JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2527-2538; doi:10.1093/jxb/eri246
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Methyl jasmonate stimulates the de novo biosynthesis of vitamin C in plant cell suspensions
1Pasteur Institute of Brussels, Engeland Street 642, B-1180 Brussels, Belgium
2Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, Technologiepark 927, 9052 Gent, Belgium
* To whom correspondence should be addressed in Brussels. Fax: +32 2 373 32 91. E-mail: bwolucka{at}pasteur.be
Received 31 January 2005; Accepted 9 June 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Vitamin C (L-ascorbic acid) is an important primary metabolite of plants that functions as an antioxidant, an enzyme cofactor, and a cell-signalling modulator in a wide array of crucial physiological processes, including biosynthesis of the cell wall, secondary metabolites and phytohormones, stress resistance, photoprotection, cell division, and growth. Plants synthesize ascorbic acid via de novo and salvage pathways, but the regulation of its biosynthesis and the mechanisms behind ascorbate homeostasis are largely unknown. Jasmonic acid and its methyl ester (jasmonates) mediate plant responses to many biotic and abiotic stresses by triggering a transcriptional reprogramming that allows cells to cope with pathogens and stress. By using 14C-mannose radiolabelling combined with HPLC and transcript profiling analysis, it is shown that methyl jasmonate treatment increases the de novo synthesis of ascorbic acid in Arabidopsis and tobacco Bright Yellow-2 (BY-2) suspension cells. In BY-2 cells, this stimulation coincides with enhanced transcription of at least two late methyl jasmonate-responsive genes encoding enzymes for vitamin C biosynthesis: the GDP-mannose 3'',5''-epimerase and a putative L-gulono-1,4-lactone dehydrogenase/oxidase. As far as is known, this is the first report of a hormonal regulation of vitamin C biosynthesis in plants. Finally, the role of ascorbic acid in jasmonate-regulated stress responses is reviewed.
Key words: Arabidopsis, ascorbic acid, BY-2 cells, hormone signalling, jasmonic acid
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Plants synthesize L-ascorbic acid (vitamin C) via de novo and salvage pathways (Valpuesta and Botella, 2004
). The de novo synthesis involves activated forms of sugars (sugar phosphates and sugar nucleotides) and requires the GDP-
-D-mannose substrate (Wheeler et al., 1998). Two distinct de novo routes for vitamin C biosynthesis in plants have been proposed: an L-galactose pathway (Wheeler et al., 1998) and an L-gulose pathway (Wolucka and Van Montagu, 2003) (Fig. 1). GDP-Man is formed from -D-mannose 1-phosphate and GTP, with a concomitant release of pyrophosphate PPi, in a reversible reaction catalysed by a GDP-Man pyrophosphorylase (mannose 1-phosphate guanylyltransferase; EC 2.7.7.13
[EC]
). The Arabidopsis genome contains at least three closely related genes encoding GDP-Man pyrophosphorylase (At2g39770, At3g55590, and At4g30570) (http://www.arabidopsis.org). Conklin and colleagues demonstrated that the At2g39770 gene product is involved in the biosynthesis of vitamin C by showing that ozone-sensitive vtc1 Arabidopsis mutants with a point mutation in the gene have reduced levels of both vitamin C and GDP-Man pyrophosphorylase activity; the authors concluded that the mutation results in an impaired supply of GDP-Man substrate for the synthesis of vitamin C (Conklin et al., 1999). However, studies on the cyt1 mutants bearing other point mutations in the same gene demonstrated severe, pleiotropic effects of such mutations (Lukowitz et al., 2001). The cyt1 mutants are deficient in cellulose, the biosynthesis of which does not require the GDP-Man substrate, and show impaired protein glycosylation and lower content of mannose and fucose in the cell walls. The At2g39770 gene product is, therefore, involved in fundamental processes of protein N-glycosylation, cell-wall formation, and the synthesis of other GDP-sugars (GDP-L-fucose and GDP-L-galactose). The exact role of the enzyme in vitamin C synthesis, its cellular localization, and biochemical characteristics are still unknown. In the same line of evidence, an antisense expression of a homologous GDP-Man pyrophosphorylase gene of Solanum tuberosum resulted in vitamin C deficiency, lower mannose content of cell-walls in leaves, and rapid senescence of potato plants (Keller et al., 1999).
|
The second step in the de novo synthesis of vitamin C, as originally proposed (Wheeler et al., 1998
), is carried out by the GDP-Man 3'',5''-epimerase (EC 5.1.3.18
[EC]
) (At5g28840) (Wolucka et al., 2001b) that catalyses a reversible conversion of GDP-D-mannose into GDP-L-galactose (Barber and Hebda, 1982). In the L-galactose pathway, the so-formed GDP-L-galactose would release L-galactose 1-phosphate by a still unknown enzymatic step. L-Galactose 1-phosphate could undergo a dephosphorylation due to the action of a recently identified phosphatase (At3g02870) that cleaves also myo-inositol 1-phosphate (Laing et al., 2004). Unfortunately, the activity of the enzyme towards other sugar 1-phosphates of L-series, such as L-gulose 1-phosphate (see below), was not tested by the authors. Free L-galactose is oxidized at the C1-position by a cytosolic L-galactose dehydrogenase (At4g33670) (Gatzek et al., 2002) to L-galactono-1,4-lactone. The latter is oxidized to L-ascorbic acid by the highly specific, mitochondrial inner membrane-associated L-galactono-1,4-lactone dehydrogenase (At3g47930) (Imai et al., 1998; Ostergaard et al., 1997). The two last enzymes, L-galactose and L-galactono-1,4-lactone dehydrogenases, are constitutively expressed, non-limiting, and, apparently, do not undergo any fine regulation (Gatzek et al., 2002; Pateraki et al., 2004), as shown by the observation that feeding with exogenous L-galactose results in a large increase in the ascorbic acid pool (Wheeler et al., 1998). Possibly the two dehydrogenases have catabolic functions in the salvage pathways for recycling and removal of potentially toxic cell wall-derived free aldoses: L-galactose and also, in the case of L-galactono-1,4-lactone dehydrogenase, D-galacturonic acid after its former reduction to L-galactono-1,4-lactone by a recently identified aldo-keto reductase (Agius et al., 2003).
Biochemical studies on the cytosolic GDP-Man 3'',5''-epimerase of A. thaliana led to the discovery of an unsuspected and novel 5''-epimerase activity responsible for the synthesis of GDP-L-gulose (Wolucka and Van Montagu, 2003). In contrast to GDP-L-galactose which serves as a donor of L-galactosyl residues for the biosynthesis of polysaccharides and glycoproteins, the presence of GDP-L-gulose is puzzling because L-gulose-containing glycoconjugates have never been found in higher plants. It was proposed that GDP-L-gulose serves as a substrate for vitamin C synthesis (the L-gulose pathway) (Wolucka and Van Montagu, 2003). L-gulose freed from GDP-L-gulose undergoes an oxidation to L-gulono-1,4-lactone by the action of the L-galactose dehydrogenase or a similar enzyme. L-gulono-1,4-lactone is not a substrate for the L-galactono-1,4-lactone dehydrogenase, and must be converted to L-ascorbic acid by one of the L-gulono-1,4-lactone dehydrogenase isozymes that are detectable in the cytosol and mitochondria (Wolucka and Van Montagu, 2003). The Arabidopsis genome contains several genes that are homologous to the rat L-gulono-1,4-lactone oxidase (At1g32300, At2g46740, At2g46750, At2g46760, At5g11540, At5g56470, At5g56490) but the gene products have not been characterized yet. Consistent with the key role of L-gulono-1,4-lactone in vitamin C synthesis, transgenic plants expressing the rat L-gulono-1,4-lactone oxidase (Gulox) gene had increased levels of vitamin C (Jain and Nessler, 2000). Furthermore, expression of the rat gene in the vtc1 Arabidopsis mutant resulted in a reversion of ascorbate content to the wild-type level (Radzio et al., 2003). These facts suggest that the reaction catalysed by an L-gulono-1,4-lactone dehydrogenase/oxidase isozyme could be a rate-limiting step in the biosynthesis of L-ascorbic acid in plants.
Two salvage pathways for vitamin C synthesis involve derivatives of uronic acids, namely D-galacturonic and D-glucuronic acids. Exogenously supplied uronic acids are poor substrates for vitamin C synthesis probably because of the lack of an efficient transport system for these intracellular intermediates. A genetic approach led to the identification of an NADPH-dependent D-galacturonic acid reductase in strawberry fruits that specifically reduces the C1 aldehyde group of D-galacturonic acid and forms L-galactonic acid or L-galactono-1,4-lactone (Agius et al., 2003). The latter serves as a substrate for the mitochondrial L-galactono-1,4-lactone dehydrogenase or another enzyme. Transgenic A. thaliana plants expressing the reductase gene were reported to have an increased vitamin C content (Agius et al., 2003). A similar reductase activity that converts D-glucuronic acid or D-glucurono-3,6-lactone to L-gulono-1,4-lactone, is thought to exist in plants. Consistent with this proposal, overexpression of the enzyme that forms D-glucuronic acid by oxidizing myo-inositol, a myo-inositol oxygenase, resulted in higher vitamin C levels in transgenic plants (Lorence et al., 2004). L-gulono-1,4-lactone is then converted to L-ascorbic acid by an L-gulono-1,4-lactone dehydrogenase/oxidase isozyme (Wolucka and Van Montagu, 2003). Thus, the salvage pathway for D-glucuronate in plants resembles the last steps of vitamin C synthesis in animals (Nishikimi and Yagi, 1996).
The regulation of ascorbate synthesis in plants is largely unknown. GDP-Man 3'',5''-epimerase may interact with a Hsc70.3 heat-shock protein and undergo a complex regulation that involves redox control and inhibition by GDP-L-fucose and by GDP (Wolucka and Van Montagu, 2003). The epimerase, therefore, could play an important role in ascorbate homeostasis in stress conditions. Ascorbic acid content of plants was observed to increase, at least transiently, in response to stress such as tobacco mosaic virus (Milo and Santilli, 1967), nematode (Arrigoni et al., 1979), and nitrogen-fixating symbiotic bacteria (Dalton et al., 1998) infections, high light (Mishra et al., 1993), chilling (Schoner and Krause, 1990), water submersion (Ushimaru et al., 1992), and exposure to SO2 and ozone (Mehlhorn et al., 1986). However, the mechanism behind stress-induced accumulation of ascorbic acid is unclear.
In plants, responses to many biotic (pathogen and pest attacks) and abiotic (mechanical wounding, heat, cold, drought, ultraviolet B, ozone, chemicals etc.) stresses are, at least in part, mediated by jasmonates that have also important roles in plant development and senescence (Creelman and Mullet, 1997; Farmer and Ryan, 1990; Liechti and Farmer, 2002). Jasmonate signalling results in a transcriptional reprogramming that allows cells to defer pathogens and respond to stress (Reinbothe et al., 1994; Turner et al., 2002). Jasmonic acid and its methyl ester (methyl jasmonate, MEJA) induce the production of a wide array of direct and indirect chemical defences such as pathogenesis-related and cellular protection molecules, including proteins involved in detoxification and redox balance, proteinase inhibitors, antimicrobial secondary metabolites, antioxidants, and toxins. However, only scarce data are available on the direct effect of jasmonates on the ascorbic acid content of plants. MEJA treatment prevented ascorbate loss in water-stressed strawberry leaves (Wang, 1999), and resulted in a slight accumulation of the ascorbic acid pool in A. thaliana leaves (Maksymiec and Krupa, 2002).
It is shown here that treatment with methyl jasmonate stimulates the de novo biosynthesis of L-ascorbic acid in N. tabacum and A. thaliana suspension cells. On the basis of transcript profiling data, it is proposed that, in tobacco BY-2 cells, this stimulation is mediated, at least in part, by enhanced transcription of MEJA-responsive genes encoding key enzymes of vitamin C synthesis.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Reagents
D-[U-14C]Mannose (specific activity 286 mCi mmol–1) was purchased from Amersham Pharmacia Biotech (Little Chalfont, Buckinghamshire, UK). Methyl jasmonate (MEJA) was purchased from Duchefa (Haarlem, The Netherlands). Murashige and Skoog basal salts with minimal organics medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and 1-naphthalene acetic acid (NAA) were purchased from Sigma-Aldrich (St Louis, MO). All reagents were of analytical grade.
Cells
Arabidopsis thaliana (L.) Heynh. ecotype Columbia cell suspensions were grown in the presence of exogenous auxin (NAA) and cytokinin (kinetin), as described (Wolucka et al., 2001a). Nicotiana tabacum L. cv. Bright Yellow-2 (BY-2) cell suspensions were grown in the dark at 26 °C on a rotary shaker (120 rpm) in Murashige and Skoog basal salts with minimal organics medium supplemented with 3% sucrose, 1.5 mM KH2PO4, 0.9 µM 2,4-D (exogenous auxin), and pH adjusted to 5.8 (Nagata et al., 1992). BY-2 cell suspensions were 100-fold diluted into fresh medium, every 7 d.
Methyl jasmonate treatment
An aliquot of a freshly prepared 100 mM solution of MEJA in DMSO at a final concentration of 50 µM, or of DMSO alone (as a control) were added to 3-d-old A. thaliana cell suspensions. After 21 h treatment, cells were labelled with D-[U-14C]mannose, as described below. Otherwise, 6-d-old BY-2 and Arabidopsis cell suspensions were diluted into appropriate fresh media with no exogenous phytohormones, and incubated for 12 h. MEJA, at a final concentration of 50 µM, or an equivalent volume of the DMSO solvent (as a control) were added, and 2 ml-samples (in duplicates) were withdrawn at times 0, 7, 24, 31, and 48 h, for the determination of the L-AA content of cells.
In vivo labelling of A. thaliana cell suspensions with D-[U-14C]Man
In vivo labelling of A. thaliana cells was performed as described by Wolucka et al. (2001a). 3-d-old cell suspensions (2 ml aliquots, in duplicates) were labelled with 1 µCi of D-[U-14C]Man for 2 h. For the determination of [14C]Man uptake, the radioactivity of the culture medium was measured at times 0 and 2 h. Cells were collected by centrifugation and L-AA was extracted with 5% metaphosphoric acid containing 2 mM DTT and 1 mM EDTA.
Ascorbic acid determination
Total L-AA (L-ascorbic and dehydro-L-ascorbic acids) was measured by the HPLC method as described by Wolucka et al. (2001a), except that the concentration of methanol in solvent A was 0.5% and the flow rate was 0.8 ml min–1.
RT-PCR analysis
RNA extraction, cDNA synthesis, and RT-PCR were performed as described (Goossens et al., 2003). The following primer pair amplifying a part of the open reading frame was designed for the GDP-Man 3'',5''-epimerase (At5g28840): 5'-CATCTCACATTGCTCGTCGT-3' and 5'-CGGAGTGAGCCTAGCTGAAC-3'. The gene encoding actin-2 (At3g18780) was used as a control, with primers 5'-GTTGCACCACCTGAAAGGAAG-3' and 5'-CAATGGGACTAAAACGCAAAA-3'. 18-amplification cycle PCR-products were visualized by ethidium bromide staining, whereas 16-amplification cycle PCR-products were visualized by chemiluminescence detection using the Gene ImagesTM detection kit (Amersham Biosciences) and purified PCR-products as probes.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Stimulation of vitamin C biosynthesis in Arabidopsis cells
It was observed that kanamycin-resistant transgenic Arabidopsis plants, expressing different, unrelated transgenes, when grown on selective media with kanamycin, contain about two times more L-ascorbic acid than the corresponding wild-type plants grown on a medium without the antibiotic (data not shown). Jasmonic acid and its volatile methyl ester are known to be involved in signal transduction in response to chemical agents such as antibiotics, and to other stresses, including wounding, pathogen and herbivore attacks, drought, and osmotic shock. Therefore, it was tested if methyl jasmonate treatment could affect the level of L-AA in Arabidopsis cell suspensions. Green, actively growing Arabidopsis cells in a medium containing exogenously added growth hormones (NAA and kinetin) were treated with 50 µM MEJA for 21 h, and the total pool of ascorbic acid in the cells was determined. As shown in Fig. 2A, the treatment resulted in a significant increase (164% of the control) of the L-AA level in MEJA-treated cells (1141 nmol g–1 FW) compared to the control (696 nmol g–1 FW). In order to determine if the observed increase of L-AA was a result of its de novo biosynthesis, the MEJA-treated and control cell suspensions were incubated with exogenous D-[U-14C]Man and, after 2 h labelling, the incorporation of the 14C-label into L-AA was determined. The amount of the radioactivity measured in L-AA extracted from MEJA-treated cells was 2 times higher (197% of the control) than that found in the control cells (Fig. 2B). About 20% of the observed rise in 14C-AA was due to a more efficient membrane transport of [14C]Man in MEJA-treated cells (119% of the control) (Table 1), possibly because of an induction of a D-Man-specific permease. Thus, the observed increase in 14C-labelled (Fig. 2B; Table 1) and cold (Fig. 2A) L-AA levels was due mainly to a MEJA-mediated induction of the de novo synthesis of L-AA.
|
|
It is worth noting that in the absence of exogenous growth regulators (auxin and cytokinin), control Arabidopsis cell suspensions were unable to grow (Fig. 3) and finally died after 4 d of incubation. The L-AA content of these cells was decreasing rapidly (Fig. 3A), and addition of exogenous MEJA did not result in any increase of L-AA level (Fig. 3B). Apparently, in Arabidopsis suspension cells, exogenous phytohormones (auxin and/or cytokinin) are necessary to support both growth and methyl jasmonate-elicited L-AA synthesis.
|
Effects of MEJA elicitation on the vitamin C content of tobacco BY-2 suspension cells
Non-photosynthetic, heterotrophic Nicotiana tabacum Bright Yellow-2 cell suspensions represent a model of choice for studying the elicitation of secondary metabolism by jasmonates (Goossens et al., 2003
). The induction of certain secondary metabolites, for example, nicotine, occurs in these cells only in the absence of exogenous auxins. The L-AA content of BY-2 cells was determined during methyl jasmonate treatment in the absence of exogenous growth regulators. Unlike A. thaliana cell suspensions, BY-2 cells were able to gain biomass in the absence of exogenous auxins, at least during the time-course of the experiment (Fig. 4A). The L-AA content of the control cells showed a slow decrease during the first 30 h, and then started to rise, parallelling the growth of the cell culture (Fig. 4A). By contrast, BY-2 cell cultures treated with MEJA did not grow (Fig. 4B) and died after about 72 h of incubation (data not shown). Methyl jasmonate treatment triggered dramatic changes in the L-AA level (Fig. 4B). Within the first 7 h of the treatment, a rapid drop in the L-AA level was observed from 287 nmol g–1 FW to 147 nmol g–1 FW, followed by a significant but transient increase to 185 nmol of L-AA g–1 FW after 24 h. Further incubation resulted in a continuous decrease of the L-AA content of cells (Fig. 4B), and after 72 h of the MEJA treatment L-AA could not be detected (data not shown). The rapid consumption of L-AA during the first 7 h of the MEJA treatment was apparently counterbalanced by an induction of L-AA synthesis, thus resulting in a transient increase of the L-AA level 24 h after the addition of the stress hormone (Fig. 4B). A similar pattern of ascorbate accumulation with a concomitant increase in cytosolic and mitochondrial ‘L-galactonolactone dehydrogenase’ activity in potato slices after wounding was reported by Ôba et al. (1994). Because the L-galactono-1,4-lactone dehydrogenase is confined to mitochondria, it is presumed that the increase of activity in wounded potato slices was due to the induction of L-gulono-1,4-lactone dehydrogenase isozymes that can also use L-galactono-1,4-lactone as a substrate (Wolucka and Van Montagu, 2003). In agreement, transcription of the mitochondrial L-galactono-1,4-lactone dehydrogenase gene was not enhanced by wounding or methyl jasmonate treatment (Pateraki et al., 2004). The de novo biosynthesis of L-AA in tobacco BY-2 cells could not be determined by in vivo labelling with [14C]Man because these cells are deficient in D-Man uptake, presumably due to the lack of an active D-mannose transporter.
|
This study's results suggest the existence of important but still not understood differences between A. thaliana and BY-2 cells regarding the role of exogenous auxins and cytokinins in the cell growth and in the cross-talk with methyl jasmonate-modulated pathways.
Transcriptional regulation of ascorbate biosynthesis in tobacco BY-2 and Arabidopsis cells
A search was made for further evidence for the induction of L-AA biosynthesis in MEJA-treated BY-2 cells by analysing the transcript profiling database of genes induced during a 24 h jasmonate treatment under identical experimental conditions as described in the present work (Goossens et al., 2003). Indeed, for two of the genes proposed to be involved in the de novo L-gulose pathway for vitamin C in plants (Wolucka and Van Montagu, 2003) (Fig. 1), a corresponding MEJA-inducible BY-2 gene tag(s) could be found: two homologues (tags C111 and C316) of a putative L-gulono-1,4-lactone oxidase/dehydrogenase (At2g46750) (Wolucka and Van Montagu, 2003), and the putative orthologue (tag T464) of the GDP-Man 3'',5''-epimerase (At5g28840) (Wolucka et al., 2001b). The above-mentioned BY-2 genes were transcriptionally up-regulated starting at 4 h following jasmonate treatment, and mRNA levels steadily increased afterwards (Fig. 5). A maximal transcript accumulation was observed at about 12 h after jasmonate elicitation, with respective inductions of 3.1-fold for GDP-Man 3'',5''-epimerase, and 2.6-fold and 54.1-fold for the two putative L-gulono-1,4-lactone oxidase/dehydrogenases (see supplementary data in Goossens et al., 2003). Preliminary data from this cDNA-AFLP analysis also suggest that gene tags corresponding to the Hsc70 heat-shock protein (At3g09440) that was proposed physically to interact with the GDP-Man 3'',5''-epimerase (At5g28840) (Wolucka and Van Montagu, 2003), and to a putative GDP-Man pyrophosphorylase, display a similar induction pattern (Fig. 5). The transcriptional activation of vitamin C biosynthetic genes in BY-2 cells (Fig. 5) corroborates this study's observation of a transient increase of the vitamin level in these cells after 24 h of MEJA treatment (Fig. 4B). The GDP-Man 3'',5''-epimerase and putative L-gulono-1,4-lactone dehydrogenase genes could, therefore, be considered as late jasmonate-responsive genes (Orozco-Cardenas and Ryan, 1999; Orozco-Cardenas et al., 2001), the induction of which results in increased de novo synthesis of vitamin C in jasmonate-elicited BY-2 cells. Induction of late MEJA-responsive genes is mediated by H2O2, and the gene products carry out protective and defensive tasks (Orozco-Cardenas and Ryan, 1999; Orozco-Cardenas et al., 2001), as in the case of the biosynthetic genes for vitamin C (Fig. 6). Accordingly, it is plausible that careful analysis of existing and future microarray and transcript profiling databases listing jasmonate-inducible genes might help unravel other, still unknown (Fig. 1) biosynthetic genes for vitamin C.
|
|
To investigate whether a similar subset of vitamin C-related genes is responsible for the increased de novo synthesis of L-AA in jasmonate-treated Arabidopsis cells, the Genevestigator microarray database (https://www.genevestigator.ethz.ch; Zimmermann et al., 2004
) was examined. Within this data set, a statistically significant (P <0.06) induction by MEJA was only observed for the GDP-Man pyrophosphorylase encoding gene (At2g39770). It should be noted, however, that these particular microarray experiments were conducted on Arabidopsis seedlings early (0–3 h) after the phytohormone addition, and that induction of vitamin C-related genes might occur later as observed in BY-2 cells (Fig. 5). Therefore, an RT-PCR analysis was performed for the GDP-Man 3'',5''-epimerase (At5g28840) of Arabidopsis suspension cells under the experimental conditions in which the de novo biosynthesis of vitamin C was enhanced (Fig. 2), i.e. in the presence of exogenous auxin and cytokinin. Transcript accumulation in Arabidopsis suspension cells was determined during the 24 h following the addition of MEJA. However, no substantial MEJA-mediated transcriptional induction of GDP-Man 3'',5''-epimerase could be observed (data not shown). These observations suggest that MEJA elicitation modulates the transcription of differential subsets of vitamin C-related genes in tobacco BY-2 versus Arabidopsis cells under the experimental conditions used. Another possibility is that, in Arabidopsis cells, the hormone might affect vitamin C gene expression post-transcriptionally. Further studies will be necessary to determine the molecular mechanism of methyl jasmonate-mediated stimulation of vitamin C synthesis in Arabidopsis cells.
Vitamin C and jasmonates
Treatment of plant cells with jasmonates induces many metabolic processes that require ascorbic acid. What are these processes? Vitamin C functions in plants as a major antioxidant and as a substrate or cofactor of many enzymes. As an antioxidant, ascorbate acts either as a direct scavenger of reactive oxygen species (ROS), including the superoxide radical anion (
), the hydroxyl radical (OH·), hydrogen peroxide (H2O2), and singlet oxygen, or indirectly, as a substrate for a highly specific ascorbate peroxidase (APX) in the detoxification of H2O2, according to the following reaction:
 |
) of the plant cell. APX expression is induced in response to many environmental stresses that result in the accumulation of ROS (Karpinski et al., 1999; Mittler, 2002). In particular, exposure to methyl jasmonate stimulated APX expression at the mRNA (Orvar et al., 1997), protein (Rakwal et al., 1999), and enzyme activity (Maksymiec and Krupa, 2002) levels. The direct product of ascorbate oxidation, the monodehydroascorbate radical, either reacts spontaneously with itself to disproportionate into ascorbate and dehydroascorbate, or undergoes a direct reduction to ascorbate. This reduction can be achieved either enzymatically by the action of NAD(P)H-dependent monodehydroascorbate reductase (MDAR) (Hossain and Asada, 1985; De Leonardis et al., 1995), or non-enzymatically with reduced ferredoxin (Miyake and Asada, 1994) or a b-type cytochrome (Navas et al., 1994), as an electron donor. Dehydroascorbate undergoes a rapid degradation unless converted back to ascorbate by a glutathione-dependent dehydroascorbate reductase (DHAR) (Shimaoka et al., 2003) and, possibly, by other proteins such as glutaredoxins and protein disulphide isomerases (Wells and Xu, 1994). Expression of key enzymes responsible for ascorbate regeneration, MDAR, and DHAR, is increased under stress conditions (Grantz et al., 1995; Mittova et al., 2003; Nishikawa et al., 2003) and upon methyl jasmonate treatment (Nishikawa et al., 2003). Preliminary cDNA-AFLP data suggest that a gene involved in ascorbate regeneration (MDAR) is also induced in MEJA-treated tobacco BY-2 cells (Fig. 5). Interestingly, the metabolism of glutathione, a compound that plays a crucial role in ascorbate regeneration by serving as a reductant for the dehydroascorbate reductase reaction, is also regulated by jasmonates (Xiang and Oliver, 1998). Jasmonic acid induced the transcription of genes for glutathione synthesis and recycling, but did not alter glutathione levels in unstressed plant cells. This observation points to a limited similarity in the regulation of ascorbate and glutathione homeostasis.
Hydrogen peroxide and other ROS are key intermediates in cellular signal transduction pathways whose function may be counterbalanced by antioxidants (Carcamo et al., 2004). H2O2 is a secondary messenger mediating responses to hormones, to biotic/abiotic environmental stresses, and to developmental cues (Neill et al., 2002). Importantly, it also acts as a secondary messenger for the induction of late defence genes in response to methyl jasmonate (Orozco-Cardenas and Ryan, 1999; Orozco-Cardenas et al., 2001). The transient increase of H2O2 levels would require a prompt generation of ROS-scavenging activity at the sites of H2O2 accumulation to neutralize its adverse effects. Therefore, ascorbic acid might be involved in the control of the strength and the propagation of the H2O2 signal between and inside cells, and of the final outcome of H2O2 signalling, i.e. cell death or tolerance. Modulation of the ascorbate pool (by increasing ascorbate levels or the ascorbate-to-dehydroascorbate ratio) may result in changes in hydrogen peroxide signalling and lead to undesired alterations in plants. For example, the H2O2-mediated, ABA-induced stomatal closure was impaired in dehydroascorbate reductase (DHAR)-overexpressing transgenic tobacco plants with an increased ascorbate-to-dehydroascorbate ratio, and resulted in enhanced transpiration and decreased drought tolerance (Chen and Gallie, 2004). Contradictory results, however, were obtained by others (Kwon et al., 2003), who reported that transgenic tobacco plants expressing human dehydroascorbate reductase showed an improved tolerance to oxidative, cold and salt stresses.
Ascorbate can serve as a cofactor of several plant-specific enzymes such as oxygenases, oxidases, de-epoxidases, and thioglucosidases. Most of the ascorbate cofactor-requiring enzymes belong to the class of non-haem ferrous- and 2-oxoglutarate-dependent dioxygenases (2-OGD), which catalyse a wide range of reactions such as hydroxylation, dehydrogenation, and epoxidation (Prescott and John, 1996). The 2-OG-dependent oxygenases are involved in important pathways leading to the biosynthesis of the cell-wall hydroxyproline-rich glycoproteins, defence-related secondary metabolites such as stress-induced phenylpropanoids and alkaloids, and also hormones. Plant prolyl 4-hydroxylases are 2-OGD enzymes requiring ascorbate and Fe2+ for the activity (Hieta and Myllyharju, 2002) that hydroxylates Pro residues post-translationally in the Golgi in a variety of hydroxyproline-rich glycoproteins (HGRPs) and polyprotein hormone precursors (Pearce et al., 2001; Pearce and Ryan, 2003). HGRPs represent a superfamily of cell-wall proteins, including extensins, repetitive proline-rich proteins, arabinogalactan proteins, some nodulins, and lectins, that play a key role in growth, development, defence, and cell signalling (Cassab, 1998). Inhibition of the peptidyl-prolyl hydroxylase activity resulted in profound changes in cell division and elongation, and also in an increased ascorbate content of onion roots, thus indicating that an important part of the ascorbate pool is devoted to hydroxyproline synthesis (De Tullio et al., 1999). The synthesis of HGRPs is induced by wounding and infection (Corbin et al., 1987; Wycoff et al., 1995). Moreover, ascorbic acid acts as a transcriptional activator of a hydroxyproline-rich glycoprotein gene in maize (Garcia-Muniz et al., 1998).
Polyproteins of the Solanaceae family are precursors of hydroxyproline-rich glycopeptide hormones that belong to the class of systemins and activate plant defence genes. The expression of hydroxyproline-rich polyprotein genes is induced by wounding, herbivory attack, and methyl jasmonate (Pearce and Ryan, 2003).
Flavonoids are a large group of stress-induced phenylpropanoids that possess an antioxidant activity and fulfil an important role in antimicrobial defence, protection against high light and oxidative stress, and signalling. The biosynthesis of flavonoids involves four closely related (Martens et al., 2003) ascorbate-dependent dioxygenases: anthocyanidin synthase (Nakajima et al., 2001), flavonol synthase (Holton et al., 1993), flavanone 3ß-hydroxylase (Britsch et al., 1992), and flavone synthase I (Britsch, 1990). Anthocyanin biosynthesis is induced by methyl jasmonate in light-grown plants (Franceschi and Grimes, 1991; Jung, 2004). Moreover, in the biosynthesis of an antifungal flavonoid sakuranetin, ascorbic acid was shown to strongly enhance the induction of the phytoalexin synthesis by jasmonate (Tamogami et al., 1997).
The biosynthesis of a tropane alkaloid scopolamine (Yun et al., 1992) involves an ascorbate-dependent 2-OGD, hyoscyamine 6ß-hydroxylase, that catalyses the hydroxylation and subsequent epoxidation of hyoscyamine to scopolamine (Hashimoto et al., 1993). A terpenoid indole alkaloid, vindoline, requires ascorbate for the hydroxylation step catalysed by desacetoxyvindoline 4-hydroxylase (De Carolis and De Luca, 1993). The latter enzyme (Hernandez-Dominguez et al., 2004; Vazquez-Flota and De Luca, 1998) and alkaloid biosynthesis in Catharanthus roseus (Aerts et al., 1994) are inducible by methyl jasmonate.
Ascorbate is a cofactor of 2-oxoacid-dependent dioxygenases responsible for the biosynthesis of gibberellins (Lange, 1997; Prescott and John, 1996), and a co-substrate in the last step of ethylene synthesis catalysed by the plant 1-aminocyclopropane-1-carboxylate oxidase (McGarvey and Christoffersen, 1992; Rocklin et al., 1999). The ascorbate-dependent ACC oxidase activity and the ethylene biosynthesis are stimulated by exogenous jasmonates during fruit ripening (Fan et al., 1998). Ascorbate is also required by dioxygenases responsible for the oxidative cleavage of carotenoids in the pathways leading to a phytohormone abscisic acid (Seo and Koshiba, 2002) and to other apocarotenoids of poorly defined function (Schwartz et al., 2001). Abscisic acid is involved in the adaptation of plants to environmental stresses. The key regulatory enzyme of the de novo biosynthesis of ABA is the ascorbate-dependent 9-cis-epoxycarotenoid dioxygenase localized in plastids (Qin and Zeevaart, 1999, 2002) that cleaves 9-cis-violaxanthin and 9'-cis-neoxanthin to xanthoxin, the precursor of abscisic acid. Moreover, in the xanthophyll cycle that play an important role in the protection of chloroplasts against photo-oxidative damage (Smirnoff, 2000), a violaxanthin derivative (all-trans form) is converted to zeaxanthin by the chloroplastic violaxanthin de-epoxidase that requires ascorbate as a reductant (Rockholm and Yamamoto, 1996). Low ascorbate levels correlated with decreased rates of violaxanthin de-epoxidation in a vitamin C-deficient mutant (vtc2) of Arabidopsis (Muller-Moule et al., 2002).
Interestingly, tocopherols (vitamin E), a family of lipophilic antioxidants that are abundant in chloroplastic membranes and function as scavengers of lipid peroxyradicals, were recently shown to accumulate in Arabidopsis plants after jasmonate treatment, apparently, due to the transcriptional activation of tyrosine aminotransferase, the first enzyme for tocopherol biosynthesis (Sandorf and Holländer-Czytko, 2002). Ascorbic acid is required not only for the regeneration of vitamin E (Navas et al., 1994) but, probably, also for its biosynthesis, as a cofactor of the hydroxyphenylpyruvate dioxygenase (Prescott and John, 1996). Thus, the biosynthesis of both interrelated vitamins (C and E) that are involved in oxidative stress responses, seems to be regulated by jasmonates.
Another enzyme that needs the ascorbate cofactor, in this case not as a reductant but to ensure base catalysis (Burmeister et al., 2000), is myrosinase of Brassicaceae, a defence-related thioglucosidase hydrolysing glucosinolates (Wittstock and Halkier, 2002).
As discussed above, the level of ROS and the expression of enzymes involved in ascorbate metabolism or requiring ascorbate as a substrate/cofactor, increase in response to stress and to jasmonate phytohormones. Moreover, jasmonate signalling itself is mediated by a ROS, H2O2 (Orozco-Cardenas et al., 2001), and must be controlled through an appropriate antioxidant response. All these processes would result in an enhanced consumption of ascorbate and, consequently, in a higher demand for its rapid synthesis and regeneration.
Consistent with the proposed role of methyl jasmonate in the regulation of vitamin C synthesis, jasmonates were reported to induce increased tolerance to ozone (Orvar et al., 1997), and also to protect tissues against ozone-induced propagation of cell-death (Tuominen et al., 2004). Furthermore, the jasmonic acid insensitive mutant jar1 is more sensitive to ozone and accumulates superoxide ions after ozone treatment (Overmyer et al., 2000). Recently, jasmonates were recognized as factors involved in the containment of the ROS-dependent propagation of lesions (Overmyer et al., 2003).
In summary, methyl jasmonate can enhance the transcription of genes involved in the de novo biosynthesis (this work) and regeneration (Nishikawa et al., 2003) of ascorbic acid. This results, in turn, in higher rates of the de novo synthesis of ascorbic acid and in an increased supply of its reduced form in order to sustain many secondary pathways and to preserve the redox status of plant cells.
|
Footnotes |
|---|
Abbreviations: AFLP, amplified fragment length polymorphism; L-AA, L-ascorbic acid; BY-2, Bright Yellow-2; 2,4-D, 2,4-dichlorophenoxyacetic acid; DMSO, dimethyl sulphoxide; MEJA, methyl ester of jasmonic acid; NAA, 1-naphthalene acetic acid.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Aerts RJ, Gisi D, Decarolis E, Deluca V, Baumann TW. 1994. Methyl jasmonate vapor increases the developmentally controlled synthesis of alkaloids in Catharanthus and Cinchona seedlings. The Plant Journal 5, 635–643.[CrossRef][ISI]
Agius F, Gonzalez-Lamothe R, Caballero JL, Munoz-Blanco J, Botella MA, Valpuesta V. 2003. Engineering increased vitamin C levels in plants by overexpression of a D-galacturonic acid reductase. Nature Biotechnology 21, 177–181.[CrossRef][ISI][Medline]
Arrigoni O, Zacheo G, Arrigoni-Liso R, Bleve-Zacheo T, Lamberti F. 1979. Relationship between ascorbic acid and resistance in tomato plants to Meloidogyne incognita. Phytopathology 69, 579–581.
Barber GA, Hebda PA. 1982. GDP-D-mannose: GDP-L-galactose epimerase from Chlorella pyrenoidosa. Methods in Enzymology 83, 522–525.[ISI][Medline]
Britsch L. 1990. Purification and characterization of flavone synthase I, a 2-oxoglutarate-dependent desaturase. Archives of Biochemistry and Biophysics 282, 152–160.[CrossRef][ISI][Medline]
Britsch L, Ruhnau-Brich B, Forkmann G. 1992. Molecular cloning, sequence analysis, and in vitro expression of flavanone 3 beta-hydroxylase from Petunia hybrida. Journal of Biological Chemistry 267, 5380–5387.
Burmeister WP, Cottaz S, Rollin P, Vasella A, Henrissat B. 2000. High resolution X-ray crystallography shows that ascorbate is a cofactor for myrosinase and substitutes for the function of the catalytic base. Journal of Biological Chemistry 275, 39385–39393.
Carcamo JM, Pedraza A, Borquez-Ojeda O, Zhang B, Sanchez R, Golde DW. 2004. Vitamin C is a kinase inhibitor: dehydroascorbic acid inhibits IkappaBalpha kinase beta. Molecular and Cell Biology 24, 6645–6652.
Cassab GI. 1998. Plant cell wall proteins. Annual Review of Plant Physiology and Plant Molecular Biology 49, 281–309.[CrossRef][ISI]
Chen Z, Gallie DR. 2004. The ascorbic acid redox state controls guard cell signalling and stomatal movement. The Plant Cell 16, 1143–1162.
Conklin PL, Norris SR, Wheeler GL, Williams EH, Smirnoff N, Last RL. 1999. Genetic evidence for the role of GDP-mannose in plant ascorbic acid (vitamin C) biosynthesis. Proceedings of the National Academy of Sciences, USA 96, 4198–4203.
Corbin DR, Sauer N, Lamb CJ. 1987. Differential regulation of a hydroxyproline-rich glycoprotein gene family in wounded and infected plants. Molecular and Cell Biology 7, 4337–4344.
Creelman RA, Mullet JE. 1997. Biosynthesis and action of jasmonates in plants. Annual Review of Plant Physiology and Plant Molecular Biology 48, 355–381.[CrossRef][ISI][Medline]
Dalton DA, Joyner SL, Becana M, Iturbe-Ormaetxe I, Chatfield JM. 1998. Antioxidant defences in the peripheral cell layers of legume root nodules. Plant Physiology 116, 37–43.
De Carolis E, De Luca V. 1993. Purification, characterization, and kinetic analysis of a 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis from Catharanthus roseus. Journal of Biological Chemistry 268, 5504–5511.
De Leonardis S, De Lorenzo G, Borraccino G, Dipierro S. 1995. A specific ascorbate free radical reductase isozyme participates in the regeneration of ascorbate for scavenging toxic oxygen species in potato tuber mitochondria. Plant Physiology 109, 847–851.[Abstract]
De Tullio MC, Paciolla C, Dalla Vecchia F, Rascio N, D'Emerico S, De Gara L, Liso R, Arrigoni O. 1999. Changes in onion root development induced by the inhibition of peptidyl-prolyl hydroxylase and influence of the ascorbate system on cell division and elongation. Planta 209, 424–34.[CrossRef][ISI][Medline]
Fan X, Mattheis JP, Fellman JK. 1998. A role for jasmonates in climacteric fruit ripening. Planta 204, 444–449.[CrossRef]
Farmer EE, Ryan CA. 1990. Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Proceedings of the National Academy of Sciences, USA 87, 7713–7716.
Franceschi VR, Grimes HD. 1991. Induction of soybean vegetative storage proteins and anthocyanins by low-level atmospheric methyl jasmonate. Proceedings of the National Academy of Sciences, USA 88, 6745–6749.
Garcia-Muniz N, Martinez-Izquierdo JA, Puigdomenech P. 1998. Induction of mRNA accumulation corresponding to a gene encoding a cell wall hydroxyproline-rich glycoprotein by fungal elicitors. Plant Molecular Biology 38, 623–632.[CrossRef][ISI][Medline]
Gatzek S, Wheeler GL, Smirnoff N. 2002. Antisense suppression of L-galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated L-galactose synthesis. The Plant Journal 30, 541–553.[CrossRef][ISI][Medline]
Goossens A, Häkkinen ST, Laakso I, et al. 2003. A functional genomics approach toward the understanding of secondary metabolism in plant cells. Proceedings of the National Academy of Sciences, USA 100, 8595–8600.
Grantz AA, Brummell DA, Bennett AB. 1995. Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiology 108, 411–418.[Abstract]
Hashimoto T, Matsuda J, Yamada Y. 1993. Two-step epoxidation of hyoscyamine to scopolamine is catalysed by bifunctional hyoscyamine 6 beta-hydroxylase. FEBS Letters 329, 35–39.[CrossRef][ISI][Medline]
Hernandez-Dominguez E, Campos-Tamayo F, Vazquez-Flota F. 2004. Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus. Biotechnology Letters 26, 671–674.[CrossRef][ISI][Medline]
Hieta R, Myllyharju J. 2002. Cloning and characterization of a low molecular weight prolyl 4-hydroxylase from Arabidopsis thaliana. Effective hydroxylation of proline-rich, collagen-like, and hypoxia-inducible transcription factor alpha-like peptides. Journal of Biological Chemistry 277, 23965–23971.
Holton TA, Brugliera F, Tanaka Y. 1993. Cloning and expression of flavonol synthase from Petunia hybrida. The Plant Journal 4, 1003–1010.[CrossRef][ISI][Medline]
Hossain MA, Asada K. 1985. Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme. Journal of Biological Chemistry 260, 12920–12926.
Imai T, Karita S, Shiratori G, Hattori M, Nunome T, Oba K, Hirai M. 1998. L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis. Plant Cell Physiology 39, 1350–1358.
Jain AK, Nessler CL. 2000. Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants. Molecular Breeding 6, 73–78.[CrossRef]
Jimenez A, Hernandez JA, Del Rio LA, Sevilla F. 1997. Evidence for the presence of the ascorbate-glutathione cycle in mitochondria and peroxisomes of pea leaves. Plant Physiology 114, 275–284.[Abstract]
Jung S. 2004. Effect of chlorophyll reduction in Arabidopsis thaliana by methyl jasmonate or norflurazon on antioxidant systems. Plant Physiology and Biochemistry 42, 225–231.[Medline]
Karpinski S, Reynolds H, Karpinska B, Wingsle G, Creissen G, Mullineaux P. 1999. Systemic signalling and acclimation in response to excess excitation energy in Arabidopsis. Science 284, 654–657.
Keller R, Renz FS, Kossmann J. 1999. Antisense inhibition of the GDP-mannose pyrophosphorylase reduces the ascorbate content in transgenic plants leading to developmental changes during senescence. The Plant Journal 19, 131–141.[CrossRef][ISI][Medline]
Kwon SY, Choi SM, Ahn YO, Lee HS, Lee HB, Park YM, Kwak SS. 2003. Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene. Journal of Plant Physiology 160, 347–353.[CrossRef][ISI][Medline]
Laing WA, Bulley S, Wright M, Cooney J, Jensen D, Barraclough D, MacRae E. 2004. A highly specific L-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis. Proceedings of the National Academy of Sciences, USA 101, 16976–16981.
Lange T. 1997. Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli. Proceedings of the National Academy of Sciences, USA 94, 6553–6558.
Liechti R, Farmer EE. 2002. The jasmonate pathway. Science 296, 1649–1650.
Lorence A, Chevone BI, Mendes P, Nessler CL. 2004. Myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis. Plant Physiology 134, 1200–1205.
Lukowitz W, Nickle TC, Meinke DW, Last RL, Conklin PL, Somerville CR. 2001. Arabidopsis cyt1 mutants are deficient in a mannose-1-phosphate guanylyltransferase and point to a requirement of N-linked glycosylation for cellulose biosynthesis. Proceedings of the National Academy of Sciences, USA 98, 2262–2267.
Maksymiec W, Krupa Z. 2002. The in vivo and in vitro influence of methyl jasmonate on oxidative processes in Arabidopsis thaliana leaves. Acta Physiologia Plantarum 24, 351–357.[ISI]
Martens S, Forkmann G, Britsch L, Wellmann F, Matern U, Lukacin R. 2003. Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley. FEBS Letters 544, 93–98.[CrossRef][ISI][Medline]
McGarvey DJ, Christoffersen RE. 1992. Characterization and kinetic parameters of ethylene-forming enzyme from avocado fruit. Journal of Biological Chemistry 267, 5964–5967.
Mehlhorn H, Seufert G, Schmidt A, Kunert KJ. 1986. Effect of SO2 and O3 on production of antioxidants in conifers. Plant Physiology 82, 336–338.
Milo Jr GE, Santilli V. 1967. Changes in the ascorbate concentration of Pinto bean leaves accompanying the formation of TMV-induced local lesions. Virology 31, 197–206.[CrossRef][ISI][Medline]
Mishra NP, Mishra RK, Singhal GS. 1993. Changes in the activities of antioxidant enzymes during exposure of intact wheat leaves to strong visible light at different temperatures in the presence of protein synthesis inhibitors. Plant Physiology 102, 903–910.[Abstract]
Mittler R. 2002. Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science 7, 405–410.[CrossRef][ISI][Medline]
Mittova V, Tal M, Volokita M, Guy M. 2003. Up-regulation of the leaf mitochondrial and peroxisomal antioxidative systems in response to salt-induced oxidative stress in the wild salt-tolerant tomato species Lycopersicon pennellii. Plant, Cell and Environment 26, 845–856.[CrossRef][Medline]
Miyake C, Asada K. 1994. Ferredoxin-dependent photoreduction of the monodehydroascorbate radical in spinach thylakoids. Plant Cell Physiology 35, 539–549.
Muller-Moule P, Conklin PL, Niyogi KK. 2002. Ascorbate deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant Physiology 128, 970–977.
Nagata T, Nemoto Y, Hasezawa S. 1992. Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. International Review of Cytology 132, 1–30.
Nakajima J, Tanaka Y, Yamazaki M, Saito K. 2001. Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. Journal of Biological Chemistry 276, 25797–25803.
Navas P, Villalba JM, Cordoba F. 1994. Ascorbate function at the plasma membrane. Biochimica et Biophysica Acta 1197, 1–13.[Medline]
Neill SJ, Desikan R, Clarke A, Hurst RD, Hancock JT. 2002. Hydrogen peroxide and nitric oxide as signalling molecules in plants. Journal of Experimental Botany 53, 1237–1247.