Plant Science Letters, 12 (1978) 371--377 371 © Elsevier/North-Holland Scientific PubLishers Ltd.

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 product (starch) by isolated pea (Pisum sativum) chloroplasts in relation to adenylates, inorganic pyrophosphate (PPi) and other compounds has been investigated in some detail. Formation of starch was favoured in the presence of PPi and catalytic amounts of 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. I t is suggested that the effect of adenylates and PPi appears as a consequence of more rapid exchange of these compounds across the chloroplast envelope in pea than that observed in spinach chloroplasts.

INTRODUCTION

Existing evidence supports the view that starch synthesis is affected by a number of photosynthet ic carbon intermediates and associated compounds 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 15- fold 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 photosynthet ic car- bon fixation as well as in the starch formation [3--5] . Low 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 movement of metaboli tes across the chloroplast envelopes. Namely, if the concentrat ion of external Pi is low it decreases the export o f soluble pro- ducts 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 of 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.

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[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 of 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 trans- locator which apparently moved ATP, although the rates of transfer were low.

It was demonstrated [ 11] tha 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 con- centration. Recent reports [12,13] as well as our previous results [ 14] showed that, when added singly to chloroplasts isolated from young pea leaves, inor- ganic 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 of 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, Insti tut 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 homo- genizer (Girmi, Iskra, Kranj, YU) in 250 ml semifrozen grinding medium con- taining 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 of cheese cloth and the brei was then filtered through 8 layers of cheese cloth + cot ton 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<lependent 02 evolu-

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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 NaH14CO3/60/~Ci / l l0 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 about 500 w • m-2. Aliquots were removed at intervals with a Gilson pipetman and added to tubes containing 1 M HC1. For the starch determinat ion 10/~1 aliquots were spot ted on Whatman No. 1 or Schleicher and Sch£ill No. 2043 a paper and fractions separated by one dimensional chromato- graphy in n-butanol-acetic acid-water [222 : 57 : 150) solvent [17] . 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 product (for details see ref. 6).

Determination of Chlorophyll Chlorophyll content was determined by the method of Arnon [20] .

RESULTS

Table I shows that total 14CO2 fixation and incorporation into insoluble product 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 concentrat ion the rate of CO2 fixation was very low but brought a relatively high rate of starch formation. When concentrations of Pi were higher, the induction period for CO2 fixation was considerably longer (data not 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 in- hibitory effect and the rates of 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 fix- ation as well as starch formation. These results were consistent in a number of experiments.

The results presented in Fig. 1 show the dependency of starch formation on ADP concentrat ions in the presence of 5 mM PPi. The optimal concen- trations of 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 noted that in the best preparations the highest rates of CO2 fixed into insoluble product were near 45/~mol/mg -~ Chl/h or abou t 30% of the total 14CO 2 fixed.

The presence o f PGA or R5P in the reaction medium brought marked

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T A B L E I

DEPENDENCY OF STARCH FORMATION ON Pi, PPi, ADP AND ATP AFTER 15 MIN OF 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 sorb i to l , 1.0 mM EDTA, 10 m M KC1, 50 mM HEPES (pH 7.6), 10 mM NaH14CO3 (60 uCi), 110 un i t s cata lase and ch lo rop las t s equ iva l en t to 100 ug Chl. Addi t ives were: 0 .25 mM Pi, 5 mM PPi, 0.2 mM ADP, 0.2 mM ATP in a f inal c o n c e n t r a t i o n . Fo r o t h e r deta i ls see Methods .

Addi t ives Tota l Inso lub le % Inso lub le

u m o l C O : - m g -1 Chl

Pi 5.1 0.6 11.8 PPi 0.8 - - - - Pi, PPi 3.1 0.2 6.4 Pi, ADP 3.8 0.4 10.5 Pi, ATP 5.8 0.8 13.8 PPi, ADP 20.0 2.2 11.0 PPi, ATP 24.9 4.1 16.4 Pi, PPi, ADP 33.3 5.5 16.5 Pi, PPi, ATP 34.4 7.6 22.1

T A B L E II

DEPENDENCY OF STARCH FORMATION ON PGA, R5P AND F-B-P AFTER 15 MIN 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 he same as in Table I e x c e p t t h a t 0 .25 mM Pi was inc luded . F ina l c o n c e n t r a t i o n s o f addi t ives were : P G A 1.0 raM, RSP 0.5 mM, F-B-P 0.5 raM. The o t h e r s as in Table I.

Additives Total Insoluble % Insoluble

~mol CO 2 °mg -1 Chl

PGA 27.0 2.1 7.8 PGA, ADP 23.2 1.9 8.2 PGA, ADP, PPi 25.4 3.6 14.1 R5P 17.9 1.1 6.0 R5P, ADP 20.8 1.4 6.7 R5P, ADP, PPi 34.1 4.4 12.9 F-B-P 6.9 0.2 2.8 F-B-P, ADP 3.9 0.1 2.5 F-B-P, ADP, PPi 15.5 1.2 7.7

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I total C02 f ixat ion

2£

> , . c (3_ 0 L. 0

E " 0

×

-5 E :3,

5

(134)

(130)

starch

(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 ADP, (-A--A-, -a--a-) 0 .5 mM ADP, ( t - e - , v---o-) 1 .0 mM ADP. Reac t ion media and c o n d i t i o n s are as descr ibed in Methods . Rates in u m o l e s • n~g -1 Chl • h LI are indicated in brackets.

s t imu la t i on o f CO2 f ixa t ion (Table I I ) b u t had l i t t le e f f ec t on s ta rch fo rm- a t ion while F-B-P also had l i t t le o r no ef fec t . Similar resul ts were ob t a ined w h e n P G A and R5P were added wi th 0.2 m M o f ADP b u t there was also a sl ight increase in the level o f s tarch . Add i t ion o f ADP and F-B-P t o g e t h e r appea r s to have an inh ib i to ry e f fec t . Again it can be seen t h a t t he highest ra tes were o b t a i n e d if ADP and PPi were p re sen t in the reac t ion mix tu re .

DISCUSSION

The p re sen t resul ts show t h a t s tarch f o r m a t i o n in isola ted ch loroplas t s f r o m y o u n g pea leaves is d e p e n d e n t o n the ex te rna l c o m p o u n d s added to the reac t ion mix t u r e . Previous results , where sp inach ch lorop las t s were used, s h o w e d t h a t s ta rch f o r m a t i o n in ch loroplas t s is s t rong ly d e p e n d e n t on Pi c o n c e n t r a t i o n and t h a t t he o p t i m a l c o n c e n t r a t i o n is l ower t han t ha t requi red

376

for maximal COs fixation [6] . In this s tudy, 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 by the concentrat ion 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 in- crease in the level o f starch formed.

The present data clearly give an additional support to the view that the exchange of adenylates and PPi across the chloroplast envelopes may be much faster in pea than 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 not 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 metaboli tes 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 of Sheffield, (U.K.) and finished at The Institute of Biology, Novi Sad, YU. I am grateful to Prof. Dr. D.A. Walker for stimulat- ing 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.

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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.

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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

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