Cloning and characterization of eight cytochrome P450 cDNAs
from chickpea (
Cicer arietinum
L.) cell suspension cultures
Stefan Overkamp, Frauke Hein, Wolfgang Barz *
Institut fu¨r Biochemie und Biotechnologie der Pflanzen,Westfa¨lische Wilhelms-Uni6ersita¨t Mu¨nster,Hindenburgplatz 55,
48143 Mu¨nster, Germany
Received 4 November 1999; received in revised form 24 January 2000; accepted 31 January 2000
Abstract
Eight different P450 sequences were isolated from a cDNA library derived from cultured chickpea cells (cultivar ILC3279) elicited with aPhytophthora sojae(formerlymegasperma) elicitor (Pmg-elicitor) by screening with heterologous and homologous probes. Screening with CYP73A1 fromHelianthus tuberosusyielded several clones with one identical sequence. A full-length clone could be isolated and this sequence was assigned CYP73A19. Heterologous expression in yeast confirmed that CYP73A19 is the
trans-cinnamic acid 4-hydroxylase of chickpea. Screening with a CYP81E2 polymerase chain reaction fragment from chickpea yielded a CYP81E2 full-length sequence and two almost identical CYP81E3 sequences, differing in only 16 out of 498 amino acids; both share more than 85% homology with the isoflavone 2%-hydroxylase from licorice (Glycyrrhiza echinata L.). Using CYP93A1 as a probe, it was possible to isolate a full-length member of the CYP93 family, CYP93C3, that shares more than 80% homology with isoflavone synthase from soybean. In addition, partial sequences CYP81E3, CYP81E4 and CYP81E5 were also found in this screening. The use of a CYP82A2 probe derived from BAC F10N7 from Arabidopsis thaliana yielded only one sequence, CYP76F1. Rescreening with CYP81E4 and CYP81E5 did not result in the isolation of any new P450 sequences. Northern blot experiments revealed that all but the CYP76F1 are induced rapidly and transiently in cell cultures upon elicitor treatment. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Cicer arietinum; Cytochrome P450; Elicitor induction; Isoflavonoids; Phytoalexins; Pterocarpans
www.elsevier.com/locate/plantsci
1. Introduction
Cytochrome P450 enzymes are found in all classes of organisms [1]. They are responsible for a wide range of oxidative reactions in the metabolism of xenobiotics, fatty acids and steroid hormones [2]. In plants, they are also involved in almost every biosynthetic pathway of secondary metabolism. Consequently, P450 sequences form a large multigene superfamily that is divided into
clans, families and subfamilies. In the plant king-dom, more than 400 sequences in 47 families from 65 species have so far been found. In the model plant Arabidopsis thaliana alone, there are today more than 200 different P450 genes, with an esti-mated total number of more than 300 [3]. The plant P450 sequences show a substantially greater diversity than those from mammals or microor-ganisms. Although there has been so much pro-gress in isolating sequences over the past 10 years, enzyme functions of the encoded proteins have been determined only in a limited number of cases. For example, it is now well established that mem-bers of the CYP73 family encode trans-cinnamic acid 4-hydroxylases (C4Hs) [4 – 6]. Other functions relevant for this study are: CYP75 encoding flavonoid 3%5%-hydroxylase [7]; CYP81E1 encoding isoflavone 2%-hydroxylase [8]; CYP93A1 encoding
The nucleotide sequence data reported in this paper have been deposited in the EMBL database under the following accession numbers: CYP73A19 (AJ007449), CYP76 F1 (AJ249799), CYP81E2 (AJ239051), CYP81E3v1 (AJ012581), CYP81E3v2 (AJ238439), CYP81E4 (AJ249801), CYP81E5 (AJ249800), CYP93C3 (AJ243804). * Corresponding author. Tel.: +49-251-8324790; Fax: + 49-251-8328371.
E-mail address:[email protected] (W. Barz)
dihydropterocarpan 6a-hydroxylase [9], CYP93B1 encoding flavanone 2-hydroxylase [10] and CYP93C1 encoding isoflavone synthase [11].
In Cicer arietinumL. cell cultures, the induction of the biosynthesis of the pterocarpan phytoalex-ins medicarpin and maackiain upon elicitor treat-ment is well described [12]. A scheme of the biosynthetic pathway is shown in Fig. 1. Starting from the amino acidL-phenylalanine, the pathway
follows the general phenylpropanoid metabolism that leads either to lignin precursors or chalcones. Then, the isoflavones are formed and several hy-droxylation and reduction steps yield the ptero-carpans. In parallel to the formation of the pterocarpan phytoalexins medicarpin and maacki-ain in chickpea, a branched pathway of 5-OH-isoflavones is activated yielding cicerin and homoferreirin [12]. In these two pathways, 11 dis-tinct reactions were shown to be dependent on cytochrome P450 mono-oxygenase activity, all of them induced by elicitor treatment [15 – 17]. Bio-chemical data did not allow one to decide whether four or six different proteins are responsible for these reactions. Due to the well-known problems with plant P450s [18], it was not possible to purify
the proteins. Therefore, we tried to isolate the cDNA sequences encoding the proteins responsi-ble for the relevant reactions. Although several other groups reported that a polymerase chain reaction (PCR)-based strategy was successful [5,19,20], in our system this approach led to the isolation of only two P450 PCR fragments (data not shown). Therefore, we had to look for an alternative strategy.
In plants, there appears to exist a group of sequences derived from a common phylogenetic plant P450 ancestor, besides sequences more re-lated to P450s from other organisms. This first group of sequences is called group A, or CLAN A [3]. All functions determined so far and correlated to sequences from this CLAN are involved in plant-specific pathways [21]. Sequence comparison identified several highly homologous motifs, espe-cially the P450 fingerprint (P/A)FGXGXRRCXG with the heme binding cysteine. All P450 se-quences showing a sequence homology greater than 40% on the amino acid level are assigned together in a certain family. Therefore, our strat-egy was to obtain interesting P450 sequences by screening a cDNA library with a set of
mologous (PCR fragments) and heterologous probes.
In this paper, the identification of eight different P450 sequences, four of them full length, is re-ported. Their rapid induction in chickpea cell cul-tures after elicitation is also demonstrated. CYP73A19 was shown to encode trans-cinnamic acid 4-hydroxylase by functional expression in yeast.
2. Material and methods
2.1. Plant material
Suspension-cultured chickpea cells of the resis-tant cultivar ILC3279 were grown in 200 ml Erlen-meyer flasks containing 40 ml PRL-4c-medium on a rotary shaker at 120 rpm at 25°C in the dark [22]. For expression experiments, 3-day-old cells (1.5 g/20 ml) were treated with 100 mg ml−1
autoclaved glucan preparation ofPhytophthora so -jae(formerlymegasperma) pv.glycinea(Pmg -elici-tor), or the same amount of distilled water (control). After indicated time intervals, cells were separated from the media by vacuum filtration, snap-frozen in liquid N2 and stored at −80°C.
2.2. cDNA library construction
A cDNA library was constructed with the lambda ZAP Kit from Stratagene according to the manufacturer’s instructions. The resistant chick-pea cultivar ILC3279 was used as source for total RNA, which was isolated as described by Wan and Wilkins [23]. For total RNA preparation, cells were harvested 1, 3, and 5 h past elicitation, the RNA isolated and the mRNA separated with the Poly-A-Tract mRNA Isolation System (Promega) according to the manufacturer’s instructions. Iso-lated mRNA from 1, 3 and 5 h was than pooled in a 25:50:25 ratio, respectively.
2.3. cDNA library screening
All probes were labelled non-radioactively with the PCR DIG labelling and detection Kit from Boehringer. Hybridization and detection was per-formed according to the manufacturer’s instruc-tions, but the prehybridization time was extended to 4 h. CDP-Star™ was used as substrate for the alkaline phosphatase.
2.4. Yeast expression
Primers for the amplification of the CYP73A19 coding sequence and introduction of Sac1 and Sma1 restriction sites were purchased from MWG Biotech (Ebersberg, Germany). Their sequences were: CY73START 5% -atatatcccgggatggatcttctcc-tattgg-3% and CYP73STOP 5% -atatatgagctcttaat-taaatgatcttggc-3%. PCR was performed using 20 ng isolated plasmid as a template, 2 U Pfu DNA polymerase (Promega) and 20 pmol of each primer (total volume, 50 ml) in the following cycling con-ditions: 3 min at 95°C, than 35 cycles for 1 min at 95°C, 45 s at 62°C and 1.5 min at 72°C.
The amplification product was purified on a 0.8% agarose gel and cloned into the pYeDP60 expression vector. Yeast (Saccharomyces cere
-6isiae) strains WAT11 and WAT21 were
trans-formed with the expression construct and the empty expression vector. Microsomes were pre-pared as described by Urban et al. [24].
2.5. C4H enzyme assay
For the C4H enzyme assay, in a total volume of
500 ml, 455−X ml microsome buffer (80 mM
Tris – HCl, 0.4 M sucrose, 40 mM ascorbat, 10
mM b-mercaptoethanol, 1 mM ethylenediamine
tetraacetic acid, 2 mM leupeptine, 0.1% (w/v) bovine serum albumen; pH 8.0), X ml microsomal preparation, equivalent to 50 mg microsomal protein, 20 ml of 20 mM NADPH and 25 ml of 2 mM tested substrate, dissolved in methanol were mixed. The samples were pre-incubated without microsomal protein for 3 min at 30°C in a water bath. The reaction was started by adding the microsomal protein. Samples were incubated for a further 30 min at 30°C and stopped by adding 60
ml of 2 N HCl. For product extraction, 800 ml
ethyl acetate was added. The samples were mixed for 30 s on a vortex and then centrifuged. Seven hundred microlitres of the upper organic phase were transferred to a new reaction tube. Open tubes were put under reduced pressure and the ethyl acetate allowed to evaporate completely overnight. The residue was resolved in 90 ml methanol and analyzed on a Waters high-perfor-mance liquid chromatography (HPLC) apparatus with a reversed-phase RP 18 column at a constant flow rate of 0.8 ml min−1with a linear gradient of
Table 1
Strategy of cDNA library screenings and the isolated clones
Clones with Hybridization
Probe Total number Isolated sequences
temperature of clones P450 sequences (°C)
55
CYP73A1 13 11 CYP73A19
(Helianthus tuberosus) 55
CYP81E2 89 61 CYP81E2, CYP81E3v1, CYP81E3v2
(Cicer arietinum)
55 70
CYP93A1 45 CYP81E2, CYP81E3v1, CYP81E3v2, CYP81E4, CYP81E5, CYP93C3
(Glycine max)
60 ca. 120 (inves- 47 CYP81E2, CYP81E3v1, CYP81E3v2, CYP93C3 CYP93C3
(Cicer arietinum) tigated 63) 60
CYP81E4 20 5 CYP81E3, CYP81E4
(Cicer arietinum)
CYP81E5 60 20 12 CYP81E5, CYP81E3, CYP81E4
(Cicer arietinum)
CYP82C2 55 12 4 CYP76 F1
(Arabidopsis thaliana)
Solvent B was 0.15% H3PO4. For detection of the
organic compounds, a Waters photodiode array detector was used and monitored at 310 nm.
2.6. Northern (RNA) analysis of expression
For Northern (RNA) blot analysis, total RNA was extracted with the single-step guanidini-umthiocyanate method [25], phenol extracted and stored at −80°C. Typically, 800 mg total RNA was recovered per gram of cells. Twenty micro-grams of total RNA were separated on a 1% denaturing agarose – formaldehyde gel by elec-trophoresis. The RNA was capillary transferred onto a positively charged nylon membrane (Ny-tran H+; Schleicher & Schuell, Germany) by the 1-h downward alkaline method [26]. Hybridization was carried out under high stringency conditions in 5×SSC, 0.1% (w/v)N-lauroyl sarcosine, 0,02% (w/v) sodium dodecyl sulfate (SDS), 2% (w/v) blocking reagent (Boehringer) and 50% (v/v) for-mamide at 68°C. Digoxygenin-UTP-labelled (Boehringer) antisense RNA probes were obtained by in vitro transcription of the cloned CYP cD-NAs with T7-RNA-Polymerase (Stratagene). Northern blots were washed at least twice at 68°C for 30 min in 0.1×SSC, 0.1×SDS and developed according to the manufacturer’s instructions (Boehringer). Signals were detected by incubation with the chemiluminescence substrate CDP-Star™ after 3 – 10 min exposition to X-ray film (Kodak).
Equal loading of RNA was checked by ethidium bromide staining of the gel.
3. Results
3.1. Screening of a chickpea cell culture cDNA library
Table 2
P450 sequences isolated from chickpea
Sequence Length (base pairs)
CYP73A19 Full length (1629) CYP76F1 Fragment (1466)
Full length (1711) CYP81E2
Full length (1668) CYP81E3v1
CYP81E3v2 Full length (1781) Fragment (798) CYP81E4
Fragment (976) CYP81E5
CYP93C3 Full length (1875)
53% identity on the amino acid level.
CYP76F1 opens a new subfamily of the CYP76 family. It is almost equally related to the other subfamilies with identities between 41% to CYP76A1 and 47% to CYP76E1. It is 46% identi-cal with CYP76D1, a cytochrome P450 previously isolated from chickpea [28]. CYP73A19 shares more than 80% identity to the other members of the CYP73 family. It is closest related to the Medicago sati6aCYP73A3, with 91% identity [29]. CYP93C3 shares 82% identity with CYP93C1 from G. max, which has recently been reported to encode isoflavone synthase [11]. All isolated se-quences belong to CLAN A of P450s, comprising sequences typical for plants [3,30,31].
3.3. Expression studies
The isolated clones were tested for their tran-scriptional activation upon elicitor treatment in chickpea cell suspension cultures. The results are shown in Fig. 3. Clone CYP76F1 is not induced by elicitation. CYP81E2,CYP81E3, CYP81E4 and CYP81E5 cannot be seen in controls; however after elicitation, their transcripts are visible al-ready after 1 – 4 h. They accumulate to their highest levels after 3, 8, and 6 h for CYP81E2, CYP81E3, CYP81E4 and CYP81E5, respectively. After 24 h, no transcript of CYP81E2 and CYP81E5, and a weak signal for CYP81E3 and CYP81E4, is detectable. CYP73A19 becomes visi-ble 2 h past elicitation, maximal transcription is observed after 3 h and, after 6 h, the transcript disappears completely. In contrast. CYP93C3 is well expressed in control cells; but the transcript level appears to increase after elicitation, with a maximum after 4 – 6 h.
3.2. Sequence comparison
Comparison with known P450 sequences re-vealed that five of the isolated clones represented new members of the CYP81 gene family. Three other clones were from families CYP73, CYP76 and CYP93, respectively (D. Nelson, personal communication). A sequence alignment of the C-terminal region of all sequences except CYP93C3 is shown in Fig. 2.
From the members of the CYP81 family, two are almost identical, showing only 16 amino acid changes in their 498 amino acids. These two
se-quences were assigned CYP81E3v1 and
CYP81E3v2 [27]. They show more than 85% ho-mology on the amino acid level with the isoflavone 2%-hydroxylase from licorice (CYP81E1), from which also two allelic sequences exist (see bib-liography of plant P450 sequences on David Nel-son’s homepage http://drnelson.utmem.edu/ bib-lioD.html). CYP81E4 and CYP81E5 share 85% identity on the amino acid level with each other, while CYP81E4 is 62% identical to CYP81E1. CYP81E2 is also closest related to CYP81E1, with
Fig. 3. Transcript accumulation of isolated chickpea P450 sequences upon treatment withPmg-elicitor in cell suspension cultures.
3.4. Heterologous expression in yeast
Full-length cDNA of CYP73A19 was intro-duced in the yeast expression vector pYeDP60 behind a galactose-inducible and a glucose-re-pressible promoter. This construct was trans-formed into transgenic yeast cells that carry the A.
thaliana NADPH:cytochrome P450 reductase gene
integrated in their genome behind the same pro-moter [32]. For testing of P450 enzymic activity, microsomes were isolated from galactose-induced yeast cells. As a control, microsomes from yeast cells transformed with the empty expression vector alone were tested. Microsomes of yeast expressing CYP73A19 were shown to be able to convert trans-cinnamic acid to para-coumaric acid, as confirmed by HPLC analysis (see Fig. 4). This reaction is strongly dependent on NADPH. The microsomes of control yeast did not show any enzyme activity. CYP73A19 was thus shown to encode trans-cinnamic acid 4-hydroxylase from chickpea. The tested microsomes were not able to metabolize structurally related substrates such as benzoic acid, m-hydroxy benzoic acid, phenylala-nine, o-coumaric acid, phenylacetic acid, o- and m-hydroxy phenylacetic acid and acetophenon (data not shown).
4. Discussion
In chickpea, the biosynthesis of the pterocarpan phytoalexins medicarpin and maackiain as well as the isoflavanones homoferreirin and cicerin is well investigated [12,17]. Eleven steps in these pathways have been shown to be dependent on the activity of cytochrome P450 mono-oxygenases [13 – 16]. These activities were previously shown to be in-duced by elicitor treatment in chickpea cell sus-pension cultures [17]. In the biosynthetic sequence leading to medicarpin and maackiain, at least six different P450-dependent reactions were clearly shown to be involved (see Fig. 1).
In this paper, the isolation of eight different cDNAs from a chickpea cell suspension culture derived cDNA library is reported. The success of the strategy shows that, with so many plant P450 sequences now reported, the use of heterologous probes is an alternative to the PCR-based ap-proach. The identified sequences are not limited to members of the CYP family of the probe, as shown by the identification of several CYP81
quences with a CYP93 probe and the identification of CYP76F1 with the CYP82 probe. But, of course, the result is limited to sequences closely related to the probes, so in our view, this strategy seems to be appropriate for those tasks where one has already some idea about the CYP families that are interesting.
The transcripts of seven of the eight sequences accumulate after elicitor treatment. One sequence (CYP93C3) is constitutively expressed and only weakly activated. Three sequences show high ho-mology to known genes of enzymes involved in phenylpropanoid metabolism: Cyp73A19 was shown to encode the C4H from chickpea by het-erologous expression in a yeast system. This en-zyme shows a high substrate specificity, being unable to metabolize structurally related com-pounds. According to our expectation, the C4H should be expressed constitutively in chickpea cells for two reasons. First, this enzyme is needed for the production of all phenylpropanoid com-pounds, including structural components for the plant cell wall. Second, we know that in chickpea cell cultures, the isoflavones formononetin and biochanin A are formed constitutively and stored as malonyl glucosides in the vacuole [33]. In agree-ment with these expectations, we have indeed ob-served a weak constitutive expression in the control cells, which is detectable only on overex-posed films (data not shown). Furthermore, there is a dramatic increase in C4H transcript accumula-tion after elicitaaccumula-tion, which fits well with the en-zyme activity data, showing that the C4H activity is induced upon elicitor treatment [17]. CYP93C3 shares 82% homology with a sequence identified as isoflavone synthase [11]. In chickpea cell cultures, the gene is constitutively expressed. As already described, it is known that the isoflavones for-mononetin and biochanin A are synthesized con-stitutively. So, like C4H, the isoflavone synthase is needed for their metabolism. It is interesting to note that one can detect a strong expression of CYP93C3 in control cells, but weak expression of CYP73A19. CYP81E3 is more than 85% ho-mologous to the isoflavone 2%-hydroxylase from Glycyrrhiza echinataL. [8]. This sequence shows a very strong induction after elicitor treatment. These results are in good agreement with our hypothesis that the 2%-hydroxylation of the isoflavone formononetin is the rate-limiting step in chickpea phytoalexin biosynthesis [15]. All other
sequences do not allow one to speculate about their function with respect to sequence similarities at this time. But, except for CYP76F1, their tran-scription is also strongly activated by elicitor treatment.
Full-length clones of CYP81E2, CYP81E3 and CYP93C3 are now subject to heterologous expres-sion in yeast in our laboratory, a method proven to be suitable for identifying the C4H activity of CYP73A19 from chickpea.
Acknowledgements
The authors would like to thank J. Ebel (Mu-nich) and D. Werck-Reichhart (Strasbourg) for providing probes CYP93A1 and CYP73A1, re-spectively, D. Nelson (Memphis) for the
assign-ment of the P450 sequences, D. Pompon
(Gif-sur-Yvette) for yeast expression vector and yeast strains, and I. Benveniste and F. Durst (Strasbourg) for substantial help with the yeast expression system. This work was financially sup-ported by the Deutsche Forschungsgemeinschaft.
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