fusicoccin and malic-acid

White lupin (Lupinus albus L.) is able to grow on soils with sparingly available phosphate (P) by producing specialized structures called cluster roots. To mobilize sparingly soluble P forms in soils, cluster roots release substantial amounts of carboxylates and concomitantly acidify the rhizosphere. The relationship between acidification and carboxylate exudation is still largely unknown. In the present work, we studied the linkage between organic acids (malate and citrate) and proton exudations in cluster roots of P-deficient white lupin. After the illumination started, citrate exudation increased transiently and reached a maximum after 5 h. This effect was accompanied by a strong acidification of the external medium and alkalinization of the cytosol, as evidenced by in vivo nuclear magnetic resonance (NMR) analysis. Fusicoccin, an activator of the plasma membrane (PM) H+-ATPase, stimulated citrate exudation, whereas vanadate, an inhibitor of the H+-ATPase, reduced citrate exudation. The burst of citrate exudation was associated with an increase in expression of the LHA1 PM H+-ATPase gene, an increased amount of H+-ATPase protein, a shift in pH optimum of the enzyme and post-translational modification of an H+-ATPase protein involving binding of activating 14-3-3 protein. Taken together, our results indicate a close link in cluster roots of P-deficient white lupin between the burst of citrate exudation and PM H+-ATPase-catalysed proton efflux.

Topics: Citric Acid; Drug Combinations; Gene Expression Profiling; Gene Expression Regulation, Plant; Glycosides; Lupinus; Malates; Oils; Phenols; Phosphates; Plant Proteins; Plant Roots; Proton-Translocating ATPases; Vanadates

White lupin (Lupinus albus L.) is able to acclimate to phosphorus deficiency by forming proteoid roots that release a large amount of citric acid, resulting in the mobilization of sparingly soluble soil phosphate in the rhizosphere. The mechanisms responsible for the release of organic acids have not been fully elucidated. In this study, we focused on the link between citrate and malate release and the release of H+ and other inorganic ions by proteoid roots of white lupin. The release of citrate was closely correlated with the release of H+, K+, Na+ and Mg2+, but not with that of Ca2+. The stoichiometric relationships between citrate release and the release of H+, K+, Na+ and Mg2+ were 1 : 1.3, 1 : 2.1, 1 : 1.5 and 1 : 0.47, respectively. Similar correlations were found between exudation of malate and cations. During 30 min incubation, fusicoccin addition stimulated H+ and malate release, but not citrate release. A concomitant stimulation of H+, malate and citrate release was measured after 60 min incubation. Vanadate inhibited the release of H+ and malate, but not that of citrate. Anthracene-9-carboxylic acid, an anion channel blocker, caused a concomitant decrease in release of citrate, malate and H+. We conclude that for export of citrate across the plasma membrane of proteoid root cells, H+ release is not strictly related to citrate release. Other cations such as K+ and Na+ can also serve as counterions for citrate release. In contrast, malate release shows a strong H+ release dependency.

Topics: Anions; Anthracenes; Biological Transport, Active; Cations; Citric Acid; Glycosides; Hydrogen-Ion Concentration; Kinetics; Lupinus; Malates; Oxidation-Reduction; Phosphorus; Plant Roots; Protons; Vanadates

Leaves regulate gas exchange through control of stomata in the epidermis. Stomatal aperture increases when the flanking guard cells accumulate K+ or other osmolytes. K+ accumulation is stoichiometric with H+ extrusion, which is compensated for by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31)-mediated malate synthesis. Plant PEPCs are regulated allosterically and by phosphorylation. Aspects of the signal-transduction network that control the PEPC phosphorylation state in guard cells are reported here. Guard cells were preloaded with [32P]orthophosphate (32Pi); then stomata were incubated with fusicoccin (FC), which activates the guard-cell plasma membrane H+-ATPase. [32P]PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. In -FC controls, stomatal size, guard-cell malate, and [32P]PEPC were low; maximum values for these parameters were observed in the presence of FC after a 90-min incubation and persisted for an additional 90 min. This high steady-state phosphorylation status resulted from continuous phosphorylation and dephosphorylation, even after the malate-accumulation phase. PEPC phosphorylation was diminished by approximately 80% when K+ uptake was associated with Cl- uptake and was essentially abolished when stomatal opening was sucrose--rather than K+--dependent. Finally, alkalinization by NH4+ in the presence of K+ did not cause PEPC phosphorylation (as it does in C4 plants). As discussed, a role for cytoplasmic protons cannot be completely excluded by this result. In summary, activation of the plasma membrane H+-ATPase was essential, but not sufficient, to cause phosphorylation of guard-cell PEPC. Network components downstream of the H+-ATPase influence the phosphorylation state of this PEPC isoform.

Topics: Cytoplasm; Glycosides; Hydrogen-Ion Concentration; Malates; Phosphates; Phosphoenolpyruvate Carboxylase; Phosphorylation; Plant Epidermis; Plant Leaves; Potassium; Proton-Translocating ATPases; Signal Transduction; Sodium Chloride; Sucrose; Vicia faba

Plants regulate water loss and CO2 gain by modulating the aperture sizes of stomata that penetrate the epidermis. Aperture size itself is increased by osmolyte accumulation and consequent turgor increase in the pair of guard cells that flank each stoma. Guard cell phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), which catalyzes the regulated step leading to malate synthesis, is crucial for charge and pH maintenance during osmolyte accumulation. Regulation of this cytosolic enzyme by effectors is well documented, but additional regulation by posttranslational modification is predicted by the alteration of PEPC kinetics during stomatal opening (FEBS Lett. 352, 45-48). In this study, we have investigated whether this alteration is associated with the phosphorylation status of this enzyme. Using sonicated epidermal peels ("isolated" guard cells) preloaded with 32PO4, we induced stomatal opening and guard cell malate accumulation by incubation with 5 microM fusicoccin (FC). In corroboratory experiments, guard cells were incubated with the FC antagonist, 10 microM abscisic acid (ABA). The phosphorylation status of PEPC was assessed by immunoprecipitation, electrophoresis, immunoblotting, and autoradiography. PEPC was phosphorylated when stomata were stimulated to open, and phosphorylation was lessened by incubation with ABA. Thus, we conclude that regulation of guard cell PEPC in vivo is multifaceted; the effects of regulatory metabolites and the activation status of the enzyme are integrated to control malate synthesis. These results, together with the coincident alteration in the kinetics of the enzyme (FEBS Lett. 352, 45-48), constitute the first unequivocal demonstration of regulatory posttranslational modification of a guard cell protein that is specifically implicated in stomatal movements.

Topics: Abscisic Acid; Enzyme Activation; Fabaceae; Glycosides; Kinetics; Malates; Phosphoenolpyruvate Carboxylase; Phosphorylation; Plant Leaves; Plants, Medicinal