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Authors personal copy ARTICLE IN PRESS Journal of Plant Physiology 167 (2010) 400–407 Contents lists available at ScienceDirect Journal of Plant Physiology journal homepage: www.elsevier.de/jplphHydrogen peroxide mediates the expression of ascorbate-related genesin response to methanol stimulation in OncidiumChin-Hui Shen, Kai-Wun Yeh nInstitute of Plant Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwana r t i c l e in f o a b s t r a c tArticle history: We investigated the signaling role of hydrogen peroxide (H2O2) in regulating the ascorbate (AsA) levelReceived 18 July 2009 after exogenous methanol (MeOH) application. The endogenous H2O2 and AsA levels as well as theReceived in revised form expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-21 October 2009 like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption ofAccepted 21 October 2009 endogenous AsA. However, a low MeOH concentration (50 mM) triggered the synthesis of H2O2 and was effective in enhancing the expression of AsA-biosynthetic genes of the Smirnoff–Wheeler andKeywords: galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by theAscorbate addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 184.108.40.206), and diphenyleneiodoniumHydrogen peroxide chloride (DPI), an inhibitor of NADPH oxidase (EC: 220.127.116.11). Thus, the H2O2 generated by MeOHMethanol application is a product of MeOH detoxiﬁcation through alcohol oxidase and NADPH oxidase activation. In this chain, H2O2 acts as a secondary messenger for the activation of AsA-related genes. Our results reveal the signaling function of H2O2 and cellular AsA homeostasis in Oncidium orchids in response to MeOH stimulation. A mechanism for the MeOH effect on AsA production is suggested. & 2009 Elsevier GmbH. All rights reserved.Introduction formic acid and CO2 to prevent damage by alcohol oxidase (Gout et al., 2000). Although the metabolism of MeOH is not completely Methanol (MeOH) is a volatile organic product, originating understood in plants, its contribution to plant physiology isfrom the demethylation of pectin by pectin methylesterase (PME; highlighted by its use in C3 plants for photosynthetic productivityEC: 18.104.22.168) for tightening of the cell wall, especially throughout (Nonomura and Benson, 1992). Methanol inﬂuences C3 plantthe early stage of leaf expansion (Fall and Benson, 1996). Some ´ growth under foliar spray or irrigation (Ramırez et al., 2006), butMeOH emissions have also been observed during changes in has no effect on C4 plants. Foliar application of MeOH causes ancell wall construction during the development of roots and fruits increase of fresh and dry weight in Arabidopsis and tobacco,(Fall and Benson, 1996). Additionally, MeOH might be produced whereas MeOH irrigation signiﬁcantly delays the growth ofand emitted in large quantities by mechanical wounding or under ´ Arabidopsis, tobacco and tomato (Ramırez et al., 2006). The ˜various stresses (Fukui and Doskey, 1998; Penuelas et al., 2005; growth promotion by foliar application was ascribed to thevon Dahl et al., 2006; Pelloux et al., 2007). Methanol accumulates increased carbon ﬁxation due to detoxiﬁcation from photore-in the intercellular air space or in the liquid pool at night, when spiration. Radiotracer 14C and 13C NMR studies revealed thatthe stomata close, and is rapidly converted to formaldehyde, MeOH is metabolized by alcohol oxidase to formaldehyde and formic acid, which are further converted to serine, methionine, purine and thymidylate (Gout et al., 2000). The CO2 produced Abbreviations: AIR, alcohol-insoluble residue; APX, ascorbate peroxidase; AsA, from the oxidization of MeOH is utilized within the Calvin–ascorbate; D-GalUA, D-galacturonate; DPI, diphenyleneiodonium chloride; GalDH, Benson cycle for glucose metabolism (Hanson and Roje, 2001).L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase; Recently, a global gene expression proﬁle resulting from 10%GalUAR, D-galacturonate reductase; GMP, GDP-D-mannose pyrophosphorylase;H2DCF-DA, 2,7-dichloroﬂuorescein diacetate; H2O2, hydrogen peroxide; L-Gal, MeOH stimulation in Arabidopsis leaves was reported (DownieL-galactose; L-GalL, L-galactono-1,4-lactone; MDHAR, monodehydroascorbate et al., 2004). Most of the genes induced by MeOH function inreductase; MeGalUA, methyl-galacturonate; MeOH, methanol; OGA, oligogalac- detoxiﬁcation and stress responses. After 1 h of MeOH treatment,turonic acid; PG, polygalacturonase; PLB, protocorm-like body; PME, Pectin the genes with the highest up-regulation are associated withmethylesterase; ROS, reactive oxygen species; SOD, superoxide dismutase n metabolism, cell communication/signal transduction processes, Corresponding author. Tel.: + 886 2 33662536; fax: + 886 2 23622703. E-mail addresses: email@example.com (C.-H. Shen), firstname.lastname@example.org defense, and RNA processing, but none are involved in(K.-W. Yeh). photosynthesis. At 24- and 72-h MeOH treatment, the genes with0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.doi:10.1016/j.jplph.2009.10.008
Authors personal copy ARTICLE IN PRESS C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 401the highest up-regulation are related to anthocyanin and and incubated at 37 1C for 1 h. The amounts of total and reducedﬂavonoid metabolism. Additionally, genes encoding detoxiﬁcation AsA were determined by monitoring the absorbance at 525 nmproteins, including cytochrome P450s (EC: 22.214.171.124), glucosyl (A525), and the amount of oxidized AsA was calculated from thetransferase (EC: 2.4.1.-) and ascorbate peroxidase (EC: 126.96.36.199), difference between the total pool and the reduced pool.were induced by MeOH. Altogether, these data revealed that The extraction and measurement of pectin content weredetoxiﬁcation and signaling pathways are predominantly acti- performed as described (Wang et al., 2008). In brief, Oncidiumvated in plants exposed to methanol. PLB cultures under different treatments were ground in 80% The modulation of gene expression by chemically inducible ethanol (5 mL/g tissue) and then boiled for 40 min. After beingsystems has attracted interest recently for its potential impact on ﬁltered, the residue was washed with 80% ethanol and dried toboth fundamental and applied plant science (Caddick et al., 1998; obtain alcohol-insoluble residues (AIRs). Starch was removedvon Dahl et al., 2006). Although many reports have described from the AIRs by suspension in 90% dimethylsulfoxide for 16 h ateffects of MeOH on metabolism and biochemistry, information on 20 1C and centrifugation at 20,000g for 20 min. The pecticthe regulatory mechanisms of gene expression and MeOH- polysaccharide was extracted from the starch-free AIRs by stirringinduced signal transduction is still limited. in 0.5% ammonium oxalate solution (25 mL/g AIRs) at 80 1C for Oncidium ‘‘Gower Ramsey’’ is an important orchid in the Asian 1 h, followed by centrifugation at 20,000g for 20 min. Theﬂoral industry. The plant requires more than one year of a supernatant was collected, and ethanol was added at ﬁve timesvegetative growth stage to develop a mature pseudobulb to start a the volume of the extract to precipitate pectic polysaccharides.phase transition. Promotion of the growth rate is a useful strategy The ﬁbrous precipitate was collected by ﬁltration through fourfor reducing the cultivation cost. In a survey of chemical layers of miracloth, vacuum dried, and weighed.stimulants, MeOH was found to be effective in growth promotion The H2O2 level of Oncidium PLB cultures under differentfor Oncidium. However, the ambiguity in physiological function treatments was measured as described (Maxwell et al., 1999)and molecular mechanism is intriguing. The present study of the with some modiﬁcations. All experimental steps were performedascorbate (AsA) metabolism of the Oncidium orchid revealed that at 4 1C. Oncidium PLB cultures were powdered in liquid nitrogenthe expression of genes involved in the AsA-biosynthetic path- and further homogenized with 100% MeOH. The supernatant wasways and AsA recycling was affected by MeOH application in PLB obtained from 20 min of centrifugation at 5000g and 4 1C, andtissues. We determined the optimal concentration of MeOH immediately frozen in liquid nitrogen until further analysis.effective in activating AsA-related genes and regulating the AsA The samples were thawed to 4 1C, and 2,7-dichloroﬂuoresceinreduction or oxidation. Our results suggest that H2O2, a byproduct diacetate (H2DCF-DA) was added to the extract at a ﬁnalof MeOH oxidation, is a secondary signal in regulating associated concentration of 5 mM. Fluorescence was measured by the usegene expression in the MeOH-induced network. of a Hitachi F2000 ﬂuorescence spectrophotometer (Tokyo, Japan) with excitation and emission wavelengths set at 488 nm and 525 nm, respectively.Materials and methodsPlant materials and chemicals RT-PCR analysis Oncidium hybrid ‘‘Gower Ramsey’’ (Oncidium Goldiana xOncidium Guinea Gold) was obtained from the Shih–Dong orchid Total RNA for one-step RT-PCR analysis was extracted fromnursery in Taiwan. Oncidium protocorm-like bodies (PLBs) were the Oncidium PLB cultures. One microgram of total RNA was usedcultured in ½ Murashige and Skoog medium (Murashige and as the template in RT-PCR with the following forward and reverseSkoog, 1962) under long-day conditions (16-h light/8-h dark primers for OgPME (ACJ02103), PME-F-50 -GCTCAAGCTT TGTTCTATcycles) at 2372 1C (Liau et al., 2003). L-Galactose (L-Gal), GGT-30 /PME-R-50 -AAAGAAAAAACAAGATAAAATATAGC-30 ; OgPGD-galacturonate (D-GalUA), methanol (MeOH), diphenyleneiodo- (A BV24998; EC: 188.8.131.52), PG-F-50 -ACGGCGGTGGCGGCAGAGGA-nium chloride (DPI) and hydroxylamine were purchased from 30 /PG-R-50 -ACACTG CCCCTGCCCTCTATAGTGCC-30 ; OgGalUARSigma Co. (St. Louis, MO). (ACJ38540; EC: 1.1.-), GalUAR-F-50 -TCC CTGCTTTACAGAAGT CCCT-30 /GalUAR-R-50 -CCTGGTTTACAAATGGAGGCA-30 ; OgGMP (FJ618566; EC: 2.7.7.-), GMP-F-50 -TTCGAGCGGCTGCCCGTCCA-30 /Methanol treatment of PLB cultures and measurement of AsA, H2O2 GMP-R-50 -GGCTGCCCGATGTCCATCCA-30 ; OgGalDH (ACJ38539; EC:and pectin 184.108.40.206), GalDH-F-50 -TACTCGGAAATTGCCTCCATG-30 /GalDH-R-50 - CCACACGATCCAAAACATATCTG-30 ; OgGalLDH (ACJ38538; EC: Treatments of MeOH and L-Gal and D-GalUA were applied to 220.127.116.11), GalLDH-F-50 -TCAAAGAGCACGGGCTTACG-30 /GalLDH-R-50 -Oncidium PLB cultures, two weeks after subculture from stock AGGGGAAACCTCCATTGTTCC-30 ; OgAPX (FJ237035), APX-F-50 - TGculture. The extraction and measurement of AsA were performed GCACTCGGCTGGGACTTACGATGT-30 /APX-R-50 -GTGGTCGGAACCTTTGas described (Gillespie and Ainsworth, 2007) with slight mod- GTAGCATCAGG-30 ; and OgMDHAR (FJ237040), MDHAR-F-50 -AGCA-iﬁcation. Oncidium PLB cultures with different treatments were GACGATGGATCGCT ATCGCCGAA-30 /MDHAR-R-50 -CGAGTTGAGGCGAhomogenized in liquid nitrogen and then mixed well with 1 mL of GTAGAGCACGTTGA-30 . A one-step RT-PCR kit was used for processing6% trichloroacetic acid. After centrifugation at 4 1C and 6000g for of all the samples (Takara, Japan). The template was reverse-15 min, the resultant supernatant acted as a reactant for assays of transcribed at 50 1C for 30 min and denatured at 94 1C for 2 min,total, reduced and oxidized AsA levels. For the total AsA assay, the followed by 12 cycles for OgPG, OgGalDH, OgGalLDH, OgAPX,reactant was mixed with 10 mM dithiothreitol to reduce the pool OgMDHAR and 18S rRNA ampliﬁcation; 15 cycles for OgPME andof oxidized AsA. After being incubated at room temperature for OgGalUAR ampliﬁcation; and 20 cycles for OgGMP for ampliﬁcation10 min, the mixture was supplemented with 0.5% N-ethylmalei- (one cycle consisted of 94 1C for 30 s for denaturation, 47–66 1C formide to remove the excess dithiothreitol. By contrast, to assay 30 s for annealing depending on the genes, and 72 1C for 30 s forreduced AsA, only deionized water was added to the reactant. elongation) and extension at 72 1C for 10 min. As a control, the RT-All mixtures were supplemented with reaction buffer (10% PCR reaction was performed for 18S rRNA with speciﬁc primers astrichloroacetic acid, 43% H3PO4, 4% a–a0 -bipyridyl and 3% FeCl3) described above.
Authors personal copy ARTICLE IN PRESS402 C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407Enzyme assays was determined from the decrease in A290 by the oxidation of AsA. One unit of APX was deﬁned as the activity that consumed 1 mmol The activities of PG, PME, GalUAR, GMP, GalDH, GalLDH and AsA min À 1 mg À 1 total protein. For SOD activity, the crude proteinAPX were assayed following the method described by Shen et al. was mixed with reaction buffer (100 mM triethanolamine–(2009). The activities of MDHAR and superoxide dismutase (SOD; diethanolamine, 7.5 mM NADH, 100 mM EDTA, 50 mM MnCl2,EC: 18.104.22.168) were measured following modiﬁed methods of pH 7.4). The SOD activity was determined from the decrease inEltayeb et al. (2007) and Paoletti et al. (1986), respectively. A340 by the oxidation of NADH. One unit of SOD was deﬁned as theOncidium PLB cultures for the PME activity assay were ground in activity that consumed 1 mmol NADH min À 1 mg À 1 total protein.extraction buffer (0.1 M citrate, 0.1 M sodium citrate, 1 MNa2HPO4 and 1 M NaCl, pH 5.0), those for PG activity assay wereground in extraction buffer (1 M NaCl, and 0.2 M Na2HPO4 in 1 M Resultscitrate buffer, pH 4.0) and those for the APX activity assays wereground in extraction buffer (2.5 mL of 25 mM potassium phos- Exogenous application of methanol stimulates AsA biosynthesis inphate buffer, pH 7.8) containing 2% polyvinylpolypyrrolidone, Oncidium PLB cultures0.4 mM EDTA and 1 mM AsA. For the other activity assays, thepseudobulbs were ground in extraction buffer (50 mM sodium To study the effect of the MeOH dosage on AsA biosynthesisphosphate buffer, pH 7.2, 2 mM EDTA, 2 mM dithiothreitol, 20% in Oncidium, 10–500 mM MeOH was applied exogenously toglycerol and 2% polyvinylpolypyrrolidone. After centrifugation for Oncidium PLB culture. The endogenous AsA level in tissues was30 min at 4 1C at 6000g, the resultant supernatant was used as the measured at 6, 12, 24 and 30 h after MeOH application. As showncrude enzyme. For the PME assay, the crude protein was mixed in Fig. 1A, application of MeOH resulted in varied AsA levels in thewith reaction buffer (0.1% esteriﬁed pectin in 0.2 M Na2HPO4 PLB cultures. In general, the AsA level preferentially decreasedbuffer, pH 6.3). After overnight incubation at 37 1C, 0.05% during the ﬁrst 6 h of incubation, then showed an irreversibleruthenium red was added and mixed before incubation for response to various concentrations of MeOH. Notably, the PLB10 min. Next, 0.6 M CaCl2 was added to precipitate the demethy- culture was lethally affected by 500 mM MeOH (Fig. 1B), and thelated pectin. The mixture was centrifuged at 14,000g for 15 min to AsA level was markedly decreased. Upon treatment with 50 mMremove the precipitate. The absorbances at 534 nm (A534) of the MeOH, the AsA level of the Oncidium PLB culture increasedsupernatants of the samples were measured. For the PG assay, the following 24 h of inoculation. Thus, a 50 mM MeOH concentrationcrude protein was mixed with reaction buffer (1% cyanoacetamide was concluded to be appropriate for signaling AsA biosynthesis inin 0.1 M borate buffer, pH 7.0) for 5 min. The PG activity was Oncidium.determined from the increase in A276 of 2-cyanoacetamide by theproduction of galacturonic acid. One unit of PG was deﬁned as the Characterization of AsA induction by MeOH stimulationactivity that produced 1 mmol of galacturonic acid min À 1 g À 1 FW.For the GalUAR assay, the crude protein was mixed with reaction To unravel the mechanism of AsA induction after 50 mMbuffer (0.1 mM NADPH and 0.1 mM galacturonic acid in 50 mM MeOH stimulation, several AsA-inducing compounds (Daveysodium phosphate buffer, pH 7.2) for 1 min. The GalUAR activity et al., 1999), such as D-galacturonate (D-GalUA) and L-galactosewas determined from the increase in A254 by the production of (L-Gal), were applied to the PLB culture and their effects wereNADP + . One unit of GalUA reductase was deﬁned as the activity compared (Fig. 2A and B). The AsA levels in the PLB culturethat oxidized 1 mM NADPH min À 1 mg À 1 total protein. For the increased by MeOH (50 mM), D-GalUA (50 mM), and L-GalGMP assay, the crude protein was mixed with reaction buffer (50 mM) treatment (Fig. 2A); however, treatment of MeOH(1 mM MgCl2, 0.4 mM glucose, 0.1 mM ADP, 0.1 mM GDP- alone signiﬁcantly decreased the AsA level during the ﬁrst 6 h ofmannose in 50 mM Tris–HCl buffer, pH 7.0). The reaction was inoculation. Interestingly, assays of the AsA redox state (reducedstarted by serially adding 12 U of hexokinase, 3 U of glucose- form AsA/oxidized form AsA) in the PLB culture showed a similar6-phosphate dehydrogenase and 1 mM sodium pyrophosphate. pattern to that of AsA level (Fig. 2B). The distinct variation of theThe GMP activity was monitored by measuring the A340 to AsA proﬁle with MeOH application suggests that MeOH ismonitor the formation of NADH. One unit of GMP was deﬁned as deleterious to Oncidium cells. The level of H2O2 signiﬁcantlythat which reduced 1 mM NAD + min À 1 mg À 1 total protein. For the increased from 27.8 to 39.1 mM with MeOH application during theGalDH assay, the crude protein was mixed with reaction buffer ﬁrst 6 h of treatment (Table 1), whereas no signiﬁcant effects(0.1 mM NAD + and 0.15 mM L-galactose in 50 mM sodium were observed by the other chemicals, such as L-Gal and D-GalUA.phosphate buffer, pH 7.2). The GalDH activity was determined Detoxiﬁcation of MeOH during the ﬁrst 6 h of treatment isfrom the increase in A340 by the formation of NADH. One unit of important for the up-regulation of AsA-related genes. Moreover,GalDH was deﬁned as the activity that reduced 1 nM NAD + MeOH has a distinct effect of H2O2 generation when applying tomin À 1 mg À 1 total protein. For the GalLDH assay, the crude Oncidium culture.protein was mixed with reaction buffer (0.2% cytochrome c and4.2 mM L-galactono-1,4-lactone in 0.01 M potassium phosphatebuffer, pH 7.8). The GalLDH activity was determined from the Methanol enhances AsA levels by up-regulating AsA-biosynthesis andincrease in A550 by the reduction of cytochrome c. One unit of defense genesGalLDH was deﬁned as the activity that oxidized 1 mmol of L-galactono-1,4-lactone min À 1 mg À 1 total protein. For MDHAR Since the application of 50 mM MeOH to Oncidium PLB cultureactivity, the crude protein was mixed with reaction buffer was effective in elevating the AsA level (Fig. 2A), we investigated(0.1 M Tris–HCl pH 7.2, 0.2 mM NADH, 2 mM AsA, 1 U AsA the effect of 50 mM MeOH on the expression level of AsA-oxidase). The MDHAR activity was determined from the decrease biosynthetic genes in the GalUA pathway, such as polygalactur-in A340 by the oxidization of NADH. One unit of MDHAR was onase (OgPG), pectin methylesterase (OgPME) and galacturonatedeﬁned as the activity that oxidized 1 nmol NADH min À 1 mg À 1 reductase (OgGalUAR), as well as those in the Smirnoff–Wheelertotal protein. For APX activity, the crude protein was mixed with pathway, such as GDP-D-mannose pyrophosphorylase (OgGMP)reaction buffer (25 mM potassium phosphate buffer, pH 7.0, and galactose dehydrogenase (OgGalDH). The RT-PCR data showed0.25 mM AsA, 0.4 mM EDTA and 0.1 mM H2O2). The APX activity that OgPG and OgPME, which are involved in pectin degradation,
Authors personal copy ARTICLE IN PRESS C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 403 0.5 50 mM ½ MS MeOH 50 mM MeOH 0.4 ½MS 10 mM MeOH µmole AsA /g F.W. 100 mM MeOH 0.3 500 mM 0.2 MeOH 500 mM MeOH 0.1 2mm 0 0 10 20 30 Time (hours)Fig. 1. (A) Effect of methanol (MeOH) doses on ascorbate (AsA) level in Oncidium protocorm-like body (PLB) cultures. Oncidium PLB cultures were incubated with 500 mM(m), 100 mM (B), 50 mM (J), and 10 mM (&) MeOH and 1/2 MS medium as a control (W) for 30 h. Vertical bars represent standard deviation of the mean obtained fromthree independent experiments. (B) The PLB culture markedly varied with MeOH dosage, with 500 mM MeOH being lethal to Oncidium PLB culture, and 50 mM having noeffects. All Oncidium PLB cultures were photographed after 12 h treatment. 0.55 12.00 50 mM L-Gal 50 mM D-GalUA + 0.50 50 mM D-GalUA + 10.00 50 mM MeOH 50 mM MeOH 50 mM L-Gal 0.45 µmole AsA/g F.W. AsA Redox ratio 50 mM D-GalUA 50 mM MeOH 8.00 50 mM D-GalUA 50 mM MeOH 0.40 CK (½ MS) CK (½ MS) 6.00 0.35 4.00 0.30 2.00 0.25 0.20 0.00 0 6 12 18 24 30 0 6 12 18 24 30 Time (hours) Time (hours)Fig. 2. The AsA level and redox state in Oncidium PLB cultures incubated with various compounds for 30 h. (A) AsA level, (B) AsA redox ratio. Vertical bars representstandard deviations of the means obtained from three independent experiments. 50 mM MeOH (&), 50 mM D-galacturonate (D-GalUA) (B), 50 mM MeOH and 50 mMD-GalUA (W), 50 mM L-galactose (L-Gal) (J) and 1/2 MS (m).were both up-regulated after 6 h of MeOH treatment. However, To further understand the proteins associated with the AsA-the expression of OgPME was decreased at 24 h. In contrast, related genes under 50 mM MeOH stimulation, their enzymaticno further changes in the expression level of OgGalUAR by MeOH activities were assayed. As shown in Fig. 4, the activities of OgPG,treatment were observed (Fig. 3). On the other hand, both OgGMP OgMDHAR, OgAPX and OgSOD were speciﬁcally enhanced from 6and OgGalDH of the Smirnoff–Wheeler pathway were up- to 12 h upon MeOH treatment, whereas other enzymes, such asregulated during the ﬁrst 6 h of MeOH treatment, effects OgGalUAR, OgGMP, OgGalDH, OgGalLDH, were not signiﬁcantlylasting for 30 h (Fig. 3). This is similar to the effect by L-Gal enhanced by MeOH stimulation, even though they were enhancedstimulation, which acts as a carbon source, similar to D-GalUA in RNA levels. These results indicated that mRNA levels of many ofin AsA-biosynthetic routes (Fig. 3; Davey et al., 1999). Finally, these genes are not correlated with enzymatic activities, whichthe expression of galactono-1,4-lactone dehydrogenase may be related to post-translational modiﬁcations.(OgGalLDH), an integrator of the AsA biosynthetic pathway, The pectin content of the Oncidium PLB culture was decreaseddisplayed an enhanced level upon MeOH treatment (Fig. 3). In in 50 mM MeOH treatment, but not in L-Gal or D-GalUA treatmentaddition, the levels of defense genes, including ascorbate (Table 1). The degradation appeared to result mainly from theperoxidase (OgAPX) and monodehydroascorbate reductase elevated activity of OgPG under MeOH stimulation (Fig. 4). In(OgMDHAR), were also increased at 6–24 h after MeOH conclusion, the AsA level was elevated in Oncidium PLB culture bytreatment (Fig. 3). Taken together, a 50 mM MeOH treatment MeOH stimulation, primarily because of the enhanced expressionwas effective to enhance the expression level of most AsA-related level and enzymatic activity of OgPG. Although the mRNA levelsgenes in the GalUA pathway, Smirnoff–Wheeler pathway and of a number of AsA-biosynthetic genes were certainly induceddefense system. and enhanced, their functional contribution in AsA biosynthesis is
Authors personal copy ARTICLE IN PRESS404 C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407unclear due to the absence of increased enzymatic activity with elevated from 27.8 to 39.1 mM in Oncidium PLB cultures inMeOH treatment. response to exogenous application of MeOH, but not D-GalUA and L-Gal, during the ﬁrst 6 h of treatment (Table 1). To unravel the source of H2O2 production, we applied hydroxylamine (1 mM)Hydrogen peroxide production in Oncidium PLB cultures through the and DPI (5 mM), inhibitors of alcohol oxidase and NADPH oxidase,activation of alcohol oxidase and NADPH oxidase under MeOH respectively, with MeOH in PLB cultures, and monitored the H2O2stimulation levels. In Oncidium PLB cultures incubated with 50 mM MeOH, an early H2O2 burst ( $45 mM) was detected during the ﬁrst 30 min, Plant cells are able to convert MeOH to formaldehyde and followed by a subsequent decrease in accumulation ( $40 mM)H2O2 by alcohol oxidase (Gout et al., 2000). The H2O2 level was that lasted for another 6 h (Fig. 5). However, this H2O2 burst was attenuated by incubation with 50 mM MeOH combinedTable 1 with 1 mM hydroxylamine or 5 mM DPI. The DPI inhibitor wasH2O2 amount and pectin concentration of PLBs incubated with various treatments. more effective in blocking H2O2 generation than the alcohol oxidase inhibitor. NADPH oxidase could play a more signiﬁcant Treatments (h) H2O2 (mM) Pectin (mg/g F.W.) role in the systemic production of H2O2 than alcohol oxidase does. CK (1/2 MS) Therefore, the stimulation of the H2O2 level by MeOH in the 0 28.2972.83 31.46 7 0.64 Oncidium culture occurs directly, through MeOH metabolism (or 6 28.5771.98 32.20 7 0.93 detoxiﬁcation) by alcohol oxidase activity, and indirectly, through 12 28.92 71.73 30.85 7 1.13 the subsequent induction of NADPH oxidase activity to amplify 24 26.50 71.24 31.65 7 1.67 30 28.68 72.32 30.45 7 1.01 H2O2 production. Moreover, the early oxidative peak of the H2O2 50 mM L-Gal level in the Oncidium culture could be largely due to the 0 28.1272.44 31.21 7 1.03 conversion of MeOH by alcohol oxidase, and the later H2O2 6 28.39 72.58 30.70 7 0.55 burst could result primarily from NADPH oxidase activation 12 28.35 71.33 29.98 7 0.51 (Fig. 5). 24 30.007 1.42 30.13 7 0.57 30 28.38 72.32 30.88 7 0.58 50 mM D-GalUA 0 26.8672.81 30.09 7 0.56 The up-regulation of AsA-related genes stimulated by MeOH is 6 28.75 71.85 30.07 7 0.56 12 28.85 72.15 30.56 7 0.93 through H2O2 signal transduction 24 27.47 72.72 31.43 7 1.16 30 27.73 71.89 30.95 7 0.16 To conﬁrm the potential signaling effects of H2O2 on the 50 mM MeOH expression of AsA-related genes, we investigated the expression 0 27.75 71.72 30.92 7 0.58 6 39.14 71.28 26.42 7 0.96 of AsA-related genes under the application of the inhibitors alone 12 33.14 71.52 25.65 7 0.58 or with MeOH. As shown in Fig. 6, the expression of AsA-related 24 31.43 71.30 26.02 7 1.45 genes did not change after 6 h with 1 mM hydroxylamine 30 30.74 71.61 27.57 7 1.00 or 5 mM DPI treatment. However, the expressional levels of 50 mM D-GalUA+ 50 mM MeOH AsA-related genes were lower with MeOH combined with 0 29.03 72.09 30.86 7 0.16 6 40.11 71.28 26.84 7 0.99 hydroxylamine or DPI than with MeOH alone. Hydroxylamine 12 34.6171.05 25.48 7 1.14 and DPI inhibited H2O2 production (Fig. 5), consequently reducing 24 32.46 72.89 26.94 7 1.58 the MeOH effect on the expression of AsA-related genes in 30 28.82 70.98 27.35 7 0.43 Oncidium PLB cultures (Fig. 6). The results suggest that H2O2PLBs= protocorm-like bodies; F.W.= fresh weight. Mean values7 S.E. were signaling is critical in up-regulating the expression of AsA-relatedobtained from three independent experiments. genes. 50 mM D-GalUA 50 mM 50 mM + GalUA pathway CK D-GalUA 50 mM MeOH 50 mM MeOH L-Gal Pectin 0 6 12 24 30 0 6 12 24 30 0 6 12 24 30 0 6 12 24 30 0 6 12 24 30h OgPG MeGalUA OgPME OgGalUAR D-GalUA OgGMP Smirnoff-Wheeler pathway OgGalDH D-Glc-6-P L-GalA OgGalLDH D-Man-1-P OgMDHAR GDP- D-Man OgAPX L-Gal L-GalL 18S rRNA Ascorbate MDHA H2O2 H 2O AsA recyclingFig. 3. Expression of AsA-related genes on treatment of Oncidium PLB cultures with various compounds. The relative amount of transcripts of OgPME, OgPG and OgGalUARin the GalUA pathway; OgGMP, OgGalDH and OgGalLDH in the Smirnoff–Wheeler pathway and OgAPX and OgMDHAR in the defense system were determined by RT-PCR.
Authors personal copy ARTICLE IN PRESS C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 405 60 1 OgPG 7 2 OgPME 1 8 3 OgGalUAR 1 50 4 OgGMP 7 5 OgGalDH 8 6 OgGalLDH 40 7 OgMDHAR Activity Unit 8 OgAPX 9 OgSOD 30 8 9 9 1 5 5 20 4 2 5 4 2 7 3 4 3 2 6 9 6 6 3 10 0 0 6 12 Time (hours)Fig. 4. Activity assays of AsA-related enzymes under 50 mM MeOH treatments. Activity units of AsA-related enzymes were deﬁned in Materials and methods, and verticalbars represent standard deviations of the means obtained from three independent experiments. 50.00 45.00 40.00 50 mM MeOH µM H2O2 50 mM MeOH + 1 mM hydroxylamine 35.00 30.00 50 mM MeOH + 5 mM DPI ½ MS + 1 mM hydroxylamine 25.00 ½ MS + 5 mM DPI CK (½ MS) 20.00 0 1 2 3 4 5 6 Time (hours)Fig. 5. The effects of hydroxylamine (inhibitor of alcohol oxidase) and diphenyleneiodonium chloride (DPI; inhibitor of NADPH oxidase) on H2O2 production in OncidiumPLB cultures for 6 h. Vertical bars represent standard deviations of the means obtained from three independent experiments.Discussion (Fig. 3). Moreover, the H2O2 level was elevated after 30 min treatment and maintained for at least 6 h (Fig. 5). By adding Methanol (MeOH) is known as a deleterious by-product hydroxylamine or DPI compounds with MeOH into the Oncidiumderived from pectin demethylation during the cell wall recon- PLB cultures, the activities of alcohol oxidase and NADPH oxidasestruction process in plants. Its effects on plant growth in Vigna were inhibited. Accordingly, H2O2 production was markedlyradiata were reported 20 years ago (Bhattacharya et al., 1985). decreased in PLB cultures by 8 to 20% (Fig. 5). In addition, NADPHAlthough the effects of MeOH on plant physiology and gene oxidase was more effective than alcohol oxidase in producingexpression have been investigated (Gout et al., 2000; Galbally and H2O2, because the inhibition of NADPH oxidase activity by DPI hadKirstine, 2002; Downie et al., 2004), its mechanism of signal a greater effect on the H2O2 level ( À20%) than inhibition oftransduction mechanism has not been elucidated. As previously alcohol oxidase activity ( À 8%) (Fig. 5). Thus, the H2O2 level wasreported in Arabidopsis, AsA-biosynthetic genes in the Smirnoff– enhanced by MeOH stimulation through two steps: MeOHWheeler pathway and AsA-recycling and pectin degradation oxidation by alcohol oxidase and systemic ampliﬁcation bygenes were all stimulated by an appropriate concentration of NADPH oxidase. In addition, the diminished H2O2 level with ´MeOH (Downie et al., 2004; Ramırez et al., 2006), but genes inhibition of alcohol oxidase and NADPH oxidase caused a reducedrelated to photosynthesis were not responsive to MeOH expression of AsA-related genes (Fig. 6). In conclusion, the resultsapplication (Downie et al., 2004). strongly suggest that H2O2 acts as a signaling messenger to In the present study, exogenous application of MeOH (50 mM) regulate AsA-related gene expression under MeOH stimulation.to Oncidium PLB cultures increased the AsA level by 30% (Figs. 1 Hydrogen peroxide is a reactive oxygen species (ROS)and 2) and AsA-related genes were markedly up-regulated produced by plants under stress conditions (Mittler et al., 2004).
Authors personal copy ARTICLE IN PRESS406 C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 50 mM MeOH 50 mM MeOH + + 1 mM 5 mM 50 mM 1 mM 5 mM GalUA pathway CK hydroxylamine DPI MeOH hydroxylamine DPI Pectin 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 h OgPG MeGalUA OgPME OgGalUAR D-GalUA Smirnoff-Wheeler OgGMP pathway OgGalDH L-GalA D-Glc-6-P OgGalLDH D-Man-1-P OgMDHAR GDP- D-Man OgAPX L-Gal L-GalL 18S rRNA Ascorbate MDHA H2O2 H2O AsA recyclingFig. 6. Expression of AsA-related genes on treatment with H2O2-producing inhibitors. Total RNA was isolated from PLBs incubated with hydroxylamine or DPI. The relativeamount of transcripts for OgPME, OgPG and OgGalUAR in the GalUA pathway; OgGMP, OgGalDH and OgGalLDH in the Smirnoff–Wheeler pathway; and OgAPX and OgMDHARin the defense system were determined by RT-PCR. Pectin PG PME MeOH + OGA + D-GalUA alcohol formaldehyde oxidase formate H2O2 GalUA pathway PM NADPH Oxidase Calvin-Benson cycle H2O2 Defense genes (APX,MDHAR, etc) Smirnoff-Wheeler pathway AsA GalUA pathway (pectin degrad. -PG,PME)Fig. 7. The proposed model of the H2O2-signaling network under MeOH stimulation in Oncidium PLB cultures. Methanol is produced along with oligogalacturonic acid(OGA) and D-GalUA during the degradation of pectin in the plant cell wall. Methanol is preferentially oxidized (detoxiﬁed) by alcohol oxidase to H2O2 and formaldehyde.Subsequently, H2O2 activates NADPH oxidase to create more H2O2, which acts as secondary messenger to induce the expression of AsA-related biosynthetic genes.In addition, OGA also enhances H2O2 production (Ridley et al., 2001). D-GalUA might be a precursor for AsA synthesis in the GalUA pathway. The products of pectindegradation involved in H2O2 signal transduction could function in elevating AsA levels in cells. A high AsA level could scavenge reactive oxygen species and protect the cellfrom stresses. APX =ascorbate peroxidase; MDHAR= monodehydroascorbate reductase.Induced H2O2 can act as a local signal for hypersensitive cell death 2009). The enzymatic activities (but not the mRNA levels)and as a diffusible signal for the induction of defense genes in of some AsA-biosynthetic genes, such as OgGMP, OgGalDH,adjacent cells (Alvarez et al., 1998). The functional roles are OgGalLDH and OgGalUAR, were not enhanced by MeOH stimula-complicated and diversiﬁed. Therefore, the induction of the plant tion, indicating regulation based on post-translational modiﬁca-defense system is tightly controlled for its production and tions. However, the AsA level is eventually increased in responsescavenging. In several model systems of plants, the oxidative to MeOH. These enzymatic activities are not critical for AsAburst and accumulation of H2O2 appear to be mediated by the synthesis in AsA-biosynthetic pathway experiencing the effects ofactivation of a membrane-bound NADPH oxidase complex (Zhang MeOH. On the other hand, the increased activity of OgPG is ¨et al., 2007; Konigshofer et al., 2008; Wen et al., 2008). In the necessary for AsA production as well as the oligogalacturonic acidOncidium system, H2O2 induction has been identiﬁed as a signal to (OGA) product, a ligand to induce H2O2 generation after MeOHinduce AsA-related genes during vegetative growth (Shen et al., treatment. In addition, AsA-recycling enzymes, OgAPX and
Authors personal copy ARTICLE IN PRESS C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 407OgMDHAR, are essential for scavenging ROS. OgSOD can function Aziz A, Heyraud A, Lambert B. Oligogalacturonide signal transduction, induction ofin dismutating superoxide (which was produced by NADPH defense-related responses and protection of grapevine against Botrytis cinerea. Planta 2004;218:767–774.oxidase) into H2O2 (Mittler et al., 2004). Their increased activity Bergey DR, Orozco-Cardenas M, de Moura DS, Ryan CA. A wound- and systemin-is indeed beneﬁcial to plant cells undergoing MeOH stimulation. inducible polygalacturonase in tomato leaves. Proc Natl Acad Sci USA Both methyl-galacturonate (MeGalUA) and D-GalUA are key 1999;96:1756–1760. Bhattacharya S, Bhattacharya NC, Bhatnagar VB. Effect of ethanol, methanol andintermediates in the GalUA pathway (Fig. 3) one of the AsA- acetone on rooting etiolated cuttings of Vigna radiata in presence of sucrosebiosynthetic routes in planta (Smirnoff, 2003). D-GalUA is converted and auxin. Ann Bot 1985;55:143–145.from MeGalUA by pectin methylesterase in plant cells, with MeOH Caddick MX, Greenland AJ, Jepson I, Krause KP, Qu N, Riddell KV, Salter MG, Schuch W, Sonnewald U, Tomsett AB. An ethanol inducible gene switch for plants usedbeing produced as a byproduct. MeGalUA, an upstream inter- to manipulate carbon metabolism. Nat Biotechnol 1998;16:177–180.mediate in the GalUA pathway, was more effective in enhancing Davey MW, Gilot C, Persiau G, Ostergaard J, Han Y, Bauw GC, Van Montagu MC.the AsA level in Arabidopsis cultures than D-GalUA, a downstream Ascorbate biosynthesis in Arabidopsis cell suspension culture. Plant Physiol 1999;121:535–543.intermediate in the GalUA pathway (Davey et al., 1999). 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