Altered activity of the P2 isoform of plastidic glucose 6‐phosphate dehydrogenase in tobacco (Nicotiana tabacum cv. Samsun) causes changes in carbohydrate metabolism and response to oxidative stress in leaves

Abstract

SummaryExpression of one speci®c isoform of plastidic glucose 6-phosphate dehydrogenase (G6PDH) was manipulated in transgenic tobacco. Antisense and sense constructs of the endogenous P2 form of G6PDH were used to transform plants under the control of the cauli¯ower mosaic virus (CaMV) 35S promotor. Recombinant plants with altered expression were taken through to homozygosity by selective screening. Northern analyses revealed substantial changes in the expression of the P2 form of G6PDH, with no apparent impact on the activity of the cytosolic isoenzyme. Analysis of G6PDH activity in chloroplasts showed that despite the large changes in expression of P2-G6PDH, the range of enzyme activity varied only from approximately 50 to 200% of the wild type, re¯ecting the presence of a second G6PDH chloroplastic isoform (P1). Although none of the transgenic plants showed any visible phenotype, there were marked differences in metabolism of both sense and antisense lines when compared with wild-type/control lines. Sucrose, glucose and fructose contents of leaves were higher in antisense lines, whereas in overexpressing lines, the soluble sugar content was reduced below that of control plants. Even more striking was the observation that contents of glucose 6-phosphate (Glc6P) and 6-phosphogluconate (6PG) changed, such that the ratio of Glc6P:6PG was some 2.5-fold greater in the most severe antisense lines, compared with those with the highest levels of overexpression. Because of the distinctive biochemical properties of P2-G6PDH, we investigated the impact of altered expression on the contents of antioxidants and the response of plants to oxidative stress induced by methyl viologen (MV). Plants with decreased expression of P2-G6PDH showed increased content of reduced glutathione (GSH) compared to other lines. They also possessed elevated contents of ascorbate and exhibited a much higher ratio of reduced:oxidised ascorbate. When exposed to MV, leaf discs of wild-type and overexpressing lines demonstrated increased oxidative damage as measured by lipid peroxidation. Remarkably, leaf discs from plants with decreased P2-G6PDH did not show any change in lipid peroxidation in response to increasing concentrations of up to 15 lM MV. The results are discussed from the perspective of the role of G6PDH in carbohydrate metabolism and oxidative stress. It is suggested that the activity of P2-G6PDH may be crucial in balancing the redox poise in chloroplasts.

NTRC links built-in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts

“…The picture is, however, not fully clear. For example, although transgenic experiments in which glucose-6-phosphate dehydrogenase was overexpressed in tobacco leaves indicate that the enzyme plays a role in determining the ratio of starch to soluble sugars (25), it is not known whether this contribution extends to amyloplasts.…”

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

“…Additionally, redox regulation and product inhibition participate in modulating G6PD activity (Wendt et al, 2000;Debnam et al, 2004;Schürmann and Buchanan, 2008). It is interesting to note that G6PD6 was shown to be inactivated by oxidation (Wakao and Benning, 2005) and that total G6PD activity was reduced in salt-stressed aska mutants.…”

“…In plants, G6PD activity is present in the plastids and cytosol (Debnam and Emes, 1999). G6PD activity has been positively correlated with environmental stresses such as salt stress (Valderrama et al, 2006;Wang et al, 2008), aluminum toxicity (Slaski et al, 2006), drought (Scharte et al, 2009), and infection by viral and fungal pathogens (Sindelár and Sindelárová, 2002;Scharte et al, 2009); by contrast, a decrease in chloroplastic G6PD activity has been shown to be beneficial for withstanding oxidative stress (Debnam et al, 2004). Thus, the role of G6PD in the stress response is currently unclear.…”

“…In addition to regulation at the transcriptional level, redox regulation and the cellular NADPH/NADP + ratio modulate the activity of the different G6PD isoforms (Wendt et al, 2000;Debnam et al, 2004;Schürmann and Buchanan, 2008). G6PD is phosphorylated in animals and plants, which has been correlated with both enhanced (Ramnanan and Storey, 2006;Dieni and Storey, 2010;Gupte et al, 2011) and reduced activity (Hauschild and von Schaewen, 2003;Xu et al, 2005;Zhang et al, 2000).…”

The GSK3/Shaggy-like kinase ASKα contributes to pattern-triggered immunity in Arabidopsis thaliana

“…G6PD activity can be regulated by the cellular redox status, the NADPH-NADP + ratio, and protein phosphorylation (Wendt et al, 2000;Debnam et al, 2004;Schürmann and Buchanan, 2008;Dal Santo et al, 2012). Under high-salinity conditions, G6PD activity is regulated by the Arabidopsis protein kinase ASKa.…”