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

To overcome nitrogen deficiency, legume roots establish symbiotic interactions with nitrogen-fixing rhizobia that are fostered in specialized organs (nodules). Similar to other organs, nodule formation is determined by a local maximum of the phytohormone auxin at the primordium site. However, how auxin regulates nodule development remains poorly understood. Here, we found that in soybean, (Glycine max), dynamic auxin transport driven by PIN-FORMED (PIN) transporter GmPIN1 is involved in nodule primordium formation. GmPIN1 was specifically expressed in nodule primordium cells and GmPIN1 was polarly localized in these cells. Two nodulation regulators, (iso)flavonoids trigger expanded distribution of GmPIN1b to root cortical cells, and cytokinin rearranges GmPIN1b polarity. Gmpin1abc triple mutants generated with CRISPR-Cas9 showed the impaired establishment of auxin maxima in nodule meristems and aberrant divisions in the nodule primordium cells. Moreover, overexpression of GmPIN1 suppressed nodule primordium initiation. GmPIN9d, an ortholog of Arabidopsis thaliana PIN2, acts together with GmPIN1 later in nodule development to acropetally transport auxin in vascular bundles, fine-tuning the auxin supply for nodule enlargement. Our findings reveal how PIN-dependent auxin transport modulates different aspects of soybean nodule development and suggest that the establishment of auxin gradient is a prerequisite for the proper interaction between legumes and rhizobia.

• MeSH
• biologický transport MeSH
• Glycine max genetika   růst a vývoj   metabolismus   MeSH
• kořenové hlízky rostlin růst a vývoj   metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• rostlinné proteiny MeSH

Polar subcellular localization of the PIN exporters of the phytohormone auxin is a key determinant of directional, intercellular auxin transport and thus a central topic of both plant cell and developmental biology. Arabidopsis mutants lacking PID, a kinase that phosphorylates PINs, or the MAB4/MEL proteins of unknown molecular function display PIN polarity defects and phenocopy pin mutants, but mechanistic insights into how these factors convey PIN polarity are missing. Here, by combining protein biochemistry with quantitative live-cell imaging, we demonstrate that PINs, MAB4/MELs, and AGC kinases interact in the same complex at the plasma membrane. MAB4/MELs are recruited to the plasma membrane by the PINs and in concert with the AGC kinases maintain PIN polarity through limiting lateral diffusion-based escape of PINs from the polar domain. The PIN-MAB4/MEL-PID protein complex has self-reinforcing properties thanks to positive feedback between AGC kinase-mediated PIN phosphorylation and MAB4/MEL recruitment. We thus uncover the molecular mechanism by which AGC kinases and MAB4/MEL proteins regulate PIN localization and plant development.

• Klíčová slova
• Arabidopsis, cell polarity, lateral diffusion, plant development, polar auxin transport, positive feedback, protein phosphorylation,
• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• biologický transport MeSH
• kořeny rostlin metabolismus   MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny genetika   MeSH
• polarita buněk MeSH
• proteiny huseníčku * genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné buňky metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• proteiny huseníčku * MeSH

Spontaneously arising channels that transport the phytohormone auxin provide positional cues for self-organizing aspects of plant development such as flexible vasculature regeneration or its patterning during leaf venation. The auxin canalization hypothesis proposes a feedback between auxin signaling and transport as the underlying mechanism, but molecular players await discovery. We identified part of the machinery that routes auxin transport. The auxin-regulated receptor CAMEL (Canalization-related Auxin-regulated Malectin-type RLK) together with CANAR (Canalization-related Receptor-like kinase) interact with and phosphorylate PIN auxin transporters. camel and canar mutants are impaired in PIN1 subcellular trafficking and auxin-mediated PIN polarization, which macroscopically manifests as defects in leaf venation and vasculature regeneration after wounding. The CAMEL-CANAR receptor complex is part of the auxin feedback that coordinates polarization of individual cells during auxin canalization.

• MeSH
• Arabidopsis enzymologie   genetika   MeSH
• biologický transport MeSH
• kyseliny indoloctové metabolismus   MeSH
• mapování interakce mezi proteiny MeSH
• membránové transportní proteiny metabolismus   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• transkripční faktory MeSH
• WRKY23 protein, Arabidopsis MeSH Prohlížeč

Directional intercellular transport of the phytohormone auxin mediated by PIN-FORMED (PIN) efflux carriers has essential roles in both coordinating patterning processes and integrating multiple external cues by rapidly redirecting auxin fluxes. PIN activity is therefore regulated by multiple internal and external cues, for which the underlying molecular mechanisms are not fully elucidated. Here, we demonstrate that 3'-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE1 (PDK1), which is conserved in plants and mammals, functions as a molecular hub that perceives upstream lipid signalling and modulates downstream substrate activity through phosphorylation. Using genetic analysis, we show that the loss-of-function Arabidopsis pdk1.1 pdk1.2 mutant exhibits a plethora of abnormalities in organogenesis and growth due to defective polar auxin transport. Further cellular and biochemical analyses reveal that PDK1 phosphorylates D6 protein kinase, a well-known upstream activator of PIN proteins. We uncover a lipid-dependent phosphorylation cascade that connects membrane-composition-based cellular signalling with plant growth and patterning by regulating morphogenetic auxin fluxes.

• MeSH
• Arabidopsis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• fosfolipidy metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny metabolismus   fyziologie   MeSH
• proteinkinasy PDK fyziologie   MeSH
• proteiny huseníčku fyziologie   MeSH
• regulátory růstu rostlin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfolipidy MeSH
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• proteinkinasy PDK MeSH
• proteiny huseníčku MeSH
• regulátory růstu rostlin MeSH

Plants, like other multicellular organisms, survive through a delicate balance between growth and defense against pathogens. Salicylic acid (SA) is a major defense signal in plants, and the perception mechanism as well as downstream signaling activating the immune response are known. Here, we identify a parallel SA signaling that mediates growth attenuation. SA directly binds to A subunits of protein phosphatase 2A (PP2A), inhibiting activity of this complex. Among PP2A targets, the PIN2 auxin transporter is hyperphosphorylated in response to SA, leading to changed activity of this important growth regulator. Accordingly, auxin transport and auxin-mediated root development, including growth, gravitropic response, and lateral root organogenesis, are inhibited. This study reveals how SA, besides activating immunity, concomitantly attenuates growth through crosstalk with the auxin distribution network. Further analysis of this dual role of SA and characterization of additional SA-regulated PP2A targets will provide further insights into mechanisms maintaining a balance between growth and defense.

• Klíčová slova
• NPR1, PIN, PP2A, auxin, auxin transport, gravitropism, immunity, phosphorylation, protein phosphatase 2A, salicylic acid,
• MeSH
• Arabidopsis růst a vývoj   fyziologie   MeSH
• imunita rostlin MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• kyselina salicylová metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteinfosfatasa 2 metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyselina salicylová MeSH
• kyseliny indoloctové MeSH
• PIN2 protein, Arabidopsis MeSH Prohlížeč
• PP2A protein, Arabidopsis MeSH Prohlížeč
• proteinfosfatasa 2 MeSH
• proteiny huseníčku 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

Auxins mediate various processes that are involved in plant growth and development in response to specific environmental conditions. Its proper spatio-temporal distribution that is driven by polar auxin transport machinery plays a crucial role in the wide range of auxins physiological effects. Numbers of approaches have been developed to either directly or indirectly monitor auxin distribution in vivo in order to elucidate the basis of its precise regulation. Herein, we provide an updated list of valuable techniques used for monitoring auxins in plants, with their utilities and limitations. Because the spatial and temporal resolutions of the presented approaches are different, their combination may provide a comprehensive outcome of auxin distribution in diverse developmental processes.

• Klíčová slova
• auxin, auxin distribution, auxin signalling, auxin transport, direct visualization, indirect visualization, receptor, sensor,
• MeSH
• Arabidopsis metabolismus   MeSH
• kyseliny indoloctové metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• vývoj rostlin fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• kyseliny indoloctové MeSH

Auxin directs plant ontogenesis via differential accumulation within tissues depending largely on the activity of PIN proteins that mediate auxin efflux from cells and its directional cell-to-cell transport. Regardless of the developmental importance of PINs, the structure of these transporters is poorly characterized. Here, we present experimental data concerning protein topology of plasma membrane-localized PINs. Utilizing approaches based on pH-dependent quenching of fluorescent reporters combined with immunolocalization techniques, we mapped the membrane topology of PINs and further cross-validated our results using available topology modeling software. We delineated the topology of PIN1 with two transmembrane (TM) bundles of five α-helices linked by a large intracellular loop and a C-terminus positioned outside the cytoplasm. Using constraints derived from our experimental data, we also provide an updated position of helical regions generating a verisimilitude model of PIN1. Since the canonical long PINs show a high degree of conservation in TM domains and auxin transport capacity has been demonstrated for Arabidopsis representatives of this group, this empirically enhanced topological model of PIN1 will be an important starting point for further studies on PIN structure-function relationships. In addition, we have established protocols that can be used to probe the topology of other plasma membrane proteins in plants.

• Klíčová slova
• Arabidopsis thaliana, auxin efflux carriers, plasma membrane protein, topology,
• MeSH
• Arabidopsis cytologie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• extracelulární prostor metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• kyseliny indoloctové metabolismus   MeSH
• membránové transportní proteiny chemie   metabolismus   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• membránové transportní proteiny MeSH
• PIN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH