JXB Advance Access originally published online on November 1, 2004
Journal of Experimental Botany 2004 55(408):2589-2597; doi:10.1093/jxb/erh262
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RESEARCH PAPER |
Localization of ascorbic acid, ascorbic acid oxidase, and glutathione in roots of Cucurbita maxima L.
1Dipartimento di Biologia e Patologia Vegetale, Università di Bari, Via E. Orabona 4, I-70125 Bari, Italia
2Istituto di Protezione delle Piante del CNR-Sezione di Torino, Dipartimentodi Biologia Vegetale dell'Università, Viale Mattioli 25, I-10125 Torino, Italia
3Dipartimento di Biologia Università di Padova, Via G. Colombo 3, I-35121 Padova, Italia
4Dipartimento di Ecologia, Università della Calabria, I-87036 Arcavacata di Rende (CS), Italia
* To whom correspondence should be addressed. Fax: +39 080 5442158. E-mail: arrigoni{at}botanica.uniba.it
Received 21 May 2004; Accepted 23 July 2004
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Abstract |
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To understand the function of ascorbic acid (ASC) in root development, the distribution of ASC, ASC oxidase, and glutathione (GSH) were investigated in cells and tissues of the root apex of Cucubita maxima. ASC was regularly distributed in the cytosol of almost all root cells, with the exception of quiescent centre (QC) cells. ASC also occurred at the surface of the nuclear membrane and correspondingly in the nucleoli. No ASC could be observed in vacuoles. ASC oxidase was detected by immunolocalization mainly in cell walls and vacuoles. This enzyme was particularly abundant in the QC and in differentiating vascular tissues and was absent in lateral root primordia. Administration of the ASC precursor L-galactono-
-lactone markedly increased ASC content in all root cells, including the QC. Root treatment with the ASC oxidized product, dehydroascorbic acid (DHA), also increased ASC content, but caused ASC accumulation only in peripheral tissues, where DHA was apparently reduced at the expense of GSH. The different pattern of distribution of ASC in different tissues and cell compartments reflects its possible role in cell metabolism and root morphogenesis. Key words: Ascorbic acid, ascorbic acid oxidase, Cucurbita maxima L., dehydroascorbic acid, glutathione, root development
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Ascorbic acid (ASC) is a compound of huge importance for plants. The activity of many ASC-dependent dioxygenases is required for the synthesis of hydroxyproline-containing proteins, gibberellins, ethylene, and ABA (Arrigoni and De Tullio, 2000
). In addition, ASC is known to operate as an antioxidant either by direct chemical interaction with reactive oxygen species, or in the reaction catalysed by ASC peroxidase in chloroplasts and other cell compartments (Shigeoka et al., 2002). All these activities cause large ASC consumption, leading to the generation of its oxidized forms, ascorbate free radical (AFR) and dehydroascorbic acid (DHA) (Liso et al., 1984; Arrigoni, 1994).
Several lines of evidence point to a specific role of ASC in root development, organization, and growth (Reid, 1941; Arrigoni, 1994; Kerk and Feldman, 1995; De Tullio et al., 1999; Arrigoni and De Tullio, 2000). Liso et al. (1984) first reported that ASC is required for cell division. When the ASC content of actively proliferating cells of the root apex was experimentally lowered, cell cycle progression was blocked in G1. Conversely, when the ASC content was experimentally raised by supplying ASC or the physiological ASC precursor L-galactono--lactone (GalL), cell division was stimulated (Citterio et al., 1994; Arrigoni et al., 1997). The administration of ASC not only stimulated cell proliferation in the meristem proper, but also induced new DNA synthesis in 80% of quiescent centre (QC) cells which are known to undergo an exceptionally long G1 phase and, therefore, divide at very low rates (Liso et al., 1988; Innocenti et al., 1990).
Recently, Feldman and co-workers reported a peculiar situation of the ASC–glutathione system in maize QC cells, including high ASC oxidase activity, low ASC, a high content of the ASC oxidized form, DHA, and a general shift towards an oxidizing environment, apparently correlated with auxin transport (Jiang et al., 2003). In an attempt to understand the function of ASC in root development, ASC distribution in roots of Cucurbita maxima and the effects of experimentally-induced changes in root ASC content were investigated.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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Plant materials and growth conditions
Seeds of Cucurbita maxima L. were surface-sterilized with sodium hypochlorite, thoroughly washed with water and grown in moist vermiculite in a dark room at 25 °C. After 4 d of germination, seedlings with roots of 3–3.5 cm were selected, carefully washed, and suspended for 24 h over beakers containing distilled water (control), 1 mM DHA, 50 µM lycorine, or 3 mM GalL. The pH of all incubation solutions was set at pH 5.8.
Histochemical localization of ASC
ASC localization was analysed as reported by Chinoy (1984). The localization procedure is based on the fact that ASC is the only reducing agent capable of reacting with silver ions in the conditions used (acidic medium and low temperature). The tissue is placed in an acidified silver nitrate solution in the dark and the black deposits of metallic silver are considered as sites of ASC localization. After seedling incubation, performed as indicated above, root tips were washed with distilled water, excised, and placed in a 5% (w/v) solution of silver nitrate dissolved in bidistilled water with 66% (v/v) absolute alcohol and 5% (v/v) glacial acetic acid. Samples were stored in the dark at 4 °C for 5 d. The reaction was stopped by washing with an alcoholic ammonia solution (5 ml of ammonia added to 95 ml of 70% ethanol). After dehydration in a t-butyl alcohol series, samples were embedded in paraffin, sectioned at 8 µm, and stained with safranin and fast green.
ASC and DHA content
ASC and DHA content in root tips were measured according to Zhang and Kirkham (1996). After the incubation of seedlings in different media, root tips (3 mm) were ground in a chilled mortar with pestle with ice-cold 5% (w/v) metaphosphoric acid. The homogenate was centrifuged for 15 min at 25 000 g and the supernatant used for determinations.
Light microscopy
Root tip segments of C. maxima from both control plants and plants treated for 24 h with DHA were fixed in 6% glutaraldehyde and processed for light microscopy as previously described (Zanchin et al., 1993). Thin sections (1 µm thick) were cut with an ultramicrotome (Ultracut Reichert-Jung, Wien, Austria), stained with 1% toluidine blue and 1% tetraborate (1:1, v/v) and observed and photographed under a light microscope (Dialux 22, Leitz, Wetzlar, Germany).
For the immunolocalization of ASC oxidase in the root tip, specific antibodies raised against a synthetic peptide of 13 amino acids highly conserved in Cucurbita ASC oxidase (the kind gift of Professor Muneharu Esaka, University of Hiroshima, Japan) were used at a 1:3000 dilution. Alkaline phosphatase-conjugated secondary antibodies and NBT/PCIP were used for detection.
Confocal microscopy
Glutathione localization was observed basically according to Sanchez-Fernandez et al. (1997). Longitudinal median sections were hand-cut from excised roots, at approximately 500 µm, and immediately transferred to a drop of 100 µM monochlorobimane (MCB) on a coverslip. The sections were mounted in a slide-coverslip sandwich, separated by additional coverslips to assemble a chamber approximately 700 µm deep. The chamber was positioned on the stage of a Leica DMRE microscope and room temperature was maintained between 18 °C and 20 °C. Roots were imaged using a Leica TCS SP2 (Spectral Confocal and Multiphoton System) confocal scanning laser microscope. The glutathione-S-bimane (GSB) conjugate was excited at 458 nm by an Ar/He/Ne laser. The fluorescence emission for GSB was collected at 514 nm using a Leica 10x0.3 NA HC PL fluotar lens. Data collection was started 3–5 min after exposure to MCB as the time necessary to assemble the chamber. Serial optical sections were collected with a mechanical focus increment of 3±0.2 µm over 75 µm depth of the root. Each optical section was averaged over 2–4 frames and sampling repeated at 20–30 s intervals for 20–60 min. Under these imaging conditions no autoflorescence was detected and, after background subtraction, the signal was related entirely to GSB conjugate fluorescence. Image processing was performed with the software Leica LCS.
Electron microscopy
For transmission electron microscopy (TEM), pumpkin root tips were fixed in 2.5% (v/v) glutaraldehyde in 10 mM Na-phosphate buffer (pH 7.2) overnight at 4 °C. After washing in the same buffer, they were postfixed in 1% (w/v) osmium tetroxide in water for 1 h, washed three times with water, and dehydrated in an ethanol series [30, 50, 70, 90, 100% (v/v); 15 min each step] at room temperature. The samples were infiltrated in 2:1 (v/v) ethanol/LR White (Polysciences, Warrington, PE, USA) for 1 h, 1:2 (v/v) ethanol/LR White resin for 2 h, 100% LR White overnight at 4 °C, and embedded in LR White resin according to Moore et al. (1991). Semi-thin sections (1 µm) were stained with 1% (w/v) toluidine blue for morphological observations. Anti-pumpkin AAO antibodies were purchased from DPC Biermann (Bad Nauheim, Germany). Immunogold labelling (dilution 1:3000–1:4000) was performed on thin sections as described by Balestrini et al. (1996) and observed with a Philips CM10 transmission electron microscope. Labelling specificity was determined by replacing the primary antibody with the buffer.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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ASC distribution in root tissues
A hstochemical procedure proposed several years ago to detect ASC in plant tissues is based on the properties of the silver ion (Ag+) as an excellent electron acceptor and of ASC as an extraordinary electron donor. When Ag+ is reduced, metallic silver is formed that precipitates as dark granules, usually forming clusters easily detectable with the light microscope (Chayen, 1952
). In principle, not only ASC, but several other cellular reducing compounds can reduce Ag+. Therefore, observing the presence of metallic silver granules in a cell does not guarantee that they were specifically reduced by ASC, rather than other reductants. This subtle problem was addressed in a series of in vitro studies (Chayen, 1952; Chinoy, 1984) that lead to the conclusion that, at low temperature, the rate of Ag+ reduction by all electron donors was slowed down, so that at 4 °C only ASC, due to its very low activation energy, was able to reduce, although at slow rate, the Ag+ ion to metallic silver.
The reliability and specificity of the protocol used under these experimental conditions was checked by comparing biochemical ASC measurements with microscopy. For this purpose, changes in root ASC content were experimentally induced. ASC content was lowered by treatment with lycorine, an inhibitor of ASC biosynthesis (Arrigoni et al., 1975; Liso et al., 1984). Conversely, ASC content was raised by adding the physiological ASC precursor L-galactono--lactone (GalL) (Smirnoff et al., 2001; Arrigoni and De Tullio, 2002). Analyses were performed in root apices of Cucurbita seedlings incubated for 24 h in water (control), 3 mM GalL or 50 µM lycorine. ASC content in roots subjected to these treatments is reported in Table 1.
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The amount of ASC measured in root tips by biochemical methods was consistent with the observed amount of deposited granules in different samples. Figure 1A shows that silver granules were clearly visible in control root sections. In GalL-treated roots, ASC content increased from 850 nmol to 2500 nmol g–1 FW and the number of detectable silver granules significantly increased (Fig. 1B). Root treatment with 50 µM lycorine caused an 85% decrease in ASC content and a severe drop in the number of metallic silver granules, such that they were barely detectable (Fig. 1C). However, some staining is still detectable, particularly in the region of the plasma membrane in what appears to be the cortical cells. This is difficult to explain and deserves further investigation. It is tempting to speculate that membrane-associated ASC could, in some way, be stabilized. ASC content detected in lycorine-treated roots (120 nmol g–1 FW) is apparently the threshold value for ASC detection with the silver nitrate method.
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In control root tips, almost all of the cells contained detectable granules; however, in border cells, namely root cap cells, the number of granules was lower than in the cells of the inner tissues (Fig. 1A). The granules were also absent in a small and delimited zone corresponding to the QC (Fig. 2A), thus confirming data reported by Kerk and Feldman (1995)
, who showed that these cells have low or undetectable levels of ASC in maize roots. Interestingly, GalL treatment also caused a marked increase in granules in the QC that therefore became indistinguishable (Fig. 2B). It is well-known that GalL availability represents a limiting factor in the ASC biosynthetic pathway (Smirnoff et al., 2001; Arrigoni and De Tullio, 2002). These data suggest that low ASC content observed in QC cells (Kerk and Feldman, 1995; Jiang et al., 2003) is apparently due to impaired ASC biosynthesis, rather than ASC oxidase activity (see below).
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Subcellular ASC distribution
Preliminary observations on the intracellular distribution of silver granules (Fig. 3A, B) show that both meristematic and differentiating root cells are surrounded by regularly distributed granules, apparently located at the plasma membrane/cell wall interface. Since ASC content is quite low in the cell walls (apoplastic fluid) of roots (Cordoba-Pedregosa et al., 2003
), these data seem to suggest that ASC is essentially located at the level of the plasma membrane.
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Granules also occurred at the surface of the nuclear membrane, and evident clusters of silver granules were often observed correspondingly in nucleoli. The presence of silver granules at the nuclear membrane and clustered granules in the nucleoli seems to confirm earlier reports (Price, 1966
; Schopfer, 1966) suggesting that ASC could be involved in RNA synthesis by interacting with nuclear proteins. The presence of ASC at the nuclear membrane and in nucleoli also supports the existence of a correlation between ASC and cell proliferation, and is possibly a key point to understand why GalL treatment promotes cell division (Arrigoni et al., 1997; Paciolla et al., 2001). The granule deposition pattern was almost the same in cells undergoing differentiation, in which a developed vacuolar system is already present (Fig. 3B). No silver granules could be detected in vacuoles (Fig. 3B), and this suggests that ASC is not present in this compartment.
The absence of granules of metallic silver in the vacuole is an intriguing point. Previous literature (Rautenkranz et al., 1994) is often cited to claim that a large amount of ASC is present in the vacuole (Griesen et al., 2004). By contrast, Rautenkrantz et al. (1994) clearly state that ASC content in vacuoles is 60-fold lower than in the cytoplasm. In addition, the method used by Rautenkranz et al. (1994) does not discriminate between ASC and its oxidized form DHA (undetectable with the silver nitrate method), and therefore ASC content is likely to be even lower.
The data reported here confirm that ASC content in vacuoles is below the detection limit of the procedure used. Immunogold EM identified, in the vacuoles of C. maxima, the presence of a protein recognized by an antibody raised against an ASC oxidase of cucurbits (see below). This finding could also be related to the absence of ASC in the vacuole.
Concerning subcellular ASC distribution, it cannot be excluded that, during the long incubation step (see Materials and methods), ASC molecules could diffuse from their original location before reacting with the silver ions. If so, the localization of silver granules in the protoplasm would not reflect the actual ASC distribution in vivo. However, if ASC diffusion occurred, one would expect to find a uniform distribution of silver granules in all cell compartments, which is not the case. In addition, these observations fit with data on the ASC content in different cell compartments obtained by subcellular fractionation (Rautenkranz et al., 1994; Cordoba-Pedregosa et al., 2003). This suggests that the intracellular ASC distribution observed with the silver nitrate method is unlikely just to be artefactual.
Localization of ASC oxidase
Immunolocalization of ASC oxidase was performed using a specific polyclonal antibody. Previous data reported that this enzyme is specifically located in the extracellular matrix (Chichiriccò et al., 1989). Immunogold EM reveals that, in meristematic cells, ASC oxidase was apparently located in a specific domain of the extracellular matrix adjacent to the plasma membrane (Fig. 4C). Gold particles were also evident in vacuoles and with the Golgi (Fig. 4), probably due to the transit of this glycoprotein to the cell wall and vacuoles. Gold particles were never observed in the nucleus, mitochondria, plastids, and the cytosol. As previously mentioned, the presence of a putative ASC oxidase in vacuoles could be correlated with the absence of ASC in this cell compartment.
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It should be considered that ASC oxidase is a complex blue copper protein which uses molecular oxygen as its only electron acceptor, but, in spite of its ‘traditional’ name, the enzyme can oxidize many other reducing agents beside ASC (Marchesini et al., 1977
). Many phenolics that potentially can act as substrates of ASC oxidase are actually present and abundant in vacuoles (Grob and Matile, 1980). The putative ASC oxidase found in vacuoles, if active, could therefore oxidize some of these compounds, generating molecules of still unknown function.
Interstingly, ASC oxidase is specifically located in a small area of the root tip, corresponding to the QC (Fig. 5A). This confirms previous observation on maize QC (Kerk and Feldman, 1995; Jiang et al., 2003) and suggests that this peculiar location of the enzyme could be a general feature of plants. Accumulation of ASC oxidase in differentiating cells of the vascular cylinder was also observed, whereas no ASC oxidase protein seems to be present in lateral root primordia (Fig. 5B).
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Feldman and co-workers suggested that ASC oxidase could be involved in the mechanism controlling cell cycle progression in QC cells (Kerk and Feldman, 1995
; Jiang et al., 2003). This mechanism is apparently related to selective auxin accumulation in QC cells and the consequent establishment of an oxidizing environment in this area (Jiang et al., 2003). High ASC oxidase activity in the QC is therefore considered as a means to decrease ASC content, switching the ASC/DHA ratio towards the more oxidized form. However, this is not consistent with data reported in the same paper by Jiang et al. (2003), showing that both ASC content and ASC oxidase activity sharply increase in the QC of roots treated with the auxin transport inhibitor NPA. This clearly indicates that ASC oxidase activity is not related to the decrease in ASC content. Recently, it has been suggested that the function of ASC oxidase is likely to be related to oxygen management, rather than ASC consumption (Arrigoni et al., 2003; De Tullio et al., 2004). More generally, up- or down-regulation of ASC oxidase expression and activity could be involved in modulating oxygen availability in different cells and tissues. This could explain why actively dividing cells (proximal meristem, root primordia) have no detectable ASC oxidase, whereas cells with lower respiratory activity/slow rate of cell division (differentiating vascular cells, QC cells) have higher ASC oxidase protein/activity. This hypothesis requires further investigation.
Contrasting effects of DHA and GalL on the distribution of newly formed ASC and on root growth
Jiang et al. (2003) reported that altered auxin transport causes changes in the cellular redox state in the root meristem, suggesting that proper redox equilibrium in different root zones could be necessary for balanced organization and growth. It has also been demonstrated that both GalL and DHA increase ASC content in root tissues, but the former stimulates root growth, whereas the latter has an inhibitory effect on growth and affects the redox state of glutathione and thiol-containing proteins in roots of Lupinus albus and Allium cepa (Paciolla et al., 2001). To understand if the contrasting effect of these compounds could be related to differences in ASC distribution, the localization of newly formed ASC in C. maxima roots in response to treatment with GalL and DHA was compared.
In DHA-treated root tips, ASC content increased from 850 nmol to 2700 nmol g–1 FW (Table 1), a value comparable to the increase induced by GalL.
As expected, the number of silver granules in roots treated with DHA was higher than in controls (Figs 1A, 6), and, although similar to that of GalL-treated roots (Fig. 1B), a clear difference in the distribution of newly formed granules was observed between the two treatments. In DHA-treated roots, increased metallic silver deposition was observed mainly in the cells of peripheral tissues (Fig. 6), apparently in connection with glutathione depletion (see below), whereas in GalL-treated root tips, granules were evenly distributed with the exception of border cells, where the amount of silver granules remained similar to that of controls (cf. Fig. 1B).
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Irregular ASC distribution occurring in DHA-treated roots seems to account for their altered growth and development. Both root length and the number of lateral roots were strongly inhibited in DHA-treated roots (Fig. 7). Sections of roots treated with 1 mM DHA for 24 h showed that the size of the root tip and the organization of the apical meristem were markedly affected by the treatment. The root tip was significantly narrower, and the dermatocalyptrogen, the distal meristem giving origin to the rhizoderm and the root cap, was not recognizable in these roots (Fig. 8).
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By analogy with data previously reported for A. cepa and L. albus roots (Paciolla et al., 2001
), root growth was also stimulated by GalL treatment in C. maxima (not shown). ASC localization in GalL-treated roots seems to reflect a general enrichment in ASC content, without altering its pattern of distribution. This indicates active GalL conversion to ASC in inner tissues, and lower conversion in border cells. On the basis of these data, it is possible to hypothesize that GalL dehydrogenase is largely present in dividing cells and during early differentiation stages, and that in these cells GalL availability is a limiting factor for GalL dehydrogenase activity and, consequently, for ASC biosynthesis (Smirnoff et al., 2001; Arrigoni and De Tullio, 2002). Conversely, the presence of a lower proportion of silver granules in root border cells of GalL-treated samples seems to suggest that GalL dehydrogenase could be less active in these cells. Considering that root cap cells are short-lived and undergo programmed cell death, it is conceivable to hypothesize that lower GalL dehydrogenase activity could also have a role in determining their fate.
Glutathione distribution in roots
Since GSH is involved in both enzymatic and non-enzymatic DHA reduction, GSH distribution in root tissues was investigated by GSH conjugation with monochlorobimane and confocal microscopy of the fluorescent adduct. The labelling pattern in Fig. 9 clearly shows that the number of fluorescent cells decreased in DHA-treated roots as compared with control roots. By contrast, the fluorescent signal progressively extended towards the apical meristem and the inner cortical cells in GalL-treated roots. As mentioned above, ASC formed by DHA reduction was largely localized in peripheral root cells (Fig. 6). Such localization of ASC in DHA-treated roots is apparently associated with a drop in GSH content, and this seems to suggest that GSH, which is known to be largely present in the root cap and cortical cells (Sanchez-Fernandez et al., 1997), is the preferential electron donor for DHA reduction in cortical root cells. Although the link between ASC, GSH, and cell proliferation is still obscure (Potters et al., 2002), it is conceivable to hypothesize that GSH depletion occurring in peripheral tissues could be involved in the inhibitory effect of DHA on C. maxima root growth.
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionsReferences |
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The data presented here show that the method proposed several years ago for the in vivo localization of ASC is very efficient and a general picture of tissue and cellular distribution of this important metabolite is achieved using the light microscope. Although this method has been used in the past to detect ASC in both animal and plant cells (Chayen, 1952
; Chinoy, 1984; Kerk and Feldman, 1995), its use has been considered with caution, in spite of many control experiments done to test its reliability. ASC is not present (or is below the threshold of detectability) in vacuoles, where a protein immunologically related to AAO is observed. It is not clear whether the putative vacuolar AAO can be responsible of the absence of ASC in this compartment.
The observation that two treatments (GalL and DHA) both increasing ASC content, but inducing opposite effects on root growth, result in a different localization of ASC in root tissues, not only confirms the reliability of the method but also suggests that the content and distribution of ASC are crucial to ensure proper root development, in connection with GSH distribution.
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Acknowledgements |
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We thank Miss Fernanda Piccarreta for her skilful assistance in the preparation of samples for histochemical analysis of ASC localization. We also thank Professor Muneharu Esaka (Hiroshima University) for kindly providing antibodies.
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References |
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