*4.2. Increasing Ascorbic Acid Improves Tolerance to Many Environmental Stresses*

Although oxygen is essential to plants, it can be highly damaging, particularly as singlet oxygen ( 1 O2) or in its reactive forms such as the superoxide anion (O2 •−), hydroxyl radical (**•** OH), or hydrogen peroxide (H2O2). ROS are detoxified through the action of antioxidants such as Asc and GSH either directly or in reactions catalyzed by SOD, APX, and CAT [69,70]. Under conditions of excess light, O2 •− is produced during photosynthesis and is converted by SOD to H2O2 which is reduced to H2O by APX as one means to maintain electron flow through the photosystems [71]. Abiotic stresses such as cold, drought, or high light increase ROS production by creating conditions

of light stress at lower light levels. H2O2 rapidly inactivates APX if Asc is limiting [72] and inhibits CO2 assimilation by inhibiting several Calvin cycle enzymes [40]. ROS can invade a plant in the form of environmental pollutants, e.g., ozone [73,74], which damages cell membranes or induces programmed cell death [75–77]. As a defense mechanism, H2O2 produced from ozone functions as a signaling intermediate in guard cells to promote stomatal closure thus limiting ozone entry into the leaf interior [78,79].

As an antioxidant, Asc would be expected to affect tolerance to environmental stress. This was first demonstrated using *vtc* mutants of *Arabidopsis* in which their reduced Asc content correlated with a reduction in tolerance to environmental ROS. With 70%–75% less Asc, the *vtc1* mutant is hypersensitive to ozone and sulfur dioxide [13,19,80] and contains a higher oxidative load relative to wild-type plants when exposed to stress conditions such as salt despite its increased GSH content [81]. The expression level of regulators of Asc biosynthesis can also affect the degree of ozone tolerance. Knockout mutants of AMR1 (for ascorbic acid mannose pathway regulator 1) resulted in up to 3-fold greater foliar Asc content in *Arabidopsis* and increased ozone tolerance [82]. In contrast, plants with increased expression of AMR1 through activation-tagging exhibited a 60% reduction in Asc and greater ozone sensitivity [82]. As AMR1 coordinately regulates transcript expression of six Smirnoff–Wheeler pathway enzyme genes to negatively regulate Asc biosynthesis, targeting regulators of biosynthetic pathways offers yet another promising approach to alter Asc content. In a second study, overexpression of the *Arabidopsis* ethylene response factor gene AtERF98 increased Asc content up to approximately 1.6-fold which was attributed primarily to an increase in the expression of genes in the Smirnoff–Wheeler pathway [83]. As AtERF98 binds to the promoter of *VTC1*, AtERF98 likely functions as a transcriptional activator of one or more genes in the Smirnoff–Wheeler pathway [83]. Increasing AtERF98 expression resulted in enhanced salt tolerance, demonstrating that increasing Asc biosynthesis improves tolerance to this abiotic stress [83].

That the endogenous level of apoplastic Asc is important in detoxifying ozone was shown in tobacco in which the level of apoplastic Asc was specifically altered [84]. Overexpressing an apoplastic-localized cucumber ascorbate oxidase (AO), which oxidizes apoplastic Asc, increased the ozone sensitivity of transgenic tobacco, correlating with the conversion of virtually all apoplastic Asc to DHA and depriving the apoplast of its ability to detoxify ozone entering the leaf interior [84]. A decrease in the cytosolic Asc redox state was also observed which would compromise the ability of a cell to detoxify ozone entering the cytosol.

Increased sensitivity to ozone following a reduction in Asc recycling was observed following loss of cytosolic DHAR expression in the *Arabidopsis AtDHAR3* mutant [58]. The lower redox state but not pool size of Asc in this mutant indicates that Asc recycling is important in preventing oxidative damage. Consistent with its role in ozone tolerance, *AtDHAR3* expression is induced by ozone [58].

If decreasing Asc content reduces tolerance to environmental ROS, increasing Asc content would be predicted to have the opposite effect, a notion supported by several studies published to date. Increasing Asc content in tobacco by increasing DHAR expression increased the Asc content of the apoplast and symplast and thus increased tolerance to ozone by reducing the oxidative load of the plant (*i.e.*, a lower level of foliar and apoplastic H2O2) which was accompanied by a lower induction of antioxidant-related enzyme activities, more chlorophyll, and a higher level of photosynthetic activity following ozone exposure [56]. This increase in tolerance occurred despite the guard cells

being less responsive to ozone as a consequence of their higher Asc content which reduces H2O2 levels [73,85]. Thus, increasing Asc content throughout a plant reduces guard cell responsiveness which permits more ozone to enter the leaf interior. The increased ozone tolerance can be understood, however, by the increased ability of every cell to detoxify ozone invading the leaf interior. Consistent with these findings, increasing Asc content 2-fold in tobacco through the expression of a cytosolic *Arabidopsis* DHAR enhanced its tolerance to ozone as well as drought, salt, or polyethylene glycol [61].

Conversely, a reduction in Asc recycling through the suppression of DHAR expression increased the responsiveness of guard cells to ozone thereby limiting ozone diffusion into the leaf interior [56]. At the same time, however, the decrease in DHAR activity lowered the Asc content of leaf cells and thus reduced their ability to detoxify any ozone that did invade [56]. Thus, increasing Asc content provides greater protection against environmental oxidative damage without compromising photosynthetic activity than does increasing guard cell responsiveness through decreasing Asc which reduces ozone entry but also reduces photosynthetic activity.

In addition to ozone, increasing Asc content provides greater tolerance to other environmental stresses. *Arabidopsis* with increased Asc content and redox state resulting from an increase in DHAR expression retained more Asc and chlorophyll with less membrane damage following exposure to high light and temperature or following treatment with paraquat [62]. *Arabidopsis* expressing a rice DHAR had greater tolerance to salt stress despite the small increases in DHAR activity and Asc achieved although no difference in cold tolerance was observed [63]. Although tobacco expressing a chloroplast-targeted human DHAR failed to increase Asc, it did increase the Asc redox state and the plants experienced less membrane damage following exposure to methyl viologen or H2O2 and had improved tolerance to low temperature and salt [60]. Combining expression of a chloroplast-localized DHAR with the expression of a chloroplast-localized CuZnSOD and APX increased the Asc and GSH redox states and the plants exhibited greater tolerance to paraquat and salt [86]. Greater tolerance to salt and cold was also observed in tobacco following the simultaneous expression of two pairs of chloroplast-localized enzymes, *i.e.*, an *E. coli* GR with either an *E. coli* glutathione-S-transferase (GST) or a rice DHAR, that increased Asc and GSH content and their redox states [64].

Because fewer studies on increasing Asc through MDAR expression have been reported and those that have been carried out have observed smaller increases in Asc content, much less is known about the effects of MDAR-mediated increases in Asc on plant growth and plant responses. However, the results to date suggest that increasing Asc through MDAR expression has similar effects to those following an increase in DHAR expression. The slight increase in Asc content and decrease in DHA content that resulted in an approximate doubling of the Asc redox state in tomato seedlings overexpressing a chloroplast-targeted tomato MDAR resulted in a reduced oxidative load (as measured by H2O2), lower thiobarbituric acid reactive substance (TBARS) content (a measure of membrane damage), a higher net photosynthetic rate, higher maximal photochemical efficiency of PSII and greater fresh weight when subjected to low or high temperature stress [51]. Reducing Asc and its redox state through the suppression of MDAR expression resulted in largely opposite phenotypes [51]. In agreement with these results, greater tolerance to ozone, reduced H2O2 levels, and increased photosynthetic activity were observed in tobacco expressing an *Arabidopsis* MDAR following salt stress [50].

ROS can also be generated during development. For example, H2O2 is produced in the peroxisome of oilseeds as a by-product of fatty acid β-oxidation during lipid catabolism that accompanies seedling growth [87,88]. Catalase in the peroxisomal matrix detoxifies H2O2 and a membrane-bound APX3 and MDAR4, encoded by *SUGAR-DEPENDENT2* (*SDP2*), together detoxify H2O2 using Asc [87,89–91]. Loss of MDAR4 expression in the *Arabidopsis sdp2* mutant is conditionally seedling-lethal as MDAR activity is needed to reduce leakage of H2O2 from peroxisomes that protects *SDP1*-encoded triacylglycerol (TAG) lipase activity and storage oil hydrolysis in the closely associated oil bodies during seedling growth [92]. Loss of MDAR4 activity results in inactivation of TAG lipase by H2O2 and a reduced ability to catabolize storage oil needed to support seedling growth [92]. Whether increasing Asc through increasing MDAR4 expression might improve seedling growth has not been examined. However, increasing APX3 expression increases tolerance against oxidative stress [93], suggesting an increase in Asc and the peroxisomal-associated APX3 and MDAR4 that use and recycle Asc may improve seedling tolerance against oxidative stress.
