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...

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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.

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 beta 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 pro-vascular cells of the funiculus. Decreased transport of auxin out of the ovule is accompanied by auxin accumulation in a 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.

The regulation of developmental pathways in cultured microspores of tobacco ( Nicotiana tabacum L) and snapdragon ( Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25 degrees C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0-8.5 for 4-6 days at 25 degrees C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of (14)C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.

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 datamining 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.

Patients in the intensive care unit often lose a considerable fraction of their gut microbiome due to exposure to broad-spectrum antibiotics and other reasons. Dysbiosis often results in prolonged diarrhea and increase occurrence of multi-drug resistant pathogens in the colon with clinical consequences not yet well understood. Restoring the microbiome by fecal microbial transplantation (FMT) is a plausible therapeutic possibility, so far only documented in case reports and case series using very heterogeneous methodologies. Before FMT with critically ill patients can be tested in randomized controlled trials, there is a burning need to describe a standardized operating procedure (SOP) of the whole process, respecting the specifics of the critically ill population, such as the risk of the disrupted intestinal barrier and time-critical nature of the procedure. We describe the SOP that has been developed for experimental use in critically ill patients by a multidisciplinary team of intensivists, gastroenterologists, and microbiologists based on feedback from regulatory authority (State Institute for Drug Control of the Czech Republic). The hallmarks of these SOPs are multi-donor freshly frozen transplants guaranteed for 2 months consisting of seven aliquots from seven unrelated healthy donors and administered by a rectal tube. In this paper we discuss the rationale for this SOP and the process of its development in detail and release the full proposed SOP is in the form of an online appendix.

Etiolated hypocotyls of exocyst mutants exhibit a defect in hypocotyl plasticity manifested as ectopic collet-like structure formation, associated with deviations in auxin signaling and starch accumulation.