14.4—
Phytochrome Physiology
The biological responses attributed to phytochrome can be usefully characterized in terms of three different but interrelated sets of criteria: (a) the nature of the involvement of light in the inductive process; (b) the temporal expression of the induced response; and (c) the type of cellular or developmental process affected.
14.4.1—
Induction-Reversion and High Irradiance Responses
Two types of light-controlled phenomena have been attributed to phytochrome—the so-called 'induction-reversion' and 'high irradiance' (HIR) responses (Mohr, 1972). This terminology arises from the irradiation conditions under which the responses are observed and suggests a fundamental difference in the manner in which the phytochrome molecule transmits the light signal to the cell in each case. It does not necessarily reflect an intrinsic property of the actual, biological parameter being monitored. Some parameters display both modes of response, others only one.
14.4.1.1—
Induction-Reversion Responses
These are the classical phytochrome responses (Borthwick, 1972). A change in the biological parameter being monitored is induced by a brief irradiation of low intensity red light and reversed by a subsequent far-red pulse. The accumulation of anthocyanin in Sinapis illustrates this point (Fig. 14.6). Another well known example is lettuce seed germination. This is repeatedly photoreversible for up to 100 alternate red and far-red irradiations (Borthwick, 1972).
Figure 14.6
Accumulation of anthocyanin in Sinapis in the dark following irradiation
treatments at time zero with 5 min red (o), 5 min far-red (Ñ ), or 5 min
red followed immediately by 5 min far-red (
Mohr et al., 1971).
This simple red/far-red photoreversibility forms the basis of the concept that Pfr is the biologically active form of the pigment whereas Pr is inactive. Attempts to quantify the relationship between the number of Pfr molecules formed and the magnitude of the induced response have been both indirect and direct.
Indirect correlations are based on the premise that observed increases in the magnitude of the response with increasing light dose are a function of the degree of photoconversion of Pr to Pfr, i.e. the more quanta, the more Pr is converted to Pfr and therefore the greater is the response. The increase in anthocyanin in response to increasing doses of red light (Table 14.1) illustrates this point (Lange et al., 1971). The so-called law of reciprocity (irradiance × time = constant) must hold for the light doses used for this interpretation to be valid (see 14.3.2). This establishes that the magnitude of the response is directly proportional to the total number of incident quanta regardless of the time or irradiance of the
irradiation providing those quanta (Table 14.1). The effects of irradiance level during the brief irradiations used in induction-reversion experiments are thus attributed entirely to the degree of photoconversion.
Biological action spectra are an extension of this principle (Fig. 14.7). The magnitude of the response at different wave-lengths is interpreted to be a function of the relative effectiveness of the quanta at those wavelengths in the phytochrome photoconversion process. The close agreement between the action spectra of several biological responses on the one hand (Fig. 14.7) and those of the phototransformation reactions of the isolated pigment on the other (Fig. 14.3) lends strong support to this notion (Borthwick, 1972; Shropshire, 1972). In these cases a seemingly good correlation exists between Pfr level and response magnitude.
Figure 14.7
Action spectra for induction and reversion of plumular hook opening in bean seedlings
(after Withrow et al., 1957).
In contrast, however, the majority of rigorous attempts to demonstrate a direct quantitative correlation between the photometrically detectable Pfr level and the relevant biological response in the same system have been unsuccessful (Hillman, 1972). The reasons for this are not understood. An apparent exception is lipoxygenase levels in Sinapis (Oelze-Karrow & Mohr, 1973).
Increases in response with increasing doses of quanta must eventually saturate. If the level of Pfr is rate-limiting the photoresponse will saturate when the photoconversion process is saturated i.e. when photoequilibrium is reached. If the response system itself is rate-limiting the response may saturate well before photoequilibrium. Examples of both extremes have been observed (Hillman, 1967). Light doses which saturate the photoconversion of Pr to Pfr do not appear to saturate the inhibition of mesocotyl lengthening in Avena (Loecher, 1966), nor the accumulation of anthocyanin in Sinapis (Drumm & Mohr, 1974). In contrast, inhibition of lipoxygenase accumulation is saturated by very low (< 3%) Pfr levels (Mohr, 1972). Other photoresponses fall between these extremes with many being saturated at less than 80% Pfr (Hillman, 1967). In addition, whereas many parameters, such as anthocyanin formation, show a graded response, others, such as lipoxygenase accumulation, respond in an all-or-none fashion to changes in Pfr level, suggesting some form of cooperative, threshold mechanism (Oelze-Karrow & Mohr, 1973).
Implicit in the far-red reversibility of an induced response is that Pfr can act in the dark. Light is strictly a trigger. The magnitude and multiplicity of the responses indicate an extensive amplification mechanism. Unlike photosynthesis where light energy is converted stoichiometrically with quantum yields of less than 1.0, the low irradiances which actuate these phytochrome responses lead to final quantum yields well in excess of unity (Galston, 1974).
14.4.1.2—
High Irradiance Responses (HIR)
If Pfr is, as postulated, the active, effector, it is clear that tripartite correlations between light dose, Pfr level and response magnitude will only be expected for irradiations terminated prior to photoequilibrium. Once photoequilibrium has been established, Pfr le vels are no longer irradiance dependent whether for short term (Eq. 14.2) or long term (Schäfer, 1975) irradiations. No further increase in the response should result therefore from further increases in irradiance after photoequilibrium.
Some parameters do, however, show a strong irradiance dependence at photoequilibrium. This effect is termed the high irradiance response (HIR) (Mohr, 1969). Such responses are observed with continuous irradiation where a phytochrome photosteady-state is rapidly established and maintained for prolonged periods. The accumulation of anthocyanin in continuous far-red light illustrates this point (Fig. 14.8) (Lange et al., 1971). The rate of accumulation is a function of the irradiance. This effect is only maintained, however, as long as the irradiation continues. The irradiance-enhanced response rate reverts rapidly
to that of the dark controls when irradiation ceases, and resumes again upon further irradiation (Fig. 14.9). Reciprocity is not demonstrable for the HIR and, in some cases e.g. lettuce hypocotyl lengthening, the variable itself does not exhibit the classical red/far-red reversible response (Hartmann, 1966). Anthocyanin accumulation, in contrast exhibits both modes of response (Figs. 14.6 and 14.8).
Figure 14.8
Accumulation of anthocyanin in Sinapis under high irradiance
conditions. Seedlings were held in continuous far-red light of
varying irradiance. All irradiances are expressed relative
to the arbitrary value of 1,000 (= 350 µW. cm–2 )
(after Lange et al., 1971).
Action spectra of HIR have peaks in the blue and far-red (Borthwick et al., 1969; Mohr, 1969). The most extensively studied response is that of the inhibition of lettuce hypocotyl lengthening (Hartmann, 1967; see also Fig. 14.10). The seedlings were irradiated continuously for 18 hours with monochromatic light of different wavelengths and irradiances. As with other HIR there is no coincidence of the observed spectrum with the absorption maxima of either Pr or Pfr.
Figure 14.9
Accumulation of ascorbate oxidase activity in Sinapis under
high irradiance conditions. Seedlings were either retained in
the dark (o) or continuous far-red
seedlings from far-red to the dark or vice versa at
various times is indicated by the arrows (̄ )
(after Drumm et al., 1972).
Figure 14.10
Action spectrum for inhibition of lettuce hypocotyl
lengthening under continuous irradiation
(after Hartmann, 1967).
The conclusion that phytochrome mediates the high irradiance effects derives from other data using the same plant system (Hartmann, 1966). Prolonged irradiations with two wavelengths which were relatively ineffective when given separately (658 nm and 766 nm) were highly effective when given simultaneously. The maximum effect with these and other wavelength pairs always occurred where the photosteady-state Pfr concentration was about 3%. This agrees well with the peak of activity observed at 720 nm with single wavelength monochromatic light (Fig. 14.10) also known to establish about 3% Pfr at photoequilibrium (Fig. 14.4). Furthermore, the effectiveness of a single wavelength irradiation at 717 nm could be nullified by simultaneous irradiations of either 658 nm or 759 nm. These wavelengths would shift the photoequilibrium away from 3% Pfr towards higher or lower values respectively.
The question remains, however, as to how both the irradiance and wavelength dependency of the HIR can be explained in terms of phytochrome. The response is unlikely to be a function of the absolute Pfr level as this is irradiance and wavelength independent with prolonged irradiations (Schäfer, 1975). The rate at which the pigment molecule oscillates between the two forms at photoequilibrium is, on the other hand, strongly irradiance and wavelength dependent. Accordingly, the HIR has been rationalized to be some function of the cycling rate of phytochrome. The effector molecule has been postulated to be some 'excited form of Pfr', denoted Pfr* (Schopfer & Mohr, 1972). No direct evidence for such a species is available, however, and its purported action has been questioned on the basis of dual wavelength experiments (Hartmann & Cohnen Unser, 1973). High levels of phytochrome intermediates are maintained at high irradiances (Kendrick & Spruit, 1973a) but the possibility that these are HIR effectors is unlikely because of the nature of the action spectra (Hartmann, 1966).
14.4.1.3—
A Unitary Model
Schäfer (1975) has recently developed a single, formalistic model which can theoretically account for both induction-reversion and HIR responses, whether graded or cooperative, in terms of phytochrome. The pigment is postulated to be a bimodal ligand with the forms Pr and Pfr which interact with receptor sites also having dual forms, X and X':
where °ks and kd are the rate constants for synthesis and destruction of phytochrome respectively; k1 and k2 rate constants for the photoconversion reactions; and k3 and k4 rate constants for the X®X' and X'®X transitions.
The irradiance and wavelength dependencies of the HIR response then become explicable in terms of the flux rates through the cycle under continuous irradiation. The basic conclusions reached are that PfrX' is the effector element in induction-reversion responses, whereas PfrX or the flux k3 . PfrX is the effector element in HIR. The molecular nature of X and the mechanism of action of the effector elements are, however, unresolved questions.
14.4.1.4—
Modes of Light Signal Transmission
Given that phytochrome mediates both induction-reversion and HIR, it is clear that the photoreceptor utilizes the incoming light signal differently in each case. Whereas induction-reversion responses are induced by a red pulse and can develop in the subsequent dark period, HIR require a continuous light energy input to sustain the response. Increases in irradiance lead to increases in response magnitude in both cases but the effect is interpreted differently for each. For induction-reversion responses the irradiance effect is only observed prior to photoequilibrium; is interchangeable with time of irradiation (reciprocity holds); and is interpreted as reflecting the effectiveness of the total light dose in determining the degree of photoconversion of Pr to Pfr. Pfr is considered the effector molecule and some form of Pfr -response stoichiometry is expected. For HIR, on the other hand, the irradiance effect is observed at photoequilibrium; is not interchangeable with time of irradiation (reciprocity does not hold); and is interpreted as being some function of the phytochrome cycling rate. The precise effector molecule or process is uncertain. For induction-reversion responses light is viewed simply as a trigger and as having no further direct role in the inductive function of Pfr. The light signal is 'stored' in the Pfr form for subsequent utilization. HIR, in contrast, require a sustained, direct interaction of the photoreceptor with the incident excitation energy. This indicates that these effects are light-driven as distinct from being light-triggered. Light appears to have a direct role in the inductive function of the pigment, the energy input being rapidly dissipated.
The suggestion that photosynthesis or cyclic photophosphorylation might in some way be responsible for HIR has been advanced but several pieces of evidence argue against this (Mohr, 1972). Speculative suggestions that phytochrome might function as a photocoupler (Quail, 1975a) or a specific, lightdriven permease (Smith, 1970) in the HIR have also been advanced but not substantiated.
14.4.2—
Response Kinetics
Phytochrome is considered to trigger, in some way, a chain of events leading sooner or later to a measurable biological response. The initial triggering of those processes necessary for the development of the response can be termed phytochrome 'action'; and the appearance of a measurable change in the
parameter being monitored can be called response 'expression'. This leads to the recognition of three categories of phenomena: (a) rapid action/rapid expression; (b) rapid action/delayed expression; and (c) delayed action/delayed expression responses. 'Rapid' here arbitrarily means
14.4.2.1—
Rapid Action/Rapid Expression Responses
As phytochrome action must either coincide with or precede expression, rapid action can be implied from the kinetics of the expression alone in these cases. A red/far-red reversible change in electric potential in Avena coleoptiles within 15 seconds of the start of irradiation is the most rapid phytochrome-mediated phenomenon thus far reported (Newman & Briggs, 1972). Similar changes have been observed in the biolelectric potential of mung bean root tips exposed to successive red and far-red irradiations (Jaffe, 1968; Racusen & Miller, 1972). Such changes had earlier been inferred from the red/far-red reversible adhesion of root tips to negatively charged glass surfaces (Tanada, 1968). Both responses are detectable within 30 seconds of the start of irradiation (Fig. 14.11). These changes in surface charge are interpreted to indicate changes in the plasmalemma. Fluorescent probe studies support this notion (Racusen, 1973). Red light also induces H+ efflux from root tips (Yunghans & Jaffe, 1972).
Phytochrome regulates leaflet movement in Mimosa, Albizia and Samanea (Satter & Galston, 1975). This movement is accompanied by an energy-dependent transfer of K+ ions between the ventral and dorsal motor cells of the pulvinus. Both effects are detectable 10 minutes after red or far-red irradiations. The changes in K+ are correlated with changes in transmembrane potential (Racusen & Satter, 1974). Furthermore, red/far-red regulated changes in surface charge similar to those of root tips are detectable 30 to 120 seconds after irradiation. These effects are also strongly indicative of phytochrome-mediated changes in plasmamembrane properties.
Phytochrome-regulated changes in the rate of plasmolysis have been detected in Mougeotia within 6 minutes of the onset of red irradiations (Wiesenseel & Smeibidl, 1973). A change in plastid orientation is also evident in this alga in less than 10 minutes after a red pulse (Haupt, 1972a). The latter effect can only be fully reversed by far-red during the first minute after red light. Thus potentiation of the response has begun within 1 minute of photoconversion. Available data indicate that the effective phytochrome is located on or near the plasmalemma. Contractile fibrils appear to be responsible for the chloroplast movement (Schönbohm, 1973).
Red light (15 seconds) induces an increase in growth rate in coleoptile tips within 60 seconds of the start of irradiation (Weintraub & Lawson, 1972). The effect is partially reversed if far-red immediately follows the red. Inhibitors of transcription and translation are without effect. As the cell must regulate wall extension through the plasmalemma, this response might also represent a
Figure 14.11
Kinetics of (A) root tip adhesion to a negatively charged glass
surface and (B) the development of a biolectric potential across
the root tip in response to irradiation with red or far-red light
(after Jaffe, 1968).
phytochrome-mediated membrane change. Rapid changes in ATP levels, induced by red and, in some cases, partially reversed by far-red, have been reported but no consistent pattern is obvious (Sandmeier & Ivart, 1972; Yunghans & Jaffe, 1972; White & Pike, 1974).
Rapid transitory increases in hormore levels in response to red light have been known for some time (Reid et al., 1968; Beevers et al., 1970; van Staden & Wareing, 1972). Unequivocal evidence of far-red reversibility has generally been lacking in the past, however, often because of poor experimental design. Recently, the level of gibberellin activity extractable from etioplast-rich preparations from grass leaves has been shown to increase 3-fold within 5 minutes of the termination of a 5 minute red irradiation of the isolated fraction (Evans, 1975; Evans & Smith, 1976a). This effect is reversed by far-red light given immediately after the red. It is postulated that phytochrome causes the movement of gibberellin across the etioplast envelope into the ambient medium.
These observations, and those made with mitochondria-rich fractions (Manabe & Furuya, 1974), are rapid, of potential physiological significance, and suggestive of in vitro phytochrome-mediated changes in the functional properties of membranes in the isolated fractions.
Lipoxygenase accumulation in Sinapis responds to changes in Pfr level in less than 5 minutes under appropriate conditions (Oelze-Karrow & Mohr, 1973). It has been concluded that the level of the enzyme in the cotyledons is controlled by phytochrome in the hypocotyl hook, through a highly cooperative threshold mechanism that responds to, and is saturated by, Pfr levels of 1–2% of the total pigment (Oelze-Karrow & Mohr, 1974). Other rapid, inter-organ transfer of phytochrome signals have also been reported (de Greef & Caubergs, 1973). Some form of biophysical transmission system has been postulated, with the membrane continuum of the plasmodesmata as a suggested candidate (Oelze-Karrow & Mohr, 1973).
14.4.2.2—
Rapid Action/Delayed Expression Responses
Rapid phytochrome action in these cases is deduced from the rate at which the response escapes susceptibility to photoreversal by far-red light following an inductive red pulse. The actual response may not be expressed for days after the irradiation although the inevitability of its appearance has long since been irreversibly established. Pfr is said to have 'potentiated' the response (Borthwick, 1972). The escape from reversibility is viewed as the Pfr -triggered reaction chain having progressed beyond those steps directly under phytochrome confrol.
The effect of a red light pulse on flowering in Pharbitis is only partially reversed by far-red given 30 seconds after the start of the red irradiation (Fredericq, 1964). After 3 minutes, far-red no longer reverses the effect. Thus although the flowering response itself is not expressed for several days, it is potentiated within seconds by Pfr. Flowering in Chenopodium album and Kalanchoe behaves similarly. A synergism between phytochrome and gibberellin in lettuce seed germination (Bewley et al., 1968) and the de-etiolation response of Pisum (Haupt, 1972a) are also in this category.
14.4.2.3—
Delayed Action/Delayed Expression Responses
These responses show a lag from irradiation to expression but are readily reversed by far-red over relatively long periods in the dark after the red pulse. Gradual escape from reversibility can occur but is relatively slow. This is viewed as indicating that the continued presence of Pfr is required over a relatively long period in the dark to maximise expression. An important feature of these responses is that dark reversion and destruction are continually and often rapidly depleting the Pfr pool during this period.
The vast majority of recorded red/far-red reversible responses, too numerous to catalogue, are included in this category (see Mitrakos & Shropshire, 1972;
Mohr, 1972; Smith, 1975). Lettuce seed irradiated for I minute with red light germinates up to 100% 24 hours later. Far-red reverses this effect up to 12 hours after the red irradiation but with decreasing effectiveness. Anthocyanin formation in Sinapis has a lag of 3 hours from irradiation to the onset of accumulation (Fig. 14.6). During this time the effect becomes decreasingly susceptible to reversal by far-red but escape is never complete. The in vitro protein synthetic capacity of 80s ribosomes from corn is enhanced by 5 minutes of red light prior to harvest (Travis et al., 1974). This effect is detectable within 30 minutes of red irradiation and escapes far-red reversibility within an hour.
14.4.3—
Response Manifestations
Phytochrome has a regulatory role in all major phases of plant growth and development. The changes in cellular biochemistry and physiology which underlie this regulation are detectable at almost any chosen level. Some responses are biophysical in nature (membrane potential), others biochemical (enzyme levels); some require protein synthesis (leaf expansion), others do not (leaf movements); some involve cell division (seed germination), others only cell expansion (plumular hook opening); some are restricted to the irradiated cells (leaf movements), others result from transmissable stimuli (floral induction); some are dependent on phytochrome-endogenous rhythm interactions (flowering), others appear independent (seed germination) (Galston, 1974; Satter & Galston, 1975).
When the nature of the responses is coupled with their kinetic properties a pattern emerges. In general, the most rapid responses are surface or membrane-associated phenomena, often physico-chemical in nature and independent of RNA and protein synthesis. Other cellular processes such as changes in enzyme levels mostly respond more slowly.