Spectral Characterization and Proteolytic Mapping of Native 120-Kilodalton Phytochrome from Cucurbita pepo L

A spectral, immunochemical, and proteolytic characterization of native 120-kilodalton (kD) phytochrome from Cucurbita pepo L. is presented and compared with that previously reported for native 124-kD phytochrome from Avena sativa. The molecule was partially purified (~200-fold) in the phytochrome—far red-absorbing form (Pfr) in the presence of the protease inhibitor, phenylmethylsulfonyl fluoride, using a modification of the procedure initially developed to purify 124-kD Avena phytochrome. The spectral properties of the preparations obtained are indistinguishable from those described for 124-kD Avena phytochrome, including a Pfr λ_max at 730 nanometers, a spectral change ratio (ΔA_r/ΔA_fr) of 1.05, and negligible dark reversion of Pfr to the red-absorbing form (Pr) in the presence or absence of sodium dithionite. This lack of dark reversion in vitro contrasts with observations that Cucurbita phytochrome, like phytochrome from most other dicotyledons, exhibits substantial dark reversion in vivo. Ouchterlony double immunodiffusion analysis with polyclonal antibodies indicates that 120-kD Cucurbita phytochrome is immunologically dissimilar to 124-kD Avena phytochrome. However, despite this dissimilarity, immunoblot analyses of proteolytic digests have identified at least three spatially separate epitopes that are common to both phytochromes. Using endogeneous protease(s), a peptide map for Cucurbita phytochrome has been constructed and the role that specific domains play in the overall structure of the photoreceptor has been examined. One domain near the NH₂ terminus is critical to the spectral integrity of the molecule indicating that this domain plays a structural role analogous to that of a domain near the NH₂ terminus of Avena phytochrome. Proteolytic removal of this domain occurs preferentially in Pr and its removal shifts the Pfr λ_max to 722 nm, increases the spectral change ratio to 1.3, and substantially enhances the dark reversion rate. The apparent conservation of this domain among evolutionarily divergent plant species and its involvement in a conformational change upon photoconversion makes it potentially relevant to the mechanism(s) of phytochrome action. Preliminary evidence from gel filtration studies suggests that the 55-kD chromophoreless COOH-terminal region of the polypeptide contains a domain responsible for dimerization of phytochrome monomers.     

Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome—far red-absorbing form (Pfr) to phytochrome—red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.     

Integral association of phytochrome with a membranous fraction fromAvena shoots: in vivo characterization and physiological significance

Phytochrome in the far-red light absorbing form (Pfr) was observed to disappear in vivo more rapidly from the non-cation-requiring pelletable phytochrome population than from the supernantant phytochrome population of oat seedlings given an increasing dark incubation after red irradiation. The amount of pelletable phytochrome in the red light absorbing form (Pr) remained relatively stable while supernatant Pr was lost. These observations indicated that supernant Pfr was subject to loss during the incubation, while pelletable Pfr was subject to both dark reversion and loss.During the incubation, the ability of far-red irradiation to reverse the red-induced increase in phytochrome pelletability was lost, with kinetics similar to those of the loss of pelletable Pfr.Far-red reversibility of the red-induced increase in coleoptile elongation correlated with the change intotal Pfr in both supernatant and pelletable phytochrome populations, but with the change in the ratio of Pfr to total phytochrome only in the pelletable phytochrome population.The possible significance of these results is discussed with reference to the action of phytochrome in the photocontrol of physiological growth responses.Abbreviations Pfr phytochrome in the far-red light absorbing form - Pr phytochrome in the red absorbing form - Ptot total phytochrome     

Unusual Spectral Properties of Bacteriophytochrome Agp2 Result from a Deprotonation of the Chromophore in the Red-absorbing Form Pr

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.     

Acceleration of dark reversion of phytochrome in vitro by calcium and magnesium

Rates of dark reversion of the far red-absorbing form of phytochrome, Pfr, to the red-absorbing form, Pr, have been determined in the presence of several salts. Low concentrations of calcium chloride and magnesium chloride (up to 3 mm) accelerated the rate of dark reversion at all stages of purification of phytochrome from etiolated rye (Secale cereale L. cv. Balbo) seedlings. The complex kinetics of the dark reversion could be resolved into two first-order components. The effect of the added divalent cations was on the relative proportion of the fast and slow reacting components, rather than on the rate constants of the two populations. It was possible to reverse the effects of the cations by adding the chelating agents ethylene-bis-(oxyethylene-nitrilo) tetraacetic acid or ethylenediaminetetraacetate. The effect of the divalent cations is not a nonspecific ionic strength effect. The relative proportion of the two populations was also affected by the degree of purity of the phytochrome samples.     

Phytochrome — all regions marked by a set of monoclonal antibodies reflect conformational changes

Monoclonal antibodies to defined locations on six regions of the phytochrome molecule (from Avena sativa L. or Zea mays L.) were each found to have a different affinity toward the farred-absorbing form of phytochrome (Pfr) and the red-absorbing form (Pr). The differences were small, but were consistently shown by antibodies which bind to the vicinity of the aminoterminus, the carboxylterminus and to sequences in between. It seems that the conformational differences between Pr and Pfr extend over the whole molecule in as far as it is represented by these regions and the antibodies binding to them.Abbreviations Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

Discrimination between the red- and far-red-absorbing forms of phytochrome from Avena sativa L. by monoclonal antibodies

A set of rat monoclonal antibodies (ARC MAC 48 to 52 and 54 to 56), raised to phytochrome from dark-grown seedlings of Avena sativa L. was tested for the ability to discriminate between the red-absorbing (Pr) and far-red-absorbing (Pfr) forms of phytochrome by indirect enzyme-linked immunosorbent assay. MAC 50 bound more strongly to Pfr and MAC 49 and 52 showed preferential binding to Pr from extracts of dark-grown Avena seedlings; MAC 50 also bound more strongly to Pfr from brushite-purified phytochrome. The remainder of the monoclonal antibodies and a rabbit polyclonal antiphytochrome preparation did not discriminate between Pr and Pfr. The results provide evidence for conformational changes in defined regions of the phytochrome apoprotein upon photoconversion.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - McAb monoclonal antibody(ies) - PBS phosphate-buffered saline - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light - PMSF phenylmethylsulphonylfluoride     

Intracellular localisation of phytochrome and ubiquitin in red-light-irradiated oat coleoptiles by electron microscopy

The intracellular localisation of phytochrome and ubiquitin in irradiated oat coleoptiles was analysed by electron microscopy. We applied indirect immunolabeling with polyclonal antibodies against phytochrome from etiolated oat seedlings or polyclonal antibodies against ubiquitin from rabbit reticulocytes, together with a goldcoupled second antibody, on serial ultrathin sections of resin-embedded material. Immediately after a 5-min pulse of red light-converting phytochrome from the red-absorbing (Pr) to the far-redabsorbing (Pfr) form-the label for phytochrome was found to be sequestered in electron-dense areas. For up to 2 h after irradiation, the size of these areas increased with increasing dark periods. The ubiquitin label was found in the same electrondense areas only after a dark period of 30 min. A 5 min pulse of far-red light, which reverts Pfr to Pr, given immediately after the red light did not cause the electron-dense structures to disappear; moreover, they contained the phytochrome label immediately after the far-red pulse. In contrast, after the reverting far-red light pulse, ubiquitin could only be visualised in the electron-dense areas after prolonged dark periods (i.e. 60 min). The relevance of these data to light-induced phytochrome pelletability and to the destruction of both Pr and Pfr is discussed.Abbreviations FR far-red light; Pfr - Pr far-red-absorbing and red-absorbing forms of phytochrome, respectively - R red light     

Agrobacterium phytochrome as an enzyme for the production of ZZE bilins

Photoconversion of phytochrome from the red-absorbing form Pr to the far-red-absorbing form Pfr is initiated by a Z to E isomerization around the ring C-ring D connecting double bond; the chromophore undergoes a ZZZ to ZZE isomerization. In vivo, phytochrome chromophores are covalently bound to the protein, but several examples of noncovalent in vitro adducts have been reported which also undergo Pr to Pfr photoconversion. We show that free biliverdin or phycocyanobilin, highly enriched in the ZZE isomer, can easily be obtained from chromophores bound in a noncovalent manner to Agrobacterium phytochrome Agp1, and used for spectral assays. Photoconversion of free biliverdin in a methanol/HCl solution from ZZE to ZZZ proceeded with a quantum yield of 1.8%, but was negligible in neutral methanol solution, indicating that this process is proton-dependent. The ZZE form of biliverdin and phycocyanobilin were tested for their ability to assemble with Agp1 and cyanobacterial phytochrome Cph1, respectively. In both cases, a Pfr-like adduct was formed but the chromophore was bound in a noncovalent manner to the protein. Agp1 Pfr undergoes dark reversion to Pr; the same feature was found for the noncovalent ZZE adduct. After dark reversion, the chromophore became covalently bound to the protein. In analogy, the PCB chromophore became covalently bound to Cph1 upon irradiation with strong far-red light which initiated ZZE to ZZZ isomerization. Agrobacterium Agp2 belongs to a yet small group of phytochromes which also assemble in the Pr form but convert from Pr to Pfr in darkness. When the Agp2 apoprotein was assembled with the ZZE form of biliverdin, the formation of the final adduct was accelerated compared to the formation of the ZZZ control, indicating that the ZZE chromophore fits directly into the chromophore pocket of Agp2.     

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.     

Molecular topography of phytochrome as deduced from the tritium-exchange method

The hydrogen-tritium-exchange measurements on phytochrome have been performed to detect the conformational differences between the red-absorbing (Pr) and the far-red absorbing (Pfr) forms of phytochrome. The large and small Pfr molecules revealed more exchangeable protons that did the corresponding Pr molecules by 96 and 70 protons, respectively. These results suggest that the Pr leads to Pfr phototransformation is accompanied by an additional exposure of the peptide chains in the Pfr molecule. Of 1682 theoretically exchangeable hydrogens in undegraded phytochrome, only 442 (26%) and 346 (21%) protons were found to be exchangeable (excluding instantaneously exchangeable protons that cannot be determined by the present method). Thus, the phytochrome protein appears to be compact and highly folded. The kinetic analyses of the tritium exchange-out curves indicate that two kinetically different groups are responsible for the conformational differences between the Pr and Pfr forms of phytochrome. These components are due to (1) the exposure of hydrogen-bonded peptide segments (alpha helix and/or beta-pleated sheet) in the chromophore vicinity of Pfr and (2) the exposure of hydrogen-bonded peptide segments on the chromophore peptide domain as well as on the chromophore-free tryptic domain of undegraded phytochrome.     

Identification with Monoclonal Antibodies of a Second Antigenic Domain on Avena Phytochrome that Changes upon Its Photoconversion

An enzyme-linked immunosorbent assay that revealed an antigenic difference between the red-absorbing and far-red-absorbing forms of phytochrome (Pr and Pfr, respectively) near its amino terminus (Cordonnier M-M, H Greppin, LH Pratt 1985 Biochemistry 24: 3246-3253) was used to screen eight additional monoclonal antibodies directed to phytochrome from etiolated oats. While six of these antibodies detected Pr and Pfr with equal affinity, two of them, designated Oat-9 and Oat-16, bound to Pfr 1.6 to 2.3 times better than to Pr. Competitive enzyme-linked immunosorbent assays indicate (a) that Oat-9 and Oat-16 probably bind to the same domain on phytochrome and (b) that this domain is at least 3.5 nanometers away from the epitope near its amino terminus that was shown earlier to change upon phototransformation. Neither the absorbance spectra of Pr and Pfr, nor the rate of dark reversion of Pfr to Pr, was influenced by the presence of Oat-9. Immunoblotting of sodium dodecyl sulfate polyacrylamide gels after electrophoretic separation of phytochrome fragments obtained by endogenous proteolytic digestion indicates that Oat-16 binds to an epitope located on the chromophore half of this chromoprotein. The observation that the epitope recognized by Oat-9 and Oat-16 is also present on at least some of the immunochemically distinct phytochrome that is obtained from green oat shoots (Shimazaki Y, LH Pratt 1985 Planta 164: 333-344), together with the evidence that this epitope undergoes a change upon photoransformation, indicates that it may play an important role in phytochrome function.     

Model for variable light sensitivity in imbibed dark-dormant seeds

The level of light-induced germination of the seed of common purslane (Portulaca oleracea L.) and curly dock (Rumex crispus L.) changes with dark incubation time prior to brief, low energy, red light treatment. The rate at which phytochrome—far red-absorbing form (Pfr) acts in the light-induced population of seeds was measured by quantitating per cent reversals of the red light effect with saturating far red light exposures at successive times after the red light exposure. A linear positive correlation was found between this rate and the final germination level. These results are compatible with a model involving changing levels, during dark incubation, of a component with which Pfr interacts. In this model, germination is initiated after attainment of a certain level of interaction between Pfr and this component. These findings also support the view that the Pfr to Pr decay rate constant and total phytochrome level are stable during dark incubation.     

Factors controlling rates of nonphotochemical transformation of Pisum phytochrome in vitro

Nonphotochemical transformations of the far-red absorbing formof phytochrome, such as its decay or dark reversion to Pr, werestudied with solutions obtained from etiolated pea epicotyltissues at various steps of purification. At pH 7.8, the rateof dark Pfr reversion became significantly faster after thecrude extract was purified by gel filtration, but that of wellpurified solutions was quite low. Decay of Pfr was not seenduring any purification step at an alkaline pH, but it occurredin the acidic range of pH even in the presence of sulfhydrylcompounds. The rate of Pfr reversion was also influenced bypH; it increased with an increasing pH. Dark reversion of Pisum Pfr was confirmed to proceed in a short,rapid initial phase followed by a slow phase. (Received September 9, 1970; )     

Phytochrome photoreversibility: Empirical test of the hypothesis that it varies as a consequence of pigment compartmentation

Phytochrome of oat (Avena sativa L., cv. Garry) coleoptile cells in the red-light-absorbing form, Pr, is diffusely distributed while after conversion to the far-red-light-absorbing form, Pfr, it is observed only in very small areas within the cell. Comparison of phytochrome photoversibility measurements to the distribution of the pigment within the cell indicates that the spectral assay is not influenced by the observed compartmentalization of the chromoprotein. However, the observed compartmentalization of phytochrome is correlated with a loss in spectrophotometrically detectable Pr.Abbreviations Pr red-absorbing form of phytochrome - Pfr farred-absorbing form of phytochrome - R red light - FR far-red light C.I.W.-D.P.B. Publication No. 622     

In vivo phytochrome reversion in immature tissue of the alaska pea seedling

Reversion of far red-absorbing phytochrome to red-absorbing phytochrome without phytochrome destruction (that is, without loss of absorbancy and photoreversibility) occurs in the following tissues of etiolated Alaska pea seedlings (Pisum sativum L.): young radicles (24 hours after start of imbibition), young epicotyls (48 hours after start of imbibition), and the juvenile region of the epicotyl immediately subjacent to the plumule in older epicotyls. Reversion occurs rapidly in the dark during the first 30 minutes following initial phototransformation of red-absorbing phytochrome to far red-absorbing phytochrome. If these tissues are illuminated continuously with red light for 30 minutes, the total amount of phytochrome remains unchanged. Beyond 30 minutes after a single phototransformation or after the start of continuous red irradiation, phytochrome destruction commences. In young radicles, sodium azide inhibits this destruction, but does not affect reversion. In older tissues in which far red-absorbing phytochrome destruction begins immediately upon phototransformation, strong evidence for simultaneous far red-absorbing phytochrome reversion is obtained from comparison of far red-absorbing phytochrome loss in the dark following a single phototransformation with far red-absorbing phytochrome loss under continuous red light.     

Phosphorylation of Phytochrome B Inhibits Light-Induced Signaling via Accelerated Dark Reversion in Arabidopsis

The photoreceptor phytochrome B (phyB) interconverts between the biologically active Pfr (λ_max = 730 nm) and inactive Pr (λ_max = 660 nm) forms in a red/far-red–dependent fashion and regulates, as molecular switch, many aspects of light-dependent development in Arabidopsis thaliana. phyB signaling is launched by the biologically active Pfr conformer and mediated by specific protein–protein interactions between phyB Pfr and its downstream regulatory partners, whereas conversion of Pfr to Pr terminates signaling. Here, we provide evidence that phyB is phosphorylated in planta at Ser-86 located in the N-terminal domain of the photoreceptor. Analysis of phyB-9 transgenic plants expressing phospho-mimic and nonphosphorylatable phyB–yellow fluorescent protein (YFP) fusions demonstrated that phosphorylation of Ser-86 negatively regulates all physiological responses tested. The Ser86Asp and Ser86Ala substitutions do not affect stability, photoconversion, and spectral properties of the photoreceptor, but light-independent relaxation of the phyB^Ser86Asp Pfr into Pr, also termed dark reversion, is strongly enhanced both in vivo and in vitro. Faster dark reversion attenuates red light–induced nuclear import and interaction of phyB^Ser86Asp-YFP Pfr with the negative regulator PHYTOCHROME INTERACTING FACTOR3 compared with phyB–green fluorescent protein. These data suggest that accelerated inactivation of the photoreceptor phyB via phosphorylation of Ser-86 represents a new paradigm for modulating phytochrome-controlled signaling.     

Characterization of the Destruction of Phytochrome in the Red-absorbing Form

Both the red-absorbing (Pr) and far red-absorbing (Pfr) forms of phytochrome undergo destruction, defined as the loss of photoreversibly detectable chromoprotein following actinic irradiation of dark-grown tissue, in 4-day-old etiolated oat seedlings. Pr and Pfr destruction follow the same time course, exhibit the same time delay after actinic irradiation when the plants are grown in sealed containers, result in a loss of antigenically detectable phytochrome, as determined by radial immunodiffusion assay, equal to the loss of spectrophotometrically detectable phytochrome, and have the same sensitivity to 2-mercaptoethanol and azide. We suggest that Pr destruction is a consequence of the same mechanism that is responsible for Pfr destruction.     

The PHY domain is required for conformational stability and spectral integrity of the bacteriophytochrome from Deinococcus radiodurans

Bacteriophytochrome from Deinococcus radiodurans (DrBphP) is a plant phytochrome homolog. To investigate the interaction of chromophore and protein structure, we purified recombinant DrBphP and performed biochemical analyses. Differences of apo- and holo-protein in electrophoretic properties in native gels and their susceptibility to trypsin indicate changes in both the conformation and surface topography of this protein as a result of chromophore assembly. Furthermore, proteolysis to Pr and Pfr conformers displayed distinctive cleavage patterns with a noticeable Pr-specific tryptic fragment. Of interest, a prolonged tryptic digestion showed a more severe impact upon the Pfr form. Most importantly, when we assessed the extent of dark reversion to evaluate the role of the cleaved part, a rapidly accelerated reversion was observed upon cleavage at residues 329-505 corresponding to the PHY domain. Our data thus show that the PHY domain is necessary for the Pfr stabilization and spectral integrity of DrBphP.     

Experimentally induced binding of phytochrome to mitochondrial and microsomal fractions in etiolated pea shoots

Summary A brief irradiation with red light of pea (Pisum sativum L.) shoot segments kept at 0° resulted in very rapid binding of both Pr and Pfr to mitochondrial and microsomal fractions. The effect was not far-red reversible. The amount of phytochrome bound to the mitochondrial fraction was proportional to the percentage of Pfr of the fraction, and the ratio of Pr and Pfr in the bound form was the same as that in 12,000 x g supernatant. After a brief exposure of the segments to red light at 0° and a subsequent dark incubation at 30° in Tris-HCL buffer containing dithiothreitol or EDTA, which bot inhibit Pfr decay, the contents of phytochrome in the mitochondrial and microsomal fractions were significantly enhanced with time. The red-light effect was reversed by far-red light. The increase of the phytochrome content in the particulate fractions continued for at least 2 h, reaching a ca. 3 times higher level in terms of (A) per mg protein.Abbreviations R red - FR far-red - Pr red-absorbing form of phytochrome - Pfr far-red-absorbing form of phytochrome