This work was supported by grants from the Belgian National Fund for Scienti&#64257;c Research (FNRS), the Interuniversity Attraction Poles Programme-Belgian Science Policy and the &#8220;Communaut&#233; fran&#231;aise de Belgique-Actions de Recherches Concert&#233;es&#8221; to FC, from the Israel Science Foundation Jerusalem (ISF) to MM (Grant # 1311/12), and to NM (Grant # 1312/12).

1. Preparation of Solutions
<ol>
	<li>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.</li>
	<li>Prepare a dry stock of &#8216;enzymatic mix&#8217; containing the following enzymes: 0.5...

<ol>
	<li>Tyerman, S. D., Niemietz, C. M., Bramley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+aquaporins:+multifunctional+water+and+solute+channels+with+expanding+roles.">Plant aquaporins: multifunctional water and solute channels with expanding roles.</a> Plant Cell Environ. 25, 173-194 (2002).</li><li>Maurel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+aquaporins:+Novel+functions+and+regulation+properties.">Plant aquaporins: Novel functions and regulation properties.</a> Febs Letters. 581, 2227 (2007).</li><li>Maurel, C., Verdoucq, L., Luu, D. T., Santoni, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+aquaporins:+Membrane+channels+with+multiple+integrated+functions.">Plant aquaporins: Membrane channels with multiple integrated functions.</a> Annual Review of Plant Biology. 59, 595 (2008).</li><li>Chaumont, F., Moshelion, M., Daniels, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+plant+aquaporin+activity.">Regulation of plant aquaporin activity.</a> Biology Of The Cell. 97, 749-764 (2005).</li><li>Ramahaleo, T., Morillon, R., Alexandre, J., Lassalles, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osmotic+water+permeability+of+isolated+protoplasts.+Modifications+during+development.">Osmotic water permeability of isolated protoplasts. Modifications during development.</a> Plant Physiology. 119, 885-896 (1999).</li><li>Suga, S., Murai, M., Kuwagata, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+aquaporin+levels+among+cell+types+of+radish+and+measurement+of+osmotic+water+permeability+of+individual+protoplasts.">Differences in aquaporin levels among cell types of radish and measurement of osmotic water permeability of individual protoplasts.</a> Plant Cell Physiol. 44, 277-286 (2003).</li><li>Shatil-Cohen, A., Attia, Z., Moshelion, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bundle-sheath+cell+regulation+of+xylem-mesophyll+water+transport+via+aquaporins+under+drought+stress:+a+target+of+xylem-borne+ABA?.">Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA?.</a> The Plant Journal. 67, 72-80 (2011).</li><li>Hachez, C., Moshelion, M., Zelazny, E., Cavez, D., Chaumont, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localization+and+quantification+of+plasma+membrane+aquaporin+expression+in+maize+primary+root:+A+clue+to+understanding+their+role+as+cellular+plumbers.">Localization and quantification of plasma membrane aquaporin expression in maize primary root: A clue to understanding their role as cellular plumbers.</a> Plant Molecular Biology. 62, 305-323 (2006).</li><li>Hachez, C., Heinen, R. B., Draye, X., Chaumont, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expression+pattern+of+plasma+membrane+aquaporins+in+maize+leaf+highlights+their+role+in+hydraulic+regulation.">The expression pattern of plasma membrane aquaporins in maize leaf highlights their role in hydraulic regulation.</a> Plant Molecular Biology. 68, 337-353 (2008).</li><li>Besserer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+regulation+of+maize+plasma+membrane+aquaporin+trafficking+and+activity+by+the+SNARE+SYP121.">Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.</a> The Plant Cell. 24, 3463-3481 (2012).</li><li>Moshelion, M., Moran, N., Chaumont, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+changes+in+the+osmotic+water+permeability+of+protoplast+plasma+membrane.">Dynamic changes in the osmotic water permeability of protoplast plasma membrane.</a> Plant Physiology. 135, 2301-2317 (2004).</li><li>Moshelion, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+water+permeability+and+aquaporin+expression+increase+during+growth+of+maize+suspension+cultured+cells.">Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells.</a> Plant, Cell &amp; Environment. 32, 1334-1345 (2009).</li><li>Sade, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+plant+stress+tolerance+and+yield+production:+is+the+tonoplast+aquaporin+SlTIP2;2+a+key+to+isohydric+to+anisohydric+conversion?.">Improving plant stress tolerance and yield production: is the tonoplast aquaporin SlTIP2;2 a key to isohydric to anisohydric conversion?.</a> New Phytologist. 181, 651-661 (2009).</li><li>Volkov, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water+permeability+differs+between+growing+and+non-growing+barley+leaf+tissues.">Water permeability differs between growing and non-growing barley leaf tissues.</a> J. Exp. Bot. 58, 377 (2007).</li><li>Locatelli, F., Vannini, C., Magnani, E., Coraggio, I., Bracale, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+transient+transformation+in+tobacco+protoplasts+is+independent+of+plasmid+amount.">Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount.</a> Plant Cell Reports. 21, 865-871 (2003).</li><li>Hosy, E., Duby, G., V&#233;ry, A. A., Costa, A., Sentenac, H., Thibaud, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+localisation+and+electrophysiological+characterisation+of+ion+channels+heterologously+expressed+in+a+plant+context.">A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.</a> Plant Methods. 19, (2005).</li><li>Shoseyov, O., Posen, Y., Grynspan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Recombinant+Type+I+Collagen+Produced+in+Plants.">Human Recombinant Type I Collagen Produced in Plants.</a> Tissue Eng Part A. 19, 1527-1533 (2013).</li></ol>

Described here is a simple and very efficient procedure for measuring the Pf of isolated plant protoplasts, applicable in principle also to other spherical cells, e.g., frog oocytes11. This method is based on measuring the Pf 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...

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 (Pf) of the cell membrane1-3.
To date, several methods have been employed in measuring the endogenous Pf of protoplast from different plant organs (i.e. roots, mesophyll, endodermis, etc., reviewed by Chaumont et al. 4). One of the approaches to measure Pf 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 Pf 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 Pf 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 Pf 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 cells8-10 or suspension cultured cells11,12. In addition, it is possible to determine Pf 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 Pf; for example, expression of tomato AQP SlTIP2;2 in Arabidopsis mesophyll protoplasts by PEG transformation and determination the SlTIP2;2-related Pf13. Finally, examination of the effect on Pf 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 Pf.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="48">
			<td height="48" style="height:48px;width:288px;">KCl</td>
			<td style="width:140px;">Chem-Impex International</td>
			<td style="width:159px;">01247-1</td>
			<td style="width:516px;"><a href="http://www.chemimpex.com/">http://www.chemimpex.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:288px;">CaCl2</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">11718006</td>
			<td><a href="http://www.merck.com/">http://www.merck.com</a> 
			Any source, anal. grade</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:288px;">2-(N-morpholine)-ethanesulphonic acid (MES)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">15152002</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:288px;">D-Sorbitol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">18032003</td>
			<td>http://www.sigmaaldrich.com 
			Any source, anal. grade</td>
		</tr>
		<tr height="54">
			<td height="54" style="height:54px;width:288px;">Cellulase</td>
			<td style="width:140px;">Worthington, Lakewood, NJ, USA</td>
			<td style="width:159px;">LS002603</td>
			<td><a href="http://www.worthingtonbiochem.com/">http://www.worthingtonbiochem.com</a></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;width:288px;">Pectolyase</td>
			<td style="width:140px;">Karlan,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Phonix, AZ, USA</td>
			<td style="width:159px;">8006</td>
			<td><a href="http://www.karlan.com/">http://www.karlan.com</a></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:288px;">Polyvinyl-pyrrolidone K 30 (PVP)</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">81420</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:288px;"><a href="https://my.labguru.com/catalog/materials/216">Bovine Serum Albumin (BSA)</a></td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">A9418-5G</td>
			<td><a href="http://www.sigmaaldrich.com/">http://www.sigmaaldrich.com</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:288px;">Protamine sulphate</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P4380</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Poly-L-Lysine</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">P8920</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Xylene cyanol</td>
			<td style="width:140px;">Sigma</td>
			<td style="width:159px;">X4126</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:288px;">Silicone vacuum grease heavy</td>
			<td style="width:140px;">Merck</td>
			<td style="width:159px;">107921</td>
			<td><a href="https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;SEO_ErrorPageOccurred=true&amp;attachments=CoA">https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&#38;SEO_ErrorPageOccurred=true&#38;attachments=CoA</a></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;">Inverted microscope&#160;</td>
			<td>Nikon</td>
			<td>Eclipse TS100/TS100F</td>
			<td><a href="http://www.nikoninstruments.com/">http://www.nikoninstruments.com</a></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">Peristaltic pump</td>
			<td>BIO-RAD</td>
			<td style="width:159px;">EP-1 Econo&#160;Pump</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;">Grayscale digital camera</td>
			<td>Scion Corporation</td>
			<td style="width:159px;">CFW-1308M</td>
			<td><a href="http://www.scioncorp.com/">http://www.scioncorp.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">CMU 1394 Camera Driver&#8217; plugin for ImageJ</td>
			<td style="width:140px;">Carnegie Mellon</td>
			<td></td>
			<td><a href="http://www.cs.cmu.edu/~iwan/1394/download.html">http://www.cs.cmu.edu/~iwan/1394/download.html</a> 
			Free software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td style="width:140px;">NIH</td>
			<td></td>
			<td><a href="http://rsb.info.nih.gov/ij/">http://rsb.info.nih.gov/ij/</a> 
			Free software</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Econo Gradient Pump Fittings Kit</td>
			<td style="width:140px;">BIO-RAD</td>
			<td style="width:159px;">731-9006</td>
			<td><a href="http://www.bio-rad.com/">http://www.bio-rad.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Connectors, manifold</td>
			<td style="width:140px;">DirectMed</td>
			<td></td>
			<td><a href="http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S">http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Burette infusion sets (columns)</td>
			<td style="width:140px;">Welford</td>
			<td style="width:159px;">IF-BR-001</td>
			<td>http://www.welfordmedical.com/content.php?id=61</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Tubing</td>
			<td style="width:140px;">TYGON</td>
			<td style="width:159px;">R-3603</td>
			<td><a href="http://www.usplastic.com/">http://www.usplastic.com</a></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Plexiglass slide etc.</td>
			<td style="width:140px;">Perspectiv</td>
			<td></td>
			<td><a href="http://www.perspectiv.co.il/index-en.html">http://www.perspectiv.co.il/index-en.html</a> 
			Our slide was custom-made, it does not appear on the web site but a copy can be remade to order as &#39;a copy of the slide already made for M. Moshelion&#39;.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">3M packaging Scotch tape 1&#39;&#39;, clear</td>
			<td style="width:140px;">Viking Industrial, UK</td>
			<td style="width:159px;">VKMONO25</td>
			<td><a href="http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8">http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8</a> 
			any clear adhesive tape (sellotape, etc.) is likely to be OK</td>
		</tr>
	</tbody>
</table>

measuring the osmotic water permeability coefficient (pf) of spherical cells: isolated plant protoplasts as an example

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 (Pf) 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 &#8216;PfFit&#8217;), to yield Pf.

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

Watch this Scientific Journal Video about Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example at JoVE.com

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

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf 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.

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

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

measuring-osmotic-water-permeability-coefficient-pf-spherical-cells

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Biology

15.9K vues.  The Hebrew University of Jerusalem.  La mesure du coefficient de perméabilité à l'eau osmotique (P f) de cellules peut aider à comprendre les mécanismes de régulation de aquaporines (AQP). P f détermination dans des protoplastes de cellules végétales sphériques présentées ici implique l'isolement de protoplastes et l'analyse numérique de leur taux initial de changement de volume à la suite d'une faute de osmotique au cours de la perfusion de bain constant....