Fig. 4. NERNST probes the NADP(H) redox status in isolated leaf protoplasts.

Expression vectors used are those described in Fig. 3a. a Representative fluorescence images of NERNST in protoplasts isolated from Arabidopsis and tobacco leaves expressing the biosensor in chloroplasts (Chlo) or the cytosol (Cyt). Panels show merge between 488-nm and chlorophyll fluorescence, and R imaging. Scale bars, 20 μm. Pseudocolor scale = R values. b NADP(H) redox potentials (E) were estimated assuming pH values of 7.3 and 8.0 for the cytosol and stroma, respectively⁶⁰. ****P ≤ 0.0001; two-way ANOVA followed by Bonferroni’s multiple comparisons test. c, d Comparison of cytosolic E_NADP(H) (c) and dynamic ranges including controls (d) between isolated Arabidopsis protoplasts stably and transiently expressing NERNST. ns, non-significant. Data are shown as means ± SD of 22-50 cells (b), 13–28 cells (c), 10-28 cells (d). Data were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test (b, d), two-tailed Unpaired t test (c). Source data, including all precise n values and exact P values, are provided as a Source Data file.