stress-signaling pathway [10], formate has also been reported to be a product of CO
2
reduction during some conditions of photosynthesis [7,11].
In barley Hordeum 6ulgare L., root FDH mRNA is induced by conditions of iron deficiency
or anaerobiosis [4]. In potato Solanum tuberosum L., leaf FDH mRNA is induced by hypoxia,
chilling, drought, mechanical wounding and pro- longed darkness [12]. The latter reference also
reported that foliar FDH mRNA was induced by spraying with 10 mM formate or 20 methanol
within 24 h of treatment.
The current report examines the degree to which the FDH enzymatic activity is induced in Ara-
bidopsis thaliana, a model plant for biochemical and genetic studies, by foliar spraying with formal-
dehyde as well as with either methanol or formate. These data indicate that FDH is induced by treat-
ment with all three one-carbon metabolites. We also cloned and sequenced the cDNA for the
Arabidopsis FDH and examined the kinetic char- acteristics of the enzyme to assess whether it may
act as a redox-sensing switch that controls whether formate is assimilated or dissimilated.
2. Methods
2
.
1
. Materials A. thaliana Columbia ecotype plants were
grown in a controlled environment chamber at 20°C, 50 relative humidity, a 16 h photoperiod
and a photon 400 – 700 nm fluence rate of : 300 mmol s
− 1
m
− 2
. Soil was subjected to steam steril- ization prior to use. In addition to regular water-
ing, plants were treated weekly with a nutrient solution containing 200 mg g
− 1
nitrogen.
2
.
2
. Plant treatments Plants were treated with a hand-held sprayer
and the indicated solution was applied until the leaf surface was wet. Experiments demonstrated
no difference between untreated plants and plants sprayed with water. Methanol and formate were
used for treatments at concentrations of 20 vv and 10 mM, respectively, based on their use at this
concentration in previous studies [12]. Formalde- hyde was also used at a concentration of 10 mM.
At no time did these concentrations appear to cause damage or stress to the plants.
2
.
3
. Enzyme assays For in vitro assays, leaf tissue was homogenized
in a Tenbroeck tissue grinder with a chilled solu- tion containing 50 mM Tris – Cl pH 8.6, 10 mM
NaCl, 1
mM MgSO
4
, 2
wv polyvinyl-
pyrrolidone and 0.1 vv 2-mercaptoethanol. The extract was filtered through Miracloth Cal-
biochem and centrifuged for 10 min at 40 000 × g to remove cell debris and insoluble material. The
supernatant fluid was fractionated by chromatog- raphy through a column of Sephadex G-25 equili-
brated with 50 mM Tris – Cl pH 8.6, 10 mM NaCl and 1 mM MgSO
4
. Formate dehydrogenase activity was measured
spectrophotometrically at 340 nm and 30°C in a volume of 1 ml containing 100 mM potassium
phosphate pH 7.0, 50 mM sodium formate and 1 mM NAD
+
. The response of activity to the con- centration of both formate and NAD
+
was hyper- bolic and followed the expected Michaelis –
Menten kinetics. To estimate the kinetic constants, the data were analyzed by a Linweaver – Burk plot.
To estimate the K
i
for NADH, the concentration of that metabolite was held constant while the
concentration of NAD
+
was varied, which pro- duced a classical pattern of competitive inhibition.
Formaldehyde dehydrogenase EC 1.2.1.1 activity was measured spectrophotometrically at 340 nm
and 30°C in a volume of 1 ml containing 50 mM Tris – Cl pH 8.6, 2 mM reduced glutathione, 1
mM MgSO
4
, 10 mM NaCl, 1.3 mM formaldehyde and 1 mM NAD
+
. For dehydrogenase enzymatic rates to be linear with time and amount of extract
added, it was necessary to use extracts chro- matographed on Sephadex G-25. The protein con-
centration in the extracts was determined using a dye-binding assay [13] with bovine serum albumin
as a standard protein.
2
.
4
. FDH purification For purification of the Arabidopsis FDH, leaf
tissue was ground in 50 mM NaPO
4
pH 7.0 buffer, centrifuged at 40 000 × g for 10 min and
the supernatant
fraction chromatographed
through Sephadex G-25. The enzyme was then chromatographed on a 5-AMP agarose column,
as previously described [14]. The specific activities of the leaf extract and the purified fraction were
1.62 and 453 nmol min
− 1
mg
− 1
of protein, respec- tively. We presume that the FDH was not purified
to homogeneity since its specific activity was sig- nificantly lower than the enzyme isolated from pea
leaves by a multi-step purification protocol [15].
2
.
5
. FDH cDNA The potato FDH sequence Accession No.
Z21493 was used to screen the Arabidopsis dbEST using BLAST [16]. Several ESTs were detected
and one was selected Clone 173L24 with homol- ogy to the N-terminal half of the potato sequence
to ensure identification of a full-length cDNA.
2
.
6
. RNA preparation and blot analysis Arabidopsis leaf tissue was rapidly frozen in
liquid nitrogen. Total RNA was extracted from 100 mg of frozen tissue using a QIAGEN
Chatsworth, CA RNeasy kit. For each experi- mental treatment, triplicate 4-mg samples of total
RNA were fractionated by electrophoresis on a formaldehyde gel [17] and blotted onto Hybond
N
+
membrane using an Ambion Austin, TX NorthernMAX-Plus kit. The membranes were
subsequently UV cross-linked. Transfer efficiency was assessed by UV shadowing of the membrane.
An identical set of samples along with a known RNA standard Sigma, St. Louis, MO was frac-
tionated on an identical formaldehyde gel. RNA was visualized by staining with ethidium bromide.
Relative abundance of 25S and 18S rRNA bands was quantitated using the public domain NIH
Image program to permit normalization of the amounts of RNA loaded.
The full-length cDNA for Arabidopsis FDH was randomly labeled with [a-
32
P]dATP. The labeled probe
2 × 10
6
dpm ml
− 1
was hybridized
overnight at 42°C to the membrane in ZIP-Hyb hybridization solution included with the Ambion
kit. The membranes were washed twice with low- stringency solution at 25°C and then washed twice
with high stringency wash solution at 50°C. Hy- bridization was detected by exposing the mem-
branes
to a
phosphor screen
Molecular Dynamics, Sunnyvale, CA. Imaging of the signal
was acquired with a computer-controlled Molecu- lar Dynamics Storm 860 at 50 micron resolution.
Quantitation of the hybridization signals was done with Image QuaNT 5.0 build 050 Molecular Dy-
namics. The intensity of the signal for each band was normalized to the ethidium bromide fluores-
cence signals as described above.
2
.
7
. Chloroplast isolation Intact chloroplasts were isolated on gradients of
Percoll and twice washed as previously described [18].
3. Results
