Planta 9 by Springer-Verlag 1977
Planta 135, 169-175 (1977)
Phenylalanine Ammonia-Lyase and Cinnamic Acid 4-Hydroxylase: Characterisation of the Concomitant Changes in Enzyme Activities in Illuminated Potato Tuber Discs C.J. Lamb University of Oxford, School of Botany, South Parks Road, Oxford OX1 3RA, U.K.
Abstract. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid 4-hydroxylase (CA4H, EC 1.14.13.11) undergo concomitant increases in activity after a 2 h lag following disc preparation and illumination. The increases in PAL and CA4H activity can be inhibited by actinomycin-D, cordycepin and cycloheximide. It is demonstrated that the concomitant increases in enzyme activity stem from the simultaneous stimulation of the respective actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps. The events between the actinomycin-D- and cordycepin-sensitive steps are rate limiting in the expression of the stimulations in terms of increased enzyme activities. Delayed transfer of discs to actinomycin-D or cordycepin' superinduces' PAL but not CA4H activity. This superinduction is related to the inhibition by cycloheximide of the subsequent decline in PAL activity and is interpreted in terms of the transcription and translation of the mRNA coding for an inactivator-protein of PAL. Key-words: Cinnamic acid 4-hydroxylase - Co-induction - Enzyme induction - Phenylalanine ammonia-lyase - S o l a n u m - Super-induction.
Introduction Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid 4-hydroxylase (CA4H, EC 1.14.13.11) catalyse the first two reactions in the biosynthesis from L-phenylalanine of a wide variety of phenylpropanoid compounds including lignin, esters of hydroxycinnamic acids and flavonoids (Stafford, 1974). The activity levels of the two enzymes increase concomitantly folAbbreviations: PAL=phenylalanine ammonia-lyase;CA4H=cin-
namic acid 4-hydroxylase
lowing illumination of buckwheat seedlings (Amrhein and Zenk, 1970), parsley cell suspension cultures (Hahlbrock et al., 1971), upon ageing of soybean cell suspension cultures (Ebel et al., 1974) and upon treatment of Jerusalem artichoke tissue with manganous ions (Durst, 1976). In potato tuber discs PAL and CA4H undergo concomitant increases in activity, and the level of PAL but not CA4H activity is further stimulated by continuous illumination (Lamb and Rubery, 1976a). The increase in enzyme activity following stimulation characteristically occurs after a lag of 1.5 to 2.5 h (Smith et al,, 1977). The effects of delayed transfer of parsley cell suspension cultures to actinomycin-D and cycloheximide following illumination has been studied to characterise the events in the lag phase before increases in PAL activity are detectable (Hahlbrock and Ragg, 1975). It was demonstrated that the actinomycin-D sensitive step in the increase in PAL activity commenced immediately following illumination whereas the cycloheximide-sensitive step commenced only at the end of the lag phase. The present paper uses the inhibitors actinomycin-D, cycloheximide and cordycepin (3'-deoxyadenosine) to characterise the sequence of events occurring in the concomitant increases in extractable activity of PAL and CA4H following potato tuber disc preparation and illumination. It is concluded that the concomitant increases in PAL and CA4H activities stem from the simultaneous stimulation of the actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps.
Materials and Methods Potatotubers (Solanum tuberosum) wereobtainedlocallyand stored in darkness for up to 2 weeks. Tubers weighingbetween 0.1 and
170
C.J. L a m b : P A L a n d Cinnamic Acid 4-Hydroxylase
0.2 kg were used for all experiments. Discs of parenchyme 20 m m diam and 2.0 m m thick were cut using a hand microtome. Discs were washed in distilled water before being placed in Petri dishes (4 discs/dish), moistened with 5 ml H 2 0 and incubated under white light at 25 ~ C (Lamb and Rubery, 1976a). Extracts of discs were prepared in 0.05 M phosphate buffer (pH 7.5,sodium as the counter ion) containing 2-mercaptoethanol (2 m M ) and treated as previously described (Lamb and Rubery, 1976 b). P A L and C A 4 H activities were assayed at 30 ~ C by stopped spectrophotometric procedures (Koukol and Conn, 1961; L a m b and Rubery, 1975).
Effect of Actinomycin-D on the Appearance of PAL and CA4H Activities The increase in enzyme activities during a 9 h incubation period are almost completely inhibited by actinomycin-D at a concentration of 80 ~tM (Fig. 2). In general, the increase in PAL activity is slightly more sensitive than the corresponding increase in CA4H activity to inhibition by actinomycin-D. To determine whether the increases in enzyme activity are equally
Results
Co-ordinate Increases in Enzyme Activities In extracts of dormant potato tuber parenchyme there is little extractable PAL or CA4H activity. Following disc preparation and illumination both PAL and CA4H activities increase after a lag of about 2 h (Fig. 1). Maximum enzyme activities are reached 1315 h after disc preparation and subsequently PAL and CA4H activities decline to significantly lower levels (Lamb and Rubery, 1976a).
100
80
o 60 :>, '7- 40
i20
[:d
0
10(3
l
I
I
0-1
1-0
I0
PAL
IActinomycin-D]
I
100
(~ M)
60 Fig. 2. The sensitivities of the appearance of P A L and CA4H activities in illuminated potato tuber discs to inhibition by actinomycin-D. Results are expressed as a percentage of enzyme activity in extracts from equivalent discs incubated on water for 9 h, for P A L ( - o - ) and CA4H ( - 9
60
40 o
.~ 20 Table 1. The effects of actinomycin-D, cordycepin and cycloheximide on the decline in P A L and C A 4 H activities from illuminated potato tuber discs "~100
Enzyme activity (% activity from equivalent discs incubated for 14 h on water)
Treatment CA4H
0, g 0
laJ
PAL
CA4H
on 0.15 m M
100 43 87
100 55 17
on 0.1 m M
17
on 1 . 0 m M
100
36
on 32 g M
50
-
on 80 p,M
63
17
60
40
20
O0
2
4 6 Time (h)
8
10
Fig. 1. Time-course of the increase in PAL and C A 4 H activities in illuminated potato tuber discs. Activities are expressed as a percentage of the increase after 9 h
14 h on water 36 h on water 14 h on w a t e r + 2 2 h cycloheximide 14 h on w a t e r + 2 2 h cordycepin 14 h on w a t e r + 2 2 h cordycepin 14 h on water + 22 h actinomycin-D 14 h on w a t e r + 2 2 h actinomycin-D
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase
171 2.0
160
1.5 140 PAL El.0
120
s
100
0'5
80
i0.0" I
2
0
I
I
4 6 Time of transfer (h)
I
8
10
Fig. 4. The ratio of PAL to CA4H activity as a function of time of transfer to actinomycin-D, data are derived from Figure 3 40 55
20
100I
"6
-~
t
I
J
f o
:~ ~
CA4H
80 o
/
80[-
~ o
u 60
.%
o
2~ 4c
~S 6C
N ~ 20 c bJ
4C
0 20
0l~
1
o-ool
O.Ol
o.1
1.o
ICordycepin] (raM) I
2
I
I
4 6 Time of transfer (h)
I
8
10
Fig. 3. The effect of delayed transfer of illuminated potato tuber discs to actinomycin-D (32 gM) on the levels of PAL and CA4H activity. Discs were incubated on water for a time and then transferred to actinomycin-D for the remainder of the 9 h incubation period before extraction and assay for PAL and CA4H activities. Enzyme activity is expressed as percentage relief of inhibition: (Activity from discs incubated t h o n ~ _ = 100 x ~water r and 9-t h on actinomycin-D ] Activity from discs incubated 9 h on water ]-
(
\
Activity from discs incubated 9 h on actinomycin-D ] Activity from discs incubated 9 h on actinomycin-D /
sensitive to inhibition by actinomycin-D throught the 9 h incubation period, discs were incubated on water for a period before transfer to actinomycin-D (32 gM) for the remainder of the 9 h period. Delay of transfer to actinomycin-D over the first 4 h following disc pre-
Fig. 5. The sensitivities of the appearance of PAL and CA4H activities in illuminated potato tuber discs to inhibition by cordycepin. Results are expressed as a percentage of the enzyme activity from equivalent discs incubated on water for 9 h, for PAL ( - o - ) and CA4H ( 9 )
paration allows an increase i n enzyme activities (expressed as a relief of inhibition compared to continuous incubation on actinomycin-D) that is proportional to the length of the delay (Fig. 3). Therefore, the actinomycin-D sensitive steps in the appearance of PAL and CA4H activities commence immediately following disc preparation and illumination. Transfer of discs to actinomycin-D 7 h after preparation and 2 h before extraction has no effect on the level of CA4H activity. This might reflect either (a) a 2 h lag between the actinomycin-D-sensitive step and expression of this event in terms of an increase in CA4H activity or (b) co-incidental cessation of the actinomycin-Dsensitive step 7 h after disc preparation. The first alternative is favoured since transfer to actinomycin-D at
172
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase
//
00 PAL
100 PAL
80
8O
60-
o
o
6O
o
4o
._c 20
"~ 20
o
o
'2 0~
0
~1o0
100 CAAH CA4H
o
80 6O
8C
4O
4C
20
2C
2
4
6
8
10
Time of t r a n s f e r (h)
Fig. 6. The effect of delayed transfer of illuminated potato tuber discs to cordycepin (0.1 m M ) on the levels of P A L and CA4H
activity. Procedure and expression of results are as described in Figure 3
later times can subsequently reduce the extractable CA4H activity relative to appropriate controls from discs incubated continuously on water (Table 1). Transfer of discs to actinomycin-D in the period 5-8 h after disc preparation produces a stimulation in the level of PAL activity relative to the activity of controls incubated on water for 9 h (Fig. 3). Actinomycin-D exhibits no tendency" to stimulate the level of CA4H activity and a plot of the ratio P A L / C A 4 H as a function of time of transfer to actinomycin-D provides an index for the paradoxical effects of actinomycin-D on the level of P A L activity (Fig. 4). Increase of the PAL/ CA4H ratio from unity is first apparent when discs are transferred to actinomycin-D 4 h after disc preparation.
Effect of Cordycepin on the Appearance of PAL and CA4H Activities The increase in PAL and CA4H activities in a 9 h incubation period is completely inhibited by 1.0 mM cordycepin (Fig. 5). The effects of delayed transfer of
0~-
2
4
6
8
10
Time of t r a n s f e r (h)
Fig. "7. The effect of delayed transfer of illuminated potato tuber discs to cycloheximide (7.5 txM) on the levels of P A L and CA4H activity. Procedure and expression of results are as described in Figure 3
discs to 0.1 m M cordycepin on the appearance of enzyme activities was investigated using the approach described above for actinomycin-D. No relief ofcordycepin inhibition of the appearance of PAL and CA4H activities was observed if transfer was delayed for up to 2 h following disc preparation (Fig. 6). Longer delays ( > 2 h) give a relief of inhibition proportional to the length of the delay minus 2 h for both PAL and CA4H. Transfer of discs to cordycepin after 8 h (ie., 1 h before extraction) causes significant inhibition of both PAL and CA4H and it is apparent that cordycepin, unlike actinomycin-D, exerts an almost immediate inhibitory effect on the appearance of PAL and CA4H activities (Fig. 6). No stimulatory effect of 0.1 m M cordycepin on PAL was observed.
Effect of Cycloheximide on the Appearance of PAL and CA4H Activities The appearance of PAL and CA4H activities in illuminated potato tuber discs is inhibited by cyclohexi-
C.J. L a m b : P A L and Cinnamic Acid 4-Hydroxylase
173
mide (Lamb and Rubery, 1976c). The appearance of PAL activity is more sensitive than the appearance of CA4H activity at concentrations of cycloheximide less than 1 gM. However, at concentrations greater than 3 laM, the appearance of both enzyme activities is completely inhibited. No relief of cyclohexidide inhibition is found for either PAL or CA4H if transfer is delayed for up to 2 h after disc preparation (Fig. 7). Relief of inhibition for both enzymes is progressively more effective, the greater the delay in transfer. Like cordycepin, but unlike actinomycin-D, cycloheximide inhibition of the appearance of enzyme activity is almost immediately effective. The profiles for the relief of cycloheximide inhibition closely resemble the timecourses for the appearance of enzyme activities (Figs. 1, 7).
Characterisation of the Decline in Enzyme Activities The decline in PAL activity from the maximum levels achieved 13-15 h after disc preparation is inhibited by high concentrations of cycloheximide (Zucker, 1968). The concomitant decline in CA4H activity is not inhibited by cycloheximide (Lamb and Rubery, 1976c). In view of the stimulation of the level of PAL activity by actinomycin-D under certain conditions, the effects of actinomycin-D and cordycepin on the decline in enzyme activity were examined. No inhibition of the decline in CA4H activity was observed following transfer of discs to actinomycin-D, cordycepin or cycloheximide after 13 h incubation on water (Table 1). Indeed, all three compounds stimulated the decline in CA4H activity suggesting that some biosynthetic activity with respect to CA4H activity is occurring at least 13 h after disc preparation. The decline in PAL activity is completely inhibited by cycloheximide (0.15 mM) and partially inhibited by actinomycin-D (32 and 80gM). Cordycepin Table 2. The effect of delayed transfer to cordycepin on the appearance of P A L and C A 4 H activities in illuminated potato tuber discs
Treatment
5 h on water 8 h on water 6 h on w a t e r + 2 h on 0.1 m M cordycepin 6 h on w a t e r + 2 h on 1.0 m M cordycepin
Enzyme activity (% activity from equivalent discs incubated for 8 h on water) PAL
CA4H
15 100 84
27 100 86
113
85
(0.1 mM) stimulates the decline in PAL activity, but 1.0 mM cordycepin completely inhibits the decline (Table 1). Discs incubated 7 h on water and then 2 h on 1.0 mM cordycepin gave a stimulation of PAL activity relative to controls incubated continuously on water (Table 2), whereas a similar transfer to 0.1 mM cordycepin gave an inhibition of the appearance of PAL activity (Fig. 6 and Table 2).
Discussion
Concomitant increases in PAL and CA4H activities following application of a stimulus occur characteristically after a lag of about 1.5 to 2.5 h (Smith et al., 1977). The results presented in this paper suggest that DNA-dependent m R N A synthesis, mRNA polyadenylation and protein biosynthesis on cytoplasmic ribosomes are involved in the concomitant increases in enzyme activity in potato tuber discs. This interpretation is based on the assumption that in this system actinomycin-D, cordycepin and cycloheximide specifically inhibit mRNA biosynthesis (Cavalieri and Nemchin, 1964; Chantrenne, 1965), mRNA polyadenylation (Mendecki et al., 1972) and cytoplasmic protein biosynthesis (Grollman, 1966). De novo synthesis of PAL in illuminated potato tuber discs has been demonstrated by deuterium incorporation (Sacher et al., 1972, Lamb, unpublished observations). CA4H is a cytochrome P-450-dependent enzyme and CA4H activity in potato tuber discs accounts for almost all the cytochrome P-450 detectable by difference spectrophotometry (Rich and Lamb, 1977). Following disc preparation and illumination, the levels of cytochrome P-450 and CA4H activity increase co-ordinately suggesting that de novo synthesis of CA4H is occurring. The close correlation between the time-courses for the appearance of enzyme activities and the time-profiles for the relief of inhibition of the appearance of enzyme activities supports the hypothesis that cycloheximide is acting as an inhibitor of protein synthesis in this system. Furthermore, the sequence for the relief of inhibition of the increase in enzyme activities of actinomycin-D before cordycepin before cycloheximide is consistent with the assumed modes of action of the inhibitors. As in the case of the light-mediated increase in PAL activity in parsley cell suspension cultures (Hahlbrock and Ragg, 1975) actinomycin-Dsensitive steps in the appearance of both PAL and CA4H activities in potato tuber tissue commence without detectable lag following disc preparation and illumination. Nuclear processing of the transcription products (i.e., the events between the actinomycin-D- and cordycepin-sensitive steps) appears to be the rate limiting process in the concomitant increases in PAL and
174 C A 4 H activities. According to this interpretation translation of the polyadenylated m R N A s is rapid once they are available to the cytoplasmic ribosomes. Thus cordycepin as well as cycloheximide exerts an almost immediate inhibitory effect on the increase in enzyme activities (Figs. 6, 7) and relief of cordycepin inhibition is obtained only if transfer to the inhibitor is delayed until the end of the lag phase in the appearance of enzyme activity (Fig. 6). The simplest model is to assume that the actinomycin-D-sensitive steps are in fact transcription of the genomes for P A L and CA4H, but in the absence of direct estimations of the m R N A for P A L and CA4H, other more complex interpretations cannot be excluded. However, these studies provide an "Operational" analysis of the events in the lag phase and it is concluded that the concomitant increases in PAL and C A 4 H activities stem from the simultaneous stimulation of the respective actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps. Recently, it has been demonstrated that the lightinduced increases in P A L activity in parsley cell suspension cultures are caused by an increased rate of de novo synthesis of the enzyme (Schr6der et al., 1976) following a light-induced increase in the amount of translatable m R N A for P A L in the polysomes (Ragg et al., 1977). However, co-ordination of the activity levels m a y be maintained by specific feed-back coarse controls ( L a m b and Rubery, 1976b; Durst, 1976) operating post-transcriptionally (Lamb, unpublished). The stimulation of the appearance of P A L but not C A 4 H activity by transfer to actinomycin-D after 58 h on water appears to be related to the cycloheximide inhibition of the decline in P A L but not C A 4 H activity. Actinomycin-D and 1.0raM cordycepjn, but not 0.1 m M cordycepin " S u p e r i n d u c e " P A L activity and inhibit the subsequent decline in P A L activity. None of the inhibitors tested inhibit the decline in C A 4 H activity or superinduce C A 4 H activity. These effects are consistent with the transcription and translation of a proteinaceous inactivator for P A L which causes the decline in activity following attainment of maxim u m levels of activity. Proteinaceous inactivators of P A L have been detected in gherkin seedlings (Smith et al., 1977) and strawberry leaf discs (Creasy, 1976). The function of these inactivators may be to control the level of P A L activity in vivo, P A L being an intrinsically stable enzyme. An inactivator for C A 4 H may not be needed if C A 4 H is intrinsically unstable in vivo. The low sensitivity of the appearance of the inactivator to inhibition by cordycepin m a y reflect a short poly (adenosine) segment in the appropriate m R N A . Polyadenylation has been considered as the rate limit-
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase ing step in the appearance of m R N A in polysomes (Perry et al., 1974) and the present study suggests that nuclear processing of the trascription products (including polyadenylation) is the rate limiting step in the increase in P A L and C A 4 H activities following stimulation of the system, Hence a short poly (adenosine) segment in the m R N A for the inactivator would allow rapid nuclear processing and translation of the m R N A for the inactivator, thus giving rapid expression of the putative regulatory role of the inactivator. I thank Dr. V.S. Butt for helpful discussions and Oxford University for an ICI Research Fellowship.
References
Amrhein, N., Zenk, M.H. : Concomitant induction of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase during illumination of excised buckwheat hypocotyls.Naturwissenschaften 57, 312 (1970) Cavalieri, L.F., Nemchin, R.C. : The mode of interaction of actinomycin-D with deoxyribonucleic acid. Biochim. Biophys. Acta 87, 641-652 (1964) Chantrenne, H.: On the use of actinomycin-D for observing the turnover of ribonucleic acid. Biochim. Biophys. Acta 95, 351-353 (1965) Creasy, L.L. : Phenylalanine ammonia-lyase inactivating system in sunflower leaves. Phytochemistry 15, 673-675 (1976) Durst, F.: The correlation of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activity changes in Jerusalem artichoke tuber tissues. Planta (Berl.) 132, 221-227 (1976) Ebel, J., Schaller-Hekeler, B., Knobloch, K.-H., Wellmann, E., Grisebach, H., Hahlbrock, K. : Co-ordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures. Biochim. Biophys. Acta 362, 417-424 (1974) Grollman, A.P. : Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between alkaloids and glutarimide derivatives. Proc. Natl. Acad. Sci. USA 56, 1867-1871 (1966) Hahlbrock, K., Ebel, J., Ortmann, R., Sutter, A., Wellmann, E., Grisebach, H.: Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension cultures of parsley (Petroselinum hortense). Biochim. Biophys. Acta 244, 7-15 (1971) Hahlbrock, K., Ragg, H. : Light-induced changes in enzymeactivities in parsley cell suspension cultures. Effects of inhibitors of RNA and protein synthesis. Arch. Biochem. Biophys. 166, 41-46 (1975) Koukol, J., Conn, E.E.: The metabolism of aromatic compounds in higher plants. IV Purification and properies of the phenylalanine deaminase of Hordeum vulgare. J. Biol. Chem. 236, 26922698 (1961) Lamb, C.J., Rubery, P.H. : A spectrophotometric assay for cinnamic acid 4-hydroxylaseactivity.Analyt. Biochem. 68, 554-561 (1975) Lamb, C.J., Rubery, P.H. : Photocontrol of chlorogenic acid biosynthesis in potato tuber discs. Phytochemistry 15, 665-668 (1976a) Lamb, C.J., Rubery, P.H. : Phenylalanine ammonia-lyaseand cinnamic acid 4-hydroxylase: Product repression of the levelof enzyme activityin potato tuber discs. Planta (Berl.) 130, 283-290 (1976b) Lamb, C.J., Rubery, P.H. : Differential effects of cycloheximide on the activity of phenylalanine ammonia-lyase and cinnamic acid
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase 4-hydroxylase in light- and dark-incubated potato tuber discs. Plant Sci. Letters 7, 33-37 (1976c) Mendecki, J., Lee, S.Y., Brawerman, G.: Characteristics of the polyadenylic acid segment associated with messenger ribonucleic acid in mouse sarcoma 180 a scites cells. Biochemistry 11,792-798 (1972) Perry, R.P., Kelley, D.E., LaTorre, J.: Synthesis and turnover of nuclear and cytoplasmic polyadenylic acid in mouse L cells. J. Mol. Bioi. 82, 315 331 (1974) Ragg, H., Schr6der, J., Hahlbrock, K.: Translation of poly(A)containing and poly(A)-free messenger RNA for PAL, a plant specific protein in a reticulocyte lysate. Biochim. Biophys. Acta 474, 226 233 (1977) Rich, P.R., Lamb, C.J. : Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochrome P450. Europ. J. Biochem. in press (1977) Sacher, J.A., Towers, G.H.N., Davies, D.D.: Effect of light and ageing on enzymes, particularly phenylalanine ammonia-lyase, in discs of storage tissue. Phytochemistry 11, 2383-2391 (1972)
175 Smith, H., Billett, E.E., Giles, A.B. : Photocontroi of gene expression in higher plants. In: The regulation of enzyme synthesis and activity in higher plants: Problems and techniques, Smith, H., ed. London-New York: Academic Press, in press 1977 Schr6der, J., Betz, B., Hahlbrock, K.: Light-induced enzyme synthesis in cell suspension cultures of Petroselinum hortense. Demonstration in a heterologous cell-free system of rapid changes in the rate of phenylalanine ammonia-lyase synthesis. Europ. J. Biochem. 67, 527 541 (1976) Stafford, H.A. : The metabolism of aromatic compounds. Ann. Rev. Plant Physiol. 25, 459486 (1974) Zucker, M. : Sequential induction of phenylaianine ammonia-lyase and a lyase-inactivating system in potato tuber discs. Plant Physiol. 43, 365-374 (1968)
Received 26 January 1977," accepted 3 March 1977
Planta 135, 169-175 (1977)
Phenylalanine Ammonia-Lyase and Cinnamic Acid 4-Hydroxylase: Characterisation of the Concomitant Changes in Enzyme Activities in Illuminated Potato Tuber Discs C.J. Lamb University of Oxford, School of Botany, South Parks Road, Oxford OX1 3RA, U.K.
Abstract. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid 4-hydroxylase (CA4H, EC 1.14.13.11) undergo concomitant increases in activity after a 2 h lag following disc preparation and illumination. The increases in PAL and CA4H activity can be inhibited by actinomycin-D, cordycepin and cycloheximide. It is demonstrated that the concomitant increases in enzyme activity stem from the simultaneous stimulation of the respective actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps. The events between the actinomycin-D- and cordycepin-sensitive steps are rate limiting in the expression of the stimulations in terms of increased enzyme activities. Delayed transfer of discs to actinomycin-D or cordycepin' superinduces' PAL but not CA4H activity. This superinduction is related to the inhibition by cycloheximide of the subsequent decline in PAL activity and is interpreted in terms of the transcription and translation of the mRNA coding for an inactivator-protein of PAL. Key-words: Cinnamic acid 4-hydroxylase - Co-induction - Enzyme induction - Phenylalanine ammonia-lyase - S o l a n u m - Super-induction.
Introduction Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid 4-hydroxylase (CA4H, EC 1.14.13.11) catalyse the first two reactions in the biosynthesis from L-phenylalanine of a wide variety of phenylpropanoid compounds including lignin, esters of hydroxycinnamic acids and flavonoids (Stafford, 1974). The activity levels of the two enzymes increase concomitantly folAbbreviations: PAL=phenylalanine ammonia-lyase;CA4H=cin-
namic acid 4-hydroxylase
lowing illumination of buckwheat seedlings (Amrhein and Zenk, 1970), parsley cell suspension cultures (Hahlbrock et al., 1971), upon ageing of soybean cell suspension cultures (Ebel et al., 1974) and upon treatment of Jerusalem artichoke tissue with manganous ions (Durst, 1976). In potato tuber discs PAL and CA4H undergo concomitant increases in activity, and the level of PAL but not CA4H activity is further stimulated by continuous illumination (Lamb and Rubery, 1976a). The increase in enzyme activity following stimulation characteristically occurs after a lag of 1.5 to 2.5 h (Smith et al,, 1977). The effects of delayed transfer of parsley cell suspension cultures to actinomycin-D and cycloheximide following illumination has been studied to characterise the events in the lag phase before increases in PAL activity are detectable (Hahlbrock and Ragg, 1975). It was demonstrated that the actinomycin-D sensitive step in the increase in PAL activity commenced immediately following illumination whereas the cycloheximide-sensitive step commenced only at the end of the lag phase. The present paper uses the inhibitors actinomycin-D, cycloheximide and cordycepin (3'-deoxyadenosine) to characterise the sequence of events occurring in the concomitant increases in extractable activity of PAL and CA4H following potato tuber disc preparation and illumination. It is concluded that the concomitant increases in PAL and CA4H activities stem from the simultaneous stimulation of the actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps.
Materials and Methods Potatotubers (Solanum tuberosum) wereobtainedlocallyand stored in darkness for up to 2 weeks. Tubers weighingbetween 0.1 and
170
C.J. L a m b : P A L a n d Cinnamic Acid 4-Hydroxylase
0.2 kg were used for all experiments. Discs of parenchyme 20 m m diam and 2.0 m m thick were cut using a hand microtome. Discs were washed in distilled water before being placed in Petri dishes (4 discs/dish), moistened with 5 ml H 2 0 and incubated under white light at 25 ~ C (Lamb and Rubery, 1976a). Extracts of discs were prepared in 0.05 M phosphate buffer (pH 7.5,sodium as the counter ion) containing 2-mercaptoethanol (2 m M ) and treated as previously described (Lamb and Rubery, 1976 b). P A L and C A 4 H activities were assayed at 30 ~ C by stopped spectrophotometric procedures (Koukol and Conn, 1961; L a m b and Rubery, 1975).
Effect of Actinomycin-D on the Appearance of PAL and CA4H Activities The increase in enzyme activities during a 9 h incubation period are almost completely inhibited by actinomycin-D at a concentration of 80 ~tM (Fig. 2). In general, the increase in PAL activity is slightly more sensitive than the corresponding increase in CA4H activity to inhibition by actinomycin-D. To determine whether the increases in enzyme activity are equally
Results
Co-ordinate Increases in Enzyme Activities In extracts of dormant potato tuber parenchyme there is little extractable PAL or CA4H activity. Following disc preparation and illumination both PAL and CA4H activities increase after a lag of about 2 h (Fig. 1). Maximum enzyme activities are reached 1315 h after disc preparation and subsequently PAL and CA4H activities decline to significantly lower levels (Lamb and Rubery, 1976a).
100
80
o 60 :>, '7- 40
i20
[:d
0
10(3
l
I
I
0-1
1-0
I0
PAL
IActinomycin-D]
I
100
(~ M)
60 Fig. 2. The sensitivities of the appearance of P A L and CA4H activities in illuminated potato tuber discs to inhibition by actinomycin-D. Results are expressed as a percentage of enzyme activity in extracts from equivalent discs incubated on water for 9 h, for P A L ( - o - ) and CA4H ( - 9
60
40 o
.~ 20 Table 1. The effects of actinomycin-D, cordycepin and cycloheximide on the decline in P A L and C A 4 H activities from illuminated potato tuber discs "~100
Enzyme activity (% activity from equivalent discs incubated for 14 h on water)
Treatment CA4H
0, g 0
laJ
PAL
CA4H
on 0.15 m M
100 43 87
100 55 17
on 0.1 m M
17
on 1 . 0 m M
100
36
on 32 g M
50
-
on 80 p,M
63
17
60
40
20
O0
2
4 6 Time (h)
8
10
Fig. 1. Time-course of the increase in PAL and C A 4 H activities in illuminated potato tuber discs. Activities are expressed as a percentage of the increase after 9 h
14 h on water 36 h on water 14 h on w a t e r + 2 2 h cycloheximide 14 h on w a t e r + 2 2 h cordycepin 14 h on w a t e r + 2 2 h cordycepin 14 h on water + 22 h actinomycin-D 14 h on w a t e r + 2 2 h actinomycin-D
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase
171 2.0
160
1.5 140 PAL El.0
120
s
100
0'5
80
i0.0" I
2
0
I
I
4 6 Time of transfer (h)
I
8
10
Fig. 4. The ratio of PAL to CA4H activity as a function of time of transfer to actinomycin-D, data are derived from Figure 3 40 55
20
100I
"6
-~
t
I
J
f o
:~ ~
CA4H
80 o
/
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~ o
u 60
.%
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o-ool
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o.1
1.o
ICordycepin] (raM) I
2
I
I
4 6 Time of transfer (h)
I
8
10
Fig. 3. The effect of delayed transfer of illuminated potato tuber discs to actinomycin-D (32 gM) on the levels of PAL and CA4H activity. Discs were incubated on water for a time and then transferred to actinomycin-D for the remainder of the 9 h incubation period before extraction and assay for PAL and CA4H activities. Enzyme activity is expressed as percentage relief of inhibition: (Activity from discs incubated t h o n ~ _ = 100 x ~water r and 9-t h on actinomycin-D ] Activity from discs incubated 9 h on water ]-
(
\
Activity from discs incubated 9 h on actinomycin-D ] Activity from discs incubated 9 h on actinomycin-D /
sensitive to inhibition by actinomycin-D throught the 9 h incubation period, discs were incubated on water for a period before transfer to actinomycin-D (32 gM) for the remainder of the 9 h period. Delay of transfer to actinomycin-D over the first 4 h following disc pre-
Fig. 5. The sensitivities of the appearance of PAL and CA4H activities in illuminated potato tuber discs to inhibition by cordycepin. Results are expressed as a percentage of the enzyme activity from equivalent discs incubated on water for 9 h, for PAL ( - o - ) and CA4H ( 9 )
paration allows an increase i n enzyme activities (expressed as a relief of inhibition compared to continuous incubation on actinomycin-D) that is proportional to the length of the delay (Fig. 3). Therefore, the actinomycin-D sensitive steps in the appearance of PAL and CA4H activities commence immediately following disc preparation and illumination. Transfer of discs to actinomycin-D 7 h after preparation and 2 h before extraction has no effect on the level of CA4H activity. This might reflect either (a) a 2 h lag between the actinomycin-D-sensitive step and expression of this event in terms of an increase in CA4H activity or (b) co-incidental cessation of the actinomycin-Dsensitive step 7 h after disc preparation. The first alternative is favoured since transfer to actinomycin-D at
172
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase
//
00 PAL
100 PAL
80
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60-
o
o
6O
o
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._c 20
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o
o
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0
~1o0
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o
80 6O
8C
4O
4C
20
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2
4
6
8
10
Time of t r a n s f e r (h)
Fig. 6. The effect of delayed transfer of illuminated potato tuber discs to cordycepin (0.1 m M ) on the levels of P A L and CA4H
activity. Procedure and expression of results are as described in Figure 3
later times can subsequently reduce the extractable CA4H activity relative to appropriate controls from discs incubated continuously on water (Table 1). Transfer of discs to actinomycin-D in the period 5-8 h after disc preparation produces a stimulation in the level of PAL activity relative to the activity of controls incubated on water for 9 h (Fig. 3). Actinomycin-D exhibits no tendency" to stimulate the level of CA4H activity and a plot of the ratio P A L / C A 4 H as a function of time of transfer to actinomycin-D provides an index for the paradoxical effects of actinomycin-D on the level of P A L activity (Fig. 4). Increase of the PAL/ CA4H ratio from unity is first apparent when discs are transferred to actinomycin-D 4 h after disc preparation.
Effect of Cordycepin on the Appearance of PAL and CA4H Activities The increase in PAL and CA4H activities in a 9 h incubation period is completely inhibited by 1.0 mM cordycepin (Fig. 5). The effects of delayed transfer of
0~-
2
4
6
8
10
Time of t r a n s f e r (h)
Fig. "7. The effect of delayed transfer of illuminated potato tuber discs to cycloheximide (7.5 txM) on the levels of P A L and CA4H activity. Procedure and expression of results are as described in Figure 3
discs to 0.1 m M cordycepin on the appearance of enzyme activities was investigated using the approach described above for actinomycin-D. No relief ofcordycepin inhibition of the appearance of PAL and CA4H activities was observed if transfer was delayed for up to 2 h following disc preparation (Fig. 6). Longer delays ( > 2 h) give a relief of inhibition proportional to the length of the delay minus 2 h for both PAL and CA4H. Transfer of discs to cordycepin after 8 h (ie., 1 h before extraction) causes significant inhibition of both PAL and CA4H and it is apparent that cordycepin, unlike actinomycin-D, exerts an almost immediate inhibitory effect on the appearance of PAL and CA4H activities (Fig. 6). No stimulatory effect of 0.1 m M cordycepin on PAL was observed.
Effect of Cycloheximide on the Appearance of PAL and CA4H Activities The appearance of PAL and CA4H activities in illuminated potato tuber discs is inhibited by cyclohexi-
C.J. L a m b : P A L and Cinnamic Acid 4-Hydroxylase
173
mide (Lamb and Rubery, 1976c). The appearance of PAL activity is more sensitive than the appearance of CA4H activity at concentrations of cycloheximide less than 1 gM. However, at concentrations greater than 3 laM, the appearance of both enzyme activities is completely inhibited. No relief of cyclohexidide inhibition is found for either PAL or CA4H if transfer is delayed for up to 2 h after disc preparation (Fig. 7). Relief of inhibition for both enzymes is progressively more effective, the greater the delay in transfer. Like cordycepin, but unlike actinomycin-D, cycloheximide inhibition of the appearance of enzyme activity is almost immediately effective. The profiles for the relief of cycloheximide inhibition closely resemble the timecourses for the appearance of enzyme activities (Figs. 1, 7).
Characterisation of the Decline in Enzyme Activities The decline in PAL activity from the maximum levels achieved 13-15 h after disc preparation is inhibited by high concentrations of cycloheximide (Zucker, 1968). The concomitant decline in CA4H activity is not inhibited by cycloheximide (Lamb and Rubery, 1976c). In view of the stimulation of the level of PAL activity by actinomycin-D under certain conditions, the effects of actinomycin-D and cordycepin on the decline in enzyme activity were examined. No inhibition of the decline in CA4H activity was observed following transfer of discs to actinomycin-D, cordycepin or cycloheximide after 13 h incubation on water (Table 1). Indeed, all three compounds stimulated the decline in CA4H activity suggesting that some biosynthetic activity with respect to CA4H activity is occurring at least 13 h after disc preparation. The decline in PAL activity is completely inhibited by cycloheximide (0.15 mM) and partially inhibited by actinomycin-D (32 and 80gM). Cordycepin Table 2. The effect of delayed transfer to cordycepin on the appearance of P A L and C A 4 H activities in illuminated potato tuber discs
Treatment
5 h on water 8 h on water 6 h on w a t e r + 2 h on 0.1 m M cordycepin 6 h on w a t e r + 2 h on 1.0 m M cordycepin
Enzyme activity (% activity from equivalent discs incubated for 8 h on water) PAL
CA4H
15 100 84
27 100 86
113
85
(0.1 mM) stimulates the decline in PAL activity, but 1.0 mM cordycepin completely inhibits the decline (Table 1). Discs incubated 7 h on water and then 2 h on 1.0 mM cordycepin gave a stimulation of PAL activity relative to controls incubated continuously on water (Table 2), whereas a similar transfer to 0.1 mM cordycepin gave an inhibition of the appearance of PAL activity (Fig. 6 and Table 2).
Discussion
Concomitant increases in PAL and CA4H activities following application of a stimulus occur characteristically after a lag of about 1.5 to 2.5 h (Smith et al., 1977). The results presented in this paper suggest that DNA-dependent m R N A synthesis, mRNA polyadenylation and protein biosynthesis on cytoplasmic ribosomes are involved in the concomitant increases in enzyme activity in potato tuber discs. This interpretation is based on the assumption that in this system actinomycin-D, cordycepin and cycloheximide specifically inhibit mRNA biosynthesis (Cavalieri and Nemchin, 1964; Chantrenne, 1965), mRNA polyadenylation (Mendecki et al., 1972) and cytoplasmic protein biosynthesis (Grollman, 1966). De novo synthesis of PAL in illuminated potato tuber discs has been demonstrated by deuterium incorporation (Sacher et al., 1972, Lamb, unpublished observations). CA4H is a cytochrome P-450-dependent enzyme and CA4H activity in potato tuber discs accounts for almost all the cytochrome P-450 detectable by difference spectrophotometry (Rich and Lamb, 1977). Following disc preparation and illumination, the levels of cytochrome P-450 and CA4H activity increase co-ordinately suggesting that de novo synthesis of CA4H is occurring. The close correlation between the time-courses for the appearance of enzyme activities and the time-profiles for the relief of inhibition of the appearance of enzyme activities supports the hypothesis that cycloheximide is acting as an inhibitor of protein synthesis in this system. Furthermore, the sequence for the relief of inhibition of the increase in enzyme activities of actinomycin-D before cordycepin before cycloheximide is consistent with the assumed modes of action of the inhibitors. As in the case of the light-mediated increase in PAL activity in parsley cell suspension cultures (Hahlbrock and Ragg, 1975) actinomycin-Dsensitive steps in the appearance of both PAL and CA4H activities in potato tuber tissue commence without detectable lag following disc preparation and illumination. Nuclear processing of the transcription products (i.e., the events between the actinomycin-D- and cordycepin-sensitive steps) appears to be the rate limiting process in the concomitant increases in PAL and
174 C A 4 H activities. According to this interpretation translation of the polyadenylated m R N A s is rapid once they are available to the cytoplasmic ribosomes. Thus cordycepin as well as cycloheximide exerts an almost immediate inhibitory effect on the increase in enzyme activities (Figs. 6, 7) and relief of cordycepin inhibition is obtained only if transfer to the inhibitor is delayed until the end of the lag phase in the appearance of enzyme activity (Fig. 6). The simplest model is to assume that the actinomycin-D-sensitive steps are in fact transcription of the genomes for P A L and CA4H, but in the absence of direct estimations of the m R N A for P A L and CA4H, other more complex interpretations cannot be excluded. However, these studies provide an "Operational" analysis of the events in the lag phase and it is concluded that the concomitant increases in PAL and C A 4 H activities stem from the simultaneous stimulation of the respective actinomycin-D-sensitive steps followed by parallel transmission of these stimuli through the cordycepin- and cycloheximide-sensitive steps. Recently, it has been demonstrated that the lightinduced increases in P A L activity in parsley cell suspension cultures are caused by an increased rate of de novo synthesis of the enzyme (Schr6der et al., 1976) following a light-induced increase in the amount of translatable m R N A for P A L in the polysomes (Ragg et al., 1977). However, co-ordination of the activity levels m a y be maintained by specific feed-back coarse controls ( L a m b and Rubery, 1976b; Durst, 1976) operating post-transcriptionally (Lamb, unpublished). The stimulation of the appearance of P A L but not C A 4 H activity by transfer to actinomycin-D after 58 h on water appears to be related to the cycloheximide inhibition of the decline in P A L but not C A 4 H activity. Actinomycin-D and 1.0raM cordycepjn, but not 0.1 m M cordycepin " S u p e r i n d u c e " P A L activity and inhibit the subsequent decline in P A L activity. None of the inhibitors tested inhibit the decline in C A 4 H activity or superinduce C A 4 H activity. These effects are consistent with the transcription and translation of a proteinaceous inactivator for P A L which causes the decline in activity following attainment of maxim u m levels of activity. Proteinaceous inactivators of P A L have been detected in gherkin seedlings (Smith et al., 1977) and strawberry leaf discs (Creasy, 1976). The function of these inactivators may be to control the level of P A L activity in vivo, P A L being an intrinsically stable enzyme. An inactivator for C A 4 H may not be needed if C A 4 H is intrinsically unstable in vivo. The low sensitivity of the appearance of the inactivator to inhibition by cordycepin m a y reflect a short poly (adenosine) segment in the appropriate m R N A . Polyadenylation has been considered as the rate limit-
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase ing step in the appearance of m R N A in polysomes (Perry et al., 1974) and the present study suggests that nuclear processing of the trascription products (including polyadenylation) is the rate limiting step in the increase in P A L and C A 4 H activities following stimulation of the system, Hence a short poly (adenosine) segment in the m R N A for the inactivator would allow rapid nuclear processing and translation of the m R N A for the inactivator, thus giving rapid expression of the putative regulatory role of the inactivator. I thank Dr. V.S. Butt for helpful discussions and Oxford University for an ICI Research Fellowship.
References
Amrhein, N., Zenk, M.H. : Concomitant induction of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase during illumination of excised buckwheat hypocotyls.Naturwissenschaften 57, 312 (1970) Cavalieri, L.F., Nemchin, R.C. : The mode of interaction of actinomycin-D with deoxyribonucleic acid. Biochim. Biophys. Acta 87, 641-652 (1964) Chantrenne, H.: On the use of actinomycin-D for observing the turnover of ribonucleic acid. Biochim. Biophys. Acta 95, 351-353 (1965) Creasy, L.L. : Phenylalanine ammonia-lyase inactivating system in sunflower leaves. Phytochemistry 15, 673-675 (1976) Durst, F.: The correlation of phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase activity changes in Jerusalem artichoke tuber tissues. Planta (Berl.) 132, 221-227 (1976) Ebel, J., Schaller-Hekeler, B., Knobloch, K.-H., Wellmann, E., Grisebach, H., Hahlbrock, K. : Co-ordinated changes in enzyme activities of phenylpropanoid metabolism during the growth of soybean cell suspension cultures. Biochim. Biophys. Acta 362, 417-424 (1974) Grollman, A.P. : Structural basis for inhibition of protein synthesis by emetine and cycloheximide based on an analogy between alkaloids and glutarimide derivatives. Proc. Natl. Acad. Sci. USA 56, 1867-1871 (1966) Hahlbrock, K., Ebel, J., Ortmann, R., Sutter, A., Wellmann, E., Grisebach, H.: Regulation of enzyme activities related to the biosynthesis of flavone glycosides in cell suspension cultures of parsley (Petroselinum hortense). Biochim. Biophys. Acta 244, 7-15 (1971) Hahlbrock, K., Ragg, H. : Light-induced changes in enzymeactivities in parsley cell suspension cultures. Effects of inhibitors of RNA and protein synthesis. Arch. Biochem. Biophys. 166, 41-46 (1975) Koukol, J., Conn, E.E.: The metabolism of aromatic compounds in higher plants. IV Purification and properies of the phenylalanine deaminase of Hordeum vulgare. J. Biol. Chem. 236, 26922698 (1961) Lamb, C.J., Rubery, P.H. : A spectrophotometric assay for cinnamic acid 4-hydroxylaseactivity.Analyt. Biochem. 68, 554-561 (1975) Lamb, C.J., Rubery, P.H. : Photocontrol of chlorogenic acid biosynthesis in potato tuber discs. Phytochemistry 15, 665-668 (1976a) Lamb, C.J., Rubery, P.H. : Phenylalanine ammonia-lyaseand cinnamic acid 4-hydroxylase: Product repression of the levelof enzyme activityin potato tuber discs. Planta (Berl.) 130, 283-290 (1976b) Lamb, C.J., Rubery, P.H. : Differential effects of cycloheximide on the activity of phenylalanine ammonia-lyase and cinnamic acid
C.J. Lamb: PAL and Cinnamic Acid 4-Hydroxylase 4-hydroxylase in light- and dark-incubated potato tuber discs. Plant Sci. Letters 7, 33-37 (1976c) Mendecki, J., Lee, S.Y., Brawerman, G.: Characteristics of the polyadenylic acid segment associated with messenger ribonucleic acid in mouse sarcoma 180 a scites cells. Biochemistry 11,792-798 (1972) Perry, R.P., Kelley, D.E., LaTorre, J.: Synthesis and turnover of nuclear and cytoplasmic polyadenylic acid in mouse L cells. J. Mol. Bioi. 82, 315 331 (1974) Ragg, H., Schr6der, J., Hahlbrock, K.: Translation of poly(A)containing and poly(A)-free messenger RNA for PAL, a plant specific protein in a reticulocyte lysate. Biochim. Biophys. Acta 474, 226 233 (1977) Rich, P.R., Lamb, C.J. : Biophysical and enzymological studies upon the interaction of trans-cinnamic acid with higher plant microsomal cytochrome P450. Europ. J. Biochem. in press (1977) Sacher, J.A., Towers, G.H.N., Davies, D.D.: Effect of light and ageing on enzymes, particularly phenylalanine ammonia-lyase, in discs of storage tissue. Phytochemistry 11, 2383-2391 (1972)
175 Smith, H., Billett, E.E., Giles, A.B. : Photocontroi of gene expression in higher plants. In: The regulation of enzyme synthesis and activity in higher plants: Problems and techniques, Smith, H., ed. London-New York: Academic Press, in press 1977 Schr6der, J., Betz, B., Hahlbrock, K.: Light-induced enzyme synthesis in cell suspension cultures of Petroselinum hortense. Demonstration in a heterologous cell-free system of rapid changes in the rate of phenylalanine ammonia-lyase synthesis. Europ. J. Biochem. 67, 527 541 (1976) Stafford, H.A. : The metabolism of aromatic compounds. Ann. Rev. Plant Physiol. 25, 459486 (1974) Zucker, M. : Sequential induction of phenylaianine ammonia-lyase and a lyase-inactivating system in potato tuber discs. Plant Physiol. 43, 365-374 (1968)
Received 26 January 1977," accepted 3 March 1977
