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.
