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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/jplph
Hydrogen peroxide mediates the expression of ascorbate-related genes
in 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 in f o a b s t r a c t
Article history: We investigated the signaling role of hydrogen peroxide (H2O2) in regulating the ascorbate (AsA) level
Received 18 July 2009 after exogenous methanol (MeOH) application. The endogenous H2O2 and AsA levels as well as the
Received 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 of
Accepted 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 and
Keywords: galacturonate (GalUA) pathways. The increased expression of these genes could be blocked by the
Ascorbate addition of hydroxylamine, an inhibitor of alcohol oxidase (EC: 1.1.3.13), and diphenyleneiodonium
Hydrogen peroxide
chloride (DPI), an inhibitor of NADPH oxidase (EC: 1.6.3.1). Thus, the H2O2 generated by MeOH
Methanol
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 is
from the demethylation of pectin by pectin methylesterase (PME; highlighted by its use in C3 plants for photosynthetic productivity
EC: 3.1.1.11) for tightening of the cell wall, especially throughout (Nonomura and Benson, 1992). Methanol influences C3 plant
the early stage of leaf expansion (Fall and Benson, 1996). Some ´
growth under foliar spray or irrigation (Ramırez et al., 2006), but
MeOH emissions have also been observed during changes in has no effect on C4 plants. Foliar application of MeOH causes an
cell 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 of
and 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 the
von 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 that
the 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 (Downie
L-galactose; L-GalL, L-galactono-1,4-lactone; MDHAR, monodehydroascorbate et al., 2004). Most of the genes induced by MeOH function in
reductase; 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 with
methylesterase; 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: f92b42021@ntu.edu.tw (C.-H. Shen), ykwbppp@ntu.edu.tw defense, and RNA processing, but none are involved in
(K.-W. Yeh). photosynthesis. At 24- and 72-h MeOH treatment, the genes with
0176-1617/$ - see front matter & 2009 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2009.10.008

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C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407 401
the highest up-regulation are related to anthocyanin and and incubated at 37 1C for 1 h. The amounts of total and reduced
flavonoid metabolism. Additionally, genes encoding detoxification AsA were determined by monitoring the absorbance at 525 nm
proteins, including cytochrome P450s (EC: 1.14.15.6), glucosyl (A525), and the amount of oxidized AsA was calculated from the
transferase (EC: 2.4.1.-) and ascorbate peroxidase (EC: 1.11.1.11), 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 were
detoxification and signaling pathways are predominantly acti- performed as described (Wang et al., 2008). In brief, Oncidium
vated 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 being
systems has attracted interest recently for its potential impact on filtered, the residue was washed with 80% ethanol and dried to
both fundamental and applied plant science (Caddick et al., 1998; obtain alcohol-insoluble residues (AIRs). Starch was removed
von Dahl et al., 2006). Although many reports have described from the AIRs by suspension in 90% dimethylsulfoxide for 16 h at
effects of MeOH on metabolism and biochemistry, information on 20 1C and centrifugation at 20,000g for 20 min. The pectic
the regulatory mechanisms of gene expression and MeOH- polysaccharide was extracted from the starch-free AIRs by stirring
induced 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. The
floral industry. The plant requires more than one year of a supernatant was collected, and ethanol was added at five times
vegetative 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 four
for 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 different
for 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 performed
ascorbate (AsA) metabolism of the Oncidium orchid revealed that at 4 1C. Oncidium PLB cultures were powdered in liquid nitrogen
the expression of genes involved in the AsA-biosynthetic path- and further homogenized with 100% MeOH. The supernatant was
ways and AsA recycling was affected by MeOH application in PLB obtained from 20 min of centrifugation at 5000g and 4 1C, and
tissues. 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-dichlorofluorescein
reduction or oxidation. Our results suggest that H2O2, a byproduct diacetate (H2DCF-DA) was added to the extract at a final
of MeOH oxidation, is a secondary signal in regulating associated concentration of 5 mM. Fluorescence was measured by the use
gene 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 methods
Plant materials and chemicals
RT-PCR analysis
Oncidium hybrid ‘‘Gower Ramsey’’ (Oncidium Goldiana x
Oncidium Guinea Gold) was obtained from the Shih–Dong orchid Total RNA for one-step RT-PCR analysis was extracted from
nursery in Taiwan. Oncidium protocorm-like bodies (PLBs) were the Oncidium PLB cultures. One microgram of total RNA was used
cultured in ½ Murashige and Skoog medium (Murashige and as the template in RT-PCR with the following forward and reverse
Skoog, 1962) under long-day conditions (16-h light/8-h dark primers for OgPME (ACJ02103), PME-F-50 -GCTCAAGCTT TGTTCTAT
cycles) at 2372 1C (Liau et al., 2003). L-Galactose (L-Gal), GGT-30 /PME-R-50 -AAAGAAAAAACAAGATAAAATATAGC-30 ; OgPG
D-galacturonate (D-GalUA), methanol (MeOH), diphenyleneiodo-
(A BV24998; EC: 3.2.1.15), PG-F-50 -ACGGCGGTGGCGGCAGAGGA-
nium chloride (DPI) and hydroxylamine were purchased from 30 /PG-R-50 -ACACTG CCCCTGCCCTCTATAGTGCC-30 ; OgGalUAR
Sigma 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 1.1.1.122), 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 1.3.2.3), GalLDH-F-50 -TCAAAGAGCACGGGCTTACG-30 /GalLDH-R-50 -
Oncidium PLB cultures, two weeks after subculture from stock AGGGGAAACCTCCATTGTTCC-30 ; OgAPX (FJ237035), APX-F-50 - TG
culture. The extraction and measurement of AsA were performed GCACTCGGCTGGGACTTACGATGT-30 /APX-R-50 -GTGGTCGGAACCTTTG
as 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 -CGAGTTGAGGCGA
homogenized in liquid nitrogen and then mixed well with 1 mL of GTAGAGCACGTTGA-30 . A one-step RT-PCR kit was used for processing
6% 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 and
of oxidized AsA. After being incubated at room temperature for OgGalUAR amplification; and 20 cycles for OgGMP for amplification
10 min, the mixture was supplemented with 0.5% N-ethylmalei- (one cycle consisted of 94 1C for 30 s for denaturation, 47–66 1C for
mide to remove the excess dithiothreitol. By contrast, to assay 30 s for annealing depending on the genes, and 72 1C for 30 s for
reduced 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 as
trichloroacetic acid, 43% H3PO4, 4% a–a0 -bipyridyl and 3% FeCl3) described above.

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Enzyme 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 protein
APX 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: 1.15.1.1) were measured following modified methods of pH 7.4). The SOD activity was determined from the decrease in
Eltayeb et al. (2007) and Paoletti et al. (1986), respectively. A340 by the oxidation of NADH. One unit of SOD was defined as the
Oncidium 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 M
Na2HPO4 and 1 M NaCl, pH 5.0), those for PG activity assay were
ground in extraction buffer (1 M NaCl, and 0.2 M Na2HPO4 in 1 M Results
citrate buffer, pH 4.0) and those for the APX activity assays were
ground in extraction buffer (2.5 mL of 25 mM potassium phos- Exogenous application of methanol stimulates AsA biosynthesis in
phate buffer, pH 7.8) containing 2% polyvinylpolypyrrolidone, Oncidium PLB cultures
0.4 mM EDTA and 1 mM AsA. For the other activity assays, the
pseudobulbs were ground in extraction buffer (50 mM sodium To study the effect of the MeOH dosage on AsA biosynthesis
phosphate buffer, pH 7.2, 2 mM EDTA, 2 mM dithiothreitol, 20% in Oncidium, 10–500 mM MeOH was applied exogenously to
glycerol and 2% polyvinylpolypyrrolidone. After centrifugation for Oncidium PLB culture. The endogenous AsA level in tissues was
30 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 shown
crude enzyme. For the PME assay, the crude protein was mixed in Fig. 1A, application of MeOH resulted in varied AsA levels in the
with reaction buffer (0.1% esterified pectin in 0.2 M Na2HPO4 PLB cultures. In general, the AsA level preferentially decreased
buffer, pH 6.3). After overnight incubation at 37 1C, 0.05% during the first 6 h of incubation, then showed an irreversible
ruthenium red was added and mixed before incubation for response to various concentrations of MeOH. Notably, the PLB
10 min. Next, 0.6 M CaCl2 was added to precipitate the demethy- culture was lethally affected by 500 mM MeOH (Fig. 1B), and the
lated pectin. The mixture was centrifuged at 14,000g for 15 min to AsA level was markedly decreased. Upon treatment with 50 mM
remove the precipitate. The absorbances at 534 nm (A534) of the MeOH, the AsA level of the Oncidium PLB culture increased
supernatants of the samples were measured. For the PG assay, the following 24 h of inoculation. Thus, a 50 mM MeOH concentration
crude protein was mixed with reaction buffer (1% cyanoacetamide was concluded to be appropriate for signaling AsA biosynthesis in
in 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 the
production of galacturonic acid. One unit of PG was defined as the
Characterization of AsA induction by MeOH stimulation
activity 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 mM
buffer (0.1 mM NADPH and 0.1 mM galacturonic acid in 50 mM
MeOH stimulation, several AsA-inducing compounds (Davey
sodium phosphate buffer, pH 7.2) for 1 min. The GalUAR activity
et al., 1999), such as D-galacturonate (D-GalUA) and L-galactose
was determined from the increase in A254 by the production of
(L-Gal), were applied to the PLB culture and their effects were
NADP + . One unit of GalUA reductase was defined as the activity
compared (Fig. 2A and B). The AsA levels in the PLB culture
that 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-Gal
GMP 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 of
mannose in 50 mM Tris–HCl buffer, pH 7.0). The reaction was
inoculation. Interestingly, assays of the AsA redox state (reduced
started by serially adding 12 U of hexokinase, 3 U of glucose-
form AsA/oxidized form AsA) in the PLB culture showed a similar
6-phosphate dehydrogenase and 1 mM sodium pyrophosphate.
pattern to that of AsA level (Fig. 2B). The distinct variation of the
The GMP activity was monitored by measuring the A340 to
AsA profile with MeOH application suggests that MeOH is
monitor the formation of NADH. One unit of GMP was defined as
deleterious to Oncidium cells. The level of H2O2 significantly
that 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 the
GalDH 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 is
from 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 to
min 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 and
4.2 mM L-galactono-1,4-lactone in 0.01 M potassium phosphate
buffer, pH 7.8). The GalLDH activity was determined from the Methanol enhances AsA levels by up-regulating AsA-biosynthesis and
increase in A550 by the reduction of cytochrome c. One unit of defense genes
GalLDH 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 culture
activity, 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 galacturonate
defined as the activity that oxidized 1 nmol NADH min À 1 mg À 1 reductase (OgGalUAR), as well as those in the Smirnoff–Wheeler
total 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 showed
0.25 mM AsA, 0.4 mM EDTA and 0.1 mM H2O2). The APX activity that OgPG and OgPME, which are involved in pectin degradation,

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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 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.
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 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).
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 enzymatic
no 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 6
and OgGalDH of the Smirnoff–Wheeler pathway were up- to 12 h upon MeOH treatment, whereas other enzymes, such as
regulated during the first 6 h of MeOH treatment, effects OgGalUAR, OgGMP, OgGalDH, OgGalLDH, were not significantly
lasting for 30 h (Fig. 3). This is similar to the effect by L-Gal enhanced by MeOH stimulation, even though they were enhanced
stimulation, which acts as a carbon source, similar to D-GalUA in RNA levels. These results indicated that mRNA levels of many of
in AsA-biosynthetic routes (Fig. 3; Davey et al., 1999). Finally, these genes are not correlated with enzymatic activities, which
the 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 decreased
displayed an enhanced level upon MeOH treatment (Fig. 3). In in 50 mM MeOH treatment, but not in L-Gal or D-GalUA treatment
addition, the levels of defense genes, including ascorbate (Table 1). The degradation appeared to result mainly from the
peroxidase (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 by
treatment (Fig. 3). Taken together, a 50 mM MeOH treatment MeOH stimulation, primarily because of the enhanced expression
was effective to enhance the expression level of most AsA-related level and enzymatic activity of OgPG. Although the mRNA levels
genes in the GalUA pathway, Smirnoff–Wheeler pathway and of a number of AsA-biosynthetic genes were certainly induced
defense system. and enhanced, their functional contribution in AsA biosynthesis is

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404 C.-H. Shen, K.-W. Yeh / Journal of Plant Physiology 167 (2010) 400–407
unclear due to the absence of increased enzymatic activity with elevated from 27.8 to 39.1 mM in Oncidium PLB cultures in
MeOH 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 H2O2
stimulation 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 combined
Table 1 with 1 mM hydroxylamine or 5 mM DPI. The DPI inhibitor was
H2O2 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 H2O2
PLBs= protocorm-like bodies; F.W.= fresh weight. Mean values7 S.E. were
signaling is critical in up-regulating the expression of AsA-related
obtained 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 recycling
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.

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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 vertical
bars 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 Oncidium
PLB 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 Oncidium
derived from pectin demethylation during the cell wall recon- PLB cultures, the activities of alcohol oxidase and NADPH oxidase
struction process in plants. Its effects on plant growth in Vigna were inhibited. Accordingly, H2O2 production was markedly
radiata were reported 20 years ago (Bhattacharya et al., 1985). decreased in PLB cultures by 8 to 20% (Fig. 5). In addition, NADPH
Although the effects of MeOH on plant physiology and gene oxidase was more effective than alcohol oxidase in producing
expression have been investigated (Gout et al., 2000; Galbally and H2O2, because the inhibition of NADPH oxidase activity by DPI had
Kirstine, 2002; Downie et al., 2004), its mechanism of signal a greater effect on the H2O2 level ( À20%) than inhibition of
transduction mechanism has not been elucidated. As previously alcohol oxidase activity ( À 8%) (Fig. 5). Thus, the H2O2 level was
reported in Arabidopsis, AsA-biosynthetic genes in the Smirnoff– enhanced by MeOH stimulation through two steps: MeOH
Wheeler pathway and AsA-recycling and pectin degradation oxidation by alcohol oxidase and systemic amplification by
genes 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 reduced
related to photosynthesis were not responsive to MeOH expression of AsA-related genes (Fig. 6). In conclusion, the results
application (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).

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406 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 recycling
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.
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 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.
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 response
scavenging. In several model systems of plants, the oxidative to MeOH. These enzymatic activities are not critical for AsA
burst and accumulation of H2O2 appear to be mediated by the synthesis in AsA-biosynthetic pathway experiencing the effects of
activation 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 acid
Oncidium system, H2O2 induction has been identified as a signal to (OGA) product, a ligand to induce H2O2 generation after MeOH
induce AsA-related genes during vegetative growth (Shen et al., treatment. In addition, AsA-recycling enzymes, OgAPX and

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The authors are grateful to the National Science Council, lacturonide-related signaling. Phytochemistry 2001;57:929–967.
Shen CH, Krishnamurthy R, Yeh KW. Decreased L-ascorbate content mediating
Taiwan, for financial support granted to Dr. Kai-Wun Yeh under bolting is mainly regulated by the galacturonate pathway in Oncidium. Plant
the project NSC-95-2317-B-002-005. The authors also thank Dr. Cell Physiol 2009;50:935–946.
Ching-Huei Kao, Department of Agronomy, National Taiwan Smirnoff N. Vitamin C booster. Nat Biotechnol 2003;21:134–136.
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