Dynamic Changes in the Osmotic Water Permeability of Protoplast Plasma Membrane  

Abstract

The osmotic water permeability coefficient (P f ) of plasma membrane of maize (Zea mays) Black Mexican Sweet protoplasts changed dynamically during a hypoosmotic challenge, as revealed using a model-based computational approach. The bestfitting model had three free parameters: initial P f , P f rate-of-change (slope Pf ), and a delay, which were hypothesized to reflect changes in the number and/or activity of aquaporins in the plasma membrane. Remarkably, the swelling response was delayed 2 to 11 s after start of the noninstantaneous (but accounted for) bath flush. The P f during the delay was #1 mm s 21 . During the swelling period following the delay, P f changed dynamically: within the first 15 s P f either (1) increased gradually to approximately 8 mm s 21 (in the majority population of low-initial-P f cells) or (2) increased abruptly to 10 to 20 mm s 21 and then decreased gradually to 3 to 6 mm s 21 (in the minority population of high-initial-P f cells). We affirmed the validity of our computational approach by the ability to reproduce previously reported initial P f values (including the absence of delay) in control experiments on Xenopus oocytes expressing the maize aquaporin ZmPIP2;5. Although mercury did not affect the P f in swelling Black Mexican Sweet cells, phloretin, another aquaporin inhibitor, inhibited swelling in a predicted manner, prolonging the delay and slowing P f increase, thereby confirming the hypothesis that P f dynamics, delay included, reflected the varying activity of aquaporins.The regulation of plant aquaporins is becoming a focus of research in an increasing number of laboratories Tyerman et al., 2002). However, the reports on the regulation of the water permeability of plant cells are still relatively few, very likely reflecting the technical difficulties inherent in such measurements.In order to quantify the permeability of a plant cell to water, one of the approaches consists of isolating protoplasts and monitoring the initial rate of change of their volume upon an osmotic challenge. If the osmotic potential of the external solution is changed instantaneously, the osmotic water permeability (termed P f or P os ) can be deduced from the initial rate of volume relaxation (e.g. Zhang et al., 1990;Verkman, 2000). There are at least two problems with this approach: (1) even when an instantaneous change of solution is possible, a systematic error is introduced, causing an underestimate of P f because, already during the initial phase of protoplast swelling, the volume, the surface area, and the internal concentration of solutes do not remain constant; and (2) instantaneous bath perfusion has technical-and physiological-limitations: unlike animal cells, isolated plant cell protoplasts (the terms protoplast and cell will be used here interchangeably) do not stick well to the chamber floor and defeat attempts of rapid (let alone, instantaneous) solution flushes. Very fast external solution exchange has been achieved by immobilizing the protoplast with a suction micropipette (e.g. Ramah...

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

“…Described here is a simple and very efficient procedure for measuring the P f of isolated plant protoplasts, applicable in principle also to other spherical cells, e.g., frog oocytes 11 . This method is based on measuring the P f in response to an osmotic challenge to the cell.…”

“…Using the P f Fit program, two more parameters, beside the P f , are obtained for the description of the protoplast responses to hypotonic challenges: delay, the time between the onset of volume change and the start of bath perfusion, and Slope Pf , the rate of change in P f during the osmotic challenge (described in detail in 11 ).…”

“…NOTE: In addition to the basic assumptions with regard to the behavior of a protoplast as a true and perfect osmometer 11 , the determination of P f rests on the presumption that P f may change with time, that this dynamics of P f underlies the time course of the cell volume change and that three parameters suffice to describe it: P fi (the initial value of P f ), Slope Pf (the rate of the linear change of P f ) and Delay (the period from the start of the bath osmolarity change till the start of the cell volume change). Different models can be tested, including different combinations of these parameters and their values, including null values 11 .…”

“…Different models can be tested, including different combinations of these parameters and their values, including null values 11 . P f Fit searches for the best combination of these parameters to yield -by calculation -the most faithful reproduction of the experimental time course of the cell volume change 11 , calculated, in turn, from the imported series of cellcontour areas (see also the Supplemental 'P f Fit User Guide').…”

Aquaporin structure–function relationships: Water flow through plant living cells

“…Harmer et al found that the mRNA level of an Arabidopsis tonoplast aquaporin, GTIP, cycled in a circadian manner [47]. The plasma membrane aquaporin in the motor cells of Samanea saman also showed a diurnal and a circadian regulation [48,49].…”