Increasing vitamin C content of plants through enhanced ascorbate recycling

Abstract

Vitamin C (ascorbic acid) is essential to prevent disease associated with connective tissue (e.g., scurvy), improves cardiovascular and immune cell functions, and is used to regenerate alpha-tocopherol (vitamin E). In contrast to most animals, humans lack the ability to synthesize ascorbic acid as a result of a mutation in the last enzyme required for ascorbate biosynthesis. Vitamin C, therefore, must be obtained from dietary sources and, because it cannot be stored in the body, it must be obtained regularly. Once used, ascorbic acid can be regenerated from its oxidized form in a reaction catalyzed by dehydroascorbate reductase (DHAR). To examine whether overexpression of DHAR in plants would increase the level of ascorbic acid through improved ascorbate recycling, a DHAR cDNA from wheat was isolated and expressed in tobacco and maize, where DHAR expression was increased up to 32- and 100-fold, respectively. The increase in DHAR expression increased foliar and kernel ascorbic acid levels 2- to 4-fold and significantly increased the ascorbate redox state in both tobacco and maize. In addition, the level of glutathione, the reductant used by DHAR, also increased, as did its redox state. These results demonstrate that the vitamin C content of plants can be elevated by increasing expression of the enzyme responsible for recycling ascorbate.

Figure 1

Isolation of DHAR and overexpression in tobacco. (A) AsA is synthesized from l-galactono-1,4-lactone. AsA is oxidized to MDHA, which is converted to AsA by MDHAR or disproportionates nonenzymatically to AsA and DHA. DHA spontaneously hydrolyzes to 2,3-diketogulonic acid unless salvaged by DHAR, which uses GSH as the reductant. Oxidized glutathione (GSSG) is reduced by glutathione reductase (GR). (B) Alignment of the amino acid sequence of wheat (Triticum aestivum) DHAR with that of rice (Oryza sativa), maize (Zea mays), tobacco (N. tabacum), tomato (Lycopersicon esculentum), and Arabidopsis. Identity with wheat DHAR is indicated by dots, whereas positions that differ are shown; dashes represent gaps introduced to maintain alignment. The absolute consensus (Con) is also shown. GenBank accession numbers are as follows: wheat, AY074784; rice, AY074786; tobacco, AY074787; Arabidopsis, AY074785; maize, AW258053 and BE552888; and tomato, AW093721. (C) Western analysis of wheat DHAR expression in T₁ tobacco (i.e., B4, C4, and D1) and a vector-only control plant (i.e., Con) is shown (Left). The presence of the His-tag wheat (TaDHAR) and endogenous tobacco (NtDHAR) proteins is indicated. Western analysis of eukaryotic elongation factor 1A (eEF1A) was used as a control. DHAR activity measured in each leaf type is represented by the histograms. The average and SDs are reported for three independent measurements. FW, fresh weight.

Figure 2

Overexpression of DHAR increases ascorbic acid and glutathione in tobacco. (A) AsA and DHA. (B) GSH and GSSG. (C) MDHAR (10⁻⁸ mol of NAPDH oxidized per min per mg of protein), APX (10⁻⁸ mol of AsA oxidized per min per mg of protein), GR (10⁻⁸ mol of NAPDH oxidized per min per mg of protein), SOD (units to inhibit nitroblue tetrazolium (NBT) photoreduction by 50%), and CAT (10⁻⁶ mol of H₂O₂ reduced per min per mg of protein) activities were measured in expanding leaves, mature leaves exhibiting maximal photosynthetic activity, presenescent leaves of control (Con), and three T₁ DHAR-overexpressing transgenics (i.e., B4, C4, and D1). The level of AsA or GSH (black histograms), DHA or GSSG (white histograms), and the enzyme activities were assayed in three independent replicates of leaves pooled from three independent plants, and the average and SD were reported. The fold increase in AsA or GSH relative to the control is indicated above the histograms. The ratio of AsA to DHA or GSH to GSSG in each line is indicated below each pair of histograms.

Figure 3

Overexpression of DHAR increases ascorbic acid and glutathione content in maize leaves. (A) DHAR activity. (B) AsA and DHA. (C) GSH and GSSG. (D) MDHAR, APX, GR, SOD, CAT, and GLDH (10⁻⁸ mol of cytochrome c reduced per min per mg of protein) activities. (E) DHAR and ribosomal protein S6 were measured in expanding leaves of 4-week-old maize. AsA or GSH (black histograms), DHA or GSSG (white histograms), and enzyme activities were assayed in three independent assays, and the average and SD were reported. For each line, leaves from syngeneic (i.e., 19C, 62C, and 65C) and DHAR-expressing (i.e., 19, 62, and 65) individuals were assayed.

Figure 4

Overexpression of DHAR increases ascorbic acid and glutathione content in maize kernels. (A) DHAR activity. (B) AsA and DHA. (C) GSH and GSSG. (D) MDHAR, APX, GR, SOD, and CAT. (E) DHAR and ribosomal protein S6 were measured in developing maize kernels. AsA or GSH (black histograms) and DHA or GSSG (white histograms) were assayed in three independent assays, and the average and SD were reported. For each line, kernels from a syngeneic line (Con) and four DHAR-expressing lines (i.e., 14, 21, 65, and 29) were assayed.