Abstract
Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (L(p)(OUT) and L(p)(IN), respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [V(o)], and hydraulic conductivity [L(p)], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). V(o) as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated V(o) values of 1.5 to 4.9 nL. For the same cell, V(o) and L(p) estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher L(p)(OUT) than L(p)(IN) (L(p)(OUT)/L(p)(IN) ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in L(p)(OUT)/L(p)(IN) over a wide range of L(p) values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex.
Highlights
Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (LpOUT and LpIN, respectively)
While the overall processes involved in automated cell pressure probe (ACPP) and cell pressure probe (CPP) operation are very similar (Fig. 1A) and repeatable values of Pcell can be obtained for sequential measurements of cells in a tissue using either system (Fig. 1B), the operator skill required to accomplish and t = 40.0 mN m21 for dimethyl silicone oil, similar in composition to the silicone oil used
Because Poil in the ACPP is automatically controlled by a proportional integral derivative algorithm to maintain either the meniscus position or the pressure at a set value, there is a tradeoff among the speed of response, overshoot or undershoot, and noise in the controlled or controlling parameters (Wong et al, 2009)
Summary
Institut des Sciences de la Vigne et du Vin, 210 Chemin de Leysotte, 33882 Villenave d’Ornon, Bordeaux, France. In the new ACPP system, both the position of the meniscus and oil pressure (Poil) are recorded frequently (typically at 10 Hz), and Poil is controlled with a resolution of 60.002 MPa. We have combined the ACPP with a new technique to reproducibly fabricate microcapillary tips of known hydraulic properties (Wada et al, 2011) in order to correct for Kh and surface tension effects in both PC and PR estimates of the water relations parameters of Tradescantia virginiana epidermal cells and have determined the relation of LpOUT to LpIN in these cells.
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