Determination of epidermal transpiration in four cultivars of Nicotiana tabacum L. using epidermal strips in a quasi-steady state system.

A quasi-steady state method is presented for quantifying epidermal transpiration of epidermal strips where simple relations between transmembrane fluxes and parameters of diffusibility of penetrating compounds hold. Contrary to most permeability studies, we did not use astomatous, enzymatically isolated, or dried cuticular membranes, because these procedures are largely responsible for the problems cited in the literature. Instead, we used freshly harvested stomatous epidermal strips, thus avoiding the sorption of lipids by the cuticular membranes during enzymatic isolation. Our approach allowed estimation of amounts and composition of intracuticular soluble lipids. Diffusion coefficients (D-values) were calculated with smaller associated standard deviations and an order of magnitude lower than previously reported; the fresh material sorption of the diffusing compound by the membrane and hydration of the cuticular pores was greatly reduced. In the present study the hold-up time (te) ranged from 66.2+/-0.3 to 110.3+/-0.9 sec. Furthermore, 0.1 microm thick membranes were used, contrary to previous studies of water permeability that used cuticles more than 2 microm thick. Because a small but constant flow of penetrant could be detected during the first half of the steady flow to te, small holes probably did not influence the reported permeability. Permeability coefficients (Pd) in the order of 0.65 x 10(-9) ms(-1) were calculated. Pd values in the order of 5.68 x 10(-3) ms(-1) were calculated when incomplete stomatal closure occurred, while when areas of mass flow were detected, Pd values in the order of 1.26 x 10(-2) ms(-1) were calculated. The degree of contamination of the epidermal strips by cellular debris was quantified and expressed as the total chlorphyll content per exposed surface area of the epidermal strip, and an average of 8.7% contamination was observed compared to the total leaf chlorophyll content. Leakage from the system was calculated to be approximately 0.18 x 10(-10) ms(-1), which represents an average 2.7% experimental variability. These results are discussed in terms of the limitations associated with using composite membranes that are stomatous and have trichomes, for possible application in drought tolerance selection.