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
Initial studies confirmed the previous report [12] that FDH was induced by wounding. Sampling of
the leaves with a leaf punch or by cutting with a razor blade caused as much FDH induction as any
of the other treatments. As a consequence, induc- tion experiments were conducted such that no
plant was sampled more than once. The data in Table 1 report the effect of spraying plants with
either 20 vv methanol, 10 mM formaldehyde or 10 mM sodium formate. This experiment was
repeated three more times and FDH specific activ- ity increases following treatment were consistently
2.1- to 2.8-fold. These data are consistent with the previous report [12] that methanol and formate
Table 1 Effect of foliar application of one-carbon metabolites on induction of leaf formate dehydrogenase and formaldehyde dehydroge-
nase specific activities
Formate dehydrogenase Formaldehyde dehydrogenase
nmol min
− 1
mg
− 1
of protein nmol min
− 1
mg
− 1
of protein Treatment h
24 6
48 24
6 48
1.5 20.1
17.5 Untreated
22.6 1.4
1.7 1.9
2.4 4.2
Methanol 17.3
18.1 15.6
17.8 22.1
19.3 3.0
1.7 2.8
Formaldehyde 1.6
3.6 4.8
18.2 19.1
Formate 17.2
Fig. 1. Induction of FDH mRNA by foliar application of methanol. Plants were either treated with 20 methanol M
or untreated − , but otherwise treated in an identical man- ner. Total RNA was prepared from leaves harvested at the
indicated time following treatment, subjected to formaldehyde agarose electrophoretic fractionation, blotted onto a nylon
membrane and hybridized with a homologous FDH cDNA.
The sequence of a cDNA clone for the FDH from A. thaliana was analyzed. Clone 173L24 was
from the lPRL2 cDNA library. This clone was sequenced Accession No. AF217195 and found
to be 1480 bp with an open reading frame encod- ing 384 amino acids. The predicted molecular
weight was 42 263. The predicted translation product exhibited : 83 identity and 86 simi-
larity to the amino acid sequences for potato, rice and barley. Most of the dissimilarity was located
at the N-terminus of the protein Fig. 2. This is the region that specifies the mitochondrial target-
ing signal for the potato, rice and barley proteins. Computer analysis using PSORT [20] predicts that
a mitochondrial targeting sequence exists in the first 27 amino acids. This analysis also predicts a
chloroplast
localization signal.
The program
ChloroP [21] predicts the same signal with a cleav- age site after amino acid 29. Such dual targeting in
Arabidopsis has been reported with ferrochelatase- I [22] as well as the methionyl- and histidyl-tRNA
synthetases [23 – 25]. The N-terminal sequences from potato, rice and barley are not identified as
chloroplast-like by either the ChloroP or PSORT analyses.
Another group has recently added two addi- tional
Arabidopsis sequences
AF208028 and
AF208029; actually the same sequence with differ- ent polyadenylation sites which are identical to
the nucleotide sequence for the coding region of the sequence that we report. A recently available
genomic sequence AP002468 is also identical in the coding regions. These data corroborate the
sequence we reported and are consistent with the presence of a single FDH sequence in Arabidopsis.
Southern blots of Arabidopsis genomic DNA also suggest a single FDH gene data not shown.
Isolated intact
chloroplasts prepared
from leaves of Arabidopsis plants using Percoll gradients
treatments increase the FDH mRNA levels in potato leaves. We additionally demonstrated that
formaldehyde, a one-carbon metabolite with an oxidation state intermediate to methanol and for-
mate, is also able to induce FDH activity in Arabidopsis leaves.
We also examined the induction of FDH mRNA by treatment of Arabidopsis plants with
methanol Fig. 1. Leaves of treated plants had increased levels of FDH mRNA. The increase
relative to nontreated plants under identical condi- tions was 3.2-fold at 6 h, 9.5-fold at 24 h and
1.5-fold at 48 h post-treatment.
In contrast to the induction of FDH by the three one-carbon metabolites, formaldehyde dehy-
drogenase activity was not significantly induced by treatment of Arabidopsis plants with methanol,
formaldehyde or formate Table 1. This is consis- tent with the previous determination that this ac-
tivity is expressed in a constitutive fashion [19]. Since the specific activity of formaldehyde dehy-
drogenase is much higher than even the induced levels of FDH, the induction of formaldehyde
dehydrogenase to higher than normal specific ac- tivities may not be an advantage for the plants.
Fig. 2. The predicted N-terminal amino acid sequences of barley, rice, potato and Arabidopsis were aligned using the program PILEUP. The underlined sequence was identified as the mitochondrial processing sequence by PSORT. The bold type indicates
the chloroplast targeting sequence identified by ChloroP.
Fig. 3. Immunoblot of proteins fractionated from leaf extract or isolated chloroplasts by SDS-PAGE. The monoclonal anti-
body used for detection was prepared against the a-subunit of the maize mitochondrial ATPase. The estimated molecular
weight from marker proteins is indicated on the left of the figure.
shown do not reveal the presence of mitochon- drial contamination.
The Arabidopsis FDH was purified : 280-fold by affinity chromatography and subjected to ki-
netic analyses. The K
m
for formate was 10 mM and the K
m
for NAD
+
was 65 mM. Because NADH is a substrate for the FDH catalyzing the
reverse reaction reduction of CO
2
to formate, we anticipated that this cofactor would function as a
competitive inhibitor of the oxidation of formate. This was indeed the case and the K
i
for NADH was : 17 mM. To demonstrate the extent to which
NADH is able to inhibit the enzymatic oxidation of formate, the rate of formate oxidation was
monitored at a variety of ratios of NAD
+
NADH, as demonstrated by the data points in Fig. 4. In this experiment, the total concentration
of NAD
+
and NADH was maintained at 1 mM, which approximates the concentration of these
metabolites within plant mitochondria [26]. The solid line in Fig. 4 represents the calculated activ-
ity based on the experimentally determined K
m
and K
i
values.
4. Discussion
