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

Eukaryotic cells rely on the accuracy and efficiency of vesicular traffic. In plants, disturbances in vesicular trafficking are well studied in quickly dividing root meristem cells or polar growing root hairs and pollen tubes. The development of the female gametophyte, a unique haploid reproductive structure located in the ovule, has received far less attention in studies of vesicular transport. Key molecules providing the specificity of vesicle formation and its subsequent recognition and fusion with the acceptor membrane are Rab proteins. Rabs are anchored to membranes by covalently linked geranylgeranyl group(s) that are added by the Rab geranylgeranyl transferase (RGT) enzyme. Here we show that Arabidopsis plants carrying mutations in the gene encoding the β-subunit of RGT (rgtb1) exhibit severely disrupted female gametogenesis and this effect is of sporophytic origin. Mutations in rgtb1 lead to internalization of the PIN1 and PIN3 proteins from the basal membranes to vesicles in provascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in tissue surrounding the growing gametophyte. In addition, female gametophyte development arrests at the uni- or binuclear stage in a significant portion of the rgtb1 ovules. These observations suggest that communication between the sporophyte and the developing female gametophyte relies on Rab-dependent vesicular traffic of the PIN1 and PIN3 transporters and auxin efflux out of the ovule.

• Klíčová slova
• Arabidopsis, PIN1, PIN3, Rab, auxin transport, female gametophyte, funiculus, ovule, rab geranylgeranyl transferase,
• MeSH
• Arabidopsis * genetika   MeSH
• kyseliny indoloctové MeSH
• proteiny huseníčku * genetika   MeSH
• pylová láčka MeSH
• vajíčko rostlin genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyseliny indoloctové MeSH
• proteiny huseníčku * MeSH

Plant growth and productivity are orchestrated by a network of signaling cascades involved in balancing responses to perceived environmental changes with resource availability. Vascular plants are divided into the shoot, an aboveground organ where sugar is synthesized, and the underground located root. Continuous growth requires the generation of energy in the form of carbohydrates in the leaves upon photosynthesis and uptake of nutrients and water through root hairs. Root hair outgrowth depends on the overall condition of the plant and its energy level must be high enough to maintain root growth. TARGET OF RAPAMYCIN (TOR)-mediated signaling cascades serve as a hub to evaluate which resources are needed to respond to external stimuli and which are available to maintain proper plant adaptation. Root hair growth further requires appropriate distribution of the phytohormone auxin, which primes root hair cell fate and triggers root hair elongation. Auxin is transported in an active, directed manner by a plasma membrane located carrier. The auxin efflux carrier PIN-FORMED 2 is necessary to transport auxin to root hair cells, followed by subcellular rearrangements involved in root hair outgrowth. This review presents an overview of events upstream and downstream of PIN2 action, which are involved in root hair growth control.

• Klíčová slova
• PIN-FORMED 2, ROP2, ROS, TOR signaling, auxin, plant adaptation, polar cell elongation, root growth, root hair growth,
• Publikační typ
• časopisecké články MeSH
• přehledy 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

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

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

Biogenesis of the plant secondary cell wall involves many important aspects, such as phenolic compound deposition and often silica encrustation. Previously, we demonstrated the importance of the exocyst subunit EXO70H4 for biogenesis of the trichome secondary cell wall, namely for deposition of the autofluorescent and callose-rich cell wall layer. Here, we reveal that EXO70H4-driven cell wall biogenesis is constitutively active in the mature trichome, but also can be activated elsewhere upon pathogen attack, giving this study a broader significance with an overlap into phytopathology. To address the specificity of EXO70H4 among the EXO70 family, we complemented the exo70H4-1 mutant by 18 different Arabidopsis (Arabidopsis thaliana) EXO70 paralogs subcloned under the EXO70H4 promoter. Only EXO70H4 had the capacity to rescue the exo70H4-1 trichome phenotype. Callose deposition phenotype of exo70H4-1 mutant is caused by impaired secretion of PMR4, a callose synthase responsible for the synthesis of callose in the trichome. PMR4 colocalizes with EXO70H4 on plasma membrane microdomains that do not develop in the exo70H4-1 mutant. Using energy-dispersive x-ray microanalysis, we show that both EXO70H4- and PMR4-dependent callose deposition in the trichome are essential for cell wall silicification.

• MeSH
• Arabidopsis účinky léků   genetika   metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• buněčná stěna účinky léků   metabolismus   MeSH
• epidermis rostlin cytologie   účinky léků   metabolismus   MeSH
• fenotyp MeSH
• flagelin farmakologie   MeSH
• glukany MeSH
• glukosyltransferasy metabolismus   MeSH
• mutace genetika   MeSH
• oxid křemičitý metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteinové domény MeSH
• proteiny huseníčku chemie   metabolismus   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• trichomy metabolismus   MeSH
• upregulace účinky léků   MeSH
• vezikulární transportní proteiny chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 1,3-beta-glucan synthase MeSH Prohlížeč
• callose MeSH Prohlížeč
• EXO70H4 protein, Arabidopsis MeSH Prohlížeč
• flagelin MeSH
• glukany MeSH
• glukosyltransferasy MeSH
• oxid křemičitý MeSH
• PMR4 protein, Arabidopsis MeSH Prohlížeč
• podjednotky proteinů MeSH
• proteiny huseníčku MeSH
• vezikulární transportní proteiny MeSH

The exocyst is a conserved vesicle-tethering complex with principal roles in cell polarity and morphogenesis. Several studies point to its involvement in polarized secretion during microbial pathogen defense. In this context, we have found an interaction between the Arabidopsis EXO70B1 exocyst subunit, a protein which was previously associated with both the defense response and autophagy, and RPM1 INTERACTING PROTEIN 4 (RIN4), the best studied member of the NOI protein family and a known regulator of plant defense pathways. Interestingly, fragments of RIN4 mimicking the cleavage caused by the Pseudomonas syringae effector protease, AvrRpt2, fail to interact strongly with EXO70B1. We observed that transiently expressed RIN4, but not the plasma membrane (PM) protein aquaporin PIP2, recruits EXO70B1 to the PM. Unlike EXO70B1, RIN4 does not recruit the core exocyst subunit SEC6 to the PM under these conditions. Furthermore, the AvrRpt2 effector protease delivered by P. syringae is able to release both RIN4 and EXO70B1 to the cytoplasm. We present a model for how RIN4 might regulate the localization and putative function of EXO70B1 and speculate on the role the AvrRpt2 protease might have in the regulation of this defense response.

• Klíčová slova
• Autophagy, EXO70B1, EXO70B2, RIN4, exocyst, plant immunity, secretion,
• MeSH
• akvaporiny genetika   metabolismus   MeSH
• Arabidopsis metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• buněčná membrána MeSH
• intracelulární signální peptidy a proteiny MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• Pseudomonas syringae metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• vezikulární transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• akvaporiny MeSH
• avrRpt2 protein, Pseudomonas syringae MeSH Prohlížeč
• bakteriální proteiny MeSH
• EXO70B1 protein, Arabidopsis MeSH Prohlížeč
• intracelulární signální peptidy a proteiny MeSH
• plasma membrane intrinsic protein 2 Arabidopsis MeSH Prohlížeč
• proteiny huseníčku MeSH
• RIN4 protein, Arabidopsis MeSH Prohlížeč
• transportní proteiny MeSH
• vezikulární transportní proteiny MeSH

Immunogold electron microscopy (EM) study of Arabidopsis root apices analyzed using specific IAA antibody and high-pressure freeze fixation technique allowed, for the first time, vizualization of subcellular localization of IAA in cells assembled intactly within plant tissues. Our quantitative analysis reveals that there is considerable portion of IAA gold particles that clusters within vesicles and membraneous compartments in all root apex cells. There are clear tissue-specific and developmental differences of clustered IAA in root apices. These findings have significant consequences for our understanding of this small molecule which is controlling plant growth, development and behavior.

• Klíčová slova
• Arabidopsis, Brefeldin A, IAA, auxin, endocytosis, polar auxin transport, roots, secretion, vesicles,
• Publikační typ
• časopisecké články MeSH