Quantification of Total and Soluble Inorganic Phosphate

Materials and Reagents

1. Plant tissues 
2. 17.5 M Glacial acetic acid
   
3. 12 M Concentrated HCl 
   
4. 16 M Concentrated HNO₃
   
5. Sodium phosphate, monobasic (NaH₂PO₄) (Bioshop, catalog number: SPM400 )
   
6. Ascorbic acid (Bioshop, catalog number: AS0704 )
   
7. Ammonium molybdate (Bioshop, catalog number: AMN333 )
   
8. Zinc acetate (Sigma-Aldrich, catalog number: Z0625 )
   
9. Quartz crucibles (Thermo Fisher Scientific, catalog number: 08-072 series)
   
10. Acid extraction solution (see Recipes)
    
11. Pi stock (for standard curve) (see Recipes)
    
12. Pi assay reagent (see Recipes)
    

Equipment

1. Crucible
   
2. Drying oven
   
3. Repeat pipetor
   
4. Isotemp Muffle Furnace (Thermo Fisher Scientific, model: 10-650-14 )
   
5. Microcentrifuge
   
6. A computer supported microplate spectrophotometer (e.g., Spectromax Plus, Molecular Devices, Sunnyvale)
   

Procedure

1. Total Pi (Hurley et al., 2010)
   1. Acid wash crucibles by incubating for at least 1 h in 0.1 N HCl at room temperature, then rinse with dH₂O and dry.
      
   2. Pre-weigh crucibles and place at least 60 mg (fresh weight) of tissue in each.
      
   3. Dry in oven at 50-80 °C for at least 16 h (e.g., overnight), and then record tissue’s dry weight (mg) in each crucible.
      
   4. Ash the tissue in the furnace using a temperature ramp program (20 min at 150 °C, 1 h at 250 °C, and 3 h at 500 °C).
      
   5. Weigh crucible and ash. Add 25 μl of acid extraction solution per mg of ash, mix well, and centrifuge at 11,000 x g for 10 min.
      
   6. Dilute the supernatant 50-fold in dH₂O.
      
   7. Assay Pi using the Drueckes et al. (1995) protocol as modified for plant tissues (Bozzo et al., 2006) by preparing a standard curve over the range 1-133 nmol of Pi using the following template.
      
      

      Well #	Vol of Pi stock (3.3 mM)	Volume of dH2O	Amount of Pi added
      	(μl)	(μl)	(nmol)
      1A	0	40	0
      1B	2	38	6.6
      1C	4	36	13.2
      1D	8	32	26.4
      1E	12	28	39.6
      1F	16	24	52.8
      1G	20	20	66.0
      1H	24	16	79.2
      2A	30	10	99.9
      2B	35	5	116.6
      2C	40	0	133.2

      
      
      1. Pipette 1-40 μl of unknown(s) into adjacent wells(s). Add dH₂O to bring each well to 40 μl final volume.
         
      2. Add 200 μl of Pi assay reagent to each well using a repeat pipetor.
         
      3. Incubate at 37 °C for 30 min.
         
      4. Measure A₆₆₀ values and use the Pi calibration (standard) curve to determine Pi content of unknowns.
         
      5. Express the data as: nmol Pi mg^-1 dry weight.
         
         
2. Soluble Pi (Bozzo et al., 2006)
   1. Extract snap-frozen tissues (1:5, w/v) with 1% (v/v) glacial acetic acid.
      
   2. Centrifuge samples at 11,000 x g for 10 min.
      
   3. Assay the supernatant for Pi as described above.
      
   4. Esterified-Pi is calculated from the difference between total and free Pi concentrations.
      

Recipes

1. Acid extraction solution
   30 ml 12 M concentrated HCl
   10 ml 16 M concentrated HNO₃
   60 ml dH₂O
   
2. Pi stock (for standard curve)
   3.3 mM NaH₂PO₄
   
3. Pi assay reagent
   
   1. Ammonium molybdate reagent
      Ammonium molybdate is added to an aqueous solution of 15 mM zinc acetate to give a 10 mM solution of molybdate. The solution is then adjusted to pH 5.0 with HCl. This solution is stored at 4 °C in the dark and is stable for several months.
      
   2. Reducing reagent
      A 10% (w/v) solution of ascorbic acid is adjusted to pH 5.0 with NaOH.
      Note: This solution must be prepared fresh daily.
      
   3. The Pi assay reagent is prepared by mixing one part of the ammonium molybdate reagent with four parts of the reducing reagent (prepare fresh daily).
      

Acknowledgments

Research in our laboratory has been generously funded by research and equipment grants from The Natural Sciences and Engineering Research Council of Canada (NSERC) and Queen’s Research Chairs program to William Plaxton.

References

1. Bozzo, G. G., Dunn, E. L. and Plaxton, W. C. (2006). Differential synthesis of phosphate‐starvation inducible purple acid phosphatase isozymes in tomato (Lycopersicon esculentum) suspension cells and seedlings. Plant Cell Environ 29(2): 303-313. 
   
2. Drueckes, P., Schinzel, R. and Palm, D. (1995). Photometric microtiter assay of inorganic phosphate in the presence of acid-labile organic phosphates. Anal Biochem 230(1): 173-177. 
   
3. Hurley, B. A., Tran, H. T., Marty, N. J., Park, J., Snedden, W. A., Mullen, R. T. and Plaxton, W. C. (2010). The dual-targeted purple acid phosphatase isozyme AtPAP26 is essential for efficient acclimation of Arabidopsis to nutritional phosphate deprivation. Plant Physiol 153(3): 1112-1122.