jurnal anggrek oncidium

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Hydrogen peroxide mediates the expression of ascorbate-related genesin response to methanol stimulation in Oncidium

Chin-Hui Shen, Kai-Wun Yeh n

Institute of Plant Biology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan

a r t i c l e i n f o

Article history:

Received 18 July 2009

Received in revised form

21 October 2009

Accepted 21 October 2009

Keywords:

Ascorbate

Hydrogen peroxide

Methanol

a b s t r a c t

We investigated the signaling role of hydrogen peroxide (H2O2) in regulating the ascorbate (AsA) level

after exogenous methanol (MeOH) application. The endogenous H2O2 and AsA levels as well as the

expression of related genes were monitored after MeOH treatment of cultures of Oncidium protocorm-

like bodies (PLB). A high MeOH concentration was deleterious and caused irreversible consumption of

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 and

galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the

addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium

chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H2O2 generated by MeOH

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

Methanol (MeOH) is a volatile organic product, originatingfrom the demethylation of pectin by pectin methylesterase (PME;EC: 3.1.1.11) for tightening of the cell wall, especially throughoutthe early stage of leaf expansion (Fall and Benson, 1996). SomeMeOH emissions have also been observed during changes incell wall construction during the development of roots and fruits(Fall and Benson, 1996). Additionally, MeOH might be producedand emitted in large quantities by mechanical wounding or undervarious stresses (Fukui and Doskey, 1998; Penuelas et al., 2005;von Dahl et al., 2006; Pelloux et al., 2007). Methanol accumulatesin the intercellular air space or in the liquid pool at night, whenthe stomata close, and is rapidly converted to formaldehyde,

formic acid and CO2 to prevent damage by alcohol oxidase (Goutet al., 2000). Although the metabolism of MeOH is not completelyunderstood in plants, its contribution to plant physiology ishighlighted by its use in C3 plants for photosynthetic productivity(Nonomura and Benson, 1992). Methanol influences C3 plantgrowth under foliar spray or irrigation (Ramırez et al., 2006), buthas no effect on C4 plants. Foliar application of MeOH causes anincrease of fresh and dry weight in Arabidopsis and tobacco,whereas MeOH irrigation significantly delays the growth ofArabidopsis, tobacco and tomato (Ramırez et al., 2006). Thegrowth promotion by foliar application was ascribed to theincreased carbon fixation due to detoxification from photore-spiration. Radiotracer 14C and 13C NMR studies revealed thatMeOH is metabolized by alcohol oxidase to formaldehyde andformic acid, which are further converted to serine, methionine,purine and thymidylate (Gout et al., 2000). The CO2 producedfrom the oxidization of MeOH is utilized within the Calvin–Benson cycle for glucose metabolism (Hanson and Roje, 2001).

Recently, a global gene expression profile resulting from 10%MeOH stimulation in Arabidopsis leaves was reported (Downieet al., 2004). Most of the genes induced by MeOH function indetoxification and stress responses. After 1 h of MeOH treatment,the genes with the highest up-regulation are associated withmetabolism, cell communication/signal transduction processes,defense, and RNA processing, but none are involved inphotosynthesis. At 24- and 72-h MeOH treatment, the genes with

ARTICLE IN PRESS

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journal homepage: www.elsevier.de/jplph

Journal of Plant Physiology

0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.

doi:10.1016/j.jplph.2009.10.008

Abbreviations: AIR, alcohol-insoluble residue; APX, ascorbate peroxidase; AsA,

ascorbate; D-GalUA, D-galacturonate; DPI, diphenyleneiodonium chloride; GalDH,

L-galactose dehydrogenase; GalLDH, L-galactono-1,4-lactone dehydrogenase;

GalUAR, D-galacturonate reductase; GMP, GDP-D-mannose pyrophosphorylase;

H2DCF-DA, 2,7-dichlorofluorescein diacetate; H2O2, hydrogen peroxide; L-Gal,

L-galactose; L-GalL, L-galactono-1,4-lactone; MDHAR, monodehydroascorbate

reductase; MeGalUA, methyl-galacturonate; MeOH, methanol; OGA, oligogalac-

turonic acid; PG, polygalacturonase; PLB, protocorm-like body; PME, Pectin

methylesterase; ROS, reactive oxygen species; SOD, superoxide dismutasen Corresponding author. Tel.: +886 2 33662536; fax: +886 2 23622703.

E-mail addresses: [email protected] (C.-H. Shen), [email protected]

(K.-W. Yeh).

Journal of Plant Physiology 167 (2010) 400–407

Author's personal copyARTICLE IN PRESS

the highest up-regulation are related to anthocyanin andflavonoid metabolism. Additionally, genes encoding detoxificationproteins, including cytochrome P450s (EC: 1.14.15.6), glucosyltransferase (EC: 2.4.1.-) and ascorbate peroxidase (EC: 1.11.1.11),were induced by MeOH. Altogether, these data revealed thatdetoxification and signaling pathways are predominantly acti-vated in plants exposed to methanol.

The modulation of gene expression by chemically induciblesystems has attracted interest recently for its potential impact onboth fundamental and applied plant science (Caddick et al., 1998;von Dahl et al., 2006). Although many reports have describedeffects of MeOH on metabolism and biochemistry, information onthe regulatory mechanisms of gene expression and MeOH-induced signal transduction is still limited.

Oncidium ‘‘Gower Ramsey’’ is an important orchid in the Asianfloral industry. The plant requires more than one year of avegetative growth stage to develop a mature pseudobulb to start aphase transition. Promotion of the growth rate is a useful strategyfor reducing the cultivation cost. In a survey of chemicalstimulants, MeOH was found to be effective in growth promotionfor Oncidium. However, the ambiguity in physiological functionand molecular mechanism is intriguing. The present study of theascorbate (AsA) metabolism of the Oncidium orchid revealed thatthe expression of genes involved in the AsA-biosynthetic path-ways and AsA recycling was affected by MeOH application in PLBtissues. We determined the optimal concentration of MeOHeffective in activating AsA-related genes and regulating the AsAreduction or oxidation. Our results suggest that H2O2, a byproductof MeOH oxidation, is a secondary signal in regulating associatedgene expression in the MeOH-induced network.

Materials and methods

Plant materials and chemicals

Oncidium hybrid ‘‘Gower Ramsey’’ (Oncidium Goldiana xOncidium Guinea Gold) was obtained from the Shih–Dong orchidnursery in Taiwan. Oncidium protocorm-like bodies (PLBs) werecultured in ½ Murashige and Skoog medium (Murashige andSkoog, 1962) under long-day conditions (16-h light/8-h darkcycles) at 2372 1C (Liau et al., 2003). L-Galactose (L-Gal),D-galacturonate (D-GalUA), methanol (MeOH), diphenyleneiodo-nium chloride (DPI) and hydroxylamine were purchased fromSigma Co. (St. Louis, MO).

Methanol treatment of PLB cultures and measurement of AsA, H2O2

and pectin

Treatments of MeOH and L-Gal and D-GalUA were applied toOncidium PLB cultures, two weeks after subculture from stockculture. The extraction and measurement of AsA were performedas described (Gillespie and Ainsworth, 2007) with slight mod-ification. Oncidium PLB cultures with different treatments werehomogenized in liquid nitrogen and then mixed well with 1 mL of6% trichloroacetic acid. After centrifugation at 4 1C and 6000g for15 min, the resultant supernatant acted as a reactant for assays oftotal, reduced and oxidized AsA levels. For the total AsA assay, thereactant was mixed with 10 mM dithiothreitol to reduce the poolof oxidized AsA. After being incubated at room temperature for10 min, the mixture was supplemented with 0.5% N-ethylmalei-mide to remove the excess dithiothreitol. By contrast, to assayreduced AsA, only deionized water was added to the reactant.All mixtures were supplemented with reaction buffer (10%trichloroacetic acid, 43% H3PO4, 4% a–a0-bipyridyl and 3% FeCl3)

and incubated at 37 1C for 1 h. The amounts of total and reducedAsA were determined by monitoring the absorbance at 525 nm(A525), and the amount of oxidized AsA was calculated from thedifference between the total pool and the reduced pool.

The extraction and measurement of pectin content wereperformed as described (Wang et al., 2008). In brief, Oncidium

PLB cultures under different treatments were ground in 80%ethanol (5 mL/g tissue) and then boiled for 40 min. After beingfiltered, the residue was washed with 80% ethanol and dried toobtain alcohol-insoluble residues (AIRs). Starch was removedfrom the AIRs by suspension in 90% dimethylsulfoxide for 16 h at20 1C and centrifugation at 20,000g for 20 min. The pecticpolysaccharide was extracted from the starch-free AIRs by stirringin 0.5% ammonium oxalate solution (25 mL/g AIRs) at 80 1C for1 h, followed by centrifugation at 20,000g for 20 min. Thesupernatant was collected, and ethanol was added at five timesthe volume of the extract to precipitate pectic polysaccharides.The fibrous precipitate was collected by filtration through fourlayers of miracloth, vacuum dried, and weighed.

The H2O2 level of Oncidium PLB cultures under differenttreatments was measured as described (Maxwell et al., 1999)with some modifications. All experimental steps were performedat 4 1C. Oncidium PLB cultures were powdered in liquid nitrogenand further homogenized with 100% MeOH. The supernatant wasobtained from 20 min of centrifugation at 5000g and 4 1C, andimmediately frozen in liquid nitrogen until further analysis.The samples were thawed to 4 1C, and 2,7-dichlorofluoresceindiacetate (H2DCF-DA) was added to the extract at a finalconcentration of 5 mM. Fluorescence was measured by the useof a Hitachi F2000 fluorescence spectrophotometer (Tokyo, Japan)with excitation and emission wavelengths set at 488 nm and525 nm, respectively.

RT-PCR analysis

Total RNA for one-step RT-PCR analysis was extracted fromthe Oncidium PLB cultures. One microgram of total RNA was usedas the template in RT-PCR with the following forward and reverseprimers for OgPME (ACJ02103), PME-F-50-GCTCAAGCTT TGTTCTATGGT-30/PME-R-50-AAAGAAAAAACAAGATAAAATATAGC-30; OgPG

(A BV24998; EC: 3.2.1.15), PG-F-50-ACGGCGGTGGCGGCAGAGGA-30/PG-R-50-ACACTG CCCCTGCCCTCTATAGTGCC-30; OgGalUAR

(ACJ38540; EC: 1.1.-), GalUAR-F-50-TCC CTGCTTTACAGAAGTCCCT-30/GalUAR-R-50-CCTGGTTTACAAATGGAGGCA-30; OgGMP

(FJ618566; EC: 2.7.7.-), GMP-F-50-TTCGAGCGGCTGCCCGTCCA-30/GMP-R-50 -GGCTGCCCGATGTCCATCCA-30; OgGalDH (ACJ38539; EC:1.1.1.122), GalDH-F-50-TACTCGGAAATTGCCTCCATG-30/GalDH-R-50-CCACACGATCCAAAACATATCTG-30; OgGalLDH (ACJ38538; EC:1.3.2.3), GalLDH-F-50-TCAAAGAGCACGGGCTTACG-30/GalLDH-R-50-AGGGGAAACCTCCATTGTTCC-30; OgAPX (FJ237035), APX-F-50- TGGCACTCGGCTGGGACTTACGATGT-30/APX-R-50-GTGGTCGGAACCTTTGGTAGCATCAGG-30; and OgMDHAR (FJ237040), MDHAR-F-50-AGCA-GACGATGGATCGCT ATCGCCGAA-30/MDHAR-R-50-CGAGTTGAGGCGAGTAGAGCACGTTGA-30. A one-step RT-PCR kit was used for processingof all the samples (Takara, Japan). The template was reverse-transcribed at 50 1C for 30 min and denatured at 94 1C for 2 min,followed by 12 cycles for OgPG, OgGalDH, OgGalLDH, OgAPX,OgMDHAR and 18S rRNA amplification; 15 cycles for OgPME andOgGalUAR amplification; and 20 cycles for OgGMP for amplification(one cycle consisted of 94 1C for 30 s for denaturation, 47–66 1C for30 s for annealing depending on the genes, and 72 1C for 30 s forelongation) and extension at 72 1C for 10 min. As a control, the RT-PCR reaction was performed for 18S rRNA with specific primers asdescribed above.

C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 401


Enzyme assays

The activities of PG, PME, GalUAR, GMP, GalDH, GalLDH andAPX were assayed following the method described by Shen et al.(2009). The activities of MDHAR and superoxide dismutase (SOD;EC: 1.15.1.1) were measured following modified methods ofEltayeb et al. (2007) and Paoletti et al. (1986), respectively.Oncidium PLB cultures for the PME activity assay were ground inextraction 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 Mcitrate buffer, pH 4.0) and those for the APX activity assays wereground in extraction buffer (2.5 mL of 25 mM potassium phos-phate buffer, pH 7.8) containing 2% polyvinylpolypyrrolidone,0.4 mM EDTA and 1 mM AsA. For the other activity assays, thepseudobulbs were ground in extraction buffer (50 mM sodiumphosphate buffer, pH 7.2, 2 mM EDTA, 2 mM dithiothreitol, 20%glycerol and 2% polyvinylpolypyrrolidone. After centrifugation for30 min at 4 1C at 6000g, the resultant supernatant was used as thecrude enzyme. For the PME assay, the crude protein was mixedwith reaction buffer (0.1% esterified pectin in 0.2 M Na2HPO4

buffer, pH 6.3). After overnight incubation at 37 1C, 0.05%ruthenium red was added and mixed before incubation for10 min. Next, 0.6 M CaCl2 was added to precipitate the demethy-lated pectin. The mixture was centrifuged at 14,000g for 15 min toremove the precipitate. The absorbances at 534 nm (A534) of thesupernatants of the samples were measured. For the PG assay, thecrude protein was mixed with reaction buffer (1% cyanoacetamidein 0.1 M borate buffer, pH 7.0) for 5 min. The PG activity wasdetermined from the increase in A276 of 2-cyanoacetamide by theproduction of galacturonic acid. One unit of PG was defined as theactivity that produced 1 mmol of galacturonic acid min�1 g�1 FW.For the GalUAR assay, the crude protein was mixed with reactionbuffer (0.1 mM NADPH and 0.1 mM galacturonic acid in 50 mMsodium phosphate buffer, pH 7.2) for 1 min. The GalUAR activitywas determined from the increase in A254 by the production ofNADP+. One unit of GalUA reductase was defined as the activitythat oxidized 1 mM NADPH min�1 mg�1 total protein. For theGMP assay, the crude protein was mixed with reaction buffer(1 mM MgCl2, 0.4 mM glucose, 0.1 mM ADP, 0.1 mM GDP-mannose in 50 mM Tris–HCl buffer, pH 7.0). The reaction wasstarted by serially adding 12 U of hexokinase, 3 U of glucose-6-phosphate dehydrogenase and 1 mM sodium pyrophosphate.The GMP activity was monitored by measuring the A340 tomonitor the formation of NADH. One unit of GMP was defined asthat which reduced 1 mM NAD+ min�1 mg�1 total protein. For theGalDH assay, the crude protein was mixed with reaction buffer(0.1 mM NAD+ and 0.15 mM L-galactose in 50 mM sodiumphosphate buffer, pH 7.2). The GalDH activity was determinedfrom the increase in A340 by the formation of NADH. One unit ofGalDH was defined as the activity that reduced 1 nM NAD+

min�1 mg�1 total protein. For the GalLDH assay, the crudeprotein 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 theincrease in A550 by the reduction of cytochrome c. One unit ofGalLDH was defined as the activity that oxidized 1 mmol of L-galactono-1,4-lactone min�1 mg�1 total protein. For MDHARactivity, the crude protein was mixed with reaction buffer(0.1 M Tris–HCl pH 7.2, 0.2 mM NADH, 2 mM AsA, 1 U AsAoxidase). The MDHAR activity was determined from the decreasein A340 by the oxidization of NADH. One unit of MDHAR wasdefined as the activity that oxidized 1 nmol NADH min�1 mg�1

total protein. For APX activity, the crude protein was mixed withreaction buffer (25 mM potassium phosphate buffer, pH 7.0,0.25 mM AsA, 0.4 mM EDTA and 0.1 mM H2O2). The APX activity

was determined from the decrease in A290 by the oxidation of AsA.One unit of APX was defined as the activity that consumed 1 mmolAsA min�1 mg�1 total protein. For SOD activity, the crude proteinwas mixed with reaction buffer (100 mM triethanolamine–diethanolamine, 7.5 mM NADH, 100 mM EDTA, 50 mM MnCl2,pH 7.4). The SOD activity was determined from the decrease inA340 by the oxidation of NADH. One unit of SOD was defined as theactivity that consumed 1 mmol NADH min�1 mg�1 total protein.

Results

Exogenous application of methanol stimulates AsA biosynthesis in

Oncidium PLB cultures

To study the effect of the MeOH dosage on AsA biosynthesisin Oncidium, 10–500 mM MeOH was applied exogenously toOncidium PLB culture. The endogenous AsA level in tissues wasmeasured at 6, 12, 24 and 30 h after MeOH application. As shownin Fig. 1A, application of MeOH resulted in varied AsA levels in thePLB cultures. In general, the AsA level preferentially decreasedduring the first 6 h of incubation, then showed an irreversibleresponse to various concentrations of MeOH. Notably, the PLBculture was lethally affected by 500 mM MeOH (Fig. 1B), and theAsA level was markedly decreased. Upon treatment with 50 mMMeOH, the AsA level of the Oncidium PLB culture increasedfollowing 24 h of inoculation. Thus, a 50 mM MeOH concentrationwas concluded to be appropriate for signaling AsA biosynthesis inOncidium.

Characterization of AsA induction by MeOH stimulation

To unravel the mechanism of AsA induction after 50 mMMeOH stimulation, several AsA-inducing compounds (Daveyet al., 1999), such as D-galacturonate (D-GalUA) and L-galactose(L-Gal), were applied to the PLB culture and their effects werecompared (Fig. 2A and B). The AsA levels in the PLB cultureincreased by MeOH (50 mM), D-GalUA (50 mM), and L-Gal(50 mM) treatment (Fig. 2A); however, treatment of MeOHalone significantly decreased the AsA level during the first 6 h ofinoculation. Interestingly, assays of the AsA redox state (reducedform AsA/oxidized form AsA) in the PLB culture showed a similarpattern to that of AsA level (Fig. 2B). The distinct variation of theAsA profile with MeOH application suggests that MeOH isdeleterious to Oncidium cells. The level of H2O2 significantlyincreased from 27.8 to 39.1 mM with MeOH application during thefirst 6 h of treatment (Table 1), whereas no significant effectswere observed by the other chemicals, such as L-Gal and D-GalUA.Detoxification of MeOH during the first 6 h of treatment isimportant for the up-regulation of AsA-related genes. Moreover,MeOH has a distinct effect of H2O2 generation when applying toOncidium culture.

Methanol enhances AsA levels by up-regulating AsA-biosynthesis and

defense genes

Since the application of 50 mM MeOH to Oncidium PLB culturewas effective in elevating the AsA level (Fig. 2A), we investigatedthe effect of 50 mM MeOH on the expression level of AsA-biosynthetic genes in the GalUA pathway, such as polygalactur-onase (OgPG), pectin methylesterase (OgPME) and galacturonatereductase (OgGalUAR), as well as those in the Smirnoff–Wheelerpathway, such as GDP-D-mannose pyrophosphorylase (OgGMP)and galactose dehydrogenase (OgGalDH). The RT-PCR data showedthat OgPG and OgPME, which are involved in pectin degradation,

C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407402


were both up-regulated after 6 h of MeOH treatment. However,the expression of OgPME was decreased at 24 h. In contrast,no further changes in the expression level of OgGalUAR by MeOHtreatment were observed (Fig. 3). On the other hand, both OgGMP

and OgGalDH of the Smirnoff–Wheeler pathway were up-regulated during the first 6 h of MeOH treatment, effectslasting for 30 h (Fig. 3). This is similar to the effect by L-Galstimulation, which acts as a carbon source, similar to D-GalUAin AsA-biosynthetic routes (Fig. 3; Davey et al., 1999). Finally,the expression of galactono-1,4-lactone dehydrogenase(OgGalLDH), an integrator of the AsA biosynthetic pathway,displayed an enhanced level upon MeOH treatment (Fig. 3). Inaddition, the levels of defense genes, including ascorbateperoxidase (OgAPX) and monodehydroascorbate reductase(OgMDHAR), were also increased at 6–24 h after MeOHtreatment (Fig. 3). Taken together, a 50 mM MeOH treatmentwas effective to enhance the expression level of most AsA-relatedgenes in the GalUA pathway, Smirnoff–Wheeler pathway anddefense system.

To further understand the proteins associated with the AsA-related genes under 50 mM MeOH stimulation, their enzymaticactivities were assayed. As shown in Fig. 4, the activities of OgPG,OgMDHAR, OgAPX and OgSOD were specifically enhanced from 6to 12 h upon MeOH treatment, whereas other enzymes, such asOgGalUAR, OgGMP, OgGalDH, OgGalLDH, were not significantlyenhanced by MeOH stimulation, even though they were enhancedin RNA levels. These results indicated that mRNA levels of many ofthese genes are not correlated with enzymatic activities, whichmay be related to post-translational modifications.

The pectin content of the Oncidium PLB culture was decreasedin 50 mM MeOH treatment, but not in L-Gal or D-GalUA treatment(Table 1). The degradation appeared to result mainly from theelevated activity of OgPG under MeOH stimulation (Fig. 4). Inconclusion, the AsA level was elevated in Oncidium PLB culture byMeOH stimulation, primarily because of the enhanced expressionlevel and enzymatic activity of OgPG. Although the mRNA levelsof a number of AsA-biosynthetic genes were certainly inducedand enhanced, their functional contribution in AsA biosynthesis is

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0 6 12 18 24 30Time (hours)

µmol

e A

sA/g

F.W

.

CK (½ MS)

50 mM L-Gal

50 mM D-GalUA

50 mM D-GalUA + 50 mM MeOH

50 mM MeOH

Time (hours)

AsA

Red

ox r

atio

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 6 12 18 24 30

CK (½ MS)

50 mM L-Gal

50 mM D-GalUA

50 mM D-GalUA +50 mM MeOH

50 mM MeOH

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 represent

standard deviations of the means obtained from three independent experiments. 50 mM MeOH (&), 50 mM D-galacturonate (D-GalUA) (B), 50 mM MeOH and 50 mM

D-GalUA (W), 50 mM L-galactose (L-Gal) (J) and 1/2 MS (m).

0

0.1

0.2

0.3

0.4

0.5

0Time (hours)

50 mM MeOH

½MS10 mM MeOH

100 mM MeOH

500 mM MeOH

½ MS

500 mMMeOH

50 mMMeOH

2mm

µmol

e A

sA /g

F.W

.

10 20 30

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 from

three 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 no

effects. All Oncidium PLB cultures were photographed after 12 h treatment.



unclear due to the absence of increased enzymatic activity withMeOH treatment.

Hydrogen peroxide production in Oncidium PLB cultures through the

activation of alcohol oxidase and NADPH oxidase under MeOH

stimulation

Plant cells are able to convert MeOH to formaldehyde andH2O2 by alcohol oxidase (Gout et al., 2000). The H2O2 level was

elevated from 27.8 to 39.1 mM in Oncidium PLB cultures inresponse to exogenous application of MeOH, but not D-GalUA andL-Gal, during the first 6 h of treatment (Table 1). To unravel thesource of H2O2 production, we applied hydroxylamine (1 mM)and DPI (5 mM), inhibitors of alcohol oxidase and NADPH oxidase,respectively, with MeOH in PLB cultures, and monitored the H2O2

levels. In Oncidium PLB cultures incubated with 50 mM MeOH, anearly H2O2 burst (�45 mM) was detected during the first 30 min,followed by a subsequent decrease in accumulation (�40 mM)that lasted for another 6 h (Fig. 5). However, this H2O2 burstwas attenuated by incubation with 50 mM MeOH combinedwith 1 mM hydroxylamine or 5 mM DPI. The DPI inhibitor wasmore effective in blocking H2O2 generation than the alcoholoxidase inhibitor. NADPH oxidase could play a more significantrole in the systemic production of H2O2 than alcohol oxidase does.Therefore, the stimulation of the H2O2 level by MeOH in theOncidium culture occurs directly, through MeOH metabolism (ordetoxification) by alcohol oxidase activity, and indirectly, throughthe subsequent induction of NADPH oxidase activity to amplifyH2O2 production. Moreover, the early oxidative peak of the H2O2

level in the Oncidium culture could be largely due to theconversion of MeOH by alcohol oxidase, and the later H2O2

burst could result primarily from NADPH oxidase activation(Fig. 5).

The up-regulation of AsA-related genes stimulated by MeOH is

through H2O2 signal transduction

To confirm the potential signaling effects of H2O2 on theexpression of AsA-related genes, we investigated the expressionof AsA-related genes under the application of the inhibitors aloneor with MeOH. As shown in Fig. 6, the expression of AsA-relatedgenes did not change after 6 h with 1 mM hydroxylamineor 5 mM DPI treatment. However, the expressional levels ofAsA-related genes were lower with MeOH combined withhydroxylamine or DPI than with MeOH alone. Hydroxylamineand DPI inhibited H2O2 production (Fig. 5), consequently reducingthe MeOH effect on the expression of AsA-related genes inOncidium PLB cultures (Fig. 6). The results suggest that H2O2

signaling is critical in up-regulating the expression of AsA-relatedgenes.

Table 1H2O2 amount and pectin concentration of PLBs incubated with various treatments.

Treatments (h) H2O2 (mM) Pectin (mg/g F.W.)

CK (1/2 MS)

0 28.2972.83 31.4670.64

6 28.5771.98 32.2070.93

12 28.9271.73 30.8571.13

24 26.5071.24 31.6571.67

30 28.6872.32 30.4571.01

50 mM L-Gal

0 28.1272.44 31.2171.03

6 28.3972.58 30.7070.55

12 28.3571.33 29.9870.51

24 30.0071.42 30.1370.57

30 28.3872.32 30.8870.58

50 mM D-GalUA

0 26.8672.81 30.0970.56

6 28.7571.85 30.0770.56

12 28.8572.15 30.5670.93

24 27.4772.72 31.4371.16

30 27.7371.89 30.9570.16

50 mM MeOH

0 27.7571.72 30.9270.58

6 39.1471.28 26.4270.96

12 33.1471.52 25.6570.58

24 31.4371.30 26.0271.45

30 30.7471.61 27.5771.00

50 mM D-GalUA+50 mM MeOH

0 29.0372.09 30.8670.16

6 40.1171.28 26.8470.99

12 34.6171.05 25.4871.14

24 32.4672.89 26.9471.58

30 28.8270.98 27.3570.43

PLBs=protocorm-like bodies; F.W.=fresh weight. Mean values7S.E. were

obtained from three independent experiments.

CK50 mM L-Gal

50 mM D-GalUA

50 mM D-GalUA

+50 mM MeOH 50 mM MeOH

OgPG

OgPME

OgGalUAR

18S rRNA

OgGMP

OgGalDH

OgGalLDH

OgMDHAR

OgAPX

D-Glc-6-P

D-Man-1-P

GDP- D-ManL-Gal

L-GalL

Ascorbate

L-GalA

MeGalUA

D-GalUA

Pectin

H2O2 H2O

MDHA

Smirnoff-Wheelerpathway

GalUA pathway

AsA recycling

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

Fig. 3. Expression of AsA-related genes on treatment of Oncidium PLB cultures with various compounds. The relative amount of transcripts of OgPME, OgPG and OgGalUAR

in 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.



Discussion

Methanol (MeOH) is known as a deleterious by-productderived from pectin demethylation during the cell wall recon-struction process in plants. Its effects on plant growth in Vigna

radiata were reported 20 years ago (Bhattacharya et al., 1985).Although the effects of MeOH on plant physiology and geneexpression have been investigated (Gout et al., 2000; Galbally andKirstine, 2002; Downie et al., 2004), its mechanism of signaltransduction mechanism has not been elucidated. As previouslyreported in Arabidopsis, AsA-biosynthetic genes in the Smirnoff–Wheeler pathway and AsA-recycling and pectin degradationgenes were all stimulated by an appropriate concentration ofMeOH (Downie et al., 2004; Ramırez et al., 2006), but genesrelated to photosynthesis were not responsive to MeOHapplication (Downie et al., 2004).

In the present study, exogenous application of MeOH (50 mM)to Oncidium PLB cultures increased the AsA level by 30% (Figs. 1and 2) and AsA-related genes were markedly up-regulated

(Fig. 3). Moreover, the H2O2 level was elevated after 30 mintreatment and maintained for at least 6 h (Fig. 5). By addinghydroxylamine or DPI compounds with MeOH into the Oncidium

PLB cultures, the activities of alcohol oxidase and NADPH oxidasewere inhibited. Accordingly, H2O2 production was markedlydecreased in PLB cultures by 8 to 20% (Fig. 5). In addition, NADPHoxidase was more effective than alcohol oxidase in producingH2O2, because the inhibition of NADPH oxidase activity by DPI hada greater effect on the H2O2 level (�20%) than inhibition ofalcohol oxidase activity (�8%) (Fig. 5). Thus, the H2O2 level wasenhanced by MeOH stimulation through two steps: MeOHoxidation by alcohol oxidase and systemic amplification byNADPH oxidase. In addition, the diminished H2O2 level withinhibition of alcohol oxidase and NADPH oxidase caused a reducedexpression of AsA-related genes (Fig. 6). In conclusion, the resultsstrongly suggest that H2O2 acts as a signaling messenger toregulate AsA-related gene expression under MeOH stimulation.

Hydrogen peroxide is a reactive oxygen species (ROS)produced by plants under stress conditions (Mittler et al., 2004).

0

10

20

30

40

50

60

1260

1

23

45

67

8

1

23 4

5

6

7

81

2

3

45

6

7

8A

ctiv

ity U

nit

Time (hours)

1

2

3

4

5

6

7

8

9

9

9 9

OgPG

OgPME

OgGalUAR

OgGMP

OgGalDH

OgGalLDH

OgMDHAR

OgAPX

OgSOD

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 vertical

bars represent standard deviations of the means obtained from three independent experiments.

Time (hours)

20.00

25.00

30.00

35.00

40.00

45.00

50.00

0 1 2 3 4 5 6

CK (½ MS)

50 mM MeOH

½ MS + 1 mM hydroxylamine½ MS + 5 mM DPI

50 mM MeOH + 1 mM hydroxylamine

50 mM MeOH + 5 mM DPI

µM H

2O2

Fig. 5. The effects of hydroxylamine (inhibitor of alcohol oxidase) and diphenyleneiodonium chloride (DPI; inhibitor of NADPH oxidase) on H2O2 production in Oncidium

PLB cultures for 6 h. Vertical bars represent standard deviations of the means obtained from three independent experiments.



Induced H2O2 can act as a local signal for hypersensitive cell deathand as a diffusible signal for the induction of defense genes inadjacent cells (Alvarez et al., 1998). The functional roles arecomplicated and diversified. Therefore, the induction of the plantdefense system is tightly controlled for its production andscavenging. In several model systems of plants, the oxidativeburst and accumulation of H2O2 appear to be mediated by theactivation of a membrane-bound NADPH oxidase complex (Zhanget al., 2007; Konigshofer et al., 2008; Wen et al., 2008). In theOncidium system, H2O2 induction has been identified as a signal toinduce AsA-related genes during vegetative growth (Shen et al.,

2009). The enzymatic activities (but not the mRNA levels)of some AsA-biosynthetic genes, such as OgGMP, OgGalDH,OgGalLDH and OgGalUAR, were not enhanced by MeOH stimula-tion, indicating regulation based on post-translational modifica-tions. However, the AsA level is eventually increased in responseto MeOH. These enzymatic activities are not critical for AsAsynthesis in AsA-biosynthetic pathway experiencing the effects ofMeOH. On the other hand, the increased activity of OgPG isnecessary for AsA production as well as the oligogalacturonic acid(OGA) product, a ligand to induce H2O2 generation after MeOHtreatment. In addition, AsA-recycling enzymes, OgAPX and

CK 1 mM

hydroxylamine5 mM

DPI

50 mM MeOH+

5 mM DPI

50 mM MeOH+

1 mM hydroxylamine

50 mM MeOH

OgPG

OgPME

OgGalUAR

18S rRNA

OgGMP

OgGalDH

OgGalLDH

OgMDHAR

OgAPX

D-Glc-6-P

D-Man-1-P

GDP- D-ManL-Gal

L-GalL

Ascorbate

L-GalA

MeGalUA

D-GalUA

Pectin

H2O2 H2O

MDHA

Smirnoff-Wheelerpathway

GalUA pathway

AsA recycling

0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 0 2 6 h

Fig. 6. Expression of AsA-related genes on treatment with H2O2-producing inhibitors. Total RNA was isolated from PLBs incubated with hydroxylamine or DPI. The relative

amount of transcripts for OgPME, OgPG and OgGalUAR in 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.

PG

Pectin

PME

MeOH D-GalUA

H2O2

NADPH Oxidase

H2O2

Defense genes (APX,MDHAR, etc)

Smirnoff-Wheeler pathway

GalUA pathway (pectin degrad. -PG,PME)AsA

GalUA pathway

PM

alcoholoxidaseformaldehyde

formate

Calvin-Benson cycle

+ OGA +

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 pectin

degradation 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 cell

from stresses. APX=ascorbate peroxidase; MDHAR=monodehydroascorbate reductase.



OgMDHAR, are essential for scavenging ROS. OgSOD can functionin dismutating superoxide (which was produced by NADPHoxidase) into H2O2 (Mittler et al., 2004). Their increased activityis indeed beneficial to plant cells undergoing MeOH stimulation.

Both methyl-galacturonate (MeGalUA) and D-GalUA are keyintermediates in the GalUA pathway (Fig. 3) one of the AsA-biosynthetic routes in planta (Smirnoff, 2003). D-GalUA is convertedfrom MeGalUA by pectin methylesterase in plant cells, with MeOHbeing produced as a byproduct. MeGalUA, an upstream inter-mediate in the GalUA pathway, was more effective in enhancingthe AsA level in Arabidopsis cultures than D-GalUA, a downstreamintermediate in the GalUA pathway (Davey et al., 1999). In ourstudy, the application of D-GalUA in Oncidium PLB cultures led toincreased AsA levels (Fig. 2) but had no effect on the expression ofAsA-related genes, such as OgGalUAR and OgGMP (Fig. 3). A possibleexplanation for the contrasting MeGalUA and D-GalUA effects isthat degradation of MeGalUA to D-GalUA can produce MeOH andinduce AsA-related gene expression, whereas D-GalUA acts only asa carbon source in AsA biosynthesis. Therefore, the MeOH effectderived from MeGalUA conversion is critical for AsA biosynthesis.

We present a model for the enhanced expression of AsA-related genes in Oncidium PLB cultures in response to MeOHstimulation (Fig. 7). Methanol is generated from pectin degra-dation of the plant cell wall through the activation of PME duringcell wall extension (Fall and Benson, 1996). The production ofpoisonous MeOH induces rapid detoxification into formaldehydeand H2O2 by alcohol oxidase. The H2O2 molecules initiallygenerated from MeOH oxidation may also have a feedbackmechanism to enhance PG and PME expression (Bergey et al.,1999). As a consequence, the levels of pectin component aredecreased in response to the MeOH effect (Table 1). In thesubsequent step of pectin degradation by PG, an OGA byproductcan also act as a ligand to enhance NADPH oxidase activity (Ridleyet al., 2001). Moreover, OGA fragments produced from cell wallscould increase the expression of many defense genes throughH2O2 signal transduction (Ridley et al., 2001; Aziz et al., 2004).Eventually, more H2O2 was produced to induce the network of theAsA-biosynthetic genes, by Smirnoff–Wheeler and GalUApathways, and defense genes, such as APX and MDHAR (Fig. 7).

In summary, we demonstrate that H2O2 is a secondarymessenger for inducing AsA-related gene expression in Oncidium

PLB cultures in response to MeOH stimulation. The induced genecascade from MeOH stimulation to AsA-related gene expression inplants is a defensive response to MeOH toxification, involving H2O2

as a critical transduction signal in this complicated genetic network.

Acknowledgments

The authors are grateful to the National Science Council,Taiwan, for financial support granted to Dr. Kai-Wun Yeh underthe project NSC-95-2317-B-002-005. The authors also thank Dr.Ching-Huei Kao, Department of Agronomy, National TaiwanUniversity (NTU), for assistance in enzymatic activity assays;and Dr. Chao-Ying Chen, Department of Plant Pathology andMicrobiology, NTU, for assistance in H2O2 measurements.

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