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

Ulrich Seitz1 1 Center of Plant Molecular Biology ZMBP, University of Tu¨bingen, Germany;2Department of Plant Biology, University of Valencia, Spain The aldo-keto reductase AKR superfami

Cloning and expression of two novel aldo-keto reductases

Isabel Gavidia1,2, Pedro Pe´rez-Bermu´dez2and H Ulrich Seitz1

1

Center of Plant Molecular Biology (ZMBP), University of Tu¨bingen, Germany;2Department 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

D4-3-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-opsisclones 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 supersub-family 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´s s/n,

46100 Burjassot, Spain.

Fax: +34 963864926, Tel.: +34 963864929,

E-mail: Isabel.gavidia@uv.es

Abbreviations: AKR, aldo-keto reductase; AR, aldose reductase.

(Received 19 November 2001, revised 18 February 2002,

accepted 12 April 2002)

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 As such

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 D4

-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 D4-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

M A T E R I A L S A N D M E T H O D S

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)1indoleacetic acid and

2 mgÆL)1 kinetin Cultures were maintained in a growth

chamber under a 14-h photoperiod at 21C 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 20/18C

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 · 106 independent clones

with an average insert size of 1.5 kb The library was

screened by plaque hybridization with a32P-labelled HindIII

fragment (500 bp) of the rat D4-3-ketosteroid 5b-reductase

cDNA as a probe Nylon filter lifts were prehybridized and

hybridized at 42C in 330 mM sodium phosphate buffer

(pH 7), 7% SDS, 1 mMEDTA 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 22C, to a tempera-ture of 4C 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 mMNaCl and samples collected after 6 and 48 h Leaves were also sampled from heat-shocked plants incubated at 41C for 2 and 4 h In all treatments the leaves were harvested and immediately immersed in liquid nitrogen

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 was used as a 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

50C in a solution containing 330 mMsodium phosphate buffer (pH 7), 7% SDS, 1% BSA, 1 mMEDTA Hybrid-ization was done overnight at 65C with a random primed

32P-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 65C 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

The cDNAs were cloned as a SphI/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 37C Gene expression was induced by the addition of isopropyl thio-b-D-galactoside to a final con-centration of 1 mM, when an D600nmof 0.6 was reached The cells were harvested, after 4 h incubation, by centrifuga-tion at 4000 g for 15 min at 4C and resuspended in buffer A (50 mMsodium phosphate buffer pH 8, 300 mM NaCl, 10 m imidazole) After the addition of lysozyme

(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 4C) and the supernatant applied

to a Ni–nitrilotriacetic acid spin column (Qiagen)

equili-brated with buffer A The buffer B (buffer A with 50 mM

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 mMimidazole)

Enzyme assays

Enzyme activity was determined in a 1-mL reaction mixture

containing 0.1M sodium phosphate buffer (pH 7.0);

150 lM NADPH, NADH, NADP or NAD; 10 mM

DL-glyceraldehyde, D-glucose or D-fructose; 10 lM

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 25C using a

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

60F254 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

110C for 10 min to visualize the steroids Substrates and

metabolites were identified by comparison with reference

steroids (Sigma)

R E S U L T S

Isolation and sequence analysis of aldose reductase (AR)

cDNAs fromD purpurea

A cDNA library from D purpurea was screened with the rat

D4-3-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 thalianaclones (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 ofD 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

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 inE 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-di(progester-one, 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 D4-3-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

D4-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.

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 D4-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 lg DNA digested with EcoRI (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)1protein) 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.

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

41C; 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 mM), 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

D I S C U S S I O N

In bile acids synthesis and steroid hormone metabolism, D4

-3-ketosteroid 5b-reductase plays an important role in

catalysing the reduction of the D4-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, D4

-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.

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 andD-glucose, as well as the ketoseD-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 D4-3-ketosteroids, but have been shown to reduce both 3- and 20-ketosteroids Thus, DpARs are ketosteroid reductases instead of D4-3-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.

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- or b-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

signal which is restricted to leav es 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

A C K N O W L E D G E M E N T S

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).

R E F E R E N C E S

1 Hartmann, T (1996) Diversity and variability of plant secondary metabolism: a mechanistic view Entomol Exp Appl 80, 177–188.

2 Zhao, J & Last, R.L (1996) Coordinate regulation of the tryp-tophan biosynthetic pathway and indolic phytoalexin accumula-tion in Arabidopsis Plant Cell 8, 2235–2244.

3 Verpoorte, R., van der Heijden, R & Memelink, J (2000) Engineering the plant cell factory for secondary metabolite pro-duction Transgenic Res 9, 323–343.

4 Pilgrim, H (1972) Cholesterol side-chain cleaving enzyme Aktivita¨t in Keimlingen und in vitro kultivierten Geweben von Digitalis purpurea Phytochemistry 11, 1725–1728.

5 Finsterbusch, A., Lindemann, P., Grimm, R., Eckerskorn, C & Luckner, M (1999) D5-3b-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh – a multifunctional enzyme in steroid metabolism? Planta 209, 478–486.

6 Ga¨rtner, D.E., Wendroth, S & Seitz, H.U (1990) A stereospecific enzyme of the putative biosynthetic pathway of cardenolides Characterisation of a progesterone 5b-reductase from leaves of Digitalis purpurea L FEBS Lett 271, 239–242.

7 Ga¨rtner, D.E & Seitz, H.U (1993) Enzyme activities in

carde-nolide-accumulating, mixotrophic shoot cultures of Digitalis

pur-purea L J Plant Physiol 141, 269–275.

8 Penning, T.M., Ma, H & Jez, J.M (2001) Engineering steroid

hormone specificity into aldo-keto reductases Chem Biol

Inter-act 130, 659–671.

9 Ga¨rtner, D.E., Keilholz, W & Seitz, H.U (1994) Purification,

characterization and partial peptide microsequencing of

prog-esterone 5b-reductase from shoot cultures of Digitalis purpurea.

Eur J Biochem 225, 1125–1132.

10 Onishi, Y., Noshiro, M., Shimosato, T & Okuda, K (1991)

Molecular cloning and sequence analysis of cDNA encoding D 4

-3-ketosteroid 5b-reductase of rat liver FEBS Lett 2, 215–218.

11 Murashige, T & Skoog, F (1962) A revised medium for rapid

growth and bioassay with tobacco tissue culture Physiol Plant.

15, 473–496.

12 Steimle, D.E., Schnitzler, J.P & Seitz, H.U (1994) Modes of

ex-pression of phenylalanine ammonia-lyase and chalcone synthase

in elicitor-treated carrot cell cultures Acta Hort 381, 206–209.

13 Dellaporta, S.L., Wood, J & Hicks, J.B (1983) A plant

DNA-minipreparation: Version II Plant Mol Biol Res 1, 19–21.

14 Bradford, M.M (1976) A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding Anal Biochem 72, 248–254.

15 Laemmli, U.K (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4 Nature 227, 680–685.

16 Oberschall, A., Dea´k, M., To¨ro¨k, K., Sass, L., Vass, I., Kova´cs, I.,

Fehe´r, A., Dudits, D & Horva´th, G.V (2000) A novel aldose/

aldehyde reductase protects transgenic plants against lipid

perox-idation under chemical and drought stresses Plant J 24, 437–446.

17 Jez, J.M., Flynn, T.G & Penning, T.M (1997) A new

nomen-clature for the aldo-keto reductase superfamily Biochem

Phar-macol 54, 639–647.

18 Tarle, I., Borhani, D.W., Wilson, D.K., Quiocho, F.A & Petrash,

J.M (1993) Probing the active site of human aldose reductase.

J Biol Chem 268, 25687–25693.

19 Barski, O.A., Gabbay, K.H., Grimshaw, C.E & Bohren, K.M.

(1995) Mechanism of human aldehyde reductase: characterization

of the activ e site pocket Biochemistry 34, 11264–11275.

20 Jez, J.M & Penning, T.M (1998) Engineering steroid

5b-reduc-tase activity into rat liver 3a-hydroxysteroid dehydrogenase

Bio-chemistry 37, 9695–9703.

21 Mundree, S.G., Whittaker, A., Thomson, J.A & Farrant, J.M.

(2000) An aldose reductase homolog from the resurrection plant

Xerophyta viscosa Baker Planta 211, 693–700.

22 Seitz, H.U & Ga¨rtner, D.E (1994) Enzymes in

cardenolide-accumulating shoot cultures of Digitalis purpurea L Plant Cell

Tiss Org Cult 38, 337–344.

23 Li, B & Foley, M.E (1995) Cloning and characterization of

dif-ferentially expressed genes in imbibed dormant and afterripened

Avena fatua embryos Plant Mol Biol 29, 823–831.

24 Bartels, D., Engelhardt, K., Roncarati, R., Schneider, K., Rotter, M & Salamini, F (1991) An ABA and GA modulated gene expressed in the barley embryo encodes an aldose reductase related protein EMBO J 10, 1037–1043.

25 Lee, S.P & Chen, T.H.H (1993) Molecular cloning of abscisic acid-responsive mRNAs expressed during the induction of freez-ing tolerance in bromegrass (Bromus inermis Leyss) suspension culture Plant Physiol 101, 1089–1096.

26 Garcı´a-Pe´rez, A., Martin, B., Murphy, H.R., Uchida, S., Murer, H., Cowley, B.D., Handler, J.S & Burg, M.B (1989) Molecular cloning of cDNA coding for kidney aldose reductase.

J Biol Chem 264, 16815–16821.

27 Bohren, K.M., Bullock, B., Wermuth, B & Gabbay, K (1989) The aldo-keto reductase superfamily J Biol Chem 264, 9547– 9551.

28 Lacy, W.R., Washenick, K.J., Cook, R.G & Dunbar, B.S (1993) Molecular cloning and expression of an abundant rabbit ovarian protein with 20a-hydroxysteroid dehydrogenase activity Mol Endocrinol 7, 58–66.

29 Warren, J.C., Murdock, G.L., Ma, Y., Goodman, S.R & Zimmer, W.E (1993) Molecular cloning of testicular 20a-hydroxysteroid dehydrogenase: identity with aldose reductase Biochemistry 32, 1401–1406.

30 Winters, C.J., Molowa, D.T & Guzelian, T.S (1990) Isolation and characterization of cloned cDNAs encoding human liver chlordecone reductase Biochemistry 29, 1080–1087.

31 Roncarati, R., Salamini, F & Bartels, D (1995) An aldose reductase homologous gene from barley: regulation and function Plant J 7, 809–822.

32 Everard, J.D., Cantini, C., Grumet, R., Plummer, J & Loescher, W.H (1997) Molecular cloning of mannose-6-phosphate reduc-tase and its developmental expression in celery Plant Physiol 113, 1427–1435.

33 Morjana, N.A & Flynn, T.G (1989) Aldose reductase from human psoas muscle Purification, substrate specificity, immuno-logical characterization, and effect of drugs and inhibitors J Biol Chem 264, 2906–2911.

34 Wilson, D.K., Bohren, K.M., Gabbay, K.H & Quiocho, F.A (1992) An unlikely sugar substrate site in the 1.65 angstrom structure of the human aldose reductase holoenzyme implicated in diabetic complications Science 257, 81–84.

35 Gui, T., Tanimoto, T., Kokai, Y & Nishimura, C (1995) Presence

of a closely related subgroup in the aldo-keto reductase family of the mouse Eur J Biochem 227, 448–453.

36 Malcolm, S & Zalucki, M (1996) Milkweed latex and cardenolide induction may resolve the lethal plant defence paradox Entomol Exp Appl 80, 193–196.

37 Saitou, N & Nei, M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees Mol Biol Evol 4, 406–425.