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

Abstract

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 ...

Hydrophobic properties of phytochrome as probed by 8-anilinonaphthalene-1-sulfonate fluorescence

“…Pike Negbi et al, 1975). The faster components can be resolved by the peeling method (Figure 8C), and as can be seen they are not significantly affected by ANS.…”

Phototransformation and dark reversion of phytochrome in deuterium oxide

“…The dark-reversion kinetics of Pfr is biphasic in aqueous solution (Negbi et al, 1975;Taylor, 1968;Boisard et al, 1970;Manabe & Furuya, 1971;Pike & Briggs, 1972). Recently, we proposed a consecutive kinetic model for the biphasic decay of Pfr (Hahn & Song, 1981):…”

“…Rates of Pr -Pfr phototransformation and Pfr -Pr photoreversion were calculated by plotting the natural logarithms of absorbances at 660 or 725 nm vs. time for the first 20 s. Kinetic parameters for the dark reversion of Pfr to Pr were determined by using the same samples (i.e., immediately prepared phytochrome or recycled samples after red and far-red irradiation cycles), according to the procedure described by Negbi et al (1975), and the kinetics were then analyzed by the "peeling" procedure to determine the relative proportions of the fast and slow reversion components as described previously (Van Liew, 1967;Hahn & Song, 1981).…”

Absorption Spectra of Phytochrome Intermediates

“…chromophore conform ation which could be frozen at -140°C). Heterogeneity of P {r has repeatedly been reported for the kinetics o f dark reversion [16][17][18] although this had been explained with partially degraded phytochrome.…”