SummaryThe exocyst is a hetero-oligomeric protein complex involved in exocytosis and has been extensively studied in yeast and animal cells. Evidence is now accumulating that the exocyst is also present in plants. Bioinformatic analysis of genes encoding plant homologs of the exocyst subunit, Exo70, revealed that three Exo70 subgroups are evolutionarily conserved among angiosperms, lycophytes and mosses. Arabidopsis and rice contain 22 and approximately 39 EXO70 genes, respectively, which can be classified into nine clusters considered to be ancient in angiosperms (one has been lost in Arabidopsis). We characterized two independent T-DNA insertional mutants of the AtEXO70A1 gene (exo70A1-1 and exo70A1-2). Heterozygous EXO70A1/exo70A1 plants appear to be normal and segregate in a 1:2:1 ratio, suggesting that neither male nor female gametophytes are affected by the EXO70A1 disruption. However, both exo70A1-1 and exo70A1-2 homozygotes exhibit an array of phenotypic defects. The polar growth of root hairs and stigmatic papillae is disturbed. Organs are generally smaller, plants show a loss of apical dominance and indeterminate growth where instead of floral meristems new lateral inflorescences are initiated in a reiterative manner. Both exo70A1 mutants have dramatically reduced fertility. These results suggest that the putative exocyst subunit EXO70A1 is involved in cell and organ morphogenesis.
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.
Root hairs are cellular protuberances extending from the root surface into the soil; there they provide access to immobile inorganic ions such as phosphate, which are essential for growth. Their cylindrical shape results from a polarized mechanism of cell expansion called tip growth in which elongation is restricted to a small area at the surface of the hair-forming cell (trichoblast) tip. Here we identify proteins that spatially control the sites at which cell growth occurs by isolating Arabidopsis mutants (scn1) that develop ectopic sites of growth on trichoblasts. We cloned SCN1 and showed that SCN1 is a RhoGTPase GDP dissociation inhibitor (RhoGDI) that spatially restricts the sites of growth to a single point on the trichoblast. We showed previously that localized production of reactive oxygen species by RHD2/AtrbohC NADPH oxidase is required for hair growth; here we show that SCN1/AtrhoGDI1 is a component of the mechanism that focuses RHD2/AtrbohC-catalysed production of reactive oxygen species to hair tips during wild-type development. We propose that the spatial organization of growth in plant cells requires the local RhoGDI-regulated activation of the RHD2/AtrbohC NADPH oxidase.
Mitogen-activated protein kinase kinase kinases (MAPKKKs) play key roles in intra-and extracellular signaling in eukaryotes. Here we report that the MAPKKK MEKK1 regulates redox homeostasis in Arabidopsis. We show that MEKK1-deficient plants are misregulated in the expression of a number of genes involved in cellular redox control and accumulate reactive oxygen species (ROS). Most strikingly, homozygous mekk1 mutant plants exhibit a lethal phenotype when developing true leaves. MEKK1 kinase activity and protein stability was regulated by H 2 O 2 in a proteasome-dependent manner and mekk1 plants were compromised in ROS-induced MAPK MPK4 activation. Whereas mpk3 and mpk6 knock out plants showed no defects in development or changes in redox control genes, mpk4 null mutant shared several phenotypic and transcript profile features with mekk1 plants. In agreement with the concept that ROS negatively regulates auxin responses in plants, mekk1 and mpk4 mutants show reduced expression of several auxin-inducible marker genes. Overall, our data defines MPK4 as downstream target of MEKK1 and show that MEKK1 functions in integrating ROS homeostasis with plant development and hormone signaling.In all eukaryotes, mitogen-activated protein kinase (MAPK) 3 pathways serve as highly conserved central regulators of growth, death, differentiation, proliferation, and stress responses (1-4). A MAPK cascade minimally consists of a MAPKKK-MAPKK-MAPK module that is linked in various ways to upstream receptors and downstream targets. Receptormediated activation of a MAPKKK can occur through physical interaction and/or phosphorylation by either the receptor itself, intermediate bridging factors or interlinking MAPKKKKs. MAPKKKs are serine/threonine kinases that activate MAPKKs through phosphorylation on two serine/threonine residues in a conserved (S/T)X 3-5 (S/T) motif (1, 2). In contrast, MAPKKs are dual-specificity kinases that phosphorylate MAPKs on threonine and tyrosine residues in the TXY motif. MAPKs are promiscuous serine/threonine kinases that phosphorylate a variety of substrates including transcription factors, protein kinases, and cytoskeleton-associated proteins (2). Specificity of MAPK cascades functioning within the same cell is generated through the presence of docking domains found in various components of MAPK modules and through a growing number of scaffold proteins (5).Recently, we have isolated OMTK1 (oxidative stress-activated MAP triple-kinase 1), a novel MAPKKK from alfalfa (Medicago sativa), which among a panel of hormones and stresses tested, was only activated by hydrogen peroxide (H 2 O 2 ) (6). Out of four MAPKs, OMTK1 specifically activated MMK3 resulting in an increased cell death rate. Pull-down analysis between recombinant proteins showed that OMTK1 directly interacts with MMK3 and that OMTK1 and MMK3 are part of a protein complex in vivo. These results indicated that OMTK1 plays a MAPK scaffolding role and functions in activation of H 2 O 2 -induced cell death in plants. Because of its autotetraploid natu...
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.
Autophagic transport to the vacuole represents an endomembrane trafficking route, which is widely used in plants, not only during stress situations, but also for vacuole biogenesis and during developmental processes. Here we report a role in autophagic membrane transport for EXO70B1 -one of 23 paralogs of Arabidopsis EXO70 exocyst subunits. EXO70B1 positive compartments are internalized into the central vacuole and co-localize with autophagosomal marker ATG8f. This internalization is boosted by induction of autophagy. Loss of function (LOF) mutations in exo70B1 cause reduction of internalized autopagic bodies in the vacuole. Mutant plants also show ectopic hypersensitive response (HR) mediated by salicylic acid (SA) accumulation, increased nitrogen starvation susceptibility and anthocyanin accumulation defects. Anthocyanin accumulation defect persists in npr1x exo70B1 double mutants with SA signaling compromised, while ectopic HR is suppressed. EXO70B1 interacts with SEC5 and EXO84 and forms an exocyst subcomplex involved in autophagy-related, Golgiindependent membrane traffic to the vacuole. We show that EXO70B1 is functionally completely different from EXO70A1 exocyst subunit and adopted a specific role in autophagic transport.
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.
Plant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.
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