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Highlights

A method to simultaneously determine membrane water permeability (L_p) and nonosmotic volume (V_b) is proposed.

Abstract. Membrane water permeability is of great importance to better understand the mechanism of water loss; however, its correct understanding and measurement is still an issue. We proposed and tested the efficacy of a numerical analytic approach for the simultaneous determination of the hydraulic conductivity (Lp) and relative osmotically inactive volume (b) of protoplasts isolated from spinach mesophyll tissue. The new approach revealed that the osmotically inactive volume was neither proportional nor constant to the protoplast size and differed for each protoplast. Usage of individually determined b for the computation provided the best fitting performance for determining the Lp, whereas collective b decreased the accuracy of regression and resulted in the underestimation of Lp. Our approach also has the advantage of a shorter period of hyperosmotic challenge, and as such, is suitable for observing fragile protoplasts. We found a gradual reduction in b and a linear increase in Lp during four days of storage at 20°C, likely due to degradation of internal components and dysfunction of the cell membrane, respectively. We also discuss the potential need to consider tissue specificity and possible damage by protoplast isolation to conduct shelf-life research of leafy vegetables.