Further quantification of the role of internal unstirred layers during the measurement of transport coefficients in giant internodes of Chara by a new stop-flow technique

Abstract

A new stop-flow technique was employed to quantify the impact of internal unstirred layers on the measurement of the solute permeability coefficient (P(s)) across the plasma membrane of internodes of the giant-celled alga Chara corallina using a cell pressure probe. During permeation experiments with rapidly permeating solutes (acetone, 2-propanol, and dimethylformamide), the solute concentration inside the cell was estimated and the external medium was adjusted to stop solute transport across the membrane, after which responses in turgor were measured. This allowed estimation of the solute concentration right at the membrane. Stop-flow experiments were also simulated with a computer. Both the stop-flow experiments and simulations provided quantitative data about internal concentration gradients and the contribution of unstirred layers to overall measured values of P(meas)(s) for the three solutes. The stop-flow experimental results agreed with stop-flow simulations assuming that solutes diffused into a completely stagnant cell interior. The effects of internal unstirred layers on the underestimation of membrane P(s) declined with decreasing P(s). They were no bigger than 37% in the presence of the most rapidly permeating solute, acetone (P(meas)(s) =4.2 x 10(-6) m s(-1)), and 14% for the less rapidly permeating dimethylformamide (P(meas)(s) =1.6x10(-6) m s(-1)). It is concluded that, even in the case of rapidly permeating solutes such as isotopic water and, even when making pessimistic assumptions about the internal mixing of solutes, an upper limit for the underestimation of P(s) due to internal unstirred layers was 37%. The data are discussed in terms of recent theoretical estimates of the effect of internal unstirred layers and in terms of some recent criticism of cell pressure probe measurements of water and solute transport coefficients. The current stop-flow data are in line with earlier estimations of the role of unstirred layers in the literature on cell water relations.