Auxin, indole-3-acetic acid (IAA), is a key phytohormone with diverse morphogenic roles in land plants, but its function and transport mechanisms in algae remain poorly understood. We therefore aimed to explore the role of IAA in a complex, streptophyte algae Chara braunii. Here, we described novel responses of C. braunii to IAA and characterized two homologs of PIN auxin efflux carriers: CbPINa and CbPINc. We determined their localization in C. braunii using epitope-specific antibodies and tested their function in heterologous land plant models. Further, using phosphoproteomic analysis, we identified IAA-induced phosphorylation events. The thallus regeneration assay showed that IAA promotes thallus elongation and side branch development. Immunolocalization of CbPINa and CbPINc confirmed their presence on the plasma membrane of vegetative and generative cells of C. braunii. However, functional assays in tobacco BY-2 cells demonstrated that CbPINa affects auxin transport, whereas CbPINc does not. The IAA is effective in the acceleration of cytoplasmic streaming and the phosphorylation of evolutionary conserved targets such as homolog of RAF-like kinase. These findings suggest that, although canonical PIN-mediated auxin transport mechanisms might not be fully conserved in Chara, IAA is involved in morphogenesis and fast signaling processes.

• Keywords
• Chara, auxin transport, indole‐3‐acetic acid, plant evolution, streptophytes,
• MeSH
• Biological Transport drug effects   MeSH
• Cell Membrane metabolism   drug effects   MeSH
• Chara * metabolism   drug effects   MeSH
• Phosphorylation drug effects   MeSH
• Indoleacetic Acids * metabolism   pharmacology   MeSH
• Membrane Transport Proteins * metabolism   MeSH
• Plant Proteins * metabolism   MeSH
• Nicotiana metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• indoleacetic acid MeSH Browser
• Indoleacetic Acids * MeSH
• Membrane Transport Proteins * MeSH
• Plant Proteins * MeSH

Microtubules of all eukaryotic cells are formed by α- and β-tubulin heterodimers. In addition to the well known cytoplasmic tubulins, a subpopulation of tubulin can occur in the nucleus. So far, the potential function of nuclear tubulin has remained elusive. In this work, we show that α- and β-tubulins of various organisms contain multiple conserved nuclear export sequences, which are potential targets of the Exportin 1/CRM1 pathway. We demonstrate exemplarily that these NES motifs are sufficient to mediate export of GFP as model cargo and that this export can be inhibited by leptomycin B, an inhibitor of the Exportin 1/CRM1 pathway. Likewise, leptomycin B causes accumulation of GFP-tagged tubulin in interphase nuclei, in both plant and animal model cells. Our analysis of nuclear tubulin content supports the hypothesis that an important function of nuclear tubulin export is the exclusion of tubulin from interphase nuclei, after being trapped by nuclear envelope reassembly during telophase.

• MeSH
• Active Transport, Cell Nucleus physiology   MeSH
• Cell Nucleus metabolism   MeSH
• Cell Line MeSH
• Cytoplasm metabolism   MeSH
• Eukaryotic Cells metabolism   MeSH
• Karyopherins metabolism   MeSH
• Humans MeSH
• Microtubules metabolism   MeSH
• Exportin 1 Protein MeSH
• Receptors, Cytoplasmic and Nuclear metabolism   MeSH
• Nicotiana metabolism   MeSH
• Protein Transport physiology   MeSH
• Tubulin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Karyopherins MeSH
• Receptors, Cytoplasmic and Nuclear MeSH
• Tubulin MeSH

The volatile two-carbon hormone ethylene acts in concert with an array of signals to affect etiolated seedling development. From a chemical screen, we isolated a quinoline carboxamide designated ACCERBATIN (AEX) that exacerbates the 1-aminocyclopropane-1-carboxylic acid-induced triple response, typical for ethylene-treated seedlings in darkness. Phenotypic analyses revealed distinct AEX effects including inhibition of root hair development and shortening of the root meristem. Mutant analysis and reporter studies further suggested that AEX most probably acts in parallel to ethylene signaling. We demonstrated that AEX functions at the intersection of auxin metabolism and reactive oxygen species (ROS) homeostasis. AEX inhibited auxin efflux in BY-2 cells and promoted indole-3-acetic acid (IAA) oxidation in the shoot apical meristem and cotyledons of etiolated seedlings. Gene expression studies and superoxide/hydrogen peroxide staining further revealed that the disrupted auxin homeostasis was accompanied by oxidative stress. Interestingly, in light conditions, AEX exhibited properties reminiscent of the quinoline carboxylate-type auxin-like herbicides. We propose that AEX interferes with auxin transport from its major biosynthesis sites, either as a direct consequence of poor basipetal transport from the shoot meristematic region, or indirectly, through excessive IAA oxidation and ROS accumulation. Further investigation of AEX can provide new insights into the mechanisms connecting auxin and ROS homeostasis in plant development and provide useful tools to study auxin-type herbicides.

• Keywords
• Arabidopsis, auxin homeostasis, chemical genetics, ethylene signaling, herbicide, quinoline carboxamide, reactive oxygen species, triple response,
• MeSH
• Amino Acids, Cyclic metabolism   MeSH
• Arabidopsis genetics   metabolism   MeSH
• Quinolones metabolism   MeSH
• Ethylenes metabolism   MeSH
• Gene Expression MeSH
• Herbicides chemistry   MeSH
• Homeostasis MeSH
• Indoleacetic Acids metabolism   MeSH
• Arabidopsis Proteins metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Seedlings metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 1-aminocyclopropane-1-carboxylic acid MeSH Browser
• Amino Acids, Cyclic MeSH
• Quinolones MeSH
• ethylene MeSH Browser
• Ethylenes MeSH
• Herbicides MeSH
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Reactive Oxygen Species MeSH

Auxin steers numerous physiological processes in plants, making the tight control of its endogenous levels and spatiotemporal distribution a necessity. This regulation is achieved by different mechanisms, including auxin biosynthesis, metabolic conversions, degradation, and transport. Here, we introduce cis-cinnamic acid (c-CA) as a novel and unique addition to a small group of endogenous molecules affecting in planta auxin concentrations. c-CA is the photo-isomerization product of the phenylpropanoid pathway intermediate trans-CA (t-CA). When grown on c-CA-containing medium, an evolutionary diverse set of plant species were shown to exhibit phenotypes characteristic for high auxin levels, including inhibition of primary root growth, induction of root hairs, and promotion of adventitious and lateral rooting. By molecular docking and receptor binding assays, we showed that c-CA itself is neither an auxin nor an anti-auxin, and auxin profiling data revealed that c-CA does not significantly interfere with auxin biosynthesis. Single cell-based auxin accumulation assays showed that c-CA, and not t-CA, is a potent inhibitor of auxin efflux. Auxin signaling reporters detected changes in spatiotemporal distribution of the auxin response along the root of c-CA-treated plants, and long-distance auxin transport assays showed no inhibition of rootward auxin transport. Overall, these results suggest that the phenotypes of c-CA-treated plants are the consequence of a local change in auxin accumulation, induced by the inhibition of auxin efflux. This work reveals a novel mechanism how plants may regulate auxin levels and adds a novel, naturally occurring molecule to the chemical toolbox for the studies of auxin homeostasis.

• MeSH
• Arabidopsis drug effects   growth & development   MeSH
• Cinnamates chemistry   metabolism   pharmacology   MeSH
• Cyclin B genetics   metabolism   MeSH
• Plants, Genetically Modified MeSH
• Isomerism MeSH
• Plant Roots drug effects   growth & development   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Bryopsida drug effects   growth & development   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Qa-SNARE Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant MeSH
• Selaginellaceae drug effects   growth & development   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cinnamates MeSH
• cinnamic acid MeSH Browser
• CycB1 protein, Arabidopsis MeSH Browser
• Cyclin B MeSH
• KNOLLE protein, Arabidopsis MeSH Browser
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Qa-SNARE Proteins MeSH

Apical dominance is one of the fundamental developmental phenomena in plant biology, which determines the overall architecture of aerial plant parts. Here we show apex decapitation activated competition for dominance in adjacent upper and lower axillary buds. A two-nodal-bud pea (Pisum sativum L.) was used as a model system to monitor and assess auxin flow, auxin transport channels, and dormancy and initiation status of axillary buds. Auxin flow was manipulated by lateral stem wounds or chemically by auxin efflux inhibitors 2,3,5-triiodobenzoic acid (TIBA), 1-N-naphtylphtalamic acid (NPA), or protein synthesis inhibitor cycloheximide (CHX) treatments, which served to interfere with axillary bud competition. Redirecting auxin flow to different points influenced which bud formed the outgrowing and dominant shoot. The obtained results proved that competition between upper and lower axillary buds as secondary auxin sources is based on the same auxin canalization principle that operates between the shoot apex and axillary bud.

• MeSH
• Biological Transport MeSH
• Pisum sativum drug effects   genetics   growth & development   MeSH
• Indoleacetic Acids pharmacology   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Growth Regulators pharmacology   MeSH
• Plant Proteins genetics   MeSH
• Plant Stems drug effects   genetics   growth & development   MeSH
• Plant Shoots drug effects   genetics   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Plant Growth Regulators MeSH
• Plant Proteins MeSH

Here we present an overview of what is known about endogenous plant compounds that act as inhibitors of hormonal transport processes in plants, about their identity and mechanism of action. We have also summarized commonly and less commonly used compounds of non-plant origin and synthetic drugs that show at least partial 'specificity' to transport or transporters of particular phytohormones. Our main attention is focused on the inhibitors of auxin transport. The urgent need to understand precisely the molecular mechanism of action of these inhibitors is highlighted.

• Keywords
• Abscisic acid, Auxin, Cell biology, Cytokinins, Inhibitors, Plant hormones, Strigolactones, Transport,
• MeSH
• Models, Biological MeSH
• Biological Transport MeSH
• Plant Growth Regulators metabolism   MeSH
• Plant Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Review MeSH
• Names of Substances
• Plant Growth Regulators MeSH
• Plant Proteins MeSH

The phenylpropanoid 3,4-(methylenedioxy)cinnamic acid (MDCA) is a plant-derived compound first extracted from roots of Asparagus officinalis and further characterized as an allelochemical. Later on, MDCA was identified as an efficient inhibitor of 4-COUMARATE-CoA LIGASE (4CL), a key enzyme of the general phenylpropanoid pathway. By blocking 4CL, MDCA affects the biosynthesis of many important metabolites, which might explain its phytotoxicity. To decipher the molecular basis of the allelochemical activity of MDCA, we evaluated the effect of this compound on Arabidopsis thaliana seedlings. Metabolic profiling revealed that MDCA is converted in planta into piperonylic acid (PA), an inhibitor of CINNAMATE-4-HYDROXYLASE (C4H), the enzyme directly upstream of 4CL. The inhibition of C4H was also reflected in the phenolic profile of MDCA-treated plants. Treatment of in vitro grown plants resulted in an inhibition of primary root growth and a proliferation of lateral and adventitious roots. These observed growth defects were not the consequence of lignin perturbation, but rather the result of disturbing auxin homeostasis. Based on DII-VENUS quantification and direct measurement of cellular auxin transport, we concluded that MDCA disturbs auxin gradients by interfering with auxin efflux. In addition, mass spectrometry was used to show that MDCA triggers auxin biosynthesis, conjugation, and catabolism. A similar shift in auxin homeostasis was found in the c4h mutant ref3-2, indicating that MDCA triggers a cross talk between the phenylpropanoid and auxin biosynthetic pathways independent from the observed auxin efflux inhibition. Altogether, our data provide, to our knowledge, a novel molecular explanation for the phytotoxic properties of MDCA.

• MeSH
• Trans-Cinnamate 4-Monooxygenase antagonists & inhibitors   metabolism   MeSH
• Arabidopsis drug effects   genetics   metabolism   MeSH
• Benzoates metabolism   pharmacology   MeSH
• Biosynthetic Pathways drug effects   MeSH
• Cinnamates chemistry   metabolism   pharmacology   MeSH
• Phenylpropionates chemistry   metabolism   pharmacology   MeSH
• Plants, Genetically Modified MeSH
• Mass Spectrometry MeSH
• Homeostasis drug effects   MeSH
• Coenzyme A Ligases antagonists & inhibitors   metabolism   MeSH
• Microscopy, Confocal MeSH
• Plant Roots drug effects   genetics   metabolism   MeSH
• Indoleacetic Acids metabolism   MeSH
• Lignin biosynthesis   MeSH
• Seedlings drug effects   genetics   growth & development   metabolism   MeSH
• Dose-Response Relationship, Drug MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• 4-coumarate-CoA ligase MeSH Browser
• Trans-Cinnamate 4-Monooxygenase MeSH
• Benzoates MeSH
• Cinnamates MeSH
• cinnamic acid MeSH Browser
• Phenylpropionates MeSH
• Coenzyme A Ligases MeSH
• Indoleacetic Acids MeSH
• Lignin MeSH
• phenylpropanoid 3,4-(methylenedioxy)cinnamic acid MeSH Browser
• piperonylic acid MeSH Browser

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.

• MeSH
• Arabidopsis genetics   metabolism   MeSH
• Biological Transport genetics   physiology   MeSH
• Indoleacetic Acids metabolism   MeSH
• Actin Cytoskeleton metabolism   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Tacrolimus Binding Proteins genetics   metabolism   MeSH
• Gene Expression Regulation, Plant genetics   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Indoleacetic Acids MeSH
• Arabidopsis Proteins MeSH
• Tacrolimus Binding Proteins MeSH
• TWD1 protein, Arabidopsis MeSH Browser

BACKGROUND: Processes of anterograde and retrograde membrane trafficking play an important role in cellular homeostasis and dynamic rearrangements of the plasma membrane (PM) in all eukaryotes. These processes depend on the activity of adenosine ribosylation factors (ARFs), a family of GTP-binding proteins and their guanine exchange factors (GEFs). However, knowledge on the function and specificity of individual ARF-GEFs for individual steps of membrane trafficking pathways is still limited in plants. RESULTS: In this work, treatments with various trafficking inhibitors showed that the endocytosis of FM 4-64 is largely dynamin-dependent and relies on proteins containing endocytic tyrosine-based internalization motif and intact cytoskeleton. Interestingly, brefeldin A (BFA), reported previously as an inhibitor of anterograde membrane trafficking in plants, appeared to be the most potent inhibitor of endocytosis in tobacco. In concert with this finding, we demonstrate that the point mutation in the Sec7 domain of the GNOM-LIKE protein1a (NtGNL1a) confers intracellular trafficking pathway-specific BFA resistance. The internalization of FM 4-64 and trafficking of PIN-FORMED1 (PIN1) auxin efflux carrier in BY-2 tobacco cells were studied to reveal the function of the ARF-GEF NtGNL1a in these. CONCLUSIONS: Altogether, our observations uncovered the role of NtGNL1a in endocytosis, including endocytosis of PM proteins (as PIN1 auxin efflux carrier). Moreover these data emphasize the need of careful evaluation of mode of action of non-native inhibitors in various species. In addition, they demonstrate the potential of tobacco BY-2 cells for selective mapping of ARF-GEF-regulated endomembrane trafficking pathways.

• MeSH
• Endocytosis MeSH
• Quaternary Ammonium Compounds metabolism   MeSH
• Pyridinium Compounds metabolism   MeSH
• Plant Cells physiology   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Nicotiana genetics   physiology   MeSH
• Protein Transport MeSH
• Guanine Nucleotide Exchange Factors genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• FM 4-64 MeSH Browser
• Quaternary Ammonium Compounds MeSH
• Pyridinium Compounds MeSH
• Plant Proteins MeSH
• Guanine Nucleotide Exchange Factors MeSH

BACKGROUND: Auxin binding protein 1 (ABP1) is a putative auxin receptor and its function is indispensable for plant growth and development. ABP1 has been shown to be involved in auxin-dependent regulation of cell division and expansion, in plasma-membrane-related processes such as changes in transmembrane potential, and in the regulation of clathrin-dependent endocytosis. However, the ABP1-regulated downstream pathway remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: Using auxin transport assays and quantitative analysis of cellular morphology we show that ABP1 regulates auxin efflux from tobacco BY-2 cells. The overexpression of ABP1can counterbalance increased auxin efflux and auxin starvation phenotypes caused by the overexpression of PIN auxin efflux carrier. Relevant mechanism involves the ABP1-controlled vesicle trafficking processes, including positive regulation of endocytosis of PIN auxin efflux carriers, as indicated by fluorescence recovery after photobleaching (FRAP) and pharmacological manipulations. CONCLUSIONS/SIGNIFICANCE: The findings indicate the involvement of ABP1 in control of rate of auxin transport across plasma membrane emphasizing the role of ABP1 in regulation of PIN activity at the plasma membrane, and highlighting the relevance of ABP1 for the formation of developmentally important, PIN-dependent auxin gradients.

• MeSH
• Arabidopsis cytology   metabolism   MeSH
• Cell Line MeSH
• Fluorescence Recovery After Photobleaching MeSH
• Microscopy, Confocal MeSH
• Indoleacetic Acids metabolism   MeSH
• Membrane Transport Modulators metabolism   MeSH
• Receptors, Cell Surface biosynthesis   metabolism   MeSH
• Plant Proteins biosynthesis   metabolism   MeSH
• Nicotiana cytology   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• auxin-binding protein 1 MeSH Browser
• endoplasmic reticulum auxin-binding protein 4, Zea mays MeSH Browser
• Indoleacetic Acids MeSH
• Membrane Transport Modulators MeSH
• Receptors, Cell Surface MeSH
• Plant Proteins MeSH