Proton-Metal Cation Exchange in the Cell Wall of Nitella flexilis

Abstract

When protons are exchanging for bivalent cations (Cu2", Zn2+, or Ca2+) on the carboxylic groups of Nitella flexilis cell wall, the values of the respective global equilibrium constants do not change up to a protonation degree of 80%. These values drastically increase at higher proton concentrations and tend to =3.4, which is the intrinsic pK value of the constitutive aD -galacturonic acid monomer. These data suggest that the electric field in the matricial polymer and the cation bridges between pairs of negative sites have disappeared. Plant cells are surrounded by a rigid cell wall usually of a pecto-cellulosic type. Fixed negative charges arise from the ioni-zation at natural pH of the constitutive pectins. They can pro-tonate under the influence of the ionic force, the pH, and the ionic composition of the bathing medium, or as a consequence of a significant proton excretion by the plasmalemma (3). The protonation of the carboxylate groups lowers the amount ofcations fixed in the cell wall, affects the ion exchange selectivity and the ion activity in the cell wall. This can alter the solute transfer from the external medium to the plasmalemma as well as the membrane resting potential (10). On the other hand, the auxin-enhanced excretion of high amounts of pectic acids from the cytoplasm to the cell wall and their ionization could act as a metabolic way of regulating the cytoplasmic pH in plants. Moreover, the neutralization by biva-lent cations of the excreted pectic acids, particularly in the inner part of the cell wall (8, 9), could be a critical factor in the growth mechanisms of the plant cell. In this study, we have measured the uronic acid protonation in the Nitella cell wall in equilibrium with metal cations and verified a global equilibrium equation describing the proton cation exchange. MATERIALS AND METHODS Biological Material. Our experiments have dealt with the giant fresh water alga Nitellaflexilis (L.) Ag. The cell wall of this alga contains an unmethylated pectin in high proportions (1, 2). The plants are grown at =22°C in their origin water renewed by half every 2 weeks. The pH is adjusted at 6.2 every 2 d by adding small amounts of diluted HCl to prevent any calcium carbonate deposit on the cell walls. The lighting is achieved through Phytor tubes, 40 wm 2 at 20 cm from the solution surface, with a 13-h photoperiod. Internodal cells longer than 2.5 cm and free from parasites are selected and their ends cut with a razor blade. Two stainless steel wires are introduced in the cylindrical cell and progressively separated from each other to obtain a flat open cell wall. The cytoplasm and the vacuole are gently removed using one of the wires, and the wall is rinsed in ethyl alcohol and ethyl ether successively. This treatment eliminates any contamination by the plasma membrane. At this stage, the cell walls which do not appear translucent are discarded. The selected walls are then put in 20-ml polyethylene vials containing the treatment solutions and stored in a thermostated room at 25 ± 0.1°C.