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The active form of phytochrome: A new hypothesis based on phytochrome pelletability studies
I. theor. Biol. (1977) 69, 581-595
The Active Form of Phytochrome: A New Hypothesis Based on Phytochrome Pelletability Studies RICHARDYU Department of Developmental Biology, Research School of Biological Sciences, The Axstralian National University, Canberra. A.C.T. 2601, Australia (Received 8 March
1977)
Difficulties arising from the current dogma that the far-red absorbing form of phytochrome (Pr,) is the only active form are discussed. A new hypothesis is proposed in which phytochrome is held to be the photoreceptor for both lowenergy(pulse) and high energy(HIR) responses. There is a common basic mechanism of action involving interaction between phytochrome and a binding site within the cell. The phytochrome involvement in low energy responses exhibits an action spectrum for binding that matches the P, absorption spectrum and reversibility by far-red irradiation. Upon prolonged irradiation the phytochrome-binding site interaction acquires different characteristics that are reminiscent of those displayed in HIR, e.g. dependence on sustained irradiation for continual binding, dependence of the degree of binding on irradiance and the similarity of the action spectrum with that of HIR action spectra, e.g. that for inhibition of lettuce hypocotyl lengthening. As expected on the basis of the new hypothesis the particulate fraction of phytochrome contains both P, and P,,. Arguments are advanced that the presence of P, in pellets of particulate phytochrome cannot be accounted for by (i) the “induced fit” hypothesis, (ii) the “pigment cycling” hypothesis, and (iii) the “open phytochrome-receptor model”. We conclude that phytochrome molecules, after being sufficiently energized can interact with their intracellular binding sites irrespective of their chromophoric configuration. 1. Introduction Phytochrome, a protein with a linear tetrapyrrole chromophore as its prosthetic group, exists in two forms: P, and Pfl. P, absorbs maximally in the red (R) region [660 nm] of the spectrum and Pf, in the far-red (FR) [730 nm] (Fig. 1). These two forms are interconvertible upon absorption of light energy. Although the exact molecular mechanism of photo-transformation is 581
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0.6 Far-red
300
I
400
500 Wavelength
600
rradloted
I
I
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FIG. 1. Absorption spectra of large rye phytochrome after saturating exposure to red and far-red light (taken from Rice, Briggs, & Jackson-White, 1973). P, and Pfr forms are the far-red and red irradiated phytochrome respectively.
not known, it is generally believed to involve photo-chemical absorption of light energy by the chromophore and a subsequent protein configurational change (Briggs & Rice, 1972). Due to the substantial overlap of their absorption spectra, a photo-equilibrium of these two forms will be established by irradiation with monochromatic light. The proportion of the total cellular phytochrome which will exist as Pfr at equilibrium has been calculated for different wavelengths (Pratt & Briggs, 1966; Hanke, Hartmann & Mohr, 1969) (Fig. 2).
Wavelength
(nm)
FIG. 2. Photostationary equilibrium (P,,/P,,,,,) established by different wavelengths of light in mustard hypocotyl hooks (taken from Hanke, Hartmann, & Mohr, 1969).
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It has been demonstrated beyond doubt that phytochrome is involved as the primary photo-receptor in photomorphogenesis of higher plants at various stages of their development and also in certain special cellular photoresponses (see Smith, 1975 for review). The range of biological displays mediated by phytochrome is vast and diverse; many hypotheses have been advanced to account for its initiator role in these diverse reactions. Those which have survived the passage of time have one feature in common, that is the postulation of an interaction of phytochrome with a receptor moleule, e.g. gene, or membrane, in the initiation of a response. By analogy with hormone action, phytochrome is often envisaged as acting as an intracellular “hormone” or “light sensor”. Depending on the duration of the irradiation, phytochrome-mediated responses have been classified as low energy (puIse) and high energy (prolonged irradiation) responses (HIR). This classification is arbitrary and demarcation is obviously difficult. Irradiation with light of about 1.5 W mm2 for a few minutes is generally considered low energy; the same irradiance used for hours, high energy. Because of the existence of in vivo dark reactions of phytochrome, prolonged irradiation of plant tissues generally results in features or characteristics of the response different from those of pulse irradiation (see Mohr, 1972; and this paper). As mentioned above, phytochrome exists as two interconvertible forms which have overlapping absorption characteristics and a mixture of the P, and Pfr forms will be established in plant tissues irradiated with monochromatic light. In considering how phytochrome functions inside the cell, it is imperative to determine whether both forms are capable of initiating biological responses or which of the two, P, or Pr,, is thus active. Current theories concerning the mechanism of action of phytochrome implicitly or explicitly assume that Pjr is the only physiological active molecule (Borthwick, 1972; Satter & Galston, 1976). This assumption has neither been unequivocally substantiated nor seriously challenged (Borthwick, 1972; Satter & Galston, 1976). It has been perpetuated nevertheless because in pulse irradiations red light is invariably used and in terms of inductive capability is the most effective. In the case of low energy phytochrome reactions, the assumption receives qualitative verification from the fact that brief irradiation with red light induces both the photo-transformation of P, -+ P,, and a biological response, and that the response can be prevented by a subsequent, immediate far-red irradiation which transforms P,-, back to P, (see Smith, 1975, for a general review). However, attempts to correlate quantitatively the extent of a biological response with the in viva content of spectrophotometrically detectable Pf, have been, in general, unsuccessful (Hillman, 1967), although it has been claimed recently that inter-organ
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control of lipoxygenase activity in the cotyledon of mustard seedlings can be precisely correlated with the physiologically-determined or theoreticallyprojected concentration of Pfl in a distant tissue of the seedling, i.e. hypocotyl hook region (Oelze-Karow & Mohr, 1973). The situation in which biological and spectrophotometric data do not agree have been called paradoxes. Typical examples are the Pisum and Zea paradoxes. In the Pisum paradox etiolated pea stem segments respond to different mixtures of R and FR (which were used to establish different percentages of Pf, at photoequilibrium) as though 20 % of the total cellular phytochrome is Pf,, although in reality spectrophotometric measurements indicate that all the phytochrome is P, (Hillman, 1965). In the Zea paradox, the alteration of sensitivity towards blue light-induced phototropism is saturated by a spectrophotometrically undetectable amount of Pfr and that alteration of sensitivity is reversed by FR which subsequently increases Psr to a detectable level (Briggs & Chon, 1966). The concepts of “bulk” (spectrophotometrically detectable but physiologically inactive) and “active” phytochrome have been proposed to rationalize the paradoxes. However, there is at present no experimental evidence to support these concepts. The paradoxical absence of detectable Pfr in tissues which nevertheless respond as though phytochrome did exist in that form suggests that it may be incorrect to say that only Prr is physiologically active. In the case of high irradiance or prolonged irradiation responses (HIR) the involvement of phytochrome has been difficult to demonstrate due to the lack of congruity between “HIR action spectra” and the phytochrome absorption spectrum (compare spectrum in Fig. 3 with spectra in Fig. 1).
I.6
Wavelength
(nm)
FIG. 3. An “action spectrum” for the high-energy response (HIR) of inhibition hypocotyl lengthening (taken from Hartmann, 1967).
of lettuce
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However, it has been plausibly argued that phytochrome is responsible, at least for the effect in the far-red region (Mohr, 1972); but difficulty is still experienced in explaining the characteristic features of the HIR: (i) Irradiance (intensity) dependence, if it is to be maintained that only Prl is responsible for the response once photo-equilibrium is established, and : (ii) The necessity for continual irradiation in order to achieve a complete response (Schopfer & Oelze-Karow, 1971; Schopfer & Mohr, 1972). The “pigment cycling hypothesis” of Hartmann & Unser (1973) which attributes the high irradiance responses to the frequency of Pf, ti P, interconversion could, in principle, account for the two features of HIR; thus higher irradiance would cause a faster, i.e. more molecules per unit time, interconversion of P, and P,, than a lower irradiance and would, therefore, generate more so-called ‘nascent PJr" at any instance of time; but there is no evidence at all to support the supposition that “nascent P,-:' is more active than “old Pf,". In fact, it appears almost impossible to test it experimentally. The hypotheses so far proposed for HIR provide no rational explanation for the occurrence of the peak in the blue in the HIR “action spectrum” (Hartmann, 1967). Indeed, some authors believe another pigment to be responsible and classify the high energy blue light responses as a separate phenomenon (see Smith, 1975, for a general review). This belief disregards the fact that both P, and Ps, forms of phytochrome absorb in the blue region of the spectrum and observations that blue light can mimic the effects of red irradiation (in, e.g. the germination of tomato seeds, Yaniv & Mancinelli, 1968; unfolding of barley leaf segments, Deutch & Deutch, 1975). Furthermore, far-red reversibility of blue light effects has been found in some systems in which the blue acts like red, such as in the photoinhibition of Pisumstem segments (Bertsch, 1963) and in the control of dormancy in Lunuluriu (Schwabe & Valio, 1970). In both cases the blue light effect is held to involve phytochrome. It has gradually become apparent that HIR is not an uncommon, isolated phenomenon; in fact, it is as widespread as the better-known low energy (pulse) reactions (Smith, 1975). A conceptual difficulty has therefore arisen in which it is customary to envisage that phytochrome sometimes mediates the same response via two separate modes of action in the same tissue (probably also in the same cell) as well as two different photoceptors mediating the same HER in the same tissue. From the foregoing discussion it appears that while a majority of phytochrome-induced phenomena is satisfactorily explicable in terms of Pf,, current dogma, which holds that Prr molecules are responsible for aZZphytochromemediated responses, is insufficient or too restrictive. Based on results of studies of phytochrome pelletability or binding to an as yet unknown intra-
cellular binding site we now propose a new model of phytochrome action which could account for both low and high irradiance responses in terms of a common mechanism of action. 2. The Hypothesis Essentially our new hypothesis can be described as follows. Radiation of any wavelength which is absorbed by phytochrome in the P, form is effective in causing it to become physiologically active. Absorption of radiant energy by the phytochrome molecule (P,) can result in activation (PI*) and/or activation plus phototransformation (P,,) depending on the quantum energy of the photon absorbed. P,* and/or Pf, can interact equivalently with the binding site or reaction partner ultimately to induce the biological display. The extent of the display or response is dependent only on the concentration of activated phytochrome molecules (P,* and/or P/J which in turn depends, within certain limits, on the amount of energy absorbed. It is obvious that for the same degree of activation, light of wavelengths less efficiently absorbed by P, than red light, e.g. far-red and blue, will require a longer duration of irradiation. (A)
SUPPORTING
EVIDENCE
Data supporting this new hypothesis were obtained from experiments on photo-induced phytochrome pelletability (or binding) in excised maize coleoptiles.? This response is unusual in that phytochrome is a component of the photo-induced product. Essentially the experimental system involves irradiation of the excised plant tissues with light and then determination of the amount of cellular phytochrome which has become bound to a particulate fraction. This determination can be made after the tissue is homogenized in a suitable extraction medium. From the dose response curves of phytochrome pelletability for a large number of wavelengths in the visible region we have constructed an action spectrum. At low energies this action spectrum matches the absorption spectrum of P, (Fuad & Yu, 1977u). It follows that phytochrome is the photo-receptor for light-induced phytochrome pelletability or binding. Because phytochrome is present in the product of the photoreaction and, immediately following irradiation, the tissue is kept on ice and the biochemical steps required to demonstrate phytochrome binding are all carried out at O”C, these facts support the view that phytochrome pelletability or binding tThe current controversy over the artefactual or non-artefactual pelletability is examined in an Appendix.
nature of phytochrome
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is an early, if not the first, consequence of irradiation (Yu, Faud & Carr, 1976; Fuad & Yu, 1977~). For low energy reactions, a classical criterion for the involvement of phytochrome in any physiological response to light is that the effect of red light shall be reversible by immediate far-red. Red-induced phytochrome binding can be demonstrated to be reversible, i.e. the “debinding” reaction occurs in uiuo during dark incubation following R/FR irradiation at 0°C but not following R irradiation (Fuad & Yu, 1977~). These facts argue for the conclusion that phytochrome binding fulfils classical criteria for phytochrome involvement in a low energy physiological response (Mohr, 1972). It is apparent from the dose-response curves used to construct the action spectrum that light of any wavelength can be effective in causing phytochrome to bind to a level similar to that induced by red light, provided a sufficient dose is given (Fuad & Yu, 1977~). This implies that there is unlikely to be any relationship between a photo-response and a photo-stationary concentration of P,,; the photo-stationary Pjr concentration is therefore irreIevant and light dose is the only relevant determinant in a given response (see following discussion). With irradiance of 1.5 W m-‘, maximal binding may require l-2 min irradiation with red, but 05-2 h with blue or far red light simply because it is less efficiently absorbed (Fuad & Yu, 19773). Under conditions of prolonged irradiation (in terms of hours) where far-red and blue can cause high levels of binding, red is comparatively less effective (for reasons, see below). The spectral dependence of binding with 4 h irradiation at a chosen light intensity (Fuad 8z Yu, 1977a) shows a remarkable resemblance to the HIR spectra of inhibition of lettuce hypocotyl lengthening (Hartmann, 1967) and anthocyanin synthesis in some plants (Smith, 1975). Implicitly this result therefore indicates that biological responses effected by prolonged irradiation (or high irradiance) and pulse irradiation (or low irradiance responses) both share the same mechanism of action, i.e. the binding of phytochrome and it further reinforces the conclusion that binding is itself a phytochrome-mediated response. The lower binding resulting from prolonged red irradiation is coincident with a more pronounced decrease of total cellular phytochrome (Fuad & Yu, 1977~2). In interpreting the observed spectral dependence of phytochrome binding induced by prolonged irradiation, we believe a modification of Hartmann’s explanation (1966) of two competing or interacting reactions, i.e. phytochrome binding and destruction of P,,, to be consistent with our data (Fig. 4) (Fuad & Yu, 1977~2).P,-r is the form held to be uniquely susceptible to destruction. Hartmann’s explanation was based on the supposition that maximum action will occur under those conditions that allow Pf,, even at very low amounts, to be present for a very long time, by balancing
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Dark
Red
PfrX
>
l
Pr Far-red Dark
\ DestructIon
FIG. 4. The “interaction hypothesis”of Hartmannfor explainingthe actionof phytochromein the high irradianceresponse (HIR) (Hartmann,1966).
the need to have a sufficiently high level of P,, to maintain action, and the opposing need to keep Pfr as low as possible to reduce destruction to a minimum. The far-red and blue “action” peaks and the troughs in the red and green regions of the HIR “action spectrum” then follow essentially from the interaction of two wavelength-dependent effects, efficiency of light absorption and susceptibility to destruction. Phytochrome pelletability induced by prolonged irradiation with far-red (or blue) shows classical features of HIR, such as dependence on irradiance and on continued irradiation. Phytochrome pelletability increases while the light is on but it decreases progessively due to release of phytochrome from the particles following the termination of irradiation (Yu et al., 1976). The extent of phytochrome binding depends on the irradiance for a chosen duration of irradiation. Hartmann’s adherence to the dogma that Prr is the only active molecule brings about a failure of his interaction hypothesis to explain the irradiance dependence characteristic of HIR. Our view, however, differs from Hartmann’s in holding that, after absorbing sufficient energy, P, can interact with the reaction partner X (using Hartmann’s terminology) and the irradiance dependence is explained simply as that, for a chosen duration of irradiation, different irradiances represent different doses delivered. It is our hypothesis that both P, and Pfr are able to bind because P, and P,, are always present in bound phytochrome (particulate phytochrome). We must now deal with the assumption generally made that Pfr is the only form of phytochrome that initially interacts with the putative receptor (binding
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site). Two hypotheses have been proposed to account for phytochrome binding, namely: (1) PJI is initially the molecule which binds. The site becomes modified so that it comes to have an affinity for P,. This is the “induced fit” hypothesis (Quail, MarmC & Schafer, 1973). This hypothesis relies on the results of experiments said to show an increase in phytochrome pelletability (over that obtained by R-irradiation alone) by FR irradiation following red irradiation (Quail et al., 1973). These results have now (Fuad & Yu, 1977c) been shown to be spurious and the product of a systematic error in method. The error arises from the release of phytochrome from the particulate fraction in uitra when the breis of iradiated tissues were left standing in ice at 0°C. If the breis of red irradiated tissues is allowed to stand in ice longer than that of R/FR irradiated, phytochrome pelletability will appear higher in R/FR than R irradiated tissues, i.e. the result of Quail et al. (1973). The hypothesis of Pz,-induced enhancement of affinity for P, is without experimental support at the present time. (2) P,.r is both initially and subsequently the only molecule which binds. Increase in pelletability is said to represent presumably accumulation of PJ,receptor complex during the period of irradiation, i.e. somehow summation occurs because the plant tissue can “count” the number of Pfr molecules generated over the period of irradiation. This is the “cycling hypothesis” (Quail & Schafer, 1974). Since the time of interconversion, i.e. cycling, (a unimolecular photochemical event) must be much shorter than the time for binding (a bimolecular thermal reaction) (Pratt & Marme, 1976) and very much shorter than the time for de-binding, a necessary corollary of this hypothesis is that accumulation of pelletable phytochrome should be time-dependant only. On the cycling hypothesis, therefore, two different light intensities, which establish the same photo-stationary state of P,.,, given for the same duration should induce the same level of binding. Alternatively, for the same dose at two different light intensities (1, and I,, where Z, > II), both of which can establish the same photostationary state of Pfr, the lower intensity irradiation, I,, should effect more binding than the higher intensity, Ii, because its duration of irradiation is necessarily longer. That this is not the case is demonstrated clearly by the data of Quail & Schafer (1974) (in which the law of reciprocity holds true), though they prefer an interpretation, i.e. the cycling hypothesis, contrary to their own data. Clearly, the data of Quail & Schafer indicate that the binding reaction is dose-dependent.In the pigment cycling concept of Hartmann & Unser (1973) it is argued that higher intensities will cause a faster rate (amount/time) of interconversion of P, and P,,, hence more phytochrome molecules will go through the P,, state and be “counted”.
But, the alleged faster rate at higher intensity lacks experimental verification. In addition, the implied mechanistic basis for “counting” has neither been defined nor experimentally demonstrated. Also, since the maximal binding and the dose required for a fixed response (say 20 % binding) are not directly related to the steady-state levels of Prr (Fuad 8z Yu, 1977u) that Pfr is the only form which initially interacts with the putative receptor is not supported. The inescapable conclusion is, then, that activated phytochrome in either of the spectrophotometric forms, P, or Prr can bind. In support of this conclusion, we have shown that following a submaximal binding using a non-saturating dose of red light, not all the Pf, molecules generated actually bind but a substantial proportion remains unbound, and the phytochrome bound cannot be adequately accounted for by Pfr molecules alone (Yu et al., 1976). However, it can be argued that even at subsaturating doses of red light, cycling of phytochrome might take place. When phytochrome binding was studied by irradiating the plant tissue with short pulses of monochromatic red light (c 1 ms) (a light pulse much shorter than the dark reaction between the excited state of P,, produced by the photon absorption, and eventual P,, formation) a plot of the percent pelletable Pfr as a function of total PJr in the extract of maize coleoptiles showed clearly that P,-r is not the only molecule which binds, especially at low levels of total Pf, (Schiifer, 197%). This conclusion is further supported by the recent kinetic data of Pratt & Marmt. They found that “at low Pfr levels, for each Prr molecule produced by red light, one additional molecule of phytochrome (although some are P,) is found in the pellett”, and “Because binding follows the irradiation period, the trivial explanation that bound P, was photochemically derived from previously bound PfF may be discarded” (Pratt & MarmC, 1976). Recently SchZfer (1975b) has presented an elegant but complicated model of phytochrome (PfJ action which is a variant of that proposed by Quail & SchXer (1974) and which is designed to explain both the pulse and the high irradiance responses (Fig. 5). His “open phytochrome receptor model”, however, is based on data which have since been found to be erroneous. Essential features of his model are therefore unfounded, for example, that P,r. X + Pfr. x’ transition is a pre-requisite for Pf, destruction (Boisard, Marme & Briggs, 1974; Schgfer, Lassig & Schopfer, 1973, and that Pfr binding to Xinduces a change of X(to X’), i.e. “induced fit”, is also incorrect (see above); moreover, Schafer’s designation of elements Pfr. X and Pf,. X’ in his “open” model as “catalytic” and “inductive” sites of action respectively (Schafer, 1975b; Kasemir, Huber & Mohr, 1976) is not empirical but purely arbitrary. Hence, Schafer’s “open” model lacks experimental support for some of its essential postulates.
ACTIVE
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“k, ---------SW
pI
PI x
OF ----3 +..----
-----+
PHYTOCHROME k,
k,
p
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Ir
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FIG. 5. The ‘open phytochrome-receptor model” of phytochrome action proposed Schafer (19756).
by
3. Conclusion Since phytochrome binding is a primary physiological reaction (see Appendix for a discussion of the data on which this conclusion is based), it follows that Pf, is not the only physiologically active molecule and photo-transformation of P, to Pf, is not a prerequisite for the phytochrome molecule to become activated for binding. If this is so, then any attempt to explain a physiological response in terms of the percentage of Pfr or amount of P,, will be unrealistic and irrational. That this is indeed the case is clearly demonstrated by the numerous reports of unsuccessful attempts in the literature. In a recent critical review on this aspect, Hillman stated that “in any phytochrome system examined with rigor, no rational, analytically useful relationship (between Pfr and response) has yet been established” and “it is less surprising to find paradoxes than the occasional appearance of rationality” (Hillman, 1972). A suggestion has been made based on the “phytochrome paradoxes” that only a small proportion of the cellular phytochrome is actually employed for physiological responses (the “active fraction”). (Hillman, 1967; Briggs & Chon, 1966). If one accepts phytochrome binding as an authentic primary physiological response of phytochrome, this suggestion cannot be maintained because over 60% of the total cellular phytochrome can become bound by irradiation. I am most grateful to Professor D. J. Carr for his critical reading of the manuscript and stimulating discussion in crystallizing ideas presented in this paper. Also, I thank Mrs. Noha Pickles-Fuad for her collaboration.
REFERENCES BERTSCH, W. F. (1963). Am. J. But. 50, 754. BOLSARD, J., MARM& D., & BRIGGS, W. R. (1974). PI. Physiol. 54, 272. B~RTHWICK, H. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), p. 16. London, New York: Academic Press. BRIGGS, W. R., & CHON, H. P. (1966). PI. Physiol. 41, 1159. BRIGOS, W. R., & RICE, H. V. (1972). A. Rev. PI. Physiol. 23, 293. DEUTCH, B., & DEUTCH, B. I. (1975). Physiologia PI. 35, 322. FUAD, N., 8c Yu, R. (1977a). Photo&em. Photobiol. 25, 491. FUAD, N., & Yu, R. (19776). 2. PpanZenphysioI. 81, 304. FUAD, N., & Yu, R. (1977~). PI. Cell Physiol. 18, 35. HANKE, J., HARTMANN, K. M., & MOHR, H. (1969). Pfanta 86, 235. HARTMANN, K. M. (1966). Photochem. photobiol. 5, 349. HARTMANN, K. M. (1967). Z. Naturfirsch. 22b, 1172. HARTMANN, K. B., & UNSER, I. C. (1973). Z. Pflanzenphysiol. 69, 109. HAUPT, W. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), pp. 553-569. London, New York: Academic Press. HILLMAN, W. S. (1965). Physiologia PI. 18, 346. HILLMAN, W. S. (1967). A. Rev. PI. Physiol. 18, 301. HILLMAN, W. S. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), pp. 573584. London, New York: Academic Press. KA~EMIR, H., HUBER, P., & MOI-IR, H. (1976). Planta, 132, 157. MARMB, D., BOISARD, J. & BRIGGS, W. R. (1973). Proc. natn. Acad. Sci., U.S.A. 70, 3861. MOHR, H. (1972). In Lectares on Photomorphogenesis, pp. 3747. New York: SpringerVerlag, Berlin : Heidelberg. OELZE-KAROW, H., & MOHR, H. (1973). Photochem. photobiol. 18, 319. PUTT, L. H., & BRIGGS, W. R. (1966). PI. Physiol. 41, 467. PRAY, L. H., & COLEMAN, R. A. (1971). Proc. natn. Acad. Sci., U.S.A., 68,2431-2425. PRATT, L. H., & MARA, D. (1976). PI. Physiol. 58, 686. QUAIL, P. H., MAR&, D., & SCH&R, E. (1973). Nature Land. 245, 189. QUAIL, P. H. (1975). Photochem. Photobiol. 22,299. QUAIL, P. H. & GRESSEL,J. (1975). In Light and Plant Deveiopment (H. Smith, ed.), p. 91. 22nd Easter School, University of Nottingham. QUAIL, P. H., and SCHKPER, E. (1974). J. membr. Biol. 15, 393. RICE, H. V., & BRIGGS, W. R. (1973). PI. Physiol. 51, 939. RICE, H. V., BRIGGS, W. R., & JACKSON-WHITE, C. J. (1973). PI. Physiol. 51, 917. RUBINSTEIN, B., DRURY, K. S., & PARK, R. B. (1969). PI. Physiol. 44, 105. Roux, S. J., & HILLMAN, W. S. (1969). Archs biochem. Biophys. 131, 423. SA~R, R. L., & GALSTON, A. W. (1976). In Chemistry and Biochemistry of Plant Pigments (‘I. W. Goodwin, ed.), Vol. 1, p. 685. London, New York: Academic Press. SCHXFER, E. (1975a). Photochem Photobiol. 21, 189. S&&R, E. (19756). J. math. Biol. 2, 41. SAFER, E., LASSIG, T.-U., & SCHOPFER, P. (1975). Photochem. Photobiol. 22, 193. SCHOPFER,P., & OELZE-KAROW, H. (1971). Planta 100, 167. SCHOPFER, P., & MOHR, H. (1972). PI. Physiol. 49, 8. SCHWABE, W. W., & VALIO, J. F. M. (1970). J. expl. Bot. 21, 122. SMITH, H. (1975). In Phytochrome and Photomurphogenesis. London: McGraw-Hill. YANIV, Z., & MANCINELLI, A. L. (1968). PI. Physiol. 43, 117. Yu, R. (1975~). Aust. J. PI. Physiol. 2, 273. Yu, R. (19756). Asst. J. PI. Physiol. 2. 281. Yu, R. (1975c). J. expl But. 25, 808. Yu, R., & CARTER, J. (1976~). J. expl. But. 27, 283. Yu, R., & CARTER, J. (19766). PI. Cell Physioi. 17, 1309. Yu, R., FUAD, N., & CARR, D. J. (1976). PI. Cell Physiol. 17, 1131.
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APPENDIX Although there is good evidence that the phenomenon of photo-induced phytochrome pelletability is a normal physiological reaction, itself an early member of the sequence of phytochrome-mediated reactions, doubts have repeatedly been raised by others concerning its reality as an intracellular process and suggestions have been made that it is an artefact because the extent of the photo-response, i.e. the degree of phytochrome pelletability or binding, can only be ascertained following tissue homogenization and biochemical fractionation. To remove these doubts let us therefore examine relevant aspects of the binding reaction in some detail since it gives us the best experimental support at present for the new hypothesis. From many physiological experiments, phytochrome is believed to interact with or be an integral part of certain cellular membranes. In unirradiated etiolated tissues, a small amount (c. 5 %) of phytochrome was found associated with a particulate (presumably the mitochondrial, Rubinstein, Drury & Park, 1969) fraction of an etiolated oat seedling. When excised dark-grown plant tissues, e.g. maize coleoptiles, are irradiated with red light, most of the phytochrome becomes pelletable upon homogenization of the plant tissues in the presence of Mg’+ whereas very little (< 10%) is pelletable either in the absence of Mg2+ or from unirradiated tissues (see Quail, 1975 for a review). An immediate question arises, in view of the nature of the biochemical methods involved in demonstrating pelletability of phytochrome. Is it an artefact? Haupt (1972) has suggested that pelletability might be due to preferential adsorption or desorption of P, or Pfr during tissue homogenization in an artificial solution environment. Indeed, Quail has suggested, from his studies on phytochrome pelletability in zucchini hypocotyl hooks, that it is an artefact resulting from an association of phytochrome (P/J with degraded ribosomal material (RNP). However, he does not explain why the artefact should not arise in non-irradiated material. We have sought to refute these criticisms by treating irradiated maize coleoptiles with bifunctional crosslinking reagents, e.g. dimethyl suberimidate, glutaraldehyde, in an attempt to immobilize phytochrome at its intracellular location by covalent linkage so as render subsequent homogenization and fractionation independent of the solution environment. In maize, following glutaraldehyde or DMS pretreatments, enhanced phytochrome pelletability is obtained following an initial red irradiation (R and R/FR) but not following FR irradiation or from unirradiated material and this is virtually independent of the solution environment during tissue homogenization (Yu, 1975a,b; Yu & Carter, 1976~). Such pre-treatments also resulted in yields of pelletable phytochrome similar
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R.
YU
to those obtained by extraction with Mg’+-containing buffer but without the crosslinking reaction (Yu & Carter, 1967b). Phytochrome in pellets obtained using bifunctional reagents showed a perfectly reversible phototransformation difference spectrum which was completely superimposable on that of phytochrome from untreated controls (Yu 8z Carter, 1967a,b). While this does not imply a total absence of degradation of phytochrome by these treatments it is evident that we are not dealing with a totally unspecific product. When coleoptiles were irradiated with R/FR and incubated in the dark before homogenization in Mg2+-containing buffer, there was a progressive loss of pelletable phytochrome but no loss of total cellular phytochrome. Crosslinking pre-treatments before homogenization in Mg”-free buffer gave yields faithfully reproducing the progressive loss of particulate phytochrome and preserving the total cellular content of phytochrome (Yu, 197%; Yu & Carter, 1976a, b). Here again we have evidence of preservation of an in uiuo state which reinforces the view that the biochemical techniques essential for the study of pelletability are not merely producing an artefact. Quail has questioned the validity of the pre-treatment approach on the ground that glutaraldehyde drastically changes phytochrome antigenic activity and causes random aggregation (Quail, 1975). This argument can be refuted for the following reasons: (1) In a thorough investigation, Rice & Briggs recently found no effect of glutaraldehyde treatment on the antigenic activity of phytochrome in P, and Pfr forms (Rice & Briggs, 1973). (2) Although Pratt and Coleman did observe a reduction of antigenic activity in glutaraldehyde-fixed tissues (cited as unpublished data in Pratt & Coleman, 1971), glutaraldehyde fixation was used subsequently in immunological studies of the localization of phytochrome (Briggs & Rice, 1972). (3) In in oitro as well as in uiuo studies on the chemical reactivity of purified and partially purified phytochrome treated with glutaraldehyde, Roux & Hillman (1969) found that the P, form of phytochrome is generally more reactive than the Pfr form. It could therefore be argued that if glutaraldehyde-enhanced pelletability were the result of a preferential reactivity of phytochrome, then dark-grown and FR-irradiated coleoptiles should be expected to yield larger amounts than red-irradiated ones. Experimentally this has been shown not to be the case (Yu, 197%). It is possible that in Quail’s experiments (which were not reported in detail) artefacts may have arisen from virtual fixation of the whole cell (Quail, 1975; Quail & Gressel, 1975). It is important to differentiate
ACTIVE
FORM
OF
PHYTOCHROME
595
between results obtained when glutaraldehyde is used as a fixative (prolonged treatment, e.g. several hours at room temperature) or as a crosslinking bifunctional reagent (20-30 min at O’C). Moreover, in our hands glutaraldehyde pretreatment affected little, if any, of the sedimentation properties of organellar membranes and their constituent enzymic properties (Yu, 1975~). Furthermore, the results we obtained with glutaraldehyde have been duplicated using milder, more selective, and defined bifunctional crosslinking reagents, e.g. DMS, (Yu & Carter, 1976~~). In our own unpublished work, glutaraldehyde pretreatment of squash hooks obviated Quail’s artefact of association of phytochrome with a RNP fraction which has been the mainspring of fears of the artefactual nature of phytochrome pelletability. Quail & Gressel (1975) have found that one of the two fractions of phytochrome pelleted from homogenates of irradiated zucchini hooks contains degraded ribosomal material (RNP). Since that fraction has been held by Quail et al. (1973) and others (MarmC, Boisard & Briggs, (1973) to consist essentially of a membrane fraction, the well-publicised result of Quail and Gressel has cast doubt on the reality of pelletability of phytochrome in other systems. However, no degraded ribosomal material is found in phytochromecontaining pellets obtained from maize (Yu, unpubl. result). Upon irradiation phytochrome evidently binds to some sites in the cell and by suitably gentle means a pellet can be obtained containing the phytochrome together with its putative binding site. This is a real phenomenon, exactly reproducible from experiment to experiment. Without irradiation no such binding takes place and the phytochrome is not pelleted. We believe that these arguments together with those given in the text favour our conclusion that phytochrome does interact intracellularly with a fraction which becomes particulate upon homogenization and that experimentally this interaction can be stabilized either intracellularly by crosslinking in a bifunctional reagent prior to disruption of cellular organization or by using Mg 2+ in the medium.
7.8.
38
The Active Form of Phytochrome: A New Hypothesis Based on Phytochrome Pelletability Studies RICHARDYU Department of Developmental Biology, Research School of Biological Sciences, The Axstralian National University, Canberra. A.C.T. 2601, Australia (Received 8 March
1977)
Difficulties arising from the current dogma that the far-red absorbing form of phytochrome (Pr,) is the only active form are discussed. A new hypothesis is proposed in which phytochrome is held to be the photoreceptor for both lowenergy(pulse) and high energy(HIR) responses. There is a common basic mechanism of action involving interaction between phytochrome and a binding site within the cell. The phytochrome involvement in low energy responses exhibits an action spectrum for binding that matches the P, absorption spectrum and reversibility by far-red irradiation. Upon prolonged irradiation the phytochrome-binding site interaction acquires different characteristics that are reminiscent of those displayed in HIR, e.g. dependence on sustained irradiation for continual binding, dependence of the degree of binding on irradiance and the similarity of the action spectrum with that of HIR action spectra, e.g. that for inhibition of lettuce hypocotyl lengthening. As expected on the basis of the new hypothesis the particulate fraction of phytochrome contains both P, and P,,. Arguments are advanced that the presence of P, in pellets of particulate phytochrome cannot be accounted for by (i) the “induced fit” hypothesis, (ii) the “pigment cycling” hypothesis, and (iii) the “open phytochrome-receptor model”. We conclude that phytochrome molecules, after being sufficiently energized can interact with their intracellular binding sites irrespective of their chromophoric configuration. 1. Introduction Phytochrome, a protein with a linear tetrapyrrole chromophore as its prosthetic group, exists in two forms: P, and Pfl. P, absorbs maximally in the red (R) region [660 nm] of the spectrum and Pf, in the far-red (FR) [730 nm] (Fig. 1). These two forms are interconvertible upon absorption of light energy. Although the exact molecular mechanism of photo-transformation is 581
582
R.
YU
0.6 Far-red
300
I
400
500 Wavelength
600
rradloted
I
I
700
800
(nml
FIG. 1. Absorption spectra of large rye phytochrome after saturating exposure to red and far-red light (taken from Rice, Briggs, & Jackson-White, 1973). P, and Pfr forms are the far-red and red irradiated phytochrome respectively.
not known, it is generally believed to involve photo-chemical absorption of light energy by the chromophore and a subsequent protein configurational change (Briggs & Rice, 1972). Due to the substantial overlap of their absorption spectra, a photo-equilibrium of these two forms will be established by irradiation with monochromatic light. The proportion of the total cellular phytochrome which will exist as Pfr at equilibrium has been calculated for different wavelengths (Pratt & Briggs, 1966; Hanke, Hartmann & Mohr, 1969) (Fig. 2).
Wavelength
(nm)
FIG. 2. Photostationary equilibrium (P,,/P,,,,,) established by different wavelengths of light in mustard hypocotyl hooks (taken from Hanke, Hartmann, & Mohr, 1969).
ACTIVE
FORM
OF
PHYTOCHROME
583
It has been demonstrated beyond doubt that phytochrome is involved as the primary photo-receptor in photomorphogenesis of higher plants at various stages of their development and also in certain special cellular photoresponses (see Smith, 1975 for review). The range of biological displays mediated by phytochrome is vast and diverse; many hypotheses have been advanced to account for its initiator role in these diverse reactions. Those which have survived the passage of time have one feature in common, that is the postulation of an interaction of phytochrome with a receptor moleule, e.g. gene, or membrane, in the initiation of a response. By analogy with hormone action, phytochrome is often envisaged as acting as an intracellular “hormone” or “light sensor”. Depending on the duration of the irradiation, phytochrome-mediated responses have been classified as low energy (puIse) and high energy (prolonged irradiation) responses (HIR). This classification is arbitrary and demarcation is obviously difficult. Irradiation with light of about 1.5 W mm2 for a few minutes is generally considered low energy; the same irradiance used for hours, high energy. Because of the existence of in vivo dark reactions of phytochrome, prolonged irradiation of plant tissues generally results in features or characteristics of the response different from those of pulse irradiation (see Mohr, 1972; and this paper). As mentioned above, phytochrome exists as two interconvertible forms which have overlapping absorption characteristics and a mixture of the P, and Pfr forms will be established in plant tissues irradiated with monochromatic light. In considering how phytochrome functions inside the cell, it is imperative to determine whether both forms are capable of initiating biological responses or which of the two, P, or Pr,, is thus active. Current theories concerning the mechanism of action of phytochrome implicitly or explicitly assume that Pjr is the only physiological active molecule (Borthwick, 1972; Satter & Galston, 1976). This assumption has neither been unequivocally substantiated nor seriously challenged (Borthwick, 1972; Satter & Galston, 1976). It has been perpetuated nevertheless because in pulse irradiations red light is invariably used and in terms of inductive capability is the most effective. In the case of low energy phytochrome reactions, the assumption receives qualitative verification from the fact that brief irradiation with red light induces both the photo-transformation of P, -+ P,, and a biological response, and that the response can be prevented by a subsequent, immediate far-red irradiation which transforms P,-, back to P, (see Smith, 1975, for a general review). However, attempts to correlate quantitatively the extent of a biological response with the in viva content of spectrophotometrically detectable Pf, have been, in general, unsuccessful (Hillman, 1967), although it has been claimed recently that inter-organ
584
R. YU
control of lipoxygenase activity in the cotyledon of mustard seedlings can be precisely correlated with the physiologically-determined or theoreticallyprojected concentration of Pfl in a distant tissue of the seedling, i.e. hypocotyl hook region (Oelze-Karow & Mohr, 1973). The situation in which biological and spectrophotometric data do not agree have been called paradoxes. Typical examples are the Pisum and Zea paradoxes. In the Pisum paradox etiolated pea stem segments respond to different mixtures of R and FR (which were used to establish different percentages of Pf, at photoequilibrium) as though 20 % of the total cellular phytochrome is Pf,, although in reality spectrophotometric measurements indicate that all the phytochrome is P, (Hillman, 1965). In the Zea paradox, the alteration of sensitivity towards blue light-induced phototropism is saturated by a spectrophotometrically undetectable amount of Pfr and that alteration of sensitivity is reversed by FR which subsequently increases Psr to a detectable level (Briggs & Chon, 1966). The concepts of “bulk” (spectrophotometrically detectable but physiologically inactive) and “active” phytochrome have been proposed to rationalize the paradoxes. However, there is at present no experimental evidence to support these concepts. The paradoxical absence of detectable Pfr in tissues which nevertheless respond as though phytochrome did exist in that form suggests that it may be incorrect to say that only Prr is physiologically active. In the case of high irradiance or prolonged irradiation responses (HIR) the involvement of phytochrome has been difficult to demonstrate due to the lack of congruity between “HIR action spectra” and the phytochrome absorption spectrum (compare spectrum in Fig. 3 with spectra in Fig. 1).
I.6
Wavelength
(nm)
FIG. 3. An “action spectrum” for the high-energy response (HIR) of inhibition hypocotyl lengthening (taken from Hartmann, 1967).
of lettuce
ACTIVE
FORM
OF
PHYTOCHROME
585
However, it has been plausibly argued that phytochrome is responsible, at least for the effect in the far-red region (Mohr, 1972); but difficulty is still experienced in explaining the characteristic features of the HIR: (i) Irradiance (intensity) dependence, if it is to be maintained that only Prl is responsible for the response once photo-equilibrium is established, and : (ii) The necessity for continual irradiation in order to achieve a complete response (Schopfer & Oelze-Karow, 1971; Schopfer & Mohr, 1972). The “pigment cycling hypothesis” of Hartmann & Unser (1973) which attributes the high irradiance responses to the frequency of Pf, ti P, interconversion could, in principle, account for the two features of HIR; thus higher irradiance would cause a faster, i.e. more molecules per unit time, interconversion of P, and P,, than a lower irradiance and would, therefore, generate more so-called ‘nascent PJr" at any instance of time; but there is no evidence at all to support the supposition that “nascent P,-:' is more active than “old Pf,". In fact, it appears almost impossible to test it experimentally. The hypotheses so far proposed for HIR provide no rational explanation for the occurrence of the peak in the blue in the HIR “action spectrum” (Hartmann, 1967). Indeed, some authors believe another pigment to be responsible and classify the high energy blue light responses as a separate phenomenon (see Smith, 1975, for a general review). This belief disregards the fact that both P, and Ps, forms of phytochrome absorb in the blue region of the spectrum and observations that blue light can mimic the effects of red irradiation (in, e.g. the germination of tomato seeds, Yaniv & Mancinelli, 1968; unfolding of barley leaf segments, Deutch & Deutch, 1975). Furthermore, far-red reversibility of blue light effects has been found in some systems in which the blue acts like red, such as in the photoinhibition of Pisumstem segments (Bertsch, 1963) and in the control of dormancy in Lunuluriu (Schwabe & Valio, 1970). In both cases the blue light effect is held to involve phytochrome. It has gradually become apparent that HIR is not an uncommon, isolated phenomenon; in fact, it is as widespread as the better-known low energy (pulse) reactions (Smith, 1975). A conceptual difficulty has therefore arisen in which it is customary to envisage that phytochrome sometimes mediates the same response via two separate modes of action in the same tissue (probably also in the same cell) as well as two different photoceptors mediating the same HER in the same tissue. From the foregoing discussion it appears that while a majority of phytochrome-induced phenomena is satisfactorily explicable in terms of Pf,, current dogma, which holds that Prr molecules are responsible for aZZphytochromemediated responses, is insufficient or too restrictive. Based on results of studies of phytochrome pelletability or binding to an as yet unknown intra-
cellular binding site we now propose a new model of phytochrome action which could account for both low and high irradiance responses in terms of a common mechanism of action. 2. The Hypothesis Essentially our new hypothesis can be described as follows. Radiation of any wavelength which is absorbed by phytochrome in the P, form is effective in causing it to become physiologically active. Absorption of radiant energy by the phytochrome molecule (P,) can result in activation (PI*) and/or activation plus phototransformation (P,,) depending on the quantum energy of the photon absorbed. P,* and/or Pf, can interact equivalently with the binding site or reaction partner ultimately to induce the biological display. The extent of the display or response is dependent only on the concentration of activated phytochrome molecules (P,* and/or P/J which in turn depends, within certain limits, on the amount of energy absorbed. It is obvious that for the same degree of activation, light of wavelengths less efficiently absorbed by P, than red light, e.g. far-red and blue, will require a longer duration of irradiation. (A)
SUPPORTING
EVIDENCE
Data supporting this new hypothesis were obtained from experiments on photo-induced phytochrome pelletability (or binding) in excised maize coleoptiles.? This response is unusual in that phytochrome is a component of the photo-induced product. Essentially the experimental system involves irradiation of the excised plant tissues with light and then determination of the amount of cellular phytochrome which has become bound to a particulate fraction. This determination can be made after the tissue is homogenized in a suitable extraction medium. From the dose response curves of phytochrome pelletability for a large number of wavelengths in the visible region we have constructed an action spectrum. At low energies this action spectrum matches the absorption spectrum of P, (Fuad & Yu, 1977u). It follows that phytochrome is the photo-receptor for light-induced phytochrome pelletability or binding. Because phytochrome is present in the product of the photoreaction and, immediately following irradiation, the tissue is kept on ice and the biochemical steps required to demonstrate phytochrome binding are all carried out at O”C, these facts support the view that phytochrome pelletability or binding tThe current controversy over the artefactual or non-artefactual pelletability is examined in an Appendix.
nature of phytochrome
ACTIVE
FORM
OF
PHYTOCHROME
587
is an early, if not the first, consequence of irradiation (Yu, Faud & Carr, 1976; Fuad & Yu, 1977~). For low energy reactions, a classical criterion for the involvement of phytochrome in any physiological response to light is that the effect of red light shall be reversible by immediate far-red. Red-induced phytochrome binding can be demonstrated to be reversible, i.e. the “debinding” reaction occurs in uiuo during dark incubation following R/FR irradiation at 0°C but not following R irradiation (Fuad & Yu, 1977~). These facts argue for the conclusion that phytochrome binding fulfils classical criteria for phytochrome involvement in a low energy physiological response (Mohr, 1972). It is apparent from the dose-response curves used to construct the action spectrum that light of any wavelength can be effective in causing phytochrome to bind to a level similar to that induced by red light, provided a sufficient dose is given (Fuad & Yu, 1977~). This implies that there is unlikely to be any relationship between a photo-response and a photo-stationary concentration of P,,; the photo-stationary Pjr concentration is therefore irreIevant and light dose is the only relevant determinant in a given response (see following discussion). With irradiance of 1.5 W m-‘, maximal binding may require l-2 min irradiation with red, but 05-2 h with blue or far red light simply because it is less efficiently absorbed (Fuad & Yu, 19773). Under conditions of prolonged irradiation (in terms of hours) where far-red and blue can cause high levels of binding, red is comparatively less effective (for reasons, see below). The spectral dependence of binding with 4 h irradiation at a chosen light intensity (Fuad 8z Yu, 1977a) shows a remarkable resemblance to the HIR spectra of inhibition of lettuce hypocotyl lengthening (Hartmann, 1967) and anthocyanin synthesis in some plants (Smith, 1975). Implicitly this result therefore indicates that biological responses effected by prolonged irradiation (or high irradiance) and pulse irradiation (or low irradiance responses) both share the same mechanism of action, i.e. the binding of phytochrome and it further reinforces the conclusion that binding is itself a phytochrome-mediated response. The lower binding resulting from prolonged red irradiation is coincident with a more pronounced decrease of total cellular phytochrome (Fuad & Yu, 1977~2). In interpreting the observed spectral dependence of phytochrome binding induced by prolonged irradiation, we believe a modification of Hartmann’s explanation (1966) of two competing or interacting reactions, i.e. phytochrome binding and destruction of P,,, to be consistent with our data (Fig. 4) (Fuad & Yu, 1977~2).P,-r is the form held to be uniquely susceptible to destruction. Hartmann’s explanation was based on the supposition that maximum action will occur under those conditions that allow Pf,, even at very low amounts, to be present for a very long time, by balancing
588
R. YU X
Dark
Red
PfrX
>
l
Pr Far-red Dark
\ DestructIon
FIG. 4. The “interaction hypothesis”of Hartmannfor explainingthe actionof phytochromein the high irradianceresponse (HIR) (Hartmann,1966).
the need to have a sufficiently high level of P,, to maintain action, and the opposing need to keep Pfr as low as possible to reduce destruction to a minimum. The far-red and blue “action” peaks and the troughs in the red and green regions of the HIR “action spectrum” then follow essentially from the interaction of two wavelength-dependent effects, efficiency of light absorption and susceptibility to destruction. Phytochrome pelletability induced by prolonged irradiation with far-red (or blue) shows classical features of HIR, such as dependence on irradiance and on continued irradiation. Phytochrome pelletability increases while the light is on but it decreases progessively due to release of phytochrome from the particles following the termination of irradiation (Yu et al., 1976). The extent of phytochrome binding depends on the irradiance for a chosen duration of irradiation. Hartmann’s adherence to the dogma that Prr is the only active molecule brings about a failure of his interaction hypothesis to explain the irradiance dependence characteristic of HIR. Our view, however, differs from Hartmann’s in holding that, after absorbing sufficient energy, P, can interact with the reaction partner X (using Hartmann’s terminology) and the irradiance dependence is explained simply as that, for a chosen duration of irradiation, different irradiances represent different doses delivered. It is our hypothesis that both P, and Pfr are able to bind because P, and P,, are always present in bound phytochrome (particulate phytochrome). We must now deal with the assumption generally made that Pfr is the only form of phytochrome that initially interacts with the putative receptor (binding
ACTIVE
FORM
OF
PHYTOCHROME
589
site). Two hypotheses have been proposed to account for phytochrome binding, namely: (1) PJI is initially the molecule which binds. The site becomes modified so that it comes to have an affinity for P,. This is the “induced fit” hypothesis (Quail, MarmC & Schafer, 1973). This hypothesis relies on the results of experiments said to show an increase in phytochrome pelletability (over that obtained by R-irradiation alone) by FR irradiation following red irradiation (Quail et al., 1973). These results have now (Fuad & Yu, 1977c) been shown to be spurious and the product of a systematic error in method. The error arises from the release of phytochrome from the particulate fraction in uitra when the breis of iradiated tissues were left standing in ice at 0°C. If the breis of red irradiated tissues is allowed to stand in ice longer than that of R/FR irradiated, phytochrome pelletability will appear higher in R/FR than R irradiated tissues, i.e. the result of Quail et al. (1973). The hypothesis of Pz,-induced enhancement of affinity for P, is without experimental support at the present time. (2) P,.r is both initially and subsequently the only molecule which binds. Increase in pelletability is said to represent presumably accumulation of PJ,receptor complex during the period of irradiation, i.e. somehow summation occurs because the plant tissue can “count” the number of Pfr molecules generated over the period of irradiation. This is the “cycling hypothesis” (Quail & Schafer, 1974). Since the time of interconversion, i.e. cycling, (a unimolecular photochemical event) must be much shorter than the time for binding (a bimolecular thermal reaction) (Pratt & Marme, 1976) and very much shorter than the time for de-binding, a necessary corollary of this hypothesis is that accumulation of pelletable phytochrome should be time-dependant only. On the cycling hypothesis, therefore, two different light intensities, which establish the same photo-stationary state of P,.,, given for the same duration should induce the same level of binding. Alternatively, for the same dose at two different light intensities (1, and I,, where Z, > II), both of which can establish the same photostationary state of Pfr, the lower intensity irradiation, I,, should effect more binding than the higher intensity, Ii, because its duration of irradiation is necessarily longer. That this is not the case is demonstrated clearly by the data of Quail & Schafer (1974) (in which the law of reciprocity holds true), though they prefer an interpretation, i.e. the cycling hypothesis, contrary to their own data. Clearly, the data of Quail & Schafer indicate that the binding reaction is dose-dependent.In the pigment cycling concept of Hartmann & Unser (1973) it is argued that higher intensities will cause a faster rate (amount/time) of interconversion of P, and P,,, hence more phytochrome molecules will go through the P,, state and be “counted”.
But, the alleged faster rate at higher intensity lacks experimental verification. In addition, the implied mechanistic basis for “counting” has neither been defined nor experimentally demonstrated. Also, since the maximal binding and the dose required for a fixed response (say 20 % binding) are not directly related to the steady-state levels of Prr (Fuad 8z Yu, 1977u) that Pfr is the only form which initially interacts with the putative receptor is not supported. The inescapable conclusion is, then, that activated phytochrome in either of the spectrophotometric forms, P, or Prr can bind. In support of this conclusion, we have shown that following a submaximal binding using a non-saturating dose of red light, not all the Pf, molecules generated actually bind but a substantial proportion remains unbound, and the phytochrome bound cannot be adequately accounted for by Pfr molecules alone (Yu et al., 1976). However, it can be argued that even at subsaturating doses of red light, cycling of phytochrome might take place. When phytochrome binding was studied by irradiating the plant tissue with short pulses of monochromatic red light (c 1 ms) (a light pulse much shorter than the dark reaction between the excited state of P,, produced by the photon absorption, and eventual P,, formation) a plot of the percent pelletable Pfr as a function of total PJr in the extract of maize coleoptiles showed clearly that P,-r is not the only molecule which binds, especially at low levels of total Pf, (Schiifer, 197%). This conclusion is further supported by the recent kinetic data of Pratt & Marmt. They found that “at low Pfr levels, for each Prr molecule produced by red light, one additional molecule of phytochrome (although some are P,) is found in the pellett”, and “Because binding follows the irradiation period, the trivial explanation that bound P, was photochemically derived from previously bound PfF may be discarded” (Pratt & MarmC, 1976). Recently SchZfer (1975b) has presented an elegant but complicated model of phytochrome (PfJ action which is a variant of that proposed by Quail & SchXer (1974) and which is designed to explain both the pulse and the high irradiance responses (Fig. 5). His “open phytochrome receptor model”, however, is based on data which have since been found to be erroneous. Essential features of his model are therefore unfounded, for example, that P,r. X + Pfr. x’ transition is a pre-requisite for Pf, destruction (Boisard, Marme & Briggs, 1974; Schgfer, Lassig & Schopfer, 1973, and that Pfr binding to Xinduces a change of X(to X’), i.e. “induced fit”, is also incorrect (see above); moreover, Schafer’s designation of elements Pfr. X and Pf,. X’ in his “open” model as “catalytic” and “inductive” sites of action respectively (Schafer, 1975b; Kasemir, Huber & Mohr, 1976) is not empirical but purely arbitrary. Hence, Schafer’s “open” model lacks experimental support for some of its essential postulates.
ACTIVE
FORM
“k, ---------SW
pI
PI x
OF ----3 +..----
-----+
PHYTOCHROME k,
k,
p
591
Ir
P,, x
FIG. 5. The ‘open phytochrome-receptor model” of phytochrome action proposed Schafer (19756).
by
3. Conclusion Since phytochrome binding is a primary physiological reaction (see Appendix for a discussion of the data on which this conclusion is based), it follows that Pf, is not the only physiologically active molecule and photo-transformation of P, to Pf, is not a prerequisite for the phytochrome molecule to become activated for binding. If this is so, then any attempt to explain a physiological response in terms of the percentage of Pfr or amount of P,, will be unrealistic and irrational. That this is indeed the case is clearly demonstrated by the numerous reports of unsuccessful attempts in the literature. In a recent critical review on this aspect, Hillman stated that “in any phytochrome system examined with rigor, no rational, analytically useful relationship (between Pfr and response) has yet been established” and “it is less surprising to find paradoxes than the occasional appearance of rationality” (Hillman, 1972). A suggestion has been made based on the “phytochrome paradoxes” that only a small proportion of the cellular phytochrome is actually employed for physiological responses (the “active fraction”). (Hillman, 1967; Briggs & Chon, 1966). If one accepts phytochrome binding as an authentic primary physiological response of phytochrome, this suggestion cannot be maintained because over 60% of the total cellular phytochrome can become bound by irradiation. I am most grateful to Professor D. J. Carr for his critical reading of the manuscript and stimulating discussion in crystallizing ideas presented in this paper. Also, I thank Mrs. Noha Pickles-Fuad for her collaboration.
REFERENCES BERTSCH, W. F. (1963). Am. J. But. 50, 754. BOLSARD, J., MARM& D., & BRIGGS, W. R. (1974). PI. Physiol. 54, 272. B~RTHWICK, H. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), p. 16. London, New York: Academic Press. BRIGGS, W. R., & CHON, H. P. (1966). PI. Physiol. 41, 1159. BRIGOS, W. R., & RICE, H. V. (1972). A. Rev. PI. Physiol. 23, 293. DEUTCH, B., & DEUTCH, B. I. (1975). Physiologia PI. 35, 322. FUAD, N., 8c Yu, R. (1977a). Photo&em. Photobiol. 25, 491. FUAD, N., & Yu, R. (19776). 2. PpanZenphysioI. 81, 304. FUAD, N., & Yu, R. (1977~). PI. Cell Physiol. 18, 35. HANKE, J., HARTMANN, K. M., & MOHR, H. (1969). Pfanta 86, 235. HARTMANN, K. M. (1966). Photochem. photobiol. 5, 349. HARTMANN, K. M. (1967). Z. Naturfirsch. 22b, 1172. HARTMANN, K. B., & UNSER, I. C. (1973). Z. Pflanzenphysiol. 69, 109. HAUPT, W. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), pp. 553-569. London, New York: Academic Press. HILLMAN, W. S. (1965). Physiologia PI. 18, 346. HILLMAN, W. S. (1967). A. Rev. PI. Physiol. 18, 301. HILLMAN, W. S. (1972). In Phytochrome (K. Mitrakos & W. Shropshire, Jr., eds), pp. 573584. London, New York: Academic Press. KA~EMIR, H., HUBER, P., & MOI-IR, H. (1976). Planta, 132, 157. MARMB, D., BOISARD, J. & BRIGGS, W. R. (1973). Proc. natn. Acad. Sci., U.S.A. 70, 3861. MOHR, H. (1972). In Lectares on Photomorphogenesis, pp. 3747. New York: SpringerVerlag, Berlin : Heidelberg. OELZE-KAROW, H., & MOHR, H. (1973). Photochem. photobiol. 18, 319. PUTT, L. H., & BRIGGS, W. R. (1966). PI. Physiol. 41, 467. PRAY, L. H., & COLEMAN, R. A. (1971). Proc. natn. Acad. Sci., U.S.A., 68,2431-2425. PRATT, L. H., & MARA, D. (1976). PI. Physiol. 58, 686. QUAIL, P. H., MAR&, D., & SCH&R, E. (1973). Nature Land. 245, 189. QUAIL, P. H. (1975). Photochem. Photobiol. 22,299. QUAIL, P. H. & GRESSEL,J. (1975). In Light and Plant Deveiopment (H. Smith, ed.), p. 91. 22nd Easter School, University of Nottingham. QUAIL, P. H., and SCHKPER, E. (1974). J. membr. Biol. 15, 393. RICE, H. V., & BRIGGS, W. R. (1973). PI. Physiol. 51, 939. RICE, H. V., BRIGGS, W. R., & JACKSON-WHITE, C. J. (1973). PI. Physiol. 51, 917. RUBINSTEIN, B., DRURY, K. S., & PARK, R. B. (1969). PI. Physiol. 44, 105. Roux, S. J., & HILLMAN, W. S. (1969). Archs biochem. Biophys. 131, 423. SA~R, R. L., & GALSTON, A. W. (1976). In Chemistry and Biochemistry of Plant Pigments (‘I. W. Goodwin, ed.), Vol. 1, p. 685. London, New York: Academic Press. SCHXFER, E. (1975a). Photochem Photobiol. 21, 189. S&&R, E. (19756). J. math. Biol. 2, 41. SAFER, E., LASSIG, T.-U., & SCHOPFER, P. (1975). Photochem. Photobiol. 22, 193. SCHOPFER,P., & OELZE-KAROW, H. (1971). Planta 100, 167. SCHOPFER, P., & MOHR, H. (1972). PI. Physiol. 49, 8. SCHWABE, W. W., & VALIO, J. F. M. (1970). J. expl. Bot. 21, 122. SMITH, H. (1975). In Phytochrome and Photomurphogenesis. London: McGraw-Hill. YANIV, Z., & MANCINELLI, A. L. (1968). PI. Physiol. 43, 117. Yu, R. (1975~). Aust. J. PI. Physiol. 2, 273. Yu, R. (19756). Asst. J. PI. Physiol. 2. 281. Yu, R. (1975c). J. expl But. 25, 808. Yu, R., & CARTER, J. (1976~). J. expl. But. 27, 283. Yu, R., & CARTER, J. (19766). PI. Cell Physioi. 17, 1309. Yu, R., FUAD, N., & CARR, D. J. (1976). PI. Cell Physiol. 17, 1131.
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APPENDIX Although there is good evidence that the phenomenon of photo-induced phytochrome pelletability is a normal physiological reaction, itself an early member of the sequence of phytochrome-mediated reactions, doubts have repeatedly been raised by others concerning its reality as an intracellular process and suggestions have been made that it is an artefact because the extent of the photo-response, i.e. the degree of phytochrome pelletability or binding, can only be ascertained following tissue homogenization and biochemical fractionation. To remove these doubts let us therefore examine relevant aspects of the binding reaction in some detail since it gives us the best experimental support at present for the new hypothesis. From many physiological experiments, phytochrome is believed to interact with or be an integral part of certain cellular membranes. In unirradiated etiolated tissues, a small amount (c. 5 %) of phytochrome was found associated with a particulate (presumably the mitochondrial, Rubinstein, Drury & Park, 1969) fraction of an etiolated oat seedling. When excised dark-grown plant tissues, e.g. maize coleoptiles, are irradiated with red light, most of the phytochrome becomes pelletable upon homogenization of the plant tissues in the presence of Mg’+ whereas very little (< 10%) is pelletable either in the absence of Mg2+ or from unirradiated tissues (see Quail, 1975 for a review). An immediate question arises, in view of the nature of the biochemical methods involved in demonstrating pelletability of phytochrome. Is it an artefact? Haupt (1972) has suggested that pelletability might be due to preferential adsorption or desorption of P, or Pfr during tissue homogenization in an artificial solution environment. Indeed, Quail has suggested, from his studies on phytochrome pelletability in zucchini hypocotyl hooks, that it is an artefact resulting from an association of phytochrome (P/J with degraded ribosomal material (RNP). However, he does not explain why the artefact should not arise in non-irradiated material. We have sought to refute these criticisms by treating irradiated maize coleoptiles with bifunctional crosslinking reagents, e.g. dimethyl suberimidate, glutaraldehyde, in an attempt to immobilize phytochrome at its intracellular location by covalent linkage so as render subsequent homogenization and fractionation independent of the solution environment. In maize, following glutaraldehyde or DMS pretreatments, enhanced phytochrome pelletability is obtained following an initial red irradiation (R and R/FR) but not following FR irradiation or from unirradiated material and this is virtually independent of the solution environment during tissue homogenization (Yu, 1975a,b; Yu & Carter, 1976~). Such pre-treatments also resulted in yields of pelletable phytochrome similar
594
R.
YU
to those obtained by extraction with Mg’+-containing buffer but without the crosslinking reaction (Yu & Carter, 1967b). Phytochrome in pellets obtained using bifunctional reagents showed a perfectly reversible phototransformation difference spectrum which was completely superimposable on that of phytochrome from untreated controls (Yu 8z Carter, 1967a,b). While this does not imply a total absence of degradation of phytochrome by these treatments it is evident that we are not dealing with a totally unspecific product. When coleoptiles were irradiated with R/FR and incubated in the dark before homogenization in Mg2+-containing buffer, there was a progressive loss of pelletable phytochrome but no loss of total cellular phytochrome. Crosslinking pre-treatments before homogenization in Mg”-free buffer gave yields faithfully reproducing the progressive loss of particulate phytochrome and preserving the total cellular content of phytochrome (Yu, 197%; Yu & Carter, 1976a, b). Here again we have evidence of preservation of an in uiuo state which reinforces the view that the biochemical techniques essential for the study of pelletability are not merely producing an artefact. Quail has questioned the validity of the pre-treatment approach on the ground that glutaraldehyde drastically changes phytochrome antigenic activity and causes random aggregation (Quail, 1975). This argument can be refuted for the following reasons: (1) In a thorough investigation, Rice & Briggs recently found no effect of glutaraldehyde treatment on the antigenic activity of phytochrome in P, and Pfr forms (Rice & Briggs, 1973). (2) Although Pratt and Coleman did observe a reduction of antigenic activity in glutaraldehyde-fixed tissues (cited as unpublished data in Pratt & Coleman, 1971), glutaraldehyde fixation was used subsequently in immunological studies of the localization of phytochrome (Briggs & Rice, 1972). (3) In in oitro as well as in uiuo studies on the chemical reactivity of purified and partially purified phytochrome treated with glutaraldehyde, Roux & Hillman (1969) found that the P, form of phytochrome is generally more reactive than the Pfr form. It could therefore be argued that if glutaraldehyde-enhanced pelletability were the result of a preferential reactivity of phytochrome, then dark-grown and FR-irradiated coleoptiles should be expected to yield larger amounts than red-irradiated ones. Experimentally this has been shown not to be the case (Yu, 197%). It is possible that in Quail’s experiments (which were not reported in detail) artefacts may have arisen from virtual fixation of the whole cell (Quail, 1975; Quail & Gressel, 1975). It is important to differentiate
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between results obtained when glutaraldehyde is used as a fixative (prolonged treatment, e.g. several hours at room temperature) or as a crosslinking bifunctional reagent (20-30 min at O’C). Moreover, in our hands glutaraldehyde pretreatment affected little, if any, of the sedimentation properties of organellar membranes and their constituent enzymic properties (Yu, 1975~). Furthermore, the results we obtained with glutaraldehyde have been duplicated using milder, more selective, and defined bifunctional crosslinking reagents, e.g. DMS, (Yu & Carter, 1976~~). In our own unpublished work, glutaraldehyde pretreatment of squash hooks obviated Quail’s artefact of association of phytochrome with a RNP fraction which has been the mainspring of fears of the artefactual nature of phytochrome pelletability. Quail & Gressel (1975) have found that one of the two fractions of phytochrome pelleted from homogenates of irradiated zucchini hooks contains degraded ribosomal material (RNP). Since that fraction has been held by Quail et al. (1973) and others (MarmC, Boisard & Briggs, (1973) to consist essentially of a membrane fraction, the well-publicised result of Quail and Gressel has cast doubt on the reality of pelletability of phytochrome in other systems. However, no degraded ribosomal material is found in phytochromecontaining pellets obtained from maize (Yu, unpubl. result). Upon irradiation phytochrome evidently binds to some sites in the cell and by suitably gentle means a pellet can be obtained containing the phytochrome together with its putative binding site. This is a real phenomenon, exactly reproducible from experiment to experiment. Without irradiation no such binding takes place and the phytochrome is not pelleted. We believe that these arguments together with those given in the text favour our conclusion that phytochrome does interact intracellularly with a fraction which becomes particulate upon homogenization and that experimentally this interaction can be stabilized either intracellularly by crosslinking in a bifunctional reagent prior to disruption of cellular organization or by using Mg 2+ in the medium.
7.8.
38