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
Plant development mediated by the phytohormone auxin depends on tightly controlled cellular auxin levels at its target tissue that are largely established by intercellular and intracellular auxin transport mediated by PIN auxin transporters. Among the eight members of the Arabidopsis PIN family, PIN6 is the least characterized candidate. In this study we generated functional, fluorescent protein-tagged PIN6 proteins and performed comprehensive analysis of their subcellular localization and also performed a detailed functional characterization of PIN6 and its developmental roles. The localization study of PIN6 revealed a dual localization at the plasma membrane (PM) and endoplasmic reticulum (ER). Transport and metabolic profiling assays in cultured cells and Arabidopsis strongly suggest that PIN6 mediates both auxin transport across the PM and intracellular auxin homeostasis, including the regulation of free auxin and auxin conjugates levels. As evidenced by the loss- and gain-of-function analysis, the complex function of PIN6 in auxin transport and homeostasis is required for auxin distribution during lateral and adventitious root organogenesis and for progression of these developmental processes. These results illustrate a unique position of PIN6 within the family of PIN auxin transporters and further add complexity to the developmentally crucial process of auxin transport.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- buněčná membrána metabolismus MeSH
- endoplazmatické retikulum metabolismus MeSH
- fylogeneze MeSH
- geneticky modifikované rostliny MeSH
- homeostáza MeSH
- kořeny rostlin růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- molekulární evoluce MeSH
- proteiny huseníčku genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The plant hormone auxin is a key regulator of plant growth and development. Auxin levels are sensed and interpreted by distinct receptor systems that activate a broad range of cellular responses. The Auxin-Binding Protein1 (ABP1) that has been identified based on its ability to bind auxin with high affinity is a prime candidate for the extracellular receptor responsible for mediating a range of auxin effects, in particular, the fast non-transcriptional ones. Contradictory genetic studies suggested prominent or no importance of ABP1 in many developmental processes. However, how crucial the role of auxin binding to ABP1 is for its functions has not been addressed. Here, we show that the auxin-binding pocket of ABP1 is essential for its gain-of-function cellular and developmental roles. In total, 16 different abp1 mutants were prepared that possessed substitutions in the metal core or in the hydrophobic amino acids of the auxin-binding pocket as well as neutral mutations. Their analysis revealed that an intact auxin-binding pocket is a prerequisite for ABP1 to activate downstream components of the ABP1 signalling pathway, such as Rho of Plants (ROPs) and to mediate the clathrin association with membranes for endocytosis regulation. In planta analyses demonstrated the importance of the auxin binding pocket for all known ABP1-mediated postembryonic developmental processes, including morphology of leaf epidermal cells, root growth and root meristem activity, and vascular tissue differentiation. Taken together, these findings suggest that auxin binding to ABP1 is central to its function, supporting the role of ABP1 as auxin receptor.
- MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- kyseliny indoloctové metabolismus MeSH
- mutageneze MeSH
- receptory buněčného povrchu genetika metabolismus MeSH
- regulátory růstu rostlin metabolismus MeSH
- rostlinné proteiny genetika metabolismus MeSH
- substituce aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Arabidopsis (Arabidopsis thaliana) leaf development relies on subsequent phases of cell proliferation and cell expansion. During the proliferation phase, chloroplasts need to divide extensively, and during the transition from cell proliferation to expansion, they differentiate into photosynthetically active chloroplasts, providing the plant with energy. The transcription factor GROWTH REGULATING FACTOR5 (GRF5) promotes the duration of the cell proliferation period during leaf development. Here, it is shown that GRF5 also stimulates chloroplast division, resulting in a higher chloroplast number per cell with a concomitant increase in chlorophyll levels in 35S:GRF5 leaves, which can sustain higher rates of photosynthesis. Moreover, 35S:GRF5 plants show delayed leaf senescence and are more tolerant for growth on nitrogen-depleted medium. Cytokinins also stimulate leaf growth in part by extending the cell proliferation phase, simultaneously delaying the onset of the cell expansion phase. In addition, cytokinins are known to be involved in chloroplast development, nitrogen signaling, and senescence. Evidence is provided that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention after dark-induced senescence, which suggests that they also cooperate to stimulate chloroplast division and nitrogen assimilation. Taken together with the increased leaf size, ectopic expression of GRF5 has great potential to improve plant productivity.
- MeSH
- Arabidopsis účinky léků genetika fyziologie MeSH
- buněčné dělení účinky léků MeSH
- chlorofyl metabolismus MeSH
- chloroplasty účinky léků metabolismus ultrastruktura MeSH
- cytokininy farmakologie MeSH
- dusík nedostatek MeSH
- fotosyntéza * účinky léků MeSH
- geneticky modifikované rostliny MeSH
- listy rostlin účinky léků růst a vývoj fyziologie ultrastruktura MeSH
- proteiny 14-3-3 genetika metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- rostlinné geny MeSH
- trans-aktivátory genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The exocyst complex, an effector of Rho and Rab GTPases, is believed to function as an exocytotic vesicle tether at the plasma membrane before soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation. Exocyst subunits localize to secretory-active regions of the plasma membrane, exemplified by the outer domain of Arabidopsis root epidermal cells. Using variable-angle epifluorescence microscopy, we visualized the dynamics of exocyst subunits at this domain. The subunits colocalized in defined foci at the plasma membrane, distinct from endocytic sites. Exocyst foci were independent of cytoskeleton, although prolonged actin disruption led to changes in exocyst localization. Exocyst foci partially overlapped with vesicles visualized by VAMP721 v-SNARE, but the majority of the foci represent sites without vesicles, as indicated by electron microscopy and drug treatments, supporting the concept of the exocyst functioning as a dynamic particle. We observed a decrease of SEC6-green fluorescent protein foci in an exo70A1 exocyst mutant. Finally, we documented decreased VAMP721 trafficking to the plasma membrane in exo70A1 and exo84b mutants. Our data support the concept that the exocyst-complex subunits dynamically dock and undock at the plasma membrane to create sites primed for vesicle tethering.
- MeSH
- Arabidopsis genetika metabolismus ultrastruktura MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- cytoplazma metabolismus ultrastruktura MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- epidermis rostlin genetika metabolismus ultrastruktura MeSH
- exocytóza MeSH
- exprese genu MeSH
- fluorescenční mikroskopie MeSH
- kořeny rostlin genetika metabolismus ultrastruktura MeSH
- proteiny huseníčku genetika metabolismus MeSH
- proteiny SNARE genetika metabolismus MeSH
- rab proteiny vázající GTP genetika metabolismus MeSH
- sekreční vezikuly metabolismus ultrastruktura MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 (pat3) mutant that we identified by an epifluorescence-based forward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFP. While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compartments (PVC) with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A (VPS35A), a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lytic vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.
- MeSH
- Arabidopsis genetika fyziologie MeSH
- endocytóza MeSH
- kompartmentace buňky * MeSH
- membránové proteiny metabolismus fyziologie MeSH
- molekulární sekvence - údaje MeSH
- mutace MeSH
- proteiny huseníčku metabolismus fyziologie MeSH
- transport proteinů MeSH
- vakuoly metabolismus MeSH
- vezikulární transportní proteiny metabolismus fyziologie MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
