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Plant Physiol. (1999) 120: 539-546
Phosphoenolpyruvate Carboxykinase Is Involved in the
Decarboxylation of Aspartate in the Bundle Sheath of Maize1
Astrid Wingler2,
Robert P. Walker,
Zhi-Hui Chen, and
Richard C. Leegood*
Robert Hill Institute and Department of Animal and Plant Sciences,
University of Sheffield, Sheffield S10 2TN, United Kingdom
 |
ABSTRACT |
We
recently showed that maize (Zea mays L.) leaves contain
appreciable amounts of phosphoenolpyruvate carboxykinase
(PEPCK; R.P. Walker, R.M. Acheson, L.I. Técsi, R.C. Leegood
[1997] Aust J Plant Physiol 24: 459-468). In the present study, we
investigated the role of PEPCK in C4 photosynthesis in
maize. PEPCK activity and protein were enriched in extracts from
bundle-sheath (BS) strands compared with whole-leaf extracts.
Decarboxylation of [4-14C]aspartate (Asp) by BS
strands was dependent on the presence of 2-oxoglutarate and
Mn2+, was stimulated by ATP, was inhibited by the
PEPCK-specific inhibitor 3-mercaptopicolinic acid, and was independent
of illumination. The principal product of Asp metabolism was
phosphoenolpyruvate, whereas pyruvate was a minor
product. Decarboxylation of [4-14C]malate was stimulated
severalfold by Asp and 3-phosphoglycerate, was only slightly
reduced in the absence of Mn2+ or in the presence of
3-mercaptopicolinic acid, and was light dependent. Our data show that
decarboxylation of Asp and malate in BS cells of maize occurs via
two different pathways: Whereas malate is mainly decarboxylated by
NADP-malic enzyme, decarboxylation of Asp is dependent on the activity
of PEPCK.
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INTRODUCTION |
C4 plants have been classified as NADP-ME,
NAD-ME, and PEPCK types, according to the major decarboxylase involved
in the decarboxylation of C4 acids in the BS
cells (Gutierrez et al., 1974 ; Hatch et al., 1975). Maize (Zea
mays) belongs to the NADP-ME subgroup of C4
plants and possesses negligible activity of PEPCK, although Gutierrez
et al. (1974) suggested that some other NADP-ME species utilize PEPCK
as an auxiliary decarboxylase. However, we showed previously that maize
leaves contain large amounts of PEPCK (Walker et al., 1997), and
Furumoto et al. (1999) identified a PEPCK gene from maize that is
specifically expressed in BS cells. Other NADP-ME species such as
Echinochloa colona, Echinochloa crus-galli,
Digitaria sanguinalis, and Paspalum notatum also
contain PEPCK, whereas PEPCK protein was not detectable in
Sorghum bicolor, Saccharum officinarum, or
Flaveria bidentis (Walker et al., 1997). The reason that the
occurrence of PEPCK in these plants has been overlooked may be due to
difficulties in measuring PEPCK activity (Walker et al., 1997) or to
the lack of an antibody.
PEPCK-type C4 species mainly use Asp as the
CO2 donor and decarboxylate the oxaloacetate
formed via the following reaction:
In NADP-ME species such as maize,
14CO2 is initially
incorporated into the C-4 position of malate and Asp (about 75% and
25%, respectively), and the C-4 of both is subsequently incorporated into other metabolites (Hatch, 1971; Morot-Gaudry and Farineau, 1978).
Using isolated BS strands, Chapman and Hatch (1981) showed that BS
cells of maize have a significant capacity to decarboxylate Asp, and
they assumed that NADP-ME was responsible. In view of the occurrence of
PEPCK in maize, the involvement of PEPCK in the decarboxylation of Asp
appears more likely, because PEPCK can directly catalyze the
decarboxylation of oxaloacetate formed from Asp without previous
reduction of oxaloacetate to malate.
To study the role of PEPCK in photosynthesis in maize, we measured the
decarboxylation of Asp and malate by BS strands under conditions
supporting the activities of either PEPCK or NADP-ME. In addition, we
determined the protein contents or activities of other enzymes
potentially involved in Asp metabolism.
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MATERIALS AND METHODS |
Plant Material
Maize (Zea mays L. cv H511) plants were grown in
high-nutrient compost (M3, Fisons, Ipswich, UK) in a growth cabinet at
a PPFD of 250 µmol m 2
s1 and a photoperiod of 12 h
d1. The temperature was 28°C during the day
and 24°C at night. For all experiments, the second and third leaves
of 10- to 15-d-old plants were used.
Isolation of BS Strands
BS strands were isolated by slicing deribbed leaves into 1- to
2-mm sections and blending them five to six times for 10 s with
150 mL of blending buffer (100 mM Bicine-KOH, pH 8.0, 0.3 M sorbitol, 0.1% [w/v] PVP-40, 0.02% [w/v] BSA, 5 mM DTT, 2 mM KH2PO4, and 5 mM K2SO4) in a
Waring blender. The strands were collected by filtration through
Miracloth (Calbiochem) and washed with 350 mL of blending buffer. Where
indicated, a digestion treatment (Hatch and Kagawa, 1974) was included
in the procedure. After the sample was blended three times for 10 s each time, the BS strands were washed with blending medium, incubated
for 25 min in 20 mM Mes-KOH (pH 5.5), 0.5 M
sorbitol, 2 mM MgCl2, 0.2% (w/v) BSA, 1% (w/v) cellulase (Onozuka R-10, Yakult, Honsha, Japan), and
0.1% (w/v) pectinase (Macerozyme R-10, Yakult), washed with blending
medium, and blended again (twice for 10 s each time).
Determination of Enzyme Activities
PEPCK was extracted in 180 mM Bicine and 20 mM 3-(cyclohexylamino)-1-propanesulfonic acid (pH 10.0)
containing 50 mM DTT, and the carboxylation reaction was
measured according to the method of Walker et al. (1995). Cytosolic
pyruvate kinase was extracted and assayed as described by Zervoudakis
et al. (1997); ADP was omitted for the assay of PEP phosphatase.
Western Analysis
Proteins were extracted in 200 mM Bicine-KOH (pH 9.5),
25 mM DTT, and 1% (w/v) SDS. Extracts were boiled for
90 s with equal volumes of solubilization buffer (62.5 mM Tris, 20% [v/v] glycerol, 2.5% [w/v] SDS, and 5%
[v/v] 2-mercaptoethanol, pH 6.8). After SDS-PAGE, the proteins were
transferred onto a PVDF membrane (Immobilon-P, Millipore) and probed
with antisera raised against PEPCK from cucumber (Walker et al., 1995),
PEPC from Amaranthus edulis, Rubisco from Brassica
napus, Ala aminotransferase from barley (Muench and Good, 1994),
Asp aminotransferase from Panicum maximum (Numuzawa et al.,
1989), and NAD-ME (Murata et al., 1989) and NADP-ME from maize
(Langdale et al., 1988). A peroxidase-conjugated secondary antibody was
used, and immunoreactive bands were visualized with an enhanced
chemiluminescence kit (Amersham).
Determination of Asp and Malate Decarboxylation
[4-14C]Aspartate and
[4-14C]malate were synthesized enzymatically
(Hatch, 1972). The reaction medium for the synthesis of
[4-14C]Asp contained 50 mM Tris-HCl
(pH 8.1), 1 mM PEP, 1 mM glutamate, 1 mM DTT, 10 mM MgCl2, 1 mM EDTA, 1 unit of PEPC (Boehringer Mannheim), 20 units of
Glu:oxaloacetate aminotransferase (Boehringer Mannheim), and 10 MBq of
NaH14CO3 (ICN) in 1 mL. The
reaction was stopped by adding 10 µL of 5 M HCl. The
[4-14C]Asp formed was purified by loading the
reaction mixture onto Dowex-50 columns, washing with 5 mL of water, and
eluting with 3 mL of 5 M NH4OH. The
eluted fractions were evaporated to dryness in a freeze drier
(Speed-Vac, Savant Instruments, Holbrook, NY), acidified with HCl,
evaporated again, and redissolved in water. [4-14C]Malate was synthesized in a medium
containing 50 mM Tris-HCl (pH 8.1), 3 mM PEP, 5 mM NADH, 1 mM DTT, 10 mM
MgCl2, 1 mM EDTA, 1 unit of PEPC, 20 units of malate dehydrogenase (Boehringer Mannheim), and 37 MBq
NaH14CO3 in a total volume
of 1 mL. The [4-14C]malate formed was purified
by loading the reaction mixture onto a Dowex-1 column, washing with 2 mL of 20 mM HCl, and eluting with 3 mL of 5 M
HCl. The eluted fractions were evaporated, redissolved in water, and
boiled for 20 min to destroy any formed
[4-14C]oxaloacetate. The identity and purity of
the labeled compounds was confirmed by TLC.
For the determination of decarboxylation rates, BS strands isolated
without the digestion step were resuspended in reaction medium
consisting of blending buffer containing 25 mM
D,L-glyceraldehyde to inhibit the reassimilation of
CO2 (Stokes and Walker, 1972) and other compounds
as indicated in the figure legends. The suspension (0.8 mL) was then
incubated with 0.2 mL of [4-14C]Asp or
[4-14C]malate solution (reaction medium
containing 62.5 mM Asp or malate with a specific
radioactivity of 0.2 MBq mmol1) in sealed
vials. After 20 min at 25°C, the reaction was stopped by injecting
0.3 mL of 1 M HCl, and the released
14CO2 was trapped on filter
paper moistened with 0.1 mL of 20% (w/v) KOH. Phaeophytin was
extracted in 80% acetone and quantified according to the method of
Vernon (1960).
Determination of PEP and Pyruvate Formation
For the determination of PEP and pyruvate formation, BS strands
were resuspended in reaction medium (blending buffer containing 10 mM 2-oxoglutarate, 1 mM
MnCl2, 1 mM
MgCl2, and different concentrations of ATP and
ADP), and 0.4 mL of the suspension was incubated with 0.1 mL of 62.5 mM Asp (dissolved in reaction medium) at 25°C. The
reaction was stopped by adding 0.5 mL of 10% (v/v)
HClO4. BS strands were extracted in a
glass-in-glass homogenizer, and metabolites were measured according to
the method of Lowry and Passonneau (1972).
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RESULTS |
PEPCK in BS Cells of Maize
Maize leaves contained substantial amounts (Fig.
1) and activities (Table
I) of PEPCK. The activity of PEPCK
recorded in maize was between one-fifth and one-half of that measured
in PEPCK C4 plants (Chapman and Hatch, 1983;
Smith and Woolhouse, 1983; Walker et al., 1997).
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| Figure 1.
A and B, Western blots for PEPC, PEPCK, and
Rubisco, and the polypeptide pattern in extracts from whole leaves (WL)
and BS strands from maize. The BS strands were isolated including (A)
or omitting (B) treatment with cellulase and pectinase. C, Western blot
for PEPCK in extracts from whole leaves (WL) and BS strands from
U. panicoides isolated after treatment with cellulase
and pectinase. Three micrograms of total protein was loaded per lane.
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Table I.
In vitro activities of PEPCK, pyruvate kinase (PK),
and PEP phosphatase (PP) in extracts of whole leaves and of BS strands,
and in vivo formation of pyruvate (Pyr) from PEP by BS strands in the
presence of 5 mM PEP, 10 mM ADP, 1 mM Mn2+, 1 mM Mg2+, and
40 mM KCl
Data are means ± SE of three plants. n.d., Not
determined.
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Because PEPCK from plants is subject to rapid proteolytic cleavage, we
first optimized the conditions for the isolation of BS strands,
checking the intactness of PEPCK protein. When the isolation procedure
included treatment with cellulase and pectinase, strands free of
mesophyll contamination, as judged by the absence of PEPC, were
obtained (Fig. 1A). Even though this treatment did not lead to general
proteolysis, it resulted in the cleavage of PEPCK. PEPCK in plant
extracts is rapidly cleaved at low pH or, in the absence of SDS, to a
62-kD form with no loss of activity (Walker et al., 1995, 1997).
However, during the isolation of BS strands from maize, PEPCK was
degraded further and sometimes no PEPCK protein was detectable on
western blots. This indicates that protease activity within intact BS
cells was stimulated by low pH. On the other hand, PEPCK in BS strands
isolated from the PEPCK-type species Urochloa panicoides was
largely intact after treatment of the BS strands with cellulase and
pectinase (Fig. 1C).
Maize BS strands isolated without the cell wall-digestion step were
contaminated with approximately 6% of the mesophyll protein, as
estimated from the distribution of PEPC and Rubisco on western blots
(Fig. 1B). PEPCK extracted from these strands was intact and enriched
compared with whole-leaf extracts, confirming the location of PEPCK in
the BS cells in maize (Walker et al., 1997). The activity of PEPCK was
also enriched in BS strands (Table I).
Decarboxylation of [4-14C]Asp
Because of the persistence of open plasmodesmata, BS cells are
permeable to metabolites and can be used to study the metabolism of
externally added substrates such as Asp. Rates of
[4-14C]Asp decarboxylation by BS strands were
independent of illumination (Fig. 2); the
reaction was completely dependent on the presence of 2-oxoglutarate.
ATP, which is a substrate of the PEPCK reaction, stimulated Asp
decarboxylation. An inhibitor of PEPCK activity, MPA (Jomain-Baum et
al., 1976), inhibited the ATP-stimulated activity. PEPCK has an
absolute requirement for Mn2+ and, accordingly,
Mg2+ did not substitute for
Mn2+ in the decarboxylation of Asp.
Mg2+ did, however, stimulate Asp decarboxylation
in the presence of Mn2+. The requirements for
Mn2+ and ATP and the inhibition by MPA are
evidence for an involvement of PEPCK. In contrast, Asp decarboxylation
by BS strands from S. bicolor, an NADP-ME species without
detectable amounts of PEPCK protein, was low (0.06 µmol
min1 mg1 phaeophytin),
was not stimulated by the addition of ATP, and was not inhibited by MPA
(data not shown).
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| Figure 2.
Decarboxylation of [4-14C]Asp (12.5 mM) by BS strands at a PPFD of 200 µmol
m2 s1 (white bars) or in the dark (black
bars). The incubation medium for the control treatment contained 10 mM 2-oxoglutarate (2-OG) and 1 mM
Mn2+. For the other treatments, 400 µM MPA,
10 mM ATP, or 1 mM Mg2+ was added
and 2-oxoglutarate or Mn2+ was omitted. Data are means ± SE of three assays.
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Metabolism of Asp
Under the conditions supporting the highest rates of Asp
decarboxylation, PEP was the main product of Asp metabolism (Fig. 3A). The rates of PEP formation (0.55 µmol min1 mg1
phaeophytin) were comparable to the rates of Asp decarboxylation (Fig.
2). Pyruvate was also formed but at a much lower rate. In the absence
of ATP, the rates of both PEP and pyruvate formation were negligible
(Fig. 3B). The predominance of PEP formation in the presence of ATP
supports the view that PEPCK, and not NADP-ME, was mainly responsible
for the decarboxylation of Asp.
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| Figure 3.
Formation of PEP ( ) and pyruvate ( ) from Asp
in a medium containing 12.5 mM Asp, 10 mM
2-oxoglutarate, 1 mM Mn2+, and 1 mM
Mg2+, with (A) or without (B) the addition of 10 mM ATP or with the addition of 1 mM ATP, 1 mM ADP, and 40 mM KCl (C). Data are means ± SE of three assays.
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To determine whether PEP could be converted into pyruvate by pyruvate
kinase, ADP (1 mM) was supplied in addition to ATP. Because
high concentrations of ATP inhibit pyruvate kinase (Baysdorfer and
Bassham, 1984), the ATP concentration was decreased to 1 mM. Under these conditions, equal amounts of PEP and
pyruvate were formed and the sum of PEP and pyruvate formation was
reduced to 0.38 µmol min1
mg1 phaeophytin (Fig. 3C). This lower rate was
probably due to the effect of a changed ATP:ADP ratio on PEPCK (Walker
et al., 1997). The accumulation of PEP, even in the presence of ADP,
shows that the activity of pyruvate kinase (plus PEP phosphatase) in BS
cells is not sufficient to convert all PEP formed by PEPCK into
pyruvate. The rate of conversion of PEP into pyruvate by BS strands
(Table I) was also lower than the rate of Asp decarboxylation. In
addition, the in vitro activities of pyruvate kinase and PEP
phosphatase in BS cells were much lower than the activity of PEPCK
(Table I).
Since PEPCK is a cytosolic enzyme, we determined whether cytosolic
aminotransferases are present in maize. Although all of the PEPCK
species we tested (U. panicoides, C. gayana, and
P. maximum) contained large amounts of cytosolic Asp and Ala
aminotransferases, these aminotransferases were not detectable in maize
or other NADP-ME species (E. crus-galli and S. officinarum; Fig. 4). In PEPCK
species, NAD-ME in combination with the respiratory chain provides ATP
for the PEPCK reaction. Although maize contained some NAD-ME protein,
the amount was only comparable to that found in
C3 species and was lower than in PEPCK species.
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| Figure 4.
Western blots for PEPCK, cytosolic Asp
aminotransferase (cAsp-AT), cytosolic Ala aminotransferase (cAla-AT),
NAD-ME, and NADP-ME in leaves of U. panicoides,
C. gayana, P. maximum, Z. mays, E. crus-galli, and S. officinarum. Equal amounts of fresh weight were loaded per
lane.
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Decarboxylation of [4-14C]Malate
In contrast to rates of Asp decarboxylation, those of
[4-14C]malate were generally low in the dark
(Fig. 5). The stimulation of malate
decarboxylation by ATP and NAD, which is needed for the oxidation of
malate to oxaloacetate, indicates some contribution of PEPCK to malate
decarboxylation in the dark. Under illumination, however, the
effect of ATP and NAD was smaller and malate decarboxylation was
strongly stimulated by Asp and 3-phosphoglycerate. Stimulation by Asp
and 3-phosphoglycerate is characteristic of malate decarboxylation catalyzed by NADP-ME (Hatch and Kagawa, 1976; Chapman and Hatch, 1979;
Boag and Jenkins, 1986). The light dependence of malate decarboxylation
also suggests the involvement of NADP-ME (Hatch and Kagawa, 1976). In
the presence of MPA or in the absence of Mn2+,
malate decarboxylation was slightly reduced, but the inhibition was
minor compared with the effect on Asp decarboxylation. In the presence
of Mn2+, Mg2+, Asp, and
3-phosphoglycerate, ATP inhibited the decarboxylation of malate,
possibly by binding the Mg2+ required for
NADP-ME.
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| Figure 5.
Decarboxylation of 12.5 mM
[4-14C]malate by BS strands at a PPFD of 200 µmol m2 s1 (white bars) or in the dark
(black bars). The incubation medium for the control treatment contained
1 mM Mn2+ and 1 mM
Mg2+. For the other treatments, 10 mM ATP, 10 mM NAD, 10 mM Asp, 5 mM
3-phosphoglycerate (PGA), or 400 µM MPA was added or, in
one treatment, Mn2+ was omitted. Data are means ± SE of three assays.
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DISCUSSION |
The Function of PEPCK in Maize
One reason that the presence of PEPCK in maize has been overlooked
may be the instability of this enzyme during the isolation of BS
strands (Fig. 1). When the procedure for maize included digestion of
the mesophyll cells with cellulase and pectinase, PEPCK protein was
almost completely degraded. In contrast, PEPCK from plants that have
been classified as PEPCK species appears to be more stable. Intactness
of the PEPCK from U. panicoides was not affected by the
digestion step, and PEPCK from P. maximum was still fully
active in BS protoplasts isolated by treatment with cellulase and
pectinase (Chapman and Hatch, 1983).
The formation of PEP from Asp (Fig. 3), the dependence of Asp
decarboxylation on Mn2+ and ATP, and the
inhibition by MPA (Fig. 2) strongly suggest that PEPCK is involved in
the metabolism of Asp in BS cells of maize. The conditions supporting
malate decarboxylation (Fig. 5) were different from those supporting
Asp decarboxylation. ATP enhanced malate decarboxylation only in the
dark, whereas Asp and 3-phosphoglycerate stimulated malate
decarboxylation in the light. Since Asp facilitates the transport of
malate into the chloroplasts (Chapman and Hatch, 1979; Boag and
Jenkins, 1986) and 3-phosphoglycerate is important for the reoxidation
of NADPH formed in the NADP-ME reaction (Hatch and Kagawa, 1976), the
stimulation by Asp and 3-phosphoglycerate confirms that malate is
mainly decarboxylated by NADP-ME and not by PEPCK.
The lack of an effect of illumination on Asp decarboxylation indicates
that the activity of PEPCK is not subject to rapid modulation by light.
In some C4 plants PEPCK is phosphorylated in the
dark and dephosphorylated under illumination (Walker and Leegood,
1996). However, PEPCK from maize is not phosphorylated (Walker et al.,
1997), and the putative phosphorylation motif (Walker and Leegood,
1995) is modified (Furumoto et al., 1999). Thus, light-dependent
activation of PEPCK by dephosphorylation probably does not occur in
maize. Expression of the PEPCK gene, on the other hand, is much higher
in the daytime than at night (Furumoto et al., 1999).
Decarboxylation of Asp not only was dependent on
Mn2+ but was stimulated when
Mg2+ was present in addition to
Mn2+. Although previous studies have shown that
in vitro activities of PEPCK from P. maximum and U. panicoides are inhibited by Mg2+ at
physiological concentrations (Burnell, 1986; Walker et al., 1997),
recent work in this laboratory has shown that
Mg2+ is an activator at physiological
concentrations of Mn2+.
Photosynthetic Metabolism in Maize
The importance of PEPCK in photosynthetic metabolism will depend
on the relative fluxes through Asp and malate during
C4 photosynthesis. In maize, substantial amounts
of Asp are labeled (Hatch, 1971; Créach et al., 1974;
Morot-Gaudry and Farineau, 1978; Chapman and Hatch, 1981), more so in
N-sufficient than in N-deficient maize (Khamis et al., 1992), but
measurements of labeling and/or pools do not give any indication of
relative fluxes. The relative capacities for Asp and malate
decarboxylation would have major energetic consequences in the BS.
Whereas malate decarboxylation by NADP-ME provides one-half of the
NADPH required for the reduction of 3-phosphoglycerate, no NADPH is
formed during Asp decarboxylation by PEPCK. Typically, BS cells of
NADP-ME-type species show little or no capacity for PSII-dependent
photoreduction of NADP. The occurrence of PEPCK in NADP-ME-type species
may be correlated with PSII activity. Species that contain PEPCK
(maize, D. sanguinalis, and Setaria lutescens;
Gutierrez et al., 1974; Walker et al., 1997) show some PSII activity in
the BS cells (Ku et al., 1974; Chapman et al., 1980), whereas BS cells
of S. bicolor and S. officinarum lack PEPCK and
have the lowest PSII activity of the C4 species tested (Ku et al., 1974).
Decarboxylation of Asp in the presence of 2-oxoglutarate shows that Asp
aminotransferase activity is present in the BS strands. In contrast to
PEPCK species, the transamination of Asp to form oxaloacetate as the
substrate for the PEPCK reaction is probably not catalyzed by cytosolic
Asp aminotransferase (Fig. 4). Chapman and Hatch (1981) showed that the
activity of Asp aminotransferase in BS cells of maize is located mainly
in the mitochondria. In addition to oxaloacetate, the PEPCK reaction
requires ATP. In PEPCK-type species, this ATP is provided by the
activity of mitochondrial NAD-ME in combination with the respiratory
chain (Hatch, 1987). In maize, some NAD-ME protein was present, but the
amount was much lower than in PEPCK-type species (Fig. 4), which is
consistent with measurements of its activity (Hatch et al., 1975). An
additional source of ATP could be the conversion of PEP to pyruvate by
pyruvate kinase. The activity of pyruvate kinase was, however, lower
than the activity of PEPCK (Table I), and in vivo rates of pyruvate formation from PEP were not sufficient for the complete conversion of
PEP into pyruvate (Fig. 3). Valle and Heldt (1991) measured similar
activities of pyruvate kinase in BS strands of maize and concluded that
these activities were not high enough to fully account for the
formation of Ala from PEP. In addition, activities of pyruvate kinase
in PEPCK-type species either are lower than rates of photosynthesis
(Smith and Woolhouse, 1983) or are not detectable (Rathnam and Edwards,
1977). Thus, PEP and not pyruvate or Ala is probably the
C3 compound mainly transported from the BS to the
mesophyll, where it can serve directly as a substrate for PEPC.
In conclusion, our results show that two distinct pathways for the
decarboxylation of malate and Asp occur in maize and probably in other
species previously classified as NADP-ME types (Fig. 6): Whereas malate is decarboxylated by
NADP-ME in the chloroplasts, resulting in the formation of pyruvate,
Asp is decarboxylated by PEPCK in the cytosol, resulting in the
formation of PEP. It would appear that NADP-ME species can be
subdivided into those with and without PEPCK, extending the biochemical
classification of C4 plants. Further studies will
show to what extent the PEPCK-dependent pathway contributes to
C4 photosynthesis under different environmental conditions and how the activity of PEPCK is regulated.
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| Figure 6.
Photosynthetic metabolism in maize leaves. The
oxaloacetate (OAA) formed by PEPC (1) in the mesophyll is either
reduced to malate or transaminated to Asp. Whereas malate is
decarboxylated to pyruvate by NADP-ME (2) in the chloroplasts of the BS
cells, oxaloacetate formed from Asp in the mitochondria is
decarboxylated to PEP by PEPCK (3) in the cytosol of the BS cells. The
CO2 produced in the decarboxylation reactions is
subsequently fixed by Rubisco (4). PGA, Phosphoglycerate.
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FOOTNOTES |
1
This research was supported by the Biotechnology
and Biological Sciences Research Council of the United Kingdom (grant
no. CO5229).
2
Present address: Botanisches Institut,
Universität Basel, Hebelstrasse 1, CH-4056 Basel, Switzerland.
*
Corresponding author; e-mail r.leegood{at}sheffield.ac.uk; fax
44-114-222-0050.
Received December 28, 1998;
accepted March 12, 1999.
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ABBREVIATIONS |
Abbreviations:
Asp, aspartate.
BS, bundle sheath.
ME, malic
enzyme.
MPA, 3-mercaptopicolinic acid.
PEPC, PEP carboxylase.
PEPCK, PEP carboxykinase.
| |
ACKNOWLEDGMENTS |
We thank A.G. Good (University of Alberta, Edmonton,
Alberta, Canada), T. Nelson (Yale University, Hartford, CT), R. Ohsugi (National Institute of Agrobiological Resources, Ibaraki, Japan), and
M. Taniguchi (Nagoya University, Japan) for providing antisera.
| |
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Créach E,
Michel JP,
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Abstract
Introduction
