Pfr Form Research Articles

Loading of quin2 into the oat protoplast and measurement of cytosolic calcium ion concentration changes by phytochrome action

The loading of quin2 into oat protoplasts was carried out in an incubation medium (0.6 M sorbitol, 1 mM CaCl 2, 5 mM Mes, 5 mM Tris, 0.05% BSA, 1 mM KCl, 1 mM MgSO 4 (pH 6.8)), in which we found the best viability of the protoplast and the highest membrane permeability of quin2/AM, compared with the results obtained from any other incubation medium we had tried to use. 50 μM of quin2/AM was added in the suspension medium containing 5 · 10 5/ml of oat protoplasts, and incubation at 4°C was performed for 24 h. From atomic absorption data, we confirmed that quin2 loading was 1.78 mmol per liter of cells. Red-light (660 nm) irradiation for 5 min caused an increase of the cytosolic Ca 2+ concentration from 30 to 193 nM. On the other hand, a subsequent irradiation with far-red light (730 nm) for 5 min decreased it by about 48 nM. Even when the extracellular Ca 2+ was completely chelated with 1 mM EDTA, red light increased the cytosolic Ca 2+ concentration by about 51 nM and far-red light decreased it to 3 nM. These results imply that the Pfr form of phytochrome functions not only in the process of influx of Ca 2+, but also in the mobilization process of Ca 2+ from the intracellular Ca 2+ pools. The fact that the Pr form of phytochrome lowers the cytosolic Ca 2+ concentration is also presented in this report.

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy applied to phytochrome and its model compounds. 2. Phytochrome and phycocyanin chromophores

Surface-enhanced resonance Raman scattering (SERRS) spectra of phytochrome at 77 K are reported. The spectra reveal significant differences between Pr and Pfr forms of phytochrome. SERRS spectra of C-phycocyanin Z,Z,Z- and Z,Z,E-chromopeptide isomers at 77 K are also reported. The phycocyanin chromopeptide studies are used to provide a basis for interpreting the phytochrome SERRS spectra. The spectra indicate that photoisomerization of chromophores from C-phycocyanin chromopeptides (from a Z,Z,Z to a Z,Z,E configuration) is detectable with SERRS.

Surface enhanced resonance Raman scattering (SERRS) as a probe of the structural differences between the Pr and Pfr forms of phytochrome

Surface enhanced resonance Raman scattering (SERRS) spectra have been obtained from the active, far-red light absorbing (Pfr) and biologically inactive (Pr) forms of phytochrome adsorbed on silver colloids. Substantial differences between the SERRS spectra of the two forms in the low and high wavenumber regions are observed using 406.7 nm wavelength excitation. These differences reinforce those seen with 413.1 nm wavelength excitation in the high wavenumber region. Simultaneously, extensive differences are observed in the SERRS obtained from the same form in the low wavenumber region using 406.7 nm, as compared with 413.1 nm wavelength excitation. The relative intensity differences observed for the two forms, and those obtained using two slightly different excitation wavelengths to illuminate the same form, suggest that some type of subtle, proteincontrolled structural variation is responsible for the spectroscopic differences. A Z → E isomerization during the Pr → Pfr phototransformation is consistent with the SERRS data, although the overall chromophore conformations are most likely conserved for the native Pr- and Pfr-phytochrome species. Slight out-of-plane ring twisting, accompanying the Pr → Pfr photoisomerization, may be responsible for the large difference in the spectroscopic properties of the native Pr and Pfr chromophores.

Differential exposure of aromatic amino acids in the red-light-absorbing and far-red-light-absorbing forms of 124-kDa oat phytochrome.

The surface topography of aromatic amino acid residues and/or other hydrophobic groups of phytochrome has been investigated by ultraviolet absorption spectra and ultraviolet circular dichroism using phytochrome-cyclodextrin inclusion complexation. Three different types of cyclodextrins (alpha, beta and gamma) with varying hydrophobic cavity sizes, were used. Complexation resulted in significant changes in the circular dichroic signals of both the red-light-absorbing (Pr) and far-red-light-absorbing (Pfr) forms of phytochrome in the ultraviolet region at 222 nm, mid-ultraviolet at 280 nm and 300 nm and in the near-ultraviolet and visible regions at 365 nm and 670 mm, respectively, alpha- and beta-Cyclodextrins were markedly (1.7-4.5-fold) more effective in reducing the mid-ultraviolet CD signal of Pr than that of Pfr, indicating a differential inclusion of the aromatic amino acid residues. gamma-Cyclodextrin did not exhibit any significant differentiation. Secondary structure analysis of the phytochrome-cyclodextrin complexes revealed a considerable increase in the alpha-helical contents of both Pr and Pfr forms. The increase in the Pfr form (17-25%) was about twice that in the Pr form (8-9%), indicating a differential effect of complexation on the conformation of the phytochrome protein. Although the photostationary-state equilibrium of the phytochrome was not affected by the cyclodextrin complexation, the Pr----Pfr phototransformation rate was significantly increased. However, the Pfr----Pr photoreversion was not affected significantly. The results suggest a differential complexation of cyclodextrins with the Pr and Pfr forms of phytochrome as a result of a difference in accessibility of aromatic amino acids in the two forms. A detailed analysis of absorption difference spectra and circular dichroic spectra around 280 nm also revealed evidence for a difference in the exposure of aromatic amino acids.

Properties of a Polycation-Stimulated Protein Kinase Associated with Purified Avena Phytochrome

ATP-dependent polycation-stimulated phosphorylation of highly purified phytochrome preparations from etiolated Avena seedlings has been reported previously (Y-S Wong, H-C Cheng, DA Walsh, JC Lagarias [1986] J Biol Chem 261: 12089-12097). In this study, we present a more detailed description of the properties of this protein kinase based on the analysis of over 30 different Avena phytochrome preparations. ATP-dependent phosphorylation of phytochrome was strongly stimulated by a wide range of polycationic molecules, including synthetic and natural polypeptides as well as nonpeptide cationic polymers. Many of the compounds known to stimulate other known protein kinases (i.e., cyclic nucleotides, Ca(2+), calmodulin, diacylglycerol, phospholipids) were either inhibitory or nonstimulatory. Among the polycations, histone H1, polylysine, and polybrene were the most effective, giving average stimulations of four- to sevenfold. Polycation-stimulated protein phosphorylation was inhibited by elevated ionic strength; of the salts examined, magnesium pyrophosphate was a particularly potent inhibitor of the kinase activity. MgATP was preferred as the phosphoryl donor to either MgGTP or magnesium pyrophosphate. The K(m) for MgATP was estimated to be 30 micromolar when histone H1 was used as a protein substrate. The Pr form of phytochrome was always a better substrate than the Pfr form regardless of the polycation present. Polylysine-stimulated, phytochrome(preparation)-dependent phosphorylation of purified maize phosphoenolpyruvate carboxylase was observed, as well as phosphorylation of a number of polypeptides in crude soluble protein extracts from etiolated Avena seedlings.

Light‐Induced Fern‐Spore Germination under Reduced Water Potential

AbstractThe phytochrome‐mediated spore germination of the fern Dryopteris filix‐mas was studied under reduced water content. Spores were sown on solutions containing polyethylene glycol (PEG), sorbitol, or mannitol as osmotically active substances, yielding water potential values (ψ) between − 500 and − 3500 kPa for most of the experiments.Our main results can be summarized as follows: The germination of the spores is completely blocked by ψ ≤ −1200 kPa (≥ 300 g/l PEG). Reduced water content of the spores, at least up to 300 g/l PEG, does not impair the phototransformation of phytochrome from its inactive form Pr to its active form Pfr. The inhibiting effect of the osmoticum is phase‐specific: the reduced water content of the spores impairs the coupling of the phytochrome system to the transduction chain. Under these conditions, Pfr is found to be stable for several days. In addition, the reduced water potential acts on the terminal response, although differently on different parameters of germination: Whereas formation of the rhizoid is completely blocked by 180 g/1 PEG, chlorophyll formation requires 300 g/1 PEG to become slightly retarded.

Partial purification and peptide mapping of ubiquitin-phytochrome conjugates from oat

Phytochrome is rapidly degraded in vivo following photoconversion from the relatively stable red light absorbing form Pr to the far red light absorbing form Pfr. In etiolated seedlings from several species, this photoconversion also induces the accumulation of ubiquitin-phytochrome conjugates (Ub-P), suggesting that Pfr is degraded via a ubiquitin-dependent proteolytic pathway. To understand why Pfr is preferentially conjugated with ubiquitin, Ub-P were partially purified and characterized with the ultimate goal of mapping ubiquitin attachment sites. Ub-P were partially purified by poly(ethylene imine) and ammonium sulfate precipitations followed by hydroxyapatite chromatography and separated from unmodified phytochrome by size-exclusion chromatography. Up-P had an apparent native molecular size of approximately 600 kDa, substantially larger than that of the unmodified Pfr dimer (365 kDa). Ub-P retained the property of spectral photoreversibility. The initial digestion patterns of Ub-P were similar to unmodified phytochrome. In an attempt to identify ubiquitin attachment site(s), Ub-P were probed with a library of anti-oat phytochrome monoclonal antibodies. Of the 16 different anti-phytochrome monoclonal antibodies tested, 3 (O76C, O19F, and O311B) poorly recognized all size classes of Ub-P, indicating that the corresponding epitopes were masked either directly or indirectly as a result of ubiquitin ligation. These epitopes are located between residuesmore » 90 and 180 (O76C), 558 and 668 (O19F), and 747 and 830 (O311B) of oat phytochrome. Because the regions recognized by O19F and O311B are near the C-terminal domain containing at least one ubiquitin attachment site, and near amino acid residues that become more accessible when the chromoprotein is in the Pfr form, these regions may be important in the Pfr-dependent ubiquitination of phytochrome.« less

Binding of 124‐kilodalton oat phytochrome to liposomes and chloroplasts

Binding of the radio‐iodinated 124‐kDa oat (Avena sativa L. cv. Garry) phytochrome to liposomes and chloroplasts was investigated as a model system in order to understand the molecular affinity of phytochrome toward cellular organelles in plants. The binding of intact (124 kDa) phytochrome to liposomes and chloroplasts is hydrophobic in nature, as in the case of the degraded (118/114 kDa) phytochrome, but electrostatic interactions play a greater role in the intact phytochrome. The physiologically active Pfr form of the intact phytochrome showed a binding preference over the inactive Pr form with neutral liposomes and chloroplasts. However, the Pfr form of intact phytochrome exhibits smaller binding preference than the Pfr form of degraded phytochrome over their respective Pr forms (see Kim, I.‐S. and Song, P.‐S. 1981, Biochemistry 20: 5482–5489, for degraded phytochrome binding). These results indicate that the 6/10 kDa N‐terminus segment, which is lost in the degraded phytochrome, plays an important role in determining the protein surface properties of the intact phytochrome. A competitive binding study on phytochrome also suggested that the Pfr form had a greater binding affinity for chloroplasts than the Pr form. However, the physiological activity of the Pfr form may not be explained simply by the observed difference in binding affinity between the two forms of phytochrome.

Binding of 124-kilodalton oat phytochrome to liposomes and chloroplasts

Binding of the radio-iodinated 124-kDa oat (Avena sativa L. cv. Garry) phytochrome to liposomes and chloroplasts was investigated as a model system in order to understand the molecular affinity of phytochrome toward cellular organelles in plants. The binding of intact (124 kDa) phytochrome to liposomes and chloroplasts is hydrophobic in nature, as in the case of the degraded (118/114 kDa) phytochrome, but electrostatic interactions play a greater role in the intact phytochrome. The physiologically active Pfr form of the intact phytochrome showed a binding preference over the inactive Pr form with neutral liposomes and chloroplasts. However, the Pfr form of intact phytochrome exhibits smaller binding preference than the Pfr form of degraded phytochrome over their respective Pr forms (see Kim, I.-S. and Song, P.-S. 1981, Biochemistry 20: 5482–5489, for degraded phytochrome binding). These results indicate that the 6/10 kDa N-terminus segment, which is lost in the degraded phytochrome, plays an important role in determining the protein surface properties of the intact phytochrome. A competitive binding study on phytochrome also suggested that the Pfr form had a greater binding affinity for chloroplasts than the Pr form. However, the physiological activity of the Pfr form may not be explained simply by the observed difference in binding affinity between the two forms of phytochrome.

The ‘end‐of‐day’ phytochrome control of internode elongation in mustard: kinetics, interaction with the previous fluence rate, and ecological implications

Abstract The ‘end‐of‐day’ phytochrome control of internode growth was characterized in Sinapis alba, seedlings previously grown under continuous white light for 13 d. The transition from white light to darkness caused a reduction in internode extension rate with a lag of less than 10 min. Following this, extension rate remained almost constant for at least 48 h. i.e. ‘re‐etiolation’ was not noticed. The phytochorme controlling the growth processes was stable in the Pfr form. The growth rate of plants receiving a red light pulse, and the growth promotion caused by a far‐red light pulse, increased with increasing fluence rate of the previous white light treatment. In far‐red treated plants a first growth rate acceleration peaked at 20–30 min after the end of white light, followed by a transient deceleration which led to a growth rate minimum at 40–60 min, followed by a final growth rate recovery yielding a more‐or‐less steady elevated rate. Pulses establishing different Pfr/P modified the extent, but not the early kinetics, of the growth response. The relative promotion of growth caused by low Pfr/P was limited by darkness as follows: (a), The growth promotion caused by far‐red directed to the internode alone was transient. (b), The promotion caused by a reduction of Pfr/P in the whole shoot persisted in darkness for at least 48 h and also persisted if, after a 3–9 h dark period, the plants were returned to continuous white light. In darkness, however, the magnitude of this growth rate promotion decreased with time, particularly when the previous white light fluence rate was low, or the pulse preceding darkness provided the lowest Pfr/P. (c), When compared over the same period in darkness, growth rate was higher in those seedlings in which Pfr/P was reduced during the continuous white light pretreatment than in those ones in which the Pfr/P was only reduced immediately before darkness. It is proposed that in the natural environment, red/far‐red signals could be more effective when provided during daytime than at the end of the photoperiod, as both the background growth rate and the relative promotion caused by low Pfr/P are reduced by darkness.

Interactions between native oat phytochrome and tetrapyrroles.

The suggestion, that the increase in the far-UV CD signal of the 124 kDa oat phytochrome upon phototransformation of the Pr to Pfr form is possibly due to the chromophore interaction with the N-terminus segment of the phytochrome protein in the Pfr from (Chai, Y.G., Song, P.S., Cordonnier, M.-M. and Pratt, L.H. (1987) Biochemistry 26, 4947-4952), has been investigated by measuring the circular dichroism in the absence of exogenous tetrapyrrolic chromophores (bilirubin, biliverdin, chlorophyllin and hemin). Open tetrapyrrolic chromophores (bilirubin and biliverdin) did not have any significant effect on the phototransformability of the far-UV CD signal of the phytochrome, whereas closed tetrapyrroles (chlorophyllin and hemin) almost completely blocked the increase in the far-UV CD signal upon Pr to Pfr phototransformation. However, closed tetrapyrroles had no effect on the decrease in the CD signal upon Pfr to Pr photoconversion. Secondary structure analysis showed that the alpha-helix content of both Pr and Pfr forms of phytochrome (with 53 and 56% alpha-helical content, respectively) increased to 62% when a 50-fold molar excess of chlorophyllin was added to them separately. Spectral phototransformation of phytochrome was not affected in the presence of tetrapyrroles, except in the case of hemin. A 50-fold molar mass of hemin caused a significant bleaching of the Pfr form of phytochrome but not that of the Pr form. These results suggest that the chromophore-protein interaction is significantly altered during the phototransformation of phytochrome.

Circadian Rhythm in the Expression of the mRNA Coding for the Apoprotein of the Light-Harvesting Complex of Photosystem II

The mRNA coding for light-harvesting complex of PSII (LHC-II) apoprotein is present in etiolated bean (Phaseolus vulgaris L.) leaves; its level is low in 5-day-old leaves, increases about 3 to 4 times in 9- to 13-day-old leaves, and decreases thereafter. A red light pulse induces an increase in LHC-II mRNA level, which is reversed by far red light, in all ages of the etiolated tissue tested. The phytochrome-controlled initial increase of LHC-II mRNA level is higher in 9- and 13-day-old than in 5- and 17-day-old bean leaves. The amount of LHC-II mRNA, accumulated in the dark after a red light pulse, oscillates rhythmically with a period of about 24 hours. This rhythm is also observed in continuous white light and in the dark following exposure to continuous white light, and persists for at least 70 hours. A second red light pulse, applied 36 hours after initiation of the rhythm, induces a phase-shift, which is prevented by far red light immediately following the second red light pulse. A persistent, but gradually reduced, far red reversibility of the red light-induced increase in LHC-II mRNA level is observed. In contrast, far red reversibility of the red light-induced clock setting is only observed when far red follows immediately the red light. It is concluded that (a) the light-induced LHC-II mRNA accumulation follows an endogenous, circadian rhythm, for the appearance of which a red light pulse is sufficient, (b) the circadian oscillator is under phytochrome control, and (c) a stable Pfr form, which exists for several hours, is responsible for sustaining LHC-II gene transcription.

Site-Selected Chromophore Oxidation of Phytochrome with Tetranitromethane

Chromophore oxidation using tetranitromethane was performed with 124 kDa, 59kDa, and 39kDa oat phytochrome in both photoreversible forms (Pr and Pfr). Kinetics and intermediates of oxidation were analyzed using a diode array spectrophotometer. A transient species (λmax = 580 and 505 nm) was observed exclusively from the Pfr (or Pbl in the case of 39 kDa phytochrome) forms of degraded phytochromes. This oxidation product was isolated by rapid size-exclusion HPLC. Comparison of its absorption spectrum with those of model compounds revealed a strong similarity to biladienes-a,b, indicating that the primary attack of tetranitromethane takes place at the methine bridge, predominantly at C-15. This is interpreted in terms of a different accessibility of carbon-15 of the chromophore in the Pr vs. Pfr form.

Detection of a Phytochrome‐like Protein in Macroalgae

AbstractA phytochrome‐like protein was detected in extracts from the red algae Corallina elongata and Gelidium sp., from the brown algae Cystoseira abiesmarina and Cystoseira tamariscifolia, and from the green algae Ulva rigida, Enteromorpha compressa and Chara hispida. Relative amounts of the photoreversible protein were determined by measurement of Δ (ΔA) values of the crude extract. SDS gel electrophoresis and immunoblotting with monoclonal antibodies directed to phytochrome from etiolated maize and oat seedlings revealed only one phytochrome‐related band with apparent molecular weight of 130 kDa. The absorption difference spectrum after partial purification showed a “normal” absorption band (λmax = 670 nm) for the Pr form but only a very weak band (λmax = 705 nm) for the “Pfr form”.

Use of bilirubin oxidase for probing chromophore topography in tetrapyrrole proteins

Bilirubin oxidase has been used to probe the surface topography of phycocyanins (C-phycocyanin and phycocyanin-645), peridinin-chlorophyll a-protein and phytochrome. The enzyme catalyzes oxidation of the tetrapyrrolic chromophores in these proteins. Relative rates of oxidation were 78.0 × 10 −6 s −1 (monitored at 617 nm) and 58.0 × 10 −6 s −1 (592 nm) for C-phycocyanin, 43.0 × 10 −6 s −1 for phycocyanin-645, 0.3 × 10 −6 s −1 (at 671 nm) and 1.3 × 10 −6 s −1 (at 480 nm) for peridinin-chlorophyll a-protein. The relative rate of free chlorophyllin a was 2.8 × 10 4 s −1 whereas upon binding to human serum albumin its rate of oxidation was reduced to 3.3 × 10 −3 s −1. Relative rates for the oxidation of Pr and Pfr forms of phytochrome were 2.9 and 19.5 s −1, respectively, which are consistent with earlier finding [(1984) Plant Physiol. 74, 755–758] that indicated a preferential exposure of tetrapyrrolic chromophore in the Pfr form. In general, k cat/ K m values derived from the Lineweaver-Burk plots followed the same trend as the relative rates of oxidation. For example, the k cat/ K m for the free chlorophyllin a was 2.8 × 10 6 M −1 s −1 but it was only 1.1 M −1s −1 for the chlorophyll a in peridinin-chlorophyll a-protein where the chlorophyll is shielded by protein. These results reflect varying degrees of protection of the tetrapyrrolic chromophores from the enzymatic oxidation and prove that bilirubin oxidase can be generally used as a probe for deducing the topography of tetrapyrrolic chromophores.

Electrophoresis and Electrofocusing of Phytochrome from Etiolated Avena sativa L.

Abstract Phytochrome from etiolated oat seedlings (Avena sativa L.) was investigated by “native” gel electrophoresis and by isoelectric focusing. At pH 8 . 8 the Pfr form migrated faster than the Pr form in electrophoresis. We assume a difference in the surface charge rather than a difference in shape for the phytochrome forms. This assumption was confirmed by isoelectric focusing which clearly showed relatively more negative charge in the Pfr form than in the Pr form. The role of the peptide region from residue 323 to 360 is discussed in this connection. It carries 9 negatively charged residues, it is exposed only in the Pfr form and it has already been described as a signal region for rapid protein degradation (PEST sequence, see Rogers et al., Science 234, 364-368, 1986). The experiments on electrofocusing revealed a microheterogeneity of phytochrome which was present in the native state as well as in the completely unfolded state. The most probable reason could be either posttranslational modification or genetic polymorphism of phytochrome in oat.

Molecular properties and biogenesis of phytochrome I and II

Previously, phytochrome was thought to consist of a single molecular species. However, physiological and spectrophotometric evidence has accumulated to indicate that there are two phytochrome pools in tissues, one of which is predominant in dark-grown tissues and rather unstable in the light, and the other present in very low concentrations but stable, even in the Pfr form, irrespective of light condition. Recently, two immunochemically distinct phytochromes I and II, PI and PII, were found in both dark- and light-grown tissues, and their comparative amino acid sequences shown to be 64% homologous. This is crucial evidence for the presence of chemically different phytochrome apoproteins in a single plant species. However, it is still an open question as to which phytochrome, PI or PII, is a component of the photolabile and photostable pools of phytochrome. Our understanding of the molecular structure of phytochrome has been greatly improved by recent, rapid progress in the cloning and characterization of phytochrome genes. The expression of PI genes is photoreversibly inhibited by the photostable Pfr pool, while that of several other genes, like Cab, appears to be induced by PI in the Pfr form. It is suggested that autoregulation of phytochrome gene expression is not so simply governed in plants as thought earlier. If there are two different phytochromes in a plant cell, the most important physiological problem to be solved is which phytochrome triggers the numerous red/far-red reversible reactions reported in the literature. Photomorphogenetic mutants and transgenic plants with engineered phytochrome genes will probably help to solve this problem in the future, and preliminary work along this line has already introduced in this article. A model of the molecular structure of pea PI dimer was proposed on the basis of small angle X-ray scattering analysis, and the model then confirmed by rotary shadowing electron microscopy. Important questions are still open, such as: what is the nature of phytochrome's partner compounds in cells (phytochrome receptor)? How is/are the phytochrome-induced signal(s) transmitted in the signal transduction chain?

A photoreversible conformational change in 124 kDa Avena phytochrome

Tryptophan (Trp) fluorescence quenching of phytochrome has been studied using anionic, cationic and neutral quenchers, I −, Cs + and acrylamide, respectively, in an effort to understand the molecular differences between the Pr and Pfr forms. The data have been analyzed using both Stern-Volmer and modified Stern-Volmer kinetic treatments. The anionic quencher, I −, was proven to be an ineffective quencher with Stern-Volmer constants, K sv, of 0.60 and 0.63 M −1, respectively, for the Pr and Pfr forms of phytochrome. The cationic quencher, Cs +, showed about a 2-fold difference in the K sv of Pr and Pfr, indicating a significant change in the fluorescent Trp environments during the Pr to Pfr phototransformation. However, only 25–37% of the fluorescent Trp residues were accessible to the cationic quencher. Most of the fluorescent Trp residues were accessible to acrylamide, but the quenching by acrylamide was indistinguishable for the Pr and Pfr forms. An additional quenching by acrylamide after a saturated quenching with Cs + showed more than 40% increase in the K sv of Pfr over Pr. These observations, along with the finding of two distinct components in the Trp fluorescence lifetime, indicate the existence of Trp residues in at least two different sets of environments in the phytochrome protein. The two components of the fluorescence had lifetimes of 1.1 ns (major) and 4.7 ns (minor) for Pr and 0.9 ns (major) and 4.6 ns (minor) for Pfr. Fluorescence quenching was found to be both static and dynamic as the Stern-Volmer constants for the steady-state fluorescence quenching were higher than for the dynamic fluorescence quenching. Based on the quenching results, in combination with the location of Trp residues in the primary structure, we conclude that the Pr to Pfr phototransformation involves a significant conformation change in the phytochrome molecule, preferentially in the 74 kDa chromophore-bearing domain.

INTERMEDIATES IN THE PHOTOCONVERSION OF FUNCTIONAL PHYTOCHROME IN FERN SPORES OF Dryopteris–II. In vivo KINETICS OF THE DECAY OF Ii700 AND OF THE FORMATION OF Pfr STUDIED WITH A DOUBLE‐LASER APPARATUS*

Abstract— Photoconversion of the red‐light‐absorbing form of phytochrome, Pr, to the far‐red‐light‐absorbing form, Pfr, was investigated in vivo at 22°C with 600 or 800 ns laser pulses of high spectral purity and induction of spore germination in Dryopteris paleacea was used as indicator for the progress of photoconversion. This reaction is initiated by a saturating R‐laser pulse of 648.5 nm, establishing an equilibrium of the photochromic system between Pr and the very early intermediates, Ii700 (Prφ Ii700)‐ The decay of Ii700 as well as the formation of Pfr was recorded by the application of a second pulse varied between 698 and 717.5 nm, which inhibits the formation of Plr being absorbed predominantly by Ii700or Pfr, respectively.The most effective inhibition for the second pulse is found up to 10 u.s after the first pulse and this is interpreted by photoreversion of Ii700 to Pr; thus reducing the formation of Pfr from Ii700. This early inhibition decreases between 10 μs to 100 ms after the R‐laser pulse, as a result of the decay of Iibl to a bleached species I,;. This decay can be described by three first order kinetics with the rate constants k12= 16830 ± 2970 s‐1, k12= 666 ± 218 s‐1,k13= 9.8 ± 0.9 s‐1. A second inhibition, due to the formation of Pfr, is found for dark intervals <100 ms and can be described by two first order kinetics with the rate constants k21= 2.9 ± 0.6 s‐1 and k22= 0.17 s‐l.

The molecular topography of phytochrome: Chromophore and apoprotein

Phytochrome serves as the photochromic receptor for a number of morphogenic and developmental responses to red light in higher plants. The photoreversible phototransformation of 124 kDa oat phytochrome involves several structural changes in the chromophore and the apoprotein, including a configurational/conformational isomerization and secondary/tertiary structural changes respectively. For example, there appears to be a specific interaction between the chromophore and the amino terminus segment in the Pfr form of phytochrome, which results in a photoreversible peptide folding of the amino terminus peptide chain. Other structural changes also accompany the phototransformation, as has been probed by peptide mapping, phosphorylation, and monoclonal antibodies.