Missense Mutation in the Amino Terminus of Phytochrome A Disrupts the Nuclear Import of the Photoreceptor    

Abstract

Phytochromes are the red/far-red photoreceptors in higher plants. Among them, phytochrome A (PHYA) is responsible for the far-red high-irradiance response and for the perception of very low amounts of light, initiating the very-low-fluence response. Here, we report a detailed physiological and molecular characterization of the phyA-5 mutant of Arabidopsis (Arabidopsis thaliana), which displays hyposensitivity to continuous low-intensity far-red light and shows reduced very-low-fluence response and high-irradiance response. Red light-induced degradation of the mutant phyA-5 protein appears to be normal, yet higher residual amounts of phyA-5 are detected in seedlings grown under low-intensity far-red light. We show that (1) the phyA-5 mutant harbors a new missense mutation in the PHYA amino-terminal extension domain and that (2) the complex phenotype of the mutant is caused by reduced nuclear import of phyA-5 under low fluences of far-red light. We also demonstrate that impaired nuclear import of phyA-5 is brought about by weakened binding affinity of the mutant photoreceptor to nuclear import facilitators FHY1 (for FAR-RED ELONGATED HYPOCOTYL1) and FHL (for FHY1-LIKE). Finally, we provide evidence that the signaling and degradation kinetics of constitutively nuclear-localized phyA-5 and phyA are identical. Taken together, our data show that aberrant nucleo/cytoplasmic distribution impairs light-induced degradation of this photoreceptor and that the amino-terminal extension domain mediates the formation of the FHY1/FHL/PHYA far-redabsorbing form complex, whereby it plays a role in regulating the nuclear import of phyA.

Comparative functional analysis of full‐length and N‐terminal fragments of phytochrome C, D and E in red light‐induced signaling

“…Six-day-old dark-grown seedlings were then subjected to various light treatments, as described in the text. The standard epifluorescence microscopy set-up and observation techniques have been described previously (Bauer et al, 2004;Viczi an & Kircher, 2010;Sokolova et al, 2012). For semiquantitative epifluorescence microscopy, etiolated seedlings were irradiated for 6 h with R light at the fluence rates given in the figure legends.…”

Arabidopsis Phytochrome A Is Modularly Structured to Integrate the Multiple Features That Are Required for a Highly Sensitized Phytochrome

“…It is well established that the nuclear localization facilitators FHY1 and FHL play a critical role in this process (Hiltbrunner et al, 2005(Hiltbrunner et al, , 2006Rösler et al, 2007;Genoud et al, 2008). The N-terminal fragment of phyA, which binds FHY1 in yeast cells (Hiltbrunner et al, 2005), accumulates in the nucleus under continuous FR (Pfeiffer et al, 2009;Wolf et al, 2011), whereas the missense mutation in the N-terminal extension impairs FR-induced nuclear accumulation (Sokolova et al, 2012). This result is also consistent with the recent model in which the association/dissociation of the phyA-FHY1/FHL complex plays an important role in FR-HIR (Rausenberger et al, 2011).…”

Effects of missense mutation on structure and function of photoreceptor

“…9 Very recently, we identified and described a mutant (phyA-5) expressing PHYA with a missense mutation. 10 This mutant line shows a fairly complex phyA-specific phenotype, including a reduced nuclear import at low intensities of FR indicating that the point mutation identified alters the FHY1/FHL-phyA Pfr association/dissociation "cycle." The fact that PHYA-5 governs wild-typelike signaling when it is fused to a nuclear localization signal (NLS), strongly supported this conclusion.…”

“…The fact that PHYA-5 governs wild-typelike signaling when it is fused to a nuclear localization signal (NLS), strongly supported this conclusion. 10 Our knowledge about the interaction of phyA with the FHY1 and FHL proteins is rather limited. Yeast two-hybrid assays showed that the Pfr form of the phyA406 (1-406 amino acids) specifically binds the FHY1/FHL transporter molecules.…”