Fig. 4

Nitrate reductase promotes NO-dependent GLB1 repression. a Time course of the GLB1 transcripts accumulation during incubation of illuminated cells in nitrite-containing medium. Chlamydomonas cells of the wild strain 6145c and its derivative mutant 305, affected in NAD(P) H-NR activity and without diaphorase-NR activity, were grown in ammonium-containing medium and transferred to medium containing 10 mM NO₂⁻ in the light for 1, 2, 3 or 4 h. Values are means ± SE of three biological replicates and three technical replicates and are given as expression level relative to a house-keeping gene RACK1 that has a value of 1. b Time course of the PII protein accumulation during incubation of illuminated cells of the strains 6145c and 305, in nitrite-containing medium. PII levels were analyzed by Western blotting in the same conditions as in (a). Each line corresponds to 10 μg of soluble proteins extracted from samples taken from cultures at the time points indicated. Quantitation of protein blots is given as proportion of signal in test variant to control variant. HSP70B signal served as a loading control. c NO production in nitrite-induced cells. Chlamydomonas cells of the strains 6145c and 305 were grown in TAP medium and transferred to nitrite-containing medium in the light for 15 min. Fluorescence intensity due to intracellular NO was determined using DAF-FM DA and was expressed as arbitrary units per μg chlorophyll . Cell autofluorescence was subtracted from the total fluorescence obtained. Fluorescence in the strain 305 is considered as control (set to 100%). Data are the means±SE from three independent experiments. Production of NO was measured by the microplate reader CLARIOstar (BMG). d NO visualization by confocal microscopy. Images of cells grown in TAP (TAP) or incubated in nitrite-containing medium (10 mM NO₂⁻) for 15 min. The left-hand panels show DAF-FM fluorescence (green color) while the right-hand panels show Chl autofluorescence (red color). Scale bar equals 10 μm