Plant Science 159 2000 205 – 212
Formate dehydrogenase in Arabidopsis thaliana: characterization and possible targeting to the chloroplast
Bradley J.S.C. Olson
a
, Maryanne Skavdahl
a
, Ha˚kon Ramberg
b
, John C. Osterman
b
, John Markwell
a,
a
Department of Biochemistry, Uni6ersity of Nebraska, Lincoln, NE
68588
-
0664
, USA
b
School of Biological Sciences, Uni6ersity of Nebraska, Lincoln, NE
68588
-
0118
, USA Received 25 April 2000; received in revised form 28 June 2000; accepted 29 June 2000
Abstract
Formate dehydrogenase E.C. 1.2.1.2 is a mitochondrial-localized NAD-requiring enzyme in green plants. The enzyme activity and corresponding mRNA in leaves of Arabidopsis thaliana are induced by treatment with one-carbon metabolites. The cDNA for
the Arabidopsis formate dehydrogenase is similar to that of other plants except for the N-terminal region, which is predicted to target chloroplasts as well as mitochondria. The specific of activity of the enzyme in isolated chloroplasts suggests it is targeted
to both mitochondria and chloroplasts in Arabidopsis. Formate dehydrogenase from Arabidopsis was partially purified and K
m
values for formate and NAD
+
were determined to be 10 mM and 65 mM, respectively; the K
i
for NADH was 17 mM. We conclude that formate dehydrogenase is normally present in Arabidopsis chloroplasts and that sensitivity to inhibition by NADH may play
a role in whether cellular formate is assimilated or dissimilated. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Formate; Formate dehydrogenase; Arabidopsis thaliana; Mitochondria; Chloroplast www.elsevier.comlocateplantsci
1. Introduction
The NAD-linked formate dehydrogenase FDH; EC 1.2.1.2 represents a family of enzymes found
in methylotrophic bacteria and yeast, as well as higher plants [1]. In methylotrophic microorgan-
isms, this enzyme plays an essential catalytic role in the final step of one-carbon metabolic oxidation
and the generation of reducing equivalents. In these organisms, FDH is inducible by growth on
one-carbon molecules. FDH activity is normally under metabolic control; when sufficient reducing
equivalents are available to the cell, the activity is inhibited to promote assimilation of carbon from
the cellular pool of formaldehyde [2]. Under con- ditions in which more NADH is required to fuel
respiration, the enzyme is active and catalyzes dissimilation by the oxidation of formate to CO
2
. FDH in plants is situated at the periphery of
one-carbon metabolism rather than in the main- stream of energy generation. Plant FDH is local-
ized in mitochondria [3] and has a homodimeric quaternary structure, with subunits : 42 000 in
molecular mass. The primary sequence is related to that of the FDH from methylotrophic yeast and
bacteria [4].
When leaves are incubated in the dark, most exogenously supplied formate is dissimilated to
CO
2
[5]. However, FDH is not locked into a dissimilatory role. It has long been known that
[
14
C]-formate is readily assimilated into metabo- lites, such as serine in plants incubated in the light
[6,7]. It has been proposed that FDH regulates the concentration of one-carbon metabolites, deter-
mining whether they are assimilated or dissimi- lated in plant tissues [3,8]. While the presence of
formate in plant cells has been proposed to result from the oxidation of glyoxylate [9] or as part of a
Corresponding author. Tel: + 1-402-4722924; fax: + 1-402- 4727842.
E-mail address
:
markwellunl.edu J. Markwell. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 3 3 7 - X
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
