Enhancing ascorbate in fruits and tubers through over‐expression of the l‐galactose pathway gene GDP‐l‐galactose phosphorylase

Abstract

Summary Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l‐galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP‐l‐galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.

Allelic Variation in Paralogs of GDP-l-Galactose Phosphorylase Is a Major Determinant of Vitamin C Concentrations in Apple Fruit

“…GGP (VTC2; Fig. 1) catalyzes the first committed step of AsA biosynthesis and has also been suggested to be the rate-limiting step for AsA biosynthesis in plants (Linster and Clarke, 2008;Bulley et al, 2009Bulley et al, , 2012. Our results here provide strong evidence that MdGGP1 is the only structural gene of AsA metabolism that is tightly linked to flesh AsA concentrations in apple and that this is independent of the environmental conditions, since significant AsA-QTL were detected over all three years of measurement.…”

“…These regions on LG 9 and LG 16 have both been shown to contain major QTL for apple fruit polyphenolic contents (Chagné et al, 2007(Chagné et al, , 2012Khan et al, 2012). We have previously reported that the sunexposed (red) side of apples has higher anthocyanin and AsA concentrations and is more resistant to both biotic and abiotic stress than the shaded (green/ yellow) side (Davey et al, , 2007, and recently, Bulley et al (2012) demonstrated that transgenic fruits from tomato and strawberry with elevated AsA contents also had an approximately 50% larger polyphenolic pool. Finally, we report here positive correlations between TAA, AsA, and totAsA concentrations in both flesh and skin tissues and colocations between flesh AsA-and TAA-QTL on LGs 6, 11, and 16.…”

“…However, apart from GGP (Bulley et al, 2012), overexpression of these structural genes has to date had limited success in altering the fruit AsA pool (Agius et al, 2003;Bulley et al, 2009;Haroldsen et al, 2011).…”

“…Genes involved in several of these mechanisms have been proposed to be key regulators of fruit AsA concentrations, including GDP-L-Gal phosphorylase (GGP) or vitamin c defective2 (VTC2) in kiwifruit (Actinidia deliciosa; Bulley et al, 2009Bulley et al, , 2012 as well as GDP-Man-3,5-epimerase (GME; Gilbert et al, 2009), L-Gal-1-Pphosphatase (GPP or VTC4; Ioannidi et al, 2009), and MDHAR (Stevens et al, 2007) in tomato. However, apart from GGP (Bulley et al, 2012), overexpression of these structural genes has to date had limited success in altering the fruit AsA pool (Agius et al, 2003;Bulley et al, 2009;Haroldsen et al, 2011).…”

Analysis of Ascorbic Acid Biosynthesis Using a Simple Transient Gene Expression System in Tomato Fruit Protoplasts

“…Cronje et al 16) reported that AsA content in GDP-mannose pyrophosphorylase (GMPase), an enzyme located at first step of the Lgalactose pathway, overexpressing tomato fruit was more increased than that in the other pathway genes, aldono-lactone oxidase and myo-inositol oxygenase, overexpressing tomato fruit. Recently, Bulley et al 17) reported that overexpression of GDP-L-galactose phosphorylase (GGPase), an upstream enzyme in the GPPase in L-galactose pathway, 18) induced a 6-fold increase in AsA contents in tomato fruit. These results indicate that the overexpression of enzyme genes in the L-galactose pathway can effectively contribute to AsA increase in tomato fruits.…”