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: 0.55 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 &#176;C.</li>
</ol>
2. Isolation of Arabidopsis Mesophyll Protoplasts
<ol>
	<li>Prepare a Petri dish (10 cm) with about 6 drops (approx. 30 &#181;l each) of isotonic solution.</li>
	<li>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.</li>
	<li>Dissolve 5.7 mg of the enzyme mix in 165 &#181;l 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.</li>
	<li>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 &#176;C.</li>
	<li>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 &#181;l pipette tip) into a 1.5 ml tube.</li>
</ol>
3. The Hypotonic Challenge Assay: Arabidopsis Mesophyll Cell Swelling
<ol>
	<li>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.</li>
	<li>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) &#8220;sticky&#8221; 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 &#8216;glue&#8217; 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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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 &#8216;CMU 1394 Camera Driver&#8217; 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).</li>
</ol>
4. Analysis of the Cell Volume Change Using ImageJ
NOTE: To analyze the series of images of a swelling cell, use the &#8216;Image Explorer&#8217; and &#8216;Protoplast Analyzer&#8217; plugins in the ImageJ software (written by Xavier Draye)14. Starting with the chosen protoplasts at their first time point, the &#8216;Protoplast Analyzer&#8217; 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).
<ol>
	<li>Start ImageJ. To open the movie, click &#8216;File&#8217; on the ImageJ panel, then, consecutively on the dropdown menus as they unfold: &#8216;Import&#8217; then &#8216;Image Explorer&#8217;. Highlight the chosen movie, then right-click on it, then left-click on &#8216;Protoplast Analyzer&#8217;. Browse through the movie (using a slider at the protoplast image bottom) to identify protoplasts that remain largely immobile during the experiment &#8211; 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 &#8216;OK&#8217; in the table of &#8216;Detection parameters&#8217; that appeared.</li>
	<li>To launch the protoplast detection algorithm, click &#8216;Local&#8217; on the protoplast image top panel, then &#8216;Process&#8217; in the dropdown menu. Examine the green circles around the selected protoplasts (Figure 2C) throughout the movie. Save the &#8216;Result&#8217; in an Excel file. Quit ImageJ. NOTE: In case a red dot appears (to indicate a bad contour fit &#8211; usually due to a poor image contrast), rerun with different parameters.</li>
	<li>To separate the lines belonging to each cell (which &#8211; if two or more cells were analyzed simultaneously &#8211; will be intertwined, because the analysis is done frame by frame), in Excel, sort the saved data by the cell number column (&#8216;object&#8217;).</li>
	<li>To determine the pixel-to-&#181;m conversion factor for obtaining the real value of Pf, 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 &#181;m2. 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 &#8216;PfFit&#8217; program.</li>
</ol>
5. Modeling the Rate of Osmolarity Change in the Experimental Chamber Using ImageJ and the Matlab Program PfFit
<ol>
	<li>Add 2 mg xylene cyanol (Table 1, below) to 100 ml of the isotonic solution (to produce the &#8216;Indicator Dye&#8217;).</li>
	<li>Prepare the perfusion system (as in 3.1) with the Indicator Dye and the non dyed hypotonic solution.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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</li>
	<li>Use the ImageJ software to analyze the video images of the Indicator Dye transmittance to obtain an averaged time course of the changing transmittance.
	<ol>
		<li>Start ImageJ, click &#8216;File&#8217;, then, &#8216;Open&#8217;, and browse for the movie. For each movie, draw a 10 pixel wide vertical rectangle anywhere on the 1st image of the movie. Click &#8216;Image&#8217; on the ImageJ main panel, then click &#8216;Crop&#8217; in the dropdown menu.</li>
		<li>To align the 60 frames (of the 60 sec movie) in one row, click again &#8216;Image&#8217;, then click consecutively in the dropdown menus as they unfold: &#8216;Stacks&#8217; and &#8216;Make Montage&#8217; (columns 60, rows 1). Draw a 1 pixel high horizontal rectangle anywhere along the whole row of images and click &#8216;Analyze&#8217; in the ImageJ main panel, then click &#8216;plot profile&#8217; in the dropdown menu. NOTE: A &#8216;Plot of Montage&#8217; 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 &#8220;time base&#8221; (the image sequential number) is 10 times longer.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Start the Matlab fitting program PfFit (the &#8216;Indicator Fit&#39; 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. PfFit is a program available for use free of charge. The &#8216; PfFit_Installer_web.exe&#8217; can be downloaded from: PfFit User Guide&#8217; with detailed explanations and definitions is accessible via Jove as a Supplemental file, which helps to familiarize the user with the PfFit program.</li>
	<li>In the &#8216;Indicator Fit&#39; panel, import the data of the mean time course of the Indicator Dye transmittance (&#8216;Indicator data file&#8217;, Figure 3A) and insert manually the current experiment parameters and the initial guesses of the parameters &#8216;width&#8217; and &#8216;t_half&#8217; describing the time course of the Indicator Dye concentration (Figure 3B. Click &#8216;Run&#8217; 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).</li>
</ol>
6. Determining the Pf using the Matlab Fitting Program PfFit
NOTE: In addition to the basic assumptions with regard to the behavior of a protoplast as a true and perfect osmometer11, the determination of Pf rests on the presumption that Pf may change with time, that this dynamics of Pf underlies the time course of the cell volume change and that three parameters suffice to describe it: Pfi (the initial value of Pf), SlopePf (the rate of the linear change of Pf) 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. PfFit searches for the best combination of these parameters to yield &#8211; by calculation &#8211; 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 &#8216;PfFit User Guide&#8217;).
<ol>
	<li>Switch to the &#8216;Volume Fit&#8217; panel (Figure 5). Choose for import the areas data file (the text file with the time course of the &#8216;areas&#8217; of the analyzed protoplasts, Figure 5A). Choose &#8216;Last Indicator Fitting&#8217; as the parameter source (Figure 5B; see the &#8216;PfFit User Guide&#8217; for alternatives). NOTE: These parameters (Figure 5D) are then used to regenerate the osmoticum change in the bath for the volumes fitting procedure.</li>
	<li>In the &#8216;Volume Fit&#8217; panel (Figure 5C), initialize (fill in the initial guesses for) the Pf parameters: Pf, SlopePf and Delay (a recommendation: start with 1, 1, and 30, respectively), Chose the model &#8216;Class&#8217; (a recommendation: start with II and mark &#8216;checks&#8217; for all three parameters to be fitted). Click &#8216;Run&#8217;, then eyeball the interim figure (Figure 5E) and adjust the Delay parameter and the length of the record, if needed.</li>
	<li>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-&#8216;Run&#8217;. NOTE: Don&#8217;t be discouraged when the program gets stuck &#8211; just restart the program!</li>
	<li>Repeat this procedure several times, starting with different combinations of initialization parameters, aiming for the lowest value of the fit error.</li>
	<li>Copy the list of the fit results directly from the screen, or find them in the PfFit-generated &#8216;_FIT_Vol_Results.txt&#8216; file.</li>
</ol>

<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>

We have nothing to disclose.

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><tbody><tr><td>KCl</td><td>Chem-Impex International</td><td>01247-1</td><td>&lt;a href=&quot;http://www.chemimpex.com/&quot;&gt;http://www.chemimpex.com&lt;/a&gt;&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>11718006</td><td>&lt;a href=&quot;http://www.merck.com/&quot;&gt;http://www.merck.com&lt;/a&gt;&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>2-(N-morpholine)-ethanesulphonic acid (MES)</td><td>Sigma</td><td>15152002</td><td>http://www.sigmaaldrich.com&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>D-Sorbitol</td><td>Sigma</td><td>18032003</td><td>http://www.sigmaaldrich.com&lt;br/&gt; Any source, anal. grade</td></tr><tr><td>Cellulase</td><td>Worthington, Lakewood, NJ, USA</td><td>LS002603</td><td>&lt;a href=&quot;http://www.worthingtonbiochem.com/&quot;&gt;http://www.worthingtonbiochem.com&lt;/a&gt;</td></tr><tr><td>Pectolyase</td><td>Karlan,&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Phonix, AZ, USA</td><td>8006</td><td>&lt;a href=&quot;http://www.karlan.com/&quot;&gt;http://www.karlan.com&lt;/a&gt;</td></tr><tr><td>Polyvinyl-pyrrolidone K 30 (PVP)</td><td>Sigma</td><td>81420</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>&lt;a href=&quot;https://my.labguru.com/catalog/materials/216&quot;&gt;Bovine Serum Albumin (BSA)&lt;/a&gt;</td><td>Sigma</td><td>A9418-5G</td><td>&lt;a href=&quot;http://www.sigmaaldrich.com/&quot;&gt;http://www.sigmaaldrich.com&lt;/a&gt;</td></tr><tr><td>Protamine sulphate</td><td>Sigma</td><td>P4380</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P8920</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Xylene cyanol</td><td>Sigma</td><td>X4126</td><td>http://www.sigmaaldrich.com</td></tr><tr><td>Silicone vacuum grease heavy</td><td>Merck</td><td>107921</td><td>&lt;a href=&quot;https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;amp;SEO_ErrorPageOccurred=true&amp;amp;attachments=CoA&quot;&gt;https://merck-chemicals.co.id/chemicals/silicone-high-vacuum-grease-heavy/MDA_CHEM-107921/p_LMib.s1Oxr4AAAEvXHg49in.?SecurePage=true&amp;amp;SEO_ErrorPageOccurred=true&amp;amp;attachments=CoA&lt;/a&gt;</td></tr><tr><td>Inverted microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse TS100/TS100F</td><td>&lt;a href=&quot;http://www.nikoninstruments.com/&quot;&gt;http://www.nikoninstruments.com&lt;/a&gt;</td></tr><tr><td>Peristaltic pump</td><td>BIO-RAD</td><td>EP-1 Econo&amp;nbsp;Pump</td><td>&lt;a href=&quot;http://www.bio-rad.com/&quot;&gt;http://www.bio-rad.com&lt;/a&gt;</td></tr><tr><td>Grayscale digital camera</td><td>Scion Corporation</td><td>CFW-1308M</td><td>&lt;a href=&quot;http://www.scioncorp.com/&quot;&gt;http://www.scioncorp.com&lt;/a&gt;</td></tr><tr><td>CMU 1394 Camera Driver&amp;rsquo; plugin for ImageJ</td><td>Carnegie Mellon</td><td></td><td>&lt;a href=&quot;http://www.cs.cmu.edu/~iwan/1394/download.html&quot;&gt;http://www.cs.cmu.edu/~iwan/1394/download.html&lt;/a&gt;&lt;br/&gt; Free software</td></tr><tr><td>ImageJ</td><td>NIH</td><td></td><td>&lt;a href=&quot;http://rsb.info.nih.gov/ij/&quot;&gt;http://rsb.info.nih.gov/ij/&lt;/a&gt;&lt;br/&gt; Free software</td></tr><tr><td>Econo Gradient Pump Fittings Kit</td><td>BIO-RAD</td><td>731-9006</td><td>&lt;a href=&quot;http://www.bio-rad.com/&quot;&gt;http://www.bio-rad.com&lt;/a&gt;</td></tr><tr><td>Connectors, manifold</td><td>DirectMed</td><td></td><td>&lt;a href=&quot;http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S&quot;&gt;http://directmed.com/main/Plastic-Medical-Tubing-Connectors.html?ACTION=S&lt;/a&gt;</td></tr><tr><td>Burette infusion sets (columns)</td><td>Welford</td><td>IF-BR-001</td><td>http://www.welfordmedical.com/content.php?id=61</td></tr><tr><td>Tubing</td><td>TYGON</td><td>R-3603</td><td>&lt;a href=&quot;http://www.usplastic.com/&quot;&gt;http://www.usplastic.com&lt;/a&gt;</td></tr><tr><td>3M packaging Scotch tape 1&#039;&#039;, clear</td><td>Viking Industrial, UK</td><td>VKMONO25</td><td>&lt;a href=&quot;http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8&quot;&gt;http://www.vikingtapes.co.uk/c-428-vkmono-mono-filament-tape.aspx#.UuvqOftdy_8&lt;/a&gt;&lt;br/&gt; 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

Research

JoVE Journal

Biology

16.0K Views.  The Hebrew University of Jerusalem. 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.

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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

This protocol details the optimized extraction of apoplast washing fluid from plant leaves, using French bean plants (Phaseolus vulgaris) as a model example.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Isolation and Transcriptome Analysis of Plant Cell Types

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, which is known as cellular heterogeneity. In recent years, single-cell and cell-type isolation combined with next-generation sequencing (NGS) techniques have become important tools for studying biological processes at single-cell resolution. However, isolating plant cells is relatively more difficult due to the presence of plant cell walls, which limits the application of single-cell approaches in plants. This protocol describes a robust procedure for fluorescence-activated cell sorting (FACS)-based single-cell and cell-type isolation with plant cells, which is suitable for downstream multi-omics analysis and other studies. Using Arabidopsis root fluorescent marker lines, we demonstrate how particular cell types, such as xylem-pole pericycle cells, lateral root initial cells, lateral root cap cells, cortex cells, and endodermal cells, are isolated. Furthermore, an effective downstream transcriptome analysis method using Smart-seq2 is also provided. The cell isolation method and transcriptome analysis techniques can be adapted to other cell types and plant species and have broad application potential in plant science.

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

In multicellular organisms, developmental programming and environmental responses can be highly divergent in different cell types or even within cells, ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

Efficient Extraction and Purification of GFP-His from N. benthamiana Leaves

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from plants is one of the most critical steps in the production process. Typically, proteins were purified from total soluble proteins (TSP), and the presence of miscellaneous intracellular proteins and cytochromes poses challenges for subsequent protein purification steps. Moreover, most therapeutic proteins like antigens and antibodies are secreted to obtain proper glycosylation, and the presence of incompletely modified proteins leads to inconsistent antigen or antibody structures. This work introduces a more effective method to obtain highly purified recombinant proteins from the plant apoplastic space. The recombinant Green fluorescent protein (GFP) is engineered to be secreted into the apoplast of Nicotiana benthamiana and is then extracted using an infiltration-centrifugation method. The GFP-His from the extracted apoplast is then purified by nickel affinity chromatography. In contrast to the traditional methods from TSP, purification from the apoplast produces highly purified recombinant proteins. This represents an important technological improvement for plant production systems.

Author Spotlight: Optimizing Apoplast Protein Extraction for Efficient Recovery of Recombinant Proteins from Plant Cells

The purification of recombinant proteins from plant systems is usually challenged by plant proteins. This work provides a method to effectively extract and purify a secreted recombinant protein from the apoplast of Nicotiana benthamiana.

Plants are a newly developing eukaryotic expression system being explored to produce therapeutic proteins. Purification of recombinant proteins from ...

Efficient Cabbage Protoplast System for Studying Flooding Tolerance Mechanisms

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia stress. To identify mechanisms of flooding tolerance in cabbages, a versatile platform for genetic functional studies is needed to overcome the transformation-recalcitrant nature of cabbages. In this study, a cabbage protoplast transient expression system and a corresponding protoplast hypoxia induction protocol were developed. This protocol achieved a high yield and integrity of protoplast isolation from cabbage leaves, with a transfection efficiency exceeding 40% using optimized enzymatic conditions. To alleviate potential hypoxic influence before treatments, the W5 solution was bubbled with oxygen gas to increase dissolved oxygen levels. Several chemicals for adjusting oxygen levels and physiological oxygen-scavenging treatments were tested, including EC-Oxyrase, OxyFluor, sodium sulfite, and an oxygen absorber pack. Dual-luciferase assays showed that promoters of anaerobic respiration response genes BoADH1 and BoSUS1L were activated in cabbage protoplasts after hypoxia treatments, with the highest induction level observed after treatment with the oxygen absorber pack. In summary, the cabbage protoplast transient expression system combined with hypoxia treatment demonstrates an efficient and convenient platform. This platform can facilitate studies of gene function and molecular mechanisms associated with hypoxia responses in cabbages.

This protocol describes an efficient cabbage mesophyll protoplast system. Various oxygen-deficient treatments were tested, and the system showed a high activation of hypoxia-responsive genes, facilitating studies of the genetic and molecular mechanisms of flooding tolerance in Brassicaceae vegetables.

As climate change brings more heavy rainfall, cabbage, a key Brassicaceae vegetable, faces significant yield losses due to flooding-induced hypoxia ...

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

Summary

Explore More Videos

Chapters in this video

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Fluorescence Activated Cell Sorting of Plant Protoplasts

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using Phaseolus vulgaris as an Example

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Isolation and Transcriptome Analysis of Plant Cell Types

Scalable Transfection of Maize Mesophyll Protoplasts

Apoplast-Extraction Based Method to Improve the Purity of Plant Produced Recombinant Protein

Development of a Cabbage Protoplast System for Studying Hypoxia Tolerance in Brassica