Phytochrome Modification and Light-enhanced, In Vivo-induced Phytochrome Pelletability

Phytochrome that was induced by red irradiation in vivo to pellet with subcellular material and that was released from the pellet by removal of divalent cations exhibited altered characteristics. Compared to phytochrome extracted in a soluble red-absorbing form from etiolated tissue, pelleted and released phytochrome, which was also assayed in the red-absorbing form even though pelleted in the far-red-absorbing form, showed 50% greater micro complement fixation activity, eluted closer to the void volume of a Sephadex G-200 column, and electrophoresed more slowly on sodium dodecyl sulfate-polyacrylamide gels. Data presented here document that phytochrome pelleted in the far-red-absorbing form differs from soluble phytochrome extracted from nonirradiated tissue. These data, however, do not permit the conclusion that there is a causal relationship between pelletability and phytochrome modification.     

Purification of 120-Kilodalton Phytochrome from Oryza Sativa L

The procedures of Grimm and Rüdiger for the purification of 120 kDa phytochrome from oat seedlings were modified to isolate native phytochrome from etiolated rice (Oryza sativa L. subsp, japonica var. nongken 58) seedlings. Approximately l kg of 6d old seedlings (the first 2 days at 33℃, the last 4 days at 27 ℃ in darkness) were frozen in liquid nitrogen and then homogenized in a modified Waring blendor with an extraction buffer, at final pH 8.45 (4 ℃). After polyethylenimine precipitation, phytochrome in extract was converted to Pfr by irradiation of the resulting supernatant for 10 min with red light. The step of ammonium sulfate precipitation was followed by resuspending of resultant pellet in buffer B with the ratio of 10 ml per phytochrome unit. The pellet precipitated with ammonium sulfate at 42% saturation from combined phytochrome cont ning fractions after hydroxyapatite chromatography was washed with 10 mmol/l phosphate buffer in 0.8 ml instead of 0.65 ml per phytochrome unit. Then it was washed successively with 200 mmol/l and 100 mmol/1 phosphate buffer (0.85 ml per phytochrome unit). Native phytochrome (120 kDa) in 12% yield was dissolved in 2 mmol/l EHPES buffer (2.2 ml per phytochrome unit, pH 7.8, containing 5 mmol/l EDTA and 14 mmol/l 2-mercaptoethanol) was proved to be pure in SDS- polyacrylamide electrophoresis and showed typical absorption spectrum as that of native oat phytochrome.     

The use and misuse of calcium carbonate as an aid to the spectrophotometric assay of phytochrome in vitro

Supernatant and resuspended pellet samples from a centrifugation of homogenised, etiolated oat seedlings were prepared and assayed spectrophotometrically for phytochrome in the presence and absence of added calcium carbonate (CaCO₃) particles under a variety of conditions. At a constant sample thickness, in the absence of CaCO₃, increasing sample concentration had no significant effect on the expected phytochrome reading. In the presence of CaCO₃, however, as sample concentration increased, the phytochrome reading was less than, expected more so in resuspended pellet samples than in supernatant samples. At a constant sample concentration in the absence of CaCO₃, increasing sample thickness gave no significant difference from the excepted phytochrome reading in supernatant samples, but led to a slight increase over the expected phytochrome reading in resuspended pellet samples. In the presence of CaCO₃, increasing sample thickness led to a drop from the expected phytochrome reading in both sample types, but more so in resuspended pellet samples. These findings show that the use of CaCO₃ as an aid to spectrophotometric phytochrome assay can lead to large artifacts in the instrument reading and that its use should be approached with caution.     

Nature of the phytochrome effect on the binding of glycolate oxidase to peroxisomes in vitro

The attachment of glycolate oxidase to the peroxisomal fraction derived from etiolated barley leaves (Hordeum vulgare L. cr. Dvir) is affected by light. The effect of red irradiation is reversed by subsequent far-red irradiation, indicating the involvement of phytochrome. This phytochrome effect is assumed to be related to phytochrome binding. Indeed, prevention by filipin (1.2·10^-6 mol g^-1 f wt) or cholesterol of phytochrome binding to membranes abolishes the effect of light on the interaction between glycolate oxidase and the peroxisomal fraction. Glycolate oxidase binding is affected by addition of quasi-ionophores such as gramicidin and filipin at a concentration of 0.6·10^-3 mol g^-1 f wt. This fact indicates that peroxisome-glycolate oxidase interaction may be affected by membrane potential. Since both ion transport and membrane potential are known to be affected by phytochrome, it is proposed that phytochrome acts in the light-induced modulation of glycolate oxidase attachment as a quasi-ionophore.Abbreviations GO glycolate oxidase - Pr and Pfr phytochrome forms absorbing in red and far-red, respectively - R and F red and far-red irradiation - Cumulative 20 Kp 20,000 g pellet obtained by centrifugation of the crude extract - 1 Kp 1,000 g pellet - 20 Kp 20,000 g pellet, obtained by centrifugation of 1 Kp supernatant - 1 Kp, 20 Kp and cumulative 20 Kp pellets obtained after density centrifugation through a sucrose cushion     

Characterization of a molecular modification of phytochrome that is associated with its conversion to the far-red-absorbing form

Phytochrome that has been photoinduced to pellet by irradiation of intact oat (cv. Garry) shoots and recovered from a pellet obtained by centrifugation of crude extracts exhibits modified behavior when compared to soluble phytochrome isolated from shoots that had never been irradiated. This modified behavior includes retarded mobility during sodium dodecyl sulfate polyacrylamide gel electrophoresis (Boeshore ML, LH Pratt 1980 Plant Physiol 66: 500-504). The electrophoretic mobility of several different kinds of phytochrome preparations were examined to study how this modification might arise.     

Effects of filipin and steroids on phytochrome pelletability

Red light given to dark-grown etiolated leaves of Hordeum vulgare L. in vivo or to crude homogenates increases the phytochrome content of the 20,000 g pellet on centrifugation. The steroids cholesterol and stigmasterol inhibit this red light-induced phytochrome pelletability. Filipin (a polyene antibiotic, which is known to combine with steroids) inhibits red light-induced phytochrome pelletability. Filipin and steroids at the appropriate concentration applied together prevent the inhibition caused by either when applied alone. These results suggest that phytochrome may bind to a steroid component of membranes. The phospholipid phosphatidyl choline dipalmitoyl has no effect on red light-induced phytochrome pelletability. Preliminary evidence demonstrates a direct association of soluble phytochrome in its active form and steroids. The physiological significance of red light-induced pelletability and the primary mechanism of phytochrome action are discussed in terms of a hypothetical steroid-binding site.     

Phytochrome Pelletability in Barley

Phytochrome pelletability in the 1000 g and 20,000 g pellet from crude homogenates of etiolated Hordeum vulgare L. cv. Ark Royal primary leaves is enhanced by red light in vivo and in vitro. Red enhanced phytochrome pelletability appears different in the 1000 g and 20,000 g pellets after red light in vivo, being irreversible by subsequent far red light in the latter. Mg²⁺ concentration in the range 1–20 mM has no effect on red enhanced phytochrome pelletability. The enhancement of pelletability is reduced by low pH and high 2-mercaptoethanol concentration, conditions which lead to a high level of pelletability of the far red absorbing form of phytochrome. Washing these pellets at high pH or low 2-mercaptoethanol concentration reveals the red enhancement of pelletability. The results are discussed in terms of a possible two point attachment of phytochrome to membranes.     

In Vivo Properties of Membrane-bound Phytochrome

After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed.     

Immunochemical detection with rabbit polyclonal and mouse monoclonal antibodies of different pools of phytochrome from etiolated and green Avena shoots

While two monoclonal antibodies directed to phytochrome from etiolated oat (Avena sativa L.) shoots can precipitate up to about 30% of the photoreversible phytochrome isolated from green oat shoots, most precipitate little or none at all. These results are consistent with a report by J.G. Tokuhisa and P.H. Quail (1983, Plant Physiol. 72, Suppl., 85), according to which polyclonal rabbit antibodies directed to phytochrome from etiolated oat shoots bind only a small fraction of the phytochrome obtained from green oat shoots. The immunoprecipitation data reported here indicate that essentially all phytochrome isolated from green oat shoots is distinct from that obtained from etiolated oat shoots. The data indicate further that phytochrome from green oat shoots might itself be composed of two or more immunochemically distinct populations, each of which is distinct from phytochrome from etiolated shoots. Phytochrome isolated from light-grown, but norflurazon-bleached oat shoots is like that isolated from green oat shoots. When light-grown, green oat seedlings are kept in darkness for 48 h, however, much, if not all, of the phytochrome that reaccumulates is like that from etiolated oat shoots. Neither modification during purification from green oat shoots of phytochrome like that from etiolated oat shoots, nor non-specific interference by substances in extracts of green oat shoots, can explain the inability of antibodies to recognize phytochrome isolated from green oat shoots. Immunopurified polyclonal rabbit antibodies to phytochrome from etiolated pea (Pisum sativum L.). shoots precipitate more than 95% of the photoreversible phytochrome obtained from etiolated pea shoots, while no more than 75% of the pigment is precipitated when phytochrome is isolated from green pea shoots. These data indicate in preliminary fashion that an immunochemically unique pool of phytochrome might also be present in extracts of green pea shoots.Abbreviation ELISA enzyme-linked immunosorbent assay - mU milliunit - Pfr far-red-absorbing form of phytochrome - Pr red-absorbing form of phytochrome     

The isolation and partial characterization of a membrane fraction containing phytochrome

If 4-day-old dark-grown zucchini squash seedlings (Cucurbita pepo L. cv. Black Beauty) are exposed briefly to red light, subsequent cell fractionation yields about 40% of the total extractable phytochrome in the far red-absorbing form bound to a particulate fraction. The amount of far red-absorbing phytochrome in the pellet is strongly dependent on the Mg concentration in the extraction medium. The apparent density of the Pfr-containing particles following sedimentation on sucrose gradients corresponds to 15% (w/w) sucrose with 0.1 mm Mg and 40% sucrose with 10 mm Mg. This particulate fraction could be readily separated from mitochondria and other particulate material by taking advantage of these apparent density changes with changes in Mg concentration. Electron microscopy of negatively stained preparations shows that with 1 mm Mg only minute particles are present. These were too small to reveal structural detail with this technique. With 3 mm Mg, separate membranous vesicles between 400 and 600 Ångstroms in diameter appear. At higher Mg concentrations, the vesicles aggregate, causing obvious turbity. The effect of Mg on vesicle formation and aggregation is completely reversible. Above 10 mm Mg, vesicle aggregation persists, but the percentage of bound Pfr decreases.     

Effects of divalent cations and EDTA on spectral properties of phytochrome in particulate fractions from etiolated pea epicotyls

The photoreversible absorbance change of phytochrome in suspensionsof a 20,000xg particulate fraction (20kP) prepared from a 1,000xgsupernatant (1kS) of etiolated pea epicotyl extracts decreasedremarkably in the presence of 5 mM Cu²⁺, Zn²⁺ and Co²⁺, butremained unchanged in 5 mM Ca²⁺, Mg²⁺, Fe²⁺ or Mn²⁺. This spectraldistortion of phytochrome was more evident in soluble preparationsand in suspensions of pellets prepared from red light (R)-irradiatedtissues than it was in suspensions of pellets prepared in thedark from etiolated tissues that received no actinic irradiation. When Cu²⁺ was added to the red-light-absorbing form of phytochrome(Pr) in resuspended pellets prepared from R-irradiated tissues,the distortion of its difference spectrum took place after irradiationwith the first actinic R. In contrast, when Cu²⁺ was added tothe far-red-light-absorbing form of phytochrome (Pfr) in thesame resuspended pellet, no distortion was seen, unless thePfr in the pellet was first photoconverted to Pr and then photoconvertedback to Pfr. Spectral distortion of Pr remained small during dark incubationat 25°C when suspensions of 20kPs were prepared and incubatedwith a buffer containing EDTA, whether the 20kP was preparedfrom nonirradiated tissue or from R-irradiated tissues. But,when EDTA was added to a suspension of 20kP prepared from 1kS,after the 1kS was irradiated with R in the presence of 10 mMCaCl₂, the spectral distortion of Pr in 20kP occurred instantaneously. (Received April 14, 1980; )     

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.     

Failure of lactoperoxidase to iodinate specifically the plasma membrane of cucurbita tissue segments

An attempt has been made to use lactoperoxidase-catalyzed iodination of excised Cucurbita hypocotyl hooks to monitor the distribution of plasma membrane fragments relative to that of phytochrome in particulate fractions from this tissue. Upon fractionation, the iodinated tissue yields a 20,000g pellet which contains 58% of the trichloroacetic acid-precipitable ¹²⁵I at a specific radioactivity 12 times that of the proteins in the supernatant. On sucrose gradients, the labeled fraction has a mean isopycnic density of 1.15 g · cm⁻³. The distribution profile is distinct from that of the total particulate protein and does not coincide with either mitochondrial or endoplasmic reticulum markers. These observations satisfy operational criteria commonly accepted in other systems as indices of selective labeling of the cell surface. The sucrose gradient profiles of the phytochrome and ¹²⁵I in the 20,000g pellets are noncoincident. In the absence of more direct evidence, this is readily interpreted to indicate a lack of association of the pigment with the plasma membrane.     

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg²⁺, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg²⁺, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg²⁺, phytochrome was associated with a “heavy” plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.     

Phytochrome radioimmunoassay

A phytochrome radioimmunoassay with a detection limit of about 2 nanograms has been developed. The radioimmunoassay does not suffer from the potential drawbacks of the commonly used spectral assay and requires less than 1 microliter of crude extract from dark-grown plants for quantitation of phytochrome. Measurement of phytochrome in crude extracts by radioimmunoassay gives values about 25% greater than those obtained by spectral assay. The amount of phytochrome detected in crude extracts of light-grown oats by radioimmunoassay is approximately 1% of that detected in comparable extracts from dark-grown oats. General interference by crude plant extracts with radioimmunoassays was also observed and corrected for.     

An unusual effect of the far-red absorbing form of phytochrome: Photoinhibition of seed germination inBromus sterilis L.

Seeds ofBromus sterilis L. germinated between 80–100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response ofB. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination.Abbreviations Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome     

The photoreceptors in the high irradiance response of plants

Several studies show that the high irradiance response (HIR) of plants is probably due to two photoreceptors. One of the photoreceptors is phytochrome, and the other is an unidentified pigment provisionally named heliochrome. One of the functions of heliochrome is the synthesis of phytochrome, using far-red and blue radiations of high intensities, to replace the phytochrome destroyed by light. Another possible function could be an interaction of heliochrome with a substance produced by phytochrome. The data presented show that heliochrome is a pigment with different properties from phytochrome. It shows a far-red/green reversibility. Heliochrome has been shown to participate with phytochrome in such HIRs as leaf movement in Albizzia and flowering in a long-day plant. The first event initiated by phytochrome and by heliochrome could be the generation of a strong positive, electrostatic charge in the cell membrane.     

Ubiquitin-phytochrome conjugates. Pool dynamics during in vivo phytochrome degradation

The plant photoreceptor chromoprotein, phytochrome, is rapidly degraded in vivo after photoconversion from a stable red light-absorbing form (Pr) to a far-red light-absorbing form (Pfr). Previously, we demonstrated that during Pfr degradation in etiolated oat seedlings, ubiquitin-phytochrome conjugates, (Ub-P), appear and disappear suggesting that phytochrome is degraded via a ubiquitin-dependent proteolytic pathway (Shanklin, J., Jabben, M., and Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 359-363). Here, we provide additional kinetic and localization data consistent with this hypothesis by exploiting the unique ability to photoregulate phytochrome degradation in vivo. An assay for the quantitation of Ub-P was developed involving immunoprecipitation of total conjugates with anti-ubiquitin antibodies, followed by the detection of Ub-P with anti-phytochrome antibodies. Using this immunoassay, we found that Ub-P will accumulate to approximately 5% of initial phytochrome during Pfr degradation induced by a saturating red light pulse. Reducing the amount of Pfr produced initially by attenuating the red light pulse, lowered the amount of phytochrome degraded in the following dark period and concomitantly reduced the maximal accumulation of Ub-P. Continuous far-red irradiations that maintained only 4% of phytochrome as Pfr induced rapid phytochrome degradation similar to that induced by a red light pulse converting 86% of Pr to Pfr. The appearance and disappearance of Ub-P were similar for each irradiation indicating that Ub-P accumulation is independent of the level of Pfr provided rapid phytochrome degradation is maintained. Pulse-chase studies employing continuous far-red light followed by darkness showed that Ub-P are continuously synthesized during phytochrome degradation and rapidly disappear once degradation ceases. Ub-P also accumulated during "cycled Pr" degradation induced by the transformation of Pr to Pfr and back to Pr. The commitment to degrade cycled Pr and form Ub-P occurred within seconds after Pfr formation making the cause(s) underlying this phenomenon one of the fastest phytochrome reactions known. Within seconds after Pfr formation, a majority of phytochrome is also known to aggregate in vivo (previously defined as sequestered or pelletable), with aggregated phytochrome preferentially lost during phytochrome degradation. In vitro analysis of aggregated phytochrome indicated that they contain most of the Ub-P. Moreover, the appearance of Ub-P in the aggregated and soluble fractions correlated with the time that phytochrome disappeared from that fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of phytochrome kinetics in light-grown Avena sativa L. seedlings

The phytochrome content, the rate of phytochrome accumulation after a light/dark transition and the rate of phytochrome destruction after a 1.5 d reaccumulation period in darkness were measured in light grown Avena sativa L. seedlings. The results using spectrophotometrical methods (Norflurazon treated seedlings) and the radio-immunoassay (RIA) (green seedlings) were almost identical. The rate of phytochrome synthesis was analysed by measuring the activity of poly(A⁺)-RNA coding for the phytochrome apoprotein. It was demonstrated that the rate of phytochrome synthesis is different in light and in dark. These results were confirmed by measuring the incorporation of radioactive label in vivo. Five minutes red (and 5 min far-red) light strongly reduces the rate of phytochrome synthesis. Even after prolonged dark periods only 50% of the initial rate of phytochrome synthesis is recovered for light and dark grown seedlings which received one red light pulse.