14.3.2—
Photoconversion Reactions

Kinetic analysis following flash excitation of phytochrome indicates that the forward reaction (Pr ®Pfr ) requires several seconds to complete, whereas the reverse transformation (Pfr ®Pr ) is apparently complete by 20 to 30 msec

(Linschitz et al., 1966; Linschitz & Kasche, 1967). Several intermediates on separate pathways for the forward and reverse reactions have been characterized spectroscopically (Kendrick & Spruit, 1973). The first photochemical intermediate on both pathways appears to result from isomerization of the chromophore only with no change in protein structure. Subsequent dark relaxations apparently involve conformational changes in the protein moeity as well as further chromophore re-arrangements. The actual mechanism involved in chromophore photo-isomerization is uncertain, although tautomerization of the pyrrole group (Fig. 14.2) plus a cross-exhange of protons between chromophore and protein is a currently favoured hypothesis (Lhoste, 1972).

The phototransformation of a static population of phytochrome molecules can be described by the expression (Butler, 1972):

where l = wavelength; I_l = intensity; t = duration of irradiation; E_rl and E_frl = the extinction coefficients at l for Pr and Pfr respectively; f_r and f_fr = the quantum yields for Pr and Pfr respectively. At t =¥ a photo-equalibrium will be established and the pigment will oscillate ('cyc'e') between the two forms at a rate which is a function of the total absorption of the two species. The ratio of Pfr to Pr will remain constant. In the absence of any net loss or gain of phytochrome molecules in the population (a 'closed' system), this ratio will be wavelength dependent but irradiance independent according to the formula:

In the living cell, however, synthesis and destruction of phytochrome (see 14.3.3.2 below) must be taken into account. This transforms the system into an 'open' one where net loss or gain of pigment molecules can and do occur. For short term irradiations (about 5 minutes), where photo-equilibrium is rapidly established, no significant change in total phytochrome (Ptot ) occurs and Eq. 14.2 is a good first approximation. Under these conditions the [Pfr ]:[Pr ] ratio in the cell will be irradiance independent but wavelength dependent. Under long term continuous irradiations, however, synthesis and destruction become significant parameters with the result that the ratio [Pfr ]:[Pr ] becomes irradiance, as well as wavelength, dependent (Schäfer & Mohr, 1974; Schäfer, 1975).

Experimentally the kinetics of phototransformation have been shown to be first order both in vivo (Schmidt et al., 1973) and in vitro (Butler, 1961). Likewise, both the rates of photoconversion (Fig. 14.3) and the short term photosteady state ratio of Pr to Pfr (Fig. 14.4) have been demonstrated to be wavelength dependent in a manner consistent with the measured absorption spectra (Butler et al., 1964; Hanke et al., 1969).

Figure 14.3
Action spectra of photochemical transformations of P r  and Pfr in
solution. The extinction coefficient Î  is in litre mol¹  cm^–1  and the
quantum yield f  is in mol Einstein^–1  (after Butler et al.,  1964).

Note that the rate and extent of photoconversion are dependent on the wavelength, irradiance and time of irradiation below photo-equilibrium (Eq. 14.1). The product of time and irradiance determines the total number of quanta or the light 'dose' administered to the system. The wavelength determines how

Figure 14.4
Proportion of phytochrome in the Pfr  form at photoequilibrium
in vivo  (Sinapis  hooks) as a function of wavelength
(after K.M. Hartmann and C.J.P. Spruit in Hanke  et al.,  1969).

efficiently the incident quanta are absorbed by the pigment (Fig. 14.1). Note also that total photoconversion of Pr to Pfr is not possible (Fig. 14.4). The maximum attainable is about 80% Pfr in the red region of the spectrum. This is because there is no wavelength where Pr absorbs and Pfr does not (Fig. 14.1). Conversely, more than 97% of the phytochrome can be converted to the Pr form by wavelengths longer than 737 nm (Hartmann & Cohnen Unser, 1973) as the Pfr absorbance exceeds that of Pr in that region of the spectrum. The photosteady state ratio of Pfr/Pr can be conveniently manipulated with monochromatic light, particularly between 660 and 750 nm (Fig. 14.4). This procedure has been used to advantage in several physiological experiments.

For many years the only quantitative assay for phytochrome has been the spectrophotometric measurement of its photoreversible absorbance changes (Fig. 14.1) (Butler et al., 1959; Spruit, 1972). This procedure can measure phytochrome both in vivo and in vitro. However, it is unsuitable for use with green tissue and provides no index of the integrity of large regions of the protein moeity. The recent successful immunocytochemical detection of phytochrome now provides a second assay for the pigment (Coleman & Pratt, 1974).