Regulation of Oxaloacetate, Aspartate, and Malate Formation in Mesophyll Protoplast Extracts of Three Types of C4 Plants

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Abstract
The use of mesophyll protoplast extracts from various C4 species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C4 photosynthesis. Conditions regulating the formation of the C4 acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C4 species, Eleusineindica, an NAD-malic enzyme C4 species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C4 species. Light-dependent induction of CO2 fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and α-ketoglutarate, or 3-phosphoglycerate induced high rates of CO2 fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C4 mesophyll cells. Induction by pyruvate or alanine and α-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO2 fixation was not.