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First published online July 17, 2003; 10.1104/pp.103.022277 Plant Physiology 132:2248-2255 (2003) © 2003 American Society of Plant Biologists Enhanced Formaldehyde Detoxification by Overexpression of Glutathione-Dependent Formaldehyde Dehydrogenase from Arabidopsis1Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad Autónoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1  ) by in vivo deletion of the SFA1 gene that
codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this
mutant confers high resistance to formaldehyde added exogenously, which
demonstrates the functional conservation of the enzyme through evolution and
supports its essential role in formaldehyde metabolism. To investigate the
role of the enzyme in plants, we have generated Arabidopsis transgenic lines
with modified levels of FALDH. Plants overexpressing the enzyme show a 25%
increase in their efficiency to take up exogenous formaldehyde, whereas plants
with reduced levels of FALDH (due to either a cosuppression phenotype or to
the expression of an antisense construct) show a marked slower rate and
reduced ability for formaldehyde detoxification as compared with the wild-type
Arabidopsis. These results show that the capacity to take up and detoxify high
concentrations of formaldehyde is proportionally related to the FALDH activity
in the plant, revealing the essential role of this enzyme in formaldehyde
detoxification.
Formaldehyde is a toxic compound produced during plant one-carbon (C1) metabolism. Most formaldehyde does not exist in vivo in a free state but is bound to endogenous nucleophiles, such as glutathione or tetrahydrofolate (Sardi and Tyihak, 1994  ;
Chen et al., 1997). The main
sources of formaldehyde in plants are dissociation of
5,10-methylenetetrahydrofolate and oxidation of methanol, derived mainly from
pectin demethylation (Fall and Benson,
1996). Formaldehyde can also arise from other oxidative
demethylation reactions (Sardi and Tyihak,
1994), from glyoxylate decarboxylation
(Prather and Sisler, 1972),
and from P-450-dependent oxidation of herbicides
(Clejan and Cederbaum, 1993).
Furthermore, formaldehyde might have an exogenous origin from industrial
waste, being a polluting agent of residual waters and of air
(Wolverton et al., 1989;
Giese et al., 1994). It is one
of the main indoor air pollutants, present in tobacco smoke, furniture,
industrial adhesives, and varnishes. It has been classified as a mutagen and
suspected carcinogen (Wippermann et al.,
1999) and as causing headaches and irritation of the mucosa.
Formaldehyde is considered a toxic compound because of its ability to react
with proteins, nucleic acids, and lipids. The World Health Organization has
established an air quality guideline of 100 µg m-3 for
formaldehyde (European Commission,
1994).
Several studies have demonstrated that exogenous formaldehyde can be
incorporated into the metabolism of photosynthetic cells and be used as a
carbon source. For example, feeding the common spider plant (Chlorophytum
comosun) with14C-formaldehyde resulted
in14C-labeled products derived from the C1 metabolism, such as Ser
and phosphatidylcholine (Giese et al.,
1994
S-formylglutathione is then hydrolyzed to formate and glutathione
by S-formylglutathione hydrolase:
Formate can give rise to C1 folates, but a quantitatively more important
fate is oxidation to CO2 mediated by formate dehydrogenase
(Cossins, 1964
In addition to formaldehyde, FALDH can also oxidize long-chain alcohols and
FALDH from Arabidopsis has been characterized, and its cDNA has been cloned
(Martínez et al.,
1996
In the present work, we have investigated the potential role of FALDH in
detoxification of exogenous formaldehyde. We have generated a yeast
(Saccharomyces cerevisiae) mutant strain (sfa1
Construction of a Yeast sfa1 Strain
To explore the physiological role of the Arabidopsis FALDH, we made
complementation experiments by transforming a yeast strain lacking FALDH
activity. This strain was constructed by in vivo deletion of the SFA1
gene, coding for the yeast glutathione-dependent FALDH
(Wehner et al., 1993
Arabidopsis FALDH cDNA was cloned into the yeast pYes2 expression vector, under the control of the GAL1-GAL10 promoter (ADH2-pYes2 plasmid), and introduced into the sfa1::HIS3 strain. The transformed strain was grown in the presence of Gal as carbon source and using His and uracil as markers. The selected colonies exhibited FALDH activity in the crude extracts, confirmed by the presence of a band of the expected size in electrofocusing gels stained by activity (Fig. 1A, lane 2), indicating both that it was correctly expressed and that it was functional in this heterologous system. To purify the enzyme from this source, we used three consecutive chromatographic steps. From 500 g of yeast cell pellet, 2.24 mg of purified protein was obtained that appeared as a single band of Mr 45,000 on SDS-PAGE (Fig. 2A). This band was recognized by an anti-rat FALDH antiserum (result not shown). The specific activity of the purified enzyme against S-hydroxymethylglutathione was found to be 15 units mg-1. Fold purification and recovery of the process are shown in Table I.
The purified enzyme was inoculated into rabbits to raise polyclonal antibodies. The antibodies recognized a single band of the expected size (45 kD) in Arabidopsis crude protein extracts that was not detected using the pre-immune sera (Fig. 2B, lanes 2 and 3, respectively). Those immunoreactions were specific because the binding of the antibodies could be competed with the protein used for immunization (Fig. 2B, lanes 4–9).
The Arabidopsis enzyme expressed in yeast showed a Km
value of 7 µM. The deduced turnover number
(Kcat) was 1,351 min-1, calculated for a
Mr = 90,000 (dimer), and the catalytic efficiency
(Kcat/Km) was 193,000
mM-1 min-1. The enzyme exhibited high
activity at pH 10 toward several plant alcohols, such as farnesol and
geraniol. However, at more physiological pH (pH 7.5), only farnesol showed a
significant activity, with a Kcat/Km
of 780 mM-1 min-1. Other medium-chain
alcohols and
The yeast sfa1::HIS3 strain could grow at the same rate as the wild type in rich medium but could not grow in the presence of 0.6 mM formaldehyde. In contrast, the wild-type yeast strain could grow in the presence of 0.6 mM formaldehyde, though after a long lag phase (data not shown). These results confirm the essential role of FALDH for yeast formaldehyde metabolism. The sfa1::HIS3 strain transformed with the ADH2-pYes2 plasmid was then grown both in the absence and in the presence of different concentrations of formaldehyde added exogenously. Figure 3A shows that both the wild type and the transformed strains grew equally well in the absence of formaldehyde, reaching saturation at around 35 h after subculturing. In contrast, at 1 mM formaldehyde, yeast wild type was unable to grow during the time of the experiment (60 h), whereas the transformed strain showed the same growth rate as in the absence of formaldehyde. At 2 mM formaldehyde, the transformed strain still grew very efficiently.
We also determined the kinetics of formaldehyde elimination from the culture medium (Fig. 3B). Our results show that exogenous formaldehyde disappeared very rapidly in the case of the transformed yeast strain and was completely metabolized in 50 to 60 h, which is consistent with the cell's growth rate shown in Figure 3A. However, in the case of the wild-type strain, the concentration of formaldehyde in the culture medium remained constant for long periods of time, which proves its inability to metabolize it efficiently and, as a consequence, to grow in the presence of this toxic compound.
We addressed the question of whether plants overexpressing FALDH might be able to cope better with moderately high concentrations of environmental formaldehyde. We generated transgenic Arabidopsis lines transformed with FALDH cDNA under the control of the 35S cauliflower mosaic virus promoter. A total of 40 independently transformed plants were obtained (T1 progeny) after infiltration of Columbia plants. The T1 plants were self-fertilized, and 16 individuals of the T2 progeny showing a Mendelian segregation of kanamycin resistance were brought to homozygosis (T3 generation). Western-blot analysis of seven individuals from the T3 generation is shown in Figure 4A. One of them is a cosuppression line (line 13), whereas the others show from a moderate to a high increase of FALDH protein levels. In the same figure, it can be seen that the increase in FALDH levels correlated well with increased values of FALDH enzymatic activity.
To test the ability of the transgenic plants to metabolize exogenous formaldehyde, we selected three of the homozygous lines (T3 generation) showing the highest expression of the transgene (lines 1, 4, and 6). Equal amounts of seeds (3 mg) were germinated and grown in liquid medium (5 mL), and formaldehyde at the desired concentration was added to 6-d-old plantlets. Subsequently, aliquots were taken to measure the variation of formaldehyde concentration during a time course. A control of liquid medium with formaldehyde but without plants was also performed to discard losses of formaldehyde by evaporation. At least three independent experiments were carried out with each line. At 2 mM formaldehyde, we observed significant differences in the rate for formaldehyde uptake (Fig. 5A). The three transgenic lines tested were able to completely detoxify the formaldehyde in 48 h, whereas Arabidopsis wild-type metabolized it at a slower rate (80% of the total in 48 h). In view of the fact that the plants could cope well with this concentration of exogenous formaldehyde (no visible defects were observed as a consequence of the addition of this compound), we decided to perform the same experiment using a concentration of 5 mM formaldehyde. As can be seen in Figure 5B, more significant differences were observed in this case. Line 1, showing the highest rate for formaldehyde uptake, could achieve a 50% decrease in formaldehyde concentration in the medium after 48 h (in contraposition to the 25% by the Arabidopsis wild type). This higher rate was evidenced very early (at 6 h, there was already a 13% differential rate), and subsequently increased (at 24 and 48 h, there was a 25% differential rate).
To corroborate the above data that suggest a correlation between the amount of FALDH expressed by the plant and its ability to detoxify exogenous formaldehyde, we performed the same experiments with transgenic lines showing reduced levels of FALDH. One line showing a cosuppression phenotype (line 13, Fig. 4A) and three lines bearing antisense constructs (lines 5a, 10a, and 17a, Fig. 4B) were incubated with 2 mM formaldehyde added exogenously. Aliquots from the culture medium were removed during a time course to measure the formaldehyde concentration. The results in Figure 5A demonstrate that the lines with reduced levels of FALDH show a 20% decrease in their ability to detoxify exogenous formaldehyde.
Plant FALDHs have been isolated from pea seeds (Uotila and Koivusalo, 1979 ;
Shafqat et al., 1996),
Arabidopsis (Martínez et al.,
1996), and maize (Zea mays;
Wippermann et al., 1999).
Their molecular and kinetic properties are remarkably similar to those found
in the homolog enzymes from mammals, invertebrates, and microorganisms,
providing evidence of the high degree of structural and functional
conservation through evolution. The enzyme shows very low expression levels in
Arabidopsis plants (Martínez et
al., 1996); thus, a system to produce a recombinant enzyme was
desirable. We have used a yeast expression system to purify the enzyme to
homogeneity and to study its functional significance in the detoxification of
exogenous formaldehyde.
Arabidopsis FALDH, purified as a recombinant enzyme from yeast, has a
specific activity of 15 units mg-1 and a Km
value of 7 µM. The polypeptide chain of the enzyme has a
molecular mass of 45 kD, calculated by SDS-PAGE, which is consistent with both
the molecular mass deduced from the cDNA sequence and with that of the enzyme
purified from Arabidopsis plants
(Martínez et al.,
1996
SFA1 deletion in yeast is not lethal but impairs its growing in
the presence of formaldehyde
(Fernández et al.,
1999 Additional support for the important role of FALDH in detoxifying formaldehyde was obtained by the generation of transgenic Arabidopsis lines. Three independent lines, exhibiting from 11- to 18-fold the wild-type FALDH activity, were used to test the capacity of plants overexpressing FALDH to take up and detoxify exogenous formaldehyde. We observed an increase in the detoxification rate of 25%, both at 2 and 5 mM formaldehyde (64 and 132.5 mL L-1, respectively). At 2 mM formaldehyde, transgenic plants could completely detoxify the exogenous formaldehyde in 48 h or less, without any visible damage. These results were corroborated by generating transgenic lines with decreased levels of FALDH enzyme (cosuppression and antisense lines). At 2 mM formaldehyde, these plants showed a 20% decrease in their ability to detoxify formaldehyde, as compared with the wild-type plants. At 5 mM formaldehyde, which is an extremely toxic concentration, the transgenic lines showed a good rate for formaldehyde uptake during the first 24 h. From this time on, we observed important phytotoxic effects, the leaves starting to display chlorotic patches that finally spread to the whole tissue. At the same time, the rate decreased significantly, and the plants could not achieve a complete detoxification of the exogenous formaldehyde. This phytotoxic effect of formaldehyde at such high concentration is not surprising and might be due to the formaldehyde itself or to the products of its oxidation. The chlorotic phenotype of the plants and the observation of leaf sections under electron microscopy (data not shown) suggest that chloroplasts are seriously damaged after long expositions to 5 mM formaldehyde. This might be due to a process of photooxidation caused by an accumulation of FALDH oxidation products that are toxic and have effects on the glutathione/redox homeostasis.
It has been reported that foliar application of methanol can stimulate
plant growth (Nonomura and Benson,
1992
In summary, our results show that overexpression of the enzyme FALDH in
plants confer a high resistance to formaldehyde, thus supporting the
"green liver" concept of plant xenobiotic metabolism
(Sandermann, 1992
Biological Material Yeast (Saccharomyces cerevisiae) W303D strain was grown at 30°C in Wickerham's medium (United States Biochemical Corp., Cleveland). Arabidopsis ecotype Columbia was grown in soil under a 16-h-light/8-h-dark regime at 22°C. For the formaldehyde treatments, plant seeds were surface sterilized and sown in six-well tissue culture clusters (Costar Corp., Cambridge, MA), containing 5 mL well-1 of sterile Murashige and Skoog media (Duchefa, Harlem, The Netherlands) supplemented with 0.5% (w/v) Suc and grown with shaking (150 rpm) under the same light regime as above.
A null mutant of the SFA1 gene, coding for the yeast FALDH, was
constructed by one-step gene replacement
(Rothstein, 1983
ADH2 cDNA was amplified using two primers based on the reported sequence
(Martínez et al.,
1996
Yeast sfa1::HIS3 strain transformed with the ADH2-pYes2 plasmid (50 L) was grown until the early stationary phase, and cell pellets were collected by centrifugation and resuspended in buffer A, containing 10 mM Tris/HCl (pH 7.5), 0.5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamidine (1:1 [w/v]). Cells were lysed with a beadbeater homogenizer (Biospec Products, Inc., Bartlesville, OK), using 0.5-mm diameter glass beads. The homogenate was centrifuged at 29,000g for 1 h, filtered through glass wool, and loaded onto a DEAE-Sepharose CL-6B column (36.5 x 4 cm) equilibrated with the buffer A. A 1,200-mL linear gradient of 0 to 0.25 M NaCl was used to eluate the bound enzyme. The active material was concentrated to 60 mL with Diaflo PM-10 membrane (Amicon, Billerica, MA), dialyzed against 10 mM KH2PO4 (pH 6.8), 0.5 mM DTT, 1 mM PMSF, and 1 mM benzamidine, and loaded onto a hydroxiapatite Bio-Gel HT (Bio-Rad Laboratories, Hercules, CA) column (16 x 2 cm) equilibrated with the same buffer. A 1,200-mL linear gradient of 10 to 400 mM KH2PO4 was applied, and the active fractions were collected, concentrated to 25 mL, dialyzed against buffer A, and loaded onto a Blue-Sepharose column (15.5 x 1.8 cm) equilibrated with the same buffer. The active material was eluted with a 600-mL linear gradient of 0 to 0.75 mM NADH, concentrated to 16 mL, and the excess of NADH was eliminated with a PD-10 gel filtration column (Amersham-Pharmacia Biotech, Uppsala) equilibrated with 10 mM Tris/HCl (pH 7.5) and 0.5 mM DTT. The active material was equilibrated with 20 mM Tris/acetate buffer (pH 6.6), 0.5 mM DTT, 1 mM PMSF, and 1 mM benzamidine, using a PD-10 gel filtration column, and loaded onto a Protein Pack Q 8HR column (Millipore, Billerica, MA) equilibrated with the same buffer. Bound proteins were eluted with a 40-mL linear gradient of 0 to 0.2 M sodium acetate. Protein concentrations were determined by the Bradford assay (Bio-Rad), using bovine serum albumin as a standard.
To prepare the sense construct, an EcoRI/XhoI restriction fragment containing the Arabidopsis FALDH coding sequence (Martínez et al., 1996 )
was purified and subcloned downstream of the cauliflower mosaic virus 35S
promoter into the SmaI site of pDH51
(Pietrzak et al., 1986). To
prepare the antisense construct, a PstI/BglII fragment (425
bp) was purified and subcloned into pDH51 cut with BamHI and
PstI. After verifying the correct orientation of the insert, the
resulting 35S::ADH2 expression cassettes were excised as
EcoRI restriction fragments and subcloned into the EcoRI
site of the Agrobacterium tumefaciens binary plasmid pBin19
(Bevan, 1984). The recombinant
plasmids were then introduced into A. tumefaciens C58CI strain by the
freeze-thaw method. Arabidopsis plants were transformed by the vacuum
infiltration method (Bechtold et al.,
1993). Transgenic plants were selected on Murashige and Skoog
solid media containing 40 mg L-1 kanamycin and later transferred to
soil for seed production. Transformants with pBin19 empty vector were also
produced to be used as controls. Segregation tests were performed with the
T2 seeds obtained by self-fertilization of independent primary
transformants. Homozygous, monogenic T3 seeds were obtained by
self-fertilization of T2 plants using kanamycin resistance as the
marker.
SDS-PAGE was performed as described
(Laemmli, 1970
Enzyme activity was determined spectrophotometrically at 25°C by
monitoring the production of NADH at 340 nm (
Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor. Received February 22, 2003; returned for revision March 24, 2003; accepted April 28, 2003.
1 This work was supported by the Dirección General de Enseñanza Superior (grant nos. PB96–1167 and PB98–0855), by the Comissionat per a Universitats i Recerca (grant no. 1999SGR 00103), and by the Commission of the European Union (grant no. BIO4–CT97–2123).  * Corresponding author; e-mail carmen.martinez{at}uab.es; fax 34–93–5811264.
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TOP
ABSTRACT
RESULTS
-hydroxy fatty acids, such as octanol and 12-hydroxydodecanoic acid.
Furthermore, it has been demonstrated recently that it is very active in the
reduction of S-nitrosoglutathione, the condensation product of
glutathione and NO (Jensen et al.,
1998
), 4
(
), and 6 (
) express high levels of FALDH. Line 13 (
) is a
cosuppression line. Lines 5a (
), 10a (
), and 17a (
) are
antisense lines. 
340 = 6.22
mM-1 cm-1) with a Hewlett-Packard 8452A diode
array spectrophotometer (Hewlett-Packard, Palo Alto, CA). In some experiments,
0.6 mM 3-acetylpyridine adenine dinucleotide (
