A role for fructose 2,6-bisphosphate in regulating carbohydrate metabolism in guard cells

Fructose 2,6-bisphosphate (Fru2,6P(2)) appears to function as a regulator metabolite in glycolysis and gluconeogenesis in animal tissues, yeast, and the photosynthetic cells of leaves. We have investigated the role of Fru2,6P(2) in guard-cell protoplasts from Vicia faba L. and Pisum sativum L. (Argenteum mutant), and in epidermal strips purified by sonication from all cells except for the guard cells. Guard-cell protoplasts were separated into fractions enriched in cytosol and in chloroplasts by passing them through a nylon net, followed by silicone oil centrifugation. The cytosol contained a pyrophosphate: fructose 6-phosphate phosphotransferase (involved in glycolysis) which was strongly stimulated by Fru2,6P(2). A cytosolic fructose 1,6-bisphosphatase (a catalyst of gluconeogenesis) was inhibited by Fru2,6P(2). There was virtually no fructose 1,6-bisphosphatase activity in guard-cell chloroplasts of V. faba. It is therefore unlikely that the starch formed in these chloroplasts originates from imported triose phosphates or phosphoglycerate.The level of Fru2,6P(2) in guard-cell protoplasts and epidermal strips was about 0.1 to 1 attomole per guard cell in the dark (corresponding to 0.05 to 0.5 nanomole per milligram chlorophyll) and increased three- to tenfold within 15 minutes in the light. Within the same time span, hexose phosphate levels in guard-cell protoplasts declined to approximately one-half, indicating that acceleration of glycolysis involved stimulation of reactions using hexose phosphates. The level of Fru2,6P(2) in guard cells appears to determine the direction in which carbohydrate metabolism proceeds.