Na+ distribution analysis
To visualize the Na+ distributions in the lateral
roots and leaf protoplasts of sitl1 and WT, a
Na+-specific fluorescent dye, CoroNa-Green AM
(Invitrogen, USA) was used (Gay & Gebicki, 2000). For the lateral root
staining, seedlings were grown under half-strength NS for 1 week and
treated with 50 mM NaCl for 3 h. Each root was detached and washed 3
times with distilled water and stained with 50 µM CoroNa-Green AM for 16
h. Samples were washed again and incubated with the lipophilic probe
FM4-64 (Thermo Fisher Scientific, USA) for 5 h and re-washed 3 times.
Images of the intercellular Na+ fluorescence and
vacuolar membrane were captured using a confocal microscope.
For the Na+ distribution in protoplasts, the leaf
protoplasts were isolated from 1-week-old rice seedlings as described
(Park, Lee, Lee, Byun, & Kim, 2009). After the enzymatic digestion, WI
buffer (0.5 M mannitol, 4 mM MES, and 20 mM KCl, pH 5.7) was used to
avoid the effect of a high concentration of Na+ in W5
buffer for protoplast incubation (Lim, Lee, & Jang, 2014). Isolated
protoplasts were incubated with WI buffer containing 0 and 50 mM NaCl
for 1 h and then washed 3 times with WI buffer. The CoroNa-Green AM was
added to the samples for a final concentration of 10 µM. After 5h, the
protoplasts were re-washed with WI buffer and the fluorescence was
captured using a confocal microscope. CoroNa-Green AM and FM4-64 were
excited at 488 nm and 543 nm with a laser, respectively, and the
fluorescence emission was collected at 510 nm (range 50nm) for
CoroNa-Green and 612 nm (rage 50 nm) for FM4-64. Average fluorescence
intensity of the CoroNa-Green in the cytosol and the vacuole was
measured using imageJ software as described (Zhang et al., 2012).