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The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens Pp EXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade., Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized Pp EXO70.3d in vivo using green fluorescent protein-tagged protein expression., Disruption of Pp EXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate., Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family. [ABSTRACT FROM AUTHOR]

• Cortical microtubules (MTs) play a major role in the patterning of secondary cell wall (SCW) thickenings in tracheary elements (TEs) by determining the sites of SCW deposition. The EXO70A1 subunit of the exocyst secretory vesicle tethering complex was implicated to be important for TE development via the MT interaction. We investigated the subcellular localization of several exocyst subunits in the xylem of Arabidopsis thaliana and analyzed the functional significance of exocyst-mediated trafficking in TE development. • Live cell imaging of fluorescently tagged exocyst subunits in TE using confocal microscopy and protein-protein interaction assays were performed to describe the role of the exocyst and its partners in TE development. • In TEs, exocyst subunits were localized to the sites of SCW deposition in an MT-dependent manner. We propose that the mechanism of exocyst targeting to MTs involves the direct interaction of exocyst subunits with the COG2 protein. We demonstrated the importance of a functional exocyst subunit EXO84b for normal TE development and showed that the deposition of SCW constituents is partially compromised, possibly as a result of the mislocalization of secondary cellulose synthase in exocyst mutants. • We conclude that the exocyst complex is an important factor bridging the pattern defined by cortical MTs with localized secretion of the SCW in developing TEs. [ABSTRACT FROM AUTHOR]

The authors discuss the effect of plant studies on the concept of behavior. They review a survey of biologists who engaged in behavioral research. The authors examine the uneasy relationship between behavior and development. They suggest that plant studies may lead to rethink the concept of behavior.

The cytoskeleton plays a central part in spatial organization of the plant cytoplasm, including the endomebrane system. However, the mechanisms involved are so far only partially understood. Formins (FH2 proteins), a family of evolutionarily conserved proteins sharing the FH2 domain whose dimer can nucleate actin, mediate the co-ordination between actin and microtubule cytoskeletons in multiple eukaryotic lineages including plants. Moreover, some plant formins contain transmembrane domains and participate in anchoring cytoskeletal structures to the plasmalemma, and possibly to other membranes. Direct or indirect membrane association is well documented even for some fungal and metazoan formins lacking membrane insertion motifs, and FH2 proteins have been shown to associate with endomembranes and modulate their dynamics in both fungi and metazoans. Here we summarize the available evidence suggesting that formins participate in membrane trafficking and endomembrane, especially ER, organization also in plants. We propose that, despite some methodological pitfalls inherent to in vivo studies based on (over)expression of truncated and/or tagged proteins, formins are beginning to emerge as candidates for the so far somewhat elusive link between the plant cytoskeleton and the endomembrane system. [ABSTRACT FROM AUTHOR]

Repetitive sequences present a challenge for genome sequence assembly, and highly similar segmental duplications may disappear from assembled genome sequences. Having found a surprising lack of observable phenotypic deviations and non-Mendelian segregation in Arabidopsis thaliana mutants in SEC10, a gene encoding a core subunit of the exocyst tethering complex, we examined whether this could be explained by a hidden gene duplication. Re-sequencing and manual assembly of the Arabidopsis thaliana SEC10 (At5g12370) locus revealed that this locus, comprising a single gene in the reference genome assembly, indeed contains two paralogous genes in tandem, SEC10a and SEC10b, and that a sequence segment of 7 kb in length is missing from the reference genome sequence. Differences between the two paralogs are concentrated in non-coding regions, while the predicted protein sequences exhibit 99% identity, differing only by substitution of five amino acid residues and an indel of four residues. Both SEC10 genes are expressed, although varying transcript levels suggest differential regulation. Homozygous T-DNA insertion mutants in either paralog exhibit a wild-type phenotype, consistent with proposed extensive functional redundancy of the two genes. By these observations we demonstrate that recently duplicated genes may remain hidden even in well-characterized genomes, such as that of A. thaliana. Moreover, we show that the use of the existing A. thaliana reference genome sequence as a guide for sequence assembly of new Arabidopsis accessions or related species has at least in some cases led to error propagation. [ABSTRACT FROM AUTHOR]

Plant pathogens are perceived by pattern recognition receptors, which are activated upon binding to pathogen-associated molecular patterns (PAMPs). Ubiquitination and vesicle trafficking have been linked to the regulation of immune signaling. However, little information exists about components of vesicle trafficking involved in immune signaling and the mechanisms that regulate them. In this study, we identified Arabidopsis thaliana Exo70B2, a subunit of the exocyst complex that mediates vesicle tethering during exocytosis, as a target of the plant U-box–type ubiquitin ligase 22 (PUB22), which acts in concert with PUB23 and PUB24 as a negative regulator of PAMP -triggered responses. We show that Exo70B2 is required for both immediate and later responses triggered by all tested PAMPs , suggestive of a role in signaling. Exo70B2 is also necessary for the immune response against different pathogens. Our data demonstrate that PUB22 mediates the ubiquitination and degradation of Exo70B2 via the 26S Proteasome. Furthermore, degradation is regulated by the autocatalytic turnover of PUB22, which is stabilized upon PAMP perception. We therefore propose a mechanism by which PUB22-mediated degradation of Exo70B2 contributes to the attenuation of PAMP -induced signaling. [ABSTRACT FROM AUTHOR]

Recently, the octameric vesicle-tethering complex exocyst was found in plants and its importance for Arabidopsis morphogenesis was demonstrated. Exo70 exocyst subunits in plants, unlike in yeasts and mammals, are represented by a multigene family, comprising 23 members in Arabidopsis. For Exo70B2 and Exo70H1 paralogues, transcriptional up-regulation was confirmed on treatment with an elicitor peptide, elf18, derived from the bacterial elongation factor. Their ability to participate in the exocyst complex formation was inferred by the interaction of both the Exo70s with several other exocyst subunits using the yeast two-hybrid system. Arabidopsis plants mutated in these two genes were used to analyse their local reaction upon inoculation with Pseudomonas syringae pv. maculicola and the fungal pathogen Blumeria graminis f. sp. hordei. The Pseudomonas sensitivity test revealed enhanced susceptibility for the two exo70B2 and one H1 mutant lines. After Blumeria inoculation, an increase in the proportion of abnormal papilla formation, with an unusual wide halo made of vesicle-like structures, was found in exo70B2 mutants. Intracellular localization of both Exo70 proteins was studied following a GFP fusion assay and Agrobacterium-mediated transient expression of the constructs in Nicotiana benthamiana leaf epidermis. GFP-Exo70H1 localizes in the vesicle-like structures, while GFP-Exo70B2 is localized mainly in the cytoplasm. It is concluded that both Exo70B2 and Exo70H1 are involved in the response to pathogens, with Exo70B2 having a more important role in cell wall apposition formation related to plant defence. [ABSTRACT FROM PUBLISHER]

Polarized deposition of cell wall pectins is a key process in Arabidopsis thaliana myxospermous seed coat development. The exocyst, an octameric secretory vesicle tethering complex, has recently been shown to be involved in the regulation of cell polarity in plants. Here, we used the Arabidopsis seed coat to study the participation of the exocyst complex in polarized pectin delivery., We characterized the amount of pectinaceous mucilage and seed coat structure in sec8 and exo70A1 exocyst mutants. Using a yeast two-hybrid screen, we identified a new interactor of the exocyst subunit Exo70A1, termed Roh1, a member of the DUF793 protein family., T-DNA insertions in SEC8, EXO70A1 caused considerable deviations from normal seed coat development, in particular reduced pectin deposition and defects in the formation of the central columella of seed epidermal cells. A gain-of-function mutation of ROH1 also caused reduced pectin deposition. Interestingly, we observed a systematic difference in seed coat development between primary and secondary inflorescences in wild-type plants: siliques from secondary branches produced seeds with thicker seed coats., The participation of exocyst subunits in mucilage deposition provides direct evidence for the role of the exocyst in polarized cell wall morphogenesis. [ABSTRACT FROM AUTHOR]

Cell reproduction is a complex process involving whole cell structures and machineries in space and time, resulting in regulated distribution of endomembranes, organelles, and genomes between daughter cells. Secretory pathways supported by the activity of the Golgi apparatus play a crucial role in cytokinesis in plants. From the onset of phragmoplast initiation to the maturation of the cell plate, delivery of secretory vesicles is necessary to sustain successful daughter cell separation. Tethering of secretory vesicles at the plasma membrane is mediated by the evolutionarily conserved octameric exocyst complex. Using proteomic and cytologic approaches, we show that EXO84b is a subunit of the plant exocyst. Arabidopsis thaliana mutants for EXO84b are severely dwarfed and have compromised leaf epidermal cell and guard cell division. During cytokinesis, green fluorescent protein–tagged exocyst subunits SEC6, SEC8, SEC15b, EXO70A1, and EXO84b exhibit distinctive localization maxima at cell plate initiation and cell plate maturation, stages with a high demand for vesicle fusion. Finally, we present data indicating a defect in cell plate assembly in the exo70A1 mutant. We conclude that the exocyst complex is involved in secretory processes during cytokinesis in Arabidopsis cells, notably in cell plate initiation, cell plate maturation, and formation of new primary cell wall. [ABSTRACT FROM AUTHOR]

Contents Summary 255 I. Introduction 256 II. Donor compartments for plant exocytotic vesicles: where do they come from? 256 III. Mechanisms of exocytotic vesicle formation: is the truth naked? 257 IV. Building the tracks for exocytotic vesicles: cytoskeleton in exocytosis and cell polarization 259 V. Manysidedness of a plant cell: delimiting functional plasmalemma domains within a cell 260 VI. Vesicle tethering, docking and fusion 262 VII. Polarity by recycling: exocytosis, endocytosis and recycling domains in plant cells 264 VIII Conclusions: constitutive or regulated secretion in plant cells? 266 Acknowledgements 267 References 267 In plants, exocytosis is a central mechanism of cell morphogenesis. We still know surprisingly little about some aspects of this process, starting with exocytotic vesicle formation, which may take place at the trans-Golgi network even without coat assistance, facilitated by the local regulation of membrane lipid organization. The RabA4b guanosine triphosphatase (GTPase), recruiting phosphatidylinositol-4-kinase to the trans-Golgi network, is a candidate vesicle formation organizer. However, in plant cells, there are obviously additional endosomal source compartments for secretory vesicles. The Rho/Rop GTPase regulatory module is central for the initiation of exocytotically active domains in plant cell cortex (activated cortical domains). Most plant cells exhibit several distinct plasma membrane domains, established and maintained by endocytosis-driven membrane recycling. We propose the concept of a ‘recycling domain’, uniting the activated cortical domain and the connected endosomal compartments, as a dynamic spatiotemporal entity. We have recently described the exocyst tethering complex in plant cells. As a result of the multiplicity of its putative Exo70 subunits, this complex may belong to core regulators of recycling domain organization, including the generation of multiple recycling domains within a single cell. The conventional textbook concept that the plant secretory pathway is largely constitutive is misleading. [ABSTRACT FROM AUTHOR]

Abstract: Keyhole limpet hemocyanin (KLH)-conjugated peptides are routinely used to raise polyclonal antibodies for biochemical or immunolocalization studies. Rats are suitable for producing antisera against plant antigens as they often lack non-specific response towards plant materials. We attempted to obtain rat antisera against peptides derived from several plant proteins. However, most antisera recognized the same background KLH-related plant antigen (KRAP) in Arabidopsis and tobacco. We characterized KRAP with respect to size and cellular localization and examined possible antigen-specific reasons for the failure of most immunizations. We also found no reports of successful use of rat anti-KLH-peptide antibodies in plant studies. We thus believe that the rat-KLH:peptide system is poorly suited for production of antibodies, especially against plant antigens, and should be used with caution, if at all. [Copyright &y& Elsevier]

The exocyst, an octameric tethering complex and effector of Rho and Rab GTPases, facilitates polarized secretion in yeast and animals. Recent evidence implicates three plant homologs of exocyst subunits (SEC3, SEC8, and EXO70A1) in plant cell morphogenesis. Here, we provide genetic, cell biological, and biochemical evidence that these and other predicted subunits function together in vivo in Arabidopsis thaliana. Double mutants in exocyst subunits (sec5 exo70A1 and sec8 exo70A1) show a synergistic defect in etiolated hypocotyl elongation. Mutants in exocyst subunits SEC5, SEC6, SEC8, and SEC15a show defective pollen germination and pollen tube growth phenotypes. Using antibodies directed against SEC6, SEC8, and EXO70A1, we demonstrate colocalization of these proteins at the apex of growing tobacco pollen tubes. The SEC3, SEC5, SEC6, SEC8, SEC10, SEC15a, and EXO70 subunits copurify in a high molecular mass fraction of 900 kD after chromatographic fractionation of an Arabidopsis cell suspension extract. Blue native electrophoresis confirmed the presence of SEC3, SEC6, SEC8, and EXO70 in high molecular mass complexes. Finally, use of the yeast two-hybrid system revealed interaction of Arabidopsis SEC3a with EXO70A1, SEC10 with SEC15b, and SEC6 with SEC8. We conclude that the exocyst functions as a complex in plant cells, where it plays important roles in morphogenesis. [ABSTRACT FROM AUTHOR]

Although angiosperm plants generally react to immunity elicitors like chitin or chitosan by the cell wall callose deposition, this response in particular cell types, especially upon chitosan treatment, is not fully understood. Here we show that the growing root hairs (RHs) of Arabidopsis can respond to a mild (0.001%) chitosan treatment by the callose deposition and by a deceleration of the RH growth. We demonstrate that the glucan synthase‐like 5/PMR4 is vital for chitosan‐induced callose deposition but not for RH growth inhibition. Upon the higher chitosan concentration (0.01%) treatment, RHs do not deposit callose, while growth inhibition is prominent. To understand the molecular and cellular mechanisms underpinning the responses to two chitosan treatments, we analysed early Ca2+ and defence‐related signalling, gene expression, cell wall and RH cellular endomembrane modifications. Chitosan‐induced callose deposition is also present in the several other plant species, including functionally analogous and evolutionarily only distantly related RH‐like structures such as rhizoids of bryophytes. Our results point to the RH callose deposition as a conserved strategy of soil‐anchoring plant cells to cope with mild biotic stress. However, high chitosan concentration prominently disturbs RH intracellular dynamics, tip‐localised endomembrane compartments, growth and viability, precluding callose deposition. [ABSTRACT FROM AUTHOR]