Planta (Berl.) 77, 49--57 (1967)
Photoinduction of Phenylalanine Deaminase in Gherkin Seedlings II. Effect of Red and Far-Red Light G. ENGELS~A Philips Research Laboratories, N. V. Philips' Gloeflampenfabrieken, Eindhoven, Netherlands Received July 3, 1967
Summary. 1. Exposure of dark-grown gherkin seedlings to red or far-red light causes changes in the level of phenylalanine deaminase (PADAse) which follow the same time course and show a similar dependence on light intensity as the changes induced in blue light. From this it is concluded that light of different regions of the visible spectrum is initiating the same sequence of dark reactions leading to enhanced production of PADAse, followed by a phase of inactivation of this enzyme and repression of its synthesis. 2. In blue light a higher PADAse level can be obtained than in red or far-red light, and only blue light is capable of inducing PADAse synthesis in excised hypocotyls. This suggests that different pigment systems are involved in the highenergy phenomena in the gherkin seedling. 3. The effects of irradiation sequences with different light qualities and intensities on the accumulation of hydroxycinnamic acids can be explained on the basis of the regulatory mechanisms which control the PADAse level. Introduction The effects of red and far-red light on the synthesis of hydroxycinnamic acids in gherkin hypocotyls are more complicated than those of blue light. On the one hand there is a response to a small dose of red light which can be abolished with far-red light. On the other hand there is the reaction caused b y prolonged irradiations with red and farred light which, similarly as with blue light, reaches saturation at much higher light doses, gives rise to a m u c h g r e a t e r effect, a n d c a n n o t be r e v e r s e d (E~GELSMA a n d Mv,IjEI~, 1965). This l a t t e r r e a c t i o n is k n o w n as t h e h i g h - e n e r g y r e a c t i o n of p h o t o m o r p h o g e n e s i s ( H E R ) . I t is g e n e r a l l y a s s u m e d t h a t t h e former t y p e of r e a c t i o n is controlled b y t h e p h o t o - r e v e r s i b l e p i g m e n t p h y t o c h r o m e , whereas t h e H E R was originally t h o u g h t to be due to a second p i g m e n t (SIEGnLMA~ a n d Hwl~I)l~ICI~S, 1957; Mom~, 1957). R e c e n t l y a h y p o t h e s i s has been p r o p o s e d which, a t least as far as t h e effects of r e d a n d far-red light are concerned, explains t h e H E R also in t e r m s of control b y p h y t o c h r o m e (I-IAI~TMANI~, 1966; see also 1VIOHI~, 1966). 4 Planta (Berl.), Bd. 77
50
G. ENGELSMA:
In the preceding paper (ENGELSNA, 1967) dealing with the effects of blue light, it was shown that the light-induced accumulation of hydroxycinnamic acids is regulated at the enzyme level and that all data can be explained on the basis of a light-stable pigment system. In this paper it will be shown that the response to prolonged irradiations with red and far-red light is determined b y the same mechanism and that there is no evidence that light-induced transformations of phytochrome are essential for the high-energy phenomena in the gherkin seedling. Methods and Materials These have been described in previous publications, namely, plants, irradiations and determination of hydroxyeinnamie acids in EN~ELS~A and M~:z~R (1965), extraction and assay of PADAse in EN(~LS~A (1967). Results
1. Synthesis o] Hydroxycinnamic Acids in Red and Far-Red Light. Current concepts about the photoreceptor involved in the response to prolonged irradiations (HER) partly originate from the results of irradiation sequences with light of different spectral regions (GR~L and VI~CE, 1965, 1966; W A G ~ and MOHR, 1966). Fig. 1 shows the results of such an experiment on the synthesis of hydroxycinnamic acids in gherkin hypocotyls. Groups of dark-grown seedlings were either irradiated for 8 hours with light of the qualities and intensities as indicated, or remained in darkness. The intensities of the red, far-red and high-intensity blue light are saturating intensities (Fig. 2; compare E~GV.LS~_~, 1967, Fig.4). After the pre-treatment each group was split into several sub-groups. One of these sub-groups was left in the same cabinet, one was transferred to darkness, and the remaining sub-groups were distributed over the other cabinets. The second light treatment lasted 16 hours. Hydroxycinnamic acids were determined at time zero and after 8 and 24 hours. The results can be summarized as follows: a) As long as the quality and intensity of the light remain unaltered, irradiation beyond 8 hours causes no further increase in phenol synthesis. b) A saturating intensity of blue light produces a higher amount of hydroxycinnamic acids than saturating intensities of red or far-red light. With the latter two light qualities, the amount produced is about the same. c) High-intensity blue light induces a rise in the synthesis of hydroxycinnamic acid in plants the phenol-synthesizing system of which has become saturated for red and far-red light. The same effect is seen with plants that have been pre-treated with low-intensity blue light.
Photoinduction of Phenylalanine Deaminase. I I
51
I n contrast, irradiation with high-intensity red or far-red light of plants the phenol-synthesizing system of which has been saturated for highintensity blue light does not lead to a further increase in hydroxycinnamic acids. However, after pre-treatment with low-intensity blue light there seems to be a small effect of high-intensity red and far-red light. d) I n all those cases where a second light t r e a t m e n t gives rise to a further enhancement in the accumulation of hydroxycinnamic acids the
2C
B
o=
~
k
Irradiation sequence
Fig. ]. Accumulation of hydroxyeirmamic acids in the hypocotyls of gherkin seedlings as a function of different light qualities and intensities (in fzW/cmZ). First light treatment lasting 8 hours (broad bars), second light treatment lasting 16 hours (narrow bars) relative effectiveness of the irradiation has been lowered by the pretreatment.
2. Induction el Phenylalanine Deaminase in Red and Far-Red Light. I n previous investigations (ENG~LSMA, 1967) dealing with the effects of blue light, a correlation was found between the level of phenylalanine deaminase (PADAse) and the rate of accumulation of hydroxycinnamic acids. Time-course studies of the changes in the PADAse level in darkgrown gherkin seedlings t h a t have been transferred to red or far-red light show that, as in blue light, the enzyme level passes through a m a x i m u m the height of which is a function of light intensity (Fig. 2). This correlates with the dependence on light intensity of the accumulation of hydroxycinnamie acids in red and far-red light (ENGv,LSMA and MExjv,~, 1965, Fig. 6). Fig. 2 further shows the effect on the PADAse level of some of the irradiation sequences used in the preceding section. The results can be summarized as follows: 4*
52
G. ENGELSMA:
a) The changes in PADAse level show a dependence on time which is independent of the spectral distribution and the intensity of the inducing light. b) I n blue light a higher enzyme level can be obtained than in red and far-red light. Saturating intensities of the latter light qualities produce maxima in the enzyme level, of about equal height.
o t~
E ~D r "E
ft.
0
2
1U 1Z 4 Time fr6om beginniSng of irradiation (hours)
14
16
Fig. 2. Levels of PADAse in the hypocotyls of gherkin seedlings as a function of irradiation sequences with different light qualities and intensities (in ~W/cm2). B blue, R red, FR far red e) High-intensity blue light induces a new m a x i m u m in the enzyme level in plants insensitive to further red and far-red irradiation. The reverse irradiation sequence, high-intensity far red after high-intensity blue, does not give rise to a second maximum. However, in plants preirradiated with low-intensity blue light a small increase in enzyme level occurs on subsequent exposure to high-intensity far-red light. d) I n all those eases where a second light treatment gives rise to a renewed induction of PADAse the relative effectiveness of the irradiation has been lowered b y the pre-treatment. F r o m the above it follows that, point by point, the effects on the accumulation of hydroxycinnamie acid can be explained in terms of PADAse induction.
Photoinduction of Phenylalanine Deaminase. II
53
3. Involvement oI Phytochrome. In comparison with prolonged highintensity red-light treatment a short irradiation with low-intensity red light results in a relatively large increase in hydroxycinnamic acids in gherkin hypocotyls (ENGELSMA and MwIJ~R, 1965). The effect of this short irradiation can be partly abolished by a subsequent exposure to far-red light, a result which suggests involvement of phytochrome. However, so far no indication could be obtained for a corresponding increase in the PADAse level induced by a short treatment with red light (Fig. 3).
darkness lO'red/~O0 btue600
M N
red2100
far red 740
Fig. 3. Effect of the cotyledons on the level of PADAse induced in the hypocotyls of gherkin seedlings by light of different spectral regions. PADAse was measured 3 hours from the beginning of the irradiation
4. E/leer o/ the Cotyledons. A difference between the effect of blue and that of red and far-red light is that only blue light is capable of inducing phenol synthesis to any appreciable extent in the hypocotyls of ecotylized seedlings (E~GELSMA and MwiJm~, 1965). Similar results have been obtained with respect to the induction of einnamic acid hydroxylase activity in hypocotyl segments (ENGELSMA, 1966). Fig. 3 shows that the induction of PADAse in gherkin hypocotyls is also more dependent on the cotyledons in red and far-red light than it is in blue. Discussion To account for the various effects of light of different spectral regions on the phenol synthesis in gherkin hypocotyls it seems necessary to assume that at least three pigment systems are involved: phytochrome and two photoreceptors that absorb maximally in the blue and far-red regions, respectively. The involvement of phytoehrome may be concluded from the red, far-red reversibility (ENGELSMA and M~IJER, 1965). Arguments that
54
G. ENGELSMA:
the effects of prolonged irradiations with blue light are mediated by a different pigment have been given in previous papers (E~o~LS~A and M ~ I J ~ , 1965; E~G~LS~A, 1966, 1967). I t was demonstrated that the response to blue light is controlled at the enzyme level and that the only function that needs to be attributed to the blue-absorbing pigment is that of initiating the sequence of dark reactions leading to enhanced enzyme production (ENG~LSM~, 1967). The effect of light intensity could be explained by assuming that the initiating reaction is a function of the mlmber of light quanta absorbed by this pigment. This implies that the high-energy phenomena due to blue light can be understood on the basis of a pigment which, unlike phytochrome, is stable in the light. The data presented in this paper show that the effects of prolonged irradiations with far-red are similar to those with blue light in t h a t the changes induced in the PADAse level follow an identical time course. This suggests that blue and far-red light initiate the same sequence of dark reactions. In Fig. 6 of the preceding paper a scheme has been designed for this reaction sequence (E~Gv.LS~A, 1967). However, there are two points of difference between the influence of blue and that of far red which make it improbable that only one pigment is mediating the effects of both spectral regions: a) 0 n l y blue light is capable of enhancing the PADAse level to any appreciable extent in the hypocotyls of eeotylized plants; b) under saturating conditions blue light induces a higher PADAse level in the hypocotyls of intact seedlings than does far-red light. The conclusion therefore seems inevitable that the effects of prolonged irradiations with far-red light are mediated by a pigment that is distinct from the blue-absorbing pigment, but in view of the similarity in dependence on light intensity we assume that both pigments function in a similar way. Increasing intensities of red light give rise to maxima in enzyme level approaching the level obtained in saturating far-red light. We therefore assume that one pigment only is responsible for the high-energy effects of the red, far-red part of the spectrum. Since in the reaction studied far-red light is more effective than red we except this pigment to have its absorption maximum at a wavelength above 700 nm. The conclusion that light from the blue and from the red, far-red region, apart from being perceived by different pigment systems, initiates the same sequence of dark reactions implies that it should be possible to interpret the results of sequential irradiations with different light qualities on the same basis as those with different intensities of blue light. The fact that, under saturating conditions, blue light produces a higher peak in enzyme level than either red or far-red light may be interpreted in two ways. The capacity of the blue-absorbing pigment system may be greater than that of the red, far-red absorbing one; or
Photminduction of Phenylalanine Deaminase. II
55
else the light-induced processes in the cotyledons and the hypocotyl that interact to induce enzyme synthesis in the latter are more favourable distributed in blue than in red and far-red light. Either assumption implies that the effect of saturating intensities of red and far-red light on the system that regulates the PADAse level should be equivalent to the effect of blue light of a particular intensity lower than its saturating intensity. In agreement with this we find that irradiation with highintensity blue light of plants pre-treated with saturating red or far-red light gives rise to a renewed increase in PADAse level, but that the height of this maximum is lower than in plants that have not been pre-treated. The same results are obtained with plants pre-trcated with low-intensity blue light (E~G~LSMA, 1967). On the same grounds we can explain the fact that irradiation with high-intensity far-red light of plants pre-treated with blue light has no effect when high-intensity blue light was used, but does have an effect after low-intensity blue light. In summary, for all irradiation sequences studied so far it seems that the changes in the responding system caused by pre-irradiation(s) determine the effect of a subsequent light treatment. The explanation given here for the H E R disagrees with a hypothesis published recently (H~mT~A~, 1966) in which H E R phenomena are interpreted on the basis of the effect of the various irradiation conditions on the concentration of the active form of phytoehrome (P730). Arguments against the supposition that the level of PT~0 is critical as regards the high-energy effects of red and far-red light on phenol synthesis in the gherkin are the following: 1. The HEI~ is due to induction of PADAse. No indication could be obtained that the red, far-red reversible effect which is generally considered as depending on the level of PTao, is achieved by the same mechanism. Similar evidence - - that is, that the phytochrome-mediated effect, and the high-energy effect depend on different mechanisms - - has been presented for anthocyanin synthesis in buckwheat seedlings (ScHERF and Z~NK, 1967). 2. According to HA~TMA~N (1966) irradiation with red light leads to accelerated destruction of phytochrome. If the response were determined by the level of P~80 we would expect a dependence on the intensity of the red light opposite to what has been actually observed (Fig. 2). 3. The results of experiments with irradiation sequences with different light qualities can be satisfactorily explained on the basis of regulation at the enzyme level. They do not provide evidence that changes caused by light at the pigment level play an important role. 4. The effect of blue light is not mediated by phytochrome, nor is there any indication that the blue-absorbing pigment has phytochrome-
56
G. E~GEnSMA:
like properties. There is no reason w h y similar effects of different spectral regions should be explained b y different mechanisms. Some support for the speculation previously made (E~c]~LSMA, 1967) - - t h a t the p r i m a r y process in the H E R is an oxidation-reduction reaction - - comes from investigations on the photoinduction of carotenoid synthesis in microorganisms (RIL~INC, 1964; RAy, 1967). I n addition to this there is evidence t h a t redox compounds play a role in the initiation of photomorphogenetic responses (KLEIN and EDSALL, 1966; SCHOPFE~, 1967). F o r the light-induced elongation of fern p r o t o n e m a t a a hypothesis has been proposed which is based on three pigment systems as well: phytoehrome, and two other photoreceptors which absorb in the blue and in the yellow to far-red region, respectively (MILLER and MZLLER, 1967). The hypothesis t h a t separate pigment systems are responsible for the high-energy p h e n o m e n a of different spectral regions could also explain the fact t h a t in some plants an action spectrum for the induction of a n t h o c y a n i n synthesis shows peaks in the blue and in the far-red region, and in other plants only in the blue region (see G]crLL and VINc]~, 1966). The author wishes to express his thanks to Miss E. M. LIN(~KENS, Miss E. C. PLAS and Mr. J. M. H. V A N BRUCG~N for technical assistance. References ENGELS~A, G. : The influence of light of different spectral regions on the synthesis of phenolic compounds in gherkin seedlings in relation to photomorphogenesis. III. Hydroxylation of cinnamic acid. Acta bet. neerl. 15, 394 ~05 (1966). - - Photoindnction of phenylalanine deaminase in gherkin seedlings. I. Effect of blue light. Planta (Berl.) 75, 207--219 (1967). --, and G. MEIZE~: The influence of light of different spectral regions on the synthesis of phenolic compounds in gherko seedlings in relation to photomorphogenesis. I. Biosynthesis of phenolic compounds. Acta bet. neerL 14, 54--72 (1965). GRILL, l~., and D. Vr~cE: Photocontrol of anthocyanin formation in turnip seedlings. II. The possible role of phytochrome in the response to prolonged irradiation with far-red or blue light. Planta (Berl.) 67, 122--135 (1965). - - - - Photoeontrol of anthoeyanin formation in turnip seedlings. III. The photoreceptors involved in the responses to prolonged irradiation. Planta (Berl.) 70, 1--12 (1966). HARTMA~, K. M.: A general hypothesis to interpret "high-energy phenomena" of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349--366 (1966). KLEIN, l~. M., and P. C. EDSALL: Substitution of redox chemicals for radiation in phytochrome-mediated morphogenesis. Plant Physiol. 41, 949--952 (1966). MILLER, J. H., and P. M. MILLE]C:Action spectra for light-induced elongation in fern protonemata. Physiol. Plant. (Kebenhavn) 20, 128--138 (1967).
Photoinduction of Phenylalanine Deaminase. II
57
Mom~, H.: Der EinfluB monochromatischer Strahlung auf das LEngenwachstum des Hypocotyls und auf die Anthoeyanbildung bei Keimlingen yon Sinapis alba L. Planta (BEE.) 49, 389--405 (1957). - - Differential gene activation as a mode of action of phytochrome 730. Photochem. Photobiol. 5, 469--483 (1966). RAy, W. : Untersuehungen fiber die liehtabhEngige Carotinoidsynthese. II. Ersatz der Liehtinduktion durch Mercuribenzoat. Planta (Ber].) 74, 263--277 (1967). I ~ I L L I I ~ G , ~I. C. " On the mechanism of photoinduction of carotenoid synthesis. Biochim. biophys. Acta (Amst.) 79, 464-475 (1964). Sct{~F, H., and M. H. ZnNK: Induction of anthocyanin and phenylalanine ammonia-lyse formation by a high energy light reaction and its control through the phy%ochrome system. Z. Pflanzenphysioh 56, 203--206 (1967). Sct{OPF~, P.: Weitere Untersuchungen zur phytochrominduzierten Akkumulation yon Ascorbins~ure beim Senfkeimling (Sinapis alba L.). Planta (Beth) 74, 210--227 (1967). SIEG~L~IA~,H.W., and S.B. HEI~DRICKS: Photocontrol of anthocyanin formation in turnip and red cabbage seedlings. Plant Physiol. 82, 393--398 (1957). WAG~E~, E., u. H. Moa~: Kinetische Studien zur Interpretation der Wirkung yon Sukzedanbestrahlungen mit Hellrot und Dunkelrot bei der Photomorphogenese (Anthoeyansynthese bei Sinapis alba L.). Planta (Berl.) 70, 34--41 (1966). Dr. G. ENGELSMA Philips Research Laboratories N. V. Philips' Gloeilampenfabrieken Eindhoven, Netherlands
Photoinduction of Phenylalanine Deaminase in Gherkin Seedlings II. Effect of Red and Far-Red Light G. ENGELS~A Philips Research Laboratories, N. V. Philips' Gloeflampenfabrieken, Eindhoven, Netherlands Received July 3, 1967
Summary. 1. Exposure of dark-grown gherkin seedlings to red or far-red light causes changes in the level of phenylalanine deaminase (PADAse) which follow the same time course and show a similar dependence on light intensity as the changes induced in blue light. From this it is concluded that light of different regions of the visible spectrum is initiating the same sequence of dark reactions leading to enhanced production of PADAse, followed by a phase of inactivation of this enzyme and repression of its synthesis. 2. In blue light a higher PADAse level can be obtained than in red or far-red light, and only blue light is capable of inducing PADAse synthesis in excised hypocotyls. This suggests that different pigment systems are involved in the highenergy phenomena in the gherkin seedling. 3. The effects of irradiation sequences with different light qualities and intensities on the accumulation of hydroxycinnamic acids can be explained on the basis of the regulatory mechanisms which control the PADAse level. Introduction The effects of red and far-red light on the synthesis of hydroxycinnamic acids in gherkin hypocotyls are more complicated than those of blue light. On the one hand there is a response to a small dose of red light which can be abolished with far-red light. On the other hand there is the reaction caused b y prolonged irradiations with red and farred light which, similarly as with blue light, reaches saturation at much higher light doses, gives rise to a m u c h g r e a t e r effect, a n d c a n n o t be r e v e r s e d (E~GELSMA a n d Mv,IjEI~, 1965). This l a t t e r r e a c t i o n is k n o w n as t h e h i g h - e n e r g y r e a c t i o n of p h o t o m o r p h o g e n e s i s ( H E R ) . I t is g e n e r a l l y a s s u m e d t h a t t h e former t y p e of r e a c t i o n is controlled b y t h e p h o t o - r e v e r s i b l e p i g m e n t p h y t o c h r o m e , whereas t h e H E R was originally t h o u g h t to be due to a second p i g m e n t (SIEGnLMA~ a n d Hwl~I)l~ICI~S, 1957; Mom~, 1957). R e c e n t l y a h y p o t h e s i s has been p r o p o s e d which, a t least as far as t h e effects of r e d a n d far-red light are concerned, explains t h e H E R also in t e r m s of control b y p h y t o c h r o m e (I-IAI~TMANI~, 1966; see also 1VIOHI~, 1966). 4 Planta (Berl.), Bd. 77
50
G. ENGELSMA:
In the preceding paper (ENGELSNA, 1967) dealing with the effects of blue light, it was shown that the light-induced accumulation of hydroxycinnamic acids is regulated at the enzyme level and that all data can be explained on the basis of a light-stable pigment system. In this paper it will be shown that the response to prolonged irradiations with red and far-red light is determined b y the same mechanism and that there is no evidence that light-induced transformations of phytochrome are essential for the high-energy phenomena in the gherkin seedling. Methods and Materials These have been described in previous publications, namely, plants, irradiations and determination of hydroxyeinnamie acids in EN~ELS~A and M~:z~R (1965), extraction and assay of PADAse in EN(~LS~A (1967). Results
1. Synthesis o] Hydroxycinnamic Acids in Red and Far-Red Light. Current concepts about the photoreceptor involved in the response to prolonged irradiations (HER) partly originate from the results of irradiation sequences with light of different spectral regions (GR~L and VI~CE, 1965, 1966; W A G ~ and MOHR, 1966). Fig. 1 shows the results of such an experiment on the synthesis of hydroxycinnamic acids in gherkin hypocotyls. Groups of dark-grown seedlings were either irradiated for 8 hours with light of the qualities and intensities as indicated, or remained in darkness. The intensities of the red, far-red and high-intensity blue light are saturating intensities (Fig. 2; compare E~GV.LS~_~, 1967, Fig.4). After the pre-treatment each group was split into several sub-groups. One of these sub-groups was left in the same cabinet, one was transferred to darkness, and the remaining sub-groups were distributed over the other cabinets. The second light treatment lasted 16 hours. Hydroxycinnamic acids were determined at time zero and after 8 and 24 hours. The results can be summarized as follows: a) As long as the quality and intensity of the light remain unaltered, irradiation beyond 8 hours causes no further increase in phenol synthesis. b) A saturating intensity of blue light produces a higher amount of hydroxycinnamic acids than saturating intensities of red or far-red light. With the latter two light qualities, the amount produced is about the same. c) High-intensity blue light induces a rise in the synthesis of hydroxycinnamic acid in plants the phenol-synthesizing system of which has become saturated for red and far-red light. The same effect is seen with plants that have been pre-treated with low-intensity blue light.
Photoinduction of Phenylalanine Deaminase. I I
51
I n contrast, irradiation with high-intensity red or far-red light of plants the phenol-synthesizing system of which has been saturated for highintensity blue light does not lead to a further increase in hydroxycinnamic acids. However, after pre-treatment with low-intensity blue light there seems to be a small effect of high-intensity red and far-red light. d) I n all those cases where a second light t r e a t m e n t gives rise to a further enhancement in the accumulation of hydroxycinnamic acids the
2C
B
o=
~
k
Irradiation sequence
Fig. ]. Accumulation of hydroxyeirmamic acids in the hypocotyls of gherkin seedlings as a function of different light qualities and intensities (in fzW/cmZ). First light treatment lasting 8 hours (broad bars), second light treatment lasting 16 hours (narrow bars) relative effectiveness of the irradiation has been lowered by the pretreatment.
2. Induction el Phenylalanine Deaminase in Red and Far-Red Light. I n previous investigations (ENG~LSMA, 1967) dealing with the effects of blue light, a correlation was found between the level of phenylalanine deaminase (PADAse) and the rate of accumulation of hydroxycinnamic acids. Time-course studies of the changes in the PADAse level in darkgrown gherkin seedlings t h a t have been transferred to red or far-red light show that, as in blue light, the enzyme level passes through a m a x i m u m the height of which is a function of light intensity (Fig. 2). This correlates with the dependence on light intensity of the accumulation of hydroxycinnamie acids in red and far-red light (ENGv,LSMA and MExjv,~, 1965, Fig. 6). Fig. 2 further shows the effect on the PADAse level of some of the irradiation sequences used in the preceding section. The results can be summarized as follows: 4*
52
G. ENGELSMA:
a) The changes in PADAse level show a dependence on time which is independent of the spectral distribution and the intensity of the inducing light. b) I n blue light a higher enzyme level can be obtained than in red and far-red light. Saturating intensities of the latter light qualities produce maxima in the enzyme level, of about equal height.
o t~
E ~D r "E
ft.
0
2
1U 1Z 4 Time fr6om beginniSng of irradiation (hours)
14
16
Fig. 2. Levels of PADAse in the hypocotyls of gherkin seedlings as a function of irradiation sequences with different light qualities and intensities (in ~W/cm2). B blue, R red, FR far red e) High-intensity blue light induces a new m a x i m u m in the enzyme level in plants insensitive to further red and far-red irradiation. The reverse irradiation sequence, high-intensity far red after high-intensity blue, does not give rise to a second maximum. However, in plants preirradiated with low-intensity blue light a small increase in enzyme level occurs on subsequent exposure to high-intensity far-red light. d) I n all those eases where a second light treatment gives rise to a renewed induction of PADAse the relative effectiveness of the irradiation has been lowered b y the pre-treatment. F r o m the above it follows that, point by point, the effects on the accumulation of hydroxycinnamie acid can be explained in terms of PADAse induction.
Photoinduction of Phenylalanine Deaminase. II
53
3. Involvement oI Phytochrome. In comparison with prolonged highintensity red-light treatment a short irradiation with low-intensity red light results in a relatively large increase in hydroxycinnamic acids in gherkin hypocotyls (ENGELSMA and MwIJ~R, 1965). The effect of this short irradiation can be partly abolished by a subsequent exposure to far-red light, a result which suggests involvement of phytochrome. However, so far no indication could be obtained for a corresponding increase in the PADAse level induced by a short treatment with red light (Fig. 3).
darkness lO'red/~O0 btue600
M N
red2100
far red 740
Fig. 3. Effect of the cotyledons on the level of PADAse induced in the hypocotyls of gherkin seedlings by light of different spectral regions. PADAse was measured 3 hours from the beginning of the irradiation
4. E/leer o/ the Cotyledons. A difference between the effect of blue and that of red and far-red light is that only blue light is capable of inducing phenol synthesis to any appreciable extent in the hypocotyls of ecotylized seedlings (E~GELSMA and MwiJm~, 1965). Similar results have been obtained with respect to the induction of einnamic acid hydroxylase activity in hypocotyl segments (ENGELSMA, 1966). Fig. 3 shows that the induction of PADAse in gherkin hypocotyls is also more dependent on the cotyledons in red and far-red light than it is in blue. Discussion To account for the various effects of light of different spectral regions on the phenol synthesis in gherkin hypocotyls it seems necessary to assume that at least three pigment systems are involved: phytochrome and two photoreceptors that absorb maximally in the blue and far-red regions, respectively. The involvement of phytoehrome may be concluded from the red, far-red reversibility (ENGELSMA and M~IJER, 1965). Arguments that
54
G. ENGELSMA:
the effects of prolonged irradiations with blue light are mediated by a different pigment have been given in previous papers (E~o~LS~A and M ~ I J ~ , 1965; E~G~LS~A, 1966, 1967). I t was demonstrated that the response to blue light is controlled at the enzyme level and that the only function that needs to be attributed to the blue-absorbing pigment is that of initiating the sequence of dark reactions leading to enhanced enzyme production (ENG~LSM~, 1967). The effect of light intensity could be explained by assuming that the initiating reaction is a function of the mlmber of light quanta absorbed by this pigment. This implies that the high-energy phenomena due to blue light can be understood on the basis of a pigment which, unlike phytochrome, is stable in the light. The data presented in this paper show that the effects of prolonged irradiations with far-red are similar to those with blue light in t h a t the changes induced in the PADAse level follow an identical time course. This suggests that blue and far-red light initiate the same sequence of dark reactions. In Fig. 6 of the preceding paper a scheme has been designed for this reaction sequence (E~Gv.LS~A, 1967). However, there are two points of difference between the influence of blue and that of far red which make it improbable that only one pigment is mediating the effects of both spectral regions: a) 0 n l y blue light is capable of enhancing the PADAse level to any appreciable extent in the hypocotyls of eeotylized plants; b) under saturating conditions blue light induces a higher PADAse level in the hypocotyls of intact seedlings than does far-red light. The conclusion therefore seems inevitable that the effects of prolonged irradiations with far-red light are mediated by a pigment that is distinct from the blue-absorbing pigment, but in view of the similarity in dependence on light intensity we assume that both pigments function in a similar way. Increasing intensities of red light give rise to maxima in enzyme level approaching the level obtained in saturating far-red light. We therefore assume that one pigment only is responsible for the high-energy effects of the red, far-red part of the spectrum. Since in the reaction studied far-red light is more effective than red we except this pigment to have its absorption maximum at a wavelength above 700 nm. The conclusion that light from the blue and from the red, far-red region, apart from being perceived by different pigment systems, initiates the same sequence of dark reactions implies that it should be possible to interpret the results of sequential irradiations with different light qualities on the same basis as those with different intensities of blue light. The fact that, under saturating conditions, blue light produces a higher peak in enzyme level than either red or far-red light may be interpreted in two ways. The capacity of the blue-absorbing pigment system may be greater than that of the red, far-red absorbing one; or
Photminduction of Phenylalanine Deaminase. II
55
else the light-induced processes in the cotyledons and the hypocotyl that interact to induce enzyme synthesis in the latter are more favourable distributed in blue than in red and far-red light. Either assumption implies that the effect of saturating intensities of red and far-red light on the system that regulates the PADAse level should be equivalent to the effect of blue light of a particular intensity lower than its saturating intensity. In agreement with this we find that irradiation with highintensity blue light of plants pre-treated with saturating red or far-red light gives rise to a renewed increase in PADAse level, but that the height of this maximum is lower than in plants that have not been pre-treated. The same results are obtained with plants pre-trcated with low-intensity blue light (E~G~LSMA, 1967). On the same grounds we can explain the fact that irradiation with high-intensity far-red light of plants pre-treated with blue light has no effect when high-intensity blue light was used, but does have an effect after low-intensity blue light. In summary, for all irradiation sequences studied so far it seems that the changes in the responding system caused by pre-irradiation(s) determine the effect of a subsequent light treatment. The explanation given here for the H E R disagrees with a hypothesis published recently (H~mT~A~, 1966) in which H E R phenomena are interpreted on the basis of the effect of the various irradiation conditions on the concentration of the active form of phytoehrome (P730). Arguments against the supposition that the level of PT~0 is critical as regards the high-energy effects of red and far-red light on phenol synthesis in the gherkin are the following: 1. The HEI~ is due to induction of PADAse. No indication could be obtained that the red, far-red reversible effect which is generally considered as depending on the level of PTao, is achieved by the same mechanism. Similar evidence - - that is, that the phytochrome-mediated effect, and the high-energy effect depend on different mechanisms - - has been presented for anthocyanin synthesis in buckwheat seedlings (ScHERF and Z~NK, 1967). 2. According to HA~TMA~N (1966) irradiation with red light leads to accelerated destruction of phytochrome. If the response were determined by the level of P~80 we would expect a dependence on the intensity of the red light opposite to what has been actually observed (Fig. 2). 3. The results of experiments with irradiation sequences with different light qualities can be satisfactorily explained on the basis of regulation at the enzyme level. They do not provide evidence that changes caused by light at the pigment level play an important role. 4. The effect of blue light is not mediated by phytochrome, nor is there any indication that the blue-absorbing pigment has phytochrome-
56
G. E~GEnSMA:
like properties. There is no reason w h y similar effects of different spectral regions should be explained b y different mechanisms. Some support for the speculation previously made (E~c]~LSMA, 1967) - - t h a t the p r i m a r y process in the H E R is an oxidation-reduction reaction - - comes from investigations on the photoinduction of carotenoid synthesis in microorganisms (RIL~INC, 1964; RAy, 1967). I n addition to this there is evidence t h a t redox compounds play a role in the initiation of photomorphogenetic responses (KLEIN and EDSALL, 1966; SCHOPFE~, 1967). F o r the light-induced elongation of fern p r o t o n e m a t a a hypothesis has been proposed which is based on three pigment systems as well: phytoehrome, and two other photoreceptors which absorb in the blue and in the yellow to far-red region, respectively (MILLER and MZLLER, 1967). The hypothesis t h a t separate pigment systems are responsible for the high-energy p h e n o m e n a of different spectral regions could also explain the fact t h a t in some plants an action spectrum for the induction of a n t h o c y a n i n synthesis shows peaks in the blue and in the far-red region, and in other plants only in the blue region (see G]crLL and VINc]~, 1966). The author wishes to express his thanks to Miss E. M. LIN(~KENS, Miss E. C. PLAS and Mr. J. M. H. V A N BRUCG~N for technical assistance. References ENGELS~A, G. : The influence of light of different spectral regions on the synthesis of phenolic compounds in gherkin seedlings in relation to photomorphogenesis. III. Hydroxylation of cinnamic acid. Acta bet. neerl. 15, 394 ~05 (1966). - - Photoindnction of phenylalanine deaminase in gherkin seedlings. I. Effect of blue light. Planta (Berl.) 75, 207--219 (1967). --, and G. MEIZE~: The influence of light of different spectral regions on the synthesis of phenolic compounds in gherko seedlings in relation to photomorphogenesis. I. Biosynthesis of phenolic compounds. Acta bet. neerL 14, 54--72 (1965). GRILL, l~., and D. Vr~cE: Photocontrol of anthocyanin formation in turnip seedlings. II. The possible role of phytochrome in the response to prolonged irradiation with far-red or blue light. Planta (Berl.) 67, 122--135 (1965). - - - - Photoeontrol of anthoeyanin formation in turnip seedlings. III. The photoreceptors involved in the responses to prolonged irradiation. Planta (Berl.) 70, 1--12 (1966). HARTMA~, K. M.: A general hypothesis to interpret "high-energy phenomena" of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349--366 (1966). KLEIN, l~. M., and P. C. EDSALL: Substitution of redox chemicals for radiation in phytochrome-mediated morphogenesis. Plant Physiol. 41, 949--952 (1966). MILLER, J. H., and P. M. MILLE]C:Action spectra for light-induced elongation in fern protonemata. Physiol. Plant. (Kebenhavn) 20, 128--138 (1967).
Photoinduction of Phenylalanine Deaminase. II
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Mom~, H.: Der EinfluB monochromatischer Strahlung auf das LEngenwachstum des Hypocotyls und auf die Anthoeyanbildung bei Keimlingen yon Sinapis alba L. Planta (BEE.) 49, 389--405 (1957). - - Differential gene activation as a mode of action of phytochrome 730. Photochem. Photobiol. 5, 469--483 (1966). RAy, W. : Untersuehungen fiber die liehtabhEngige Carotinoidsynthese. II. Ersatz der Liehtinduktion durch Mercuribenzoat. Planta (Ber].) 74, 263--277 (1967). I ~ I L L I I ~ G , ~I. C. " On the mechanism of photoinduction of carotenoid synthesis. Biochim. biophys. Acta (Amst.) 79, 464-475 (1964). Sct{~F, H., and M. H. ZnNK: Induction of anthocyanin and phenylalanine ammonia-lyse formation by a high energy light reaction and its control through the phy%ochrome system. Z. Pflanzenphysioh 56, 203--206 (1967). Sct{OPF~, P.: Weitere Untersuchungen zur phytochrominduzierten Akkumulation yon Ascorbins~ure beim Senfkeimling (Sinapis alba L.). Planta (Beth) 74, 210--227 (1967). SIEG~L~IA~,H.W., and S.B. HEI~DRICKS: Photocontrol of anthocyanin formation in turnip and red cabbage seedlings. Plant Physiol. 82, 393--398 (1957). WAG~E~, E., u. H. Moa~: Kinetische Studien zur Interpretation der Wirkung yon Sukzedanbestrahlungen mit Hellrot und Dunkelrot bei der Photomorphogenese (Anthoeyansynthese bei Sinapis alba L.). Planta (Berl.) 70, 34--41 (1966). Dr. G. ENGELSMA Philips Research Laboratories N. V. Philips' Gloeilampenfabrieken Eindhoven, Netherlands
