Annick Graziana

Organization of cytoskeleton controls the changes in cytosolic calcium of cold-shocked Nicotiana plumbaginifolia protoplasts

Using Nicotiana plumbaginifolia constitutively expressing the recombinant bioluminescent calcium indicator, aequorin, it has been previously demonstrated that plant cells react to cold-shock by an immediate rise in cytosolic calcium. Such an opportune system has been exploited to address the regulatory pathway involved in the calcium response. For this purpose, we have used protoplasts derived from N. plumbaginifolia leaves that behave as the whole plant but with a better reproducibility. By both immunodetecting cytoskeletal components on membrane ghosts and measuring the relative change in cytosolic calcium, we demonstrate that the organization of the cytoskeleton has profound influences on the calcium response. The disruption of the microtubule meshwork by various active drugs, such as colchicin, oryzalin and vinblastin, leads to an important increase in the cytosolic calcium (up to 400 nM) in cold-shocked protoplasts over control. beta-Lumicolchicin, an inactive analogue of colchicin, is ineffective either on cytoplasmic calcium increase or on microtubule organization. A microfilament disrupting drug, cytochalasin D, exerts a slight stimulatory effect, whereas the simultaneous disruption of microtubule and microfilament meshworks results in a dramatic increase in the calcium response to cold-shock. The results described in the present paper illustrate the role of the intracellular organization and, more specifically, the role of cytoskeleton in controlling the intensity of calcium response to an extracellular stimulus.

Activation of plasma membrane voltage-dependent calcium-permeable channels by disruption of microtubules in carrot cells

Plasma membrane‐bound voltage‐dependent calcium channels may couple the perception of an initial stimulus to a regulated pathway for calcium influx. The activities of these channels have been shown to be very low and highly unstable but may be recruited by large‐predepolarizing pulses, according to a process referred to as recruitment. By combining pharmacological and electrophysiological approaches, we demonstrate in the present paper that the cytoskeleton plays an important role in the regulation of the activity and stability of voltage‐dependent calcium channels during whole‐cell patch‐clamp experiments on carrot protoplasts. Whereas drugs affecting the organization of the microfilament network have no measurable effect, the manipulation of the microtubule network elicits important changes. Thus, the addition of colchicine or oryzalin, which are known to disrupt microtubule organization, leads to a 6–10‐fold increase in calcium channel activities and half‐life. In contrast, stabilization of the microtubules by taxol has no effect on any of these parameters. The data obtained suggest that interactions of microtubules and voltage‐dependent calcium channels by either direct or indirect mechanisms inhibit channel activities and decrease their half‐life. In contrast, the disruption of the network overcomes such an inhibitory effect and allows the activation of calcium channels. It is speculated that under normal physiological conditions these protein‐protein interactions may work in a reversible manner and contribute to signal transduction in higher plants.

Provoked changes in cellular calcium controlled protein phosphorylation and activity of quinate:NAD+ oxidoreductase in carrot cells

Quinate:NAD+ oxidoreductase activity decreased when carrot cell‐suspension cultures were supplemented with the Ca2+‐ionophore, A‐23187 and EGTA. The protein phosphorylation pattern changed as judged by autoradiography. The loss in enzyme activity was correlated with the Ca2+ efflux. Addition of Ca2+ to protein extracts in combination with calmodulin or not had no effect. Initial quinate:NAD+ oxidoreductase activity was partially recovered only after preincubation with ATP‐Mg2+ and Ca2+. The reactivation was abolished by EGTA or fluphenazine. It is concluded that cellular Ca2+ controls the enzyme activity by affecting its degree of phosphorylation in vivo.

The reversible association of quinate:NAD+ oxidoreductase from carrot cells with a putative regulatory subunit depends on light conditions

The transfer of carrot cell‐suspension cultures from light to dark conditions provoked considerable and reversible changes in the regulatory and structural properties of quinate:NAD+ oxidoreductase. Thus, the enzyme became directly activatable by Ca2+ and its M r shifted from 42 kDa to 110 kDa. The analysis of the dark enzyme, purified to homogeneity, showed that an additional subunit of 60–63 kDa was associated to QORase. The homogeneous native enzyme retained the sensitivity to calcium. Preincubation with trypsin had no effect on sensitivity to Ca2+ while a chaotropic agent mimicked the action of Ca2+. It is concluded that light—dark transitions provoke the association of QORase with a putative regulatory subunit which may be a calciprotein.

A Novel Plant Calciprotein as Transient Subunit of Enzymes

It is generally accepted that the conversion of a stimulus into biological/biochemical responses involves a multistep process that includes the variation in the amounts of second messenger(s) and the transduction of the chemical message through sensing molecules (Cohen, 1982). Such a scheme, inferred from animal cell physiology has been shown to be essentially valid for plants. Thus, several lines of evidence have established that calcium controls different biochemical/biological processes which are being discussed during this workshop; especially calcium is implicitly recognized as the main second messenger in plants.

Putative L-Type Calcium Channels in Plants: Biochemical Properties and Subcellular Localisation

It is generally recognised that many essential cellular responses are regulated by the cytoplasmic free calcium ion concentration (Poovaiah and Reddy, 1987). Changes in [Ca] levels are detected by proteins and enzymes whose activities are modified in response to altered calcium levels (Hepler and Wayne, 1985: Ranjeva and Boudet, 1987); this results in the control of metabolism, gene expression and integrated functions such as exocytosis (Braam and Davis, 1990; Steer, 1988). For these reasons, calcium is considered as an important second messenger in eukaryotic organisms.

The In Vitro Reversible Association of the Regulatory and Catalytic Subunits of Quinate: NAD + Oxidoreductase

Quinate: NAD+ oxidoreductase (QORase) occurs as a heterodimeric protein in dark-grown cell suspension cultures of carrot. The enzyme contains a catalytic (42 kDa) and a regulatory (65 kD) subunit that behaves as a calciprotein and renders QORase dependent upon calcium ions. Upon illumination, the enzyme dissociates and becomes independent upon calcium.