CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to (14)CO2 in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO2 photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of (14)CO2 both in the light and in the dark. Presumably, the (14)CO2 is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with (14)CO2 showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-(14)C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant (14)CO2 fixation. However, adding PEP to an homogenate stimulated (14)CO2 uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO2 and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.