Fig. 3: CRY2 inhibits phosphorylation of PRR9 in a light intensity-dependent manner.
From: Aschoff’s rule on circadian rhythms orchestrated by blue light sensor CRY2 and clock component PRR9
a Immunoblot showing the band shift of PRR9-HA was eliminated upon λPPase treatment. Total PRR9-HA tobacco protein lysates were treated with λPPase or phosphatase inhibitors NaF and Na3VO4, as noted. Red open brace and green arrow indicated phosphorylated and unphosphorylated forms of PRR9, respectively. CBB staining was used as a loading control. b Band shift of PRR9-GFP was largely reduced when co-expressing with CRY2-HA. Total tobacco protein lysates were treated as in a. c Immunoblot showing dynamic phosphorylation of PRR9 was regulated by blue light intensity. Ten days PRR9:PRR9-GFP transgenic plants were exposed to 80, 20 or 1 μmol m−2 s−1 blue light and harvested at indicated time points. Lysates were analyzed by immunoblot with GFP, CRY2, and Actin antibody. Red and green arrows indicated phosphorylated and unphosphorylated form of PRR9, respectively. d Transcriptional expression level of PRR9 of seedlings in c by RT-qPCR. Data were mean ± s.d., n = 3, technical repeats. **p < 0.01, ***p < 0.001, n.s. means no significant difference compared to 80 μmol m−2 s−1 by two-tailed student’s t-test. e Schematic overview of PRR9 immunoprecipitation-liquid chromatography-mass spectrometry (IP-MS) workflow. Two-week-old PRR9:PRR9-GFP seedlings grown in LD condition were exposed to 40 μmol m−2 s−1 blue light for 5 h before harvesting. ***p < 0.001, **p < 0.01, *p < 0.05 by two-tailed student’s t-test. f Representative mass spectrogram of PRR9 phosphopeptide. Phosphosites-S267/S269 were indicated. g Schematic diagram showing domain structure of PRR9 and position of phosphosites identified by IP-MS. The phospho-serine (S) or threonine (T) residues are highlighted with red letters, and mutation to alanine (A) or aspartic acid (D) were indicated with blue letters. h PRR9-GFP, PRR99A-GFP and PRR99D-GFP were transiently expressed with or without CRY2-HA in N. benthamiana leaves. Total lysates were analyzed by immunoblot probed with GFP and HA antibodies, respectively. Representative figures of a–d and g from three biological repeats.
