Phytochrome B (phyB) is the major informational photoreceptor in light-grown plants. The phyB polypeptide is folded into two domains, the N-terminal domain and the C-terminal domain. The N-terminal domain covalently binds to the chromophore via a particular cysteine residue, which allows the holoprotein to absorb light and undergo a photoreversible conformational change. The N-terminal domain of phyB interacts with transcription factors, such as PIF3 (PHYTOCHROME-INTERACTING FACTOR 3), to transduce the light signal to downstream components. Since substitution of the chromophore attachment site, Cys357, with alanine (C357A) abolishes the biological activity of Arabidopsis phyB, the covalent attachment with the chromophore is widely assumed to be necessary for phyB signal transduction. In this study, we show that Arabidopsis phyB is capable of transducing signals with a non-covalently retained chromophore. Substituting the Tyr276 residue of phyB with histidine (Y276H) is known to confer constitutive phyB signaling. PhyB containing both Y276H and C357A substitutions exhibited light-independent biological activity in transgenic Arabidopsis plants in a chromophore-dependent manner. Spectrophotometric analysis showed that the N-terminal domain of phyB containing just the C357A substitution could retain the chromophore non-covalently. The N-terminal domain containing both the Y276H and C357A substitutions interacted with PIF3 in a light-independent but chromophore-dependent fashion in yeast two-hybrid assays. From these results, we conclude that the constitutive phyB signaling conferred by Y276H requires the chromophore, but that the chromophore does not need to be covalently bonded to phyB.
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