Autoradiography of Pi Distribution in Barley Seedlings

Materials and Reagents

1. Barley seedlings
   
2. Radioisotopes ³²P or ³³P labeled NaH₂PO₄ dissolved in water (MP Biomedicals, PerkinElmer or American Radiolabeled Chemicals)
   
3. 5 mM CaSO₄ solution
   
4. Hydroponic culture solution (see Recipes)
   

Equipment

1. Cling film
   
2. 1.5 ml plastic tubes
   
3. 15 ml plastic tubes (1.5 ml tube is fitted by opening a hole in the lid) (Figure 1)
   
4. Cotton
   
5. Plastic sponge
   
6. Imaging plate (FCR Imaging Plate for general purpose) (Fujifilm Corporation) and plate cassette (FCR standard cassette) (Fujifilm Corporation)
7. Imaging analyzer (GE Healthcare, model: Typhoon 9400 or other Radioisotope imaging analyzers)
   

Procedure

1. Barley plants are germinated on moist filter paper for 2-3 days and then seedlings are grown in hydroponic culture for 7-8 days.
   
2. Cotton is put in a 1.5 ml tube from which the cap has been removed, then a radioisotope medium consisting of 600 µl of 0.2  mM NaH₂³²PO₄ (specific activity 3.7 MBq nmol⁻¹) in 5 mM CaSO₄ is added. Cotton is put enough to absorb all 600 µl NaH₂³²PO₄, But, do not put too much cotton to keep it moisture.
   
3. Into the 15 ml tube, an appropriate amount of incubation medium (5 mM CaSO₄) is added. A barley plant sandwiched with sponge is put into the medium, and the barley leaf is manipulated into position against the plastic sponge separated from solution, such that when the smaller tube containing the cotton is mounted into a hole in the cap of the 15 ml tube, the tip of the leaf comes into contact with the cotton soaked in radioactive medium (Figure 1).
   
   
   Figure 1. Setup for the radioisotopic labelling of a leaf tip
   
   
4. After an appropriate labelling period (about 2 h) at 25 degrees, the sample is washed with water several times and wrapped with cling film. The sample is then set on an imaging plate and exposed (Figure 2).
   
   
   Figure 2. Setup of the radiolabeled sample on an imaging plate
   
   To avoid contamination of imaging plate with ³²P, samples are wrapped by cling film. Wrapped sample is attached to the imaging plate by using binding case to expose.
   
   
5. Examine the imaging plate with an imaging analyzer (Figure 3). ³²P concentration is indicated by pseudo-color.
   
   
   Figure 3. Autoradiogram of ³²P distribution in a barley plant which was radiolabeled from the leaf tip
   
   
6. Following imaging, plants can continue to be incubated in 5 mM CaSO₄ solutions under the same light conditions for 1 or 2 d without cap in order to examine the movement of the radioisotope. In Figure 3 it can be seen that NaH₂³²PO₄ absorbed at the leaf tip was translocated to the root after one day of incubation.
   Figure 4 shows autoradiograms taken every 2 days following the initial incubation of roots in a Pi-deficient barley plant with an appropriate amount of incubation medium mixed NaH₂³²PO₄ (Mimura et al., 1996)
   
   
   Figure 4. Autoradiogram of ³²P distribution in 10 days old barley plant grown in a Pi-deficient medium, which was radiolabeled from the root. The number put at the leaves in order of development.
   

Notes

1. Resolution and clarity of images are strongly dependent on the radioisotope contents of plants and exposure time on the imaging plate.

Recipes

1. Hydroponic culture solution
   9 mM KNO₃
   6 mM Ca (NO₃)₂
   3  mM MgSO₄
   1.5  mM KH₂PO₄
   0.125 mM Fe-EDTA
   Micronutrients: 10 µM MnSO₄, 1 µM CuSO₄, 1 µM ZnSO₄, 30 µM H₃BO₃, 30 µM (NH₄)₆Mo₇O₂₄, 0.1  µM CoCl₂
   

Acknowledgments

This protocol was adapted from the following publications, Nagai et al. (2013) and Mimura et al. (1996). This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology and Japan Society for the Promotion of Science (JSPS), CREST of JST (Japan Science and Technology Corporation) and in part by Hyogo Science and Technology Association. The authors also thank Yokogawa Analytical Systems Inc. for use of ion chromatography equipment.

References

1. Mimura, T., Sakano, K. and Shimmen, T. (1996). Studies on the distribution, re‐translocation and homeostasis of inorganic phosphate in barley leaves. Plant Cell Environ 19(3): 311-320.
   
2. Nagai, M., Ohnishi, M., Uehara, T., Yamagami, M., Miura, E., Kamakura, M., Kitamura, A., Sakaguchi, S., Sakamoto, W., Shimmen, T., Fukaki, H., Reid, R. J., Furukawa, A. and Mimura, T. (2013). Ion gradients in xylem exudate and guttation fluid related to tissue ion levels along primary leaves of barley. Plant Cell Environ 36(10): 1826-1837.