JXB Advance Access originally published online on December 6, 2004
Journal of Experimental Botany 2005 56(412):605-611; doi:10.1093/jxb/eri036
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RESEARCH PAPER |
A red beet (Beta vulgaris) UDP-glucosyltransferase gene induced by wounding, bacterial infiltration and oxidative stress
1Instituto de Biotecnología, UNAM, Apartado Postal 510-3, Cuernavaca 62250 Morelos, México
2Centro de Desarrollo de Productos Bióticos-IPN, PO Box No. 24, Yautepec 62731 Morelos, México
* To whom correspondence should be addressed. Fax: +52 777 3 17 23 88. E-mail: rocha{at}ibt.unam.mx
Received 10 July 2004; Accepted 22 September 2004
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Abstract |
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Mechanical wounding, infiltration with P. syringae or A. tumefaciens, and exposure to an H2O2-generating system (Glc/Glc oxidase) induce betacyanin synthesis in red beet (Beta vulgaris) leaves. These conditions also induced the expression of BvGT, a gene encoding a glucosyltransferase (GT) from Beta vulgaris. BvGT has a high similarity to Dorotheanthus bellidiformis betanidin-5 GT involved in betacyanin synthesis. Furthermore, the transient expression of a BvGT antisense construct resulted in the reduction of BvGT transcript accumulation and betanin synthesis, suggesting a role for this gene product in betacyanin glucosylation. In addition, the NADPH oxidase inhibitor, diphenylene iodonium (DPI), inhibited the accumulation of the BvGT transcript in response to infiltration with Agrobacterium tumefaciens. Hence, this result suggests that ROS produced by a plasma membrane NADPH oxidase may act as a signal to induce BvGT expression, necessary for betanin synthesis after wounding and bacterial infiltration.
Key words: Bacterial infiltration, betacyanin, Beta vulgaris, glucosyltransferase, red beet, wounding
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Wounding and pathogens induce transcription of genes encoding UDP-glucosyltransferases that glucosylate hormones and secondary metabolites (O'Donnell et al., 1998
; Roberts et al., 1999). This glucosylation reaction is one of the mechanisms that allows plants to maintain metabolic homeostasis. Secondary metabolites are often converted to their glyco-conjugates and then accumulated and compartmentalized in vacuoles or specialized plastids. Furthermore, glucosylation of low-molecular-weight molecules, like harmful metabolites or environmental compounds, allows the solubilization of these compounds in water for detoxification and modulation of their biological activity (Jones and Vogt, 2001). Oxidative stress and conditions that promote cell death induce the expression of glucosyltransferase genes and the production of transportable glucosides which function as reactive oxygen species (ROS) scavengers (Mazel and Levine, 2002). ROS, such as hydrogen peroxide (H2O2), superoxide (
) hydroxyl radicals (
), and singlet oxygen (1O2) act as early messengers in signalling cascades activated by diverse external stimuli (Lamb and Dixon, 1997). In plant–pathogen interactions, ROS may exacerbate tissue damage or signal the activation of defence responses. In the first case, ROS can limit the spread of pathogen infection by strengthening the plant cell wall or killing pathogens directly. Regarding the activation of defence responses, ROS are known to induce expression of pathogenesis-related (PR) protein genes, regulating accumulation of secondary metabolites, and genes encoding ROS detoxifying enzymes (Dat et al., 2000; Grant and Loake, 2000).
Among the ROS-scavenging pathways, the production of antioxidants such as ascorbic acid and glutathione may be essential to keep ROS levels low (Mittler, 2002). Secondary metabolites such as terpenoids and phenolic compounds, whose antioxidant properties have been evaluated in vitro, may also participate in the modulation of ROS levels (Grassmann et al., 2002; Mittler, 2002).
In red beet (Beta vulgaris), betacyanins (red–violet pigments) constitute a class of secondary metabolites where the main betacyanin, betanin, is found in a high concentration in the store root (0.5 g of betanin kg–1) (Strack et al., 2003). Betacynin biosynthesis (Fig. 1) starts by the oxidation of tyrosine to dopa by a tyrosine hydroxylase (1) (Yamamoto et al., 2001). Dopa is then converted to dopaquinone in a reaction carried out by a tyrosinase or a polyphenol oxidase (2) (Steiner et al., 1999, Yamamoto et al., 2001). Recently, a gene for a 4,5-extradiol dioxygenase (4) involved in betalamic acid synthesis was described (Laurent et al., 2004). Glucosylation is known to be a key reaction in the formation of the red–violet pigment betanin, but the precise mechanism involved, however, remains to be elucidated. Betanidin and cyclo-dopa have been proposed as possible targets for sugar attachment in the formation of betanin. In vitro glucosylation of betanidin by a betanidin 5-O-glucosyltransferase (betanidin 5-GT, 7), present in cell suspension cultures of Dorotheanthus bellidiformis, takes place after the condensation of cyclo-dopa with betalamic acid (Heuer et al., 1996). A cDNA, corresponding to the betanidin 5-GT, has been cloned (Vogt et al., 1999). On the other hand, the glucosylation of cyclo-dopa (8) is also controversial. This possibility was first suggested by the detection of cyclo-dopa glucoside in young B. vulgaris plants (Wyler et al., 1984). However, it was not possible to detect an enzyme or a recombinant gene product capable of catalysing cyclo-dopa glucosylation and only traces of this compound, presumably generated by betanin hydrolysis were found in young red beet plants (Strack et al., 2003). Recently, the glucosylation of cyclo-dopa was observed in crude extracts of Miriabilis jalapa L. and five other plants producing betacyanin (Sasaki et al., 2004).
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Previously, it was found that betacyanins are induced by wounding, bacterial infiltration, and a H2O2 generating system (Glc/GlcO) and a role for betacyanins as ROS scavengers was suggested (Sepúlveda-Jiménez et al., 2004
). In this report, data are provided that suggest the participation of a GT from B. vulgaris (BvGT), in betalain synthesis in response to wounding, infiltration with P. syringae or A. tumefaciens, and exposure to H2O2. These data also suggest that ROS act as a signal to induce BvGT expression, necessary for the betanin synthesis during wounding or bacterial infiltration.
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Materials and methods |
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Plant material and bacteria strains
Seeds of red beet (B. vulgaris. var. ‘Crosby Egyptian’) were germinated in the dark between wet paper sheets at 25 °C, and after 8 d, seedlings were transferred to pots with sterile Metromix:Peatmoss substrate. Seedlings were grown in a greenhouse at 25 °C under natural daylight conditions and the first two true leaves of 6-week-old plants were used in the experiments described. Pseudomonas syringae pv. tabaci was grown overnight at 28 °C in King's B liquid medium (King et al., 1954
). Agrobacterium tumefaciens was grown for 18 h at 28 °C in Luria B liquid medium supplemented with antibiotics (50 µg ml–1 rifampicin and 100 µg ml–1 kanamycin). For inoculations, the concentration of P. syringae and A. tumefaciens was adjusted to 1.0x107 cfu cm–3 in 10 mM MgSO4 solution.
Wounding treatment and leaf infiltration with bacteria or Glc/GlcO system
Mechanical wounding was done by rubbing once with sandpaper on the axial side of red beet leaves. Leaves were infiltrated with bacteria or MgSO4 using a 1.0 ml syringe without a needle. Wounded or infiltrated areas (control or treatments) were identified immediately after treatment, delineating the limits of wounding or liquid spread with a felt-tip marker. At 12, 24, 48, 72, 96, and 120 h after treatment 0.5 cm diameter leaf discs were collected from the marked areas.
The H2O2 generating system, Glc/GlcO (Sigma, Aldrich Chemicals, St Louis), was prepared and used immediately for plant treatment. GlcO (20, 50, 100 U ml–1) was added to 10 mM Glc in 20 mM sodium phosphate buffer (pH 6.5) and infiltrated with a 1.0 ml syringe without a needle. For NADPH oxidase inhibition, 5 or 10 µM of diphenyl-iodonium (DPI) (Sigma, Aldrich Chemicals, St Louis) was co-infiltrated with the bacterial suspension.
Determination of betanin
Plant material was extracted with 80% methanol, and pigments were analysed by reverse phase, high-performance liquid chromatography (HPLC), using the solvent system described by Heuer et al. (1996). A HPLC Waters modular system (Waters, Milford, MA, USA) equipped with a 5 µm Nucleosil C18 column (250x4 mm, id, Macherey-Nagel, Düren, Germany), a system controller 600E, an injector U6K, and a UV-visible variable wavelength detector 486 were used along with a chromatography data management system (Maxima 800). Betanin was detected at 536 nm and the concentration of betanin content was estimated using the molar extinction coefficient 65x106 cm2 mol–1. Betanin was purified from beet root and used as a standard (Schwartz and von Elbe, 1980; Pourrat et al., 1988).
Amplification by PCR of BvGT
Genomic DNA was extracted from 3 g of red beet leaves as described by Saghai-Maroof et al. (1984) and used to isolate a fragment of a glucosyltransferase by PCR with the forward primer: 5'-CCHGAYATGTTYYTNCCHTGG-3' and the reverse primer 5'-RTAWGAWGAWCCWCCYTCYTC-3'. These primers were designed based on amino acid consensus sequence from a multiple alignment of glucosyltransferases of Dorotheanthus bellidiformis (accession number Y18871), Nicotiana tabacum (accession number U32643), and Lycopersicum esculentum (accession number X85138). The alignment was constructed with the PileUp program (Wisconsin Package, Version 10.2, Genetics Computer Group, Madison, WI). PCR reaction was carried out in 50 µl PCR reaction mixtures with 0.2 mM dNTPs, 0.2 U Taq polymerase (Gibco-BRL, Life Technologies, Rockville, MD), 0.2 µM of each primer, and 2 µg of genomic DNA. PCR conditions were as follows: 3 min initial heating at 94 °C, followed by 35 three-step cycles of 1 min denaturation at 94 °C, 30 s annealing at 61 °C, and 20 s elongation at 68 °C, followed by a final 7 min elongation step at 72 °C in a GeneAmp PCR system 9700 (Perkin Elmer Life Sciences, Boston). The expected size DNA fragment of 1.1 kb was recovered from an agarose gel using the GFX PCR purification kit (Amersham, Biosciences AB, Uppsala), and cloned into the pMos blue vector (Amersham, Biosciences AB, Uppsala).
Sequencing and analysis of DNA
Sequencing of the genomic BvGT clone was carried out using a thermo-sequenase radiolabelled terminator cycle sequencing kit (Amersham Pharmacia Biotech, USA), following the instructions of the manufacturer with fluorescence dideoxynucleotides, and using a 377-18 DNA sequencer (PE Applied Biosystems, USA). The protein alignment was constructed with the ClustalW (http://www.ebi.ac.uk/clustalw/) and MacBoxshade (Version 2.01) programs.
Transient expression assay of an antisense construct of BvGT
A 1036 bp BvGT fragment digested with SalI and KpnI restriction enzymes was inserted between the same sites of pBin 3X35S-NOS vector in the antisense orientation. This construct (AsBvGT) or only the vector was introduced into A. tumefaciens and red beet leaves were infiltrated with the bacterium carrying the AsBvGT contruct or the pBin3X35S-NOS vector.
Expression analysis by RT-PCR
Total RNA was extracted from infiltrated leaf fragments according to the method described by Logemann et al. (1987). RNAs were treated with 0.5 U RNase-free DNase (Sigma, Aldrich Chemicals, St Louis), precipitated with ethanol, and resuspended in water. RNA yield was determined spectrophotometrically and its quality was determined by electrophoresis in an agarose/formaldehyde gel followed by ethidium bromide staining and UV light visualization. Two µg of RNA were used as a template for RT-PCR reactions using the Mo-MLV RT enzyme (Gibco-BRL, Life Technologies, Rockville, MD) and 20 pmol of BvGT specific primers (reverse primer 5'-CTCAATGGAATGCACCGCTT-3', and forward primer 5'-GCTGAAGAAAAGGCACGAAAAG-3'). For endogenous BvGT mRNA analysis, the first strand was synthesized using the reverse primer. For the antisense transcript amplification, the first strand cDNA was synthesized using the forward primer instead of the reverse primer. PCR reactions were carried out in 50 µl PCR reaction mixtures with 0.2 mM dNTPs, 0.2 U Taq polymerase (Gibco-BRL, Life Technologies, Rockville, MD), 0.2 µM of each primer, and 2 µl of cDNA. PCR conditions were as follows: 3 min initial heating at 94 °C, followed by 35 three-step cycles of 1 min denaturation at 94 °C, 30 s annealing at 63 °C, and 20 s elongation at 68 °C, followed by a final 7 min elongation step at 72 °C as described above. Amplification of 18S rRNA as an internal standard was performed using the ‘Quantum RNA 18S’ kit (Ambion, Inc). PCR products were analysed on 1.2% agarose gels stained with ethidium bromide.
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Results |
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BvGT encodes a GT from B. vulgaris
Based on amino acid sequence data, an alignment was carried out to find consensus sequences for plant UDP-glucose:glucosyltransferases and used to design primers for amplification by PCR of a DNA encoding a GT from B. vulgaris (accession number T525033). The expected 1036 bp product was obtained and its identity as a GT was supported by sequencing. The deduced amino acid sequence showed a region between residues 226 and 269 (underlined in Fig. 2) that corresponds to the UDP-binding domain and a highly conserved region among plant GTs known as a plant secondary product GT signature (PSPG box) (Bairoch, 1992
). The BvGT encoded by the genomic fragment is 63.8% identical (71.8% similar) to the betanidin-5 GT from D. bellidiformis and 57.3% and 56.5% identical (67.3% and 67.4% similar) with flavonoid GTs from N. tabacum and L. esculentum, respectively (Fig. 2).
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Reduction of BvGT expression leads to a decrease in betanin content induced by infiltration with A. tumefaciens
To assess the possibility that the BvGT gene product may be implicated in betacyanin glucosylation, the expression of the BvGT was down-regulated by its antisense sequence using an in planta Agrobacterium-mediated transient expression assay and the betanin content was evaluated. Previous studies have shown that the betacyanin synthesis in red beet leaves was induced by A. tumefaciens infiltration after 48 h (Sepúlveda-Jiménez et al., 2004
). Likewise, in this study the appearance of the red–violet pigmentation was observed in red beet leaves infiltrated with A. tumefaciens carrying only the vector after 48 h. However, the infiltrated leaves with A. tumefaciens carrying the AsBvGT construct showed the absence of this visible red–violet pigmentation (Fig. 3A).
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To determine the effect of the A. tumefaciens-infiltration with AsBvGT on BvGT transcript abundance, RT-PCR assays were conducted to evaluate the accumulation of sense (endogenous) or antisense (transgene) transcripts. As an RNA loading control for the PCR assay, the attenuated product of 18S rRNA of 315 bp was obtained using specific primers, and related competimers. Control leaves were infiltrated with 10 mM MgSO4 solution, and only a weak BvGT transcript accumulation between 12 and 96 h post-infiltration was observed (Fig. 4A). In leaves infiltrated with A. tumefaciens, BvGT mRNA was accumulated between 12 h and 96 h reaching the highest level between 48 h and 72 h after treatment (Fig. 4B). The accumulation of endogenous BvGT mRNA, observed between 48 h and 72 h post-infiltration, was absent in A. tumefaciens-infiltrated leaves with the AsBvGT construct (compare Fig. 4B and C). By contrast, the BvGT antisense transcript (tBvGT) progressively accumulated at 48 h and begun to decline 120 h after A. tumefaciens infiltration (Fig. 4D).
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To determine whether betanin content was affected by the transient expression of the AsBvGT construct, HPLC profiles were obtained from A. tumefaciens-infiltrated leaves 12, 24, 48, 72, 96, and 120 h post-infiltration. This allowed detection and quantification of betanin under the conditions tested. Treatment of plants with A. tumefaciens carrying the vector induced a rise in pigment content from 28.6 nmol betanin disc–1 at 48 h to 56.2 nmol betanin disc–1 at 120 h after treatment. By contrast, in A. tumefaciens-infiltrated plants with the AsBvGT construct, the amount of pigment detected was 7.3 nmol betanin disc–1 at 48 h and of 10.3 nmol betanin disc–1 at 120 h post-infiltration. As a control, the time-course of betanin accumulation, in leaves infiltrated with MgSO4 solution, was also obtained. This treatment induced a weak increment of betanin content after 48 h (3.2 nmol betanin disc–1) that rose up to 11.2 nmol betanin disc–1 120 h after infiltration (Fig. 3B).
The reduction of betanin accumulation caused by the transient expression of the AsBvGT construct indicates that the BvGT participates in the betanin synthesis pathway in red beet leaves.
BvGT mRNA accumulates in response to P. syringae and ROS
In this study, the expression of BvGT in response to infiltration with the same bacteria that induced betacyanin accumulation was analysed. Red beet leaves were infiltrated with P. syringae and samples were collected at 12 h intervals up to 120 h after treatments, and total RNA was obtained for RT-PCR analysis. Results in Fig. 4E show that BvGT mRNA accumulated between 12 h and 96 h in leaves infiltrated with P. syringae, reaching the highest level 48 h after treatment. Together these results showed that infiltration with P. syringae or A. tumefaciens, conditions that lead to betacyanin accumulation, also induced BvGT transcript accumulation.
To determine whether the Glc/GlcO system was also able to induce BvGT mRNA accumulation, red beet leaves were infiltrated with 10 mM Glc and with three concentrations of GlcO (20, 50, and 100 U ml–1). Figure 5A shows that the Glc/GlcO treatment induced BvGT transcript accumulation, reaching its highest level when 100 U ml–1 of enzyme were used. Low levels of the BvGT transcript were detected in leaves infiltrated with Glc alone. In addition, the NADPH oxidase inhibitor diphenylene iodonium (DPI) at 10 µM, blocked BvGT mRNA accumulation induced by the infiltration of A. tumefaciens for 48 h (Fig. 5B). These data suggest that NADPH oxidase is involved in ROS production that, in turn, may act as a signal to induce the expression of BvGT.
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Discussion |
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Higher plants have developed self-defence mechanisms that protect them against environmental stress factors. Some of these mechanisms involve the synthesis of secondary metabolites, such as phenylpropanoids, terpenoids, and alkaloids with antimicrobial properties or antioxidant activities (Wink, 1999
). However, due to their cellular toxicity, many of these compounds are converted to their glyco-conjugates and then accumulated in vacuoles or specialized plastids. Thus, the glucosylation reaction is one of the mechanisms that modulates the biological activity of secondary metabolites and allows the metabolic homeostasis of plants to be maintained (Jones and Vogt, 2001). Previous work has shown that wounding and pathogens induced the transcription of genes that encode glucosyltransferases that can glucosylate secondary metabolites and hormones (O'Donnell et al., 1998; Roberts et al., 1999). In this work, some evidence is presented that BvGT encodes a glucosyltransferase related to betacyanin synthesis during defence responses. The accumulation of BvGT mRNA was induced by wounding, bacterial infiltration, and ROS, the same conditions that induced betacyanin accumulation (Sepúlveda-Jiménez et al., 2004). Here, betanin synthesis induced by infiltration with A. tumefaciens was reduced when the transient expression and accumulation of the AsBvGT construct was A. tumefaciens-infiltrated. Furthermore, the similarity observed between the BvGT and the betanidin 5-GT gene of D. bellidiformis, suggests that BvGT encodes an enzyme involved in betanin synthesis. To confirm this idea, however, it will be necessary to analyse the substrate specificity of the BvGT product. The biochemical studies of the BvGT enzyme will be facilitated by the use of recombinant protein produced in an E. coli expression system.
Agrobacterium alters the expression of plant defence-related genes commonly triggered by abiotic stress factors and by non-pathogenic bacteria in cell cultures of Ageratum conyzoides. These genes encode a pathogenesis-related protein (PR), a glucosyltransferase involved in the biosynthesis of phenylpropanoids, and peroxidases (Ditt et al., 2001). Similar to the defence response of A. conyzoides cell culture, these results showed that, in reed beet leaves, A. tumefaciens induced the synthesis of betanin and the expression of a glucosyltransferase that may be involved in the defence response.
Recent studies indicate that ROS play an important role in plant cells as signalling molecules involved in the regulation of gene expression during stress or pathogen infection (Dat et al., 2000). Here it is shown that treatment with an artificial H2O2-generating system induced BvGT expression, and treatments with DPI blocked its expression induced by the A. tumefaciens infiltration. These data suggest that ROS accumulation is required to induce BvGT expression that, in turn, is necessary for betacyanin synthesis. In this context, the production of betacyanins in response to wounding or bacterial infiltration has been evaluated previously, suggesting that the pigment produced acts as a ROS scavenger, limiting the damage caused by these stress conditions (Sepúlveda-Jiménez et al., 2004). Similarly, the treatment of tobacco cell suspension cultures with a fungal elicitor resulted in the induction of a glucosyltransferase for the production of transportable phenylpropanoid glucosides, followed by the release of free antioxidant phenolics into the extracellular medium and subsequent H2O2 scavenging by phenolic compounds (Chong et al., 1999).
In plants, diverse mechanisms have been implicated in the oxidative burst, including a plasma membrane located NADPH oxidase, a cell wall peroxidase, and apoplastic amine oxidase-type enzymes (Grant and Loake, 2000). Observations in the laboratory showed that treatment with DPI reduced the betacyanin synthesis induced for the A. tumefaciens infiltration (Sepúlveda-Jiménez et al., 2004). In addition, the same NADPH oxidase inhibitor blocked BvGT expression, suggesting that an NADPH oxidase is implicated in ROS production in red beet leaves infiltrated with bacteria. However, a number of important questions remain unanswered. The analysis of antisense red beet lines would help to assess the potential role of ROS in the production of betacyanins.
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Acknowledgements |
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We thank Dra. Alejandra A Covarrubias (IBT-UNAM) and Dr Mario Rodríguez Monroy (CeProBi-IPN) for critical reading of the manuscript. We also thank MC Rene Hernández Vargas for automatic sequencing and Dr Paul Gaytan Colín and MC Eugenio López Bustos for oligonucleotide synthesis. Gabriela Sepúlveda-Jiménez thanks COFAA-IPN for the fellowship awarded.
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Footnotes |
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Abbreviations: DPI, diphenylene iodonium; Glc, glucose; GlcO, glucose oxidase; GT, glucosyltransferase; ROS, reactive oxygen species.
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