Regulation of cellular Ca2+ is an essential cell function that is accomplished by a complex of processes collectively called the Ca2+ homeostat. This review discusses some of the progress that has been made in identifying the compo­ nents of the Ca2+ homeostat in plants-pumps, secondary transporters, and ion channels-as well as progress in characterizin g the operation of the homeostat

Calcium Regulation in Plant Cells and its Role in Signaling

Today no one questions the assertion that Ca2+ is a crucial regulator of growth and development in plants. The myriad processes in which this ion participates is large and growing and involves nearly all aspects of plant development (recent reviews in [Harper et al., 2004][1]; [Hetherington and

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Calcium: A Central Regulator of Plant Growth and Development

Regulation, recognition and cell signaling involve the coordinated actions of many players. To achieve this coordination, each participant must have a valid identification (ID) that is easily recognized by the others. For proteins, these IDs are often within intrinsically disordered (also ID) regions. The functions of a set of well-characterized ID regions from a diversity of proteins are presented herein to support this view. These examples include both more recently described signaling proteins, such as p53, alpha-synuclein, HMGA, the Rieske protein, estrogen receptor alpha, chaperones, GCN4, Arf, Hdm2, FlgM, measles virus nucleoprotein, RNase E, glycogen synthase kinase 3beta, p21(Waf1/Cip1/Sdi1), caldesmon, calmodulin, BRCA1 and several other intriguing proteins, as well as historical prototypes for signaling, regulation, control and molecular recognition, such as the lac repressor, the voltage gated potassium channel, RNA polymerase and the S15 peptide associating with the RNA polymerase S-protein. The frequent occurrence and the common use of ID regions in important protein functions raise the possibility that the relationship between amino acid sequence, disordered ensemble and function might be the dominant paradigm for the molecular recognition that serves as the basis for signaling and regulation by protein molecules.

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling.

VIVO 273 EFFECTS OF FC IN VITRO: MODEL OF MECHANISM OF ACTION 275 FC AND THE INTERDEPENDENCE OF TRANSPORT PROCESSES 277 METABOLIC CHANGES ASSOCIATED WITH FC-INDUCED H+ EXTRUSION .... 279 MECHANISM OF FC ACTION ON CELL ENLARGEMENT 280 FC AND THE REGULATION OF CELL TURGOR: THE EFFECT ON STOMATAL OPENING 282 FC, CELL MULTIPLICATION, AND THE BREAKING OF SEED DORMANCY ... .. .. 282 FC AS A TOOL TO UNDERSTAND HORMONE ACTION 283 CONCLUSIONS 284

Fusicoccin: A Tool in Plant Physiology

Most transport proteins in plant cells are energized by electrochemical gradients of protons across the plasma membrane. The formation of these gradients is due to the action of plasma membrane H+ pumps fuelled by ATP. The plasma membrane H+-ATPases share a membrane topography and general mechanism of action with other P-type ATPases, but differ in regulatory properties. Recent advances in the field include the identification of the complete H+-ATPase gene family in Arabidopsis, analysis of H+-ATPase function by the methods of reverse genetics, an improved understanding of the posttranslational regulation of pump activity by 14-3-3 proteins, novel insights into the H+ transport mechanism, and progress in structural biology. Furthermore, the elucidation of the three-dimensional structure of a related Ca2+ pump has implications for understanding of structure-function relationships for the plant plasma membrane H+-ATPase.

PLANT PLASMA MEMBRANE H+-ATPases: Powerhouses for Nutrient Uptake.

Evidence for the coupling of proton extrusion to K+ uptake in pea internode segments treated in fusicoccin or auxin

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca(2+) upon addition of MgATP. MgATP-dependent Ca(2+) uptake co-migrates with the plasma membrane H(+)-ATPase on a sucrose gradient. Ca(2+) uptake is insensitive to oligomycin, inhibited by vanadate (IC(50) 40 micromolar) and erythrosin B (IC(50) 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca(2+) uptake is insensitive to protonophores. These results indicate that Ca(2+) transport in these microsomal vesicles is catalyzed by a Mg(2+)-dependent ATPase localized on the plasma membrane. Ca(2+) strongly reduces DeltapH generation by the plasma membrane H(+)-ATPase and increases MgATP-dependent membrane potential difference (Deltapsi) generation. These effects of Ca(2+) on DeltapH and Deltapsi generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca(2+) uptake into plasma membrane vesicles. The Ca(2+)-induced increase of Deltapsi is collapsed by permeant anions, which do not affect Ca(2+)-induced decrease of DeltapH generation by the plasma membrane H(+)-ATPase. The rate of decay of MgATP-dependent DeltapH, upon inhibition of the plasma membrane H(+)-ATPase, is accelerated by MgATP-dependent Ca(2+) uptake, indicating that the decrease of DeltapH generation induced by Ca(2+) reflects the efflux of H(+) coupled to Ca(2+) uptake into plasma membrane vesicles. It is therefore proposed that Ca(2+) transport at the plasma membrane is mediated by a Mg(2+)-dependent ATPase which catalyzes a nH(+)/Ca(2+) exchange.

The Ca2+-Transport ATPase of Plant Plasma Membrane Catalyzes a nH+/Ca2+ Exchange

The effect of fusicoccin on Mg:ATP-dependent H+-pumping in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings was investigated by measuring the initial rate of decrease in the absorbance of the ΔpH probe acridine orange. Fusicoccin stimulated Mg:ATP-dependent H+-pumping when the pH of the assay medium was in the range 7.0 to 7.6 while no effect of fusicoccin was detected between pH 6.6 and pH 6.0. Both basal and fusicoccin-stimulated H+-pumping were completely inhibited by vanadate and almost unaffected by nitrate. Fusicoccin did not change membrane permeability to protons and fusicoccin-induced stimulation of Mg:ATP-dependent H+-pumping was not affected by changes in the buffer capacity of the incubation medium. Deacetylfusicoccin stimulated H+-pumping as much as fusicoccin, while the physiologically inactive derivative 8-oxo-9-epideacetylfusicoccin did not. Stimulation of H+-pumping was saturated by 100 nanomolar fusicoccin. These data indicate that fusicoccin activates the plasma membrane H+-ATPase by acting at the membrane level independently of the involvement of other cell components. The percent stimulation by fusicoccin was the same at all ATP concentrations tested (0.5-5.0 millimolar), thus suggesting that with fusicoccin there is an increase in Vmax of the plasma membrane H+-ATPase rather than a decrease in its apparent Km for Mg:ATP.

H+-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

Correlation between proton extrusion and stimulation of cell enlargement effects of fusicoccin and of cytokinins on leaf fragments and isolated cotyledons

Fusicoccin, a toxin stimulating cell enlargement and inducing proton extrusion in various plant tissues, has been shown to replace kinetin, gibberellic acid and red light in breaking seed dormancy. It also removes the inhibitory effect of abscisic acid.



The present data also show that the stimulating effect of fucicoccin on embryo growth of decoated radish (Raphanus sativus L.) and maize (Zea mays) seeds and on the development of maize embryos is accompanied by an early, significant acidification of the medium. Acidification of the medium is also observed when fusicoccin reverses the abscisic acid-induced inhibition of germination. These results support the hypothesis that the mode of action of fusicoccin in promoting germination involves, as in stimulation of cell enlargement, the activation at the cell membrane level of proton extrusion processes.



The physiological significance of fusicoccin-induced release of protons at the onset of germination is discussed in comparison with the results concerning the mechanism of action of fusicoccin on cell enlargement in other plant materials.

Franca Rasi-Caldogno

Papers

Evidence for the coupling of proton extrusion to K+ uptake in pea internode segments treated in fusicoccin or auxin

The Ca2+-Transport ATPase of Plant Plasma Membrane Catalyzes a nH+/Ca2+ Exchange

H+-pumping driven by the plasma membrane ATPase in membrane vesicles from radish: stimulation by fusicoccin

Correlation between proton extrusion and stimulation of cell enlargement effects of fusicoccin and of cytokinins on leaf fragments and isolated cotyledons

Acidification of the Medium Associated with Normal and Fusicoccin-Induced Seed Germination