Figure 8. Genetics analyses of PPK-dependent CRY2 phosphorylation.

(a) 27-day-old plants of indicted genotypes, including the ppk123 and ppk124 mutants, and a amiR^4k-expressing line, grown in LD photoperiods (16 h light, 8 h dark) are shown. (b,c) Flowering time of the indicated genotypes measured as ‘leaf number' (b) or ‘Days to flowering' (c), with the s.d. shown (n>20). ** in (b,c) indicate P<0.01. (d) Immunoblot showing the lack of blue light-dependent CRY2 phosphorylation and degradation in the ppk123 and ppk124 mutants, and an amiR^4k-expressing line. Etiolated seedlings of the indicated genotypes were exposed to blue light (5 μmol m⁻² s⁻¹) for the indicated time (0–120 min) before sample collection. Proteins were analysed by immunoblot probed with the anti-CRY2 (CRY2) or anti-HSP82 (HSP82) antibody, respectively. (e) Relative abundance of FT mRNA in 12-day-old plants of indicated genotypes grown in LD photoperiods. Relative expression unit (REU) was calculated by normalization to the reference gene IPP2 (AT3G02780). S.d. (n=3) are shown. (f) A model depicting the molecular basis of the blue light-dependent phosphorylation and function of CRY2. Photoexcited CRY2 undergoes homodimerization and PPK-catalysed phosphorylation. Light regulation of light regulated genes (LRGs, such as FT) is mediated by the partially active unphosphorylated CRY2 dimer (thin arrow) and the fully active phosphorylated CRY2 dimer (thick arrow). Phosphorylated CRY2 is ubiquitinated by E3 ligases and degraded by the 26S proteasome.