Differential interactions of phytochrome A (Pr vs. Pfr) with monoclonal antibodies probed by a surface plasmon resonance technique.

Phytochromes are red- and far-red light-reversible photoreceptors for photomorphogenesis in plants. Phytochrome A is a dimeric chromopeptide that mediates very low fluence and high irradiance responses. To analyze the surface properties of phytochrome A (phyA), the epitopes of 21 anti-phyA monoclonal antibodies were determined by variously engineered recombinant phyA proteins and the dissociation constants of seven anti-phyA monoclonal antibodies with phyA were measured using a surface plasmon resonance (SPR)-based resonant mirror biosensor (IAsys). Purified oat phyA was immobilized on the sensor surface using a carboxymethyl dextran cuvette in advance, and the interactions of each chosen monoclonal antibody against phyA in either red light absorbing form (Pr) or far-red light absorbing form (Pfr) at different concentrations were monitored. The binding profiles were analyzed using the FAST Fit program of IAsys. The resultant values of dissociation constants clearly demonstrated the differential affinities between the phyA epitopes and the monoclonal antibodies dependent upon Pr vs. Pfr conformations. Monoclonal antibody mAP20 preferentially recognized the epitope at amino acids 653-731 in the Pr form, whereas mAA02, mAP21 and mAR07/mAR08 displayed preferential affinities for the Pfr's surfaces at epitopes 494-601 (the hinge region between the N- and C-terminal domains), 601-653 (hinge in PASI domain), and 772-1128 (C-terminal domain), respectively. The N-terminal extension (1-74) was not recognized by mAP09 and mAP15, suggesting that the N-terminal extreme is not exposed in the native conformation of phyA. On the other hand, the C-terminal domain becomes apparently exposed on Pr-to-Pfr phototransformation, suggesting an inter-domain cross-talk. The use of surface plasmon resonance spectroscopy offers a new approach to study the surface properties of phytochromes associated with the photoreversible structural changes, as well as for the study of protein-protein interactions of phytochromes with their interacting proteins involved in light signaling events in plants.     

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor

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 Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 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, Delta6-12 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 Delta6-12 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 Delta6-12 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.     

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.     

Photoresponses of transgenic Arabidopsis overexpressing the fern Adiantum capillus-veneris PHY1.

The phytochrome gene (PHY1) cDNA from the fern Adiantum capillus-veneris encodes an amino acid sequence that shows equal similarity (50-60%) to all five Arabidopsis phytochromes (PHYA-E). The A. capillus-veneris PHY1 cDNA was transformed into Arabidopsis ecotype Landsberg erecta to investigate its activity in angiosperms. Three of the resulting lines contained at least 8 times more spectrally active phytochrome than the wild type, indicating that A. capillus-veneris phytochrome can incorporate the chromophore of the host plants. Hypocotyl growth inhibition of these transgenic lines was investigated under red and far-red light. The results indicated dominant negative activity of A. capillus-veneris phy1 on the phytochrome A response in the host plants under continuous far-red light. However, the fern phytochrome did not interfere with the red-light repression of hypocotyl growth mediated by endogenous phytochrome B, and it failed to complement a phyB mutant phenotype. These observations suggest that the phy1 phytochrome molecule is too diverged from those of Arabidopsis to be fully functional.     

Action spectra for the inhibition of growth in radish hypocotyls

In etiolated seedlings of Raphanus sativus L. the inhibition of hypocotyl elongation by continuous light showed a major bimodal peak of action in the red and far-red, and two minor peaks in the blue regions of the spectrum. It is argued that, under conditions of prolonged irradiation, phytochrome is the pigment controlling the inhibition of hypocotyl elongation by red and far-red light, but that its mode of action in far-red is different from that in red. A distinct pigment is postulated for blue light.Abbreviations B blue - FR far red - G green - R red - HIR high irradiance reaction - P_r and P_fr red and far red absorbing forms of phytochrome - R red     

Arabidopsis phytochrome a is modularly structured to integrate the multiple features that are required for a highly sensitized phytochrome

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.     

An amino-terminal deletion of rice phytochrome A results in a dominant negative suppression of tobacco phytochrome A activity in transgenic tobacco seedlings

Overexpression of phytochrome A results in an increased inhibition of hypocotyl elongation under red and far-red light. We used this approach to assay for the function of N-terminal mutations of rice (Oryza sativa L.) phytochrome A. Transgenic tobacco seedlings that express the wild-type rice phytochrome A (RW), a rice phytochrome A lacking the first 80 amino acids (NTD) or a rice phytochrome A with a conversion of the first 10 serines into alanine residues (S/A) were compared with untransformed wild-type tobacco (Nicotiana tabacum L. cv. Xanthi) seedlings. Experiments under different fluence rates showed that RW and, even more strongly, S/A increased the response under both red and far-red light, whereas NTD decreased the response under far-red light but hardly altered the response under red light. These results indicate that NTD not only lacks residues essential for an increased response under red light but also distorts the wild-type response under far-red light. Wild-type rice phytochrome A and, even more so, S/A mediate an enhanced phytochrome A as well as phytochrome B function, whereas NTD interferes with the function of endogenous tobacco phytochrome A as well as that of rice phytochrome A when co-expressed in a single host. Experiments with seedlings of different ages and various times of irradiation under far-red light demonstrated that the effect of NTD is dependent on the stage of development. Our results suggest that the lack of the first 80 amino acids still allows a rice phytochrome A to interact with the phytochrome transduction pathway, albeit nonproductively in tobacco seedlings.Abbreviations HIR high-irradiance response - NTD N-terminal deletion mutant of rice phytochrome A - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - RW rice wild-type phytochrome A - S/A serine-to-alanine mu-tant of rice phytochrome A - wNTD weakly expressing NTD line - XAN wild-type tobacco cv. Xanthi We thank Masaki Furuya (Adv. Research Laboratory, Hitachi, Saitama, Japan) and Akira Nagatani (RIKEN Institute, Saitama, Japan) for providing the monoclonal antibodies mAP5 and mAR14. The work was supported by a grant from the Human Frontier Science Program. K.E. was a recipient of a Landesgraduiertenförderung fellowship.     

Phytochrome E Controls Light-Induced Germination of Arabidopsis

Germination of Arabidopsis seeds is light dependent and under phytochrome control. Previously, phytochromes A and B and at least one additional, unspecified phytochrome were shown to be involved in this process. Here, we used a set of photoreceptor mutants to test whether phytochrome D and/or phytochrome E can control germination of Arabidopsis. The results show that only phytochromes B and E, but not phytochrome D, participate directly in red/far-red light (FR)-reversible germination. Unlike phytochromes B and D, phytochrome E did not inhibit phytochrome A-mediated germination. Surprisingly, phytochrome E was required for germination of Arabidopsis seeds in continuous FR. However, inhibition of hypocotyl elongation by FR, induction of cotyledon unfolding, and induction of agravitropic growth were not affected by loss of phytochrome E. Therefore, phytochrome E is not required per se for phytochrome A-mediated very low fluence responses and the high irradiance response. Immunoblotting revealed that the need of phytochrome E for germination in FR was not caused by altered phytochrome A levels. These results uncover a novel role of phytochrome E in plant development and demonstrate the considerable functional diversification of the closely related phytochromes B, D, and E.     

Photochemical characterization of phytochrome missense mutants

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses

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.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase ( chs ) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA*, phyB*) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as β-glucuronidase (GUS) expression mediated by light-regulated promoters ( chs , chlorophyll a/b binding protein ( lhcb1 ) and ferredoxin NADP+ oxidoreductase ( fnr )). Microinjection of phyA* under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs —GUS, lhcb1 —GUS and fnr —GUS expression. Microinjection of phyB* under identical conditions induced chlorophyll accumulation and mediated lhcb1 —GUS and fnr —GUS expression but neither anthocyanin accumulation nor chs —GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB* injections under red light conditions indicated that phyB* needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.     

Influence of the Dietary Protein Deficiency on the Protein Synthetic Activity of Microsome and Soluble Fraction from Muscle and Liver of Rats

Phytochromes are photoreceptors that regulate many aspects of plant growth and development in response to red/far-red light signals from the environment. In this study, we analyzed chromophore ligation and photochromism of missense phytochrome mutants in the Per-Arnt-Sim (PAS)-related domain (PRD). Among the 14 mutants analyzed, the Gly768Asp mutant of Avena phytochrome A showed aberrant photochromism and dark reversion, suggesting that amino acid residues in the C-terminal domain affect the photochemical properties of the photosensory N-terminal domain.     

Characterization of a novel non-constitutive photomorphogenic cop1 allele

A specific light program consisting of multiple treatments with alternating red and far-red light pulses was used to isolate mutants in phytochrome A-dependent signal transduction in Arabidopsis seedlings. Because of their phenotype, the mutants were called eid (empfindlicher im dunkelroten Licht, which means hypersensitive in far-red light). One of the isolated mutants, eid6, is a novel recessive allele of the COP1 gene (constitutive photomorphogenic 1) that carries an amino acid transition in a conserved histidine residue of the RING finger domain. Mutant seedlings exhibited an extreme hypersensitivity towards all tested light qualities, but in contrast to known cop1 alleles, no constitutive photomorphogenic phenotype was detectable in darkness. Thus, the novel cop1eid6 allele seems to encode for a protein whose remaining activity is sufficient for the suppression of photomorphogenesis in dark-grown plants. In adult cop1eid6 plants, the development of the Cop1 phenotype is dominated by phytochrome B. Comparison of the phenotype of the novel cop1eid6 and the weak cop1-4 allele under continuous far-red light indicates that the RING finger and coiled-coil domains of COP1 are sufficient for some specific regulatory function in phytochrome A-dependent high irradiance responses.     

FAR-RED INSENSITIVE219 modulates CONSTITUTIVE PHOTOMORPHOGENIC1 activity via physical interaction to regulate hypocotyl elongation in Arabidopsis

FAR-RED INSENSITIVE219 (FIN219) in Arabidopsis (Arabidopsis thaliana) is involved in phytochrome A-mediated far-red (FR) light signaling. Previous genetic studies revealed that FIN219 acts as an extragenic suppressor of CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1). However, the molecular mechanism underlying the suppression of COP1 remains unknown. Here, we used a transgenic approach to study the regulation of COP1 by FIN219. Transgenic seedlings containing ectopic expression of the FIN219 amino (N)-terminal domain in wild-type Columbia (named NCox for the expression of the N-terminal coiled-coil domain and NTox for the N-terminal 300-amino acid region) exhibited a dominant-negative long-hypocotyl phenotype under FR light, reflected as reduced photomorphogenic responses and altered levels of COP1 and ELONGATED HYPOCOTYL5 (HY5). Yeast two-hybrid, pull-down, and bimolecular fluorescence complementation assays revealed that FIN219 could interact with the WD-40 domain of COP1 and with its N-terminal coiled-coil domain through its carboxyl-terminal domain. Further in vivo coimmunoprecipitation study confirms that FIN219 interacts with COP1 under continuous FR light. Studies of the double mutant fin219-2/cop1-6 indicated that HY5 stability requires FIN219 under darkness and FR light. Moreover, FIN219 levels positively regulated by phytochrome A can modulate the subcellular location of COP1 and are differentially regulated by various fluence rates of FR light. We conclude that the dominant-negative long-hypocotyl phenotype conferred by NCox and NTox in a wild-type background was caused by the misregulation of COP1 binding with the carboxyl terminus of FIN219. Our data provide a critical mechanism controlling the key repressor COP1 in response to FR light.     

Expression of an N-terminal fragment of COP1 confers a dominant-negative effect on light-regulated seedling development in Arabidopsis.

CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is an essential regulatory gene that plays a role in light control of seedling development in Arabidopsis. The COP1 protein possesses three recognizable structural domains: a RING finger zinc binding domain near the N terminus, followed by a coiled-coll domain and a domain with WD-40 repeats in the C-terminal half. To determine whether COP1 acts specifically as a light-inactivable repressor of photomorphogenic development and to elucidate the functional roles of the specific structural domains, mutant cDNAs encoding the N-terminal 282 amino acids (N282) of COP1 were expressed and analyzed in transgenic plants. High-level expression of the N282 fragment caused a dominant-negative phenotype similar to that of the loss-of-function cop1 mutants. The phenotypic characteristics include hypersensitivity of hypocotyl elongation to inhibition by white, blue, red, and far-red light stimuli. In the dark, N282 expression led to pleiotropic photomorphogenic cotyledon development, including cellular differentiation, plastid development, and gene expression, although it has no significant effect on the hypocotyl elongation. However, N282 expression had a minimal effect on the expression of stress- and pathogen-inducible genes. These observations support the hypothesis that COP1 is directly involved in the light control of seedling development and that it acts as a repressor of photomorphogenesis. Further, the results imply that the N282 COP1 fragment, which contains the zinc binding and colled-coil domains, is capable of interacting with either downstream targets or with the endogenous wild-type COP1, thus interfering with normal regulatory processes. The fact the N282 is able to interact with N282 and full-length COP1 in yeast provided evidence for the latter possibility.     

Phytochrome A regulates red-light induction of phototropic enhancement in Arabidopsis.

Phytochrome A (phyA) and phytochrome B photoreceptors have distinct roles in the regulation of plant growth and development. Studies using specific photomorphogenic mutants and transgenic plants overexpressing phytochrome have supported an evolving picture in which phyA and phytochrome B are responsive to continuous far-red and red light, respectively. Photomorphogenic mutants of Arabidopsis thaliana that had been selected for their inability to respond to continuous irradiance conditions were tested for their ability to carry out red-light-induced enhancement of phototropism, which is an inductive phytochrome response. We conclude that phyA is the primary photoreceptor regulating this response and provide evidence suggesting that a common regulatory domain in the phyA polypeptide functions for both high-irradiance and inductive phytochrome responses.     

Interaction of a plant 14-3-3 protein with the signal peptide of a thylakoid-targeted chloroplast precursor protein and the presence of 14-3-3 isoforms in the chloroplast stroma

The fhy3 mutation of Arabidopsis impairs phytochrome A (phyA)-mediated inhibition of hypocotyl growth without affecting the levels of phyA measured spectrophotometrically or immunochemically. We investigated whether the fhy3-1 mutation has similar effects on very low fluence responses (VLFR) and high irradiance responses (HIR) of phyA. When exposed to hourly pulses of far-red light, etiolated seedlings of the wild type or of the fhy3-1 mutant showed similar inhibition of hypocotyl growth, unfolding of the cotyledons, anthocyanin synthesis, and greening upon transfer to white light. In the wild type, continuous far-red light was significantly more effective than hourly far-red pulses (at equal total fluence). In the fhy3-1 mutant, hourly pulses were as effective as continuous far-red light, i.e. the failure of reciprocity typical of HIR was not observed. Germination was similarly promoted by continuous or pulsed far-red in wild-type and fhy3-1 seeds. Thus, for hypocotyl growth, cotyledon unfolding, greening, and seed germination, the fhy3-1 mutant retains VLFR but is severely impaired in HIR. These data are consistent with the idea that VLFR and HIR involve divergent signaling pathways of phyA.