Phytochrome A (phyA) plays an important role during germination and early seedling development. Because phyA is the primary photoreceptor for the high-irradiance response and the very-lowfluence response, it can trigger development not only in red and far-red (FR) light but also in a wider range of light qualities. Although phyA action is generally associated with translocation to the nucleus and regulation of transcription, there is evidence for additional cytoplasmic functions. Because nuclear accumulation of phyA has been shown to depend on far-red-elongated hypocotyl 1 (FHY1) and FHL (FHY1-like), investigation of phyA function in a double fhl/fhy1 mutant might be valuable in revealing the mechanism of phyA translocation and possible cytoplasmic functions. In fhl/fhy1, the FR-triggered nuclear translocation of phyA could no longer be detected but could be restored by transgenic expression of CFP:FHY1. Whereas the fhl/fhy1 mutant showed a phyA phenotype in respect to hypocotyl elongation and cotyledon opening under high-irradiance response conditions as well as a typical phyA germination phenotype under very-low-fluence response conditions, fhl/fhy1 showed no phenotype with respect to the phyAdependent abrogation of negative gravitropism in blue light and in red-enhanced phototropism, demonstrating clear cytoplasmic functions of phyA. Disturbance of phyA nuclear import in fhl/fhy1 led to formation of FR-induced phyA:GFP cytoplasmic foci resembling the sequestered areas of phytochrome. FHY1 and FHL play crucial roles in phyA nuclear translocation and signaling. Thus the double-mutant fhl/fhy1 allows nuclear and cytoplasmic phyA functions to be separated, leading to the novel identification of cytoplasmic phyA responses.cytoplasmic signaling ͉ far-red-elongated hypocotyl 1 ͉ localization

SummaryPhytochrome A (phyA) plays a primary role in initiating seedling de-etiolation and is the only plant photoreceptor known to be activated by far-red light (FR). The signaling intermediate FHY1 appears to either participate directly in relaying the phyA signal or to positively regulate a critical signaling event(s) downstream of phyA activation. Here we identify a homolog of FHY1 named FHL (FHY1-like) as a novel signaling factor essential for complete responsiveness to phyA. FHL possesses functional nuclear localization and nuclear export signals. Lines in which FHL function was abolished by insertional mutagenesis or attenuated by RNAimediated suppression displayed a weaker hyposensitivity to continuous FR than fhy1 null mutants and most reported phyA signaling mutants. However, hypocotyl elongation assays indicated that suppression of FHL expression in fhy1-3 caused an insensitivity of hypocotyl elongation to FR and blue light (B) indistinguishable from that seen in phyA. Real-time PCR indicates that in FR, FHY1 transcripts are approximately 15-fold more abundant than FHL transcripts. Although both FHY1 and FHL are capable of homo-and hetero-interaction via their C-termini, the ability of FHL overexpression to restore wild-type (WT) morphological and molecular phenotypes to fhy1-3 seedlings suggests that the extreme insensitivity to FR associated with suppression of FHL expression in fhy1-3 cannot be accounted for by a critical role for FHY1-FHL heterodimers in phyA signal transmission. Rather, we suggest that the relative abundances of FHY1 and FHL in WT plants account for the differences in the severity of fhy1 and fhl mutations. As for FHY1, FHL transcript accumulation is dependent on FHY3 and is decreased after exposure to FR, R or B light. These findings reiterate the prevalence of partial degeneracy in plant signaling networks that regulate responses crucial to survival.

Phytochromes are red/far-red photochromic photoreceptors central to regulating plant development. Although they are known to enter the nucleus upon light activation and, once there, regulate transcription, this is not the complete picture. Various phytochrome effects are manifested much too rapidly to derive from changes in gene expression, whereas others seem to occur without phytochrome entering the nucleus. Phytochromes also guide directional responses to light, excluding a genetic signaling route and implying instead plasma membrane association and a direct cytoplasmic signal. However, to date, no such association has been demonstrated. Here we report that a phytochrome subpopulation indeed associates physically with another photoreceptor, phototropin, at the plasma membrane. Yeast two-hybrid methods using functional photoreceptor molecules showed that the phytochrome steering growth direction in Physcomitrella protonemata binds several phototropins specifically in the photoactivated Pfr state. Split-YFP studies in planta showed that the interaction occurs exclusively at the plasma membrane. Coimmunoprecipitation experiments provided independent confirmation of in vivo phy-phot binding. Consistent with this interaction being associated with a cellular signal, we found that phytochrome-mediated tropic responses are impaired in Physcomitrella phot − mutants. Split-YFP revealed a similar interaction between Arabidopsis phytochrome A and phototropin 1 at the plasma membrane. These associations additionally provide a functional explanation for the evolution of neochrome photoreceptors. Our results imply that the elusive phytochrome cytoplasmic signal arises through binding and coaction with phototropin at the plasma membrane.

The plant photoreceptor phytochrome plays an important role in the nucleus as a regulator of transcription. Numerous studies imply, however, that phytochromes in both higher and lower plants mediate physiological reactions within the cytoplasm. In particular, the tip cells of moss protonemal filaments use phytochrome to sense light direction, requiring a signaling system that transmits the directional information directly to the microfilaments that direct tip growth. In this work we describe four canonical phytochrome genes in the model moss species Physcomitrella patens, each of which was successfully targeted via homologous recombination and the distinct physiological functions of each gene product thereby identified. One homolog in particular mediates positive phototropism, polarotropism, and chloroplast movement in polarized light. This photoreceptor thus interacts with a cytoplasmic signal͞response system. This is our first step in elucidating the cytoplasmic signaling function of phytochrome at the molecular level.