Plant a
Planta (1982) 156:545-552
9 Springer-Verlag 1982
Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants Bartolomeo Lercari, Francesco Sodi and Cristina Fastami Istituto di Orticoltura e Floricoltura, Universitfi di Pisa, Viale delle Piagge 23, 1-56100 Pisa, Italy
Abstract. Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-rain red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings. Key words: Cotyledon - Lycopersicon (enzyme induction) - Phenylalanine ammonia-lyase - Phytochrome (enzyme induction).
Introduction The nature of the photoreceptors involved in the control by light of growth and differentiation (photomorphogenesis) is still under debate. It is well Abbreviations: Norflurazon = N F = 4-chloro-5-(methylamino)-
2-(~,c~,c~,-trifluoro-m-tolyl)-3 (2 H) pyridazinone; P A L = p h e nylalanine ammonia-lyase; ~0z= phytochrome photoequilibrium Pfr/Pto~. ; Pf~= far-red absorbing form of phytochrome; P~ =red absorbing form of phytochrome; Ptot total phytochrome: Pr + Pfr =
0032-0935/82/0156/0546/$01.40
known that "many plant photomorphogenic responses are fully expressed only under prolonged exposures to high irradiance of light" (Mancinelli and Rabino 1978). It has been suggested that more than one pigment is involved in the action of longterm light treatments (Meijer 1968; Black and Shuttleworth 1974; Thomas and Dickinson 1979; Drumm and Mohr 1978; Drumm-Herrel and Mohr 1982). Most researchers agree that phytochrome can be considered the only photoreceptor involved in controlling most of the photomorphogenetic responses at wavelengths > 600 nm (Hartmann 1966; Mohr 1972, Schfifer 1975, Mancinelli and Rabino 1978), whereas the effects of blue and near UV light have been ascribed to other photoreceptor(s). Tomato (Lycopersicon eseulentum) plants are sensitive to both phytochrome and blue light c o n t r o l (Thomas and Dickinson 1979, Rabino etal. 1977, Drumm-Herrel and Mohr 1982). Therefore, we have selected the tomato system to investigate the nature of the photoreceptor(s) involved in the photomorphogenesis of this dicotyledonous seedling. Preliminary work of Lercari and Fastami (1981) showed that in the tomato the level of phenylalanine ammonia-lyase (PAL) responds rapidly to light treatments which produce or remove the far red absorbing form of phytochrome (Pf~). In the present paper we have, therefore, chosen PAL induction as a reliable probe for the light response. In this paper we describe a series of experiments performed with the aim of clarifying whether the blue light mediated increase of assayable PAL activity is phytochrome dependent or not. The results reported here are part of a long-term research project aimed at achieving more information on the photoreceptor(s) involved in light-mediated development, and therefore productivity, of tomato
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
plants grown during the winter in greenhouse where light is the limiting factor. Material and methods Seedlings. Seeds from Lycopersicon esculentum Mill. cv Chico III were purchased from Raci Sementi (Parma, Italy). Thirty seeds were germinated in darkness in glass boxes on four layers of filter paper (Omnia-filtra, Supervelox, 10cm diameter) soaked with 10 ml distilled water. The seedlings were grown at 25•176 C in the dark, and experiments were started 96 h after sowing. In some experiments the seedlings were germinated on filter paper with 5.10-6M Norflurazon, which produces photosynthetically incompetent plants (Herbicide handbook 1974). Irradiation. White light was produced using Philips TLM 40 W/ 33 RS fluorescent tubes. The standard fluence rate of this "white light" was 24 W m - z . The sources for broad band red, far red, and blue light were the same as described in Lercari 1982. Fluence rates from red and far red were usually 2.3 and 3.5 W m -z, respectively. The fluence rate from blue light was usually 0.3 or 2.2 W m -z. The cyclic light treatments (6 rain light + 54 rain dark) were performed with different fluence rates, thus, the seedlings were given the same total energy fluence as well as being under continuous light during the same period. Red light pulses (6 min) were performed in a standard red light field with a high fluence rate of 15 W m -2 (red 10) or 1.5 W m -2 (red), the fluence rate from continuous red light being 1.5 W m -2. Far-red light pulses (6 rain) were performed in a standard far-red field at a high fluence rate of 6.5 W m -2 and the fluence rate from continuous far red was lowered to 0,65 W m -2 by introducing neutral density wratten gelatine filters (Eastman Kodak Co., Rochester, USA). Blue light pulses (6 rain) were performed in a standard blue light field with a fluence rate of 0.4 m -2, the fluence rate of continuous blue light was lowered to 0.04 W m - 2 as described above for far red light. The choice of fluence rates under cyclic light treatments was due to maximum fluence rates obtainable with different light sources. Dichromatic irradiations with red plus blue light were performed by adding standard red (1.5 W m -2 or 0.05 W m -2) to standard blue light at a fluence rate of 0.3 W m -2, In another dichromatic experiment the fluence rates were 1.8 and 0.05 W m - z, respectively. One set of dichromatic irradiations with red plus far red light was performed by adding standard red light at a fluence rate of 1.0 W m 2 to standard far red light 3.5 W m-2. The reason for using different energy fluence rates, with the dichromatic irradiation technique from Hartmann (1966), is the following: under continuous light it is possible to shift the ~0 by the application of appropriate values of the fluence rate of two light beams of known quality. Thus, the extent of the response will change provided that the response is dependent on Per. For monochromatic light experiments, Leitz Prado projectors were used as light sources in combination with the following Schott interference filters: Dil 665 nm (0.6 W m - z ) ; Depil 698 nm (1.8 W m - Z ) ; Dil 708 nm (1.9 W m-Z); Depil 714 nan (1.8 W m - 2 ) ; Depil 724 nm (1.7 W m-2); A1 758 nm (6.1 W m-2); and a Balzer interference filter 456 nm (1.4 W m-z). A 5-rain irradiation saturates the PAL induction at all wavelengths used. The spectral emission curves and the spectral fluence rate were measured with an ISCO SR spectroradiometer, previously calibrated with an ISCO SRC spectroradiometer calibrator. The spectral emission curves were shown previously in detail (Lercari 1982).
547
PAL extraction and assay. Twenty pairs of cotyledons were ground for 3 rain at 2 ~ C with 1.5 g quartz sand in 6 ml 0.1 M borate buffer, pH 8.8. The homogenate was centrifuged at 39.000 g for 20 rain. The filtrate after a Sephadex G 25 "filtration" was used for the enzyme assay which was performed at 25 ~ C and pH 8.8, essentially according to the procedure used by Schopfer and Mohr (1972). Increase in absorbance at 290 nm against a control without phenylalanine was recorded over a period of at least 8 h, at 30-min or 1-h intervals, with a Perk• 124 spectrophotometer. The rate of appearance of transeinnamic acid was taken as a measure of the enzyme activity. Enzyme activity is expressed as pkat (i.e., pmol transcinnamic acid s-1) per pair of cotyledons. Mean values and standard errors are based on 8 16 experiments. In this paper, the terms "decrease or increase in PAL activity" are used without " a priori" implications regarding the mechanism of this phenomenon (activation, synthesis, degradation).
Results Pulse treatments. Tomato seedlings were grown in
the dark for 4 d (96 h). At this time seedlings were given 5-rain light pulses of monochromatic light. Following irradiation, the seedlings were placed in the dark, PAL activity was assayed 100 h after sowing, i.e., 4 h after the pulse. As can be seen in Table 1, a 5-rain irradiation at 665 nm was very effective in inducing P A L activity. In addition, irradiation at 456 nm gave a significant increase over dark control. However, if 5 rain at 665 nm or at 456 nm are
followed by 5 min at 758 nm, the increases in PAL activity are completely reversed. This sort of response is generally accepted as proof of phytochrome mediation in light response (Mohr 1972). By using different wavelenghts, establishing various photostationary states, it could be shown that the extent of induced PAL activity is determined by the level of Per (Table 2). Table 1. Induction-reversion experiments to demonstrate the involvement of phytochrome in the control of phenylalanine ammonia-lyase (PAL) level. PAL activity of 100 h old tomato seedlings as a function of light pulse treatments given at 96 h after sowing Irradiation programme
PAL activity (pkat. pair of cotyledons - 1)
96hd 96hd+4hd 96 h d + 5 min 665 96 h d + 5 rain 665 +230 rain d 96 h d + 5 rain 456 96 h d + 5 rain 456 +230 m i n d 96 h d + 5 min 758
n m + 2 3 5 rain d n m + 5 min 758
0.048 • 0.001 0.044 • 0.001 0.126+_0.005 0.058 • 0.006
nm+235 mind n m + 5 min 758 nm
0.088 • 0.001 0.060 • 0.005
n m + 2 3 5 rain d
0.060 + 0.003
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
548 Table 2. PAL activity of 100 h hold tomato seedlings as a function of light pulse treatments given at 96 h after sowing Irradiation programme
96 96 96 96 96 96 96 96 100
h h h h h h h h h
d + 5 min d + 5 min d + 5 min d + 5 min d + 5 min d + 5 min d + 5 rain dark dark
665 698 456 708 714 724 758
(0~"
nm+235 nm+235 n m + 235 nm+235 nm + 235 nm+235 n m + 235
min d min d mind mind min d min d rain d
PAL activity (pkat" pair of cotyledons- 1) 0.126+0.005 0.090 4- 0.005 0.092 4- 0.003 0.079 _+0.007 0.069 • 0.004 0.066+0.010 0.054 • 0.003 0.048 ___0.001 0.044 • 0.001
0.80 0.32 0.30 0.15 0.039 0.013 0.001
a Data about photostationary state of phytochrome from Schfifer et al. (1975)
0,3
o
Irradiation programme
PAL activity (pkat- pair of cotyledons- 1)
96hd 96 h d (San 5"I0-6M) 96 h d + 4 h white light 96 h d + 4 h white light (San 5" 10-6 M)
0.048 i 0.001 0.050 • 0.001 0.345_+0.014 0.324_ 0.015
ferent light qualities, the peaks of the PAL levels under continuous illuminations were in all cases found at about 4 h after the beginning of the light treatments. The results shown in Fig. I also indicate that the spectral sensitivity in tomato cotyledons under prolonged irradiation depends upon the length of exposure. Under 4 h irradiation, the blue and far red are more effective than red irradiations. After 24 h the effectiveness of the three wavebands becomes more similar and the red light is slightly more effective than far red and blue light.
Induction by continuous white light in seedlings treated with Norflurazon. Table 3 shows that the
far red (3,5Wm-:
0,~
Table 3. The influence of Norfluorazon (San) 5' 10-6M on the levels of PAL in the cotyledons of tomato seedlings in the dark and after 4 h white light
PAL response is exactly the same in tomato seedlings photobleached by the herbicide Norflurazon (5.10- 6 M) and in untreated green plants. Therefore, that photosynthesis has role in the light-mediated increase of PAL can be ruled out.
"8 ( 2 Wm -2)
\
Induction by continuous red light. The question of
o 0,1
# dark control
1
96
~
8
100
[
102
,5'
)
120
Time after sowing [ h i
Fig. 1. Time courses of PAL levels in tomato seedling cotyledons in darkness and under continuous white, blue, far red and red light. Irradiations started at 96 h after sowing. The vertical bars represent the standard error
Kinetics of PAL under continuous light of different quality. Seedlings grown for 96 h in the dark were placed in continuous red, far red, blue, or white light. Light-induced changes in the activity of PAL in tomato cotyledons occurred after an apparent initial lag period of about i h (Fig. 1). The rates of change in PAL activity were different under dif-
whether or not continuous red light operates through phytochrome was studied using cyclic red irradiations (Ku and Mancinelli 1972). Our results (Fig. 2) indicate that cyclic irradiations are as effective as continuous light, providing that the seedlings are given the same total energy fluence covering the same period. In fact, the cyclic irradiation 6 min redlo + 54 min dark can substitute for continuous red irradiation. While the cyclic irradiation 6 min red + 54 min dark can only partially (about 50%) substitute for continuous red irradiation. In such case equal fluence rates are compared, but the continuous red irradiation has ten times m o r e fluence than the cyclic irradiation. The reversibility of the effect of red light pulses by 758 nm pulses is incomplete (Fig. 2).
Induction by continuous far red or blue light. Hourly light pulses of the same total fluence do not produce the same effect as continuous far red or continuous blue light, as shown in Figs. 3 and 4.
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
549
(6min redlo +5/,min dark) .nh continuous red
0,2 em
0,~ (6min redlo +Smin 758nm+49 min dark).nh
o
"S
blue 2,20Wm -2 ff blue O,30Wm -2
(6min red+ 54min dark).nh
Q-
BO, (Smin758nm+ 55min dark).nh
i
o
%. 0,2
.>
*d
dark control
blue O,040Wm-2
"5 ?-
[76 min blue (0,40Wm-z ) +54min dark ].nh
iii
,;0 Time after sowing [ h ]
Fig. 2. Action of continuous and intermittent red irradiations on the levels of PAL activity. Reversion experiments of intermittent red irradiations are shown. Red~o means a fluence rate of 15 W na -2 whereas red without subscript was given with 1.5 W m -z. Irradiations started at 96 h after sowing
>-,
_> ~6 ( 5min 758nm+55min dark ).nh LU
\
E 6min blue ( O,z,OWm-2)+Smin 758nm +54 min dark].nh 9
w
dark control
0,3
far red (0,65Win -2) I
I
96
98
I
100
Time after sowing [ h ]
Fig. 4. Action of continuous (at different fluence rates) and intermittent blue irradiations on the level of PAL activity. Reversion experiments of intermittent blue irradiations are shown. Irradiations started at 96 h after sowing
rm 'T O
0,2 u
~5 El
r red (6,50W m-2)+ 54min dark] .nh
/
> *d
The response induced by continuous blue light is fluence rate dependent. The reversibility of the blue light pulses was incomplete.
eJ O,
LU
.~ dark control
.J
96
I
I
98
100
Time after sowing [ hi
Fig. 3. Action of continuous and intermittent far red irradiations on the level of PAL activity. Irradiations started at 96 h after sowing
Dichromatic irradiation. The question of whether or not continuous blue light operates through phytochrome was studied using dichromatic irradiation (Hartmann /966). Table 4 shows that, with respect to PAL induction, a substantial increase in the Pfr level by the simultaneous application of red (r red=0.8) and blue light ((o blue=0.3) leads to a decrease of the effectiveness of blue light. Furthermore, the response to blue light can be simulated by a dichromatic irradiation with far red plus red, which produces a Pfr/Ptot ratio similar to that found in blue light.
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
550 Table 4. Increase of PAL levels between 96 and 100 h after
sowing in tomato seedling cotyledons. Onset of light 96 h after sowing. At this point the seedlings were transferred for 2 or 4 h to different light conditions as indicated in the table. (These experiments are independent of and therefore do not exactly agree with those of Figs. 1-4) Light treatments
Blue light (1.8 W m -z)
~0~a
0.30
PAL activity (pkat" pair of cotyledons - 3) 2h
4h
0.159+_0.008
0.267+_0.015
Blue light (0.3 W m -2)
0.30
0.144+0.009
0.257_+0.009
Blue light (1.80 W m -2) plus red light (0.05 W m -2)
0.40
0.143_+0.010
0.245_+0.015
Blue light (0.3 W m -g) plus red light (0.05 W m -2)
0.45
0.128+_0.010
0.227-t-0.004
F a r r e d l i g h t ( 3 . S W m -2) 0,30 plus red light
0.147_+0.009
0.257+__0.017
(1.0 W m -2) Blue light (0.3 W m-2) plus red light
0.80 0.099+0.005 0.173+0.006
(1.5 W m -2) Red light (1.5 W m -2)
0.80
0.094___0.005 0.135_+0.008
Red light (1.0 W m -2)
0.80
0.094-+0.005
0.145_+0.01
Red light (0.05 W m -2)
0.80
0.091-+0.003
0.145-+0.006
Far red light (3.5 W m -z) 0.02
0.136-+0.011
0.248_+0,02
a
Calculated from Hartmann's equation (1966)
Discussion
Induction conditions. The data from Table 1 show that the increase in PAL promoted by blue and red pulses in cotyledons of tomato seedlings are reversed by 758 nm irradiation immediately following. Therefore, the operational criteria for the involvement of phytochrome are fulfilled (Mohr 1972). Furthermore, the data from Table 2 show that the increase in PAL in the dark is determined by the level of Pfr established by saturating short light pulses producing various photostationary states, ~0x. Tong and Schopfer (1978) have found a similar relationship between the photostationary state produced by 5-min light pulses and the level of induced PAL activity in cotyledons of Sinapis alba. Blue light established a photoequilibrium of about 30% Pfr as a 698 nm irradiation does, and both pulses induce the same level of PAL activity. These experimental results therefore indicate that the effect of light in the photocontrol of PAL activity, under "induction conditions", depends on phytochrome action, even in blue.
Continuous light. Under continuous illumination, the light-mediated increase in PAL activity shows the characteristic features of high irradiance responses in the form of irradiance dependence (blue) and changes in spectral sensitivity dependent on the length of irradiation, in combination with a multiple induction effect of phytochrome in the : red and blue part of the spectrum (Figs. 1 ~ , Table 4). The red waveband. It is widely accepted that the action of long-term red light is exerted through phytochrome (Hartmann 1966; Mohr 1972; Sch/ifer 1975; Mancinelli and Rabino 1978). The possible involvement of chlorophyll can be rejected as long as the PAL response under white light is exactly the same in tomato seedlings photobleached by the herbicide Norflurazon and in untreated green plants. Furthermore, hourly red light pulses can completely substitute continuous red irradiation (Fig. 2). The experimental results obtained under continuous red irradiation could, therefore, be explained in terms of a multiple induction effect (i.e., the response to a light pulse in the following dark period) of phytochrome. Similar results and conclusions have been obtained by Sch/ifer et al. (1981) with respect to light mediated anthocyanin synthesis in cotyledons and inhibition of hypocotyl elongation in Sinapis alba seedlings. Accordingly, the effect of continuous red is independent of the fluence rate as long as saturation is reached within a few minutes (cfr. Table 4). On the other hand, the reversibility of the effect of the red light pulses by 758 nm pulses is incomplete (Fig. 2). In cotyledons of Sinapis alba, Acton et al. (1980) Showed that the light-mediated increase in PAL synthesis is preceded by a lag-phase of not more than a few minutes. They argued that the apparent lag-phase preceding the light-mediated increase in PAL activity could be due to an experimental incapacity to detect small differences in enzyme levels. The incomplete reversion of multiple red light pulses could be explained by assuming that, to a small extent, the stimulus-response chain may have been entirely, and therefore irreversibly, completed before Pfr was removed from the cotyledons by a saturating pulse with 758 nm. The outcomes of each induction-reversion treatment can thus result in the production of a small, undetectable amount of enzyme. Consequently, the multiple hourly induction-reversion treatments could produce a significant increase in enzyme activity. Furthermore, it is well known that if enzyme induction is performed with more light pulses, the effectiveness of phytochrome (Pfr) is
B. Lercari et al. : Light mediatedinductionof PAL in Lycopersicon considerably increased by the previous pulses (Tong and Schopfer 1978). The far red waveband. It has been widely accepted that phytochrome may be the only photoreceptor involved in high irradiance responses in the far red region (Hartmann 1966; Mohr 1972; Sch/ifer 1975; Mancinelli and Rabino 1978). In tomato cotyledons, continuous far red light is very effective in mediating an increase in PAL activity. Hourly light pulses of the same total fluence do not produce the same effect as continuous far red light, as shown in Fig. 3. The low effectiveness of cyclic far red irradiations cannot be interpreted in terms of a multiple induction response. The high effectiveness of far red waveband can be explained on the basis of the high irradiance responses of phytochrome (Sch/ifer et al. 1981). The blue wavebands. The nature of the photoreceptors, absorbing in the blue part of the spectrum, which are involved in controlling plant growth has long been a source of controversy (Thomas and Dickinson 1979). Although dichromatic irradiations (Table 4) cannot rule out the action of a blue light photoreceptor which might require the presence of Pfr for its expression (Drumm and Mohr 1978), our experimental data (Table 4) indicate that the action of l o n ~ t e r m blue light in tomato cotyledons, with respect to PAL levels, is very probably exerted through phytochrome. Indeed, the light mediated increase in PAL activity shows a dependence of the response on ~0 (Table 4). Under experimental conditions which determine a large range of PAL levels (see blue, red and blue, plus red irradiations, Table 4), an increase in the ~0 compared to blue light, by the simultaneous application of red light, leads to a substantial decrease in the effectiveness of blue light. The critical experiment done mixing red and far red light to obtain ~0=0.3 (like blue light) is not conclusive because the results are not significantly different from the far red control (Table 4). However, in these experiments the response (PAL level) is restricted to a small range between the blue and far red treatments (Table 4), as a consequence the dichromatic irradiation red plus far red ((o=0.3) can induce only a small change in the PAL level, compared to far red, if the response to blue and far red light is dependent on Pfr level. Table 4 shows that these sort of results have been found. There is clearly a good correlation between ~0,
551 at least in the range of between 0.8 and 0.3, and the extent of the response. If the Per level established by blue light (0.3 W m-2) is increased by the simultaneous application of increasing fluence rates of red light, the PAL level is decreased. This indicates that the influence of total fluence rate on the phytochrome system is less pronounced than the effect exerted through increasing (0. The PAL response to blue light appears to be fluence-rate dependent (Fig. 4). On the other hand, a relevant (about 80%) part of the continuous blue light effect can be substituted for by hourly 6-min light pulses of the same light quality (Fig. 4). One can conclude, according to Sch/ifer et al. (1981), that the continuous blue light response is a combination of a multiple induction response and of a high irradiance response of phytochrome. Conclusion
The present investigation was carried out in an attempt to examine in detail the light-mediated phenylalanine-ammoniolyase induction in cotyledons of tomato seedlings. The results obtained with continuous light and with pulse treatments can be interpreted in terms of phytochrome action without implicating a specific blue light photoreceptor, while other laboratories have found strong and specific blue light effects in tomato (Drumm-Herrel and Mohr 1982; Thomas and Dickinson 1979). However, these latter data refer to photomorphogenetic responses in hypocotyls. It is well known that the photomorphogenetic behavior of the two organs, cotyledons and hypocotyl, is different. In particular, the ligh~mediated anthocyanin synthesis in tomato hypocotyl differs substantially in comparison to the light-mediated PAL induction in tomato cotyledons: Single light pulses have no effect on anthocyanin synthesis in the hypocotyl (Drumm-Herrel and Mohr 1982), whereas a significant reversible response appears in cotyledons with respect to PAL induction (Table 1). In different organs of tomato plants, light operating via phytochrome controls the synthesis of anthocyanins (Rabino et al. 1977; Drumm-Herrel and Mohr 1982) and of yellow flavonoids (Piringer and Heinze 1954). PAL catalyzes the first reaction in biosynthesis from phenylalanine to both anthocyanins and yellow flavonoids. The differences in the mechanism ofphotoregulation in PAL levels and of anthocyanin synthesis in different organs of tomato seedlings may indi-
552
cate that the increase in PAL levels in cotyledons could or could not be related to anthocyanin synthesis, depending on irradiation conditions (pulse conditions or continuous light). This is indeed the case in mustard cotyledons: PAL induction and anthocyanin synthesis by light pulses is largely restricted to the lower epidermis, while the synthesis of PAL and yellow flavonoids mediated by continuous light is confined to the upper epidermis (Wellmann 1974). Further work is required to establish whether or not PAL induction and anthocyanin synthesis are differently photoregulated in tomato seedlings. Supported by a grant from Consiglio Nazionale delle Ricerche to B.L. We thank Dr. F. Lenci and Dr. G. Colombetti for stimulating discussions and for the critical reading of the manuscript. We also are most grateful to Dr. Merten Jabben for the generous gift of Sandoz 9789 (Norflurazon).
References Acton, G.J., Fischer, W., Schopfer, P. (1980) Lag-phase and rate of synthesis in phytochrome-mediated induction of phenylalanine ammonia-lyase in mustard (Sinapis alba L.) cotyledons. Planta 150, 53-57 Black, M., Shuttleworth, J.E. (1974) The role of cotyledons in the photocontrol of hypocotyl extension in Cueumis sativusL. Planta 117, 57-66 Drumm, H., Mohr, H. (1978) The mode of interaction between blue (UV) light photoreceptor and phytochrome in anthocyanin formation of the Sorghum seedling. Photochem. Photobiol. 27, 214-218 Drumm-Herrel, H., Mohr, H. (1982) The effect of prolonged light exposure on the effectiveness of phytochrome in anthocyanin synthesis in tomato seedlings. Photochem. Photobiol. 35, 233-236 Hartmann, K.M. (1966) A general hypothesis to interpret "high energy phenomena" of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349-366 Herbicide handbook (1974) Weed Science Society of America, Champaign, Ill. Ku, P.K., Mancinelli, A.L. (1972) Photocontrol of anthocyanin synthesis. I. Action of short, prolonged, and intermittent irradiation on the formation of anthocyanin in cabbage, mustard, and turnip seedlings. Plant Physiol. 49, 212-217
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon Lercari, B. (1982) The promoting effect of far-red light on bulb formation in the long day plant Allium cepa L. Plant Sci. Lett. 27, 243-254 Lercari, B., F astami, C. (1981) The influence of light o f different spectral regions on the PAL activity in tomato cotyledons. XXI Congresso Societ/t Ital. Fisiol. Veg. Pistoia. (Abstr.) 29 Mancinelli, A.L., Rabino, I. (1978) The high irradiance responses of plant photomorphogenesis. Bot. Rev. 44, 129-180 Meijer, G. (1968) Rapid growth inhibition of gherkin hypocotyls in blue light. Acta. Bot. Need. 17, 9-14 Mohr, H. (1972) Lectures on photomorphogenesis. Springer, Berlin Heidelberg New York Piringer, A.A., Heinze, P.H. (1954) Effect of light on the formation of a pigment in the tomato fruit cuticle. Plant Physiol. 29, 467-472 Rabino, I., Mancinelli, A.L., Kuzmanoff, K.M. (1977) Photocontrol of anthocyanin synthesis. VI. Spectral sensitivity, irradiance dependence, and recyprocity relationship. P l a n t Physiol. 59, 569-573 Schgfer, E. (1975) A new approach to explain the "high irradiance response" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 Sch~ifer, E., Lassig, T.U., Schopfer, P. (1975) Photocontrol of phytochrome destruction in grass seedlings. The influence of wavelength and irradiance. Photochem. Photobiol. 22, 193-202 Sch~ifer, E., Beggs, C.J., Fukshansky, L., Holmes, M.G., Jabben, M. (1981) A comparative study of the responsivity of Sinapis alba L. seedlings to pulsed and continuous irradiation. Planta 153, 258-261 Schopfer, P., Mohr, H. (1972) Phytochrome mediated induction of phenylanine ammonia-lyase in mustard seedlings. Plant Physiol. 49, 8-10 Thomas, B., Dickinson, H.G. (1979) Evidence for two photoreceptors controlling growth in de-etiolated seedlings. Planta 146, 545-550 Tong, W.F., Schopfer, P. (1978) Absence of Pfr destruction in the modulation of phenylalanine ammonia-lyase synthesis of mustard cotyledons. Plant Physiol. 61, 59-61 Wellmann, E. (1974) Gewespezifische Kontrolle von Enzymen des Flavonoid-Stoffwechsels durch Phytochrom in Kotyledonen des Senfkeimlings (Sinapis alba L.). Ber. Dtsch. Bot. Ges. 87, 275-279
Received 30 December 1981; accepted 20 September 1982
Planta (1982) 156:545-552
9 Springer-Verlag 1982
Phytochrome-mediated induction of phenylalanine ammonia-lyase in the cotyledons of tomato (Lycopersicon esculentum Mill.) plants Bartolomeo Lercari, Francesco Sodi and Cristina Fastami Istituto di Orticoltura e Floricoltura, Universitfi di Pisa, Viale delle Piagge 23, 1-56100 Pisa, Italy
Abstract. Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5.) induction in cotyledons from 96-h dark-grown Lycopersicon esculentum Mill. was studied in response to continuous light and hourly light pulses (blue, red, far red). The increases of PAL promoted by blue and red pulses are reversed completely by immediately following 758 nm irradiations. The response to continuous red light could be substituted for by hourly 6-rain red light pulses. The effect of continuous red treatments is mainly due to a multiple induction effect of phytochrome. In contrast to red light, hourly light pulses with far red and blue light can only partially substitute for continuous irradiation. The continuous blue response could be due to a combination of a multiple induction response and of a high irradiance response of phytochrome. The continuous far red response could represent a high irradiance response of phytochrome. Dichromatic irradiations indicate that phytochrome is the photoreceptor controlling the light response (PAL) in tomato seedlings. Key words: Cotyledon - Lycopersicon (enzyme induction) - Phenylalanine ammonia-lyase - Phytochrome (enzyme induction).
Introduction The nature of the photoreceptors involved in the control by light of growth and differentiation (photomorphogenesis) is still under debate. It is well Abbreviations: Norflurazon = N F = 4-chloro-5-(methylamino)-
2-(~,c~,c~,-trifluoro-m-tolyl)-3 (2 H) pyridazinone; P A L = p h e nylalanine ammonia-lyase; ~0z= phytochrome photoequilibrium Pfr/Pto~. ; Pf~= far-red absorbing form of phytochrome; P~ =red absorbing form of phytochrome; Ptot total phytochrome: Pr + Pfr =
0032-0935/82/0156/0546/$01.40
known that "many plant photomorphogenic responses are fully expressed only under prolonged exposures to high irradiance of light" (Mancinelli and Rabino 1978). It has been suggested that more than one pigment is involved in the action of longterm light treatments (Meijer 1968; Black and Shuttleworth 1974; Thomas and Dickinson 1979; Drumm and Mohr 1978; Drumm-Herrel and Mohr 1982). Most researchers agree that phytochrome can be considered the only photoreceptor involved in controlling most of the photomorphogenetic responses at wavelengths > 600 nm (Hartmann 1966; Mohr 1972, Schfifer 1975, Mancinelli and Rabino 1978), whereas the effects of blue and near UV light have been ascribed to other photoreceptor(s). Tomato (Lycopersicon eseulentum) plants are sensitive to both phytochrome and blue light c o n t r o l (Thomas and Dickinson 1979, Rabino etal. 1977, Drumm-Herrel and Mohr 1982). Therefore, we have selected the tomato system to investigate the nature of the photoreceptor(s) involved in the photomorphogenesis of this dicotyledonous seedling. Preliminary work of Lercari and Fastami (1981) showed that in the tomato the level of phenylalanine ammonia-lyase (PAL) responds rapidly to light treatments which produce or remove the far red absorbing form of phytochrome (Pf~). In the present paper we have, therefore, chosen PAL induction as a reliable probe for the light response. In this paper we describe a series of experiments performed with the aim of clarifying whether the blue light mediated increase of assayable PAL activity is phytochrome dependent or not. The results reported here are part of a long-term research project aimed at achieving more information on the photoreceptor(s) involved in light-mediated development, and therefore productivity, of tomato
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
plants grown during the winter in greenhouse where light is the limiting factor. Material and methods Seedlings. Seeds from Lycopersicon esculentum Mill. cv Chico III were purchased from Raci Sementi (Parma, Italy). Thirty seeds were germinated in darkness in glass boxes on four layers of filter paper (Omnia-filtra, Supervelox, 10cm diameter) soaked with 10 ml distilled water. The seedlings were grown at 25•176 C in the dark, and experiments were started 96 h after sowing. In some experiments the seedlings were germinated on filter paper with 5.10-6M Norflurazon, which produces photosynthetically incompetent plants (Herbicide handbook 1974). Irradiation. White light was produced using Philips TLM 40 W/ 33 RS fluorescent tubes. The standard fluence rate of this "white light" was 24 W m - z . The sources for broad band red, far red, and blue light were the same as described in Lercari 1982. Fluence rates from red and far red were usually 2.3 and 3.5 W m -z, respectively. The fluence rate from blue light was usually 0.3 or 2.2 W m -z. The cyclic light treatments (6 rain light + 54 rain dark) were performed with different fluence rates, thus, the seedlings were given the same total energy fluence as well as being under continuous light during the same period. Red light pulses (6 min) were performed in a standard red light field with a high fluence rate of 15 W m -2 (red 10) or 1.5 W m -2 (red), the fluence rate from continuous red light being 1.5 W m -2. Far-red light pulses (6 rain) were performed in a standard far-red field at a high fluence rate of 6.5 W m -2 and the fluence rate from continuous far red was lowered to 0,65 W m -2 by introducing neutral density wratten gelatine filters (Eastman Kodak Co., Rochester, USA). Blue light pulses (6 rain) were performed in a standard blue light field with a fluence rate of 0.4 m -2, the fluence rate of continuous blue light was lowered to 0.04 W m - 2 as described above for far red light. The choice of fluence rates under cyclic light treatments was due to maximum fluence rates obtainable with different light sources. Dichromatic irradiations with red plus blue light were performed by adding standard red (1.5 W m -2 or 0.05 W m -2) to standard blue light at a fluence rate of 0.3 W m -2, In another dichromatic experiment the fluence rates were 1.8 and 0.05 W m - z, respectively. One set of dichromatic irradiations with red plus far red light was performed by adding standard red light at a fluence rate of 1.0 W m 2 to standard far red light 3.5 W m-2. The reason for using different energy fluence rates, with the dichromatic irradiation technique from Hartmann (1966), is the following: under continuous light it is possible to shift the ~0 by the application of appropriate values of the fluence rate of two light beams of known quality. Thus, the extent of the response will change provided that the response is dependent on Per. For monochromatic light experiments, Leitz Prado projectors were used as light sources in combination with the following Schott interference filters: Dil 665 nm (0.6 W m - z ) ; Depil 698 nm (1.8 W m - Z ) ; Dil 708 nm (1.9 W m-Z); Depil 714 nan (1.8 W m - 2 ) ; Depil 724 nm (1.7 W m-2); A1 758 nm (6.1 W m-2); and a Balzer interference filter 456 nm (1.4 W m-z). A 5-rain irradiation saturates the PAL induction at all wavelengths used. The spectral emission curves and the spectral fluence rate were measured with an ISCO SR spectroradiometer, previously calibrated with an ISCO SRC spectroradiometer calibrator. The spectral emission curves were shown previously in detail (Lercari 1982).
547
PAL extraction and assay. Twenty pairs of cotyledons were ground for 3 rain at 2 ~ C with 1.5 g quartz sand in 6 ml 0.1 M borate buffer, pH 8.8. The homogenate was centrifuged at 39.000 g for 20 rain. The filtrate after a Sephadex G 25 "filtration" was used for the enzyme assay which was performed at 25 ~ C and pH 8.8, essentially according to the procedure used by Schopfer and Mohr (1972). Increase in absorbance at 290 nm against a control without phenylalanine was recorded over a period of at least 8 h, at 30-min or 1-h intervals, with a Perk• 124 spectrophotometer. The rate of appearance of transeinnamic acid was taken as a measure of the enzyme activity. Enzyme activity is expressed as pkat (i.e., pmol transcinnamic acid s-1) per pair of cotyledons. Mean values and standard errors are based on 8 16 experiments. In this paper, the terms "decrease or increase in PAL activity" are used without " a priori" implications regarding the mechanism of this phenomenon (activation, synthesis, degradation).
Results Pulse treatments. Tomato seedlings were grown in
the dark for 4 d (96 h). At this time seedlings were given 5-rain light pulses of monochromatic light. Following irradiation, the seedlings were placed in the dark, PAL activity was assayed 100 h after sowing, i.e., 4 h after the pulse. As can be seen in Table 1, a 5-rain irradiation at 665 nm was very effective in inducing P A L activity. In addition, irradiation at 456 nm gave a significant increase over dark control. However, if 5 rain at 665 nm or at 456 nm are
followed by 5 min at 758 nm, the increases in PAL activity are completely reversed. This sort of response is generally accepted as proof of phytochrome mediation in light response (Mohr 1972). By using different wavelenghts, establishing various photostationary states, it could be shown that the extent of induced PAL activity is determined by the level of Per (Table 2). Table 1. Induction-reversion experiments to demonstrate the involvement of phytochrome in the control of phenylalanine ammonia-lyase (PAL) level. PAL activity of 100 h old tomato seedlings as a function of light pulse treatments given at 96 h after sowing Irradiation programme
PAL activity (pkat. pair of cotyledons - 1)
96hd 96hd+4hd 96 h d + 5 min 665 96 h d + 5 rain 665 +230 rain d 96 h d + 5 rain 456 96 h d + 5 rain 456 +230 m i n d 96 h d + 5 min 758
n m + 2 3 5 rain d n m + 5 min 758
0.048 • 0.001 0.044 • 0.001 0.126+_0.005 0.058 • 0.006
nm+235 mind n m + 5 min 758 nm
0.088 • 0.001 0.060 • 0.005
n m + 2 3 5 rain d
0.060 + 0.003
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
548 Table 2. PAL activity of 100 h hold tomato seedlings as a function of light pulse treatments given at 96 h after sowing Irradiation programme
96 96 96 96 96 96 96 96 100
h h h h h h h h h
d + 5 min d + 5 min d + 5 min d + 5 min d + 5 min d + 5 min d + 5 rain dark dark
665 698 456 708 714 724 758
(0~"
nm+235 nm+235 n m + 235 nm+235 nm + 235 nm+235 n m + 235
min d min d mind mind min d min d rain d
PAL activity (pkat" pair of cotyledons- 1) 0.126+0.005 0.090 4- 0.005 0.092 4- 0.003 0.079 _+0.007 0.069 • 0.004 0.066+0.010 0.054 • 0.003 0.048 ___0.001 0.044 • 0.001
0.80 0.32 0.30 0.15 0.039 0.013 0.001
a Data about photostationary state of phytochrome from Schfifer et al. (1975)
0,3
o
Irradiation programme
PAL activity (pkat- pair of cotyledons- 1)
96hd 96 h d (San 5"I0-6M) 96 h d + 4 h white light 96 h d + 4 h white light (San 5" 10-6 M)
0.048 i 0.001 0.050 • 0.001 0.345_+0.014 0.324_ 0.015
ferent light qualities, the peaks of the PAL levels under continuous illuminations were in all cases found at about 4 h after the beginning of the light treatments. The results shown in Fig. I also indicate that the spectral sensitivity in tomato cotyledons under prolonged irradiation depends upon the length of exposure. Under 4 h irradiation, the blue and far red are more effective than red irradiations. After 24 h the effectiveness of the three wavebands becomes more similar and the red light is slightly more effective than far red and blue light.
Induction by continuous white light in seedlings treated with Norflurazon. Table 3 shows that the
far red (3,5Wm-:
0,~
Table 3. The influence of Norfluorazon (San) 5' 10-6M on the levels of PAL in the cotyledons of tomato seedlings in the dark and after 4 h white light
PAL response is exactly the same in tomato seedlings photobleached by the herbicide Norflurazon (5.10- 6 M) and in untreated green plants. Therefore, that photosynthesis has role in the light-mediated increase of PAL can be ruled out.
"8 ( 2 Wm -2)
\
Induction by continuous red light. The question of
o 0,1
# dark control
1
96
~
8
100
[
102
,5'
)
120
Time after sowing [ h i
Fig. 1. Time courses of PAL levels in tomato seedling cotyledons in darkness and under continuous white, blue, far red and red light. Irradiations started at 96 h after sowing. The vertical bars represent the standard error
Kinetics of PAL under continuous light of different quality. Seedlings grown for 96 h in the dark were placed in continuous red, far red, blue, or white light. Light-induced changes in the activity of PAL in tomato cotyledons occurred after an apparent initial lag period of about i h (Fig. 1). The rates of change in PAL activity were different under dif-
whether or not continuous red light operates through phytochrome was studied using cyclic red irradiations (Ku and Mancinelli 1972). Our results (Fig. 2) indicate that cyclic irradiations are as effective as continuous light, providing that the seedlings are given the same total energy fluence covering the same period. In fact, the cyclic irradiation 6 min redlo + 54 min dark can substitute for continuous red irradiation. While the cyclic irradiation 6 min red + 54 min dark can only partially (about 50%) substitute for continuous red irradiation. In such case equal fluence rates are compared, but the continuous red irradiation has ten times m o r e fluence than the cyclic irradiation. The reversibility of the effect of red light pulses by 758 nm pulses is incomplete (Fig. 2).
Induction by continuous far red or blue light. Hourly light pulses of the same total fluence do not produce the same effect as continuous far red or continuous blue light, as shown in Figs. 3 and 4.
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
549
(6min redlo +5/,min dark) .nh continuous red
0,2 em
0,~ (6min redlo +Smin 758nm+49 min dark).nh
o
"S
blue 2,20Wm -2 ff blue O,30Wm -2
(6min red+ 54min dark).nh
Q-
BO, (Smin758nm+ 55min dark).nh
i
o
%. 0,2
.>
*d
dark control
blue O,040Wm-2
"5 ?-
[76 min blue (0,40Wm-z ) +54min dark ].nh
iii
,;0 Time after sowing [ h ]
Fig. 2. Action of continuous and intermittent red irradiations on the levels of PAL activity. Reversion experiments of intermittent red irradiations are shown. Red~o means a fluence rate of 15 W na -2 whereas red without subscript was given with 1.5 W m -z. Irradiations started at 96 h after sowing
>-,
_> ~6 ( 5min 758nm+55min dark ).nh LU
\
E 6min blue ( O,z,OWm-2)+Smin 758nm +54 min dark].nh 9
w
dark control
0,3
far red (0,65Win -2) I
I
96
98
I
100
Time after sowing [ h ]
Fig. 4. Action of continuous (at different fluence rates) and intermittent blue irradiations on the level of PAL activity. Reversion experiments of intermittent blue irradiations are shown. Irradiations started at 96 h after sowing
rm 'T O
0,2 u
~5 El
r red (6,50W m-2)+ 54min dark] .nh
/
> *d
The response induced by continuous blue light is fluence rate dependent. The reversibility of the blue light pulses was incomplete.
eJ O,
LU
.~ dark control
.J
96
I
I
98
100
Time after sowing [ hi
Fig. 3. Action of continuous and intermittent far red irradiations on the level of PAL activity. Irradiations started at 96 h after sowing
Dichromatic irradiation. The question of whether or not continuous blue light operates through phytochrome was studied using dichromatic irradiation (Hartmann /966). Table 4 shows that, with respect to PAL induction, a substantial increase in the Pfr level by the simultaneous application of red (r red=0.8) and blue light ((o blue=0.3) leads to a decrease of the effectiveness of blue light. Furthermore, the response to blue light can be simulated by a dichromatic irradiation with far red plus red, which produces a Pfr/Ptot ratio similar to that found in blue light.
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon
550 Table 4. Increase of PAL levels between 96 and 100 h after
sowing in tomato seedling cotyledons. Onset of light 96 h after sowing. At this point the seedlings were transferred for 2 or 4 h to different light conditions as indicated in the table. (These experiments are independent of and therefore do not exactly agree with those of Figs. 1-4) Light treatments
Blue light (1.8 W m -z)
~0~a
0.30
PAL activity (pkat" pair of cotyledons - 3) 2h
4h
0.159+_0.008
0.267+_0.015
Blue light (0.3 W m -2)
0.30
0.144+0.009
0.257_+0.009
Blue light (1.80 W m -2) plus red light (0.05 W m -2)
0.40
0.143_+0.010
0.245_+0.015
Blue light (0.3 W m -g) plus red light (0.05 W m -2)
0.45
0.128+_0.010
0.227-t-0.004
F a r r e d l i g h t ( 3 . S W m -2) 0,30 plus red light
0.147_+0.009
0.257+__0.017
(1.0 W m -2) Blue light (0.3 W m-2) plus red light
0.80 0.099+0.005 0.173+0.006
(1.5 W m -2) Red light (1.5 W m -2)
0.80
0.094___0.005 0.135_+0.008
Red light (1.0 W m -2)
0.80
0.094-+0.005
0.145_+0.01
Red light (0.05 W m -2)
0.80
0.091-+0.003
0.145-+0.006
Far red light (3.5 W m -z) 0.02
0.136-+0.011
0.248_+0,02
a
Calculated from Hartmann's equation (1966)
Discussion
Induction conditions. The data from Table 1 show that the increase in PAL promoted by blue and red pulses in cotyledons of tomato seedlings are reversed by 758 nm irradiation immediately following. Therefore, the operational criteria for the involvement of phytochrome are fulfilled (Mohr 1972). Furthermore, the data from Table 2 show that the increase in PAL in the dark is determined by the level of Pfr established by saturating short light pulses producing various photostationary states, ~0x. Tong and Schopfer (1978) have found a similar relationship between the photostationary state produced by 5-min light pulses and the level of induced PAL activity in cotyledons of Sinapis alba. Blue light established a photoequilibrium of about 30% Pfr as a 698 nm irradiation does, and both pulses induce the same level of PAL activity. These experimental results therefore indicate that the effect of light in the photocontrol of PAL activity, under "induction conditions", depends on phytochrome action, even in blue.
Continuous light. Under continuous illumination, the light-mediated increase in PAL activity shows the characteristic features of high irradiance responses in the form of irradiance dependence (blue) and changes in spectral sensitivity dependent on the length of irradiation, in combination with a multiple induction effect of phytochrome in the : red and blue part of the spectrum (Figs. 1 ~ , Table 4). The red waveband. It is widely accepted that the action of long-term red light is exerted through phytochrome (Hartmann 1966; Mohr 1972; Sch/ifer 1975; Mancinelli and Rabino 1978). The possible involvement of chlorophyll can be rejected as long as the PAL response under white light is exactly the same in tomato seedlings photobleached by the herbicide Norflurazon and in untreated green plants. Furthermore, hourly red light pulses can completely substitute continuous red irradiation (Fig. 2). The experimental results obtained under continuous red irradiation could, therefore, be explained in terms of a multiple induction effect (i.e., the response to a light pulse in the following dark period) of phytochrome. Similar results and conclusions have been obtained by Sch/ifer et al. (1981) with respect to light mediated anthocyanin synthesis in cotyledons and inhibition of hypocotyl elongation in Sinapis alba seedlings. Accordingly, the effect of continuous red is independent of the fluence rate as long as saturation is reached within a few minutes (cfr. Table 4). On the other hand, the reversibility of the effect of the red light pulses by 758 nm pulses is incomplete (Fig. 2). In cotyledons of Sinapis alba, Acton et al. (1980) Showed that the light-mediated increase in PAL synthesis is preceded by a lag-phase of not more than a few minutes. They argued that the apparent lag-phase preceding the light-mediated increase in PAL activity could be due to an experimental incapacity to detect small differences in enzyme levels. The incomplete reversion of multiple red light pulses could be explained by assuming that, to a small extent, the stimulus-response chain may have been entirely, and therefore irreversibly, completed before Pfr was removed from the cotyledons by a saturating pulse with 758 nm. The outcomes of each induction-reversion treatment can thus result in the production of a small, undetectable amount of enzyme. Consequently, the multiple hourly induction-reversion treatments could produce a significant increase in enzyme activity. Furthermore, it is well known that if enzyme induction is performed with more light pulses, the effectiveness of phytochrome (Pfr) is
B. Lercari et al. : Light mediatedinductionof PAL in Lycopersicon considerably increased by the previous pulses (Tong and Schopfer 1978). The far red waveband. It has been widely accepted that phytochrome may be the only photoreceptor involved in high irradiance responses in the far red region (Hartmann 1966; Mohr 1972; Sch/ifer 1975; Mancinelli and Rabino 1978). In tomato cotyledons, continuous far red light is very effective in mediating an increase in PAL activity. Hourly light pulses of the same total fluence do not produce the same effect as continuous far red light, as shown in Fig. 3. The low effectiveness of cyclic far red irradiations cannot be interpreted in terms of a multiple induction response. The high effectiveness of far red waveband can be explained on the basis of the high irradiance responses of phytochrome (Sch/ifer et al. 1981). The blue wavebands. The nature of the photoreceptors, absorbing in the blue part of the spectrum, which are involved in controlling plant growth has long been a source of controversy (Thomas and Dickinson 1979). Although dichromatic irradiations (Table 4) cannot rule out the action of a blue light photoreceptor which might require the presence of Pfr for its expression (Drumm and Mohr 1978), our experimental data (Table 4) indicate that the action of l o n ~ t e r m blue light in tomato cotyledons, with respect to PAL levels, is very probably exerted through phytochrome. Indeed, the light mediated increase in PAL activity shows a dependence of the response on ~0 (Table 4). Under experimental conditions which determine a large range of PAL levels (see blue, red and blue, plus red irradiations, Table 4), an increase in the ~0 compared to blue light, by the simultaneous application of red light, leads to a substantial decrease in the effectiveness of blue light. The critical experiment done mixing red and far red light to obtain ~0=0.3 (like blue light) is not conclusive because the results are not significantly different from the far red control (Table 4). However, in these experiments the response (PAL level) is restricted to a small range between the blue and far red treatments (Table 4), as a consequence the dichromatic irradiation red plus far red ((o=0.3) can induce only a small change in the PAL level, compared to far red, if the response to blue and far red light is dependent on Pfr level. Table 4 shows that these sort of results have been found. There is clearly a good correlation between ~0,
551 at least in the range of between 0.8 and 0.3, and the extent of the response. If the Per level established by blue light (0.3 W m-2) is increased by the simultaneous application of increasing fluence rates of red light, the PAL level is decreased. This indicates that the influence of total fluence rate on the phytochrome system is less pronounced than the effect exerted through increasing (0. The PAL response to blue light appears to be fluence-rate dependent (Fig. 4). On the other hand, a relevant (about 80%) part of the continuous blue light effect can be substituted for by hourly 6-min light pulses of the same light quality (Fig. 4). One can conclude, according to Sch/ifer et al. (1981), that the continuous blue light response is a combination of a multiple induction response and of a high irradiance response of phytochrome. Conclusion
The present investigation was carried out in an attempt to examine in detail the light-mediated phenylalanine-ammoniolyase induction in cotyledons of tomato seedlings. The results obtained with continuous light and with pulse treatments can be interpreted in terms of phytochrome action without implicating a specific blue light photoreceptor, while other laboratories have found strong and specific blue light effects in tomato (Drumm-Herrel and Mohr 1982; Thomas and Dickinson 1979). However, these latter data refer to photomorphogenetic responses in hypocotyls. It is well known that the photomorphogenetic behavior of the two organs, cotyledons and hypocotyl, is different. In particular, the ligh~mediated anthocyanin synthesis in tomato hypocotyl differs substantially in comparison to the light-mediated PAL induction in tomato cotyledons: Single light pulses have no effect on anthocyanin synthesis in the hypocotyl (Drumm-Herrel and Mohr 1982), whereas a significant reversible response appears in cotyledons with respect to PAL induction (Table 1). In different organs of tomato plants, light operating via phytochrome controls the synthesis of anthocyanins (Rabino et al. 1977; Drumm-Herrel and Mohr 1982) and of yellow flavonoids (Piringer and Heinze 1954). PAL catalyzes the first reaction in biosynthesis from phenylalanine to both anthocyanins and yellow flavonoids. The differences in the mechanism ofphotoregulation in PAL levels and of anthocyanin synthesis in different organs of tomato seedlings may indi-
552
cate that the increase in PAL levels in cotyledons could or could not be related to anthocyanin synthesis, depending on irradiation conditions (pulse conditions or continuous light). This is indeed the case in mustard cotyledons: PAL induction and anthocyanin synthesis by light pulses is largely restricted to the lower epidermis, while the synthesis of PAL and yellow flavonoids mediated by continuous light is confined to the upper epidermis (Wellmann 1974). Further work is required to establish whether or not PAL induction and anthocyanin synthesis are differently photoregulated in tomato seedlings. Supported by a grant from Consiglio Nazionale delle Ricerche to B.L. We thank Dr. F. Lenci and Dr. G. Colombetti for stimulating discussions and for the critical reading of the manuscript. We also are most grateful to Dr. Merten Jabben for the generous gift of Sandoz 9789 (Norflurazon).
References Acton, G.J., Fischer, W., Schopfer, P. (1980) Lag-phase and rate of synthesis in phytochrome-mediated induction of phenylalanine ammonia-lyase in mustard (Sinapis alba L.) cotyledons. Planta 150, 53-57 Black, M., Shuttleworth, J.E. (1974) The role of cotyledons in the photocontrol of hypocotyl extension in Cueumis sativusL. Planta 117, 57-66 Drumm, H., Mohr, H. (1978) The mode of interaction between blue (UV) light photoreceptor and phytochrome in anthocyanin formation of the Sorghum seedling. Photochem. Photobiol. 27, 214-218 Drumm-Herrel, H., Mohr, H. (1982) The effect of prolonged light exposure on the effectiveness of phytochrome in anthocyanin synthesis in tomato seedlings. Photochem. Photobiol. 35, 233-236 Hartmann, K.M. (1966) A general hypothesis to interpret "high energy phenomena" of photomorphogenesis on the basis of phytochrome. Photochem. Photobiol. 5, 349-366 Herbicide handbook (1974) Weed Science Society of America, Champaign, Ill. Ku, P.K., Mancinelli, A.L. (1972) Photocontrol of anthocyanin synthesis. I. Action of short, prolonged, and intermittent irradiation on the formation of anthocyanin in cabbage, mustard, and turnip seedlings. Plant Physiol. 49, 212-217
B. Lercari et al. : Light mediated induction of PAL in Lycopersicon Lercari, B. (1982) The promoting effect of far-red light on bulb formation in the long day plant Allium cepa L. Plant Sci. Lett. 27, 243-254 Lercari, B., F astami, C. (1981) The influence of light o f different spectral regions on the PAL activity in tomato cotyledons. XXI Congresso Societ/t Ital. Fisiol. Veg. Pistoia. (Abstr.) 29 Mancinelli, A.L., Rabino, I. (1978) The high irradiance responses of plant photomorphogenesis. Bot. Rev. 44, 129-180 Meijer, G. (1968) Rapid growth inhibition of gherkin hypocotyls in blue light. Acta. Bot. Need. 17, 9-14 Mohr, H. (1972) Lectures on photomorphogenesis. Springer, Berlin Heidelberg New York Piringer, A.A., Heinze, P.H. (1954) Effect of light on the formation of a pigment in the tomato fruit cuticle. Plant Physiol. 29, 467-472 Rabino, I., Mancinelli, A.L., Kuzmanoff, K.M. (1977) Photocontrol of anthocyanin synthesis. VI. Spectral sensitivity, irradiance dependence, and recyprocity relationship. P l a n t Physiol. 59, 569-573 Schgfer, E. (1975) A new approach to explain the "high irradiance response" of photomorphogenesis on the basis of phytochrome. J. Math. Biol. 2, 41-56 Sch~ifer, E., Lassig, T.U., Schopfer, P. (1975) Photocontrol of phytochrome destruction in grass seedlings. The influence of wavelength and irradiance. Photochem. Photobiol. 22, 193-202 Sch~ifer, E., Beggs, C.J., Fukshansky, L., Holmes, M.G., Jabben, M. (1981) A comparative study of the responsivity of Sinapis alba L. seedlings to pulsed and continuous irradiation. Planta 153, 258-261 Schopfer, P., Mohr, H. (1972) Phytochrome mediated induction of phenylanine ammonia-lyase in mustard seedlings. Plant Physiol. 49, 8-10 Thomas, B., Dickinson, H.G. (1979) Evidence for two photoreceptors controlling growth in de-etiolated seedlings. Planta 146, 545-550 Tong, W.F., Schopfer, P. (1978) Absence of Pfr destruction in the modulation of phenylalanine ammonia-lyase synthesis of mustard cotyledons. Plant Physiol. 61, 59-61 Wellmann, E. (1974) Gewespezifische Kontrolle von Enzymen des Flavonoid-Stoffwechsels durch Phytochrom in Kotyledonen des Senfkeimlings (Sinapis alba L.). Ber. Dtsch. Bot. Ges. 87, 275-279
Received 30 December 1981; accepted 20 September 1982
