Measurement of ³³P-PO₄ Absorption Capacity and Root-to-leaf Transfer in Arabidopsis

Materials and Reagents

1. Plastic 12-well plates (Denmark, Nunc)
   Note: One for the absorption step, one for the desorption step for each treatment (plant type or culture condition).
   
2. Plastic 20 ml vials for radioactivity measurement (Ratiolab GmbH, Dreieich)
   Note: You will need one vial per plant, or 2 vials per plant if you want to quantify ³³P in both roots and leaves. The vials should be numbered from 1 to N before you start the experiment. They also should be placed in order in appropriate racks (PerkinElmer) adapted to the beta counter.
   
3. Tips
   
4. Young in vitro plantlets
   
5. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
   
6. CaCl₂ (Sigma-Aldrich)
   
7. KH₂PO₄ (Sigma-Aldrich)
   
8. ³³P-PO₄ 5 mCi/ml (40-158 Ci/mg, 1.48-5.84 TBq/mg, >99% isotopically pure, less than 0.5 μM Pi) (PerkinElmer)
   
9. Scintillation cocktail (PerkinElmer, Ultima Gold^TM)
   
10. MgSO₄
    
11. NH₄NO₃
    
12. KNO₃
    
13. NaH₂PO₄
    
14. KI
    
15. FeCl₂
    
16. MnSO₄
    
17. ZnSO₄
    
18. CuSO₄
    
19. CoCl₂
    
20. Na₂MoO₄
    
21. Thiamine
    
22. Pyridoxine
    
23. Nicotinic acid
    
24. Inositol
    
25. Sucrose
    
26. Agar
    
27. MS/10 medium (see Recipes)
    
28. Stock solution (see Recipes)
    
29. Incubation medium (see Recipes)
    
30. Desorption medium (see Recipes)
    

Equipment

1. Experiments should be performed on a bench or under a hood illuminated with white light (150 -180 μE m^-2 s^-1) during the incubation step
   
2. Liquid scintillation counter (PerkinElmer, Packard Instrument Company, model: TRI-CARB )
   
3. Ice-containing large boxes for the desorption step (all 12-well plates will be placed horizontally on ice for 2 h)
   
4. Tweezers for handling the plantlets
   
5. If necessary, a razor to separate roots and aerial parts
   
6. Micropipets
   
7. Shield for protection against radiations (plexiglass)
   
8. Scanner or camera (Epson America, model: Perfection V850Pro or Canon, model: Powershot SX130 ), respectively but other devices from other manufacturers could suit perfectly
   

Software

1. ImageJ version 1.46r with NeuronJ plugin (http://imagej.nih.gov/ij)

Procedure

1. Preparation of plant materials
   9 to 11 day-old plantlets are convenient for the experiment.
   
   1. When plating, space the seeds in order to maintain root systems independent for every single plantlet; this will help when measuring the length of the root system (see below). 6 to 10 seeds are sown per plate depending on the growth medium (respectively with high or low Pi, see below). 2 to 3 plates will be necessary per plant type (genotype or treatment depending on the experiment).
      
   2. Plants are grown vertically in 12 x 12 cm Petri plates in modified MS/10 medium.
      
   3. Number the plants on each Petri plate, in order to identify each root system individually.
      
   4. 10 to 12 replicates per treatment or genotype will be necessary plus 5 to 10 plants for blanks.
      
   5. Scan or photograph the plates (in black and white and jpg format). Use graph paper as scale bar for measurement of the root length. Scan should be 300 dpi or photos should be about 1,000 x 800 pixels (this is recommended by ImageJ).
      
   6. Root length measurement and calculation: We use ImageJ version 1.46r with NeuronJ plugin (http://imagej.nih.gov/ij). Figure 1 shows a photo of a plate, and how root length is measured with imageJ. The scale (1 cm using the graph paper) is measured (Figure 1A), and then with NeuronJ plugin the primary and lateral roots are traced (pink trace, Figure 1B). Values (in cm) are transferred in an Excel file. Total root length is calculated by adding primary and lateral root lengths (see detailed protocol in Figure 1 legend).
      
      
      Figure 1. Root length measurement. A. Set the scale: with ImageJ, trace a 1 cm scale on the graph paper (1, 2) then ‘set scale’ (3). This gives pixels/cm (4). B. Measurement of the root length: with NeuronJ plugin, load the image file (5) then set scale (as defined in A). Add tracings (6) by drawing the along the roots (pink trace, 7) then measure tracings (RUN, 8). Results of root length in cm (9) can be copied to an excel file. A and B are screenshots of the process.
   
   
2. Incubation
   
   1. This step should be performed behind a shield for protection against radiation.
      
   2. Before starting a series of experiments, you must check that Pi absorption is linear in your conditions (plant specificity, temperature, light). To do that, a time course is performed between 30 min and 2 h following the protocol as described below. In our conditions, Pi absorption was always linear.
      
   3. Prepare 12-well plates, one for each plant treatment. Number the wells: 1 to 12.
      
   4. Add 4 ml of incubation medium per well. Place 1 plant per well with tweezers (Figure 2), the roots should be immersed and the leaves outside the well. To avoid excessive dehydration of the young plantlets during the incubation, carefully put a cover on the plate avoiding the immersion of the leaves in the solution (Figure 2B). Incubate for 2 h at room temperature (22-24 °C) under white light (150-180 μE m^-2 s^-1). Discard plants that are fully immersed in the liquid, if any.
      
      
      Figure 2. Incubation experiment. A. Photo of a 12-well plate with plants numbered 1 to 12 in the incubation medium. The cover plate has been removed for clarity. Please note that rosettes are out of the medium and roots are fully immersed. B. Photo of the plate and the cover (side view) showing how the cover is placed onto the 12-well plate.
      
      
   5. In order to evaluate Pi adsorption on the root (mechanical or chemical adsorption can occur on cells but in that case, Pi does not enter inside the root cells), blank samples are treated as follows: The plant root is dipped in the incubation medium for 2 sec (use tweezers) and directly transferred in the desorption medium for 2 h (see below for the desorption procedure).
      
   
   
3. Desorption (see Video 1)
   
   
   
   Video 1. Desorption
   
   
   1. Prepare new 12-well plates, with 3 ml of cold desorption medium per well. Place the plates on ice.
      
   2. Rinse the incubated plants in water (1-2 sec).
      
   3. With the help of tweezers, transfer the plants (in the same order you put them in the incubation solution, 1 plant/well) with both leaves and roots immersed in the desorption medium for 2 h on ice.
      
   4. Transfer each plant in a counting vial (use tweezers). Alternatively, separate the rosette and roots with a sharp razor and put each part of the plant in a separate vial. It is convenient to put the vials in the counting racks as soon as you harvest the plants (in order to avoid mismatches).
   
   
4. Dry the plants in an oven at 50 °C overnight
   
   
5. Radioactivity measurement
   
   1. Under a chemical hood, add 2 ml of scintillation cocktail and tighten the top on each counting vial. Then place the vials in racks adapted to the counter.
      
   2. With a beta counter, count ³³P in each sample (Cs) and in blanks (Cb is a mean of the blank replicates) and also in the incubation medium (C₁₀, 10 μl of incubation medium are placed in a counting vial and 2 ml scintillation cocktail are added). In the scintillation cocktail, beta radiations are transformed in photons that are detected by the beta counter. The measurement is in cpm (count per min).
      
   3. Data analysis:
      The amount of PO₄ absorbed per hour per root cm (V in nmol/h/cm) is calculated as follows:
      V = (Cs-Cb) * (S *10^-3)* B / (T * L_root * C₁₀)
      Cs: Radioactivity in the sample (cpm)
      Cb: Mean value of radioactivity in the blanks (cpm)
      S: Pi concentration in the incubation medium (S=50 μM Pi).
      S*10^-3=0.05: Pi content (nmol/μl) in the incubation medium (S=50 μM).
      C₁₀: Radioactivity in 10 μl of the incubation solution (cpm). C₁₀/(S*10^-2) is the specific activity of ³³P in the incubation medium
      B=10: Volume of the incubation solution for measurement of radioactivity before incubation of the plants (10 μl is convenient).
      T=2: Incubation is 2 h
      L_root: Root length (cm)
      In addition, using the same equation as above, but omitting L_root, you can calculate Pi absorption per plant (Cs corresponds to 33P in the whole plant), per rosette tissue or root tissue (Cs corresponds to 33P in the leaves or the root system, respectively).
      It is also possible to calculate the ratio of ³³P (Cs in leaves in cpm) measured in the leaves compared to total ³³P absorbed by the whole plant [Cs in leaves + Cs in roots (cpm)].

Representative data

Pi uptake per hour and per root cm for plants grown on low or high Pi (Table 1). Two genotypes are presented (WT and mutant) in order to show the range of variation of Pi uptake between plants and the distribution of Pi in the plant.

Table 1. Pi uptake and distribution in WT and mutant plantlets grown on low or high Pi. Pi uptake capacity is expressed as nmol Pi per hour per root cm (mean±SD). Pi distribution is calculated from the ratio of ³³P in leaves compared to ³³P in the whole plant.

Recipes

1. MS/10 medium
   10x time diluted Murashige and Skoog medium containing:
   0.15 mM MgSO₄
   2.1 mM NH₄NO₃
   1.9 mM KNO₃
   0.5 (high Pi) or 0.005 (low Pi) mM NaH₂PO₄
   0.34 mM CaCl₂
   0.5 μM KI
   10 μM FeCl₂
   10 μM H₃BO₃
   10 μM MnSO₄
   3 μM ZnSO₄
   0.1 μM CuSO₄
   0.1 μM CoCl₂
   1 μM Na₂MoO₄
   5.9 μM thiamine
   4.9 μM pyridoxine
   8.1 μM nicotinic acid
   55 μM inositol
   3.4 mM MES
   0.5% sucrose and 0.8% agar at pH 5.7
   
2. Stock solution
   0.1 mM CaCl₂ in 5 mM MES, adjusted at pH 5.5
   For 1 L (water): 0.976 g MES + 0.0147 g CaCl₂, adjust pH to 5.5 with 10 N NaOH
   
3. 1 M KH₂PO₄
   For 1 L (water): 136 g KH₂PO₄
   
4. Incubation medium (4 ml/plant are necessary)
   50 µM KH₂PO₄ in stock solution: 50 µl 1 M KH₂PO₄ in 1 L stock solution
   Add 5,550 Bq ³³P/ml (0.15 µCi ³³P/ml)
   
5. Desorption medium (3 ml/plant are necessary)
   1 mM KH₂PO₄ in stock solution: 1 ml 1 M KH₂PO₄ in 1 L stock solution
   

Acknowledgments

This protocol was adapted from the previously published studies: Narang et al. (2000) and Misson et al. (2004) and it was performed by Ayadi et al. (2015). This work was supported by the Commissariat à l’Energie Atomique et aux Energies Alternatives.

References

1. Ayadi, A., David, P., Arrighi, J. F., Chiarenza, S., Thibaud, M. C., Nussaume, L. and Marin, E. (2015). Reducing the genetic redundancy of Arabidopsis PHOSPHATE TRANSPORTER1 transporters to study phosphate uptake and signaling. Plant Physiol 167(4): 1511-1526.
   
2. Misson, J., Thibaud, M. C., Bechtold, N., Raghothama, K. and Nussaume, L. (2004). Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants. Plant Mol Biol 55(5): 727-741.
   
3. Narang, R. A., Bruene, A. and Altmann, T. (2000). Analysis of phosphate acquisition efficiency in different Arabidopsis accessions. Plant Physiol 124(4): 1786-1799.