Mesophyll Chloroplasts of Some C4 Plants1 - NCBI

Plant Physiol. (1990) 94, 950-959 0032-0889/90/94/0950/1 0/$01 .00/0

Received for publication March 12, 1990 Accepted June 4, 1990

Involvement of Na+ in Active Uptake of Pyruvate in Mesophyll Chloroplasts of Some C4 Plants1 Na+/Pyruvate Cotransport Jun-ichi Ohnishi*, Ulf-Ingo Flugge2, Hans W. Heldt, and Ryuzi Kanai Department of Biochemistry, Faculty of Science, Saitama University, Urawa 338, Japan (J.O., R.K.); and Institut fur Biochemie der Pflanze, Universitat Gottingen, Untere Karspule 2, 3400 Gottingen, Federal Republic of Germany (U.-/.F., H. W.H.) ABSTRACT

for pyruvate, which was predominantly found in mesophyll cells, i.e. the gradient seemed to be against the supposed flow (28). This apparent contradiction could be circumvented if pyruvate in mesophyll cells is compartmented in subcellular organelle(s) making its cytosolic concentration lower. In fact, recent findings have demonstrated light-driven active uptake of pyruvate into mesophyll chloroplasts of maize (a NADPmalic enzyme type) (6) and Panicum miliaceum (a NADmalic enzyme type) (17, 18). Huber and Edwards (12) first showed the existence of a pyruvate carrier in the envelope of mesophyll chloroplasts of Digitaria sanguinalis, a NADP-malic enzyme type C4 species. Since they studied the transport only in the dark, the internal pyruvate concentration was always less than the external one. However, pyruvate uptake in C4 mesophyll chloroplasts was enhanced by illumination resulting in an active accumulation of pyruvate as noted above. The energy source of this lightdependent uptake seemed to be the pH gradient across the envelope, because light enhancement of pyruvate uptake correlated to some extent with the light-dependent stromal alkalization (18). However, an artificial pH gradient across the envelope did not enhance pyruvate uptake in mesophyll chloroplasts of P. miliaceum. In the course of such experiments, we found that an abrupt increase of [Na+]ex3 (a Na+ jump) enhanced pyruvate uptake remarkably in the dark, partially mimicking the light effect (19). Thus, a Na+ gradient across the envelope could be an alternative energy source for the active transport of pyruvate. In this paper we have examined 22Na' and pyruvate uptake in mesophyll chloroplasts of several species from all the three subgroups of C4 plants. Further evidence is presented to support Na+/pyruvate cotransport in mesophyll chloroplasts of P. miliaceum and some other species. MATERIALS AND METHODS

An artificial Na' gradient across the envelope (Na+ jump) enhanced pyruvate uptake in the dark into mesophyll chloroplasts of a C4 plant, Panicum miliaceum (NAD-malic enzyme type) (J Ohnishi, R Kanai [1987] FEBS Lett 219: 347). In the present study, 22Na and pyruvate uptake were examined in mesophyll chloroplasts of several species of C4 plants. Enhancement of pyruvate uptake by a Na+ jump in the dark was also seen in mesophyll chloroplasts of Urochloa panicoides and Panicum maximum (phosphoenolpyruvate carboxykinase types) but not in Zea mays or Sorghum bicolor (NADP-malic enzyme types). In mesophyll chloroplasts of P. miliaceum and P. maximum, pyruvate in tum enhanced Na+ uptake in the dark when added together with Na+. When flux of endogenous Na+ was measured in these mesophyll chloroplasts preincubated with 22Na+, pyruvate addition induced Na+ influx, and the extent of the pyruvate-induced Na+ influx positively correlated with that of pyruvate uptake. A Na+/H+ exchange ionophore, monensin, nullified all the above mutual effects of Na+ and pyruvate in mesophyll chloroplasts of P. miliaceum, while it accelerated Na' uptake and increased equilibrium level of chloroplast 22Na+. Measurements of initial uptake rates of pyruvate and Na+ gave a stoichiometry close to 1:1. These results point to Na+/pyruvate cotransport into mesophyll chloroplasts of some C4 plants.

The operation of C4 photosynthesis requires extensive metabolite flow, both intercellularly between mesophyll and bundle sheath cells, and intracellularly across the organelle membranes (see ref. 5 for review). The intercellular flow is supposed to occur by diffusion through plasmodesmata and to be driven by concentration gradients between the two cell types. Such concentration gradients were demonstrated in vivo in maize leaves for several transport metabolites except

Chloroplast Isolation

' Supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and also by grants from the Ministry of Agriculture, Forestry and Fishery, from Itoh Science Foundation, Japan, and from Deutsche Forschungsgemeinschaft, FRG. 2 Present address: Institut fur Botanik und pharmazeutische Biologie, Universitat Wurzburg, Mittlerer Dallenbergweg 64, 8700 Wurzburg, FRG.

Mesophyll protoplasts were isolated enzymatically using 1% Sumizyme C and 0.2% Macerozyme R-10 from young 3Abbreviations: [Na+Iex and [Na+]in, Na+ concentration in the medium and chloroplasts; PEP, phosphoenolpyruvate; DMO, 5,5dimethyloxazolidine-2,4-dione; CCCP, carbonylcyanide-m-chlorophenylhydrazone; pCMS, p-chloromercuriphenylsulfonate; [Pyr]in, stromal concentration of pyruvate. 950

Na+/PYRUVATE COTRANSPORT IN C4 MESOPHYLL CHLOROPLASTS

leaves of C4 plants, Panicum miliaceum (NAD-malic enzyme type), Zea mays, Sorghum bicolor (NADP-malic enzyme type), Panicum maximum and Urochloa panicoides (PEP carboxykinase type), and intact chloroplasts obtained therefrom were purified by centrifugation through a Percoll layer (16, 17). The chloroplasts were suspended in 0.35 M sorbitol, 50 mM Hepes-KOH (pH 7.8) and 5 mM EDTA (free base or K salt). This medium normally contained 50-150 uM Na+, although the purest reagents available were used. Therefore, the supernatant of chloroplast suspension usually contained 100 to 200 juM Na+ ([Na+]ex) due to the carry over by the chloroplasts. [Na+]ex was measured in each experiment by flame photometry and indicated in the legend. Assay of Pyruvate and Na+ Uptake

Uptake of pyruvate or Na+ into the 'sorbitol-impermeable space' of chloroplasts (9) was determined at 4°C unless stated otherwise. Two types of silicone oil layer filtering centrifugation methods were employed: one is the ordinary single-layer system (9, 16, 17) for incubation times of 7 s and longer, and the other is the two-aqueous-layer system (originally developed by Howitz and McCarty (1 1) and hereafter referred to as the double-layer system) for the shortest incubation time within 2 s. In the single-layer system, incubation was started with the addition of radioactive substance (0.1 tCi/tube), 22Na+ (1 or 2 mM) or ['4C]pyruvate (0.2 mM) together with [3H]sucrose (0.2 ,Ci/tube), to 200 juL of chloroplast suspension and terminated by centrifugation for 30 s. In the doublelayer system, the tube contained from the bottom, 20 ,L of 1 M HC104, 70 ,uL of normal silicone oil (p = 1.04), 100 ,uL of uptake layer of suspending medium (containing radioisotopes and half of its sorbitol being replaced by sucrose), 30 ,uL of low density (p = 0.96) silicone oil, and 100 ,uL of chloroplast layer. The composition of the uptake layer was such that the layer is heavier than the chloroplast layer but still lighter than the lower silicone oil layer. The use of 10% Percoll as the uptake layer in the original method was abandoned, because the Percoll solution contained a very high concentration of Na+ and even repeated dialysis could not reduce the Na+ concentration below 10 mM. In this double-layer system the upper oil layer floated to the top at the start of centrifugation and the chloroplasts were centrifuged down through the uptake layer in very short time. The effective incubation time of chloroplasts with radioactive substances in the uptake layer was estimated in the original method using 10% Percoll to be between 1 and 2 s (7, 1 1). The absolute time was not further investigated in our system but would not be much different from these values. Complete medium exchange, when chloroplasts were centrifuged through the uptake layer, was ascertained according to Howitz and McCarty (1 1). The amount of ['4C]pyruvate or 22Na' in the chloroplast pellet was corrected in each tube for the contribution of the 'sorbitol space' (adhering medium plus intermembrane space of double membranes of envelope, in this case measured as sucrose space) using the count of [3H]sucrose in the pellet. This correction method gave reproducible and reliable results, so that sample duplication was unnecessary. The sorbitol space and sorbitolimpermeable space of chloroplasts from all the species ranged 20 to 25 and 25 to 30 ,uL/mg Chl, respectively.

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Uptake or flux of a y-nuclide 22Na' was estimated in two types of experiments. The first method ('uptake experiments') measured Na+ uptake by adding 5 ,lL solution of22NaCl (final concentration 1 or 2 mM) to the chloroplast suspension in the single-layer system or by inclusion of 22NaCl in the uptake layer in the double-layer system. This method should detect not only the net flux but also the isotope exchange leading to equilibration of 22Na' between inside and outside of the chloroplasts. In the second method ("preequilibration experiments"), chloroplasts were preincubated with carrier-free 22Na' for a period longer than 10 min. Then pyruvate was added or the light condition was changed to measure the net flux due to this treatment. Chloroplast Na+ concentration ([Na+]i,) shown in "Results" was calculated from the specific radioactivity of the added 22NaCl solution (in uptake experiments) or the preexisting medium Na+ (in preequilibration experiments), supposing that the 22Na' detected in the pellet (corrected for the contribution of the sorbitol space) was uniformly distributed in the internal space. This naturally leads to overestimation of stromal concentration of free Na+, when a part of the Na+ is bound to membrane or protein, or selectively sequestered into the thylakoid lumen. For counting 22Na, samples were processed as in the experiments measuring uptake of beta nuclide-labeled substrates (9) and the radioactivity in the supernatant and in an aliquot of the water extract of the pellet was determined with liquid scintillation counting with the correction for the spillover of 22Na count in the 3H window. Complete extraction of 22Na' was ascertained in a control experiment by a direct counting of the pellet and its water extract with an Aloka Autowell Gamma System ARC 300. In a few experiments, [3H]sucrose was not included, and 22Na of the pellet was directly counted with the above gamma counter and corrected for the sorbitol space from parallel incubations with 3H20 and ['4C]sorbitol. Other Assays Stromal pH was determined by measuring the distribution of the weak acid [2-'4C]DMO (9). Chl was determined according to Wintermans and De Mots (29). Materials

Radioactive substances, 22NaC1, [1-'4C]pyruvate, 3H20, [U-'4C]sorbitol, [6,6'(n)-3H]sucrose, and [2-'4C]DMO were obtained from Amersham. Monensin, CCCP, and pCMS were from Sigma, and Percoll was from Pharmacia. Sumizyme C was a kind gift from Shin-Nihon Chemical Co., Ltd. and Macerozyme R- 10 was obtained from Yakult Pharmaceutical Industry Co., Ltd. RESULTS

Correlation of Pyruvate Uptake and Na+ Uptake in C4 Mesophyll Chloroplasts In the previous report (19) we showed that the addition of Na+ together with [14C]pyruvate to the suspension of mesophyll chloroplasts of Panicum miliaceum enhanced pyruvate uptake in the dark leading to accumulation of pyruvate in the

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Table I. Effect of a Na+ Jump on Pyruvate Uptake and Stromal pH of Mesophyll Chloroplasts of C4 Plants in the Dark In pyruvate uptake measurements, chloroplast suspension (100 gL) was injected into 100 ,uL of medium containing [1-14C]pyruvate (0.2 mm final) and Na-gluconate (35 mm final) partially replacing sorbitol and incubated for 7 s. In stromal pH measurements, 50 RL of 0.175 M Na-gluconate, 5 mm EDTA, and 50 mm HEPES-KOH (pH 7.8) were injected into 150 ,L of chloroplast suspension containing [2-14C]DMO (final Na+ concentration was 44 mM). All the experiments were done at 4°C except for the one shown in parentheses, done at 200C. [Na+]0x was not measured in these experiments but is supposed to be about 200 jlM judging from the later experiments using a medium made from the same reagents. Stromal pH

Pyruvate Uptake

Species

-Na+

After Na+ addition

Before Na+ addition addition

+Na+

30 30ss

2 min 2m

nmol/mg Chl * h

7.34 7.34 7.45 3.7 6.90 7.00 7.00 4.5 ND ND ND b 1.7a o.9a ND ND ND 0.3 0.4 (7.37) (7.34) (7.25) (0.3) (0.3) 7.43 7.50 7.45 2.3 2.3 Sorghum bicolor a Determined as described in "Materials and Methods" with and without a 2 mm Na+ jump. determined. Panicum miliaceum Urochloa panicoides Panicum maximum Zea mays

0.4 1.6

stroma. This effect was specific to Na+ among various monoand divalent cations. In other words, an artificial Na+ gradient across the envelope enhanced pyruvate uptake in the dark and partially mimicked the light effect. We first investigated whether such Na+-induced pyruvate uptake could also be seen in mesophyll chloroplasts of other C4 plants. Enhancement of pyruvate uptake by simultaneous addition of Na+ was also seen in two species of PEP carboxykinase type, U. panicoides and P. maximum but not in two species of NADP-malic enzyme type, maize or sorghum (Table I). The addition of Na+ did not significantly change the stromal pH in either species (see "Discussion"). 22Na' uptake in the dark was investigated in mesophyll chloroplasts of P. miliaceum and maize, the former representing the species of positive Na+ effect and the latter those of no Na+ effect. Figure 1 shows the time courses of 22Na+ uptake at 1 mm with and without 0.2 mM pyruvate (top) and those of [14C]pyruvate uptake at 0.2 mM with and without 1 mM NaCl (bottom). Uptake of Na+ consisted of an initial fast phase and a slow phase, and pyruvate apparently enhanced the initial phase in P. miliaceum (see also Table III and the accompanying explanation in the text for shorter incubation time). Similar results were also obtained in mesophyll chloroplasts of P. maximum (data not shown). Na+ in turn enhanced pyruvate uptake in P. miliaceum as already shown in Table I and in the previous report (19). On the other hand, in maize, where no effect of Na+ on pyruvate uptake was seen, pyruvate had no effect on Na+ uptake. In a preequilibration experiment shown in Figure 2, mesophyll chloroplasts of P. miliaceum were first preincubated with a tracer amount of 22Na' in the dark for 10 min. It was confirmed in a separate experiment that equilibration was complete within 10 min (data not shown). On illumination of the chloroplasts starting at zero time, [Na+]i. decreased significantly, suggesting an efflux of Na+ and light dependent

z

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x N

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15 30 0 Incubation Time ( s)

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Figure 1. Effect of simultaneous addition of pyruvate (0.2 mM) and NaCI (1 mM) on the uptake of 22Na+ (top) and [14C]pyruvate (bottom), respectively, in mesophyll chloroplasts of Panicum miliaceum and maize in the dark. [Pyr]ji and [Na+]m, represent the calculated chloroplast concentration of transported pyruvate and Na+, respectively. The dashed line shows the external concentration of each added substrate. The initial medium concentration of Na+ ([Na+]e.) prior to substrate addition was 100 AM in P. miliaceum and 120 jM in maize. The pyruvate/Na+ ratio (see the text for the definition) in 7 s incubation in P. miliaceum was 0.6.


30 min) 0 15 Time in the Light (s) Figure 2. Changes of [Na+],n during dark to light transition and

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Neither an uncoupler CCCP nor a sulfhydryl reagent pCMS affected the mutual enhancement by pyruvate and Na+ of the uptake of each other (Fig. 5). Pyruvate uptake with and without Na+ was partially inhibited by pCMS. Partial inhibition of dark pyruvate uptake by SH reagents, pCMS and Mersalyl, was already noted in our previous report (17). Figure 6 shows the effect of CCCP and pCMS on pyruvate uptake and Na+ flux in a preequilibration experiment. Chloroplasts were preincubated with 22Na' in the light with or without inhibitors or in the dark and pyruvate was added at zero time (right). Pyruvate uptake was also measured in parallel incubations (left). CCCP and pCMS in the light inhibited both light-enhanced pyruvate uptake and accompanying Na+ influx almost to the level seen in the dark. Even in the dark small Na+ influx accompanied low residual pyruvate uptake. The results so far described suggest a mechanism of Na+/ pyruvate cotransport at the envelope of mesophyll chloroplasts of some C4 species including P. miliaceum (further experiments were done only in mesophyll chloroplasts of P. miliaceum). If this is true, it follows that when the Na+

0.4

on

addition of 0.2 mm pyruvate in mesophyll chloroplasts of P. miliaceum. [Na+]ex was 50 zlM. Chloroplasts were preincubated with a tracer amount of 22Na+ in the dark for 10 min. The insert shows the time course of [14C]pyruvate uptake in a parallel experiment. The pyruvate/ Na+ ratio in 7 s incubation was 6.8.

formation of a Na+ gradient across the envelope. This efflux was reproducible, although small. Thereafter the addition of 0.2 mM pyruvate induced Na+ influx similarly to the time course of pyruvate uptake in a parallel incubation (the insert of Fig. 2). Similar induction of Na+ influx by pyruvate was also seen in mesophyll chloroplasts of P. maximum but not

E E

in those of maize (Fig. 3). Effects of Other Anions and Cations The induction of Na+ influx was specific to pyruvate in mesophyll chloroplasts of P. miliaceum; anion substrates of other metabolite translocators of chloroplast envelope, Pi and malate, had no effect on the Na+ status in a preequilibration experiment (Fig. 4). The effects of the inclusion of divalent cations, Mg2' and Ca2+ (instead of EDTA) in the medium on pyruvate and Na+ uptake into mesophyll chloroplasts of P. miliaceum were investigated in experiments similar to those in Figures 1 and 2 (Table II). The divalent cations had practically no effects not only on light pyruvate uptake but also on the mutual enhancement by pyruvate and Na+ of the uptake of each other in the dark (Experiment A). The inclusion of the divalent cations had also no effects on pyruvate-induced Na+ flux in the light in a preequilibration experiment (B). Effects of Inhibitors Inhibitors of light-dependent pyruvate uptake (6, 17, 18) were tested for the Na+ and pyruvate uptake in the dark.

C

0

z

0.7

Incubation Time (s ) Figure 3. Changes of [Na+]i, on addition of pyruvate in mesophyll chloroplasts of maize and P. maximum in the light. The system was the same as in Figure 2. [Na+Ie. was 0.15 mm in maize and 0.5 mm in P. maximum.

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0.4 0

*

+Pyr E 0.2 c

z 6-s

i ~ ~Mal +Pi

t I -,

.-

30 Incubation Time (s)

60

Figure 4. Effect of substrate addition on [Na+]i, in mesophyll chloroplasts of P. miliaceum in the light. Chloroplasts were preincubated with a tracer amount of 22NaCI in the light and substrate, pyruvate (0.2 mM), malate (Mal, 1 mM), or Pi (1 mM), was added at zero time. [Na+]. was 110 uM.


gradient across the envelope is dissipated, no active uptake of pyruvate should occur. As expected, monensin, a Na+/H+ exchange ionophore (22), inhibited light-dependent pyruvate uptake and pyruvate-induced Na+ influx ([Na]i, increase on pyruvate addition at zero time, Fig. 7). Meanwhile, monensin increased [Na+]i. far higher than the control level (see "Discussion" for the possible cause of this increased Na+ level). However, this alone is not proof that the Na+ gradient is the driving force of pyruvate uptake, because monensin at the same time nullified light-induced stromal alkalization (data not shown) apparently via Na+ (or K+)/H+ exchange and, therefore, it is not known whether monensin inactivated pyruvate uptake directly through dissipation of the Na+ gradient or indirectly through stromal acidification. The apparently correlated inhibition of stromal alkalization and active pyruvate uptake was already noted in the experiment using CCCP (a proton ionophore) in our previous report (18). In a further uptake experiment shown in Figure 8, monensin also inhibited the effect of a Na+ jump on pyruvate uptake. Monensin not only nullified the Na+-enhancement of pyruvate uptake but also was inhibitory to the dark uptake without Na+ (bottom). It enhanced the initial fast phase of Na+ uptake but nullified pyruvate-enhancement of Na+ uptake (note the same time courses in the presence of monensin with and without pyruvate, top). Monensin seems to have collapsed not only the artificial Na+ gradient induced by a Na+ jump but also the preexisting Na+ gradient. Thus, monensin nullified the mutual effects of pyruvate and Na+ both in the light and dark and in this aspect differs from CCCP (compare Figs. 7

Table II. Effects of Divalent Cations on Pyruvate and Na+ Uptake in Mesophyll Chloroplasts of P. miliaceum Chloroplasts were initially suspended in suspending medium without EDTA, then supplemented with 1 mM MgCI2 or CaCl2 where indicated and preincubated more than 10 min. The systems in experiments A and B were the same as in Figures 1 and 2, respectively, except that 2 mM Na+ was added in A and 0.5 mM pyruvate in B. Results are the means of duplicate samples. The initial [Na+]ex was 0.2 mm. Internal Concentration after 7 s Incubation

Experiment A Substrate

Conditions

Control

+MgCI2

+CaCI2

mM

Pyruvate

Na+

Light Dark No addition Na+ jump Dark No addition +Pyruvate Difference

0.35 0.10 0.32 0.83 1.24 0.41

1.31 0.41

0.44

0.10 0.35 1.09 1.56 0.47

[Na+]1,

Experiment B Condition

0.40 0.08 0.35 0.90

Time after pyruvate addition

Control

+MgCI2

0s 7s

0.30 1.1 1.2

0.26 1.1 1.3

+CaCI2

mM

Light

30 s

0.31 0.90 1.5


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c

z

Figure 5. Effect of CCCP and pCMS on pyruvate (bottom) and Na+ (top) uptake in the dark -

in mesophyll chloroplasts of P. miliaceum. The experimental system was the same as in Fig. 1, except that chloroplasts were preincubated with or without the inhibitors. The initial [Na+]x was 80 AM. The pyruvate/Na+ ratios in 7 s incubations were 1.1 (control), 1.5 (+CCCP) and 1.3

,-.,

c

0-..

(+pCMS).

Incubation Time

(s)

and 8 with Figs. 5 and 6). The effects of monensin strongly suggest that pyruvate uptake is performed by the mechanism of cotransport with Na+.

Stoichiometry of Na+ and Pyruvate The double-layer system (effective incubation time less than 2 s as noted in "Materials and Methods") was adopted to measure the initial rate of pyruvate and Na+ uptake in the light and dark (Table III). As noted in "Materials and Methods," the uptake of exogenous Na+ in uptake experiments should contain two components, net flux and isotope exchange. The net Na+ flux accompanying pyruvate uptake should be the difference of Na+ uptake with and without pyruvate. This Na+ flux is to be compared with the pyruvate uptake with Na+. In preliminary experiments, chloroplasts were preincubated with the same concentration and the same specific radioactivity of 22Na+ as in the uptake layer. The difference of Na+ uptake with and without pyruvate, which is here supposed to be the net Na+ flux accompanying pyruvate uptake, was the same with and without preincubation (data not shown), which justifies the above supposition. As shown in Table III, light enhanced both pyruvate uptake and the accompanying Na+ uptake. Inclusion of extra Na+ in the uptake layer (Na+ jump, series B) also enhanced both. The effects of light and a Na+ jump on pyruvate uptake were additive as already noted in our previous paper (19). Most results are consistent with a 1:1 stoichiometry of pyruvate and Na+, except for a few cases where the standard deviation is too large or uptake is very small. The results in Table III also supplement and support those in Figure 1, where the initial rise curves were in a sense arbitrarily drawn. The dark uptake of Na+ with and without pyruvate in Figure 1 correspond to that in series B (+Pyr and -Pyr), respectively, and dark pyruvate uptake with and without Na+ to that in series B and A, respectively.

[Pyr]in

[Na]lin

Pyr Incubation Time

(S)

Figure 6. Effect of light, 10 Mm CCCP and 0.4 mM pCMS on pyruvate uptake (left) and pyruvate-induced Na+ flux (right) in mesophyll chloroplasts of P. miliaceum. Chloroplasts were preincubated with or without the inhibitors and with a tracer amount of 22NaCl (right panel), and cold or labeled pyruvate was added at zero time. [Na+]e. was 80 AM. The pyruvate/Na+ ratios in 7 s incubations were 1.1 (dark), 3.9 (light control), 1.6 (+CCCP) and 2.6 (+pCMS).

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24

Fg *

g

Mon

o

1

0°

c

Control

z

I

0

I

1.0


8; Tables IIA and III) or flux of endogenous Na+ preequilibrated with 22Na (preequilibration experiments: Figs. 2, 3, 4, 6, and 7; Table IIB). Special attention was drawn to the enhancement of Na+ uptake by pyruvate and pyruvate-induced Na+ influx, respectively. All the Na+ detected in chloroplasts by the present method is not necessarily free in the stroma as already stated in "Materials and Methods." (See below for the results suggesting the presence of multiple pools of chloroplast Na+.) The most serious problem in the present study would be caused by a nonspecific binding of 22Na' to the outer surface of the envelope membranes. We have, therefore, checked the effect of including 1 mM Mg2+ or Ca2+ instead of EDTA in the medium, which is supposed to suppress Na+ binding to membranes through electrostatic effects. Both divalent cations affected neither 22Na' uptake (or flux) nor pyruvate uptake (Table II). The result supports that the observed Na+ uptake and influx are the real flux across the envelope. The existence of a Na+/pyruvate cotransport system in the envelope of mesophyll chloroplasts of P. miliaceum (and also

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0.5

2

ont rol

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c

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0

>>o~~~n 0't 0

7

15 Incubation Time (s)

,

30

Figure 7. Effect of monensin on pyruvate uptake and pyruvateinduced Na+ flux in mesophyll chloroplasts of P. miliaceum in the light. The experimental system was the same as in Figure 2, except that chloroplasts were preincubated with or without 5 Mm monensin. [Na+]ex was in this experiment exceptionally high (0.5 mM). The pyruvate/Na+ ratio in 7 s incubation was 1.3 (without monensin).

E

Q-

DISCUSSION The first suggestion of the involvement of a Na+ gradient the envelope in pyruvate uptake in mesophyll chloroplasts of P. miliaceum (NAD-malic enzyme type) (19) has now been extended to two species from PEP carboxykinase type, P. maximum and U. panicoides. In these three species, a Na+ jump enhanced pyruvate uptake in the dark without affecting the stromal pH (Table I). It seems, therefore, that a Na+ jump affects pyruvate transport system rather directly not through the pH gradient across the envelope. In the present study, we have also measured uptake of exogenously added 22Na (uptake experiments: Figs. 1, 5, and

across

Incubation Time

(s)

Figure 8. Effect of monensin on pyruvate and Na+ uptake in mesophyll chloroplasts of P. miliaceum in the dark. The experimental system was the same as in Fig. 1, except that chloroplasts were preincubated with or without 5 uM monensin and the concentration of added Na+ was 2 mm. The initial [Na+]ex was 0.2 mm. The pyruvate/ Na+ ratio in 7 s incubation was 1.1 (without monensin).


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Table Ill. Initial Uptake of Pyruvate and Na+ in Mesophyll Chloroplast of P. miliaceum Measured with the Double-Layer System Results are the means of duplicate or triplicate samples ± SD. Medium Na+ concentration of the chloroplast layer was 0.12 mm in all the experiments and that in the uptake layer was either the same (0.12 mM) in the series designated as A or 1 mm in series B. The tubes contained a constant amount of chloroplasts (12-16 ,g Chl) in each experiment. Exp. No. Na+ and Pyruvate (D) DIC Condition -Pyr + Pyr Difference (C) pmol Exp. IA Dark 26 ± 2 40 ± 3 14 ± 5 12 ±1 0.9 19 ± 1 56 ± 4 Light 37 ± 5 46 ± 2 1.2 Exp. IIA Dark 14 ± 1 42 ± 3 28 ± 4 14 ± 2 0.5 15 ± 1 69 ± 8 Light 54 ± 8 65 ± 4 1.2 Exp. llB 52 ± 1 Dark 106 ± 36 152 ± 14 46 ± 50 1.1 Light 153 ± 25 304 ± 22 151 ± 47 176 ± 5 1.2 Exp. lIlA 21 ± 2 Dark 38 ± 5 17 ± 7 11 ± 1 0.6 24 ± 1 Light 54 ± 6 30 ± 7 43 ± 4 1.4 Exp. IIIB 177 ± 24 54 ± 32 Dark 123 ± 8 45 ± 4 0.8 Light 179 ± 8 252 ± 39 73 ± 47 89 ± 8 1.2

the two species of PEP carboxykinase type) is supported by the following observations. 1) Pyruvate and Na+ mutually enhanced the uptake of each other in the dark (Figs. 1 and 5, Tables II and III); 2) Na+ influx always accompanied pyruvate uptake under various conditions (Figs. 2 and 6); 3) Na+ was pumped out on illumination (Fig. 2) suggesting light-dependent formation of a Na+ gradient across the envelope; 4) monensin, a Na+/H+ exchange ionophore, nullified mutual effects of Na+ and pyruvate (Figs. 7 and 8). In addition, pyruvate uptake in mesophyll chloroplasts of P. miliaceum in the light was inhibited by amiloride (50% at 1 mM) and harmaline (90% inhibition at 5 mM) (J. Ohnishi, unpublished results), both inhibitors of Na+/H+ antiporters and/or Na+ cotransporters ( 13, 25). The measured ratios of the initial uptake of pyruvate and Na+ were close to or at least consistent with a 1 to 1 stoichiometry (Table III). The ratios of transported pyruvate and Na+ were also calculated from the measurements of 7 s incubation in each experiment shown in the figures and indicated in the legends. Dark uptake experiments gave values of 0.6 to 1.5, rather consistent with the results in Table III. Preequilibration experiments gave stoichiometry ranging from 1 in Figure 5 (in the dark) to higher values up to 7 in Figure 2 (in the light), the ratio being higher in the light than in the dark. These higher values in the light could be explained by supposing a simultaneous operation of a Na+ pump (or a Na+/H+ exchanger as discussed below), which exports Na+ to develop and maintain an effective Na+ gradient across the envelope. Such a Na+ efflux was really observed on illumination (Fig. 2). Na+/metabolite cotransport systems are found in mammalian epithelial transport of nutrients (30) and also in microorganisms (27). However, such a role of Na+ has never

been found in higher plants (23) and the Na+/pyruvate cotransport presumed here is rather unique. Na+ is usually found in plant cells at a high concentration but seems to be excreted through plasma membrane or sequestered into vacuole via Na+/H+ antiport (1, 24). Positive role(s) of Na+ was first suggested by Brownell and coworkers as an essential micronutrient in some C4 plants (3) and further confirmed (2, 21). However, its exact role has not yet been clarified (2), although recent reports have suggested its role in NO3- uptake and metabolism (20, 21) or metabolite transport (2). The Na+/ pyruvate cotransport demonstrated here in mesophyll chloroplasts of several C4 species could be one of the physiological roles of Na+ in C4 plants. It should be added that a significant discrepancy exists in the distribution of positive Na+ effects among C4 species between the present in vitro study of pyruvate transport and in vivo study on growth (2, 3, 21). The growth of P. miliaceum did not respond to the Na+ level in culture medium (21), while the growth of two NADP-malic

enzyme type species, Kochia childsii and Portulaca grandi-

flora, responded to the Na+ level (3). Further efforts should be directed to a comparative study. The mechanism responsible for the formation of the Na+ gradient across the envelope (or the mechanism of lowering stromal free Na+ concentration), which is here implicated in active pyruvate uptake into C4 mesophyll chloroplasts, could be theoretically primary or secondary. Primary Na+ pumps driven by electron transport or ATPases have been detected in bacteria and mammals (27). However, neither electron transport activity nor Na+-ATPase has been demonstrated in the envelope membranes of either C3 or C4 chloroplasts. A secondary Na+ pump, namely Na+/H+ exchange would be an alternative mechanism. Na+/H+ antiporters have been reported in cell membranes, tonoplast and mitochondrial mem-

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branes (1, 14, 27) and could also be postulated on the envelope membranes of C4 mesophyll chloroplasts. Light dependent stromal alkalization and, therefore, the pH gradient across the envelope could drive the Na+/H+ exchange across the envelope and lower the stromal Na+ concentration. It should be added that this presumed exchange seems to be irreversible within a short time range, because a Na+ jump in the dark did not significantly change the stromal pH (Table I). As for the cation flux across the envelope, light-dependent K+/H+ exchange leading to K+ influx has been reported in C3 chloroplasts (4). Maury et al. (15) postulated a Mg2+-activated reversible K+/H+ exchange. In these studies, it was assumed without any direct measurement of Na+ flux that Na+ also behaved like K+. Light-dependent stromal alkalization could also drive Na+/ H+ exchange across the thylakoid (Na+ sequesterization into the thylakoid lumen). Cations, especially Mg2+, are reported to be transported out from thylakoid lumen to compensate for the H+ accumulation in the lumen in C3 chloroplasts (10). Although K+ and Na+ were shown to be rather immobile in C3 thylakoid preparations (10), a possibility still remains that Na+/H+ exchange occurs on the thylakoid of C4 chloroplasts. No study has been so far directed to the cation status in C4 chloroplasts. This is needed to confirm that a Na+ gradient really exists across the envelope by measuring the concentration of free Na+ in the stroma and cytosol (or medium). Preliminary measurements of Na+ content of water extracts of P. miliaceum mesophyll chloroplasts by flame photometry and atomic absorption spectroscopy (J. Ohnishi, unpublished results) gave up to 10 times higher values in a millimolar range than those in a submillimolar range obtained in the present study by isotope exchange (Figs. 2, 3, 4, 6, and 7). This result together with the high Na+ content (2 mM) measured in the presence of the Na+ ionophore monensin (Fig. 7) suggests the presence of multiple pools of chloroplast Na+. Na+ measurements in C3 chloroplasts also gave values in a millimolar range (24). It seems, therefore, that we have measured in the present study only limited pools of chloroplast Na+ (including the free pool), which is easily exchangeable with external 22Na+. In vitro 23Na-NMR study (26) or a microfluorometric study using a Na+-indicator (8) could give a clue to clarify the Na+ status in C4 mesophyll chloroplasts in vivo and in vitro. In the present study no mutual effects of Na+ and pyruvate were found (Table I, Figs. 1 and 3) in mesophyll chloroplasts of two species of NADP-malic enzyme type C4 plants, maize and sorghum (Table I, Figs. 1 and 3), although active pyruvate uptake in the light is also a character of mesophyll chloroplasts of these species (6). In a preliminary study which will be

published elsewhere, a sudden decrease of medium pH (pH jump) enhanced pyruvate uptake into mesophyll chloroplasts of maize and sorghum (J. Ohnishi, R. Kanai, unpublished data). This finding together with the present results leads to the idea that pyruvate is transported by cotransporters in the envelope of C4 mesophyll chloroplasts diffenng in the specificity of the co-ion, utilizing either H+ or Na+. ACKNOWLEDGMENT The research stay of J.O. in FRG was supported by a fellowship from Alexander von Humboldt-Stiftung, FRG. The experiments


using 22Na were performed in Isotope Center (Zentrales Isotopenlabor) of Universitat Gottingen in FRG and in RIKEN Institute in Japan. Thanks are due to the staffs of the former institute and also to Dr. Y. Inoue, Dr. T. Ogawa and Dr. Y. Ikeuchi of Solar Energy Research Group, RIKEN Institute for their support. We are also obliged to Dr. R. Hedrich of Pflanzenphysiologisches Institut, Universitat Gottingen, and Dr. I. Ike of RIKEN Institute for allowing us to use the flame photometer and the gamma counter, respectively. LITERATURE CITED 1. Blumwald E, Poole RJ (1985) Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris. Plant Phys-

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