Oat Phytochrome Research Articles

Cis-acting elements involved in photoregulation of an oat phytochrome promoter in rice.

Phytochrome negatively regulates the transcription of its own phyA genes. High levels of Pfr, the active, far-red-light absorbing form of phytochrome, repress phyA transcription; low Pfr levels result in derepression. We have utilized microprojectile-mediated gene transfer to identify regions of an oat phyA3 gene involved in this autoregulation. Chimeric constructs containing various deletion and sequence substitution mutants of the oat phyA3 gene fused to a chloramphenicol acetyltransferase reporter (phyA3/CAT) have been introduced into etiolated rice seedlings by particle bombardment. Low Pfr concentrations induce high phyA3/CAT expression, whereas high Pfr represses activity to near basal levels. Removal of phyA3 sequences 3' to the transcription start site reduces expression about fivefold, suggesting that intron 1 of the phyA3 gene may be required for high activity. The degree of high-Pfr-imposed repression is unaffected by any of a series of deletions or sequence substitutions in the phyA3 promoter, thus providing no evidence of any Pfr-activated negative elements. In contrast, 5' and internal deletions identify a minimum of three major positive promoter elements, designated PE1 [-381 base pairs (bp) to -348 bp], PE2 (-635 bp to -489 bp), and PE3 (-110 bp to -76 bp) that are necessary for high-level expression in low-Pfr cells. The data indicate that PE1 and PE2 are functionally redundant, but that PE3 is required in conjunction with either PE1 or PE2 for activity. PE3 contains a sequence element that is highly conserved between monocot phyA promoters, indicative of a critical role in phyA expression.

Cis-Acting Elements Involved in Photoregulation of an Oat Phytochrome Promoter in Rice

cis-Acting Elements Involved in Photoregulation of an Oat Phytochrome Promoter in Rice

A differential molecualr topography of the Pr and Pfr forms of native oat phytochrome as probed by fluoresence quenching

Tryptophan (Trp) surface topography of the red- and far-red-absorbing forms of phytochrome (Pr, Pfr) ofAvena sativa L. has been investigated by analyzing quenching of the two components of Trp fluorescence decay, in order to understand the differences in the two forms at the molecular level. Stern-Volmer kinetic analysis of the quenching data for two cationic surface quenchers, Cs(+) and Tl(+), showed strong quenching of the short component of the Pr fluorescence (Stern-Volmer constants,K sv , 27.2 and 21.4 M(-1), respectively) relative to that of Pfr fluorescenceK sv , 10.4 and 12.3 M(-1), respectively). The long component of the Trp fluorescence was quenched differentially by Cs(+) and Tl(+), withK sv of 9.0 and 19.8 M(-1), respectively, for the Pr fluorescence andK sv of 13.7 and 8.7 M(-1), respectively, for the Pfr fluorescence. The results indicate that the phytochrome Trp residues with short fluorescence lifetime are more accessible to the cationic surface quenchers than those with long fluorescence lifetime. The data, taken together with our earlier study (Singh et al. 1988, Biochim, Biophys. Acta936, 395-405), indicate that most, if not all the ten Trp residues of phytochrome, are fluorescent and exist in distinct groups differing in their topography and microenvironment, and the peptide segment containing Trp-774 and Trp-778 within the 55-kilodalton C-terminal domain of phytochrome also undergoes a subtle alteration in its surface topography during Pr→Pfr phototransformation.

Phosphopeptide mapping of Avena phytochrome phosphorylated by protein kinases in vitro

We previously demonstrated that protein kinases are useful probes of conformational changes that occur upon photoconversion of phytochrome [Wong, Y.-S., Cheng, H.-C., Walsh, D. A., & Lagarias, J. C. (1986) J. Biol. Chem. 261, 12089-12097]. Here we present phosphopeptide analyses of oat phytochrome phosphorylated by three mammalian protein kinases and by a polycation-stimulated, phytochrome-associated protein kinase. Phosphorylation of the Pr form by the cAMP-dependent protein kinase occurs predominantly on Ser17 while Ser598 is the preferred phosphorylation site on Pfr. The cGMP-dependent and Ca2(+)-activated, phospholipid-dependent protein kinases, which phosphorylate only the Pr form of phytochrome, recognize the same region on the phytochrome polypeptide as the cAMP-dependent protein kinase. Polycation-stimulated phytochrome phosphorylation reveals that, in contrast to the mammalian enzymes, the plant kinase recognizes the serine-rich, blocked N-terminus of phytochrome. The potential regulatory role of phytochrome phosphorylation, particularly in the structurally conserved serine/threonine-rich N-terminal region of the phytochrome polypeptide, is suggested by these results.

Fluorescence studies of phytochrome in the cells of etiolated oat seedlings and 124-kDa phytochrome isolated from them

Fluorescence properties of the red phytochrome form (P r) in etiolated oat seedlings and 124-kDa oat phytochrome in vitro (A 666/A 280=0.93) (P r′) were directly compared for the first time. Using a number of parameters, P r′ was found to be quite close to P r. The maxima were located in the low-temperature (85 K) emission spectrum at 685 nm and 684 nm, and in the excitation spectrum at 673 and 672 nm for P r and P r′, respectively. With the rise in temperature from 85 K to 293 K the fluorescence yield (ϕ F) decreased 30 – 40 times in both cases. The decreases in fluorescence connected with the P r⇄P fr phototransformation are also practically identical, 0.80 – 0.82. However, other pigment characteristics undergo changes after its isolation. There are differences in the ϕ F temperature dependence and, correspondingly, in the activation energy ( E a) of the fluorescence decay; in P r E a = 3.9 – 4.1 kJ mol −1 at temperatures of 218 K and below and E a = 23.9 kJ mol −1 at 218 K and above. Within the mentioned temperature intervals E a is 2.7 – 3.0 kJ mol −1 and 17 – 20 kJ mol −1 for P r′. The variable fluorescence connected with irreversible and reversible phototransformations of the pigment into the photoproduct (lumi-R) at low temperatures (below or equal to 200 K) was 2 – 3 times lower in P r′ than in P r. The data are interpreted in terms of several native species of phytochrome whose relative concentrations and, probably, properties undergo some changes during isolation of the pigment.

Chromophore topography and secondary structure of 124-kilodalton Avena phytochrome probed by zinc-induced chromophore modification

The relative extent of chromophore exposure of the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of 124-kDa oat phytochrome and the secondary structure of the phytochrome apoprotein have been investigated by using zinc-induced modification of the phytochrome chromophore. The absence of bleaching of Pr in the presence of a 1:1 stoichiometric ratio of zinc ions, in contrast to extensive spectral bleaching of the Pfr form, confirms previous reports of differential exposure of the Pfr chromophore relative to the Pr chromophore [Hahn et al. (1984) Plant Physiol. 74, 755-758]. The emission of orange fluorescence by zinc-chelated Pfr indicates that the Pfr chromophore has been modified from its native extended/semi-extended conformation to a cyclohelical conformation. Circular dichroism (CD) analyses of native phytochrome in 20 mM Tris buffer suggests that the Pr-to-Pfr phototransformation is accompanied by a photoreversible change in the far-UV region consistent with an increase in the alpha-helical folding of the apoprotein. The secondary structure of phytochrome in Tris buffer, as determined by CD, differs slightly from that of phytochrome in phosphate buffer, suggesting that phytochrome is a conformationally flexible molecule. Upon the addition of a 1:1 molar ratio of zinc ions to phytochrome, a dramatic change in the CD of the Pfr form is observed, while the CD spectrum of the Pf form is unaffected. Analysis of the bleached Pfr CD spectrum by the method of Chang et al. (1978) reveals that chelation with zinc ions significantly alters the secondary structure of the phytochrome molecule, specifically by increasing the beta-sheet content primarily at the expense of alpha-helical folding.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular localisation of phytochrome in oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome in oat (Avena sativa L. cv. Garry Oat) coleoptiles was analysed by electron microscopy. Serial ultrathin sections of resin-embedded material were indirectly immunolabeled with polyclonal antibodies against phytochrome together with a gold-coupled second antibody. The limits of detectability of sequestered areas of phytochrome (SAPs) were analysed as a function of light pretreatments and amounts of the far-red absorbing form of phytochrome (Pfr) established. In 5-d-old dark-grown Avena coleoptiles SAPs were not detectable if less than 13 units of Pfr - compared with 100 units total phytochrome of 5-d-old dark-grown seedlings - were established by a red light pulse. In other sets of experiments, seedlings were preirradiated either with a non-saturating red light pulse to allow destruction to occur or with a saturating red followed by a far-red light pulse to induce first SAP formation and then its disaggregation. These preirradiations resulted in an increase of the limit of detectability of SAP formation after a second red light pulse to 38-41 and 19-23 units Pfr, respectively. We conclude that with respect to Pfr-induced SAP formation an adaptation process exists and that our data indicate that SAP formation is not a simple self-aggregation of newly formed Pfr.

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.

Oat Phytochrome Is Biologically Active in Transgenic Tomatoes.

To determine the functional homology between phytochromes from evolutionarily divergent species, we used the cauliflower mosaic virus 35S promoter to express a monocot (oat) phytochrome cDNA in a dicot plant (tomato). Immunoblot analysis shows that more than 50% of the transgenic tomato plants synthesize the full-length oat phytochrome polypeptide. Moreover, leaves of light-grown transgenic plants contain appreciably less oat phytochrome than leaves from dark-adapted plants, and etiolated R1 transgenic seedlings have higher levels of spectrally active phytochrome than wild-type tomato seedlings in direct proportion to the level of immunochemically detectable oat polypeptide present. These data suggest that the heterologous oat polypeptide carries a functional chromophore, allowing reversible photoconversion between the two forms of the molecule, and that the far-red absorbing form (Pfr) is recognized and selectively degraded by the Pfr-specific degradative machinery in the dicot cell. The overexpression of oat phytochrome has pleiotropic, phenotypic consequences at all major phases of the life cycle. Adult transgenic tomato plants expressing high levels of the oat protein tend to be dwarfed, with dark green foliage and fruits. R1 transgenic seedlings have short hypocotyls with elevated anthocyanin contents. We conclude that a monocot phytochrome can be synthesized and correctly processed to a biologically active form in a dicot cell, and that the transduction pathway components that interact with the photoreceptor are evolutionarily conserved.

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.

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.

Red Light-Induced Accumulation of Ubiquitin-Phytochrome Conjugates in Both Monocots and Dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.

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.

Expression of a functional monocotyledonous phytochrome in transgenic tobacco

A chimeric oat phytochrome structural gene with an uninterrupted coding region was constructed for expression of the monocot protein in transgenic plants. The structural gene was placed under the transcriptional control of either a light-regulated oat phytochrome promoter or the constitutively active cauliflower mosaic virus 35S promoter. These genes were then introduced into Nicotiana tabacum and N.plumbaginifolia. None of the regenerated plants showed expression of oat phytochrome RNA when transcription was controlled by the oat promoter. In contrast, RNA was obtained in plants when the structural gene was functionally linked to the 35S promoter. Transformants expressing oat phytochrome RNA produced a full length 124-kd polypeptide that was recognized by oat-specific anti-phytochrome monoclonal antibodies. The oat protein was a substrate for chromophore addition in tobacco as judged by its red/far-red photoreversible sensitivity to trypsin degradation. Production of oat phytochrome in transgenic plants gave rise to increased phytochrome spectral activity in both light- and dark-grown plants. This increased phytochrome content resulted in phenotypic changes in transformed plants, including semi-dwarfism, darker green leaves, increased tillering and reduced apical dominance. The possible significance of expressing a biologically active phytochrome in transgenic plants is discussed.

Laser-flash photolysis of 124 kDa oat phytochrome: Studies concerning the late steps of P fr formation

Phototransformation kinetics of 124 kDa oat phytochrome at 298 K after red (660 nm) laser-flash excitation were recorded at different wavelengths under various conditions. Only the last steps of the phototransformation were investigated. On changing the environment of the apoprotein, especially by increasing the microviscosity, the formation of meta-R c and P fr, which presumably involve rearrangement of the apoprotein, was retarded. In contrast, the decay of the intermediate meta-R a was not retarded. Using phytochrome specifically labelled by covalent attachment of fluorescein mercuric acetate acting as a reporter group, it was possible to trace changes in the apoprotein conformation. In this way, it was demonstrated that major changes on exposed sites of the protein take place at the very last step of phototransformation, i.e. during formation of P fr from the preceding meta-R c. The data are discussed in terms of molecular aspects, such as conformational changes of the protein.

The rice phytochrome gene: structure, autoregulated expression, and binding of GT-1 to a conserved site in the 5' upstream region.

We have isolated and characterized both cDNA and genomic clones encoding the apoprotein of rice phytochrome. The mRNA produced from this gene is expressed at a low level in etiolated leaves. Following a flash of red light, the steady-state mRNA level decreases within 15 minutes, and is barely detectable after 2 hours. This effect is partially reversed by far red light demonstrating autoregulation of phytochrome mRNA levels. Nuclear run-on experiments show that this effect is exerted on transcription of the phytochrome gene. In etiolated plants, phytochrome mRNA is twofold higher in leaves than in roots, whereas the reverse is true in fully green plants where phytochrome mRNA accumulates despite illumination of the leaves. DNA gel blots and screening of libraries indicate the presence of only a single gene, allowing convenient study of the autoregulatory phenomenon for a specific phytochrome gene. Gel retardation analysis using a fragment from the 5' upstream region reveals that GT-1 is present in nuclear extracts of etiolated rice leaves and binds to sites conserved between rice and oat phytochrome genes.

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.

Polyclonal antibodies raised to phycocyanins contain components specific for the red-absorbing form of phytochrome

Polyclonal antibodies raised in rabbits to a mixture of sodium-dodecyl-sulphate-denatured C- and allo-phycocyanin, isolated from Anabaena cylindrica, cross-react with 124-kilodalton (kDa) phytochrome from etiolated oats, in enzyme-linked immunosorbent assays and on Western blots. The component(s) of the anti-phycocyanin serum that cross-reacts with phytochrome appears to be specific for the red-absorbing form of phytochrome (Pr). These antibodies can be detached from Pr by irradiation with red light, and thus show photoreversible binding. This property has been used to immunopurify the anti-phytochrome component from the antiserum using red light as the eluting agent. Competition assays and epitope-mapping studies indicate that the anti-phytochrome component may bind to a site located between 6 and 10 kDa from the amino-terminus of etiolated oat phytochrome.