Light-Induced Phosphorylation and Degradation of the Negative Regulator PHYTOCHROME-INTERACTING FACTOR1 fromArabidopsisDepend upon Its Direct Physical Interactions with Photoactivated Phytochromes

Abstract

The phytochrome (phy) family of photoreceptors regulates changes in gene expression in response to red/far-red light signals in part by physically interacting with constitutively nucleus-localized phy-interacting basic helix-loop-helix transcription factors (PIFs). Here, we show that PIF1, the member with the highest affinity for phys, is strongly sensitive to the quality and quantity of light. phyA plays a dominant role in regulating the degradation of PIF1 following initial light exposure, while phyB and phyD and possibly other phys also influence PIF1 degradation after prolonged illumination. PIF1 is rapidly phosphorylated and ubiquitinated under red and far-red light before being degraded with a half-life of ;1 to 2 min under red light. Although PIF1 interacts with phyB through a conserved active phyB binding motif, it interacts with phyA through a novel active phyA binding motif. phy interaction is necessary but not sufficient for the light-induced phosphorylation and degradation of PIF1. Domain-mapping studies reveal that the phy interaction, light-induced degradation, and transcriptional activation domains are located at the N-terminal 150-amino acid region of PIF1. Unlike PIF3, PIF1 does not interact with the two halves of either phyA or phyB separately. Moreover, overexpression of a light-stable truncated form of PIF1 causes constitutively photomorphogenic phenotypes in the dark. Taken together, these data suggest that removal of the negative regulators (e.g., PIFs) by light-induced proteolytic degradation might be sufficient to promote photomorphogenesis.

CUL4 forms an E3 ligase with COP1 and SPA to promote light-induced degradation of PIF1

“…The slow migrating form of PIF1 has previously been shown as the phosphorylated forms of PIF1 (ref. 17). These data suggest that COP1-SPA complexes preferentially recruit the phosphorylated forms of PIF1 in the CUL4 COP1-SPA E3 ubiquitin ligase for ubiquitylation and subsequent degradation in response to light.…”

“…The phyB plays a minor role under these conditions ( Supplementary Fig. 3) 17 . To identify which SPA protein is necessary for this response, we performed immunoblots for PIF1 levels in spa1, spa2, spa12, spa123 and spa124 mutants compared with wild type.…”

“…The light-induced degradation of PIFs is UPS-mediated and involves phosphorylation and polyubiquitylation of PIFs 16,17,20,23 . We examined whether the phosphorylation or ubiquitylation of PIF1 is affected in the cop1-4, spaQ and cul4cs seedlings.…”

“…This difference is most likely due to the strong sensitivity of native PIF1 to light exposure as the degradation rate of native PIF1 is much faster than that of TAP-PIF1 (ref. 17). Moreover, these experiments were performed under standard seedling growth conditions where seeds are plated in white light and exposed to prolonged saturated white light for inducing complete germination.…”

“…Recent data showed that direct physical interaction with phytochromes and rapid light-induced phosphorylation of PIFs are necessary for the UPS-mediated degradation [16][17][18] . Two kinases have been shown to phosphorylate PIFs in vitro 19 .…”