Physiologia Plantarum 126: 343355.

2006

Copyright Physiologia Plantarum 2006, ISSN 0031-9317

R E VI EW

Progress in manipulating ascorbic acid biosynthesis and accumulation in plants

Takahiro Ishikawa1, John Dowdle and Nicholas Smirnoff*
School of Biosciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter EX4 4QD, UK 1 Permanent address: Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane 690-8504, Japan

Correspondence *Corresponding author, e-mail: n.smirnoff@exeter.ac.uk Received 19 September 2005; revised 2 November 2005 doi: 10.1111/j.1399-3054.2006.00640.x

L-Ascorbic acid (vitamin C) is synthesized from hexose sugars. It is an antioxidant and redox buffer, as well as an enzyme cofactor, so it has multiple roles in metabolism and in plant responses to abiotic stresses and pathogens. Plant-derived ascorbate also provides the major source of vitamin C in the human diet. An understanding of how ascorbate metabolism is controlled should provide a basis for engineering or otherwise manipulating its accumulation. Biochemical and molecular genetic evidence supports synthesis from GDP-D-mannose via L-galactose (D-Man/L-Gal pathway) as a significant source of ascorbate. More recently, evidence for pathways via uronic acids has been obtained: overexpression of myo-inositol oxygenase, D-galacturonate reductase and L-gulono-1,4-lactone oxidase all increase leaf ascorbate concentration. Interestingly, this has proved more effective in pathway engineering than overexpressing various D-Man/L-Gal pathway genes. Ascorbate oxidation generates the potentially unstable dehydroascorbate, and the overexpression of glutathione-dependent dehydroascorbate reductase has resulted in increased ascorbate. Ascorbate is catabolized to products such as oxalate, L-threonate and L-tartrate. The enzymes involved have not been identified, so catabolism is not yet amenable to manipulation. In the examples of pathway engineering so far, the increase in ascorbate has been modest on an absolute or proportional basis. Therefore, a deeper understanding of ascorbate metabolism is needed to achieve larger increases. Identifying genes that control ascorbate accumulation by techniques such as analysis of quantitative trait loci (QTL) or activation tagging may hold promise, particularly if regulatory genes can be identified.

Introduction
L-Ascorbic

acid (vitamin C) has the status of a vitamin in some cases because primates, and a number of other animals, have lost the ability to synthesize this

multifunctional enzyme cofactor and antioxidant. Fruit and vegetables form the major part of the vitamin C supply in the diet, so the factors that control their ascorbate content are of interest. It seems that relatively few people

Abbreviations ABA, abscisic acid; AO, ascorbate oxidase; CaMV, cauliflower mosaic virus; DHA, dehydroascorbate; Gal, galactose; GaIL, galactono-1, 4-lactone; GDP, guanosine diphosphate; GME, GDP-Mannose-30 , 50 -epimerase; GMP, GDPmannose pyrophosphorylase; MDH, malate dehydrogenase; Man, mannose; MDHA, monodehydroascorbate; NAT, nucleobase-ascorbate transporter; PMI, phosphomannose isomerase; PMM, phosphomannose mutase; QTL, quantitative trait locus; SVCT; sodium-dependent vitamin C transporter; TCA, tricarboxylic acid.
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suffer from vitamin C deficiency and also, considering that the evidence for benefits of increased intake above the currently recommended levels is very mixed, there is currently not a strong incentive for publicly funded agencies to invest in efforts to increase the vitamin C content of crops. Private companies may nevertheless be interested in the possibility of producing ascorbate-enhanced plants for niche markets. There are also other reasons for understanding ascorbate metabolism. First, it is particularly abundant in plants, where it has a role as a redox buffer and enzyme cofactor. Its roles in photoprotection are well established (Muller-Moule et al. 2002, 2004), whereas new roles in redox processes related to cell growth, hormone responses (Pignocchi and Foyer 2003), programmed cell death, senescence and pathogen responses (Barth et al. 2004, Chen and Gallie 2004, Conklin 2001, Pastori et al. 2003, Vacca et al. 2004) are becoming apparent. As will be reviewed below, ascorbate metabolism is also strongly associated with photosynthesis and respiration. The second reason is that an understanding of plant ascorbate metabolism could be useful to efforts to improve current manufacturing methods based on microbial transformations (Hancock and Viola 2002, Running et al. 2004). Ascorbate is a high-volume but low-cost commodity used as a food additive and in the vitamin supplement market, so the development of a more efficient process would be profitable. We will assess recent progress in manipulating plant ascorbate concentration after first reviewing what is known about the pathways and control of ascorbate metabolism in plants and the potential roles of intracellular and long-distance transport as factors determining ascorbate concentration.

Ascorbate biosynthesis
The ascorbate biosynthetic pathway in mammals has been known since the 1950s and was based on in vivo radiolabelling and feeding experiments in rats. UDPD-glucose derived from glycogen is considered to be the main substrate for the de novo synthesis of ascorbate, and intermediates include D-glucuronate, L-gulonate and L-gulono-1,4-lactone. The pathway has been localized to the cytosol except for the final steps, which are microsomal (ul-Hassan and Lehninger 1956). Early evidence from radiolabelling studies indicated that the biosynthetic pathway in plants was different from that in animals (Loewus 1999), and subsequently, it was proposed that ascorbate is synthesized in plants by oxidation of L-galactose (L-Gal) (Wheeler et al. 1998). This is produced from GDPD-mannose (GDP-D-Man) via GDP-L-Gal (Fig. 1; Smirnoff and Gatzek 2004, Smirnoff et al. 2001, Wheeler et al. 1998). The enzymatic steps and evidence for their occurrence are summarized below.
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Phosphomannose isomerase (PMI) catalyses the first step in directing hexose phosphates into D-Man metabolism. PMI has not been purified from plants although two putative genes can be identified in Arabidopsis thaliana (At3g02570 and At1g67070) based on sequence homology. At1g67070 is a dark inducible gene (din9), which has undetectable transcript levels in photosynthesizing leaves but substantially increased levels in plants removed from the light for more than 24 h (Fujiki et al. 2001). PMI from Escherichia coli has been used as a selectable marker for plant transformation, because it renders plants mannose resistant (Datta et al. 2003). However, A. thaliana plants expressing E. coli PMI do not have increased ascorbate (J. Dowdle and N. Smirnoff, unpublished results). Conversion of D-Man 6-P to D-Man 1-P is catalysed by phosphomannose mutase (PMM). As with PMI, little is known about this enzyme in plants. Based on sequence homology, At2g45790 is a candidate A. thaliana PMM gene, and we have demonstrated that it has PMM activity when overexpressed in E. coli (J. Dowdle, S. Gatzek and N. Smirnoff, unpublished data). GDP-D-Man synthesis from D-Man 1-P and GTP is catalysed by GDPD-Man pyrophosphorylase (GMP). The low-ascorbate A. thaliana mutant vtc1 has reduced GMP activity (Conklin et al. 1999). VTC1 (At2g39770) encodes one of two predicted A. thaliana GMPs based on sequence homology. The function of the other gene (At3g55590) has not been established, but a null mutation of the CYT1 gene, which encodes the same protein as VTC1, is lethal (Lukowitz et al. 2001), suggesting that At3g55590 cannot substitute for CYT1/VTC1. Antisense suppression of GMP in potato also reduces ascorbate content (Keller et al. 1999). GDP-D-Man is converted to GDP-L-Gal by a reversible double epimerization, catalysed by GDP-D-Man-3,5-epimerase (GME) that was first identified in Chlorella (Hebda et al. 1979), pea and A. thaliana (Wheeler et al. 1998). This enzyme has recently been purified and cloned from A. thaliana (At5g28840) (Wolucka et al. 2001) and purified from the alga Prototheca (Running et al. 2004). As well as being involved in ascorbate synthesis, GDP-D-Man and GDP-L-Gal are substrates for polysaccharide synthesis and protein glycoslyation. In particular, L-Gal is a component of the pectin rhamnogalacturonan II. This pectin is essential for proper plant development (ONeill et al. 2004). The steps subsequent to GDP-L-Gal are likely to be dedicated to ascorbate synthesis. GDP-L-Gal is initially broken down to L-Gal 1-P, which is subsequently hydrolysed to L-Gal (Smirnoff and Gatzek 2004). Enzymes catalysing these steps have been recently purified and characterized. GDP-L-Gal is converted to L-Gal 1-P and GDP by a novel and highly specific phosphatePhysiol. Plant. 126, 2006

Fig. 1. The network of proposed biosynthetic pathways for ascorbate in plants. A combination of radiolabelling, mutant analysis and transgenic manipulation provides evidence for multiple pathways of ascorbate biosynthesis and is described in the text. The relative fluxes through the pathways and possible variations in different tissue types have not been established. Enzymes catalysing reactions 1113 have not been purified. It is assumed that enzymes 25 also catalyse the conversion of GDP-L-Gul to L-GulL. The role of the unnumbered reactions in ascorbate biosynthesis is uncertain. Enzymes: 1, GDP-D-Man pyrophosphorylase; 2, GDP-Man-30 ,50 -epimerase; 3, GDP-L-Gal phosphorylase (GDP-L-Gal:orthophosphate guanylyltransferase; S. Gatzek, J. Dowdle, T. Ishikawa and N. Smirnoff, unpublished data); 4, L-Gal 1-phosphate phosphatase; 5, L-Gal dehydrogenase; L-GalL dehydrogenase; 7, GDPD-mannose-4,6-dehydratase; 8, GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase; 9, D-galacturonate reductase; 10, myo-inositol oxygenase; 11, D-glucuronate reductase; 12, aldonolactonase; 13, L-GulL oxidase or dehydrogenase. L-Fuc, L-fucose; L-Gal, L-galactose; L-GalL, L-galactono1,4-lactone; GDP, guanosine diphosphate; L-Gul, L-gulose; L-GulL, L-gulono-1,4-lactone; D-Man, D-mannose; UDP, uridine diphosphate.

dependent GDP-L-Gal phosphorylase (J. Dowdle, T. Ishikawa, S. Gatzek and N. Smirnoff, unpublished results). An A. thaliana gene (At3g02870), previously annotated as an inositol monophosphatase, has high affinity and specificity for L-Gal 1-P, hydrolysing it to L-Gal and inorganic phosphate (Laing et al. 2004). We have now shown that the low-ascorbate A. thaliana mutant vtc4-1 has a point mutation in At3g02870, providing positive evidence that it is involved in ascorbate synthesis (P. L. Conklin, S. Gatzek, J. Dowdle and N. Smirnoff, manuscript submitted). The released L-Gal is then oxidized in two steps, first by a cytosolic NAD-dependent L-Gal dehydrogenase (L-GalDH) at C1 to form L-galactono-1,4lactone (L-GalL) (Gatzek et al. 2002, Wheeler et al. 1998) and then by L-GalL dehydrogenase (L-GalLDH) at C2/C3 resulting in the production of ascorbate. The final
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oxidative step occurs on the inner mitochondrial membrane where L-GalLDH uses cytochrome c as an electron acceptor (Bartoli et al. 2000, Millar et al. 2003, Siendones et al. 1999). Although a proteomic analysis of mitochondria membrane indicated that L-GalLDH is associated with complex I of the mitochondrial electron transport chain, the possibility that the enzyme is linked to electron transport between complexes III and IV is not ruled out (Bartoli et al. 2000, Millar et al. 2003). L-GalLDH has been characterized from several sources including sweet potato (Imai et al. 1998, Oba et al. 1995), cauliflower (Ostergaard et al. 1997), spinach (Mutsuda et al. 1995) and tobacco (Yabuta et al. 2000), and it appears to be highly specific for L-GalL. The antisense suppression of LGalDH and L-GalLDH decreases ascorbate concentration (Gatzek et al. 2002, Tabata et al. 2001).
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Although the D-Man/L-Gal pathway appears to be the predominant pathway, there is some suggestion that other biosynthetic pathways via uronic acid intermediates (Fig. 1) contribute to the ascorbate content of plant tissues and that these may be developmentally regulated. A. thaliana cells supplied with exogenous D-glucuronolactone and the methyl esters of D-glucuronic and D-galacturonic acid were shown to have increased concentrations of ascorbate (Davey et al. 1999), and radiolabelled D-galacturonic acid was also shown to be metabolized to ascorbate by an inversion pathway in detached strawberry fruit (Loewus and Kemp 1961). Molecular evidence for a D-galacturonic acid pathway was provided by the recent cloning and characterization of a D-galacturonic acid reductase from strawberry fruit (Agius et al. 2003). Overexpression of the strawberry gene in A. thaliana led to two- to three-fold increase in the ascorbate content of foliar tissue (Agius et al. 2003). The authors proposed that D-galacturonic acid derived from pectin was reduced to L-galactonic acid, which in turn is readily converted to ascorbate. Early experiments, in which radiolabelled myo-inositol was supplied to ripening strawberries and parsley leaves, resulted in no radiolabelled ascorbate and revealed that most of the radiolabel was incorporated into pectic polysaccharide residues (Loewus and Kelly 1963, Loewus et al. 1962). Recently, a myo-inositol oxygenase gene from A. thaliana (A4g26260) has been cloned which catalyses the oxidation of myo-inositol into D-glucuronate (Lorence et al. 2004). Constitutive expression of this gene resulted in a two- to three-fold increase in the ascorbate content of A. thaliana leaves compared with controls, suggesting that myo-inositol could be a potential precursor for ascorbate synthesis. The contradiction between this result and the radiolabelling evidence against myo-inositol remains to be resolved. It could be caused by label randomization masking label incorporation into ascorbate, but it should also be noted that the transgenic approach does not provide direct evidence that this pathway operates in wild-type plants. A difference between the observed and predicted equilibrium constant of the GME enzyme led to the identification of GDP-L-gulose as the 50 epimerization product of the reaction (Wolucka and Van Montagu 2003). The authors proposed that the hydrolysis of GDP-L-gulose would result in the production of L-gulose which could be converted to L-gulono-1,4-lactone by L-GalDH and subsequently into ascorbate by the L-gulono-1,4-lactone dehydrogenase activity known to exist in plants (Fig. 1). The conversion of L-gulose to ascorbate in whole tissue has been demonstrated in cress (Isherwood et al. 1954), tobacco (Jain and Nessler 2000), strawberry and bean (Baig et al. 1970).
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Control of ascorbate biosynthesis

It is now well known that ascorbate content in higher plants is influenced by light (Smirnoff 2000a, Tamaoki et al. 2003) and varies during development, for example during senescence (Bartoli et al. 2000), germination (Pallanca and Smirnoff 1999) and fruit ripening (Agius et al. 2003, Jimenez et al. 2002, Pateraki et al. 2004). This suggests that there are regulatory mechanisms that control ascorbate pool size. Although in an unlikely position for pathway control, the final enzyme, L-GalLDH, has been the most widely studied. For instance, in melon (Cucumis melo L), the transcript level of L-GalLDH was much higher in ascorbate-rich photosynthetic tissues such as leaves and stems than in ascorbate-poor tissues such as roots and seeds (Pateraki et al. 2004). On the other hand, in A. thaliana, the expression level of the L-GalLDH gene in roots was much higher than that in mature leaves, although the ascorbate level in the root was markedly lower than in the mature leaves (Tamaoki et al. 2003). On the contrary, L-GalLDH activity in A. thaliana roots was lower than that in the mature leaves (Tamaoki et al. 2003). It is possible that the activity of L-GalLDH is post-transcriptionally regulated. In spite of such a situation, in young leaves and developing tissue and cells, the expression level of L-GalLDH is relatively well correlated with their ascorbate concentration. In A. thaliana young rosette leaves, the expression pattern of both transcripts and enzyme activity of L-GalLDH were parallel to the diurnal change of ascorbate contents (Tamaoki et al. 2003). The transcript levels of L-GalLDH in melon fruit correlated with the increase in the tissue ascorbate content during ripening (Pateraki et al. 2004). Moreover, the melon L-GalLDH transcript level was markedly increased after germination, and the high expression was sustained in the light. The exposure of melon seedlings to various hormones did not affect the L-GalLDH expression level (Pateraki et al. 2004). In the case of tobacco BY-2 cells, the cellular ascorbate levels were temporarily increased at the beginning of their exponential phase, and the expression pattern of L-GalLDH gene was well correlated with the alterations in the quantity of ascorbate (Tabata et al. 2002). Furthermore, the addition of L-GalL to hypocotyls of kidney bean caused time-dependent increase in L-GalLDH activity (Siendones et al. 1999), whereas feeding L-GalL or ascorbate to tobacco BY-2 cells resulted in the suppression of gene expression (Tabata et al. 2002), and the inhibition of activity by a high concentration of L-GalL was also reported in potato tubers (Tudela et al. 2003). In summary, the enzyme activity and transcription level of L-GalLDH generally tend to correlate with the tissue
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ascorbate content, although there is species-to-species variation. Considering the localization of L-GalLDH in the mitochondrial inner membrane and the utilization of cytochrome c as an electron acceptor for the reaction, a relationship between ascorbate biosynthesis and respiration is possible. L-GalLDH in A. thaliana is associated with complex I, and the electron flow through the complex I affects the ascorbate biosynthesis rate (Millar et al. 2003). Also, the biosynthesis requires oxidized cytochrome c, and the biosynthesis rate is dependent on the redox balance of cytochrome c (Bartoli et al. 2000). The mechanism is unknown, but subtle changes in respiratory chain capacity, membrane potential, substrate supply and various antioxidant levels including ascorbate occur during senescence. In addition, complex I is potentially a source of reactive oxygen species generation. In fact, ascorbate concentration decreases in older leaves as they approach senescence, as does the activity of L-GalLDH (Bartoli et al. 2000). The relationship between ascorbate synthesis and respiration deserves more attention. Interestingly, it is reported that the antisense suppression of mitochondrial malate dehydrogenase and aconitase mutants in tomato have higher ascorbate content, suggesting a link between tricarboxylic acid (TCA) cycle activity and ascorbate synthesis (Nunes-Nesi et al. 2005). L-GalDH, the enzyme preceding L-GalLDH in the D-Man/L-Gal pathway, is likely to be a cytosolic enzyme. Its transcripts and activity in A. thaliana were not affected by high light exposure, whereas the ascorbate accumulation and the rate of L-Gal synthesis were increased (Gatzek et al. 2002). In the case of spinach L-GalDH, the transcript and activity levels were not affected by illumination (Mieda et al. 2004). These results indicate that L-GalDH is constitutively expressed in plant tissue regardless of ascorbate concentration. However, reversible inhibition of L-GalDH activity by ascorbate has been observed, indicating possible feedback regulation of ascorbate synthesis (Mieda et al. 2004). The inhibition kinetics showed a linear-competitive inhibition with a Ki of 133 mM. There is evidence that ascorbate biosynthesis could be regulated by feedback inhibition. The rate of ascorbate biosynthesis from [U-14C]D-glucose decreased linearly with the increase in the pool size of ascorbate in embryonic pea seedlings (Pallanca and Smirnoff 2000). Although the contribution of other enzymes upstream of L-GalDH to feedback regulation cannot be ruled out at the present moment, L-GalDH could be one of the strong possibilities. It is not clear whether the feedback inhibition observed in vitro occurs in vivo because L-Gal feeding rapidly increases ascorbate content (Wheeler et al. 1998). It has been assumed that GME is a rate-limiting enzyme of the pathway because of its low Vmax (Wolucka and Van
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Montagu 2003). Weak inhibition of the recombinant A. thaliana enzyme by ascorbate and L-GalL was reported, but the mechanism was not investigated. Additionally, GME copurified with heat-shock protein 70, but the possible role of this chaperone in regulating GME activity or the production of GDP-L-Gal vs GDP-L-Gul was not investigated (Wolucka and Van Montagu 2003). Overall, at the present moment, it is not possible from the limited information available to explain the control of ascorbate biosynthesis via the D-Man/L-Gal pathway. Not enough is known about the uronic acid pathways to consider their control or their relative contribution to the ascorbate pool. Nevertheless, it is clear that there are intriguing relationships between ascorbate biosynthesis, respiration and photosynthesis that remain to be unravelled. There is also a possible link to wound responses, because methyl jasmonate stimulates the synthesis of ascorbate from radiolabelled mannose in cell suspension cultures. Interestingly, transcripts for GMP, GME and a putative L-gulonolactone oxidase/dehydrogenase were also induced by methyl jasmonate (Wolucka et al. 2005). In addition to biosynthesis, it is very likely that ascorbate pool size is influenced by the extent of catabolism. The ascorbate pool turns over appreciably in some tissues (Conklin et al. 1997, Pallanca and Smirnoff 2000) and forms products such as oxalate (Kostman et al. 2001) and tartrate (Loewus 1999). Recently, it has been shown that apoplastic ascorbate is converted to L-threonate and L-tartrate via a novel metabolite, 4-O-oxalyl-L-threonate, using a combination of enzyme catalysed and non-enzymic reactions (Green and Fry 2005; Fig. 2). Furthermore, the efficiency of recycling of monodehydroascorbate (ascorbate-free radical; MDHA) and dehydroascorbate (DHA) back to ascorbate (Fig. 2) could influence ascorbate pool size. The overexpression of DHA (see Engineering ascorbate accumulation section) and glutathione reductase (Foyer et al. 1995) both increase ascorbate pool size.

Ascorbate transport
Ascorbate is freely mobile in plants, so intra- and intercellular transport could impact on pool size. Therefore, a consideration of transport mechanisms is relevant to understanding ascorbate pool size. Intracellular transport After biosynthesis of ascorbate on the inner membrane of mitochondria, it is distributed to all subcellular compartments including the apoplast. Ascorbate does not readily permeate lipid bilayers because of its size and negative charge at physiological pH (pK values of 4.17 and 11.57). Although DHA is a neutral molecule and
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Fig. 2. Diagrammatic summary of the redox reactions and catabolism of ascorbate in plants. Ascorbate is oxidized by a number of enzyme-catalysed and non-enzymatic reactions (all shown on the same reaction path for the ease of representation), the primary product generally being monodehydroascorbate. Two monodehydroascorbate molecules can disproportionate giving rise to ascorbate and dehydroascorbate. Ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, which co-operate to regenerate ascorbate, are found in most subcellular compartments and are each encoded by multiple genes. Ascorbate oxidase is a cell wall and, in some cases, a vacuolar enzyme. A number of compounds are produced by ascorbate catabolism in the cell wall and intracellularly, although no enzymes have been identified. Enzymes: 1. Ascorbate peroxidase; 2, ascorbate oxidase; 3, monodehydroascorbate reductase; 4, dehydroascorbate reductase; 5, glutathione reductase. GSH, glutathione; GSSG, glutathione disulphide; M, transition metal; R*, a free radical. Ascorbate can donate electrons to a wide range of radicals, including alkoxyl, peroxyl and tocopheroxyl radicals (Buettner and Schafer 2004).

more hydrophobic than ascorbate, simple diffusion of DHA across lipid bilayers is generally considered to be negligible because the oil : water distribution coefficient of DHA is smaller than that of mannitol, which is excluded from lipid bilayers (Rose 1987). Therefore, both the ascorbate and DHA transport systems are mainly mediated by facilitated diffusion or active transport systems. The ascorbate transport system in higher plants has not been elucidated yet, although substantial investigations of ascorbate uptake using isolated intact organelles or protoplasts indicated its existence in higher plants. For example, Beck et al. (1983) and Anderson et al. (1983) showed that ascorbate uptake by intact spinach chloroplasts followed Michaelis Menten kinetics. However, the Km values (the range of 2040 mM) are very high. Similarly, low affinities occur for ascorbate uptake by mitochondria isolated from tobacco BY-2 cells (Szarka et al. 2004). Barley
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protoplasts took up ascorbate with high affinity (Km 5 90 mM) (Rautenkranz et al. 1994), whereas the Km for ascorbate/DHA uptake from the apoplast of Betula pendula leaf was 12.8 mM (Kollist et al. 2001). On the other hand, the uptake of ascorbate into vacuoles does not have saturation kinetics, indicating the lack of a specific transporter (Rautenkranz et al. 1994). In contrast to the situation in higher plants, mammal ascorbate transporters (sodium-dependent vitamin C transporters, SVCT1 and SVCT2) have been identified and well characterized as an active ascorbate transport system (Daruwala et al. 1999, Tsukaguchi et al. 1999). SVCT1 and SVCT2 transport ascorbate by a sodiumdependent cotransport mechanism with high ascorbate affinities (Km 5 10250 mM). These proteins belong to the nucleobase-ascorbate transporter (NAT) family, which is ubiquitous from bacteria to higher plants and animals (de Koning and Diallinas 2000). Recent

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computer-assisted homology searches indicated that at least 12 NAT orthologous genes are present in the A. thaliana genome, and one of them (MQB2.190, At5g62890) contained highly a conserved region with mammal SVCTs (Li and Schultes 2002). Further investigation is needed to determine whether they function as plant (sodium-dependent) ascorbate-specific transporters. In contrast to ascorbate, DHA tends to be more effectively transported across plant membranes, with higher affinity and capacity, than ascorbate (Horemans et al. 1998a, Rautenkranz et al. 1994, Szarka et al. 2004). Incubation of tobacco suspension cells with DHA resulted in a remarkable increase of cellular ascorbate contents, also supporting the presence of a DHA transport system on the plasma membrane (de Pinto et al. 1999, Potters et al. 2000). Although the substantial contribution of glucose transporters for DHA uptake in animal cells has been elucidated (Wilson 2004), a potential role of the transporter in DHA transport is inconclusive in higher plants (Horemans et al. 2000a). The uptake of DHA via glucose transporters in animal cells is characterized by its competitive inhibition by a simultaneous addition of glucose and specific inhibitors such as phloretin, phlorizin and cytochalasin B (Wilson 2004). On the other hand, although cytochalasin B and genistein inhibited DHA and glucose uptake in plant mitochondria isolated from tobacco BY-2 cells, phloretin and phlorizin had no effect on the glucose uptake (Szarka et al. 2004). A similar phenomenon was observed in the experiment using tobacco BY-2 protoplast (Horemans et al. 1998b). Horemans et al. (2000b) have proposed the presence of a plasma membrane ascorbate/DHA exchange carrier, which takes up apoplastic DHA in exchange for cytosolic ascorbate. Unfortunately, the protein or gene associated with this transport activity has not been identified. Long-distance transport Ascorbate occurs in almost all plant tissues. It tends to be more concentrated in photosynthetic tissues, fruits and meristems than in non-photosynthetic tissues such as roots (Davey et al. 2000). Until recently, there has been no information on the extent of long-distance ascorbate transport. In pioneering work, Franceschi and Tarlyn (2002) have shown ascorbate transport via the phloem from source leaves to sink tissues such as root tips, shoots and floral organs. Autoradiography showed the movement of radiolabelled ascorbate in the phloem of Medicago sativa and A. thaliana. The same phenomenon was also observed in potato source leaf phloem and sink tubers (Tedone et al. 2004). The
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ascorbate content of phloem exudates in both leaf and tuberizing stolons of potato showed significant diurnal changes, indicating that ascorbate accumulation in sink tissue is affected by source leaf ascorbate biosynthesis. Hancock et al. (2003) have also suggested that ascorbate biosynthesis occurs in phloem via the D-Man/L-Gal pathway. Long-distance ascorbate transport may be required to supplement in situ ascorbate synthesis by sink tissues. Ascorbate demand in developing sink tissues is high, because ascorbate is probably required for cell cycle and cell division modulation and cell elongation affecting cell-wall construction (Smirnoff 2000b). The high content of ascorbate in certain fruits could be achieved by a combination of transport and in situ synthesis. D-Galacturonic acid derived from pectin breakdown during fruit ripening could also be a source of ascorbate (Fig. 1), and a strawberry D-galacturonic acid reductase gene is expressed only during fruit ripening (Agius et al. 2003). Manipulation of ascorbate transport in the phloem may provide a useful approach to increase the ascorbate content of fruits and tubers.

Engineering ascorbate accumulation

As was reviewed above, very little is known about the control of ascorbate biosynthesis and the determination of its pool size in plant tissues and or subcellular compartments. Ascorbate pool size is influenced not only by its biosynthesis but also by recycling. Combined with the possibility of the existence of multiple biosynthesis pathways, these facts make the situation difficult to predict and engineer. Three distinct approaches have been taken: overexpression of biosynthesis enzymes; overexpression of recycling enzymes (DHA reductase) and antisense suppression of ascorbate oxidase (AO). AO is an apoplastic enzyme that affects the redox state of extracellular ascorbate (Pignocchi et al. 2003, Sanmartin et al. 2003). The results of these engineering experiments are summarized in Table 1. Other lesstargeted approaches are also being taken, but the investigations are at early stages of development. These include the use of QTL analysis and molecular markers to identify novel genes associated with high ascorbate and screening activation-tagged A. thaliana lines for high ascorbate (N. Smirnoff, unpublished results). Ascorbate biosynthesis Changes in L-GalLDH activity and transcription levels are associated with changes in ascorbate content during leaf senescence, changes in light intensity and fruit development (Pateraki et al. 2004, Smirnoff 2000a). The rate of L-Gal synthesis is also increased by high light intensity,
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Table 1. Metabolic engineering for ascorbate accumulation in higher plants. The effect of transgenic manipulation of various enzymes on ascorbate pool size is summarized. AO, ascorbate oxidase; DHAR, dehydroascorbate reductase; L-GalDH, L-galactose dehydrogenase; L-GalLDH, L-galactonolactone dehydrogenase; L-GulLOx, L-gulonolactone oxidase; D-GalUAR, D-galacturonic acid reductase; G6PDH, glucose 6-P dehydrogenase; MDH, malate dehydrogenase; myo-inositol ox, myo-inositol oxygenase. Plant species engineered Increase in Gene regulation ascorbate pool (fold) Ascorbate/DHA ratio Reference Up Up Up Up Up Up Up Up Up Up Up Down Up Down Down Down Not changed 1.52.0 (7 days) Approximately 2.0 2.0 7.0 2.03.0 2.03.0 2.23.9 Approximately 1.9 Not changed 2.4 (in apoplast) 1.9 (in apoplast) Not changed Not changed Not changed Approximately 5.7 Gatzek et al. (2002) Tokunaga et al. (2005) Radzio et al. (2003) Jain and Nessler (2000) Jain and Nessler (2000) Lorence et al. (2004) Agius et al. (2003) Chen et al. (2003) Chen et al. (2003) Kwon et al. (2003) Pignocchi et al. (2003) Pignocchi et al. (2003) Yamamoto et al. (2005) Yamamoto et al. (2005) Debnam et al. (2004) Nunes-Nesi et al. (2005)

Gene source Enzyme Arabidopsis Tobacco Rat Rat Rat Arabidopsis Strawberry Wheat Wheat Human Pumpkin Tobacco Tobacco Tobacco Tobacco Tomato
L-GalDH

Tobacco Tobacco BY-2 cells L-GulLOx Arabidopsis L-GulLOx Lettuce L-GulLOx Tobacco myo-Inositol ox Arabidopsis D-GalUAR Arabidopsis DHAR Tobacco (to chloroplasts) DHAR Maize (to chloroplasts) DHAR Tobacco (to cytosol) AO Tobacco AO Tobacco AO Tobacco AO Tobacco G6PDH Tobacco (to chloroplasts) MDH Tomato (to mitochondria)
L-GalLDH

Increase Increase Increase Decrease Increase Decrease Increase Increase

suggesting that the early steps of the D-Man/L-Gal pathway have increased flux (Gatzek et al. 2002). Therefore, it was expected that altering the expression levels of the enzymes related to this pathway would affect the accumulation of ascorbate. However, in general, the overexpression of enzymes in the D-Man/L-Gal pathway has not resulted in increased ascorbate concentration. For example, although the overexpression of an L-GalDH in tobacco plants resulted in a three-fold increase in the activity, no effect on ascorbate levels was observed (Gatzek et al. 2002). On a more positive note, Tokunaga et al. (2005) using tobacco suspension cells overexpressing L-GalLDH demonstrated an increase in ascorbate content. The cells overexpressing L-GalLDH under the control of CaMV 35S promoter showed a moderate (approximately two-fold) enhancement of total ascorbate content during stationary phase and still sustained significant ascorbate levels after that, whereas stationary-phase wild-type cells showed a decrease in ascorbate level. In contrast to attempts to engineer the D-Man/L-Gal pathway, the expression of genes that encode proposed enzymes of the uronic acid pathways results in moderate increases in ascorbate contents. With respect to D-galacturonate reductase, the overexpression of the enzyme from strawberry fruit (Fragaria ananassa) in A. thaliana resulted in a two- to three-fold increase of total ascorbate content (Agius et al. 2003). The overexpression of myo-inositol oxygenase gene (miox4), another enzyme assumed to take part in glucuronic acid pathway, in transgenic A. thaliana also produced
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a moderate enhancement of ascorbate content in leaves (Lorence et al. 2004). On the other hand, A. thaliana mutants disrupted in other MIOX genes homologues (MIOX2 or MIOX5) were not affected in their leaf ascorbate content (Kanter et al. 2005), possibly indicating that homologues are not involved in ascorbate biosynthesis in leaves. L-GulL is the predicted precursor of ascorbate in the glucuronic acid pathway. The ectopic expression of rat L-GulL oxidase in transgenic A. thaliana resulted in a moderate increase in total ascorbate level compared with wild-type plants, suggesting the presence of L-GulL as a penultimate precursor of the uronic acid pathway in higher plants. Both glucuronic acid and galacturonic acid are major components of cell-wall biomass derived from UDPglucuronic acid and UDP-galacturonic acid as their precursors (Seifert 2004). However, the biosynthetic pathway of UDP-glucuronic acid is still incompletely understood. The identification of MIOX in higher plants now opens the window on understanding the regulation of the poorly understood inositol oxygenation pathway. Increasing the flux of this pathway towards the production of uronic acids by metabolic engineering might be expected to affect the accumulation of ascorbate in higher plants. Ascorbate recycling Because the rate of ascorbate turnover is relatively fast, 13% of the pool per h in pea seedlings (Pallanca and
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Smirnoff 2000) and 40% per 22 h in A. thaliana leaves (Conklin et al. 1997), it might be expected that the enhancement of the ascorbate recycling pathway or the downregulation of ascorbate oxidation would also affect the ascorbate accumulation. This may not be true of all tissues. For example, potato leaves have a low turnover rate (Imai et al. 1999). Transgenic tobacco and maize overexpressing cytosolic DHA reductase from wheat resulted in a two- to four-fold increase in total ascorbate contents and decreased the proportion of DHA in both leaves and maize kernel (Chen et al. 2003). Unlike the overexpression of DHA reductase in cytosol, the overexpression of human DHA reductase in the chloroplasts of transgenic tobacco plants showed no significant difference in total ascorbate content, whereas an increase in the ascorbate/DHA ratio was observed (Kwon et al. 2003). Interestingly, transgenic tobacco with the suppressed activity of one of the chloroplastic glucose 6-phosphate dehydrogenase (P2-G6PDH) isoforms by antisense expression of the gene showed a significant increase in the cellular ascorbate/DHA ratio, but not in total ascorbate content (Debnam et al. 2004). The suppression of G6PDH activity would be expected to reduce NADPH content, so this result cannot be easily explained. The overexpression of MDHA reductase might both affect ascorbate accumulation and increase the redox status of ascorbate towards reduction, because MDHA reductase functions upstream of DHA reductase in the ascorbate recycling pathway. However, there are no reports of the effects of MDHA reductase overexpression. Ascorbate oxidase AO is located in the apoplast (Pignocchi et al. 2003). It is possible that the modulation of AO activity would lead to the alteration of ascorbate accumulation. However, sense and antisense reduction of AO in transgenic tobacco had no effect on total leaf ascorbate contents (Pignocchi et al. 2003), because most of the ascorbate is in the cytosol, not in the apoplast. However, the ascorbate status in apoplast of these transgenics showed a significant increase in ascorbate contents and altered redox balance of ascorbate than those in wild-type plants. The total ascorbate content of the apoplast was increased by up to 2.4-fold compared with wild-type in sense and antisense plants. In the antisense plants, the proportion of the reduced form in the apoplast was markedly increased (66%) compared with the wild-type plants (40%), whereas in the sense plants, it was significantly decreased (3%). Similar results have also been reported by another research group using transgenic tobacco expressing AO gene in sense and
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antisense orientation (Yamamoto et al. 2005). The proportion of ascorbate/DHA in both whole leaf and apoplast was statistically higher in antisense tobacco plants and lower in sense plants than that in wild-type plants, while apoplastic total ascorbate contents of these plants were not influenced by AO manipulations. Taken together, the engineering of ascorbate recycling or oxidation pathway is also potentially useful to the improvement of ascorbate accumulation and its redox status. In addition, it might be important to consider targeting gene products to specific cellular compartments. Other approaches Recently, unexpected results were obtained when a mitochondrial malate dehydrogenase was suppressed in transgenic tomato (Lycopersicon esculentum cv. Money Maker). Gas chromatography-mass spectrometry analysis of antisense mitochondrial MDH lines showed up to 5.7fold increased levels of ascorbate in their leaves than those of wild-type tomato (Nunes-Nesi et al. 2005). Higher ascorbate level was also reported in a mitochondrial aconitase mutant tomato (Aco1, Lycopersicon pennellii) (Nunes-Nesi et al. 2005). In the antisense MDH plants, there was no effect on mitochondrially located L-GalLDH activity or on the concentration of galacturonic acid and glucuronic acid. Although the reason for the phenomenon is not fully apparent, it suggests that TCA cycle activity can influence ascorbate metabolism.

Conclusions
Considering the research so far, the attempts to alter ascorbate accumulation in plants have clearly shown that ascorbate metabolism in higher plants is regulated by complex control networks that are affected by multiple factors. Although the engineering of ascorbate accumulation has at least been demonstrated in principal, engineering plants with substantially increased ascorbate pool through enhanced flux using single enzymes, particularly in the D-Man/L-Gal pathway, has been unsuccessful. Much remains to be learned about the contribution of alternative ascorbate biosynthetic pathways in each plant tissue and the regulation of the metabolic fluxes, and many important genes still have not yet been identified. In addition, the manipulation of other antioxidants could affect ascorbate accumulation, and it will be important to learn more about these relationships. For example, a double mutant for the simultaneous loss of tocopherol and glutathione had a significant increase in ascorbate content, whereas the overproduction of these two antioxidants tends to cause a decrease in ascorbate accumulation (Kanwischer et al. 2005). The mechanism of
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ascorbate transport across cellular compartment and transportation from source to sink organs are also largely unknown. The increase in the apoplastic DHA content of AO-overexpressing tobacco (Pignocchi et al. 2003) suggests that it might be useful to improve DHA transport efficiency. Because ascorbate is one of the major redox buffers of the plant cell, along with glutathione (Noctor and Foyer 1998), understanding the effect of the enhanced levels of ascorbate on all aspects of plant function is also of particular importance. Altering ascorbate levels may affect other aspects of physiology, such as abscisic acid synthesis (Pastori et al. 2003), control of cell division and growth (Potters et al. 2000, 2004), cellular H2O2 concentration and gene expression profiles (Pastori et al. 2003). It is important to consider possible deleterious effects as well. For example, DHAR overexpression reduces stomatal closure in response to water stress, because improved H2O2 scavenging interferes with its role in ABA signalling (Chen and Gallie 2004). Also, higher ascorbate content could reduce basal resistance to pathogen infection (Barth et al. 2004). A combination of metabolic profiling and gene expression profiling could reveal much about how plants react to engineered changes. Finally, as more knowledge of ascorbate metabolism becomes available, the strategies available to address our goal of increasing its concentration in a rational manner will certainly increase.

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Edited by C. H. Foyer

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