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.     

A role for ABCB19‐mediated polar auxin transport in seedling photomorphogenesis mediated by cryptochrome 1 and phytochrome B

During seedling establishment, blue and red light suppress hypocotyl growth through the cryptochrome 1 (cry1) and phytochrome B (phyB) photosensory pathways, respectively. How these photosensory pathways integrate with growth control mechanisms to achieve the appropriate degree of stem elongation was investigated by combining cry1 and phyB photoreceptor mutations with genetic manipulations of a multidrug resistance‐like membrane protein known as ABCB19 that influenced auxin distribution within the plant, as evidenced by a combination of reporter gene assays and direct auxin measurements. Auxin signaling and ABCB19 protein levels, hypocotyl growth rates, and apical hook opening were measured in mutant and wild‐type seedlings exposed to a range of red and blue light conditions. Ectopic/overexpression of ABCB19 (B19OE) greatly increased auxin in the hypocotyl, which reduced the sensitivity of hypocotyl growth specifically to blue light in long‐term assays and red light in high‐resolution, short‐term assays. Loss of ABCB19 partially suppressed the cry1 hypocotyl growth phenotype in blue light. Hypocotyl growth of B19OE seedlings in red light was very similar to phyB mutants. Altered auxin distribution in B19OE seedlings also affected the opening of the apical hook. The cry1 and phyB photoreceptor mutations both increased ABCB19 protein levels at the plasma membrane, as measured by confocal microscopy. The B19OE plant proved to be a useful tool for determining aspects of the mechanism by which light, acting through cry1 or phyB, influences the auxin transport process to control hypocotyl growth during de‐etiolation.     

Clathrin regulates blue light‐triggered lateral auxin distribution and hypocotyl phototropism in Arabidopsis

Phototropism is the process by which plants grow towards light in order to maximize the capture of light for photosynthesis, which is particularly important for germinating seedlings. In Arabidopsis, hypocotyl phototropism is predominantly triggered by blue light (BL), which has a profound effect on the establishment of asymmetric auxin distribution, essential for hypocotyl phototropism. Two auxin efflux transporters ATP‐binding cassette B19 (ABCB19) and PIN‐formed 3 (PIN3) are known to mediate the effect of BL on auxin distribution in the hypocotyl, but the details for how BL triggers PIN3 lateralization remain poorly understood. Here, we report a critical role for clathrin in BL‐triggered, PIN3‐mediated asymmetric auxin distribution in hypocotyl phototropism. We show that unilateral BL induces relocalization of clathrin in the hypocotyl. Loss of clathrin light chain 2 (CLC2) and CLC3 affects endocytosis and lateral distribution of PIN3 thereby impairing BL‐triggered establishment of asymmetric auxin distribution and consequently, phototropic bending. Conversely, auxin efflux inhibitors N‐1‐naphthylphthalamic acid and 2,3,5‐triiodobenzoic acid affect BL‐induced relocalization of clathrin, endocytosis and lateralization of PIN3 as well as asymmetric distribution of auxin. These results together demonstrate an important interplay between auxin and clathrin function that dynamically regulates BL‐triggered hypocotyl phototropism in Arabidopsis.     

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle‐tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss‐of‐function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin–A compartments is delayed after the brefeldin‐A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin‐A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin‐A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA‐a1‐labelled early endosomes or the trans‐Golgi network, but are RAB‐A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.     

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.     

Clathrin light chains regulate hypocotyl elongation by affecting the polarization of the auxin transporter PIN3 in Arabidopsis

PIN-FORMED (PIN)-dependent directional auxin transport is crucial for plant development. Although the redistribution of auxin mediated by the polarization of PIN3 plays key roles in modulating hypocotyl cell expansion, how PIN3 becomes repolarized to the proper sites within hypocotyl cells is poorly understood. We previously generated the clathrin light chain clc2-1 clc3-1 double mutant in Arabidopsis thaliana and found that it has an elongated hypocotyl phenotype compared to the wild type. Here, we performed genetic, cell biology, and pharmacological analyses combined with live-cell imaging to elucidate the molecular mechanism underlying the role of clathrin light chains in hypocotyl elongation. Our analyses indicated that the defects of the double mutant enhanced auxin maxima in epidermal cells, thus, promoting hypocotyl elongation. PIN3 relocated to the lateral sides of hypocotyl endodermal cells in clc2-1 clc3-1 mutants to redirect auxin toward the epidermal cell layers. Moreover, the loss of function of PIN3 largely suppressed the long hypocotyl phenotype of the clc2-1 clc3-1 double mutant, as did treatment with auxin transport inhibitors. Based on these data, we propose that clathrin modulates PIN3 abundance and polarity to direct auxin flux and inhibit cell elongation in the hypocotyl, providing novel insights into the regulation of hypocotyl elongation.     

Functional expression and characterization of Arabidopsis ABCB,AUX 1 and PIN auxin transporters in Schizosaccharomyces pombe

Heterologous expression systems based on tobacco BY‐2 cells, Arabidopsis cell cultures, Xenopus oocytes, Saccharomyces cerevisiae, and human HeLa cells have been used to express and characterize PIN, ABCB (PGP), and AUX/LAX auxin transporters from Arabidopsis. However, no single system has been identified that can be used for effective comparative analyses of these proteins. We have developed an accessible Schizosaccharomyces pombe system for comparative studies of plant transport proteins. The system includes knockout mutants in all ABC and putative auxin transport genes and Gateway^®‐compatible expression vectors for functional analysis and subcellular localization of recombinant proteins. We expressed Arabidopsis ABCB1 and ABCB19 in mam1pdr1 host lines under the inducible nmt41 promoter. ABCB19 showed a higher ³H‐IAA export activity than ABCB1. Arabidopsis PIN proteins were expressed in a mutant lacking the auxin effluxer like 1 (AEL1) gene. PIN1 showed higher activity than PIN2 with similar protein expression levels. Expression of AUX1 in a permease‐deficient vat3 mutant resulted in increased net auxin uptake activity. Finally, ABCB4 expressed in mam1pdr1 displayed a concentration‐dependent reversal of ³H‐IAA transport that is consistent with its observed activity in planta. Structural modelling suggests that ABCB4 has three substrate interaction sites rather than the two found in ABCB19, thus providing a rationale for the observed substrate activation. Taken together, these results suggest that the S. pombe system described here can be employed for comparative analyses and subsequent structural characterizations of plant transport proteins.     

The AGC kinase,PINOID, blocks interactive ABCB/PIN auxin transport

Plant growth and development is determined by intracellular and intercellular auxin gradients that are controlled at first hand by auxin efflux catalysts of the ABCB/PGP and PIN families. ABCB transport activity was shown to be counter-actively regulated by protein phosphorylation by the AGC protein kinase, PINOID (PID), that is coordinated by interaction with the immunophilin-like FKBP42, TWISTED DWARF1 (TWD1). In contrast, PID was shown to determine PIN polarity, however, the direct impact of PID on PIN activity has yet not been tested. Co-expression in yeast indicates that PID had no effect on PIN1,2 alone but specifically inhibits interactive ABCB1-PIN1/PIN2 auxin efflux in an action that is dependent on its kinase activity. PIN1-PID co-transfection in N. benthamiana revealed that PID blocks PIN1-mediated auxin efflux without changing PIN1 location. In summary, these data provide evidence that PID phosphorylation does not only determine PIN polarity but also has a direct impact on transport activity of the activity of the binary PIN-ABCB1 complex.     

Regulation of ABCB1/PGP1-catalysed auxin transport by linker phosphorylation

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. In-vitro 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.     

The putative auxin efflux carrier OsPIN3t is involved in the drought stress response and drought tolerance

The phytohormone auxin plays a critical role in plant growth and development, and its spatial distribution largely depends on the polar localization of the PIN‐FORMED (PIN) auxin efflux carrier family members. In this study, we identify a putative auxin efflux carrier gene in rice, OsPIN3t, which acts in auxin polar transport but is also involved in the drought stress response in rice. We show that OsPIN3t–GFP fusion proteins are localized in plasma membranes, and this subcellular localization changes under 1‐N‐naphthylphthalamic acid (NPA) treatment. The tissue‐specific expression patterns of OsPIN3t were also investigated using a β‐glucuronidase (GUS) reporter, which showed that OsPIN3t was mainly expressed in vascular tissue. The GUS activity in OsPIN3tpro::GUS plants increased by NAA treatment and decreased by NPA treatment. Moreover, knockdown of OsPIN3t caused crown root abnormalities in the seedling stage that could be phenocopied by treatment of wild‐type plants with NPA, which indicated that OsPIN3t is involved in the control of polar auxin transport. Overexpression of OsPIN3t led to improved drought tolerance, and GUS activity significantly increased when OsPIN3tpro::GUS plants were subjected to 20% polyethylene glycol stress. Taken together, these results suggest that OsPIN3t is involved in auxin transport and the drought stress response, which suggests that a polar auxin transport pathway is involved in the regulation of the response to water stress in plants.     

ABCB19‐mediated polar auxin transport modulates Arabidopsis hypocotyl elongation and the endoreplication variant of the cell cycle

Elongation of the Arabidopsis hypocotyl pushes the shoot‐producing meristem out of the soil by rapid expansion of cells already present in the embryo. This elongation process is shown here to be impaired by as much as 35% in mutants lacking ABCB19, an ATP‐binding cassette membrane protein required for polar auxin transport, during a limited time of fast growth in dim white light beginning 2.5 days after germination. The discovery of high ectopic expression of a cyclin B1;1‐based reporter of mitosis throughout abcb19 hypocotyls without an equivalent effect on mitosis prompted investigations of the endoreplication variant of the cell cycle. Flow cytometry performed on nuclei isolated from upper (growing) regions of 3‐day‐old hypocotyls showed ploidy levels to be lower in abcb19 mutants compared with wild type. CCS52A2 messenger RNA encoding a nuclear protein that promotes a shift from mitosis to endoreplication was lower in abcb19 hypocotyls, and fluorescence microscopy showed the CCS52A2 protein to be lower in the nuclei of abcb19 hypocotyls compared with wild type. Providing abcb19 seedlings with nanomolar auxin rescued their low CCS52A2 levels, endocycle defects, aberrant cyclin B1;1 expression, and growth rate defect. The abcb19‐like growth rate of ccs52a2 mutants was not rescued by auxin, placing CCS52A2 after ABCB19‐dependent polar auxin transport in a pathway responsible for a component of ploidy‐related hypocotyl growth. A ccs52A2 mutation did not affect the level or pattern of cyclin B1;1 expression, indicating that CCS52A2 does not mediate the effect of auxin on cyclin B1;1.     

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.     

ABCB19/PGP19 stabilises PIN1 in membrane microdomains in Arabidopsis

Auxin transport is mediated at the cellular level by three independent mechanisms that are characterised by the PIN-formed (PIN), P-glycoprotein (ABCB/PGP) and AUX/LAX transport proteins. The PIN and ABCB transport proteins, best represented by PIN1 and ABCB19 (PGP19), have been shown to coordinately regulate auxin efflux. When PIN1 and ABCB19 coincide on the plasma membrane, their interaction enhances the rate and specificity of auxin efflux and the dynamic cycling of PIN1 is reduced. However, ABCB19 function is not regulated by the dynamic cellular trafficking mechanisms that regulate PIN1 in apical tissues, as localisation of ABCB19 on the plasma membrane was not inhibited by short-term treatments with latrunculin B, oryzalin, brefeldin A (BFA) or wortmannin--all of which have been shown to alter PIN1 and/or PIN2 plasma membrane localisation. When taken up by endocytosis, the styryl dye FM4-64 labels diffuse rather than punctuate intracellular bodies in abcb19 (pgp19), and some aggregations of PIN1 induced by short-term BFA treatment did not disperse after BFA washout in abcb19. Although the subcellular localisations of ABCB19 and PIN1 in the reciprocal mutant backgrounds were like those in wild type, PIN1 plasma membrane localisation in abcb19 roots was more easily perturbed by the detergent Triton X-100, but not other non-ionic detergents. ABCB19 is stably associated with sterol/sphingolipid-enriched membrane fractions containing BIG/TIR3 and partitions into Triton X-100 detergent-resistant membrane (DRM) fractions. In the wild type, PIN1 was also present in DRMs, but was less abundant in abcb19 DRMs. These observations suggested a rationale for the observed lack of auxin transport activity when PIN1 is expressed in a non-plant heterologous system. PIN1 was therefore expressed in Schizosaccharomyces pombe, which has plant-like sterol-enriched microdomains, and catalysed auxin transport in these cells. These data suggest that ABCB19 stabilises PIN1 localisation at the plasma membrane in discrete cellular subdomains where PIN1 and ABCB19 expression overlaps.     

phot1 inhibition of ABCB19 primes lateral auxin fluxes in the shoot apex required for phototropism

It is well accepted that lateral redistribution of the phytohormone auxin underlies the bending of plant organs towards light. In monocots, photoreception occurs at the shoot tip above the region of differential growth. Despite more than a century of research, it is still unresolved how light regulates auxin distribution and where this occurs in dicots. Here, we establish a system in Arabidopsis thaliana to study hypocotyl phototropism in the absence of developmental events associated with seedling photomorphogenesis. We show that auxin redistribution to the epidermal sites of action occurs at and above the hypocotyl apex, not at the elongation zone. Within this region, we identify the auxin efflux transporter ATP-BINDING CASSETTE B19 (ABCB19) as a substrate target for the photoreceptor kinase PHOTOTROPIN 1 (phot1). Heterologous expression and physiological analyses indicate that phosphorylation of ABCB19 by phot1 inhibits its efflux activity, thereby increasing auxin levels in and above the hypocotyl apex to halt vertical growth and prime lateral fluxes that are subsequently channeled to the elongation zone by PIN-FORMED 3 (PIN3). Together, these results provide new insights into the roles of ABCB19 and PIN3 in establishing phototropic curvatures and demonstrate that the proximity of light perception and differential phototropic growth is conserved in angiosperms.     

Modulation of P-glycoproteins by auxin transport inhibitors is mediated by interaction with immunophilins

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.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild‐type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss‐of‐function mutations, or in a dominant (gain‐of‐function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.