In plants, membrane compartmentalization requires vesicle trafficking for communication among distinct organelles. Membrane proteins involved in vesicle trafficking are highly dynamic and can respond rapidly to changes in the environment and to cellular signals. Capturing their localization and dynamics is thus essential for understanding the mechanisms underlying vesicular trafficking pathways. Quantitative mass spectrometry and imaging approaches allow a system-wide dissection of the vesicular proteome, the characterization of ligand-receptor pairs and the determination of secretory, endocytic, recycling and vacuolar trafficking pathways. In this review, we highlight major proteomics and imaging methods employed to determine the location, distribution and abundance of proteins within given trafficking routes. We focus in particular on methodologies for the elucidation of vesicle protein dynamics and interactions and their connections to downstream signalling outputs. Finally, we assess their biological applications in exploring different cellular and subcellular processes.

• Klíčová slova
• Golgi, endocytosis, exocytosis, microscopy, proteomics, vesicle,
• MeSH
• biologický transport MeSH
• endocytóza MeSH
• hmotnostní spektrometrie metody   MeSH
• proteom * analýza   metabolismus   MeSH
• proteomika * metody   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• proteom * MeSH

Plant phospholipase Ds (PLDs), essential regulators of phospholipid signaling, function in multiple signal transduction cascades; however, the mechanisms regulating PLDs in response to pathogens remain unclear. Here, we found that Arabidopsis (Arabidopsis thaliana) PLDδ accumulated in cells at the entry sites of the barley powdery mildew fungus, Blumeria graminis f. sp hordei Using fluorescence recovery after photobleaching and single-molecule analysis, we observed higher PLDδ density in the plasma membrane after chitin treatment; PLDδ also underwent rapid exocytosis. Fluorescence resonance energy transfer with fluorescence lifetime imaging microscopy showed that the interaction between PLDδ and the microdomain marker AtREMORIN1.3 (AtREM1.3) increased in response to chitin, indicating that exocytosis facilitates rapid, efficient sorting of PLDδ into microdomains upon pathogen stimulus. We further unveiled a trade-off between brefeldin A (BFA)-resistant and -sensitive pathways in secretion of PLDδ under diverse conditions. Upon pathogen attack, PLDδ secretion involved syntaxin-associated VAMP721/722-mediated exocytosis sensitive to BFA. Analysis of phosphatidic acid (PA), hydrogen peroxide, and jasmonic acid (JA) levels and expression of related genes indicated that the relocalization of PLDδ is crucial for its activation to produce PA and initiate reactive oxygen species and JA signaling pathways. Together, our findings revealed that the translocation of PLDδ to papillae is modulated by exocytosis, thus triggering PA-mediated signaling in plant innate immunity.plantcell;31/12/3015/FX1F1fx1.

• MeSH
• Arabidopsis genetika   imunologie   metabolismus   mikrobiologie   MeSH
• Ascomycota patogenita   MeSH
• brefeldin A imunologie   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• chitin imunologie   metabolismus   MeSH
• cyklopentany metabolismus   MeSH
• exocytóza účinky léků   imunologie   MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• kyseliny fosfatidové metabolismus   MeSH
• nemoci rostlin imunologie   mikrobiologie   MeSH
• oxylipiny metabolismus   MeSH
• peroxid vodíku metabolismus   MeSH
• přirozená imunita * účinky léků   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny Qa-SNARE metabolismus   MeSH
• proteiny R-SNARE metabolismus   MeSH
• proteiny SNARE genetika   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• signální transdukce imunologie   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• brefeldin A MeSH
• chitin MeSH
• cyklopentany MeSH
• fosfolipasa D MeSH
• jasmonic acid MeSH Prohlížeč
• kyseliny fosfatidové MeSH
• oxylipiny MeSH
• PEN1 protein, Arabidopsis MeSH Prohlížeč
• peroxid vodíku MeSH
• phospholipase D delta MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny Qa-SNARE MeSH
• proteiny R-SNARE MeSH
• proteiny SNARE MeSH
• reaktivní formy kyslíku MeSH
• REM1 protein, Arabidopsis MeSH Prohlížeč
• VAMP721 protein, Arabidopsis MeSH Prohlížeč
• VAMP722 protein, Arabidopsis MeSH Prohlížeč

The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling, governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana) NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• dusičnany metabolismus   MeSH
• fosforylace MeSH
• kořeny rostlin růst a vývoj   MeSH
• kyseliny indoloctové metabolismus   MeSH
• proteiny huseníčku metabolismus   MeSH
• proteiny přenášející anionty genetika   metabolismus   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vápníková signalizace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ANR1 protein, Arabidopsis MeSH Prohlížeč
• dusičnany MeSH
• kyseliny indoloctové MeSH
• NRT1.1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny přenášející anionty MeSH
• rostlinné proteiny MeSH
• transkripční faktory MeSH

Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein-protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced protein-like 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant-pathogen interactions, water transport and intracellular trafficking.

• Klíčová slova
• Arabidopsis flotillin 2, immunopurification, intracellular trafficking, mass spectrometry, plant–pathogen interaction, protein–protein interactions, split-ubiquitin yeast system, water transport,
• Publikační typ
• časopisecké články MeSH