Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.

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Phytochrome A (phyA) is a versatile plant photoreceptor that mediates responses to brief light exposures (very low fluence responses, VLFR) as well as to prolonged irradiation (high irradiance responses, HIR). We identified the phyA-303 mutant allele of Arabidopsis thaliana bearing an R384K substitution in the GAF subdomain of the N-terminal half of phyA. phyA-303 showed reduced phyA spectral activity, almost normal VLFR, and severely impaired HIR. Recombinant N-terminal half oat of PHYA bearing the phyA-303 mutation showed poor incorporation of chromophore in vitro, despite the predicted relatively long distance (>13 Å ) between the mutation and the closest ring of the chromophore. Fusion proteins bearing the N-terminal domain of oat phyA, ␤-glucuronidase, green fluorescent protein, and a nuclear localization signal showed physiological activity in darkness and mediated VLFR but not HIR. At equal protein levels, the phyA-303 mutation caused slightly less activity than the fusions containing the wild-type sequence. Taken together, these studies highlight the role of the N-terminal domain of phyA in signaling and of distant residues of the GAF subdomain in the regulation of phytochrome bilin-lyase activity.Plant photoreceptors monitor the cues provided by the light environment and trigger modifications that adjust growth and development to the prevailing conditions (1, 2). Phytochromes are sensors of red and far-red (FR) 3 light that bind an open chain tetrapyrrole chromophore (3, 4). Arabidopsis thaliana bears five phytochrome apoprotein genes (PHYA through PHYE) (5). phyA is a versatile photoreceptor that induces seed germination in response to brief exposures to light (6, 7), which the seeds may experience during soil labor. phyA is also required to perceive the prolonged exposures to FR that the seedlings experience when they emerge from the soil under dense plant canopies (8). These two photoresponses mediated by phyA are of the types called very low fluence responses (VLFR) and high irradiance responses (HIR), respectively (9). Some physiological processes (e.g. inhibition of hypocotyl growth, unfolding of the cotyledons) exhibit both VLFR and HIR as two discrete phases of response, where VLFR saturates with infrequent FR pulses and HIR requires very frequent or continuous FR (10). HIR requires cis-acting elements at target gene promoters (11) and domains of the phyA molecule itself (12) that are dispensable for VLFR.The N-terminal domain of phytochromes bears the chromophore attachment site (13) and provides differential spectral selectivity to phyA (more active under FR than red light) compared with phyB (more active under red light than FR) (14). The C-terminal domain contains a histidine kinase-related sequence motif that is able to mediate phosphorylation (15, 16), two PAS (domain named after Per, Arnt, and Sim) motifs important for downstream signaling in the context of the full molecule (17), and residues necessary for dimerization (18,19) and targeting to the nucleus upon light activation (20, 21)...

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