Ascorbate (ASC) is a major soluble redox molecule with pivotal roles in allowing several metabolic pathways to work properly. ASC regenerates other metabolites, among which tocopherols, from oxidative damages and protects the catalytic site of a number of enzymes (e.g., hydroxylases) from irreversible oxidation, possibly caused by reactive oxygen species (ROS) in both animal and plant cells. It can be used as substrate or enzyme cofactor in various biological reactions (Lodge, 2008; De Gara et al., 2010). ASC is synthesized by fungi, protozoa, plants, and animals, even if by means of different biosynthetic pathways (Bleeg and Christensen, 1982; Banhegyi et al., 1997; Wheeler et al., 1998; Logan et al., 2007). For few animal species, among which guinea pig, some birds, humans, and primates in general, ASC is a vitamin (vitamin C), since during their evolution, they have lost the capability to synthesize it. In human this was caused by the loss of functionality of gulono-1,4 γ-lactone oxidase (GuLO), the enzyme catalyzing the last step of animal ASC biosynthesis (Nishikimi et al., 1992, 1994; Figure 1). Although ASC is considered essential for aerobic life (De Gara et al., 2010; Gest et al., 2013), it is worth noting that in fungi, protozoa, and animals the last reaction of its biosynthesis also produces hydrogen peroxide (H₂O₂), a putative toxic species (Banhegyi et al., 1997; Figure 1). Therefore, in animals consuming foods that ensure sufficient ASC intake, the loss of ASC biosynthetic capability may be done an evolutionary acquisition leading to an ameliorated control of redox homeostasis in the cells of these organisms. It is interesting to notice that plants, which produce a great amount of ASC in almost all their tissues (ASC reaches several tens of millimolar concentrations in green tissues), have evolved a synthesizing pathway with the last step catalyzed by a dehydrogenase (L-Galactono-1,4-γ-lactone dehydrogenase GaLDH) that does not produce H₂O₂ (Figure 1).

Plant-derived food are the main dietary source for vitamin C (Table 1). Vitamin C is also present in some meats, such as cow liver (liver and kidney are ASC synthesizing organs in animals, Kanfer et al., 1959), but they are irrelevant in supplying vitamin C because of their limited use in human nutrition and the consistent lost of ASC content caused by food processing, mainly due to ASC thermal instability (Munyaka et al., 2010).

Severe vitamin C deficiency causes scurvy, a disease discovered in the sailors of 15th and 16th century that could not consume fresh plant-derived food for months (Baron, 2009). Scurvy has been considered one of the most important disease derived from nutrient deficiency in the history of humanity (Magiorkinis et al., 2011). Scurvy symptoms consists in generalized edema, skin hemorrhages, swollen, bleeding gum and, if prolonged, can cause death (Magiorkinis et al., 2011). It is generally accepted that it is due to an impairment of collagen formation (Peterkofsky, 1991). Indeed ASC participates to collagen cross-linking reactions as cofactor of prolyl hydroxilases. The role of ASC in these reactions is to maintain the iron present into the enzymatic catalytic site in the reduced state and thus converting back the inactivated form of the enzymes into the active one (Gorres and Raines, 2010). Today scurvy is rare in developed as well as developing countries, since it requires a severe and prolonged deficiency in vitamin C in order to become evident; however, recent epidemiological studies underline that even in western populations sub-optimal vitamin C intake is widespread (Troesch et al., 2012).

Recommended dietary allowance (RDA) for vitamin C is a controversial matter, since different countries provide different advice; for example RDAs for adult men are 40 mg/day in UK; 90 mg/day in USA; 100 mg/day in Germany; 70 mg/day in Netherland (Troesch et al., 2012). Moreover, in order to enhance health benefits due to vitamin C intake, the scientific community is suggesting to increase its RDA to 200 mg/day (Frei et al., 2012). Epidemiologic studies have actually revealed that ASC intake over the current RDA has a significant impact in reducing the risk of diseases such as respiratory tract infections, cardio-vascular diseases and cancer (Schlueter and Johnston, 2011). With the exception of special population groups, as for example people suffering for kidney stones (whose formation could be promoted by oxalate, a catabolic derivate of ASC in mammals; Linster and Van Schaftingen, 2007), adverse effects caused by vitamin C over-ingestion, such as diarrhea, only occurs when the intake overcomes 2000 mg/day (Schlueter and Johnston, 2011). Moreover, pro-oxidant effects of vitamin C was only reported for daily intake higher than 500 mg (Podmore et al., 1998).

In human, vitamin C uptake is controlled by specific Na-dependent active transporters probably present in all the cells accumulating ASC (Savini et al., 2008). In animal tissues, the oxidized form of vitamin C, dehydroascorbate (DHA), is less efficiently taken up on glucose transporters. Within cells DHA is reduced back to ASC (the active form of the vitamin C) by enzymes using glutathione (GSH) and pyridine nucleotides as electron donor (Rumsey et al., 1997; Linster and Van Schaftingen, 2007; Figure 2). According to literature data, 200 mg/day is the intake of vitamin C that leads to the saturation of renal clearance for reabsorption of vitamin C, since a dose-dependent vitamin C release is observed in urine starting from an intake higher that this dose (Levine et al., 1996, 2001). In this perspective all the current RDAs for vitamin C (see above), assessed for avoiding scurvy and mild deficiency, are starting to be considered suboptimal in regards to the potential health benefits triggered by this vitamin (Frei et al., 2012).

In developed countries vitamin C supplementation is largely adopted especially for preventing/reducing cold related diseases. Actually the industrial production of vitamin C represents a low efficient and expensive technology (Hancock and Viola, 2002). Recently, it has also been demonstrated that vitamin C from plant-derived food (i.e., kiwifruit) is more bio-available than the chemically synthesize or purified molecule used in supplementation (Vissers et al., 2011). This could be the consequence of the presence in food matrixes of plant origin of several molecules with antioxidant/redox properties, which can have a synergic effects with ASC or be able to preserve vitamin C in its active reduced state (Villanueva and Kross, 2012). The possibility that other biological molecules could stabilize ASC is supported by DHA/ASC redox potential (estimated around as 90 mV; Noctor, 2006) that makes ASC a good reductant and, at the same time, its oxidized form DHA reducible in cellular metabolic conditions (Szarka et al., 2012).

Another aspect that makes interesting to increase vitamin C level in the edible plant tissues is the fact that this metabolite improves the post-harvesting properties of several vegetables. Indeed, its addition to several food matrix is used for extending their shelf life, as well as for improving specific technological properties, such as the kneading of wheat flour and dough rheological properties (Paradiso et al., 2006).

On these bases, ASC bio-fortification of the plants utilized for food production is becoming an important nutritional claims also for having promising technological implications. The plethora of roles played by ASC in plant metabolism increases the complexity of this goal. Here an overview of the results obtained in vitamin C bio-fortification is given with particular attention to the results obtained on crops and on the reasons why vitamin C bio-fortification still remains an ambitious target.

Enzymatic ASC oxidation mainly occurs in plants through the reactions catalyzed by ascorbate oxidase (AOX) and ASC peroxidase (APX), two typical plant enzymes. AOX is an apoplastic enzyme involved in cell elongation (Takahama and Oniki, 1994). The down-regulation of this enzyme causes a shift in the apoplastic ASC pool toward its reduced state; it also increases plant yield during water deficit, through a carbon flux re-allocation, but does not determine a significant ASC increase in the investigated tissues (Garchery et al., 2013).

APX down-regulation is not a feasibly strategy for preserving ASC in plant, since this enzyme is a key player in many plant defense responses. A decrease in its activity might enhance plant susceptibility to stress (Örvar and Ellis, 1997; de Pinto et al., 2006). Indeed, the appearance of various APX isoforms during evolution can be considered a specific acquisition for promoting survival of these sessile organisms by using a molecule that plants can produce by themselves (Ishikawa and Shigeoka, 2008). APX uses ASC as electron donor to scavenge H₂O₂ normally produced in aerobic metabolism and over-produced during abiotic and biotic stresses (Karpinski et al., 1997; Mittler et al., 1999; Paradiso et al., 2008; De Gara et al., 2010; Figure 2). It has been suggested that transcriptional and post-transcriptional regulation of APX is a signaling strategy able to finely regulate the H₂O₂ level into the cell, switching the role of this molecule from toxic compound to signaling molecule (de Pinto et al., 2012). Actually, being the only ROS able to cross cell membranes, H₂O₂ can work as messenger in the transduction pathway activated as consequence of different stimuli (Foyer and Noctor, 2005; Bienert et al., 2007). Indeed H₂O₂ has been supposed to regulate gene expression during plant defense response playing a major role in tolerance acquisition against stress (Miller et al., 2008). For example, in thermal acclimation, H₂O₂ is reported to regulate the expression of heat shock proteins and of a thermostable APX isoenzyme (Banzet et al., 1998; Lee et al., 2000; Suzuki and Mittler, 2006; Volkov et al., 2006).

A general enhancement of antioxidant systems are involved in plant acclimation to stress. This has relevant implications in post-harvest procedures, since post-harvest controlled stress, such as moderate temperature, are able to increase antioxidant shield in plant tissues thus improving food quality during storage (Cisneros-Zevallos, 2003).

As emerging from data reported above, ASC is involved in a wide net of metabolic reactions controlling growth and development as well as stress responses of plants (as reviewed by Foyer and Noctor, 2011). Indeed a plethora of different reactions depend on ASC and affect its level (from ROS removal to the synthesis of secondary metabolites and phyto-hormones, or to prolyl hydroxylation). This is probably why bio-engineering of a single gene involved in ASC biosynthesis or recycle often led to unsatisfactory results (see above). Actually, ASC level in plant organs and tissues can be considered a quantitative complex trait.

For this reason, in order to obtain plant-derived foods with a consistent enrichment in vitamin C, systemic approaches have been recently used. At this purpose a quantitative trait loci (QTL) analysis have been implemented in order to identify polygenic traits able to enhance ASC in edible crops. It is expected that these findings can ameliorate breeding strategies for increasing nutritional value of plant-derived foods.

Tomato has been mainly investigated, as a model crop (Stevens et al., 2007). A number of tomato wild accessions accumulating higher amounts of ASC (up to fivefold) than the cultivated lines have been identified (Di Matteo et al., 2010). An introgression line (IL) has been obtained by using the QTL identified in a wild progenitor and correlated to high ASC accumulation in mature tomato fruits. Di Matteo et al. (2010) demonstrated that the rise of ASC occurring during ripening in this IL depends on an increased flux of ASC precursors not involved in the S-W pathway. In particular, pectine degradation seems to be pivotal for feeding ASC de novo synthesis during tomato fruit ripening.

It is known that domestication often caused the loss of characters able to promote plant fitness and competitiveness in natural environment. In this perspective, it is possible that human selection on plants has caused a reduction in the synthesis/storage of precious metabolites. This could be occurred with vitamin C during tomato domestication. Indeed tomato varieties have been selected in the past for high yield and it probably caused ASC loss by oxidation. As already discussed, ASC oxidation promotes plant cell elongation (Takahama and Oniki, 1994; Fry et al., 2001). Moreover, the selection of tomato cultivars having prolonged fruit shelf life, and therefore prolonged flesh firmness, might lead to the selection of lines having low ASC as a consequence of a reduced or delayed pectine degradation which is responsible for fruit softening (Dumville and Fry, 2003).

However, different species can require different strategies for optimizing the post-harvest properties of their edible organs. In apples and pears the ASC level has been reported to be correlated with post-harvest quality (Veltman et al., 2000; Davey et al., 2007). In apple fruits, ASC level depends on harvest time and, as a consequence, it affects susceptibility to phytopathogens during post-harvest storage (Davey and Keulemans, 2004; Davey et al., 2007). Co-localized QTL for wound-related flesh browning and DHA content were recently identified in apple (Davey et al., 2006). ASC oxidation occurring during storage conditions has been also hypothesized to be mainly involved in post-harvest diseases of pear (Cascia et al., 2013). QTL analysis have recently suggested that candidate genes regulating ASC level and post-harvest quality in apple flesh are a paralog of GGP and MDHAR3, respectively (Mellidou et al., 2012b). An allele of MDHAR has been also proposed as major candidate gene for high ASC level in tomato fruit (Stevens et al., 2008); whereas in strawberry candidate genes for stable QLT correlated to high ASC have identified in alternative biosynthesis pathways, such as GalUR e MIOX (Zorrilla-Fontanesi et al., 2011). All these findings are supported by the role of high levels of ASC and related redox enzymes in protecting plant from stress conditions. Indeed, ASC production is enhanced by several injuries. It is in fact reported that jasmonate and its derivates produced in plant as consequence of wounding, promptly induces ASC biosynthesis (Suza et al., 2010). Consistently Arabidopsis vtc mutants, containing about 30% of ASC compared to wt, were identified for their increased susceptibility to ozone and then characterized for their slow growth phenotype (Conklin et al., 1996; Veljovic-Jovanovic et al., 2001). Moreover, pathogenesis related genes were up-regulated in vtc1, thus suggesting an impairment in the defense responses of this ASC-deficient mutant (Pastori et al., 2003; Pavet et al., 2005).

Another omic approach that has recently given information on metabolic networks responsive for ASC accumulation in fruit is trascriptomic analysis. Such analysis has been carried on in tomato IL showing reduced fruit ASC accumulation in comparison with its cultivated parental line. The main differences between the two lines have been identified in the steady state of mRNA related to oxidative and antioxidant pathways. In particular, this tomato IL showed an accelerated oxidative metabolism and decreased antioxidant systems compared to the parental line (Di Matteo et al., 2012). The accelerated oxidative metabolism could explain the low ASC amount in tomato IL by a reduction of sugar flux toward ASC biosynthesis. It has been suggested that ASC plays a protective role in climacteric fruits contrasting ROS rise during ripening (Jimenez et al., 2002). Therefore in the mentioned tomato IL the increased oxidative metabolism probably increases ROS, thus causing a further ASC consumption.

As emerging above, so far scientific studies have failed in identifying a single master regulator responsible for ASC accumulation in plant -derived foods. This makes vitamin C bio-fortification a real challenge of plant science research. It is clear that ASC level of plant edible tissues depends on several cross-talking factors acting at different physiological levels. Within cells, competition for hexose fluxes between vitamin C biosynthesis and other metabolic pathways, as well as the balance between ASC consuming and recycling reactions are pivotal for ASC storing. This is further complicated by the fact that ASC is used in almost all cellular compartments which are characterized by the presence of diversely regulated isoenzymes of the Foyer-Halliwell-Asada cycle and of different ASC-utilizing enzymes. At organ level, the fluxes, between source and sink tissues, of ASC precursors and ASC itself could be a critical aspect for increasing ASC level in certain tissues or organs. These fluxes are developmentally regulated but they can also be altered by specific stresses or environmental conditions that diversely affect various organs of the plant. Therefore the strategies adopted to increase ASC in plant edible tissues or organs have to take into account all these considerations in order to obtain plants with an increased nutritional value and with the opportune productivity and resistance against adverse environmental conditions.

Since fruits can be considered the best dietary sources for vitamin C being consumed raw, they are the main target of vitamin C bio-fortification. In this perspective, the identification of the most efficient strategy for increasing vitamin C in fruits is further complicated by the variety of ASC accumulation trends showed during ripening of fruits from different species (Figure 5). In particular, depending on the specie, ASC level is reported to decrease during fruit ripening (i.e., peach; Imai et al., 2009), to remain almost constant during fruit ripening after reaching a maximum level during early fruit development (i.e., kiwifruit; Li et al., 2010a) or increasing during ripening (i.e., tomato, Ioannidi et al., 2009). These trends can show further intra-species variations at least in terms of the timing of different development and ripening stages as well as they can be affected by agronomic and environmental conditions. All these aspects make clear why ASC manipulation of biosynthesis or recycle level not always has led to a consistent vitamin C bio-fortification (Zhang et al., 2010; Cronje et al., 2012; Gest et al., 2012).

A novel systemic approach is promising in skipping difficulties possibly derived by single gene bio-engineering. Indeed it is emerging the concept that specific allelic forms of genes directly involved in ASC metabolism, or positively correlated with ASC storage, can differently affect ASC level in plant edible tissues. In particular, the identification of the QTL common or positively correlated to both ASC level and plant defense responses are opening new perspectives. In this context the comparison of wild progenitors and the derived cultivated lines seems to be very useful for identifying strategies adopted by plants during evolution for increasing their fitness. In a near future, these strategies could be implemented in crop by introgression through “classical” breeding techniques or multiple genes bio-engineering. In this perspective, the discovery of the main genetic mechanisms controlling ASC level in different plant species is a prerequisite that can allow scientists to identify successful strategies for vitamin C bio-fortification.

It is also necessary to move toward a novel and more efficient concept of biofortification, that at the same time takes into account the increase in nutritional value and plant stress tolerance as a unique goal of the improving strategy. Indeed, improvement of plant tolerance to adverse environmental conditions has also a direct positive effect on human health, as an example by possibly reducing the use of pesticides during field grown and post-harvest storage with an expectable effect on both environmental pollution and human health. Even if the level of pesticides are strictly controlled by law, a number of studies reports carcinogenic effects at high doses that can be also reaches for a low but prolonged exposure of toxic molecule (Alavanja and Bonner, 2012). Moreover, producing “fortified” crops in this novel perspective can also increase the availability of plant foods for the increasing world population. This is particularly challenging for reducing harvest and post-harvest crop losses and consequently food costs in an era of climatic changes increasing the geographic areas subjected to water and thermal stresses.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.