Photosynthesis Research 11:161-171 (1987) © Martinus Nijhoff Publishers, Dordrecht - - Printed in the Netherlands
161
Regular paper
Regulation of the photosynthetic electron transport during dark-light transitions by activation of the ferredoxin-NADP+-oxidoreductase in higher plants* W. RI~HLE, R. PSCHORN and A. WILD Institut fiir Allgemeine Botanik, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany
(Received." 24 February 1986; in revisedform: 9 May 1986)
Keywords:cytochrome f, electron transport,
ferredoxin-NADP ÷ -oxidoreductase, light ac-
tivation, Sinapis alba, Valerianella locusta Abstract. Absorbance changes associated with the oxidation and reduction of cytochrome f
belong to the classical observations about the interaction of the two photosystems. A complex induction pattern of cytochrome f oxidation results, if both photosystems are excited simultaneously. This indicates a light-modulated regulation of the photosynthetic electron transport, which we examined for intact biological systems of decreasing complexity. The ferredoxin-NADP + -oxidoreductase (FNR) is suggested to be activated by light and inactivated in the dark. This is pointed out by the kinetics of variable fluorescence and by the influence of different artificial electron acceptors on the cytochrome f kinetics, The photoreduction of NADP ÷ by carefully prepared thylakoids demonstrates the activation process directly.
Introduction In the photosynthetic electron transport chain c y t o c h r o m e f is located between both photosystems [8]. A b s o r b a n c e changes o f cytochrome f indicate its redox-state, whose time-course can be well used as an indicator for the activities o f photosystem I and 1I. Intact leaves show a complex induction pattern o f c y t o c h r o m e f oxidation during the first few seconds o f excitation [18]. A similar induction p h e n o m e n o n was described for intact chloroplasts from the siphonal green alga Bryopsis [21, 23]. Isolated thylakoids, however, only exhibit a m o n o p h a s i c oxidation to the steadystate level. The induction o f the c y t o c h r o m e f oxidation (Fig. l a) is divided into one phase o f quick oxidation, a second one o f reduction and a third glow oxidation phase. It was discussed that the induction kinetics o f Bryopsis chloroplasts reflect a light activation o f the f e r r e d o x i n - N A D P ÷oxidoreductase ( F N R , EC 1.18.1.2) [21] which was directly measured by the rate o f N A D P ÷ -photoreduction [22]. Properties o f the F N R concerning its conformation, mobility and the binding o f N A D P ÷ were studied * This work was supported by the Deutsche Forschungsgemeinschaft.
162 with the isolated enzyme from spinach [6, 24] and in a reconstituted system [27, 26]. In this study we examine the cytochrome f signals of higher plants on the level of intact leaves, protoplasts, intact chloroplasts and thylakoids and try to correlate it to the regulation of electron flow. Materials and methods
Plants of Valerianella locusta and Sinapis alba were grown as described previously [18], and 25 days old leaves of Valerianella were taken for the measurement- and isolation-procedures. Isolations of protoplasts and intact CO2-fixating chloroplasts (class A) were prepared by enzymatic digestion [17]. The intact chloroplasts were centrifuged only at 150 g and measured in an assay medium according to [16] containing 0.33M sorbitol, 0.05M HEPES/KOH pH7.6, l mM MnCIE, 5mM MgC12, 2mM EDTA, 5raM NaaP2OT, 10raM NaHCO3, 1 mM 3-phosphoglycerine acid and 1 mM oxaloacetate. The intactness was about 90% as determined by the assay of [I0] with a Clark-type oxygen electrode. Osmotically shocked choroplasts (class B) were revealed by transferring class A chloroplasts into distilled water for 60 s and addition of double concentrated assay medium. Isolation of thylakoids (class C chloroplasts) was described earlier [28], whereas the centrifugation procedures (1400 g) were carried out at 0 °C. An improved activity of the thylakoids was achieved by isolating them in a slurry of half frozen isolation media. The measurements of the cytochrome f kinetics were carried out with an AMINCO-DW 2 double-wavelength spectrophotometer in the dualwavelength-mode (560--554 nm). Excitation light was filtered through a 4 mm RG 645 (Schott, Germany), and the photomultiplier was shielded against actinic light by a 4 mm BG 18 filter. The signals obtained during a defined dark-light schedule were recorded in a VUKO-VK-12 transientrecorder, and several kinetics were averaged in a CBM-4032 computer. Kinetics of special wavelengths (e.g. 535 nm) and the difference-spectra were taken in a similar way using the split-beam-mode. Variable fluorescence was measured under the same conditions of excitation as the cytochrome f kinetics. As the fluorescence emission overlays with the excitation of PS I a flash system was used [25]. The measuring flash from an Argon/Hydrogen flash lamp (Impulsphysik GmbH, Germany) was triggered at variable times during the excitation. A 4 mm BG 28 colour filter removed all red components from the flash, and the photomultiplier was shielded against the actinic flash by a 3 mm RG 660 (Schott, Germany). The fluorescence signal with a halftime of 0.5 ms was separated from the DC signal, which was due to the PS I excitation, by a high pass filter of 200 Hz. It was stored and averaged in the system described above.
163 The rate of the NADP + -photoreduction was recorded at 366 nm using a BG 28 filter as a shield against the same excitation light as above. 1 mM NADP + and 15/~g. ml-1 ferredoxin were added to the final sample containing class B thylakoids of 150 ktg Chl. m1-1 . The absorbance changes after different durations of excitation were recorded, and the rate of NADP + -photoreduction was calculated using an extinction coefficient for NADP + ofe366 = 3.3cm2"/~mol i. Chlorophyll was determined according to [30]. Ferredoxin was isolated by the method of Buchanan and Arnon [4].
Results
Figure 1 displays the typical response ofa cytochrome finduction kinetics. A first quick phase of oxidation is followed by a temporary reduction phase. During a third phase cytochrome f is oxidized once more until a steady state is reached. Such a signal could be found for several species of higher plants, such as Sinapis alba, Valerianella locusta, Helianthus annuus and Viburnum rhytidiophyllum. The spectra of the signal, computed at different times after onset of excitation, are presented in Figure lb. The ~-peak at 554 nm demonstrates that all phases of the kinetics are attributed to absorbance changes of cytochrome f. Furthermore the spectra point out the light-scattering signal at 535 nm, which is due to the formation of the proton gradient [12, 13], and the electrochromic shift at 520 nm [29]. Interferences of other components are small compared to measurements of the cytochrome f signals in the soret band at 422 nm. Figure 2 compares the induction signals in systems of decreasing complexity from the intact leaf (a), protoplast (b), intact chloroplast (c), and the isolated thylakoid (d). The three phases observed in the leaf can be recognized also in protoplasts and intact chloroplasts [21] although they are expressed to a lower extend. This is due to a decrease of the photosynthetic rate by the isolation procedure. The amplitude of the signal in the leaf is bigger because of the increased optical path [19]. Thylakoids with an artificial electron acceptor exhibit phase 1 only, which demonstrates that the induction phenomenon is lost at this stage. Figure 3 compares the kinetics of the fluorescence induction, the lightscattering signal at 535 nm and the electrochromic shift at 520 nm to the cytochrome f kinetics. The peak of the variable fluorescence coincides with the minimum in the cytochrome f kinetics at the end of phase 2. Phase 3 is accompanied by a parallel decline of the fluorescence signal [see also 24]. The electrochromic shift (Figure 3) has already reached the steady state at the beginning of phase 3. There is a clear coincidence between phase 3 of the cytochrome f signal and the increase of the proton gradient
164
Light
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III
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d
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520
530
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550
560
570
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165 although both traces are not completely parallel. Addition of N H } prevents the induction effect (Figure 4 b) and produces monophasic oxidation kinetics of cytohrome f. Hill reagents like NO2 and SOl- which withdraw electrons after photosystem I in the vicinity of ferredoxin suppress phases 2 and 3 in a similar way. This leads to the conclusion that phase 2 is a consequence of accumulated electrons due to a block behind photosystem I. Phase 3 would then reflect the time which is necessary to remove this block. The ferredoxin-NADP ÷ -oxidoreductase is the sole component between ferredoxin and N A D P ÷ and therefore should represent the site of activation. In order to substantiate this aspect we tried to measure N A D P ÷ reduction in thylakoid systems. Addition of ferredoxin restored the electron transport to N A D P ÷ in class C chloroplasts but phase 3 of the cytochrome f signal was not visible. This may be due to the easy detachment of the F N R from the membrane [5]. Intact chloroplasts still showed the complete cytochrome f signal. We used them to prepare class B thylakoids by gentle disruption of the envelope. The cytochrome f signal of these thylakoids is depicted in Figure 5a and does not differ from the kinetics of intact chloroplasts. Furthermore the trace of the N A D P ÷ photoreduction under the same conditions (Fig. 5 b) demonstrates an activation of the F N R which coincides with phase 3 of the cytochrome f signal. Discussion
The induction of the cytochrome f oxidation is divided into one phase of quick oxidation, a second one of reduction and a third slow oxidation phase. The first phase is best pronounced if the plastoquinone-pool is in an oxidized state. This can either be achieved by preillumination with photosystem I-light or by long dark intervals [18, 20]. The second phase reflects an higher activity of photosystem II compared to photosystem I, which is inverted during the third phase. Several possibilities of explanation exist as far as this inversion is concerned: - - changes in the membranal energy distribution by the phosphorylat]on of the light-harvesting complex [2]; - - the decrease of the cytochrome f reduction rate by the formation of the proton gradient [11]; -activation of photosystem I by stimulation of the ferredoxin-NADP ÷oxidoreductase [21, 7].
Figure 1. a. Typical cytochromef kinetics (560-554nm) of a Sinapis leaf during excitation with 15W.m-2 RG 645 light. Averages of 10 measurementswith a dark interval of 60s. b. Light minus dark differencespectra computed from split beam kinetics at the different moments which are marked by I-VI in figure la. The spectra are displaced on the ordinate for the sake of clearness.
166
Light
on
off
.
L
a
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. ,
.~-
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Figure 2. comparison of the cytochrome f signals of an intact leaf (a), protoplasts (b), intact (class A) chloroplasts (c), and thylakoids (d) from Valerianella locusta. Excitation with 15 W. m-2 R G 645 light. Averages of 10 measurements (in a, b, d) and 36 measurements (c) with a dark interval of 60 s.
167
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161
Regular paper
Regulation of the photosynthetic electron transport during dark-light transitions by activation of the ferredoxin-NADP+-oxidoreductase in higher plants* W. RI~HLE, R. PSCHORN and A. WILD Institut fiir Allgemeine Botanik, Saarstr. 21, D-6500 Mainz, Federal Republic of Germany
(Received." 24 February 1986; in revisedform: 9 May 1986)
Keywords:cytochrome f, electron transport,
ferredoxin-NADP ÷ -oxidoreductase, light ac-
tivation, Sinapis alba, Valerianella locusta Abstract. Absorbance changes associated with the oxidation and reduction of cytochrome f
belong to the classical observations about the interaction of the two photosystems. A complex induction pattern of cytochrome f oxidation results, if both photosystems are excited simultaneously. This indicates a light-modulated regulation of the photosynthetic electron transport, which we examined for intact biological systems of decreasing complexity. The ferredoxin-NADP + -oxidoreductase (FNR) is suggested to be activated by light and inactivated in the dark. This is pointed out by the kinetics of variable fluorescence and by the influence of different artificial electron acceptors on the cytochrome f kinetics, The photoreduction of NADP ÷ by carefully prepared thylakoids demonstrates the activation process directly.
Introduction In the photosynthetic electron transport chain c y t o c h r o m e f is located between both photosystems [8]. A b s o r b a n c e changes o f cytochrome f indicate its redox-state, whose time-course can be well used as an indicator for the activities o f photosystem I and 1I. Intact leaves show a complex induction pattern o f c y t o c h r o m e f oxidation during the first few seconds o f excitation [18]. A similar induction p h e n o m e n o n was described for intact chloroplasts from the siphonal green alga Bryopsis [21, 23]. Isolated thylakoids, however, only exhibit a m o n o p h a s i c oxidation to the steadystate level. The induction o f the c y t o c h r o m e f oxidation (Fig. l a) is divided into one phase o f quick oxidation, a second one o f reduction and a third glow oxidation phase. It was discussed that the induction kinetics o f Bryopsis chloroplasts reflect a light activation o f the f e r r e d o x i n - N A D P ÷oxidoreductase ( F N R , EC 1.18.1.2) [21] which was directly measured by the rate o f N A D P ÷ -photoreduction [22]. Properties o f the F N R concerning its conformation, mobility and the binding o f N A D P ÷ were studied * This work was supported by the Deutsche Forschungsgemeinschaft.
162 with the isolated enzyme from spinach [6, 24] and in a reconstituted system [27, 26]. In this study we examine the cytochrome f signals of higher plants on the level of intact leaves, protoplasts, intact chloroplasts and thylakoids and try to correlate it to the regulation of electron flow. Materials and methods
Plants of Valerianella locusta and Sinapis alba were grown as described previously [18], and 25 days old leaves of Valerianella were taken for the measurement- and isolation-procedures. Isolations of protoplasts and intact CO2-fixating chloroplasts (class A) were prepared by enzymatic digestion [17]. The intact chloroplasts were centrifuged only at 150 g and measured in an assay medium according to [16] containing 0.33M sorbitol, 0.05M HEPES/KOH pH7.6, l mM MnCIE, 5mM MgC12, 2mM EDTA, 5raM NaaP2OT, 10raM NaHCO3, 1 mM 3-phosphoglycerine acid and 1 mM oxaloacetate. The intactness was about 90% as determined by the assay of [I0] with a Clark-type oxygen electrode. Osmotically shocked choroplasts (class B) were revealed by transferring class A chloroplasts into distilled water for 60 s and addition of double concentrated assay medium. Isolation of thylakoids (class C chloroplasts) was described earlier [28], whereas the centrifugation procedures (1400 g) were carried out at 0 °C. An improved activity of the thylakoids was achieved by isolating them in a slurry of half frozen isolation media. The measurements of the cytochrome f kinetics were carried out with an AMINCO-DW 2 double-wavelength spectrophotometer in the dualwavelength-mode (560--554 nm). Excitation light was filtered through a 4 mm RG 645 (Schott, Germany), and the photomultiplier was shielded against actinic light by a 4 mm BG 18 filter. The signals obtained during a defined dark-light schedule were recorded in a VUKO-VK-12 transientrecorder, and several kinetics were averaged in a CBM-4032 computer. Kinetics of special wavelengths (e.g. 535 nm) and the difference-spectra were taken in a similar way using the split-beam-mode. Variable fluorescence was measured under the same conditions of excitation as the cytochrome f kinetics. As the fluorescence emission overlays with the excitation of PS I a flash system was used [25]. The measuring flash from an Argon/Hydrogen flash lamp (Impulsphysik GmbH, Germany) was triggered at variable times during the excitation. A 4 mm BG 28 colour filter removed all red components from the flash, and the photomultiplier was shielded against the actinic flash by a 3 mm RG 660 (Schott, Germany). The fluorescence signal with a halftime of 0.5 ms was separated from the DC signal, which was due to the PS I excitation, by a high pass filter of 200 Hz. It was stored and averaged in the system described above.
163 The rate of the NADP + -photoreduction was recorded at 366 nm using a BG 28 filter as a shield against the same excitation light as above. 1 mM NADP + and 15/~g. ml-1 ferredoxin were added to the final sample containing class B thylakoids of 150 ktg Chl. m1-1 . The absorbance changes after different durations of excitation were recorded, and the rate of NADP + -photoreduction was calculated using an extinction coefficient for NADP + ofe366 = 3.3cm2"/~mol i. Chlorophyll was determined according to [30]. Ferredoxin was isolated by the method of Buchanan and Arnon [4].
Results
Figure 1 displays the typical response ofa cytochrome finduction kinetics. A first quick phase of oxidation is followed by a temporary reduction phase. During a third phase cytochrome f is oxidized once more until a steady state is reached. Such a signal could be found for several species of higher plants, such as Sinapis alba, Valerianella locusta, Helianthus annuus and Viburnum rhytidiophyllum. The spectra of the signal, computed at different times after onset of excitation, are presented in Figure lb. The ~-peak at 554 nm demonstrates that all phases of the kinetics are attributed to absorbance changes of cytochrome f. Furthermore the spectra point out the light-scattering signal at 535 nm, which is due to the formation of the proton gradient [12, 13], and the electrochromic shift at 520 nm [29]. Interferences of other components are small compared to measurements of the cytochrome f signals in the soret band at 422 nm. Figure 2 compares the induction signals in systems of decreasing complexity from the intact leaf (a), protoplast (b), intact chloroplast (c), and the isolated thylakoid (d). The three phases observed in the leaf can be recognized also in protoplasts and intact chloroplasts [21] although they are expressed to a lower extend. This is due to a decrease of the photosynthetic rate by the isolation procedure. The amplitude of the signal in the leaf is bigger because of the increased optical path [19]. Thylakoids with an artificial electron acceptor exhibit phase 1 only, which demonstrates that the induction phenomenon is lost at this stage. Figure 3 compares the kinetics of the fluorescence induction, the lightscattering signal at 535 nm and the electrochromic shift at 520 nm to the cytochrome f kinetics. The peak of the variable fluorescence coincides with the minimum in the cytochrome f kinetics at the end of phase 2. Phase 3 is accompanied by a parallel decline of the fluorescence signal [see also 24]. The electrochromic shift (Figure 3) has already reached the steady state at the beginning of phase 3. There is a clear coincidence between phase 3 of the cytochrome f signal and the increase of the proton gradient
164
Light
on
off
•
• V,.
CO f
a
0 TI
i
5 s
it O0 I 0
III
~')
d
b
,q
I
a
I
~
|
I
!
510
520
530
540
550
560
570
nm
165 although both traces are not completely parallel. Addition of N H } prevents the induction effect (Figure 4 b) and produces monophasic oxidation kinetics of cytohrome f. Hill reagents like NO2 and SOl- which withdraw electrons after photosystem I in the vicinity of ferredoxin suppress phases 2 and 3 in a similar way. This leads to the conclusion that phase 2 is a consequence of accumulated electrons due to a block behind photosystem I. Phase 3 would then reflect the time which is necessary to remove this block. The ferredoxin-NADP ÷ -oxidoreductase is the sole component between ferredoxin and N A D P ÷ and therefore should represent the site of activation. In order to substantiate this aspect we tried to measure N A D P ÷ reduction in thylakoid systems. Addition of ferredoxin restored the electron transport to N A D P ÷ in class C chloroplasts but phase 3 of the cytochrome f signal was not visible. This may be due to the easy detachment of the F N R from the membrane [5]. Intact chloroplasts still showed the complete cytochrome f signal. We used them to prepare class B thylakoids by gentle disruption of the envelope. The cytochrome f signal of these thylakoids is depicted in Figure 5a and does not differ from the kinetics of intact chloroplasts. Furthermore the trace of the N A D P ÷ photoreduction under the same conditions (Fig. 5 b) demonstrates an activation of the F N R which coincides with phase 3 of the cytochrome f signal. Discussion
The induction of the cytochrome f oxidation is divided into one phase of quick oxidation, a second one of reduction and a third slow oxidation phase. The first phase is best pronounced if the plastoquinone-pool is in an oxidized state. This can either be achieved by preillumination with photosystem I-light or by long dark intervals [18, 20]. The second phase reflects an higher activity of photosystem II compared to photosystem I, which is inverted during the third phase. Several possibilities of explanation exist as far as this inversion is concerned: - - changes in the membranal energy distribution by the phosphorylat]on of the light-harvesting complex [2]; - - the decrease of the cytochrome f reduction rate by the formation of the proton gradient [11]; -activation of photosystem I by stimulation of the ferredoxin-NADP ÷oxidoreductase [21, 7].
Figure 1. a. Typical cytochromef kinetics (560-554nm) of a Sinapis leaf during excitation with 15W.m-2 RG 645 light. Averages of 10 measurementswith a dark interval of 60s. b. Light minus dark differencespectra computed from split beam kinetics at the different moments which are marked by I-VI in figure la. The spectra are displaced on the ordinate for the sake of clearness.
166
Light
on
off
.
L
a
I |
. ,
.~-
:.
co !
oT - -
L I
!
!
5s
d
Figure 2. comparison of the cytochrome f signals of an intact leaf (a), protoplasts (b), intact (class A) chloroplasts (c), and thylakoids (d) from Valerianella locusta. Excitation with 15 W. m-2 R G 645 light. Averages of 10 measurements (in a, b, d) and 36 measurements (c) with a dark interval of 60 s.
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