Figure 3.
COL12 is degraded in darkness in a COP1-dependent fashion. A and B, COL12 protein levels are strongly reduced in dark-grown seedlings. Homozygous 35S::HA-COL12 seedlings (line #16) were grown in continuous white light (Wc, 80 μmol m−2 s−1) for 10 d and subsequently transferred to darkness (D) for 24 h (A) or the indicated time (B). A and b in A indicate two biological replicates. C, COL12 is degraded by the 26S-proteasome. Homozygous 35S::HA-COL12 seedlings (line # 20) were grown in Wc for 16 d then treated with MG132 and subsequently kept in Wc or transferred to darkness (D) for 14 h. D, Degradation of COL12 in darkness requires COP1. The 35S::HA-COL12 transgene (line # 8) was crossed into cop1-4. Homozygous 35S::HA-COL12 seedlings in the COP1 wild-type or cop1-4 mutant background were grown in Wc for 10 d and subsequently transferred to darkness for 14 h. Nuclear proteins were isolated. COL12-HA protein was detected using α-HA antibodies. Histone H3 served as a loading control.