A Constitutively Active Allele of Phytochrome B Maintains Circadian Robustness in the Absence of Light  

Abstract

The sensitivity of the circadian system to light allows entrainment of the clock, permitting coordination of plant metabolic function and flowering time across seasons. Light affects the circadian system via both photoreceptors, such as phytochromes and cryptochromes, and sugar production by photosynthesis. In the present study, we introduce a constitutively active version of phytochrome B-Y276H (YHB) into both wild-type and phytochrome null backgrounds of Arabidopsis (Arabidopsis thaliana) to distinguish the effects of photoreceptor signaling on clock function from those of photosynthesis. We find that the YHB mutation is sufficient to phenocopy red light input into the circadian mechanism and to sustain robust rhythms in steady-state mRNA levels even in plants grown without light or exogenous sugars. The pace of the clock is insensitive to light intensity in YHB plants, indicating that light input to the clock is constitutively activated by this allele. Mutation of YHB so that it is retained in the cytoplasm abrogates its effects on clock function, indicating that nuclear localization of phytochrome is necessary for its clock regulatory activity. We also demonstrate a role for phytochrome C as part of the red light sensing network that modulates phytochrome B signaling input into the circadian system. Our findings indicate that phytochrome signaling in the nucleus plays a critical role in sustaining robust clock function under red light, even in the absence of photosynthesis or exogenous sources of energy.

Sucrose and Ethylene Signaling Interact to Modulate the Circadian Clock

“…S3 and S7), which is essentially the opposite phenotype to gi mutants in these conditions. A similar phenotype has been reported in seedlings harboring a constitutively active allele of a red-light photoreceptor, phytochrome B (Jones et al, 2015). These observations indicate that loss of transcriptional circadian rhythms in continuous dark is not simply a consequence of limiting energy availability but is the result of specific regulatory or signaling events.…”

New Constitutively Active Phytochromes Exhibit Light-Independent Signaling Activity

“…For example, YHA was used to elucidate the FR light signaling mechanisms of phyA (Rausenberger et al, 2011), and YHB was used to investigate the molecular function of HEMERA, which is known as an essential component for both phyB localization to nuclear bodies and the degradation of PIFs (Chen et al, 2010;Galvão et al, 2012). In addition, YHB was used to prove that a noncovalently attached chromophore can mediate phyB signaling (Oka et al, 2011) and also to study the phyB function on the circadian clock (Jones et al, 2015). Therefore, the constitutively active mutants are useful tools for studying the molecular functions of phytochromes in plant light signaling.…”

A Tightly Regulated Genetic Selection System with Signaling-Active Alleles of Phytochrome B

“…Protein extraction and immunoblot analysis were performed as described previously with some modifications (Su and Lagarias, 2007;Jones et al, 2015). Immunoblot visualization of FLAG-tagged proteins, actin, and tubulin were accomplished using the fluorescence-based Odyssey infrared imaging system as described previously, and PIF3 and YHB/PHYB proteins were detected by the ECL method using the GE ImageQuant LAS4000 system.…”