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

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.

Several differences between these species representing the three C₄ groups were observed. Substrate induction of CO₂ fixation in mesophyll protoplast extracts of D. sanguinalis gave malate as a major product; only by an apparent exchange reaction with cold aspartate did substantial label appear in aspartate (up to 53% of labeled products). In contrast, aspartate was a major product when alanine and α-ketoglutarate served as inducing substrates with E. indica (up to 57%) and U. panicoides (up to 86%). With induction by pyruvate or 3-phosphoglycerate, mesophyll preparations of U. panicoides and E. indica were less effective in forming malate (up to 31% of products) than D. sanguinalis (up to 87% of products). After 2 seconds of whole leaf ¹⁴CO₂ fixation, malate was the major labeled product (57%) with D. sanguinalis, whereas with E. indica and U. panicoides aspartate was the predominant product (73% and 76%, respectively).

With mesophyll protoplast extracts of D. sanguinalis, aspartate inhibited CO₂ fixation (about 50% at 0.6 mm), while malate was relatively uninhibitory at comparable concentrations. CO₂ fixation by mesophyll protoplast extracts of E. indica was inhibited by malate (about 50% at 0.6 mm), while aspartate was relatively uninhibitory. With mesophyll preparations of U. panicoides, malate or aspartate (2 mm) caused only slight inhibition of CO₂ fixation. The regulation of aspartate and malate synthesis in C₄ mesophyll cells is discussed relative to initial products of photosynthesis in C₄ species in vivo and species differences in the mechanisms of C₄ photosynthesis.