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.
Highlights
Phys are head-to-head homodimers with each polypeptide harboring an N-terminal photosensory module (PSM) that binds the bilin followed by a C-terminal output module (OPM) that supports dimerization and often promotes signal transmission to downstream effectors[1,8]
To provide further insights into how various modular features within Phys influence their dimeric structure and photointerconversion between red light (Pr)/Pfr interconversion, we first studied a series of C-terminal truncation mutants (1–908, 1–624, and 1–450) of Arabidopsis PhyB that sequentially eliminated the histidine kinase-related domain (HKRD), the pair of internal PAS domains, and the PHY domain, respectively, from the full-length 1172-residue bili-protein (Fig. 1a)
Analysis of the GAF domain by itself was not attempted given that its intimate connection to the N-terminal PAS domain through the figure-ofeight knot appears to be required for proper folding[21]
Summary
Phys are head-to-head homodimers with each polypeptide harboring an N-terminal photosensory module (PSM) that binds the bilin followed by a C-terminal output module (OPM) that supports dimerization and often promotes signal transmission to downstream effectors[1,8]. Given how sensitive Phy signaling is to Pr/Pfr interconversion, it is not surprising that multiple features both proximal and distal to the bilin are crucial to facilitate or modify this exchange This is especially evident for plant Phys, some of which must be delicately balanced to perceive both light and temperature[3,4]. Manipulating the contact points between the hairpin and GAF domain either substantially promotes or dampens Pfr → Pr thermal reversion[21,26,27], whereas the NTE in plant Phys is important for stabilizing the Pfr state and subsequent signaling once formed[21,28,29,30]. Like other plant Phys, the PSM of Arabidopsis PhyB is followed by a PAS-PAS and HKRD region that helps promote dimerization, nuclear import, and possibly signaling through interaction with a family of PHYTOCHROME-INTERACTING FACTOR (PIF) transcriptional repressors[21,37]
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