Materials and Reagents

9-cm-diameter Petri dish
Surgical blade (Swann Morton Surgical Scalpel Blade No. 20)
1.7 ml and 2 ml Eppendorf tubes
Dialysis tubing (Sigma-Aldrich, catalog number: D9777 )
50 ml Falcon tube
Whatman #1 filter paper (GE Healthcare, catalog number: 1001-125 )
Pipette tips
Arabidopsis seeds
Triton X-100 (Sigma-Aldrich, catalog number: X100 )
30% acrylamide/Bis solution (Solarbio, catalog number: A1010 )
TEMED (Sigma-Aldrich, catalog number: T22500 )
Agar (Sigma-Aldrich, catalog number: A1296 )
Bovine serum albumin (BSA)*
95% ethanol*
Ammonium persulfate (NH₄)₂S₂O₈*
Sodium hypochlorite solution (NaClO)*
Sodium hydroxide (NaOH)*
Sodium acetate (NaAc)*
Phosphoric acid (H₃PO₄)*
Hydrochloric acid (HCl)*
Ammonium nitrate (NH₄NO₃)*
Potassium nitrate (KNO₃)*
Calcium chloride (CaCl₂)*
Magnesium sulfate (MgSO₄)*
Potassium dihydrogen phosphate (KH₂PO₄)*
Potassium sulfate (K₂SO₄)*
Potassiu iodide (KI)*
Orthoboric acid (H₃BO₃)*
Manganese sulfate (MnSO₄)*
Zinc sulfate (ZnSO₄)*
Sodium molybdate (Na₂MoO₄)*
Cobalt(II) chloride (CoCl₂)*
Copper sulfate (CuSO₄)*
Fe-EDTA*
Myo Inositol*
Nicotinic acid*
Pyridoxine HCl*
Thiamine HCl*
Glycine*
Sucrose*
Bromophenol blue*
Coomassie Brilliant Blue G-250*
MES (AMRESCO, catalog number: E169 )
β-naphthyl acid phosphate (Sigma-Aldrich, catalog number: N7375 )
Fast Black K (Sigma-Aldrich, catalog number: F7253 )
pNPP (Sigma-Aldrich, catalog number: P4744 )
BCIP (Gold Bio, catalog number: B-500-10 )
Tris base (NOVON SCIENTIFIC, catalog number: ZZ02531 )
DTT (Sigma-Aldrich, catalog number: DTT-RO )
SDS (AMRESCO, catalog number: 0227 )
DMSO (AMRESCO, catalog number: 0231 )
PMSF (Sigma-Aldrich, catalog number: 52332 )
Liquid nitrogen
Murashige and Skoog (MS) medium (P+ and P- MS medium) (see Recipes, Murashige and Skoog, 1962)
APase activity profiling detection buffer, pH 4.9 (see Recipes)
Reaction buffer, pH 4.9 (see Recipes)
pNPP solution (1.0 mg/ml) (see Recipes)
BCIP stock solution (100x) (see Recipes)
Bradford solution (see Recipes)
5x protein loading buffer (see Recipes)

*Note: Similar reagents from any qualified company are suitable for this experiment.

Equipment

Pipettes
Forceps (Fisher Scientific, catalog number: 22-327379 )
Spectrophotometer (WPA, Biowave)
250 ml Erlenmeyer (conical) flask
Orbital shaker (Qilinbeier, model: TS-1 )
Freeze dryer (Beijing Songyuanhuaxing, model: LGJ-10 )
Microfuge (Eppendorf, model: 5415 D )
Microwave oven
Small mortar and pestle
Water bath (Shanghai Yiheng, model: DK-8D )**
Analytical balance (Mettler Toledo, model: PB153-L)**
pH meter (Mettler Toledo, model: FE20 )**
Mini-PROTEAN^® Tetra Vertical Electrophoresis Cell (Bio-Rad Laboratories, model: Mini-PROTEAN^® Tetra Vertical Electrophoresis Cell , catalog number: 1658004)

**Note: Similar equipment from any qualified company is suitable for this experiment.

Software

SPSS Statistics

Procedure

Quantification of root-associated APase activity and the activity of APases released into the growth media
1. Quantification of root-associated APase activity
  1. Arabidopsis seeds are suspended with 5% sodium hypochlorite solution/0.1% Triton X-100 and incubated for 10 min at room temperature with occasionally inverting for surface-sterilization.
  2. Remove the liquid and wash the seeds with sterilized ddH₂O for 4 times.
  3. Place 20-30 surface-sterilized Arabidopsis seeds horizontally on the upper part of each 9-cm-diameter Petri dish containing full-strength or half-strength Murashige and Skoog (MS) medium (P+ or P- medium). For each biological sample, use three replicates.
  4. Keep the Petri dishes at 4 °C for 2 days for stratification.
  5. Place the Petri dishes vertically in a controlled environment growth room and allow the seedlings to grow for 6-7 days under a 16-h light/8-h dark regime (90 µmol photons m^-2 sec^-1) at 22-23 °C.
  6. Excise roots with a razor blade and measure root length. The average root length of the Arabidopsis seedlings grown on P+ medium is 4.0-5.5 cm and that grown on P- medium is 2.5-4.0 cm.
  7. Immerse two roots with forceps into 650 µl of pre-warmed (37 °C) reaction buffer in a 1.7 ml Eppendorf tube.
  8. Quickly add 50 µl of pNPP solution.
  9. Incubate at 37 °C for 1-4 h.
  10. Add 100 µl of 0.4 N NaOH and shake vigorously by hand to stop the reaction.
  11. Remove the roots with forceps and measure the absorbance at 410 nm (A₄₁₀) in the remaining solution with a spectrophotometer.
  12. Calculate the root-associated APase activity as A₄₁₀/cm root/h (Figure 1A).
    
    Figure 1. Analysis of APase activity secreted by Arabidopsis seedlings of WT and various single, double, and triple pap (purple acid phosphatase) mutants grown in or on P+ or P- medium. Root-associated APase activities (A) and APase activities in the medium (B) were determined with pNPP as the substrate. Values are means with SE. A one-way ANOVA analysis was carried out for the whole data set, and post hoc comparisons were conducted using the SPSS Tukey HSD test at P < 0.05. Within each of the four plots, values with different letters are significantly different. Adapted from Wang et al. (2014).
2. Concentrating the proteins in the liquid growth medium
  1. Transfer about 150 9-day-old seedlings that were grown vertically on P+ solid medium with autoclaved forceps to a 250 ml autoclaved conical flask containing 100 ml of P+ or P- liquid medium.
  2. Place the flasks on an orbital shaker (40 rpm/min) and allow the seedlings to grow for 6-7 days under a 16-h light/8-h dark regime (90 µmol photons m^-2 sec^-1) at 22-23 °C.
  3. Remove all of the seedlings from the flasks and freeze the liquid medium at -20 °C.
  4. Put the medium in the holder of a pre-cooled freeze dryer, cover the lid, and lyophilize the medium for 3-4 days.
  5. Thoroughly wash the bottom of the flask using 5-6 ml of ddH₂O and transfer the washing solution to dialysis tubing.
  6. Dialyze the sample for 24 h at 4 °C against 4 L of pre-cooled ddH₂O and change the ddH₂O every 6 h.
  7. Transfer the dialyzed sample to a 2 ml tube and centrifuge for 5 min at 1,500 x g.
  8. Transfer the supernatant to a 50 ml Falcon tube and freeze the liquid at -20 °C, then lyophilize for 2 days as described in step A2d.
  9. Dissolve the lyophilized sample in 200 µl of ddH₂O.
  10. Measure the protein concentration using the Bradford method as described in step A3.
  11. The concentrated, secreted proteins can be used immediately or can be stored at -80 °C for further analysis.
3. Determination of protein concentration
  1. Pipette 0, 2, 4, 8, 12, 16, or 20 μl of 1 μg/μl BSA into 1.7 ml Eppendorf tubes and add ddH₂O to a final volume of 20 μl.
  2. Add 980 μl of Bradford solution to each tube and mix well.
  3. Incubate at room temperature for 5 min.
  4. Measure the absorbance at 595 nm (A₅₉₅) with a spectrophotometer.
  5. Make a Bradford standard curve by plotting BSA content and A₅₉₅.
  6. Add 20 μl of concentrated secreted proteins to 980 μl of Bradford solution.
  7. Mix well and incubate at room temperature for 5 min.
  8. Measure the A₅₉₅ with a spectrophotometer.
  9. Compare the resulting A₅₉₅ value with the Bradford standard curve to determine the protein concentration of various samples.
4. Quantification of APase activity in the growth medium
  1. Add 10 µl of concentrated proteins (about 2.5 µg) collected from the liquid growth medium to 590 µl of reaction buffer in a 1.7 ml Eppendorf tube. For each sample, make a least three replicates.
  2. Add 80 μl of pNPP solution to the reaction mixture.
  3. Incubate at 37 °C for 1 h.
  4. Add 120 μl of 0.4 N NaOH to terminate the reaction.
  5. Measure the A₄₁₀ with a spectrophotometer.
  6. Calculate the APase activity in the growth medium as A₄₁₀/mg protein/min (Figure 1B).
Analysis of root-associated APase activity using histochemical staining
1. Grow seedlings vertically on P+ or P- MS medium for 6-7 days as described in steps A1a-A1e.
2. Add 100 ml of ddH₂O and 0.5 g agar to a 250 ml conical flask, gently shake the flask, and then heat the flask in a microwave oven until the agar is completely dissolved.
3. Add 1 ml of the BCIP stock solution (100x) and shake briefly by hand.
4. Keep the BCIP/agar solution at room temperature or use flowing water to cool the solution for a few minutes to about 40 °C.
5. Keep the BCIP/agar solution in a 40 °C water bath.
6. Uniformly overlay about 5 ml of the BCIP/agar solution on one row of the Arabidopsis roots and incubate at room temperature for about 24 h (Figure 2).
  
  Figure 2. A demonstration of how to overlay the BCIP/agar solution on the root surface of Arabidopsis seedlings grown on Petri dish
7. The roots of Arabidopsis grown on P- medium will be stained blue, while these grown on P+ medium will remain unstained (Figure 3).
  
  Figure 3. Histochemical staining of root-associated APase activity. Upper row: The morphologies of 7-day-old Arabidopsis seedlings grown on MS P+ and P- medium without BCIP staining. Bottom row: Seedlings whose roots were stained with a 0.01% BCIP/0.5% agar solution for 24 h. In the lower row, root-associated APase activity (as indicated by the blue color) was detected on roots of seedlings grown on P- medium (bottom right) but not on roots of seedlings grown on P+ medium (bottom left).
8. APase histochemical staining is greatly affected by the BCIP concentration. A high concentration of BCIP (0.08%-0.16%) can cause over-staining, which may mask differences in the level of secreted APase activity (Figure 4).
  
  Figure 4. The effect of different concentrations of BCIP on staining of root-associated APase activity of 7-day-old Arabidopsis seedlings grown on MS P- medium. The concentrations of BCIP used are indicated at the left bottom corner. nop1-1 and nop1-2 are Arabidopsis mutants that have mutations in the AtPAP10 gene. With the increase in BCIP concentration, the activities of other root-associated APases, mainly AtPAP12 and AtPAP26, are also detected. Adapted from Wang et al. (2011).

Analysis of profiles of APase isoforms released into the growth medium (in-gel assay)
The isozyme profile of APases released into the growth medium is visualized by separating protein samples on SDS-PAGE (denaturing gel) or Native-PAGE (non-denaturing gel), which are then stained with the chromogenic substrates β-NAP and fast black K. For Native-PAGE analysis, omit SDS from the gel and running buffer.
Prepare a 10% separating gel and a 5% stacking gel (SDS-PAGE) according to the following Table 1.

Table 1. Recipes for separating gel and stacking gel

Stock solution	5% stacking gel (5 ml)	10% separating gel (10 ml)
30% Acrylamide/Bis solution	0.83 ml	3.3 ml
1.5 M Tris-HCl, pH 8.8	-	2.5 ml
1 M Tris-HCl, pH 6.8	0.63 ml	-
10% SDS	0.05 ml	0.1 ml
10% (NH₄)₂S₂O₈	0.05 ml	0.1 ml
TEMED	0.005 ml	0.004 ml
ddH₂O	3.40 ml	4.0 ml

Set up the SDS-PAGE gel running system according to the Bio-Rad instructions (http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf).
Add 1x protein running buffer that was pre-cooled to 4 °C.
Thoroughly mix 2.5 μg of secreted proteins with 1/5 volume of 5x protein loading buffer and load the sample on the SDS-PAGE gel.
Note: Do not heat the sample.
Separate proteins at 60 V for 30 min followed by 120 V for 150 min (both at 4 °C).
Carefully transfer the gel to pre-cooled (4 °C) ddH₂O in a container and gently shake for 10 min to remove SDS from the gel.
Change pre-cooled (4 °C) ddH₂O and repeat 4 times.
Add 50 ml of reaction buffer and shake gently for 15 min at 4 °C.
Change reaction buffer and repeat 4 times.
Add 50 ml of APase activity profiling detection buffer and incubate at 37 °C for 1-3 h with gentle agitation. The isozyme profile of APases is visualized as the red-brown bands on the gel (Figure 5).

Figure 5. Profiles of APase activity in the growth medium. A 2.5-μg quantity of secreted protein was separated by SDS-PAGE (A) or Native-PAGE (B) and stained with 0.3 mg/ml β-NAP and 0.5 mg/ml Fast Black K. Protein molecular markers are on the left side of the gel in (A). The numbers on the right of each panel indicate different APase isoforms. Adapted from Wang et al. (2014).

Notes

The temperature of BCIP/agar solution is important for detecting root-associated APase activity. If the solution is too hot (above 60 °C), it will damage Arabidopsis roots and inactivate the APase on the root surface.
Removal of SDS is critical for obtaining a clear profile of APase isozymes.

Recipes

Murashige and Skoog (MS) medium (P+ and P- MS medium)
P+ MS medium is the full-strength MS medium (Murashige and Skoog, 1962). The recipe for full-strength MS medium is shown below. For P- MS medium, the 1.25 mM KH₂PO₄ in P+ MS medium is replaced with 1.25 mM K₂SO₄. Autoclave P+ and P- medium at 121 °C for 10 min, and store the medium at room temperature

NH₄NO₃	20.62 mM	CuSO₄	0.1 µM
KNO₃	18.79 mM	Fe-EDTA	100 µM
CaCl₂	2.99 mM	Myo inositol	0.55 µM
MgSO₄	1.5 mM	Nicotinic acid	4.1 µM
KH₂PO₄	1.25 mM	Pyridoxine HCl	2.4 µM
K₂SO₄	0.625 mM	Thiamine HCl	0.3 µM
KI	5 µM	Glycine	30 µM
H₃BO₃	100 µM
MnSO₄	92.5 µM	Sucrose	10 g/L
ZnSO₄	30 µM	MES	5.1 g/L
NaMoO₄	1 µM	pH	5.8
CoCl₂	0.1 µM	Agar	10 g/L

APase activity profiling detection buffer, pH 4.9 (freshly prepared)
0.3 mg/ml β-naphtyl acid phosphate
0.5 mg/ml Fast Black K
10 mM MgCl₂
50 mM NaAc
Reaction buffer, pH 4.9
10 mM MgCl₂
50 mM NaAc
Store at 4 °C
pNPP solution (1.0 mg/ml)
A 10-mg quantity of pNPP is dissolved in 10 ml of reaction buffer
Store aliquots at -20 °C
BCIP stock solution (100x)
The concentration of the BCIP stock solution is 10 g/L, and that of the working solution is 0.1 g/L
To prepare the BCIP stock solution:

Dissolve 0.1 g of BCIP powder in 8 ml of ddH₂O and then add approximately 14 drops of 1 N NaOH until the BCIP power is totally dissolved
Add ddH₂O to a total volume of 10 ml and then filter the solution to remove un-dissolved BCIP particles
Store aliquots at -20 °C

Bradford solution
Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol
Add 100 ml of 85% H₃PO₄ and then add ddH₂O to 1,000 ml
Pass through a Whatman #1 filter paper, and store the filtrate at 4 °C in a brown bottle
5x protein loading buffer
250 mM Tris-HCl, pH 6.8
10 mM DTT
10% SDS
30% (v/v) glycerol
0.05% (w/v) bromophenol blue
Store the buffer at room temperature

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant Nos. 31370290 and 30670170).

References

Lloyd, J. C., Zakhleniuk, O. V. and Raines, C. A. (2001). Identification of mutants in phosphorus metabolism. Annals Applied Biol 138: 111-115.
Murashige, T and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum 15(3): 473-497.
Tomscha, J. L., Trull, M. C., Deikman, J., Lynch, J. P. and Guiltinan, M. J. (2004). Phosphatase under-producer mutants have altered phosphorus relations. Plant Physiol 135(1): 334-345.
Tran, H. T., Hurley, B. A. and Plaxton, W. C. (2010). Feeding hungry plants: The role of purple acid phosphatases in phosphate nutrition. Plant Sci 179: 14-27.
Trull, M. C. and Deikman, J. (1998). An Arabidopsis mutant missing one acid phosphatase isoform. Planta 206(4): 544-550.
Vicki, K. and William, P. (2013). Protein extraction, acid phosphatase activity assays, and determination of soluble protein concentration. Bio-protocol e889.
Wang, L., Li, Z., Qian, W., Guo, W., Gao, X., Huang, L., Wang, H., Zhu, H., Wu, J. W., Wang, D. and Liu, D. (2011). The Arabidopsis purple acid phosphatase AtPAP10 is predominantly associated with the root surface and plays an important role in plant tolerance to phosphate limitation. Plant Physiol 157(3): 1283-1299.
Wang, L., Lu, S., Zhang, Y., Li, Z., Du, X. and Liu, D. (2014). Comparative genetic analysis of Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation. J Integr Plant Biol 56(3): 299-314.