Elevating vitamin C content via overexpression of myo-inositol oxygenase and l-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses

Abstract

l-Ascorbic acid (vitamin C) is an abundant metabolite in plant cells and tissues. Ascorbate functions as an antioxidant, as an enzyme cofactor, and plays essential roles in multiple physiological processes including photosynthesis, photoprotection, control of cell cycle and cell elongation, and modulation of flowering time, gene regulation, and senescence. The importance of this key molecule in regulating whole plant morphology, cell structure, and plant development has been clearly established via characterization of low vitamin C mutants of Arabidopsis, potato, tobacco, tomato, and rice. However, the consequences of elevating ascorbate content in plant growth and development are poorly understood. Here we demonstrate that Arabidopsis lines over-expressing a myo-inositol oxygenase or an l-gulono-1,4-lactone oxidase, containing elevated ascorbate, display enhanced growth and biomass accumulation of both aerial and root tissues. To our knowledge this is the first study demonstrating such a marked positive effect in plant growth in lines engineered to contain elevated vitamin C content. In addition, we present evidence showing that these lines are tolerant to a wide range of abiotic stresses including salt, cold, and heat. Total ascorbate content of the transgenic lines remained higher than those of controls under the abiotic stresses tested. Interestingly, exposure to pyrene, a polycyclic aromatic hydrocarbon and known inducer of oxidative stress in plants, leads to stunted growth of the aerial tissue, reduction in the number of root hairs, and inhibition of leaf expansion in wild type plants, while these symptoms are less severe in the over-expressers. Our results indicate the potential of this metabolic engineering strategy to develop crops with enhanced biomass, abiotic stress tolerance, and phytoremediation capabilities.

Keywords: Vitamin C; ascorbic acid; phytoremediation; plant growth; stress tolerance.

Figure 1

Pathways involved in ascorbate biosynthesis and regeneration in plants: The d-mannose/l-galactose (Man/Gal) route, the l-gulose (Gul) shunt, the d-galacturonate (GalU) pathway, and the myo-inositol (MI) route. A purple acid phosphatase with phytase activity has been shown to channel phytate to the MI pathway, while VTC4 has been shown to also use l-myo-inositol-1 phosphate and contribute to both myo-inositol and ascorbate metabolisms. The enzymes participating in the Man/Gal route are: Phosphoglucose isomerase (EC 5.3.1.9); phosphomannose isomerase (PMI, EC 5.1.3.1.8); phosphomannose mutase (PMM, EC 5.4.2.8); GDP-mannose pyrophosphorylase (VTC1, EC 2.7.7.13); GDP-mannose-3’,5’-epimerase (GME, EC 5.1.3.18); GDP-galactose phosphorylase (VTC2, EC 2.7.7.B2); l-galactose-1-phosphate phosphatase (VTC4); l-galactose dehydrogenase (GalDH, EC 1.1.1.48); l-galactono-1,4-lactone dehydrogenase (GLDH, EC 1.3.2.3). The enzymes in the GalU pathway are: d-galacturonate reductase (GalUR) and gluconolactonase (EC 3.1.1.17). The enzymes in the MI pathway are: Inositol phosphate phosphatase (EC 3.1.3.25); myo-inositol oxygenase (MIOX, EC 1.13.99.1); glucuronate reductase (GlcUR, EC 1.1.1.19); gluconolactonase (GNL, EC 3.1.1.17), and l-gulono-1,4-lactone oxidase (GLOase, EC 1.1.3.8). The enzymes involved in AsA recycling are: Monodehydroascorbate reductase (MDHAR, EC. 1.6.5.4), and dehydroascorbate reductase (DHAR, EC 1.8.5.1). Where omitted EC number have not been assigned. GLDH is a known mitochondrial enzyme. In this study we analyzed in detail Arabidopsis lines constitutively expressing MIOX and GLOase, enzymes in the MI pathway (underlined).

Figure 2

Arabidopsis lines with elevated foliar AsA content (MIOX4 and GLOase over-expressers) show enhanced biomass when grown in soil under normal conditions. (a) Total ascorbate content (µmol g⁻¹ FW), (b) dry weight of aerial tissue (mg), (c) inflorescence height (cm), and (d) rosette diameter (cm). The proportion of reduced ascorbate was 95% or higher of the total AsA. Data represent the means ± standard deviation (SD) of 40 replicates. *Indicates significant differences between WT and high-AsA lines as determined by t-test (P=0.05).

Figure 3

Arabidopsis lines with elevated foliar AsA content show enhanced biomass of both aerial and root tissues when grown in liquid culture. Ten-day old seedlings were inoculated into 125 ml flasks, containing 35 ml liquid MS media. Biomass was harvested after 2 wk growth in liquid culture. Comparison of aerial tissue biomass of WT (a), pCAMBIA (b), MIOX4 (c), and GLOase (d) lines. Comparison of root biomass of WT (e), pCAMBIA (f), MIOX4 (g), and GLOase (h) lines. Dry weight measurements corresponding of WT, pCAMBIA, MIOX4 and GLOase plants grown in liquid culture. Data represent the means ± SD of 6 replicates (i). *Indicates statistically significant differences between WT and high-AsA lines (t-test, P=0.05).

Figure 4

Effect of salt stress on the growth and AsA content of MIOX4 and GLOase over-expressers compared to WT controls. (a) Effect of NaCl on root length of the wild type and transgenic lines grown on agar plates plus NaCl for 19 d. Data are means ± SD of 50 biological replicates. (b) Total AsA content of seedlings grown on plates plus NaCl. Data are means ± SD of 5 biological replicates (each replicate containing five seedlings). (c) Phenotype of individual plants under NaCl stress after 2 wk of stress treatment. (d) Effect of salt stress on a population of 6-wk-old plantlets. (e) Total AsA content of soil-grown plants watered with NaCl until they reached the end of the vegetative growth (developmental stage 5.1). Data are means ± SD of 5 biological replicates. The proportion of reduced ascorbate was 95% or higher of the total AsA. *Indicates statistically significant differences between WT and high-AsA lines (t-test, P=0.05).

Figure 5

High-AsA Arabidopsis lines are tolerant to cold stress (16°C) and maintain elevated AsA under this stress. (a) Dry weight of aerial tissue (mg), (b) inflorescence height (cm), and (c) rosette diameter (cm). Data represent the means ± SD of 40 replicates. (d) Total AsA content of soil-grown plants under cold stress. Leaf tissue was collected at the end of the vegetative growth. Data are means ± SD of 5 biological replicates. The proportion of reduced ascorbate was 95% or higher of the total AsA. *Indicates significant differences between WT and high-AsA lines as determined by t-test (P=0.05).

Figure 6

Effect of heat shock (29°C) on the phenotype and AsA content of GLOase, MIOX4, and control plants at10 d after heat stress. Wild type (WT) displayed chlorosis (loss of chlorophyll) after heat exposure (a), while MIOX4 and GLOase retained their chlorophyll level (b). Data represent the means ± SD of 3 replicates. (c) Total AsA content of seedlings grown under heat stress. Data are means ± SD of 5 biological replicates (each replicate containing five seedlings). The proportion of reduced ascorbate was 95% or higher of the total AsA. *Indicates significant differences between WT and high-AsA lines (t-test, P=0.05).

Figure 7

Elevated ascorbate protects Arabidopsis from stress-induced morphogenetic responses caused by pyrene (PYR). (a) Reduction of the growth of the aerial tissue in WT, while high-AsA lines grow normal in the presence of PYR. PYR inhibits leaf expansion (b) and reduces the amount of root hairs (c) in WT, but does not affect high-AsA lines (GLOase data shown here). Seeds were germinated on MS medium and then transferred to MS plus PYR for 14 d. Images were collected 3 wk after sowing. (d) Effect of PYR on root length of the wild type and transgenic lines grown on agar plates plus PYR for 19 d. Data are means ± SD of 50 biological replicates. (e) Total AsA content of seedlings grown under PYR stress. Data are means ± SD of 5 biological replicates (each replicate containing five seedlings). The proportion of reduced ascorbate was 95% or higher of the total AsA. *Indicates significant differences between WT and high-AsA lines (t-test, P=0.05).