Plant Cell Physiol. 38(11): 1217-1225 (1997)
JSPP © 1997
Reappraisal of the Role of Sodium in the Light-Dependent Active Transport
of Pyruvate into Mesophyll Chloroplasts of C4 Plants
Naohiro Aoki' and Ryuzi Kanai
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, Urawa, 338 Japan
The mechanism of light-dependent active transport
of pyruvate in C4 mesophyll chloroplasts has not been
clarified, particularly in Na+-type C4 species, in which the
pyruvate uptake into mesophyll chloroplasts is enhanced
by illumination or by making a Na+ gradient (Na+-jump)
across the envelope in the dark. We re-investigated here the
effect of Na + on the active transport of pyruvate in mesophyll chloroplasts of Panicum miliaceum, a Na+-type C4
species, by comparing the rate of pyruvate uptake at various external pHs under four conditions; in the light and
dark together with/without Na+-jump: (1) At neutral pH,
the rate of pyruvate uptake in the dark was enhanced by
Na+-jump but scarcely by illumination. (2) While the enhancement effect by Na+-jump was independent of external pH, that by illumination increased greatly at pH over
7.4, and the effects of light and Na+ at the alkaline pH were
synergistic. (3) The light-enhanced pyruvate uptake was related to stromal alkalization induced by illumination. In
fact, pyruvate uptake was induced by H+-jump in the medium from pH 8.0 to 6.7. (4) Stromal pH was lowered by the
addition of K+-pyruvate and more by Na+-pyruvate into
the medium at pH 7.8 in the light. (5) However, the pH and
ATP levels in the stroma were not affected by Na+-jump.
Thus, we discussed possibility that besides pyruvate/
Na + cotransport at neutral pH in the medium, pyruvate/
H + cotransport enhanced by the presence of Na + operates
in mesophyll chloroplasts of Na+-type C4 species at alkaline medium.
Key words: Active pyruvate transport — C4 photosynthesis
— Mesophyll chloroplasts — Panicum miliaceum L. —
Pyruvate/H + cotransport — Pyruvate/Na + cotransport.
The operation of C4 dicarboxylic acid pathway requires both intercellular and intracellular transports of metabolites. Particularly, pyruvate uptake into mesophyll
chloroplasts is an essential process in all subtypes of C4
plants. Huber and Edwards (1977) first showed the exAbbreviations: CAM, crassulacean acid metabolism; DMO,
5,5-dimethyloxazolidine-2,4-dione.
1
Present address: Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS),
Ministry of Agriculture, Forestry and Fisheries, 2-1 Ohwashi,
Tsukuba, 305 Japan.
istence of a pyruvate carrier in the envelope of mesophyll
chloroplasts of a C4 plant, Digitaria sanguinalis. They
measured pyruvate uptake only in the dark, and suggested
that major mode of pyruvate uptake would be the pyruvate
anion uniport. Thereafter, light-dependent active pyruvate
uptake into C4 mesophyll chloroplasts was demonstrated in
Zea mays (Fliigge et al. 1985) and Panicum miliaceum
(Ohnishi and Kanai 1987a). This activity was demonstrated
in mesophyll chloroplasts of all the C4 species tested (Aoki
et al. 1992) and of CAM-induced Mesembryanthemum crystallinum (Kore-eda et al. 1996), but not in bundle sheath
chloroplasts of P. miliaceum nor in C3 chloroplasts (Ohnishi
and Kanai 1987a).
As to a substitute for the light effect of the active pyruvate uptake, it has been shown to date that a cation (Na +
or H + ) gradient formed across the envelope (Na + - or H + jump) enhances pyruvate uptake in the dark (Ohnishi and
Kanai 1987c, 1990). C4 species thus far tested could be divided into two groups, Na+-type and H + -type, due to the
cation specificity (Aoki et al. 1992).
For the mechanism of the active pyruvate uptake into
C4 mesophyll chloroplasts in the light, it was suggested that
driving force of the active pyruvate uptake was H + gradient formed by illumination between cytosol and chloroplast stroma across the envelope (Ohnishi and Kanai
1987b). In H + -type C4 species such as maize, in fact, pyruvate is cotransported with H + in the ratio of one only in the
light (Aoki et al. 1994). The H + gradient in the light is maintained by H + export accompanied with transport of phosphoeholpyruvate which is made from pyruvate by pyruvate
Pi dikinase in the chloroplast (Aoki and Kanai 1995).
On the other hand, in Na+-type C4 species such as
P. miliaceum, it has been thought that the driving force for
light-dependent pyruvate uptake is a Na + gradient formed
across the envelope by illumination because cotransport of
pyruvate and Na + was shown in mesophyll chloroplasts of
P. miliaceum (Ohnishi et al. 1990). However, no conclusive
evidence has been obtained for the formation and maintenance of Na + gradient across the envelope in the light.
In this report, we made further extension of the previous investigations, in order to obtain more insight for the
mechanism of light-dependent active transport of pyruvate
in the mesophyll chloroplasts of Na + -type C4 plants.
1217
1218
Pyruvate transport in C4 mesophyll chloroplasts
Materials and Methods
Plant materials—Seeds of Zea mays L. and Panicum miliaceum L. were sown in soil and grown in a growth chamber (Koito
3S-1O3A) under natural light supplemented with incandescent
lamps, in which night/day temperatures were maintained at 25/
30°C. Young expanding leaves (2 to 3 weeks after sowing) were
used for experiments.
Isolation of mesophyll chloroplasts from leaves of Ct plants
—Mesophyll protoplasts were obtained by enzymatic digestion of
C4 leaves and suspended in a medium containing 0.3S M sorbitol,
2 mM EDTA and 50 mM HEPES-KOH (pH 7.0). Mesophyll chloroplasts were isolated from the protoplasts according to Aoki et
al. (1994). Intact chloroplasts of P. miliaceum were resuspended
in the suspending medium adjusted pH at 7.8. The suspending medium still contained 50 to 150 pM Na + ions, measured by flame
photometry, in spite of using chemicals of minimum impurities
(Ohnishi et al. 1990).
Chlorophyll content was determined by the method of Wintermans and De Mots (1965).
Determination of pyruvate uptake and stromal pH—[l*C]Pyruvate uptake into mesophyll chloroplasts was determined by a
silicone oil filtering centrifugation method as described by Heldt
(1980). Two types of the system were employed: one is a single
layer system for incubation times of 7 s or longer (Heldt 1980,
Ohnishi and Kanai 1987a), and the other is a double layer system
for short incubation time (Howitz and McCarty 1985, Ohnishi
and Kanai 1990). Figure 1 summarizes the reaction system of the
two silicone oil centrifugation methods. The incubation was done
on ice. Na + - (or H + -) jump in the single layer system was accomplished by adding a small volume of 0.5 M NaCl (or 0.2 M
HC1) together with [14C]pyruvate and [3H]sorbitol to the chloroplast layer. In the double layer system, NaCl was added to the uptake layer for Na + -jump. Reaction time in the double layer system
was estimated to be 1 s, previously (Aoki et al. 1994). However,
re-estimation of the reaction time according to Gross et al. (1990)
for the present study proved to be 2 s, probably due to declining
power of the centrifuge apparatus (Beckman Microfuge B).
Stromal pH was calculated from the uptake of [UC]DMO
into the [3H]sorbitol-impermeable space, measured by the single
layer system of silicone oil centrifugation (Heldt 1980).
NadfarNa*-jump S j
orHCtfbrH'itsnp
/
(14qpyiuva»
and (3H]«ort«o(
4 200pi or 100fil- •
Chloroplast
Layer
.30 pi of Silicone oil
(p = 0.96)
-100 pi of Uptake Layer
rr
70plof
/>
oontsininQ
["qpyruvats and [3H]9orbitot
plus NaCl tor Na'-jump
Silicone oil— %,
^ — SHI
2
(P .1.04)
20 pi of
MHCIO4~
Double Layer
Single Layer
System
System
Fig. 1 Two silicone oil filtering centrifugation methods for the
measurement of pyruvate uptake into mesophyll chloroplasts.
Determination of A TP content—Chloroplasts precipitated
by the single layer silicone oil centrifugation were used for the
determination of ATP. The precipitate in HC1O4 layer was neutralized with 0.1 M KOH, centrifuged and the supernatant was
diluted to 10 times with distilled water. ATP content in the samples was determined by a luciferin-luciferase method (Strehler
1974, Kobayashi et al. 1979) using an ATP photometer (Monolight 401, Analytical Luminescence Laboratory Inc.).
Radiochemicals—Sodium salt of [l-14C]pyruvic acid was obtained from Du Pont. To remove the Na + ion, [l4C]pyruvate in
distilled water (300 pi) was mixed with 15 mg of a cation exchange
resin in H + form (AR 50W-X8, 200-400 mesh, Bio-Rad) and centrifuged. The supernatant was used for the assay.
[2-14C]DMO and D-[l-3H]sorbitol were obtained from American Radiolabeled Chemicals Inc. (ARC). D-[U-'4C]Sorbitol and
3
H2O for the measurement of sorbitol-impermeable space were obtained from ARC and New England Nuclear, respectively.
Results
Difference between Na+-type and H^-type C4 species
in the pyruvate-uptake rate and stromal pH of the mesophyll chloroplasts in the medium of pH from 7.0 to 8.6—
Figure 2A-1 and 2B-1 compare the effects of external pH on
the rate of pyruvate uptake into the isolated mesophyll chloroplasts of P. miliaceum and Z. mays, respectively.
In Z. mays (Fig. 2B), a H + -type C4 species, pyruvate
uptake was induced by illumination, but not by Na + -jump
of 10 mM both in the light and dark (Fig. 2B-1). Light-dependent activity was highest at pH 7.0, declined with increasing external pH and the least activity was detectable at
pH 8.6. These results confirm that Na + is not effective for
pyruvate transport in mesophyll chloroplasts of H + -type
C« species (Ohnishi and Kanai 1990, Aoki et al. 1992). The
pH dependency of light-dependent pyruvate uptake corresponds quite well to the pH difference between the stroma and the external medium [-dpH(In—Out)] in the light,
as well as the light-induced pH change in the stroma
U4pH in (L-D)] (Fig. 2B-3). The results indicate that H + gradient formed in the light is the driving force of pyruvate
transport in the H + -type C4 mesophyll chloroplast.
In P. miliaceum (Fig. 2A), a Na + -type C4 species, the
rate of pyruvate uptake in the light was greatly increased at
pH from 7.4 to 8.6 (Fig. 2A-1), as reported by Ohnishi and
Kanai (1987a), whereas pyruvate uptake induced by Na + jump in the dark was shown to be independent of external
pH from 7.0 to 8.6 (Fig. 2A-1) in the present study. The
light-dependent activities were further enhanced by concomitant Na + - jump in all pH ranges (Fig.2A-l).
Compared with the results of maize, stromal pH in the
mesophyll chloroplasts of P. miliaceum was much lower
than the external pH in the dark (Fig. 2A-2); therefore, the
JpH(In—Out) reached to a value lower than — 1 , when
mesophyll chloroplasts were suspended in the medium at
alkaline pH in the dark (Fig. 2A-3). This may suggest that
mesophyll chloroplasts of P. miliaceum retain H + due to
low permeability of the envelope against H + .
Pyruvate transport in C4 mesophyll chloroplasts
A. Panicum miliacaum (Na»-type)
1219
B. Zaa mays (H*-type)
Exp.1
Bcp.2
Exp. 3
Bcp.4
40
Iu
I-
&p. 5
•
20
0
X
&
•
+
A
•
o
•
a
Oar*
Uatt
Na»+mn Control
••
•
4
I
A •*
K°X
O
o
y./
J/
y
-
a
1
a
-
•
7.0
6.0
.
a
o
•+^
•
•
10
/
8.0
9.0
Stromal pH
Fig. 3 The relationship between stromal pH and the rates of pyruvate uptake induced by illumination or Na + -jump in the dark in
mesophyll chloroplasts of P. miliaceum. Data from Figure 2A-1
(Exp. 1) and the other four experiments.
8.0
External pH
9.0
Fig. 2 Changes in the rate of pyruvate uptake and stromal pH in
mesophyll chloroplasts of P. miliaceum and Z. mays. Mesophyll
chloroplasts were suspended in the medium of various pH.
HEPES-buffer was used for pH 7.0, 7.4 and 7.8, and Bicine-buffer
for pH 7.8, 8.2 and 8.6. The rate of pyruvate uptake was measured under four conditions; in the light (c and d) and dark (a and
b) together with (b and d)/without (a and c) Na + -jump. pH difference between the medium and stroma [JpH (In minus Out)] and
change in stromal pH by illumination [JpH in (Light minus Dark)]
were calculated from the measurements of stromal pH in the
absence of Na + . NaCl of 10 mM at final concentration was contained in the uptake layer in the Na + -jump experiment.
As the rate of light-induced pyruvate uptake increased
roughly in parallel with <dpHin(L — D), but not ^lpH(In —
Out) in the light (Fig.2A), all results obtained from five
independent experiments were summarized in Figure 3. It is
clear that high light-dependent activities are induced when
stromal pH reaches 7.8 or more, while the uptake activity
caused by Na + -jump is not affected by stromal pH (Fig. 3)
and external pH (Fig. 2A-1).
Figure 4 shows an analysis of the rates of pyruvate uptake from the data in Figure 2A-1 by subtracting the dark
activity (Curve a in Fig. 2A-1) from the others (Curves b, c
and d). The 'Na + -jump' effect was independent of external
pH from 7.0 to 8.6, while the 'Light' enhancement became
remarkable at pH 7.8 or more. Noteworthy is the fact that
the combined effect of 'Na + -jump plus Light' was larger
than the sum of 'Na + -jump' and 'Light' effects at alkaline
pH from 7.4 to 8.2. The synergistic phenomena were seen
in all five independent experiments, although a preliminary
result had been obtained by Ohnishi and Kanai (1987c).
These results indicate that illumination and Na + -jump
have a synergistic effect on the rate of pyruvate uptake in
the alkaline pH range.
Effect of exogenous Na+ on the rate of pyruvate uptake into mesophyll chloroplasts of P. miliaceum—Figure
Hi^•Na'-jump' sftoct
40
a
•
UgMVNa •Hump'/
o«oca(o • « ) > v
1/
/I
I-
- • 1
I 10
a.
%
S
&
o
0
7.0
J
Ugfif Affect
.-—«
°
'Na*Hump'effea
•
•
.
8.0
9.0
External pH
Fig. 4 Analysis of pyruvate uptake into mesophyll chloroplasts
of P. miliaceum. From the data in Figure 2A-1 (a to d), pyruvate
uptake activity in the dark (a) was subtracted from the others (b, c
and d). Asterisks indicate sum of values of 'Light' and 'Na + jump' effects.
1220
Pyruvate transport in C4 mesophyll chloroplasts
so
B. pH 7.8
A. pH 7.0
SO
40
30
30
Ugnt Na'-jump
i *>
20
--^-^
o
S.
10
Dart, Na'+jmp
'
10
. Ugnt, In ma p r a a n o al Na •
y/.Danc. In ma presenca o( Na •
flr—J—fc
i—A—*
A4
5
10
NaCI (inlkl)
5
10
NaCI (mM)
Fig. 5 Effect of exogenous Na + concentration on the rate of pyruvate uptake by mesophyll chloroplasts of P. miliaceum. The rate of
pyruvate uptake was measured by the double layer system both in the dark (closed symbols) and in the light (open symbols) at external
pH of 7.0 (A) or 7.8 (B). In Na + -jump experiments (circles), Na + was added to the uptake layer. In experiments in the presence of Na +
(triangles), N a + was added to both the uptake layer and the chloroplast layer about 10 min before centrifugation. For details, see
Materials and Methods and Figure 1.
5 shows the dependency of exogenously added Na + concentration on the rate of pyruvate uptake at external pH of 7.0
or 7.8, measured by the double layer system. In the experiments of Na + -jump, NaCI was added only in the layer containing radioisotopes (the uptake layer) while in the experiments in the presence of Na + , NaCI was contained both in
the uptake layer and in the chloroplast layer (see the double
layer system in Fig. 1) and preincubated for about 10 min
before centrifugation. Pyruvate uptake in the dark was
very slow by the preincubation with NaCI (closed triangles)
at pH 7.0 (Fig. 5A) and at pH 7.8 (Fig. 5B), while the activity was induced by Na + -jump (closed circles). At pH 7.8,
pyruvate uptake was remarkably enhanced by illumination
in the experiments either in Na + -jump or in the presence of
Na + (open symbols in Fig. 5B), although a very little effect
of light was observed at pH 7.0 (open symbols in Fig. 5A).
The enhancement effects induced by Na + -jump and/or illumination showed similar pattern against the NaCI concentration, and the rate reached to maximum at about 5 mM.
At pH 7.8, it is clear that the synergistic effect of light and
Na + is obtained not only by Na + -jump but also by preincubation with Na + , although the effect was observed without exogenous addition of Na + in the light (open squares in
Fig. 5B). Endogenous Na + in the medium may contribute
considerably because the chloroplast suspension from
P. miliaceum usually contained 100 to 200 fiM Na + (Ohnishi
et al. 1990).
Changes in the capacity of pyruvate uptake after pre-illumination or Na+-jump in the mesophyll chloroplasts of
P. miliaceum—Figure 6A shows time courses of the decay
of the light-induced activity of the pyruvate uptake into
mesophyll chloroplasts of P. miliaceum in the medium of
pH 7.8 after 10 min preillumination. Similar to the previous report (Ohnishi and Kanai 1987b), the rate of pyruvate
uptake in the dark declined to about 40% within 30 s, and
the uptake capacity formed by preillumination disappeared
after 2 min (Fig. 6A). This decay pattern of the capacity
was not affected by the presence of 10 mM NaCI which
were added to both the chloroplast layer and the uptake
layer, although the capacity was remarkably enhanced by
Na + .
Using mesophyll chloroplasts isolated from the same
protoplast preparation, the result from preillumination experiment was compared with those from a pre-Na + -jump
experiment. The decay of the uptake capacity after preNa + -jump was very fast; much faster than that after preillumination. Pyruvate uptake due to Na + -jump disappeared
completely within 30 s in the dark and light (crosses in
Fig.6B). In consistent with the experiment in the presence
of Na + in Figure 5B, when 10 mM NaCI were also added to
the uptake layer, the pyruvate uptake activity decreased
only 40% in 30 s after pre-Na + -jump in the dark and did
not decline in the light (triangles in Fig. 6B). Thus, the
decay of the capacity for pyruvate uptake in the presence
of Na + after pre-Na + -jump in the dark (closed triangles in
Fig. 6B) was equal to or even slower than that after preillumination (Fig. 6A). The results may indicate that Na + gradient formed across the envelope would not be the sole driving force for light-dependent active uptake of pyruvate at
pH7.8.
Effect of H+-jump on pyruvate uptake into mesophyll
chloroplasts of P. miliaceum—In the previous studies
Pyruvate transport in C4 mesophyll chloroplasts
A. Pre-illumination
1221
B. Pre-Na* -jump
50
COO)
'ft
o
r
40
«
30
I
40
30
*
A
"
II
>
UgnttNaa in me Uptake Layer
I
-
• (18)1
(100)
T
(100)*
1 *>
20
-
)
* 10 mM NaCl
Oark^NaO In the Uptake Layer
(34)
8 (0). 10
(20)
^irr—8(0)
y
Da*
,
n
0
60
120
600
0
' Tims In the dark (sac)
,
||
•
60
120
600
Time after NaCI-addltlon
to the Chloroplast Layer (sec)
Fig. 6 Decays of the rate of pyruvate uptake after preillumination and pre-Na + -jump in mesophyll chloroplasts of P. miliaceum. All
experiments were done at the external pH of 7.8. In the experiment of preillumination (A), mesophyll chloroplasts placed in a tube with
the double layer system were preincubated for 10 min in the light. After giving darkness for the time indicated on the axis of abscissa, the
rates of pyruvate uptake were determined in the absence (o) or the presence (®) of 10 mM Na + . In the experiment of pre-Na+-jump (B),
NaCl (10 mM at final concentration) was added to the chloroplast layer of the double layer system at zero time in the dark (x and A) or in
the light (+ and A). After the incubation with time indicated, the uptake activities were measured in the absence (x and +) or the presence (A and A) of Na + in the uptake layer. Numbers in parentheses indicate the relative activities.
(Ohnishi and Kanai 1990, Aoki et al. 1992), dark H + -jump
(lowering the external pH from 7.8 to 6.8) in the mesophyll
chloroplasts from Na + -type C4 species did not induce any
pyruvate uptake. However, as shown in Figure 2A-2,
stromal pH of the mesophyll chloroplasts of P. miliaceum
was about 7.0 in the dark when the external pH was 7.8.
Thus, lowering external pH from 7.8 to 6.8 would not
achieve enough H + gradient across the envelope in meso-
phyll chloroplasts of P. miliaceum. To obtain large H + gradient across the envelope of P. miliaceum in the dark, we investigated the effect of H + -jump from pH 8.0 to pH 6.7 on
pyruvate uptake activities and actual changes in stromal
pH of the chloroplasts. Table 1 shows the effects of light
with/without Na + or H + -jump on pyruvate uptake for 10 s
into mesophyll chloroplasts of P. miliaceum, measured by
the single layer system.
Table 1 H + -jump experiments in mesophyll chloroplasts of P. miliaceum, a Na + -type C4
species
[pyrL"
(mM)
Stromal pH
(In-Out)
Dark
Control
Na + -jump (10 mM)
H + -jump (pH 8.0 to 7.4)
H + -jump (pH 8.0 to 6.7)
0.09±0.01
0.85 ±0.02
0.12±0.01
0.27 ±0.02
7.20±0.01
7.16±0.03
7.14±0.01
6.99±0.02
-0.8
-0.8
-0.3
+ 0.3
Light
Control
Na + -jump (10 mM)
H + -jump (pH 8.0 to 7.4) •
H + -jump (pH 8.0 to 6.7)
1.08 ±0.02
3.13±O.O8
0.83 ±0.02
0.73±0.04
8.32±0.01
8.28±0
8.01±0
7.66±0
+0.3
+0.3
+0.6
+ 1.0
Mesophyll chloroplasts were suspended in a medium containing 5 mM HEPES-buffer (pH 8.0) and
their pyruvate uptake for 10 s was measured by the single layer system. Pyruvate added to the chloroplasts was 1.0 mM at final concentration. Na + - or H + -jump was achieved by adding 1 M NaCl or 0.2
M HC1, respectively, to the chloroplast layer (see Fig. 1).
" Pyruvate concentration in stroma space of the chloroplasts.
1222
Pyruvate transport in Ct mesophyll chloroplasts
When pH of the medium was lowered from 8.0 to 7.4
in the dark, the pH difference between stroma and external
medium [zlpH(In — Out)] was —0.3 and no pyruvate uptake was induced. Lowering external pH from 8.0 to 6.7,
by which 4pH(In — Out) resulted in +0.3, induced some
pyruvate uptake in the dark, although the activity was
much lower than that induced by illumination or Na + jump. Pyruvate uptake into mesophyll chloroplasts was
also observed when the H + -jumps were performed in the
light (Table 1), although it was less than light effect. These
results may suggest that besides the Na + gradient, a H + gradient formed across the envelope [ 4 p H ( I n - O u t ) ^ + 0 . 3 ]
contributes in some extent to the pyruvate uptake in the
alkaline pH range of the medium.
Effect of Na* -jump on stromal pH of mesophyll chloroplasts of P. miliaceum—In mesophyll chloroplasts of H + type C4 species such as maize, pH of the stroma decreased
by 0.25 to 0.3 units within 15 s after the addition of K + -salt
of pyruvate, due to pyruvate/H + cotransport (Aoki et al.
1994, 1995).
Figure 7 shows the changes of stromal pH when Na + or K + -salt of pyruvic or gluconic acid (each 5 mM at final
concentration) were added to the mesophyll chloroplasts
of P. miliaceum in the medium of pH 7.8. No changes in
stromal pH occurred either in the light (Fig. 7A) or in the
dark (Fig. 7B) by adding Na + - or K+-salt of gluconic acid,
which is known to have low permeability to the chloroplast
envelope. Similar results were obtained by using NaCl or
KC1, instead of gluconates (data not shown). When K + pyruvate was added to the mesophyll chloroplasts in the
light, stromal pH decreased by about 0.2 units within 15 s
and thereafter recovered slowly (Fig. 7A). This stromal
acidification was less in the dark (Fig. 7B) and in the light
and dark at pH 7.0 (data not shown). The result indicates
that H + influx due to pyruvate uptake occurs even in mesophyll chloroplasts of Na + -type C4 species such as P. miliaceum, as shown in those of H + -type C4 species (Aoki et al.
A. Ugh!
8.1
Before addHtao
O
O
8.0
OsIHadnalec
Na*-glucona!e
•\v
\
7.9
7.8
^_______K'^iymvau
'
^
V—-
7.7
a Dark
7.1
^
+
Na+^iynjvata
1.0
K'-pynivae
&
0
20
40
60
Incubation time (sec)
Fig. 7 Changes in stromal pH on addition of Na + - and K+-salt
of pyruvic or gluconic acid to mesophyll chloroplasts of P. miliaceum in the medium of pH 7.8. Mesophyll chloroplasts (200/il)
were preincubated with [I4C]DMO and [3H]sorbitol for 5 min in
the light (A) or in the dark (B). Then, 2 pi of 0.5 M pyruvates, 0.5
M gluconates or distilled water were added at zero time and centrifuged at the time indicated.
1994). Furthermore, this pyruvate-induced stromal acidification in mesophyll chloroplasts of P. miliaceum was
enlarged by using Na + -salt, instead of K + -salt, at pH 7.8
only in the light (Fig. 7A).
Effects of illumination and Na*-jump on ATP content
in mesophyll chloroplasts from P. miliaceum—Change in
ATP content by illumination or Na + -jump was investigated to check possibilities whether Na + release from meso-
Table 2 Effect of illumination and Na + -jump on the ATP level in mesophyll chloroplasts of P. miliaceum
ATP content [nmol (mg Chi)"1]
Condition
Exp. 1
Exp. 2
Exp. 3
pH7.0
pH7.8
pH8.6
pH7.8
pH7.8, +5mMMgCl 2
pH7.8
Light
Dark
Control
Na -jump"
Control
Na + -jump"
4.8
4.5
4.8
4.6
4.5
28.6
25.0
24.7
n.d.
n.d.
n.d.
4.5
+
5.2
5.2
4.8
4.7
20.9
20.2
n.d.
n.d.
3.9
3.9
21.2
19.8
The precipitate of mesophyll chloroplasts after silicone oil centrifugation was used for the assay of ATP.
" 10 mM NaCl at final concentration was added,
n.d.; not determined.
Pyruvate transport in C4 mesophyll chloroplasts
phyll chloroplasts could be driven by ATP formed in the
stroma by illumination or Na + addition to the medium in
the dark (Na + -jump); the latter might induce ATP synthesis by a mechanism of Na + pump found in some bacteria
(Heefner and Harold 1980a, b). In fact, ATP content in the
mesophyll chloroplasts of P. miliaceum was increased from
about 5 to 20nmol (mgChl)" 1 by illumination (Table 2),
which is comparable to spinach chloroplasts (from about
15 to 40nmol (mgChir 1 , Kobayashi et al. 1979). However, Na + -jump in the dark did not alter the ATP level in
mesophyll chloroplasts of P. miliaceum at the external pH
of 7.0, 7.8 and 8.6 (Table 2). Similar results were obtained,
in the light or in the presence of MgCl2 which was generally
required for the reaction of ATP synthesis (Table 2).
Discussion
Light-dependent pyruvate uptake into C4 mesophyll
chloroplasts is correlated with stromal alkalization by illumination—By the comparison of pyruvate uptake capacity
formed by preillumination and light-dependent changes of
stromal pH as well as ATP content in mesophyll chloroplasts from P. miliaceum, Ohnishi and Kanai (1987b) concluded that the active pyruvate uptake in the light is primarily driven by the H + gradient across the envelope, but
not by ATP in the stroma. In mesophyll chloroplasts of
H + -type C4 species such as maize, in fact, stromal pH was
shifted from 7 to 8 by illumination at pH 7.0 of the medium (Fig. 2B-2). The light-dependent pyruvate uptake
was decreased by increasing external pH in parallel with
stromal alkalization by illumination [ApHia(L — D)] and
H + gradient across the envelope [^lpH(In —Out)] in the
light. Therefore, H + gradient formed across the envelope
by illumination is the sole driving force of active pyruvate
uptake (pyruvate/H + cotransport) into mesophyll chloroplasts of H + -type C4 species.
In mesophyll chloroplasts of P. miliaceum, on the
contrary, the change in the activity of light-dependent
pyruvate uptake under various pHs was more related
with 4 p H i n ( L - D ) , but not with-dpH(In-Out) in the light
(Fig. 2A). Furthermore, a high activity of pyruvate uptake
was achieved only when stromal pH was alkalized to about
7.8 or more in the light (Fig. 3). These results suggest that
light-dependent pyruvate uptake into mesophyll chloroplasts of Na + -type C4 species is induced mainly by stromal
alkalization due to the photosynthetic electron transport activity of the thylakoid, but less by H + gradient formed
across the envelope in the light.
Is Na+ essential for the light-dependent pyruvate uptake into mesophyll chloroplasts of Na^-type C4 species?—
In Na + -type C4 species such as P. miliaceum, it has been
considered that the active pyruvate uptake into the mesophyll chloroplasts in the light is due to a Na + gradient formed across the envelope, because light-dependent pyruvate
1223
uptake is mimicked by Na + -jump in the dark (Ohnishi and
Kanai 1987c) and pyruvate is cotransported with Na + in
the ratio of one (Ohnishi et al. 1990). By use of ^Na4", they
also showed that Na + level in the mesophyll chloroplasts of
P. miliaceum was declined in some extent by illumination,
although the lowered level was still higher than that in external medium (cf. Figure 2 in Ohnishi et al. 1990). As shown
in Figure 5, pyruvate-uptake activities were dependent on
Na + concentration and remarkably enhanced by illumination of the chloroplasts in the medium of pH 7.8, while
only Na + -jump was effective at external pH of 7.0. Thus,
the presence of Na + is essential for light-dependent pyruvate uptake in alkaline medium as well as a Na + gradient,
if any, formed by illumination across the chloroplast envelope.
Possible mechanisms for the formation of Na+ gradient across the envelope of mesophyll chloroplasts in the
light—Figure 2A-2 and Table 2 show that stromal pH and
the amount of ATP in mesophyll chloroplasts of P. miliaceum were increased by illumination and that stromal
alkalization by illumination is related to light-dependent pyruvate uptake. These will be candidates for the formation
of Na + gradient.
On the other hand, to form Na + gradient across the
envelope of the mesophyll chloroplasts, the stromal Na +
should be either released to the cytosol or taken up into the
thylakoid lumen. However, the latter cannot explain continuous maintenance of the Na + gradient across the envelope
during photosynthesis in vivo. Furthermore, Hind et al.
(1974) reported that the distribution of Na + is essentially
unaffected by illumination in isolated thylakoid from spinach, a C3 plant, using a Na + electrode.
A Na + transport system in the envelope is requisite
for the former mechanism. N a + / H + antiporters are documented in membranes of various organisms, which uses
electrochemical potential of H + (4^H + ) for the driving
force in exchange of Na + with H + (Padan and Schuldiner
1994). If such a N a + / H + antiporter is present in the envelope of mesophyll chloroplasts of Na + -type C4 species,
<dpH between stroma and cytosol would be a candidate to
supply driving energy for Na + export. However, it is unlikely that a N a + / H + antiporter plays a major role in the formation of Na + gradient across the envelope for light-dependent active pyruvate uptake in mesophyll chloroplasts
of P. miliaceum, a Na + -type C4 species, since the following
results were obtained in this study (Fig. 2A): (i) The external pH dependency of light-dependent pyruvate uptake
was not correspondent with that of ApH(ln — Out) in the
light, (ii) The pyruvate uptake activity caused by Na + -jump
in the dark was totally independent of external pH. (iii) In
mesophyll chloroplasts of P. miliaceum, the maximum
value of ^lpH(In —Out) in the light was only about 0.5
units in the range of external pH from 7.0 to 8.6, while it
was more than 1.0 unit at pH 7.0 in maize (Fig. 2B). (iv) No
1224
Pyruvate transport in C4 mesophyll chloroplasts
H + release from the stroma was observed on addition of
Na + -gluconate at pH 7.8 (Fig. 7).
Nevertheless, some role of N a + / H + antiporter would
not be negated, since H + -jump induced some pyruvate uptake into mesophyll chloroplasts of P. miliaceum in the
light (Table 1). In addition, the membrane potential of the
envelope would be another candidate to drive pyruvate uptake. Actually, Ohnishi and Kanai (1987b) reported that pyruvate uptake into mesophyll chloroplasts of P. miliaceum
was partly inhibited by 5 mM tetraphenylphosphonium,
which reduce the membrane potential. This may suggest
some contribution of the membrane potential to the formation of Na + gradient across the envelope.
Secondly, as light-dependent pyruvate uptake in mesophyll chloroplasts of P. miliaceum was remarkably enhanced in alkaline medium (Fig. 2A-1), it was expected
that Na + might be released across the envelope by a Na + translocating adenosine-5'-triphosphatase (Na + -ATPase),
which is present in the plasma membrane of an enterobacterium, Enterococcus hirae {Streptococcus faecalis, Heefner
and Harold 1980a, b). This organism has both N a + / H +
antiporter and Na + -ATPase. Both systems have been implied in Na + excretion from the bacterium; the N a + / H +
antiporter works in the culture medium from acidic to neutral pH where AfM + is sufficient, whereas the Na + -ATPase
works at alkaline pH where AfiH+ is limiting. So far, the artificial Na + gradient formed across the envelope did not induce ATP synthesis in mesophyll chloroplasts of P. miliaceum (Table 2); the result would negate participation of a
Na + -ATPase in mesophyll chloroplasts of Na + -type C4 species. Still remaining to study is the contribution of other
ATPases, for example, Na + /K + -ATPase which is present
in the plasma membrane of various animal cells, although
pyruvate uptake into mesophyll chloroplasts of P. miliaceum was not enhanced by the addition of K + together
with pyruvate in the dark (Ohnishi and Kanai 1987c).
Pyruvate/H+ cotransport in mesophyll chloroplasts
of Na*-type C4 species at alkaline pH in the light—We previously reported that, in mesophyll chloroplasts of all Na + type C4 species tested, pyruvate uptake in the light was not
accompanied with pH change in the medium of pH 7.0
(Aoki et al. 1994). However, results in Figure 7 may indicate
that H + and pyruvate cotransport into mesophyll chloroplasts of P. miliaceum at pH 7.8 in the light. In fact, a H +
gradient which is made across the envelope by H + -jump
induced some pyruvate uptake into mesophyll chloroplasts
of P. miliaceum in the light and dark (Table 1), although
the amount of pyruvate uptake was not proportional with
,dpH(In — Out). Therefore, in an alkaline pH range, lightdependent active uptake of pyruvate into mesophyll chloroplasts of Na + -type C4 species would consist of two components; one is pyruvate/Na + cotransport driven by Na +
gradient across the envelope formed by unknown mechanism^) in the light, and the other is pyruvate/H + cotrans-
port driven by H + gradient or electrochemical potential
formed across the envelope by illumination. Furthermore,
the latter could be accelerated by the presence of Na + in the
cytosol, since larger stromal acidification in the light was induced by the addition of Na+-pyruvate than K + -pyruvate
(Fig. 7). The fact may suggest a catalytic or stimulative role
of Na + in pyruvate/H + cotransport in addition to pyruvate/Na + cotransport and explain the synergistic effect of illumination and Na + at alkaline pH.
Problems remained to be studied for understanding
the mechanism of light-dependent active transport of pyruvate in mesophyll chloroplasts of Na*-type C4 species in
vivo—It is vital to know in vivo pH values of the cytosol in
mesophyll cells of Na + -type C4 species such as P. miliaceum, although it is generally thought to be nearly neutral. At the external pH of 7.0, the rate of light-dependent
pyruvate uptake in mesophyll chloroplasts of H + -type C4
plants such as maize was highest. However, the rate in
those of P. miliaceum was rather low at neutral pH of the
medium, while a high activity and a synergistic effect of
light and Na + were seen at alkaline pH (Fig. 4). This makes
difficult to implicate the physiological significance of lightdependent active pyruvate uptake at alkaline pH by mesophyll chloroplasts of Na + -type C4 species. Although pyruvate uptake was induced by Na + -jump in the light and dark
even at pH 7.0 (Fig. 2A-1 and 5A), it remains to clarify the
mechanism in relation to light-dependent formation of
Na + gradient across the envelope for driving pyruvate/
Na + cotransport. To solve this and other problems, further
experiments are required to estimate the actual change
in the stromal Na + concentration by illumination and to
know the cytosolic pH in vivo.
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(Received June 7, 1997; Accepted September 2, 1997)