OXI1 kinase is necessary for oxidative burst-mediated signalling in Arabidopsis

Active oxygen species (AOS) generated in response to stimuli and during development can function as signalling molecules in eukaryotes, leading to specific downstream responses. In plants these include such diverse processes as coping with stress (for example pathogen attack, wounding and oxygen deprivation), abscisic-acid-induced guard-cell closure, and cellular development (for example root hair growth). Despite the importance of signalling via AOS in eukaryotes, little is known about the protein components operating downstream of AOS that mediate any of these processes. Here we show that expression of an Arabidopsis thaliana gene (OXI1) encoding a serine/threonine kinase is induced in response to a wide range of H2O2-generating stimuli. OXI1 kinase activity is itself also induced by H2O2 in vivo. OXI1 is required for full activation of the mitogen-activated protein kinases (MAPKs) MPK3 and MPK6 after treatment with AOS or elicitor and is necessary for at least two very different AOS-mediated processes: basal resistance to Peronospora parasitica infection, and root hair growth. Thus, OXI1 is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses.     

An Arabidopsis hAT-like transposase is essential for plant development

A significant proportion of the genomes of higher plants and vertebrates consists of transposable elements and their derivatives. Autonomous DNA type transposons encode a transposase that enables them to mobilize to a new chromosomal position in the host genome by a cut-and-paste mechanism. As this is potentially mutagenic, the host limits transposition through epigenetic gene silencing and heterochromatin formation. Here we show that a transposase from Arabidopsis thaliana that we named DAYSLEEPER is essential for normal plant growth; it shares several characteristics with the hAT (hobo, Activator, Tam3) family of transposases. DAYSLEEPER was isolated as a factor binding to a motif (Kubox1) present in the upstream region of the Arabidopsis DNA repair gene Ku70. This motif is also present in the upstream regions of many other plant genes. Plants lacking DAYSLEEPER or strongly overexpressing this gene do not develop in a normal manner. Furthermore, DAYSLEEPER overexpression results in the altered expression of many genes. Our data indicate that transposase-like genes can be essential for plant development and can also regulate global gene expression. Thus, transposases can become domesticated by the host to fulfil important cellular functions.     

拟南芥bHLH家族转录因子DYT1在花药发育过程中调控胼胝质降解(英文)

胼胝质的合成和降解是雄配子体减数分裂过程中的一个重要特征,对后期花粉成熟有重要作用.在此研究中,分离到了一个雄性不育突变体msl57,该突变体的绒毡层分化及胼胝质降解过程出现异常,导致花粉败育.图位克隆和遗传分析表明:MSl57基因与bHLI-I家族转录因子DYTI(At4g21330)是同一基因.因此,将ms157突变体改名为dyt1-2.反式激活作用实验揭示了DYTI的激活功能域位于基因的250 ～504bp之间.通过酵母双杂交实验发现DYT1蛋白在体内可以形成同源二聚体来执行其功能.RT-PCR及定量PCR分析表明胼胝质酶相关基因A6的表达在突变体背景下严重下调.因此,DVF1通过调控胼胝质的降解来影响花药发育过程.     

MOR1 is essential for organizing cortical microtubules in plants

Microtubules orchestrate cell division and morphogenesis, but how they disassemble and reappear at different subcellular locations is unknown. Microtubule organizing centres are thought to have an important role, but in higher plants microtubules assemble in ordered configurations even though microtubule organizing centres are inconspicuous or absent. Plant cells generate highly organized microtubule arrays that coordinate mitosis, cytokinesis and expansion. Inhibiting microtubule assembly prevents chromosome separation, blocks cell division and impairs growth polarity. Microtubules are essential for the formation of cell walls, through an array of plasma-membrane-associated cortical microtubules whose control mechanisms are unknown. Using a genetic strategy to identify microtubule organizing factors in Arabidopsis thaliana, we isolated temperature-sensitive mutant alleles of the MICROTUBULE ORGANIZATION 1 (MOR1) gene. Here we show that MOR1 is the plant version of an ancient family of microtubule-associated proteins. Point mutations that substitute single amino-acid residues in an amino-terminal HEAT repeat impart reversible temperature-dependent cortical microtubule disruption, showing that MOR1 is essential for cortical microtubule organization.     

Bmi1 is essential for cerebellar development and is overexpressed in human medulloblastomas

Overexpression of the polycomb group gene Bmi1 promotes cell proliferation and induces leukaemia through repression of Cdkn2a (also known as ink4a/Arf) tumour suppressors. Conversely, loss of Bmi1 leads to haematological defects and severe progressive neurological abnormalities in which de-repression of the ink4a/Arf locus is critically implicated. Here, we show that Bmi1 is strongly expressed in proliferating cerebellar precursor cells in mice and humans. Using Bmi1-null mice we demonstrate a crucial role for Bmi1 in clonal expansion of granule cell precursors both in vivo and in vitro. Deregulated proliferation of these progenitor cells, by activation of the sonic hedgehog (Shh) pathway, leads to medulloblastoma development. We also demonstrate linked overexpression of BMI1 and patched (PTCH), suggestive of SHH pathway activation, in a substantial fraction of primary human medulloblastomas. Together with the rapid induction of Bmi1 expression on addition of Shh or on overexpression of the Shh target Gli1 in cerebellar granule cell cultures, these findings implicate BMI1 overexpression as an alternative or additive mechanism in the pathogenesis of medulloblastomas, and highlight a role for Bmi1-containing polycomb complexes in proliferation of cerebellar precursor cells.     

Vanilloid receptor-1 is essential for inflammatory thermal hyperalgesia

The vanilloid receptor-1 (VR1) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. VR1 responds to noxious stimuli including capsaicin, the pungent component of chilli peppers, heat and extracellular acidification, and it is able to integrate simultaneous exposure to these stimuli. These findings and research linking capsaicin with nociceptive behaviours (that is, responses to painful stimuli in animals have led to VR1 being considered as important for pain sensation. Here we have disrupted the mouse VR1 gene using standard gene targeting techniques. Small diameter dorsal root ganglion neurons isolated from VR1-null mice lacked many of the capsaicin-, acid- and heat-gated responses that have been previously well characterized in small diameter dorsal root ganglion neurons from various species. Furthermore, although the VR1-null mice appeared normal in a wide range of behavioural tests, including responses to acute noxious thermal stimuli, their ability to develop carrageenan-induced thermal hyperalgesia was completely absent. We conclude that VR1 is required for inflammatory sensitization to noxious thermal stimuli but also that alternative mechanisms are sufficient for normal sensation of noxious heat.     

arrow encodes an LDL-receptor-related protein essential for Wingless signalling

The Wnt family of secreted molecules functions in cell-fate determination and morphogenesis during development in both vertebrates and invertebrates (reviewed in ref. 1). Drosophila Wingless is a founding member of this family, and many components of its signal transduction cascade have been identified, including the Frizzled class of receptor. But the mechanism by which the Wingless signal is received and transduced across the membrane is not completely understood. Here we describe a gene that is necessary for all Wingless signalling events in Drosophila. We show that arrow gene function is essential in cells receiving Wingless input and that it acts upstream of Dishevelled. arrow encodes a single-pass transmembrane protein, indicating that it may be part of a receptor complex with Frizzled class proteins. Arrow is a low-density lipoprotein (LDL)-receptor-related protein (LRP), strikingly homologous to murine and human LRP5 and LRP6. Thus, our data suggests a new and conserved function for this LRP subfamily in Wingless/Wnt signal reception.     

Hair cell synaptic ribbons are essential for synchronous auditory signalling

Hearing relies on faithful synaptic transmission at the ribbon synapse of cochlear inner hair cells (IHCs). At present, the function of presynaptic ribbons at these synapses is still largely unknown. Here we show that anchoring of IHC ribbons is impaired in mouse mutants for the presynaptic scaffolding protein Bassoon. The lack of active-zone-anchored synaptic ribbons reduced the presynaptic readily releasable vesicle pool, and impaired synchronous auditory signalling as revealed by recordings of exocytic IHC capacitance changes and sound-evoked activation of spiral ganglion neurons. Both exocytosis of the hair cell releasable vesicle pool and the number of synchronously activated spiral ganglion neurons co-varied with the number of anchored ribbons during development. Interestingly, ribbon-deficient IHCs were still capable of sustained exocytosis with normal Ca2+-dependence. Endocytic membrane retrieval was intact, but an accumulation of tubular and cisternal membrane profiles was observed in ribbon-deficient IHCs. We conclude that ribbon-dependent synchronous release of multiple vesicles at the hair cell afferent synapse is essential for normal hearing.     

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone--an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.     

Sphingolipid signalling in Arabidopsis guard cells involves heterotrimeric G proteins

In animals, the sphingolipid metabolite sphingosine-1-phosphate (S1P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S1P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S1P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S1P production, sphingosine kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S1P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA1 (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S1P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S1P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.     

An Arabidopsis ctpA homologue is involved in the repair of photosystem II under high light

A T-DNA insertion mutant AtctpA1 was identified to study the physiological roles of a carboxyl-terminal processing protease (CtpA) homologue in Arabidopsis. Under normal growth conditions, disruption of AtctpA1 did not result in any apparent alterations in growth rate and thylakoid membrane protein components. However the mutant plants exhibited increased sensitivity to high irradiance. Degradation of PSII reaction center protein D1 was accelerated in the mutant during photoinhibition. These results demostrated that AtctpA1 was required for efficient repair of PSII in Arabidopsis under high irradiance.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis

Localized attack by a necrotizing pathogen induces systemic acquired resistance (SAR) to subsequent attack by a broad range of normally virulent pathogens. Salicylic acid accumulation is required for activation of local defenses, such as pathogenesis-related protein accumulation, at the initial site of attack, and for subsequent expression of SAR upon secondary, distant challenge. Although salicylic acid moves through the plant, it is apparently not an essential mobile signal. We screened Agrobacterium tumefaciens transfer DNA (tDNA) tagged lines of Arabidopsis thaliana for mutants specifically compromized in SAR. Here we show that Defective in induced resistance 1-1 (dir1-1) exhibits wild-type local resistance to avirulent and virulent Pseudomonas syringae, but that pathogenesis-related gene expression is abolished in uninoculated distant leaves and dir1-1 fails to develop SAR to virulent Pseudomonas or Peronospora parasitica. Petiole exudate experiments indicate that dir1-1 is defective in the production or transmission from the inoculated leaf of an essential mobile signal. DIR1 encodes a putative apoplastic lipid transfer protein and we propose that DIR1 interacts with a lipid-derived molecule to promote long distance signalling.     

Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection

Ebola virus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that antiviral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the amino-terminal domain, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit. Thus, NPC1 is essential for EboV entry and a target for antiviral therapy.