Phytochrome Stability <i>in Vitro</i>

Lisansky, Stephen G.; Galston, Arthur W.

doi:10.1104/pp.53.3.352

Cited by 26 publications

(14 citation statements)

References 16 publications

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“…The pH of the phytochrome at the taneously preclude correcting the present reversion results for ,es was not adjusted prior to the determination of the loss of phytochrome. Lisansky and Galston (10), on the other netics, since it has been shown that the kinetic param-hand, report that miagnesium and calcium have no effect on tvary over a pH range of 6.2 to 8.2 (16). All manipu-"dark destruction"; they utilized the cations at concentrations r to phototransformation were carried out at 5 C lower than used in the present studies.…”

Section: W F Thompson (Personal Communication) Noted That Caci2mentioning

confidence: 83%

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Acceleration of Dark Reversion of Phytochrome in Vitro by Calcium and Magnesium

Negbi

Hopkins²,

Briggs³

1975

Plant Physiol.

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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-(oxyethylenenitrilo)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.Dark reversion of Pfr to Pr was suggested by physiological studies years before phytochrome was detected in vivo or isolated and studied in vitro (2). Since then there have been numerous studies of dark reversion using the photoreversible absorbance property as an assay of phytochrome (for a comprehensive review see 6). Dark reversion has been detected in vivo in gymnosperms (7,15,19) and in most dicots, except in some Centrospermae (8) and Cucurbita (1). Most studies have shown no dark reversion in monocots (for a complete review, see 4).In vitro dark reversion proceeds in two phases, a short, rapid phase followed by a slower one (5,11,16,18 Phytochrome Measurements. Assays of phytochrome in rye, pea, and pine tissue were made with the Ratiospect R-2 dual wavelength difference spectrophotometer (12, 16). Phytochrome measurements were performed with a Zeiss PMQII spectrophotometer or Gilford 2000 automatic spectrophotometer. Actinic red or far red light was provided by a 500-w projector with filters (Baird-Atomic interference filter, 646 nm peak wavelength, 10 nm half-band-width, or Rohm-Haas broad-band plastic, transmitting wavelengths above about 715 nm).Phytocbrome Samples. Rye phytochrome samples were purified by the method of Briggs et al. (3). Briefly, phytochrome was purified by treatment with 10 mM CaCl2, brushite column chromatography, precipitation with 33%-saturated ammonium sulfate at pH 7, and solution of the precipitate and dialysis against 50 mM tris-Cl pH 7.8. Phytochrome was further purified by DEAEcellulose chromatography; dark reversion was measured in the elution buffer, 50 ms tris and 190 mi KC1, pH 7.4. Further purification was achieved by ammonium sulfate precipitation (as above) and hydroxylapatite column chromatography; dark reversion was measured in the elution buffer, 100 mm KCI with approximately 50 mm potassium phosphate, pH 7.5. Ammonium sulfate fractionation and application to an agarose 1.5-m (8% gel) column yielded highly purified phytochrome, in 100 mm sodium phosphate, pH 7.5. The ...

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Section: W F Thompson (Personal Communication) Noted That Caci2mentioning

confidence: 83%

“…Rye phytochrome samples were purified by the method of Briggs et al (3). Briefly, phytochrome was purified by treatment with 10 Figure 1. CaCd2af-fects the proportion of fast and slow reacting components of phytochrome dark reversion (Table I).…”

Section: W F Thompson (Personal Communication) Noted That Caci2mentioning

confidence: 99%

Acceleration of Dark Reversion of Phytochrome in Vitro by Calcium and Magnesium

Negbi

Hopkins²,

Briggs³

1975

Plant Physiol.

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“…In contrast to the above listed cations, transition metals that form chelate complexes with large stability constants, such as Cd 2., Co 2 § Cu 2 § and Zn 2*, cause spectral denaturation of Pfr (100) and enhance its rate of degradation (57). Treatment of seedlings with EDTA, sulfhydryl compounds, azide, cyanide or carbon monoxide inhibits degradation of Pfr in vivo (9,21).…”

Section: Applicability Of Hypothesis To Phytochromementioning

confidence: 99%

“Light-derepressible” genes are regulated by metal-protein complexes: A hypothesis

Hoober

1988

Carlsberg Res. Commun.

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The hypothesis is presented that genes whose expression is sensitive to light are regulated by metal-protein complexes. This hypothesis is based on induction of a gene for an Mr 21,000 polypeptide in the prokaryote Arthrobacter sp. by photodynamic conditions, chelating agents, or a decrease in pH. It is proposed that these conditions promote dissociation of a regulatory metal-protein complex by photooxidation of the metal-binding site, lowering the concentration of free metal ion, or protonation of amino acid side-chains in the metal-binding site, respectively. Repression of this gene is restored when cells are returned to the dark, metal ions are added in excess of chelating agents or the pH of the medium is returned to neutral. Restoration of repression is inhibited by chloramphenicol and novobiocin, which suggests that this process requires resynthesis of a regulatory protein and reassociation of the complex with DNA. The hypothesis predicts a number of characteristics of the regulatory system, most of which are found among the effects ofphytochrome. The well-documented effects of transition metal ions on the Pfr form ofphytochrome suggest that changes in gene expression in plants mediated by photoisomerisation of this chromoprotein are initiated by a chelating activity of Pfr-

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“…In an earlier paper (11), we drew attention to the parallels between some of the observed effects of metal ions on phytochrome-mediated physiological responses and their effects on phytochrome photoreversibility in vitro. At the time, there were a number of possible mechanisms to account for the disappearance of photoreversibility in v'ivo after irradiation.…”

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confidence: 99%

“…In addition, the relation between phytochrome destruction and metal ions (9,11), sulfhydryl reagents (5), chelating agents (14,17), and plant components such as "'killer factor" (5,6) is poorly understood. These experiments were undertaken to clarify the apparent contradiction between the very low levels of phytochrome found in green tissue and the many phytochrome-mediated responses found in green plants. If phytochrome is produced in meristematic cells, as is indicated by the profiles of phytochrome titer in etiolated plants (2) and the correlation between phytochrome synthesis and cell division in tissue cultures (20), then loss of photoreversibility should occur when the meristematic region becomes and extracts of apical and immediately subapical green tissue of light-grown peas.…”

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confidence: 99%