Radiotracer methods have been employed to study some aspects of the mechanism of the C4-dicarboxylic acid pathway of photosynthesis and its distribution in plant species. This pathway which is characterized by the primary fixation of 14CO2 into the C4-dicarboxylic acids, malate, aspartate and oxaloacetate was shown to occur in sugar cane leaves over a wide range of physiological conditions, light intensity, CO2-concentrations and leaf maturities.
Using a pulse-chase procedure, the movement of radiocarbon from the C-4 of the C4-dicarboxylic acid-pool to the C-1 of 3-phosphoglycerate was demonstrated. By a quantitative analysis of the data, it was shown that this pathway accounted for all radiocarbon entering 3-phosphoglycerate and products. Evidence was obtained that unlabelled intracellular bicarbonate pools were small and therefore would not obscure any path of direct fixation of 14CO2 into 3-phosphoglycerate. Further, a comparison of the rate of photosynthetic CO2-fixation by sugar cane leaves and the rate of carbon movement through the C4-dicarboxylic acid pool demonstrated that this is the major route of carbon flow accounting for at least 83% of CO2 fixed.
A pulse-chase procedure with sorghum leaves was employed to detect minor labelled intermediates and products. Dihydroxyacetone phosphate, fructose diphosphate and ribulose mono- and diphosphates were identified by paper chromatographic procedures as intermediates of the C4-pathway. The amino acids, serine, glycine, alanine and glutamate were identified as side products. Phosphopyruvate was detected as a labelled compound only after longer exposures to 14CO2 and apparently exists as a very small pool.
An analysis of the behaviour of labelled intermediates during a CO2-free air treatment provided evidence that ribulose diphosphate or a C2-compound derived from this pentose was the acceptor for carbon transfer from the C4-dicarboxylic acid-pool to 3-phosphoglycerate. The movement of carbon from the C4-acids was virtually halted in the dark and the decline in the pool of ribulose diphosphate was slow. The implications arising from this data are discussed.
A study of the distribution of the C4-pathway of photosynthesis amongst plant species revealed that this pathway is operative in species which are generally located in tropical regions of high temperatures and seasonal dry periods. Such species include members of the Panicoideae and Chloridoideae subfamilies of the Gramineae, species of the genus Cyperus (Cyperaceae), and species from the dicotyledonous genera Amaranthus and Gomphrena (Amaranthaceae), Atriplex (Chenopodiaceae) and Portulaca (Portulacaceae). Not all species of a family, subfamily or even a genus necessarily utilized the same photosynthetic pathway. The results are discussed in relation to physiological and anatomical characteristics associated with C4-pathway-plants, taxonomy within the Gramineae, and evolution of plant species.
A study of several enzymes involved in the operation of the C4-pathway of photosynthesis has provided information concerning the mechanism and control of this pathway. The partial purification and properties of NADP-malic enzyme and NADP-malate dehydrogenase from maize leaves are reported. The catalytic properties of maize leaf NADP-malic enzyme are similar to those reported for this enzyme from a wide variety of sources. Maize leaf NADP-malate dehydrogenase is physically distinct from both NAD-malate dehydrogenase and NADP-malic enzyme, and exists as three molecular weight forms. These forms may be monomeric, dimeric and tetrameric species of the same enzyme. The catalytic properties of NADP-malate dehydrogenase are similar to the NAD enzyme, but the activity is dependent on the presence of a thiol. NADP-malate dehydrogenase is rapidly inactivated in the dark and reactivated in the light and this control mechanism is discussed. Preliminary data was obtained on inactivation and activation requirements of this enzyme in leaf extracts.
An analysis of the activities and distribution of NADP-malic enzyme, NADP-malate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in the leaves of several C4-pathway-plants has indicated that differences may exist between species concerning fine points in the mechanism of the C4-pathway. Malate is considered the C4-donor acid in the Panicoid grasses, providing the C-4 carboxyl as CO2 for fixation by ribulose diphosphate carboxylase. Evidence suggests that aspartate may be the C4-donor acid in the Chloridoid grasses and dicotyledons. The significance of the proposed differences in the mechanism of the C4-pathway in relation to ultrastructural characteristics of chloroplasts and environmental localities of the plants is discussed.