ABSTRACT
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.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins , Arabidopsis/drug effects , Calcium/metabolism , Cell Membrane/drug effects , Plant Proteins/genetics , rab GTP-Binding Proteins , Arabidopsis/cytology , Arabidopsis/genetics , Biological Transport/drug effects , Blotting, Northern , Cell Membrane/metabolism , Dose-Response Relationship, Drug , Extracellular Space/drug effects , Extracellular Space/metabolism , Gene Expression Regulation, Plant/drug effects , RNA, Plant/drug effects , RNA, Plant/genetics , RNA, Plant/metabolism , Time FactorsABSTRACT
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.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins , Arabidopsis/physiology , Gene Expression/drug effects , Ion Channels/physiology , Plant Proteins/genetics , rab GTP-Binding Proteins , Anions , Barium Compounds/pharmacology , Chlorides/pharmacology , Electric Conductivity , Ion Channels/antagonists & inhibitors , Patch-Clamp Techniques , Potassium Channel Blockers , Potassium Channels/physiology , Signal Transduction , Tetraethylammonium/pharmacology , Zinc Sulfate/pharmacologyABSTRACT
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.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins , Arabidopsis/drug effects , Arabidopsis/genetics , Plant Proteins/genetics , Potassium Channels/drug effects , rab GTP-Binding Proteins , Abscisic Acid/chemistry , Abscisic Acid/metabolism , Animals , Arabidopsis/metabolism , Cattle , Gene Expression Regulation, Plant/drug effects , Genes, Plant , Hydrogen-Ion Concentration , Serum Albumin, Bovine , StereoisomerismABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.