Diversification of SEC15a and SEC15b isoforms of an exocyst subunit in seed plants is manifested in their specific roles in Arabidopsis sporophyte and male gametophyte.

The exocyst complex is an octameric evolutionarily conserved tethering complex engaged in the regulation of polarized secretion in eukaryotic cells. Here, we focus on the systematic comparison of two isoforms of the SEC15 exocyst subunit, SEC15a and SEC15b. We infer that SEC15 gene duplication and diversification occurred in the common ancestor of seed plants (Spermatophytes). In Arabidopsis, SEC15a represents the main SEC15 isoform in the male gametophyte, and localizes to the pollen tube tip at the plasma membrane. Although pollen tubes of sec15a mutants are impaired, sporophytes show no phenotypic deviations. Conversely, SEC15b is the dominant isoform in the sporophyte and localizes to the plasma membrane in root and leaf cells. Loss-of-function sec15b mutants exhibit retarded elongation of hypocotyls and root hairs, a loss of apical dominance, dwarfed plant stature and reduced seed coat mucilage formation. Surprisingly, the sec15b mutants also exhibit compromised pollen tube elongation in vitro, despite its very low expression in pollen, suggesting a non-redundant role for the SEC15b isoform there. In pollen tubes, SEC15b localizes to distinct cytoplasmic structures. Reciprocally to this, SEC15a also functions in the sporophyte, where it accumulates at plasmodesmata. Importantly, although overexpressed SEC15a could fully complement the sec15b phenotypic deviations in the sporophyte, the pollen-specific overexpression of SEC15b was unable to fully compensate for the loss of SEC15a function in pollen. We conclude that the SEC15a and SEC15b isoforms evolved in seed plants, with SEC15a functioning mostly in pollen and SEC15b functioning mostly in the sporophyte.

SEC6 exocyst subunit contributes to multiple steps of growth and development of Physcomitrella (Physcomitrium patens).

Spatially directed cell division and expansion is important for plant growth and morphogenesis and relies on cooperation between the cytoskeleton and the secretory pathway. The phylogenetically conserved octameric complex exocyst mediates exocytotic vesicle tethering at the plasma membrane. Unlike other exocyst subunits of land plants, the core exocyst subunit SEC6 exists as a single paralog in Physcomitrium patens and Arabidopsis thaliana genomes. Arabidopsis SEC6 (AtSEC6) loss-of-function (LOF) mutation causes male gametophytic lethality. Our attempts to inactivate the P. patens SEC6 gene, PpSEC6, using targeted gene replacement produced two independent partial LOF ('weak allele') mutants via perturbation of the PpSEC6 gene locus. These mutants exhibited the same pleiotropic developmental defects: protonema with dominant chloronema stage; diminished caulonemal filament elongation rate; and failure in post-initiation gametophore development. Mutant gametophore buds, mostly initiated from chloronema cells, exhibited disordered cell file organization and cross-wall perforations, resulting in arrested development at the eight- to 10-cell stage. Complementation of both sec6 moss mutant lines by both PpSEC6 and AtSEC6 cDNA rescued gametophore development, including sexual organ differentiation. However, regular sporophyte formation and viable spore production were recovered only by the expression of PpSEC6, whereas the AtSEC6 complementants were only rarely fertile, indicating moss-specific SEC6 functions.

Regulation of Exocyst Function in Pollen Tube Growth by Phosphorylation of Exocyst Subunit EXO70C2.

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.

Protein Prenylation in Plant Stress Responses.

Protein prenylation is one of the most important posttranslational modifications of proteins. Prenylated proteins play important roles in different developmental processes as well as stress responses in plants as the addition of hydrophobic prenyl chains (mostly farnesyl or geranyl) allow otherwise hydrophilic proteins to operate as peripheral lipid membrane proteins. This review focuses on selected aspects connecting protein prenylation with plant responses to both abiotic and biotic stresses. It summarizes how changes in protein prenylation impact plant growth, deals with several families of proteins involved in stress response and highlights prominent regulatory importance of prenylated small GTPases and chaperons. Potential possibilities of these proteins to be applicable for biotechnologies are discussed.

Developmental plasticity of Arabidopsis hypocotyl is dependent on exocyst complex function.

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.

The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

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 PpEXO70.3d (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 PpEXO70.3d in vivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d 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.

EXO70C2 Is a Key Regulatory Factor for Optimal Tip Growth of Pollen.

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.

Heterologous expression in budding yeast as a tool for studying the plant cell morphogenesis machinery.

The budding yeast (Saccharomyces cerevisiae) can serve as a unique experimental system for functional studies of heterologous genes, allowing not only complementation of readily available yeast mutations but also generation of overexpression phenotypes and in some cases also rescue of such phenotypes. Here we summarize the main considerations that have to be taken into account when using the yeast expression system for investigating the function of plant genes participating in cell morphogenesis; outline the strategies of experiment planning, yeast strain selection (or construction), and expression vector choice; and provide detailed protocols for yeast transformation, transformant selection, and phenotype evaluation.

The exocyst complex contributes to PIN auxin efflux carrier recycling and polar auxin transport in Arabidopsis.

In land plants polar auxin transport is one of the substantial processes guiding whole plant polarity and morphogenesis. Directional auxin fluxes are mediated by PIN auxin efflux carriers, polarly localized at the plasma membrane. The polarization of exocytosis in yeast and animals is assisted by the exocyst: an octameric vesicle-tethering complex and an effector of Rab and Rho GTPases. Here we show that rootward polar auxin transport is compromised in roots of Arabidopsis thaliana loss-of-function mutants in the EXO70A1 exocyst subunit. The recycling of PIN1 and PIN2 proteins from brefeldin-A compartments is delayed after the brefeldin-A washout in exo70A1 and sec8 exocyst mutants. Relocalization of PIN1 and PIN2 proteins after prolonged brefeldin-A treatment is largely impaired in these mutants. At the same time, however, plasma membrane localization of GFP:EXO70A1, and the other exocyst subunits studied (GFP:SEC8 and YFP:SEC10), is resistant to brefeldin-A treatment. In root cells of the exo70A1 mutant, a portion of PIN2 is internalized and retained in specific, abnormally enlarged, endomembrane compartments that are distinct from VHA-a1-labelled early endosomes or the trans-Golgi network, but are RAB-A5d positive. We conclude that the exocyst is involved in PIN1 and PIN2 recycling, and thus in polar auxin transport regulation.

Evolution of the land plant exocyst complexes.

Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits - Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 - were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single "Exocyst complex" - instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.

The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction.

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.

The Arabidopsis exocyst complex is involved in cytokinesis and cell plate maturation.

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.

Arabidopsis RAB geranylgeranyl transferase beta-subunit mutant is constitutively photomorphogenic, and has shoot growth and gravitropic defects.

RAB GTPases are important directional regulators of intracellular vesicle transport. Membrane localization of RAB GTPases is mediated by C-terminal double geranylgeranylation. This post-translational modification is catalyzed by the alpha-beta-heterodimer catalytic core of RAB geranylgeranyl transferase (RAB-GGT), which cooperates with the RAB escort protein (REP) that presents a nascent RAB. Here, we show that RAB-geranylgeranylation activity is significantly reduced in two homozygous mutants of the major Arabidopsis beta-subunit of RAB-GGT (AtRGTB1), resulting in unprenylated RAB GTPases accumulation in the cytoplasm. Both endocytosis and exocytosis are downregulated in rgtb1 homozygotes defective in shoot growth and morphogenesis. Root gravitropism is normal in rgtb1 roots, but is significantly compromised in shoots. Mutants are defective in etiolation and show constitutive photomorphogenic phenotypes that cannot be rescued by brassinosteroid treatment, similarly to the det3 mutant that is also defective in the secretory pathway. Transcriptomic analysis revealed an upregulation of specific RAB GTPases in etiolated wild-type plants. Taken together, these data suggest that the downregulation of the secretory pathway is interpreted as a photomorphogenic signal in Arabidopsis.

Exocytosis and cell polarity in plants - exocyst and recycling domains.

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.

Plant antigens cross-react with rat polyclonal antibodies against KLH-conjugated peptides.

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.

An exocyst complex functions in plant cell growth in Arabidopsis and tobacco.

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.

A specific feature of the angiosperm Rab escort protein (REP) and evolution of the REP/GDI superfamily.

Rab GTPases participating in the regulation of vesicle trafficking in eukaryotes are geranylgeranylated by the Rab geranylgeranyl transferase (RabGGTase) in complex with the Rab escort protein (REP). Here, we describe basic properties of the Arabidopsis thaliana REP (AthREP), first REP outside yeasts or metazoans to be characterized. GFP-tagged AthREP, as well as the geranylgeranylation activity, were localized predominantly to the cytoplasm. Recombinant AthREP interacted with yeast 6His-Ypt1, tobacco 6His-RabA1a, and Arabidopsis RabA2a in vitro preferring the GDP-bound form of the latter. Recombinant AthREP with C-terminal but not N-terminal tags stimulated geranylgeranylation of various Rab GTPases in Arabidopsis extracts in vitro. Neither recombinant AthREP protein exhibited activity in yeast extracts, while recombinant yeast REP (6His-SceMrs6) stimulated Rab geranylgeranylation in all extracts tested. We found that a conserved arginine residue, R195, known to be crucial for yeast REP function, is substituted by an asparagine or threonine residue in angiosperm REPs. A point mutant allele of AthREP with arginine at this position complemented the yeast REP mutation, while wild-type AthREP did not. Based on phylogenetic analysis of REP and GDP dissociation inhibitor (GDI) sequences from a broad range of eukaryotic lineages, we propose a new view on evolution of the REP/GDI superfamily with a bi-functional REP/GDI protein as a direct ancestor.