Spectral properties of soluble and pelletable phytochrome from epicotyls of etiolated pea seedlings

The red-light(R)-absorbing form of phytochrome (Pr) was detected spectrophotometrically in a 20,000 g particulate fraction prepared from a 1,000 g supernatant fraction from epicotyl tissue of pea (Pisum sativum L.) seedlings grown in the dark and only briefly exposed to dim green light. The difference spectrum of phytochrome in this fraction was essentially the same as that of soluble phytochrome from the same tissue. When the non-irradiated 20,000 g particulate fraction was incubated in the dark at 25° C, an absorbance change (decrease) of Pr after actinic red irradiation was found only in the far-red (FR) region. When the 20,000 g particulate fraction was irradiated with R and then incubated in the dark, the FR-absorbing form of phytochrome (Pfr) disappeared spectrally at a rate about half that in the soluble fraction, and the difference spectrum of the Pr which became detectable after dark incubation of the 20,000 g particulate fraction was markedly distorted. In contrast, Pfr in a 20,000 g particulate fraction prepared from tissues irradiated with R did not change optically during dark incubation at 25° C for 60 min, while Pfr in the soluble fraction from the same tissue disappeared in the dark. No dissociation of either Pr or Pfr from the 20,000 g particulate fraction was indicated during a 60-min dark incubation at 25° C, but Pfr in a 20,000 g particulate fraction prepared in vitro from R-irradiated 1,000 g supernatant fraction in the presence of CaCl₂ disappeared spectrally and the difference spectrum of Pr in the 20,000 g particulate fraction became quite distorted during the dark incubation.Abbreviations Pr red-light-absorbing form of phytochrome - Pfr far-red-light-absorbing form of phytochrome - FR far-red light - FR1 first actinic far-red light - FR2 second actinic far-red light - R red light - R1 first actinic red light - 1kS 1,000 g supernatant fraction - 20kS 20,000 g supernatant fraction - 20kP 20,000 g particulate fraction     

The influence of light quality on the phytochrome content of light grown Sinapis alba L. and Phaseolus aureus Roxb.

The effect on the phytochrome system of light regimes establishing a range of photoequilibria was studied in two light grown dicotyledonous plants, both of which were treated with the herbicide SAN 9789 to prevent chlorophyll accumulation. In Sinapis alba L. cotyledons the results are comparable with phytochrome behaviour in etiolated mustard seedlings; the level of Pfr becomes independent of wave-length whereas the total phytochrome level is wave-length dependent. Contrasting properties are exhibited in Phaseolus aureus Roxb. leaves in which total phytochrome is unaffected by light quality; consequently the Pfr level is dependent on wavelength. Nevertheless, the amount of phytochrome in mung leaves increased after transfer to darkness suggesting that light still has a profound influence on the phytochrome system, even though light quality during the light period and prior to darkness does not.Abbreviations FR far-red light - WL white light - PAR photosynthetically active radiation - Pfr far-red light absorbing form of phytochrome - Pr red light absorbing form of phytochrome - Ptot total phytochrome level (=Pr+Pfr) - Pfr/Pfr+Pr - SAN 9789 4-chloro-5-(methylamino) 2(,, trifluoro-m tolyl)-3(2H)-pyridazinone     

Effect of malformin on phytochrome reactions in Vigna and Avena

The rate of destruction of the far red absorbing form of phytochrome(Pfr) in green or etiolated cuttings of Vigna radiata was slowerin the presence of malformin than in its absence. Malforminhad no effect on the accumulation of total phytochrome in thedark, or on the reaccumulation of phytochrome after destructionin red light. The amount of photoconversion of the red absorbingform of phytochrome (Pr) to Pfr or Pfr to Pr by given dosesof red or far red radiation was slightly but consistently lessin malformin-treated cuttings of V. radiata than in controls.Malformin had no effect on the rate of destruction or photoconversionof phytochrome in etiolated shoots of Avena sativa. The decreasein destruction rate of Pfr by malformin in V. radiata may contributeto the inhibition of dark abscission by malformin after lighttreatment. (Received October 3, 1979; )     

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.     

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; )     

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.     

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     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

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.Abbreviations ELISA enzyme-linked immunosorbent assay - kDa kilodaton - FR far-red light - Pfr far-red-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - R red light - SDS sodium dodecyl sulphate     

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     

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.     

Phytochrome Pelletability Induced by Irradiation in Vivo: TEST FOR IN VITRO BINDING OF ADDED [S]PHYTOCHROME

Undegraded, highly purified [³⁵S]phytochrome was immunoaffinity-purified either from dark control oat (cv. Garry) shoots or from etiolated oat shoots that were previously irradiated first with red and then with far-red light so that, if proper extraction conditions had been utilized, about 60% of the total phytochrome would have been pelletable. When [³⁵S]phytochrome was added to extraction buffer immediately prior to homogenization of etiolated oat shoots, pelletability assays indicated that there was no preferential binding of [³⁵S]phytochrome regardless of (a) whether it was purified from dark control or irradiated shoots, (b) whether it was added as phytochrome-red-absorbing form or phytochrome-far-red-absorbing form, or (c) whether it was added to dark control or red-irradiated shoots. Similarly, binding of [³⁵S]phytochrome to resuspended pellets obtained from crude oat extracts was not specific for the source of [³⁵S]phytochrome, for its form, or for the irradiation treatment given to intact shoots used to prepare the resuspended pellets. No evidence was obtained to support the hypothesis that phytochrome binds with specificity to particulate material in vitro under conditions used to assay for light-enhanced, in vivo-induced phytochrome pelletability.     

Isolation and characterization of adenosine triphosphatase from Salicornia pacifica var. utahensis

Membrane-bound ATPase associated with plasma membrane and solubleATPase associated with the cytoplasm were prepared from shootsof Salicornia pacifica var. utahensis by sucrose density gradientcentrifugation. The isolated ATPases were tolerant to high concentrationsof NaCl. The Km for membrane-bound ATPase was 1.75 mM and forsoluble ATPase, it was 1.4 mM. The relative effectiveness ofdivalent cations for stimulation of membrane-bound ATPase wasMg²⁺>Fe²⁺>Mn²⁺>Co²⁺>Cu²⁺. Soluble ATPase activitywas stimulated by Ba²⁺>Ca²⁺>Mg²⁺ and was inhibited byCu²⁺, Zn²⁺, Co²⁺ and Fe²⁺. The compounds N,N¹-dicyclohexylcarbodiimide,NaF and ADP, did inhibit the ATPases but ouabain, triphenyltinhydroxide, sodium azide, indoleacetic acid and abscisic aciddid not inhibit the ATPases from s. pacifica var. utahensis. ¹ Present address: Department of Biology, Kong-Ju National College,Kong-Ju, Korea. (Received April 1, 1980; )     

Native Cadmium-Metallothionein from the Yeast Saccharomyces cerevisiae: Its Primary Structure and Function in Heavy-Metal Resistance

A Cd-resistant strain of yeast (Saccharomyces cerevisiae, strain30IN) accumulated Cd with the concomitant synthesis of a Cd-bindingprotein of low molecular weight when grown in Cd²⁺-containingmedium. Analysis of the amino acid composition, N-terminal sequenceand immunological properties of the protein revealed its structuralhomology to Cu-metallothionein (Cu-MT) in S. cerevisiae 2186,a Cu-resistant strain of yeast (Winge et al. 1985). The synthesisof MT in Cu-resistant strains of yeast is known to be underthe strict control of Cu²⁺ ions, while that in 301N was inducedboth by Cd²⁺ and Cu²⁺ ions. When 301N was precultured for 48h in the presence of 1 mM CuSO₄, its resistance to Cu²⁺ andthe synthesis of MT in response to Cu²⁺ were enhanced whileanalogous responses to Cd²⁺ were conversely reduced. These resultssuggest that the synthesis of MT is controlled by Cd²⁺ and Cu²⁺in a counteractive manner in strain 301N and, therefore, theregulation of the synthesis of MT plays a role in the adaptationof this strain to conditions when either metal is present. (Received November 1, 1990; Accepted February 22, 1991)     

Modifications of Sulfhydryl Groups on Phytochrome and Their Influence on Physicochemical Differences between the Red- and Far-Red-Absorbing Forms

Phytochrome extracted from shoots of dark-grown rye (Secale cereale cv Rymin) and oat (Avena sativa cv Garry) as the far-red-form (Pfr) and/or under conditions conducive to oxidation exhibited a blue shift in the visible absorption maximum of its red-light-absorbing form (Pr) relative to that measured in vivo. This spectral alteration could not be reversed but could be prevented by inclusion of 10 millimolar diethyldithiocarbamate and 140 millimolar 2-mercaptoethanol in homogenization buffers. Similar blue shifts were induced in purified rye phytochrome by addition of the sulfhydryl-modifying reagent, 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). In spectrally normal phytochrome (i.e., no detectable blue shift), Pfr had three to four more sulfhydryls available for rapid reaction with DTNB than did Pr. This difference was maintained over a 2.5-hour time course. Phytochrome purified under conditions resulting in a blue-shifted Pr absorption maximum exhibited a decreased short-term reactivity of Pfr to DTNB. Comparison of the binding and elution of altered and unaltered phytochrome from agarose-immobilized Cibacron blue 3GA confirmed that the Pfr form of spectrally normal phytochrome had a greater affinity for the dye than did the Pr form but that spectral alteration of phytochrome was accompanied by a loss of this difference as evidenced by an increased binding of Pr to the dye. It was concluded that phytochrome has highly reactive sulfhydryl residues located on the portion of the protein that undergoes conformational changes on interconversion of Pr and Pfr and that these residues require rigorous protection in order to extract the native form of the protein from plant tissue.     

Accumulation of Heavy Metals in Cell Walls of Polygonum cuspidatum Roots from Metalliferous Habitats

The distribution and accumulation of Cu²⁺, Zn²⁺ and Cd²⁺ ionsin the roots of Polygonum cuspidatum, collected from metalliferousand non-metalliferous habitats, were examined. About 90% ofthe metal ions was located in the cell wall fraction of rootsof plants grown in either type of habitat. The metal-ion exchangecapacity of the cell walls is not specific to the plants frommetalliferous habitats, and there were no significant differencesamong plants from the various habitats. The capacity for Cu²⁺ions was greater than that for Zn²⁺ or Cd²⁺ ions. Cu²⁺ ionshave a high affinity for the cell wall and, thus, it appearsthat the cell wall is a barrier for the transportation of Cu²⁺into the cytoplasm. (Received March 10, 1988; Accepted March 17, 1989)     

The kinetics of type 1 phytochrome in green,light-grown wheat (Triticum aestivum L.)

The kinetics of type 1 phytochrome were investigated in green, light-grown wheat. Phytochrome was measured by a quantitative sandwich enzyme-linked immunosorbent assay using monoclonal antibodies. The assay was capable of detecting down to 150 pg of phytochrome. In red light, rapid first-order destruction of the far-red-light-absorbing form of phytochrome (Pfr) with a half-life of 15 min was observed. Following white light terminated by red, phytochrome synthesis was delayed in darkness by about 15 h compared to plants given a terminal far-red treatment. Synthesis of the red-light-absorbing form of phytochrome (Pr) was zero-order in these experiments. Phytochrome synthesis in far-red light was approximately equal to synthesis in darkness in wheat although net destruction occurred in light-grown Avena sativa tissues in continuous far-red light, as has been reported for other monocotyledons. In wheat, destruction of Pfr apparently did not occur below a certain threshold level of Pfr or Pfr/total phytochrome. These results are consistent with an involvement of type 1 phytochrome in the photoperiodic control of flowering in wheat and other long-day plants.Abbreviations ELISA enzyme-linked immunosorbent assay - FR far-red light - HIR high-irradiance response - Pfr farred-light-absorbing form of phytochrome - Pr red-light-absorbing form of phytochrome - Ptot total phytochrome (Pr + Pfr) - R red light The authors wish to thank Prof. Daphne Vince-Prue (University of Reading) for many helpful discussions regarding this work. Hugh Carr-Smith was supported by a Science and Engineering Research Council studentship and Chris Plumpton by an Agricultural and Food Research Council (AFRC) studentship. B. Thomas and G. Butcher were supported by the AFRC.     

INTRACELLULAR PHYTOCHROME DISTRIBUTION AS A FUNCTION OF ITS MOLECULAR FORM AND OF ITS DESTRUCTION

The immunocytochemically observed intracellular redistribution of phytochrome as a function of its molecular form is described by utilizing color photomicrography. The reversible change from a diffuse to a discretely localized distribution following photoconversion of the red-absorbing Pr form to the far-red-absorbing Pfr form observed with etiolated oat (Avena sativa L., cv. Garry) coleoptile parenchyma cells is not seen with etiolated wheat (Triticum sativum L., cv. unknown), barley (Hordeum vulgare L., cv. Harrison), or rye (Secale cereale L., cv. Balbo). Whether redistribution in these latter cases does not occur or is below the limit of detection is not known. Upon continuous actinic irradiation, phytochrome, which is discretely localized as Pfr, rapidly disappears by both immunocytochemical and spectral assay. However, after about 90 min irradiation, a new association of phytochrome with nuclei is evident which is more pronounced after 4 or 8 h of irradiation. With longer irradiation times there is a total loss of antigenically detectable phytochrome at the resolution employed in these experiments.