RESUMEN

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/enzimología , Péptido Hidrolasas/metabolismo , Péptido Sintasas/metabolismo , Proteínas de Plantas/metabolismo , Complejo de la Endopetidasa Proteasomal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Modelos Moleculares , Datos de Secuencia Molecular , Péptido Hidrolasas/química , Péptido Sintasas/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Proteínas Ligasas SKP Cullina F-box , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transactivadores/metabolismo

RESUMEN

Marine macrolides latrunculins are highly specific toxins which effectively depolymerize actin filaments (generally F-actin) in all eukaryotic cells. We show that latrunculin B is effective on diverse cell types in higher plants and describe the use of this drug in probing F-actin-dependent growth and in plant development-related processes. In contrast to other eukaryotic organisms, cell divisions occurs in plant cells devoid of all actin filaments. However, the alignment of the division planes is often distorted. In addition to cell division, postembryonic development and morphogenesis also continue in the absence of F-actin. These experimental data suggest that F-actin is of little importance in the morphogenesis of higher plants, and that plants can develop more or less normally without F-actin. In contrast, F-actin turns out to be essential for cell elongation. When latrunculin B was added during germination, morphologically normal Arabidopsis and rye seedlings developed but, as a result of the absence of cell elongation, these were stunted, resembling either genetic dwarfs or environmental bonsai plants. In conclusion, F-actin is essential for the plant cell elongation, while this F-actin-dependent cell elongation is not an essential feature of plant-specific developmental programs.

Asunto(s)

Actinas/fisiología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Plantas/efectos de los fármacos , Tiazoles/farmacología , División Celular/efectos de los fármacos , Ácidos Indolacéticos/farmacología , Mitosis , Desarrollo de la Planta , Tiazolidinas

RESUMEN

Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/genética , Proteínas de Plantas/análisis , Rhizobium/genética , Arabidopsis/metabolismo , Western Blotting , ADN Bacteriano/genética , Epítopos , Técnica del Anticuerpo Fluorescente , Genes Reporteros , Vectores Genéticos , Glucuronidasa/genética , Glucuronidasa/metabolismo , Intrones , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformación Genética

RESUMEN

The effects of jasmonic acid (JA) applied in the medium and its methylester (MeJA) applied either in the medium or as a vapour, on shoot growth and microtuber formation were evaluated in three important food yam species (Dioscorea alata, D. cayenensis, and D. rotundata). Single nodes with leaves, derived from in vitro-multiplied material, were used as explants. When delivered at higher concentrations (10 or 50 µM) both JA and MeJA suppressed node formation. Microtuberisation was supported in all three species by adding either JA or MeJA to the medium. Significant promotory effects were observed only when photoperiod, salt compositions and sucrose concentrations known to favour microtuberisation processes in yams were used. MeJA applied as a vapour strongly inhibited microtuber differentiation in D. alata on all media tested but in D. rotundata and D. cayenensis yams MeJA, also applied in the vapour phase, exhibited slight promotory effects on microtuberisation.

RESUMEN

Embryogenic cultures were established from silver fir (Abies alba Mill.) female megagametliophytes with developing embryos and from excised mature embryos after pollination with Abies cephalonica Lond. or Abies numidica DeLann pollea The frequency of embryogenic callus formation was dependent on genotype, collection time, medium and explants used. The embryogenic callus initiation potential of megagamethophytes with developing embryos in both hybrids was higher in early July and dropped as the zygotic embryos matured. Excised cotyledonary embryos were less suitable for induction of embryogenic cultures. SH medium supplemented with 1mg/l BAP was the most efficient for callus induction and maintenance. Cultures were composed of early somatic embryos with an embryonal mass formed of highly cytoplasmic cells, rich in cell organelles and a suspensor built up by vacuolated, strongly elongated cells. Maturation of embryos was detected with the formation of bipolar structures with shoot and root apices. Nutrition reserves were observed in cells of embryos cultured on DCR medium containing 1 or 10 mg/l ABA. Cotyledon formation, hypocotyl elongation and low frequency germination occured following transfer of the embryos to the same medium without ABA.