Planta 143, 41-49 (1978)
Pl~)ll~ 9 by Springer-Verlag 1978
Regulation of Photosynthetic Electron Transport and Photophosphorylation in Intact Chloroplasts and Leaves of Spinacia oleracea L. U. Heber, H. Egneus*, U. Hanck, M. Jensen, and S. K6ster Botanisches Institut der Universitfit Dfisseldorf, UniversitS.tsstraBe1, D-4000 D/isseldorf, Federal Republic of Germany
Abstract. Oxygen ist reduced by the electron transport chain of chloroplasts during CO2 reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO2-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO2 reduction by electron flow to N A D P and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.
Key words: Cyclic photophosphorylation - Electron transport - Light scattering - Photosystem 1/II Regulation of electron flow. * P r e s e n t address," Botaniska Institutionen, Avd. f. Fysiologisk Botanik, Carl Skottsbergs Gata 22, S-41319 G6teborg, Sweden
Introduction It is still a matter of debate, how much ATP is synthesized in photosynthesis, when two electrons are transferred from water to an acceptor molecule such as NADP. The corresponding number is called the ATP/2 e ratio. Most published values vary between 0,9 and 2 (Hall, 1976). Robinson and Wiskich (1976) observed recently ATP/2 e ratios approaching 2, while Chain and Arnon (1977) reported values close to l. These data were obtained with broken chloroplasts which had lost the capability to photoreduce CO> In photosynthetically competent chloroplasts, calculated ATP/2 e ratios ranged between 1.1-1.4 (Heber and Kirk, 1975). CO2 reduction needs somewhat more ATP than that. The ATP/2 e requirement of the Calvin cycle is 1.5. Intact chloroplasts can also photoreduce glycerate (Heber et al., 1974). Its reduction to dihydroxyacetone phosphate needs 2 molecules of ATP per 2 electrons, but since phosphoglycerate (PGA) is an intermediate which is easily !o st from the chloroplasts by counterexchange with phosphate, the actual ATP/2 e requirement is higher than 2. It is obvious that, when the ATP requirement of a chloroplast reaction cannot be met by the ATP synthesized when 2 electrons travel from water to NADP, extra ATP must be produced by another photoreaction. We have shown previously (Egneus et al., 1975) that intact spinach chloroplasts can reduce oxygen to hydrogen peroxide during CO 2 reduction. During PGA reduction, no hydrogen peroxide formation was observed. As CO2 reduction has a higher ATP requirement than PGA reduction (the ATP/2 e requirement is 1.0 for PGA reduction), we proposed that electron transport to oxygen supplied extra ATP needed for CO2 reduction at least at low light intensities. Similar results obtained with different types of chloroplasts and intact plants have later been 0032-0935/78/0143/0041/$ 01.80
42
u. Heber et al.: Electron Transport and Photophosphorylation
p r e s e n t e d by others ( H u b e r a n d E d w a r d s , 1975; Glidewell a n d R a v e n , 1975; G i v a n , 1976; F o r t i a n d G e r o l a , 1977; Jennings a n d F o r t i , 1975). The q u e s t i o n o f h o w m u c h A T P can be s u p p l i e d by electron transp o r t to o x y g e n in intact c h l o r o p l a s t s d u r i n g CO2 red u c t i o n r e m a i n e d u n a n s w e r e d . The rate o f o x y g e n u p t a k e in the M e h l e r - r e a c t i o n (Mehler, 1951) is slow in the a b s e n c e of an a d d e d e l e c t r o n a c c e p t o r ( F o r t i a n d J a g e n d o r f , 1961). In this p a p e r we p r e s e n t evidence t h a t in CO2r e d u c i n g i n t a c t s p i n a c h c h l o r o p l a s t s which are n o t sufficiently well c o u p l e d to satisfy the A T P - d e m a n d by linear e l e c t r o n t r a n s p o r t , cyclic e l e c t r o n t r a n s p o r t takes p a r t in A T P p r o d u c t i o n . The affinity o f elect r o n s to different electron carriers a n d r e d o x c o n t r o l (poising) regulate the d i s t r i b u t i o n of electrons between N A D P , o x y g e n a n d the p r i m a r y a c c e p t o r o f the cyclic electron t r a n s p o r t p a t h w a y .
Materials and Methods
Intact chloroplasts capable of high rates of CO2-dependent oxygen evolution were isolated from greenhouse-or field-grown spinach as described previously (Egneus et al., 1975; Heber, 1973). Preparations used in this investigation contained more than 80% (up to 98%) chloroplasts which had retained their envelopes during isolation as measured routinely by the ferricyanide method (Heber and Santarius, 1970). COz-dependent oxygen evolution was measured by a Clark electrode, the quenching of 9-aminoacridine fluorescence (concentration 5 DM) by a photomultiplier. Rates of CO2reduction ranged between 130 and 280 gmoles (rag chlorophyll)- 1 h- 1 Light source and filter arrangements were described by Tillberg, Giersch and Heber (1977). Chloroplast suspensions were made and kept anaerobic by adding 10 mM glucose and some glucose oxidase. H=O 2 formed during glucose oxidation was decomposed by an excess of catalase (usually about 1,300 international enzyme units ml-*). Transient oxygenation of anaerobic chloroplast suspensions was brought about by injecting known amounts of HzO 2. Light absorption by chloroplasts was determined in an Ulbricht sphere. Scattering of a measuring beam of 535 nm light by leaves or isolated chloroplasts was measured usually in transmission (Heber, 1969), but occasionally also as 70~ backscattering. Leaves were kept in a stream of CO2-free air or nitrogen during the measurements. The flow rate was between 40 and 60 1 h- 1 Mass spectrometric experiments were performed as described by Egneus et al. (1975),
Table 1. Oxygen exchange by intact spinach chloroplasts under different light intensities as measured with a mass spectrometer. Substrate: 2 or 4 mM HCO~
Incident intensity of red light (Win-2)
COz-dependent oxygen exchange, [in pmol (mg chl)- 1 h ~]
9 12 84 120
16 34 67 70
% intact chloroplasts in preparation
net 02 evolution 02 uptake 6 4 7 11
93 87 93 72
ing glycolate p r o d u c t i o n in the r i b u l o s e b i s p h o s p h a t e o x y g e n a s e r e a c t i o n was m i n i m i z e d by using a s a t u r a t ing b i c a r b o n a t e c o n c e n t r a t i o n . G l y c o l a t e synthesis is c o m p e t i t i v e l y i n h i b i t e d b y CO2. M o s t o f the 180 2 u p t a k e m e a s u r e d is therefore caused by oxygen reduction by the electron t r a n s p o r t chain which is c o u p l e d to A T P f o r m a t i o n (Egneus et al., 1975), The results p r e s e n t e d in T a b l e 1 s h o w that, as the light intensity is increased, the r e d u c t i o n o f o x y g e n does n o t increase p r o p o r t i o n a l l y to the C O 2 - d e p e n d e n t oxygen evolution, i.e. o x y g e n r e d u c t i o n d u r i n g p h o t o s y n t h e s i s appears to be s a t u r a t e d at r a t h e r low light intensities. This suggests t h a t at high light intensities electron t r a n s p o r t to o x y g e n c a n n o t c o n t r i b u t e all the A T P n e e d e d for p h o t o s y n t h e s i s in insufficiently well-coup l e d c h l o r o p l a s t s . W e w a n t e d to k n o w w h e t h e r cyclic p h o t o p h o s p h o r y l a t i o n also p r o v i d e s A T P for p h o t o synthesis. A s cyclic p h o t o p h o s p h o r y l a t i o n is k n o w n to be sensitive to a n t i m y c i n A ( T a g a w a et al., 1963; % 100-
b
o
12 Wm -z red tight
50-
X.+§ 245Wm-2 red tight
Results
1. Electron Flow to Oxygen and Cyclic Electron Transport during of Isolated Spinach Chloroplasts
CO 2
Reduction
T a b l e 1 shows p h o t o s y n t h e t i c o x y g e n e v o l u t i o n during CO2 r e d u c t i o n by intact c h l o r o p l a s t s a n d simultan e o u s o x y g e n u p t a k e at different light intensities as m e a s u r e d in a m a s s s p e c t r o m e t e r . O x y g e n u p t a k e dur-
I
2,5 Antimycin A
5pM
Fig. 1. CO2-dependent oxygen evolution by intact chloroplasts as a function of the concentration of antimycin A. Photosynthetic rates are expressed as percent of control rates observed in the absence of antimycin A. In 3 different experiments, control rates at 245Wm -z were 131,210 and 258 pmot (mgchl) lh-1, at 12 Wm -2 17, 36 and 42 gmol (rag chI) lh-i
U, Heber etal, : Electron Transport and Photophosphorylation
fo
9 l m M OAA
o
~
o-.~
~
without substrate
40-
2 LT.
Pl~)ll~ 9 by Springer-Verlag 1978
Regulation of Photosynthetic Electron Transport and Photophosphorylation in Intact Chloroplasts and Leaves of Spinacia oleracea L. U. Heber, H. Egneus*, U. Hanck, M. Jensen, and S. K6ster Botanisches Institut der Universitfit Dfisseldorf, UniversitS.tsstraBe1, D-4000 D/isseldorf, Federal Republic of Germany
Abstract. Oxygen ist reduced by the electron transport chain of chloroplasts during CO2 reduction. The rate of electron flow to oxygen is low. Since antimycin A inhibited CO2-dependent oxygen evolution, it is concluded that cyclic photophosphorylation contributes ATP to photosynthesis in chloroplasts which cannot satisfy the ATP requirement of CO2 reduction by electron flow to N A D P and to oxygen. Inhibition of photosynthesis by antimycin A was more significant at high than at low light intensities suggesting that cyclic photophosphorylation contributes to photosynthesis particularly at high intensities. Cyclic electron flow in intact chloroplasts is under the control of electron acceptors. At low light intensities or under far-red illumination it is decreased by substrates which accept electrons from photosystem I such as oxaloacetate, nitrite or oxygen. Obviously, the cyclic electron transport pathway is sensitive to electron drainage. In the absence of electron acceptors, cyclic electron flow is supported by far-red illumination and inhibited by red light. The inhibition by light exciting photosystem II demonstrated that the cyclic electron transport pathway is accessible to electrons from photosystem II. Inhibition can be relieved by oxygen which appears to prevent over-reduction of electron carriers of the cyclic pathway and thus has an important regulatory function. The data show that cyclic electron transport is under delicate redox control. Inhibition is caused both by excessive oxidation and by over-reduction of electron carriers of the pathway.
Key words: Cyclic photophosphorylation - Electron transport - Light scattering - Photosystem 1/II Regulation of electron flow. * P r e s e n t address," Botaniska Institutionen, Avd. f. Fysiologisk Botanik, Carl Skottsbergs Gata 22, S-41319 G6teborg, Sweden
Introduction It is still a matter of debate, how much ATP is synthesized in photosynthesis, when two electrons are transferred from water to an acceptor molecule such as NADP. The corresponding number is called the ATP/2 e ratio. Most published values vary between 0,9 and 2 (Hall, 1976). Robinson and Wiskich (1976) observed recently ATP/2 e ratios approaching 2, while Chain and Arnon (1977) reported values close to l. These data were obtained with broken chloroplasts which had lost the capability to photoreduce CO> In photosynthetically competent chloroplasts, calculated ATP/2 e ratios ranged between 1.1-1.4 (Heber and Kirk, 1975). CO2 reduction needs somewhat more ATP than that. The ATP/2 e requirement of the Calvin cycle is 1.5. Intact chloroplasts can also photoreduce glycerate (Heber et al., 1974). Its reduction to dihydroxyacetone phosphate needs 2 molecules of ATP per 2 electrons, but since phosphoglycerate (PGA) is an intermediate which is easily !o st from the chloroplasts by counterexchange with phosphate, the actual ATP/2 e requirement is higher than 2. It is obvious that, when the ATP requirement of a chloroplast reaction cannot be met by the ATP synthesized when 2 electrons travel from water to NADP, extra ATP must be produced by another photoreaction. We have shown previously (Egneus et al., 1975) that intact spinach chloroplasts can reduce oxygen to hydrogen peroxide during CO 2 reduction. During PGA reduction, no hydrogen peroxide formation was observed. As CO2 reduction has a higher ATP requirement than PGA reduction (the ATP/2 e requirement is 1.0 for PGA reduction), we proposed that electron transport to oxygen supplied extra ATP needed for CO2 reduction at least at low light intensities. Similar results obtained with different types of chloroplasts and intact plants have later been 0032-0935/78/0143/0041/$ 01.80
42
u. Heber et al.: Electron Transport and Photophosphorylation
p r e s e n t e d by others ( H u b e r a n d E d w a r d s , 1975; Glidewell a n d R a v e n , 1975; G i v a n , 1976; F o r t i a n d G e r o l a , 1977; Jennings a n d F o r t i , 1975). The q u e s t i o n o f h o w m u c h A T P can be s u p p l i e d by electron transp o r t to o x y g e n in intact c h l o r o p l a s t s d u r i n g CO2 red u c t i o n r e m a i n e d u n a n s w e r e d . The rate o f o x y g e n u p t a k e in the M e h l e r - r e a c t i o n (Mehler, 1951) is slow in the a b s e n c e of an a d d e d e l e c t r o n a c c e p t o r ( F o r t i a n d J a g e n d o r f , 1961). In this p a p e r we p r e s e n t evidence t h a t in CO2r e d u c i n g i n t a c t s p i n a c h c h l o r o p l a s t s which are n o t sufficiently well c o u p l e d to satisfy the A T P - d e m a n d by linear e l e c t r o n t r a n s p o r t , cyclic e l e c t r o n t r a n s p o r t takes p a r t in A T P p r o d u c t i o n . The affinity o f elect r o n s to different electron carriers a n d r e d o x c o n t r o l (poising) regulate the d i s t r i b u t i o n of electrons between N A D P , o x y g e n a n d the p r i m a r y a c c e p t o r o f the cyclic electron t r a n s p o r t p a t h w a y .
Materials and Methods
Intact chloroplasts capable of high rates of CO2-dependent oxygen evolution were isolated from greenhouse-or field-grown spinach as described previously (Egneus et al., 1975; Heber, 1973). Preparations used in this investigation contained more than 80% (up to 98%) chloroplasts which had retained their envelopes during isolation as measured routinely by the ferricyanide method (Heber and Santarius, 1970). COz-dependent oxygen evolution was measured by a Clark electrode, the quenching of 9-aminoacridine fluorescence (concentration 5 DM) by a photomultiplier. Rates of CO2reduction ranged between 130 and 280 gmoles (rag chlorophyll)- 1 h- 1 Light source and filter arrangements were described by Tillberg, Giersch and Heber (1977). Chloroplast suspensions were made and kept anaerobic by adding 10 mM glucose and some glucose oxidase. H=O 2 formed during glucose oxidation was decomposed by an excess of catalase (usually about 1,300 international enzyme units ml-*). Transient oxygenation of anaerobic chloroplast suspensions was brought about by injecting known amounts of HzO 2. Light absorption by chloroplasts was determined in an Ulbricht sphere. Scattering of a measuring beam of 535 nm light by leaves or isolated chloroplasts was measured usually in transmission (Heber, 1969), but occasionally also as 70~ backscattering. Leaves were kept in a stream of CO2-free air or nitrogen during the measurements. The flow rate was between 40 and 60 1 h- 1 Mass spectrometric experiments were performed as described by Egneus et al. (1975),
Table 1. Oxygen exchange by intact spinach chloroplasts under different light intensities as measured with a mass spectrometer. Substrate: 2 or 4 mM HCO~
Incident intensity of red light (Win-2)
COz-dependent oxygen exchange, [in pmol (mg chl)- 1 h ~]
9 12 84 120
16 34 67 70
% intact chloroplasts in preparation
net 02 evolution 02 uptake 6 4 7 11
93 87 93 72
ing glycolate p r o d u c t i o n in the r i b u l o s e b i s p h o s p h a t e o x y g e n a s e r e a c t i o n was m i n i m i z e d by using a s a t u r a t ing b i c a r b o n a t e c o n c e n t r a t i o n . G l y c o l a t e synthesis is c o m p e t i t i v e l y i n h i b i t e d b y CO2. M o s t o f the 180 2 u p t a k e m e a s u r e d is therefore caused by oxygen reduction by the electron t r a n s p o r t chain which is c o u p l e d to A T P f o r m a t i o n (Egneus et al., 1975), The results p r e s e n t e d in T a b l e 1 s h o w that, as the light intensity is increased, the r e d u c t i o n o f o x y g e n does n o t increase p r o p o r t i o n a l l y to the C O 2 - d e p e n d e n t oxygen evolution, i.e. o x y g e n r e d u c t i o n d u r i n g p h o t o s y n t h e s i s appears to be s a t u r a t e d at r a t h e r low light intensities. This suggests t h a t at high light intensities electron t r a n s p o r t to o x y g e n c a n n o t c o n t r i b u t e all the A T P n e e d e d for p h o t o s y n t h e s i s in insufficiently well-coup l e d c h l o r o p l a s t s . W e w a n t e d to k n o w w h e t h e r cyclic p h o t o p h o s p h o r y l a t i o n also p r o v i d e s A T P for p h o t o synthesis. A s cyclic p h o t o p h o s p h o r y l a t i o n is k n o w n to be sensitive to a n t i m y c i n A ( T a g a w a et al., 1963; % 100-
b
o
12 Wm -z red tight
50-
X.+§ 245Wm-2 red tight
Results
1. Electron Flow to Oxygen and Cyclic Electron Transport during of Isolated Spinach Chloroplasts
CO 2
Reduction
T a b l e 1 shows p h o t o s y n t h e t i c o x y g e n e v o l u t i o n during CO2 r e d u c t i o n by intact c h l o r o p l a s t s a n d simultan e o u s o x y g e n u p t a k e at different light intensities as m e a s u r e d in a m a s s s p e c t r o m e t e r . O x y g e n u p t a k e dur-
I
2,5 Antimycin A
5pM
Fig. 1. CO2-dependent oxygen evolution by intact chloroplasts as a function of the concentration of antimycin A. Photosynthetic rates are expressed as percent of control rates observed in the absence of antimycin A. In 3 different experiments, control rates at 245Wm -z were 131,210 and 258 pmot (mgchl) lh-1, at 12 Wm -2 17, 36 and 42 gmol (rag chI) lh-i
U, Heber etal, : Electron Transport and Photophosphorylation
fo
9 l m M OAA
o
~
o-.~
~
without substrate
40-
2 LT.
