TWISTED DWARF1, a unique plasma membrane-anchored immunophilin-like protein, interacts with Arabidopsis multidrug resistance-like transporters AtPGP1 and AtPGP19

Null-mutations of the Arabidopsis FKBP-like immunophilin TWISTED DWARF1 (TWD1) gene cause a pleiotropic phenotype characterized by reduction of cell elongation and disorientated growth of all plant organs. Heterologously expressed TWD1 does not exhibit cis-trans-peptidylprolyl isomerase (PPIase) activity and does not complement yeast FKBP12 mutants, suggesting that TWD1 acts indirectly via protein-protein interaction. Yeast two-hybrid protein interaction screens with TWD1 identified cDNA sequences that encode the C-terminal domain of Arabidopsis multidrug-resistance-like ABC transporter AtPGP1. This interaction was verified in vitro. Mapping of protein interaction domains shows that AtPGP1 surprisingly binds to the N-terminus of TWD1 harboring the cis-trans peptidyl-prolyl isomerase-like domain and not to the tetratrico-peptide repeat domain, which has been shown to mediate protein-protein interaction. Unlike all other FKBPs, TWD1 is shown to be an integral membrane protein that colocalizes with its interacting partner AtPGP1 on the plasma membrane. TWD1 also interacts with AtPGP19 (AtMDR1), the closest homologue of AtPGP1. The single gene mutation twd1-1 and double atpgp1-1/atpgp19-1 (atmdr1-1) mutants exhibit similar phenotypes including epinastic growth, reduced inflorescence size, and reduced polar auxin transport, suggesting that a functional TWD1-AtPGP1/AtPGP19 complex is required for proper plant development.     

Immunophilin-like TWISTED DWARF1 modulates auxin efflux activities of Arabidopsis P-glycoproteins

The immunophilin-like protein TWISTED DWARF1 (TWD1/FKBP42) has been shown to physically interact with the multidrug resistance/P-glycoprotein (PGP) ATP-binding cassette transporters PGP1 and PGP19 (MDR1). Overlapping phenotypes of pgp1/pgp19 and twd1 mutant plants suggested a positive regulatory role of TWD1 in PGP-mediated export of the plant hormone auxin, which controls plant development. Here, we provide evidence at the cellular and plant levels that TWD1 controls PGP-mediated auxin transport. twd1 and pgp1/pgp19 cells showed greatly reduced export of the native auxin indole-3-acetic acid (IAA). Constitutive overexpression of PGP1 and PGP19, but not TWD1, enhanced auxin export. Coexpression of TWD1 and PGP1 in yeast and mammalian cells verified the specificity of the regulatory effect. Employing an IAA-specific microelectrode demonstrated that IAA influx in the root elongation zone was perturbed and apically shifted in pgp1/pgp19 and twd1 roots. Mature roots of pgp1/pgp19 and twd1 plants revealed elevated levels of free IAA, which seemed to account for agravitropic root behavior. Our data suggest a novel mode of PGP regulation via FK506-binding protein-like immunophilins, implicating possible alternative strategies to overcome multidrug resistance.     

AtMRP2, an Arabidopsis ATP binding cassette transporter able to transport glutathione S-conjugates and chlorophyll catabolites: functional comparisons with Atmrp1.

Three ATP binding cassette (ABC) transporter-like activities directed toward large amphipathic organic anions have recently been identified on the vacuolar membrane of plant cells. These are the Mg-ATP-energized, vanadate-inhibitable vacuolar accumulation of glutathione S-conjugates (GS conjugates), chlorophyll catabolites, and bile acids, respectively. Although each of these activities previously had been assigned to distinct pumps in native plant membranes, we describe here the molecular cloning, physical mapping, and heterologous expression of a gene, AtMRP2, from Arabidopsis thaliana that encodes a multispecific ABC transporter competent in the transport of both GS conjugates and chlorophyll catabolites. Unlike its isoform, AtMRP1, which transports the model Brassica napus chlorophyll catabolite transporter substrate Bn-NCC-1 at low efficiency, heterologously expressed AtMRP2 has the facility for simultaneous high-efficiency parallel transport of GS conjugates and Bn-NCC-1. The properties of AtMRP2 therefore establish a basis for the manipulation of two previously identified plant ABC transporter activities and provide an explanation for how the comparable transporter in native plant membranes would be systematically mistaken for two distinct transporters. These findings are discussed with respect to the functional organization of AtMRP2, the inability of AtMRP2 and AtMRP1 to transport the model bile acid transporter substrate taurocholate (despite the pronounced sensitivity of both to direct inhibition by this agent), the differential patterns of expression of their genes in the intact plant, and the high capacity of AtMRP2 for the transport of glutathionated herbicides and anthocyanins.     

The Arabidopsis ATP-binding Cassette Protein AtMRP5/AtABCC5 Is a High Affinity Inositol Hexakisphosphate Transporter Involved in Guard Cell Signaling and Phytate Storage

Arabidopsis possesses a superfamily of ATP-binding cassette (ABC) transporters. Among these, the multidrug resistance-associated protein AtMRP5/AtABCC5 regulates stomatal aperture and controls plasma membrane anion channels of guard cells. Remarkably, despite the prominent role of AtMRP5 in conferring partial drought insensitivity upon Arabidopsis, we know little of the biochemical function of AtMRP5. Our phylogenetic analysis showed that AtMRP5 is closely related to maize MRP4, mutation of which confers a low inositol hexakisphosphate kernel phenotype. We now show that insertion mutants of AtMRP5 display a low inositol hexakisphosphate phenotype in seed tissue and that this phenotype is associated with alterations of mineral cation and phosphate status. By heterologous expression in yeast, we demonstrate that AtMRP5 encodes a specific and high affinity ATP-dependent inositol hexakisphosphate transporter that is sensitive to inhibitors of ABC transporters. Moreover, complementation of the mrp5-1 insertion mutants of Arabidopsis with the AtMRP5 cDNA driven from a guard cell-specific promoter restores the sensitivity of the mutant to abscisic acid-mediated inhibition of stomatal opening. Additionally, we show that mutation of residues of the Walker B motif prevents restoring the multiple phenotypes associated with mrp5-1. Our findings highlight a novel function of plant ABC transporters that may be relevant to other kingdoms. They also extend the signaling repertoire of this ubiquitous inositol polyphosphate signaling molecule.     

Disruption of AtMRP4, a guard cell plasma membrane ABCC-type ABC transporter, leads to deregulation of stomatal opening and increased drought susceptibility

ATP-binding cassette (ABC) transporters are membrane proteins responsible for cellular detoxification processes in plants and animals. Recent evidence shows that this class of transporters may also be involved in many other cellular processes. Because of their homology with human multidrug resistance-associated proteins (MRP), cystic fibrosis transmembrane conductance regulator (CFTR) and sulfonylurea receptor (SUR), some plant ABC transporters have been implicated in the regulation of ion channel activities. This paper describes an investigation of the AtMRP4 gene and its role in stomatal regulation. Reporter gene studies showed that AtMRP4 is highly expressed in stomata and that the protein is localized to the plasma membrane. Stomatal aperture in three independent atmrp4 mutant alleles was larger than in wild-type plants, both in the light and in the dark, resulting in increased water loss but no change in the photosynthetic rate. In baker's yeast, AtMRP4 shows ATP-dependent, vanadate-sensitive transport of methotrexate (MTX), an antifolate and a substrate of mammalian MRPs. Treatment with MTX reduced stomatal opening in wild-type plants, but had no effect in atmrp4 mutants. These results indicate the involvement of AtMRP4 in the complex regulation of stomatal aperture.     

Transport of antimony salts by Arabidopsis thaliana protoplasts over-expressing the human multidrug resistance-associated protein 1 (MRP1/ABCC1)

ABC transporters from the multidrug resistance-associated protein (MRP) subfamily are glutathione S-conjugate pumps exhibiting a broad substrate specificity illustrated by numerous xenobiotics, such as anticancer drugs, herbicides, pesticides and heavy metals. The engineering of MRP transporters into plants might be interesting either to reduce the quantity of xenobiotics taken up by the plant in the context of “safe-food” strategies or, conversely, in the development of phytoremediation strategies in which xenobiotics are sequestered in the vacuolar compartment. In this report, we obtained Arabidopsis transgenic plants overexpressing human MRP1. In these plants, expression of MRP1 did not increase plant resistance to antimony salts (Sb(III)), a classical glutathione-conjugate substrate of MRP1. However, the transporter was fully translated in roots and shoots, and targeted to the plasma membrane. In order to investigate the functionality of MRP1 in Arabidopsis, mesophyll cell protoplasts (MCPs) were isolated from transgenic plants and transport activities were measured by using calcein or Sb(III) as substrates. Expression of MRP1 at the plasma membrane was correlated with an increase in the MCPs resistance to Sb(III) and a limitation of the metalloid content in the protoplasts due to an improvement in Sb(III) efflux. Moreover, Sb(III) transport was sensitive to classical inhibitors of the human MRP1, such as MK571 or glibenclamide. These results demonstrate that a human ABC transporter can be functionally introduced in Arabidopsis, which might be useful, with the help of stronger promoters, to reduce the accumulation of xenobiotics in plants, such as heavy metals from multi-contaminated soils.     

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.     

Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the completion of the sequencing of the entire genome of Arabidopsis thaliana (L.) Heynh., the exact determination of each single gene and its function remains an open question. This is especially true for multigene families. An approach that combines analysis of genomic structure, expression data and functional genomics to ascertain the role of the members of the multidrug-resistance-related protein ( MRP) gene family, a subfamily of the ATP-binding cassette (ABC) transporters from Arabidopsis is presented. We used cDNA sequencing and alignment-based re-annotation of genomic sequences to define the exact genic structure of all known AtMRP genes. Analysis of promoter regions suggested different induction conditions even for closely related genes. Expression analysis for the entire gene family confirmed these assumptions. Phylogenetic analysis and determination of segmental duplication in the regions of AtMRP genes revealed that the evolution of the extraordinarily high number of ABC transporter genes in plants cannot solely be explained by polyploidisation during the evolution of the Arabidopsis genome. Interestingly MRP genes from Oryza sativa L. (rice; OsMRP) show very similar genomic structures to those from Arabidopsis. Screening of large populations of T-DNA-mutagenised lines of A. thaliana resulted in the isolation of AtMRP insertion mutants. This work opens the way for the defined analysis of a multigene family of important membrane transporters whose broad variety of functions expands their traditional role as cellular detoxifiers.     

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.     

<Emphasis Type="Italic">AtMRP6</Emphasis>/<Emphasis Type="Italic">AtABCC6</Emphasis>, an ATP-Binding Cassette transporter gene expressed during early steps of seedling development and up-regulated by cadmium in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

ABC proteins constitute one of the largest families of transporters found in all living organisms. In Arabidopsis thaliana, 120 genes encoding ABC transporters have been identified. Here, the characterization of one member of the MRP subclass, AtMRP6, is described.     

Enhanced multispecificity of arabidopsis vacuolar multidrug resistance-associated protein-type ATP-binding cassette transporter, AtMRP2

Recent investigations have established that Arabidopsis thaliana contains a family of genes encoding ATP-binding cassette transporters belonging to the multidrug resistance-associated protein (MRP) family. So named because of the phenotypes conferred by their animal prototypes, many MRPs are MgATP-energized pumps active in the transport of glutathione (GS) conjugates and other bulky amphipathic anions across membranes. Here we show that Arabidopsis MRP2 (AtMRP2) localizes to the vacuolar membrane fraction from seedlings and is not only competent in the transport of GS conjugates but also glucuronate conjugates after heterologous expression in yeast. Based on the stimulatory action of the model GS conjugate 2,4-dinitrophenyl-GS (DNP-GS) on uptake of the model glucuronide 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG) and vice versa, double-label experiments demonstrating that the two substrates are subject to simultaneous transport by AtMRP2 and preloading experiments suggesting that the effects seen result from cis, not trans, interactions, it is inferred that some GS conjugates and some glucuronides reciprocally activate each other's transport via distinct but coupled binding sites. The results of parallel experiments on AtMRP1 and representative yeast and mammalian MRPs indicate that these properties are specific to AtMRP2. The effects exerted by DNP-GS on AtMRP2 are not, however, common to all GS conjugates and not simulated by oxidized glutathione or reduced glutathione. Decyl-GS, metolachlor-GS, and oxidized glutathione, although competitive with DNP-GS, do not promote E(2)17betaG uptake by AtMRP2. Reduced glutathione, although subject to transport by AtMRP2 and able to markedly promote E(2)17betaG uptake, neither competes with DNP-GS for uptake nor is subject to E(2)17betaG-promoted uptake. A multisite model comprising three or four semi-autonomous transport pathways plus distinct but tightly coupled binding sites is invoked for AtMRP2.     

Requirement of the N-terminal extension for vacuolar trafficking and transport activity of yeast Ycf1p,an ATP-binding cassette transporter

Ycf1p is the prototypical member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette (ABC) transporters. Ycf1p resides in the vacuolar membrane and mediates glutathione-dependent transport processes that result in resistance to cadmium and other xenobiotics. A feature common to many MRP proteins that distinguishes them from other ABC transporters is the presence of a hydrophobic N-terminal extension (NTE), whose function is not clearly established. The NTE contains a membrane spanning domain (MSD0) with five transmembrane spans and a cytosolic linker region (L0). The goal of this study was to determine the functional significance of the NTE of Ycf1p by examining the localization and functional properties of Ycf1p partial molecules, expressed either singly or together. We show that MSD0 plays a critical role in the vacuolar membrane trafficking of Ycf1p, whereas L0 is dispensable for localization. On the other hand, L0 is required for transport function, as determined by monitoring cadmium resistance. We also examine an unusual aspect of Ycf1p biology, namely, the posttranslational proteolytic processing that occurs within a lumenal loop of Ycf1p. Processing is shown to be Pep4p dependent and thus serves as a convenient marker for proper vacuolar localization. The processed fragments associate with each other, suggesting that these natural cleavage products contribute together to Ycf1p function.     

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

Although the vacuole is the most important final store for toxic heavy metals like cadmium (Cd²⁺), our knowledge on how they are transported into the vacuole is still insufficient. It has been suggested that Cd²⁺ can be transported as phytochelatin‐Cd²⁺ by an unknown ABC transporter or in exchange with protons by cation/proton exchanger (CAX) transporters. To unravel the contribution of vacuolar transporters to Cd²⁺ detoxification, a quantitative proteomics approach was performed. Highly purified vacuoles were isolated from barley plants grown under minus, low (20 μM), and high (200 μM) Cd²⁺ conditions and protein levels of the obtained tonoplast samples were analyzed using isobaric tag for relative and absolute quantitation (iTRAQ?). Although 56 vacuolar transporter proteins were identified, only a few were differentially expressed. Under low‐Cd²⁺ conditions, an inorganic pyrophosphatase and a γ‐tonoplast intrinsic protein (γ‐TIP) were up‐regulated, indicating changes in energization and water fluxes. In addition, the protein ratio of a CAX1a and a natural resistance‐associated macrophage protein (NRAMP), responsible for vacuolar Fe²⁺ export was increased. CAX1a might play a role in vacuolar Cd²⁺ transport. An increase in NRAMP activity leads to a higher cytosolic Fe²⁺ concentration, which may prevent the exchange of Fe²⁺ by toxic Cd²⁺. Additionally, an ABC transporter homolog to AtMRP3 showed up‐regulation. Under high Cd²⁺ conditions, the plant response was more specific. Only a protein homologous to AtMRP3 that showed already a response under low Cd²⁺ conditions, was up‐regulated. Interestingly, AtMRP3 is able to partially rescue a Cd²⁺‐sensitive yeast mutant. The identified transporters are good candidates for further investigation of their roles in Cd²⁺ detoxification.     

The Twisted Dwarf's ABC: How Immunophilins Regulate Auxin Transport

There is increasing evidence that immunophilins function as key regulators of plant development. One of the best investigated members, the multi-domain FKBP TWISTED DWARF1 (TWD1)/FKBP42, has been shown to reside on both the vacuolar and plasma membranes where it interacts in mirror image with two pairs of ABC transporters, MRP1/ MRP2 and PGP1/PGP19(MDR1), respectively. Twisted dwarf1 and pgp1/pgp19 mutants display strongly overlapping phenotypes, including reduction and disorientation of growth, suggesting functional interaction.In a recent work using plant and heterologous expression systems, TWD1 has been demonstrated to modulate PGP-mediated export of the plant hormone auxin, which controls virtually all plant developmental processes. Here we summarize recent molecular models on TWD1 function in plant development and PGP-mediated auxin tranport and discuss open questions.Key Words: Twisted Dwarf1, plant development, auxin, immunophilin, P-glycoprotein, ABC transporterFK506-binding Proteins (FKBPs), together with unrelated cyclophilins, belong to the immunophilins, an ancient and ubiquitous protein family.^1,4,5 They were first described as receptors for immunosuppressive drugs in animal and human cells, FK506 and cyclosporin A, respectively.¹ All FKBP-type immunophilins share a characteristic peptidyl-prolyl cis-trans isomerase domain (PPIase domain or FKBD, Fig. 2A) making protein folding a key feature among immunophilins.² The best investigated example, the human cytosolic single-domain FKBP12, modulates Ca²⁺ release channels^6,7 and associates with the cell cycle regulator TGF-β.⁸ Furthermore, the human FKBP12/FK506 complex is known to bind and inhibit calcineurin activity,⁹ leading to immune response inhibition. However, not all single- and multiple-domain FKBPs own folding activity and, interestingly, many form distinct protein complexes with diverse functions.^3–5Open in a separate windowFigure 2Model of TWISTED DWARF 1 interacting proteins. (A) Domain structure of TWD1 and putative interacting proteins. FKBD, FK506-binding domain: TPR, tetratricopeptide repeat; CaM(-BD, calmodulin-binding domain; MA, membrane anchor. For details, see text. (B) Functional TWD1-ABC transporter complexes on both the vacuolar and plasma membrane. While for TWD1/PGP pairs, the positive regulatory role on auxin transport was demonstrated,¹⁸ the modulation of MRP-mediated vacuolar import of glutathion conjugates (GS-X) was established using mammalian test substrates¹⁷ because the in vivo substrates are unknown. Note that C-terminal nucleotide binding folds of MRP- and PGP-like ABC transporters interact with distinct functional domains of TWD1, the TPR and FKBD, respectively. The native auxin, IAAH, gets trapped by deprotonization upon uptake into the cell. Export is catalyzed by secondary active export via PIN-like efflux carriers¹⁵ and/or by primary active, ATP-driven P-glycoproteins (PGPs, right panel); loss-of TWD1 function abolishes PGP-mediated auxin export (left panel).     

Arabidopsis TWISTED DWARF1 Functionally Interacts with Auxin Exporter ABCB1 on the Root Plasma Membrane

Plant architecture is influenced by the polar, cell-to-cell transport of auxin that is primarily provided and regulated by plasma membrane efflux catalysts of the PIN-FORMED and B family of ABC transporter (ABCB) classes. The latter were shown to require the functionality of the FK506 binding protein42 TWISTED DWARF1 (TWD1), although underlying mechanisms are unclear. By genetic manipulation of TWD1 expression, we show here that TWD1 affects shootward root auxin reflux and, thus, downstream developmental traits, such as epidermal twisting and gravitropism of the root. Using immunological assays, we demonstrate a predominant lateral, mainly outward-facing, plasma membrane location for TWD1 in the root epidermis characterized by the lateral marker ABC transporter G36/PLEIOTROPIC DRUG-RESISTANCE8/PENETRATION3. At these epidermal plasma membrane domains, TWD1 colocalizes with nonpolar ABCB1. In planta bioluminescence resonance energy transfer analysis was used to verify specific ABC transporter B1 (ABCB1)–TWD1 interaction. Our data support a model in which TWD1 promotes lateral ABCB-mediated auxin efflux via protein–protein interaction at the plasma membrane, minimizing reflux from the root apoplast into the cytoplasm.     

Vacuolar import of phosphatidylcholine requires the ATP-binding cassette transporter Ybt1

ATP-binding cassette (ABC) transporters are well known for their roles as multidrug resistance determinants but also play important roles in regulation of lipid levels. In the yeast Saccharomyces cerevisiae, the plasma membrane ABC transporter proteins Pdr5 and Yor1 are required for normal rates of transport of phosphatidyethanolamine to the surface of the cell. Loss of these ABC transporters causes a defect in phospholipid asymmetry across the plasma membrane and has been linked with slowed rates of trafficking of other membrane proteins. Four ABC transporter proteins are found on the limiting membrane of the yeast vacuole and loss of one of these vacuolar ABC transporters, Ybt1, caused a major defect in the normal delivery of the phosphatidylcholine (PC) analog NBD-PC (7-nitro-2,1,3-benzoxadiazol-PC) to the lumen of the vacuole. NBD-PC accumulates on cytosolic membranes in an ybt1Δ strain. We demonstrated that Ybt1 is required to import NBD-PC into vacuoles in the presence of ATP in vitro. Loss of Ybt1 prevented vacuolar remodeling of PC analogs. Turnover of Ybt1 was reduced under conditions in which function of this vacuolar remodeling pathway was required. Our data describe a novel vacuolar route for lipid remodeling and reutilization in addition to previously described enzymatic avenues in the cytoplasm.     

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.     

Comparative mutant analysis of Arabidopsis ABCC-type ABC transporters: AtMRP2 contributes to detoxification, vacuolar organic anion transport and chlorophyll degradation

The enormous metabolic plasticity of plants allows detoxificationof many harmful compounds that are generated during biosyntheticprocesses or are present as biotic or abiotic toxins in theirenvironment. Derivatives of toxic compounds such as glutathioneconjugates are moved into the central vacuole via ATP-bindingcassette (ABC)-type transporters of the multidrug resistance-associatedprotein (MRP) subfamily. The Arabidopsis genome contains 15AtMRP isogenes, four of which (AtMRP1, 2, 11 and 12) clustertogether in one of two major phylogenetic clades. We isolatedT-DNA knockout alleles in all four highly homologous AtMRP genesof this clade and subjected them to physiological analysis toassess the function of each AtMRP of this group. None of thesingle atmrp mutants displayed visible phenotypes under controlconditions. In spite of the fact that AtMRP1 and AtMRP2 hadbeen described as efficient ATP-dependent organic anion transportersin heterologous expression experiments, the contribution ofthree of the AtMRP genes (1, 11 and 12) to detoxification ismarginal. Only knockouts in AtMRP2 exhibited a reduced sensitivitytowards 1-chloro-2,4-dinitrobenzene, but not towards other herbicides.AtMRP2 but not AtMRP1, 11 and 12 is involved in chlorophylldegradation since ethylene-treated rosettes of atmrp2 showedreduced senescence, and AtMRP2 expression is induced duringsenescence. This suggests that AtMRP2 is involved in vacuolartransport of chlorophyll catabolites. Vacuolar uptake studiesdemonstrated that transport of typical MRP substrates was reducedin atmrp2. We conclude that within clade I, only AtMRP2 contributessignificantly to overall organic anion pump activity in vivo.