Metabolic regulation of carbon flux during C4 photosynthesis : II. In situ evidence for reffixation of photorespiratory CO2 by C 4 phosphoenolpyruvate carboxylase.

The potential for glycolate and glycine metabolism and the mechanism of refixation of photorespiratory CO2 in leaves of C4 plants were studied by parallel inhibitor experiments with thin leaf slices, different leaf cell types and isolated mitochondria of C3 and C4 Panicum species. CO2 evolution by leaf slices of P. bisulcatum, a C3 species, fed glycolate or glycine was light-independent and O2-sensitive. The C4 P. maximum and P. miliaceum leaf slices fed glycolate or glycine evolved CO2 in the dark but not in the light. In C4 species, dark CO2 evolution was abolished by the addition of phosphoenolpyruvate (PEP)(4). The addition of maleate, a PEP carboxylase inhibitor, resulted in photorespiratory CO2 efflux by C4 leaf slices in the light also. However, PEP and maleate had no effect on either glycolate-dependent O2 uptake by the C4 leaf slices or on glycolate and glycine metabolism in C3 leaf slices. The rate of photorespiratory CO2 evolution in the C3 Panicum species was 3 times higher than that observed with the C4 species. The ratio of glycolate-dependent CO2 evolution to O2 uptake in both groups was 1:2. Isolated C4 mesophyll protoplasts or their mitochondria did not metabolize glycolate or glycine. However, both C3 mesophyll protoplasts and C4 bundle sheath strands readily metabolized glycolate and glycine in a light-independent, O2-sensitive manner, and the addition of PEP or maleate had no effect. C4 bundle sheath- and C3-mitochondria were capable of oxidizing glycine. This oxidation was linked to the mitochondrial electron transport chain, was coupled to three phosphorylation sites and was sensitive to electron transport inhibitors. C4 bundle sheath- and C3-mitochondrial glycine decarboxylation was stimulated by oxaloacetate and NAD had no effect. In marked contrast, mitochondria isolated from C4 mesophyll cells were incapable of oxidizing or decarboxylating added glycine. The results suggest that in leaves of C4 plants bundle sheath cells are the primary site of O2-sensitive photorespiratory CO2 evolution and the PEP carboxylase present in the mesophyll cells has the Potential for efficiently refixing CO2 before it escapes out of the leaf. The relative role of the PEP carboxylase mediated CO2 pump and reassimilation of photorespiratory CO2 are discussed in relation to the apparent lack of photorespiration in leaves of C4 species.