Glucitol metabolism in plants: Regulation of key enzymes by post-translational modification

FIGUEROA CM; HARTMAN MD; PIATTONI CV; IGLESIAS AA

Göttingen

Congreso; Trends in Enzymology 2012: Going beyond frontiers; 2012

Georg-August-Universität Göttingen

In addition to sucrose and starch, glucitol (Gol) is a major photosynthetic product in plants from the Rosaceae family. In mature leaves, glucose-6P (Glc-6P) is converted into Gol-6P by a NADPH-dependent aldose 6 phosphate reductase (Ald-6PRase, EC 1.1.1.200). Subsequently, the phosphate group is hydrolyzed by a specific phosphatase. Gol is then transferred to sink tissues, where it is oxidized to fructose (Fru) by a NAD-dependent Gol dehydrogenase (GolDHase, EC 1.1.1.14). Despite the importance Ald-6PRase and GolDHase for carbon partitioning in these plants, little is known about their regulation. In this work, we show the recombinant expression, one-step purification, and kinetic/ regulatory characterization of Ald-6PRase and GolDHase from peach leaves and fruits, respectively. It is widely known that enzyme activity could be regulated by different mechanisms, including post-translational modifications like phosphorylation and oxidation/reduction. In the case of Ald-6PRase from peach leaves, we found that it can be recovered in a Fe3+ affinity chromatography, which suggests that this enzyme could be phosphorylated in vivo. Utilizing [32P]ATP, we showed that recombinant Ald-6PRase is phosphorylated in vitro by a protein extract from peach leaves. Thereafter, a Mg2+- and Ca2+-dependent protein kinase (PKase) was partially purified from peach leaves and characterized. We found that the PKase was inhibited by pyrophosphate, Fru 1,6 bisP, phosphoenolpyruvate, Gol-6P and Glc-6P (I0.5 values were 0.49, 1.9, 2.1, 3.3 and 7.7 mM, respectively). As intracellular levels of these metabolites fluctuate along the day, we propose that PKase could be regulated by them in dependence on the day/night cycle. Considering that Ald-6PRase activity was found to change during the photoperiod, with our results it is tempting to speculate that the enzyme could be light/dark modulated in vivo through protein phosphorylation. On the other hand, we found that GolDHase is highly inhibited by Cu2+ and Hg2+ (I0.5 values were 31 and 96 nM, respectively). In addition, loss of activity was observed when GolDHase was incubated with oxidizing reagents like diamide, H2O2 and oxidized glutathione (k´´ values were 12.3, 6.93 and 0.086 M-1s-1, respectively). The activity of the oxidized enzyme was recovered by reduction with DTT, reduced glutathione and recombinant thioredoxin from peach fruits. Considering that Gol accumulation has been related with abiotic stress tolerance, we hypothesize that redox regulation of GolDHase could be a mechanism involved in maintaining levels of Gol high enough to exert a protective role as a hydroxyl-radical scavenger. As a whole, our results suggest that in peach (and probably also in other Rosaceae plants) Ald-6PRase and GolDHase could be key targets for in vivo post-translational regulation.