Báo cáo Y học: Cloning and expression of two novel aldo-keto reductases from Digitalis purpurea leaves potx

Cloning and expression of two novel aldo-keto reductases
from
Digitalis purpurea
leaves
Isabel Gavidia
1,2
, Pedro Pe
´
rez-Bermu
´
dez
2
and H. Ulrich Seitz
1
1
Center of Plant Molecular Biology (ZMBP), University of Tu
¨
bingen, Germany;
2
Department of Plant Biology, University of
Valencia, Spain
The aldo-keto reductase (AKR) superfamily comprises
proteins that catalyse mainly the reduction of carbonyl
groups or carbon–carbon double bonds of a wide variety of
substrates including steroids. Such types of reactions have
been proposed to occur in the biosynthetic pathway of the
cardiac glycosides produced by Digitalis plants. Two cDNAs
encoding leaf-specific AKR proteins (DpAR1 and DpAR2)
were isolated from a D. purpurea cDNA library using the rat
D
4

-3-ketosteroid 5b-reductase clone. Both cDNAs encode
315 amino acid proteins showing 98.4% identity. DpAR
proteins present high identities (68–80%) with four Arabid-
opsis clones and a 67% identity with the aldose/aldehyde
reductase from Medicago sativa. A molecular phylogenetic
tree suggests that these seven proteins belong to a new sub-
family of the AKR superfamily. Southern analysis indicated
that DpARs are encoded by a family of at most five genes.
RNA-blot analyses demonstrated that the expression of
DpAR genes is developmentally regulated and is restricted
to leaves. The expression of DpAR genes has also been
induced by wounding, elevated salt concentrations, drought
stress and heat-shock treatment. The isolated cDNAs were
expressed in Escherichia coli and the recombinant proteins
purified. The expressed enzymes present reductase activity
not only for various sugars but also for steroids, preferring
NADH as a cofactor. These studies indicate the presence of
plant AKR proteins with ketosteroid reductase activity. The
function of the enzymes in cardenolide biosynthesis is dis-
cussed.
Keywords: aldo-keto reductases; cardenolide biosynthesis;
Digitalis purpurea; gene expression.
Plants produce a wide variety of secondary metabolites,
which, in contrast with primary metabolites, appear to be
dispensable for plant growth and development but indis-
pensable for the survival of a plant population [1].
Biosynthesis of secondary metabolites requires precursors
from primary metabolic pathways. Although coordinate
regulation between both processes has been reported [2], the
regulatory mechanisms have not been elucidated.

Many of these natural products have been shown to have
important ecological functions, comprising resistance
against diseases (phytoalexins) and herbivore (proteinase
inhibitors, bitter and toxic deterrents, etc.). Besides this,
plant secondary metabolism is the source for many fine
chemicals such as drugs, dyes, flavours and fragrances, all of
increasing commercial importance. Therefore, the possibil-
ities to alter the production of secondary metabolites are of
great interest, but the limited knowledge of the biosynthetic
routes, often based only on feeding experiments and/
or theoretical considerations, is a major constraint in this
field [3].
One group of natural products of major interest in the
pharmaceutical industry is cardiac glycosides from Digitalis
species, as they are widely prescribed for the treatment of
congestive heart failure. Cardiac glycosides possess a basic
skeleton, a steroid genin, namely digitoxigenin, digoxigenin
or gitoxigenin. Different studies using labelled and unla-
belled precursors have led to a hypothetical pathway for
cardenolide biosynthesis, but knowledge about the forma-
tion of the aglycon is not well established. The first steps of
this route basically resemble those of cholesterol metabolism
towards steroid hormones in animals. Upstream of digit-
oxigenin, only four reactions have been described: the
transformation of cholesterol to pregnenolone [4], prog-
esterone formation from pregnenolone [5], the sequential
reductions of progesterone to 5b-pregnane-3,20-dione [6]
and 5b-pregnan-3b-ol-20-one [7]. In animal tissues all of
these reactions of the steroid metabolism, except the
cholesterol side-chain-cleaving reaction, are catalysed by

enzymes of the aldo-keto reductase (AKR) superfamily [8].
The members of the AKR superfamily are cytosolic,
monomeric proteins that catalyse mainly the NAD(P)H-
dependent reduction of a wide variety of carbonyl com-
pounds; some enzymes function also as carbon–carbon
double bond reductases. Within the range of substrates of
AKRs are different steroids that are metabolized by
hydroxysteroid dehydrogenases and some stereospecific
double bond reductases. An enzyme belonging to this last
group, progesterone 5b-reductase, has been proposed to
have a key function in the cardenolide pathway [6]
producing the required 5b-configured natural products.
One goal of our studies on cardenolide biosynthesis
was to clone the gene that encodes the progesterone
5b-reductase. In order to achieve this goal, two cloning
strategies were used. The first approach is based on
Correspondence to I. Gavidia, Department of Plant Biology,
Fac. Pharmacy, University of Valencia, Avenue V.A. Estelle
´
ss/n,
46100 Burjassot, Spain.
Fax: +34 963864926, Tel.: +34 963864929,
E-mail:
Abbreviations: AKR, aldo-keto reductase; AR, aldose reductase.
(Received 19 November 2001, revised 18 February 2002,
accepted 12 April 2002)
Eur. J. Biochem. 269, 2842–2850 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02931.x
progesterone 5b-reductase purification from D. purpurea
plants according to the protocol of Ga
¨

rtner et al. [9] and the
subsequent amino acid sequencing.
The second strategy is based on the use of orthologous
genes for screening a cDNA library of D. purpurea.Assuch
a type of steroid stereospecific enzyme has never been cloned
in plants, and considering that some aspects of the steroid
metabolism in higher plants cannot be separated from
corresponding ones in animals, the cDNA encoding D
4
-3-
ketosteroid 5b-reductase of rat liver [10] was used as a
probe. The results obtained in this second experimental
approach are described in the present paper, which reports
on the cloning and expression of two AKR genes from
D. purpurea. Both proteins reduce the ketone group of
steroid structures but they are not active on the D
4
-double
bond of the steroids assayed. It is worth noting that this is
the first report for such activity on steroids from a plant
AKR enzyme.
MATERIALS AND METHODS
Plant materials
Shoot cultures of D. purpurea were established as described
previously [7]. Every 3 weeks newly developed shoots were
transferred to fresh liquid nutrient medium [11] supple-
mented with 3% glucose, 1 mgÆL
)1
indoleacetic acid and
2mgÆL

)1
kinetin. Cultures were maintained in a growth
chamber under a 14-h photoperiod at 21 °C on a rotary
shaker (90 r.p.m.). Alternatively, complete plantlets were
transplanted into soil and acclimatized to ex vitro condi-
tions. Plants were grown under standard greenhouse
conditions at a day/night temperature regime of 20/18 °C.
Tissue samples were harvested from in vitro or mature
plants as indicated, frozen in liquid nitrogen and stored at
)80 °C until use.
Construction and screening of cDNA library
Total RNA was extracted from 11-day-old leaves of
D. purpurea shoot cultures according to the procedure
described by Steimle et al.[12].Poly(A)
+
RNA was
prepared from total RNA using oligo(dT) cellulose
(Boehringer). A directional cDNA library was constructed
using 5 lg poly(A)
+
RNA according to the instructions for
a Stratagene cDNA synthesis kit (Uni-ZAP XR). The
resultant library contained  2 · 10
6
independent clones
with an average insert size of 1.5 kb. The library was
screened by plaque hybridization with a
32
P-labelled HindIII
fragment (500 bp) of the rat D

4
-3-ketosteroid 5b-reductase
cDNA as a probe. Nylon filter lifts were prehybridized and
hybridized at 42 °Cin330m
M
sodium phosphate buffer
(pH 7), 7% SDS, 1 m
M
EDTA and 1% BSA. The positive
clones were isolated, their cDNA inserts in vivo excised, then
subcloned into pBluescript SK(–).
DNA sequence analysis
Restriction analysis was used to classify the clones.
cDNA clones were subjected to nucleotide sequencing
by the dideoxy chain termination method, using a DNA
sequencing kit (PE Biosystems) on an ABI 310 Genetic
Analyser (PerkinElmer). Complete nucleotide sequences
were determined for both strands of the cDNAs and
analysed by the
DNASTAR
program package (Lasergene)
and
CLUSTALW
.
Plant treatments
To determine the effects of several stress conditions on gene
expression, D. purpurea plants were grown in a greenhouse
for 4 months. For mechanical wounding experiments, holes
of 1 mm diameter were made across the lamina, which
effectively damaged approximately 5% of the leaf area.

Samples were collected 1, 2 and 3 h following treatment.
For dehydration experiments, the plants were left on
Whatman 3MM paper until the appearance of clear wilting
symptoms (24 h). Low temperature treatment was carried
out by exposing the plants, grown at 22 °C, to a tempera-
ture of 4 °C under dim light (16 h a day). Leaves were
harvested after 2 and 4 days. Plants subjected to salt stress
were watered with a solution of 250 m
M
NaCl and samples
collected after 6 and 48 h. Leaves were also sampled from
heat-shocked plants incubated at 41 °Cfor2and4h.Inall
treatments the leaves were harvested and immediately
immersedinliquidnitrogen.
Nucleic acid isolation and analysis
For Northern analyses, 20 lg total RNA were separated on
1.2% formaldehyde–agarose gels and then capillary trans-
ferred to a Hybond-N
+
membrane (Amersham Pharmacia
Biotech) following the manufacturer’s protocol. To check
the integrity of the samples, RNAs were visualized by
adding ethidium bromide to the sample before loading. The
cDNA encoding actin from D. purpurea wasusedasa
loading control. Genomic DNA was isolated from leaves of
adult plants according to Dellaporta et al.[13].Twenty
micrograms genomic DNA were digested with restriction
endonucleases, separated by electrophoresis in a 1% agarose
gel and immobilized on nylon membranes (Hybond-N
+

).
RNA and DNA gel blots were prehybridized for 3 h at
50 °C in a solution containing 330 m
M
sodium phosphate
buffer (pH 7), 7% SDS, 1% BSA, 1 m
M
EDTA. Hybrid-
ization was done overnight at 65 °C with a random primed
32
P-labelled cDNA probe (SmaI fragment of 900 bp from
DpAR1). The membranes were finally washed in 2 · NaCl/
Cit, 0.1% SDS for 20 min at 65 °C. Autoradiography of the
filters was obtained on X-OMAT AR films (Kodak) using
an intensifying screen at )80 °C.
Expression and purification of recombinant
DpAR proteins
ThecDNAswereclonedasaSphI/BglII fragment into
pQE70 QIAexpress vector (Qiagen). Both recombinant
plasmids were transfected into Escherichia coli strain M15/
PREP4. Cells were grown in Luria–Bertani media supple-
mented with 100 lgÆmL
)1
ampicillin and 25 lgÆmL
)1
kanamycin at 37 °C. Gene expression was induced by the
addition of isopropyl thio-b-
D
-galactoside to a final con-
centration of 1 m

M
,whenanD
600nm
of 0.6 was reached. The
cells were harvested, after 4 h incubation, by centrifuga-
tion at 4000 g for 15 min at 4 °C and resuspended in
buffer A (50 m
M
sodium phosphate buffer pH 8, 300 m
M
NaCl, 10 m
M
imidazole). After the addition of lysozyme
Ó FEBS 2002 Aldo-keto reductases in D. purpurea leaves (Eur. J. Biochem. 269) 2843
(1 mgÆmL
)1
) and a further 30 min incubation on ice, the
cells were disrupted by sonication (3 · 30 s with 70 W
Micro Tip Sonifier B12; Branson, Danbury, Connecticut,
USA). Cell debris were then precipitated by centrifugation
(10 000 g for 20 min at 4 °C) and the supernatant applied
to a Ni–nitrilotriacetic acid spin column (Qiagen) equili-
brated with buffer A. The buffer B (buffer A with 50 m
M
imidazole) was used for washing the columns from
contaminant proteins. The elution of the His-tagged pro-
teins was carried out using buffer C (buffer A containing
250 m
M
imidazole).

Enzyme assays
Enzyme activity was determined in a 1-mL reaction mixture
containing 0.1
M
sodium phosphate buffer (pH 7.0);
150 l
M
NADPH, NADH, NADP or NAD; 10 m
M
DL
-glyceraldehyde,
D
-glucose or
D
-fructose; 10 l
M
prog-
esterone, 5b-pregnan-3,20-dione, 17a-hydroxiprogesterone
or 5b-pregnan-3b-ol-20-one. The reaction was initiated by
the addition of the protein, and monitored at 25 °Cusinga
Uvicon 930 spectrophotometer (Kontron Instruments,
Germany). The activity was determined by measuring
NADPH, NADH oxidation or NADP, NAD reduction
from the decrease or increase in absorbance at 340 nm,
respectively. Steroids were dissolved in ethanol, which did
not exceed 5% of the total volume. The appropriate blank
was subtracted from each determination to correct nonspe-
cific oxidation or reduction of cosubstrate. One unit of
enzyme activity was defined as the amount that oxidized
1 lmol NAD(P)HÆmin

)1
. Assays were run in triplicate. The
protein concentration was determined by the method of
Bradford [14]. The electrophoretic separation of proteins
was performed on 12% polyacrylamide gels according to
Laemmli [15].
The steroids were extracted from the reaction mixture
according to Ga
¨
rtner and Seitz [7], applied to Silica gel
60F
254
TLC plates (Merck) and then separated in a
hexane/ethyl acetate (65 : 35, v/v) solvent system. Plates
were dried at room temperature and photographed under
UV illumination. The plates were also developed by
spraying anisaldehyde reagent (Sigma) and heating at
110 °C for 10 min to visualize the steroids. Substrates and
metabolites were identified by comparison with reference
steroids (Sigma).
RESULTS
Isolation and sequence analysis of aldose reductase (AR)
cDNAs from
D. purpurea
A cDNA library from D. purpurea was screened with the rat
D
4
-3-ketosteroid 5b-reductase cDNA [10] as probe. After
three rounds we isolated two positive clones of 1324 and
1319 bp, designated DpAR1 and DpAR2, respectively. The

nucleotide sequences of DpAR1 and DpAR2 have been
submitted to the EMBL database and are available under
accession numbers AJ309822 and AJ309823, respectively.
DpAR1 and DpAR2 contain 948 bp long ORFs encoding
315 amino acids of a calculated molecular mass 34 898 and
34 883 Da, respectively. Their nucleotide sequences exhibit
97.8% identity, and their amino-acid sequences show a
98.4% identity.
The sequence comparison analysis revealed the significant
homology of DpAR1 and DpAR2 to the AKR protein
superfamily. Comparison of DpAR1 and DpAR2 amino-
acid sequences with those of plants (Fig. 1) revealed that
these proteins present 80, 73, 70 and 68% identity with four
Arabidopsis thaliana clones (accession numbers AAC23647,
AAD32792, AAC23646 and CAB88350, respectively) and
67% with the alfalfa aldose/aldehyde reductase (accession
number X97606). Furthermore, we found a 45–47%
conservation in amino-acid residues with AKR4 proteins
from plants and 40–42% with AKR1 proteins from human
and animals.
Out of the four Arabidopsis clones, only one (CAB88350)
has been appointed as a putative AKR, whereas the other
three are postulated to be alcohol dehydrogenases. Never-
theless, the high degree of homology of their amino-acid
sequences and those of the alfalfa and D. purpurea ARs
suggests that all these proteins belong to a plant AKR
subfamily. Our results are in agreement with the conclusions
of Oberschall et al. [16] based on the comparison of only
two Arabidopsis clones with the AKR sequence of Medicago
sativa.

In order to prove this hypothesis, a molecular phylo-
genetic tree of the amino-acid sequences of the most related
AKR proteins from plants, animals and human with the
DpARs was designed (Fig. 2). This analysis suggests that
DpAR proteins phylogenically belong to a new subfamily of
the AKR4 family. This clearly differentiated cluster from
dicotyledons species comprises four Arabidopsis enzymes,
the alfalfa and DpAR proteins. The rest of the plant AKR4
forms two distinct clusters, one of which includes enzymes
from four monocotyledonous species, while the other, with
a deep branching of the internal modes, comprises proteins
from both mono- and di-cotyledonous plants.
An analysis of the primary structure of DpAR1 and
DpAR2 showed three consensus patterns specific for this
family of proteins. One is located in the N-terminus (42–59
numbered according to DpARs); the second signature is at
150–160 amino acids, and the third pattern is located in the
C-terminus (256–266). Alignment of AKR sequences shows
that 10 residues are invariant. In DpARs these amino acids
are G42, D47, E55, G59, K81, P116, G156, P176, Q180 and
S257. Besides these residues, Y52, H114 and K256 are
almost strictly conserved in the different AKR members.
Some of these amino acids are involved in catalysis [17].
Thus, it has been postulated that oxidoreductases of the
AKR superfamily present a common reaction mechanism
by using a tetrad of amino acids (D, Y, K, H) at the active
centre, where Y is the proton donor [18–20]. In DpAR1 and
DpAR2 these four residues are D47, Y52, K81 and H114.
In relation to the cosubstrate binding site, the amino acids
K256, S257, R262 and N266 of DpARs would be involved

in NAD(P)H binding. The residues K256 and S257 are part
of a typical AKR motif (IPKS) having cosubstrate binding
functions [21]. Although this motif is highly conserved, all
residues are not invariant, as happens within the subfamily
proposed (see Fig. 1) where the residue I changes to L.
Genomic organization of
D. purpurea
AR genes
The molecular organization of the AR genes in D. purpurea
was determined by Southern blot analysis of genomic DNA
digested with EcoRI, BamHI and HindIII. There were no
2844 I. Gavidia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
BamHI or HindIII restriction sites in any of the DpAR
cDNAs, and only one EcoRI site in the DpAR2 clone. The
900-bp cDNA fragment of DpAR1 was used as a probe.
After washing the blotting membrane under high-stringent
conditions, five bands were detected for cuts by BamHI or
HindIII enzymes while up to 10 bands were found when
genomic DNA was digested with EcoRI (Fig. 3). These
results indicate that a small multigene family of at most five
genes is encoding ARs in the genome of D. purpurea.
Heterologous expression of DpARs in
E. coli
The cDNAs of DpAR1 and DpAR2 were over-expressed in
E. coli as fusion proteins with His-tag (pQAR1 and
pQAR2). The recombinant DpARs were purified by affinity
chromatography from the extracts of bacteria transformed
with pQAR1 or pQAR2, using a His-binding resin column.
The purified proteins were visualized as a single band of
35 kDa after SDS/PAGE. To test the cofactor specificity of

the recombinant proteins NADPH, NADP, NADH or
NAD were used as cofactors in the spectrophotometric
assays. The substrate specificity was analysed using different
sugars and steroids. The DpAR enzymes work in the
direction of reduction of such substrates; they do not react
with NADP or NAD. As shown in Table 1, both enzymes
can metabolize aldose and ketose substrates, as well as
steroids, in the presence of NADH cofactor. However,
using NADPH as coenzyme, only
DL
-glyceraldehyde was
reduced; no activity was detected with the other substrates
tested. The purified DpAR1 reduced
DL
-glyceraldehyde,
D
-glucose and
D
-fructose with a similar specific activity
(1.8 UÆmg
)1
) and threefold higher than that observed with
DpAR2 ( 0.6 UÆmg
)1
). All the steroids tested (progester-
one, 5b-pregnan-3,20-dione, 5b-pregnan-3b-ol-20-one and
17a-hydroxyprogesterone) served as substrates for both
enzymes, although their reaction rates also differed. These
results demonstrate that both DpAR1 and DpAR2 are
members of the AKR family with a broad spectrum of

substrates including steroids.
Preliminary results indicated that these enzymes work by
reducing progesterone and 17a-hydroxyprogesterone; mol-
ecules having 20-one and D
4
-3-one structures, which are
susceptible for such reduction. TLC analysis of the steroids
extracted from the enzymatic reactions, using progesterone
as a substrate, showed a lack of fluorescence with respect to
the control (Fig. 4); this could be due to the reduction of the
D
4
-double bond and/or the ketone group at position 3. To
determine the specific function of DpAR recombinant
enzymes, different steroids lacking one or more of such
structures were assayed. 5b-pregnan-3,20-dione (having
3- and 20-one structures) served as substrate with a reaction
rate almost identical to that obtained for progesterone.
Fig. 1. Alignment of the amino-acid sequences
of proteins within a closely related AKR family.
Sequences aligned are DpAR1 and DpAR2,
D. purpurea (this study); AAC23647,
AAD32792, CAB88350 and AAC2346,
Arabidopsis thaliana;Medicago,M. sativa
[16]. The amino-acid residues identical to the
DpAR1 sequence are indicated by dots. Gaps
are introduced to optimize the alignment.
Ó FEBS 2002 Aldo-keto reductases in D. purpurea leaves (Eur. J. Biochem. 269) 2845
However, 5b-pregnan-3b-ol-20-one (having only 20-one
structure) showed lower specific activity (Table 1). Com-

paring the specific activities of these proteins for those
substrates, we can conclude that both proteins reduce the
ketone structures, but are not active on the D
4
-double bond
Fig. 2. Molecular phylogenetic tree of the amino-acid sequences of the
most related plant AKR proteins. Xerophyta, Xerophyta viscosa
(AAD22264); Bromus, Bromus inermis (JQ2253); Hordeum, Hordeum
vulgare (P23901); Avena, Avena fatua (S61421); Sesbania, Sesbania
rostrata (CAA11226); Oryza, Oryza sativa (AAK52545); Medicago,
M. sativa (X97606). AR proteins from mammals were used as the
outgroup: human (P14550), cow (P16116), rat (P0794), mouse
(P45376). The tree was constructed using the neighbour-joining
method [37] and the program
CLUSTAL W
to create the multiple
sequence alignment. Scale bar: p distance, which is approximately
equal to the number of nucleotide substitutions per site.
Fig. 3. Southern blot analysis of genomic DNA from D. purpurea.
Samples of 20 lgDNAdigestedwithEcoRI (E), HindIII (H) or
BamHI (B) were loaded onto each lane. The blot was hybridized with
the 900-bp cDNA probe isolated from DpAR1.
Table 1. Enzymatic activity of DpAR1 and DpAR2 recombinant pro-
teins. Data are mean values (± SD) of triplicate assays.
Substrate
Enzymatic activity (UÆmg
)1
protein)
DpAR1 DpAR2
Glyceraldehyde 1.81 ± 0.11 0.68 ± 0.03

Glucose 1.80 ± 0.09 0.57 ± 0.05
Fructose 1.83 ± 0.15 0.60 ± 0.05
Progesterone 1.77 ± 0.16 1.49 ± 0.09
5b-Pregnane-3,20-dione 1.70 ± 0.10 1.45 ± 0.11
5b-Pregnan-3b-ol-20-one 1.32 ± 0.08 1.04 ± 0.12
Fig. 4. TLC analysis of steroids visualized under UV-light. Lane 1,
authentic progesterone (P); lane 2, reaction mixture with DpAR1
recombinant protein and NADH as cosubstrate; lane 3, reaction
mixture without NADH; lane 4, reaction mixture without protein.
2846 I. Gavidia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
of the steroids assayed. It is worth noting that this is the first
report for such steroid activity from a plant AKR enzyme.
Expression of DpAR genes
The size of the DpAR transcripts and their expression
profile were determined by Northern hybridization analysis
of total RNA. As shown in Fig. 5, a single mRNA species
with a size of  1.4 kb was detected with the 900 bp
DpAR1 cDNA probe. The organ-specific expression of
DpAR genes in mature D. purpurea plants (1 year old) is
shown in Fig. 5A. A highly specific expression profile was
obtained, as the hybridization signal was restricted to the
leaf blade. No signals were detected in the petiole, stem or
roots even after over-exposure of the film. The transcription
level was also examined during in vitro development of
D. purpurea shoot cultures, leaf samples being taken at
different time points. DpAR expression slightly increased
along the culture time course, although at the end of the
experiment (3 months) the transcription level of the in vitro
plants was clearly weaker than in mature field plants
(Fig. 5B).

We also analysed the effect of physical treatments on the
expression of DpAR genes. Four-month-old D. purpurea
plants were subjected to different stress factors. Northern
analysis of total RNA isolated from leaves showed that the
expression of DpAR genes was triggered by heat, salt and
drought treatment and wounding (Fig. 6). Cold tempera-
tures significantly decreased the transcription level after
2 days of treatment, but after 4 days the level increased,
being similar to that of control plants (Fig. 6A). The
DpAR genes are induced after heat shock treatment at
41 °C; the increased transcription level was detectable
following 2 h of treatment with further progressive mRNA
accumulation, as shown in Fig. 6B. When D. purpurea
plants were subjected to desiccation and elevated NaCl
concentration (250 m
M
), leaves also responded by
increased levels of DpAR mRNA during the treatment
(Fig. 6C,D). However, DpAR genes show transient accu-
mulation of the transcripts after wounding, reaching the
maximum level after 1 h and then starting to decline. The
time by which expression returned to the control level was
approximately 3 h. This temporal expression pattern
suggests that these genes function in the rather early stages
of the wound response.
DISCUSSION
In bile acids synthesis and steroid hormone metabolism, D
4
-
3-ketosteroid 5b-reductase plays an important role in

catalysing the reduction of the D
4
-double bond to give A/
B-cis conformation [10]. A similar reaction, reduction of
progesterone to 5b-pregnane-3,20-dione, catalysed by prog-
esterone 5b-reductase, has been considered as the stereo-
specific starting point of the cardenolide pathway leading to
digitoxigenin [22]. Thus, a heterologous AKR clone, D
4
-3-
ketosteroid 5b-reductase [10] from rat, has been used for
screening a D. purpurea cDNA library. Although the aldo
and keto groups of the substrate are not involved chemically
in the reaction, this enzyme is classified as a member of the
AKR superfamily because it shares  50% homology and
the typical signatures with other members of this family,
including the ARs.
Following this cloning strategy, we isolated and
sequenced two full-length cDNAs from D. purpurea leaves
that encode DpAR1 and DpAR2, two new members of the
AKR superfamily; specifically, the amino-acid sequences of
DpARs show relatively high levels of similarity to mam-
malian ARs. The highest identities were obtained with
several Arabidopsis proteins of unknown function and the
aldose-aldehyde reductase of M. sativa [16]. Lower levels of
similarity, within the plant AKRs, were found with the AR
proteins from the monocotyledoneous Avena fatua [23],
Hordeum vulgare [24], Bromus inermis [25] and Xerophyta
viscosa [21].
Fig. 5. Expression of DpAR genes. For all Northern analysis, 20 lg

total RNA were loaded per lane. The blot was hybridized with the
32
P-labelled DpAR1 cDNA probe. (A) RNA gel blot analysis of
DpARs transcript levels in various organs from mature plants (R,
root; S, stem; P, petiole; L, leaf). (B) Expression of DpARs in leaves at
different ages, 1 month (1), 3 months (3) and mature plants (M).
Hybridization with an actin probe was used as a control of sample
loading.
Ó FEBS 2002 Aldo-keto reductases in D. purpurea leaves (Eur. J. Biochem. 269) 2847
The phylogenetic relationships among the most related
plant AKRs indicated that DpARs belong to the same
subfamily as the alfalfa enzyme [16] and form a separate
cluster from the other plant AKR4 (Fig. 2). Southern blot
analysis revealed that DpARs are encoded by a family of at
most five genes.
A characteristic function of the ARs is the catalysis of the
first reaction in the polyol pathway, the reduction of glucose
to sorbitol. In mammals they play a role in cellular osmotic
regulation [26] and are associated with diabetic complica-
tions [27]. Furthermore, ARs are also involved in the
metabolism of steroid hormones [28,29] and xenobiotics
[30]. In plants, AR proteins are also associated with osmotic
stress or desiccation tolerance in barley [24,31], avena [23]
and Xerophyta viscosa [21], or protect against freezing in
bromegrass [25]. Interestingly, the aldose-aldehyde reduc-
tase identified in alfalfa seems to be an important factor in
the defence system of stressed plants. Oberschall et al.[16]
observed that the activity of the enzyme is linked to an
increased resistance to oxidative agents, salt, heavy metals
and drought. Nevertheless, to date, plant AR proteins have

not been linked to steroid metabolism.
As has been reported for both animal and plant AKRs,
the corresponding protein expressed in bacteria has the
same properties as the in vivo protein [32]. Purification of the
recombinant DpARs from E. coli allowed us to show that
these enzymes are capable of reacting with sugars and
steroids. The typical aldose substrates
DL
-glyceraldehyde
and
D
-glucose, as well as the ketose
D
-fructose, were reduced
in the presence of NADH by both enzymes with similar
activities. Two important differences have been observed
when comparing DpARs with the AR from barley and
alfalfa, as these proteins use NADPH as cosubstrate, and
their activities with glyceraldehyde were clearly higher than
with glucose. When reacting with steroids, DpAR1 and
DpAR2 cannot reduce the carbon–carbon double bond
in D
4
-3-ketosteroids, but have been shown to reduce both
3- and 20-ketosteroids. Thus, DpARs are ketosteroid
reductases instead of D
4
-3-ketosteroid 5b-reductases. As
inferred from the results of their enzymatic activities,
DpAR1 and DpAR2 may be two isoforms with different

substrate specificities.
In contrast with plants, it is well established that ARs
from animals, as other members of the AKR superfamily,
participate in steroid metabolism. The catalytic efficiency of
AR varies widely for different substrates, but shows a
marked preference for hydrophobic compounds [33]. This is
in accordance with the presence of a highly hydrophobic
active-site pocket, which greatly favours apolar substrates
over highly polar monosaccharides [34]. Warren and
coworkers [29] reported for the first time that progesterone
and 17a-OH-progesterone are substrates for ARs; they
Fig. 6. DpAR gene expression under several
stress conditions. Details of the filters are the
same as in Fig. 5. (A) Cold treatment. (B)
Heat shock. (C) Drought. (D) NaCl
(250 m
M
). (E) Mechanical wounding.
Hybridization with an actin probe was used as
a control of sample loading.
2848 I. Gavidia et al. (Eur. J. Biochem. 269) Ó FEBS 2002
found that the 20-ketosteroid reductase (20a-HSD) is the
previously named bovine lens AR with enzymatic activity
on different sugars. However, some ARs lack catalytic
activities for steroid substrates; this fact has been attributed
to a subtle difference in the amino-acid residues lining the
active-site pocket [35]. In other AKRs a single amino acid
substitution has determined a new enzyme activity, i.e. the
change of activity from 3a-HSD to 5b-reductase by the
modification of a catalytic residue [20]. With regard to

DpARs, we found slight differences between their substrate
specificity, which may be related to the variation of certain
amino acids, and it can be also assumed that the natural
substrates for these DpARs have not been detected in our
system. A problem commonly connected with AKR
enzymes is their broad substrate specificity, which makes
it difficult to determine the physiologically used substrate(s)
and consequently the physiological role(s) of a particular
enzyme of this family. Thus, in our case both enzymes
function not only as typical ARs, but also as ketosteroid
reductases; this suggests their involvement in steroid meta-
bolism. DpAR1 and DpAR2 show enzymatic activity with
some intermediate products of the pregnane metabolism.
Accordingly, both proteins may participate in the formation
of a-orb-pregnane derivatives. The latter case would imply
their involvement in the pathway of cardenolide biosynthe-
sis as b-configured pregnanes are the putative precursors of
these natural products.
Northern analysis revealed the tissue-specific expression
of DpAR genes in D. purpurea plants, showing a specific
signalwhichisrestrictedtoleaves.Furthermore,the
transcription level increased with plant development as
mature leaves exhibited higher expression levels than those
of young plantlets. The lack of specific probes for each
gene did not permit determination of whether both
DpAR genes exhibit differential expression associated to
the plant developmental stage. These results allow us to
establish interesting correlations between the enzymatic
activity on steroids, the organ-specific and developmen-
tally regulated expression of the genes, and the specific

biosynthesis and accumulation of cardenolides in mature
Digitalis leaves. Once again our results suggest that
DpARs not only participate in the general steroid
metabolism, but could also be particularly involved in
cardenolide biosynthesis.
Many of the roles of plant secondary metabolites
remain unknown, although it is widely accepted that, in
the context of ecological interactions, plant protection is a
major function of natural products. Cardenolides taste
bitter and are extremely toxic to most insects and higher
animals, therefore it is likely that the presence of these
products in leaves serve as deterrents to herbivores. Based
on this idea, we determined whether leaf damage resulted
in enhanced expression of DpAR1 and DpAR2 genes.
Leaves of D. purpurea were damaged mechanically and
then sampled at various intervals to measure changes in
the transcription level. These experiments showed clearly
that wounding provokes a transient over-expression of
DpARs, and the pattern obtained suggests that these
genes may function in the early stages of the wound
response. Interestingly, an identical response to wounding
has been observed in the gene encoding the progesterone
5b-reductase, a key enzyme for cardenolide biosynthesis
(Gavidia and Seitz, unpublished data). Both findings are in
accordance with observations of Malcolm & Zalucki [36],
who reported a transient increased production of carde-
nolides in response to damage caused by feeding of the
monarch butterfly larvae in leaves of Asclepias syriaca.
The expression of DpARs genes has also been induced by
elevated salt concentrations, drought-generated osmotic

stress and heat-shock treatment. The response of plant AR
enzymes to a wide range of stresses was also observed in
M. sativa [16], wherein a physiological role in plant defence
has been attributed; the authors suggested that such
resistance might primarily be due to detoxification of toxic
aldehydes. The stimulation of AR synthesis under stress
conditions points to a physiological role of these enzymes in
plants exposed to environmental stresses.
In conclusion, we have isolated two cDNA clones and
determined the primary structure of two AKRs from
D. purpurea. These proteins share considerable similarities
not only with plant ARs but also with the mammalian
AKRs having a role in steroid metabolism. This observation
is the first report that biologically active steroids are
substrates for plant AKRs. These results, besides others
mentioned above, suggest that DpARs are involved in
cardenolide biosynthesis. Nevertheless, the limited know-
ledge on the intermediates and enzymes of this biosynthetic
pathway, besides the broad substrate specificity of these
AKRs, are major restrictions to elucidate the physiological
role of these proteins in D. purpurea. Work is underway to
determine the precise role of DpARs in plant steroid
metabolism in general and in cardenolide biosynthesis in
particular. More experiments will be necessary to determine
their link with stress tolerance. This knowledge would be of
great importance not only for these plant processes but also
for a comparison of multifunctional roles of AKRs in plants
and mammals.
ACKNOWLEDGEMENTS
The authors thank Dr Temp. M. Noshiro (Hiroshima University) for

providing the rat D
4
-3-ketosteroid 5b-reductase cDNA clone and
Dr Temp. J. A. Rossello
´
for help with the phylogenetic studies. The
European Commission supported this work with postdoctoral grants to
I.G. (Contracts BIO4-CT97-5019 and QLK3-CT-1999-51296).
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