14.3—
The Phytochrome Molecule
14.3.1—
Molecular Properties
The purified phytochrome molecule is a water-soluble chromoprotein containing less than 4% carbohydrate (Briggs & Rice, 1972). The native monomer is a polypeptide of 120,000 daltons but can form higher molecular weight aggregates. The chromophore is thought to be a linear tetrapyrrole with some evidence suggesting that there is one chromophore per monomer (Tobin & Briggs, 1973). The proposed chromophore structures, their postulated linkages to the protein and a possible photo-isomerization mechanism are shown in Fig. 14.2 (Rüdiger, 1972).
Figure 14.2
Proposed structure for the phytochrome chromophore, its linkage to the
protein and possible phototransformation mechanism (after Rüdiger, 1972).
The 'blue' form is thought to correspond to P r and the 'green-yellow' form to Pfr.
Information on differences in the molecular organization of the Pr and Pfr species has been sought in the hope that this might provide some insight into potential reaction mechanisms. Differences in the chromophore environment are evident from the absorption (Fig. 14.1), circular dichroism and optical rotatory dispersion spectra in the visible region (Kroes, 1970). Changes in protein conformation during photoconversion are also implied from low temperature and freeze-dry studies (Spruit & Kendrick, 1973; Kendrick, 1974). However, the post-conversion differences in the Pr and Pfr protein configurations are apparently only quite small as revealed by a variety of spectral and chemical methods (Briggs & Rice, 1972). Differences in surface residues are suggested by the ultraviolet difference spectrum (Tobin & Briggs, 1973); the differential reactivity of Pr and Pfr toward glutaraldehyde (Roux, 1972), and N -ethy maleimide (Gardner et al., 1974); and the differential electrostatic binding of Pr and Pfr to ribosomal material in plant extracts (Quail, 1975b).
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; Il = intensity; t = duration of irradiation; Erl and Efrl = the extinction coefficients at l for Pr and Pfr respectively; fr and ffr = 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).
14.3.3—
Dark Reactions
'Dark reversion' and 'destruction' are the so-called dark reactions of phytochrome. Neither the molecular bases nor the physiological significance of these processes is well understood.
14.3.3.1—
Dark Reversion
Pr is thermodynamically stable and can only be converted to Pfr by light (Lhoste, 1972). Pfr, in contrast, is metastable and can therefore revert thermally to Pr in the dark.
In vivo, dark reversion occurs in most dicotyledons but not monocotyledons, whereas phytochrome from both sources reverts in vitro (Frankland, 1972; Briggs & Rice, 1972). The process in vivo appears to be first order and rapid. In several plants reversion is complete within 30 minutes at 20ºC although only 15% to 20% of the Pfr molecules are involved. The remainder continue to undergo 'destruction' for a considerable period after reversion has ceased. Separate 'reversion' and 'destruction' pools of Pfr have been postulated to account for this apparent anomaly (Schäfer & Schmidt, 1974). Discontinuities in Arrhenius plots of the extent of reversion suggest that the process might be membrane associated (Schäfer & Schmidt, 1974).
14.3.3.2—
Synthesis and Destruction
Dry seeds contain phytochrome (Spruit & Mancinelli, 1969). Rapid, early increases in the photometrically detectable pigment (Ptot ) during imbibition are apparently due to rehydration of the molecule rather than synthesis (Tobin et al.,
1973). The pigment can be stored as either Pr or Pfr and rehydrated in the stored form (Vidaver & Hsiao, 1972). This suggests an explanation for the appearance of Pfr in the dark, sometimes observed in seeds (Rollin, 1972). Further increases in Ptot during the growth of etiolated seedlings result from de novo synthesis of new molecules in the Pr form (Quail et al., 1973b). The pigment accumulates to high levels in the dark ultimately reaching a plateau (Fig. 14.5).
'Destruction' is the disappearance of photometrically detectable Pfr without the concomitant appearance of equimolar quantities of Pr (Frankland, 1972). This decrease in photoactivity is paralleled by a loss of immunologically detectable phytochrome (Coleman & Pratt, 1974). Since no recycling of the protein moeity occurs, 'destruction' would appear to be a genuine degradative process (Quail et al., 1973b). Some evidence suggests that this process may be enzymatic (Kidd & Pratt, 1973). Destruction is temperature dependent, but the absence of discontinuities in Arrhenius plots suggests that in contrast to dark reversion, it is not membrane associated (Schäfer & Schmidt, 1974). Destruction is observed in both monocotyledons and dicotyledons with half-times ranging from 20 minutes to 4 hours (Frankland, 1972; Schäfer et al., 1973; Kidd & Pratt, 1973).
Destruction of Pfr occurs both in the dark following brief irradiations and in continuous light. In the dark, destruction rapidly removes all unreverted Pfr leading to a short-term decline in Ptot. The reduced pigment levels are replenished, however, as the dark period proceeds by de novo synthesis of new Pr molecules (Quail et al., 1973b). In continuous light, initially high Ptot levels decline at a rate proportional to the photosteady-state Pfr concentration (Frankland, 1972) ultimately reaching a new plateau (Fig. 14.5). This new
Figure 14.5
Total phytochrome levels in Sinapis cotyledons as function of time in the dark
(o); continuous far-red (
48 h dark ® far-red (
plateau represents a steady-state equilibrium between synthesis and degradation (Schäfer et al., 1972; Quail et al., 1973b). Pr synthesis appears to be a continuous zero order process, itself unaffected by light. The Ptot level is then regulated against this background by the disparate first order degradation rate constants for Pr and Pfr.
The plateau level of Ptot established at the steady-state by prolonged irradiations is a function of both wavelength and irradiance (Schäfer & Mohr, 1974; Schäfer, 1975). The Pfr level, in direct contrast however, is independent of both irradiance and wavelength under these conditions. This has the extremely important consequence that, whereas the ratio of [Pfr ]:[Ptot ] will change depending on wavelength and irradiance, the absolute level of Pfr will be the same under all continuous irradiation conditions once the steady-state has been established. This has important implications for the interpretation of the effects of prolonged irradiations.
14.3.3.3—
Physiological Significance of Dark Reactions
The dark reactions provide a mechanism for the light independent removal of Pfr and thereby the potential for a dark period to reverse light-induced responses which require the sustained presence of the effector. Furthermore, as the disappearance of Pfr is a time dependent process the system has the potential for measuring time. In principle, therefore, phytochrome should enable the plant to distinguish between light and dark and to time the dark period. Initially it was thought that this so-called 'hour-glass' principle was the basis for phytochrome-controlled photoperiodism (see Vince, 1972). Supporting evidence is scant, however, and this theory has now fallen into disrepute. Some systems do, nevertheless, respond to the timed disappearance of Pfr in the dark. An example is accumulation of the enzyme lipoxygenase (Oelse-Karrow & Mohr, 1973).
During prolonged irradiations, synthesis and destruction appear to have the additional role of maintaining a constant absolute level of Pfr irrespective of wavelength (Schäfer, 1975). This implies that Pfr per se is not the effector of high irradiance responses (see 14.4.1.2).
14.3.4—
Localization
The distribution of phytochrome is highly specific at the tissue and cellular levels as determined photometrically (Briggs & Siegelman, 1965) and immunocytochemically (Pratt & Coleman, 1971; 1974). In dicotyledons, the highest pigment levels are in the apical regions. In etiolated grass shoots, large quantities occur in parenchyma cells near the tip of the coleoptile and in the rapidly differentiating tissue near the shoot apex. High levels of the pigment are also found in root cap cells.
Attempts to establish the subcellular localization of phytochrome fall into two categories: (a) measurement of phytochrome-induced responses having a
spatial or vectorial component from which the photoreceptor location can be inferred; (b) direct measurements of the pigment itself, either in situ or in subcellular fractions.
The pattern of chloroplast movement in the alga Mougeotia in response to polarized red and far-red microbeams has been taken as evidence that phytochrome located and orienled on or near the plasmalemma controls this response (Haupt, 1972b). The directional growth of the germ tubes of Dryopteris in polarized light has been interpreted similarly (Etzold, 1965). The change in ion flux associated with phytochrome mediated leaflet movement (Satter & Galston, 1973), root tip adhesion to glass surfaces (Tanada, 1968), and changes in bioelectric potentials (Newman & Briggs, 1972) are also indicative of phytochromecontrolled changes in surface properties but not necessarily that the pigment is a permanent membrane component. A report of phytochrome-controlled development of isolated etioplasts in vitro (Wellburn & Wellburn, 1973) implies the presence of functionally active pigment in or on the organelles. More recently a rapid, phytochrome-mediated change in the level of gibberellin extractable from an etioplast-rich fraction in response to in vitro irradiations has been demonstrated (Evans, 1975; Evans & Smith, 1976a; Cooke & Saunders, 1975). A red/far-red reversible reduction of NADP in vitro in response to irradiation of a mitochondria-rich fraction has also been reported (Manabe & Furuya, 1974). Furthermore, phytochrome has been detected spectrophotometrically in both etioplast- (Evans & Smith, 1975) and mitochondria-rich fractions (Manabe & Furuya, 1974).
Both spectrophotometric and immunological techniques have been used for direct, in situ measurements of the intracellular distribution of phytochrome. The presence of the pigment in the nucleus has been claimed on the basis of microspectrophotometric scans of cells (Galston, 1968) but these data have been challenged (Kendrick & Spruit, 1972; Tobin et al., 1973). The cytochemical visualization of phytochrome antibody in non-irradiated tissue sections indicates a general distribution of the photoreceptor throughout the cytoplasm in addition to an association with nuclei and plastids (Pratt & Coleman, 1971). Brief red irradiation prior to fixation causes the pigment in some tissues to concentrate in discrete, as yet unidentified regions of the cytoplasm (Mackenzie et al., 1974). Non-saturating irradiations of maize coleoptile segments with red and far-red light polarized normal to the longitudinal axis were found to photoconvert about 20% more phytochrome than when polarized parallel to this axis (Marmé & Schäfer, 1972). This was interpreted as indicating that phytochrome is located and oriented in the plasmalemma. In considering the locational and orientational rigidity of phytochrome implied from polarized light studies the known rapid and highly fluid lateral and rotational diffusion of other membrane proteins should be borne in mind (Cone, 1972; Singer, 1974).
Cell fractionation procedures have also been used in attempts to localize phytochrome in subcellular components. The well-known precipitation of the pigment protein at low pHs (
has been overlooked in some studies leading to claims of associations of phytochrome with mitochondria (Gordon, 1961) and plasmalemma (Marmé et al., 1971). Little of the pigment (< 10%) sediments from homogenates of nonirradiated tissue at neutral pH at forces up to 144,000 × g (Rubinstein et al., 1969; Siegelman & Butler, 1965). Red irradiation prior to extraction, however, substantially enhances the level of phytochrome subsequently associated with pelletable material (Quail et al., 1973a). Irradiation of extracts from dark grown material has a similar effect (Marmé et al., 1973). Initially claims were made of the isolation of a phytochrome-containing membrane fraction that could be reversibly 'solubilized' by withdrawal of Mg2+ (Marmé et al., 1973; 1974). More recently, however, the pigment in this fraction has been shown to be associated with degraded ribonucleoprotein (RNP) material, probably of ribosomal origin (Quail, 1975b). This association apparently results from the preferential electrostatic adsorption of Pfr onto ribosomal material—either free or membrane-bound in the endoplasmic reticulum (Williamson et al., 1975). Whether such an association is artefactual or biologically meaningful is yet to be established. A recent promising variation on this approach is the use of glutaraldehyde in an attempt to immobilize the pigment in the cell prior to extraction (Yu, 1975.)
The existence of meaningful phytochrome-membrane interactions are by no means excluded by the above findings. It has been shown, for example, that phytochrome can mediate photoreversible conductance changes in artificial lipid membranes (Roux & Yguerabide, 1973). The suggestion that phytochrome might function as a stereospecific protein ligand capable of interaction with cellular membranes has been made (Quail & Schäfer, 1974; Boisard et al., 1974).