Light-regulated nuclear import and degradation of Arabidopsis phytochrome-A N-terminal fragments.

The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ∼30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.

A cell-free system for light-dependent nuclear import of phytochrome.

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum. These nuclei are up to 100 mum in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana. In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.

Treatment of painful osteoporotic compression and burst fractures using kyphoplasty: a prospective observational design.

OBJECT: The aim of this study was to test the hypothesis that kyphoplasty is an effective treatment in painful osteoporotic vertebral fractures, even with involvement of the posterior cortical wall. METHODS: Between December 2001 and May 2004, 74 consecutive patients were treated with kyphoplasty for 118 painful osteoporotic compression (38%) or burst (62%) fractures. Additional decompression of the spinal canal was performed in six patients, internal fixation in three. Data were collected in a prospective observational design until May 2005. The preoperative workup included neuroimaging (plain x-ray films, densitometry, short tau inversion recovery magnetic resonance imaging, and computed tomography scanning) and clinical parameters (general and neurological examinations, visual analog scale [VAS], Karnofsky Performance Scale [KPS], and 36-Item Short Form Health Survey [SF]-36). At predefined time intervals (at discharge and 6 weeks and 3, 6, 12, and 24 months posttherapy) the patients were evaluated (x-ray films, neurological status, VAS, KPS, and SF-36). Kyphoplasty led to a significant reduction in kyphotic deformity (mean +/- standard error of the mean, sagittal index: preoperative 10 +/- 1 degrees, postoperative 5 +/- 1 degrees), and an improvement in pain (VAS: preoperative 70 +/- 3, postoperative 23 +/- 2), activity (KPS score: preoperative 51 +/- 3, postoperative 71 +/- 2), and mental and physical health (SF-36, mental status: preoperative 43, postoperative 58; SF-36, physical status: preoperative 24, postoperative 35). No secondary narrowing of the spinal canal by the retropulsed posterior wall was observed after the procedure. Clinical improvement was durable (mean follow up 15 +/- 1.1 months), although the VAS score secondarily increased slightly. All patients, who suffered from a compression-induced motor deficit, recovered completely during the follow-up interval. The main procedural complications consisted of one symptomatic extravertebral cement leakage (permanent monoparesis) requiring open revision, two nerve root contusions (transient radiculopathy), and one wound infection. CONCLUSIONS: Kyphoplasty is effective in the treatment of painful osteoporotic vertebral compression and burst fractures, at least under medium-term conditions. The potential complication of procedure-related secondary narrowing of the spinal canal by the retropulsed posterior wall in burst fractures appears to be more of a theoretical than an actual risk.

A short amino-terminal part of Arabidopsis phytochrome A induces constitutive photomorphogenic response.

Phytochrome A (phyA) is the dominant photoreceptor of far-red light sensing in Arabidopsis thaliana. phyA accumulates at high levels in the cytoplasm of etiolated seedlings, and light-induced phyA signaling is mediated by a complex regulatory network. This includes light- and FHY1/FHL protein-dependent translocation of native phyA into the nucleus in vivo. It has also been shown that a short N-terminal fragment of phyA (PHYA406) is sufficient to phenocopy this highly regulated cellular process in vitro. To test the biological activity of this N-terminal fragment of phyA in planta, we produced transgenic phyA-201 plants expressing the PHYA406-YFP (YELLOW FLUORESCENT PROTEIN)-DD, PHYA406-YFP-DD-NLS (nuclear localization signal), and PHYA406-YFP-DD-NES (nuclear export signal) fusion proteins. Here, we report that PHYA406-YFP-DD is imported into the nucleus and this process is partially light-dependent whereas PHYA406-YFP-DD-NLS and PHYA406-YFP-DD-NES display the expected constitutive localization patterns. Our results show that these truncated phyA proteins are light-stable, they trigger a constitutive photomorphogenic-like response when localized in the nuclei, and neither of them induces proper phyA signaling. We demonstrate that in vitro and in vivo PHYA406 Pfr and Pr bind COP1, a general repressor of photomorphogenesis, and co-localize with it in nuclear bodies. Thus, we conclude that, in planta, the truncated PHYA406 proteins inactivate COP1 in the nuclei in a light-independent fashion.

Phytochrome A-specific signaling in Arabidopsis thaliana.

Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling de-etiolation. Until now more then ten positive and some negative components acting downstream of phyA have been identified. However, their site of action and hierarchical relationships are not completely understood yet.