Figure 3.

Kinetics of Blue Light–Dependent Degradation of Endogenous CRY2 in Wild-Type Seedlings or LUC-CRY2 in Transgenic Seedlings.

(A) Immunoblot showing endogenous CRY2 in etiolated wild-type seedlings exposed to blue light (16 μmol m⁻² s⁻¹) for the indicated time. Five-day-old etiolated seedlings were treated with cycloheximide (CHX) for 4 h before transferring to blue light for the indicated duration. Protein samples were extracted, fractioned by 10% SDS-PAGE, and blotted. The immunoblot was probed with anti-CRY2, stripped, and reprobed with anti-CRY1 antibodies. The relative level of CRY2 (including both phosphorylated upper bands and unphopsphorylated lower band) is calculated as described (see Methods) and presented as CRY2^blue (level of CRY2 after blue light treatment)/CRY2^dark (level of CRY2 before blue light treatment).

(B) Change in luminescence of LUC-CRY2 seedlings in response to blue light. LUC-CRY2 seedlings were grown on MS medium (∼150 seedlings per Petri dish) in far-red light for 5 d and exposed to blue light of the fluence rates and durations indicated. The luminescence of untreated and blue light–treated seedlings was measured using a CCD camera, and the relative luminescence per seedling is presented as the percentage of LUC-CRY2^blue (luminescence after blue light treatment)/LUC-CRY2^far-red (luminescence before blue light treatment).

(C) and (D) Immunoblot showing endogenous CRY2 in etiolated wild-type seedlings exposed to blue light with the fluence rates indicated for 30 min (C) or 2 h (D). The relative level of CRY2 is calculated as in (A). Arrows indicate the fluence rates by which ∼70% of CRY2 was degraded.