Tuesday, June 10, 2014

Electrolyte Leakage Assay

*Note - I had tried to schedule this to self post yesterday but apparently it did not... soo here it is a little late :) * 

Most people when they hear the word electrolytes they think Gatorade. Most people have seen the commercials and know that after working out you should replace the electrolytes lost from exercise. Gatorade has electrolytes, thus you should drink Gatorade, right? Well... yes and no. Yes, you do lose electrolytes when you workout via sweat. But, you have to be sweating a lot for Gatorade to actually be better than water. Gatorade has calories, and honestly we get a lot of salt (one of the electrolytes) in our diet in the U.S. anyway, so water is sufficient if you are exercises for an hour-ish. Now if you are running a marathon or playing a full game of football/volleyball/soccer etc., you should turn to Gatorade or other sports drinks to replace the electrolytes in a quick and efficient manner.

So now the question is, what do electrolytes have to do with my research topic of cold stress in plants? Glad you asked, let me explain!

I am learning a new technique in lab right now, the electrolyte leakage assay. It is a technique I see a lot in various articles that I read, not to mention one that other labmates have mentioned in their presentations. But I never knew what it involves, and honestly, I only had a vague idea of how it works. Before I volunteered to do this for my PI while on vacation, I knew that high numbers are bad and mean that the plant is not coping with the cold.  Now that I am actually doing this technique I decided that I needed to know more about it.

In the lab, my PI explained the basics of how I collect the data, and then how I plot the data. It is fairly simple. The plants were either acclimated (had been in cold temperatures) or nonacclimated (had been at normal temperatures) for a few days. Then we cut leaf discs out of them and put them in test tubes.
Acclimated plants must stay cold, so sampling must occur in the
walk in fridge! So, so cold!

These test tubes were then put in a cold water/glycerol bath (the glycerol is needed to keep the water in the bath from freezing solid). Leave them at various sub-zero temperatures for 2 hours, this can take all day depending on how many temperatures you use. We had 5 different temperatures, that's 10 hours of removing tubes and shifting down the temperature.
90 tubes.. we had 90 tubes!
Then put them in the fridge overnight. The next day, add 3 milliliters of water and shake them at room temperature for 3 hours. After which, it is finally time to measure them. We use a conductivity meter, which measures the electrical potential of the water. The electrical potential of the water increases as the ion concentration increases. And since electrolytes are ions, this provides a measure of the electrolytes in the solution.



Simply, read the leaves/water mixture three times per tube, all 90 tubes (this step takes a while). Then remove and save the water (the labeling on this is a nightmare), freeze the leaves for at least a day. Next time point all the water is put back in the tubes and repeat the conductivity readings. This provides a total leakage point to compare the original samples with to obtain a percent leakage.

Time-consuming but easy. Ok, great. But what do electrolyte levels have to do with cold stress? This technique of using the expressed vs total leakage was recommended by Stuart in a 1939 journal article(Stuart NW, 1939. Comparative cold hardiness of scion roots from fifty apple varieties. Proc. Am. Soc. Hort.). That's almost 100 years of usage, must be a good method! Electrolytes are present in cells and can pass through the cell membrane. When a cell is healthy, a small amount of electrolytes can pass between the membrane. But as stressors increase, the membrane can change permeability or be damaged and this allows more electrolytes to be released. The more damage to the membrane, the higher electrolyte leakage. It gives a easy, cheap way to evaluate how much stress the plant was experiencing.

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