Novel ABA- and dehydration-inducible aldehyde
dehydrogenase genes isolated from the resurrection plant
Craterostigma plantagineum and Arabidopsis thaliana
Hans-Hubert Kirch
*
, Ambili Nair and Dorothea Bartels
Institute of Botany, University of Bonn, Kirschallee 1, 53115 Bonn, Germany
Received 2 May 2001; revised 24 August 2001; accepted 6 September 2001.
*
For correspondence (fax+49 228 732689; e-mail hhkirch@uni-bonn.de).
Summary
In order to identify genes that are critical for the ABA-dependent stress response in the resurrection
plant Craterostigma plantagineum, a gene was isolated with homology to class 3 variable substrate
aldehyde dehydrogenases (ALDH). The C. plantagineum gene Cp-ALDH constitutes a novel class of plant
ALDHs. In a search for corresponding genes from Arabidopsis thaliana, Ath-ALDH3 and Ath-ALDH4 were
isolated, showing 70% and 80% similarity to Cp-ALDH. Phylogenetically, the Cp- and Ath-ALDH3 and
-ALDH4 proteins are closely related to aldehyde dehydrogenases from bacteria and mammalian species
and are separated from known plant ALDHs and betaine-aldehyde dehydrogenases (BADH). Cp-ALDH
transcript and polypeptide are up-regulated in vegetative tissues and callus in response to dehydration
or ABA-treatment. Ath-ALDH3 expression was induced in response to dehydration and ABA treatment,
while Ath-ALDH4 is constitutively expressed at a low level. Recombinant Cp-ALDH protein oxidizes
nonanal, propionaldehyde and acetaldehyde, with K
m
values of 2.2 mM, 0.27 mM and 3.23 mM,
respectively, in an NAD-dependent manner. Immunogold electron microscopy shows that Cp-ALDH is
localized in plastids.
Keywords: abscisic acid, aldehyde dehydrogenase, Arabidopsis thaliana, Craterostigma plantagineum,
dehydration, enzyme activity.
Introduction
Aldehydes are a ubiquitous class of highly reactive mol-
ecules involved in different physiological processes. Many
aldehydes are oxidized by the large family of NAD(P)
+
-
dependent aldehyde dehydrogenases [aldehyde: NAD(P)
+
oxidoreductases, EC 1.2.1]. This group of enzymes consists
of diverse subfamilies with four main different functions:
detoxi®cation, intermediary metabolism, osmotic protec-
tion and NADPH generation (Perozich et al., 1999).
Substrate speci®c aldehyde dehydrogenases are distin-
guished from variable substrate aldehyde dehydrogenases
(ALDH), which catalyze the irreversible oxidation of a
broad spectrum of aliphatic and aromatic aldehydes in a
NAD-dependent (EC 1.2.1.3) reaction. Only a few ALDHs
use NADP as coenzyme (EC 1.2.1.5) (Dickinson, 1989).
ALDHs have been studied in various organisms from
bacteria to mammals. ALDH genes have been character-
ized in detail especially in yeast and in humans (Navarro-
Avin
Ä
o et al., 1999; Yoshida et al., 1998). At least two main
classes of variable substrate ALDHs can be distinguished.
Class 1/2 ALDHs are cytosolic or mitochondrial tetrameric
enzymes involved in detoxi®cation or associated with
susceptibility to ethanol-related diseases. Class 3 ALDHs
are cytosolic or microsomal dimeric enzymes oxidizing
aromatic aldehydes and fatty aldehydes (medium-chain
aliphatic aldehydes) and are associated with carcinogen-
esis and severe genetic disorders (Yoshida et al., 1998).
In plants ®ve different ALDH genes, all belonging to the
mitochondrial and cytosolic class 1/2 isozymes have been
analyzed up to now. The ®rst identi®ed ALDH gene (Rf2)
represents a maize nuclear restorer gene of cytoplasmic
male sterility (Cui et al., 1996). Two tobacco mitochondrial
ALDH genes, TobALDH2a and TobALDH2b were expressed
in reproductive tissues and exhibited a high acetaldehyde
oxidizing activity in vitro. TobALDH activity might be
crucial for pollen tube growth, functioning in biosynthesis
and energy production (Op Den Camp and Kuhlemeier,
The Plant Journal (2001) 28(5), 555±567
ã 2001 Blackwell Science Ltd 555

1997). Recently, two rice acetaldehyde-oxidizing ALDHs,
ALDH1a and ALDH2a, were characterized. ALDH1a is a
cytosolic enzyme, while ALDH2a seems to be localized in
mitochondria (Li et al., 2000; Nakazono et al., 2000).
Recombinant ALDH2a ef®ciently oxidized acetaldehyde
in vitro, suggesting a function in plant tolerance under
anaerobic conditions (Nakazono et al., 2000).
Expression of ALDH-related genes in response to high
salinity and dehydration stress has been reported in
several plant species. One class are the substrate speci®c
betaine aldehyde dehydrogenases (BADH), which are
induced under saline or water de®cit conditions and
catalyze the oxidation of betaine-aldehyde to glycine-
betaine. BADH genes have been characterized in several
plant species and BADH proteins show about 40%
homology to variable substrate ALDHs (Op Den Camp
and Kuhlemeier, 1997; Wood et al., 1996). ALDH-like genes
also include the so-called turgor-responsive genes, which
are induced in response to water stress or fruit ripening
(Guerrero et al., 1990; Stroeher et al., 1995; Yamada et al.,
1999). These exhibit a remarkable homology to antiquitin
from humans (Lee et al., 1994) and display about 30%
identity to various ALDHs, but their function is unknown.
The resurrection plant Craterostigma plantagineum
(Scrophulariaceae) is able to tolerate extreme desiccation.
Vegetative tissues can withstand almost complete water
loss and recover within hours after rehydration. This is a
complex trait involving the expression of many genes.
Despite a detailed molecular characterization of the dehy-
dration-responsive genes, only few can be related to a
function (Chandler and Bartels, 1999).
Callus from the resurrection plant Craterostigma planta-
gineum requires ABA to become desiccation tolerant
(Bartels et al., 1990). ABA analogs are a possible tool to
uncouple the expression of dehydration stress associated
genes from the physiological stress response in
Craterostigma plantagineum (Chandler et al., 1997). To
identify genes that are important for desiccation tolerance
we analyzed mRNA populations from untreated, ABA and
ABA analog treated callus by subtractive suppression
hybridization. Here we report on the characterization and
functional analysis of an ABA- and water stress-inducible
ALDH-gene from C. plantagineum and of two homologues
from Arabidopsis thaliana, belonging to a novel class of
plant aldehyde dehydrogenases.
Results
Isolation of Cp-ALDH from C. plantagineum
A differentially expressed cDNA fragment was obtained
from a subtractive hybridization experiment. This frag-
ment showed a high homology to aldehyde dehydro-
genases from various species. A full length cDNA clone,
termed Cp-ALDH, was isolated and contained an insert of
1646 bp, comprising a coding region of 1440 bp, a 39-
nucleotide 5¢ leader sequence and a 3¢ untranslated region
of 168 nucleotides. The open reading frame encodes a
polypeptide of 479 amino acids with a predicted molecular
mass of 52.7 kDa and a pI of 7.25. The deduced protein
sequence is homologous to ALDHs from various organ-
isms (Figure 1a) with the highest similarities to two
putative aldehyde dehydrogenases from A. thaliana, Ath-
ALDH3 (57% identity, 72% similarity; see below) and Ath-
ALDH4 (63% identity, 76% similarity; see below), a tumor-
associated ALDH from rat (44% identity, 56% similarity;
Jones et al., 1988), an ALDH from Synechococcus (43%
identity, 57% similarity; Kashiwagi et al., 1994), the dioxin-
inducible cytosolic aldehyde dehydrogenase-3 from
mouse (44% identity, 56% similarity; Vasiliou et al., 1993)
and an ALDH-like protein from rice (44% identity, 59%
similarity; Yau et al., 1999). Several functionally important
residues of ALDHs are conserved in the amino acid
sequence of Cp-ALDH: Gly-190 is integral to the coen-
zyme-binding site (aa-position 190±195) and Phe-343 prob-
ably interacts with the nicotinamide portion of NAD(P).
Cys-247 is part of the catalytic site and Asn-117 may co-
ordinate the carbonyl oxygen atom of the substrate
aldehyde. Gly-244 is required for the correct positioning
of the Cys-residue and Glu-212, embedded into a highly
conserved motif (aa-position 209±218), is involved in the
catalytic mechanism (Hurley and Weiner, 1999; Perozich
et al., 1999).
Genomic DNA blot analysis indicated that Cp-ALDH is
represented by one or two copies in the genome (data not
shown).
Isolation of Ath-ALDH3 and Ath-ALDH4, two homologs
from Arabidopsis thaliana
Database searches revealed at least 10 different putative
Arabidopsis ALDH-genes. Two genes displayed a high
similarity to the Cp-ALDH sequence (Figure 1) and are
designated here as Ath-ALDH3 (gene bank accession no.
AL035521.1, CAB36701) and Ath-ALDH4 (gene bank acces-
sion no. AC074228.7, AAG30995.1). A gene speci®c 547 bp
PCR fragment of Ath-ALDH3 was used to isolate full-length
transcripts (Gosti et al., 1995). One cDNA clone (Ath-
ALDH3) of 1908 bp encompasses an open reading frame
of 1653 bp, which encodes a polypeptide of 550 amino
acids with a predicted molecular mass of 60.2 kDa and a pI
of 8.74. The full-length transcript contains a 36-nucleotide
5¢ leader sequence and a 219-nucleotide 3¢ untranslated
region. Sequence analysis of the polypeptide predicted a
putative chloroplast transit peptide of 60 aa. The deduced
protein sequence displays similarities to other ALDHs
(Figure 1a) in approximately the same range (55±58%) as
determined for the Cp-ALDH polypeptide. Ath-ALDH3 does
556 Hans-Hubert Kirch et al.
ã Blackwell Science Ltd, The Plant Journal, (2001), 28, 555±567