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: Isabel.gavidia@uv.es 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. 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