Over-expression of l-gulono-γ-lactone oxidase (GLOase) gene leads to ascorbate accumulation with enhanced abiotic stress tolerance in tomato

Abstract

Vitamin C (ascorbic acid, AsA) is an essential component for collagen biosynthesis and also for correct functioning of the cardiovascular system in humans. Plants and several animals can synthesize ascorbic acid, whereas humans lack the gene essential for ascorbic acid biosynthesis and must acquire the vitamin from their diet. In the present study, we developed a transgenic tomato plant (Lycopersicon esculentum Mill.) over-expressing the rat L-gulono-γ-lactone oxidase (GLOase) gene driven by CaMV35S constitutive promoter. The overexpression of GLOase resulted in a 1.5-fold increase in AsA levels in transgenic tomato fruits, which was positively correlated with increased GLOase activity. The Southern and reverse transcriptase polymerase chain reaction results revealed the stable integration of the transgene in the progeny of the engineered plants. The transgenic plants with increased vitamin C content in the fruit showed enhanced tolerance to abiotic stresses induced by methyl viologen, NaCl, and mannitol as compared with wild-type plants. The leaf disc senescence assay showed better tolerance in transgenic plants by retaining higher chlorophyll content compared with wild-type plants. The T 1 plants survived under salt stress (200 mM NaCl) whereas wild-type plants were unable to survive such high salt concentrations. This study suggests that the increased accumulation of AsA could upregulate the antioxidant system which imparts improved tolerance against various abiotic stresses in transgenic tomato plants as compared with wild-type plants.

Ectopic expression of FaGalUR leads to ascorbate accumulation with enhanced oxidative stress, cold, and salt tolerance in tomato

“…In this study, the over-expression of FaGalUR led to elevated ascorbic acid content in tomato and improved tolerance to oxidative (75 lM MV), salt (200 mM), and cold (4°C) stresses. Similar results were obtained in transgenic plants expressing ascorbate biosynthesis-related genes, including tomato GDP-Mannose 3 0 ,5 0 -epimerase gene, strawberry Dgalacturonic acid reductase gene and rat L-gulono-c-lactone oxidase gene that exhibited better survival under various abiotic stresses (Hemavathi et al 2009;Zhang et al 2011a;Lim et al 2012;Hemavathi et al 2010).…”

“…Consequently, the compound plays a very important role in resistance to a number of environmental stresses. The increasing evidence suggests that elevating ascorbate content could enhance tolerance to various abiotic stresses, such as oxidative (induced by MV), salt, mannitol, ozone, osmosis and chilling stresses, under in vitro conditions (Lim et al 2012;Hemavathi et al 2009;Zhang et al 2011a;Eltayeb et al 2007;Duan et al 2012a;Li et al 2010;Sun et al 2010). In this study, the over-expression of FaGalUR led to elevated ascorbic acid content in tomato and improved tolerance to oxidative (75 lM MV), salt (200 mM), and cold (4°C) stresses.…”

“…Overexpressing a yeast-derived GDP-mannose pyrophosphorylase (GMP) with constitutive CaMV 35S promoter increased ascorbic acid content by up to 50 and 35 % in green and red fruit, respectively (Cronje et al 2012); Overexpression of GDP-D-mannose 3,5-epimerase (GME) genes under the control of CaMV35S promoter exhibited a significant increase in total ascorbic acid in leaves and red fruit in tomato (Zhang et al 2011a), leading to a 1.42-fold in leaves, and 1.60-fold in red ripe fruits; over-expressing the kiwifruit GGP, under the control of the 35S promoter, had significantly raised ascorbic acid concentration in fruit by threefold to sixfold increase (Bulley et al 2012); Over-expressing L-gulono-clactone oxidase (GLOase) gene resulted in a 1.5-fold increase in AsA levels in transgenic tomato fruits, which correlated with increased GLOase activity (Lim et al 2012). Suppressed expression of ascorbate oxidase gene (AO) (Zhang et al 2011c) and mitochondrial ascorbate peroxidase (mitAPX) (Zhang et al 2011b) gene modulate ascorbic acid content in tomato fruit, leading to 1.5-fold and 2.2-fold increase in ascorbic acid levels compared with wild-type control, respectively.…”

Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis

“…On the other hand, RnGulLO may not undergo such a post-transcriptional regulation as plant GulLOs undergo, because RnGulLO was able to use the available L-GulL pool, as evidenced by an increase in AsA content in several plant species when RnGulLO was overexpressed [21‒23], Figure 10). Although RnGulLO can use both L-GalL and L-GulL as substrates [12], it is conceivable that RnGulLO uses L-GulL for two reasons.…”

“…Rat GulLO (RnGulLO) can use both L-GalL and L-gulono-1,4-lactone (L-GulL) as substrates [12, 13, 15], while plant GLDHs are very specific to L-GalL and have limited activity or none towards L-GulL [16‒20]. An increase in AsA content was observed by the overexpression of RnGulLO in tobacco [21], potato [22], tomato [23] and Arabidopsis [24], whereas no increased AsA was observed in tobacco when GLDH was overexpressed [25]. This suggests that the flux through L-GulL can also be used to increase AsA in plants irrespective of the availability of the L-GalL pool.…”