Carboxylic Acid Cycle Are Present in the Green Alga by steepslope9876


									Plant Physiol. (1992) 98, 535-539                                                                    Received for publication June 27, 1991
0032-0889/92/98/0535/05/$01 .00/0                                                                             Accepted September 5, 1991

           Some Enzymes and Properties of the Reductive
         Carboxylic Acid Cycle Are Present in the Green Alga
                  Chiamydomonas reinhardtii F-60'
                                          Changguo Chen and Martin Gibbs*
       Institute for Photobiology of Cells and Organelles, Brandeis University, Waltham, Massachusetts 02254

                           ABSTRACT                                           There are no reports in the literature to support the exis-
   The reductive carboxylic acid cycle, the autotrophic pathway            tence of the reductive carboxylic acid cycle in a eukaryotic
Of CO2 assimilation in prokaryotes (photosynthetic and nonpho-             cell. The F-60 mutant of the green alga Chlamydomonas
tosynthetic autotrophic bacteria), was investigated in Chiamydo-           reinhardtii seems appropriate to probe for this pathway in a
monas reinhardtii F-60, an algal mutant lacking a complete pho-            eukaryote because the mutant lacks phosphoribulokinase, and
tosynthetic carbon reduction pathway (C3) due to a deficiency in           acetate enhances the rate of CO2 photoassimilation by this
phosphoribulokinase. Evidence was obtained consistent with the             cell (10).
presence of the reductive carboxylic acid cycle in F-60. This
conclusion is based on the fact that: (a) acetate approximately
doubled CO2 fixation in whole cells (4 micromoles per milligram
chlorophyll per hour) and in chloroplasts (32 nanomoles per                               MATERIALS AND METHODS
milligram chlorophyll per hour); and (b) pyruvate synthase, a-
ketoglutarate synthase, and ATP-citrate lyase, three indicators of
                                                                           Algal Growth Condition
the cycle, were found in cell-free extracts.                                  Chlamydomonas reinhardtii (Dangeard) F-60 was obtained
                                                                           from Dr. R. K. Togasaki, Indiana University. Cells were
                                                                           grown in batch cultures in an acetate-supplemented medium
                                                                           (7) at 25°C on a rotary shaker under a continuous light
  The reductive pentose-P pathway (C3), the C4 pathway, and
                                                                           intensity of 8 W/m2. Cells were grown for 4 d until they
                                                                           reached the end of the exponential phase. These cells were
CAM are the three well-documented photosynthetic pathways                  harvested for experiments with intact cells and for the isola-
whereby higher plants assimilate CO2. The C3 pathway is also               tion of chloroplasts.
established in algae. Demonstrated in photosynthetic (3, 6)                   For the preparation of cell-free extracts, F-60 cells were
and in autotrophic aerobic hydrogen-oxidizing (14) and sul-                grown in the same medium in synchronous cultures with a
fate-reducing (13) bacteria is the lesser-known reductive car-             12 h: 12 h light-dark cycle. Cells were inoculated into 1 L of
boxylic acid cycle (reductive citric acid cycle).                          culture medium through which filtered air was bubbled. Cells
  The reductive carboxylic acid cycle first reported in the                were harvested after 58 h of growth at 25°C and contained
photosynthetic green sulfur bacterium Chlorobium thiosulfa-                about 1 mg Chl.
tophilum (6) is in effect a reversal of the oxidative citric acid
cycle of Krebs, and one complete cycle yields one molecule
of oxaloacetate from four molecules of CO2. A variant of the
cycle is the conversion of two molecules of CO2 to acetic acid.            F-60 Chloroplast Isolation
In both pathways, reduced Fd is needed to form pyruvate                       The procedures for isolating intact chloroplasts from F-60
from acetyl-CoA and CO2 and a-ketoglutarate from succinyl-                 cells followed Klein et al. (8) as modified by Willeford and
CoA and CO2 catalyzed by pyruvate synthase and a-ketoglu-                  Gibbs (17).
tarate synthase, respectively.
  One reason for concluding that this cycle is the major
pathway for the incorporation of CO2 in C. thiosulfatophilum               Preparation of Cell-Free Extracts
was the absence of Rubisco and phosphoribulokinase (4).
Another basis was the product distribution of '4CO2 fixation.                 F-60 cells from 1 L of culture medium were harvested,
The CO2 fixation patterns of Buchanan et al. (4) and of                    washed twice in 100 mm potassium phosphate buffer (pH
Sirevag and Omerod (15) indicated that isotope was incor-                  6.5), and resuspended in the same buffer (40 mL). Cells were
porated into amino acids, principally glutamic acid, rather                placed in 15 mL stainless steel centrifuge tubes, which were
than into glycerate 3-P or the dicarboxylic acids. These results           then covered with serum rubber stoppers. The tubes were
ruled out the C3, C4, and CAM pathways.                                    evacuated and refilled with N2 for five cycles. The cells were
                                                                           frozen in liquid N2 and thawed in a 35°C water bath five
  'Supported by U.S. Department of Energy DE-FG02-86                       times. The ruptured cells were spun down at 12,000g at 4°C
ER 1 3486.                                                                 for 10 min to remove cell debris. The supernatant was either
536                                                             CHEN AND GIBBS                                     Plant Physiol. Vol. 98, 1992

                                                                           binowitz (12). The phenylhydrazones were collected by filtra-
                                                                           tion through a 0.45 ,um Millipore filter. After drying overnight
                                                                           at room temperature, radioactivity was determined with a
                                                                           gas-flow counter.
                                                                           Protein Assay
           0                                                                 Protein was measured according to Bradford (2).

           0                                                               Chi Determination
                                                                             Chl was measured according to Arnon (1).
                     0.0      1.0     2.0      3.0     4.0
                                    Time (h)                               Reagents and Other Materials
Figure 1. Time course of C02 fixation by C. reinhardtii F-60 cells in         Spinach and C. reinhardtii F-60 Fd was purified according
the presence or absence of acetate and DCMU under light and dark.          to Buchanan and Arnon (3). Additional C. reinhardtii Fd was
The 1 mL (final volume) reaction medium contained 50 Mmol Mes-Tris         a gift of Dr. Rene Gfeller. Clostridium pasteurianum Fd and
buffer (pH 6.5), 10 Mmol NaH14CO3 (20 MCi), cells containing 36.4 ,g       other reagents were purchased from Sigma. High-grade puri-
Chi, and, where indicated, 5 Mmol acetate, 10 nmol DCMU. Experi-           fied H2 and N2 were supplied by Matheson.
ments were carried out in glass vials sealed with rubber stoppers
under 100% H2 at 250C. Darkness was obtained by wrapping the
glass vials with two layers of aluminum foil. The light intensity was
230 W/m2. Samples from each bottle were transferred with a syringe                         RESULTS AND DISCUSSION
at time intervals to scintillation counting vials that contained 0.4 mL    C02 Fixation by Whole Cells
of 0.5 N HCI. After shaking the vials for at least 2 h, 0.3 mL of 0.5 N
NaHCO3 and 0.3 mL of 0.5 N NaOH were added to neutralize the                 In Figure 1, we confirm the earlier report of Moll and
sample solution. ACS (9 mL) was added to each vial. 14CO2 fixation         Levine (1 1) that in F-60 cells the rate of CO2 assimilation was
was determined by a scintillation counter. Plus acetate, light (0); plus   enhanced by the addition of acetate. When the optimum pH
acetate, dark (0); minus acetate, light (A); minus acetate, dark (A);      of 6 was applied, CO2 assimilation was doubled in the presence
plus acetate, plus DCMU, light (El); plus acetate, plus DCMU, dark         of acetate (Fig. 2). The highest rate was possibly due to an
(U); minus acetate, plus DCMU, light (K); minus acetate, plus DCMU,
dark (*).

directly used or was the source of partially purified pyruvate                                                              Ac
synthase.                                                                                 4.5                   .Q

Determination of Enzymatic Activities                                                 C3.5-
   Pyruvate synthase, C02-pyruvate exchange, and a-ketoglu-
tarate synthase were determined by procedures developed by                           0
Buchanan and Arnon (3).                                                                   2.5-
   Pyruvate synthase and a-ketoglutarate synthase assays uti-
lize PSII-inactivated spinach thylakoids to generate reduced
Fd with reduced DCIP2 as donor. Chloroplasts were prepared
from 10-week-old plants by the method of Mill and Joy (10).                               1.5                 ,       .       ,
To inactivate PSII activity, the intact chloroplasts were heated                                5.5          6.5             7.5
at 55°C for 5 min, spun down at 6000g for 5 min, and the                                                      pH
thylakoids were suspended in 100 mm K-phosphate buffer
(pH 6.5).                                                                  Figure 2. Influence of pH and of acetate on the rate of CO2 fixation
   To measure ['4C]pyruvate and [14C]a-ketoglutarate gener-                by C. reinhardtii F-60 cells. Experiments were carried out in glass
ated in the assays, the reactions were terminated by addition              vials sealed with rubber stoppers under 100% H2 at 300C and 230
of 0.5 mL of 12 N HCI. The resulting precipitate was removed               W/m2 light intensity for 30 min. The 1 mL reaction medium contained
by centrifugation and 1 mL of the supernatant was used to                  25 Mmol MeS-Hepes buffer (pH 6.0-7.5, adjusted with Tris); 10 ,mol
produce the 2,4-dinitrophenylhydrazones as described by Ra-                NaH14C03 (20 MCi); 5 ,umol Na-acetate (where indicated), and 25.7
                                                                           Mg Chi. Aliquots of 100 ML were taken at time intervals and placed
                                                                           on planchets that contained one layer of lens paper and 0.4 mL of
  'Abbreviations: DCIP, 2,6-dichlorophenolindophenol; TPP, thia-           0.5 N HCI. The planchets were dried ovemight at room temperature.
mine pyrophosphate; PTA, phosphotransacetylase.                            14C02 fixation was determined with a gas flow counter.
                       PROPERTIES OF THE REDUCTIVE CARBOXYLIC ACID CYCLE IN CHLAMYDOMONAS                                                537

increased level of undissociated acetic acid resulting from the   Table I. Requirements for Pyruvate and a-Ketoglutarate Synthesis
lower pH rather than an effect on reactions within the plasma     by Cell-Free Extracts after DEAE-Sephadex G-25 Column
membrane. Inasmuch as anaerobicity enhanced CO2 fixation,         Chromatography
the experiments were performed in an atmosphere of H2. This          All steps of this experiment were performed under strictly anaero-
property was not studied further.                                 bic (N2) conditions. Crude cell-free extract was subjected to chro-
   Fixation of CO2 was light-dependent. Furthermore, addi-        matography primarily to remove Fd. One gram of DEAE-Sephadex
tion of DCMU increased CO2 fixation, but only when acetate        G-25 resins was placed overnight in 20 mm K-phosphate buffer (pH
was available (Fig. 1). Clearly, DCMU would block PSII            6.5). The resins were loaded into a 10 mL plastic syringe to make a
activity, eliminate H20 as the electron donor, and prevent        3 x 1.5 cm column. Cell-free extract prepared from 1 L of culture
the evolution of 02. A decrease in O2 would protect pyruvate      medium was passed through the column with the 20 mm K-phosphate
synthase and a-ketoglutarate synthase from aerobic inactiva-      buffer (pH 6.5). For pyruvate synthesis, the complete reaction mixture
tion and reduced Fd from oxidation by the Mehler reaction,        of 1.5 mL contained 150 Amol K-phosphate buffer (pH 6.5), 0.1 umol
                                                                  DCIP, 10 ,Amol Na-ascorbate, 25 Amol acetyl-P (lithium salt), 0.25
resulting in the higher observed rate. Thus, in the presence of   Amol CoA, 45 gg Fd, 25 zmol semicarbazide (neutralized with KOH),
DCMU, H2 would reduce Fd catalyzed by the anaerobically           5 IAg PTA, 1.5 Amol MgC12, 15 ,imol NaH14CO3 (30 MCi), 50 zg TPP,
induced hydrogenase (9) and the function of light would be        heated spinach chloroplasts (300 ,4g Chi), and extract (1.5 mg pro-
restricted to the generation of ATP.                              tein). For a-ketoglutarate synthesis, the complete reaction mixture of
                                                                  1.5 mL contained 150 ,mol K-phosphate buffer (pH 6.5), 0.024 ,Amol
                                                                  DCIP, 10 ,4mol Na-ascorbate, 3 lmol Na4-EDTA, 1.5 ,mol MgC12, 50
Pyruvate Synthase                                                 /mol K-succinate, 2.5 Amol ATP, 0.25 iAmol CoA, 15 /tmol NaH14C03
                                                                  (30 IiCi), 45 jug Fd, heated spinach chloroplasts (300 ugg Chl), and
   The properties of F-60 pyruvate synthase are identical to      extract (1.5 mg protein). The experiments were carried out in light
those reported for the C. thiosulfatophilum enzyme (3) with a     (230 W/m2) under N2 and at 300C for 60 min. The reactions were
requirement for reduced Fd, CoA, TPP, and a pH optimum            terminated by the addition of 0.5 mL of 12 N HCI. Determination of
of 6.4 (Table I, Fig. 3). Again similar to the photosynthetic     ['4C]pyruvate and [14C]a-ketoglutarate is given in "Materials and
green bacterial enzyme, C. pasteurianum Fd was as effective       Methods.'
as F-60 Fd, whereas spinach Fd was about 20% less effective                  Treatment            Pyruvate Synthesis       a-KetSynutarate
(data not shown). A preparation of pyruvate synthase purified
threefold by (NH4)2SO4 fractionation in an atmosphere of N2                                                nmol/mg protein. h
synthesized pyruvate with a specific activity of 21.3 nmol/mg        Complete                            8.8                     5.4
protein * h and at a linear rate up to 1 h (data not shown).         -Fd                                 3.0                     1.7
                                                                     -CoA                                1.6
                                                                     -PTA                                3.6
                                                                     -Acetyl-P                           2.9
C02-Pyruvate Exchange                                                -Succinate                                                  1.8
                                                                     -TPP                                3.4
  All preparations of pyruvate synthase examined so far              Complete, boiled extract            0                       0
catalyze an exchange between CO2 and the carboxyl group of           Dark                                1.4
pyruvate, and CoA is a requirement (3). F-60 pyruvate syn-
thase catalyzed a '4C02-pyruvate exchange that was depen-
dent upon CoA (Table II). Hydroxypyruvate and lactate did
not substitute for pyruvate. The pH optimum for exchange                                                                   450
was more alkaline than that for synthesis (Fig. 3), a finding               0
consistent with the photosynthetic green bacterial enzyme (3).                                                                    2
Our preparation catalyzed an exchange reaction at a linear                  0
rate up to 1 h (data not shown).                                                                                           300 (0
                                                                            -. 10.0
a-Ketoglutarate Synthase                                                    0~                                             150       P
                                                                            C,    5.0
   The crude preparation of F-60 catalyzed the synthesis of a-
ketoglutarate from succinate and showed a requirement for                   0
reduced Fd (Table I). A pH optimum at 6.5 (Fig. 3) is identical             E
                                                                            c     n  n
                                                                                  v..v                                     0
to that reported for the C. thiosulfatophilum enzyme (3).
                                                                                         6                             8
Compartmentation of Pyruvate Synthase                             Figure 3. Effect of pH on pyruvate synthase, C02-pyruvate ex-
                                                                  change, and a-ketoglutarate synthase. The reaction mixtures and
   Isolated intact chloroplast preparations fixed CO2 and, sim-   experimental conditions for pyruvate synthase and a-ketoglutarate
ilar to the cells' uptake, was enhanced by acetate (Table III).   synthase are described in Table I and for C02-pyruvate exchange in
The increase in CO2 fixation on the addition of acetate and       Table Il; pH was maintained by K-phosphate buffer. (0) Pyruvate
detection of the C02-pyruvate exchange (Table III) is taken       synthase; () a-ketoglutarate synthase; (A) C02-pyruvate exchange.
538                                                         CHEN AND GIBBS                                     Plant Physiol. Vol. 98, 1992

Table II. Requirements for C02-Pyruvate Exchange                      Table Ill. Anaerobic Metabolism of C02 by Intact Chloroplasts
   The complete reaction medium of 1.5 mL contained 150 jsmol K-         The complete reaction medium of 1.5 mL (final volume) for C02
phosphate buffer (pH 6.5), 0.075 MAmol CoA, 75 ,umol pyruvate, 15     fixation contained 37.5 Mmol Mes-Tris buffer (pH 6.5), 180 Mimol
jAmol NaH'4CO3 (30 MCi), crude extract containing 1.4 mg protein,     mannitol, 1.5 Mmol MgCI2, 1.5 Mmol MnCl2, 3 ltmol Na4-EDTA, 15
and, where indicated, 75 ,umol hydroxypyruvate and 75 IAmol DL-       Mimol NaH14C03 (30 MCi), 45 ug Chl, and 7.5 Mmol acetate. The
lactate. The experiment was carried out at 300C under N2 and in the   reaction was performed at 300C in the light (230 W/m2) under H2.
dark for 60 min. The reaction was terminated by the addition of 0.5   For C02-pyruvate exchange, the reaction was carried out in the same
mL of 12 N HCI. The precipitated protein was spun down and 1 mL       medium containing 82.5 Mmol pyruvate under H2 for 30 min and in
of the supematant was analyzed for [14C]pyruvate as described in      the dark. After 30 min, an aliquot of 200 MAL was placed on a planchet
"Materials and Methods."                                              that contained one layer of lens paper and 400 ML of 0.5 N HCI. The
              Addition          Rate of C02-Pyruvate Exchange         sample was dried ovemight at room temperature to remove unfixed
                                                                      14CO2. Radioactivity was determined with a gas flow counter.
                                       nmolmg protein.h
        + Pyruvate                                                        Addition       CO2 Fixation      Addition       C02-Pyruvate
        + CoA                               367.9
        + Pyruvate                                                                     nmoil/mg Chi -h                   prnol/mg Chl -h
        -CoA                                220.9                          Acetate          32.1            Pyruvate          75.1
        - Pyruvate                                                       - Acetate          13.6         - Pyruvate            5.5
        + CoA                                 30.8
        - Pyruvate
        -CoA                                  22.1
        + Hydroxypyruvate                                             where H20 is the electron donor (8). Enzymic activity data
        + CoA                                  2.7                    reported here for pyruvate synthase, a-ketoglutarate synthase,
        + Lactate                                                     and ATP-citrate lyase when compared with the enzymes
        + CoA                                 23.1                    catalyzing the C3 pathway in Chlamydomonas (17) or the
        + Pyruvate                                                    reductive carboxylic acid cycle in Chlorobium (4) and Desul-
        + CoA                                                         fobacter hydrogenophilus (13) are consistent with the observed
        + Boiled extract                       1.9                    rates of C02 fixation by each route. Therefore, in the prokar-
                                                                      yotes (4, 13, 14) the reductive carboxylic acid cycle is the
                                                                      autotrophic pathway of C02 fixation, whereas this cycle pre-
                                                                      sumably plays an auxiliary role to the C3 pathway in the
as evidence for the presence of pyruvate synthesis within the         eukaryotic cell. However, in an anaerobic environment rich
chloroplast. Conversion of acetate to acetyl-CoA the substrate        with acetate and C02 documented in the raw sewage lagoons
of the pyruvate synthase reaction confirms the earlier report         in the Antelope Valley of the California Mojave Desert where
of chloroplast-localized aceto-CoA kinase reaction (17).              the rapid growth of nearly unialgal cultures of Chlamydomo-
                                                                       nas has been investigated (5), the reductive carboxylic acid
                                                                      cycle may be an important pathway for C02 photoassimila-
                         CONCLUSION                                   tion. Acetate would be in abundance in this environment
   In this communication, we have characterized two key
                                                                      because it is a major product of starch fermentation by the
enzymes,   pyruvate synthase and a-ketoglutarate synthase, of
                                                                       Chlamydomonas cell (7).
the reductive carboxylic acid cycle in broken cell preparations
of C. reinhardtii F-60. Another indicative enzyme is ATP-
citrate lyase, which is able to regenerate oxaloacetate from                                  LITERATURE CITED
citrate and to release acetyl-CoA for the pyruvate synthase            1. Arnon DI (1949) Copper enzymes in isolated chloroplasts. Poly-
reaction. ATP-citrate lyase was assayed in the crude cell-free              phenoloxidases in Beta vulgaris. Plant Physiol 24: 1-14
extracts by the method of Takeda et al. (16) and was found             2. Bradford MM (1976) A rapid and sensitive method for the
to have a specific activity of 16 nmol acetyl-CoA formed/mg                 quantitation of microgram quantities of protein utilizing the
protein * min. The enzymic data are consistent with the oper-               principle of protein-dye binding. Anal Biochem 72: 248-254
ation of a reductive carboxylic acid cycle as a route of CO2           3. Buchanan BB, Arnon DI (1969) The reductive carboxylic acid
                                                                             cycle. Methods Enzymol 28: 170-181
fixation in C. reinhardtii F-60.                                       4. Buchanan BB, Schurmann P, Shanmugam KT (1972) Role of the
   Inasmuch as the isolated chloroplasts fixed CO2 in the                   reductive carboxylic acid cycle in a photosynthetic bacterium
presence of acetate, and pyruvate synthesis and a-ketoglutar-               lacking ribulose 1,5-diphosphate carboxylase. Biochim Biophys
ate synthesis depend on reduced Fd, it is reasonable to con-                Acta 283: 135-145
sider that the compartmention of the reductive carboxylic              5. Eppley RW, Maciasr FM (1962) Rapid growth of sewage lagoon
acid cycle must be in the chloroplast, which is the only proven             Chlamydomonas with acetate. Physiol Plant 15: 72-79
site of reduced Fd in this organism. In addition to pyruvate           6. Evans MCW, Buchanan BB, Arnon DI (1966) A new ferredoxin-
synthase (Table III), two other enzymes, aceto-CoA kinase                   dependent carbon reduction cycle in photosynthetic bacte-
                                                                            rium. Proc Natl Acad Sci USA 55: 928-934
and malate dehydrogenase, of the cycle have been reported to           7. Gfeller RP, Gibbs M (1984) Fermentative metabolism of Chlam-
be localized in the C. reinhardtii chloroplast (17).                        ydomonas reinhardtii. I. Analysis of fermentative products
   Our intact cells of F-60 primed with acetate fixed CO2 at a              from starch in dark and light. Plant Physiol 75: 212-218
rate about 2 to 3% of that of wild-type Chlamydomonas                  8. Klein U, Chen C, Gibbs M, Platt-Aloia KA (1983) Cellular
                          PROPERTIES OF THE REDUCTIVE CARBOXYLIC ACID CYCLE IN CHLAMYDOMONAS                                                539

        fractionation of Chlamydomonas reinhardtii with emphasis on       13. Schauder R, Widdel F, Fuchs G (1987) Carbon-assimilation in
        the isolation of the chloroplast. Plant Physiol 72: 481-487             sulfate-reducing bacteria. II. Enzymes of a reductive citric acid
 9.   Maione TE, Gibbs M (1986) Hydrogenase-mediated activities in              cycle in the autotrophic Desulfobacter hydrogenophilus. Arch
        isolated chloroplasts of Chlamydomonas reinhardtii. Plant               Microbiol 148: 218-225
        Physiol 80: 360-363                                               14. Shiba H, Kanasumi T, Igarashi Y, Kodma T, Minoda Y (1985)
10.   Mill WR, Joy KW (1980) A rapid method for isolation ofpurified            The CO2 assimilation via the tricarboxylic acid cycle in an
        physiologically active chloroplasts, used to study the intra-           obligately autotrophic aerobic hydrogen-oxidizing bacterium,
        cellular distribution of amino acids in pea leaves. Planta 148:         Hydrogenobacter thermophilus. Arch Microbiol 141: 198-203
        75-83                                                             15. Sirevag R, Omerod JG (1970) Carbon dioxide fixation in Chlo-
11.   Moll B, Levine RP (1970) Characterization of a photosynthetic             robium. Biochem J 120: 399-408
        mutant strain of Chlamydomonas reinhardtii deficient in phos-     16. Takeda Y, Suzuki F, Inoue H (1969) ATP citrate lyase [citrate-
        phoribulokinase activity. Plant Physiol 46: 576-580                     cleavage enzyme]. Methods Enzymol 13: 153-160
12.   Rabinowitz JC (1960) Factor B and other compounds related to        17. Willeford KO, Gibbs M (1989) Localization of the enzymes
        vitamin B12 in pyruvic acid-CO2 exchange. J Biol Chem                   involved in the photoevolution of H2 from acetate in Chlam-
        235: 50                                                                 ydomonas reinhardtii. Plant Physiol 90: 788-791

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