Photosensing and Thermosensing by Phytochrome B Require Both Proximal and Distal Allosteric Features within the Dimeric Photoreceptor

Abstract

Phytochromes (Phys) encompass a diverse collection of bilin-containing photoreceptors that help plants and microorganisms perceive light through photointerconversion between red light (Pr) and far-red light (Pfr)-absorbing states. In addition, Pfr reverts thermally back to Pr via a highly enthalpic process that enables temperature sensation in plants and possibly other organisms. Through domain analysis of the Arabidopsis PhyB isoform assembled recombinantly, coupled with measurements of solution size, photoconversion, and thermal reversion, we identified both proximal and distal features that influence all three metrics. Included are the downstream C-terminal histidine kinase-related domain known to promote dimerization and a conserved patch just upstream of an N-terminal Period/Arnt/Sim (PAS) domain, which upon removal dramatically accelerates thermal reversion. We also discovered that the nature of the bilin strongly influences Pfr stability. Whereas incorporation of the native bilin phytochromobilin into PhyB confers robust Pfr → Pr thermal reversion, that assembled with the cyanobacterial version phycocyanobilin, often used for optogenetics, has a dramatically stabilized Pfr state. Taken together, we conclude that Pfr acquisition and stability are impacted by a collection of opposing allosteric features that inhibit or promote photoconversion and reversion of Pfr back to Pr, thus allowing Phys to dynamically measure light, temperature, and possibly time.

PCH1 regulates light, temperature, and circadian signaling as a structural component of phytochrome B-photobodies in Arabidopsis

“…Detailed protocols for plant growth conditions and plasmid constructs, in vitro phyB thermal reversion assay (37), hypocotyl elongation assay and statistical analysis, fixation of seedlings and confocal microscopy, protein extraction and Western blot experiments, in vitro co-IP/binding assay and yeast two-hybrid analysis, and LUC-based circadian clock assays are described in SI Appendix, Materials and Methods. All primers used for cloning are listed in SI Appendix, Table S1.…”

Differential phosphorylation of the N‐terminal extension regulates phytochrome B signaling

“…Removal of the NTE from the phyB protein accelerated thermal reversion of phyB PSM fragments in vitro and abolished phyB localization to PBs in Arabidopsis (Chen et al, 2005;Burgie et al, 2014;Burgie et al, 2017). In general, the NTE of phyB is assumed to be important for thermal stability of the Pfr form.…”

Light‐controllable Transcription System by Nucleocytoplasmic Shuttling of a Truncated Phytochrome B

“…In an Arabidopsis thaliana PhyB truncation study, PhyB (1-450 aa) failed to associate with PIF6 reversibly (22). A recent study demonstrated that PhyB (1-450 aa) truncation has an absorption maximum at 693 nm (Pfr) when PCB was used as a chromophore, although full-length PhyB shows maximum absorption at 714 nm (Pfr) (17). The reversible control of the truncated PhyB-PIF6 association we demonstrated might be Figure 2.…”

“…In an Arabidopsis thaliana PhyB truncation study, PhyB (1–450 aa) failed to associate with PIF6 reversibly . A recent study demonstrated that PhyB (1–450 aa) truncation has an absorption maximum at 693 nm (Pfr) when PCB was used as a chromophore, although full‐length PhyB shows maximum absorption at 714 nm (Pfr) . The reversible control of the truncated PhyB‐PIF6 association we demonstrated might be attributable to the difference of irradiation conditions: we irradiated with 735 nm light for the dissociation of PhyB‐PIF6 binding, whereas photoirradiation with light that was >750 nm was applied using a near‐IR glass filter in an earlier study .…”

“…The proteins including 2 × NLS (PIF6_VP16 and PIF6_VP64) alone allow for nucleocytoplasmic shuttling because of the low molecular mass (PIF6_VP16; 24.94 kDa and PIF6_VP64; 23.52 kDa, respectively). Reportedly, Arabidopsis thaliana full‐length PhyB forms a homodimer or heterodimer with PhyC‐E , whereas a truncation of PhyB (1–450 aa) remains a monomer . Gal4 (50–94 aa) is known as a complete dimerization domain .…”