*3.2. Targeting DHAR Expression to Increase Ascorbic Acid*

If MDHA is not enzymatically reduced by Fd or MDAR, it will undergo spontaneous disproportionation to Asc and DHA, the rate of which is dependent on the pH, such as in the thylakoid lumen where Fd and MDAR are absent and the pH is low during light exposure. Disproportionation of MDHA can also occur in other cellular compartments if not enzymatically reduced. The DHA produced can be reduced to Asc by dehydroascorbate reductase (DHAR) using glutathione (GSH) as the reductant [53,54] (Figure 2). If it is not rapidly reduced, DHA undergoes irreversible hydrolysis to 2,3-diketogulonic acid and, as this is unable to be converted to Asc, it is lost to the Asc pool. Increasing the level of DHAR activity, therefore, limits DHA degradation by improving its recycling back into Asc before it is lost. As DHAR activity determines the relative levels of DHA and Asc and the enzyme is expressed in rate-limiting amounts in plants, it serves as a major regulator of the Asc redox state [5,55–57].

**Figure 2.** L-Ascorbic acid recycling through DHAR and MDAR. Following Asc synthesis from L-galactono-1,4-lactone by L-galactono-1,4-lactone dehydrogenase (GLDH) and oxidization to monodehydroascorbate (MDHA), monodehydroascorbate reductase (MDAR) can reduce MDHA to Asc. Alternatively, two MDHA molecules can disproportionate non-enzymatically to Asc and dehydroascorbate (DHA). Dehydroascorbate reductase (DHAR) can reduce DHA to Asc using glutathione (GSH) as the reductant. Oxidized glutathione (GSSG) is reduced by glutathione reductase (GR) to GSH using NADPH as the reductant. DHA will spontaneously hydrolyze to 2,3-diketogulonic acid if not reduced by DHAR.

DHAR is encoded by three gene members in *Arabidopsis*: (*AtDHAR1*; At5g16710), (*AtDHAR3*; At1g75270), and (*AtDHAR5*; At1g19570) [44]. Microarray expression analysis suggests another gene, At5g36270 (*AtDHAR2*), is likely a pseudogene as it may not be expressed [44]. A fifth gene, At1g19550 (*AtDHAR4*), is smaller than other DHAR paralogs due to multiple deletions throughout the polypeptide. AtDHAR3 is likely cytoplasmic while AtDHAR5 and AtDHAR1 are targeted to the mitochondria and chloroplast, respectively [58]. No DHAR isoform is transported to the apoplast. Consequently, any Asc transported to the apoplast is quickly oxidized, disproportionates, and the resulting DHA is either degraded or transported to the cytoplasm for recycling into Asc.

Because DHAR is a major recycler of Asc, a number of studies have focused on increasing the expression of this enzyme as a means to increase Asc content in plants, which has achieved success in several species. Although ectopic expression of a human DHAR in tobacco chloroplasts failed to increase Asc despite a 2-fold increase in DHAR activity [59,60], expression of a cytosolic wheat DHAR in tobacco did increase Asc content up to 4-fold as well as the redox state (*i.e.*, an increase in the Asc to DHA ratio) with a simultaneous increase in Asc and a decrease in DHA [5]. Similar results were obtained when this cytosolic wheat DHAR was expressed in leaves and developing kernels of maize [5], demonstrating that changes in Asc can be made in photosynthetic and non-photosynthetic organs. Because Asc is transported from the cytoplasm to the apoplast and apoplastic DHA is transported back to the cytoplasm, expression of the cytosolic wheat DHAR not only increased the Asc content of the cytosol but the apoplast as well when measured from the apoplastic fluid [55], demonstrating that cytosolic DHAR regulates the symplastic and apoplastic Asc pool size and redox

state. No change in Asc biosynthesis was observed following the increase in DHAR expression indicating that the synthesis of Asc and its recycling are independently controlled. The increase in DHAR expression and Asc recycling was accompanied by an increase in the GSH pool size and redox state [5]. As GSH is used by DHAR as the reductant, this suggests that the GSH pool size is affected by changes in DHAR activity. The extent to which the increase in GSH contributed to any physiological changes in these plants was not examined.

As the Asc pool size is determined by the rate of its synthesis and decay, the ability of DHAR to increase Asc content is a consequence of its recycling function that reduces DHA before it is lost through decay. As increasing DHAR expression results in improved Asc recycling and higher Asc levels, the endogenous level of DHAR is likely rate-limiting. Whether this is generally true throughout plant species is unknown. Consequently, the potential to increase Asc through increased DHAR expression will be greatest for species in which DHAR expression is rate-limiting. The strategy of increasing Asc content through increased DHAR expression, however, has been validated by subsequent studies that increased DHAR expression in the cytosol or in the chloroplast of a variety of species. Two studies that expressed a cytosolic DHAR from *Arabidopsis* in tobacco reported increases in Asc content by nearly 2-fold [52,61] whereas expression of an *Arabidopsis* cytosolic DHAR in *Arabidopsis* increased foliar Asc by 2 to 4.25-fold [62]. Expression of a rice cytosolic DHAR in *Arabidopsis* resulted in a slight increase in Asc content [63] as did expression of a rice DHAR in transformed tobacco chloroplasts [64]. Further increases in Asc content were observed when chloroplasts were transformed with glutathione reductase (GR) and DHAR [64].

Grains represent the most important food group supporting the global population either directly or indirectly as use in animal feed. Improving the nutritional value of grains offers the greatest potential for improving the diet of many and recent research has focused on engineering increasing multiple vitamins and micronutrients in grains as an efficient delivery mechanism for those whose diets are deficient in several vitamins. Although Asc content in grains is typically low, it is present during grain development but undergoes progressive oxidation during late development and is largely present as DHA by maturity [65]. The relationship between increased DHAR expression and increased Asc content in cereals was first shown in developing maize grain [5]. This was followed by a combinatorial approach to increase the levels of Asc, folate and β-carotene in maize grain using a barley D-hordein promoter to drive expression of a rice DHAR and an *E. coli* GTP cyclohydrolase (*folE*) to increase the level of Asc and folate, respectively, and a wheat LMW glutenin promoter to drive expression of maize phytoene synthase (*psy1*) and the D-hordein promoter to drive expression of *Pantoea ananatis* carotene desaturase (*crtI*) in order to increase β-carotene content [7]. These transgenes resulted in a 6-fold increase in Asc, a 2-fold increase in folate, and a 169-fold increase in β-carotene [7], demonstrating that an increase in Asc content can be combined with increases in the level of other vitamins to improve substantially the nutritional value of a fundamentally important staple. Increasing Asc content in other important, non-grain foods has been reported. Expression of a potato cytosolic DHAR from the CaMV 35S promoter increased foliar Asc content by more than 1.6-fold and in tubers by more than 1.2-fold which correlated with its expression where it is expressed higher in tubers than in leaves [66]. Expression of a chloroplast-localized potato DHAR increased foliar Asc content up to 1.5-fold but not in tubers which also correlated with its expression in leaves but a lack of expression in tubers [66]. Therefore, the strategy of increasing Asc content

through increased Asc recycling through chloroplast-targeting of DHAR expression is likely to be limited to photosynthetically active tissues whereas increasing Asc content in non-photosynthetic organs will likely require expression of a cytosolic isoform of DHAR. Supporting this conclusion were results from the expression of a sesame DHAR under the control of a patatin promoter in potato. Just as the patatin promoter is active in tubers but not in leaves, expression of the sesame DHAR increased Asc 1.1 to 1.3-fold in tubers but no increase was observed in leaves. In contrast, expression of sesame DHAR under the constitutively active CaMV 35S promoter increased Asc content in leaves by 1.5-fold and 1.6-fold in tubers [67]. Overexpressing a cytosolic tomato DHAR from a constitutive promoter in tomato (var. Micro-Tom) increased Asc content in mature green and red ripe fruit by 1.6-fold in plants grown under low light [48]. In this example, however, no increase in foliar Asc was observed. The increase in Asc and GSH observed during the initial phases of embryogeny in Norway spruce following overexpression of the class I homeobox of knox 3 gene, HBK3, was attributed to increased activities of DHAR, GR, and ascorbate free radical reductase [68], suggesting that DHAR may also contribute to regulating Asc content in gymnosperm species.
