The analysis of dynamic cellular processes such as plant cytokinesis stands and falls with live-cell time-lapse confocal imaging. Conventional approaches to time-lapse imaging of cell division in Arabidopsis root tips are tedious and have low throughput. Here, we describe a protocol for long-term time-lapse simultaneous imaging of multiple root tips on a vertical-stage confocal microscope with automated root tracking. We also provide modifications of the basic protocol to implement this imaging method in the analysis of genetic, pharmacological or laser ablation wounding-mediated experimental manipulations. Our method dramatically improves the efficiency of cell division time-lapse imaging by increasing the throughput, while reducing the person-hour requirements of such experiments.

• Klíčová slova
• Arabidopsis, Automation, Confocal microscopy, Cytokinesis, Laser ablation, Root meristem, Time-lapse imaging,
• MeSH
• Arabidopsis * MeSH
• buněčné dělení MeSH
• časosběrné zobrazování MeSH
• konfokální mikroskopie MeSH
• lidé MeSH
• meristém MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Polarized exocytosis is essential for many vital processes in eukaryotic cells, where secretory vesicles are targeted to distinct plasma membrane domains characterized by their specific lipid-protein composition. Heterooctameric protein complex exocyst facilitates the vesicle tethering to a target membrane and is a principal cell polarity regulator in eukaryotes. The architecture and molecular details of plant exocyst and its membrane recruitment have remained elusive. Here, we show that the plant exocyst consists of two modules formed by SEC3-SEC5-SEC6-SEC8 and SEC10-SEC15-EXO70-EXO84 subunits, respectively, documenting the evolutionarily conserved architecture within eukaryotes. In contrast to yeast and mammals, the two modules are linked by a plant-specific SEC3-EXO70 interaction, and plant EXO70 functionally dominates over SEC3 in the exocyst recruitment to the plasma membrane. Using an interdisciplinary approach, we found that the C-terminal part of EXO70A1, the canonical EXO70 isoform in Arabidopsis, is critical for this process. In contrast to yeast and animal cells, the EXO70A1 interaction with the plasma membrane is mediated by multiple anionic phospholipids uniquely contributing to the plant plasma membrane identity. We identified several evolutionary conserved EXO70 lysine residues and experimentally proved their importance for the EXO70A1-phospholipid interactions. Collectively, our work has uncovered plant-specific features of the exocyst complex and emphasized the importance of the specific protein-lipid code for the recruitment of peripheral membrane proteins.

• Klíčová slova
• EXO70A1, cell polarity, exocyst, phospholipids, plasma membrane,
• MeSH
• Arabidopsis metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• exocytóza MeSH
• fosfolipidy metabolismus   MeSH
• polarita buněk MeSH
• proteiny huseníčku metabolismus   MeSH
• proteomika metody   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• fosfolipidy MeSH
• proteiny huseníčku MeSH

Localized delivery of plasma-membrane and cell-wall components is a crucial process for plant cell growth. One of the regulators of secretory-vesicle targeting is the exocyst tethering complex. The exocyst mediates first interaction between transport vesicles and the target membrane before their fusion is performed by SNARE proteins. In land plants, genes encoding the EXO70 exocyst subunit underwent an extreme proliferation with 23 paralogs present in the Arabidopsis (Arabidopsis thaliana) genome. These paralogs often acquired specialized functions during evolution. Here, we analyzed functional divergence of selected EXO70 paralogs in Arabidopsis. Performing a systematic cross-complementation analysis of exo70a1 and exo70b1 mutants, we found that EXO70A1 was functionally substituted only by its closest paralog, EXO70A2. In contrast, none of the EXO70 isoforms tested were able to substitute EXO70B1, including its closest relative, EXO70B2, pointing to a unique function of this isoform. The presented results document a high degree of functional specialization within the EXO70 gene family in land plants.

• Klíčová slova
• Arabidopsis, EXO70, EXO70A1, EXO70B1, exocyst complex, polar exocytosis,
• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• exocytóza MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• transportní vezikuly metabolismus   MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH

Pollen development, pollen grain germination, and pollen tube elongation are crucial biological processes in angiosperm plants that need precise regulation to deliver sperm cells to ovules for fertilization. Highly polarized secretion at a growing pollen tube tip requires the exocyst tethering complex responsible for specific targeting of secretory vesicles to the plasma membrane. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) EXO70A2 (At5g52340) is the main exocyst EXO70 isoform in the male gametophyte, governing the conventional secretory function of the exocyst, analogous to EXO70A1 (At5g03540) in the sporophyte. Our analysis of a CRISPR-generated exo70a2 mutant revealed that EXO70A2 is essential for efficient pollen maturation, pollen grain germination, and pollen tube growth. GFP:EXO70A2 was localized to the nucleus and cytoplasm in developing pollen grains and later to the apical domain in growing pollen tube tips characterized by intensive exocytosis. Moreover, EXO70A2 could substitute for EXO70A1 function in the sporophyte, but not vice versa, indicating partial functional redundancy of these two closely related isoforms and higher specificity of EXO70A2 for pollen development-related processes. Phylogenetic analysis revealed that the ancient duplication of EXO70A, one of which is always highly expressed in pollen, occurred independently in monocots and dicots. In summary, EXO70A2 is a crucial component of the exocyst complex in Arabidopsis pollen that is required for efficient plant sexual reproduction.

• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• exocytóza genetika   fyziologie   MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genotyp MeSH
• pylová láčka genetika   růst a vývoj   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné geny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis (Arabidopsis thaliana). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121ΔC, which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding.

• MeSH
• Arabidopsis cytologie   genetika   růst a vývoj   metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• exocytóza fyziologie   MeSH
• geneticky modifikované rostliny MeSH
• mutace MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• proteiny Qa-SNARE genetika   metabolismus   MeSH
• proteiny R-SNARE genetika   metabolismus   MeSH
• proteiny SNARE genetika   metabolismus   MeSH
• rezonanční přenos fluorescenční energie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EXO70A1 protein, Arabidopsis MeSH Prohlížeč
• PEN1 protein, Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• proteiny Qa-SNARE MeSH
• proteiny R-SNARE MeSH
• proteiny SNARE MeSH
• VAMP721 protein, Arabidopsis MeSH Prohlížeč

The heterooctameric vesicle-tethering complex exocyst is important for plant development, growth, and immunity. Multiple paralogs exist for most subunits of this complex; especially the membrane-interacting subunit EXO70 underwent extensive amplification in land plants, suggesting functional specialization. Despite this specialization, most Arabidopsis exo70 mutants are viable and free of developmental defects, probably as a consequence of redundancy among isoforms. Our in silico data-mining and modeling analysis, corroborated by transcriptomic experiments, pinpointed several EXO70 paralogs to be involved in plant biotic interactions. We therefore tested corresponding single and selected double mutant combinations (for paralogs EXO70A1, B1, B2, H1, E1, and F1) in their two biologically distinct responses to Pseudomonas syringae, root hair growth stimulation and general plant susceptibility. A shift in defense responses toward either increased or decreased sensitivity was found in several double mutants compared to wild type plants or corresponding single mutants, strongly indicating both additive and compensatory effects of exo70 mutations. In addition, our experiments confirm the lipid-binding capacity of selected EXO70s, however, without the clear relatedness to predicted C-terminal lipid-binding motifs. Our analysis uncovers that there is less of functional redundancy among isoforms than we could suppose from whole sequence phylogeny and that even paralogs with overlapping expression pattern and similar membrane-binding capacity appear to have exclusive roles in plant development and biotic interactions.

• Klíčová slova
• Arabidopsis thaliana, EXO70, biotic stress, exocyst, gene expression, lipid binding, redundancy, root hairs,
• Publikační typ
• časopisecké články MeSH

Exocyst is a heterooctameric protein complex crucial for the tethering of secretory vesicles to the plasma membrane during exocytosis. Compared to other eukaryotes, exocyst subunit EXO70 is represented by many isoforms in land plants whose cell biological and biological roles, as well as modes of regulation remain largely unknown. Here, we present data on the phospho-regulation of exocyst isoform EXO70C2, which we previously identified as a putative negative regulator of exocyst function in pollen tube growth. A comprehensive phosphoproteomic analysis revealed phosphorylation of EXO70C2 at multiple sites. We have now performed localization and functional studies of phospho-dead and phospho-mimetic variants of Arabidopsis EXO70C2 in transiently transformed tobacco pollen tubes and stably transformed Arabidopsis wild type and exo70C2 mutant plants. Our data reveal a dose-dependent effect of AtEXO70C2 overexpression on pollen tube growth rate and cellular architecture. We show that changes of the AtEXO70C2 phosphorylation status lead to distinct outcomes in wild type and exo70c2 mutant cells, suggesting a complex regulatory pattern. On the other side, phosphorylation does not affect the cytoplasmic localization of AtEXO70C2 or its interaction with putative secretion inhibitor ROH1 in the yeast two-hybrid system.

• Klíčová slova
• Exo70, exocyst, membrane trafficking, phosphorylation, pollen tube, secretion inhibitor, tip-growth,
• Publikační typ
• časopisecké články MeSH

The collet (root-hypocotyl junction) region is an important plant transition zone between soil and atmospheric environments. Despite its crucial importance for plant development, little is known about how this transition zone is specified. Here we document the involvement of the exocyst complex in this process. The exocyst, an octameric tethering complex, participates in secretion and membrane recycling and is central to numerous cellular and developmental processes, such as growth of root hairs, cell expansion, recycling of PIN auxin efflux carriers and many others. We show that dark-grown Arabidopsis mutants deficient in exocyst subunits can form a hair-bearing ectopic collet-like structure above the true collet, morphologically resembling the true collet but also retaining some characteristics of the hypocotyl. The penetrance of this phenotypic defect is significantly influenced by cultivation temperature and carbon source, and is related to a defect in auxin regulation. These observations provide new insights into the regulation of collet region formation and developmental plasticity of the hypocotyl.

• Klíčová slova
• Arabidopsis thaliana, auxin, collet, etiolated hypocotyl, exocyst, root–hypocotyl junction, starch accumulation,
• MeSH
• Arabidopsis genetika   růst a vývoj   MeSH
• hypokotyl genetika   růst a vývoj   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• proteiny huseníčku MeSH

The exocyst, a eukaryotic tethering complex, coregulates targeted exocytosis as an effector of small GTPases in polarized cell growth. In land plants, several exocyst subunits are encoded by double or triple paralogs, culminating in tens of EXO70 paralogs. Out of 23 Arabidopsis thaliana EXO70 isoforms, we analyzed seven isoforms expressed in pollen. Genetic and microscopic analyses of single mutants in EXO70A2, EXO70C1, EXO70C2, EXO70F1, EXO70H3, EXO70H5, and EXO70H6 genes revealed that only a loss-of-function EXO70C2 allele resulted in a significant male-specific transmission defect (segregation 40%:51%:9%) due to aberrant pollen tube growth. Mutant pollen tubes grown in vitro exhibited an enhanced growth rate and a decreased thickness of the tip cell wall, causing tip bursts. However, exo70C2 pollen tubes could frequently recover and restart their speedy elongation, resulting in a repetitive stop-and-go growth dynamics. A pollen-specific depletion of the closest paralog, EXO70C1, using artificial microRNA in the exo70C2 mutant background, resulted in a complete pollen-specific transmission defect, suggesting redundant functions of EXO70C1 and EXO70C2. Both EXO70C1 and EXO70C2, GFP tagged and expressed under the control of their native promoters, localized in the cytoplasm of pollen grains, pollen tubes, and also root trichoblast cells. The expression of EXO70C2-GFP complemented the aberrant growth of exo70C2 pollen tubes. The absent EXO70C2 interactions with core exocyst subunits in the yeast two-hybrid assay, cytoplasmic localization, and genetic effect suggest an unconventional EXO70 function possibly as a regulator of exocytosis outside the exocyst complex. In conclusion, EXO70C2 is a novel factor contributing to the regulation of optimal tip growth of Arabidopsis pollen tubes.

• MeSH
• Arabidopsis genetika   růst a vývoj   metabolismus   MeSH
• geneticky modifikované rostliny MeSH
• konfokální mikroskopie MeSH
• kořeny rostlin genetika   metabolismus   MeSH
• mutace MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• pyl genetika   růst a vývoj   metabolismus   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin * MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• vývojová regulace genové exprese * MeSH
• zelené fluorescenční proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EXO70C2 protein, Arabidopsis MeSH Prohlížeč
• protein - isoformy MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH
• zelené fluorescenční proteiny MeSH

The vesicle-tethering complex exocyst is one of the crucial cell polarity regulators. The EXO70 subunit is required for the targeting of the complex and is represented by many isoforms in angiosperm plant cells. This diversity could be partly responsible for the establishment and maintenance of membrane domains with different composition. To address this hypothesis, we employed the growing pollen tube, a well-established cell polarity model system, and performed large-scale expression, localization, and functional analysis of tobacco (Nicotiana tabacum) EXO70 isoforms. Various isoforms localized to different regions of the pollen tube plasma membrane, apical vesicle-rich inverted cone region, nucleus, and cytoplasm. The overexpression of major pollen-expressed EXO70 isoforms resulted in growth arrest and characteristic phenotypic deviations of tip swelling and apical invaginations. NtEXO70A1a and NtEXO70B1 occupied two distinct and mutually exclusive plasma membrane domains. Both isoforms partly colocalized with the exocyst subunit NtSEC3a at the plasma membrane, possibly forming different exocyst complex subpopulations. NtEXO70A1a localized to the small area previously characterized as the site of exocytosis in the tobacco pollen tube, while NtEXO70B1 surprisingly colocalized with the zone of clathrin-mediated endocytosis. Both NtEXO70A1a and NtEXO70B1 colocalized to different degrees with markers for the anionic signaling phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidic acid. In contrast, members of the EXO70 C class, which are specifically expressed in tip-growing cells, exhibited exocytosis-related functional effects in pollen tubes despite the absence of apparent plasma membrane localization. Taken together, our data support the existence of multiple membrane-trafficking domains regulated by different EXO70-containing exocyst complexes within a single cell.

• MeSH
• buněčná membrána metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• exocytóza genetika   MeSH
• fylogeneze MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• konfokální mikroskopie MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• protein - isoformy genetika   metabolismus   MeSH
• proteomika metody   MeSH
• pylová láčka genetika   růst a vývoj   metabolismus   MeSH
• regulace genové exprese u rostlin MeSH
• rostlinné proteiny klasifikace   genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• tabák genetika   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protein - isoformy MeSH
• rostlinné proteiny MeSH