumen, the electrophysiological evidence shows clearly that some ABP must reach the outer face of the plasma membrane. One possible mechanism is suggested by our earlier demonstration that the ABP C-terminus recognised by MAC 256 undergoes an auxin-induced conformational change, masking the KDEL epitope and it is of interest that this C-terminal region appears to be important in auxin signalling [22]. So far we have been unable to detect the secretion of ABP into the medium of maize cell (bms) cultures reported by Jones and Herman [7]. However, recent silver enhanced immunogold studies on maize protoplasts have succeeded in visualizing ABP at the cell surface, as well as auxin-specific clustering of the signal induced within 30 minutes. The function of ABP in the ER, as well as the mechanisms of auxin signal transduction both at plasma membrane and gene levels remain to be elucidated.     

A Novel Little Membrane Protein Confers Salt Tolerance in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.