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A phytochrome-mediated increase in the level of phenylalanine ammonialyase activity in the terminal buds of Pisum sativum
BIOCtIIMICA E.T BIOPIIYSICA ACTA
PRELIMINARY
805
NOTES
BBA 21198
A phytochrome-mediated increase in the level of phenylalanine ammonialyase activity in the terminal buds of P/sum sativum Previous investigations have shown that flavonoid biosynthesis in the terminal buds of etiolated seedlings of Pisum sativum vat. Alaska is stimulated by the photoactivation of phytochrome l-a. We have investigated the effect of irradiation with red light on the levels of certain enzymes known to be involved in the biosynthesis of tile flavonoids, and preliminary results indicate that the photoconversion of the red-absorbing form of phytochrome (Pr) to the active far-red-absorbing form (Pfr) brings about a specific increase in the level or activity of L-phenylalanine ammonialyase (EC4.3.I.5), previously called phenylalanine deaminase, an enzyme which catalyzes the deamination of L-phenylalanine to trans-cinnamic acid and which was first found in higher plants b y KOUKOL AND CONN~. The kinetics of this response are consistent with an induction of enzyme synthesis b y phytochrome being involved. Growth of plant material, the nature of the light sources, and the method of harvesting were as previously described 3, and measurements of phenylalanine ammonia-lyase activity in crude enzyme preparations were made by a modification of a method of KOUKOLAND CONN4. Terminal buds (2oo-4oo per treatment) were excised under dim green light and homogenized in a Griffiths tube with o.2 M borate buffer (pH 9.o), containing o.I ffmole/ml reduced glutathione. The homogenates were centrifuged at 26ooo × g for 15 min at 2 ° and the supernatant made up to I.O ml corresponding to o.I g fresh weight with buffered glutathione. The enzyme preparations were incubated for 6 h at 35 ° with 15o ffmoles L-phenylalanine, 6o ffmoles reduced
x 5CZ
O! rO k
b o
2.0 .,c: 1.6
& 1.2 0.8
0.4
/
O, 12
16
o
20 Time
(h)
Fig. I. K i n e t i c s of increase in p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y e x t r a c t a b l e f r o m t h e t e r m i n a l b u d s a f t e r i r r a d i a t i o n of i n t a c t , 8-day-old etiolated seedlings w i t h 2oo k e r g s / c m ~ red light. T h e light m e d i a t e d increase in e n z y m e a c t i v i t y (i.e., a c t i v i t y of e x t r a c t f r o m t r e a t e d p l a n t minus a c t i v i t y of e x t r a c t f r o m d a r k - c o n t r o l plant) is p l o t t e d as (a) A e n z y m e u n i t s / m g protein, a n d as (b) zt e n z y m e u n i t s / g fresh wt.
Biochim. Biophys. Acta, 148 (1967) 805-807
806
PRELIMINARY
NOTES
glutathione, and 1.2 mmoles borate buffer (pH 9.o), in a totM volume of 15.o m!. The reaction was stopped by the addition of o.6 ml 6 M HCI and the cinnamic acid produced extracted into ether. The amount of cinnamate produced was measured spectrophotometrically in ether, using an observed molar extinction coefficien± at 27 ° n m of 219oo. An enzyme unit is arbitrarily defined as that amount of enzyme which will produce o.I/,mo!e cinnalnaie per h from phenylalanine under the above conditions. Kinetic analysis shows that in seedlings which were irradiated with red light (2oo kergs/cm 2) and returned to darkness, an increase in phenylalanine ammonia-lyase activity occurred, reaching a peak at 6-1o h and declining somewhat between io and 20 h (Fig. 1). Enzyme activity is presented both as units/g fresh weight and as units/mg protein showing quaiitatively similar responses. T h a t this response to light is typical phytochrome-mediaied response is shown by its low energy requirement and its logarithmic dose-response relationship (Fig. 2), and by its photoreversibility
~
b
J
f
/ J
z O0
260
400
60C U ~4 k ergs/cm 2
40
4(50
F i g . 2. D o s e - r e s p o n s e r e l a ' c i o n s h i p of r e d l i g h t m e d i a t e d i n c r e a s e i n p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y ; (a) l i n e a r p l o t , (b) s e m i - l o g a r i t h m i c p l o t . 8 - d a y - o l d e t i o l a t e d s e e d l i n g s w e r e u s e d a n d enzyme extracts were made io h after the irradiation treatments.
b y far red light (Table I). The time course of the increase in phenylalanine ammonialyase activity suggests that Pfr may be acting in this system, directly or indirectly. by inducing the synthesis of the enzyme. It is also possible that the decrease in level of the enzyme after zo h may be due to the feedback regression of the synthesis of the enzyme by one of the products of its action. BOTTOMLEY.SlVIITHAND GALSTON~ have shown that the synthesis of the acylated kaempferol and quercetin triglucosides TABLE
I O F RED LIGHT-INDUCED INCREASE iN PHENYLALANIN]~ F A R - R E D LIGHT
PHOTOREVERSIBILIT¥ ACTIVITY
BY
AMMONIA~LYASE
8-day-old etiolated seedlings were exposed to 5 ° kergs/cm ~ red light (6oo-69o m # ) followed immediately b y 15oo kergs/cm ~ far-red light 735-13oo m ~ I~ed -- far-redl; or to the red light treatment alone (Red) ; or to far red alone (Far-red) ; or nnirradiated as contro]s (Dark]. E n z y m e preparations were m a d e io h after irradiation.
ExpL No.
I 2
Phe~ylala~i*ae ~mmo~aia-lyase acEivity tu~its/g fl,esh wa. J Dark
Red
Red q-far-red
Far-rod
o.42 0.44
1.52 1.43
I.O 4 0.99
o. 78 0.80
Biochim. Biophys. Acta, 148 (x967) 8 0 5 - 8 0 7
PRELIMINARY
807
NOTES
two of the final products of the p a t h w a y , occurs at a m a x i m u m rate between 6 and IO h after red light treatment, coinciding with the peak in activity of the enzyme. There is as yet no further evidence t h a t induction or feedback repression are involved, b u t experiments are in progress to investigate these possibilities. Other investigators have reported light-mediated induction of phenylalanine ammonia-lyase 6-n. I n none of these cases has a response to a single photoactivation of p h y t o c h r o m e been proved. Thus the results reported here appear to be the first demonstration of an unequivocal p h y t o c h r o m e - m e d i a t e d increase in this enzyme. We have also investigated the effects of photoactivation of p h y t o c h r o m e on the l[evels of s h i k i m a t e : N A D P oxidoreductase (EC 1.I.1.25), a further enzyme involved in the biosynthesis of the flavonoids, using the methods of BALINSKY AND DAV:EESTM.This enzyme is present at 40-80 times the level of phenylalanine ammonialyase, but its level is not affected at all b y red light treatment. This suggests t h a t p h y t o c h r o m e is not acting b y bringing about an enhanced synthesis of all the enzymes in the general p a t h w a y of flavonoid biosynthesis, and t h a t its controlling effect could be exerted t h r o u g h the control of the single enzyme, phenylalanine ammonia-lyase. Investigations into possible changes in other enzymes of flavonoid biosynthesis are proceeding. T h a t Pfr a p p a r e n t l y acts in this system b y bringing about increased syn£hesis of an enzyme is in accordance with the hypothesis t h a t p h y t o c h r o m e acts t h r o u g h the control of e n z y m e synthesis, b u t it constitutes no proof t h a t direct control of gene activation is the p r i m a r y role of p h y t o c h r o m e as has been recently suggested 13. A n y hypothesis a d v a n c e d to explain these results m u s t take into account the fact t h a t e n z y m e synthesis proceeds at a low rate in darkness, and t h a t irradiation treatm e n t only has a quantitative effect on the rate of e n z y m e synthesis. This work was supported b y a grant from the University of L o n d o n Central Research Fund. T. H. A. is a holder of a Science Research Council Research Studentship. Department of Botany, Queen M a r y College, London, E . L (Great Britain) i 2 3 4 5 6 7 8 9 IO ii 12 13
T. H. ATTRIDGE HARRY SMITH
F. E. MUMFORD,D. H. SMITHAND J. R. CASTLE,Plant Physiol., 36 (1961) 752. ~[. FURUYAAND R. G. THOMAS,Plant Physiol., 39 (1964) 634. V?. BOTTOMLEY,H. SMITHAND A. W. GALSTON,Phytochemistry, 5 (1966) 117. J- KOVKOLAND E. E. CONN,J. Biol. Chem., 236 (1961) 2692. V~. BOTTOML•Y, H. SMITHAND A. W. GALSTON,Nature, 207 (1965) 1311. M. ZUCKER,Plant Physiol., 4° (1965) 779. C. NITSCHAND J. P. NITSClL Compt. Rend., 262 (1966) 11o2. G. ENGELSMA,Planta, 75 (1967) 207G. ENGELSMA,Planta, in the press. 12~.DURSTAND H. MOI-IR,Naturwissenschaften, 53 (1966) 531. ~[. SCHERFAI,IDM. H. ZEI,IK,Z. Pflanzenphysiol., 56 (1967) 203. D. BALINSKYAND D. D. DAVI~S,Biochem. J., 80 (1961) 292. H. MOHR, Photochem. Photobiol., 5 (1966) 469.
Received September xlth, 1967 Biochim. Biophys. Acta, 148 (1967) 805-807
PRELIMINARY
805
NOTES
BBA 21198
A phytochrome-mediated increase in the level of phenylalanine ammonialyase activity in the terminal buds of P/sum sativum Previous investigations have shown that flavonoid biosynthesis in the terminal buds of etiolated seedlings of Pisum sativum vat. Alaska is stimulated by the photoactivation of phytochrome l-a. We have investigated the effect of irradiation with red light on the levels of certain enzymes known to be involved in the biosynthesis of tile flavonoids, and preliminary results indicate that the photoconversion of the red-absorbing form of phytochrome (Pr) to the active far-red-absorbing form (Pfr) brings about a specific increase in the level or activity of L-phenylalanine ammonialyase (EC4.3.I.5), previously called phenylalanine deaminase, an enzyme which catalyzes the deamination of L-phenylalanine to trans-cinnamic acid and which was first found in higher plants b y KOUKOL AND CONN~. The kinetics of this response are consistent with an induction of enzyme synthesis b y phytochrome being involved. Growth of plant material, the nature of the light sources, and the method of harvesting were as previously described 3, and measurements of phenylalanine ammonia-lyase activity in crude enzyme preparations were made by a modification of a method of KOUKOLAND CONN4. Terminal buds (2oo-4oo per treatment) were excised under dim green light and homogenized in a Griffiths tube with o.2 M borate buffer (pH 9.o), containing o.I ffmole/ml reduced glutathione. The homogenates were centrifuged at 26ooo × g for 15 min at 2 ° and the supernatant made up to I.O ml corresponding to o.I g fresh weight with buffered glutathione. The enzyme preparations were incubated for 6 h at 35 ° with 15o ffmoles L-phenylalanine, 6o ffmoles reduced
x 5CZ
O! rO k
b o
2.0 .,c: 1.6
& 1.2 0.8
0.4
/
O, 12
16
o
20 Time
(h)
Fig. I. K i n e t i c s of increase in p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y e x t r a c t a b l e f r o m t h e t e r m i n a l b u d s a f t e r i r r a d i a t i o n of i n t a c t , 8-day-old etiolated seedlings w i t h 2oo k e r g s / c m ~ red light. T h e light m e d i a t e d increase in e n z y m e a c t i v i t y (i.e., a c t i v i t y of e x t r a c t f r o m t r e a t e d p l a n t minus a c t i v i t y of e x t r a c t f r o m d a r k - c o n t r o l plant) is p l o t t e d as (a) A e n z y m e u n i t s / m g protein, a n d as (b) zt e n z y m e u n i t s / g fresh wt.
Biochim. Biophys. Acta, 148 (1967) 805-807
806
PRELIMINARY
NOTES
glutathione, and 1.2 mmoles borate buffer (pH 9.o), in a totM volume of 15.o m!. The reaction was stopped by the addition of o.6 ml 6 M HCI and the cinnamic acid produced extracted into ether. The amount of cinnamate produced was measured spectrophotometrically in ether, using an observed molar extinction coefficien± at 27 ° n m of 219oo. An enzyme unit is arbitrarily defined as that amount of enzyme which will produce o.I/,mo!e cinnalnaie per h from phenylalanine under the above conditions. Kinetic analysis shows that in seedlings which were irradiated with red light (2oo kergs/cm 2) and returned to darkness, an increase in phenylalanine ammonia-lyase activity occurred, reaching a peak at 6-1o h and declining somewhat between io and 20 h (Fig. 1). Enzyme activity is presented both as units/g fresh weight and as units/mg protein showing quaiitatively similar responses. T h a t this response to light is typical phytochrome-mediaied response is shown by its low energy requirement and its logarithmic dose-response relationship (Fig. 2), and by its photoreversibility
~
b
J
f
/ J
z O0
260
400
60C U ~4 k ergs/cm 2
40
4(50
F i g . 2. D o s e - r e s p o n s e r e l a ' c i o n s h i p of r e d l i g h t m e d i a t e d i n c r e a s e i n p h e n y l a l a n i n e a m m o n i a - l y a s e a c t i v i t y ; (a) l i n e a r p l o t , (b) s e m i - l o g a r i t h m i c p l o t . 8 - d a y - o l d e t i o l a t e d s e e d l i n g s w e r e u s e d a n d enzyme extracts were made io h after the irradiation treatments.
b y far red light (Table I). The time course of the increase in phenylalanine ammonialyase activity suggests that Pfr may be acting in this system, directly or indirectly. by inducing the synthesis of the enzyme. It is also possible that the decrease in level of the enzyme after zo h may be due to the feedback regression of the synthesis of the enzyme by one of the products of its action. BOTTOMLEY.SlVIITHAND GALSTON~ have shown that the synthesis of the acylated kaempferol and quercetin triglucosides TABLE
I O F RED LIGHT-INDUCED INCREASE iN PHENYLALANIN]~ F A R - R E D LIGHT
PHOTOREVERSIBILIT¥ ACTIVITY
BY
AMMONIA~LYASE
8-day-old etiolated seedlings were exposed to 5 ° kergs/cm ~ red light (6oo-69o m # ) followed immediately b y 15oo kergs/cm ~ far-red light 735-13oo m ~ I~ed -- far-redl; or to the red light treatment alone (Red) ; or to far red alone (Far-red) ; or nnirradiated as contro]s (Dark]. E n z y m e preparations were m a d e io h after irradiation.
ExpL No.
I 2
Phe~ylala~i*ae ~mmo~aia-lyase acEivity tu~its/g fl,esh wa. J Dark
Red
Red q-far-red
Far-rod
o.42 0.44
1.52 1.43
I.O 4 0.99
o. 78 0.80
Biochim. Biophys. Acta, 148 (x967) 8 0 5 - 8 0 7
PRELIMINARY
807
NOTES
two of the final products of the p a t h w a y , occurs at a m a x i m u m rate between 6 and IO h after red light treatment, coinciding with the peak in activity of the enzyme. There is as yet no further evidence t h a t induction or feedback repression are involved, b u t experiments are in progress to investigate these possibilities. Other investigators have reported light-mediated induction of phenylalanine ammonia-lyase 6-n. I n none of these cases has a response to a single photoactivation of p h y t o c h r o m e been proved. Thus the results reported here appear to be the first demonstration of an unequivocal p h y t o c h r o m e - m e d i a t e d increase in this enzyme. We have also investigated the effects of photoactivation of p h y t o c h r o m e on the l[evels of s h i k i m a t e : N A D P oxidoreductase (EC 1.I.1.25), a further enzyme involved in the biosynthesis of the flavonoids, using the methods of BALINSKY AND DAV:EESTM.This enzyme is present at 40-80 times the level of phenylalanine ammonialyase, but its level is not affected at all b y red light treatment. This suggests t h a t p h y t o c h r o m e is not acting b y bringing about an enhanced synthesis of all the enzymes in the general p a t h w a y of flavonoid biosynthesis, and t h a t its controlling effect could be exerted t h r o u g h the control of the single enzyme, phenylalanine ammonia-lyase. Investigations into possible changes in other enzymes of flavonoid biosynthesis are proceeding. T h a t Pfr a p p a r e n t l y acts in this system b y bringing about increased syn£hesis of an enzyme is in accordance with the hypothesis t h a t p h y t o c h r o m e acts t h r o u g h the control of e n z y m e synthesis, b u t it constitutes no proof t h a t direct control of gene activation is the p r i m a r y role of p h y t o c h r o m e as has been recently suggested 13. A n y hypothesis a d v a n c e d to explain these results m u s t take into account the fact t h a t e n z y m e synthesis proceeds at a low rate in darkness, and t h a t irradiation treatm e n t only has a quantitative effect on the rate of e n z y m e synthesis. This work was supported b y a grant from the University of L o n d o n Central Research Fund. T. H. A. is a holder of a Science Research Council Research Studentship. Department of Botany, Queen M a r y College, London, E . L (Great Britain) i 2 3 4 5 6 7 8 9 IO ii 12 13
T. H. ATTRIDGE HARRY SMITH
F. E. MUMFORD,D. H. SMITHAND J. R. CASTLE,Plant Physiol., 36 (1961) 752. ~[. FURUYAAND R. G. THOMAS,Plant Physiol., 39 (1964) 634. V?. BOTTOMLEY,H. SMITHAND A. W. GALSTON,Phytochemistry, 5 (1966) 117. J- KOVKOLAND E. E. CONN,J. Biol. Chem., 236 (1961) 2692. V~. BOTTOML•Y, H. SMITHAND A. W. GALSTON,Nature, 207 (1965) 1311. M. ZUCKER,Plant Physiol., 4° (1965) 779. C. NITSCHAND J. P. NITSClL Compt. Rend., 262 (1966) 11o2. G. ENGELSMA,Planta, 75 (1967) 207G. ENGELSMA,Planta, in the press. 12~.DURSTAND H. MOI-IR,Naturwissenschaften, 53 (1966) 531. ~[. SCHERFAI,IDM. H. ZEI,IK,Z. Pflanzenphysiol., 56 (1967) 203. D. BALINSKYAND D. D. DAVI~S,Biochem. J., 80 (1961) 292. H. MOHR, Photochem. Photobiol., 5 (1966) 469.
Received September xlth, 1967 Biochim. Biophys. Acta, 148 (1967) 805-807