Figure 4.

Peredox-mCherry Status in Different Tissues of an Arabidopsis Seedling.

(A) tS and mC fluorescence of 5-d-old seedlings was recorded by confocal laser scanning microscopy, and the ratio is plotted in false colors (sequential excitation, 405 and 570 nm; emission recorded at 517.5 ± 7.5 and 612.5 ± 7.5 nm, respectively), where high tS/mC (red) corresponds to reduced NADH/NAD⁺ steady state levels. The seedlings were kept in the dark for 120 min before image acquisition to avoid potential effects of active photosynthesis. A representative seedling of line #3 is shown. In the close-ups, tS/mC ratios were analyzed in the indicated ROIs, with 1 out of 10 randomly distributed circular ROIs across a cotyledon shown to facilitate visibility of the actual differences. Enlarged are I, cotyledon; II, hypocotyl and shoot apical meristem (SAM) region; III, root; and IV, root tip. Note that the SAM region was not exposed in the etiolated seedlings and was hence not analyzed to avoid mechanically invasive sample preparation. Bars = 1 mm. cotyl., cotyledon; epid., epidermis; hypoc., hypocotyl; vasc., vasculature.

(B) Comparison of the tS/mC ratios of light-grown (A) versus etiolated seedlings in the dark (C), and comparison of tissue-specific differences in the seedlings under each condition. Seedlings of line #3 were used for the analysis; n = 6, whiskers represent minimum and maximum values. Statistical analysis with a two-way ANOVA followed by the Tukey test; different letters indicate significant differences between ROIs of seedlings under the indicated growth condition (P ≤ 0.05), asterisks indicate significant differences between light-grown versus etiolated seedlings for the indicated ROI (***P ≤ 0.001; Supplemental Data Set 1F).

(C) Confocal microscopy analysis of a representative 5-d-old seedling etiolated in the dark. For statistical comparison of the NAD redox state to light-grown seedlings (B), ROIs were selected analogously to (A). Bars = 1 mm.