A Novel Molecular Recognition Motif Necessary for Targeting Photoactivated Phytochrome Signaling to Specific Basic Helix-Loop-Helix Transcription Factors[W]

Abstract

The phytochrome (phy) family of sensory photoreceptors (phyA to phyE) in Arabidopsis thaliana control plant developmental transitions in response to informational light signals throughout the life cycle. The photoactivated conformer of the photoreceptor Pfr has been shown to translocate into the nucleus where it induces changes in gene expression by an unknown mechanism. Here, we have identified two basic helix-loop-helix (bHLH) transcription factors, designated PHYTOCHROME-INTERACTING FACTOR5 (PIF5) and PIF6, which interact specifically with the Pfr form of phyB. These two factors cluster tightly with PIF3 and two other phy-interacting bHLH proteins in a phylogenetic subfamily within the large Arabidopsis bHLH (AtbHLH) family. We have identified a novel sequence motif (designated the active phytochrome binding [APB] motif) that is conserved in these phy-interacting AtbHLHs but not in other noninteractors. Using the isolated domain and site-directed mutagenesis, we have shown that this motif is both necessary and sufficient for binding to phyB. Transgenic expression of the native APB-containing AtbHLH protein, PIF4, in a pif4 null mutant, rescued the photoresponse defect in this mutant, whereas mutated PIF4 constructs with site-directed substitutions in conserved APB residues did not. These data indicate that the APB motif is necessary for PIF4 function in light-regulated seedling development and suggest that conformerspecific binding of phyB to PIF4 via the APB motif is necessary for this function in vivo. Binding assays with the isolated APB domain detected interaction with phyB, but none of the other four Arabidopsis phys. Collectively, the data suggest that the APB domain provides a phyB-specific recognition module within the AtbHLH family, thereby conferring photoreceptor target specificity on a subset of these transcription factors and, thus, the potential for selective signal channeling to segments of the transcriptional network.

Phytochrome‐mediated inhibition of shade avoidance involves degradation of growth‐promoting bHLH transcription factors

“…It has previously been proposed that the APB domain of PIF4 is essential for function in vivo (Khanna et al, 2004). It was thus surprising to notice that a high proportion of primary transformants containing 35S:DN-PIF4-HA and 35S:DN-PIF5-HA also displayed a strong constitutive shadeavoidance phenotype (Supplementary Figure S1, and data not shown).…”

“…Such plants had considerably elongated hypocotyls and displayed what may be regarded as a constitutive shade-avoidance phenotype (Supplementary Figure S1). This phenotype correlated with strong expression of the HA-tagged PIF proteins, and was similar but often more extreme than the phenotype reported for PIF4-or PIF5-overexpressing plants (Fujimori et al, 2004;Khanna et al, 2004). We therefore analyzed lines with moderate phenotypes, and concentrated our analysis on PIF5-expressing plants because plants expressing high levels of PIF4 were difficult to maintain.…”

“…It has been proposed that the interaction of phyB with PIF4 is essential for the function of this transcription factor (Khanna et al, 2004). On the other hand, PIF1 inhibits several light responses, and phytochrome activation of these responses occurs by inhibition of this negative regulator Oh et al, 2004).…”

“…Members of the PIF family control phytochrome responses including seed germination, seedling de-etiolation and shade avoidance (Huq and Quail, 2002;Huq et al, 2004;Kim et al, 2003;Oh et al, 2004;Penfield et al, 2005;Salter et al, 2003;Shin et al, 2007). Interaction between phyB and the PIFs is mediated by the active phytochrome binding (APB) domain that is present at the N-terminus of these proteins (Khanna et al, 2004). Some PIF proteins, such as PIF1, interact with both phyB and phyA, while others, such as PIF3, PIF4 and PIF5, interact preferentially with phyB.…”

“…Prior to photo-excitation, phyB is a soluble cytoplasmic protein, but, in its Pfr conformation, phyB accumulates in the nucleus where it has been suggested to regulate gene expression (Jiao et al, 2007). In its light-activated state (Pfr), phyB can physically interact with a group of related bHLH transcription factors known as phytochrome interacting proteins (PIFs), suggesting a short signal transduction cascade from light sensing to regulated gene expression (Jiao et al, 2007;Khanna et al, 2004;Martinez-Garcia et al, 2000). Members of the PIF family control phytochrome responses including seed germination, seedling de-etiolation and shade avoidance (Huq and Quail, 2002;Huq et al, 2004;Kim et al, 2003;Oh et al, 2004;Penfield et al, 2005;Salter et al, 2003;Shin et al, 2007).…”