IS-2248-94

Publications

Achievements

Fructose 2,6-Bisphosphate and Citrate: Their Roles in the Regulation of Carbohydrate Metabolism in Citrus Fruits and Leaves	Budget	Duration
	$300,000	3 years

	Goren, R.	Hebrew U
	Exteberria, E.	U Florida
	Zehavi, U.	Hebrew U

Final Report Abstract
Citrus fruits contain large quantities of sugars and organics which constitute the principal parameters of fruit quality and value.The flow of carbon between sugars and acids within cells is finely regulated primarily at the level of fructose-6-P and fructose-1,6-bisphosphate inter-conversion controlled by ATP and PPi dependent phosphofructokineses (PFK and PFP, respectively) and fructose-1,6-bisphosphate. The objectives of the present work were to determine the kinetic parameters of the three enzymes involved in the regulatory step mentioned above. In addition we investigated the regulatory effect of citrate on the above mentioned enzymes, cytosolic citrate concentration and its sequestration in the vacuole of juice sac cells. Kinetics of the regulatory enzymes were determined and found that some TCA organic acids (mainly citrate) inhibit glycolysis (both PFP andPFK) but not gluconeogenesis. Fructose-2,6-P2 was quantified in juice cell protoplasts. Studies on citrate compartmentation and transport revealed that a unique tonoplast-bound ATPase enables the formation of electrochemical conditions that allow for total compartmentation of citrate in the vacuole. The tonoplast-bound ATPase was not inhibited by Cl or bafilomycin, but showed strong inhibition in the presence of valnadate. Gel filtration showed the enzyme having similar molecular weight than that from sweet lime which showed conventional patterns to endomembrane ATPase inhibitors. In addition, the tonoplast of acidic citrus fruits appeared to be impermeable to protons since tonoplast vesicles retain the imposed artificial H+ gradient after termination of ATPase activity. Feeding experiments demonstrated that fructose and glucose (products of sucrose hydrolysis) are phosphorylated by a membrane bound hexokinase and used for respiration. In spite the inhibitory potential of citrate on glycolys it was shown that its cytosolic concentration is too low to influence the direction of carbon flow in juice sacs cells.