Planta
Planta 133, 295-302 (1977)
9 by Springer-Verlag 1977
The Capacity of Chlorophyll-a Biosynthesis in the Mustard Seedling Cotyledons as Modulated by Phytochrome and Circadian Rhythmicity H. Gehring, H. Kasemir, and H. M o h r Biologisches Institut II, Universiffit Freiburg, Sch~inzlestrafle9-11, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Within the temporal pattern of " p r i m a r y differentiation" the capacity of c h l o r o p h y l l - a biosynthesis in the cotyledons of Sinapis alba L. seedlings is controlled by phytochrome (in continuous light) or by releasing the circadian rhythm either with lightdark cycles or by a light - , dark transition. The sensor pigment for this process is phytochrome. It is very probable that in continuous light as well as under conditions under which the circadian rhythm plays the major part, the capacity of chlorophyll a biosynthesis is limited by the capacity of the biosynthetic step which produces 5-aminolaevulinate. Key words: 5-Aminolaevulinic acid - Chlorophyll synthesis -- Circadian rhythm -- Phytochrome -Sinapis alba L.
tion about the steps of Chl biosynthesis was recently published by Schneider (1975). The present paper is concerned with the regulation of Chl biosynthesis. We pose the question of what factors determine the actual capacity of the Chl synthesizing anabolic channel in the intact plant. By " c a p a c i t y " we mean the m a x i m u m rate of Chl synthesis as it is observed under light conditions which saturate the PChl ~ Chl photoconversion (Koski et al., 1951 ; Smith, 1960). It was found previously (Kasemir et al., 1973) that phytochrome influences the capacity for Chl synthesis in the mustard seedling. In the present paper we wanted to measure the time course of the capacity in the mustard seedling cotyledons and to identify those factors which determine the capacity during development. It will be shown that besides phytochrome the temporal pattern o f " primary differentiation" (see Mohr, 1972) and the circadian rhythm (see Biinning, 1973) must be considered.
Introduction The biosynthetic pathway leading to chlorophyll a (Chl) is known in principle (see A r o n o f f and Ellsworth, 1968; Rebeiz and Castelfranco, 1973; Bogorad, 1976) even though the mode of formation of 5-aminolaevulinate (ALA) is not yet fully established (see Beale and Castelfranco, 1974; Meller et al., 1975). There is good evidence that Chl synthesis takes place in the plas.tid c o m p a r t m e n t (Wellburn and Wellburn, 1971; Rebeiz et al., 1973). However, the enzymes involved are very probably being synthesized exclusively in the cytoplasm (see B6rner, 1973; Galling et al., 1973). A thorough review of the informaAbbreviations: Chl=chlorophyll(ide) a; ALA=5-aminolaevulihate; LA=laevulinate; PChI=protochlorophyll(ide); ALAD= aminolaevutinate dehydratase (EC 4.2.1.24); cpx=[PfrJ/[Ptot], photoequilibrium of the phytochrome system at the wavelength 2, whereby [P,oJ=[Pr] +[Pfr]. Pfr is the physiologically active, far-red absorbing form of the phytochrome system.
Materials and Methods Standard Techniques Standard techniques for photomorphogenic research with mustard seedlings were used (Mohr, 1966, 1972). The seeds (Sinapis alba L.) were purchased in 1971 from Asgrow Company (Hamburg). The seedlings were grown at 25.0 +0.2~ in the dark. Preirradiations were performed either with standard red (emission maximum at 656 nm, band width 15 nm, irradiance 0.675 Wm 2) or standard far-red (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm-2) sources (Mohr etal., 1964; Mohr, 11966) or with monochromatic 756 nm light obtained from a modified Leitz projector Prado 500 (Mohr and Schoser, 1959) equipped with a AL interference filter from Schott, Mainz (2m,~: 756 rim; band width 21 rim; irradiance 7 Wm-2). Standard white light was obtained from fluorescent white light tubes (Osram, alternating L40 W/15 and L40 W/25) at an illuminance of 7,000 lx. The characteristics of the standard light sources have been described repeatedly (e.g. Mohr, 1966). Continuous f~ir-red light is consMered to operate exclusivelythrough phytochrome (Hartmann, 1966; Mohr,
296
H. Gehring et al. : Chlorophyll Synthesis, Phytochrome and Circadian Rhythms
1972; Drumm et al., 1975; Sch~ifer, 1975). The use of the biological unit " p a i r of cotyledons" as the suitable system of reference for Chl was justified previously (see Mohr, 1972). Determination of Chlorophyll a and 5-aminolaevulinate Extraction and determination of Chl were performed after Ziegler and Egle (1965) as described by Kasemir and Mohr (1967). While the presence of chlorophyll b was considered in the determination of the chlorophyll a values, chlorophyll b per se was not followed in the present investigation. Inhibition of A L A D with LA and determination of ALA was performed according to Masoner and Kasemir (1975) using a molar extinction coefficient of 64 x 10311 mo1-1 cm -1] (Urata and Granick, 1963). The LA required to inhibit A L A D was purchased from Fluka A.G., Buchs, Switzerland.
2 and 5 h after the onset of the standard white light. Therefore the capacity of fully etiolated seedlings is probably slightly over estimated in the present paper as compared to seedlings which have already received a phytochrome pretreatment. Since this effect has no bearing on the principal results we are presenting and discussing in this paper, the experiments with dichromatic irradiation will not be described in detail. The pertinent information is available elsewhere (Gehring, 1976). Means. The mean values as given in the figures are based on at least 4 (sometimes up to 12) independent parallels. The estimated standard error of the means is of the order of 4~5%.
Results
1. Development of the Capacity under Constant Environmental Conditions
Determination of the Capacity of the Chl Synthesizing Channel Figure 1 shows the time course of Chl accumulation under continuous standard white light (7,000 lx). The illuminance of the standard white light suffices to saturate the PChl ~ Chl photoconversion under all circumstances. It was shown that no detectable photodestruction occurs at an illuminance of 7,000 ix (see Gehring, 1973). Figure 1 also shows the time course of Chl accumulation under repeated white l i g h t - d a r k cycles (12 h d a r k - 1 2 h light). Since there is no PChl ~ Chl photoconversion in the dark, Chl accumulation takes place step wise. The fact that there is no significant decrease of the Chl contents during the dark period indicates that Chl turnover does not play a role during the period of our experimentation. The capacity of the Chl forming channel is defined as
Figure 3 shows the development of the capacity in the dark, under continuous standard far-red light (i.e. far-red light was given until the onset of white light to determine the capacity in agreement with the procedure described in Fig. 2) and under continuous white light (see Fig. 1). We notice that there is a similar time course of the capacity under all conditions : continuous increase starting shortly after 24 h after sowing, plateau, continuous decrease. Light stimulates the capacity dramatically but cannot prevent the decrease. The decrease of the capacity is neither related to the lack of inorganic ions (the application of Hoag-
A Chl - [nmol Chl pair of cotyledons- 1 h - 1] At under white light (7,000 lx) which saturates the PChl ~ Chl photoconversion. To determine the capacity in continuous white light and under 12 h l i g h t - 12 h dark cycles the first derivatives of the Chl accumulation curves were determined graphically (Fig. 1). The capacity at a given time in etiolated seedlings and in seedlings pretreated with phytochrome (operationally, with light pulses or with longterm far-red light) was measured by determining the slope of the Chl accumulation curve during the linear phase of Chl synthesis in white light after the initial lag-phase (2 h) was overcome (Fig. 2). The linear regression line was calculated with a program from Diehl-Combitron 5, archive no. 10376. The slope was considered as "capacity". The capacity of the ALA forming system was determined analogously from the slope of the linear ALA accumulation curve (see Fig. 2). The possibility that the Pfr formed in white light will affect the capacity of etiolated or pretreated seedlings during the first 5 h after the onset of white light (see Fig. 2) was thoroughly investigated using dichromatic irradiation (Hartmann, 1966). High irradiance far-red light obtained with a Xenon arc and a RG9 glass filter fi'om Schott, Mainz, which establishes a very low photoequilibrium of the phytochrome system (q~rr0.5). It was found that even a strong decrease of the photoequilibrium (~ofr+wh40.5, approaching q~fr) has no detectable influence on Chl accumulation during the first 5 h in the case of pre-irradiated seedlings whereas in the case of fully etiolated seedlings, which had not received any light treatment prior to the onset of white light, the rate of Chl accumulation was somewhat reduced by the simultaneous application of high irradiance (380 Wm-Z) far-red light between
50
4O w
?-
30
g o
E ,_% 17
2O ChL o
O
O, 0 J 2/,h a f t e r
d 12
24
time
l
J
i
J _ _ _ J ~
36
1,8
60
72
a f t e r onset of w h i t e
[In]
84
Light
sawing
Fig. 1. Time course of Chl accumulation in continuous standard white light (7,000 Ix) ( - - o - - ) and under light-dark cycles (--- 12 h white light ( w h ) - 1 2 h dark (d)--, o - - ) . Onset of light: 24h after sowing
H. Gehring et al. : Chlorophyll Synthesis, Phytochrome and Circadian Rhythms
~ALA
5 -
ly following far-red light pulse; see Kasemir et al., 1973) we may conclude that continuous far-red light operates exclusively through phytochrome (Pfr)There are strong theoretical arguments in favor of this conclusion (see Materials and Methods). The very similar development of the capacity in far-red and in white light (Fig. 3) indicates that even in continuous white light the capacity is determined mainly by phytochrome (Pfr). In any case, photosynthesis has no positive effect on the capacity, and there is no feedback from actual Cht synthesis to the capacity for Chl synthesis. In the far-red light the rate of Chl synthesis is less than 1% of the rate in white light and the plastids from far-red and from white light grown mustard seedlings are very different (Frosch et al., 1976). Nevertheless the development of the capacity in continuous far-red and white light is almost the same. It is concluded that the pattern of development of the capacity is determined by endogenous factors (" developmental homeostasis ", "primary differentiation", see Mohr, 1972) whereas the actual extent of the capacity is determined by phytochrome (Pf,). Similar conclusions with regard to the developmental homeostasis of temporal patterns in photomorphogenesis were drawn previously (Brfining et al., 1975; Frosch et al., 1976; Mohr, 1976) in connection with the induction of Calvin cycle enzymes by phytochrome.
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Planta 133, 295-302 (1977)
9 by Springer-Verlag 1977
The Capacity of Chlorophyll-a Biosynthesis in the Mustard Seedling Cotyledons as Modulated by Phytochrome and Circadian Rhythmicity H. Gehring, H. Kasemir, and H. M o h r Biologisches Institut II, Universiffit Freiburg, Sch~inzlestrafle9-11, D-7800 Freiburg i.Br., Federal Republic of Germany
Abstract. Within the temporal pattern of " p r i m a r y differentiation" the capacity of c h l o r o p h y l l - a biosynthesis in the cotyledons of Sinapis alba L. seedlings is controlled by phytochrome (in continuous light) or by releasing the circadian rhythm either with lightdark cycles or by a light - , dark transition. The sensor pigment for this process is phytochrome. It is very probable that in continuous light as well as under conditions under which the circadian rhythm plays the major part, the capacity of chlorophyll a biosynthesis is limited by the capacity of the biosynthetic step which produces 5-aminolaevulinate. Key words: 5-Aminolaevulinic acid - Chlorophyll synthesis -- Circadian rhythm -- Phytochrome -Sinapis alba L.
tion about the steps of Chl biosynthesis was recently published by Schneider (1975). The present paper is concerned with the regulation of Chl biosynthesis. We pose the question of what factors determine the actual capacity of the Chl synthesizing anabolic channel in the intact plant. By " c a p a c i t y " we mean the m a x i m u m rate of Chl synthesis as it is observed under light conditions which saturate the PChl ~ Chl photoconversion (Koski et al., 1951 ; Smith, 1960). It was found previously (Kasemir et al., 1973) that phytochrome influences the capacity for Chl synthesis in the mustard seedling. In the present paper we wanted to measure the time course of the capacity in the mustard seedling cotyledons and to identify those factors which determine the capacity during development. It will be shown that besides phytochrome the temporal pattern o f " primary differentiation" (see Mohr, 1972) and the circadian rhythm (see Biinning, 1973) must be considered.
Introduction The biosynthetic pathway leading to chlorophyll a (Chl) is known in principle (see A r o n o f f and Ellsworth, 1968; Rebeiz and Castelfranco, 1973; Bogorad, 1976) even though the mode of formation of 5-aminolaevulinate (ALA) is not yet fully established (see Beale and Castelfranco, 1974; Meller et al., 1975). There is good evidence that Chl synthesis takes place in the plas.tid c o m p a r t m e n t (Wellburn and Wellburn, 1971; Rebeiz et al., 1973). However, the enzymes involved are very probably being synthesized exclusively in the cytoplasm (see B6rner, 1973; Galling et al., 1973). A thorough review of the informaAbbreviations: Chl=chlorophyll(ide) a; ALA=5-aminolaevulihate; LA=laevulinate; PChI=protochlorophyll(ide); ALAD= aminolaevutinate dehydratase (EC 4.2.1.24); cpx=[PfrJ/[Ptot], photoequilibrium of the phytochrome system at the wavelength 2, whereby [P,oJ=[Pr] +[Pfr]. Pfr is the physiologically active, far-red absorbing form of the phytochrome system.
Materials and Methods Standard Techniques Standard techniques for photomorphogenic research with mustard seedlings were used (Mohr, 1966, 1972). The seeds (Sinapis alba L.) were purchased in 1971 from Asgrow Company (Hamburg). The seedlings were grown at 25.0 +0.2~ in the dark. Preirradiations were performed either with standard red (emission maximum at 656 nm, band width 15 nm, irradiance 0.675 Wm 2) or standard far-red (emission maximum at 740 nm, band width 123 nm, irradiance 3.5 Wm-2) sources (Mohr etal., 1964; Mohr, 11966) or with monochromatic 756 nm light obtained from a modified Leitz projector Prado 500 (Mohr and Schoser, 1959) equipped with a AL interference filter from Schott, Mainz (2m,~: 756 rim; band width 21 rim; irradiance 7 Wm-2). Standard white light was obtained from fluorescent white light tubes (Osram, alternating L40 W/15 and L40 W/25) at an illuminance of 7,000 lx. The characteristics of the standard light sources have been described repeatedly (e.g. Mohr, 1966). Continuous f~ir-red light is consMered to operate exclusivelythrough phytochrome (Hartmann, 1966; Mohr,
296
H. Gehring et al. : Chlorophyll Synthesis, Phytochrome and Circadian Rhythms
1972; Drumm et al., 1975; Sch~ifer, 1975). The use of the biological unit " p a i r of cotyledons" as the suitable system of reference for Chl was justified previously (see Mohr, 1972). Determination of Chlorophyll a and 5-aminolaevulinate Extraction and determination of Chl were performed after Ziegler and Egle (1965) as described by Kasemir and Mohr (1967). While the presence of chlorophyll b was considered in the determination of the chlorophyll a values, chlorophyll b per se was not followed in the present investigation. Inhibition of A L A D with LA and determination of ALA was performed according to Masoner and Kasemir (1975) using a molar extinction coefficient of 64 x 10311 mo1-1 cm -1] (Urata and Granick, 1963). The LA required to inhibit A L A D was purchased from Fluka A.G., Buchs, Switzerland.
2 and 5 h after the onset of the standard white light. Therefore the capacity of fully etiolated seedlings is probably slightly over estimated in the present paper as compared to seedlings which have already received a phytochrome pretreatment. Since this effect has no bearing on the principal results we are presenting and discussing in this paper, the experiments with dichromatic irradiation will not be described in detail. The pertinent information is available elsewhere (Gehring, 1976). Means. The mean values as given in the figures are based on at least 4 (sometimes up to 12) independent parallels. The estimated standard error of the means is of the order of 4~5%.
Results
1. Development of the Capacity under Constant Environmental Conditions
Determination of the Capacity of the Chl Synthesizing Channel Figure 1 shows the time course of Chl accumulation under continuous standard white light (7,000 lx). The illuminance of the standard white light suffices to saturate the PChl ~ Chl photoconversion under all circumstances. It was shown that no detectable photodestruction occurs at an illuminance of 7,000 ix (see Gehring, 1973). Figure 1 also shows the time course of Chl accumulation under repeated white l i g h t - d a r k cycles (12 h d a r k - 1 2 h light). Since there is no PChl ~ Chl photoconversion in the dark, Chl accumulation takes place step wise. The fact that there is no significant decrease of the Chl contents during the dark period indicates that Chl turnover does not play a role during the period of our experimentation. The capacity of the Chl forming channel is defined as
Figure 3 shows the development of the capacity in the dark, under continuous standard far-red light (i.e. far-red light was given until the onset of white light to determine the capacity in agreement with the procedure described in Fig. 2) and under continuous white light (see Fig. 1). We notice that there is a similar time course of the capacity under all conditions : continuous increase starting shortly after 24 h after sowing, plateau, continuous decrease. Light stimulates the capacity dramatically but cannot prevent the decrease. The decrease of the capacity is neither related to the lack of inorganic ions (the application of Hoag-
A Chl - [nmol Chl pair of cotyledons- 1 h - 1] At under white light (7,000 lx) which saturates the PChl ~ Chl photoconversion. To determine the capacity in continuous white light and under 12 h l i g h t - 12 h dark cycles the first derivatives of the Chl accumulation curves were determined graphically (Fig. 1). The capacity at a given time in etiolated seedlings and in seedlings pretreated with phytochrome (operationally, with light pulses or with longterm far-red light) was measured by determining the slope of the Chl accumulation curve during the linear phase of Chl synthesis in white light after the initial lag-phase (2 h) was overcome (Fig. 2). The linear regression line was calculated with a program from Diehl-Combitron 5, archive no. 10376. The slope was considered as "capacity". The capacity of the ALA forming system was determined analogously from the slope of the linear ALA accumulation curve (see Fig. 2). The possibility that the Pfr formed in white light will affect the capacity of etiolated or pretreated seedlings during the first 5 h after the onset of white light (see Fig. 2) was thoroughly investigated using dichromatic irradiation (Hartmann, 1966). High irradiance far-red light obtained with a Xenon arc and a RG9 glass filter fi'om Schott, Mainz, which establishes a very low photoequilibrium of the phytochrome system (q~rr0.5). It was found that even a strong decrease of the photoequilibrium (~ofr+wh40.5, approaching q~fr) has no detectable influence on Chl accumulation during the first 5 h in the case of pre-irradiated seedlings whereas in the case of fully etiolated seedlings, which had not received any light treatment prior to the onset of white light, the rate of Chl accumulation was somewhat reduced by the simultaneous application of high irradiance (380 Wm-Z) far-red light between
50
4O w
?-
30
g o
E ,_% 17
2O ChL o
O
O, 0 J 2/,h a f t e r
d 12
24
time
l
J
i
J _ _ _ J ~
36
1,8
60
72
a f t e r onset of w h i t e
[In]
84
Light
sawing
Fig. 1. Time course of Chl accumulation in continuous standard white light (7,000 Ix) ( - - o - - ) and under light-dark cycles (--- 12 h white light ( w h ) - 1 2 h dark (d)--, o - - ) . Onset of light: 24h after sowing
H. Gehring et al. : Chlorophyll Synthesis, Phytochrome and Circadian Rhythms
~ALA
5 -
ly following far-red light pulse; see Kasemir et al., 1973) we may conclude that continuous far-red light operates exclusively through phytochrome (Pfr)There are strong theoretical arguments in favor of this conclusion (see Materials and Methods). The very similar development of the capacity in far-red and in white light (Fig. 3) indicates that even in continuous white light the capacity is determined mainly by phytochrome (Pfr). In any case, photosynthesis has no positive effect on the capacity, and there is no feedback from actual Cht synthesis to the capacity for Chl synthesis. In the far-red light the rate of Chl synthesis is less than 1% of the rate in white light and the plastids from far-red and from white light grown mustard seedlings are very different (Frosch et al., 1976). Nevertheless the development of the capacity in continuous far-red and white light is almost the same. It is concluded that the pattern of development of the capacity is determined by endogenous factors (" developmental homeostasis ", "primary differentiation", see Mohr, 1972) whereas the actual extent of the capacity is determined by phytochrome (Pf,). Similar conclusions with regard to the developmental homeostasis of temporal patterns in photomorphogenesis were drawn previously (Brfining et al., 1975; Frosch et al., 1976; Mohr, 1976) in connection with the induction of Calvin cycle enzymes by phytochrome.
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