SUMMARY Background An important contributing factor to the success of terrestrial flowering plants in colonizing the land was the evolution of a developmental strategy, termed skotomorphogenesis, whereby post-germinative seedlings emerging from buried seed grow vigorously upward in the subterranean darkness toward the soil surface. Results Here we provide genetic evidence that a central component of the mechanism underlying this strategy is the collective repression of premature photomorphogenic development in dark-grown seedlings by several members of the phytochrome (phy)-interacting factor (PIF) subfamily of bHLH transcription factors (PIF1, PIF3, PIF4 and PIF5). Conversely, evidence presented here and elsewhere, collectively indicates that a significant component of the mechanism by which light initiates photomorphogenesis upon first exposure of dark-grown seedlings to irradiation involves reversal of this repression by rapid reduction in the abundance of these PIF proteins, through degradation induced by direct interaction of the photoactivated phy molecule with the transcription factors. Conclusions We conclude that bHLH transcription factors PIF1, PIF3, PIF4 and PIF5 act as constitutive repressors of photomorphogenesis in the dark, action that is rapidly abrogated upon light exposure by phy-induced proteolytic degradation of these PIFs, allowing the initiation of photomorphogenesis to occur.

No 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.

Phytochrome interacting factors (PIFs) are nuclear basic helix-loop-helix (bHLH) transcription factors that negatively regulate photomorphogenesis both in the dark and in the light in Arabidopsis. The phytochrome (phy) family of photoreceptors induces the rapid phosphorylation and degradation of PIFs in response to both red and far-red light conditions to promote photomorphogenesis. Although phys have been shown to function under blue light conditions, the roles of PIFs under blue light have not been investigated in detail. Here we show that PIF1 negatively regulates photomorphogenesis at the seedling stage under blue light conditions. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12 hr light/12 hr dark) blue light conditions. Double-mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1, and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Blue light induced the rapid phosphorylation, polyubiquitination, and degradation of PIF1 through the ubi/26S proteasomal pathway. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the blue light-induced degradation of PIF1. phyA played a dominant role under pulses of blue light, while phyA, phyB, and phyD induced the degradation of PIF1 in an additive manner under prolonged continuous blue light conditions. Interestingly, the absence of cry1 and cry2 enhanced the degradation of PIF1 under blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis under blue light conditions and that blue light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis.

Phytochrome Interacting Factor 1 (PIF1), a basic helix-loop-helix (bHLH) protein, functions as a negative regulator of various facets of photomorphogenesis. To indentify PIF1-interacting proteins, we performed yeast two-hybrid screening using PIF1 as a bait and identified a group of proteins including PIF1 itself, PIF3 and long hypocotyl in far-red 1 (HFR1), an atypical HLH protein. Directed yeast two-hybrid interaction assays showed that PIF1 can form heterodimers with all other PIFs as well as with HFR1. PIF1 and PIF3 interacted with each other in both in vitro and in vivo co-immunoprecipitation assays. PIF1-PIF3 heterodimer also bound to a G-box DNA sequence element in vitro. To understand the biological significance of these interactions, a pif1pif3 double mutant was obtained and characterized. Analyses of the single and double mutants showed that PIF3 plays a prominent role in repressing photomorphogenesis under continuous blue light conditions. pif1 and pif3 showed additive phenotypes more prominently under discontinuous blue light conditions. Similar to PIF1, PIF3 was also rapidly phosphorylated, poly-ubiquitylated and degraded in response to blue light. PIF3 also interacted with phytochromes in response to blue light. A PIF3 mutant defective in interaction with both phyA and phyB displayed reduced degradation under blue light, suggesting that phy-interaction was necessary for the blue light-induced degradation of PIF3. Taken together, these data suggest a combinatorial control of photomorphogenesis by bHLH proteins in response to light in Arabidopsis.