Planta (Berl.) 125, 63--67 (1975) 9 by Springer-Verlag 1975

Formation of C-4 Dicarboxylic Acids by Intact Spinach Chloroplasts 1%. Scheibe and E. Beck Botanisches Institut der Universit~t Mfinchen, Menzinger Str. 67, D-8000 Miinchen, Federal Republic of Germany Received 27 March; accepted 5 May 1975

Summary. It is concluded from 14C labelling kinetics as well as from enzyme analysis that formation of malate and aspartate from glycerate-3P via carboxylation of P-enolpyruvate occurs in isolated photosynthesizing spinach (Spinacia oleracea L.) chloroplasts. Carbon incorporation into both dicarboxylic acids was about 1% of the total CO2 fixation. Both acids are suggested to act as substrates for counter exchange for glutamate and ~.-ketoglutarate. Introduction W h e n isolated i n t a c t spinach chloroplasts were allowed t o fix 14C02, a b o u t 1% of the i n c o r p o r a t e d 14C was regularly found in i n m a t e a n d aspartate. I n addition, 14C-labelled P E P could be d e t e c t e d occasionally. G l u t a m a t e , e-ketog l u t a r a t e and citrate, however, were n e v e r labelled. The e x p l a n a t i o n s for these findings could be either t h a t the plastids h a d been c o n t a m i n a t e d by cytoplasmic enzymes, or t h a t t h e y were able to form t h e carbon skeleton of C-4 d i c a r b o x y l i c acids (but not t h a t of C-5 a n d C-6 diearboxylie acids). The present c o m m u n i c a t i o n will d e m o n s t r a t e the l a t t e r of the two possibilities. F o r this purpose chloroplasts were p r e p a r e d free of a n y cytoplasmic c o n t a m i n a t i o n . E n z y m e analysis of these chloroplasts r e v e a l e d the r e q u i r e d e n z y m e activities for the f o r m a t i o n of m a l a t e and a s p a r t a t e f r o m P E P (or via P G A ) a n d CO 2.

Materials and Methods

Preparation o] the Chloroplasts. Washed intact chloroplasts were isolated from fresh spinach (Vital R) leaves according to the method of Beck etal. (1971) and further purified by discontinuous sucrose density centrifugation before enzymatic assay. :For this purpose the method of Miflin (1974) was slightly modified by using different sucrose concentrations. Two significant green bands appeared at the interfaces between the 32% (w/v) and 50% (w/v) sucrose, and on top of the 60% (w/v) sucrose layer, respectively. A third weak band could be observed on top of the 75% (w/v) sucrose cushion which contained cell fragments. The first green band consisted of ruptured, the second (at 1.23 spec. dens.) of intact chloroplasts, as identification by phase contrast microscopy revealed. The suspension of the intact chloroplasts was diluted with 0,3 M sucrose solution in 10 rnM tricine buffer pH 7.6, centrifuged at 3000 X g, and the pellet was resuspended in the same medium to lower the sucrose concentration. Based on their different specific densities, mitochondria, ER and microbodies could be expected in interfaces on top of the 60% (w/v) sucrose (1.192 g/cm3)

Abbreviations: P GA = glycerate-3 P; PEP = P-enolpyruvate; DTE = dithioerythritol; I~uDP = ribulose-l,5 P~; OAA = oxMoacetate; G6P ~ glucose-6 P; GAP = glycerMdehyde-3 P.

64

R. Scheibe and E. Beck o. . . . . o g l y c e r a t e o. . . .

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a sugar- monophosphates ~ sugar-diphosphates

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maLate (xlO)

9 ..... 9 aspartate (xlO)

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aianine(xlO} CO 2 f i x a t i o n

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(lamol/mg chlorophyll)

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Fig. 1. CO2 fixation and 14C-labelling kinetics of various assimilation products of intact spinach chloroplasts

a n d the 75% (w/v) sucrose (1.287g/cm3), respectively. All solutions for isolation and purification were 0.001 3/[ with respect to DTE. 14C02-Fixation by Isolated Chloroplasts. I n t a c t chloroplasts prepared according to the method of Beck et al. (1971) were suspended in H E P E S - b u f f e r p i t 7.6 (Jensen and Bassham, 1966) and preilluminated for 3 minutes in a rotating flask. Then NaH14CO3 (8 mM) was added while the illumination was continued and aliquot samples were withdrawn and killed b y injection into boiling methanol at the intervals given in Fig. 1. The chloroplast extract was analyzed b y the chromatographic methods described elsewhere (Beck et al., 1971). 14Cdistribution was determined from the paper ehromatograms using a methane flow counter. Enzyme Assays. Carboxylation of P~uDP and P E P were measured upon lysis of the chloroplasts in hypotonie buffers according to the method of B a h r a n d Jensen (1974). adC incorporation into acid-stable products was determined. The other enzyme activities were d e t e r m i n e d in a chloroplast extract prepared b y hypotonie t r e a t m e n t a t 0 ~ for 5 min (Douce et al., 1973). After removal of all membrane particles by centrifugation a t 23000 x g the clear s u p e r n a t a n t was used. NAD-Malate Dehydrogenase (E.C. 1.1.1.37). The reaction mixture consisted of tris buffer p H 8.0, 0.1 M; MgCl 2 0.01 M; N A D H 0.2 raM; and chloroplast extract. The reaction was started with OAA (2 m)/[) a n d followed b y the decrease of optical density at 360 nm. Control assays were r u n without OAA. NADP-Malate Dehydrogenase. For activation the chloroplast extract was i n c u b a t e d for 1 h a t 25 ~ C with D T E (5 mM) (Ahmadi and Ting, 1973). Then the same test was used as for the NAD-dependent malate dehydrogenase. N A D H was replaced b y N A D P t t . Phosphatase (E.C. 3.1.3.). To decide whether the NADPH-linked activity of malate dehydrogenase was an artifact caused b y the dephosphorylation of N A D P H through a phosphatase in the chloroplast extract (as shown by Wells a n d t t a g e m a n n (1974) for NADPlinked nitrate reductase), the malate dehydrogenase-test was performed with the NADdependent enzyme (from Boehringer) a n d N A D P H which was generated enzymatieally from glucose-6P b y glucose-6P dehydrogenase and NADP.

Formation of Dicarboxylie Acids by Chloroplasts

65

Upon the addition of chloroplast extract no effect of fluoride (10 raM) could be observed, suggesting the absence of 3'-phosphatase and thus the presence of NADP-linked malate dehydrogenase. Glutamate Oxaloacetate Transaminase (E.C. 2.6.1.1.). GOT-activity was determined by the method of Bergmeyer and Bernt (1970).

3-PGA Mutase (E.C. 6.4.2.1), 2-PGA Enolase (E.C.d.2.1.11) and Pyruvate Kinase (E.C. 2.7.1.40). All 3 enzymes were assayed together in the following reaction mixture consisting of tris-buffer pH 7.0 (0.1M); MgSOd.7H20 (7.5raM); ADP (0.01 M); NADH (0.2 raM); lactate dehydrogenase (10 units); and chloroplast extract. The reaction was started with PGA (15 raM). Results

1. ~C-incorporation into C-4 Dicarboxylie Acids by Photosynthesizing Intact Spinach Chloroplasts 14Cincorporation into malate and aspartate by photosynthesizing intact chloroplasts was carried out for 20 min. The results of a typical experiment are shown in Fig. 1. The data indicate a small but siguificant and constant de novo synthesis of malate and aspartate during the linear phase of CO2 fixation. In contrast to the total CO S fixation, the incorporation of ~4C into both acids did not decline after 12 min, but actually increased slightly. [14C]alanine was formed, too, the labelling of which paralleled that of both C-4 dicarboxylic acids. 2. Purity Proo/ o/ the Chloroplast Suspension Since the synthesis of malate and aspartate is known to occur preferentially in the cytoplasm, the observation of 14C labelling of both acids could be explained in terms of a cytoplasmic impurity of the chloroplast preparation. Thus, prior to enzyme analysis, the purity of the chloroplasts had to be confirmed. For this purpose the washed isolated chloroplasts were further purified by discontinuous sucrose density centrifugation followed by a second washing step in 0.3 M sucrose. Cytoplasmic contamination of this preparation could be excluded since activity of non-reversible glyeeraldehyde dehydxogenase was not detectable. This enzyme can be distinguished from the reversible chloroplastic glyceraldehyde dehydrogenase by the inhibitory effect of L-GAP and further by the omission of arsenate or phosphate from the reaction mixture; it was shown by Kelly and Gibbs (1973) to occur only in the cytoplasm.

3. Analysis o/Enzyme Activities with Respect to C-4 Dicarboxylic Acid Formation The activity of enzymes which could be responsible for the formation of both C-4 dicarboxylic acids was assayed after osmotic rupture of the chloroplasts. The results of these assays are listed in Table 1. The data show that all enzymes which are necessary for the formation of malate and aspartate from PGA and CO~ via carboxylation of P E P and reduction or transamination of oxaloacetate were present in the chloroplasts. I t is obvious that the rate-limiting enzyme is P E P carboxylase the activity of which corresponds to the amount of C-4 dicarboxylic acids found upon CO 2 fixation of the intact chloroplasts. The specific activity of this enzyme is of the same order of magnitude as described by Gibbs 5 Planta (Berl.), Vol. 125

66

R. Seheibe and E. Beck

Table 1. Enzyme activities (tzmolper mg chlorophyll per hour) determined after rupture of the purified chloroplasts Enzyme PEP Carboxylase Glutamate oxaloaeetate transaminase

[~mol.mgCh1-1,h-1 0.3 99.0

NADH-malate dehydrogenase NADPH-malate dehydrogenase 3-PGA Mutase ] 2-PGA Enolase ~ Pyruvate Kinase ]

118.0 11.0

RuDP-Carboxylase PEP Carboxykinase Non-reversible glyeeraldehyde dehydrogenase

360.0 0 0

5.1

et al. (1970) 1. P E P carboxykinase could not be detected. A comparison between the P E P carboxylase activity of a crude leaf protein extract and t h a t from the chloroplasts on a chlorophyll basis suggested t h a t 10% of the total P E P carboxylase was located within the chloroplasts. However, activity found in the leaf extract could be increased threefold by chromatography on Sephadex G 25 whereas t h a t from the chloroplasts remained unchanged upon this treatment. Therefore, when the low P E P carboxylase activity of the crude leaf extract was due to an artificial inhibition by a compound of low molecular weight which could be removed by gel chromatography, only 3% of the total P E P earboxylase activity was found within the chloroplasts. The limiting step in the formation of alanine seems to be the trans~mination of p y r u v a t e (see Table 1).

Discussion The low rate of carbon incorporation into the C-4 dicarboxylic acids b y photosynthesizing chloroplasts suggests t h a t P E P carboxylation should have another function t h a n an additional (inefficient) type of CO 2 fixation. According to Heldt et al. (1974) a very efficient translocator is supposed to be situated in the chloroplast envelope catalyzing a counter exchange of dicarboxylie acids and, with less efficiency, an uptake of these acids b y the chloroplast (the latter might be due to the artificial environment of the isolated plastids). The physiological importance of this translocating system has not been elucidated so far, it might, however, be involved in a transport of reducing equivalents from the chloroplast to the cytoplasm. I t is unlikely, though, t h a t the small amounts of diearboxylie acids produced b y the chloroplast itself can play such a role. 1 However, in contrast to the data of these authors the activity of the enzymes leading from PGA to PEP was found to be significantly higher.

Formation of Dicarboxylic Acids by Chloroplasts

67

Since with the purified chloroplast preparations formation of [14C]glutamate (and ~-ketoglutarate) could never be observed, it is concluded t h a t the chloroplast gains this aminogroup donor or accepter via a counter-exchange (Heldt et al., 1974) against dicarboxylic acids produced b y the plastid itself. I n order to overcome the small concentrations of dicarboxylic acids within the chloroplast with respect to an efficient counter exchange a v e r y high affinity of the translocator for its substrates as well as a high efficiency would be necessary. The translocating system described by Heldt would meet such a demand. The authors are very much indebted to Dr. G. J. Kelly, Abt. Chem. Pflanzenphysiologie, Techn. Universit~t Miinchen, Weihenstephan, for helpful advice with respect to the nonreversible glyceraldehyde dehydrogenase and to Dr. P. Dittrich, ]3otan. Institut der Universit~it Miinchen, for encouraging discussions.

References

Ahmadi, N., Ting, I. P.: Activation of NADP-malate-dehydrogenase by lipoate. PI. Sci. Letters 1, 11-14 (1973) ]3ahr, J.T., Jensen, R. G. : Ribulose diphosphate carboxylase from freshly ruptured spinach chloroplasts having an in rive Km [Cos]. Plant Physiol. 58, 39-44 (1974) Beck, E., Stransky, H., Fiirbringer, M.: Synthesis of hamamelose-diphosphate by isolated spinach chloroplasts. FE]3S Letters 13, 229-234 (1971) ]3ergmeyer, H.U., ]3ernt, E.: Glutamat-Oxalacetat-Transaminase: UV-Test mit MalatDehydrogenase als Indikatorenzym. In: iKethoden der enzymatischen Analyse, S. 685-690, ]3ergmeyer, H. U. ed. Weinheim: Verlag Chemic 1970 Deuce, R., Holtz, R. ]3., ]3cnson, A. A.: Isolation and properties of the envelope of spinach chloroplasts. J. biol. Chem. 248, 7215-7222 (1973) Gibbs, M., Latzko, E., O'Neal, D., Hew, Ch. S.: Photosynthetic carbon fixation by isolated maize chloroplasts. ]3iochem. biophys. Res. Commun. 40, 1356-1361 (1970) Heldt, H.W., Fliege, R., Lehner, K., Milovancev, M., Werdan, K. : Metabolite movement and COs-fixation in spinach chloroplasts. Prec. 3rd Int. Congress of Photesynth. Res., M. Avron, ed., 1974 (in press) Jensen, R. G., ]3assham, J. A. : Photosynthesis by isolated chloroplasts. Prec. nat. Aead. Sci. (Wash.) 56, 1095-1100 (1966) Kelly, G.J., Gibbs, 1K.: Non-reversible D-glyceraldehyde 3-phosphate dehydrogenase of plant tissues. Plant Physiol. 52, 111-118 (1973) Lea, P.J., Miflin , ]3. J.: Alternative route for nitrogen assimilation in higher plants. Nature (Lend.) 251, 614-616 (1974) Miflin, ]3. J.: The location of nitrite reductase and other enzymes related to amino acid biosynthesis in the plastids of root and leaves. Plant Physiol. 54, 550-555 (1974) Wells, G. N., Hageman, R. H. : Specificity for nicotine amide adenine dinucleotide by nitrate reductase from leaves. Plant Physiol. 54, 136-141 (1974)

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Formation of C-4 dicarboxylic acids by intact spinach chloroplasts.

It is concluded from (14)C labelling kinetics as well as from enzyme analysis that formation of malate and aspartate from glycerate-3P via carboxylati...
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