Abstract
Ascorbic acid (AsA) has high antioxidant activities, and its biosynthesis has been well studied by engineering of a single structural gene (SG) in staple crops, such as tomato (Solanum lycopersicum). However, engineering the AsA metabolic pathway by multi-SG for biofortification remains unclear. In this study, pyramiding transgenic lines including GDP-Mannose 3′,5′-epimerase (GME) × GDP-d-mannose pyrophosphorylase (GMP), GDP-l-Gal phosphorylase (GGP) × l-Gal-1-P phosphatase (GPP) and GME × GMP × GGP × GPP, were obtained by hybridization of four key genes to get over-expression transgenic plants (GME, GMP, GGP, and GPP) in tomato. Pyramiding lines exhibited a significant increase in total ascorbate in leaves and red fruits except for GGP × GPP. Expression analysis indicated that increased accumulation of AsA in pyramiding transgenic lines is due to multigene regulation in AsA biosynthesis. Substrate feeding in leaf and fruit suggested that AsA biosynthesis was mainly contributed by the d-Man/l-Gal pathway in leaves, while alternative pathways may contribute to AsA accumulation in tomato fruit. Pyramiding lines showed an enhanced light response, stress tolerance, and AsA transport capacity. Also, fruit shape, fruit size, and soluble solids were slightly affected by pyramiding. This study provides the first comprehensive analysis of gene pyramiding for ascorbate biosynthesis in tomato. SGs pyramiding promotes AsA biosynthesis, which in turn enhances light response and oxidative stress tolerance. Also, the data revealed an alternative ascorbate biosynthesis pathway between leaves and fruit of tomato.
Highlights
L-Ascorbic acid (AsA, vitamin C) is a highly abundant and essential organic acid for plants and animals
The GDP-D-mannose pyrophosphorylase (GMP) × GME and GDP-L-Gal phosphorylase (GGP) × Gal-1-P phosphatase (GPP) pyramiding lines were generated by conventional crossing of GMP with GME and GGP with GPP, respectively
The total AsA and reduced AsA levels in GGP overexpression line and GMP × GME, GMP × GME × GGP × GPP pyramiding lines showed a 2-fold increase compared with wild-type, and significant increase was observed in GMP, GME, GPP, GGP × GPP lines (Figure 1A)
Summary
L-Ascorbic acid (AsA, vitamin C) is a highly abundant and essential organic acid for plants and animals. As an antioxidant and enzyme cofactor, AsA plays a crucial role in various plant physiological processes, including removing reactive oxygen species (ROS) [1,2], enhancing oxidative stress tolerance [3], premature senescence and programmed cell death (PCD) [4,5], and cell elongation and division in plants [6]. It has been shown that 30% to 40% of AsA in plant cells is located in the chloroplast, which plants use to produce ROS under light respiration and photosynthesis, suggesting. AsA plays a vital role in protecting against side effects of photosynthesis in plant [7,8]. Fresh vegetables and fruits including tomato are considered as the primary sources of vitamin C in the human diet
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