*3.1. Targeting MDAR Expression to Increase Ascorbic Acid*

Once used in enzymatic or non-enzymatic reactions, Asc is oxidized to monodehydroascorbate (MDHA). Asc can be regenerated from MDHA through reduction by several means. If MDHA is produced in the chloroplast stroma, it can be recycled to Asc by ferredoxin (Fd), which is part of the photosynthetic electron transport chain, or by monodehydroascorbate reductase (MDAR) in the stroma [40]. Other MDAR isoforms are present in the cytosol, peroxisome, and mitochondria which reduce MDHA produced in those compartments. As no MDAR isoform is in the thylakoid lumen, MDHA cannot be reduced by MDAR or by Fd, which lies on the stromal side of the thylakoid membrane. As a result, this short-lived radical spontaneously disproportionates rapidly to Asc and DHA, particularly when the pH of the thylakoid lumen is low which occurs during the light driven transport of protons across the thylakoid membrane from the stroma into the lumen [40,41]. Under these conditions, the high pH of the stroma slows the disproportionation of MDHA and it undergoes reduction primarily through Fd or MDAR. Once photoreduced by PsaC in the PSI complex, Fd reduces MDHA directly or alternatively reduces NADP<sup>+</sup> to NADPH as catalyzed by Fd-NADP<sup>+</sup> reductase (FNR) which MDAR uses (or NADH instead of NADPH) to reduce MDHA to Asc [42,43]. Fd reduces MDHA at a rate that is 34-fold greater than the rate of photoreduction of NADP<sup>+</sup> so that MDHA is likely reduced through Fd as part of the thylakoid scavenging system rather than by stromal MDAR when it is produced proximal to the thylakoid membrane [42]. MDAR, however, is

available to reduce any stromal MDHA produced distal to the thylakoid membrane as part of the stromal scavenging system.

The multiple isoforms of MDAR are encoded by a five member nuclear gene family in *Arabidopsis* (referred to as *AtMDAR1* through *AtMDAR5*) that are targeted to the cytosol, chloroplast, mitochondria, and peroxisomes [44]. Dual targeting of MDAR to chloroplasts and mitochondria results from the use of at least two transcription start sites which produce a seven amino acid extension in the mitochondrial-targeted form of the protein [45]. The 47-kDa AtMDAR1 and 54-kDa AtMDAR4 isoforms contain a *C*-terminal sequence that targets them to the peroxisomal matrix (PTS1) and peroxisomal membrane, respectively [46]. MDAR isoforms targeted to peroxisomes, chloroplasts, or mitochondria typically function together with ascorbate peroxidase (APX) to scavenge H2O2 [47] through the transfer of electrons from two molecules of Asc to H2O2 to form water and two molecules of MDHA. Disproportionation of H2O2 is also catalyzed by catalase (CAT) when present, e.g., in the peroxisome.

Increasing Asc content by targeting MDAR expression has achieved only limited success. Expression of a cytosolic tomato MDAR from a constitutive promoter in tomato (var. Micro-Tom) resulted in a reduction in Asc in mature green tomato fruits but unaltered foliar Asc content [48] although it may improve the chilling tolerance of fruit [49]. Improved tolerance against salt and osmotic stresses was also observed following an increase in MDAR expression in tobacco [50]. Increasing expression of a tomato chloroplast-targeted MDAR in tomato increased Asc marginally (1.2-fold) but was accompanied by a decrease in DHA, resulting in an approximate doubling of the Asc redox state [51]. Similar results were obtained following the expression of an *Arabidopsis*  cytosolic MDAR in tobacco [52]. The little work that has been reported to date suggests that increasing MDAR expression may achieve only minor increases in Asc content.
