To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans-Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress.

• Klíčová slova
• Arabidopsis thaliana, ARF, GEF, endocytosis, flippase, osmotic stress, protein trafficking, secretion,
• MeSH
• Arabidopsis * metabolismus   MeSH
• biologický transport MeSH
• buněčná membrána metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• osmotický tlak MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• membránové proteiny MeSH
• proteiny huseníčku * MeSH

Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.

• Klíčová slova
• PIN1, dimerization, hydrophilic hoop, intrinsic disorder, subcellular trafficking,
• MeSH
• Arabidopsis * metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• vnitřně neuspořádané proteiny * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku * MeSH
• vnitřně neuspořádané proteiny * MeSH

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.

• MeSH
• Arabidopsis cytologie   genetika   metabolismus   MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána metabolismus   MeSH
• cytokininy chemie   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• fluorescenční barviva chemie   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• meristém cytologie   metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• receptory buněčného povrchu genetika   metabolismus   MeSH
• rekombinantní fúzní proteiny genetika   metabolismus   MeSH
• signální transdukce účinky léků   MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• brefeldin A MeSH
• cytokininy MeSH
• fluorescenční barviva MeSH
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH
• rekombinantní fúzní proteiny MeSH
• WOL protein, Arabidopsis MeSH Prohlížeč
• zelené fluorescenční proteiny MeSH

Directional transport of the phytohormone auxin is a versatile, plant-specific mechanism regulating many aspects of plant development. The recently identified plant hormones, strigolactones (SLs), are implicated in many plant traits; among others, they modify the phenotypic output of PIN-FORMED (PIN) auxin transporters for fine-tuning of growth and developmental responses. Here, we show in pea and Arabidopsis that SLs target processes dependent on the canalization of auxin flow, which involves auxin feedback on PIN subcellular distribution. D14 receptor- and MAX2 F-box-mediated SL signaling inhibits the formation of auxin-conducting channels after wounding or from artificial auxin sources, during vasculature de novo formation and regeneration. At the cellular level, SLs interfere with auxin effects on PIN polar targeting, constitutive PIN trafficking as well as clathrin-mediated endocytosis. Our results identify a non-transcriptional mechanism of SL action, uncoupling auxin feedback on PIN polarity and trafficking, thereby regulating vascular tissue formation and regeneration.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• heterocyklické sloučeniny tricyklické metabolismus   MeSH
• hrách setý genetika   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• laktony metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin genetika   fyziologie   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GR24 strigolactone MeSH Prohlížeč
• heterocyklické sloučeniny tricyklické MeSH
• kyseliny indoloctové MeSH
• laktony MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.

• MeSH
• ADP-ribosylační faktory metabolismus   MeSH
• Arabidopsis účinky léků   metabolismus   MeSH
• biologický transport účinky léků   MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• genetická epistáze účinky léků   MeSH
• GTP-fosfohydrolasy metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• mutace genetika   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející fosfolipidy metabolismus   MeSH
• tabák metabolismus   MeSH
• trans-Golgiho síť účinky léků   metabolismus   MeSH
• vazba proteinů účinky léků   MeSH
• výměnné faktory guaninnukleotidů metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ADP-ribosylační faktory MeSH
• brefeldin A MeSH
• GTP-fosfohydrolasy MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku MeSH
• proteiny přenášející fosfolipidy MeSH
• výměnné faktory guaninnukleotidů MeSH

The trafficking of subcellular cargos in eukaryotic cells crucially depends on vesicle budding, a process mediated by ARF-GEFs (ADP-ribosylation factor guanine nucleotide exchange factors). In plants, ARF-GEFs play essential roles in endocytosis, vacuolar trafficking, recycling, secretion, and polar trafficking. Moreover, they are important for plant development, mainly through controlling the polar subcellular localization of PIN-FORMED transporters of the plant hormone auxin. Here, using a chemical genetics screen in Arabidopsis thaliana, we identified Endosidin 4 (ES4), an inhibitor of eukaryotic ARF-GEFs. ES4 acts similarly to and synergistically with the established ARF-GEF inhibitor Brefeldin A and has broad effects on intracellular trafficking, including endocytosis, exocytosis, and vacuolar targeting. Additionally, Arabidopsis and yeast (Saccharomyces cerevisiae) mutants defective in ARF-GEF show altered sensitivity to ES4. ES4 interferes with the activation-based membrane association of the ARF1 GTPases, but not of their mutant variants that are activated independently of ARF-GEF activity. Biochemical approaches and docking simulations confirmed that ES4 specifically targets the SEC7 domain-containing ARF-GEFs. These observations collectively identify ES4 as a chemical tool enabling the study of ARF-GEF-mediated processes, including ARF-GEF-mediated plant development.

• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• brefeldin A farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• chromony chemie   farmakologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endocytóza účinky léků   MeSH
• geneticky modifikované rostliny MeSH
• membránové glykoproteiny genetika   metabolismus   MeSH
• membránové transportní proteiny genetika   metabolismus   MeSH
• mutace MeSH
• proteinové domény MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae účinky léků   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• transkripční faktory genetika   metabolismus   MeSH
• transport proteinů účinky léků   MeSH
• výměnné faktory guaninnukleotidů chemie   genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ARF1 protein, Arabidopsis MeSH Prohlížeč
• brefeldin A MeSH
• chromony MeSH
• DNA vazebné proteiny MeSH
• GNL1 protein, Arabidopsis MeSH Prohlížeč
• GNOM protein, Arabidopsis MeSH Prohlížeč
• membránové glykoproteiny MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• SEC12 protein, S cerevisiae MeSH Prohlížeč
• transkripční faktory MeSH
• výměnné faktory guaninnukleotidů MeSH

Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterized compared with that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing tandem affinity purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologs of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in Arabidopsis caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like1/2 loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the ongoing characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in Arabidopsis.

• MeSH
• Arabidopsis genetika   metabolismus   MeSH
• endocytóza genetika   fyziologie   MeSH
• klathrin genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• semenáček genetika   metabolismus   MeSH
• transport proteinů MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• klathrin MeSH
• proteiny huseníčku MeSH

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
• endocytóza MeSH
• kvartérní amoniové sloučeniny metabolismus   MeSH
• pyridinové sloučeniny metabolismus   MeSH
• rostlinné buňky fyziologie   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• tabák genetika   fyziologie   MeSH
• transport proteinů MeSH
• výměnné faktory guaninnukleotidů genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• FM 4-64 MeSH Prohlížeč
• kvartérní amoniové sloučeniny MeSH
• pyridinové sloučeniny MeSH
• rostlinné proteiny MeSH
• výměnné faktory guaninnukleotidů 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 cytologie   metabolismus   MeSH
• buněčné linie MeSH
• FRAP MeSH
• konfokální mikroskopie MeSH
• kyseliny indoloctové metabolismus   MeSH
• modulátory membránového transportu metabolismus   MeSH
• receptory buněčného povrchu biosyntéza   metabolismus   MeSH
• rostlinné proteiny biosyntéza   metabolismus   MeSH
• tabák cytologie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• auxin-binding protein 1 MeSH Prohlížeč
• endoplasmic reticulum auxin-binding protein 4, Zea mays MeSH Prohlížeč
• kyseliny indoloctové MeSH
• modulátory membránového transportu MeSH
• receptory buněčného povrchu MeSH
• rostlinné proteiny MeSH

The phytohormone auxin is transported through the plant body either via vascular pathways or from cell to cell by specialized polar transport machinery. This machinery consists of a balanced system of passive diffusion combined with the activities of auxin influx and efflux carriers. Synthetic auxins that differ in the mechanisms of their transport across the plasma membrane together with polar auxin transport inhibitors have been used in many studies on particular auxin carriers and their role in plant development. However, the exact mechanism of action of auxin efflux and influx inhibitors has not been fully elucidated. In this report, the mechanism of action of the auxin influx inhibitors (1-naphthoxyacetic acid (1-NOA), 2-naphthoxyacetic acid (2-NOA), and 3-chloro-4-hydroxyphenylacetic acid (CHPAA)) is examined by direct measurements of auxin accumulation, cellular phenotypic analysis, as well as by localization studies of Arabidopsis thaliana L. auxin carriers heterologously expressed in Nicotiana tabacum L., cv. Bright Yellow cell suspensions. The mode of action of 1-NOA, 2-NOA, and CHPAA has been shown to be linked with the dynamics of the plasma membrane. The most potent inhibitor, 1-NOA, blocked the activities of both auxin influx and efflux carriers, whereas 2-NOA and CHPAA at the same concentration preferentially inhibited auxin influx. The results suggest that these, previously unknown, activities of putative auxin influx inhibitors regulate overall auxin transport across the plasma membrane depending on the dynamics of particular membrane vesicles.

• MeSH
• biologický transport účinky léků   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buňky MeSH
• fenylacetáty farmakologie   MeSH
• glykoláty farmakologie   MeSH
• kyseliny indoloctové metabolismus   MeSH
• tabák účinky léků   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1-naphthoxyacetic acid MeSH Prohlížeč
• 2-naphthoxyacetic acid MeSH Prohlížeč
• 3-chloro-4-hydroxyphenylacetic acid MeSH Prohlížeč
• fenylacetáty MeSH
• glykoláty MeSH
• kyseliny indoloctové MeSH