Phytochrome A and its Functional Manifestations in Etiolated and Far-red Light-grown Seedlings of the Wild-type Rice and its Hebiba and Cpm2 Mutants Deficient in the Defense-related Phytohormone Jasmonic Acid.

Interaction between phytochromes and hormones is becoming one of the major issues in plant photophysiology. In this work, effects of defense-related jasmonic acid (JA) on phytochrome A (phyA) were investigated by fluorescence spectroscopy making use of two JA biosynthesis mutants of rice: cpm2 with the inactivated gene allene oxide cyclase and hebiba with additional genes deleted. Constant far-red light (FRc) mediated by phyA reduced its content in the wild type (WT) and mutants, and brought about domination of its light-stable pool (phyA″) in WT and light-labile pool (phyA') in the mutants. Pulsed FRp was much less effective. This FR effect classifies as primarily HIR with a low fluence threshold; it comprises inhibition of phyA biosynthesis, stimulation of phyA″→phyA' transformation and phyA' destruction. In the mutants, phyA suppresses [Pchlide] under FRp (VLFR) and stimulates it under FRc (HIR); these effects are lacking in WT. Similarly, phyA suppresses roots'growth under FRp in the mutants but not in WT. These JA mutant features suggest that JA reduces the phyA functional activity primarily in its phyA″ form mediating HIR. This modulating JA action on phyA functions under FR limiting their extreme manifestations may have contributed to the evolutionary advances of the land plants.

Hinge region of Arabidopsis phyA plays an important role in regulating phyA function.

Phytochrome A (phyA) is the only plant photoreceptor that perceives far-red light and then mediates various responses to this signal. Phosphorylation and dephosphorylation of oat phyA have been extensively studied, and it was shown that phosphorylation of a serine residue in the hinge region of oat phyA could regulate the interaction of phyA with its signal transducers. However, little is known about the role of the hinge region of Arabidopsis phyA. Here, we report that three sites in the hinge region of Arabidopsis phyA (i.e., S590, T593, and S602) are essential in regulating phyA function. Mutating all three of these sites to either alanines or aspartic acids impaired phyA function, changed the interactions of mutant phyA with FHY1 and FHL, and delayed the degradation of mutant phyA upon light exposure. Moreover, the in vivo formation of a phosphorylated phyA form was greatly affected by these mutations, while our data indicated that the abundance of this phosphorylated phyA form correlated well with the extent of phyA function, thus suggesting a pivotal role of the phosphorylated phyA in inducing the far-red light response. Taking these data together, our study reveals the important role of the hinge region of Arabidopsis phyA in regulating phyA phosphorylation and function, thus linking specific residues in the hinge region to the regulatory mechanisms of phyA phosphorylation.

Biological activity and dimerization state of modified phytochrome A proteins.

To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc) epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity. All the tagged phyA proteins migrate as dimers on native gels and co-immunoprecipitation reveals no binding interaction of phyA to any of the type II phys in the dark or under continuous far-red light. Dimers of the phyA 1-615 amino acid N-terminal photosensory domain (NphyA), generated in vivo with a yeast GAL4 dimerization domain and attached to a constitutive nuclear localization sequence, are expressed at a low level and, although they cause a cop phenotype in darkness and mediate a very low fluence response to pulses of FR, have no activity under continuous FR. It is concluded that type I phyA in its Pr form is present in plants predominantly or exclusively as a homodimer and does not stably interact with type II phys in a dimer-to-dimer manner. In addition, its activity in mediating response to continuous FR is sensitive to modification of its N- or C-terminus.

Streptophyte phytochromes exhibit an N-terminus of cyanobacterial origin and a C-terminus of proteobacterial origin.

BACKGROUND: Phytochromes are red light-sensitive photoreceptors that control a variety of developmental processes in plants, algae, bacteria and fungi. Prototypical phytochromes exhibit an N-terminal tridomain (PGP) consisting of PAS, GAF and PHY domains and a C-terminal histidine kinase (HK). RESULTS: The mode of evolution of streptophyte, fungal and diatom phytochromes from bacteria is analyzed using two programs for sequence alignment and six programs for tree construction. Our results suggest that Bacteroidetes present the most ancient types of phytochromes. We found many examples of lateral gene transfer and rearrangements of PGP and HK sequences. The PGP and HK of streptophyte phytochromes seem to have different origins. In the most likely scenario, PGP was inherited from cyanobacteria, whereas the C-terminal portion originated from a proteobacterial protein with multiple PAS domains and a C-terminal HK. The plant PhyA and PhyB lineages go back to an early gene duplication event before the diversification of streptophytes. Fungal and diatom PGPs could have a common prokaryotic origin within proteobacteria. Early gene duplication is also obvious in fungal phytochromes. CONCLUSIONS: The dominant question of the origin of plant phytochromes is difficult to tackle because the patterns differ among phylogenetic trees. We could partially overcome this problem by combining several alignment and tree construction algorithms and comparing many trees. A rearrangement of PGP and HK can directly explain the insertion of the two PAS domains by which streptophyte phytochromes are distinguished from all other phytochromes.

Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean.

BACKGROUND: Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean, to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses. RESULTS: We found two novel dysfunctional alleles in accessions originally considered to have a dominant E3 allele according to known DNA markers. The E3 locus, together with E1 and E4, contained multiple dysfunctional alleles. We identified 15 multi-locus genotypes, which we subdivided into 6 genotypic groups by classifying their alleles by function. Of these, the e1-as/e3/E4 genotypic group required an additional novel gene (different from E1, E3, and E4) to condition photoperiod insensitivity. Despite their common pre-flowering photoperiod insensitivity, accessions with different multi-locus genotypes responded differently to the post-flowering photoperiod. Cultivars carrying E3 or E4 were sensitive to photoperiod for post-flowering characteristics, such as reproductive period and stem growth after flowering. The phytochrome A-regulated expression of the determinate growth habit gene Dt1, an ortholog of Arabidopsis TERMINAL FLOWER1, was involved in the persistence of the vegetative activity at the stem apical meristem of flower-induced plants under long-day conditions. CONCLUSIONS: Diverse genetic mechanisms underlie photoperiod insensitivity in soybean. At least three multi-locus genotypes consisting of various allelic combinations at E1, E3, and E4 conferred pre-flowering photoperiod insensitivity to soybean cultivars but led to different responses to photoperiod during post-flowering vegetative and reproductive development. The phyA genes E3 and E4 are major controllers underlying not only pre-flowering but also post-flowering photoperiod responses. The current findings improve our understanding of genetic diversity in pre-flowering photoperiod insensitivity and mechanisms of post-flowering photoperiod responses in soybean.

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.

Solid-state NMR spectroscopic study of chromophore-protein interactions in the Pr ground state of plant phytochrome A.

Despite extensive study, the molecular structure of the chromophore-binding pocket of phytochrome A (phyA), the principal photoreceptor controlling photomorphogenesis in plants, has not yet been successfully resolved. Here, we report a series of two-dimensional (2-D) magic-angle spinning solid-state NMR experiments on the recombinant N-terminal, 65-kDa PAS-GAF-PHY light-sensing module of phytochrome A3 from oat (Avena sativa), assembled with uniformly 13C- and 15N-labeled phycocyanobilin (u-[13C,15N]-PCB-As.phyA3). The Pr state of this protein was studied regarding the electronic structure of the chromophore and its interactions with the proximal amino acids. Using 2-D 13C-13C and 1H-15N experiments, a complete set of 13C and 15N assignments for the chromophore were obtained. Also, a large number of 1H-13C distance restraints between the chromophore and its binding pocket were revealed by interfacial heteronuclear correlation spectroscopy. 13C doublings of the chromophore A-ring region and the C-ring carboxylate moiety, together with the observation of two Pr isoforms, Pr-I and Pr-II, demonstrate the local mobility of the chromophore and the plasticity of its protein environment. It appears that the interactions and dynamics in the binding pocket of phyA in the Pr state are remarkably similar to those of cyanobacterial phytochrome (Cph1). The N-terminus of the region modeled (residues 56-66 of phyA) is highly mobile. Differences in the regulatory processes involved in plant and Cph1 phytochromes are discussed.

On the collective nature of phytochrome photoactivation.

The red/far-red-sensing biological photoreceptor phytochrome is a paradigmatic two-state signaling system. The two thermally stable states are interconverted via a photoreaction of the covalently bound tetrapyrrole chromophore. Applying recently developed solid-state nuclear magnetic resonance, we study both the chromophore and its protein pocket in the Pr (red-absorbing) and Pfr (far-red-absorbing) states. The observations show that the phototransformation combines local chemical reactions with a mesoscopic transition of order. Both the chromophore and its binding pocket are quasi-liquid and disordered in Pr, yet quasi-solid and ordered in Pfr. Possible biochemical implications are discussed.

Structure of the chromophore binding pocket in the Pr state of plant phytochrome phyA.

A homology structural model was generated for plant phytochrome phyA utilizing the crystal structure of the sensory module of cyanobacterial phytochrome Cph1 (Cph1Δ2). As chromophores, either the native phytochromobilin cofactor (PΦB) or phycocyanobilin (PCB), the natural cofactor in Cph1, was incorporated. These homology models were further optimized by molecular dynamics (MD) simulations revealing a satisfying overall agreement with the crystal structure of Cph1Δ2. Notable differences in the PΦB adduct of phyA result from a restructuring of the small helical segment α(7) that leads to displacements of a few amino acids away from the cofactor. This repositioning of residues also include aspartate 218 such that, instead of its carbonyl function as in Cph1Δ2, an additional water molecule forms hydrogen bonds with the ring B and C NH groups. To validate the phyA structural model in the chromophore binding pocket, Raman spectra of the cofactor were calculated by means of the quantum mechanics/molecular mechanics (QM/MM) hybrid methodology and compared with the experimental resonance Raman (RR) spectra. The satisfactory overall agreement between calculated and experimental spectra is taken as an indication for the good quality of the structural model. Moreover, the methine bridge stretching modes and the effects of isotopic labeling at selected positions of the chromophore are very well reproduced to allow confirming even details of the methine bridge geometry as predicted by the homology model. Specifically, it is demonstrated that the experimental RR spectra are consistent with a torsional angle of ring D with respect to ring C that is distinctly higher for phyA-PCB (45°) and phyA-PΦB (42°) than for Cph1Δ2 (30°). Raman spectra calculated from different points of the MD trajectory display variations of the mode frequencies and intensities reflecting the structural fluctuations from snapshot to snapshot. The snapshot spectrum of the lowest energy structure and the sum of all snapshot spectra afford an equally good description of the experimental data. Particularly large variations between the snapshots are noted for the N-H in-plane bending mode of the pyrrole rings B and C, which reflect alterations of the hydrogen bond interactions brought about by fluctuations of water molecules in the cofactor cavity. This overestimation of the water molecule mobility is a consequence of the deficiency of the current QM/MM methodology that, due to the lack of appropriate protein force fields, cannot adequately account for the electrostatics in the cofactor pocket.

Kinome profiling reveals an interaction between jasmonate, salicylate and light control of hyponastic petiole growth in Arabidopsis thaliana.

Plants defend themselves against infection by biotic attackers by producing distinct phytohormones. Especially jasmonic acid (JA) and salicylic acid (SA) are well known defense-inducing hormones. Here, the effects of MeJA and SA on the Arabidopsis thaliana kinome were monitored using PepChip arrays containing kinase substrate peptides to analyze posttranslational interactions in MeJA and SA signaling pathways and to test if kinome profiling can provide leads to predict posttranslational events in plant signaling. MeJA and SA mediate differential phosphorylation of substrates for many kinase families. Also some plant specific substrates were differentially phosphorylated, including peptides derived from Phytochrome A, and Photosystem II D protein. This indicates that MeJA and SA mediate cross-talk between defense signaling and light responses. We tested the predicted effects of MeJA and SA using light-mediated upward leaf movement (differential petiole growth also called hyponastic growth). We found that MeJA, infestation by the JA-inducing insect herbivore Pieris rapae, and SA suppressed low light-induced hyponastic growth. MeJA and SA acted in a synergistic fashion via two (partially) divergent signaling routes. This work demonstrates that kinome profiling using PepChip arrays can be a valuable complementary ∼omics tool to give directions towards predicting behavior of organisms after a given stimulus and can be used to obtain leads for physiological relevant phenomena in planta.

The histidine kinase-related domain of Arabidopsis phytochrome a controls the spectral sensitivity and the subcellular distribution of the photoreceptor.

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.

Mapping the interaction sites of Aspergillus nidulans phytochrome FphA with the global regulator VeA and the White Collar protein LreB.

Aspergillus nidulans senses red and blue-light and employs a phytochrome and a Neurospora crassa White Collar (WC) homologous system for light perception and transmits this information into developmental decisions. Under light conditions it undergoes asexual development and in the dark it develops sexually. The phytochrome FphA consists of a light sensory domain and a signal output domain, consisting of a histidine kinase and a response regulator domain. Previously it was shown that the phytochrome FphA directly interacts with the WC-2 homologue, LreB and another regulator, VeA. In this paper we mapped the interaction of FphA with LreB to the histidine kinase and the response regulator domain at the C-terminus in vivo using the bimolecular fluorescence complementation assay and in vitro by co-immunoprecipitation. In comparison, VeA interacted with FphA only at the histidine kinase domain. We present evidence that VeA occurs as a phosphorylated and a non-phosphorylated form in the cell. The phosphorylation status of the protein was independent of the light receptors FphA, LreB and the WC-1 homologue LreA.

FTIR study of the photoinduced processes of plant phytochrome phyA using isotope-labeled bilins and density functional theory calculations.

Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global (13)C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE-->ZZZ), which involves only small protein structural changes.

A rice phytochrome A in Arabidopsis: The Role of the N-terminus under red and far-red light.

The phytochrome (phy)A and phyB photoreceptors mediate three photobiological response modes in plants; whereas phyA can mediate the very-low-fluence response (VLFR), the high-irradiance response (HIR) and, to some extent, the low fluence response (LFR), phyB and other type II phytochromes only mediate the LFR. To investigate to what level a rice phyA can complement for Arabidopsis phyA or phyB function and to evaluate the role of the serine residues in the first 20 amino acids of the N-terminus of phyA, we examined VLFR, LFR, and HIR responses in phyB and phyAphyB mutant plants transformed with rice PHYA cDNA or a mutant rice PHYA cDNA in which the first 10 serine residues were mutated to alanines (phyA SA). Utilizing mutants without endogenous phyB allowed the evaluation of red-light-derived responses sensed by the rice phyA. In summary, the WT rice phyA could complement VLFR and LFR responses such as inhibition of hypocotyl elongation under pulses of FR or continuous R light, induction of flowering and leaf expansion, whereas the phyA SA was more specific for HIR responses (e.g. inhibition of hypocotyl elongation and anthocyanin accumulation under continuous far-red light). As the N-terminal serines can no longer be phosphorylated in the phyA SA mutant, this suggests a role for phosphorylation discriminating between the different phyA-dependent responses. The efficacy of the rice phyA expressed in Arabidopsis was dependent upon the developmental age of the plants analyzed and on the physiological response, suggesting a stage-dependent downstream modulation of phytochrome signaling.

Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants.

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.

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 region of Arabidopsis phytochrome A is required for protein stability.

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6-12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation.

Time-resolved detection of conformational changes in oat phytochrome A: time-dependent diffusion.

Conformational changes in oat phytochrome A (phy) in solution after photoexcitation of the red-absorbing form (Pr) were studied in time-domain by the pulsed laser-induced transient grating technique. It was found that the diffusion coefficient (D) of far-red-absorbing form (Pfr) of large phy (1.3 x 10(-11) m(2) s(-1)) is markedly reduced compared with that of Pr (5.8 x 10(-11) m(2) s(-1)). This large reduction indicates that the conformation of Pfr is significantly changed from that of Pr, so that the intermolecular interaction with water molecules increases. This change completes within 1 ms after the photoexcitation. On the other hand, D of Pr of intact phy (4.1 x 10(-11) m(2) s(-1)) first decreases upon photoexcitation to 0.89 x 10(-11) m(2) s(-1) within 1 ms and then gradually increases with a time constant of 100 ms to the value of Pfr, 1.7 x 10(-11) m(2) s(-1). This slower phase suggests that the conformation of the N-terminal region changes with 100 ms to decrease the intermolecular interaction with water after a global change in the large phy region. The increase of D was interpreted in terms of alpha-helix formation in the Pfr form from the random coil structure in the Pr form.