Agronomic breeding practices for grapevines (Vitis vinifera L.) include the application of growth regulators in the field. Brassinosteroids (BRs) are a family of sterol-derived plant hormones that regulate several physiological processes and responses to biotic and abiotic stress. In grapevine berries, the production of biologically active BRs, castasterone and 6-deoxocastasterone, has been reported. In this work, key BR genes were identified, and their expression profiles were determined in grapevine. Bioinformatic homology analyses of the Arabidopsis genome found 14 genes associated with biosynthetic, perception and signaling pathways, suggesting a partial conservation of these pathways between the two species. The tissue- and development-specific expression profiles of these genes were determined by qRT-PCR in nine different grapevine tissues. Using UHPLC-MS/MS, 10 different BR compounds were pinpointed and quantified in 20 different tissues, each presenting specific accumulation patterns. Although, in general, the expression profile of the biosynthesis pathway genes of BRs did not directly correlate with the accumulation of metabolites, this could reflect the complexity of the BR biosynthesis pathway and its regulation. The development of this work thus generates a contribution to our knowledge about the presence, and diversity of BRs in grapevines.

• Klíčová slova
• UHPLC-MS/MS, brassinosteroids, development, grapevine,
• MeSH
• Arabidopsis * metabolismus   MeSH
• brassinosteroidy * metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin genetika   metabolismus   MeSH
• šlechtění rostlin MeSH
• tandemová hmotnostní spektrometrie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• brassinosteroidy * MeSH
• regulátory růstu rostlin MeSH

Brassinosteroid (BR) hormones are indispensable for root growth and control both cell division and cell elongation through the establishment of an increasing signalling gradient along the longitudinal root axis. Because of their limited mobility, the importance of BR distribution in achieving a signalling maximum is largely overlooked. Expression pattern analysis of all known BR biosynthetic enzymes revealed that not all cells in the Arabidopsis thaliana root possess full biosynthetic machinery, and that completion of biosynthesis relies on cell-to-cell movement of hormone precursors. We demonstrate that BR biosynthesis is largely restricted to the root elongation zone, where it overlaps with BR signalling maxima. Moreover, optimal root growth requires hormone concentrations to be low in the meristem and high in the root elongation zone, attributable to increased biosynthesis. Our finding that spatiotemporal regulation of hormone synthesis results in local hormone accumulation provides a paradigm for hormone-driven organ growth in the absence of long-distance hormone transport in plants.

• MeSH
• Arabidopsis růst a vývoj   metabolismus   fyziologie   MeSH
• brassinosteroidy biosyntéza   metabolismus   MeSH
• kořeny rostlin růst a vývoj   metabolismus   MeSH
• meristém metabolismus   MeSH
• metabolické sítě a dráhy MeSH
• regulace genové exprese u rostlin MeSH
• regulátory růstu rostlin metabolismus   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• brassinosteroidy MeSH
• regulátory růstu rostlin 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

The asymmetric localization of proteins in the plasma membrane domains of eukaryotic cells is a fundamental manifestation of cell polarity that is central to multicellular organization and developmental patterning. In plants, the mechanisms underlying the polar localization of cargo proteins are still largely unknown and appear to be fundamentally distinct from those operating in mammals. Here, we present a systematic, quantitative comparative analysis of the polar delivery and subcellular localization of proteins that characterize distinct polar plasma membrane domains in plant cells. The combination of microscopic analyses and computational modeling revealed a mechanistic framework common to diverse polar cargos and underlying the establishment and maintenance of apical, basal, and lateral polar domains in plant cells. This mechanism depends on the polar secretion, constitutive endocytic recycling, and restricted lateral diffusion of cargos within the plasma membrane. Moreover, our observations suggest that polar cargo distribution involves the individual protein potential to form clusters within the plasma membrane and interact with the extracellular matrix. Our observations provide insights into the shared cellular mechanisms of polar cargo delivery and polarity maintenance in plant cells.

• Klíčová slova
• lateral diffusion, polar recycling, polar secretion, protein clustering, protein dynamics modeling, protein trafficking,
• Publikační typ
• časopisecké články MeSH