- Email: [email protected]
Starch formation in isolated pea chloroplasts
Plant Science Letters, 12 (1978) 371--377 © Elsevier/North-Holland Scientific PubLishers Ltd.
371
STARCH FORMATION IN ISOLATED PEA CHLOROPLASTS ~IVKO SAVA STANKOVI(~ Institute o f Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, (Yugoslavia) (Received October 16th, 1977) (Accepted February 27th, 1978)
SUMMARY
Incorporation of 14CO: into an insoluble p r o d u c t (starch) b y isolated pea
(Pisum sativum) chloroplasts in relation to adenylates, inorganic pyrophosphate (PPi) and other c o m p o u n d s has been investigated in some detail. Formation of starch was favoured in the presence o f PPi and catalytic amounts o f adenylates (ADP or ATP) while 3-phosphoglyceric acid (PGA), ribose-5-phosphate (R5P), or fructose 1,6-biphosphate (F-B-P) had zero or very little effect. It is suggested that the effect of adenylates and PPi appears as a consequence of more rapid exchange of these c o m p o u n d s across the chloroplast envelope in pea than that observed in spinach chloroplasts.
INTRODUCTION
Existing evidence supports the view that starch synthesis is affected b y a number of photosynthetic carbon intermediates and associated c o m p o u n d s such as Pi, ATP, PGA, F-6-P, F-B-P [1]. It was shown that leaf ADP-glucose pyrophosphatase, a key enzyme in starch synthesis, was activated 5 to 15fold by PGA. The stimulation was also observed with F-6-P and F-B-P but to a lesser extent [ 2 ] . Pi appears to play an important role in photosynthetic carbon fixation as well as in the starch formation [3--5]. L o w concentrations of external Pi stimulate starch synthesis and conversely high concentrations of Pi have an ~nhibitory effect [3,4,6]. This appears to be a consequence of the role of Pi in the m o v e m e n t of metabolites across the chloroplast envelopes. Namely, if the concentration of external Pi is low it decreases the export o f soluble products o f CO2 fixation such as DHAP and PGA from the stroma to the cytosol and also favours allosteric activation of ADP-glucose pyrophosphorylase. Conversely, high concentrations o f external Pi will favour the translocation of metabolites, in exchange for triosephosphates, and supress CO2 fixation Abbreviations: BSA, bovine serum albumin, fraction V; F-B-P, fructose 1,6-biphosphate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PGA, 3-phosphoglyceric acid; PPi, inorganic pyrophosphate; R5P, ribose-5-phosphate.
372 [7,8]. Starch synthesis will then be lowered as well, partly as a consequence of diminished substrate and partly as a result of an unsuitable environment for the most effective operation o f the pyrophosphorylase. This inhibition by Pi could be reversed by PGA and triosephosphates [4,5]. According to Heldt and Rapley [9] this transport occurs via a phosphate translocator as a specific carrier. Heldt [ 10] also reported the presence of an adenine nucleotide translocator which apparently moved ATP, although the rates of transfer were low. It was demonstrated [ 11] t h a t the rate of photosynthesis falls to a very low value in phosphorus deficient chloroplasts from spinach and could be restored by addition of exogenous Pi or PPi which had the same effect at half the concentration. Recent reports [12,13] as well as our previous results [ 14] showed that, when added singly to chloroplasts isolated from young pea leaves, inorganic PPi inhibits photosynthesis, but when added together with small quantities of ADP or ATP they cause a marked stimulation. On the basis of those results it has been postulated that the exchange o f adenylates and PPi across the chloroplast envelope may be more rapid in chloroplasts from young pea leaves than in chloroplasts from spinach. In this study experiments were undertaken to investigate in some detail starch formation in isolated pea chloroplasts in relation to adenylates, inorganic PPi and other compounds. MATERIALS AND METHODS Pea (Pisum sativum var. Mali Provansalac, Institut za povrtarstvo, Smederevska Palanka, YU) seeds were germinated in vermiculite and grown in a glasshouse. Var. Feltham First, Suttons Seeds Ltd. Early, Reading, U.K. was employed in some experiments. Except that the temperature was kept constant at 20°C other factors (light intensity, humidity and photoperiod) were similar to those in the field. The seedlings used for chloroplast isolation were 11--14 days old [14].
Isolation of Chloroplasts Chloroplasts Were prepared as before [14] with slight modifications. 70--80 g of freshly harvested leaves and shoots were ground twice for 3--4 s in a homogenizer (Girmi, Iskra, Kranj, YU) in 250 ml semifrozen grinding medium containing 0.33 M sorbitol, 0.1% (w/v) NaC1, 0.1% bovine serum albumin (BSA), 0.2% D-isoascorbate and 0.1 M 2/N-morpholino/ethanesulfonic acid at pH 6.5. The homogenate was squeezed through two layers o f cheese cloth and the brei was then filtered through 8 layers o f cheese cloth + c o t t o n wool and spun in a Janetzki K24 centrifuge for 90 s from rest to rest. The surface o f chloroplast pellet was washed once with a medium containing 0.33 M sorbitol, 0.1% BSA, 0.08 mM EDTA, 0.8 mM KC1 and 4 mM HEPES buffer pH 7.6. The pellet was then resuspended in a medium containing 0.33 M sorbitol, 1.0 mM EDTA, 10 mM KC1, 0.4% BSA and 50 mM HEPES at pH 7.6 and kept in cold before use.
Assay Procedures Chloroplast intactness was determined by ferricyanide
373 tion [15] in a Clark-type electrode apparatus [16] purchased from Hansatech Ltd., Hardwich Industrial Estate, Kings Lynn, Norfolk, (U.K).
C02 Fixation Aliquots of chloroplasts (equivalent to 100/~g chlorophyll} were added to tubes containing resuspending medium plus 10 mM N a H 1 4 C O 3 / 6 0 / ~ C i / l l 0 units catalase and additives as indicated (see l~esults) in a final volume of I ml. The assay tubes were placed in a water bath at 25°C and illuminated with two quartz-iodine projector lamps on both sides of the tubes. The light was passed through coloured perspex filters to give light mostly in the range of 580--750 nm. The irradiation was a b o u t 500 w • m-2. Aliquots were removed at intervals with a Gilson pipetman and added to tubes containing 1 M HC1. For the starch determination 10/~1 aliquots were spotted on Whatman No. 1 or Schleicher and Sch£ill No. 2043 a paper and fractions separated by one dimensional chromatography in n-butanol-acetic acid-water [222 : 57 : 150) solvent [ 1 7 ] . The paper was cut on strips and counted either by a Nuclear Chicago Actigraph III Scanner [18] or in scintillation vials containing toluene-PPO-POPOP [19]. Starch was determined as labeled acid-stable insoluble p r o d u c t (for details see ref. 6).
Determination of Chlorophyll Chlorophyll c o n t e n t was determined b y the m e t h o d o f Arnon [ 2 0 ] . RESULTS Table I shows that total 14CO2 fixation and incorporation into insoluble p r o d u c t b y isolated chloroplasts depended on Pi, PPi and adenylates added to the reaction mixture. It can be seen that when Pi was added singly at low concentration the rate o f CO2 fixation was very low b u t brought a relatively high rate of starch formation. When concentrations of Pi were higher, the induction period for CO2 fixation was considerably longer (data n o t shown), the rates of photosynthesis lower and starch formation showed a sharp decrease. If PPi or ADP were added singly or with Pi they caused an inhibitory effect and the rates o f CO2 fixation and into insoluble product were lower than with Pi added singly. Finally, when Pi, PPi and catalytic amounts of ADP (or ATP) were added together they caused high rates of CO2 fixation as well as starch formation. These results were consistent in a n u m b e r o f experiments. The results presented in Fig. 1 show the dependency o f starch formation on ADP concentrations in the presence o f 5 mM PPi. The optimal concentrations o f ADP for maximal rates for starch synthesis ranged between 0.2 and 0.5 mM and up to 1 mM for photosynthesis. It should be n o t e d that in the best preparations the highest rates o f CO2 fixed into insoluble product were near 45/~mol/mg -~ Chl/h or a b o u t 30% o f the total 14CO 2 fixed. The presence o f PGA or R5P in the reaction medium brought marked
374 TABLE I
DEPENDENCY OF STARCH FORMATION ON Pi, PPi, ADP A N D ATP A F T E R 15 MIN O F CO: F I X A T I O N R e a c t i o n m i x t u r e c o n t a i n e d 0.33 M s o r b i t o l , 1.0 m M E D T A , 10 m M KC1, 50 m M HEPES (pH 7.6), 10 m M NaH14CO3 ( 6 0 uCi), 110 u n i t s catalase a n d c h l o r o p l a s t s e q u i v a l e n t t o 100 ug Chl. A d d i t i v e s were: 0.25 m M Pi, 5 m M PPi, 0.2 m M ADP, 0.2 m M A T P in a final c o n c e n t r a t i o n . F o r o t h e r details see M e t h o d s .
Additives
Total
Insoluble
umolCO: Pi PPi Pi, PPi Pi, ADP Pi, A T P PPi, A D P PPi, A T P Pi, PPi, A D P Pi, PPi, A T P
5.1 0.8 3.1 3.8 5.8 20.0 24.9 33.3 34.4
% Insoluble
- m g -1 Chl 0.6 -0.2 0.4 0.8 2.2 4.1 5.5 7.6
11.8 -6.4 10.5 13.8 11.0 16.4 16.5 22.1
T A B L E II
DEPENDENCY OF STARCH FORMATION ON PGA, R5P A N D F-B-P A F T E R 15 M I N O F CO 2 F I X A T I O N R e a c t i o n m i x t u r e was t h e s a m e as in T a b l e I e x c e p t t h a t 0.25 m M Pi was i n c l u d e d . F i n a l c o n c e n t r a t i o n s o f a d d i t i v e s w e r e : P G A 1.0 raM, R S P 0.5 m M , F-B-P 0.5 raM. T h e o t h e r s as in T a b l e I.
Additives
Total
Insoluble
% Insoluble
~ m o l CO 2 ° m g -1 Chl PGA PGA, ADP PGA, ADP, PPi R5P R5P, ADP R5P, ADP, PPi F-B-P F-B-P, ADP F-B-P, ADP, PPi
27.0 23.2 25.4 17.9 20.8 34.1 6.9 3.9 15.5
2.1 1.9 3.6 1.1 1.4 4.4 0.2 0.1 1.2
7.8 8.2 14.1 6.0 6.7 12.9 2.8 2.5 7.7
375
I
total C02 fixation
2£ >, .c (3_ 0 L. 0
(134) (130)
E "0 ×
starch -5 E :3, 5
(lZ..5)
3
5
8 12 15 Time (Min) Fig. 1. Effect of different concentrations of ADP on rates of CO 2 fixation and starch formation in the presence of 0.25 mM Pi and 5 mM PPi. (-o---o-, o---~-) 0.2 mM A D P , (-A--A-, -a--a-) 0.5 mM A D P , ( t - e - , v---o-) 1.0 m M ADP. R e a c t i o n m e d i a and c o n d i t i o n s are as described in Methods. Rates in u m o l e s • n~g -1 Chl • h LI are indicated in brackets.
s t i m u l a t i o n o f CO2 f i x a t i o n (Table II) b u t h a d little e f f e c t o n s t a r c h f o r m a t i o n while F-B-P also h a d little o r n o e f f e c t . Similar results w e r e o b t a i n e d w h e n P G A a n d R 5 P w e r e a d d e d w i t h 0.2 m M o f A D P b u t t h e r e was also a slight increase in t h e level o f starch. A d d i t i o n o f A D P a n d F-B-P t o g e t h e r a p p e a r s t o h a v e an i n h i b i t o r y e f f e c t . Again it can b e seen t h a t t h e highest r a t e s w e r e o b t a i n e d if A D P a n d PPi were p r e s e n t in t h e r e a c t i o n m i x t u r e . DISCUSSION T h e p r e s e n t results s h o w t h a t starch f o r m a t i o n in isolated c h l o r o p l a s t s f r o m y o u n g p e a leaves is d e p e n d e n t o n t h e e x t e r n a l c o m p o u n d s a d d e d t o t h e r e a c t i o n m i x t u r e . Previous results, w h e r e s p i n a c h c h l o r o p l a s t s w e r e used, s h o w e d t h a t s t a r c h f o r m a t i o n in c h l o r o p l a s t s is s t r o n g l y d e p e n d e n t o n Pi c o n c e n t r a t i o n a n d t h a t t h e o p t i m a l c o n c e n t r a t i o n is l o w e r t h a n t h a t r e q u i r e d
376
for maximal COs fixation [6]. In this study, in which pea chloroplasts isolated in a similar or modified media (see Methods) and similar reaction mixture were used, it was found that the rates of starch formation were rather modest in respect to those reported for spinach. However, when PPi and catalytic amounts of adenylates (ADP or ATP) were added together to the reaction mixture, high rates of ~4CO2 fixation and incorporation into insoluble product were obtained. It becomes evident that starch formation in pea chloroplasts is affected not only by Pi concentrations but also b y the concentration of adenylates, too. To overcome the possible effect of Pi and to support this view, PGA was added externally (to keep high PGA/Pi ratio) to the reaction mixture; the rate of COs incorporation into starch was not affected under the experimental conditions. Similar results were obtained by adding R5P. Again in the presence of ADP or ATP and PPi there was an increase in the level o f starch formed. The present data clearly give an additional support to the view that the exchange o f adenylates and PPi across the chloroplast envelopes may be much faster in p e a t h a n in spinach [13,14] ;this being the case,the behaviour of pea chloroplasts is in marked contrast to those of spinach. Although the role of PPi has n o t been completely clarified, the results seem to be consistent with previous results which indicate that PPi may interfere with the operation of the Pi translocator and lead to the retention of metabolites in the stroma which would otherwise be lost to the medium [8,11,14]. ACKNOWLEDGEMENTS
The experiments in this investigation were undertaken at the Department of Botany, University o f Sheffield, (U.K.) and finished at The Institute of Biology, Novi Sad, YU. I am grateful to Prof. Dr. D.A. Walker for stimulating discussions and suggestions. REFERENCES 1 J. Preiss, H.P. Ghosh and J. Wittkop, Regulation of the biosynthesis of starch in spinach leaf chloroplasts, in T.W. Goodwin (Ed.), Biochemistry of Chloroplasts, Vol. II, Academic Press, New York, 1967, p. 131. 2 G.G. Sanwal, E. Greenberg, J. Hardie, E.C. Cameron and J. Preiss, Plant Physiol., 43 (1968) 417. 3 H.P. Chosh and J. Preiss, Biochemistry, 4 (1965) 1354. 4 G.G. Sanwal and J. Preiss, Arch. Biochem. Biophys., 119 (1967) 454. 5 W. Cockburn, D.A. Walker and C.W. Baldry, Biochem. J., 107 (1968) 89. 6 H.W. Heldt, C.J. Chon, D. Maronde, A. Herold, Z.S. Stankovic, D.A. Walker, A. Kraminer, M.R. Kirk and U. Heber, Plant Physiol., 59 (1977) 1146. 7 U. Heber, Ann. Rev. Plant Physiol., 25 (1974) 393. 8 D.A. Walker, CO~ fixation by intact chloroplasts: Photosynthetic induction and its relation to transport phenomena and control mechanisms, in J. Barber (Ed.), The Intact Chloroplasts, Elsevier, Amsterdam, 1976, p. 235.
377 9 H.W. Heldt and L. Rapley, FEBS Lett., 10 (1970} 143. 10 H.W. Heldt, FEBS Lett., 5 (1969) 11. 11 W. Cockburn, D.A. Walker and C.W. Baldry, Plant Physiol., 43 (1968) 1415. 12 S.P. Robinson and J.T. Wiskich, Plant Physiol., 58 (1976) 156. 13 S.P. Robinson and J.T. Wiskich, Plant Physiol., 59 (1977} 422. 14 Z.S. Stankovic and D.A. Walker, Plant Physiol., 59 (1977) 428. ] 5 R. McC. Lilley, M.P. Fitzgerald, K.G. Rienits and D.A. Walker, New Phytol., 75 (1975) 1. 16 T. Delieu and D.A. Walker, New Phytol., 71 (1972) 201. 17 C.W. Baldry, C. Bucke and D.A. Walker, Nature, 210 (1966) 793. 18 S.H. Chen-She, D.H. Lewis and D.A. Walker, New Phytol., 74 (1975) 383. 19 E.A. Davidson, Techniques for paper strip counting in a scintillation spectrometer, Packard Technical Bull., No 4, 1970. 20 D.I. Aron, Plant Physiol., 24 (1949} 1.
371
STARCH FORMATION IN ISOLATED PEA CHLOROPLASTS ~IVKO SAVA STANKOVI(~ Institute o f Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, (Yugoslavia) (Received October 16th, 1977) (Accepted February 27th, 1978)
SUMMARY
Incorporation of 14CO: into an insoluble p r o d u c t (starch) b y isolated pea
(Pisum sativum) chloroplasts in relation to adenylates, inorganic pyrophosphate (PPi) and other c o m p o u n d s has been investigated in some detail. Formation of starch was favoured in the presence o f PPi and catalytic amounts o f adenylates (ADP or ATP) while 3-phosphoglyceric acid (PGA), ribose-5-phosphate (R5P), or fructose 1,6-biphosphate (F-B-P) had zero or very little effect. It is suggested that the effect of adenylates and PPi appears as a consequence of more rapid exchange of these c o m p o u n d s across the chloroplast envelope in pea than that observed in spinach chloroplasts.
INTRODUCTION
Existing evidence supports the view that starch synthesis is affected b y a number of photosynthetic carbon intermediates and associated c o m p o u n d s such as Pi, ATP, PGA, F-6-P, F-B-P [1]. It was shown that leaf ADP-glucose pyrophosphatase, a key enzyme in starch synthesis, was activated 5 to 15fold by PGA. The stimulation was also observed with F-6-P and F-B-P but to a lesser extent [ 2 ] . Pi appears to play an important role in photosynthetic carbon fixation as well as in the starch formation [3--5]. L o w concentrations of external Pi stimulate starch synthesis and conversely high concentrations of Pi have an ~nhibitory effect [3,4,6]. This appears to be a consequence of the role of Pi in the m o v e m e n t of metabolites across the chloroplast envelopes. Namely, if the concentration of external Pi is low it decreases the export o f soluble products o f CO2 fixation such as DHAP and PGA from the stroma to the cytosol and also favours allosteric activation of ADP-glucose pyrophosphorylase. Conversely, high concentrations o f external Pi will favour the translocation of metabolites, in exchange for triosephosphates, and supress CO2 fixation Abbreviations: BSA, bovine serum albumin, fraction V; F-B-P, fructose 1,6-biphosphate; HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PGA, 3-phosphoglyceric acid; PPi, inorganic pyrophosphate; R5P, ribose-5-phosphate.
372 [7,8]. Starch synthesis will then be lowered as well, partly as a consequence of diminished substrate and partly as a result of an unsuitable environment for the most effective operation o f the pyrophosphorylase. This inhibition by Pi could be reversed by PGA and triosephosphates [4,5]. According to Heldt and Rapley [9] this transport occurs via a phosphate translocator as a specific carrier. Heldt [ 10] also reported the presence of an adenine nucleotide translocator which apparently moved ATP, although the rates of transfer were low. It was demonstrated [ 11] t h a t the rate of photosynthesis falls to a very low value in phosphorus deficient chloroplasts from spinach and could be restored by addition of exogenous Pi or PPi which had the same effect at half the concentration. Recent reports [12,13] as well as our previous results [ 14] showed that, when added singly to chloroplasts isolated from young pea leaves, inorganic PPi inhibits photosynthesis, but when added together with small quantities of ADP or ATP they cause a marked stimulation. On the basis of those results it has been postulated that the exchange o f adenylates and PPi across the chloroplast envelope may be more rapid in chloroplasts from young pea leaves than in chloroplasts from spinach. In this study experiments were undertaken to investigate in some detail starch formation in isolated pea chloroplasts in relation to adenylates, inorganic PPi and other compounds. MATERIALS AND METHODS Pea (Pisum sativum var. Mali Provansalac, Institut za povrtarstvo, Smederevska Palanka, YU) seeds were germinated in vermiculite and grown in a glasshouse. Var. Feltham First, Suttons Seeds Ltd. Early, Reading, U.K. was employed in some experiments. Except that the temperature was kept constant at 20°C other factors (light intensity, humidity and photoperiod) were similar to those in the field. The seedlings used for chloroplast isolation were 11--14 days old [14].
Isolation of Chloroplasts Chloroplasts Were prepared as before [14] with slight modifications. 70--80 g of freshly harvested leaves and shoots were ground twice for 3--4 s in a homogenizer (Girmi, Iskra, Kranj, YU) in 250 ml semifrozen grinding medium containing 0.33 M sorbitol, 0.1% (w/v) NaC1, 0.1% bovine serum albumin (BSA), 0.2% D-isoascorbate and 0.1 M 2/N-morpholino/ethanesulfonic acid at pH 6.5. The homogenate was squeezed through two layers o f cheese cloth and the brei was then filtered through 8 layers o f cheese cloth + c o t t o n wool and spun in a Janetzki K24 centrifuge for 90 s from rest to rest. The surface o f chloroplast pellet was washed once with a medium containing 0.33 M sorbitol, 0.1% BSA, 0.08 mM EDTA, 0.8 mM KC1 and 4 mM HEPES buffer pH 7.6. The pellet was then resuspended in a medium containing 0.33 M sorbitol, 1.0 mM EDTA, 10 mM KC1, 0.4% BSA and 50 mM HEPES at pH 7.6 and kept in cold before use.
Assay Procedures Chloroplast intactness was determined by ferricyanide
373 tion [15] in a Clark-type electrode apparatus [16] purchased from Hansatech Ltd., Hardwich Industrial Estate, Kings Lynn, Norfolk, (U.K).
C02 Fixation Aliquots of chloroplasts (equivalent to 100/~g chlorophyll} were added to tubes containing resuspending medium plus 10 mM N a H 1 4 C O 3 / 6 0 / ~ C i / l l 0 units catalase and additives as indicated (see l~esults) in a final volume of I ml. The assay tubes were placed in a water bath at 25°C and illuminated with two quartz-iodine projector lamps on both sides of the tubes. The light was passed through coloured perspex filters to give light mostly in the range of 580--750 nm. The irradiation was a b o u t 500 w • m-2. Aliquots were removed at intervals with a Gilson pipetman and added to tubes containing 1 M HC1. For the starch determination 10/~1 aliquots were spotted on Whatman No. 1 or Schleicher and Sch£ill No. 2043 a paper and fractions separated by one dimensional chromatography in n-butanol-acetic acid-water [222 : 57 : 150) solvent [ 1 7 ] . The paper was cut on strips and counted either by a Nuclear Chicago Actigraph III Scanner [18] or in scintillation vials containing toluene-PPO-POPOP [19]. Starch was determined as labeled acid-stable insoluble p r o d u c t (for details see ref. 6).
Determination of Chlorophyll Chlorophyll c o n t e n t was determined b y the m e t h o d o f Arnon [ 2 0 ] . RESULTS Table I shows that total 14CO2 fixation and incorporation into insoluble p r o d u c t b y isolated chloroplasts depended on Pi, PPi and adenylates added to the reaction mixture. It can be seen that when Pi was added singly at low concentration the rate o f CO2 fixation was very low b u t brought a relatively high rate of starch formation. When concentrations of Pi were higher, the induction period for CO2 fixation was considerably longer (data n o t shown), the rates of photosynthesis lower and starch formation showed a sharp decrease. If PPi or ADP were added singly or with Pi they caused an inhibitory effect and the rates o f CO2 fixation and into insoluble product were lower than with Pi added singly. Finally, when Pi, PPi and catalytic amounts of ADP (or ATP) were added together they caused high rates of CO2 fixation as well as starch formation. These results were consistent in a n u m b e r o f experiments. The results presented in Fig. 1 show the dependency o f starch formation on ADP concentrations in the presence o f 5 mM PPi. The optimal concentrations o f ADP for maximal rates for starch synthesis ranged between 0.2 and 0.5 mM and up to 1 mM for photosynthesis. It should be n o t e d that in the best preparations the highest rates o f CO2 fixed into insoluble product were near 45/~mol/mg -~ Chl/h or a b o u t 30% o f the total 14CO 2 fixed. The presence o f PGA or R5P in the reaction medium brought marked
374 TABLE I
DEPENDENCY OF STARCH FORMATION ON Pi, PPi, ADP A N D ATP A F T E R 15 MIN O F CO: F I X A T I O N R e a c t i o n m i x t u r e c o n t a i n e d 0.33 M s o r b i t o l , 1.0 m M E D T A , 10 m M KC1, 50 m M HEPES (pH 7.6), 10 m M NaH14CO3 ( 6 0 uCi), 110 u n i t s catalase a n d c h l o r o p l a s t s e q u i v a l e n t t o 100 ug Chl. A d d i t i v e s were: 0.25 m M Pi, 5 m M PPi, 0.2 m M ADP, 0.2 m M A T P in a final c o n c e n t r a t i o n . F o r o t h e r details see M e t h o d s .
Additives
Total
Insoluble
umolCO: Pi PPi Pi, PPi Pi, ADP Pi, A T P PPi, A D P PPi, A T P Pi, PPi, A D P Pi, PPi, A T P
5.1 0.8 3.1 3.8 5.8 20.0 24.9 33.3 34.4
% Insoluble
- m g -1 Chl 0.6 -0.2 0.4 0.8 2.2 4.1 5.5 7.6
11.8 -6.4 10.5 13.8 11.0 16.4 16.5 22.1
T A B L E II
DEPENDENCY OF STARCH FORMATION ON PGA, R5P A N D F-B-P A F T E R 15 M I N O F CO 2 F I X A T I O N R e a c t i o n m i x t u r e was t h e s a m e as in T a b l e I e x c e p t t h a t 0.25 m M Pi was i n c l u d e d . F i n a l c o n c e n t r a t i o n s o f a d d i t i v e s w e r e : P G A 1.0 raM, R S P 0.5 m M , F-B-P 0.5 raM. T h e o t h e r s as in T a b l e I.
Additives
Total
Insoluble
% Insoluble
~ m o l CO 2 ° m g -1 Chl PGA PGA, ADP PGA, ADP, PPi R5P R5P, ADP R5P, ADP, PPi F-B-P F-B-P, ADP F-B-P, ADP, PPi
27.0 23.2 25.4 17.9 20.8 34.1 6.9 3.9 15.5
2.1 1.9 3.6 1.1 1.4 4.4 0.2 0.1 1.2
7.8 8.2 14.1 6.0 6.7 12.9 2.8 2.5 7.7
375
I
total C02 fixation
2£ >, .c (3_ 0 L. 0
(134) (130)
E "0 ×
starch -5 E :3, 5
(lZ..5)
3
5
8 12 15 Time (Min) Fig. 1. Effect of different concentrations of ADP on rates of CO 2 fixation and starch formation in the presence of 0.25 mM Pi and 5 mM PPi. (-o---o-, o---~-) 0.2 mM A D P , (-A--A-, -a--a-) 0.5 mM A D P , ( t - e - , v---o-) 1.0 m M ADP. R e a c t i o n m e d i a and c o n d i t i o n s are as described in Methods. Rates in u m o l e s • n~g -1 Chl • h LI are indicated in brackets.
s t i m u l a t i o n o f CO2 f i x a t i o n (Table II) b u t h a d little e f f e c t o n s t a r c h f o r m a t i o n while F-B-P also h a d little o r n o e f f e c t . Similar results w e r e o b t a i n e d w h e n P G A a n d R 5 P w e r e a d d e d w i t h 0.2 m M o f A D P b u t t h e r e was also a slight increase in t h e level o f starch. A d d i t i o n o f A D P a n d F-B-P t o g e t h e r a p p e a r s t o h a v e an i n h i b i t o r y e f f e c t . Again it can b e seen t h a t t h e highest r a t e s w e r e o b t a i n e d if A D P a n d PPi were p r e s e n t in t h e r e a c t i o n m i x t u r e . DISCUSSION T h e p r e s e n t results s h o w t h a t starch f o r m a t i o n in isolated c h l o r o p l a s t s f r o m y o u n g p e a leaves is d e p e n d e n t o n t h e e x t e r n a l c o m p o u n d s a d d e d t o t h e r e a c t i o n m i x t u r e . Previous results, w h e r e s p i n a c h c h l o r o p l a s t s w e r e used, s h o w e d t h a t s t a r c h f o r m a t i o n in c h l o r o p l a s t s is s t r o n g l y d e p e n d e n t o n Pi c o n c e n t r a t i o n a n d t h a t t h e o p t i m a l c o n c e n t r a t i o n is l o w e r t h a n t h a t r e q u i r e d
376
for maximal COs fixation [6]. In this study, in which pea chloroplasts isolated in a similar or modified media (see Methods) and similar reaction mixture were used, it was found that the rates of starch formation were rather modest in respect to those reported for spinach. However, when PPi and catalytic amounts of adenylates (ADP or ATP) were added together to the reaction mixture, high rates of ~4CO2 fixation and incorporation into insoluble product were obtained. It becomes evident that starch formation in pea chloroplasts is affected not only by Pi concentrations but also b y the concentration of adenylates, too. To overcome the possible effect of Pi and to support this view, PGA was added externally (to keep high PGA/Pi ratio) to the reaction mixture; the rate of COs incorporation into starch was not affected under the experimental conditions. Similar results were obtained by adding R5P. Again in the presence of ADP or ATP and PPi there was an increase in the level o f starch formed. The present data clearly give an additional support to the view that the exchange o f adenylates and PPi across the chloroplast envelopes may be much faster in p e a t h a n in spinach [13,14] ;this being the case,the behaviour of pea chloroplasts is in marked contrast to those of spinach. Although the role of PPi has n o t been completely clarified, the results seem to be consistent with previous results which indicate that PPi may interfere with the operation of the Pi translocator and lead to the retention of metabolites in the stroma which would otherwise be lost to the medium [8,11,14]. ACKNOWLEDGEMENTS
The experiments in this investigation were undertaken at the Department of Botany, University o f Sheffield, (U.K.) and finished at The Institute of Biology, Novi Sad, YU. I am grateful to Prof. Dr. D.A. Walker for stimulating discussions and suggestions. REFERENCES 1 J. Preiss, H.P. Ghosh and J. Wittkop, Regulation of the biosynthesis of starch in spinach leaf chloroplasts, in T.W. Goodwin (Ed.), Biochemistry of Chloroplasts, Vol. II, Academic Press, New York, 1967, p. 131. 2 G.G. Sanwal, E. Greenberg, J. Hardie, E.C. Cameron and J. Preiss, Plant Physiol., 43 (1968) 417. 3 H.P. Chosh and J. Preiss, Biochemistry, 4 (1965) 1354. 4 G.G. Sanwal and J. Preiss, Arch. Biochem. Biophys., 119 (1967) 454. 5 W. Cockburn, D.A. Walker and C.W. Baldry, Biochem. J., 107 (1968) 89. 6 H.W. Heldt, C.J. Chon, D. Maronde, A. Herold, Z.S. Stankovic, D.A. Walker, A. Kraminer, M.R. Kirk and U. Heber, Plant Physiol., 59 (1977) 1146. 7 U. Heber, Ann. Rev. Plant Physiol., 25 (1974) 393. 8 D.A. Walker, CO~ fixation by intact chloroplasts: Photosynthetic induction and its relation to transport phenomena and control mechanisms, in J. Barber (Ed.), The Intact Chloroplasts, Elsevier, Amsterdam, 1976, p. 235.
377 9 H.W. Heldt and L. Rapley, FEBS Lett., 10 (1970} 143. 10 H.W. Heldt, FEBS Lett., 5 (1969) 11. 11 W. Cockburn, D.A. Walker and C.W. Baldry, Plant Physiol., 43 (1968) 1415. 12 S.P. Robinson and J.T. Wiskich, Plant Physiol., 58 (1976) 156. 13 S.P. Robinson and J.T. Wiskich, Plant Physiol., 59 (1977} 422. 14 Z.S. Stankovic and D.A. Walker, Plant Physiol., 59 (1977) 428. ] 5 R. McC. Lilley, M.P. Fitzgerald, K.G. Rienits and D.A. Walker, New Phytol., 75 (1975) 1. 16 T. Delieu and D.A. Walker, New Phytol., 71 (1972) 201. 17 C.W. Baldry, C. Bucke and D.A. Walker, Nature, 210 (1966) 793. 18 S.H. Chen-She, D.H. Lewis and D.A. Walker, New Phytol., 74 (1975) 383. 19 E.A. Davidson, Techniques for paper strip counting in a scintillation spectrometer, Packard Technical Bull., No 4, 1970. 20 D.I. Aron, Plant Physiol., 24 (1949} 1.