Osmotic Water Permeability of Isolated Protoplasts. Modifications during Development1

Abstract

A transference chamber was developed to measure the osmotic water permeability coefficient (P os ) in protoplasts 40 to 120 m in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. P os was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 m s ؊1 for the various types of protoplasts tested. The precision for P os was <40%, and within this limit, no asymmetry in the water fluxes was observed. Measurements on protoplasts isolated from 2-to 5-d-old roots revealed a dramatic increase in P os during root development. A shift in P os from 10 to 500 m s ؊1 occurred within less than 48 h. This phenomenon was found in maize (Zea mays), wheat (Triticum aestivum), and rape (Brassica napus) roots. These results show that early developmental processes modify water-transport properties of the plasma membrane, and that the transference chamber is adapted to the study of water-transport mechanisms in native membranes.The balance between water loss and water absorption in plants is one of the oldest physiological problems. It was realized early that osmotic and turgor pressures can determine the dynamics of water movement through the different compartments of the plant or tissue. The rate of water flow across membrane systems was modeled by Dainty (1963) using the nonequilibrium thermodynamic equations of Kedem and Katchalsky (1958). Each membrane can be characterized by a parameter, the hydraulic conductivity (L p ) or the related osmotic coefficient (P os ). In higher plants this parameter has been estimated mostly using the cell pressure-probe technique. In animal cells this parameter can be deduced from the kinetics of cellular volume change, as determined by light scattering or fluorescence quenching (for a general review, see Verkman et al., 1996); such measurements require a large population of cells and an elaborate mechanical and electronic apparatus. Recently, the P os of single cells in epithelial sheets was measured by nonimaging interferometry (Farinas and Verkman, 1996). Light microscopy is the most straightforward method for determining individual cell membrane properties. It is based on the recording of time variations in the cell dimensions, induced by an osmoticum change in the external medium. Measurements of P os by standard light microscopy have been performed on epidermal cells (Url, 1971; Stadelmann and Lee-Stadelmann, 1989) and more recently on mouse oocytes (Gao et al., 1994(Gao et al., , 1996.When individual cells cannot be easily observed in the tissue, it may be of interest to work on isolated protoplasts. In this paper we describe a new method for measuring P os on protoplasts or vacuoles, 40 to 120 m in diameter, isolated from higher-plant cells. The accuracy of the method was examined and the permeabilities obtained were compared with those from other techniques. Measurements were performed on root, hypocotyl, and leaf protoplasts from se...

Does calcium ameliorate the negative effect of NaCl on melon root water transport by regulating aquaporin activity?

“…n et al, 1997). Other recent reports showed that rape (Brassica napus) protoplasts, fixed through suction applied to a glass pipette, had a linear variation for c. 10 s (Ramahaleo et al, 1999). In our experiments the mixing time was c. 3 s, therefore we still were in the linear part of the curve as shown in Fig.…”

“…The fact that a strong decrease in P f was observed in NaCltreated protoplasts compared to controls, and that there was practically no Hg inhibition, led us to consider that aquaporins were inactivated. Ramahaleo et al (1999) suggested that mercury could be damaging the protoplasts in their experiments, since the reduction in P f could be interpreted as the induction of leakage of osmoticum across the membrane. However, in our experiments the fact that Hg was present in the protoplast media for only 2 min and was then removed could explain why damage to the cells was greatly reduced.…”

Water Transport by Aquaporins in the Extant Plant Physcomitrella patens

“…Isolated protoplasts were resuspended in a storing solution (mannitol, 500 mmol/kg; Ca(No 3 ) 2 , 1 mM; bovine serum albumin, 0.05% w/v; MES, 5 mM, buffered with Tris to pH 5.5), and P os was measured at room temperature according to a technique previously described (Ramahaleo et al, 1999); the value of P os was deduced from the initial rate of swelling of the protoplast, when transferred from the stock solution to a solution with lower osmoticum (mannitol, 100-300 mmol/kg; Ca(No 3 ) 2 , 1 mM; bovine serum albumin, 0.05% w/v; MES, 5 mM, buffered with Tris to pH 5.5).…”

“…6), as previously described (Ramahaleo et al, 1999). The data are presented on a logarithmic scale because of the wide range of P os values obtained.…”

Measuring the Osmotic Water Permeability Coefficient (P_f) of Spherical Cells: Isolated Plant Protoplasts as an Example

“…Two different methods were previously described based on this approach, both based on an instantaneous exchange of solutions. The first one consists of immobilizing the protoplast with a suction micropipette and switching the solution flow 5 and the second one of transferring the protoplast from one solution to another using a micropipette 6 . These micropipette suction and micropipette transferring methods, which allow image acquisition at the very start of the fast solution exchange (to capture the early linear phase of volume change), likely involve a physical stress to protoplasts and require specialized equipment and expert micromanipulation.…”