progress in manipulating ascorbic acid biosynthesis and accumulation in plants

REVIEW

Progress in manipulating ascorbic acid biosynthesis andaccumulation in plantsTakahiro Ishikawa1, John Dowdle and Nicholas Smirnoff*

School of Biosciences, Geoffrey Pope Building, University of Exeter, Stocker Road, Exeter EX4 4QD, UK1Permanent address: Faculty of Life and Environmental Sciences, Shimane University, Matsue, Shimane 690-8504, Japan

Correspondence

*Corresponding author,

e-mail: [email protected]

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 anti-oxidant and redox buffer, as well as an enzyme cofactor, so it has multipleroles in metabolism and in plant responses to abiotic stresses and pathogens.Plant-derived ascorbate also provides the major source of vitamin C in thehuman diet. An understanding of how ascorbate metabolism is controlledshould provide a basis for engineering or otherwise manipulating its accu-mulation. Biochemical and molecular genetic evidence supports synthesisfrom GDP-D-mannose via L-galactose (D-Man/L-Gal pathway) as a significantsource of ascorbate. More recently, evidence for pathways via uronic acidshas been obtained: overexpression of myo-inositol oxygenase, D-galacturo-nate reductase and L-gulono-1,4-lactone oxidase all increase leaf ascorbateconcentration. Interestingly, this has proved more effective in pathway engi-neering than overexpressing various D-Man/L-Gal pathway genes. Ascorbateoxidation generates the potentially unstable dehydroascorbate, and the over-expression of glutathione-dependent dehydroascorbate reductase hasresulted in increased ascorbate. Ascorbate is catabolized to products suchas oxalate, L-threonate and L-tartrate. The enzymes involved have not beenidentified, so catabolism is not yet amenable to manipulation. In the exam-ples of pathway engineering so far, the increase in ascorbate has beenmodest on an absolute or proportional basis. Therefore, a deeper under-standing of ascorbate metabolism is needed to achieve larger increases.Identifying genes that control ascorbate accumulation by techniques suchas analysis of quantitative trait loci (QTL) or activation tagging may holdpromise, particularly if regulatory genes can be identified.

Introduction

L-Ascorbic acid (vitamin C) has the status of a vitamin insome cases because primates, and a number of otheranimals, have lost the ability to synthesize this

multifunctional enzyme cofactor and antioxidant. Fruitand vegetables form themajor part of the vitamin C supplyin the diet, so the factors that control their ascorbatecontent 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, GDP-

mannose pyrophosphorylase; MDH, malate dehydrogenase; Man, mannose; MDHA, monodehydroascorbate; NAT, nucleo-

base-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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Physiologia Plantarum 126: 343–355. 2006 Copyright � Physiologia Plantarum 2006, ISSN 0031-9317

suffer from vitamin C deficiency and also, considering thatthe evidence for benefits of increased intake above thecurrently recommended levels is very mixed, there iscurrently not a strong incentive for publicly funded agen-cies to invest in efforts to increase the vitamin C content ofcrops. Private companies may nevertheless be interestedin the possibility of producing ascorbate-enhanced plantsfor niche markets. There are also other reasons for under-standing ascorbate metabolism. First, it is particularlyabundant in plants, where it has a role as a redox bufferand enzyme cofactor. Its roles in photoprotection are wellestablished (Muller-Moule et al. 2002, 2004), whereasnew roles in redox processes related to cell growth,hormone responses (Pignocchi and Foyer 2003), pro-grammed 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 becomingapparent. As will be reviewed below, ascorbate metabol-ism is also strongly associated with photosynthesis andrespiration. The second reason is that an understanding ofplant ascorbate metabolism could be useful to efforts toimprove current manufacturing methods based on micro-bial transformations (Hancock and Viola 2002, Runninget al. 2004). Ascorbate is a high-volume but low-costcommodity used as a food additive and in the vitaminsupplement market, so the development of a more effi-cient process would be profitable. We will assess recentprogress in manipulating plant ascorbate concentrationafter first reviewing what is known about the pathwaysand control of ascorbate metabolism in plants and thepotential roles of intracellular and long-distance transportas factors determining ascorbate concentration.

Ascorbate biosynthesis

The ascorbate biosynthetic pathway in mammals hasbeen known since the 1950s and was based on in vivoradiolabelling and feeding experiments in rats. UDP-D-glucose derived from glycogen is considered to be themain substrate for the de novo synthesis of ascorbate, andintermediates include D-glucuronate, L-gulonate andL-gulono-1,4-lactone. The pathway has been localized tothe cytosol except for the final steps, which are microso-mal (ul-Hassan and Lehninger 1956). Early evidence fromradiolabelling studies indicated that the biosynthetic path-way in plants was different from that in animals (Loewus1999), and subsequently, it was proposed that ascorbate issynthesized in plants by oxidation of L-galactose (L-Gal)(Wheeler et al. 1998). This is produced from GDP-D-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 evidencefor their occurrence are summarized below.

Phosphomannose isomerase (PMI) catalyses the firststep in directing hexose phosphates into D-Man meta-bolism. PMI has not been purified from plants althoughtwo putative genes can be identified in Arabidopsisthaliana (At3g02570 and At1g67070) based onsequence homology. At1g67070 is a dark induciblegene (din9), which has undetectable transcript levels inphotosynthesizing leaves but substantially increasedlevels in plants removed from the light for more than24 h (Fujiki et al. 2001). PMI from Escherichia coli hasbeen used as a selectable marker for plant transforma-tion, because it renders plants mannose resistant (Dattaet al. 2003). However, A. thaliana plants express-ing 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 byphosphomannose mutase (PMM). As with PMI, little isknown about this enzyme in plants. Based on sequencehomology, At2g45790 is a candidate A. thaliana PMMgene, and we have demonstrated that it has PMM activ-ity when overexpressed in E. coli (J. Dowdle, S. Gatzekand N. Smirnoff, unpublished data). GDP-D-Man synth-esis from D-Man 1-P and GTP is catalysed by GDP-D-Man pyrophosphorylase (GMP). The low-ascorbateA. thaliana mutant vtc1 has reduced GMP activity(Conklin et al. 1999). VTC1 (At2g39770) encodes oneof two predicted A. thaliana GMPs based on sequencehomology. The function of the other gene (At3g55590)has not been established, but a null mutation of theCYT1 gene, which encodes the same protein as VTC1,is lethal (Lukowitz et al. 2001), suggesting thatAt3g55590 cannot substitute for CYT1/VTC1. Antisensesuppression of GMP in potato also reduces ascorbatecontent (Keller et al. 1999). GDP-D-Man is converted toGDP-L-Gal by a reversible double epimerization, cata-lysed by GDP-D-Man-3,5-epimerase (GME) that was firstidentified in Chlorella (Hebda et al. 1979), pea andA. thaliana (Wheeler et al. 1998). This enzyme has recentlybeen purified and cloned from A. thaliana (At5g28840)(Wolucka et al. 2001) and purified from the algaPrototheca (Running et al. 2004). As well as being involvedin ascorbate synthesis, GDP-D-Man and GDP-L-Gal aresubstrates for polysaccharide synthesis and protein gly-coslyation. In particular, L-Gal is a component of thepectin rhamnogalacturonan II. This pectin is essentialfor proper plant development (O’Neill et al. 2004).

The steps subsequent to GDP-L-Gal are likely to bededicated to ascorbate synthesis. GDP-L-Gal is initiallybroken down to L-Gal 1-P, which is subsequently hydro-lysed to L-Gal (Smirnoff and Gatzek 2004). Enzymescatalysing these steps have been recently purified andcharacterized. GDP-L-Gal is converted to L-Gal 1-Pand GDP by a novel and highly specific phosphate-

344 Physiol. Plant. 126, 2006

dependent GDP-L-Gal phosphorylase (J. Dowdle, T.Ishikawa, S. Gatzek and N. Smirnoff, unpublishedresults). An A. thaliana gene (At3g02870), previouslyannotated as an inositol monophosphatase, has highaffinity and specificity for L-Gal 1-P, hydrolysing it toL-Gal and inorganic phosphate (Laing et al. 2004). Wehave now shown that the low-ascorbate A. thalianamutant vtc4-1 has a point mutation in At3g02870, provid-ing positive evidence that it is involved in ascorbate synth-esis (P. L. Conklin, S. Gatzek, J. Dowdle and N. Smirnoff,manuscript submitted). The released L-Gal is thenoxidizedin two steps, first by a cytosolic NAD-dependent L-Galdehydrogenase (L-GalDH) at C1 to form L-galactono-1,4-lactone (L-GalL) (Gatzek et al. 2002, Wheeler et al. 1998)and then by L-GalL dehydrogenase (L-GalLDH) at C2/C3resulting in the production of ascorbate. The final

oxidative step occurs on the inner mitochondrial mem-brane where L-GalLDH uses cytochrome c as an electronacceptor (Bartoli et al. 2000, Millar et al. 2003, Siendoneset al. 1999). Although a proteomic analysis of mitochon-driamembrane indicated that L-GalLDH is associatedwithcomplex I of the mitochondrial electron transport chain,the possibility that the enzyme is linked to electron trans-port between complexes III and IV is not ruled out (Bartoliet al. 2000, Millar et al. 2003). L-GalLDH has been char-acterized 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 behighly specific for L-GalL. The antisense suppression of L-GalDH and L-GalLDH decreases ascorbate concentration(Gatzek et al. 2002, Tabata et al. 2001).

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 11–13 have not been purified. It is assumed that

enzymes 2–5 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, GDP-

D-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-galactono-

1,4-lactone; GDP, guanosine diphosphate; L-Gul, L-gulose; L-GulL, L-gulono-1,4-lactone; D-Man, D-mannose; UDP, uridine diphosphate.


Although the D-Man/L-Gal pathway appears to be thepredominant pathway, there is some suggestion thatother biosynthetic pathways via uronic acid intermedi-ates (Fig. 1) contribute to the ascorbate content of planttissues and that these may be developmentally regu-lated. A. thaliana cells supplied with exogenousD-glucuronolactone and themethyl esters of D-glucuronicand D-galacturonic acid were shown to have increasedconcentrations of ascorbate (Davey et al. 1999), andradiolabelled D-galacturonic acid was also shown tobe metabolized to ascorbate by an inversion pathwayin detached strawberry fruit (Loewus and Kemp 1961).Molecular evidence for a D-galacturonic acid pathwaywas provided by the recent cloning and characterizationof a D-galacturonic acid reductase from strawberry fruit(Agius et al. 2003). Overexpression of the strawberrygene in A. thaliana led to two- to three-fold increase inthe ascorbate content of foliar tissue (Agius et al. 2003).The authors proposed that D-galacturonic acid derivedfrom pectin was reduced to L-galactonic acid, which inturn is readily converted to ascorbate. Early experi-ments, in which radiolabelled myo-inositol was sup-plied to ripening strawberries and parsley leaves,resulted in no radiolabelled ascorbate and revealedthat most of the radiolabel was incorporated into pecticpolysaccharide residues (Loewus and Kelly 1963,Loewus et al. 1962). Recently, a myo-inositol oxygenasegene from A. thaliana (A4g26260) has been clonedwhich catalyses the oxidation of myo-inositol intoD-glucuronate (Lorence et al. 2004). Constitutive expres-sion of this gene resulted in a two- to three-fold increasein the ascorbate content of A. thaliana leaves comparedwith controls, suggesting that myo-inositol could be apotential precursor for ascorbate synthesis. The contra-diction between this result and the radiolabelling evi-dence against myo-inositol remains to be resolved. Itcould be caused by label randomization masking labelincorporation into ascorbate, but it should also be notedthat the transgenic approach does not provide directevidence that this pathway operates in wild-type plants.A difference between the observed and predicted

equilibrium constant of the GME enzyme led to theidentification of GDP-L-gulose as the 50 epimerizationproduct of the reaction (Wolucka and Van Montagu2003). The authors proposed that the hydrolysis ofGDP-L-gulose would result in the production of L-gulosewhich could be converted to L-gulono-1,4-lactone byL-GalDH and subsequently into ascorbate by theL-gulono-1,4-lactone dehydrogenase activity known toexist in plants (Fig. 1). The conversion of L-gulose toascorbate in whole tissue has been demonstrated incress (Isherwood et al. 1954), tobacco (Jain andNessler 2000), strawberry and bean (Baig et al. 1970).

Control of ascorbate biosynthesis

It is now well known that ascorbate content in higherplants is influenced by light (Smirnoff 2000a, Tamaokiet al. 2003) and varies during development, for exampleduring senescence (Bartoli et al. 2000), germination(Pallanca and Smirnoff 1999) and fruit ripening (Agiuset al. 2003, Jimenez et al. 2002, Pateraki et al. 2004).This suggests that there are regulatory mechanisms thatcontrol ascorbate pool size. Although in an unlikelyposition for pathway control, the final enzyme,L-GalLDH, has been the most widely studied. Forinstance, in melon (Cucumis melo L), the transcriptlevel of L-GalLDH was much higher in ascorbate-richphotosynthetic tissues such as leaves and stems than inascorbate-poor tissues such as roots and seeds (Paterakiet al. 2004). On the other hand, in A. thaliana, theexpression level of the L-GalLDH gene in roots wasmuch higher than that in mature leaves, although theascorbate level in the root was markedly lower than inthe mature leaves (Tamaoki et al. 2003). On the con-trary, L-GalLDH activity in A. thaliana roots was lowerthan that in the mature leaves (Tamaoki et al. 2003). It ispossible that the activity of L-GalLDH is post-transcrip-tionally regulated. In spite of such a situation, in youngleaves and developing tissue and cells, the expressionlevel of L-GalLDH is relatively well correlated with theirascorbate concentration. In A. thaliana young rosetteleaves, the expression pattern of both transcripts andenzyme activity of L-GalLDH were parallel to the diur-nal change of ascorbate contents (Tamaoki et al. 2003).The transcript levels of L-GalLDH in melon fruit correl-ated with the increase in the tissue ascorbate contentduring ripening (Pateraki et al. 2004). Moreover, themelon L-GalLDH transcript level was markedlyincreased after germination, and the high expressionwas sustained in the light. The exposure of melon seed-lings to various hormones did not affect the L-GalLDHexpression level (Pateraki et al. 2004). In the case oftobacco BY-2 cells, the cellular ascorbate levels weretemporarily increased at the beginning of their exponen-tial phase, and the expression pattern of L-GalLDH genewas well correlated with the alterations in the quantityof ascorbate (Tabata et al. 2002). Furthermore, the add-ition of L-GalL to hypocotyls of kidney bean causedtime-dependent increase in L-GalLDH activity(Siendones et al. 1999), whereas feeding L-GalL orascorbate to tobacco BY-2 cells resulted in the suppres-sion of gene expression (Tabata et al. 2002), and theinhibition of activity by a high concentration of L-GalLwas also reported in potato tubers (Tudela et al. 2003).In summary, the enzyme activity and transcription levelof L-GalLDH generally tend to correlate with the tissue


ascorbate content, although there is species-to-speciesvariation. Considering the localization of L-GalLDH inthe mitochondrial inner membrane and the utilization ofcytochrome c as an electron acceptor for the reaction, arelationship between ascorbate biosynthesis and respira-tion is possible. L-GalLDH in A. thaliana is associated withcomplex I, and the electron flow through the complex Iaffects the ascorbate biosynthesis rate (Millar et al. 2003).Also, the biosynthesis requires oxidized cytochrome c, andthe biosynthesis rate is dependent on the redox balance ofcytochrome c (Bartoli et al. 2000). The mechanism isunknown, but subtle changes in respiratory chain capa-city, membrane potential, substrate supply and variousantioxidant levels including ascorbate occur during senes-cence. In addition, complex I is potentially a source ofreactive oxygen species generation. In fact, ascorbate con-centration decreases in older leaves as they approachsenescence, as does the activity of L-GalLDH (Bartoliet al. 2000). The relationship between ascorbate synthesisand respiration deserves more attention. Interestingly, it isreported that the antisense suppression of mitochondrialmalate dehydrogenase and aconitase mutants in tomatohave higher ascorbate content, suggesting a link betweentricarboxylic acid (TCA) cycle activity and ascorbate synth-esis (Nunes-Nesi et al. 2005).

L-GalDH, the enzyme preceding L-GalLDH in theD-Man/L-Gal pathway, is likely to be a cytosolicenzyme. Its transcripts and activity in A. thaliana werenot affected by high light exposure, whereas the ascorb-ate accumulation and the rate of L-Gal synthesis wereincreased (Gatzek et al. 2002). In the case of spinachL-GalDH, the transcript and activity levels were notaffected by illumination (Mieda et al. 2004). Theseresults indicate that L-GalDH is constitutively expressedin plant tissue regardless of ascorbate concentration.However, reversible inhibition of L-GalDH activity byascorbate has been observed, indicating possible feed-back regulation of ascorbate synthesis (Mieda et al.2004). The inhibition kinetics showed a linear-competi-tive inhibition with a Ki of 133 mM. There is evidencethat ascorbate biosynthesis could be regulated by feed-back inhibition. The rate of ascorbate biosynthesis from[U-14C]D-glucose decreased linearly with the increasein the pool size of ascorbate in embryonic pea seedlings(Pallanca and Smirnoff 2000). Although the contributionof other enzymes upstream of L-GalDH to feedbackregulation cannot be ruled out at the present moment,L-GalDH could be one of the strong possibilities. It is notclear whether the feedback inhibition observed in vitrooccurs in vivo because L-Gal feeding rapidly increasesascorbate content (Wheeler et al. 1998).

It has been assumed that GME is a rate-limiting enzymeof the pathway because of its low Vmax (Wolucka and Van

Montagu 2003). Weak inhibition of the recombinantA. 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 pos-sible role of this chaperone in regulating GME activity orthe production of GDP-L-Gal vs GDP-L-Gul was not inves-tigated (Wolucka and Van Montagu 2003). Overall, at thepresent moment, it is not possible from the limited infor-mation available to explain the control of ascorbate bio-synthesis via the D-Man/L-Gal pathway. Not enough isknown about the uronic acid pathways to consider theircontrol or their relative contribution to the ascorbate pool.Nevertheless, it is clear that there are intriguing relation-ships between ascorbate biosynthesis, respiration andphotosynthesis that remain to be unravelled. There is alsoa possible link to wound responses, because methyl jas-monate stimulates the synthesis of ascorbate from radiola-belled mannose in cell suspension cultures. Interestingly,transcripts for GMP, GME and a putative L-gulonolactoneoxidase/dehydrogenase were also induced by methyl jas-monate (Wolucka et al. 2005).

In addition to biosynthesis, it is very likely that ascorbatepool size is influenced by the extent of catabolism. Theascorbate pool turns over appreciably in some tissues(Conklin et al. 1997, Pallanca and Smirnoff 2000) andforms products such as oxalate (Kostman et al. 2001) andtartrate (Loewus 1999). Recently, it has been shown thatapoplastic ascorbate is converted to L-threonate andL-tartrate via a novel metabolite, 4-O-oxalyl-L-threonate,using a combination of enzyme catalysed and non-enzy-mic 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 ascorbatepool size. The overexpression of DHA (see Engineeringascorbate 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 inter-cellular transport could impact on pool size. Therefore,a consideration of transport mechanisms is relevant tounderstanding ascorbate pool size.

Intracellular transport

After biosynthesis of ascorbate on the inner membraneof mitochondria, it is distributed to all subcellular com-partments including the apoplast. Ascorbate does notreadily permeate lipid bilayers because of its size andnegative charge at physiological pH (pK values of 4.17and 11.57). Although DHA is a neutral molecule and


more hydrophobic than ascorbate, simple diffusion ofDHA across lipid bilayers is generally considered to benegligible because the oil : water distribution coeffi-cient of DHA is smaller than that of mannitol, which isexcluded from lipid bilayers (Rose 1987). Therefore,both the ascorbate and DHA transport systems aremainly mediated by facilitated diffusion or active trans-port systems. The ascorbate transport system in higherplants has not been elucidated yet, although substantialinvestigations of ascorbate uptake using isolated intactorganelles or protoplasts indicated its existence inhigher plants. For example, Beck et al. (1983) andAnderson et al. (1983) showed that ascorbate uptakeby intact spinach chloroplasts followed Michaelis–Menten kinetics. However, the Km values (the range of20–40 mM) are very high. Similarly, low affinities occurfor ascorbate uptake by mitochondria isolated fromtobacco BY-2 cells (Szarka et al. 2004). Barley

protoplasts took up ascorbate with high affinity(Km 5 90 mM) (Rautenkranz et al. 1994), whereas theKm for ascorbate/DHA uptake from the apoplast ofBetula pendula leaf was 12.8 mM (Kollist et al. 2001).On the other hand, the uptake of ascorbate intovacuoles does not have saturation kinetics, indicatingthe lack of a specific transporter (Rautenkranz et al.1994). In contrast to the situation in higher plants, mam-mal ascorbate transporters (sodium-dependent vitaminC transporters, SVCT1 and SVCT2) have been identifiedand well characterized as an active ascorbate transportsystem (Daruwala et al. 1999, Tsukaguchi et al. 1999).SVCT1 and SVCT2 transport ascorbate by a sodium-dependent cotransport mechanism with high ascorbateaffinities (Km 5 10–250 mM). These proteins belong tothe nucleobase-ascorbate transporter (NAT) family,which is ubiquitous from bacteria to higher plants andanimals (de Koning and Diallinas 2000). Recent

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 monodehy-

droascorbate. 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).


computer-assisted homology searches indicated that atleast 12 NAT orthologous genes are present in theA. thaliana genome, and one of them (MQB2.190,At5g62890) contained highly a conserved region withmammal SVCTs (Li and Schultes 2002). Further investi-gation is needed to determine whether they functionas plant (sodium-dependent) ascorbate-specifictransporters.

In contrast to ascorbate, DHA tends to be more effect-ively transported across plant membranes, with higheraffinity and capacity, than ascorbate (Horemans et al.1998a, Rautenkranz et al. 1994, Szarka et al. 2004).Incubation of tobacco suspension cells with DHAresulted in a remarkable increase of cellular ascorbatecontents, also supporting the presence of a DHA trans-port system on the plasma membrane (de Pinto et al.1999, Potters et al. 2000). Although the substantial con-tribution of glucose transporters for DHA uptake in ani-mal cells has been elucidated (Wilson 2004), a potentialrole of the transporter in DHA transport is inconclusivein higher plants (Horemans et al. 2000a). The uptake ofDHA via glucose transporters in animal cells is charac-terized by its competitive inhibition by a simultaneousaddition of glucose and specific inhibitors such as phlor-etin, phlorizin and cytochalasin B (Wilson 2004). Onthe other hand, although cytochalasin B and genisteininhibited DHA and glucose uptake in plant mitochon-dria isolated from tobacco BY-2 cells, phloretin andphlorizin had no effect on the glucose uptake (Szarkaet al. 2004). A similar phenomenon was observed in theexperiment using tobacco BY-2 protoplast (Horemanset al. 1998b). Horemans et al. (2000b) have proposedthe presence of a plasma membrane ascorbate/DHAexchange carrier, which takes up apoplastic DHA inexchange for cytosolic ascorbate. Unfortunately, theprotein or gene associated with this transport activityhas not been identified.

Long-distance transport

Ascorbate occurs in almost all plant tissues. It tends tobe more concentrated in photosynthetic tissues, fruitsand meristems than in non-photosynthetic tissues suchas roots (Davey et al. 2000). Until recently, there hasbeen no information on the extent of long-distanceascorbate transport. In pioneering work, Franceschiand Tarlyn (2002) have shown ascorbate transport viathe phloem from source leaves to sink tissues such asroot tips, shoots and floral organs. Autoradiographyshowed the movement of radiolabelled ascorbate inthe phloem of Medicago sativa and A. thaliana. Thesame phenomenon was also observed in potato sourceleaf phloem and sink tubers (Tedone et al. 2004). The

ascorbate content of phloem exudates in both leaf andtuberizing stolons of potato showed significant diurnalchanges, indicating that ascorbate accumulation in sinktissue is affected by source leaf ascorbate biosynthesis.Hancock et al. (2003) have also suggested that ascor-bate biosynthesis occurs in phloem via the D-Man/L-Galpathway. Long-distance ascorbate transport may berequired to supplement in situ ascorbate synthesis bysink tissues. Ascorbate demand in developing sink tis-sues is high, because ascorbate is probably required forcell cycle and cell division modulation and cell elonga-tion affecting cell-wall construction (Smirnoff 2000b).The high content of ascorbate in certain fruits could beachieved by a combination of transport and in situsynthesis. D-Galacturonic acid derived from pectinbreakdown during fruit ripening could also be a sourceof ascorbate (Fig. 1), and a strawberry D-galacturonicacid reductase gene is expressed only during fruit ripen-ing (Agius et al. 2003). Manipulation of ascorbate trans-port in the phloem may provide a useful approach toincrease the ascorbate content of fruits and tubers.

Engineering ascorbate accumulation

As was reviewed above, very little is known about thecontrol of ascorbate biosynthesis and the determinationof its pool size in plant tissues and or subcellular com-partments. Ascorbate pool size is influenced not only byits biosynthesis but also by recycling. Combined withthe possibility of the existence of multiple biosynthesispathways, these facts make the situation difficult to pre-dict and engineer. Three distinct approaches have beentaken: overexpression of biosynthesis enzymes; overex-pression of recycling enzymes (DHA reductase) andantisense suppression of ascorbate oxidase (AO). AO isan apoplastic enzyme that affects the redox state ofextracellular ascorbate (Pignocchi et al. 2003,Sanmartin et al. 2003). The results of these engineeringexperiments are summarized in Table 1. Other less-targeted approaches are also being taken, but the inves-tigations are at early stages of development. Theseinclude the use of QTL analysis and molecular markersto identify novel genes associated with high ascorbateand screening activation-tagged A. thaliana lines forhigh ascorbate (N. Smirnoff, unpublished results).

Ascorbate biosynthesis

Changes in L-GalLDH activity and transcription levels areassociated with changes in ascorbate content during leafsenescence, changes in light intensity and fruit develop-ment (Pateraki et al. 2004, Smirnoff 2000a). The rate ofL-Gal synthesis is also increased by high light intensity,


suggesting that the early steps of the D-Man/L-Gal pathwayhave increased flux (Gatzek et al. 2002). Therefore, it wasexpected that altering the expression levels of theenzymes related to this pathway would affect the accu-mulation of ascorbate. However, in general, the overex-pression of enzymes in the D-Man/L-Gal pathway has notresulted in increased ascorbate concentration. For exam-ple, although the overexpression of an L-GalDH intobacco plants resulted in a three-fold increase in theactivity, no effect on ascorbate levels was observed(Gatzek et al. 2002). On a more positive note, Tokunagaet al. (2005) using tobacco suspension cells overexpres-sing L-GalLDH demonstrated an increase in ascorbatecontent. The cells overexpressing L-GalLDH under thecontrol of CaMV 35S promoter showed a moderate(approximately two-fold) enhancement of total ascorbatecontent during stationary phase and still sustained signifi-cant ascorbate levels after that, whereas stationary-phasewild-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 proposedenzymes of the uronic acid pathways results in moder-ate increases in ascorbate contents. With respect toD-galacturonate reductase, the overexpression of theenzyme from strawberry fruit (Fragaria ananassa) inA. thaliana resulted in a two- to three-fold increase oftotal ascorbate content (Agius et al. 2003). The over-expression of myo-inositol oxygenase gene (miox4),another enzyme assumed to take part in glucuronicacid pathway, in transgenic A. thaliana also produced

a moderate enhancement of ascorbate content in leaves(Lorence et al. 2004). On the other hand, A. thalianamutants disrupted in other MIOX genes homologues(MIOX2 or MIOX5) were not affected in their leafascorbate content (Kanter et al. 2005), possibly indicat-ing that homologues are not involved in ascorbate bio-synthesis in leaves. L-GulL is the predicted precursor ofascorbate in the glucuronic acid pathway. The ectopicexpression of rat L-GulL oxidase in transgenicA. thaliana resulted in a moderate increase in totalascorbate level compared with wild-type plants, sug-gesting the presence of L-GulL as a penultimate precur-sor of the uronic acid pathway in higher plants. Bothglucuronic acid and galacturonic acid are majorcomponents of cell-wall biomass derived from UDP-glucuronic acid and UDP-galacturonic acid as theirprecursors (Seifert 2004). However, the biosyntheticpathway of UDP-glucuronic acid is still incompletelyunderstood. The identification of MIOX in higher plantsnow opens the window on understanding the regulationof the poorly understood inositol oxygenation pathway.Increasing the flux of this pathway towards the produc-tion of uronic acids by metabolic engineering might beexpected to affect the accumulation of ascorbate inhigher plants.

Ascorbate recycling

Because the rate of ascorbate turnover is relatively fast,13% of the pool per h in pea seedlings (Pallanca and

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-galactono-

lactone 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.

Gene source Enzyme

Plant species

engineered Gene regulation

Increase in

ascorbate pool (fold) Ascorbate/DHA ratio Reference

Arabidopsis L-GalDH Tobacco Up Not changed Gatzek et al. (2002)

Tobacco L-GalLDH Tobacco BY-2 cells Up 1.5–2.0 (7 days) Tokunaga et al. (2005)

Rat L-GulLOx Arabidopsis Up Approximately 2.0 Radzio et al. (2003)

Rat L-GulLOx Lettuce Up 2.0 Jain and Nessler (2000)

Rat L-GulLOx Tobacco Up 7.0 Jain and Nessler (2000)

Arabidopsis myo-Inositol ox Arabidopsis Up 2.0–3.0 Lorence et al. (2004)

Strawberry D-GalUAR Arabidopsis Up 2.0–3.0 Agius et al. (2003)

Wheat DHAR Tobacco (to chloroplasts) Up 2.2–3.9 Increase Chen et al. (2003)

Wheat DHAR Maize (to chloroplasts) Up Approximately 1.9 Increase Chen et al. (2003)

Human DHAR Tobacco (to cytosol) Up Not changed Increase Kwon et al. (2003)

Pumpkin AO Tobacco Up 2.4 (in apoplast) Decrease Pignocchi et al. (2003)

Tobacco AO Tobacco Down 1.9 (in apoplast) Increase Pignocchi et al. (2003)

Tobacco AO Tobacco Up Not changed Decrease Yamamoto et al. (2005)

Tobacco AO Tobacco Down Not changed Increase Yamamoto et al. (2005)

Tobacco G6PDH Tobacco (to chloroplasts) Down Not changed Increase Debnam et al. (2004)

Tomato MDH Tomato (to mitochondria) Down Approximately 5.7 Nunes-Nesi et al. (2005)


Smirnoff 2000) and 40% per 22 h in A. thaliana leaves(Conklin et al. 1997), it might be expected that theenhancement of the ascorbate recycling pathway orthe downregulation of ascorbate oxidation would alsoaffect the ascorbate accumulation. This may not be trueof all tissues. For example, potato leaves have a lowturnover rate (Imai et al. 1999). Transgenic tobacco andmaize overexpressing cytosolic DHA reductase fromwheat resulted in a two- to four-fold increase in totalascorbate contents and decreased the proportion ofDHA in both leaves and maize kernel (Chen et al.2003). Unlike the overexpression of DHA reductase incytosol, the overexpression of human DHA reductase inthe chloroplasts of transgenic tobacco plants showed nosignificant difference in total ascorbate content, whereasan increase in the ascorbate/DHA ratio was observed(Kwon et al. 2003). Interestingly, transgenic tobaccowith the suppressed activity of one of the chloroplasticglucose 6-phosphate dehydrogenase (P2-G6PDH) iso-forms by antisense expression of the gene showed asignificant increase in the cellular ascorbate/DHAratio, but not in total ascorbate content (Debnam et al.2004). The suppression of G6PDH activity would beexpected to reduce NADPH content, so this result can-not be easily explained. The overexpression of MDHAreductase might both affect ascorbate accumulation andincrease the redox status of ascorbate towards reduc-tion, because MDHA reductase functions upstream ofDHA reductase in the ascorbate recycling pathway.However, there are no reports of the effects of MDHAreductase overexpression.

Ascorbate oxidase

AO is located in the apoplast (Pignocchi et al. 2003). Itis possible that the modulation of AO activity wouldlead to the alteration of ascorbate accumulation.However, sense and antisense reduction of AO in trans-genic tobacco had no effect on total leaf ascorbatecontents (Pignocchi et al. 2003), because most of theascorbate is in the cytosol, not in the apoplast.However, the ascorbate status in apoplast of these trans-genics showed a significant increase in ascorbate con-tents and altered redox balance of ascorbate than thosein wild-type plants. The total ascorbate content of theapoplast was increased by up to 2.4-fold compared withwild-type in sense and antisense plants. In the antisenseplants, the proportion of the reduced form in the apo-plast was markedly increased (66%) compared with thewild-type plants (40%), whereas in the sense plants, itwas significantly decreased (3%). Similar results havealso been reported by another research group usingtransgenic tobacco expressing AO gene in sense and

antisense orientation (Yamamoto et al. 2005). The pro-portion of ascorbate/DHA in both whole leaf and apo-plast was statistically higher in antisense tobacco plantsand lower in sense plants than that in wild-type plants,while apoplastic total ascorbate contents of these plantswere not influenced by AO manipulations. Takentogether, the engineering of ascorbate recycling or oxi-dation pathway is also potentially useful to the improve-ment of ascorbate accumulation and its redox status. Inaddition, it might be important to consider targetinggene products to specific cellular compartments.

Other approaches

Recently, unexpected results were obtained when a mito-chondrial malate dehydrogenase was suppressed intransgenic tomato (Lycopersicon esculentum cv. MoneyMaker). Gas chromatography-mass spectrometry analysisof antisense mitochondrial MDH lines showed up to 5.7-fold increased levels of ascorbate in their leaves thanthose of wild-type tomato (Nunes-Nesi et al. 2005).Higher ascorbate level was also reported in a mitochon-drial aconitase mutant tomato (Aco1, Lycopersicon pen-nellii) (Nunes-Nesi et al. 2005). In the antisense MDHplants, there was no effect on mitochondrially locatedL-GalLDH activity or on the concentration of galacturonicacid and glucuronic acid. Although the reason for thephenomenon is not fully apparent, it suggests that TCAcycle activity can influence ascorbate metabolism.

Conclusions

Considering the research so far, the attempts to alterascorbate accumulation in plants have clearly shownthat ascorbate metabolism in higher plants is regulatedby complex control networks that are affected by multi-ple factors. Although the engineering of ascorbate accu-mulation has at least been demonstrated in principal,engineering plants with substantially increased ascorbatepool through enhanced flux using single enzymes, parti-cularly in the D-Man/L-Gal pathway, has been unsuccess-ful. Much remains to be learned about the contribution ofalternative ascorbate biosynthetic pathways in each planttissue and the regulation of the metabolic fluxes, andmany important genes still have not yet been identified.In addition, the manipulation of other antioxidants couldaffect ascorbate accumulation, and it will be important tolearn more about these relationships. For example, adouble mutant for the simultaneous loss of tocopheroland glutathione had a significant increase in ascorbatecontent, whereas the overproduction of these two anti-oxidants tends to cause a decrease in ascorbate accumu-lation (Kanwischer et al. 2005). The mechanism of


ascorbate transport across cellular compartment andtransportation from source to sink organs are also largelyunknown. The increase in the apoplastic DHA content ofAO-overexpressing tobacco (Pignocchi et al. 2003) sug-gests that it might be useful to improve DHA transportefficiency. Because ascorbate is one of the major redoxbuffers of the plant cell, along with glutathione (Noctorand Foyer 1998), understanding the effect of theenhanced levels of ascorbate on all aspects of plantfunction is also of particular importance. Altering ascor-bate levels may affect other aspects of physiology, suchas abscisic acid synthesis (Pastori et al. 2003), control ofcell division and growth (Potters et al. 2000, 2004),cellular H2O2 concentration and gene expression profiles(Pastori et al. 2003). It is important to consider possibledeleterious effects as well. For example, DHAR overex-pression reduces stomatal closure in response to waterstress, because improved H2O2 scavenging interfereswith its role in ABA signalling (Chen and Gallie 2004).Also, higher ascorbate content could reduce basal resis-tance to pathogen infection (Barth et al. 2004). A com-bination of metabolic profiling and gene expressionprofiling could reveal much about how plants react toengineered changes. Finally, as more knowledge ofascorbate metabolism becomes available, the strategiesavailable to address our goal of increasing its concentra-tion in a rational manner will certainly increase.

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Edited by C. H. Foyer


progress in manipulating ascorbic acid biosynthesis and accumulation in plants

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