Article Figures & Data
Figures
- Fig. 1.
Measurements of H2O2levels during development of field-grown cotton fibers. Flowers of field-grown cotton were tagged at anthesis and bolls were collected at the indicated times postanthesis. A, H2O2 was determined in detached fibers according to the method of Snell and Snell (1949). B, H2O2 production was followed in whole locules by incubating them in DCFDA for 60 min. 2′,7′-Dichlorofluorescein, the H2O2-dependent oxidation product, was measured at indicated times at 485-nm excitation and 525-nm emission wavelengths. Catalase (x) was added to the 19-DPA sample. 9 DPA (♦), 19 DPA (▴), and 26 DPA (▪). d.w., Dry weight.
- Fig. 2.
Microscopic observation of H2O2 generation during development of field-grown cotton fibers. Bolls were opened and placed in a solution of dihydrorhodamine-123, as described in Methods. Fibers that were still attached to ovules were analyzed by fluorescence microscopy. a, Primary walls (10 DPA); b, transition period (15 DPA); c, massive secondary wall synthesis (20 DPA); and d, mature fibers (26 DPA).
- Fig. 3.
Birefringence of cultured cotton fibers following either addition of exogenous H2O2 or inhibition of H2O2 generation by DPI. Cotton fibers were cultured with their associated fertilized ovules in 24-well plates at 30°C. a and b, Controls 15 DPA (a) or 16 DPA (b) to which only 0.1% DMSO was added 12 DPA. c and d, Fibers 15 DPA (c) or 16 DPA (d) to which 50 μm H2O2 was added once daily 13 and 14 DPA. e and f, Fibers 15 DPA (e) or 16 DPA (f) that had been cultured in 1 μm DPI/0.1% DMSO since 12 DPA. a, c, e, and f were viewed at ×250 magnification, whereas b and d were at ×100.
- Fig. 4.
The effects of H2O2-regulating compounds on cellulose content and the amount of H2O2 in cultured fibers. A, Total cellulose content was measured 16 DPA after the following compounds were added 12 DPA: Control (no additions) 1 μmDPI, 0.5 mm Tempo, 1 mm 4-hydroxy-tempo, 5 mm SHAM, and 50 μmH2O2. The control value was 282 ± 6 μg cellulose/mg dry weight of fibers. The sd of samples was <7%. B, Dihydrorhodamine-123 staining of 15-DPA cotton fibers cultured from 12 DPA in the presence of 0.1% DMSO as control (a), 1 mm 4-hydroxy-tempo (4H-TEMPO; b), 0.5 mm Tempo (c), 1 μm DPI (d), and 5 mm SHAM (e).
- Fig. 5.
The effects of transient Rac expression on H2O2 production in soybean and Arabidopsis cells. Soybean (A) and Arabidopsis (B) cells were transformed withA. tumefaciens carrying the specific Rac constructs cloned into a binary vector. H2O2 generation was measured with the DCFDA dye, added directly to the medium of cultured cells, for 10 min as described in the Figure 1 legend. Fluorescence emitted from cells transformed in parallel with theGUS gene served as the control. C, Representative field of GUS-transformed (control) cells stained with 5-bromo-4-chloro-3-indoyl-β-d-glucuronic acid. soy, Soybean.
