Ascorbate function and associated transport systems in plants

Plant Physiol. Biochem. 38 (2000) 531−540 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S0981942800007828/REV

Ascorbate function and associated transport systems in plants Nele Horemansa*, Christine H. Foyerb, Geert Pottersa, Han Asarda a

University of Antwerp (RUCA), Department of Biology, Groenenborgerlaan 171, B-2020 Antwerp, Belgium

b

IACR-Rothamsted, Department of Biochemistry and Physiology, Harpenden, Herts, UK

Received 15 December 1999; accepted 11 May 2000 Abstract – Ascorbate is present in different cell compartments of higher plant cells. At a physiological level, the best-studied phenomena involving ascorbate is its participation in an oxygen scavenging pathway in the chloroplast known as the ascorbate-glutathione cycle. In addition, evidence is emerging that ascorbate fulfils essential roles in growth, development and defence outside the chloroplast. Despite its importance in plant biology, the pathway of ascorbate biosynthesis has only recently been elucidated. From the site of synthesis in the mitochondria, ascorbate must be transported to other cellular compartments where it accumulates to high concentrations. Translocation of ascorbate through the plasmalemma and chloroplast membrane is mediated by specific carriers. Initial observations indicate that carriers for both ascorbate and its oxidised form dehydroascorbate are present in plant membranes. Regulation of ascorbate transport systems may be central in the regulation of different physiological processes including progression through the cell cycle, expansion of the cell wall and defence against abiotic and biotic threats. © 2000 Éditions scientifiques et médicales Elsevier SAS antioxidant / ascorbate / chloroplast membrane / dehydroascorbate / plasma membrane / transport / vitamin C Asc, ascorbate / AOS, active oxygen species / BY-2, Bright Yellow 2 / CCCP, carbonylcyanide-3-chlorophenylhydrazone / DHA, dehydroascorbate / MDHA, monodehydroascorbate

1. INTRODUCTION Ascorbate (Asc) is one of the best characterised components contributing to the nutritive value of green vegetables and fruits. Since the elucidation of its key role in the prevention of scurvy, Asc has been shown to fulfil many other essential functions in animals and plants. For example, epidemiological evidence for reduced risk of some cancers and cardiovascular disease as well as prevention of age-related degenerative ill health and problems common to obese or diabetic people has led to the understanding that consumption of green vegetables and fruits provides important health benefits beyond simply providing essential nutrients [59]. In plants, Asc acts as an antioxidant protecting cells against oxidative stress. It also plays a pivotal role in the synthesis of hydroxyproline-rich proteins (analogous to roles in collagen formation) which are components of the plant cell wall. In addition functions in * Correspondence and reprints: fax +32 3 218 04 17 e-mail [email protected]

plant cell growth and division have been described. The biosynthetic pathways of Asc in plants, which has only recently been elucidated [78] seems, however, to be rather different from that in animals. This update presents an overview on the function of Asc in plant cells with special emphasis on the importance of the translocation through different cell membranes by specific carriers.

2. ASCORBATE METABOLISM 2.1. A common constituent of plant cells In most plant tissues, Asc is present in millimolar concentrations. However, Asc contents vary considerably between tissues and depend on the physiological status of the plant as well as on environmental factors [44, 67]. Photosynthetic tissues have high concentrations of Asc as do fruits and other storage organs [26, 45]. Asc contents are generally higher in younger tissues than in older ones and Asc accumulates in actively growing tissues such as meristems [16, 47]. The onset of senescence triggers loss of tissue Asc

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Figure 1. Schematic drawing of a plant cell with different cell organelles and compartments indicated (V, vacuole; M, mitochondrion; C, chloroplast; P, peroxisome). The relative occurrence of ascorbate (Asc) in these different compartments is given as a percentage. Since no separate data are available, the peroxisomal and mitochondrial Asc concentration is included in the percentage of the cytosol. The final step of Asc biosynthesis is indicated as the activity of L-galactono-γ-lactone (GAL) dehydrogenase, on the inner mitochondrial membrane, converting GAL to Asc. Asc translocation through the different membranes is presented as follows: identified Asc or dehydroascorbate (DHA) carrier systems, ; transport that is not carrier mediated, ; and translocation that has not been studied so far, . For the plasma membrane, the possible presence of different Asc and DHA transporters as well as the possibility of carrier working as a DHA/Asc exchange carrier is indicated.

while Asc loss is retarded in stay-green mutants [7, 42]. Dry seeds [4, 73] and dormant Malus sp. buds [76] have little or no Asc. Germination and bud breakage are marked by a sharp increase in both tissue Asc and Asc-metabolising enzymes. Cells in the quiescent centre of Zea mays roots have negligible Asc and can be induced to divide upon the addition of exogenous Asc [41]. Asc is present in most, if not all, cell compartments. For example, Asc accumulates in chloroplasts to high concentrations, where it can amount from 12 to 30 % of the total Asc content of the leaves [21, 25]. Substantial amounts of Asc are also found in the apoplast [74, 75] cytosol and vacuole [57]. The cytosolic Asc pool has been calculated to be approximately 20 mM [21]. The ultimate step of Asc synthesis catalysed by L-galactono-γ-lactone dehydrogenase occurs in the mitochondria and isolated mitochondria

produce large amounts of Asc in the presence of the precursor, L-galactono-γ-lactone [5]. The Asc pool of the intracellular compartments is largely reduced except under conditions of oxidative stress [17]. In marked contrast, in the extracellular matrix of plant cells, the redox status of Asc (i.e. the relative occurrence of reduced and oxidised Asc forms) depends on the age of the tissue, the degree of stress to which a plant is subjected and appears also to be species-dependent [17]. The relative distribution of Asc through the plant cell is given in figure 1.

2.2. Ascorbate biosynthesis Advance in the understanding of the biosynthetic pathway of Asc in plants has only been made recently studying Pisum sativum and Arabidopsis plants [78]. These authors clearly showed that one way that plants synthesise Asc involves D-glucose-6-P, D-fructose-

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6-P, D-mannose-6-P, D-mannose-1-P, GDP-Dmannose, GDP-L-galactose, L-galactose-1-P, and L-galactose as intermediates in the production of L-galactono-γ-lactone, the immediate precursor of Asc. Apparently, ozone sensitive Arabidopsis mutant vtc1 (formerly soz1) is deficient in the biosynthesis of Asc [11]. Since Asc levels in this mutant still amount up to 30 % of wild type, the possibility of other Asc biosynthetic pathways is not excluded. The subcellular localisation of the different steps in the Asc biosynthesis remains to be elucidated but the enzyme L-galactono-γ-lactone-dehydrogenase catalysing the final conversion of L-galactono-γ-lactone to Asc, is situated on the inner mitochondrial membrane [51, 64]. The pathway of Asc biosynthesis proposed by Wheeler et al. [78] together with the identification of new mutants in the pathway of Asc biosynthesis [12] will be invaluable in the further characterisation of the pathway and in the determination of its relationship to the carbohydrate metabolism of plant cells. The pathway has a number of key roles in plant metabolism because it provides not only intermediates for Asc biosynthesis but also supplies intermediates for cell wall biosynthesis and protein glycosylation [12].

2.3. Ascorbate metabolism and degradation Asc is generally involved as an electron donor transferring electrons in two sequential steps. The first step results in the formation of Asc free radical (monodehydroascorbate or MDHA) and the second in the fully oxidised dehydroascorbate (DHA). Both oxidation products are relatively unstable in aqueous solution. The MDHA concentration of cells is generally low because of the rapid disproportionation of the radicals to Asc and DHA (k2 (pH 7) = 105 to 2.8·106 M–1·s–1; [22, 66]). MDHA does not readily interact with other molecules such as lipids and is therefore relatively harmless. DHA undergoes spontaneous and irreversible hydrolysis to 2,3-diketogulonic acid [77]. The site of Asc degradation in plant cells is unknown but Asc has been shown to be involved in the synthesis of tartrate and oxalate (for review see Loewus and Loewus [45], De Gara and Tommasi [16]). Asc turnover is remarkably slow, in the order of 2 % turnover of the total pool per hour in darkness [38]. Efficient cycling between oxidised and reduced forms of Asc thus minimises DHA degradation and decreases the requirement for high rates of de novo synthesis. Feedback inhibition of Asc on the pathway of its own biosynthesis is strongly indicated [38, 50].

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3. ASCORBATE FUNCTIONS 3.1. Asc as an antioxidant The importance of Asc in various physiological phenomena of higher plants has extensively been reviewed elsewhere [1, 16, 53, 55, 67]. We will therefore consider these functions in view of the recently discovered need for transport of Asc and the subcellular compartmentation of these processes. By far the best studied function of Asc in plant cells is its role in the protection of the chloroplast against oxidative damage. Active oxygen species (AOS) are inevitably formed during photosynthesis and photorespiration [23, 55]. The scavenging of O2– and H2O2 produced in the chloroplast is catalysed by stromal and thylakoid-bound superoxide dismutases and Asc peroxidases [24, 49]. MDHA formed by the action of the latter enzyme can be directly reduced by ferredoxin [24, 49]. This series of reactions occurs close to the thylakoid membrane and is known as the Mehlerperoxidase reaction sequence. In addition, a second enzymatic cascade (commonly known as Ascglutathione [20]) is important in the regeneration of Asc from DHA. The presence of two Asc regeneration systems guarantees the complete regeneration of reduced Asc at the expense of glutathione and NAD(P)H [20]. While the Mehler-peroxidase sequence is restricted to the chloroplast, the Asc-glutathione cycle is found in several compartments of the plant cell. Asc not only functions as a major antioxidant in the soluble phase of plant cells but also supports essential membrane-bound antioxidants in their role as cellular protectants. For example, carotenoid pigments (carotenes and xanthophylls) and α-tocopherol present in the chloroplast membranes quench triplet chlorophyll and singlet oxygen and remove AOS as they are formed within the membrane environment. These membrane-associated antioxidants protect against lipid peroxidation, and depend on Asc in two ways. First, the de-epoxidation of violaxanthin, which produces zeaxanthin, an essential component of the protective xanthophyll cycle, is dependent on Asc [54]. Second, Asc acts as a secondary antioxidant involved in the direct regeneration of reduced α-tocopherol from its oxidised form. Asc is also involved in the protection of other cell compartments against oxidative damage. Asc peroxidases are found in the cytosol and in the peroxisomes. In these peroxisomes, Asc peroxidase may support catalase in the removal of the large amounts of H2O2 produced in the photorespiratory glycollate pathway. Considerable attention has recently focused on Asc in

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the cell wall and the role of Asc in ozone-scavenging in the apoplast [18, 40]. There is apparently an inverse relationship between total foliar Asc concentration and sensitivity to ozone [13]. Exposure to oxidative stress increases tissue Asc accumulation and results in an enhancement of Ascdependent detoxification processes. The Arabidopsis thaliana mutant, vtc1, which has only 30 % of the Asc present in the wild type, shows increased sensitivity to ozone and to oxidative stress caused by exposure to UVB irradiation or SO2 pollution [12]. Taken together, these observations support the view that Asc has a general role in defence reactions in most, if not all, compartments of the plant cell. The subcellular localisation of the rise in Asc or Asc-dependent enzymes in these stress conditions has not been investigated in detail so far.

enzyme is controlled by auxins [41]. Cell division in A. thaliana root tips is also modulated by Asc [63]. However, in this system both reduced glutathione and Asc are implicated in the regulation of mitosis [48]. Observations, such as described above, have led to the hypothesis that the redox status of low molecular weight antioxidants such as Asc and glutathione may modulate the degree of cell proliferation. In animal systems, a similar hypothesis was recently proposed for animal fibroblasts [37]. Regulation of cell growth by the redox status of antioxidants could be an important strategy for survival in a constantly fluctuating environment [63]. This is rationalised in terms of prevention of replication of damaged DNA in conditions of extreme oxidative stress [48].

3.2. Asc modulates cell division

In addition to cell division, several lines of evidence seem to indicate a specific role for Asc in cell elongation. For example, the elongation of A. cepa root cells is stimulated by the addition of Asc or generation of MDHA [28, 31]. Different Asc-mediated reactions may be important in this process. First, as a co-factor of the cytosolic enzyme prolyl-hydroxylase Asc is essential for the biosynthesis of hydroxyprolinerich proteins important components of the cell wall structure. Second, the apoplastic Asc concentration or its redox status may alter properties of the plasma membrane that could lead to enhanced cell elongation such as plasma membrane hyperpolarisation and cell wall acidification [29] or increased nutrient uptake by the plasma membrane [32]. Third, Asc has a stimulating effect on cell elongation by inhibiting peroxidasecatalysed cross-linking of hydroxyproline-rich glycoproteins with phenolic acids [14, 62, 71]. This crosslinking contributes to cell wall secondary thickening and is negatively correlated with cell growth [80]. Asc does not directly inhibit the cell wall peroxidases but will re-reduce their reaction products, phenolic acid radicals, and thereby prevent cross-linking. Since the net reaction will be reduction of H2O2 by Asc, this system may, in addition to its regulatory function in cell elongation, also serve as an antioxidant system preventing oxidative damage [62, 70, 71].

A significant role of Asc in the control of cell growth has been presented (initially proposed by Reid [58]; for recent reviews, see Arrigoni [3], GonzálezReyes et al. [28] Navas and Gómez-Díaz [52]). Current evidence suggests a two-fold action of Asc: first, by modifying the cell cycle and stimulating quiescent cells into division; and second, by accelerating cell expansion and elongation. For example, it was shown that addition of exogenous Asc accelerates the onset of cell proliferation in root primordia cells of Allium cepa and Pisum sativum [10, 15]. In cells treated with lycorine (a proposed inhibitor of galactonolactone dehydrogenase) to lower endogenous Asc levels, the cell cycle is arrested in the G1 phase [43]. The presence of Asc appears to be necessary for progression through the cell cycle but it will not by itself induce cell proliferation in noncompetent cells [10]. Work with tobacco BY-2 cells recently showed that Asc contents increased during cell division whereas DHA levels decreased [17, 39]. The absence of reduced Asc in the quiescent centre of maize roots was correlated with the absence of cell division in an extended G1 phase [41]. These observations emphasise the importance of the Asc redox status in these processes. Moreover, since in the quiescent cells Asc oxidase activity is high, it is possible that this enzyme may control the cell cycle through regulation of the cellular Asc [41]. Alternatively, the expression and activity of Asc oxidase and the consequent production of DHA may be controlled by the cell cycle [39]. Recent evidence suggests that Asc oxidase may be located exclusively in the apoplast, and that the activity and expression of this

3.3. Asc and cell elongation

4.. ASCORBATE TRANSPORT 4.1. Subcellular transport of Asc Once synthesised in the mitochondria, Asc needs to be translocated to other cell compartments. Rapid

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Table I. Overview of Asc transport systems in plants. Cell-type organelle Spinach chloroplasts Pea chloroplasts Pea thylakoid Barley vacuole Barley protoplasts

Redox state

Km

Transport mechanism

Asc Asc Asc

20 mM 18–45 mM

Asc; DHA

90 µM; 20 µM

Asc; no DHA

6.7 mM

Tobacco BY-2 protoplasts

DHA

139 µM

Bean plasma membrane vesicles

DHA

14–79 µM

Pea protoplasts

24 µM TBY-2 plasma membrane vesicles

changes in Asc concentration are observed in stress conditions [9, 46, 47, 68]. On the other hand, almost no Asc is found in certain tissues or cell types, such as in the quiescent centre of roots [41] and Zea mays mesophyll cells [19]. As Asc is negatively charged at physiologically relevant pH levels, it will not freely diffuse through membranes. In addition, it has been demonstrated that the electroneutral DHA molecules are also insufficiently lipophilic to cross the lipid membrane barrier by simple diffusion (see Rose [60]). These observations suggest the presence of regulated transport systems for specifically enhancing or excluding Asc from different cell compartments according to the requirements of physiology, development and metabolism. At present, translocation of Asc through chloroplast, thylakoid, tonoplast and plasma membranes has been investigated but no data are yet available for transport through other membrane systems such as those of the mitochondria or peroxisomes. An overview of the different Asc transport systems is given in table I and figure 1. Since the last Asc biosynthetic step occurs on the inner mitochondrial membrane [51, 64], it is conceivable that transport of Asc from the intermembrane space of the mitochondria to the cytosol occurs by simple diffusion. Asc transport from the cytosol into the chloroplast stroma is a carrier-mediated process with a rather low but physiologically relevant affinity for Asc [2, 6, 21]. The uptake into intact chloroplasts shows cis-inhibition and trans-stimulation by DHA and is suggested to be driven by facilitated exchange diffusion. In contrast to these results, translocation of Asc through thylakoid

Facilitated diffusion Facilitated diffusion Facilitated diffusion, glucose independent No saturable transport found No saturable transport found Driven by a protonelectrochemical gradient Facilitated diffusion, glucose independent Facilitated diffusion, glucose independent Facilitated diffusion, glucose independent Counterflow Counterflow

Reference [2] [6] [21] [21] [57] [57] [21] [36] [33–35]

Horemans, unpubl. results

membranes [21] or the tonoplast membrane [57] is low, non-saturable and not affected by light treatment or ATP addition. This indicates that although Asc is present in the thylakoid lumen in a concentration of around 4 mM, no carrier-mediated translocation of Asc occurs into the thylakoid lumen [21]. In addition, the low pH values found in the thylakoid lumen in the light will favour diffusion of ascorbic acid (pK1 = 4.25) into the stroma over movement in the opposite direction. This could therefore lead to a restriction of the amount of Asc available to violaxanthin de-epoxidase. It is at present not known how the plant cell copes with this problem.

4.2. Transport through the plant cell membrane Initial evidence for movement of Asc through the plant plasma membrane was indicated by Mozafar and Oertli [50] who studied uptake of radioactively labelled Asc into intact leaves and roots from soybean. More detailed studies, using protoplasts isolated from Hordeum vulgare leaves [57] or P. sativum leaves [21] or from Nicotiana tabacum BY-2 cell cultures [36], have subsequently confirmed the presence of Asc and DHA transporters. Transport mechanisms have also been studied using highly purified Phaseolus vulgaris plasma membrane vesicles [33–35]. Addition of [14C]-Asc to the bathing medium of protoplasts or plasma membrane vesicles results in time- and concentrationdependent uptake of label showing Michealis-Menten kinetics. Compared to uptake through the chloroplast membrane [2, 6], the plasma membrane-associated carrier shows high affinities for Asc and DHA (table I).

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Since in animal cells Asc and DHA specific transporters have been described (see review Goldenberg and Schweinzer [27]), studies have been performed to determine which species of Asc is transported through the plant plasma membrane. Uptake of Asc, as well as DHA, was found in H. vulgare protoplasts with higher affinities for DHA uptake than Asc ([57], table I). Horemans and co-workers showed a clear preference for uptake of the oxidised DHA molecules by P. vulgaris plasma membrane vesicles or N. tabacum protoplasts [34, 36]. These findings demonstrate the existence of a system transporting oxidised Asc molecules from the apoplast to the cytoplasmic compartment of H. vulgare, P. vulgaris and N. tabacum cells. In addition to a DHA-carrier, an independent uptake system for reduced Asc from the extracellular to the intracellular compartment probably exists. For example, Foyer and Lelandais [21] showed that Asc is effectively transported into P. sativum protoplasts and in H. vulgare protoplasts, transport of both Asc and DHA was detected, with apparent competition of the two species for the same binding site on the carrier [57]. Such a system may be necessary to recover Asc diffusing from the cytosol to the apoplast. Translocation of Asc from the cytosol to the apoplast has not yet been investigated in detail. However, the air pollutant ozone accelerates Asc movement from the cytosol to the apoplast in a manner that is not associated with increased membrane leakiness [47]. The mechanism driving uptake of Asc or DHA through the plant plasma membrane remains to be elucidated. Horemans et al. [33, 35] suggested that DHA transport in P. vulgaris plasma membrane vesicles was driven by facilitated diffusion. In contrast, in metabolically active protoplasts, a protonelectrochemical gradient seems to be involved as a driving force [36, 57]. Takahama [69] reported that apoplastic contents of Asc and DHA in Vigna angularis changed upon the addition of CCCP suggesting that the proton-electrochemical gradient was an important factor determining the concentration difference of Asc and DHA across the plasma membrane. An interesting property of Asc transport observed in purified plasma membrane vesicles is the mechanism of trans-stimulation. Bean or tobacco BY-2 plasma membrane vesicles artificially loaded with Asc showed highly stimulated DHA uptake rates and accumulation (Horemans et al. [35] and Horemans unpubl. results). Moreover, addition of DHA to bean membrane vesicles loaded with [14C]-labelled Asc resulted in a significant release of radioactive Asc molecules from these vesicles [35]. Based on these results, an Asc/DHA exchange

mechanism for Asc transport through the plasma membrane was suggested. Interestingly, a similar exchange mechanism has also been suggested for the operation of an Asc transporter in brush border membranes of rat and guinea pig [65, 72]. In figure 1, a schematic overview is given of the different Asc and/or DHA transport systems, including DHA/Asc exchange, as has been studied so far.

4.3. Physiological role of Asc transport through the chloroplast membrane As discussed above, transport of Asc through the chloroplast envelope is probably mediated by a facilitated diffusion carrier [2, 6, 21]. Although the affinity of the carrier for Asc is low (5 mM), it lies in the range of the Asc concentration in the cytosol (20 mM) and could therefore be capable of transporting Asc into the chloroplast stroma at sufficiently high rates. Interestingly, the chloroplast carrier also shows transstimulation by DHA [6]. Therefore it is possible that, similar to the Asc carrier present on the plasma membrane, the chloroplastic Asc carrier could be driven by an Asc/DHA exchange mechanism. While this hypothesis lacks experimental support, it opens the possibility that the chloroplastic Asc carrier could actively transport DHA out of the chloroplast stroma for re-reduction in the cytosol if re-reduction in the chloroplast becomes limiting. In light, when reducing power is plentiful, MDHA and DHA may be reduced mainly within the stroma. The stroma lacks DHA reductase and it may be necessary to reduce some DHA (arising from the chloroplast) in the cytosol in the light. In the dark, reducing power derived from respiration will be required to drive DHA reduction in the cytosol. Foyer and Lelandais [21] showed that transport of Asc through the thylakoid membrane is not carriermediated and proposed simple diffusion of the uncharged Asc acid molecules depending on the existing pH and concentration gradient. In the thylakoid lumen, Asc has an important function in the synthesis of zeaxanthin since Asc is a co-factor of the enzyme violaxanthin de-epoxidase [79]. An important question therefore is whether sufficient Asc could be transported into the thylakoid lumen to sustain the needs of zeaxanthin synthesis. In this context, it was demonstrated that the actual substrate for violaxanthin de-epoxidase is not Asc but rather the Asc acid [8] and that the enzyme’s activity is highly dependent on pH [30]. Based on these observations, it is suggested that changes in Asc acid availability via limitation of transport may be of regulatory influence for zeaxan-

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thin formation [21]. Asc plays a central role in reactions regulating the transthylakoid proton gradient and electron transport in the chloroplasts [22]. It is also worth noting that the last enzyme of Asc biosynthesis is located on the inner mitochondrial membrane and donates electrons directly to the respiratory electron transport chain [5].

4.4. Physiological importance of Asc transport through the plant plasma membrane The apoplast of leaf cells can contain up to 10 % of the total foliar Asc pool ([74, 75]; figure 1). Since no extracellular Asc biosynthesis exists, transport from the cytoplasm to the apoplast is essential. Recent evidence suggests that there is also a need for transport in the reverse direction; that is, the uptake of DHA and/or Asc from the apoplast, as well as for a strict control of exchange and transport [46, 71]. For example, rapid changes in apoplastic Asc and DHA in response to stress conditions are most easily explained in terms of modified transport of Asc through the plasma membrane [9, 68]. Although some glutathione reductase and DHA reductase are found in the apoplast, Asc regeneration via the Asc-glutathione cycle cannot occur in this compartment since it does not contain NAD(P)H or glutathione [9, 46, 56, 74, 75]. Therefore, most of the reduction of DHA to Asc must occur in the cytosol following transport through the plasma membrane. Fumigation of Spinacia oleracea leaves with ozone resulted in the oxidation of apoplastic Asc and cytoplasmic glutathione [47]. In Fagus sylvatica leaves, an increase in both foliar DHA and oxidised glutathione was demonstrated, correlating with the ozone concentration [46]. These experiments provide indirect evidence in support of the hypothesis that DHA is taken up into the cytosol for re-reduction and point towards a direct function of the plasma membrane DHA transporter in the maintenance of the external Asc pool in oxidative stress conditions. The activity of cytosolic glutathione reductase, as well as the system regulating transport of Asc and DHA across the membrane, differ between species and may play a key role in determining the ozone susceptibility of different plants [46]. As described above, Asc has a dual effect on cell wall production. During cell expansion, Asc is required for extensin biosynthesis while it inhibits cross-linking processes. This implies that in order to allow lignification of the cell wall, apoplastic Asc should be completely oxidised. It was therefore suggested by Takahama and Oniki [71] that a strict control of the concentration as well as the redox status of Asc

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molecules exists in the cell wall. Although no hard evidence is available yet, the Asc carrier present in the plasma membrane is ideally placed to fulfil this role. Studying cell cycle progression in tobacco BY-2 cell cultures in media enriched with Asc or DHA led de Pinto et al. [17] to the conclusion that an increased uptake of DHA into the cells affects cell proliferation. In the assessment of Asc transport systems and their role in plant metabolism, it is important to recognise that even adjacent cells in tissues may have different functions and metabolism. The bundle sheath cells and mesophyll cells of the C4 plant maize, for example, show differential compartmentation of enzymes involved in Asc oxidation and re-reduction as well as of the presence of reduced or oxidised Asc [19]. Under optimal growth conditions and in leaves of plants grown at sub-optimal temperatures, glutathione reductase and DHA reductase were exclusively localised in the mesophyll cells whereas Asc and Asc peroxidase were predominantly present in the bundle sheath cells, which lack photosystem II and so possess only a limited capacity for reductant generation via the photosynthetic electron transport chain. To facilitate regeneration of Asc, therefore, cycling of Asc and DHA between the two cell types was proposed [19]. In conclusion, physiological experiments point towards a role of Asc/DHA translocators in different processes such as defence against oxidative stress and cell wall lignification, cell division as well as in the distribution of Asc and DHA between different plant cell types. Specifically, transport of DHA seems a necessary condition to allow DHA reduction which is not possible in all cell compartments and thereby keeps the Asc pool in the reduced state.

5. CONCLUSION AND PERSPECTIVES Asc is an essential constituent of higher plants that plays key roles in antioxidative defence, cell division and cell elongation. The vtc1 A. thaliana mutant, which has only 30 % of the Asc of the wild type, is extremely stress-sensitive. Asc is synthesised in the mitochondria from where it is transported to all the other compartments of plant cells to fulfil essential functions. The different membranes bounding the various cellular compartments have different Asc transport systems. Whereas specific carriers have been identified on the chloroplast and plasma membrane, transport through thylakoid and tonoplast membranes seems to be driven by simple diffusion alone. Most of the information so far available is on transport of Asc

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through the plasma membrane which has been studied in in vitro and in vivo systems. Different Asc transporters occur on the plasma membrane; they can be distinguished by the redox form of Asc that is preferentially transported (Asc or DHA). Current concepts of Asc transport systems in plants are derived solely from kinetic studies. The glucose transporters, GLUT1 and GLUT3, in animal cells transport DHA but not Asc [61]. Plant hexose transporters on the plasma membrane do not appear to transport DHA [21] but there are many members of this family of transporters, which may vary in their substrate specificities. To date, no Asc or DHA transporters have been purified and no genes for these transporters have been identified. Only when mutants and transformed plants modified in Asc transport systems become available will the relative importance of each of the transport systems in optimal and stress situations become apparent. Furthermore, Asc transport between cells, tissues and organs may facilitate or limit plant growth and development as well as influencing Asc biosynthesis rates in different cell types. A central question here is how do rates of transport compare to: (a) rates of biosynthesis; and (b) rates of redox cycling. This must be known if the influence of transport on contents and redox state is to be elucidated. The possibilities for exploitation of Asc transporters to improve tolerance to gaseous pollutants and to control the cell cycle may provide additional benefits to plant breeding. A general increase in the accumulation of this essential vitamin is already a key commercial target for many plant organs used in human nutrition. Acknowledgments. H.A., N.H. and G.P. are Research Associate and Doctoral Worker and Aspirant, respectively, at the Fund for Scientific Research - Flanders (Belgium) (FWO). REFERENCES [1] Alcain F.J., Buron M.I., Ascorbate on cell growth and differentiation, J. Bioenerg. Biomemb. 26 (1994) 393–398. [2] Anderson J.W., Foyer C.H., Walker D.A., Lightdependent reduction of dehydroascorbate and uptake of exogenous ascorbate by spinach chloroplasts, Planta 158 (1983) 442–450. [3] Arrigoni O., Ascorbate system in plant development, J. Bioenerg. Biomemb. 26 (1994) 407–419. [4] Arrigoni O., De Gara L., Tommasi F., Liso R., Changes in the ascorbate system during seed development of Vicia faba L, Plant Physiol. 99 (1992) 235–238.

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