Protocols

Leaf phosphorus assay protocol (adapted from Improving the stability of the malachite green method for the determination of phosphate using Pluronic F68 (Martínez Gache et al. 2020).

Materials:

Reagents:

Warning: Perchloric acid is extremely hazardous and should be treated with appropriate caution. Please consult MSDS before attempting this procedure. Perchloric acid should be handled in the hood with appropriate PPE (e.g., thick nitrile gloves and safety goggles)

Glasswear

(All glasswear should be very clean to avoid phosphorus contamination. This can be accomplished by acid-washing glasswear in 0.1 N HCl).

Preparation of extraction solvent (2% acetic acid)

(Some of the older bottles of acetic acid in the acid cabinet are somehow contaminated with phosphorus. Use the newest bottle and be careful to avoid contamination).

  1. Add ~ 800 ml of milliQ water to a very clean 1 L volumetric flask.
  2. Add 20 ml of glacial acetic acid to the flask.
  3. Fill to the line with milliQ water.
Preparation of Pluronic F68 stock solution (0.17 g / ml)
  1. Weigh 5 g of Pluronic F68.
  2. Heat 20 ml of milliQ water on a hot plate.
  3. Slowly add pluronic F68 to hot water while stirring.
  4. When beads are fully dissolved fill beaker to 30 ml.
  5. Continue stirring as the solutions cools or it will start to solidify on top.
  6. Transfer solution to a clean 50 ml Falcon tube for storage.
Preparation of 5x color reagent (25 ml):
  1. Weigh 0.44 g of ammonium heptamolybdate tetrahydrate.
  2. Add 10 ml milliQ water to a beaker with a small stir bar.
  3. While stirring, slowly add the ammonium molybdate into the beaker.
  4. Add 6 ml of 70% perchloric acid.
  5. Slowly add 0.048 g of malachite green while stirring.
  6. Immediately add 1.5 ml of pluronic F68 solution (0.17 g/ ml).
  7. Carefully pour the solution into a 25 ml volumetric flask and fill to 25 ml with milliQ water.

The color reagent can be stored for at least one year at room temperature.

Preparation of phosphorus standards
Leaf extraction

(Samples should be kept on ice throughout the extraction procedure (e.g., in between homogenizations).).

  1. Weigh 20 mg of oven-dried leaf material or 40 mg of flash frozen leaf material into a fast-prep tube.
  2. Add one scoop of zirconium beads.
  3. Add 500 µL of ice-cold extraction solvent (2% acetic acid in milliQ water).
  4. Homogenize leaves on fast-prep (15 s at ).
  5. Sonicate in ice-water for 6 minutes.
  6. Homogenize leaves on fast-prep (15 s at ).
  7. Centrifuge at ~ 20,000 RCF for 5 minutes.
  8. Pipette supernatant into fresh eppendorf tube.
  9. Centrifuge at ~ 20,000 RCF for 5 minutes.
  10. Pipette supernatant into fresh eppendorf tube.

Phosphorus assay

The supernatant from the extractions must be diluted into the linear range for the assay using the extraction solvent (2% acetic acid). This may require some trial and error as you don’t know the concentrations of your samples ahead of time. It may be helpful to make a test plate with a representative subset of samples at different dilutions. It is easiest to make the dilutions by pipetting a small volume of each sample directly into the well after adding an appropriate volume of extraction solvent. At least one set of standards should be run on each plate. For greater reproducibility, two sets of standards can be run on each plate. One set of standards requires 8 wells, so you can plan to run 88 samples per plate with one set of standards or 80 samples with two sets of standards. It is probably wise to also prepare a blank that is extracted with beads without any plant material to control for phosphorous contamination in the extraction process (e.g., due to dirty beads).

Load phosphate standards onto the plate

I’ve found that reproducibility is fine making the dilutions directly on the plate (e.g., by making 2-fold serial dilutions starting from 80 µM). Make sure the volume of the standards matches the volume of your samples (i.e., in this case 80 µL).

  1. Start with 160 µL of the undiluted (80 µM) standard in the first well.
  2. Add 80 µL of extraction buffer to the next 7 wells.
  3. Remove 80 µL from the first well and transfer it to the second well, mix by pipetting up and down at least three times.
  4. Repeat this process for the next 5 wells.
  5. Make sure to discard 80 µL from the final well so that all volumes are equal. The 8th well should be pure extraction buffer (0 µM).
  6. If preparing two sets of standards on the plate, repeat this process in another non-contiguous set of wells.

This procedure should achieve the following concentrations: 80, 40, 20, 10, 5, 2.5, 1.25, 0 µM PO4. Alternatively, each standard can be prepared in a larger volume and 80 µL can be pipetted into each well, but I have found that this does not increase reproducibility.

Load samples onto the plate
  1. Add 79 µL of 2% acetic acid solution into as many wells as you have samples.
  2. Add 1 µL of supernatant for each sample (for a 1.25% dilution). These numbers can be adjusted as needed to bring the samples into the linear range. As you add each sample, make sure to mark its location on the plate in a notebook.
  3. After all samples are loaded, add 20 µL of 5x color reagent to each well. Make sure to change tips between wells. (You do not want to contaminate the color reagent!)
  4. After adding the color reagent to all of the wells, allow the plate to incubate at room temperature for 30 minutes.
  5. Read the plate on the plate reader at 630 nm.