- © 1975 American Society of Plant Biologists
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.