Michal Hála

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.

The Arabidopsis Exocyst Complex Is Involved in Cytokinesis and Cell Plate Maturation

The plant cell cytokinesis is driven from the onset by highly organized vesicle fusion resulting in cell plate and new cell wall formation separating daughter cells. The evolutionarily conserved exocyst complex regulating exocytic vesicle binding to the plasma membrane is involved in both the final separation of cells as in animals and also in the initiation of cell plate in plant cells. 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.

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 exocyst complex in plants

Plant cell morphogenesis requires precise regulation of localized cell expansion and cell division. Vectorial exocytosis is a major morphogenetic process in plant cells, intimately interwoven with the dynamics of the cytoskeleton. Small GTPases of the Ras superfamily act as molecular switches participating in the control of both vesicle trafficking (Rab GTPases) and cytoskeleton dynamics (Rho GTPases). In tip-growing plant cells, such as the pollen tubes, wall formation is confined to the tip. The cytoplasm is highly polarized and grows by exocytosis in the apex, which makes pollen tubes an excellent model to study a large variety of dynamic events: targeted vesicle transport and docking, cell wall formation, guidance and maintenance of polarity, organelle movement, membrane recycling. We have previously shown that Rab homologues (especially those involved in the delivery of secretory vesicles to the plasma membrane) are the most abundant small GTPases in tobacco pollen tubes (Zarsky and Cvrckova, 1997) and cloned a tobacco pollen Rop (Rho of plants) homologue (Cvrckova and Zarsky, 1999). We have also shown the importance of GTPases (possibly Rop) in polarized pollen tube growth by microinjection of GTP/GDP analogues into growing pollen tubes (Elias et al., 2001). Both Rab and Rho GTPases are potential interactors of the exocyst (Sec6/8) complex, a multisubunit protein assembly involved in exocytosis that has been characterized in Saccharomyces cerevisiae and mammalian cells (reviewed in Hsu et al., 1999). The exocyst is composed of eight distinct subunits, referred to in yeast as Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p. The mammalian exocyst contains homologues of all these proteins. The complex is critical for specification of the site of vesicle docking and fusion and probably acts as a tethering complex before the steps mediated by SNARE and associated proteins. The yeast exocyst is known to interact directly with both Rab (Sec4) and Rho GTPases. Importantly, the Rho family GTPases Rho1 and Cdc42 are involved in the regulation of intracellular localization of the exocyst (Guo et al., 2001). Since the exocyst seems to be well conserved among distinct lineages of eukaryotes, it is relevant to ask whether plants also contain a similar module involved in exocytosis and secretion. Attempts to clone plant genes homologous to SEC15 via complementation of a yeast mutant with an Arabidopsis cDNA library were unsuccessful (Matsuda and Nakano, 1998). However, our searches through public sequence databases did identify genes significantly similar to all exocyst subunits (Cvrckova et al., 2001, and our unpublished data). The genome of Arabidopsis thaliana seems to code for two highly similar genes homologous to SEC3, SEC5, one homologue of SEC6, SEC8 and SEC10 each, two paralogues related to SEC15 and three copies of a putative EXO84. Surprisingly, there are as many as 23 potential Arabidopsis genes related to EXO70, although Saccharomyces, Drosophila, Caenorhabditis and perhaps also mammals possess only one copy of the EXO70 gene. This suggests that the plant exocyst might have some special features compared to other eukaryotes (Fig. 1). We have started molecular characterization of the putative Arabidopsis exocyst subunits. First we isolated a cDNA clone of the AtSec15b gene using a PCR-based library screen, and obtained cDNA clones potentially containing complete coding sequence of other exocyst * Corresponding author. Tel.: +420-2-21953179; fax: +420-2-21953306. E-mail address: zarsky@ueb.cas.cz (V. Zarsky). Cell Biology International 27 (2003) 199–201 Cell Biology International

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.

Arabidopsis RAB geranylgeranyl transferase β-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.

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

EXO70C2, from the family of exocyst subunits, is a novel factor regulating pollen tube tip growth in Arabidopsis, and its paralog EXO70C1 has a partially redundant function. 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.

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.

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.