ACTTON SPECTRA OF PHYTOCHROME IN VITRO*

Abstract

ATTENTION to phytochrome and phytochrome-controlled physiological responses has been devoted chiefly to the red and far-red regions of the visible spectrum. Radiation of shorter wavelengths, however, also has an influence on plant development through its action on phytochrome.('. ' ) The absorption spectra of the two forms of phytochrome show, in addition to the major absorption bands in the red and far-red regions, minor bands in the blue and near u.v.@$ which are also effective in the photoconversions of phytochrome, PR s PFR. The present paper presents quantitative data on the effectiveness of monochromatic light in the spectral region between 300 and 800 nm for the transformations of PR and PFR in purified solutions.The photochemical transformations of phytochrome are first order in both directions.(Z) The rate equations for the transformations of PR and PFR by monochromatic light of wavelength Xare:[PR] and [PFd are the mole fractions of PR and PFR at time I, [PR m] X and [PFR a] X are the mole fractions at the photostationary state ( t = a) in monochromatic light of wavelength A, [PRO], and [PFRo] are the mole fractions at time t = 0 and KA is the first-order rate constant. Eh is the intensity of monochromatic light (Einstein cm-2 sec-l), eRA and cFRA are the extino tion coefficients of PR and PFR at h (cm2mole-',to the base 10) and C$R and,C$FR are the quantum yields for the transformations of PR and PFR (moles Einstein-l). Equation (1) requires the absorption of radiation by the sample to be small so that the intensity of radiation at any point in the sample can be assumed to be equal to the incident intensity. The action, defined as the product €4, can be determined at any wavelength from the value of KAr provided EA and [PR m ]~ or [PFR a]/\ are known. Previous (2, 5, calculations of the €4 products were based on the assumption that the red actinic light converted essentially all of the PR to PFR. This was assumed because difference spectra showed that the same maximal degree of transformation was achieved by irradiation

Comparison of Spectral Properties of Phytochromes from Different Preparations

“…The spectral parameters of purified phytochrome determined in recent studies (3,8,15,17,18) are different from those reported by Butler et al (2). Two factors are probably responsible for these differences.…”

“…Two factors are probably responsible for these differences. The phytochrome samples used in the old studies (1,2) contained a high proportion of proteolytically degraded 60 kD phytochrome, while most recent preparations consist mainly of undegraded ('native') 124 kD phytochrome (8,17,18). Recent preparations are purer, as suggested by values of the A665/A280 specific absorbance ratio for Pr much higher than that reported for the old phytochrome preparations (1,2,14).…”

“…Since the first isolation ofphytochrome, reported by Siegelman and Firer (14) in 1964, and the study of its photoconversion kinetics by Butler (1) and Butler et al (2), the techniques for phytochrome purification have improved considerably. The spectral parameters of purified phytochrome determined in recent studies (3,8,15,17,18) are different from those reported by Butler et al (2).…”

“…A full comparison is given for the properties ofthree phytochrome preparations from Avena (1,2,8,17,18) and a partial comparison for the properties of a Secale phytochrome preparation (WO Smith, personal communication) and for Cucurbita phytochrome in vivo (12). The basic spectral properties of these five phytochromes are summarized in Table I. MATERIALS AND METHODS Photoconversion coefficients and Pfr/P ratios at photoequilibrium (refer to Table II for definitions and symbols of spectral parameters) at different wavelengths for BHS and S and S phytochrome are those reported in Table I of the paper by Butler et aL (2) and in Table 1 and Figure 4 of the paper by Seyfried and Schifer (12). Photoconversion coefficients and Pfr/P ratios at photoequilibrium for K and L phytochrome were calculated using the extinction coefficients of Pr and Pfr reported in Tables 3 and 4 ofthe supplement for the microfilm edition ofthe paper by Kelly and Lagarias (8) and the quantum efficiencies for Pr to Pfr and Pfr to Pr photoconversion reported in Table I.…”

“…The phytochrome samples used in the old studies (1,2) contained a high proportion of proteolytically degraded 60 kD phytochrome, while most recent preparations consist mainly of undegraded ('native') 124 kD phytochrome (8,17,18). Recent preparations are purer, as suggested by values of the A665/A280 specific absorbance ratio for Pr much higher than that reported for the old phytochrome preparations (1,2,14). Despite the availability of spectral parameters for undegraded phytochrome, many researchers still use the old data, as one can easily see by a survey of recent literature, one example being a recent paper on the distortion of action spectra of phytochrome-mediated responses (7).…”