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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. 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 ...

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Uptake of [I-Wlpyruvateinto the sorbitol-impermeable space of mesophyll chloroplasts of a C, plant, Panicum miliaceum L., was investigated using a silicone oil filtering centrifugation method. An abrupt change of Na+ concentration in the suspending medium of the chloroplasts in the dark induced accumulation of pyruvate in the stroma, similar to the case of light-driven active uptake. The effect was specific to Na+ among various mono-and divalent cations. The apparent K,,, for Na+ was in the range 2-5 mM. The K,,, for pyruvate was about 0.7 mM, which was similar to the value obtained in light-driven uptake. The possible role of Na+ symport in active pyruvate uptake by C, mesophyll chloroplasts is discussed.

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