19 research outputs found

    H+/Ca2+ exchange driven by the plasma membrane Ca2+-ATPase of Arabidopsis thaliana reconstituted in proteoliposomes after calmodulin-affinity purification

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    AbstractThe plasma membrane Ca2+-ATPase was purified from Arabidopsis thaliana cultured cells by calmodulin (CaM)-affinity chromatography and reconstituted in proteoliposomes by the freeze-thaw sonication procedure. The reconstituted enzyme catalyzed CaM-stimulated 45Ca2+ accumulation and H+ ejection, monitored by the increase of fluorescence of the pH probe pyranine entrapped in the liposomal lumen during reconstitution. Proton ejection was immediately reversed by the protonophore FCCP, indicating that it is not electrically coupled to Ca2+ uptake, but it is a primary event linked to Ca2+ uptake in the form of countertransport

    Phosphorylation of serine residues in the N-terminus modulates the activity of ACA8, a plasma membrane Ca2+-ATPase of Arabidopsis thaliana

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    ACA8 is a plasma membrane-localized isoform of calmodulin (CaM)-regulated Ca2+-ATPase of Arabidopsis thaliana. Several phosphopeptides corresponding to portions of the regulatory N-terminus of ACA8 have been identified in phospho-proteomic studies. To mimic phosphorylation of the ACA8 N-terminus, each of the serines found to be phosphorylated in those studies (Ser19, Ser22, Ser27, Ser29, Ser57, and Ser99) has been mutated to aspartate. Mutants have been expressed in Saccharomyces cerevisiae and characterized: mutants S19D and S57D—and to a lesser extent also mutants S22D and S27D—are deregulated, as shown by their low activation by CaM and by tryptic cleavage of the N-terminus. The His-tagged N-termini of wild-type and mutant ACA8 (6His-1M-I116) were expressed in Escherichia coli, affinity-purified, and used to analyse the kinetics of CaM binding by surface plasmon resonance. All the analysed mutations affect the kinetics of interaction with CaM to some extent: in most cases, the altered kinetics result in marginal changes in affinity, with the exception of mutants S57D (KD ∼10-fold higher than wild-type ACA8) and S99D (KD about half that of wild-type ACA8). The ACA8 N-terminus is phosphorylated in vitro by two isoforms of A. thaliana calcium-dependent protein kinase (CPK1 and CPK16); phosphorylation of mutant 6His-1M-I116 peptides shows that CPK16 is able to phosphorylate the ACA8 N-terminus at Ser19 and at Ser22. The possible physiological implications of the subtle modulation of ACA8 activity by phosphorylation of its N-terminus are discussed

    Dissecting the susceptibility/resistance mechanism of Vitis vinifera for the future control of downy mildew

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    23CO.NA.VI. 2020 – 8° Convegno Nazionale di Viticoltura, Udine, Italy, July 5-7, 2021openInternationalBothThe Eurasian grapevine (Vitis vinifera), a species cultivated worldwide for high-quality wine production, is extremely susceptible to the agent of downy mildew, Plasmopara viticola. Nevertheless, germplasm from Georgia (Southern Caucasus, the first grapevine domestication centre), characterized by a high genetic variability, showed resistance traits to P. viticola. The cultivar Mgaloblishvili exhibited the most promising phenotype in terms of resistance against P. viticola. Its defence response results in: i) low disease intensity; ii) low sporulation; iii) damaged mycelium; iv) production of antimicrobial compounds such as volatile organic compounds (VOCs), whose effectiveness on the pathogen was evaluated by leafdisc assays. At the transcriptomic level, its resistance mechanism is determined by the differential expression of both resistance and susceptible genes. The resistance genes are related to: i) pathogen recognition through PAMP, DAMP and effector receptors; ii) ethylene signalling pathway; iii) synthesis of antimicrobial compounds (VOCs) and fungal wall degrading enzymes; iv) development of structural barriers (cell wall reinforcement). The first putative susceptible gene was the transcription factor VviLBDIf7 gene, whose validation was carried out by dsRNA (double-stranded RNA) assay. In this work, these unique results on plant-pathogen interaction are reviewed with the aim of developing new strategies to control the disease.openRicciardi, Valentina; Marcianò, Demetrio; Sargolzaei, Maryam; Marrone Fassolo, Elena; Fracassetti, Daniela; Brilli, Matteo; Moser, Mirko; Vahid, Shariati J.; Tavakole, Elahe; Maddalena, Giuliana; Passera, Alessandro; Casati, Paola; Pindo, Massimo; Cestaro, Alessandro; Costa, Alex; Bonza, Maria Cristina; Maghradze, David; Tirelli, Antonio; Failla, Osvaldo; Bianco, Piero Attilio; Quaglino, Fabio; Toffolatti, Silvia Laura; De Lorenzis, GabriellaRicciardi, V.; Marcianò, D.; Sargolzaei, M.; Marrone Fassolo, E.; Fracassetti, D.; Brilli, M.; Moser, M.; Vahid, S.J.; Tavakole, E.; Maddalena, G.; Passera, A.; Casati, P.; Pindo, M.; Cestaro, A.; Costa, A.; Bonza, M.C.; Maghradze, D.; Tirelli, A.; Failla, O.; Bianco, P.A.; Quaglino, F.; Toffolatti, S.L.; De Lorenzis, G

    La Ca2+ -aptasi del plasmalemma delle piante superiori: purificazione dell'enzima ed isolamento del corrispondente cDNA

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    Dottorato di ricerca in biologia vegetale. 12. ciclo. Docente guida Maria Ida De Michelis. Coordinatore Francesco SalaConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

    Purification of the Plasma Membrane Ca 2+

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    Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps*

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    ACA8 is a type 2B Ca2+-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu252 to Asn345 sequence. Mutation to Ala of any of six tested acidic residues (Glu252, Asp273, Asp291, Asp303, Glu302, or Asp332) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca2+-ATPase plays a role in the attainment of the auto-inhibited state
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