On the in vitro Phosphorylation of Aldose-6-phosphate Reductase from Apple and Peach Leaves

C.M. FIGUEROA; A.A. IGLESIAS

Montreal

Congreso; Joint Annual Meeting of the American Society of Plant Biologists and the Canadian Society of Plant Physiologists; 2010

American Society of Plant Biologists

On the in vitro phosphorylation of aldose-6-phosphate reductase from apple and peach leaves.Figueroa, Carlos M.-presenter carfigue@fbcb.unl.edu.ar (a) Piattoni, Claudia (a) Iglesias, Alberto (a)In addition to sucrose and starch, many plant species synthesize glucitol (Gol) as a main photosynthetic product. Gol synthesis occurs by the action of a NADPH-dependent aldose-6-phosphate reductase (A6PRase, EC 1.1.1.200), which converts glucose-6P (Glc6P) into Gol6P, followed by the hydrolysis of the phosphate group by a specific phosphatase.Gol is then transferred to sink tissues, where it can be converted into fructose by a NAD-dependent Gol dehydrogenase. Despite the importance of these enzymes for carbon partitioning in plants from the Rosaceae family (i.e., apple and peach), few studies deal with their characterization. In this work, we show the recombinant expression, one-step purification and kinetic characterization of A6PRase from apple and peach leaves. The purified enzymes exhibited kinetic properties similar to those reported for A6PRase purified from the leaves of apple and loquat. Utilizing [32P]ATP, we found that the recombinant enzymes can be in vitro phosphorylated by extracts from leaves of the two plant species. Using A6PRase as a substrate, a Mg2+- and Ca2+-dependent protein kinase (PKase) was partially purified from apple and peach leaves. In our hands, the PKase was inhibited by ribose-5P, PPi, phosphoenolpyruvate, fructose-1,6bisP, Gol6P and Glc6P. As intracellular levels of these metabolites fluctuate along the day, it is proposed that the PKase could be regulated with dependence on the day/night cycle. Considering that the activity of A6PRasewas found to change during the photoperiod, it is tempting to speculate that the enzyme could be light/dark modulated in vivo through protein phosphorylation.