Name: Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin (Video) | JoVE Journal
Uploaded: 2009-03-11T00:00:00.000+00:00
Duration: 9 min 51 s
Description: Learn to measure plant cell wall extension using a constant-force extensometer with cucumber hypocotyls. This method demonstrates acid-induced creep and expansin activity for studying plant growth mechanics.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

The techniques shown here were developed with funding by grants from the Department of Energy (Bioenergy Sciences) and the National Science Foundation.

Part 1: Growing and storing suitable plant material
<ol>
<li>In our experience, young hypocotyls from etiolated cucumber seedlings serve as a convenient source of cell wall material for these experiments. Cucumber seeds are sown on wet paper in a light-proof box, which is keep in a darkened cabinet in a constant temperature room set at 26 °C. The exact temperature is not critical, as anything between 22° and 30 °C should be fine, but the temperature will determine how fast the seedlings reach an appropriate stage of development. The warmer the temperature, the faster the seedlings will develop. We typically use seedlings when they have grown to about 5 cm in length, which is reached 3-4 days after sowing. It is important that the seedling be grown in the dark, as even small amounts of light affect both the rate of seedling development and the cell wall properties that we measure with this technique. On day 3 you can peek in the box, using a dim green filtered light, to check on seedling development.</li>
<li>Seedlings are rapidly cut and packed in small plastic boxes, 100-150 seedlings per box, and stored at -80 °C. At this temperature they remain useful for weeks.</li>
</ol>
Part 2: Preparing cell wall samples
<ol>
<li>Small groups (8-10) of frozen cut seedlings are transferred from the freezer to an insulated container containing a -80 freezer block.</li>
<li>The cuticle covering the hypocotyl is abraded with carborundum. This is done by repeatedly drawing the hypocotyl between the thumb and forefinger, which are coated with a thick slurry of wet carborundum powder. It takes a little experience to know the correct amount of pressure to use: too much pressure and the epidermal layer begins to be shredded and torn; too little pressure and the cuticle will not be permeabilized. One also has to work quickly because as the frozen hypocotyl thaws, it becomes flaccid and hard to manage.</li>
<li>The abraded hypocotyl is dipped in ice water to remove most of the adhering carborundum and then stored on ice water while the remaining hypocotyls are prepared in a similar manner.</li>
<li>The hypocotyls are cut to the desired length, usually 1.2 cm, with a new single edged razor blade and then aligned on a glass slide.</li>
<li>Now we need to flatten the walls to remove cell sap and to facilitate clamping. A second glass slide is placed on top of the group of 8-10 samples, forming a sandwich. A weight (400-500 g) is placed on top of the glass slide for 5 min. For the weight we routinely use a beaker containing a suitable amount of water.</li>
<li>Optional step: Depending on the experiment, the hypocotyls may be inactivated with a brief heat treatment at this point. To do this we bind the glass slides together with a pair of rubber bands, put the assembly in a container with 100 mL of de-ionized water at room temperature and place it in a microwave oven at full power. With our microwave oven the water starts to boil at about 50 s and we stop the microwave 15 s after boiling begins. The hot water is quickly poured off and replaced with cold water to stop denaturation. You may have to vary these timings, as your microwave may be different than ours. With excessive heating the samples become weak and break easily. With insufficient heating the endogenous expansin is not inactivated and the wall samples retain some responsiveness to acidic pH.</li>
</ol>
Part 3: Extensometer setup
<ol>
<li>The wall samples are now clamped in a constant force extensometer. This is a custom-built device that consists of a Plexiglas cuvette for holding the basal end of the wall sample and a moveable clamp attached to the apical end of the sample. The moveable clamp is mounted on the end of a rod that passes through the open coils of a position sensor, an LVDT or 'Linear Variable Differential Transformer', that electronically detects the position of a small metal cylinder, or 'core', that is attached to the rod. The upper end of the rod is linked to a lever with an adjustable counterweight. This lever exerts an adjustable amount of upward force on the wall sample. The force is adjusted by adding or removing calibrated metal weights to the far end of the lever.</li>
<li>Back to the wall sample - the basal end of the hypocotyl sample is picked up with fine forceps and ~2-3 mm of the apical end is placed in between the open jaws of the moveable clamp. This clamp is a spring-loaded alligator clamp whose metal jaws are coated with plastic to prevent direct contact between the metal surface and the buffer solution or the wall sample. In our experience metal ions can leach from the clamp and inhibit the wall's ability to extend, and so we keep the metal covered.</li>
<li>Holding the moveable clamp assembly in one hand, the basal end of the wall specimen is now maneuvered in between the two hinged pieces of the Plexiglas cuvette and the cuvette pieces are brought together and screwed tight, thereby locking the bottom end of the wall sample in the cuvette.</li>
<li>The moveable clamp assembly is now gently released, allowing the full force of the counterweights to be transferred to the wall specimen. We routinely use a total counterweight of 20 g for wall samples prepared from cucumber hypocotyls. Other wall materials or experiments might require different weights.</li>
<li>The cuvette is filled with buffer (200 uL) and the position of the cuvette moved up or down with an adjustment screw, so that the moveable clamp is brought to the lower end of the LVDT measuring range. Our LVDT is connected to a data acquisition unit of a microcomputer, and so we monitor LVDT position through the computer. We have eight LVDT assemblies connected in parallel with one computer, which allows us to run 8 wall samples simultaneously. The computer records the position of each of the LVDT assemblies once every 30 s.</li>
</ol>
Part 4: Measuring extension response to acid pH or expansin - Representative Results
<ol>
<li>[First Experiment] For measurement of acid-induced extension, we start with native wall samples (that is, not inactivated with heat) and neutral buffer is added to the cuvette. The wall samples extend for a few minutes, in response to the added tension, but the extension decays to a low rate after a few minutes. Our computer lets us monitor either the change in length of the sample (that is, the change in position of the moveable clamp, after the start of the experiment) or we can monitor the time derivative of position, in other words the rate of extension. The extension rate stabilizes to a low value with time.</li>
<li>After ~20 min, the neutral buffer is removed. We use a thin metal pipe, made from a large gauge hypodermic needle, connected to a vacuum pump to remove the buffer quickly, with minimal disturbance of the wall or the mechanical assembly. Acidic buffer is then added, sometimes with 1-2 quick exchanges to insure the complete exchange to acidic buffer. Then we sit back and watch the response of the wall. Typically we can detect the faster rate of extension in a few minutes. After 60 min we usually have enough information to assess the extension response, although in some cases the measurements may extend to longer periods.</li>
<li>[Second Experiment] For measurement of expansin-induced cell wall extension, we start with heat-inactivated wall samples and acidic buffer is added to the cuvette. As in the First Experiment, the rate of cell wall extension gradually stabilizes to a low value because of the lack of functional expansin.</li>
<li>After ~20 min, expansin protein is added to the cuvette by 'spiking' with the buffer in the cuvette with 10-20 uL of an expansin solution. The expansin rapidly penetrates the cell wall sample and within a few minutes we see that the extension rate has increased. This extension response can be followed for an hour or more.</li>
</ol>
<img src="/files/ftp_upload/1263/fig1.png" alt="Etiolated cucumber seedling growth and extension measurement process diagram with extensometer setup." width="600" height="469" data-altupdatedat="1787630408000"> Figure 1: Outline of procedures for preparing cell walls to assess acid-induced or expansin-induced wall extension.

<ol><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+long-term+extension+of+isolated+cell+walls+from+growing+cucumber+hypocotyls%5BTitle%5D)+AND+%22Planta%22%5BJournal%5D)">Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls.</a> Planta. 177, 121-130 (1989).</li>
<li>Rayle, D. L., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Acid+Growth+Theory+of+auxin-induced+cell+elongation+is+alive+and+well%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">The Acid Growth Theory of auxin-induced cell elongation is alive and well.</a> Plant Physiol. 99, 1271-1274 (1992).</li>
<li>Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Auxin-induced+growth+of+Avena+coleoptiles+involves+two+mechanisms+with+different+pH+optima%5BTitle%5D)+AND+%22Plant+Physiol%22%5BJournal%5D)">Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.</a> Plant Physiol. 99, 1556-1561 (1992).</li>
<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loosening+of+plant+cell+walls+by+expansins%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Loosening of plant cell walls by expansins.</a> Nature. 407, 321-326 (2000).</li>
<li>McQueen-Mason, S., Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Two+endogenous+proteins+that+induce+cell+wall+expansion+in+plants%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">Two endogenous proteins that induce cell wall expansion in plants.</a> Plant Cell. 4, 1425-1433 (1992).</li>
</ol>

For this demonstration we used cell walls from cucumber hypocotyls because they have proved to be a reliable source of wall samples that are easy to handle and that respond with good sensitivity. We have also had good success with walls from other seedlings as well as some materials from the supermarket, such as young spinach leaves and celery stalks. Basically, young, soft, rapidly growing plant tissues are likely to be easily measured with this technique, but tough, old, oxidized or nongrowing plant tissues are unlike...

measuring plant cell wall extension (creep) induced by acidic ph and by alpha-expansin

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The plant hormone auxin rapidly stimulates cell elongation in young stems and similar tissues at least in part by an acid-growth mechanism 2, 3. Auxin activates a H+ pump in the plasma membrane, causing acidification of the cell wall solution. Wall acidification activates expansins, which are endogenous cell wall-loosening proteins 4, causing the cell wall to yield to the wall tensions created by cell turgor pressure. As a result, the cell begins to enlarge rapidly. This 'acid growth' phenomenon is readily measured in isolated (nonliving) cell wall specimens. The ability of cell walls to undergo acid-induced extension is not simply the result of the structural arrangement of the cell wall polysaccharides (e.g. pectins), but depends on the activity of expansins 5. Expansins do not have any known enzymatic activity and the only way to assay for expansin activity is to measure their induction of cell wall extension. This video report details the sources and preparation techniques for obtaining suitable wall materials for expansin assays and goes on to show acid-induced extension and expansin-induced extension of wall samples prepared from growing cucumber hypocotyls.
 To obtain suitable cell wall samples, cucumber seedlings are grown in the dark, the hypocotyls are cut and frozen at -80 &deg;C. Frozen hypocotyls are abraded, flattened, and then clamped at constant tension in a special cuvette for extensometer measurements. To measure acid-induced extension, the walls are initially buffered at neutral pH, resulting in low activity of expansins that are components of the native cell walls. Upon buffer exchange to acidic pH, expansins are activated and the cell walls extend rapidly. We also demonstrate expansin activity in a reconstitution assay. For this part, we use a brief heat treatment to denature the native expansins in the cell wall samples. These inactivated cell walls do not extend even in acidic buffer, but addition of expansins to the cell walls rapidly restores their ability to extend.

We demonstrate the use of a constant-force extensometer to measure long-term extension (creep) of plant cell wall specimens induced by acidic buffers and expansin protein.

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

Here we demonstrate methods for measuring expanse and activity by cell wall extens geometry. Suitable wall specimens can be prepared from the hypo coddles of E TATed cucumber seed lanes to measure acid in induced extension, which is catalyzed by endogenous expansions bound to native cell walls. We clamp the walls at neutral buffer and an extensometer upon switch to acidic pH expansions are activated and the cell walls extend rapidly.We also demonstrate an expansion reconstitution assay. For this part, we use a brief heat treatment to denature endogenous expansions. Heat inactivated cell walls do not extend even an acidic buffer, but addition of expansion rapidly restores wall extensibility.Hi, I'm Dan De Roko from the laboratory of Dr.Daniel Cosgrove in the biology department at Penn State University. Today we will show you a procedure for measuring irreversible extension or creep of isolated plant cell walls. So let's get started.In our experience, young hypo avocados from et elated cucumber seedlings serve as a convenient source of cell wall material. For these experiments, cucumber seeds are sewn on wet paper in a light proof box, which is kept in a darkened cabinet and a constant temperature room set at 26 degrees Celsius. Temperatures between 22 and 30 degrees Celsius will also work, but it is important to note that the temperature will determine the rate of growth.The warmer the temperature, the faster the seedlings will develop. Maintaining a dark growth environment is critical as even small amounts of light effect both the rate of seedling development and the cell wall properties that we measure with this technique. On day three, you can peek in the box using a dim green filtered light to check on seedling development.Usually when peeking into the box on day three or four, you will find seedlings grown to about five centimeters in length, which is typically what we used for our experiments. These seedlings can be frozen at negative 80 degrees Celsius before use To begin preparing cell wall samples, small groups of eight to 10 of frozen cut seedlings are transferred from the freezer to an insulated container containing a negative 80 degree Celsius freezer block. Using a thick slurry of carbo run powder to coat the thumb and forefinger the cuticle covering of the hypo coddle is removed by repeatedly drying the hypo coddle between the thumb and forefinger.While performing this technique, it is critical to use the correct amount of pressure that will ensure permeation of the cuticle without damaging the epidermal layer. One should also work quickly because as the frozen hypo coddle thaws, it becomes flad and hard to manage to remove most of the adhering carum. The abraded hypo coddle is dipped and then stored on ice water while the remaining hypo coddles are being prepared.Following cuticle removal on all seedlings, they are cut to the desired length, usually 1.2 centimeters with a new single ledge razor blade, and then aligned on a glass slide after alignment. A second glass slide is placed on top of the group of eight to 10 sample, and a weight 400 to 500 grams is placed on top of the glass slide for five minutes. We typically use a beaker filled with a suitable amount of water.The weight of the beaker will flatten the cell walls to remove cell sap and to facilitate clamping. Depending on the experiment, the hypo coddles may be inactivated with a brief heat treatment at this point. To do this, we bind the glass slides together with a pair of rubber bands, put the assembly in a container with a hundred milliliters of deionized water at room temperature and place it in a microwave oven at full power with our microwave oven.The water starts to boil at about 50 seconds and we stop the microwave 15 seconds after boiling begins, the hot water is quickly poured off and replaced with cold water to stop denaturation. Optimal heating will mean that samples will lack any responsiveness to low pH, while at the same time not become weak and break easily. The wall samples are now clamped in a constant force X extensometer.This is a custom built device that consists of a plexiglass cubit for holding the basal end of the wall sample and a movable clamp attached to the APIC end of the sample. The movable clamp is mounted on the end of a rod that passes through the open coils of a position sensor an LVDT or linear variable differential transformer. That electronically detects the position of a small metal cylinder or core that is attached to the rod.The upper end of the rod is linked to a lever with an adjustable counterweight. This lever exerts an adjustable amount of upward force on the wall sample. The force is adjusted by adding or removing calibrated metal weights to the far end of the lever to prepare samples for measurement of cell wall expansion.The basal end of the hypo cotto sample is picked up with fine forceps in approximately two to three millimeters of the APIC end is placed in between the open jaws of the movable clamp. This clamp is a spring loaded alligator clamp. The metal jaws are coated with plastic to prevent direct contact between the metal surface and the buffer solution or the wall sample.In our experience, metal ions can leach from the clamp and inhibit the wall's ability to extend, and so we keep metal covered holding the movable clamp assembly in one hand, the basal end of the wall specimen is now maneuvered in between the two hinge pieces of the plexiglass vete and the vete pieces are brought together and screwed tight, thereby locking the bottom end of the wall sample in the vete. The movable clamp assembly is now gently released allowing the full force of the counterweights to be transferred to the wall specimen. We routinely use a total counterweight of 20 grams for wall samples prepared from cucumber, hypo coddles.Other wall materials or experiments might require different weights. The qve is filled with buffer and the position of the QVE moved up or down with an adjustment screw so that the movable clamp is brought to the lower end of the LVDT measuring range. Our LVDT is connected to a data acquisition unit of the computer.We have eight LVDT assemblies connected in parallel with one computer, which allows us to run eight wall samples simultaneously. The computer records the position of each of the LVDT assemblies once every 30 seconds for measurement of acid induced extension. We start with native wall samples that is not inactivated with heat and neutral buffer is added to the vet.The wall samples extend for a few minutes in response to the added tension, but the extension decays to a low rate. After a few minutes, our computer lets us monitor either the change in length of the sample that is the change in position of the movable clamp after the start of the experiment, or we can monitor the time derivative of position. In other words, the rate of extension.The extension rate stabilizes to a low value with time. After approximately 20 minutes, the neutral buffer is removed using a thin metal pipe made from a large gauge hypodermic needle, which is connected to a vacuum pump. To remove the buffer quickly.The setup allows buffer to be removed with minimal disturbance of the wall or the mechanical assembly. Acidic buffer is then added sometimes with one to two quick exchanges to ensure the complete exchange to acidic buffer. Then we sit back and watch the response of the wall.Typically, we can detect the faster rate of extension in a few minutes. After 60 minutes, we usually have enough information to assess the extension response, although in some cases the measurements may extend to longer periods. For measurement of expansion induced cell wall extension, we start with heat inactivated wall samples and acidic buffer is added to the qve.As in the first experiment, the rate of cell wall extension gradually stabilizes to a low value because of the lack of functional expansion. After approximately 20 minutes expansion, protein is added to the Q vet by spiking with the buffer in the Q vet with 10 to 20 microliters of an expansion solution. Given that the cuticle has been removed, the expansion rapidly penetrates the cell wall sample, and within a few minutes we see that the extension rate has increased.This extension response can be followed for an hour or more. We've just shown you how to measure cell wall extension due to endogenous expansions and due to addition of expansions. When doing this procedure, it's important to remember to keep mechanical vibrations and temperature fluctuations to a minimum as they will generate noise in the extension curves.So that's it. Thanks for watching and good luck with your extension assays.

measuring-plant-cell-wall-extension-creep-induced-acidic-ph-alpha

Research

JoVE Journal

Biology

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

A novel imaging protocol was developed using a custom motor-driven mechanical actuator to allow the measurement of real time responses to mechanical strain in live cells. Relevant to mechanobiology, the system can apply strains up to 20% while allowing near real-time imaging with confocal or atomic force microscopy.

Live Cell Imaging during Mechanical Stretch

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging.  Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA).  Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Here, we present atomic force microscopy (AFM), operated as a nano- and micro-indentation tool on cells and tissues. The instrument allows the simultaneous acquisition of 3D surface topography of the sample and its mechanical properties, including cell wall Young's modulus as well as turgor pressure.

Use of Atomic Force Microscopy to Measure Mechanical Properties and Turgor Pressure of Plant Cells and Plant Tissues

Here we describe a protocol for live cell imaging of the cortical microtubule cytoskeleton at the shoot apical meristem and monitoring its response to changes in physical forces.

Live Cell Imaging of Microtubule Cytoskeleton and Micromechanical Manipulation of the Arabidopsis Shoot Apical Meristem

The atomic force microscopy indentation protocol offers the possibility to dissect the role of the physical properties of the cell wall of a particular cell of a tissue or organ during normal or constrained growth (i.e., under water deficit).

Atomic Force Microscopy to Study the Physical Properties of Epidermal Cells of Live Arabidopsis Roots

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

This manuscript presents a detailed protocol to image the 3-D cell wall dynamics of living moss tissue, allowing the visualization of the detachment of cell walls in ggb mutants and thickening cell wall patterns in the wild type during development over a long period.

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens

This report describes the fundamental methods used to culture and experimentally manipulate the unicellular streptophyte alga, Penium margaritaceum. It also provides fundamental protocols of microscopy-based imaging, including live cell labeling with monoclonal antibodies and other fluorescent probes and scanning electron microscopy.

Experimental Screening Protocols, Immunocytochemistry and Microscopy-based Imaging Techniques for Penium margaritaceum

Learn to measure plant cell wall extension using a constant-force extensometer with cucumber hypocotyls. This method demonstrates acid-induced creep and expansin activity for studying plant growth mechanics.

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

Explore More Videos