Fig. 1: CRY2 integrates light and temperature signals to regulate flowering and CRY2 interacts with CIS1 in a blue light-dependent manner.
From: CRY2 interacts with CIS1 to regulate thermosensory flowering via FLM alternative splicing
a Number of rosette leaves at the time of flowering for cry2-1 at 22 and 16 °C under LD (16-h light, 8-h dark) or SD (8-h light, 16-h dark) photoperiods. Blue and red circles indicate the data from individual plants. Error bars represent standard deviation (s.d., n ≥ 10). Lowercase letters indicate statistically significant differences, as determined by one-way ANOVA with Tukey’s multiple comparisons test (P < 0.05). Two-sided Student’s t-test was used to calculate P values between groups. b β-gal assays of yeast cells grown at –LT medium 28 °C in darkness (D) or exposed to blue light (B, 30 μmol m–2 s–1) for the indicated times. Three biological replicates are listed. CRY2D387A is a site-specific mutant of CRY2 that cannot be activated by blue light. CRY2W374A is a constitutively active site-specific mutant of CRY2. c Co-localization of CRY2 and CIS1 in nuclear speckles in N. benthamiana leaves. mCherry served as a negative control. BF, brightfield. Merge and overlay the YFP, and brightfield images. Scale bar = 5 μm. d BiFC assay showing in vivo protein interaction between CRY2 and CIS1. CIS1(ΔC)-cCFP and nYFP-CRY2D387A were used as negative controls. N. benthamiana was co-infiltrated with the indicated constructs. Scale bar = 20 μm. e Co-IP assays showing that CRY2 interacts with CIS1 in a blue light-dependent manner in plant cells. 22 °C LD-grown 7-day-old Col-0, cry2-1, and cis1-1 seedlings were pretreated in darkness for 24 h, then treated with 25 μM MG132 in the dark for 2 h and exposed to blue light (B, 30 μmol m–2 s–1) for 20 min. Input: immunoblots showing the abundance of CIS1 and CRY2 in the total protein extracts. CIS1 immunoprecipitation (IP): IP products precipitated by the anti-CIS1 antibody. Total proteins (Input) or IP products of CIS1-beads (CIS1 IP) were probed in immunoblots with an anti-CIS1 or anti-CRY2 antibody. In c–e, three independent experiments were performed with similar results.
