Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor
Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan,
We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (−)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (−)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosyntheis of (−)-phytocassanes in rice.
Mohan et al. (1996) reported that enzyme extracts from chitin-elicited, suspension-cultured rice cells converted ent-copalyl diphosphate (ent-CDP) to ent-sandaracopimara-8(14),15-diene, a putative diterpene hydrocarbon precursor of oryzalexins A–F. In addition, syn-CDP was converted to 9β-pimara-7,15-diene and stemar-13-ene, putative diterpene hydrocarbon precursors of momilactones A and B, and oryzalexin S, respectively. (−)-Phytocassanes A–E were identified as putative derivatives of ent-cassa-12,15-diene (Koga et al., 1995, 1997; Yajima and Mori, 2000). By analogy with known biosynthetic pathways of polycyclic diterpenes such as gibberellins (Hedden and Kamiya, 1997), geranylgeranyl diphosphate (GGDP) is postulated to be sequentially cyclized via ent-CDP, to ent-cassa-12,15-diene and ent-sandaracopimara-8(14),15-diene, and via syn-CDP, to 9β-pimara-7,15-diene and stemar-13-ene (Figure 1). Diterpene cyclases catalyzing conversion of ent-CDP or syn-CDP to the four diterpene hydrocarbons therefore play key roles in the biosynthesis of rice diterpenoid phytoalexins. cDNAs encoding diterpene cyclases involved in the biosynthesis of rice diterpenoid phytoalexins are useful tools for the understanding of biosynthetic regulation. To isolate diterpene cyclase cDNAs, we performed reverse transcription-polymerase chain reaction (RT-PCR) using primers designed based on conserved motifs of diterpene cyclases such as ent-kaurene synthase (ent-KS), ent-copalyl diphosphate synthase (ent-CPS), and abietadiene synthase (AS; Ait-Ali et al., 1997; Bensen et al., 1995; Hedden and Kamiya, 1997; Richman et al., 1999; Vogel et al., 1996; Yamaguchi et al., 1996, 1998). Here, we show the isolation and characterization of a cDNA encoding a novel diterpene cyclase tentatively named OsDTC1 from suspension-cultured rice cells treated with a chitin elicitor. This enzyme functioned as ent-cassa-12,15-diene synthase that possibly plays a key role in the biosynthesis of (−)-phytocassanes. We also identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (−)-phytocassanes, as an endogenous compound in the chitin-elicited rice cells and ultraviolet (UV)-irradiated rice leaves.
Results and discussion
Isolation of a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor
We prepared poly(A)+ RNA from suspension-cultured rice (Oryza sativa L. cv. BL-1) cells 8 h after adding the chitin elicitor N-acetylchitoheptaose because the putative diterpene hydrocarbon precursors of rice phytoalexins accumulated in the chitin-elicited rice cells (unpublished data). RT-PCR, using a combination of primers designed based on the conserved amino acid sequences AYDTAWV and SPS(T/S/A)TA, yielded a specific cDNA fragment of the anticipated size (549 bp). The predicted amino acid sequence showed significant similarity to that of plant diterpene cyclases (Ait-Ali et al., 1997; Bensen et al., 1995; Richman et al., 1999; Vogel et al., 1996; Yamaguchi et al., 1996, 1998). These results suggested that the 549-bp fragment is a part of a cDNA encoding a putative diterpene cyclase.
To isolate a full-length cDNA containing the 549-bp PCR-amplified fragment, we performed 5′ and 3′ rapid amplification of cDNA ends (RACE). Double-stranded cDNA prepared from the chitin-elicited rice cells was ligated with EcoRI/NotI adaptors (Stratagene, La Jolla, CA, USA) and used as a template. 3′-RACE was carried out using a forward primer based on the nucleotide sequence of the 549-bp PCR-amplified fragment and a poly(T)-end adaptor primer to yield a 2.1-kbp PCR-amplified fragment. Contrary to our expectation, the 2.1-kbp and 549-bp fragments were found to be derived from different mRNA species. Thus, at least two putative diterpene cyclase genes were expressed in the elicited rice cells. Diterpene cyclase genes containing the 2.1-kbp and 549-bp cDNA fragments were tentatively named OsDTC1 and OsDTC2, respectively.
To isolate the 5′-region of OsDTC1 cDNA, we performed 5′-RACE to amplify a RACE product of the anticipated size (approximately 0.8 kbp). The nucleotide sequence of the amplified cDNA fragment was identical to the original cDNA fragment of 2.1 kbp in the overlapped region (84 bp). The nucleotide sequence of a full-length cDNA encoding OsDTC1 was thus determined (the nucleotide sequence data of OsDTC1 cDNA reported in this paper will appear in the Genome Sequence Data Base (GSDB), DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and National Center for Biotechnology Information (NCBI) nucleotide sequence databases with the Accession number AB089272). We are now cloning the full-length OsDTC2 cDNA.
The OsDTC1 cDNA is 2772 bp in length, with an open-reading frame (ORF) encoding 830 amino acid residues. In OsDTC1, the first 59 N-terminal amino acids are rich in serine and threonine (20%), and the estimated pI of this region is 11. The entire amino acid sequence was calculated to have a pI of 5.44. These features of OsDTC1 are common characteristics of transit peptides, which target proteins to plastids (Keegstra et al., 1989). This information was also supported by our analysis using signalp V. 1.1 (Center for Biological Sequence Analysis, Technical University of Denmark, Denmark). The above results strongly suggest that OsDTC1 is localized in plastids, as are the plant diterpene cyclases such as ent-CPS and ent-KS involved in gibberellin biosynthesis (Aach et al., 1995; Railton et al., 1984).
Functional analysis of OsDTC1 protein
The OsDTC1 protein was overexpressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-OsDTC1). E. coli BL21(DE3) harboring a plasmid for the expression of GST-OsDTC1 (pGEX-OsDTC1) was cultured, and its expression was induced by the addition of 1 mm isopropyl-1-thio-β-d-galactoside (IPTG). The fusion protein was affinity-purified on Glutathione Sepharose 4B (Amersham Bioscience, Piscataway, NJ, USA) affinity column chromatography and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). In the affinity-purified fraction of extracts of E. coli BL21, harboring pGEX-OsDTC1, a 118-kDa protein (GST-OsDTC1) was detected. Also, the E. coli BL21 harboring the control plasmid pGEX-c yielded a 26-kDa protein (GST). The N-terminal five-amino acid sequence of the 118-kDa GST fusion protein was the same as that of GST (Smith and Johnson, 1988). We used the affinity-purified GST-OsDTC1 fusion protein for enzyme assays.
Enzyme assays of the recombinant OsDTC1 for diterpene cyclase activity were performed with GGDP, ent-CDP or syn-CDP as a substrate. When the recombinant OsDTC1 was incubated with GGDP, no reaction product was detected by gas chromatography–mass spectrometry (GC–MS; data not shown). On the other hand, incubation of the recombinant OsDTC1 with ent-CDP gave the unknown diterpene hydrocarbon-like compound X as a major product and a trace amount of pimara-8,15-diene (Figure 2a). As previously described, all the rice diterpene phytoalexins characterized so far are oxygenated derivatives of the four structurally distinct types of polycyclic diterpene hydrocarbons: 9β-pimara-7,15-diene, ent-sandaracopimara-8(14),15-diene, stemar-13-ene, and ent-cassa-12,15-diene (Figure 1). Three of the diterpene hydrocarbons were identified from rice (Mohan et al., 1996). However, as ent-cassa-12,15-diene is a hypothetical biosynthetic intermediate leading to (−)-phytocassanes, no authentic sample was available. Considering that ent-CDP is a possible precursor of ent-cassa-12,15-diene, ent-cassa-12,15-diene might be a candidate for compound X. This speculation is supported by GC–MS, showing that compound X accumulated in the chitin elicitor-treated rice cells (Figure 2c). We therefore synthesized ent-cassa-12,15-diene from (R)-Wieland-Mischer ketone (Yajima et al., in press). GC–MS analysis indicated that the mass spectrum and GC retention time of the synthesized sample corresponded to those of compound X (Figure 2a,c,d).
On the other hand, when racemic syn-CDP was used as a substrate, the recombinant OsDTC1 protein converted the substrate into pimara-8,15-diene and aphidicol-15-ene, but their yields were quite low (less than 1%; Figure 2b). Both products were minor diterpene hydrocarbons in the chitin-elicited rice cells (Figure 2c), and their metabolites have not been identified as rice phytoalexins. It is thus concluded that OsDTC1 functions as ent-cassa-12,15-diene synthase in rice cells, although OsDTC1 might be involved in biosynthesis of the minor diterpene hydrocarbons.
Sequence comparison with other plant diterpene cyclases
Expression levels of OsDTC1 (ent-cassa-12,15-diene synthase) mRNA
To analyze the expression levels of OsDTC1 mRNA in chitin-elicited suspension-cultured rice cells, we performed RT-PCR using a pair of gene-specific primers. Levels of OsDTC1 mRNA began to increase from 6 h after addition of the elicitor, and the level reached the maximum after 8 h, and gradually decreased (Figure 4a). On the other hand, ent-cassa-12,15-diene began to accumulate in suspension-cultured rice cells 8 h after addition of the elicitor, and the levels continued to increase for at least 16 h (unpublished data).
Rice leaves produce diterpenoid phytoalexins after UV irradiation as well as infection with the blast fungus M. grisea (Kato et al., 1993, 1994). Ultraviolet irradiation is a convenient method for obtaining tissues for investigating rice phytoalexin biosynthesis. The expression levels of OsDTC1 mRNA in rice leaves after UV irradiation were also investigated by Northern blot analysis. As shown in Figure 4(b), OsDTC1 mRNA was clearly induced in rice leaves after UV irradiation. In non-irradiated rice leaves (control), the level of OsDTC1 was below the limit of detection. These results were supported by RT-PCR using gene-specific primers and poly(A)+ RNA prepared from the UV-irradiated and control rice leaves (data not shown). GC–MS analysis indicated that the production of ent-cassa-12,15-diene was clearly induced in the rice leaves after UV irradiation (Figure 5). It was thus indicated that OsDTC1 functions as ent-cassa-12,15-diene synthase not only in the elicited suspension-cultured rice cells but also in the UV-irradiated rice leaves.
In this study, we isolated a cDNA encoding a diterpene cyclase from the chitin-elicited suspension-cultured rice cells, and indicated that the gene product functioned as ent-cassa-12,15-diene synthase in the elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The ent-cassa-12,15-diene synthase cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of the diterpenoid phytoalexins (−)-phytocassanes in rice. In our search of the rice genome database recently opened to the public (http://RiceBLAST.dna.affrc.go.jp/), the possibility was indicated that at least 10 diterpene cyclase genes exist in rice including OsDTC1 and OsDTC2. Cloning and functional analysis of the respective cDNAs are now under way.
Calli of O. sativa L. cv. BL-1 were kindly supplied by Mr Masahiro Kobayashi of Chugai Pharmaceutical Co. (Tokyo, Japan) and maintained on gerangum (0.3%) plates using modified N6 medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 1 mg l−1) as described previously by Nojiri et al. (1996). For suspension cultures, approximately 1-ml aliquots of the calli were transferred to 500-ml Erlenmeyer flasks containing 150 ml each of N6 medium with 2,4-D (1 mg l−1) and cultured on a rotary shaker (120 r.p.m.) at 25°C in the dark. The suspension-cultured cells were collected every week and filtered through a 20-mesh screen to make fine aggregates. At the time of each collection, a 1-ml aliquot of the resultant cells was transferred to a new 500-ml Erlenmeyer flask containing 150 ml of the above medium and cultured further as described above. The cells were used for elicitor-treatment experiments 6 days after the transfer.
Preparation of poly(A)+ RNA from the elicited suspension-cultured rice cells
Suspension-cultured cells were treated with the elicitor N-acetylchitoheptaose at a concentration of 10 p.p.m. as described previously by Nojiri et al. (1996). The cells were filtered and homogenized to fine powder, and then total RNA was extracted by SDS-phenol methods (Ausubel et al., 1999). poly(A)+ RNA was isolated from total RNA using Oligotex™-MAG mRNA purification kit according to the manufacturer's instructions (TaKaRa, Ontsu, Japan).
RT-PCR to isolate a cDNA fragment encoding a diterpene cyclase
Reverse transcription was performed with SuperScript II RT (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instruction using two degenerate primers, 5′-GCITA(C/T)GA(C/T)ACIGCITGGGT-3′ (forward primer) and 5′-IGCIGTIGHIGANGGNGA-3′ (reverse primer), and poly(A)+ RNA (1 µg) prepared from suspension-cultured rice cells 8 h after addition of the elicitor. PCR was carried out in a 50-µl reaction volume using Ex Taq DNA polymerase (TaKaRa) with the following program: 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 56°C, and 1 min at 72°C, followed by 7 min at 72°C, followed by cooling down to 4°C, resulting in amplification of a cDNA fragment of 549 bp. The PCR product, which appeared as a single band on an agarose gel electrophoresis, was cloned into pT7-Blue T-vector (Novagen, Darmstadt, Germany), and the nucleotide sequence was determined using DSQ-2000 L (Shimadzu, Kyoto, Japan).
Isolation of a full-length OsDTC1 cDNA
To isolate the full-length cDNA containing the 549-bp PCR-amplified fragment, we performed 5′- and 3′-RACE. Double-stranded cDNA was synthesized with the SuperScript system (Invirogen) from 1 µg of poly(A)+ RNA obtained from suspension-cultured rice cells 8 h after adding the elicitor. The cDNA was ligated to EcoRI/NotI adaptors (Stratagene) and used as a template. To isolate the 3′-region of the cDNA fragment, we performed PCR with a combination of the forward primer (5′-GGACTGAGTTTCATTGGAAGAAAT-3′; corresponding to GLSFIGRN), and reverse poly(T)-end adaptor primer (5′-AAAAGAATTCGCGGCCGCTTTTTTTTTTTTTTTTT-3′, EcoRI and NotI sites are underlined) with the same program as described above. The resulting 2.1-kbp PCR-amplified fragment was ligated into pT7/Blue-vector (Novagen), and the nucleotide sequence was determined. To isolate the 5′-terminus, we performed PCR according to the above program with a combination of the forward 5′-end adaptor primer (5′-TATGAATTCGCGGCCGCT-3′, EcoRI and NotI site are underlined) and the reverse gene-specific primer (5′-TGGGAAAGTGATGCCGAAACCTAT-3′; corresponding to IGFGITFP) designed based on the nucleotide sequence of the 2.1-kbp cDNA fragment. The resulting 0.8-kbp PCR-amplified fragment was ligated into pT7/Blue-vector (Novagen), and the nucleotide sequence was determined. For functional expression, we performed PCR according to the above program with a pair of primers based on the sequences from the RACE products, 5′BamHI/OsDTC1 (5′-AAAAGGATCCGGCAGCAGCAGCGGCATGATGCTGCTAGGTTCCCCT-3′, BamHI site is underlined) and 3′NotI/OsDTC1 (5′-AAATATTGCGGCCGCTTACAATAATCTGAGTTGAAG-3′, NotI site is underlined), to amplify OsDTC1 ORF. The PCR product was digested with BamHI and NotI and subcloned into the BamHI–NotI-digested pGEX-6P-2 vector (Amersham Bioscience), resulting in preparation of pGEX-OsDTC1, a plasmid for the expression of a GST fusion protein.
Expression of OsDTC1 cDNA in E. coli
pGEX-OsDTC1 was transformed into E. coli BL21 to yield BL21/pGEX-OsDTC1, which was grown in 2× tryptone 16 g l−1, yeast extract 10 g l−1, NaCl 5 g l−1; pH 7.0 (YT) medium (Sambrook et al., 1989) containing ampicillin (50 µg ml−1) at 37°C. When the optical density at 600 nm reached 0.6, IPTG was added to a final concentration of 1 mm, and the cells were incubated at 30°C for another 10 h and then harvested by centrifugation. The resultant cells were washed with 20 mm Tris–HCl buffer (pH 7.5), re-suspended in the same buffer, and disrupted by mild sonication on ice. After centrifugation at 15 000 g for 30 min, the supernatant was purified by Glutathione Sepharose 4B (Amersham Bioscience, Piscataway, NJ, USA) affinity column chromatography with the elution buffer containing 10 mm glutathione (Sigma, Aldrich, St Louis, MO, USA) according to the manufacturer's instructions. As a control, E. coli BL21 (DE3) harboring a plasmid for the expression of GST (pGEX-c) was similarly cultured, and the bacterial lysate was subjected to affinity column chromatography. The affinity-purified GST-OsDTC1 was analyzed by 10% SDS–PAGE with Coomassie Brilliant Blue staining (Laemmli, 1970), and the N-terminal five-amino acid sequence was determined by a Procise™· cLc protein sequencing system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions.
Geranylgeranyl diphosphate was purchased from Sigma-Aldrich ent-Coparol and (±)-syn-coparol were prepared by the methods of Toshima et al. (2000, 2002). These diterpene alcohols were converted to the corresponding diphosphate esters according to the method described previously by Mohan et al. (1996).
Enzyme assays of the GST–OsDTC1 fusion protein
The assay solution consisted of 2 µg of the substrate (GGDP, ent-CDP, or (±)-syn-CDP) in a solution of dithiothreitol (2 mm), EDTA (0.5 mm), proteinase inhibitor cocktail (1/100 tablet; Complete™·, Roche, Basel, Switzerland), MgCl2·6H2O (5 mm), and Tris–HCl buffer (100 mm, pH 7.4). The total volume of the enzyme assay solution was 0.5 ml. After affinity-purified protein (4 µg of OsDTC1–GST or 5 µg of GST) was added to the assay solution, the assay mixture was incubated at 30°C for 1 h. After 500 µl of dH2O was added to the reaction mixture, the solution was extracted with n-hexane (4 ml). The n-hexane extract was evaporated to dryness under a gentle nitrogen flow and subjected to GC–MS analysis and purified as described previously by Ogawa et al. (1996).
Expression analysis of OsDTC1 mRNA in the elicited suspension-cultured rice cells
RT-PCR was performed using poly(A)+ RNA (1 µg) prepared from suspension-cultured rice cells 0, 3, 6, 8, 10, and 12 h after adding the elicitor and a pair of the gene-specific primers used to amplify OsDTC1 ORF. As an internal standard, the rice actin gene ACT1 was amplified by RT-PCR using the gene-specific primers ACT1-P1 (5′-CATGCTATCCCTCGTCTCGACCT-3′) and ACT1-P2 (5′-CGCACTTCATGATGGAGTTGTAT-3′; Li et al., 2000).
Expression analysis of OsDTC1 mRNA in the UV-irradiated rice leaves
Rice plants (O. sativa L. cv. Nipponbare) were cultured in a greenhouse. At the sixth-leaf stage, the fourth and fifth leaves were detached. The detached leaves were placed on wet paper and UV-irradiated for 20 min using a Toshiba GL 15 lamp (254 nm) at a distance of 20 cm from the leaf surface, and then incubated at 30°C in a moist box at high humidity in the dark for 48 h and successively under white light for 24 h. After the incubation, the UV-irradiated leaves were frozen by liquid N2 (UV+) and stored at −80°C until use. Control rice leaves (UV−) were handled similarly except that they were not exposed to UV irradiation. Total RNA was extracted by SDS-phenol method. Ten micrograms of total RNA was separated on a 1% (w/v) agarose/2.2 m formaldehyde gel, and blotted on to a nylon membrane (Hybond N+ Amersham Bioscience) using standard blotting techniques (Sambrook et al., 1989). The membrane was hybridized with a [32P]-labeled 654-bp cDNA fragment (+466 to +1019) in a rapid hybridization buffer (Amersham Bioscience) at 68°C for 18 h, and finally washed with 0.2× SSC containing 0.1% SDS at 68°C for 1 h. Radioactivity was recorded on an imaging plate using a Bio-Imaging Analyzer (Fujix BAS1500, Tokyo, Japan).
Gas chromatography–mass spectrometry was conducted using an Agilent 6890 N GC-5973 N MSD mass selective detector system (ionization voltage 70 eV) fitted with a fused silica chemically bonded capillary column (DB-WAX; 0.25 mm in diameter, 60 m long, 0.25 µm film thickness; J&W Scientific Inc., Folsom, CA, USA). Each sample was injected onto the column at 80°C in the splitless mode. After a 2-min isothermal hold at 80°C, the column temperature was increased by 5 or 2°C min−1 to 250°C with a 10-min isothermal hold at 250°C. The flow rate of the helium carrier gas was 1 ml min−1. For GC–MS analyses of the diterpene hydrocarbons, 9.9 g (FW) of the suspension-cultured rice cells (elicitor treated for 48 h) or 5.0 g (FW) each of UV-irradiated or control rice leaves were extracted with methanol and purified (Ogawa et al., 1996).
We thank Drs Eiichi Minami and Takeshi Yamaguchi of the National Institute of Agrobiological Resources, Tsukuba, Ibaraki 305-8602, Japan, for help in the culture of suspension-cultured rice cells. We also thank Mr Tetsuya Chujo of Department of Biotechnology, The University of Tokyo, for his assistance in amino acid sequencing of GST-OsDTC1. This work was supported in part by a Grant-in-Aid for Scientific Research (No. 12460051) to H.Y. from the Ministry of Education, Science, Culture, and Sports of Japan.