TWISTED DWARF1 Research Articles

A transcriptome-wide identification of ATP-binding cassette (ABC) transporters revealed participation of ABCB subfamily in abiotic stress management of Glycyrrhiza glabra L

Transcriptome-wide survey divulged a total of 181 ABC transporters in G. glabra which were phylogenetically classified into six subfamilies. Protein–Protein interactions revealed nine putative GgABCBs (-B6, -B14, -B15, -B25, -B26, -B31, -B40, -B42 &-B44) corresponding to five AtABCs orthologs (-B1, -B4, -B11, -B19, &-B21). Significant transcript accumulation of ABCB6 (31.8 folds), -B14 (147.5 folds), -B15 (17 folds), -B25 (19.7 folds), -B26 (18.31 folds), -B31 (61.89 folds), -B40 (1273 folds) and -B42 (51 folds) was observed under the influence of auxin. Auxin transport-specific inhibitor, N-1-naphthylphthalamic acid, showed its effectiveness only at higher (10 µM) concentration where it down regulated the expression of ABCBs, PINs (PIN FORMED) and TWD1 (TWISTED DWARF 1) genes in shoot tissues, while their expression was seen to enhance in the root tissues. Further, qRT-PCR analysis under various growth conditions (in-vitro, field and growth chamber), and subjected to abiotic stresses revealed differential expression implicating role of ABCBs in stress management. Seven of the nine genes were shown to be involved in the stress physiology of the plant. GgABCB6, 15, 25 and ABCB31 were induced in multiple stresses, while GgABCB26, 40 & 42 were exclusively triggered under drought stress. No study pertaining to the ABC transporters from G. glabra is available till date. The present investigation will give an insight to auxin transportation which has been found to be associated with plant growth architecture; the knowledge will help to understand the association between auxin transportation and plant responses under the influence of various conditions.

Arabidopsis TWISTED DWARF1 regulates stamen elongation by differential activation of ABCB1,19-mediated auxin transport.

Despite clear evidence that a local accumulation of auxin is likewise critical for male fertility, much less is known about the components that regulate auxin-controlled stamen development. In this study, we analyzed physiological and morphological parameters in mutants of key players of ABCB-mediated auxin transport, and spatially and temporally dissected their expression on the protein level as well as auxin fluxes in the Arabidopsis stamens. Our analyses revealed that the FKBP42, TWISTED DWARF1 (TWD1), promotes stamen elongation and, to a lesser extent, anther dehiscence, as well as pollen maturation, and thus is required for seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1-as a described ABCB chaperone-is a positive regulator of ABCB1- and ABCB19-mediated auxin transport. However, reduced stamen number was dependent on TWD1 but not on investigated ABCBs, suggesting additional players downstream of TWD1. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls stamen development by differential activation of ABCB1,19-mediated auxin transport in the stamen.

The mechanism of auxin transport in lateral root spacing

The mechanism of auxin transport in lateral root spacing

Non-steroidal Anti-inflammatory Drugs Target TWISTED DWARF1-Regulated Actin Dynamics and Auxin Transport-Mediated Plant Development

The widely used non-steroidal anti-inflammatory drugs (NSAIDs) are derivatives of the phytohormone salicylic acid (SA). SA is well known to regulate plant immunity and development, whereas there have been few reports focusing on the effects of NSAIDs in plants. Our studies here reveal that NSAIDs exhibit largely overlapping physiological activities to SA in the model plant Arabidopsis. NSAID treatments lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the protein phosphatase 2A (PP2A), NSAIDs also exhibit PP2A-independent effects. Cell biological and biochemical analyses reveal that many NSAIDs bind directly to and inhibit the chaperone activity of TWISTED DWARF1, thereby regulating actin cytoskeleton dynamics and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants and provide insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds.

NSAIDs Target TWISTED DWARF1-Regulated Actin Dynamics and Auxin Transport-Mediated Plant Development

The widely used non-steroidal anti-inflammatory drugs (NSAIDs), such as aspirin and ibuprofen, are derivatives of the phytohormone salicylic acid (SA). In plants, SA is well-known to regulate immunity as well as development, whereas there was no study focusing on the effects and mechanism of action of NSAIDs in plants. Our studies here in the model plant Arabidopsis thaliana reveal that NSAIDs exhibit largely overlapping physiological activities to SA. NSAID treatments in Arabidopsis seedlings lead to shorter and agravitropic primary roots and inhibited lateral root organogenesis. Notably, in addition to the SA-like action, which in roots involves binding to the Protein Phosphatase 2A (PP2A), NSAIDs exhibit also PP2A-independent effects. Cell biological and biochemical analyses unravel that many NSAIDs bind directly to and inhibit the chaperone activity of the FKBP42, TWISTED DWARF1, thereby regulating the dynamics of actin cytoskeleton and subsequent endosomal trafficking. Our findings uncover an unexpected bioactivity of human pharmaceuticals in plants, and provide novel insights into the molecular mechanism underlying the cellular action of this class of anti-inflammatory compounds.

A Critical View on ABC Transporters and Their Interacting Partners in Auxin Transport.

Different subclasses of ATP-binding cassette (ABC) transporters have been implicated in the transport of native variants of the phytohormone auxin. Here, the putative, individual roles of key members belonging to the ABCB, ABCD and ABCG families, respectively, are highlighted and the knowledge of their assumed expression and transport routes is reviewed and compared with their mutant phenotypes. Protein-protein interactions between ABC transporters and regulatory components during auxin transport are summarized and their importance is critically discussed. There is a focus on the functional interaction between members of the ABCB family and the FKBP42, TWISTED DWARF1, acting as a chaperone during plasma membrane trafficking of ABCBs. Further, the mode and relevance of functional ABCB-PIN interactions is diagnostically re-evaluated. A new nomenclature describing precisely the most likely ABCB-PIN interaction scenarios is suggested. Finally, available tools for the detection and prediction of ABC transporter interactomes are summarized and the potential of future ABC transporter interactome maps is highlighted.

TWISTED DWARF1 Mediates the Action of Auxin Transport Inhibitors on Actin Cytoskeleton Dynamics.

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

Immunophilin-like FKBP42/TWISTED DWARF1 Interacts with the Receptor Kinase BRI1 to Regulate Brassinosteroid Signaling in Arabidopsis

Mutation of the immunophilin-like FK506-binding protein TWISTED DWARF1 (FKBP42/TWD1) causes dwarf and twisted-organ phenotypes in Arabidopsis. However, the function of FKBP42 is not fully understood at the molecular level. Using genetic, physiological, and immunological experiments, we show here that FKBP42/TWD1 is necessary for brassinosteroid (BR) signal transduction. The twd1 mutant showed reduced BR sensitivity in growth responses and activation of the BZR1 transcription factor. However, twd1 showed normal responses to an inhibitor of BIN2/GSK3, suggesting that twd1 has a defect upstream of BIN2 in the BR signaling pathway. In vitro and in vivo assays showed that TWD1 interacts physically with the kinase domains of the BR receptor kinases BRI1 and BAK1. TWD1 is not required for normal localization of BRI1-GFP to the plasma membrane or for activation of the flagellin receptor kinase FLS2. Our results suggest that FKBP42/TWD1 plays a specific role in the activation of BRI1 receptor kinase.

TWISTED DWARF 1 Associates with BRASSINOSTEROID-INSENSITIVE 1 to Regulate Early Events of the Brassinosteroid Signaling Pathway

A genome-wide screen for mutants showing altered brassinosteroid (BR) sensitivity or bri1-like phenotypes resulted in the identification of two new mutant alleles of TWISTED DWARF 1 (TWD1), twd1-4, and twd1-5. Previous studies indicated that TWD1, also named as ULTRACURVATA 2 or FKBP42, associates with auxin efflux transporters and is essential for their biological functions. Although earlier reports showed that BR signaling is downregulated in twd1, how TWD1 is integrated in BR signaling has not been elucidated. Here, we provide genetic and biochemical evidence demonstrating that TWD1 interacts with the BR receptor BRI1 in vivo in a BR-independent manner. Further analyses indicated that TWD1 modulates the BR signal transduction not by altering ER quality control or protein abundance of BRI1; instead, TWD1 appears to be critical in BR-induced interaction of BRI1 and its co-receptor BAK1, as well as BR-induced phosphorylation of these two proteins. These results provide better understanding of the early events of the BR signaling pathway.

Effect of T‐DNA insertions on mRNA transcript copy numbers upstream and downstream of the insertion site in Arabidopsis thaliana explored by surface enhanced Raman spectroscopy

We report the effect of a T-DNA insertion on the expression level of mRNA transcripts of the TWISTED DWARF 1 (TWD1) gene upstream and downstream of the T-DNA insertion site in Arabidopsis. A novel approach based on surface-enhanced Raman spectroscopy (SERS) was developed to detect and quantify the effect of a T-DNA insertion on mRNA transcript accumulation at 5'- and 3'-ends of the TWD1 gene. A T-DNA insertion mutant in the TWD1 gene (twd1-2) was chosen to test the sensitivity and the feasibility of the approach. The null mutant of the FK506-like immunophilin protein TWD1 in Arabidopsis shows severe dwarfism and strong disoriented growth of plant organs. A spontaneous arising suppressor allele of twd1-2 called twd-sup displayed an intermediate phenotype between wild type and the knockout phenotype of twd1-2. Both twd1 mutant alleles have identical DNA sequences at the TWD1 locus including the T-DNA insertion in the fourth intron of the TWD1 gene but they show clear variability in the mutant phenotype. We present here the development and application of SERS-based mRNA detection and quantification using the expression of the TWD1 gene in wild type and both mutant alleles. The hallmarks of our SERS approach are a robust and fast assay to detect up to 0.10 fm of target molecules including the ability to omit in vitro transcription and amplification steps after RNA isolation. Instead we perform direct quantification of RNA molecules. This enables us to detect and quantify rare RNA molecules at high levels of precision and sensitivity.

Expression of TWISTED DWARF1 lacking its in‐plane membrane anchor leads to increased cell elongation and hypermorphic growth

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN-FORMED (PIN) and ATP-binding cassette protein subfamily B/phosphor-glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C-terminal in-plane membrane anchor of TWD1 in the regulation of ABCB-mediated auxin transport. In contrast with dwarfed twd1 loss-of-function alleles, TWD1 gain-of-function lines that lack a putative in-plane membrane anchor (HA-TWD1-Ct ) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA-TWD1-Ct . As a consequence, HA-TWD1-Ct displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin-induced cell elongation rates. Our data highlight the importance of C-terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB-mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1-mediated export into the apoplast, which is required for auxin-mediated cell elongation.

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.

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 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.

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.

The Twisted Dwarf's ABC

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.

Solid-state NMR characterization of the putative membrane anchor of TWD1 from Arabidopsis thaliana

Structure and membrane interaction of a 31 amino acid residue fragment of the membrane bound FKBP-like protein twisted dwarf 1 (TWD1) from Arabidopsis thaliana was investigated by solid-state NMR spectroscopy. The studied peptide TWD1(335-365) contained the putative membrane anchor of the protein (residues 339-357) that was previously predicted by sequence hydrophobicity analysis. The TWD1 peptide was synthesized by standard solid phase peptide synthesis and contained three uniformly (13)C- and (15)N-labelled residues (Phe 340, Val 350, Ala 364). The peptide was incorporated into either multilamellar vesicles or oriented planar membranes composed of an equimolar ternary phospholipid mixture (POPC, POPE, POPG), where the POPC was sn-1 chain-deuterated. (31)P NMR spectra of the membrane in the absence and in the presence of the peptide showed axially symmetric powder patterns indicative of a lamellar bilayer phase. Further, the addition of peptide caused a decrease in the lipid hydrocarbon chain order as indicated by reduced quadrupolar splittings in the (2)H NMR spectra of the POPC in the membrane. The conformation of TWD1(335-365) was investigated by (13)C cross-polarization magic-angle spinning NMR spectroscopy. At a temperature of -30 degrees C all peptide signals were resolved and could be fully assigned in two-dimensional proton-driven (13)C spin diffusion and (13)C single quantum/double quantum correlation experiments. The isotropic chemical shift values for Phe 340 and Val 350 exhibited the signature of a regular alpha-helix. Chemical shifts typical for a random coil conformation were observed for Ala 364 located close to the C-terminus of the peptide. Static (15)N NMR spectra of TWD1(335-365) in mechanically aligned lipid bilayers demonstrated that the helical segment of TWD1(335-365) adopts an orientation perpendicular to the membrane normal. At 30 degrees C, the peptide undergoes intermediate time scale motions.

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.

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.