Good day scholar,
How do I measure kinetic influx of potassium into plant root. I read a couple of papers on potassium influx. A paper" Varietal differences in potassium uptake by barley" (Anthony and Glass, 1980) gave me an insight into what i could expect. However i still have some reservations. I want to outline the details have arrived at so far, and the challenges am been faced with.
PROCEDURE FOR POTASSIUM INFLUX
1. In a container, add 50ml of CaSO4in 50ml of 0.01mM of HCl in a container and repeat the same process for 0.05, 0.1, and 0.2mM of HCl solution.
2. Each medium solution will be aerated continuously during the 10minutes influx period and maintained at 30oC within a water bath.
3. Roots (grouped into 3 or 4) are immersed in these solutions and subsequently desorbed in ice-cold non-labeled influx media for 5min.
4· Roots will be separated from the shoot, spun (what is the meaning of spun?) for 20s in a basket centrifuge.
5· Weigh into scintillation vials and ash for 3hrs at 500OC.
6· Ashes will be dissolved in 10ml of distilled water
7.The radioactivity will be obtained by Cerenkov counting in a Searle Isocap 300 scintillation spectrophotometer.
Please scholars in answers to the following questions (Influx media preparation) :
1. In point 2/ point 3, do I need to aerate while the roots are these mediums or I put the roots in after aerating the medium.
2. “Desorbing in ice-cold non-labeled influx media” does translate to putting the samples in a cold –ice or another non-labeled should be prepared.
3. Will I use oven to ash the samples at 500 degree?
Thanks for your apt response @ Paul Milham . Please can you share the procedure or send a related article? Thank you.
This is not my field. Look at papers by Zhonghua Chen et al. from Western Sydney University
Is that the correct procedure for potassium influx? Step 1 mentions Calcium Sulphate, but not potassium, and doesn't suggest how much radioactivity might be needed. Is the HCl for pH adjustment or some other reason? I would be considering what pH the roots would be exposed to in soil and adjusting with a buffer rather than just tossing in an unbuffered mass of acid.
Step 2 is pretty typical for hydroponic solutions, bubble oxygen or air through a tube to agitate the solution and keep the system oxygenated.
Step 3 requires the same concentrations of non-labelled solution, close to 0degC, to wash off any labelled tracer that is not absorbed by the roots, (but might be adsorbed onto the cell surfaces). Step 4 is literally spinning in a basket centrifuge (or a domestic salad spinner) to remove the washing solutions from step 3.
Ashing is usually in a muffle furnace which is a dedicated lab oven that gets a lot hotter than the typical drying ovens in labs. I don't know what sort of scintillation counter you have, so the remaining steps are somewhat more site-specific.
Thanks so much for this insightful comment @Matthew Wheal . I am trying to get a proper procedure before moving on with the influx experiment. Can ICP also be used for potassium influx?
To respond to you followup question, it depends on the scale of your measurements: ICP will accurately measure mg/kg concentrations of potassium in roots. To accurately observe this change will take maybe hours or days of uptake, or many replicates of plants. In comparison, tracer studies can measure uptake rates over minutes to hours, but may not extrapolate as well across the lifespan of the plant.
What is the question you wish to examine: rapid uptake or long term accumulation?
A good way to estimate the kinetics of Potassium influx may be using the "two electrodes voltage clamp" (TEVC) protocol, using protoplast, applying increasing concentrations of KCl and KNO3, comparing it to increasing concentrations of NaCl and NaNO3. If you need more information, I can find it and attach it here if you wish, or we can talk by PM.
Matthew Wheal Thanks so much for this great contribution. I want to measure the uptake rate of potassium. But i don't know how to go about it. Please can you send me the protocol?
J.D. Franco-Navarro Thanks for recommending voltage clamp method. I will like to learn more about this. Can I PM you for more details? Thanks
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