Some Spectral Properties of Pea Phytochrome in Vivo and in Vitro

Abstract

The transformation difference spectrum for phytochrome (Pr spectrum minus Pfr spectrum) in pea tissue is determined below 560 nanometers and compared with similar data on phytochrome in vitro The difference spectrum in vivo between phytochrome intermediates and Pfr is also shown for comparison with the data on phytochrome solu. tions. These comparisons show that the peaks in the spectra occurring in the blue wave lengths are shifted to shorter wave lengths and are much enhanced when phytochrome is extracted from the cell and placed in solution. The results indicate that the physicochemical state of phytochrome in the cell may be different from that of the extracted pigment. about 0.1 OD per A (A OD) of phytochrome. A typical tissue sample for spectrophotometric observation could contain 0.2 A (A OD); thus the change in optical density to be expected is of the order of 0.02 OD in a region where the total optical density is between 2 and 3.Since we had available a spectrophotometer designed to measure small absorbancy changes in dense tissue samples, we attempted to observe absorbancy changes related both to phytochrome transformation and to the long-lived phytochrome intermediates described by Briggs and Fork (3,4) at wave lengths below 560 nm. Such observations might provide information on the state of phytochrome in vivo and could partially explain the difference in the relative effectiveness of blue light for transformation in vivo and in vitro. MATERUILS AND METHODSIn many physiological responses mediated by phytochrome, it has been shown that blue light can evoke reactions similar to those caused by red and far red light (1, 2, 13). Such observations led to the hypothesis that both forms of phytochrome, Pr and Pfr, absorb blue light (1, 2). With the development of methods for extracting phytochrome, it was indeed found that the pigment has absorption maxima in the blue spectral regions -Pr at about 370 nm and Pfr at about 400 nm (7,11,16 Plant Material. Peas (Piswn sativum L. cv Alaska) were soaked in tap water for 4 hr and planted on moist cellulose packing material, Kimpak 6223 (Kimberly-Clark), in plastic refrigerator boxes. The plants were grown in complete darkness at 25 C and 80% relative humidity for 3 days, although occasionally 2-or 4-day-old plants were used. To prepare a sample, the apical hook regions (5 mm) were harvested under dim green light, the plumules were removed, and the sections were placed in a beaker on ice. Approximately 150 such sections were chopped into pieces about 1 mm3 and placed in a chilled cuvette 1.6 cm in diameter. The sections were gently packed so that light attenuation through the sample was uniform. Total phytochrome content uncorrected for incomplete photoconversion by red light was measured from absorption spectra of the samples after saturating red and far red irradiation. The average photoreversible phytochrome content of such samples varied between 0.15 and 0.20 A (A OD).Phytochrome Isolation. Phytochrome from peas was obtained by minor modification of met...

“Disaggregation” of Phytochrome in Vitro—A Consequence of Proteolysis

“…The calcium pectate precipitate was removed by centrifugation and the supernatant was applied to a brushite (calcium phosphate gel [37,42]) column which had been equilibrated with 2 column volumes of 10 mm KPB, pH 7.0, and 2 additional volumes of the same buffer containing 0.7% 2-ME. Approximately 1 liter of brushite was used per 'Abbreviations: 2-ME: 2-mercaptoethanol; KPB 7.0, containing 0.5% 2-ME, and 1 volume of 15 mm KPB, pH 7.0 (without 2-ME). The 2-ME was absent from all further buffers in the procedure.…”

Partial Characterization of Oat and Rye Phytochrome

“…Extensive information on phototransformation intermediates has been gathered from low temperature (20,45,46), flash photolysis (33,34), and other transformation studies (3,10,11,14,22,37,39,42,47) performed on oat phytochrome preparations of varying purity. Recently, circular dichroic measurements (4,28,31) have been made on more highly purified oat phytochrome.…”

Phytochrome Properties and the Molecular Environment

“…This difference with respect to reversion in vivo and in vitro is not the only change that results upon extraction. The peak wavelength of Pfr absorbance shifts from 732 nm in vivo to 725 nm in vitro (7). Another reaction until now only detected in vivo, and in particular in seeds, is the so-called "inverse dark reversion," which is the production of Pfr in darkness from Pr after far red light (2,18,30).…”