Fig. 3

In vivo analysis of the NADP(H) redox state in the cytosol and chloroplasts of leaf cells and roots.

a Top, constructs for stable NERNST expression in the cytosol or chloroplasts of plants under control of the P_35S promoter. TP: plastid-targeting sequence; L1: 30-aa linker. Bottom, estimation of E_NADP(H) in the chloroplasts and cytosol of illuminated Arabidopsis and N. tabacum leaf cells. Potentials were calculated assuming pH of 7.3 and 8.0 for cytosol and stroma, respectively⁶⁰. Data shown are means ± SD of 131–253 cells. ****P ≤ 0.0001; two-way ANOVA, Bonferroni’s multiple comparisons test. b Light response of NERNST in tobacco leaf cells illuminated (Light) or not (Dark) at 200 μmol quanta m⁻² s⁻¹ for 3 h. E_NADP(H) values were calculated assuming stromal pH of 7.2 and 8.0 for dark and light conditions, respectively^61,62. Data shown are means ± SD of 12-363 cells. *P ≤ 0.05, ****P ≤ 0.0001; two-tailed Mann–Whitney test (Chloroplast) or Unpaired t test (Cytosol). c Drought responses of NERNST in tobacco leaf cells. Excised leaves were air-dried for 3 h, prior to NERNST imaging. Data shown are means ± SD of 12-356 cells. ****P ≤ 0.0001; two-tailed Mann–Whitney test (Chloroplast) or Unpaired t test (Cytosol). d Salt responses of NERNST-expressing tobacco leaf discs exposed to the indicated concentrations of NaCl for 16 h. Data shown are means ± SD of 12-229 cells. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001; ns, non-significant; one-way ANOVA, Dunn’s multiple comparisons test (Chloroplast) or Bonferroni’s multiple comparisons test (Cytosol). e NERNST imaging of tobacco leaves inoculated with P. carotovorum at two distances from the infiltration site. Data shown are means ± SD of 10-175 cells. **P ≤ 0.01, ****P ≤ 0.0001; ns, non-significant; one-way ANOVA, Dunn’s multiple comparisons test. f Ratiometric images (R values) of roots from 14-day-old tobacco plants grown in 0.5×MS-agar. g Transiently-expressed NERNST in the chloroplasts and cytosol of N. benthamiana leaf cells. Right panels: merge between 488-nm and chlorophyll fluorescence. Representative images and data shown were produced in at least two independent experiments. Scale bars, 50 μm. b–f Pseudocolor scales = R values. Source data, including all precise n values and exact P values, are provided as a Source Data file.