Functional Analyses of Cytosolic Glucose-6-Phosphate Dehydrogenases and Their Contribution to Seed Oil Accumulation in Arabidopsis

Abstract

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for biochemical reactions and in modulating the redox state of cells. In plants, identification of its role is complicated due to the presence of several isoforms in the cytosol and plastids. Here we focus on G6PDHs in the cytosol of Arabidopsis (Arabidopsis thaliana) using single and double mutants disrupted in the two cytosolic G6PDHs. Only a single G6PDH isoform remained in the double mutant and was present in chloroplasts, consistent with a loss of cytosolic G6PDH activity. The activities of the cytosolic isoforms G6PD5 and G6PD6 were reciprocally increased in single mutants with no increase of their respective transcript levels. We hypothesized that G6PDH plays a role in supplying NADPH for oil accumulation in developing seeds in which photosynthesis may be light limited. G6PDH activity in seeds derived from G6PD6 and a plastid G6PDH isoform and showed a similar temporal activity pattern as oil accumulation. Seeds of the double mutant but not of the single mutants had higher oil content and increased weight compared to those of the wild type, with no alteration in the carbon to nitrogen ratio or fatty acid composition. A decrease in total G6PDH activity was observed only in the double mutant. These results suggest that loss of cytosolic G6PDH activity affects the metabolism of developing seeds by increasing carbon substrates for synthesis of storage compounds rather than by decreasing the NADPH supply specifically for fatty acid synthesis.

LEAFY COTYLEDON1Is a Key Regulator of Fatty Acid Biosynthesis in Arabidopsis

“…These results are somewhat expected, because fatty acid biosynthesis appears to be controlled by a coordinated regulatory mechanism. This coregulation mechanism is not only pertinent to major steps of the fatty acid and lipid metabolism pathways but also requires the coordination of key components in carbohydrate metabolism, in particular the regulation of Suc and hexose flux (Rawsthorne, 2002;Ruuska et al, 2002;Wakao and Benning, 2005;Baud et al, 2007b;Wakao et al, 2008). Thus, in addition to the manipulation of key regulatory reactions such as ACCase, coregulation of other key genes is essential for an increased flux of the entire pathway.…”

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling

“…Moreover, irrespective of the exact proportion of the oxidative PPP flux occurring in the cytosol, two recent studies demonstrate the importance of the cytosolic activity. First, disrupting the expression of the two genes encoding cytosolic isozymes of Glc-6-P dehydrogenase (G6PD5 and G6PD6) in Arabidopsis through insertional inactivation alters seed lipid content (Wakao et al, 2008). Second, supplementation or replacement of cytosolic Glc-6-P dehydrogenase with an alternative isoform improves the resistance of tobacco (Nicotiana tabacum) leaves to Phytophthora infestans, and this is attributed to changes in the provision of NADPH and stimulation of the hypersensitive defense response (Scharte et al, 2009).…”

Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis

“…We also analyzed a previously described G6PD6 T-DNA insertion line, which was unaffected by salt stress (Wakao et al, 2008). In this T-DNA insertion line, G6PD6 is disrupted downstream of the ASKa phosphorylation site Thr-467, underlining the significance of Thr-467 for adaptive regulation of G6PD.…”

“…The major G6PD activity in leaves originates from cytosolic G6PD isoforms (Debnam and Emes, 1999). The lack of a potential plastid targeting sequence in ASKa prompted us to focus our analyses on the two cytosolic G6PD isoforms, G6PD5 and G6PD6 (Wakao et al, 2008).…”