Characterization of the requirements for localization of phytochrome B to nuclear bodies

Abstract

Phytochromes are red- and far-red-sensing photoreceptors that detect the quantity, quality, and duration of light throughout the entire life cycle of plants. Phytochromes accumulate in the cytoplasm in the dark. As one of the earliest responses after light illumination, phytochromes localize to the nucleus where they become associated with discrete nuclear bodies (NBs). Here, we describe the steady-state dynamics of Arabidopsis phytochrome B (phyB) localization in response to different light conditions and define four phyB subnuclear localization patterns: diffuse nuclear localization, small and numerous NBs only, both small and large NBs, and large NBs only. We show that phyB nuclear import is not sufficient for phyB NB formation. Rather, phyB accumulation in NBs is mainly determined by the percentage of the total amount of phyB protein that is in the active phyB conformer, with large NBs always correlating with strong phyB responses. A genetic screen to identify determinants required for subnuclear localization of phyB resulted in several phyB mutants, mutants deficient in phytochrome chromophore biosynthesis, and mutations in at least one previously uninvestigated locus. This study lays the groundwork for future investigations to identify the molecular mechanisms of light-regulated partitioning of plant photoreceptors to discrete subnuclear domains.

Fig. 1.

Characterization of phyB::GFP NB patterns in response to changing fluence rates of R light. (A) Steady-state phyB::GFP localization patterns in hypocotyl cells of 4-day-grown PBG seedlings under different fluence rates of R with different percentages of Pfr in the total. See text for details. (B) Hypocotyl length measurements of 4-day-grown PBG seedlings under different fluence rates of R. (C) NB pattern of phyB::GFP in hypocotyl cells growing in 8 μmol·m^-2·s^-1 of R and 8 μmol·m^-2·s^-1 of FR was similar to that of cells growing in 1 μmol·m^-2·s^-1 of R only. (Bar = 10 μm.) The number labels represent fluence rates with a unit of μmol·m^-2·s^-1.

Fig. 2.

Mutants with defective phyB apoproteins do not form NBs under R. (A) phyB mutations were found throughout the PHYB gene. (B) Evenly distributed phyB::GFP pattern in phyB mutants under 8 μmol·m^-2·s^-1 of R. PhyB-28::YFP showed small NBs under 8 μmol·m^-2·s^-1 of R. (C) Hypocotyl length of phyB mutants under the same fluence rates of R. (D) PhyB::GFP levels were determined by Western blot analysis with a GFP antibody. (Bar = 10 μm.)

Fig. 3.

Phytochrome chromophore mutants contain smaller NBs in high-fluence rates of R. (A-D) PhyB::GFP patterns in R/FR hyposensitive lines under 8 μmol·m^-2·s^-1 of R. These mutants have a similar number of NBs; however, the size of the NBs was much smaller than that of the PBG line. (E) Hypocotyl-length measurements under 8 μmol·m^-2·s^-1 ofRor16 μmol·m^-2·s^-1 of FR. (F) Mutant hy1-201 is rescued by BV feeding. (Bar = 10 μm.)

Fig. 4.

dsf mutants with big and small NBs define extragenic factors required for phyB's localization to stage IV NBs. (A) phyB::GFP NB pattern in dsf mutants grown in 8 μmol·m^-2·s^-1 of R. (B) dsf mutants were specifically less sensitive to R. (C) phyB::GFP levels were similar to that of the PBG line by Western blot analysis with GFP antibodies. (Bar = 10 μm.)

Fig. 5.

Model of Arabidopsis phyB-regulated translocation. Red-light-induced phyB subnuclear localization comprises two steps, nuclear import and localization to NBs. Whereas PfrPr is sufficient for nuclear import, a higher percent of Pfr (likely PfrPfr) is required for phyB localization to NBs. NB formation may serve as a desensitization mechanism to control the amount of active nucleoplasmic phyB. This process could also be feedback-enhanced by phyB responses. DSFs may be components in the phyB-signaling pathways leading to the feedback loop. In dsf mutants, defective phyB NB formation could be due to reduced phyB responses.