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

Summary

Measuring the osmotic water permeability coefficient (P_f) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). P_f determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

Abstract

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (P_f) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the ‘PfFit’), to yield P_f.

Introduction

Water uptake and flow across cellular membranes is a fundamental requirement for plant existence at both the cellular and the whole plant levels. At the cellular level, aquaporins (AQPs) play a key role in the regulation of the osmotic water permeability coefficient (P_f) of the cell membrane^1-3.

To date, several methods have been employed in measuring the endogenous P_f of protoplast from different plant organs (i.e. roots, mesophyll, endodermis, etc., reviewed by Chaumont et al. ⁴). One of the approaches to measure P_f is to expose the protoplasts to an osmotic challenge and to monitor the initial rate of its volume change (i.e., the slope of the early linear phase of the volume change). 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 flow⁵ and the second one of transferring the protoplast from one solution to another using a micropipette⁶. 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.

The method described here minimizes the disturbance to the cells, involves no micromanipulation and permits derivation of P_f when the bath perfusion is not instantaneous.

After the enzymatic digestion, the protoplasts, submerged in an isotonic solution, are immobilized on the coverslip-glass bottom of a Plexiglass (aka Lucite or perspex) chamber by charge interaction. Then, during a constant bath perfusion, the isotonic solution is flushed away by a hypotonic solution generating a hypoosmotic challenge to the protoplasts. The swelling of the protoplast is video recorded and then, by combining the information about the time course of the bath perfusion and the time course of the cell swelling, the P_f is determined by image processing and curve fitting procedures.

The advantages of this method are that the experiment is very efficient, i.e. it is possible to monitor a few cells simultaneously in a single assay, and that it does not require special equipment or particular micromanipulation skills. Several applications for this method are possible. For example, determination of the native P_f of a variety of cells from different tissues and plants, such as mesophyll and bundle sheath cells from Arabidopsis leaf⁷, maize leaf mesophyll or root cortex cells^8-10 or suspension cultured cells^11,12. In addition, it is possible to determine P_f of spherical animal cells such as oocyte cells¹¹. Another example involves examination of AQP activity by transient expression of their gene in the protoplasts (or any other genes which may affect them; e.g., genes of kinases) and determination of their contribution to P_f; for example, expression of tomato AQP SlTIP2;2 in Arabidopsis mesophyll protoplasts by PEG transformation and determination the SlTIP2;2-related P_f¹³. Finally, examination of the effect on P_f of different molecules/substances (drugs, hormones, etc.) added to the solutions can also be examined, for example of the AQP blocker HgCl₂⁷.

The following protocol describes the isolation of protoplasts of Arabidopsis mesophyll cells and determination of their P_f.

Protocol

1. Preparation of Solutions

1. Prepare isotonic (600 mOsm) and hypotonic (500 mOsm) solutions containing 10 mM KCl, 1 mM CaCl₂, and 8 M 2-(N-morpholine)-ethanesulphonic acid (MES), pH 5.7 and adjust osmolarity with the appropriate amounts of D-sorbitol: 540 mM for the isotonic and 440 mM for the hypotonic solution. Verify the osmolarity of the solution (within 3 % of the target value) using an osmometer.
2. Prepare a dry stock of ‘enzymatic mix’ containing the following enzymes: 0.55 g cellulase, 0.1 g pectolyase, 0.33 g polyvinylpyrrolidone K 30, 0.33 g BSA (see Table 1 below), mix the dry powder by vortex, make 5.7 mg aliquots and store at -20 °C.

2. Isolation of Arabidopsis Mesophyll Protoplasts

1. Prepare a Petri dish (10 cm) with about 6 drops (approx. 30 µl each) of isotonic solution.
2. Peel the abaxial (lower) Arabidopsis leaf epidermis, cut the peeled leaf into squares of about 4 x 4 mm², then place the squares on the isotonic solution drops with the exposed abaxial side down, touching the solution.
3. Dissolve 5.7 mg of the enzyme mix in 165 µl isotonic solution (3.3 % w/w) in a 1.5 ml tube, mix gently by pipetting for a minute or so until dissolved, and place several similar drops of the enzymatic solution in the same Petri dish.
4. Transfer the leaf pieces onto the enzymatic solution drops, close the dish sealing the lid with one round of parafilm and incubate for 20 min, floating the dish in a water bath set to 28 °C.
5. Add several more drops of the isotonic solution to the dish (2 drops per each enzyme solution drop). Transfer each leaf piece to a new isotonic solution drop, then, sequentially, to a second drop (to wash the enzymatic solution away). Lift the piece by its edge using forceps, shake it in the second drop (like a tea bag) to release the protoplasts. Collect the drops with the protoplasts (using a clipped off 100 µl pipette tip) into a 1.5 ml tube.

3. The Hypotonic Challenge Assay: Arabidopsis Mesophyll Cell Swelling

1. Prepare the perfusion system (Figure 1A) by filling one column with the isotonic solution and another column with the hypotonic solution. Open the valve, let some solution flow (first the hypotonic, then the isotonic) to fill the tubing all the way down to the inlet manifold (Figure 1B). Ensure there are no trapped air bubbles, and then close the valve.
2. Seal a coverslip, using silicone grease (Table 1), to make a bottom for the chamber within the plexiglass slide (Figure 1B; see also the schematics of the chamber in Figure 1C). To make the chamber bottom (the upward facing exposed surface of the coverslip within the grease ring) “sticky” for protoplasts, coat it with positive-charge-bearing protamine sulphate (1 % in water; Table 1) or poly-L-Lysine (0.1 % in water; Table 1). Spread this ‘glue’ over the coverslip using a pipette tip, wait for 1 - 2 min, rinse 3 - 4 times with the isotonic solution and shake away the remaining solution.
3. Fill the chamber up with the isotonic solution. Then, add a drop of protoplasts containing solution to the chamber, using a clipped off pipette tip and wait 3 - 4 min for the protoplasts to settle. Cover the chamber with a transparent cover (Figures 1D, 1E) touching the solution surface (avoid trapping air bubbles beneath).
4. Place the slide (gently!) on an inverted microscope table, connect it to the perfusion system and the pump (guarding against air bubbles in the tubing!) and turn on the isotonic solution flow for constant perfusion at 1 ml/min (faster rates can be used, up to 4 ml/min).
5. For recording volume changes, an inverted microscope is used, with a 20X objective and with a CCD video camera connected to a PC computer. Use the ‘CMU 1394 Camera Driver’ plugin of the ImageJ software (see the Table of Specific Materials for the download addresses of these two software pieces) to record a 60 sec video movie of selected immobile protoplasts (presumably, those stuck to the bottom) at a rate of 1 image /sec (1 Hz). Start the recording with a 15 sec wash of the isotonic solution (this constitutes the baseline), switch to the hypotonic solution for 45 sec (to complete a total 60 sec from the start of perfusion). Save the movie in TIF format. NOTE: Choose a view field with as many cells as possible, fulfilling the following criteria: spherical in shape and with a well-focused cell contour at their largest perimeter (Figure 2A).

4. Analysis of the Cell Volume Change Using ImageJ

NOTE: To analyze the series of images of a swelling cell, use the ‘Image Explorer’ and ‘Protoplast Analyzer’ plugins in the ImageJ software (written by Xavier Draye)¹⁴. Starting with the chosen protoplasts at their first time point, the ‘Protoplast Analyzer’ plugin will detect automatically the protoplasts edges (contours) and calculate the time course of their areas during the experiment (the plugins are available with the PfFit analysis program, below).

1. Start ImageJ. To open the movie, click ‘File’ on the ImageJ panel, then, consecutively on the dropdown menus as they unfold: ‘Import’ then ‘Image Explorer’. Highlight the chosen movie, then right-click on it, then left-click on ‘Protoplast Analyzer’. Browse through the movie (using a slider at the protoplast image bottom) to identify protoplasts that remain largely immobile during the experiment – these will be analyzed. Back on the first image, using the mouse, draw circles (picked from the ImageJ drawing tools) around the selected protoplasts (Figure 2B), then click ‘OK’ in the table of ‘Detection parameters’ that appeared.
2. To launch the protoplast detection algorithm, click ‘Local’ on the protoplast image top panel, then ‘Process’ in the dropdown menu. Examine the green circles around the selected protoplasts (Figure 2C) throughout the movie. Save the ‘Result’ in an Excel file. Quit ImageJ. NOTE: In case a red dot appears (to indicate a bad contour fit – usually due to a poor image contrast), rerun with different parameters.
3. To separate the lines belonging to each cell (which – if two or more cells were analyzed simultaneously – will be intertwined, because the analysis is done frame by frame), in Excel, sort the saved data by the cell number column (‘object’).
4. To determine the pixel-to-µm conversion factor for obtaining the real value of P_f, snap an image of a micrometer ruler via the same 20X microscope objective. Drag a line (picked from the ImageJ drawing tools) along the ruler image and read the pixel number equivalent to the ruler length at the bottom of the ImageJ main panel. Convert the arbitrary pixel area values in the Excel file into µm². Save the areas time course (for each cell separately) as a text file (two columns of numbers only). NOTE: This will be an input to the volume-fitting ‘PfFit’ program.

5. Modeling the Rate of Osmolarity Change in the Experimental Chamber Using ImageJ and the Matlab Program P_fFit

1. Add 2 mg xylene cyanol (Table 1, below) to 100 ml of the isotonic solution (to produce the ‘Indicator Dye’).
2. Prepare the perfusion system (as in 3.1) with the Indicator Dye and the non dyed hypotonic solution.
3. Seal a cover slip using silicon grease to the bottom of the Plexiglass chamber, then gently fill the chamber with the Indicator Dye, cover it with a cover slip (as with the protoplasts before) and place it on the microscope stage.
4. Connect the chamber to the perfusion system and the pump, and turn on the Indicator Dye flow for a constant perfusion at l ml/min.
5. Record a 60 sec movie at the rate of 1 Hz. Start the recording with 15 sec of Indicator Dye, switch to the hypotonic solution for 45 sec. Stop filming. Flush with the Indicator Dye (at least for 30 sec), then start a new movie. Repeat about 5 - 6 times and save all the movies
6. Use the ImageJ software to analyze the video images of the Indicator Dye transmittance to obtain an averaged time course of the changing transmittance.
   1. Start ImageJ, click ‘File’, then, ‘Open’, and browse for the movie. For each movie, draw a 10 pixel wide vertical rectangle anywhere on the 1^st image of the movie. Click ‘Image’ on the ImageJ main panel, then click ‘Crop’ in the dropdown menu.
   2. To align the 60 frames (of the 60 sec movie) in one row, click again ‘Image’, then click consecutively in the dropdown menus as they unfold: ‘Stacks’ and ‘Make Montage’ (columns 60, rows 1). Draw a 1 pixel high horizontal rectangle anywhere along the whole row of images and click ‘Analyze’ in the ImageJ main panel, then click ‘plot profile’ in the dropdown menu. NOTE: A ‘Plot of Montage’ window will appear (not shown), and a list of transmittance data can be opened from its menu. Each image of the movie is represented in this list by 10 transmittance values originating in its 10 pixel wide rectangle and consequently the “time base” (the image sequential number) is 10 times longer.
   3. Copy the lists of the transmittance data (one list per movie) to an Excel file. Average the transmittance time courses obtained from the several movies of the Indicator Dye flushes. Generate a real time base by multiplying the image sequential number by 0.1. Save the averaged time course (two columns) to a text file. NOTE: Before averaging, if desired, plot the individual time courses, to reject any irregularities. Ensure that the movie includes at least 5 final sec of steady-state transmittance of the Indicator Dye.
7. Start the Matlab fitting program P_fFit (the ‘Indicator Fit' panel, Figure 3) to compute the various parameters of the osmolarity time course. NOTE: based on the known initial and final concentrations of the solution in the bath, the time course of the changing osmotic concentration of the solution is calculated from the concentration time course (calculated, in turn, from the Indicator Dye transmittance), assuming it follows the same dynamics as the dye concentration. P_fFit is a program available for use free of charge. The ‘ PfFit_Installer_web.exe’ can be downloaded from: P_fFit User Guide’ with detailed explanations and definitions is accessible via Jove as a Supplemental file, which helps to familiarize the user with the P_fFit program.
8. In the ‘Indicator Fit' panel, import the data of the mean time course of the Indicator Dye transmittance (‘Indicator data file’, Figure 3A) and insert manually the current experiment parameters and the initial guesses of the parameters ‘width’ and ‘t_half’ describing the time course of the Indicator Dye concentration (Figure 3B. Click ‘Run’ to view the plots of the time courses of the Indicator Dye concentration (real data and fit, Figure 4A), and of the modeled (calculated) bath osmolarity (Figure 4B). NOTE: a good fit to the data is essential(a recommendation: start with the values shown in Figure 3).

6. Determining the P_f using the Matlab Fitting Program P_fFit

NOTE: In addition to the basic assumptions with regard to the behavior of a protoplast as a true and perfect osmometer¹¹, 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 values¹¹. P_fFit 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 change¹¹, calculated, in turn, from the imported series of cell-contour areas (see also the Supplemental ‘P_fFit User Guide’).

1. Switch to the ‘Volume Fit’ panel (Figure 5). Choose for import the areas data file (the text file with the time course of the ‘areas’ of the analyzed protoplasts, Figure 5A). Choose ‘Last Indicator Fitting’ as the parameter source (Figure 5B; see the ‘P_fFit User Guide’ for alternatives). NOTE: These parameters (Figure 5D) are then used to regenerate the osmoticum change in the bath for the volumes fitting procedure.
2. In the ‘Volume Fit’ panel (Figure 5C), initialize (fill in the initial guesses for) the P_f parameters: P_f, Slope_Pf and Delay (a recommendation: start with 1, 1, and 30, respectively), Chose the model ‘Class’ (a recommendation: start with II and mark ‘checks’ for all three parameters to be fitted). Click ‘Run’, then eyeball the interim figure (Figure 5E) and adjust the Delay parameter and the length of the record, if needed.
3. Examine the results graph (Figure 6) to evaluate the fit quality and record the fit error. Change the initializing parameters a few fold each, and re-‘Run’. NOTE: Don’t be discouraged when the program gets stuck – just restart the program!
4. Repeat this procedure several times, starting with different combinations of initialization parameters, aiming for the lowest value of the fit error.
5. Copy the list of the fit results directly from the screen, or find them in the PfFit-generated ‘_FIT_Vol_Results.txt‘ file.

Results

In order to determine the P_f and compare the activity of different AQPs, mesophyll protoplasts from Arabidopsis leaf are used. These protoplasts were found to have low basal (background) P_f levels ⁷ and can serve as a functional-expression system to enable reproducible P_f measurements.

Protoplasts from a mature leaf from a 6 week old Arabidopsis plant were isolated and three gene constructs with AQP genes from Arabidopsis (AtPIP2;1) and maiz...

Discussion

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 oocytes¹¹. This method is based on measuring the P_f in response to an osmotic challenge to the cell. In contrast to the other methods based on this approach, however, the change of solutions, i.e., of the osmolarity, is not instantaneous, but gradual, during a constant bath perfusion, starting with t...

Materials

Name	Company	Catalog Number	Comments
KCl	Chem-Impex International	01247-1	http://www.chemimpex.com Any source, anal. grade
CaCl2	Merck	11718006	http://www.merck.com Any source, anal. grade
2-(N-morpholine)-ethanesulphonic acid (MES)	Sigma	15152002	http://www.sigmaaldrich.com Any source, anal. grade
D-Sorbitol	Sigma	18032003	http://www.sigmaaldrich.com Any source, anal. grade
Cellulase	Worthington, Lakewood, NJ, USA	LS002603	http://www.worthingtonbiochem.com
Pectolyase	Karlan,               Phonix, AZ, USA	8006	http://www.karlan.com
Polyvinyl-pyrrolidone K 30 (PVP)	Sigma	81420	http://www.sigmaaldrich.com
Bovine Serum Albumin (BSA)	Sigma	A9418-5G	http://www.sigmaaldrich.com
Protamine sulphate	Sigma	P4380	http://www.sigmaaldrich.com
Poly-L-Lysine	Sigma	P8920	http://www.sigmaaldrich.com
Xylene cyanol	Sigma	X4126	http://www.sigmaaldrich.com
Silicone vacuum grease heavy	Merck	107921	https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&SEO_ErrorPageOccurred=true&attachments=CoA
Inverted microscope	Nikon	Eclipse TS100/TS100F	http://www.nikoninstruments.com
Peristaltic pump	BIO-RAD	EP-1 Econo Pump	http://www.bio-rad.com
Grayscale digital camera	Scion Corporation	CFW-1308M	http://www.scioncorp.com
CMU 1394 Camera Driver’ plugin for ImageJ	Carnegie Mellon		http://www.cs.cmu.edu/~iwan/1394/download.html Free software
ImageJ	NIH		http://rsb.info.nih.gov/ij/ Free software
Econo Gradient Pump Fittings Kit	BIO-RAD	731-9006	http://www.bio-rad.com
Connectors, manifold	DirectMed		http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S
Burette infusion sets (columns)	Welford	IF-BR-001	http://www.welfordmedical.com/content.php?id=61
Tubing	TYGON	R-3603	http://www.usplastic.com
Plexiglass slide etc.	Perspectiv		http://www.perspectiv.co.il/index-en.html Our slide was custom-made, it does not appear on the web site but a copy can be remade to order as 'a copy of the slide already made for M. Moshelion'.
3M packaging Scotch tape 1'', clear	Viking Industrial, UK	VKMONO25	http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8 any clear adhesive tape (sellotape, etc.) is likely to be OK