Materials and Reagents

Arabidopsis thaliana seeds (Col-0)
Eppendorf tubes (1.5 ml)
Commercial Bleach
Pipettes and tips (1 and 5 ml)
Silicon caps (or aluminum foil)
Square Petri dishes
Plastic wrap
Spatula and forceps
Flasks (200 ml)
Plastic wrap
Glass culture tubes
Tween-20 (Sigma-Aldrich, catalog number: P-1379 )
Murashige and Skoog medium (MS) (PhytoTechnology Laboratories^®, catalog number: M524 )
Sucrose (VWR International, catalog number: BDH-0308 )
Potassium Hydroxide (KOH) (Thermo Fisher Scientific, catalog number: P-250 )
Agar-agar (Sigma-Aldrich, catalog number: A-1296 )
Sterile distilled water
MES hydrate (Sigma-Aldrich, catalog number: M-8250 )
Seed sterilization solution (see Recipes)
MS solid (see Recipes)
MS liquid (see Recipes)
Assay solution (see Recipes)

Equipment

Autoclave
Rotary shaker
Flow hood
Fridge (4 °C)
Plant growth chamber (Conviron, catalog number: ATC26 )
Magnetic stirrer and stirring bars
pH meter (Beckman Coulter)
Note: pH probe should be capable of measuring pH in samples with volumes <= 3 ml.
Balance

Procedure

Seed sterilization
1. Put 5 mg (200-300 seeds) of Arabidopsis seeds into an Eppendorf tube and add 750 µl sterilization solution.
2. Vortex for 15 min at room temperature.
3. Decant the sterilization solution (with sterile tips) under flow hood and add 750 µl sterile water.
4. Vortex briefly.
5. Repeat steps c and d 3 times.
6. Stratify the seeds in 750 µl sterile water at 4 °C for 2 d in the dark.
Plating seeds (under flow hood)
1. Sow seeds (about 10) on sterile solid MS plates. Keep a density of approximately one seed per cm². Crowded plates produce unhealthy seedlings.
2. Close and seal plates with plastic wrap. Use only one layer of plastic wrap. More than one layer prevents gaseous exchange and healthy plant growth.
3. Incubate plates in a vertical position in a growth chamber at 21 °C with a 12-h-light/12-h-dark cycle (150 μmol m^-2 s^-1) (Forde, 2000). Vertical growth reduces root stress and prevents agar transfer when transplanting seedlings.
4. Grow seedlings for 5-7 d.
Plant growth in liquid media
1. Prepare 200 ml flasks with 4 ml liquid MS each.
2. Close flasks with a “silicon” cap (or with aluminum foil).
3. Autoclave flasks at 121 °C for 20 min.
4. Let flasks cool in flow hood.
5. Transfer 10 seedlings into each flask. Put seedlings in the bottom with the help of sterile spatula and forceps. Be sure that the seedlings stay in contact with the media.
6. Close flasks with a layer of plastic wrap. Transparent plastic wrap favors transmission of light, promotes uniform plant growth, and prevents media evaporation.
7. Grow plants on a rotary shaker at 50 rpm in chamber at 21 °C with a 12-h-light/12-h-dark cycle (150 μmol m^-2 s^-1) for 2 weeks.
8. Pay attention: usually seedlings consume all the liquid media before the 2-week period. In this case, add 4 ml sterile liquid MS.
Acidification assay
1. Empty flasks and wash roots with the assay solution (5 ml) for 5 min.
2. Empty flasks again and add fresh assay solution (3 ml).
3. Prepare a control flask with assay solution (3 ml), but without any plants.
4. Close flasks with plastic (transparent) foil and keep them in the growth chamber for 6 h.
5. Transfer all the 3 ml of assay solution from flasks to a glass tube and proceed to pH measurements.
6. Take out plants from flasks and check the number of plants in each flask.
7. Dry plants with paper towels, cut roots from shoots, and immediately weigh to avoid plant dehydration, and the concomitant weight loss. Root and shoot weights are important values because we can either calculate protons released per gram of root or seedling.
Calculation
1. Use pH values from each flask to compute [H⁺] (mole/L) in sample and control using the equation pH=-log [H⁺].
2. Calculate the number of H⁺ moles in 3 ml of experimental and control samples [mole/ 3 ml].
3. Compute crude H⁺ released to the medium by subtracting the average values of H⁺_control from H⁺_sample. A typical experiment requires from 3 to 5 independent replicates.
4. Relativize average number of H⁺ moles to either plant number, or to root or plant fresh weight. The units are going to be either mole/plant or mole/g. These relativized values allow us to calculate an average from several flasks and also to compare flasks with different plant numbers or plant lines.

Representative data

Figure 1. Protons released from the roots during day and night hours by Col-0 (empty bars) and transgenic plants overexpressing the type I H⁺-PPase AVP1 (AVP1-1; Pizzio et al., 2015) (black bars) plants grown in liquid media (mean ± SE; n= 6 pools of 10 plants per line, per time of day, per trial; two independent trials)

Recipes

Seed sterilization solution
30% (v/v) commercial bleach
0.05% (v/v) Tween-20
Murashige and Skoog (MS) solid
One-half-strength MS
1% [w/v] Sucrose
pH 5.7 with KOH
1% [w/v] agar
MS liquid
One-half-strength MS⁺
1% [w/v] Sucrose (pH 5.7 with KOH)
Assay solution
One-quarter-strength MS
2 mM MES buffer (pH 6.8 with KOH). Low MES concentration assures similar starting pH for all plants.

Acknowledgments

RAG, GAP, and KR were supported by the National Science Foundation (grant no. IOS-1122148). This protocol was adapted from Pizzio et al. (2015).

References

Forde, B. G. (2000). Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta 1465(1-2): 219-235.
Hawes, M. C., Bengough, G., Cassab, G. and Ponce, G. (2003). Root caps and rhizosphere. J Plant Growth Regul 21: 352-367.
Miller, A. J. and Smith, S. J. (1996). Nitrate transport and compartmentation in cereal root cells. Journal of Experimental Botany, Vol. 47, No. 300, pp. 843-854.
Pizzio, G. A., Paez-Valencia, J., Khadilkar, A. S., Regmi, K., Patron-Soberano, A., Zhang, S., Sanchez-Lares, J., Furstenau, T., Li, J., Sanchez-Gomez, C., Valencia-Mayoral, P., Yadav, U. P., Ayre, B. G. and Gaxiola, R. A. (2015). Arabidopsis type I proton-pumping pyrophosphatase expresses strongly in phloem, where it is required for pyrophosphate metabolism and photosynthate partitioning. Plant Physiol 167(4): 1541-1553.
Shen, J., Li, C., Mi, G., Li, L., Yuan, L., Jiang, R. and Zhang, F. (2013). Maximizing root/rhizosphere efficiency to improve crop productivity and nutrient use efficiency in intensive agriculture of China. J Exp Bot 64(5): 1181-1192.