Involvement of Na⁺ in Active Uptake of Pyruvate in Mesophyll Chloroplasts of Some C₄ Plants

Abstract

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 ...

Sodium as nutrient and toxicant

“…In C 4 species engaging in the 'MEP' (methyl-erythritol-phosphate) pathway, pyruvate import into chloroplasts, across the envelope, is pivotal to the C 4 habit, and it is specifically here where Na + -coupling is now established (Weber and von Caemmerer 2010). By contrast, at the functional level, the Na + -coupled mechanism is not found in chloroplasts of corn, offering at least a partial explanation why only some C 4 species rely on sodium as a nutrient (Ohnishi et al 1990;Aoki et al 1992;Weber and von Caemmerer 2010). Recently, in a benchmark study, Furumoto et al (2011) characterized the Na + -pyruvate cotransport mechanism at the molecular level, by identification of the BASS2 gene in Flaveria and Cleome, and showing the protein's localization to the chloroplast envelope and the facilitation of pyruvate influx.…”

“…This was first demonstrated physiologically in Panicum miliaceum, where pyruvate uptake into chloroplasts was found to be proportional to the concentrations of Na + supply, indeed following a 1:1 ratio for Na + :pyruvate in the transport function (Ohnishi and Kanai 1987). This suggested the existence of a Na + -dependent pyruvate cotransport system in chloroplasts, possibly driven by a light-stimulated, H + -coupled, Na + efflux pump (Ohnishi et al 1990;Furumoto et al 2011). Pyruvate is central to the CO 2 -concentrating mechanism in C 4 species, and it serves as a precursor to several major biochemical pathways in all plants, such as fatty acid synthesis and isoprenoid metabolism; furthermore, as the end product of glycolysis, it is positioned at a key intersection point of both primary and secondary metabolism (Schwender et al 2004).…”

“…The paradigm that C 4 species benefit particularly substantially, and indeed in many, albeit not all, cases require Na + as an essential nutrient, is now widely accepted (Harmer and Benne 1945;Barbier and Chambannes 1951;Brownell and Wood 1957;Brownell 1965;Alekseev and Abdurakhamanov 1966;Brownell and Jackman 1966;Brownell and Crossland 1972;Marschner 1995;Pessarakli and Marcum 2000;Pessarakli 2001;Subbarao et al 2003). Important exceptions include the leading C 4 crops corn and sorghum, which have been shown not to benefit from Na + addition (Ohta et al 1988;Ohnishi et al 1990;Subbarao et al 2003). In C 4 species, it is thought that Na + facilitates the conversion of pyruvate into phosphoenolpyruvate (PEP), which occurs in mesophyll cells, prior to the Calvin cycle (Johnston et al 1988).…”

Interactions of C4 Subtype Metabolic Activities and Transport in Maize Are Revealed through the Characterization of DCT2 Mutants

“…Pyruvate import into the M cell plastids in C 4 plants could involve Na + or H + (Flugge et al, 1985;Kanai, 1987a, 1987b). In maize, Ohnishi et al (1990) suggest the driving force is a H + gradient, as reviewed by Heldt (2002). The presence of a pyruvate transporter in both M and BS cells was described (Furumoto et al, 2011), though the mechanism of pyruvate export from BS plastids remains unknown (Weber and von Caemmerer, 2010).…”

Filtering Centrifugation Through Two Layers of Silicone Oil: A Method for the Kinetic Analysis of Rapid Metabolite Transport in Organelles.

“…Because it is physically difficult to obtain data before the steady state of transport is reached, the method is not suitable for measuring initial transport rates when transport is completed in a few seconds. Many investigators have sought alternative methods of filter centrifugation in which transport could be stopped after short incubation periods (1,3,5,6,9,14,15). Most of these methods require the addition of specific transport inhibitors or provide a single incubation period with no guarantee that it is short enough to yield an initial transport rate.…”