Figure 5.—

Blue light induces rapid phosphorylation and ubiquitination prior to degradation of PIF1. (A) Blue light induces phosphorylation of PIF1. TAP-PIF1 was immunoprecipitated from protein extracts prepared using 4-day-old dark-grown 35S:TAP-PIF1 seedlings kept in the dark or exposed to Bp (30 μmol m⁻² sec⁻¹ × 2 min = 3600 μmol m⁻²) followed by dark incubation. The immunoprecipitated pellets from the Bp-exposed samples were dissolved in buffer and incubated without (−) or with (+) native calf intestine alkaline phosphatase (CIAP) or with boiled CIAP (+B). Samples were then separated on 6.5% SDS–PAGE gels and Western blots probed with anti-MYC antibody. Asterisks denote cross-reacting bands. (B) Blue light induces ubiquitination of PIF1. TAP-PIF1 was immunoprecipitated from protein extracts prepared using 4-day-old dark-grown seedlings either kept in the dark (Dk) or exposed briefly to Bp light (30 μmol m⁻² sec⁻¹ × 2 min = 3600 μmol m⁻²). The immunoprecipitated samples were then separated on 6.5% SDS–PAGE gels and probed with anti-ubiquitin (Ubi) or anti-MYC antibodies. Arrows indicate ubiquitinated forms of PIF1.