Evidence that phytochrome functions as a protein kinase in plant light signalling

Abstract

It has been suggested that plant phytochromes are autophosphorylating serine/threonine kinases. However, the biochemical properties and functional roles of putative phytochrome kinase activity in plant light signalling are largely unknown. Here, we describe the biochemical and functional characterization of Avena sativa phytochrome A (AsphyA) as a potential protein kinase. We provide evidence that phytochrome-interacting factors (PIFs) are phosphorylated by phytochromes in vitro. Domain mapping of AsphyA shows that the photosensory core region consisting of PAS-GAF-PHY domains in the N-terminal is required for the observed kinase activity. Moreover, we demonstrate that transgenic plants expressing mutant versions of AsphyA, which display reduced activity in in vitro kinase assays, show hyposensitive responses to far-red light. Further analysis reveals that far-red light-induced phosphorylation and degradation of PIF3 are significantly reduced in these transgenic plants. Collectively, these results suggest a positive relationship between phytochrome kinase activity and photoresponses in plants.

Phytochromes and Phytochrome Interacting Factors

“…However, direct in vivo evidence showing phytochrome function as a kinase was lacking until recently. Interestingly, a recent study showed that both oat (Avena sativa) and Arabidopsis phytochromes function as Ser/Thr kinases for PIFs in vitro (Shin et al, 2016). These authors have mapped the kinase domain within the PAS-GAF-PHY domains located at the N terminus of phytochromes and identified critical residues that are necessary for ATP binding.…”

Shining Light on the Function of NPH3/RPT2-Like Proteins in Phototropin Signaling

“…PKS1 was identified originally as a kinase substrate for phytochrome A (phyA; Fankhauser et al, 1999). Whether phyA is bona fide protein kinase is still debated, although recent work has offered renewed support for its role as a protein kinase in regulating photomorphogenesis (Shin et al, 2016). While PKS proteins are required for phytochrome signaling (Lariguet et al, 2003;Schepens et al, 2008), some of the phenotypes associated with pks mutants resemble those of nph3, rpt2, and phot-deficient mutants.…”

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity

“…Full-length phytochrome cDNAs of wild-type AsphyA and the AsYVA mutant fused with a 10-amino acid streptavidin affinity-tag at the C terminus were cloned into pPIC3.5K (Invitrogen), and recombinant phytochrome proteins were prepared using the Pichia protein expression system (Invitrogen) and streptavidin affinity chromatography (IBA), as described previously (Kim et al, 2004;Shin et al, 2014Shin et al, , 2016. The QuickChange Site-Directed Mutagenesis System (Stratagene) was used to generate AsYVA with the mutagenic primers shown in Supplemental Table S1.…”

“…Among PIFs, PIF3 is the founding member that interacts with phytochromes in a Pfr-specific manner and negatively regulates phytochrome signaling (Ni et al, 1998;Kim et al, 2003). Moreover, the physical interaction of phytochromes with PIF3 is known to lead to the latter's phosphorylation and subsequent degradation via the 26S proteasome (AlSady et al, 2006;Ni et al, 2014;Shin et al, 2016), which eventually regulates the transcription of various photoresponsive genes for photomorphogenesis in plants (Bae and Choi, 2008;Chen and Chory, 2011;Xu et al, 2015).…”