SummaryDirectional 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.

The plant signalling molecule auxin provides positional information in a variety of developmental processes by means of its differential distribution (gradients) within plant tissues. Thus, cellular auxin levels often determine the developmental output of auxin signalling. Conceptually, transmembrane transport and metabolic processes regulate the steady-state levels of auxin in any given cell. In particular, PIN auxin-efflux-carrier-mediated, directional transport between cells is crucial for generating auxin gradients. Here we show that Arabidopsis thaliana PIN5, an atypical member of the PIN gene family, encodes a functional auxin transporter that is required for auxin-mediated development. PIN5 does not have a direct role in cell-to-cell transport but regulates intracellular auxin homeostasis and metabolism. PIN5 localizes, unlike other characterized plasma membrane PIN proteins, to endoplasmic reticulum (ER), presumably mediating auxin flow from the cytosol to the lumen of the ER. The ER localization of other PIN5-like transporters (including the moss PIN) indicates that the diversification of PIN protein functions in mediating auxin homeostasis at the ER, and cell-to-cell auxin transport at the plasma membrane, represent an ancient event during the evolution of land plants.

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.

The immunophilin-like FKBP42 TWISTED DWARF1 (TWD1) has been shown to control plant development via the positive modulation of ABCB/P-glycoprotein (PGP)-mediated transport of the plant hormone auxin. TWD1 functionally interacts with two closely related proteins, ABCB1/PGP1 and ABCB19/PGP19/MDR1, both of which exhibit the ability to bind to and be inhibited by the synthetic auxin transport inhibitor N-1-naphylphtalamic acid (NPA). They are also inhibited by flavonoid compounds, which are suspected modulators of auxin transport. The mechanisms by which flavonoids and NPA interfere with auxin efflux components are unclear. We report here the specific disruption of PGP1-TWD1 interaction by NPA and flavonoids using bioluminescence resonance energy transfer with flavonoids functioning as a classical established inhibitor of mammalian and plant PGPs. Accordingly, TWD1 was shown to mediate modulation of PGP1 efflux activity by these auxin transport inhibitors. NPA bound to both PGP1 and TWD1 but was excluded from the PGP1-TWD1 complex expressed in yeast, suggesting a transient mode of action in planta. As a consequence, auxin fluxes and gravitropism in twd1 roots are less affected by NPA treatment, whereas TWD1 gain-of-function promotes root bending. Our data support a novel model for the mode of drug-mediated P-glycoprotein regulation mediated via protein-protein interaction with immunophilin-like TWD1.Bioactive flavonoids derived from plant secondary metabolism serve as important nutraceuticals (1). They have healthpromoting effects, including antioxidant, anticarcinogenic, antiviral, and anti-inflammatory activities; however, the cellular targets of the in vivo protein remain largely unknown (1, 2). In plants, among other functions, flavonoids such as quercetin, kaempferol, and other aglycone molecules have been shown to inhibit cell-to-cell/polar auxin transport (PAT) 3 and consequently to enhance localized auxin accumulation (1, 3-6). During PAT, the plant hormone auxin, which determines many aspects of plant physiology and development, is moved directionally in a cell-to-cell mode (7-9).The regulatory impact of flavonoids on PAT initially was based on their ability to compete with N-1-naphtylphtalamic acid (NPA), a synthetic auxin transport inhibitor (ATI) (4, 10 -12) and herbicide (naptalam, alanap), for transporter binding sites. This concept is further supported by auxin-related phenotypes of Arabidopsis mutants with altered flavonoid levels (1, 3, 13), although fundamental physiological processes occur in the absence of flavonoids. Currently the flavonoids are seen as transport regulators or modulators (14); nevertheless, the mechanisms by which flavonoids interfere with auxin efflux components are not yet clear (1).The auxin efflux complex is thought to regulate PAT on the molecular level and consists of at least two proteins: a membrane integral transporter and an NPA-binding protein (NBP) regulatory subunit (11,(15)(16)(17). Recently, ABCB/P-glycoprotein (PGP)/multidrug resistance (MDR) proteins, members o...

Polar transport of the plant hormone auxin is controlled by PIN-and ABCB/PGP-efflux catalysts. PIN polarity is regulated by the AGC protein kinase, PINOID (PID), while ABCB activity was shown to be dependent on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Using co-immunoprecipitation (co-IP) and shotgun LC-MS/MS analysis, we identified PID as a valid partner in the interaction with TWD1. Invitro and yeast expression analyses indicated that PID specifically modulates ABCB1-mediated auxin efflux in an action that is dependent on its kinase activity and that is reverted by quercetin binding and thus inhibition of PID autophosphorylation. Triple ABCB1/PID/TWD1 co-transfection in tobacco revealed that PID enhances ABCB1-mediated auxin efflux but blocks ABCB1 in the presence of TWD1. Phospho-proteomic analyses identified S634 as a key residue of the regulatory ABCB1 linker and a very likely target of PID phosphorylation that determines both transporter drug binding and activity. In summary, we provide evidence that PID phosphorylation has a dual, counter-active impact on ABCB1 activity that is coordinated by TWD1-PID interaction.

Differential distribution of the plant hormone auxin within tissues mediates a variety of developmental processes. Cellular auxin levels are determined by metabolic processes including synthesis, degradation, and (de)conjugation, as well as by auxin transport across the plasma membrane. Whereas transport of free auxins such as naturally occurring indole-3-acetic acid (IAA) is well characterized, little is known about the transport of auxin precursors and metabolites. Here, we identify a mutation in the ABCG37 gene of Arabidopsis that causes the polar auxin transport inhibitor sensitive1 (pis1) phenotype manifested by hypersensitivity to auxinic compounds. ABCG37 encodes the pleiotropic drug resistance transporter that transports a range of synthetic auxinic compounds as well as the endogenous auxin precursor indole-3-butyric acid (IBA), but not free IAA. ABCG37 and its homolog ABCG36 act redundantly at outermost root plasma membranes and, unlike established IAA transporters from the PIN and ABCB families, transport IBA out of the cells. Our findings explore possible novel modes of regulating auxin homeostasis and plant development by means of directional transport of the auxin precursor IBA and presumably also other auxin metabolites.PDR9 | PDR8 | IBA transport | auxin synthesis P lants have evolved outstanding capacities to adapt their metabolism and development to respond to their environment. Changes in the availability and distribution of endogenous signaling molecules-plant hormones-play important roles in these responses (1). The phytohormone auxin, perceived by TIR1/AFB receptor proteins and interpreted by downstream nuclear signaling pathway, is an important signal that mediates transcriptional developmental reprogramming (reviewed in refs. 2 and 3). The differential distribution of auxin within tissues is essential for many adaptive responses including embryo and leaf patterning, root and stem elongation, lateral root initiation, and leaf expansion (4). Differential distribution of the major active auxin, IAA, depends on its intercellular transport and metabolic processes that involve biosynthesis by several pathways and release from storage forms including amide-or ester-linked conjugates with amino acids, peptides, and sugars (reviewed in ref. 5). The role of another endogenously occurring auxinic compound IBA is still unclear. It has been proposed that IBA acts independently of IAA (6), but a number of recent genetic findings suggest that IBA functions as an important precursor to IAA during conversion resembling peroxisomal fatty acid β-oxidation (5, 7). Besides metabolism, a crucial process controlling cellular auxin levels is the directional, intercellular auxin transport that depends on specialized influx and efflux carriers (reviewed in ref. 8). IAA transporters include amino acid permeases-like AUXIN RESISTANT1 (AUX1) mediating auxin influx (9-11), the PIN-FORMED (PIN) efflux carriers (12)(13)(14), and the MULTIDRUG RESISTANCE/P-GLYCOPROTEIN (PGP) class of ATP-Binding Cassette (ABC) auxin trans...

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.

Increasing evidence indicates that volatile compounds emitted by bacteria can influence the growth of other organisms. In this study, the volatiles produced by three different strains of Burkholderia ambifaria were analysed and their effects on the growth of plants and fungi, as well as on the antibiotic resistance of target bacteria, were assessed. Burkholderia ambifaria emitted highly bioactive volatiles independently of the strain origin (clinical environment, rhizosphere of pea, roots of maize). These volatile blends induced significant biomass increase in the model plant Arabidopsis thaliana as well as growth inhibition of two phytopathogenic fungi (Rhizoctonia solani and Alternaria alternata). In Escherichia coli exposed to the volatiles of B. ambifaria, resistance to the aminoglycoside antibiotics gentamicin and kanamycin was found to be increased. The volatile blends of the three strains were similar, and dimethyl disulfide was the most abundant compound. Sulfur compounds, ketones, and aromatic compounds were major groups in all three volatile profiles. When applied as pure substance, dimethyl disulfide led to increased plant biomass, as did acetophenone and 3-hexanone. Significant fungal growth reduction was observed with high concentrations of dimethyl di- and trisulfide, 4-octanone, S-methyl methanethiosulphonate, 1-phenylpropan-1-one, and 2-undecanone, while dimethyl trisulfide, 1-methylthio-3-pentanone, and o-aminoacetophenone increased resistance of E. coli to aminoglycosides. Comparison of the volatile profile produced by an engineered mutant impaired in quorum-sensing (QS) signalling with the corresponding wild-type led to the conclusion that QS is not involved in the regulation of volatile production in B. ambifaria LMG strain 19182.