Functional Analysis of a 450–Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis

Abstract

Phytochrome, a major photoreceptor in plants, consists of two domains: the N-terminal photosensory domain and the C-terminal domain. Recently, the 651-amino acid photosensory domain of phytochrome B (phyB) has been shown to act as a functional photoreceptor in the nucleus. The phytochrome (PHY) domain, which is located at the C-terminal end of the photosensory domain, is required for the spectral integrity of phytochrome; however, little is known about the signal transduction activity of this domain. Here, we have established transgenic Arabidopsis thaliana lines expressing an N-terminal 450-amino acid fragment of phyB (N450) lacking the PHY domain on a phyB-deficient background. Analysis of these plants revealed that N450 can act as an active photoreceptor when attached to a short nuclear localization signal and b-glucuronidase. In vitro spectral analysis of reconstituted chromopeptides further indicated that the stability of the N450 Pfr form, an active form of phytochrome, is markedly reduced in comparison with the Pfr form of full-length phyB. Consistent with this, plants expressing N450 failed to respond to intermittent light applied at long intervals, indicating that N450 Pfr is short-lived in vivo. Taken together, our findings show that the PHY domain is dispensable for phyB signal transduction but is required for stabilizing the Pfr form of phyB.

Photobody Localization of Phytochrome B Is Tightly Correlated with Prolonged and Light-Dependent Inhibition of Hypocotyl Elongation in the Dark

“…6, C and D), the Pfr of PBG lasts for about 18 h compared with only 6 h for the Pfr of NGB. Because PBG and NGB have similar dark reversion rates in vitro (Oka et al, 2004), our data suggest that the life of the Pfr of PBG is extended three times longer than that of NGB in vivo. This dramatic decrease in the capacity for hypocotyl growth inhibition in PBG between the 10 mmol m 22 s 21 R-to-D transition (Fig.…”

“…One possible explanation could come from differences in the stability of the Pfr of phyB. Although the dark reversion rate of NGB is similar to that of full-length phyB in vitro (Oka et al, 2004), the dark reversion rate of full-length phyB in vivo is much slower; it has been proposed that, in vivo, photobodies can stabilize the Pfr form of phyB (Rausenberger et al, 2010). To test this hypothesis, we treated PBG and NGB seedlings with a 15-min FR pulse to convert PBG and NGB to their respective Pr before transferring them to darkness.…”

Evolutionary Studies Illuminate the Structural-Functional Model of Plant Phytochromes

“…The C-terminal regions of plant PHY and of those prokaryotic PHY that have been sequenced are not homologous (Lamparter, 2004;Mathews, 2006); in green plants, this region comprises two PAS and single His kinase and ATPase domains ( Figure 3B), the latter two domains form the core of the His kinase-related domain (HKRD; Rockwell et al, 2006). The PAS repeats and HKRD contain sites necessary for dimerization and nuclear localization (Quail, 1997;Chen et al, 2003) and for modulating phytochrome signaling (Krall and Reed, 2000;Matsushita et al, 2003;Oka et al, 2004;Mü ller et al, 2009). Sequence conservation among the photosensory cores of plant and Synechocystis 6803 Cph1 phytochromes facilitates their alignment (Essen et al, 2008), and the alignment serves as a preliminary basis for relating positions in plant phytochromes to Cph1 structural elements ( Figure 3C).…”

“…Currently, high-resolution structures of the photosensory core of prokaryotic phytochromes are available; this region is homologous with the sensory module of plant phytochromes (Montgomery and Lagarias, 2002;Lamparter, 2004;Karniol et al, 2005) and consists of PAS, GAF, and PHY domains ( Figure 3B). It is necessary for photoreversibility (Rockwell et al, 2006) and is sufficient for signaling when fused with dimerization and nuclear localization signals (Matsushita et al, 2003;Oka et al, 2004). The N-terminal extension ( Figure 3B) is unique to phytochromes in embryophytes and their algal relatives; it is present in the phytochromes of zygnemetalean green alga, which diverge from the tree before Coleochaetales and Charales (the closest algal relatives of embryophytes; Delwiche et al, 2004) but is not present in prokaryotic phytochromes, and no crystal structure for this region is available.…”

The Structure of Phytochrome: A Picture Is Worth a Thousand Spectra

“…The C-terminal regulatory domains of plant phytochromes have been shown to mediate homodimerization and light-modulated nuclear targeting, both of which are required for signal transmission (Matsushita et al, 2003;Chen et al, 2005). Signaling by plant phytochromes also involves protein-protein interactions with the N-terminal part of the protein (Ni et al, 1999;Oka et al, 2004), although it is not yet known whether these interactions are actually mediated via the conserved photosensory core (P2-P3-P4), via the N-terminal Ser/Thr-rich extension specific to plant phytochromes, or both. In more primitive plants and algae, atypical phytochromes have been described in which the C-terminal region has been replaced by fortuitous gene fusions with phototropins and other eukaryotic Ser/Thr kinases (Thü mmler et al, 1992;Nozue et al, 1998;Suetsugu et al, 2005).…”