Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the ⌬6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. ⌬6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, ⌬6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and ⌬6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by ⌬6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.Phytochromes (phy) comprise a family of photoreceptors that help adjust plant growth and development to the ambient light environment. These photoreceptors sense red light (R) and far-red light (FR) through photo-interconversion between two stable conformations, an R-absorbing Pr form and an FRabsorbing Pfr form. In seed plants such as Arabidopsis, as many as five phy isoforms are present (Mathews and Sharrock, 1997). One of the more influential is phyA, the most abundant isoform in darkgrown seedlings (Hirschfeld et al., 1998). phyA helps perceive (a) the brief light pulses that can promote seed germination (Botto et al., l996; Shinomura et al., 1996), (b) the difference between darkness and the FR-rich environment that initiates de-etiolation beneath dense canopies (Yanovsky et al., 1995), (c) the changes in irradiance associated with the presence of neighboring vegetation (Yanovsky et al., 1998), and (d) the duration of the photoperiod (Johnson et al., 1994).phyA can initiate two photobiologically distinct responses, the very-low-fluence responses (VLFRs) and the high-irradiance responses (HIRs). The VLFR can be achieved by short intermittent pulses of R or FR. For example, the VLFR that inhibits hypocotyl growth can be saturated in Arabidopsis by a 3-min pulse of FR every 2 h with the half-maximal effect requiring 0.1 mol m Ϫ2 s Ϫ1 of FR (Casal et al., 2000). In contra...
SummaryPhotoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal
Zn viva low-temperature (85 K) fluorescence spectroscopy has defined two phytochrome A (phyA) subpopulations, designated phyA ' and phyA", in etiolated seedlings (V. A. Sineshchekov, J. Photochem. Photobwl. 28, 53-55, 1995). Phytochrome A' is the more abundant but Light-labile species characterized by longer wavelength emissiodabsorption maxima (687/673 nm) and by a higher extent of the photoconversion of its red-absorbing form (Pr) into photoproduct (I&-R) at 85 K ( y , = 0.5). Phytochrome A" i s the minor but relatively light-stable species, characterized by shorter wavelength maxima (6824668 nm) and by a lower y , (c0.05). To help define domains within phyA responsible for these differences, the low-temperature spectral properties of transgenic tobacco expressing full-length (FL) oat phyA and C-and N-terminally truncated versions (CD [A78611291 and NA [A7-69], respectively) were compared. Oat phytochrome expression was more pronounced than that of tobacco in the basal section of etiolated seedlings following 2 h irradiation with white Light. Seedlings expressing FL and CD phyA had spectral properties for phyA' and phyA that were indistinguishable from that of wild-type tobacco. Conversely, expression of NA phyA generated an abundant phy species that behaved like phyA". From this we conclude that the N-terminal domain of phyA is involved in determining the photochemical and spectroscopic distinctions between the native phyA' and phyA" species.
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