On the Mechanism of Hyperacidification in Lemon

Abstract

Lemon fruit vacuoles acidify their lumens to pH 2.5, 3 pH units lower than typical plant vacuoles. To study the mechanism of hyperacidification, the kinetics of ATPdriven proton pumping by tonoplast vesicles from lemon fruits and epicotyls were compared. Fruit vacuolar membranes were less permeable to protons than epicotyl membranes. H ؉ pumping by epicotyl membranes was chloride-dependent, stimulated by sulfate, and inhibited by the classical vacuolar ATPase (V-ATPase) inhibitors nitrate, bafilomycin, N-ethylmaleimide, and N,N-dicyclohexylcarbodiimide. In addition, the epicotyl H ؉ pumping activity was inactivated by oxidation at room temperature, and oxidation was reversed by dithiothreitol. Cold inactivation of the epicotyl V-ATPase by nitrate ( 100 mM) was correlated with the release of V 1 complexes from the membrane. In contrast, H ؉ pumping by the fruit tonoplast-enriched membranes was chloride-independent, largely insensitive to the VATPase inhibitors, and resistant to oxidation. Unlike the epicotyl H ؉ -ATPase, the fruit H In animal cells, different compartments of the endocytotic pathway have characteristic lumenal pHs, ranging from pH 6.5 in the coated vesicles to pH 5.0 in the lysosomes, suggesting that the lumenal pH of each organelle is tightly regulated (6). A number of observations suggest that the pH of plant vacuoles is also regulated. In plants with crassulacean acid metabolism for example, the vacuolar pH of the leaves varies diurnally, from pH 3 at night to pH 6 in the day (7). In stomatal guard cells, the vacuolar pH is 4.5 in the dark when the stomata are closed, and 6 in the light when the stomata are open (8). During fruit development the vacuolar pH often changes, becoming either more or less acidic as ripening progresses. Such fluctuations indicate that the vacuolar pH is under metabolic and developmental control. However, even in the case of vacuoles with a constant pH the V-ATPase may be continually regulated, inasmuch as the typical steady state ⌬pH across the tonoplast appears to be considerably less than the theoretical maximum. From the H ϩ /ATP stoichiometry of the pump (n), the membrane potential (⌬), the Faraday constant (F), and the ⌬G ATP , the maximum ⌬pH at equilibrium can be calculated according to the equation:Bennett and Spanswick (9) determined an H ϩ /ATP stoichiometry of 2 for the plant V-ATPase, which was confirmed by Guern et al. (10). Schmidt and Briskin (11) extended these studies to the H ϩ -PPase and included estimates of internal buffering capacity. They confirmed an n value of 2 for the V-ATPase and calculated a maximum possible ⌬pH across the tonoplast of 5.0 -5.4 units when the membrane potential is ϩ20 mV. The authors concluded that the V-ATPase normally functions far from equilibrium and is regulated by factors other than energy supply. Mechanisms that have been proposed to regulate the V-ATPase include "slip" (12), cytosolic activators or inhibitors (13-15), chloride (12, 16), cytosolic pH (17), and oxidation/reduction (18,19). As yet, none of these mech...

Gibberellic Acid Induces Vacuolar Acidification in Barley Aleurone.

“…pH v also varies according to cell function. In specialized cells, such as the lemon fruit juice sac, pH v values are as low as 2.2, whereas in epicotyl cells of lemon, the pH of vacuoles is ~5.5 (Muller et al, 1996). The pH v in cells undergoing crassulacean acid metabolism can range from as low as 3.5 at night when organic acids are accumulated to pH 6 during the day when organic acids are metabolized (Liittge, 1987).…”

“…It has been postulated that phosphorylation of the B subunit of the V1 domain of the V-ATPase plays a role in regulating the activity of the enzyme (Martiny-Baron et al, 1992). Other mechanisms proposed to regulate the V-ATPase are cytosolic pH (Davies et al, 1994) and the redox state of the cytosol (Feng and Forgac, 1992;Muller et al, 1996).…”

Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+exchange

“…23,29 The electrogenic H + pumps V-H + -ATPase and V-H + -PPase are major components of the vacuolar membrane of plant cells. 23,26 With the noticeable exception of lemon, where H + -PPase can be ruled out as the primary proton pump, 30 all plant species from which vacuolar membranes were studied exhibit V-H + -PPase activity in addition to V-H + -ATPase activity. The V-H + -ATPase is universally present in the membranes of different internal acidic organelles in eukaryotic cells and has an intricate structure: a peripheral V 1 sector which contains three copies of the A-and B-subunits, responsible for the catalytic activity, and the subunits C-H which form a central stalk linking the V 1 to the hydrophobic membrane-embedded V o sector.…”

Analyses of Expression Patterns of Ethylene Receptor Genes in Apple (Malus domestica Borkh.) Fruits Treated with or without 1-Methylcyclopropene (1-MCP)

“…Since respiration is suppressed by 1-MCP, it has been assumed that suppression of the reduction of acidity in 1-MCPtreated fruit is caused by the suppression of respiration (Defilippi et al, 2004). Some enzymes or proteins that control acidity or organic acid metabolism in ripening fruit have been identified (Etienne et al, 2002a(Etienne et al, , 2002bMüller et al, 1996;Terrier et al, 2001). The activity of some of these enzymes or proteins might be regulated by ethylene, but no correlation has been reported.…”