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Vitamin C in Plants: From Functions to Biofortification

Department of Biology, University of Bari “Aldo Moro”, Via E. Orabona 4, 70125 Bari, Italy
Author to whom correspondence should be addressed.
Antioxidants 2019, 8(11), 519;
Received: 2 October 2019 / Revised: 25 October 2019 / Accepted: 26 October 2019 / Published: 29 October 2019
(This article belongs to the Special Issue Phytochemical Antioxidants and Health)


Vitamin C (l-ascorbic acid) is an excellent free radical scavenger, not only for its capability to donate reducing equivalents but also for the relative stability of the derived monodehydroascorbate radical. However, vitamin C is not only an antioxidant, since it is also a cofactor for numerous enzymes involved in plant and human metabolism. In humans, vitamin C takes part in various physiological processes, such as iron absorption, collagen synthesis, immune stimulation, and epigenetic regulation. Due to the functional loss of the gene coding for l-gulonolactone oxidase, humans cannot synthesize vitamin C; thus, they principally utilize plant-based foods for their needs. For this reason, increasing the vitamin C content of crops could have helpful effects on human health. To achieve this objective, exhaustive knowledge of the metabolism and functions of vitamin C in plants is needed. In this review, the multiple roles of vitamin C in plant physiology as well as the regulation of its content, through biosynthetic or recycling pathways, are analyzed. Finally, attention is paid to the strategies that have been used to increase the content of vitamin C in crops, emphasizing not only the improvement of nutritional value of the crops but also the acquisition of plant stress resistance.
Keywords: ascorbate; antioxidant; biofortification; light; plant growth; reactive oxygen species; vitamin C ascorbate; antioxidant; biofortification; light; plant growth; reactive oxygen species; vitamin C

1. Introduction

Vitamin C (l-ascorbic acid) was isolated from the adrenal cortex by Albert Szent-Györgyi in 1928. Szent-Györgyi demonstrated that this compound, which can act as a powerful reducing agent, indicated with the empirical formula of C6H8O6, had a molecular mass of 178 ± 3 and was a lactone with an acidic hydrogen atom. Due to its similarity to simple sugars and its acidic properties, Szent-Györgyi called this compound “hexuronic acid” [1]. In 1932, Charles Glen King isolated an antiscorbutic compound from lemon juice that was recognized as the hexuronic acid found by Szent-Györgyi [2,3]. At the same time, Szent-Györgyi showed that 1 mg/day of hexuronic acid provided ample protection against scurvy [4]. The definitive structure of vitamin C, which is a hexonic acid aldono-1,4-lactone with an enediol group on C2 and C3, was achieved by Norman Haworth in 1933 [5]. The evidence that this compound was able to prevent scurvy led to it being renamed from hexuronic acid to ascorbic acid [6].
Vitamin C is the most abundant water-soluble compound working in one-electron reactions, and it is an essential micronutrient and a key element for the metabolism of almost all living organisms. In humans, vitamin C has numerous functions, mainly acting as an antioxidant and a cofactor for mono-oxygenases and dioxygenases [7].
The roles of vitamin C as an antioxidant in humans has been established based on a large body of scientific evidence. Vitamin C, by scavenging free radicals, protects DNA, proteins, and lipids from oxidative damages [8]. Vitamin C is used as an antioxidant throughout the body but may have specific roles in some organs. For instance, vitamin C is required in the eyes at a millimolar concentration to guarantee protection from oxidative damage due to solar radiation [9]. Vitamin C inhibits the synthesis of the carcinogenic nitrosamines, which can be synthesized in the intestine or absorbed with food [10], and reduces tetrahydrobiopterin, the cofactor of nitric oxide synthase, which catalyzes the synthesis of nitric oxide [11]. Vitamin C is also important for iron bioavailability, reducing non-heme iron from the ferric (Fe3+) to the ferrous (Fe2+) form, which is more easily absorbed in the intestine; for this reason, this vitamin is indirectly needed to protect against anemia [12]. Vitamin C also influences iron metabolism through the stimulation of ferritin synthesis and the inhibition of ferritin degradation [13].
Vitamin C, as a cofactor of peptidyl-glycine alpha-amidating monooxygenase, is involved in the biosynthesis of many signaling peptides, such as oxytocin, vasopressin, cholecystokinin, and calcitonin [14,15,16]. Vitamin C functions as a cofactor for many dioxygenases, reducing the iron in the active site of these enzymes to Fe2+. Vitamin C contributes to the correct formation of collagen through post-translational modifications of procollagen. In particular, this vitamin acts as a cofactor for the reaction catalyzed by prolyl 3-hydroxylase, prolyl 4-hydroxylase, and lysyl hydroxylase, which are involved in the hydroxylation of lysine and proline and permit the formation of the stable structure of collagen [17,18,19]. Vitamin C, as a cofactor of hydroxylase, is used for the synthesis of norepinephrine and carnitine [20,21,22]. Vitamin C intervenes in many cytochrome-P450-dependent hydroxylation reactions, such as the transformation of cholesterol into bile acids, the degradation of exogenous substances such as pollutants and drugs, and the synthesis of steroid hormones [23].
Recently, vitamin C has been identified as a cofactor for the methylcytosine dioxygenases ten-eleven translocation (TET), which is involved in DNA demethylation and JmjC-domain-containing proteins, which catalyze the demethylation of histones [24]. As a result of vitamin C deficiency, especially in the nucleus, the requirements of TETs or some JmjC-domain-containing histone demethylases may not be met, leading to alterations in the methylation–demethylation dynamics of DNA and histones, which can subsequently contribute to phenotypic alterations or even diseases. By regulating the epigenome, vitamin C can be involved in embryonic development, postnatal development and aging, and cancer and other diseases [24]. Being able to modulate the epigenome, vitamin C has been proposed as an effective molecule in anticancer therapies [25].
Vitamin C is considered a vitamin only for a few vertebrate species, including humans, that are unable to synthesize it [26]. Indeed, vitamin C can be synthesized by plants and most animals [27]. Primates, guinea pigs, bats, some species of birds, insects, invertebrates, and fish are examples of species not able to synthesize this vitamin [26,27]. The inability of humans to synthesize vitamin C lies in the functional loss of the gene coding l-gulonolactone oxidase, the last enzyme involved in the animal biosynthetic pathway of this vitamin [26].
The best way for humans to obtain vitamin C is by diet, and plant foods represent the primary source of this vitamin. Although synthetic vitamin C is chemically indistinguishable from the plant-derived vitamin, fruits and vegetables have different micronutrients and phytochemicals that can affect its bioavailability [28]. Many studies conducted on vitamin-C-deficient animals have shown that vitamin C in plant foods has greater bioavailability than that found in drugs or supplements [28]. For instance, in homozygote Gulo mice, the uptake and tissue distribution of vitamin C were higher when the vitamin was furnished by kiwifruit gel than when it was added as a synthetic supplement in drinking water [29]. Nevertheless, studies conducted on humans have not shown significant differences in bioavailability between synthetic and plant-derived vitamin C [28]. Despite the comparable dosage and bioavailability, it has been shown that orange juice and not synthetic vitamin C drink protects leukocytes from oxidative DNA damage [30]. Probably, the improved effects of vitamin C dispensed with fruits and vegetables is due to the interaction with other micronutrients, such as vitamin E [31] and iron [32]. Consistently, plant-derived vitamin C is related to reduced occurrence of different chronic diseases [33].
The loss of the capability to synthesize vitamin C in our ancestors would not have been a disadvantage with a diet rich in vegetables and fruits, which could have provided enough vitamin C [34]. On the contrary, since l-gulonolactone oxidase produces the potentially toxic H2O2, this loss could have been an evolutionary improvement in the control of redox homeostasis [35].
Nowadays, very low levels of vitamin C are present in main crops, with the consequence that diet does not provides enough intake of this vitamin. Thus, obtaining plant foods with enhanced vitamin C content represents an important goal for human health. In-depth knowledge of the metabolism and functions of vitamin C in plants is needed to achieve biofortification.

2. Vitamin C as an Antioxidant

Vitamin C is an essential element of plant and animal antioxidant systems, which can be defined as complex redox networks, including metabolites and enzymes, with mutual interactions and synergistic effects [36]. Antioxidants can spontaneously provide electrons to free radicals, alleviating the oxidative cellular environments caused by aerobic metabolism.
Chemically, vitamin C is a dibasic acid with an enediol group on C2 and C3 of a heterocyclic lactone ring. At physiological pH, the hydroxyl group at C3 is deprotonated, giving a monovalent anion, which is indicated as ascorbate (ASC) [37]. The enediol group permits the donation of one or two electrons, forming monodehydroascorbate (MDHA) and dehydroascorbate (DHA), respectively [36]. The ASC redox potential ranges from +0.40 to +0.50 V [38,39]; thus, the molecule can directly donate electrons to reactive oxygen species (ROS), such as singlet oxygen, superoxide anions, and hydroxyl radicals, as well as to tocopheroxyl radicals (Figure 1) [36]. Being able to reduce tocopheroxyl radicals, ASC is indirectly involved in the scavenging of lipid peroxides and radicals, contributing to the decrease of lipid peroxidation and, consequently, to the protection of membranes [40]. Due to the fast reduction of ROS by ASC, the damage of biomolecules can be prevented before the activation of antioxidant enzymes.
ASC can also reduce metals such as copper and iron, leading to the formation of ROS through the Haber-Weiss and Fenton reactions [41]. Thus, in some cases, ASC, acting as a reducing agent, will generate oxidants. This can occur in cell culture media, within the physiologic concentration of ASC, in presence of metals or in vivo in humans only when plasma and extracellular fluids contain millimolar concentrations of ASC [42].
Due to resonance stabilization of unpaired electrons, MDHA, derived from the loss of one electron, has very low reactivity with other radicals and, consequently, has very low toxicity. Two molecules of MDHA can spontaneously dismutate in ASC and DHA (Figure 1) [43]. DHA, if not rapidly reduced to ASC, will be permanently hydrolyzed to threonate, oxalate, oxalyl threonate, or tartrate [44].
ASC has low direct reactivity with H2O2, but in plants, ASC works as a specific electron donor for ascorbate peroxidase (APX), a heme peroxidase which catalyzes the conversion of H2O2 to H2O and O2, giving MDHA (Figure 1). APX has a high affinity for H2O2 and removes this ROS, even at low concentrations [36]. In plants, different APX isoenzymes have been identified in cytosol, mitochondria, peroxisomes, and chloroplasts, but all are coded by nuclear genes. The plant model system Arabidopsis thaliana possesses six APX genes coding for six isoenzymes, two of which are targeted to the cytosol (APX1 and 2), two to peroxisomes (APX3 and 5), one to the thylakoid membrane (tAPX), and one that is dual-targeted to chloroplast stroma and mitochondrial matrix (sAPX) [45]. The crop species rice and tomato possess seven and eight APX isozymes, respectively [46].
APX is part of the ASC-glutathione (GSH) cycle (Figure 1), which is involved in ASC regeneration [47]. MDHA is reduced to ASC by MDHA reductase (MDHAR), which is a flavin enzyme that utilizes NAD(P)H as electron donors [43]. Many cell compartments possess MDHAR activity. Arabidopsis has five genes coding for MDHAR2 and 3, localized in the cytosol; MDHAR1 and 4, in peroxisomes and membranes; and MDHAR6 in chloroplasts and mitochondria [48]. DHA can be reduced to ASC by DHA reductase (DHAR), which utilizes GSH as an electron donor, leading to the formation of glutathione disulphide (GSSG). DHAR has an important role in maintaining the reduced ASC in order to avoid DHA degradation [49]. DHAR activity has been identified in cytosol, chloroplasts, mitochondria, and peroxisomes [48]. Arabidopsis has three genes coding for DHAR localized differently in the cells: DHAR1 localized in cytosol and peroxisomes, DHAR2 localized only in the cytosol, and DHAR3 targeted to chloroplasts [50]. In the ASC-GSH cycle, GSSG is reduced to GSH by the NADPH-dependent glutathione reductase (GR). GR plays a pivotal role in maintaining the correct balance between reduced GSH and ASC pools [51]. GR activity has been detected in chloroplasts, cytosol, mitochondria, and peroxisomes [52]. In Arabidopsis, two genes encode for GR in plants: GR1 is predicted to code a cytosolic isoenzyme and GR2 encodes for a dual-targeted plastidic/mitochondrial protein [53].
Owing to its high antioxidant properties and to the presence of an effective system for redox regeneration, in plants, vitamin C plays a significant role in the defense against oxidative stress, which arises in response to biotic or abiotic stresses [54]. The important role of vitamin C in the tolerance to several stresses is underlined by the increase in the enzymes involved in biosynthesis and recycling, observed in the presence of adverse environmental conditions [49,55]. Interestingly, feeding with l-galactono-1,4-lactone, which enhances the vitamin C content, can increase resistance to various kinds of stress [56,57,58,59].

3. Multiple Roles of Vitamin C in Plants

A significant part of accessible glucose (about 1%) is used for vitamin C production, which is present at high concentration in plants [60]. Vitamin C was found in all cell compartments, including the apoplast (the cell wall and extracellular space), reaching a concentration of 20 mM in chloroplasts [61]. However, the vitamin C content significantly differs among plant species and in the same species between diverse cultivars [62]. Moreover, the vitamin C content varies among different tissues and organs, usually being high in leaves, meristematic tissues, flowers, or young fruits and low in non-photosynthetic organs such as stems and roots [54,62]. Only seeds that reach maturity in a stage of strong dehydration (orthodox seeds) contain little vitamin C, which is essentially in the oxidized form [63,64]. In the same organ or tissue, vitamin C content is influenced by the plant developmental stage and environmental changes [65,66,67,68]. Light is one of the most significant environmental signals involved in the regulation of vitamin C levels [69,70].
As in humans, vitamin C favors iron uptake in plants. The ASC efflux in the apoplast contributes to the reduction of Fe3+, catalyzed by the ferric chelate reductase plasma membrane enzyme. Arabidopsis mutants having low vitamin C content (vtc mutants) show a decrease in Fe3+ reducing capability and a consequent reduction of iron accumulation in the seeds [71].
Vitamin C, having different functions in chloroplasts, is essential for the correct functionality of photosynthesis. Firstly, ASC has a key role in the direct scavenging of ROS and in the removal of H2O2 through the water-water cycle [72,73]. ASC also participates in the xanthophyll cycle, which is needed to protect photosystem II (PSII) from photoinhibition. In this cycle, ASC is the cofactor of violaxanthin de-epoxidase, which converts violaxanthin in zeaxanthin, the xanthophyll responsible for dissipating excess excitation energy in the light harvesting complexes of PSII [74]. Finally, ASC can donate electrons to both photosystems, especially when they are damaged by stress conditions [75,76]. Changes in vitamin C content significantly modify the expression of genes linked to photosynthesis [77]. The lowering of the ASC content, through the suppression of DHAR expression, leads to the loss of chlorophyll a, the reduction of the RUBISCO large subunit, and a decrease in CO2 assimilation [78]. Consistently, vitamin C-deficient Arabidopsis mutants enter senescence earlier than wild-type [79]. Thus, vitamin C, by preserving photosynthetic functioning and limiting ROS-mediated damage, slows down leaf senescence [78,79,80,81].
Vitamin C is involved in the synthesis of the plant hormone ethylene, acting as a cofactor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase, the enzyme that catalyzes the last biosynthetic step. Indeed, ASC contributes to the ring opening of ACC by supplying the electron to the active site of the enzyme [62,82]. Being a cofactor of dioxygenases, vitamin C could also be involved in the synthesis of abscisic acid and gibberellins, as well as in the catabolism of auxins [62].
A complex interplay between vitamin C and hormone signaling intervenes in different phases of plant growth and development, as well as in plant response to the environment and pathogens [83,84]. In particular, vitamin C involvement in the defense response against pathogens is strictly dependent on pathogen lifestyles [84]. It is known that defense against biotrophic pathogens is mediated by salicylic acid signaling, whereas defense against necrotrophic pathogens is mediated by jasmonic acid and ethylene signaling [85]. Arabidopsis mutants with low vitamin C levels show an increase in salicylic acid, pathogenesis-related proteins, and camalexin and are more resistant to Pseudomonas syringae and Peronospora parasitica [79,86,87,88]. On the contrary, the same mutants are more susceptible to the necrotrophic ascomycete Alternaria brassicicola [89]. Nevertheless, exogenous addition of ASC acts as an inducer of disease resistance in different plant-pathogen interactions [89,90,91,92].
In plants, vitamin C can control the division, elongation, and differentiation of cells, as well as programmed cell death (PCD). Vitamin C plays a significant role in the control of cell division. This metabolite in the meristematic cells of root meristems can shorten the G1 phase and stimulate entry into the S phase [93,94]. In the quiescent center of the root meristem, where cells are not dividing, low levels of ASC, linked to a significant increase of the ASC-consuming enzyme ASC oxidase (AOX), are responsible for the arrest of the cell cycle in the G1 phase [95]. In tobacco BY-2 cells, a peak in ASC, as well as l-galactono-1,4-lactone dehydrogenase (GLDH), activity overlaps with the peak in the mitotic index [96,97]. Moreover, cells enriched with ASC show stimulation of cell division, whereas enrichment with DHA leads to a reduction in cell division, suggesting that the ASC redox state is fundamental to cell cycle progression [96]. Low ASC levels and an altered redox state negatively affect cell cycle progression in the root meristem of Arabidopsis, with a consequent decrease in the number of cells in the proliferation zone [98]. An ASC increase has also been shown during cell divisions in developing embryos [99]. The stimulation of division in the apical meristem by ASC seems to be principally due to the inhibition of peroxidase involved in the crosslink of cell wall components. Inhibition of vitamin C biosynthesis leads to abortion of the meristem [100].
The stimulation of cell elongation is due to the expression of AOX, the activity of which determines an increase in oxidized forms of vitamin C in the apoplast [101,102]. Indeed, apoplastic MDHA participates in transmembrane electron transfer, accepting electrons from cytochrome b. This process induces plasma membrane hyperpolarization and activation of H+-ATPase, with an acidification of the apoplast that favors cell wall relaxing [103]. The parallel oxidation of NADH acidifies the cytoplasm and activates vacuolar H+-ATPase, increasing vacuolization and cell expansion [103,104,105]. In the apoplast, ASC in the presence of Cu2+ can exert its pro-oxidant role, producing H2O2, which induces degradation of polysaccharides [106,107]. Moreover, by reducing lignin precursors utilized by peroxidases, ASC delays cell wall lignification [108]. With the transition from meristematic to differentiated cells, ASC levels significantly decrease, permitting the activity of secretory peroxidases and, consequently, cell wall stiffening and lignification, occurring during differentiation [109].
Vitamin C is also involved in the control of PCD. A. thaliana mutants, with low vitamin C content, spontaneously trigger localized cell death like that which occurs during hypersensitive response, a plant-defense mechanism activated to block pathogen invasion [87]. An ASC decrease is necessary for PCD induced by H2O2 and heat shock (HS) in tobacco BY-2 cells [110,111,112,113]. The decrease in ASC during HS-induced PCD is due to inactivation of the last enzyme of the vitamin C biosynthetic pathway [114]. Low levels of ASC during PCD are parallel to a decrease in the level of transcript, protein, and activity of APX [111,112,115]. The impairment in ASC and APX is needed to increase ROS, which are essential for PCD induction [116,117]. Interestingly, the increase in vitamin C biosynthesis by the supply of l-galactono-1,4-lactone delays PCD occurring during kernel maturation in durum wheat, with a consequent postponement of dehydration and improvement in kernel filling [118].
Vitamin C, regulating the abovementioned processes at molecular and cellular levels, is therefore involved in different phases of plant growth and development, such as seed maturation and germination, flowering, fruit ripening, and senescence [119].

4. Vitamin C Biosynthesis in Plants

Vitamin C biosynthesis in higher plants has been the subject of dispute for many years. The first investigations related to vitamin C biosynthesis in plants date back to 1950 [120]. A definitive mechanism was formulated only 40 years later [60]. Unlike the animal pathway, in plants, no carbon inversion occurs in the biosynthesis of vitamin C, and the C1 in the d-glucose molecule remains as C1 after conversion. The Smirnoff–Wheeler pathway, in which vitamin C is synthesized from d-mannose and l-galactose (d-mannose/l-galactose pathway), represents the major route of vitamin C biosynthesis in plants [62,121]. Three other routes have been proposed for vitamin C biosynthesis: the gulose pathway, the myoinositol pathway, and the galacturonate pathway (Figure 2) [122,123,124].

4.1. d-Mannose/l-Galactose Pathway

In the Smirnoff-Wheeler pathway, d-glucose-6-phosphate is transformed into d-fructose-6-phosphate by phosphoglucose isomerase (PGI) and then is directed into d-mannose metabolism by phosphomannose isomerase (PMI), which produces d-mannose-6-phosphate. In Arabidopsis, PMI1 expression increases concomitantly with vitamin C levels under continuous light, and knockdown pmi1 plants showed decreased levels of this metabolite [125]. d-Mannose-6-phosphate is then converted into d-mannose-1-phosphate by phosphomannose mutase (PMM). Genetic evidence for the involvement of PMM in vitamin C biosynthesis has been obtained in Nicotiana benthamiana and Arabidopsis [126,127].
GDP-d-mannose pyrophosphorylase (GMP) transfers guanosine monophosphate from GTP to give GDP-d-mannose. In Arabidopsis, GMP is coded by VTC1; the vtc1 mutants accumulate ~25–30% of vitamin C levels of wild type and are hypersensitive to ozone [128,129]. vtc1 mutants also have altered sensitivity to other ROS-generating conditions, including H2O2, UV-B, SO2, and combined high light and salt stress [61]. Additional support for the involvement of GMP in vitamin C biosynthesis was obtained in potato plants constitutively expressing the antisense GMP gene. These plants showed a significant decrease in the activity of the enzyme and a significant reduction of vitamin C in leaves and tubers [130].
GDP-l-galactose is produced directly by GDP-d-mannose through a 3′5′ epimerization catalyzed by GDP-d-mannose epimerase (GME). GME has been characterized in Chlorella, flax, and Arabidopsis [131,132]. The enzyme belongs to the extended short-chain dehydratase/reductase protein family, with a modified NAD+ binding Rossman fold domain [133]. GME is also able to catalyze the 5′ epimerization of GDP-mannose, giving GDP-l-gulose, which is the precursor of a possible side-branch biosynthetic pathway (the gulose pathway) for vitamin C synthesis [122,132,134].
GDP-d-mannose and GDP-l-galactose are substrates for the synthesis of glycoproteins and polysaccharides of cell walls [135,136]. Thus, the first dedicated step for vitamin C synthesis in the d-mannose/l-galactose pathway is the conversion of GDP-l-galactose into l-galactose-1-phosphate, catalyzed by GDP-l-galactose-phosphorylase (GGP) [137].
In Arabidopsis, GGP is encoded by the VTC2 and VTC5 genes [121]. The VTC2 expression levels are significantly higher (100–1000 times) than that of VTC5; moreover, T-DNA insertion mutants of VTC2 and VTC5 have 20% and 80% of the vitamin C content of wild-type plants, respectively. The double vtc2 vtc5 mutants are unable to grow after cotyledon expansion, unless there is feeding of galactose or ASC, suggesting that at least in Arabidopsis, the d-mannose/l-galactose pathway is the substantial font of vitamin C [121]. The key role of GGP as a control point in vitamin C biosynthesis has been shown not only in Arabidopsis but also in tobacco, tomato, kiwifruit, strawberry, potato, citrus, and blueberry [138,139,140,141,142,143]. The transcript levels of VTC2 and VTC5 strongly correlate with the vitamin C content and the increase with light irradiation [121,144]. Moreover, VTC2, as well as other orthologue genes, can be controlled at the translation level by a noncanonical upstream open reading frame (uORF). In the presence of a high amount of ASC, the uORF encodes for a peptide, which acts as an inhibitor of translation, whereas under a low amount of ASC, the uORF is bypassed and GGP is translated [145].
l-Galactose-1-phosphate is converted into l-galactose by l-galactose-1-phosphate phosphatase (GPP), which is encoded by VTC4 in Arabidopsis [146,147]. However, vtc4 mutants have a partial decrease of GPP activity and vitamin C content [147,148]. Accordingly, in Arabidopsis, the reaction can also be catalyzed by the purple acid phosphatase AtPAP15 [149]. l-galactose dehydrogenase (GDH) is the NAD-dependent enzyme catalyzing the conversion of l-galactose into l-galactono-1,4-lactone [60]. This step of ASC biosynthesis is not limiting. Indeed, in Arabidopsis, the overexpression of GDH does not change the vitamin C content, and antisense plants show vitamin C decrease only under high light [150].
The last step of the Smirnoff-Wheeler pathway is catalyzed by GLDH, a flavoprotein which converts l-galactono-1,4-lactone into l-ascorbate, transferring electrons to cytochrome c [27,120,151]. An important observation is that GLDH does not release H2O2, as happens with l-gulonolactone oxidase in animals, and therefore the production of vitamin C in plants does not affect the redox state of the cell [152]. Unlike the other enzymes of the d-mannose/l-galactose pathway, which are all localized in the cytosol, GLDH is an integral protein of the mitochondrial inner membrane [97,153,154]. Specifically, GLDH has been detected in complex I and acts as an essential plant-specific factor for complex I assembly [155,156,157,158]. Due to the GLDH localization, vitamin C biosynthesis is very sensitive to stresses that cause impairments of electron flux [111,114,159]. On the other hand, an increase in respiration, like that observed during tomato ripening, is associated with an enhancement of vitamin C content [160]. The different steps of the Smirnoff-Wheeler pathway are schematized in Figure 2 and Figure 3.

4.2. Other Vitamin C Biosynthetic Pathways

As reported above, the gulose pathway starts from the 3′ epimerization of the GDP-d-mannose catalyzed by GME, with the formation of GDP-l-gulose (Figure 2) [122]. In this pathway, GDP-l-gulose is successively converted into l-guolse-1P, l-gulose, and l-gulono-1,4-lactone. l-gulono-1,4-lactone has been detected in plant extracts [161]. Moreover, external supplementation of l-gulono-1,4-lactone causes an increase in vitamin C content in Arabidopsis cells and tobacco leaves but is less efficient than feeding with l-galactono-1,4-lactone [162,163,164]. Activity of gulonolactone oxidase (GulLO) has been found in potato [163], and more recently, two genes coding for GulLOs have been identified in Arabidopsis. These enzymes are dehydrogenases with specificity for l-gulono-1,4-lactone and differ from plant GLDHs and mammalian GulLOs. The Arabidopsis GulLOs seem to be regulated post-transcriptionally and the limited enzyme availability could explain the slow utilization of the substrate [165].
l-Gulono-1,4-lactone is also the last precursor of vitamin C biosynthesis in the myoinositol pathway (Figure 2). In this pathway, myoinositol is converted into d-glucuronate by myoinositol oxygenase (MIOX). The other two steps producing l-gulonic acid and l-gulono-1,4-lactone are respectively catalyzed by glucuronate reductase and aldono lactonase [166]. A myoinositol oxygenase (MIOX4) has been identified in Arabidopsis [123]. However, the effective contribution of myoinositol to vitamin C synthesis in vivo is strongly debated [167,168,169].
The galacturonate pathway is also known as the salvage pathway, since it utilizes sugars provided by the breakdown of cell walls [124]. The degradation of pectin releases methyl-galacturonate, which is converted into d-galacturonate by methyl esterase and successively into l-galactonate by d-galacturonate reductase (GalUR). An aldono lactonase converts l-galactonate into l-galactono-1,4-lactone, which is the last precursor of vitamin C in the Smirnoff–Wheeler pathway (Figure 2) [27]. A gene encoding GalUR was initially identified in strawberry [124,170]. This pathway seems to be active mainly during fruit ripening in some species [124,170,171,172,173]. In tomato, vitamin C synthesis in immature green fruit is enhanced only by the supply of l-galactose, whereas in red ripened fruits by feeding with both l-galactose and d-galacturonate [170]. Moreover, the high vitamin C content found in tomato introgression lines IL12–4 compared with the parental M82 seems to be due to a higher expression of a pectinesterase and two polygalacturonases [171].

5. Light-Dependent Vitamin C Accumulation in Plants

Several papers have reported that plant exposure to light significantly increases vitamin C content [139,174,175]. Consistently, probably due to a reduction of irradiance levels, plants grown in greenhouses show lower levels of vitamin C compared with plants cultivated in the field [176]. However, the enhancement of vitamin C in plants seems to be dependent on the total amount of incident global radiation, which can be regulated by modulating light intensity or the duration of plant’s exposure to light. The use of continuous light for 48 and 72 h after a period of darkness causes a great increase of vitamin C levels in lettuce and Arabidopsis, respectively [70,177]. However, the continuous exposure of lettuce plants to very high irradiation causes a loss of vitamin C, whereas continuous low irradiation improves the content of this metabolite [178]. Shen et al. [179] found that in lettuce, continuous illumination with red-blue light emitting diodes (LEDs) increases the vitamin C content in relation to the exposure time. Interestingly, in the same plants cultivated for 15 days under continuous red-blue LEDs exposure, the maximum peak of vitamin C content was found after nine days from the beginning of light exposure [180].
The quality of light can also influence the vitamin C pool. The regulatory effects of monochromatic lights of the UV-Vis spectrum on the modulation of vitamin C content have been studied in different species, but the obtained results are often contradictory, suggesting that different species can respond differently to specific wavelengths. High-red/far-red ratios enhanced vitamin C levels in the leaves of Phaseolus vulgaris [67]. Lettuce plants illuminated with single or combined blue and red lights showed higher vitamin C contents than plants grown in white light [181]. In Chinese kale, different lights, except for blue, used during sprout growth improved the vitamin C content, and the highest concentration of ASC was found in shoots exposed to white LEDs [182]. The influence of light on the vitamin C content was also evaluated in numerous fruits, such as apple [183], tomato [175,184], Satsuma mandarin, Valencia orange, and Lisbon lemon [185]. In these three citrus varieties, the enhancement of vitamin C content in the fruits was greater with the increasing intensity of blue LEDs. Furthermore, continuous irradiation with blue LEDs is more effective than pulsed irradiation and is related to increased expression of genes involved in the modulation of the vitamin C pool, suggesting a control at the transcriptional level [185]. On the contrary, accumulation of vitamin C in lettuce grown under continuous light is mainly due to changes in activity, rather than in expression, of the enzymes involved in vitamin C biosynthesis and oxidation [180].
Light treatments have also been tested in the postharvest, but also in this case, contradictory results have been reported. In cabbages stored for 15 days at low temperatures, the content of vitamin C was higher in the presence of blue light [186], unlike what was observed in asparagus stored at 4 °C, in which the vitamin C content after six days of blue light did not differ from the control in the dark [187]. In broccoli as well, blue light had no positive effects on the vitamin C content, whereas green, red, and yellow lights, probably stimulating metabolic and physiological activity, permitted de novo vitamin C synthesis [188].
In addition to visible light radiation, plants are also exposed in nature to UV radiation, which constitutes about 7% of solar radiation [189]. UV was shown to improve vitamin C content in soybean sprouts [190]. Increased levels of vitamin C in cucumber plants illuminated with UV-B have been linked to a significant increase in the activity of MIOX, GLDH, and enzymes of the ASC-GSH cycle [191].

Light Regulation of Vitamin C Accumulation

The pivotal role of light-dependent vitamin C accumulation in green tissues is due to photosynthesis. In Arabidopsis leaves and tomato green fruits, the photosynthetic inhibitor DCMU blocks vitamin C accumulation by reducing the expression of genes involved in its biosynthesis [70,170]. Moreover, photosynthesis increases the amount of soluble carbohydrates, which are biosynthetic precursors of vitamin C. Three genes involved in carbohydrate accumulation and translocation have been related to a quantitative trait locus (QTL) associated with a 1.4-fold vitamin C increase in tomato [192]. On the other hand, Ntagkas et al. [193] found no correlation between vitamin C content and levels of soluble carbohydrates.
Light can control vitamin C accumulation through different types of regulation (Figure 3).
Light-dependent accumulation of vitamin C in plants seems to be principally due to the enhanced expression of different genes involved in the d-mannose/l-galactose biosynthetic pathway [70,121,142,175]. It has been shown that in rice, GLDH and GPP contain light-responsive cis-elements (GT1 box and TGACG motif) in their promoters [194]. Concerning GLDH, light beyond regulating its expression can induce changes in respiration, indirectly modulating enzymatic activity [69,159]. Light also influences the expression of genes involved in ASC recycling. During germination, corn seeds exposed to high light exhibited a higher DHAR expression along with an increased vitamin C content (Figure 3) [195].
In tomato, light induces the expression of the transcription factor HZ24, which in turn activates GMP transcription in leaves and immature fruits [196]. High light also induces a decrease in the expression of AMR1, a transcription factor acting as a negative regulator of the last six genes of the d-mannose/l-galactose biosynthetic pathway, consequently increasing vitamin C biosynthesis (Figure 3) [197]. Finally, in the dark, CSN5B promotes GMP degradation via 26S proteasome, reducing vitamin C levels [198]. Additionally, darkness promotes vitamin C catabolism [44].
The complexity of light-dependent vitamin C regulation (Figure 3) highlights that obtaining vitamin-C-biofortified plants by light treatments requires in-depth knowledge of the metabolic and physiological processes involved [199].

6. Vitamin C Biofortification

Increasing the vitamin C content in plants can have a triple-positive effect: producing food with a high content of vitamin C for human health, increasing the postharvest shelf life, and, not less important, increasing the resistance of plants to various kinds of stress. The different strategies adopted for vitamin C biofortification (Figure 4) are discussed below.

6.1. Manipulation of d-Mannose/l-Galactose Pathway

Several genes of the d-mannose/l-galactose pathway have been overexpressed in different crops in order to enhance vitamin C levels, but not all have given good results [34,35]. It has been proved that overexpression of GGP, which represents the bottleneck of vitamin C biosynthesis [137], is a good strategy for biofortification [200]. For instance, in Arabidopsis, transient overexpression of GGP leads to a 2.5-fold increase in vitamin C content, whereas the overexpression of the other genes involved in the same pathway does not cause relevant differences in terms of vitamin C [142]. Similar results were obtained in rice where, among different transgenic lines overexpressing six Arabidopsis genes involved in vitamin C biosynthesis, the highest vitamin C content was found in the line overexpressing GGP [201]. A kiwi gene coding for GGP, initially tested with good results in Arabidopsis [138], has been overexpressed in three crops, leading to a vitamin C increase of six-fold in tomato, three-fold in potato, and two-fold in strawberry; however, in tomato, GGP overexpression has led to some morphological fruit alterations, such as seed loss [140]. The GGP gene of acerola, a well-known crop with high vitamin C content, under the control of a leaf-specific promoter, has been overexpressed in rice, increasing the foliar content up to 2.5-fold, which did not cause morphological changes and conferred multistress tolerance [202]. Interestingly, also, the editing of the uORF, which controls the translation of the GGP2 gene in lettuce and tomato, increases the vitamin C content by 150% and confers tolerance to oxidative stress, providing a good strategy to obtain transgene-free lines with improved vitamin C [203,204]. Moreover, in apple, three paralogs of GGP, colocated in ASC-QTL clusters and, specifically, the GGP1 allele, play a key role in the regulation of vitamin C content in fruits. This suggests that a single-nucleotide polymorphism of this allele is an excellent candidate for breeding in order to improve vitamin C levels in fruits [205].
The multigenic approach, based on the coexpression of genes of the d-mannose/l-galactose pathway, represents an interesting strategy to obtain high levels of vitamin C in crops. The transient coexpression of GGP and GME in tobacco leaves caused a seven-fold increase in vitamin C content [138]. In Arabidopsis, GGP overexpressing lines had a 2.9-fold enhancement of vitamin C, whereas the double-gene transformation with GGP-GPP and GGP-GLDH led to an up to 4.1-fold vitamin C increase [206]. The contemporary overexpression of acerola GGP, GMP, and GME genes in tomato protoplasts caused an increase in vitamin C content, which was approximately four-fold higher than in wild type [207]. A stable transformation with GME, GMP, GGP, and GPP was obtained in tomato through pyramiding, which is a conventional hybridization that is technically achievable and generates stable inherited target genes [208,209]. Pyramiding transgenic lines GME × GMP and GME × GMP × GGP × GPP showed a substantial increase in vitamin C content in leaves and fruits. Moreover, in these lines, vitamin C transport capability, fruit shape and size, as well as stress tolerance were significantly ameliorated [209].

6.2. Manipulation of Other Biosynthetic Pathways

The overexpression of genes of the alternative biosynthetic pathways have also given good results in terms of vitamin C content in different crops. Regarding the gulose pathway, positive results have been obtained with the expression of rat cDNA encoding GulLO, the enzyme involved in the final step of the animal vitamin C biosynthetic pathway [163,210,211]. Lettuce and tobacco plants constitutively expressing this gene showed four- and seven-fold increases in vitamin C levels, respectively [163]. Transgenic potato plants, overexpressing the same gene, show improved vitamin C accumulation in tubers and increased tolerance to several abiotic stresses [210]. In the same way, Arabidopsis lines overexpressing this GulLO contained high vitamin C contents and exhibited improved growth and enhanced biomass of shoots and roots, as well as higher tolerance to diverse abiotic stresses [211].
Interesting results have also been obtained by manipulating the galacturonate pathway. Overexpression of the strawberry FaGalUR led to a two-fold increase in vitamin C content in potato, and this enhancement allowed for an increase in tolerance to abiotic stresses in the transgenic lines [212]. Similar positive results have been reported for tomato, where, although there was a moderate increase in vitamin C content, an increase in total antioxidants occurred that was linked to redox state regulation [213]; moreover, tomato plants overexpressing FaGalUR were found to be more tolerant to abiotic stresses [214]. Interestingly, in the tomato introgression line IL12-4-SL, the genes encoding for pectin methylesterase, polygalacturonase, and UDP-d-glucuronic-acid-4-epimerase, which are involved in pectin degradation, have been identified as candidate genes for a QTL associated with high vitamin C content, suggesting that marker-assisted selection could be a good strategy to enhance vitamin C accumulation [215].

6.3. Manipulation of Recycling Genes

Vitamin C enhancement in crops can also be achieved by manipulating the genes coding for MDHAR and DHAR, which are the enzymes involved in the reduction of MDHA and DHA, respectively. Several papers have reported that ASC regeneration by DHAR overexpression could represent an efficient method of vitamin C biofortification in different species, such as corn [216], tomato [217], and blueberry [143]. Recently, a cytosolic DHAR identified in the woody plant Liriodendron chinense was overexpressed in Arabidopsis, which led not only to vitamin C enhancement but also to an improvement of growth under stress conditions [218]. In apple, colocation between DHAR3-3 and a QTL for browning has been found, showing a relationship between ASC redox state and fruit vulnerability to browning [205].
Research conducted on MDHAR shows discordant results depending on the species. The overexpression in tobacco of the Arabidopsis cytosolic isoform of MDHAR enhances vitamin C content [219]. Similarly, the acerola MDHAR, overexpressed in tobacco, led to a two-fold increase in vitamin C content and a better tolerance to salt stress [220]. On the other hand, overexpression of the cytosolic-targeted tomato MDHAR caused a 0.7-fold reduction in vitamin C content in tomato fruits [217]. Another study conducted on tomato indicates that transgenic lines overexpressing MDHAR display a reduction in vitamin C content in leaves, while lines with silencing of MDHAR show an increase of vitamin C in both fruits and leaves [221]. The enhancement of vitamin C in silenced MDHAR lines could be due to a decrease in degradation [44]. In cherry tomato, the suppression of AOX was also found to increase vitamin C, lycopene, and carotene contents of the fruits and to confer tolerance to salt stress [222].

6.4. Manipulation of Regulatory Networks

Despite the many results obtained by overexpressing vitamin C-related genes, limited success has been reported in most species. In light of this, attention has shifted to the manipulation of components of the regulatory network, such as transcription and regulator factors. The overexpression of ERF98, which is a positive regulator of GMP, GGP, and GLDH genes, enhanced vitamin C content and increased tolerance to salt stress in Arabidopsis [223]. Similarly, tomato plants overexpressing HZ24, a transcriptional factor that binds the promoters of GMP, GME2, and GGP, showed increased vitamin C levels and reduced sensitivity to oxidative stress [196]. Overexpression of the regulator factors KONJAC1 and 2, which are two nucleotide sugar pyrophosphorylase-like proteins that modulate GMP activity, led to an increase in vitamin C content in Arabidopsis [224]. Arabidopsis and tomato plants, overexpressing the regulator factor SlZF3, showed inhibition of GMP degradation by COP9 signalosome, with a consequent enhancement of vitamin C content and tolerance to salt stress [225]. A new transcription factor bHLH59, which can activate the transcription of PMI, PMM, and GMP 2–4 and colocalize with the vitamin C QTL TFA9, has been identified in tomato. The overexpression of bHLH59 causes vitamin C accumulation and increases oxidative stress tolerance. The differences in vitamin C accumulation within different tomato accessions is ascribed to nucleotide differences in the promoter region of HLH59. This finding could be used to plan breeding strategies for vitamin C improvement [226].

7. Conclusions

In humans, different physiological processes require vitamin C as an antioxidant or a cofactor of mono-oxygenases and dioxygenases, whereby low vitamin C levels prevent optimal functioning. An increase in vitamin C intake through food is surely beneficial for human physiology [227,228]. The recommended daily intake (RDI) of vitamin C is 75–90 mg/day [229]. Nevertheless, 100 g of potatoes and tomatoes have about one-fourth of the RDI and cereal grains contain very low, almost undetectable, quantities of vitamin C [230]. Assuming their potential to make available adequate vitamin C levels, biofortified crops could be decisive in the elimination of vitamin C deficiency on a worldwide scale. Apart from having beneficial effects on human health, vitamin C biofortification also has the potential to improve plant tolerance to various stresses, which is a prominent target to guarantee crop productivity in an era of global climate change.
However, vitamin C accumulation in different plant organs is dependent on multiple metabolic processes, such as biosynthesis, recycling, degradation, and transport. Moreover, the vitamin C content is influenced by endogenous stimuli and environmental factors, among which light is of primary importance. Thus, as for other micronutrients, comprehensive knowledge of the genetic, biochemical, and molecular networks that govern vitamin C levels is mandatory to obtain vitamin-C-biofortified crops [231].
Considerable progresses on the understanding of the multiple roles of vitamin C and its interaction with other antioxidants, as well as with signal transduction pathways of hormones and ROS have been made. However, little is known about the influence that increased vitamin C levels may have on the different physiological processes of plants. Since changes in vitamin C levels greatly modify gene expression, and in particular, the transcript levels of genes involved in photosynthesis and the defense response to pathogens [77], the possibility of undesirable consequences resulting from the altered vitamin C content has to be carefully considered. Thus, efforts to obtain vitamin-C-biofortified plants necessitate an in-depth investigation into how these changes can alter plant growth, development, and responses to biotic and abiotic stresses under field conditions. To limit the possible unplanned consequences, targeted approaches altering vitamin C levels in specific tissues or organs are required.
Particular attention must be also paid to the choice of methodology utilized for vitamin C biofortification. The multigenic approach, obtained with co-expression or pyramiding, has led to a significant increase in vitamin C content. However, considering that genetically modified organisms are not always easily accepted by public opinion, methodologies avoiding the use of transgenes must be taken in consideration. For instance, the editing of the uORF on the promoter of genes coding for GGP is a good method to obtain non transgenic plants enriched with vitamin C. The identification of candidate genes in QTL associated with high vitamin C content could also allow for obtaining vitamin-C-biofortified plants by marker-assisted selection, thus avoiding the use of transgenes.
Thoroughly understanding the regulatory mechanisms involved in vitamin C accumulation, which can differ between crops and growth phases, together with the choice of better approaches to be utilized to improve vitamin C levels are important goals for developing more efficient strategies for vitamin C biofortification.

Author Contributions

Writing—original draft preparation, C.P., S.F., N.D., A.P., S.D.L., L.M., and M.C.d.P.; writing—review and editing, C.P. and M.C.d.P.; visualization, A.P. and S.D.L.; supervision, M.C.d.P.


This research was funded by University of Bari Aldo Moro, grant number H95E10000710005.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Szent-Gyorgyi, A. Observations on the function of peroxidase systems and the chemistry of the adrenal cortex: Description of a new carbohydrate derivative. Biochem. J. 1928, 22, 1387–1409. [Google Scholar] [CrossRef] [PubMed]
  2. King, C.G.; Waugh, W.A. The chemical nature of vitamin C. Science 1932, 75, 357–358. [Google Scholar] [CrossRef] [PubMed]
  3. Svirbely, J.L.; Szent-Gyorgyi, A. The chemical nature of vitamin C. Biochem. J. 1932, 26, 865–870. [Google Scholar] [CrossRef] [PubMed][Green Version]
  4. Svirbely, J.L.; Szent-Gyorgyi, A. Hexuronic acid as the antiscorbutic factor. Nature 1932, 129, 690. [Google Scholar] [CrossRef]
  5. Haworth, W.H.; Hirst, E.L. Synthesis of ascorbic acid. J. Soc. Chem. Ind. 1933, 52, 645–646. [Google Scholar] [CrossRef]
  6. Szent-Györgyi, A.; Haworth, W.H. Hexuronic acid (ascorbic acid) as the antiscorbutic factor. Nature 1933, 131, 24. [Google Scholar] [CrossRef]
  7. Padayatty, S.J.; Levine, M. Vitamin C: The known and the unknown and Goldilocks. Oral Dis. 2016, 22, 463–493. [Google Scholar] [CrossRef]
  8. Turck, D.; Bresson, J.L.; Burlingame, B.; Dean, T.; Fairweather-Tait, S.; Heinonen, M.; Hirsch-Ernst, K.I.; Mangelsdorf, I.; McArdle, H.J.; Naska, A.; et al. Scientific opinion on Vitamin C and protection of DNA, proteins and lipids from oxidative damage: Evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006. EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). EFSA J. 2017, 15, 4685. [Google Scholar] [CrossRef]
  9. Brubaker, R.F.; Bourne, W.M.; Bachman, L.A.; McLaren, J.W. Ascorbic acid content of human corneal epithelium. Invest. Ophthalmol. Vis. Sci. 2000, 41, 1681–1683. [Google Scholar]
  10. Tannenbaum, S.R.; Wishnok, J.S.; Leaf, C.D. Inhibition of nitrosamine formation by ascorbic acid. Am. J. Clin. Nutr. 1991, 53, 247S–250S. [Google Scholar] [CrossRef]
  11. Huang, A.; Vita, J.A.; Venema, R.C.; Keaney, J.F. Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J. Biol. Chem. 2000, 275, 17399–17406. [Google Scholar] [CrossRef] [PubMed]
  12. Lane, D.J.; Richardson, D.R. The active role of vitamin C in mammalian iron metabolism: Much more than just enhanced iron absorption! Free Radic. Biol. Med. 2014, 75, 69–83. [Google Scholar] [CrossRef] [PubMed]
  13. Lane, D.J.; Bae, D.H.; Merlot, A.M.; Sahni, S.; Richardson, D.R. Duodenal cytochrome b (DCYTB) in iron metabolism: An update on function and regulation. Nutrients 2015, 7, 2274–2296. [Google Scholar] [CrossRef] [PubMed]
  14. Eipper, B.A.; Mains, R.E. The role of ascorbate in the biosynthesis of neuroendocrine peptides. Am. J. Clin. Nutr. 1991, 54, 1153S–1156S. [Google Scholar] [CrossRef] [PubMed]
  15. Prigge, S.T.; Mains, R.E.; Eipper, B.A.; Amzel, L.M. New insights into copper monooxygenases and peptide amidation: Structure, mechanism and function. Cell. Mol. Life Sci. 2000, 57, 1236–1259. [Google Scholar] [CrossRef] [PubMed]
  16. Kumar, D.; Mains, R.E.; Eipper, B.A. 60 YEARS OF POMC: From POMC and alpha-MSH to PAM, molecular oxygen, copper, and vitamin C. J. Mol. Endocrinol. 2016, 56, T63–T76. [Google Scholar] [CrossRef]
  17. Peterkofsky, B. Ascorbate requirement for hydroxylation and secretion of procollagen: Relationship to inhibition of collagen synthesis in scurvy. Am. J. Clin. Nutr. 1991, 54, 1135S–1140S. [Google Scholar] [CrossRef]
  18. Pekkala, M.; Hieta, R.; Kursula, P.; Kivirikko, K.I.; Wierenga, R.K.; Myllyharju, J. Crystallization of the proline-rich-peptide binding domain of human type I collagen prolyl 4-hydroxylase. Acta Crystallogr. D Biol. Crystallogr. 2003, 59, 940–942. [Google Scholar] [CrossRef]
  19. Mandl, J.; Szarka, A.; Banhegyi, G. Vitamin C: Update on physiology and pharmacology. Br. J. Pharmacol. 2009, 157, 1097–1110. [Google Scholar] [CrossRef]
  20. Fleming, P.J.; Kent, U.M. Cytochrome b561, ascorbic acid, and transmembrane electron transfer. Am. J. Clin. Nutr. 1991, 54, 1173S–1178S. [Google Scholar] [CrossRef]
  21. Dunn, W.A.; Rettura, G.; Seifter, E.; Englard, S. Carnitine biosynthesis from gamma-butyrobetaine and from exogenous protein-bound 6-N-trimethyl-L-lysine by the perfused guinea pig liver. Effect of ascorbate deficiency on the in situ activity of gamma-butyrobetaine hydroxylase. J. Biol. Chem. 1984, 259, 10764–10770. [Google Scholar] [PubMed]
  22. Rebouche, C.J. Ascorbic acid and carnitine biosynthesis. Am. J. Clin. Nutr. 1991, 54, 1147S–1152S. [Google Scholar] [CrossRef] [PubMed]
  23. Kobayashi, M.; Hoshinaga, Y.; Miura, N.; Tokuda, Y.; Shigeoka, S.; Murai, A.; Horio, F. Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats. Biosci. Biotechnol. Biochem. 2014, 78, 1060–1066. [Google Scholar] [CrossRef] [PubMed][Green Version]
  24. Camarena, V.; Wang, G. The epigenetic role of vitamin C in health and disease. Cell. Mol. Life Sci. 2016, 73, 1645–1658. [Google Scholar] [CrossRef] [PubMed][Green Version]
  25. Blaszczak, W.; Barczak, W.; Masternak, J.; Kopczynski, P.; Zhitkovich, A.; Rubis, B. Vitamin C as a modulator of the response to cancer therapy. Molecules 2019, 24, 453. [Google Scholar] [CrossRef] [PubMed]
  26. Linster, C.L.; Van Schaftingen, E.; Vitamin, C. Biosynthesis, recycling and degradation in mammals. FEBS J. 2007, 274, 1–22. [Google Scholar] [CrossRef]
  27. Smirnoff, N.; Conklin, P.L.; Loewus, F.A. Biosynthesis of ascorbic acid in plants: A renaissance. Ann. Rev. Plant. Physiol. Plant. Mol. Biol. 2001, 52, 437–467. [Google Scholar] [CrossRef]
  28. Carr, A.C.; Vissers, M.C. Synthetic or food-derived vitamin C—Are they equally bioavailable? Nutrients 2013, 5, 4284–4304. [Google Scholar] [CrossRef]
  29. Vissers, M.C.; Bozonet, S.M.; Pearson, J.F.; Braithwaite, L.J. Dietary ascorbate intake affects steady state tissue concentrations in vitamin C-deficient mice: Tissue deficiency after suboptimal intake and superior bioavailability from a food source (kiwifruit). Am. J. Clin. Nutr. 2011, 93, 292–301. [Google Scholar] [CrossRef]
  30. Tanaka, K.; Hashimoto, T.; Tokumaru, S.; Iguchi, H.; Kojo, S. Interactions between vitamin C and vitamin E are observed in tissues of inherently scorbutic rats. J. Nutr. 1997, 127, 2060–2064. [Google Scholar] [CrossRef]
  31. Guarnieri, S.; Riso, P.; Porrini, M. Orange juice vs vitamin C: Effect on hydrogen peroxide-induced DNA damage in mononuclear blood cells. Br. J. Nutr. 2007, 97, 639–643. [Google Scholar] [CrossRef] [PubMed]
  32. Beck, K.; Conlon, C.A.; Kruger, R.; Coad, J.; Stonehouse, W. Gold kiwifruit consumed with an iron-fortified breakfast cereal meal improves iron status in women with low iron stores: A 16-week randomised controlled trial. Br. J. Nutr. 2011, 105, 101–109. [Google Scholar] [CrossRef] [PubMed]
  33. Carr, A.C.; Frei, B. Toward a new recommended dietary allowance for vitamin C based on antioxidant and health effects in humans. Am. J. Clin. Nutr. 1999, 69, 1086–1107. [Google Scholar] [CrossRef] [PubMed][Green Version]
  34. Macknight, R.C.; Laing, W.A.; Bulley, S.M.; Broad, R.C.; Johnson, A.A.; Hellens, R.P. Increasing ascorbate levels in crops to enhance human nutrition and plant abiotic stress tolerance. Curr. Opin. Biotechnol. 2017, 44, 153–160. [Google Scholar] [CrossRef] [PubMed]
  35. Locato, V.; Cimini, S.; Gara, L.D. Strategies to increase vitamin C in plants: From plant defense perspective to food biofortification. Front. Plant. Sci. 2013, 4, 152. [Google Scholar] [CrossRef] [PubMed]
  36. Paciolla, C.; Paradiso, A.; de Pinto, M.C. Cellular redox homeostasis as central modulator in plant stress. In Redox State as a Central Regulator of Plant-Cell Stress Responses; Gupta, D.K., Palma, J.M., Corpas, F.J., Eds.; Springer: Cham, Switzerland, 2016; pp. 1–23. [Google Scholar] [CrossRef]
  37. Tripathi, R.P.; Singh, B.; Bisht, S.S.; Pandey, J. L-Ascorbic acid in organic synthesis: An overview. Curr. Org. Chem. 2009, 13, 99–122. [Google Scholar] [CrossRef]
  38. Zhang, L.; Dong, S.J. The electrocatalytic oxidation of ascorbic acid on polyaniline film synthesized in the presence of camphorsulfonic acid. J. Electroanal. Chem. 2004, 568, 189–194. [Google Scholar] [CrossRef]
  39. Matsui, T.; Kitagawa, Y.; Okumura, M.; Shigeta, Y. Accurate standard hydrogen electrode potential and applications to the redox potentials of vitamin C and NAD/NADH. J. Phys. Chem. A 2015, 119, 369–376. [Google Scholar] [CrossRef]
  40. Szarka, A.; Tomasskovics, B.; Banhegyi, G. The ascorbate-glutathione-alpha-tocopherol triad in abiotic stress response. Int. J. Mol. Sci. 2012, 13, 4458–4483. [Google Scholar] [CrossRef]
  41. Buettner, G.R.; Jurkiewicz, B.A. Catalytic metals, ascorbate and free radicals: Combinations to avoid. Radiat. Res. 1996, 145, 532–541. [Google Scholar] [CrossRef]
  42. Parrow, N.L.; Leshin, J.A.; Levine, M. Parenteral ascorbate as a cancer therapeutic: A reassessment based on pharmacokinetics. Antioxid. Redox Signal. 2013, 19, 2141–2156. [Google Scholar] [CrossRef] [PubMed]
  43. Hossain, M.A.; Asada, K. Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. J. Biol. Chem. 1985, 260, 12920–12926. [Google Scholar] [PubMed]
  44. Truffault, V.; Fry, S.C.; Stevens, R.G.; Gautier, H. Ascorbate degradation in tomato leads to accumulation of oxalate, threonate and oxalyl threonate. Plant J. 2017, 89, 996–1008. [Google Scholar] [CrossRef] [PubMed]
  45. Maruta, T.; Sawa, Y.; Shigeoka, S.; Ishikawa, T. Diversity and evolution of ascorbate peroxidase functions in chloroplasts: More than just a classical antioxidant enzyme? Plant Cell Physiol. 2016, 57, 1377–1386. [Google Scholar] [CrossRef]
  46. Teixeira, F.K.; Menezes-Benavente, L.; Margis, R.; Margis-Pinheiro, M. Analysis of the molecular evolutionary history of the ascorbate peroxidase gene family: Inferences from the rice genome. J. Mol. Evol. 2004, 59, 761–770. [Google Scholar] [CrossRef]
  47. Foyer, C.H.; Noctor, G. Ascorbate and glutathione: The heart of the redox hub. Plant Physiol. 2011, 155, 2–18. [Google Scholar] [CrossRef]
  48. Sano, S. Molecular and functional characterization of monodehydroascorbate and dehydroascorbate reductases. In Ascorbic Acid in Plant Growth, Development and Stress Tolerance; Hossain, M.A., Munnè-Bosch, S., Burritt, D.J., Diaz-Vivancos, P., Fujita, M., Lorence, A., Eds.; Springer: Cham, Switzerland, 2017; pp. 129–156. [Google Scholar] [CrossRef]
  49. Gallie, D.R. The role of L-ascorbic acid recycling in responding to environmental stress and in promoting plant growth. J. Exp. Bot. 2013, 64, 433–443. [Google Scholar] [CrossRef]
  50. Noshi, M.; Yamada, H.; Hatanaka, R.; Tanabe, N.; Tamoi, M.; Shigeoka, S. Arabidopsis dehydroascorbate reductase 1 and 2 modulate redox states of ascorbate-glutathione cycle in the cytosol in response to photooxidative stress. Biosci. Biotechnol. Biochem. 2017, 81, 523–533. [Google Scholar] [CrossRef][Green Version]
  51. Ding, S.; Lu, Q.; Zhang, Y.; Yang, Z.; Wen, X.; Zhang, L.; Lu, C. Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state. Plant Mol. Biol. 2009, 69, 577–592. [Google Scholar] [CrossRef]
  52. Harshavardhan, V.T.; Wu, T.M.; Hong, C.Y. Glutathione reductase and abiotic stress tolerance in plants. In Glutathione in Plant Growth, Development, and Stress Tolerance; Hossain, M.A., Mostofa, M.G., Diaz-Vivancos, P., Burritt, D.J., Fujita, M., Tram, L.S.P., Eds.; Springer: Cham, Switzerland, 2017; pp. 265–286. [Google Scholar] [CrossRef]
  53. Chew, O.; Rudhe, C.; Glaser, E.; Whelan, J. Characterization of the targeting signal of dual-targeted pea glutathione reductase. Plant Mol. Biol 2003, 53, 341–356. [Google Scholar] [CrossRef]
  54. Gest, N.; Gautier, H.; Stevens, R. Ascorbate as seen through plant evolution: The rise of a successful molecule? J. Exp. Bot. 2013, 64, 33–53. [Google Scholar] [CrossRef] [PubMed]
  55. Holler, S.; Ueda, Y.; Wu, L.; Wang, Y.; Hajirezaei, M.R.; Ghaffari, M.R.; von Wiren, N.; Frei, M. Ascorbate biosynthesis and its involvement in stress tolerance and plant development in rice (Oryza sativa L.). Plant Mol. Biol. 2015, 88, 545–560. [Google Scholar] [CrossRef] [PubMed]
  56. Maddison, J.; Lyons, T.; Plochl, M.; Barnes, J. Hydroponically cultivated radish fed L-galactono-1,4-lactone exhibit increased tolerance to ozone. Planta 2002, 214, 383–391. [Google Scholar] [CrossRef] [PubMed]
  57. Paradiso, A.; Berardino, R.; de Pinto, M.C.; Sanita di Toppi, L.; Storelli, M.M.; Tommasi, F.; De Gara, L. Increase in ascorbate-glutathione metabolism as local and precocious systemic responses induced by cadmium in durum wheat plants. Plant Cell Physiol. 2008, 49, 362–374. [Google Scholar] [CrossRef]
  58. Wang, S.D.; Zhu, F.; Yuan, S.; Yang, H.; Xu, F.; Shang, J.; Xu, M.Y.; Jia, S.D.; Zhang, Z.W.; Wang, J.H.; et al. The roles of ascorbic acid and glutathione in symptom alleviation to SA-deficient plants infected with RNA viruses. Planta 2011, 234, 171–181. [Google Scholar] [CrossRef]
  59. Sgobba, A.; Paradiso, A.; Dipierro, S.; De Gara, L.; de Pinto, M.C. Changes in antioxidants are critical in determining cell responses to short- and long-term heat stress. Physiol. Plant. 2015, 153, 68–78. [Google Scholar] [CrossRef]
  60. Wheeler, G.L.; Jones, M.A.; Smirnoff, N. The biosynthetic pathway of vitamin C in higher plants. Nature 1998, 393, 365–369. [Google Scholar] [CrossRef]
  61. Smirnoff, N. Ascorbic acid: Metabolism and functions of a multi-facetted molecule. Curr. Opin. Plant Biol. 2000, 3, 229–235. [Google Scholar] [CrossRef]
  62. Smirnoff, N. Ascorbic acid metabolism and functions: A comparison of plants and mammals. Free Radic. Biol. Med. 2018, 122, 116–129. [Google Scholar] [CrossRef]
  63. De Gara, L.; de Pinto, M.C.; Arrigoni, O. Ascorbate synthesis and ascorbate peroxidase activity during the early stage of wheat germination. Physiol. Plant. 1997, 100, 894–900. [Google Scholar] [CrossRef]
  64. Tommasi, F.; Paciolla, C.; de Pinto, M.C.; De Gara, L. A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds. J. Exp. Bot. 2001, 52, 1647–1654. [Google Scholar] [CrossRef] [PubMed]
  65. Veljovic-Jovanovic, S.D.; Pignocchi, C.; Noctor, G.; Foyer, C.H. Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. Plant Physiol. 2001, 127, 426–435. [Google Scholar] [CrossRef] [PubMed]
  66. Kukavica, B.; Jovanovic, S.V. Senescence-related changes in the antioxidant status of ginkgo and birch leaves during autumn yellowing. Physiol. Plant. 2004, 122, 321–327. [Google Scholar] [CrossRef]
  67. Bartoli, C.G.; Tambussi, E.A.; Diego, F.; Foyer, C.H. Control of ascorbic acid synthesis and accumulation and glutathione by the incident light red/far red ratio in Phaseolus vulgaris leaves. FEBS Lett. 2009, 583, 118–122. [Google Scholar] [CrossRef]
  68. Heyneke, E.; Luschin-Ebengreuth, N.; Krajcer, I.; Wolkinger, V.; Muller, M.; Zechmann, B. Dynamic compartment specific changes in glutathione and ascorbate levels in Arabidopsis plants exposed to different light intensities. BMC Plant Biol. 2013, 13, 104. [Google Scholar] [CrossRef]
  69. Bartoli, C.G.; Yu, J.; Gomez, F.; Fernandez, L.; McIntosh, L.; Foyer, C.H. Inter-relationships between light and respiration in the control of ascorbic acid synthesis and accumulation in Arabidopsis thaliana leaves. J. Exp. Bot. 2006, 57, 1621–1631. [Google Scholar] [CrossRef]
  70. Yabuta, Y.; Mieda, T.; Rapolu, M.; Nakamura, A.; Motoki, T.; Maruta, T.; Yoshimura, K.; Ishikawa, T.; Shigeoka, S. Light regulation of ascorbate biosynthesis is dependent on the photosynthetic electron transport chain but independent of sugars in Arabidopsis. J. Exp. Bot. 2007, 58, 2661–2671. [Google Scholar] [CrossRef][Green Version]
  71. Grillet, L.; Ouerdane, L.; Flis, P.; Hoang, M.T.; Isaure, M.P.; Lobinski, R.; Curie, C.; Mari, S. Ascorbate efflux as a new strategy for iron reduction and transport in plants. J. Biol. Chem. 2014, 289, 2515–2525. [Google Scholar] [CrossRef]
  72. Asada, K. THE WATER-WATER CYCLE IN CHLOROPLASTS: Scavenging of active oxygens and dissipation of excess photons. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999, 50, 601–639. [Google Scholar] [CrossRef]
  73. Awad, J.; Stotz, H.U.; Fekete, A.; Krischke, M.; Engert, C.; Havaux, M.; Berger, S.; Mueller, M.J. 2-Cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions. Plant Physiol. 2015, 167, 1592–1603. [Google Scholar] [CrossRef]
  74. Saga, G.; Giorgetti, A.; Fufezan, C.; Giacometti, G.M.; Bassi, R.; Morosinotto, T. Mutation analysis of violaxanthin de-epoxidase identifies substrate-binding sites and residues involved in catalysis. J. Biol. Chem. 2010, 285, 23763–23770. [Google Scholar] [CrossRef] [PubMed]
  75. Toth, S.Z.; Nagy, V.; Puthur, J.T.; Kovacs, L.; Garab, G. The physiological role of ascorbate as photosystem II electron donor: Protection against photoinactivation in heat-stressed leaves. Plant Physiol. 2011, 156, 382–392. [Google Scholar] [CrossRef] [PubMed]
  76. Ivanov, B.N. Role of ascorbic acid in photosynthesis. Biochemistry (Moscow) 2014, 79, 282–289. [Google Scholar] [CrossRef] [PubMed]
  77. Kiddle, G.; Pastori, G.M.; Bernard, S.; Pignocchi, C.; Antoniw, J.; Verrier, P.J.; Foyer, C.H. Effects of leaf ascorbate content on defense and photosynthesis gene expression in Arabidopsis thaliana. Antioxid. Redox Signal. 2003, 5, 23–32. [Google Scholar] [CrossRef] [PubMed]
  78. Chen, Z.; Gallie, D.R. Dehydroascorbate reductase affects leaf growth, development, and function. Plant Physiol. 2006, 142, 775–787. [Google Scholar] [CrossRef] [PubMed]
  79. Barth, C.; Moeder, W.; Klessig, D.F.; Conklin, P.L. The timing of senescence and response to pathogens is altered in the ascorbate-deficient Arabidopsis mutant vitamin c-1. Plant Physiol. 2004, 134, 1784–1792. [Google Scholar] [CrossRef] [PubMed]
  80. Barth, C.; De Tullio, M.; Conklin, P.L. The role of ascorbic acid in the control of flowering time and the onset of senescence. J. Exp. Bot. 2006, 57, 1657–1665. [Google Scholar] [CrossRef] [PubMed]
  81. Gallie, D.R. Increasing Vitamin C content in plant foods to improve their nutritional value-successes and challenges. Nutrients 2013, 5, 3424–3446. [Google Scholar] [CrossRef]
  82. Murphy, L.J.; Robertson, K.N.; Harroun, S.G.; Brosseau, C.L.; Werner-Zwanziger, U.; Moilanen, J.; Tuononen, H.M.; Clyburne, J.A.C. A simple complex on the verge of breakdown: Isolation of the elusive cyanoformate ion. Science 2014, 344, 75–78. [Google Scholar] [CrossRef]
  83. Akram, N.A.; Shafiq, F.; Ashraf, M. Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Front. Plant Sci. 2017, 8, 613. [Google Scholar] [CrossRef]
  84. Boubakri, H. The role of ascorbic acid in plant-pathogen interactions. In Ascorbic Acid in Plant Growth, DEVELOPMENT and stress Tolerance; Hossain, M.A., Munnè-Bosch, S., Burritt, D.J., Diaz-Vivancos, P., Fujita, M., Lorence, A., Eds.; Springer: Cham, Switzerland, 2017; pp. 255–268. [Google Scholar] [CrossRef]
  85. Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 2005, 43, 205–227. [Google Scholar] [CrossRef] [PubMed]
  86. Pastori, G.M.; Kiddle, G.; Antoniw, J.; Bernard, S.; Veljovic-Jovanovic, S.; Verrier, P.J.; Noctor, G.; Foyer, C.H. Leaf vitamin C contents modulate plant defense transcripts and regulate genes that control development through hormone signaling. Plant Cell 2003, 15, 939–951. [Google Scholar] [CrossRef] [PubMed]
  87. Pavet, V.; Olmos, E.; Kiddle, G.; Mowla, S.; Kumar, S.; Antoniw, J.; Alvarez, M.E.; Foyer, C.H. Ascorbic acid deficiency activates cell death and disease resistance responses in Arabidopsis. Plant Physiol. 2005, 139, 1291–1303. [Google Scholar] [CrossRef] [PubMed]
  88. Mukherjee, M.; Larrimore, K.E.; Ahmed, N.J.; Bedick, T.S.; Barghouthi, N.T.; Traw, M.B.; Barth, C. Ascorbic acid deficiency in Arabidopsis induces constitutive priming that is dependent on hydrogen peroxide, salicylic acid, and the NPR1 gene. Mol. Plant Microbe 2010, 23, 340–351. [Google Scholar] [CrossRef]
  89. Botanga, C.J.; Bethke, G.; Chen, Z.; Gallie, D.R.; Fiehn, O.; Glazebrook, J. Metabolite Profiling of Arabidopsis Inoculated with Alternaria brassicicola reveals that ascorbate reduces disease severity. Mol. Plant Microbe 2012, 25, 1628–1638. [Google Scholar] [CrossRef] [PubMed]
  90. Egan, M.J.; Wang, Z.Y.; Jones, M.A.; Smirnoff, N.; Talbot, N.J. Generation of reactive oxygen species by fungal NADPH oxidases is required for rice blast disease. Proc. Natl. Acad. Sci. USA 2007, 104, 11772–11777. [Google Scholar] [CrossRef][Green Version]
  91. Fujiwara, A.; Shimura, H.; Masuta, C.; Sano, S.; Inukai, T. Exogenous ascorbic acid derivatives and dehydroascorbic acid are effective antiviral agents against Turnip mosaic virus in Brassica rapa. J. Gen. Plant Pathol. 2013, 79, 198–204. [Google Scholar] [CrossRef]
  92. Li, J.Y.; Trivedi, P.; Wang, N. Field evaluation of plant defense inducers for the control of citrus Huanglongbing. Phytopathology 2016, 106, 37–46. [Google Scholar] [CrossRef]
  93. Liso, R.; Calabrese, G.; Bitonti, M.B.; Arrigoni, O. Relationship between ascorbic acid and cell division. Exp. Cell Res. 1984, 150, 314–320. [Google Scholar] [CrossRef]
  94. Liso, R.; Innocenti, A.M.; Bitonti, M.B.; Arrigoni, O. Ascorbic acid-induced progression of quiescent center cells from G1-phase to S-phase. New Phytol. 1988, 110, 469–471. [Google Scholar] [CrossRef]
  95. Kerk, N.M.; Feldman, L.J. A Biochemical-model for the initiation and maintenance of the quiescent center—Implications for organization of root-meristems. Development 1995, 121, 2825–2833. [Google Scholar]
  96. de Pinto, M.C.; Francis, D.; De Gara, L. The redox state of the ascorbate-dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells. Protoplasma 1999, 209, 90–97. [Google Scholar] [CrossRef] [PubMed]
  97. de Pinto, M.C.; Tommasi, F.; De Gara, L. Enzymes of the ascorbate biosynthesis and ascorbate-glutathione cycle in cultured cells of Tobacco Bright Yellow-2 s. Plant Physiol. Biochem. 2000, 38, 541–550. [Google Scholar] [CrossRef]
  98. de Simone, A.; Hubbard, R.; de la Torre, N.V.; Velappan, Y.; Wilson, M.; Considine, M.J.; Soppe, W.J.J.; Foyer, C.H. redox changes during the cell cycle in the embryonic root meristem of Arabidopsis thaliana. Antioxid. Redox Sign. 2017, 27, 1505–1519. [Google Scholar] [CrossRef]
  99. Stasolla, C.; Yeung, E.C. Ascorbic acid metabolism during white spruce somatic embryo maturation and germination. Physiol. Plant. 2001, 111, 196–205. [Google Scholar] [CrossRef]
  100. Stasolla, C.; Yeung, E.C. Cellular ascorbic acid regulates the activity of major peroxidases in the apical poles of germinating white spruce (Picea glauca) somatic embryos. Plant Physiol. Biochem. 2007, 45, 188–198. [Google Scholar] [CrossRef]
  101. Pignocchi, C.; Fletcher, J.M.; Wilkinson, J.E.; Barnes, J.D.; Foyer, C.H. The function of ascorbate oxidase in tobacco. Plant Physiol. 2003, 132, 1631–1641. [Google Scholar] [CrossRef]
  102. Li, R.; Xin, S.; Tao, C.C.; Jin, X.; Li, H.B. Cotton ascorbate oxidase promotes cell growth in cultured tobacco bright yellow-2 cells through generation of apoplast oxidation. Int. J. Mol. Sci. 2017, 18, 1346. [Google Scholar] [CrossRef]
  103. Gonzalez-Reyes, J.A.; Alcain, F.J.; Caler, J.A.; Serrano, A.; Cordoba, F.; Navas, P. Relationship between apoplastic ascorbate regeneration and the stimulation of root-growth in Allium-Cepa L. Plant Sci. 1994, 100, 23–29. [Google Scholar] [CrossRef]
  104. Horemans, N.; Foyer, C.H.; Asard, H. Transport and action of ascorbate at the plant plasma membrane. Trends Plant Sci. 2000, 5, 263–267. [Google Scholar] [CrossRef]
  105. Horemans, N.; Foyer, C.H.; Potters, G.; Asard, H. Ascorbate function and associated transport systems in plants. Plant Physiol. Biochem. 2000, 38, 531–540. [Google Scholar] [CrossRef]
  106. Fry, S.C. Oxidative scission of plant cell wall polysaccharides by ascorbate-induced hydroxyl radicals. Biochem. J. 1998, 332, 507–515. [Google Scholar] [CrossRef] [PubMed][Green Version]
  107. Schopfer, P. Hydroxyl radical-induced cell-wall loosening in vitro and in vivo: Implications for the control of elongation growth. Plant J. 2001, 28, 679–688. [Google Scholar] [CrossRef] [PubMed]
  108. Padu, E. Apoplastic peroxidases, ascorbate and lignification in relation to nitrate supply in wheat stem. J. Plant Physiol. 1999, 154, 576–583. [Google Scholar] [CrossRef]
  109. de Pinto, M.C.; De Gara, L. Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation. J. Exp. Bot. 2004, 55, 2559–2569. [Google Scholar] [CrossRef]
  110. de Pinto, M.C.; Tommasi, F.; De Gara, L. Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells. Plant Physiol. 2002, 130, 698–708. [Google Scholar] [CrossRef]
  111. Vacca, R.A.; de Pinto, M.C.; Valenti, D.; Passarella, S.; Marra, E.; De Gara, L. Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism are early events in heat shock-induced programmed cell death in tobacco bright-yellow 2 cells. Plant Physiol. 2004, 134, 1100–1112. [Google Scholar] [CrossRef]
  112. de Pinto, M.C.; Paradiso, A.; Leonetti, P.; De Gara, L. Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death. Plant J. 2006, 48, 784–795. [Google Scholar] [CrossRef]
  113. Locato, V.; Gadaleta, C.; De Gara, L.; De Pinto, M.C. Production of reactive species and modulation of antioxidant network in response to heat shock: A critical balance for cell fate. Plant Cell Environ. 2008, 31, 1606–1619. [Google Scholar] [CrossRef]
  114. Valenti, D.; Vacca, R.A.; de Pinto, M.C.; De Gara, L.; Marra, E.; Passarella, S. In the early phase of programmed cell death in Tobacco Bright Yellow 2 cells the mitochondrial adenine nucleotide translocator, adenylate kinase and nucleoside diphosphate kinase are impaired in a reactive oxygen species-dependent manner. BBA Biochim. Biophys. Acta 2007, 1767, 66–78. [Google Scholar] [CrossRef][Green Version]
  115. de Pinto, M.C.; Locato, V.; Sgobba, A.; Romero-Puertas, M.D.; Gadaleta, C.; Delledonne, M.; De Gara, L. S-Nitrosylation of ascorbate peroxidase is part of programmed cell death signaling in Tobacco Bright Yellow-2 cells. Plant Physiol. 2013, 163, 1766–1775. [Google Scholar] [CrossRef] [PubMed]
  116. de Pinto, M.C.; Locato, V.; De Gara, L. Redox regulation in plant programmed cell death. Plant Cell Environ. 2012, 35, 234–244. [Google Scholar] [CrossRef] [PubMed]
  117. Locato, V.; Paradiso, A.; Sabetta, W.; De Gara, L.; de Pinto, M.C. Nitric Oxide and Reactive Oxygen Species in PCD Signaling. Adv. Bot. Res. 2016, 77, 165–192. [Google Scholar] [CrossRef]
  118. Paradiso, A.; de Pinto, M.C.; Locato, V.; De Gara, L. Galactone-gamma-lactone-dependent ascorbate biosynthesis alters wheat kernel maturation. Plant Biol. 2012, 14, 652–658. [Google Scholar] [CrossRef] [PubMed]
  119. Ortiz-Espín, A.; Sánchez-Guerrero, A.; Sevilla, F.; Jiménez, A. The role of ascorbate in plant growth and development. In Ascorbic Acid in Plant Growth, Development and Stress Tolerance; Hossain, M.A., Munnè-Bosch, S., Burritt, D.J., Diaz-Vivancos, P., Fujita, M., Lorence, A., Eds.; Springer: Cham, Switzerland, 2017; pp. 25–45. [Google Scholar] [CrossRef]
  120. Hancock, R.D.; Viola, R. Biosynthesis and catabolism of L-ascorbic acid in plants. Crit. Rev. Plant Sci. 2005, 24, 167–188. [Google Scholar] [CrossRef]
  121. Dowdle, J.; Ishikawa, T.; Gatzek, S.; Rolinski, S.; Smirnoff, N. Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability. Plant J. 2007, 52, 673–689. [Google Scholar] [CrossRef] [PubMed]
  122. Wolucka, B.A.; Van Montagu, M. GDP-mannose 3’,5’-epimerase forms GDP-L-gulose, a putative intermediate for the de novo biosynthesis of vitamin C in plants. J. Biol. Chem. 2003, 278, 47483–47490. [Google Scholar] [CrossRef]
  123. Lorence, A.; Chevone, B.I.; Mendes, P.; Nessler, C.L. Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiol. 2004, 134, 1200–1205. [Google Scholar] [CrossRef]
  124. Agius, F.; Gonzalez-Lamothe, R.; Caballero, J.L.; Munoz-Blanco, J.; Botella, M.A.; Valpuesta, V. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nat. Biotechnol. 2003, 21, 177–181. [Google Scholar] [CrossRef]
  125. Maruta, T.; Yonemitsu, M.; Yabuta, Y.; Tamoi, M.; Ishikawa, T.; Shigeoka, S. Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis. J. Biol. Chem. 2008, 283, 28842–28851. [Google Scholar] [CrossRef]
  126. Qian, W.; Yu, C.; Qin, H.; Liu, X.; Zhang, A.; Johansen, I.E.; Wang, D. Molecular and functional analysis of phosphomannomutase (PMM) from higher plants and genetic evidence for the involvement of PMM in ascorbic acid biosynthesis in Arabidopsis and Nicotiana benthamiana. Plant J. 2007, 49, 399–413. [Google Scholar] [CrossRef] [PubMed]
  127. Hoeberichts, F.A.; Vaeck, E.; Kiddle, G.; Coppens, E.; van de Cotte, B.; Adamantidis, A.; Ormenese, S.; Foyer, C.H.; Zabeau, M.; Inze, D.; et al. A Temperature-sensitive mutation in the Arabidopsis thaliana phosphomannomutase gene disrupts protein glycosylation and triggers cell death. J. Biol. Chem. 2008, 283, 5708–5718. [Google Scholar] [CrossRef] [PubMed]
  128. Conklin, P.L.; Williams, E.H.; Last, R.L. Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc. Natl. Acad. Sci. USA 1996, 93, 9970–9974. [Google Scholar] [CrossRef] [PubMed]
  129. Conklin, P.L.; Norris, S.R.; Wheeler, G.L.; Williams, E.H.; Smirnoff, N.; Last, R.L. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proc. Natl. Acad. Sci. USA 1999, 96, 4198–4203. [Google Scholar] [CrossRef][Green Version]
  130. Keller, R.; Renz, F.S.; Kossmann, J. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. Plant J. 1999, 19, 131–141. [Google Scholar] [CrossRef]
  131. Barber, G.A. Observations on the mechanism of the reversible epimerization of GDP-D-mannose to GDP-L-galactose by an enzyme from Chlorella pyrenoidosa. J. Biol. Chem. 1979, 254, 7600–7603. [Google Scholar]
  132. Wolucka, B.A.; Persiau, G.; Van Doorsselaere, J.; Davey, M.W.; Demol, H.; Vandekerckhove, J.; Van Montagu, M.; Zabeau, M.; Boerjan, W. Partial purification and identification of GDP-mannose 3”,5”-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway. Proc. Natl. Acad. Sci. USA 2001, 98, 14843–14848. [Google Scholar] [CrossRef]
  133. Fenech, M.; Amaya, I.; Valpuesta, V.; Botella, M.A. Vitamin C content in fruits: Biosynthesis and regulation. Front. Plant Sci. 2019, 9, 2006. [Google Scholar] [CrossRef]
  134. Maruta, T.; Ichikawa, Y.; Mieda, T.; Takeda, T.; Tamoi, M.; Yabuta, Y.; Ishikawa, T.; Shigeoka, S. The contribution of Arabidopsis homologs of L-gulono-1,4-lactone oxidase to the biosynthesis of ascorbic acid. Biosci. Biotechnol. Biochem. 2010, 74, 1494–1497. [Google Scholar] [CrossRef]
  135. Lukowitz, W.; Nickle, T.C.; Meinke, D.W.; Last, R.L.; Conklin, P.L.; Somerville, C.R. Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proc. Natl. Acad. Sci. USA 2001, 98, 2262–2267. [Google Scholar] [CrossRef]
  136. Reiter, W.D.; Vanzin, G.F. Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol. Biol. 2001, 47, 95–113. [Google Scholar] [CrossRef] [PubMed]
  137. Bulley, S.; Laing, W. The regulation of ascorbate biosynthesis. Curr. Opin. Plant Biol. 2016, 33, 15–22. [Google Scholar] [CrossRef] [PubMed]
  138. Bulley, S.M.; Rassam, M.; Hoser, D.; Otto, W.; Schunemann, N.; Wright, M.; MacRae, E.; Gleave, A.; Laing, W. Gene expression studies in kiwifruit and gene over-expression in Arabidopsis indicates that GDP-L-galactose guanyltransferase is a major control point of vitamin C biosynthesis. J. Exp. Bot. 2009, 60, 765–778. [Google Scholar] [CrossRef] [PubMed][Green Version]
  139. Li, M.; Ma, F.; Liang, D.; Li, J.; Wang, Y. Ascorbate biosynthesis during early fruit development is the main reason for its accumulation in kiwi. PLoS ONE 2010, 5, e14281. [Google Scholar] [CrossRef]
  140. Bulley, S.; Wright, M.; Rommens, C.; Yan, H.; Rassam, M.; Lin-Wang, K.; Andre, C.; Brewster, D.; Karunairetnam, S.; Allan, A.C.; et al. Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase. Plant Biotechnol. J. 2012, 10, 390–397. [Google Scholar] [CrossRef]
  141. Alos, E.; Rodrigo, M.J.; Zacarias, L. Differential transcriptional regulation of L-ascorbic acid content in peel and pulp of citrus fruits during development and maturation. Planta 2014, 239, 1113–1128. [Google Scholar] [CrossRef]
  142. Yoshimura, K.; Nakane, T.; Kume, S.; Shiomi, Y.; Maruta, T.; Ishikawa, T.; Shigeoka, S. Transient expression analysis revealed the importance of VTC2 expression level in light/dark regulation of ascorbate biosynthesis in Arabidopsis. Biosci. Biotechnol. Biochem. 2014, 78, 60–66. [Google Scholar] [CrossRef]
  143. Liu, F.; Wang, L.; Gu, L.; Zhao, W.; Su, H.; Cheng, X. Higher transcription levels in ascorbic acid biosynthetic and recycling genes were associated with higher ascorbic acid accumulation in blueberry. Food Chem. 2015, 188, 399–405. [Google Scholar] [CrossRef]
  144. Gao, Y.; Badejo, A.A.; Shibata, H.; Sawa, Y.; Maruta, T.; Shigeoka, S.; Page, M.; Smirnoff, N.; Ishikawa, T. Expression analysis of the VTC2 and VTC5 genes encoding GDP-L-galactose phosphorylase, an enzyme involved in ascorbate biosynthesis, in Arabidopsis thaliana. Biosci. Biotechnol. Biochem. 2011, 75, 1783–1788. [Google Scholar] [CrossRef]
  145. Laing, W.A.; Martinez-Sanchez, M.; Wright, M.A.; Bulley, S.M.; Brewster, D.; Dare, A.P.; Rassam, M.; Wang, D.; Storey, R.; Macknight, R.C.; et al. An upstream open reading frame is essential for feedback regulation of ascorbate biosynthesis in Arabidopsis. Plant Cell 2015, 27, 772–786. [Google Scholar] [CrossRef]
  146. Laing, W.A.; Bulley, S.; Wright, M.; Cooney, J.; Jensen, D.; Barraclough, D.; MacRae, E. A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proc. Natl. Acad. Sci. USA 2004, 101, 16976–16981. [Google Scholar] [CrossRef] [PubMed]
  147. Conklin, P.L.; Gatzek, S.; Wheeler, G.L.; Dowdle, J.; Raymond, M.J.; Rolinski, S.; Isupov, M.; Littlechild, J.A.; Smirnoff, N. Arabidopsis thaliana VTC4 encodes L-galactose-1-P phosphatase, a plant ascorbic acid biosynthetic enzyme. J. Biol. Chem. 2006, 281, 15662–15670. [Google Scholar] [CrossRef] [PubMed]
  148. Torabinejad, J.; Donahue, J.L.; Gunesekera, B.N.; Allen-Daniels, M.J.; Gillaspy, G.E. VTC4 is a bifunctional enzyme that affects myoinositol and ascorbate biosynthesis in plants. Plant Physiol. 2009, 150, 951–961. [Google Scholar] [CrossRef] [PubMed]
  149. Zhang, W.; Gruszewski, H.A.; Chevone, B.I.; Nessler, C.L. An Arabidopsis purple acid phosphatase with phytase activity increases foliar ascorbate. Plant Physiol. 2008, 146, 431–440. [Google Scholar] [CrossRef] [PubMed]
  150. Gatzek, S.; Wheeler, G.L.; Smirnoff, N. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. Plant J. 2002, 30, 541–553. [Google Scholar] [CrossRef]
  151. Leferink, N.G.H.; van den Berg, W.A.M.; van Berkel, W.J.H. L-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis. FEBS J. 2008, 275, 713–726. [Google Scholar] [CrossRef]
  152. Wheeler, G.; Ishikawa, T.; Pornsaksit, V.; Smirnoff, N. Evolution of alternative biosynthetic pathways for vitamin C following plastid acquisition in photosynthetic eukaryotes. Elife 2015, 4, e06369. [Google Scholar] [CrossRef][Green Version]
  153. Siendones, E.; Gonzalez-Reyes, J.A.; Santos-Ocana, C.; Navas, P.; Cordoba, F. Biosynthesis of ascorbic acid in kidney bean. L-galactono-gamma-lactone dehydrogenase is an intrinsic protein located at the mitochondrial inner membrane. Plant Physiol. 1999, 120, 907–912. [Google Scholar] [CrossRef]
  154. Bartoli, C.G.; Pastori, G.M.; Foyer, C.H. Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol. 2000, 123, 335–343. [Google Scholar] [CrossRef]
  155. Heazlewood, J.L.; Howell, K.A.; Millar, A.H. Mitochondrial complex I from Arabidopsis and rice: Orthologs of mammalian and fungal components coupled with plant-specific subunits. BBA Biochim. Biophys. Acta 2003, 1604, 159–169. [Google Scholar] [CrossRef]
  156. Pineau, B.; Layoune, O.; Danon, A.; De Paepe, R. L-Galactono-1,4-lactone dehydrogenase is required for the accumulation of plant respiratory complex I. J. Biol. Chem. 2008, 283, 32500–32505. [Google Scholar] [CrossRef] [PubMed]
  157. Schertl, P.; Sunderhaus, S.; Klodmann, J.; Grozeff, G.E.G.; Bartoli, C.G.; Braun, H.P. L-Galactono-1,4-lactone dehydrogenase (GLDH) forms part of three subcomplexes of mitochondrial complex I in Arabidopsis thaliana. J. Biol. Chem. 2012, 287, 14412–14419. [Google Scholar] [CrossRef] [PubMed]
  158. Schimmeyer, J.; Bock, R.; Meyer, E.H. L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis. Plant Mol. Biol. 2016, 90, 117–126. [Google Scholar] [CrossRef] [PubMed]
  159. Millar, A.H.; Mittova, V.; Kiddle, G.; Heazlewood, J.L.; Bartoli, C.G.; Theodoulou, F.L.; Foyer, C.H. Control of ascorbate synthesis by respiration and its implications for stress responses. Plant Physiol. 2003, 133, 443–447. [Google Scholar] [CrossRef]
  160. Ioannidi, E.; Kalamaki, M.S.; Engineer, C.; Pateraki, I.; Alexandrou, D.; Mellidou, I.; Giovannonni, J.; Kanellis, A.K. Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions. J. Exp. Bot. 2009, 60, 663–678. [Google Scholar] [CrossRef][Green Version]
  161. Wagner, C.; Sefkow, M.; Kopka, J. Construction and application of a mass spectral and retention time index database generated from plant GC/EI-TOF-MS metabolite profiles. Phytochemistry 2003, 62, 887–900. [Google Scholar] [CrossRef]
  162. Davey, M.W.; Gilot, C.; Persiau, G.; Ostergaard, J.; Han, Y.; Bauw, G.C.; Van Montagu, M.C. Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol. 1999, 121, 535–543. [Google Scholar] [CrossRef]
  163. Jain, A.K.; Nessler, C.L. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Mol. Breed. 2000, 6, 73–78. [Google Scholar] [CrossRef]
  164. Imai, T.; Niwa, M.; Ban, Y.; Hirai, M.; Oba, K.; Moriguchi, T. Importance of the L-galactonolactone pool for enhancing the ascorbate content revealed by L-galactonolactone dehydrogenase-overexpressing tobacco plants. Plant Cell Tissue Organ Cult. 2009, 96, 105–112. [Google Scholar] [CrossRef]
  165. Aboobucker, S.I.; Suza, W.P.; Lorence, A. Characterization of two Arabidopsis l-gulono-1,4-lactone oxidases, AtGulLO3 and AtGulLO5, involved in ascorbate biosynthesis. React. Oxyg. Spec. (Apex) 2017, 4, 389–417. [Google Scholar] [CrossRef]
  166. Valpuesta, V.; Botella, M.A. Biosynthesis of L-ascorbic acid in plants: New pathways for an old antioxidant. Trends Plant Sci. 2004, 9, 573–577. [Google Scholar] [CrossRef] [PubMed]
  167. Endres, S.; Tenhaken, R. Myoinositol oxygenase controls the level of myoinositol in Arabidopsis, but does not increase ascorbic acid. Plant Physiol. 2009, 149, 1042–1049. [Google Scholar] [CrossRef] [PubMed]
  168. Endres, S.; Tenhaken, R. Down-regulation of the myo-inositol oxygenase gene family has no effect on cell wall composition in Arabidopsis. Planta 2011, 234, 157–169. [Google Scholar] [CrossRef] [PubMed][Green Version]
  169. Ivanov Kavkova, E.; Blöchl, C.; Tenhaken, R. The Myo -inositol pathway does not contribute to ascorbic acid synthesis. Plant Biol. 2018, 21, 95–102. [Google Scholar] [CrossRef] [PubMed]
  170. Badejo, A.A.; Wada, K.; Gao, Y.; Maruta, T.; Sawa, Y.; Shigeoka, S.; Ishikawa, T. Translocation and the alternative D-galacturonate pathway contribute to increasing the ascorbate level in ripening tomato fruits together with the D-mannose/L-galactose pathway. J. Exp. Bot. 2012, 63, 229–239. [Google Scholar] [CrossRef]
  171. Di Matteo, A.; Sacco, A.; Anacleria, M.; Pezzotti, M.; Delledonne, M.; Ferrarini, A.; Frusciante, L.; Barone, A. The ascorbic acid content of tomato fruits is associated with the expression of genes involved in pectin degradation. BMC Plant Biol. 2010, 10. [Google Scholar] [CrossRef]
  172. Melino, V.J.; Soole, K.L.; Ford, C.M. Ascorbate metabolism and the developmental demand for tartaric and oxalic acids in ripening grape berries. BMC Plant Biol. 2009, 9. [Google Scholar] [CrossRef]
  173. Cruz-Rus, E.; Amaya, I.; Sanchez-Sevilla, J.F.; Botella, M.A.; Valpuesta, V. Regulation of L-ascorbic acid content in strawberry fruits. J. Exp. Bot. 2011, 62, 4191–4201. [Google Scholar] [CrossRef]
  174. Tabata, K.; Takaoka, T.; Esaka, M. Gene expression of ascorbic acid-related enzymes in tobacco. Phytochemistry 2002, 61, 631–635. [Google Scholar] [CrossRef]
  175. Massot, C.; Stevens, R.; Genard, M.; Longuenesse, J.J.; Gautier, H. Light affects ascorbate content and ascorbate-related gene expression in tomato leaves more than in fruits. Planta 2012, 235, 153–163. [Google Scholar] [CrossRef]
  176. Massot, C.; Genard, M.; Stevens, R.; Gautier, H. Fluctuations in sugar content are not determinant in explaining variations in vitamin C in tomato fruit. Plant Physiol. Biochem. 2010, 48, 751–757. [Google Scholar] [CrossRef] [PubMed][Green Version]
  177. Zhou, W.L.; Liu, W.K.; Yang, Q.C. Quality changes in hydroponic lettuce grown under pre-harvest short-duration continuous light of different intensities. J. Hortic. Sci. Biotechem. 2012, 87, 429–434. [Google Scholar] [CrossRef]
  178. Riga, P.; Benedicto, L.; Gil-Izquierdo, A.; Collado-Gonzalez, J.; Ferreres, F.; Medina, S. Diffuse light affects the contents of vitamin C, phenolic compounds and free amino acids in lettuce plants. Food Chem. 2019, 272, 227–234. [Google Scholar] [CrossRef] [PubMed]
  179. Shen, Y.Z.; Guo, S.S.; Ai, W.D.; Tang, Y.K. Effects of illuminants and illumination time on lettuce growth, yield and nutritional quality in a controlled environment. Life Sci. Space Res. 2014, 2, 38–42. [Google Scholar] [CrossRef]
  180. Zha, L.Y.; Zhang, Y.B.; Liu, W.K. Dynamic responses of ascorbate pool and metabolism in lettuce to long-term continuous light provided by red and blue LEDs. Environ. Exp. Bot. 2019, 163, 15–23. [Google Scholar] [CrossRef]
  181. Ohashi-Kaneko, K.; Takase, M.; Kon, N.; Fujiwara, K.; Kurata, K. Effect of light quality on growth and vegetable quality in leaf lettuce, spinach and komatsuna. Environ. Control Biol. 2007, 45, 189–198. [Google Scholar] [CrossRef]
  182. Qian, H.; Liu, T.; Deng, M.; Miao, H.; Cai, C.; Shen, W.; Wang, Q. Effects of light quality on main health-promoting compounds and antioxidant capacity of Chinese kale sprouts. Food Chem. 2016, 196, 1232–1238. [Google Scholar] [CrossRef]
  183. Li, M.J.; Ma, F.W.; Shang, P.F.; Zhang, M.; Hou, C.M.; Liang, D. Influence of light on ascorbate formation and metabolism in apple fruits. Planta 2009, 230, 39–51. [Google Scholar] [CrossRef]
  184. Ntagkas, N.; Woltering, E.; Nicole, C.; Labrie, C.; Marcelis, L.F.M. Light regulation of vitamin C in tomato fruit is mediated through photosynthesis. Environ. Exp. Bot. 2019, 158, 180–188. [Google Scholar] [CrossRef]
  185. Zhang, L.C.; Ma, G.; Yamawaki, K.; Ikoma, Y.; Matsumoto, H.; Yoshioka, T.; Ohta, S.; Kato, M. Regulation of ascorbic acid metabolism by blue LED light irradiation in citrus juice sacs. Plant Sci. 2015, 233, 134–142. [Google Scholar] [CrossRef][Green Version]
  186. Lee, Y.J.; Ha, J.Y.; Oh, J.E.; Cho, M.S. The effect of LED irradiation on the quality of cabbage stored at a low temperature. Food Sci. Biotechnol. 2014, 23, 1087–1093. [Google Scholar] [CrossRef]
  187. Mastropasqua, L.; Tanzarella, P.; Paciolla, C. Effects of postharvest light spectra on quality and health-related parameters in green Asparagus officinalis L. Postharvest Biol. Technol. 2016, 112, 143–151. [Google Scholar] [CrossRef]
  188. Loi, M.; Liuzzi, V.C.; Fanelli, F.; De Leonardis, S.; Maria Creanza, T.; Ancona, N.; Paciolla, C.; Mule, G. Effect of different light-emitting diode (LED) irradiation on the shelf life and phytonutrient content of broccoli (Brassica oleracea L. var. italica). Food Chem. 2019, 283, 206–214. [Google Scholar] [CrossRef]
  189. Frohnmeyer, H.; Staiger, D. Ultraviolet-B radiation-mediated responses in plants. Balancing damage and protection. Plant Physiol. 2003, 133, 1420–1428. [Google Scholar] [CrossRef] [PubMed]
  190. Xu, M.J.; Dong, J.F.; Zhu, M.Y. Effects of germination conditions on ascorbic acid level and yield of soybean sprouts. J. Sci. Food Agric. 2005, 85, 943–947. [Google Scholar] [CrossRef]
  191. Liu, P.; Li, Q.; Gao, Y.; Wang, H.; Chai, L.; Yu, H.; Jiang, W. A New perspective on the effect of UV-B on L-ascorbic acid metabolism in cucumber seedlings. J. Agric. Food Chem. 2019, 67, 4444–4452. [Google Scholar] [CrossRef] [PubMed]
  192. Calafiore, R.; Aliberti, A.; Ruggieri, V.; Olivieri, F.; Rigano, M.M.; Barone, A. Phenotypic and molecular selection of a superior Solanum pennellii introgression sub-line suitable for improving quality traits of cultivated tomatoes. Front. Plant Sci. 2019, 10, 190. [Google Scholar] [CrossRef]
  193. Ntagkas, N.; Woltering, E.; Bouras, S.; de Vos, R.C.; Dieleman, J.A.; Nicole, C.C.; Labrie, C.; Marcelis, L.F. Light-induced vitamin c accumulation in tomato fruits is independent of carbohydrate availability. Plants 2019, 8, 86. [Google Scholar] [CrossRef]
  194. Fukunaga, K.; Fujikawa, Y.; Esaka, M. Light regulation of ascorbic acid biosynthesis in rice via light responsive cis-elements in genes encoding ascorbic acid biosynthetic enzymes. Biosci. Biotechem. Biochem. 2010, 74, 888–891. [Google Scholar] [CrossRef]
  195. Liu, F.Y.; Nan, X.; Jian, G.H.; Yan, S.J.; Xie, L.H.; Brennan, C.S.; Huang, W.J.; Guo, X.B. The manipulation of gene expression and the biosynthesis of Vitamin C, E and folate in light-and dark-germination of sweet corn seeds. Sci. Rep.-UK 2017, 7, 7484. [Google Scholar] [CrossRef]
  196. Hu, T.; Ye, J.; Tao, P.; Li, H.; Zhang, J.; Zhang, Y.; Ye, Z. The tomato HD-Zip I transcription factor SlHZ24 modulates ascorbate accumulation through positive regulation of the D-mannose/L-galactose pathway. Plant J. 2016, 85, 16–29. [Google Scholar] [CrossRef] [PubMed]
  197. Zhang, W.Y.; Lorence, A.; Gruszewski, H.A.; Chevone, B.I.; Nessler, C.L. AMR1, an Arabidopsis gene that coordinately and negatively regulates the mannose/L-galactose ascorbic acid biosynthetic pathway. Plant Physiol. 2009, 150, 942–950. [Google Scholar] [CrossRef]
  198. Wang, J.; Yu, Y.; Zhang, Z.; Quan, R.; Zhang, H.; Ma, L.; Deng, X.W.; Huang, R. Arabidopsis CSN5B interacts with VTC1 and modulates ascorbic acid synthesis. Plant Cell 2013, 25, 625–636. [Google Scholar] [CrossRef] [PubMed]
  199. Ntagkas, N.; Woltering, E.J.; Marcelis, L.F.M. Light regulates ascorbate in plants: An integrated view on physiology and biochemistry. Environ. Exp. Bot. 2018, 147, 271–280. [Google Scholar] [CrossRef]
  200. Mellidou, I.; Kanellis, A.K. Genetic control of ascorbic acid biosynthesis and recycling in horticultural crops. Front. Chem. 2017, 5, 50. [Google Scholar] [CrossRef] [PubMed]
  201. Zhang, G.Y.; Liu, R.R.; Zhang, C.Q.; Tang, K.X.; Sun, M.F.; Yan, G.H.; Liu, Q.Q. Manipulation of the rice L-galactose pathway: Evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance. PLoS ONE 2015, 10, e0125870. [Google Scholar] [CrossRef]
  202. Ali, B.; Pantha, S.; Acharya, R.; Ueda, Y.; Wu, L.B.; Ashrafuzzaman, M.; Ishizaki, T.; Wissuwa, M.; Bulley, S.; Frei, M. Enhanced ascorbate level improves multi-stress tolerance in a widely grown indica rice variety without compromising its agronomic characteristics. J. Plant Physiol. 2019, 240, 152998. [Google Scholar] [CrossRef]
  203. Zhang, H.; Si, X.; Ji, X.; Fan, R.; Liu, J.; Chen, K.; Wang, D.; Gao, C. Genome editing of upstream open reading frames enables translational control in plants. Nat. Biotechnol. 2018, 36, 894–898. [Google Scholar] [CrossRef]
  204. Li, T.D.; Yang, X.P.; Yu, Y.; Si, X.M.; Zhai, X.W.; Zhang, H.W.; Dong, W.X.; Gao, C.X.; Xu, C. Domestication of wild tomato is accelerated by genome editing. Nat. Biotechnol. 2018, 36, 1160–1163. [Google Scholar] [CrossRef]
  205. Mellidou, I.; Chagne, D.; Laing, W.A.; Keulemans, J.; Davey, M.W. Allelic variation in paralogs of GDP-l-galactose phosphorylase is a major determinant of vitamin C concentrations in apple fruit. Plant Physiol. 2012, 160, 1613–1629. [Google Scholar] [CrossRef]
  206. Zhou, Y.; Tao, Q.C.; Wang, Z.N.; Fan, R.; Li, Y.; Sun, X.F.; Tang, K.X. Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation. Biol. Plant. 2012, 56, 451–457. [Google Scholar] [CrossRef]
  207. Suekawa, M.; Fujikawa, Y.; Inoue, A.; Kondo, T.; Uchida, E.; Koizumi, T.; Esaka, M. High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits. Biosci. Biotechnol. Biochem. 2019, 83, 1713–1716. [Google Scholar] [CrossRef] [PubMed]
  208. Saltzman, A.; Birol, E.; Bouis, H.E.; Boy, E.; De Moura, F.F.; Islam, Y.; Pfeiffer, W.H. Biofortification: Progress toward a more nourishing future. Glob. Food Secur. 2013, 2, 9–17. [Google Scholar] [CrossRef]
  209. Li, X.; Ye, J.; Munir, S.; Yang, T.; Chen, W.; Liu, G.; Zheng, W.; Zhang, Y. Biosynthetic gene pyramiding leads to ascorbate accumulation with enhanced oxidative stress tolerance in tomato. Int. J. Mol. Sci. 2019, 20, 1558. [Google Scholar] [CrossRef] [PubMed]
  210. Hemavathi, *!!! REPLACE !!!*; Upadhyaya, C.P.; Akula, N.; Young, K.E.; Chun, S.C.; Kim, D.H.; Park, S.W. Enhanced ascorbic acid accumulation in transgenic potato confers tolerance to various abiotic stresses. Biotechnol. Lett. 2010, 32, 321–330. [Google Scholar] [CrossRef] [PubMed]
  211. Lisko, K.A.; Torres, R.; Harris, R.S.; Belisle, M.; Vaughan, M.M.; Jullian, B.; Chevone, B.I.; Mendes, P.; Nessler, C.L.; Lorence, A. Elevating vitamin C content via overexpression of myo-inositol oxygenase and L-gulono-1,4-lactone oxidase in Arabidopsis leads to enhanced biomass and tolerance to abiotic stresses. In Vitro Cell. Dev. Biol. Plant 2013, 49, 643–655. [Google Scholar] [CrossRef]
  212. Hemavathi; Upadhyaya, C.P.; Young, K.E.; Akula, N.; Kim, H.S.; Heung, J.J.; Oh, O.M.; Aswath, C.R.; Chun, S.C.; Kim, D.H.; et al. Over-expression of strawberry D-galacturonic acid reductase in potato leads to accumulation of vitamin C with enhanced abiotic stress tolerance. Plant Sci. 2009, 177, 659–667. [Google Scholar] [CrossRef]
  213. Amaya, I.; Osorio, S.; Martinez-Ferri, E.; Lima-Silva, V.; Doblas, V.G.; Fernandez-Munoz, R.; Fernie, A.R.; Botella, M.A.; Valpuesta, V. Increased antioxidant capacity in tomato by ectopic expression of the strawberry D-galacturonate reductase gene. Biotechnol. J. 2015, 10, 490–500. [Google Scholar] [CrossRef]
  214. Lim, M.Y.; Jeong, B.R.; Jung, M.; Harn, C.H. Transgenic tomato plants expressing strawberry D-galacturonic acid reductase gene display enhanced tolerance to abiotic stresses. Plant Biotechnol. Rep. 2016, 10, 105–116. [Google Scholar] [CrossRef]
  215. Rigano, M.M.; Lionetti, V.; Raiola, A.; Bellincampi, D.; Barone, A. Pectic enzymes as potential enhancers of ascorbic acid production through the D-galacturonate pathway in Solanaceae. Plant Sci. 2018, 266, 55–63. [Google Scholar] [CrossRef]
  216. Naqvi, S.; Zhu, C.; Farre, G.; Ramessar, K.; Bassie, L.; Breitenbach, J.; Perez Conesa, D.; Ros, G.; Sandmann, G.; Capell, T.; et al. Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc. Natl. Acad. Sci. USA 2009, 106, 7762–7767. [Google Scholar] [CrossRef] [PubMed][Green Version]
  217. Haroldsen, V.M.; Chi-Ham, C.L.; Kulkarni, S.; Lorence, A.; Bennett, A.B. Constitutively expressed DHAR and MDHAR influence fruit, but not foliar ascorbate levels in tomato. Plant Physiol. Biochem. 2011, 49, 1244–1249. [Google Scholar] [CrossRef] [PubMed][Green Version]
  218. Hao, Z.; Wang, X.; Zong, Y.; Wen, S.; Cheng, Y.; Li, H. Enzymatic activity and functional analysis under multiple abiotic stress conditions of a dehydroascorbate reductase gene derived from Liriodendron Chinense. Environ. Exp. Bot 2019, 167, 103850. [Google Scholar] [CrossRef]
  219. Yin, L.N.; Wang, S.W.; Eltayeb, A.E.; Uddin, M.I.; Yamamoto, Y.; Tsuji, W.; Takeuchi, Y.; Tanaka, K. Overexpression of dehydroascorbate reductase, but not monodehydroascorbate reductase, confers tolerance to aluminum stress in transgenic tobacco. Planta 2010, 231, 609–621. [Google Scholar] [CrossRef] [PubMed]
  220. Eltelib, H.A.; Fujikawa, Y.; Esaka, M. Overexpression of the acerola (Malpighia glabra) monodehydroascorbate reductase gene in transgenic tobacco plants results in increased ascorbate levels and enhanced tolerance to salt stress. S. Afr. J. Bot. 2012, 78, 295–301. [Google Scholar] [CrossRef]
  221. Gest, N.; Garchery, C.; Gautier, H.; Jimenez, A.; Stevens, R. Light-dependent regulation of ascorbate in tomato by a monodehydroascorbate reductase localized in peroxisomes and the cytosol. Plant Biotechnol. J. 2013, 11, 344–354. [Google Scholar] [CrossRef]
  222. Abdelgawad, K.F.; El-Mogy, M.M.; Mohamed, M.I.A.; Garchery, C.; Stevens, R.G. Increasing ascorbic acid content and salinity tolerance of cherry tomato plants by suppressed expression of the ascorbate oxidase genes. Agronomy 2019, 9, 51. [Google Scholar] [CrossRef]
  223. Zhang, Z.J.; Wang, J.; Zhang, R.X.; Huang, R.F. The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis. Plant J. 2012, 71, 273–287. [Google Scholar] [CrossRef]
  224. Sawake, S.; Tajima, N.; Mortimer, J.C.; Lao, J.; Ishikawa, T.; Yu, X.; Yamanashi, Y.; Yoshimi, Y.; Kawai-Yamada, M.; Dupree, P.; et al. KONJAC1 and 2 are key factors for GDP-Mannose generation and affect L-ascorbic acid and glucomannan biosynthesis in Arabidopsis. Plant Cell 2015, 27, 3397–3409. [Google Scholar] [CrossRef]
  225. Li, Y.; Chu, Z.N.; Luo, J.Y.; Zhou, Y.H.; Cai, Y.J.; Lu, Y.G.; Xia, J.H.; Kuang, H.H.; Ye, Z.B.; Ouyang, B. The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnol. J. 2018, 16, 1201–1213. [Google Scholar] [CrossRef]
  226. Ye, J.; Li, W.F.; Ai, G.; Li, C.X.; Liu, G.Z.; Chen, W.F.; Wang, B.; Wang, W.Q.; Lu, Y.G.; Zhang, J.H.; et al. Genome-wide association analysis identifies a natural variation in basic helix-loop-helix transcription factor regulating ascorbate biosynthesis via D-mannose/L-galactose pathway in tomato. PLoS Genet. 2019, 15, e1008149. [Google Scholar] [CrossRef] [PubMed]
  227. Johnston, C.S.; Corte, C.; Swan, P.D. Marginal vitamin C status is associated with reduced fat oxidation during submaximal exercise in young adults. Nutr. Metab. 2006, 3, 35. [Google Scholar] [CrossRef] [PubMed]
  228. Johnston, C.S.; Barkyoumb, G.M.; Schumacher, S. Vitamin C supplementation slightly improves physical activity levels and reduces cold incidence in men with marginal vitamin C status: A randomized controlled trial. Nutrients 2014, 6, 2572–2583. [Google Scholar] [CrossRef] [PubMed]
  229. Monsen, E.R. Dietary reference intakes for the antioxidant nutrients: Vitamin C, vitamin E, selenium, and carotenoids. J. Am. Diet. Assoc. 2000, 100, 637–640. [Google Scholar] [CrossRef]
  230. Food Data Central. Available online: (accessed on 16 September 2019).
  231. Bhullar, N.K.; Gruissem, W. Nutritional enhancement of rice for human health: The contribution of biotechnology. Biotechnol. Adv. 2013, 31, 50–57. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Redox-dependent reactions of vitamin C. ASC can donate electrons directly to reactive oxygen species, metals, and tocopheroxyl radicals. The reduction of H2O2 by ASC occurs via APX. MDHA can undergo dismutation (green arrows), providing ASC and DHA. The reduction of oxidized forms occurs through the ASC-GSH cycle. MDHA and DHA are reduced by MDHAR and DHAR, respectively, whereas the reduced glutathione is recovered by GR (more details are provided in the text). Abbreviations: ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulphide.
Figure 1. Redox-dependent reactions of vitamin C. ASC can donate electrons directly to reactive oxygen species, metals, and tocopheroxyl radicals. The reduction of H2O2 by ASC occurs via APX. MDHA can undergo dismutation (green arrows), providing ASC and DHA. The reduction of oxidized forms occurs through the ASC-GSH cycle. MDHA and DHA are reduced by MDHAR and DHAR, respectively, whereas the reduced glutathione is recovered by GR (more details are provided in the text). Abbreviations: ASC, ascorbate; APX, ascorbate peroxidase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; MDHA, monodehydroascorbate; MDHAR, monodehydroascorbate reductase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione disulphide.
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Figure 2. Schematic representation of vitamin C biosynthetic pathways. Different colors indicate different pathways. In grey, the myoinositol pathway; in green, the gulose pathway; in yellow, the d-mannose/l-galactose pathway; in orange, the galacturonate pathway. Represented in blue are the initial steps leading to GDP-d-mannose, which is a common precursor to the d-mannose/l-galactose and gulose pathways. A question mark indicates enzymes not identified in plants (more details are provided in the text). Abbreviations: AL, aldono lactonase; GalUR, d-galacturonate reductase; GDH, l-galactose dehydrogenase; GGP, GDP-l-galactose-phosphorylase; GGulP, GDP-l-gulose pyrophosphatase; GLDH, l-galactono-1,4-lactone dehydrogenase; GluUR, glucuronate reductase; GME, GDP-d-mannose epimerase; GMP, GDP-d-mannose pyrophosphorylase; GPP, l-galactose-1-phosphate phosphatase; GulDH, l-gulose dehydrogenase; GulPP, l-gulose-1-phosphate phosphatase; GulLO, gulonolactone oxidase; MIOX, myoinositol oxygenase; PGI, phosphoglucose isomerase; PME, pectin methyl esterase; PMI, phosphomannose isomerase; PMM, phosphomannose mutase.
Figure 2. Schematic representation of vitamin C biosynthetic pathways. Different colors indicate different pathways. In grey, the myoinositol pathway; in green, the gulose pathway; in yellow, the d-mannose/l-galactose pathway; in orange, the galacturonate pathway. Represented in blue are the initial steps leading to GDP-d-mannose, which is a common precursor to the d-mannose/l-galactose and gulose pathways. A question mark indicates enzymes not identified in plants (more details are provided in the text). Abbreviations: AL, aldono lactonase; GalUR, d-galacturonate reductase; GDH, l-galactose dehydrogenase; GGP, GDP-l-galactose-phosphorylase; GGulP, GDP-l-gulose pyrophosphatase; GLDH, l-galactono-1,4-lactone dehydrogenase; GluUR, glucuronate reductase; GME, GDP-d-mannose epimerase; GMP, GDP-d-mannose pyrophosphorylase; GPP, l-galactose-1-phosphate phosphatase; GulDH, l-gulose dehydrogenase; GulPP, l-gulose-1-phosphate phosphatase; GulLO, gulonolactone oxidase; MIOX, myoinositol oxygenase; PGI, phosphoglucose isomerase; PME, pectin methyl esterase; PMI, phosphomannose isomerase; PMM, phosphomannose mutase.
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Figure 3. Light-dependent mechanisms involved in vitamin C accumulation (details are provided in the text). Abbreviations: PMM, phosphomannose mutase; GMP, GDP-d-mannose pyrophosphorylase; GME, GDP-d-mannose epimerase; GGP, GDP-l-galactose-phosphorylase; GPP, l-galactose-1-phosphate phosphatase; GDH, l-galactose dehydrogenase; GLDH, l-galactono-1,4-lactone dehydrogenase; APX, ascorbate peroxidase; MDHA, monodehydroascorbate; MDHAR monodehydroascorbate reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase.
Figure 3. Light-dependent mechanisms involved in vitamin C accumulation (details are provided in the text). Abbreviations: PMM, phosphomannose mutase; GMP, GDP-d-mannose pyrophosphorylase; GME, GDP-d-mannose epimerase; GGP, GDP-l-galactose-phosphorylase; GPP, l-galactose-1-phosphate phosphatase; GDH, l-galactose dehydrogenase; GLDH, l-galactono-1,4-lactone dehydrogenase; APX, ascorbate peroxidase; MDHA, monodehydroascorbate; MDHAR monodehydroascorbate reductase; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase.
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Figure 4. Strategies for vitamin C biofortification. Modulation of light intensity and quality can be used to obtain vitamin C enrichment in crops. The overexpression of single or multiple genes belonging to the biosynthetic and recycling pathways, as well as to the regulatory network, represents a good tool for vitamin C biofortification. Vitamin C accumulation can be controlled by modulating translation by the editing of the uORF in the GGP gene. Finally, vitamin C biofortification can be obtained by plant breeding that exploits candidate genes in the quantitative trait locus (QTL) associated with high vitamin C content (more details are provided in the text).
Figure 4. Strategies for vitamin C biofortification. Modulation of light intensity and quality can be used to obtain vitamin C enrichment in crops. The overexpression of single or multiple genes belonging to the biosynthetic and recycling pathways, as well as to the regulatory network, represents a good tool for vitamin C biofortification. Vitamin C accumulation can be controlled by modulating translation by the editing of the uORF in the GGP gene. Finally, vitamin C biofortification can be obtained by plant breeding that exploits candidate genes in the quantitative trait locus (QTL) associated with high vitamin C content (more details are provided in the text).
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