Planta
Planta 136, 1 8 1 - I86 (1977)
9 by Springer-Verlag 1977
Regulation of Enzyme Levels by Phytochrome in Mustard Cotyledons: Multiple Mechanisms? S. Frosch, H. Drumm, and H. Mohr Biologisches Institut II, UniversitO.t Freiburg, Schfinzlestr. 1, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of Pf~, e.g. due to the liberation of a "second messenger". We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL) § and ribulosebisphosphate carboxylase (Carboxylase) +. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25~ The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of Pfr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m-2 red light and 67.5 mW m -2 red light lead to the same time course), PAL induction is a graded response over a wide range of Per doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome. The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome Abbreviations: Pfr=the far-red absorbing, physiologically active
form of phytochrome; Pr=the red absorbing physiologically inactive form of phytochrome ; Ptotal-- [Pr] + [Pfr] ; PAL = phenylalanine ammonia-lyase (EC 4.3.1.5) ; Carboxylase=ribulosebisphosphate carboxylase (EC 4.1.1.39)
even though the realization of the pattern of development can only occur in the presence of phytochrome (Pfr). It seems that there is no feedback from pattern realization to pattern specification. Key words: Enzyme Regulation - Sinapis alba Phytochrome - Phenylalanine ammonia-lyase -- Ribulosebisphosphate carboxylase.
Introduction
Photoregulation of enzyme levels in plants by the photochromic sensor pigment, phytochrome, is a well-established phenomenon (see Mohr, 1974; Schopfer, 1977). However, the details of the mechanism involved are still under intense debate (see Smith, 1975). A major issue at present is whether or not the multiplicity of phytochrome-mediated effects on enzyme levels can be traced back to the same initial action of phytochrome. The present investigation was undertaken to study the question of whether the same mechanism is involved in the modulation by phytochrome of different enzyme levels within the same plant organ, the attached mustard cotyledon. The usefulness of the mustard cotyledons as a system of reference for enzyme studies was justified previously (e.g. Mohr, 1974). M6singer (personal communication) has confirmed previous results by Weidner (1967) that during the period of our experimentation mustard seedling cotyledons do not increase in cell number and DNA content. In fact, photomorphogenesis of this organ involves a cell population already present in the seed. Phytochrome controls the appearance of many enzymes in the mustard seedling cotyledons (see Schopfer, 1977). The problem has been whether the modulatory effect of phytochrome on the appearance of en-
182
zymes in this organ can be traced back to a common initial action of Per. We have chosen PAL and Carboxylase in our present investigation for the following reasons : 1. The appearance of both enzymes is strongly modulated by light in the mustard cotyledons (Durst and Mohr, 1966; Brfining et al., 1975). It is very probable that only phytochrome is involved as a photoreceptor iri this light effect. 2. It is very probable that in both cases the light-induced increase of the assayable enzyme activity is due to synthesis de novo of enzyme molecules (Kleinkopf et al., 1970; Peterson et al., 1973; Tong and Schopfer, 1976; Schopfer, 1977). 3. The two enzymes are not related functionally. While PAL is a major enzyme of phenylpropanoid biogenesis, located in the cytoplasm, Carboxylase is a major Calvin cycle enzyme, only occurring within the plastid compartment. However, it was shown previously (Frosch et al., 1976) that the light mediated rise of Carboxylase levels is neither related to the greening process nor to organizational (ultrastructural) changes of the plastid compartment. It was suggested (Ellis, 1975; Frosch etal., 1976) that the level of the functional enzyme in the plastid compartment is determined by the availability of the small subunit (which is encoded in the nuclear genome and synthesized on cytoplasmic ribosomes) and that phytochrome determines the rate at which the small subunit is synthesized. 4. It was found previously (Peter and Mohr, 1974) that PAL synthesis responds to phytochrome at approximately 27 h after sowing of the mustard seeds whereas Carboxylase synthesis responds to phytochrome only at 42 h after sowing under identical growth conditions (Brtining et al., 1975). Thus a temporal pattern of responsivity to phytochrome had to be inferred (Mohr, 1976). In the present paper we deal with the following questions: a. Why do the mustard cotyledons respond to phytochrome with regard to PAL synthesis at 27 h \ after sowing and with regard to Carboxylase at 42 h after sowing? b. Is the "mechanism" of the modulation of PAL synthesis by phytochrome the same as in the case of modulation of Carboxylase synthesis? Or rather, is the fact that the two modulations are widely separated in time indicative that different mechanisms of phytochrome action are involved?
Materials and Methods Standard techniques for photomorphogenic research with mustard seedlings were used (Mohr, 1966). The seed of Sinapis alba L. was purchased in 1971 from Asgrow Company (Freiburg-Ebnet).
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome The seeds were selected and the seedlings were grown at 25.0+0.2~ according to the previously described procedure (Mohr, 1977). The selected seed material is normally distributed with respect to seed weight. For light treatments a standard far-red source (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm 2) and a standard red source (emission maximum at 656 nm, band width 15 nm, irradiance 0.675 Wm 2) were used (see Mohr, 1966). Monochromatic 756 nm-light was obtained from a Leitz-Projector (Prado 500) modified after Mohr and Schoser (1959) und equipped with a Schott (Mainz, Germany) AL interference filter •max 756 rim; band width 20 nm; irradiance 7 Wm-Z). The photostationary state (=photoequilibrium) of the phytochrome system in the cotyledons of the mustard seedling established by standard red light is close to 0.8 (~0rea=[Pfr]/[P~o~]=0.8). Cpra~.rcd is of the order of a few %, see Sch/ifer et al. (1973). The photoequilibrium at 756 nm is very low (q?756 nm .c
300
300
2 ~, 200
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sowing
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r 36
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Duration of red light
Fig. 2. Time courses of Carboxylase levels in mustard seedling cotyledons in darkness and under continuous red or far-red light. Irradiations from time of sowing. The enzyme level in the dark at 72 h is taken as reference (100%)
Fig. 4. Action of continuous red light of different durations a n d of 756 nm-light pulses on Carboxylase levels in mustard seedling cotyledons 72 h after sowing. The dark level at 72 h is taken as reference (100%). The value for red at time zero was obtained by a 5 min red light pulse given immediately after sowing
3. Reversibility of Red Light Induction
receptor might have occurred). Figure 3 shows that in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to approximately 27 h after sowing, i.e. up to the starting point. This means that up to this point the red light signal remains localized within the phytochrome-(receptor-) system (as Prr or PfrX'; see Schfifer, 1975) and can thus be fully reversed by a 756 nm-light pulse. On the other hand with Carboxylase (Fig. 4) full reversibility in continuous red light is lost at approximately 15 h after sowing, i.e. about 27 h before the starting point for Carboxylase. This means that the original light signal must be stored for approximately 27 h outside
In this paragraph we study the onset of the initial action of phytochrome with regard to the modulation of PAL and Carboxylase synthesis. The term "initial action" was introduced previously (Jabben and Mohr, 1975) to operationally define the coupling of the phytochrome-(receptor-) system to cell fnnctions. The onset of "initial action" is defined by the loss of full reversibility by a saturating pulse with long wavelength (756 nm) light. As long as a light effect is fully reversible by a 756 nm-light pulse, the inductive light signal is still retained within the phylachrome system (even though the binding of Per to a
184
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome
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Fig. 5. Action of continuous far-red light of different durations and of 756 nm-light pulses on PAL levels 48 h after sowing. Onset of far-red light at time of sowing. The dark level at 48 h after sowing is indicated by the shaded bar
9
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72
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Fig. 7. Increase of Carboxylase levels within 24 h in the mustard seedling cotyledons under different light qualities and fluence rates. Onset of light: 48 h after sowing. The dark level at 72 h is taken as reference (100%)
sowing
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Fig. 6. Action of continuous far-red light of different durations and of 756 nm-light pulses on Carboxylase levels 72 h after sowing. Onset of far-red light at time of sowing. The dark level at 72 h is taken as reference (100%)
Fig. 8. Increase of PAL levels within 6 h in the mustard Seedling cotyledons under different light qualities and fluence rates. Onset of light : 36 h after sowing
the phytochrome system. Some kind of a longlived " t r a n s m i t t e r " is clearly implied, while in the case of PAL this hypothetical element is not required.
point. Up to 32 h after sowing the induction effect of continuous far-red light is not influenced by a 756 nm-light pulse. We will consider this penomenon at the beginning of the discussion.
4. Induction by Continuous Far-red Light Figure 5 shows that the effect of continuous far-red light (given from the time of sowing) becomes detectable in the case of PAL only at approximately 27 h after sowing while an inductive effect of the far-red light on Carboxylase synthesis is detectable even 6 h after the onset of far-red light (Fig. 6). Again a "transmitter-concept" (some long-lived intermediate which is already separated from the phytochrome system) is required in the case of Carboxylase while it is not required with PAL since no effect of continuous far-red light is detectable up to the starting
5. Dependency of the Continuous Light Effects on Prr Level and on the Fluence Rate (Irradiance) The modulation of Carboxylase synthesis is already saturated at a very low level of Pfr (Fig. 7). As an example, continuous 756 nm-light saturates the response even though ~07s6 nm is less than 0.1 per cent (see Materials and Methods). The effect of red light does not depend on fluence rate (irradiance) within the range we have tested. As an example, continuous 675 m W m - 2 red light and 67.5 m W m - : red light lead to the same time course. On the other hand, the mad-
s. Erosch et al. : Regulation of EnzymeLevelsby Phytochrome ulation of PAL synthesis is a graded response over a wide range of Pf~ doses and depends on the fluence rate (irradiance) (Fig. 8).
Discussion
It was found (Figs. 5, 6) that up to approximately 32 h after sowing the induction effect of continuous far-red light is not modified at all by a 756 nm-light pulse. This fact remains to be explained. Hartmann (1966) advanced and justified the concept of interpreting the effect of long-term red and far-red light on the basis of phytochrome. Schfifer (1975) has developed an open phytochrome-receptor-model which explains, in quantitative terms, the action of continuous far-red light. According to Schfifer's model farred light exerts predominantly a catalytic action which can no longer be caoncelled by returning Per back to Pr at the end of the light period. Only a small proportion of the effect of far-red light is exerted by Pfr acting in the dark after the light is turned off. This small proportion becomes only measurable (operationally, reversible with 756 nm-light) once a relatively high level of total phytochrome (Ptot) is reached. This is the case only beyond approximately 32 h after sowing. Since the non-reversibility of the effect of continuous high irradiance far-red light was explained in detail previously (see Steinitz et al., 1976; Kasemir et al., 1976) this brief statement should suffice in the present context. As far as the question (b) of the Introduction is concerned, we conclude from our results that modulations by phytochrome of PAL and Carboxylase synthesis in the mustard seedling cotyledons are not only separated in time but differ in every regard except that both responses are mediated by phytochrome. Smith (1975) has suggested that phytochrome initially acts through a single "second messenger" released from a membrane-bound storage compartment by phytochrome. The "second messenger" would then react with a multiplicity of different reaction partners with the consequence of multiple responses. We have previously described evidence (Drumm and Mohr, 1974; Mohr and Oelze-Karow, 1976) strongly suggesting that there is no common primary reaction of the phytochrome system in responses such as anthocyanin accumulation and suppression of lipoxygenase accumulation. The comparative investigation of protochlorophyll resynthesis and shortening of the duration of the Shibata shift has previously led to the suggestion that there is no common initial action of the phytochrome system in these two responses (Jabben and Mohr, 1975). We now conclude that the data of the present paper dealing with the modula-
185 tion of the synthesis of two enzymes within the same organ are difficult to reconcile with the notion of a common initial action of phytochrome leading to the liberation of a "second messenger". To make the concept of a " second messenger" compatible with our data this would require drastic auxiliary assumptions, e.g. an extremely rapid turnover of the "second messenger" to account for the PAL and anthocyanin (Steinitz et al., 1976) data. Kinnersley and Davies (1976) have studied phytochrome-mediated anthocyanin synthesis, hair formation, and the synthesis of ascorbate oxidase in the hypocotyl of mustard. A careful comparison of these three responses showed that in certain respects they differ greatly in their response to the light treatment. The authors conclude that "the difference in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ". We have emphasized previously (Steinitz et al., 1976; Kasemir etal., 1976) that the starting points for phytochrome-mediated responses are determined by developmental homeostasis which is independent of light. In the photomorphogenetic, phytochromedependent development of a seedling, we must keep two different steps apart: pattern specification and pattern realization. The specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (Pfr). From our studies about suppression of lipoxygenase synthesis by phytochrome we know that there is no feedback from pattern realization to pattern specification (see Mohr and OelzeKarow, 1976). As another example, it was found that the onset and the rate of Carboxylase synthesis in the mustard cotyledons is always the same irrespective of the photomorphogenetic light treatment the seedling has received prior to the starting point which is at 42 h after sowing at 25~ (Brfining et al., 1975; Frosch et al., 1976). While these different light treatments between sowing and 42 h after sowing lead to very different time courses of PAl levels, the starting point and the rate of Carboxylase synthesis are not at all affected. In brief, pattern specification with regard to Carboxylase synthesis is not influenced by the extent of pattern realization with regard to PAL. So far approaches to analyze the causalities of pattern specification have not really been successful, neither in plants nor in animals. Therefore, a "molecular" answer to question (a) in the Introduction is not possible at present. Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 46). We thank Birgit Haas and Dagmar Brinschwitzfor competent technical assistance.
186
References Brtining, K., Drumm, H., Mohr, H. : On the role of phytochrome in controlling enzyme levels in plastids. Biochem. Physiol. Pflanzen 168, 141-156 (1975) Drumm, H., Mohr, H. : The dose response curve in phytochromemediated anthocynin synthesis in the mustard seedling. Photochem. Photobiol. 20, 151-157 (1974) Durst, F., Mohr, H. : Phytochrome-mediated induction of enzyme synthesis in mustard seedlings (Sinapis alba L.). Naturwissenschaften 53, 531-532 (1966) Ellis, R.J. : Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide. Phytochemistry 14, 89-93 (1975) Frosch, S., Bergfeld, R., Mohr, H. : Light control of plastogenesis and ribulose bisphosphate carboxylase levels in mustard seedling cotyledons. Planta 133, 53-56 (1976) Hartmann, K.M.: A general hypothesis to interpret "high energy phenomena " of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349-366 (1966) Jabben, M., Mohr, H. : Stimulation of the Shibata shift by phytochrome in the cotyledons of the mustard seedling. Photochem. Photobiol. 22, 55-58 (1975) Kasemir, H., Huber, P., Mohr, H.: Timing of the initial action of phytochrome with regard to protochlorophyll synthesis in the mustard seedling. Planta (Bert.) 132, 157-160 (1976) Kinnersley, A.M., Davies, P.J. : Comparison of three phytochromemediated processes in the hypocotyl of mustard. Plant Physiol. 58, 777 782 (1976) Kleinkopf, G.E., Huffaker, R.C., Matheson, H. : Light -induced de novo synthesis of ribulose 1,5-diphosphate carboxylase in greening leaves of barley. Plant Physiol. 46, 416-418 (1970) Mohr, H. : Untersuchungen zur phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.). Z. Pflanzenphysiol. 54, 63-83 (1966) Mohr, H.: The role of phytochrome in controlling enzyme levels in plants. In: MTP international reviews of science. Biochemistry series one. Biochemistry of cell differentiation, Vol. 9, pp. 37 81. Paul, J., ed. London: Butterworths 1974 Mohr, H.: Zur Zielsetzung der Entwicklungsbiologie. Biologie in unserer Zeit 6, 161-168 (1976)
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome Mohr, H., Oelze-Karow, H.: Phytochrome action as a threshold phenomenon. In: Light and plant development~ pp. 257 284. Smith, H. ed. London-Boston: Butterworths 1976 Mohr, H., Schoser, G.: Eine Interferenzfilter-Monochromatoranlage ffir photobiologische Zwecke. Planta (Bed.) 53, 1-17 (1959) Peter, K., Mohr, H. : Control of phenylalanine ammonialyase and ascorbate oxidase in the mustard seedling by light and Hoagland's nutrient solution. Z. Naturforsch. 29c, 222-228 (1974) Peterson, L.W., Kleinkopf, G.E., Huffaker, R.C.: Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves. Plant Physiol. 51, 1042-1045 (1973) Sch~ifer, E. : A new approach to explain the "high irradiance responses" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 (1975) Sch/ifer, E., Lassig, T.-U., Schopfer, P. : Photocontrol of phytochrome destruction in grass seedlings. The influence of wavelength and irradiance. Photochem. Photobiol. 22, 193-202 (1975) Sch~fer, E., Schmidt, W., Mohr, H.: Comparative measurements of phytochrome in cotyledons and hypocotyl hook of mustard (Sinapis alba L.). Photochem. Photobiol. 18, 331-334 (1973) Schopfer, P.: Phytochrome control of enzymes. Ann. Rev. Plant Physiol. 28, 223~52 (1977) Schopfer, P., Mohr, H.: Phytochrome-mediated induction of phenylalanine ammonialyase in mustard seedlings. Plant Physiol. 49, 8 10 (1972) Smith, H.: The mechanism of action and the function of phytochrome. In : Light and plant development, pp. 63-65. Proceedings of 22nd Easter School in Agricultural Science, University of Nottingham 1975 Steinitz, B., Drumm, H., Mohr, H. : The appearance of competence for phytochrome-mediated anthocyanin synthesis in the cotyledons of Sinapis alba L. Planta )Bed.) 130, 23-31 (1976) Tong, W.F., Schopfer, P.: Phytochrome-mediated de novo synthesis of phenylalanine ammonialyase: A new approach using preinduced mustard seedlings. Proc. Nat. Acad. Sci. (Wash.) 73, 4017-4021 (1976) Weidner, M.: I)er DNS-Gehalt yon Kotyledonen und Hypokotyl des Senfkeimlings (Sinapis alba L.) bei der phytochromgesteuerten Photomorphogenese. Planta (Berl.) 75, 94-98 (1967)
Received 11 May; accepted 27 May 1977
E r ra t u m I n the article in the last issue (Vol. 136, N o . 1, 1977, pp. 4 5 - 4 8 ) e n t i t l e d " E v i d e n c e f o r a P h o t o i n d u c e d S y n t h e s i s o f P o l y ( A ) C o n t a i n i n g m R N A in Fusarium aquaeductuum" by E . L . S c h r o t t a n d W. R a u , line 4 o n p a g e 46, left c o l u m n , s h o u l d r e a d as f o l l o w s : 10 min later 0.8 #Ci/ml [3H]uridine was added. Line 7 should read:
amount o f [14C]uridine (0.2 #Ci/ml), were collected by vacuum filtration
Planta 136, 1 8 1 - I86 (1977)
9 by Springer-Verlag 1977
Regulation of Enzyme Levels by Phytochrome in Mustard Cotyledons: Multiple Mechanisms? S. Frosch, H. Drumm, and H. Mohr Biologisches Institut II, UniversitO.t Freiburg, Schfinzlestr. 1, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of Pf~, e.g. due to the liberation of a "second messenger". We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL) § and ribulosebisphosphate carboxylase (Carboxylase) +. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25~ The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of Pfr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m-2 red light and 67.5 mW m -2 red light lead to the same time course), PAL induction is a graded response over a wide range of Per doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome. The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome Abbreviations: Pfr=the far-red absorbing, physiologically active
form of phytochrome; Pr=the red absorbing physiologically inactive form of phytochrome ; Ptotal-- [Pr] + [Pfr] ; PAL = phenylalanine ammonia-lyase (EC 4.3.1.5) ; Carboxylase=ribulosebisphosphate carboxylase (EC 4.1.1.39)
even though the realization of the pattern of development can only occur in the presence of phytochrome (Pfr). It seems that there is no feedback from pattern realization to pattern specification. Key words: Enzyme Regulation - Sinapis alba Phytochrome - Phenylalanine ammonia-lyase -- Ribulosebisphosphate carboxylase.
Introduction
Photoregulation of enzyme levels in plants by the photochromic sensor pigment, phytochrome, is a well-established phenomenon (see Mohr, 1974; Schopfer, 1977). However, the details of the mechanism involved are still under intense debate (see Smith, 1975). A major issue at present is whether or not the multiplicity of phytochrome-mediated effects on enzyme levels can be traced back to the same initial action of phytochrome. The present investigation was undertaken to study the question of whether the same mechanism is involved in the modulation by phytochrome of different enzyme levels within the same plant organ, the attached mustard cotyledon. The usefulness of the mustard cotyledons as a system of reference for enzyme studies was justified previously (e.g. Mohr, 1974). M6singer (personal communication) has confirmed previous results by Weidner (1967) that during the period of our experimentation mustard seedling cotyledons do not increase in cell number and DNA content. In fact, photomorphogenesis of this organ involves a cell population already present in the seed. Phytochrome controls the appearance of many enzymes in the mustard seedling cotyledons (see Schopfer, 1977). The problem has been whether the modulatory effect of phytochrome on the appearance of en-
182
zymes in this organ can be traced back to a common initial action of Per. We have chosen PAL and Carboxylase in our present investigation for the following reasons : 1. The appearance of both enzymes is strongly modulated by light in the mustard cotyledons (Durst and Mohr, 1966; Brfining et al., 1975). It is very probable that only phytochrome is involved as a photoreceptor iri this light effect. 2. It is very probable that in both cases the light-induced increase of the assayable enzyme activity is due to synthesis de novo of enzyme molecules (Kleinkopf et al., 1970; Peterson et al., 1973; Tong and Schopfer, 1976; Schopfer, 1977). 3. The two enzymes are not related functionally. While PAL is a major enzyme of phenylpropanoid biogenesis, located in the cytoplasm, Carboxylase is a major Calvin cycle enzyme, only occurring within the plastid compartment. However, it was shown previously (Frosch et al., 1976) that the light mediated rise of Carboxylase levels is neither related to the greening process nor to organizational (ultrastructural) changes of the plastid compartment. It was suggested (Ellis, 1975; Frosch etal., 1976) that the level of the functional enzyme in the plastid compartment is determined by the availability of the small subunit (which is encoded in the nuclear genome and synthesized on cytoplasmic ribosomes) and that phytochrome determines the rate at which the small subunit is synthesized. 4. It was found previously (Peter and Mohr, 1974) that PAL synthesis responds to phytochrome at approximately 27 h after sowing of the mustard seeds whereas Carboxylase synthesis responds to phytochrome only at 42 h after sowing under identical growth conditions (Brtining et al., 1975). Thus a temporal pattern of responsivity to phytochrome had to be inferred (Mohr, 1976). In the present paper we deal with the following questions: a. Why do the mustard cotyledons respond to phytochrome with regard to PAL synthesis at 27 h \ after sowing and with regard to Carboxylase at 42 h after sowing? b. Is the "mechanism" of the modulation of PAL synthesis by phytochrome the same as in the case of modulation of Carboxylase synthesis? Or rather, is the fact that the two modulations are widely separated in time indicative that different mechanisms of phytochrome action are involved?
Materials and Methods Standard techniques for photomorphogenic research with mustard seedlings were used (Mohr, 1966). The seed of Sinapis alba L. was purchased in 1971 from Asgrow Company (Freiburg-Ebnet).
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome The seeds were selected and the seedlings were grown at 25.0+0.2~ according to the previously described procedure (Mohr, 1977). The selected seed material is normally distributed with respect to seed weight. For light treatments a standard far-red source (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm 2) and a standard red source (emission maximum at 656 nm, band width 15 nm, irradiance 0.675 Wm 2) were used (see Mohr, 1966). Monochromatic 756 nm-light was obtained from a Leitz-Projector (Prado 500) modified after Mohr and Schoser (1959) und equipped with a Schott (Mainz, Germany) AL interference filter •max 756 rim; band width 20 nm; irradiance 7 Wm-Z). The photostationary state (=photoequilibrium) of the phytochrome system in the cotyledons of the mustard seedling established by standard red light is close to 0.8 (~0rea=[Pfr]/[P~o~]=0.8). Cpra~.rcd is of the order of a few %, see Sch/ifer et al. (1973). The photoequilibrium at 756 nm is very low (q?756 nm .c
300
300
2 ~, 200
200
o
sowing
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~
o
~oo,
r 36
h after
9 red light red light+5 rain 756nm Light o 6 rain 756nm Light only
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Time after sowing
--+---o- . . . 2_--_~_ . . . . . . . . . . . . .o. . . .
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o
i
6
I
9
l
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i
115 18
211
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319
Duration of red light
Fig. 2. Time courses of Carboxylase levels in mustard seedling cotyledons in darkness and under continuous red or far-red light. Irradiations from time of sowing. The enzyme level in the dark at 72 h is taken as reference (100%)
Fig. 4. Action of continuous red light of different durations a n d of 756 nm-light pulses on Carboxylase levels in mustard seedling cotyledons 72 h after sowing. The dark level at 72 h is taken as reference (100%). The value for red at time zero was obtained by a 5 min red light pulse given immediately after sowing
3. Reversibility of Red Light Induction
receptor might have occurred). Figure 3 shows that in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to approximately 27 h after sowing, i.e. up to the starting point. This means that up to this point the red light signal remains localized within the phytochrome-(receptor-) system (as Prr or PfrX'; see Schfifer, 1975) and can thus be fully reversed by a 756 nm-light pulse. On the other hand with Carboxylase (Fig. 4) full reversibility in continuous red light is lost at approximately 15 h after sowing, i.e. about 27 h before the starting point for Carboxylase. This means that the original light signal must be stored for approximately 27 h outside
In this paragraph we study the onset of the initial action of phytochrome with regard to the modulation of PAL and Carboxylase synthesis. The term "initial action" was introduced previously (Jabben and Mohr, 1975) to operationally define the coupling of the phytochrome-(receptor-) system to cell fnnctions. The onset of "initial action" is defined by the loss of full reversibility by a saturating pulse with long wavelength (756 nm) light. As long as a light effect is fully reversible by a 756 nm-light pulse, the inductive light signal is still retained within the phylachrome system (even though the binding of Per to a
184
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome
Assay: r--~ c
3O
25
48h
after
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I 24
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I 30
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I 32
D u r a t i o n of far-red
I 3/.
0
Fig. 5. Action of continuous far-red light of different durations and of 756 nm-light pulses on PAL levels 48 h after sowing. Onset of far-red light at time of sowing. The dark level at 48 h after sowing is indicated by the shaded bar
9
far-red
72
h after
I 18
onset
of
Light
after sowing
Fig. 7. Increase of Carboxylase levels within 24 h in the mustard seedling cotyledons under different light qualities and fluence rates. Onset of light: 48 h after sowing. The dark level at 72 h is taken as reference (100%)
sowing
Light
I
o far-red Light +5min 756nm Light o 5min 756nm t i g h t only
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,
o
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~I r_h]
6
Time a f t e r onset of Light 36h a f t e r sowing
Fig. 6. Action of continuous far-red light of different durations and of 756 nm-light pulses on Carboxylase levels 72 h after sowing. Onset of far-red light at time of sowing. The dark level at 72 h is taken as reference (100%)
Fig. 8. Increase of PAL levels within 6 h in the mustard Seedling cotyledons under different light qualities and fluence rates. Onset of light : 36 h after sowing
the phytochrome system. Some kind of a longlived " t r a n s m i t t e r " is clearly implied, while in the case of PAL this hypothetical element is not required.
point. Up to 32 h after sowing the induction effect of continuous far-red light is not influenced by a 756 nm-light pulse. We will consider this penomenon at the beginning of the discussion.
4. Induction by Continuous Far-red Light Figure 5 shows that the effect of continuous far-red light (given from the time of sowing) becomes detectable in the case of PAL only at approximately 27 h after sowing while an inductive effect of the far-red light on Carboxylase synthesis is detectable even 6 h after the onset of far-red light (Fig. 6). Again a "transmitter-concept" (some long-lived intermediate which is already separated from the phytochrome system) is required in the case of Carboxylase while it is not required with PAL since no effect of continuous far-red light is detectable up to the starting
5. Dependency of the Continuous Light Effects on Prr Level and on the Fluence Rate (Irradiance) The modulation of Carboxylase synthesis is already saturated at a very low level of Pfr (Fig. 7). As an example, continuous 756 nm-light saturates the response even though ~07s6 nm is less than 0.1 per cent (see Materials and Methods). The effect of red light does not depend on fluence rate (irradiance) within the range we have tested. As an example, continuous 675 m W m - 2 red light and 67.5 m W m - : red light lead to the same time course. On the other hand, the mad-
s. Erosch et al. : Regulation of EnzymeLevelsby Phytochrome ulation of PAL synthesis is a graded response over a wide range of Pf~ doses and depends on the fluence rate (irradiance) (Fig. 8).
Discussion
It was found (Figs. 5, 6) that up to approximately 32 h after sowing the induction effect of continuous far-red light is not modified at all by a 756 nm-light pulse. This fact remains to be explained. Hartmann (1966) advanced and justified the concept of interpreting the effect of long-term red and far-red light on the basis of phytochrome. Schfifer (1975) has developed an open phytochrome-receptor-model which explains, in quantitative terms, the action of continuous far-red light. According to Schfifer's model farred light exerts predominantly a catalytic action which can no longer be caoncelled by returning Per back to Pr at the end of the light period. Only a small proportion of the effect of far-red light is exerted by Pfr acting in the dark after the light is turned off. This small proportion becomes only measurable (operationally, reversible with 756 nm-light) once a relatively high level of total phytochrome (Ptot) is reached. This is the case only beyond approximately 32 h after sowing. Since the non-reversibility of the effect of continuous high irradiance far-red light was explained in detail previously (see Steinitz et al., 1976; Kasemir et al., 1976) this brief statement should suffice in the present context. As far as the question (b) of the Introduction is concerned, we conclude from our results that modulations by phytochrome of PAL and Carboxylase synthesis in the mustard seedling cotyledons are not only separated in time but differ in every regard except that both responses are mediated by phytochrome. Smith (1975) has suggested that phytochrome initially acts through a single "second messenger" released from a membrane-bound storage compartment by phytochrome. The "second messenger" would then react with a multiplicity of different reaction partners with the consequence of multiple responses. We have previously described evidence (Drumm and Mohr, 1974; Mohr and Oelze-Karow, 1976) strongly suggesting that there is no common primary reaction of the phytochrome system in responses such as anthocyanin accumulation and suppression of lipoxygenase accumulation. The comparative investigation of protochlorophyll resynthesis and shortening of the duration of the Shibata shift has previously led to the suggestion that there is no common initial action of the phytochrome system in these two responses (Jabben and Mohr, 1975). We now conclude that the data of the present paper dealing with the modula-
185 tion of the synthesis of two enzymes within the same organ are difficult to reconcile with the notion of a common initial action of phytochrome leading to the liberation of a "second messenger". To make the concept of a " second messenger" compatible with our data this would require drastic auxiliary assumptions, e.g. an extremely rapid turnover of the "second messenger" to account for the PAL and anthocyanin (Steinitz et al., 1976) data. Kinnersley and Davies (1976) have studied phytochrome-mediated anthocyanin synthesis, hair formation, and the synthesis of ascorbate oxidase in the hypocotyl of mustard. A careful comparison of these three responses showed that in certain respects they differ greatly in their response to the light treatment. The authors conclude that "the difference in the response of the three reactions to light suggest that the phytochrome-mediated reactions which control their development also differ". We have emphasized previously (Steinitz et al., 1976; Kasemir etal., 1976) that the starting points for phytochrome-mediated responses are determined by developmental homeostasis which is independent of light. In the photomorphogenetic, phytochromedependent development of a seedling, we must keep two different steps apart: pattern specification and pattern realization. The specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (Pfr). From our studies about suppression of lipoxygenase synthesis by phytochrome we know that there is no feedback from pattern realization to pattern specification (see Mohr and OelzeKarow, 1976). As another example, it was found that the onset and the rate of Carboxylase synthesis in the mustard cotyledons is always the same irrespective of the photomorphogenetic light treatment the seedling has received prior to the starting point which is at 42 h after sowing at 25~ (Brfining et al., 1975; Frosch et al., 1976). While these different light treatments between sowing and 42 h after sowing lead to very different time courses of PAl levels, the starting point and the rate of Carboxylase synthesis are not at all affected. In brief, pattern specification with regard to Carboxylase synthesis is not influenced by the extent of pattern realization with regard to PAL. So far approaches to analyze the causalities of pattern specification have not really been successful, neither in plants nor in animals. Therefore, a "molecular" answer to question (a) in the Introduction is not possible at present. Supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 46). We thank Birgit Haas and Dagmar Brinschwitzfor competent technical assistance.
186
References Brtining, K., Drumm, H., Mohr, H. : On the role of phytochrome in controlling enzyme levels in plastids. Biochem. Physiol. Pflanzen 168, 141-156 (1975) Drumm, H., Mohr, H. : The dose response curve in phytochromemediated anthocynin synthesis in the mustard seedling. Photochem. Photobiol. 20, 151-157 (1974) Durst, F., Mohr, H. : Phytochrome-mediated induction of enzyme synthesis in mustard seedlings (Sinapis alba L.). Naturwissenschaften 53, 531-532 (1966) Ellis, R.J. : Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide. Phytochemistry 14, 89-93 (1975) Frosch, S., Bergfeld, R., Mohr, H. : Light control of plastogenesis and ribulose bisphosphate carboxylase levels in mustard seedling cotyledons. Planta 133, 53-56 (1976) Hartmann, K.M.: A general hypothesis to interpret "high energy phenomena " of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349-366 (1966) Jabben, M., Mohr, H. : Stimulation of the Shibata shift by phytochrome in the cotyledons of the mustard seedling. Photochem. Photobiol. 22, 55-58 (1975) Kasemir, H., Huber, P., Mohr, H.: Timing of the initial action of phytochrome with regard to protochlorophyll synthesis in the mustard seedling. Planta (Bert.) 132, 157-160 (1976) Kinnersley, A.M., Davies, P.J. : Comparison of three phytochromemediated processes in the hypocotyl of mustard. Plant Physiol. 58, 777 782 (1976) Kleinkopf, G.E., Huffaker, R.C., Matheson, H. : Light -induced de novo synthesis of ribulose 1,5-diphosphate carboxylase in greening leaves of barley. Plant Physiol. 46, 416-418 (1970) Mohr, H. : Untersuchungen zur phytochrominduzierten Photomorphogenese des Senfkeimlings (Sinapis alba L.). Z. Pflanzenphysiol. 54, 63-83 (1966) Mohr, H.: The role of phytochrome in controlling enzyme levels in plants. In: MTP international reviews of science. Biochemistry series one. Biochemistry of cell differentiation, Vol. 9, pp. 37 81. Paul, J., ed. London: Butterworths 1974 Mohr, H.: Zur Zielsetzung der Entwicklungsbiologie. Biologie in unserer Zeit 6, 161-168 (1976)
S. Frosch et al. : Regulation of Enzyme Levels by Phytochrome Mohr, H., Oelze-Karow, H.: Phytochrome action as a threshold phenomenon. In: Light and plant development~ pp. 257 284. Smith, H. ed. London-Boston: Butterworths 1976 Mohr, H., Schoser, G.: Eine Interferenzfilter-Monochromatoranlage ffir photobiologische Zwecke. Planta (Bed.) 53, 1-17 (1959) Peter, K., Mohr, H. : Control of phenylalanine ammonialyase and ascorbate oxidase in the mustard seedling by light and Hoagland's nutrient solution. Z. Naturforsch. 29c, 222-228 (1974) Peterson, L.W., Kleinkopf, G.E., Huffaker, R.C.: Evidence for lack of turnover of ribulose 1,5-diphosphate carboxylase in barley leaves. Plant Physiol. 51, 1042-1045 (1973) Sch~ifer, E. : A new approach to explain the "high irradiance responses" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 (1975) Sch/ifer, E., Lassig, T.-U., Schopfer, P. : Photocontrol of phytochrome destruction in grass seedlings. The influence of wavelength and irradiance. Photochem. Photobiol. 22, 193-202 (1975) Sch~fer, E., Schmidt, W., Mohr, H.: Comparative measurements of phytochrome in cotyledons and hypocotyl hook of mustard (Sinapis alba L.). Photochem. Photobiol. 18, 331-334 (1973) Schopfer, P.: Phytochrome control of enzymes. Ann. Rev. Plant Physiol. 28, 223~52 (1977) Schopfer, P., Mohr, H.: Phytochrome-mediated induction of phenylalanine ammonialyase in mustard seedlings. Plant Physiol. 49, 8 10 (1972) Smith, H.: The mechanism of action and the function of phytochrome. In : Light and plant development, pp. 63-65. Proceedings of 22nd Easter School in Agricultural Science, University of Nottingham 1975 Steinitz, B., Drumm, H., Mohr, H. : The appearance of competence for phytochrome-mediated anthocyanin synthesis in the cotyledons of Sinapis alba L. Planta )Bed.) 130, 23-31 (1976) Tong, W.F., Schopfer, P.: Phytochrome-mediated de novo synthesis of phenylalanine ammonialyase: A new approach using preinduced mustard seedlings. Proc. Nat. Acad. Sci. (Wash.) 73, 4017-4021 (1976) Weidner, M.: I)er DNS-Gehalt yon Kotyledonen und Hypokotyl des Senfkeimlings (Sinapis alba L.) bei der phytochromgesteuerten Photomorphogenese. Planta (Berl.) 75, 94-98 (1967)
Received 11 May; accepted 27 May 1977
E r ra t u m I n the article in the last issue (Vol. 136, N o . 1, 1977, pp. 4 5 - 4 8 ) e n t i t l e d " E v i d e n c e f o r a P h o t o i n d u c e d S y n t h e s i s o f P o l y ( A ) C o n t a i n i n g m R N A in Fusarium aquaeductuum" by E . L . S c h r o t t a n d W. R a u , line 4 o n p a g e 46, left c o l u m n , s h o u l d r e a d as f o l l o w s : 10 min later 0.8 #Ci/ml [3H]uridine was added. Line 7 should read:
amount o f [14C]uridine (0.2 #Ci/ml), were collected by vacuum filtration
