RESUMEN
Pretreatment of Arabidopsis thaliana suspension cells with impermeant calcium chelator EGTA inhibited the ABA-induced RAB18 gene expression. However, extracellular calcium alone, up to 10 mM, did not trigger RAB18 expression. Spectrofluorimetric extracellular Ca(2+) measurement with Fluo-3 showed a fast, within 1 min, Ca(2+) influx associated with outer plasmalemma ABA perception. In the presence of the Ca(2+) blockers Cd(2+) and Ni(2+), RAB18 expression was suppressed. Pimozide and fluspirilene inhibited Ca(2+) influx and ABA-induced RAB18 expression. Thus we demonstrated the involvement of specific Ca(2+) influx in the ABA signaling sequence leading to RAB18 expression.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis , Arabidopsis/efectos de los fármacos , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas de Unión al GTP rab , Arabidopsis/citología , Arabidopsis/genética , Transporte Biológico/efectos de los fármacos , Northern Blotting , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , ARN de Planta/efectos de los fármacos , ARN de Planta/genética , ARN de Planta/metabolismo , Factores de TiempoRESUMEN
The abscissic acid (ABA) transduction cascade following the plasmalemma perception was analyzed in intact Arabidopsis thaliana suspension cells. In response to impermeant ABA, anion currents were activated and K(+) inward rectifying currents were inhibited. Anion current activation was required for the ABA specific expression of RAB18. By contrast, specific inhibition of K(+) channels by tetraethylammonium or Ba(2+) did not affect RAB18 expression. Thus, outer plasmalemma ABA perception triggered two separated signaling pathways.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis , Arabidopsis/fisiología , Expresión Génica/efectos de los fármacos , Canales Iónicos/fisiología , Proteínas de Plantas/genética , Proteínas de Unión al GTP rab , Aniones , Compuestos de Bario/farmacología , Cloruros/farmacología , Conductividad Eléctrica , Canales Iónicos/antagonistas & inhibidores , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Transducción de Señal , Tetraetilamonio/farmacología , Sulfato de Zinc/farmacologíaRESUMEN
Important progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches (Leung and Giraudat, 1998). However, we do not yet know the mechanism of ABA perception. Conflicting results concerning the site of ABA perception have been published. The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist. In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA-BSA conjugate to mimic permeant free ABA effects. We show that purified ABA-BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (+)-ABA enantiomer. The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells. Using the voltage-clamp technique we show that ABA-BSA, similarly to ABA, evokes a membrane depolarization and activates time- and voltage-dependent outward rectifying currents (ORC). We demonstrate that these ORC are due to a K+ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba2+. These observations provide evidence in favour of an extracellular site for ABA perception.
Asunto(s)
Ácido Abscísico/farmacología , Proteínas de Arabidopsis , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Plantas/genética , Canales de Potasio/efectos de los fármacos , Proteínas de Unión al GTP rab , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Animales , Arabidopsis/metabolismo , Bovinos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Concentración de Iones de Hidrógeno , Albúmina Sérica Bovina , EstereoisomerismoRESUMEN
A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl(2) in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids.
RESUMEN
The synthesis of alpha-tocopherol from 2,3-dimethylphytylquinol and S-adenosyl-l-methionine was achieved using Capsicum annuum fruit chromoplasts. The enzymes involved in the cyclization (2,3-dimethyl-phytylquinol cyclase) and methylation (S-adenosyl methionine:gamma-tocopherol methyl-transferase) are both localized in the chromoplast membrane fraction (envelopes and/or a-chlorophyll lamellae), in contrast to the stroma fraction.
RESUMEN
Capsicum chromoplasts incubated with isopentenyl diphosphate actively synthesize carotenoids. The enzymes involved in these reactions are compartmentalized: the stroma is the site of phytoene synthesis, the first colourless carotenoid, while desaturation and cyclization of the latter leading to coloured carotenoids, occur in the membrane fraction (chromoplast envelope + achlorophyll lamellae derived in part from the inner envelope membrane). Phytoene synthetase could be used as a marker of chromoplast stroma.
