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First published online June 17, 2005; 10.1104/pp.105.059386 Plant Physiology 138:1322-1333 (2005) © 2005 American Society of Plant Biologists Surrogate Splicing for Functional Analysis of Sesquiterpene Synthase Genes1,[w]Plant Physiology, Biochemistry and Molecular Biology Program, Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546–0312 (S.W., M.A.S., B.T.G., S.T., S.L., J.C.); and Department of Chemistry, University of Illinois, Urbana, Illinois 61801 (R.M.C.)
A method for the recovery of full-length cDNAs from predicted terpene synthase genes containing introns is described. The approach utilizes Agrobacterium-mediated transient expression coupled with a reverse transcription-polydeoxyribonucleotide chain reaction assay to facilitate expression cloning of processed transcripts. Subsequent expression of intronless cDNAs in a suitable prokaryotic host provides for direct functional testing of the encoded gene product. The method was optimized by examining the expression of an intron-containing  -glucuronidase gene agroinfiltrated into petunia (Petunia hybrida) leaves, and its utility was demonstrated by defining the function of two previously uncharacterized terpene synthases. A tobacco (Nicotiana tabacum) terpene synthase-like gene containing six predicted introns was characterized as having 5-epi-aristolochene synthase activity, while an Arabidopsis (Arabidopsis thaliana) gene previously annotated as a terpene synthase was shown to possess a novel sesquiterpene synthase activity for  -barbatene, thujopsene, and -chamigrene biosynthesis.
Terpene synthases are a class of enzymes that catalyze the conversion of prenyl diphosphates to mono-, sesqui-, and diterpenoid compounds (Chappell, 1995  ; Davis and Croteau, 2000). The reactions catalyzed by many of these enzymes are stereospecific and complex, often generating one to upwards of 30 reaction products (Steele et al., 1998), many of which may contain one or more ring structures. Terpene synthases are of particular interest to phytochemists for their role in the synthesis of natural products, including flavors and fragrances, such as nootkatone and patchouli alcohol and antimicrobial phytoalexins like capsidiol (Chappell, 1995). Sesquiterpene synthases, a subset of terpene synthases catalyzing the biosynthesis of products derived from farnesyl diphosphate, have been characterized from many plant species including tobacco (Nicotiana tabacum; Facchini and Chappell, 1992), tomato (Lycopersicon esculentum; Colby et al., 1998), Hyoscyamus (Back and Chappell, 1995), cotton (Gossypium hirsutum; Chen et al., 1995), Arabidopsis (Arabidopsis thaliana; Chen et al., 2003), and maize (Zea mays; Degenhardt and Gershenzon, 2000; Schnee et al., 2002).
Many of the initial molecular studies of sesquiterpene synthases were based on the identification of cDNA clones encoding for enzymes catalyzing the biosynthesis of particular sesquiterpene compounds (Facchini and Chappell, 1992
Annotation of putative terpene synthase genes frequently occurs via the initial identification of the open reading frame by gene prediction software (Pavy et al., 1999 As part of our ongoing investigation into the structure and catalytic function of terpene synthases, the objective of this work was to develop a strategy for terpene synthase cDNA recovery in which a priori knowledge of gene expression patterns was not necessary, and a method in which the number of molecular manipulations was kept to a minimum before functional expression of the resulting cDNA. Our approach was to combine Agrobacterium-mediated transient expression in infiltrated leaf mesophyll (agroinfiltration) with a reverse transcription (RT)-PCR assay that facilitated cloning into expression vectors (Scheme I). In this way, we should be able to dictate the conditions of gene expression in a plant cell, exploit native genetic machinery to produce fully processed mRNA, and recover cognate cDNA. We refer to this strategy as surrogate splicing for functional genomics and demonstrate its utility by examining the function of two previously uncharacterized sesquiterpene synthase-like genes, one from tobacco and the other from Arabidopsis.
Optimization of the Surrogate Splicing Method
We initially assessed the surrogate splicing method using a previously constructed
The time course for petunia mesophyll cells taking up and expressing T-DNA-borne transgenes following agroinfiltration was determined in leaf discs collected at daily intervals and tested quantitatively for GUS enzyme activity (Fig. 1A). Detached petunia leaves were infiltrated with a suspension of Agrobacterium tumefaciens carrying the intron-containing GUS gene (GUSi) driven by the cassava vein mosaic virus (CsVMV-GUSi) promoter, a promoter previously characterized for its ability to direct strong constitutive expression in leaf tissue (Verdaguer et al., 1998
Recovery of a full-length cDNA from petunia leaf tissue agroinfiltrated with the GUSI gene was used to assess further the utility of this system for the generation of properly processed transcripts (Fig. 1B). Total RNA was isolated using a standard isolation procedure and 5 µg used for first-strand cDNA synthesis with an oligo(dT) primer. An aliquot of the first-strand synthesis reaction was then used in combination with primers designed to bracket the start and stop codons of the GUS gene and containing convenient restriction sites for future insertion of the PCR fragments into suitable prokaryotic expression vectors. Single-primer, RNA-only, and templateless controls showed no amplification products (lanes 2–5), while the complete experimental reaction yielded a reaction product that was approximately 190 bp smaller than the positive control product amplified directly from the GUSi gene (compare lane 6 to lane 7). The amplification product of lane 6 was subsequently cloned and sequenced. The sequence revealed that it was identical to the original GUS gene minus the 189-bp artificial intron (Vancanneyt et al., 1990 ) that had been properly removed at the 5' (TAC/GTAA) and 3' (GCAG/CT) splice sites.
General applicability of the surrogate splicing method for functional analysis of an unknown gene containing several predicted introns was initially assessed using a putative tobacco terpene synthase genomic clone referred to as g110 (Fig. 2). This genomic clone, along with approximately 30 other clones, was obtained when a tobacco cv Xanthi genomic library was screened with a probe corresponding to the first two exons of the 5-epi-aristolochene synthase 4 gene (EAS4; Facchini and Chappell, 1992
Leaf discs of detached petunia leaves infiltrated with A. tumefaciens harboring the CsVMV-110 construct were collected after 4 d of incubation. RT-PCR using RNA from these leaves resulted in the amplification of a product (Fig. 2B, lane7) significantly smaller than the positive size control of the g110 construct (2,327 bp, lane 8), but comparable in size to the amplification product from the authentic EAS4 cDNA (1,647 bp, lane 6). Control amplifications using single primers, RNA without RT, or no template, gave no comparable products (lanes 1, 3–5). The product of the experimental reaction (lane 7) was cloned and sequenced. The sequence of the recovered amplification product was identical to g110 except six intervening sequences had been removed accurately at the previously predicted intron-exon junctions.
The processed cDNA, now termed RT110, was digested with suitable restriction enzymes and ligated into the pET28a expression vector (Novagen, Madison, WI), which adds a poly-His tag at the amino terminus of the expressed protein. Lysate of isopropylthio-
To evaluate the general applicability of the surrogate splicing methodology to genes from nonsolanaceous plants, Arabidopsis genes annotated as terpene synthases were considered. Of the 34 to 40 sequences annotated (Aubourg et al., 2002
The At5g44630 gene was first PCR amplified from genomic DNA using PCR primers bracketing the putative (Fig. 3) start and stop codons, and recombined into a pBI121 vector modified with a suitable recombination cloning cassette (Hartley et al., 2000
The isolated RT-At5g44630 cDNA was inserted into the pET28a expression vector and transformed into E. coli host cells supplemented with additional tRNA genes considered limiting for eukaryotic gene expression (Rossetta cells, Novagen, Madison, WI). Lysates of IPTG-induced E. coli bearing the pET28a-RT-At5g44630 cDNA construct were recovered after sonication and centrifugation and initially assessed for total monoterpene, sesquiterpene, and diterpene synthase activities. Significant hexane-extractable, radioactive products were observed from incubations with high specific activity [1-3H]FPP but not with either [1-3H]GPP or [1-3H]GGPP (Fig. 3C). Unfortunately, further attempts to purify the Arabidopsis sesquiterpene synthase via an amino-terminal fused His-tag failed. Enzyme activity was completely lost, indicative of the labile nature of this enzyme and/or these preparations. Hence, the sesquiterpene reaction products were initial verified by comparison of lysates from noninduced and IPTG-induced bacteria cultures incubated with nonradioactive FPP and the organic solvent extractable products were evaluated by gas chromatography-mass spectrum (GC-MS; Supplemental Fig. 3). Approximately 16 unique sesquiterpene hydrocarbon reaction products were reproducibly observed in association with extracts from the induced cell extracts, but not the extracts from noninduced cultures. To more fully document the reaction products, we partially purified the synthase activity by rapid anion-exchange chromatography method (Vogeli et al., 1990 ) and following incubation with FPP, the organic solvent extractable reaction products purified by silica gel chromatography before examining them by GC-MS (Fig. 3, D–G). The three dominant peaks were identified by MS (and quantified by total ion chromatography [TIC]) as -barbatene (constituting 27.3% of the total reaction products), thujopsene (17.8% of the total products), and -chamigrene (9.9% of the total products). We have no evidence at this time concerning the absolute configuration of the three sesquiterpenes products. Both enantiomers of thujopsene and -chamigrene have been reported to occur naturally. However, it seems reasonable to suppose that they are the same as those established recently for the same compounds detected in volatile emissions from Arabidopsis flowers (Chen et al., 2003; Scheme II).
The remaining 13 reaction products were qualified as sesquiterpenes by having parent ions of 204 and fragmentation patterns typical for sesquiterpenes and individually accounted for 0.25% to 5% of the total products. Given the dominant -barbatene reaction product, the At5g44630 locus can now be referred to as an Arabidopsis -barbatene synthase (AtBS) gene.
Validation of the Surrogate Splicing Method
Agroinfiltration or in planta transient expression have been used in several novel ways recently to investigate various aspects of the transformation process and gene function. In studies evaluating the contribution of the Ti-plasmid vir genes to the T-DNA transfer process, Narasimhulu et al. (1996)
Splicing of RNA transcripts in plant cells comprises several distinct steps, reviewed by Brown and Simpson (1998)
Special instances are likely in which surrogate splicing may not result in the generation of mRNAs that are reflective of the native expressed forms. Such could be the case for transcripts that are alternatively spliced to produce distinct mRNA isoforms (Li and Howe, 2001
There are many methods for obtaining expressible cDNA clones for putative genomic genes including construction and screening of cDNA libraries and PCR screens for expressed mRNAs. The surrogate splicing process described here represents another relatively rapid method that only requires 4 to 5 d from infiltration to RT-PCR recovery of the cDNA. The greater part of the effort before and after these steps entails the manipulation of genomic or cDNA clones into various sequencing and expression vectors. The current method has also been improved by use of a recombination-based cloning system (Hartley et al., 2000
Several studies have demonstrated how difficult gene identification based solely on sequence identity may be (Lehfeldt et al., 2000
These results ascribe catalytic function to two genomic clones. While identification of the tobacco g110 gene as an epi-aristolochene synthase is not unexpected given its high degree of similarity to EAS and the existence of 12 to 16 related terpene synthase genes in the tobacco genome (Facchini and Chappell, 1992
While precedence for the generation of multiple reaction products of by single terpene synthases has been firmly established (Steele et al., 1998
Plant Materials and Agroinfiltration Petunia (Petunia x hybrida) plants were started from commercially available seed. Plants were grown in a greenhouse with supplemental light provided by sodium vapor lamps. Prior to experimentation, a population of plants was generated by crossing two parental lines chosen for broad rosette leaves suitable for infiltration. Seeds generated from this cross were collected and subsequently maintained as population A. Leaves for experimental infiltration were chosen on the basis of size with a 5-cm width minimum. Leaves were either left on the plant or cut from plants and rinsed in tap water to remove any adhering debris. This brief submersion was also helpful to promote hydration of the leaf, as fully turgid leaves were preferable for infiltration. Immediately prior to infiltration, detached leaves were placed on dampened paper towels in plastic boxes on the lab bench. Agrobacterium tumefaciens strain GV3850 was transformed by electroporation and maintained under kanamycin and rifampicin selection. Overnight cultures for infiltration were concentrated by centrifugation, resuspended in a 10% Suc solution, reconcentrated, and finally resuspended in 10% Suc to a final concentration of OD600 equal to 0.5±0.05. Addition of 20 mM acetosyringone 3 h prior to infiltration enhanced in planta expression, but was not necessary. Petunia leaves were nicked on the lower leaf surface, and the bacterial suspension introduced using a needle-less syringe. Infiltrated plants were maintained in the greenhouse while infiltrated leaves were maintained in an open plastic container on wet paper towels for up to 1 week.
Leaf discs for GUS enzyme assays (Gallagher, 1992
GUSi (Vancanneyt et al., 1990 RT of RNA to generate a first strand for PCR was performed as follows: Total RNA (5 µg) was combined with water and first strand primer, T (27) or T(27)V, 20 pmol, to a total volume of 12 µL. This mixture was heated to 70°C for 10 min, then allowed to cool to room temperature, and the remaining reagents added: 10 units RNase inhibitor, 100 mM Tris-HCl, pH 8.3, 40 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 1.25 mM each dNTP, 4 mM sodium pyrophosphate, and 200 units of reverse transcriptase (Superscript II, Invitrogen) in a total volume of 20 µL. The reaction was incubated at 42°C for 1 h, then placed on ice. PCR reactions (50 µL) used 2 µL of first strand cDNA as template and included 20 pmol of specific forward and reverse primers, 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 80 µM each dNTP, and 1 unit Taq polymerase (Invitrogen). Typical amplification conditions were 95°C, 1 min; 55°C to 60°C, 1 min; 72°C, 2 min, for 30 cycles. Primers for the amplification of the GUSi gene, for initial transgene construction, and for RT-PCR recovery were (forward, incorporating a BamHI site) 5'-AAG GAT CCT ATG TTA CGT CCT GTA GAA ACC-3', and (reverse, incorporating a SstI site) 5'-AAG AGC TCA TTG TTT GCC TCC CTG CT-3'. Primers for the initial amplification and recovery of the g110 clones were (forward, incorporating a BamHI site) 5'-AAG GAT CCA TGG CCT CAG CAG CAG TTG CA-3' and (reverse, incorporating a SstI site) 5'-AAA GAG CTC GCA GCT CAA ATT TTG ATG GA-3'. Restoration of the g110 reading frame employed a forward primer 5'-GGA TCC ATG GCC TCA GCA GCA GTT GCA AAC TAT GAA GAA GA-3' that incorporates a deleted "A" (underlined) at position +21 relative to the start codon. Primers for the initial amplification of the At5g44630 genomic clone were (forward, incorporating an attB1 recombination site) 5'-GGGG ACA AGT TTG TAC AAA AAA GCA GGC TCC ATG GGC AGC AGC CAT CAT CAT CAT CAT CAC ATG GAA GCA TTA GGA AAC TTT GAT TAC-3' and (reverse primer, incorporating an attB2 site) 5'-GGGG CAC CAC TTT GTA CAA GAA AGC TGG GTC AAG AAG TAT AGG ATC TAC GAG-3'. Primers for the RT-PCR recovery of the RT-At5g44630 clone were (forward, incorporating a SstI site) 5'-CGC GAG CTC ATG GAA GCA TTA GGA AAC TTT GAT TAC G-3' and (reverse, incorporating a XhoI) 5'-GCC GCT CGA GCT AAA GTA TAG GAT CTA CGA GCA AAA G-3'. RT-PCR amplification products were digested with appropriate restriction enzymes as necessary and ligated into one of several available cloning vectors (pGEM-T Easy, Promega, Madison, WI), pBS (Strategene, San Diego) for DNA sequence analysis. All the DNA sequencing reactions were performed using the BigDye Terminator Cycle sequencing kit (Perkin-Elmer, Wellesley, MA) with the sequences read on an automated ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).
The RT-110 BamHI-SstI digestion fragment was ligated into the corresponding sites within the pET-28a vector in-frame with an N-terminal hexahistidyl tag (Novagen, Madison, WI). This construct was transformed into BL21 (DE3) competent Escherichia coli cells (Novagen), which were grown to an OD600 equal to 1.0 at 37°C, induced with 0.1 mM IPTG, and incubated for an additional 5 h at 28°C. Cells were collected by centrifugation, the pellets were frozen overnight at –80°C and then sonicated three times for 30 s, and the lysate was recovered after a 20-min centrifugation at 38,000g. The lysate was filtered through a 0.45-micron filter and the His-tagged protein purified by Ni2+ affinity chromatography as per Mathis et al. (1997)
The RT-At5g44630 SstI-XhoI digestion fragment was similarly ligated into the pET28a vector in-frame with the N-terminal histidyl tag, except transformed into BL21 Rosetta competent E. coli cells (Novagen). Where indicated, cell cultures were grown to an OD600 equal to 0.6 at 37°C before addition of 0.4 mM IPTG and continued incubation overnight at 28°C. Cells were collected, stored, and extracted as described above. Because enzyme activity was lost upon Ni2+ affinity chromatography, synthase activity was partially purified by anion-exchange chromatography (Vogeli et al., 1990 Protein concentrations were estimated by the Bradford method using IgG as the standard (Bio-Rad, Hercules, CA).
Initial terpene synthase activities were measured with radioactive [3H]GPP (ARC, St. Louis, 20 Ci/mmol), [3H]FPP (Perkin-Elmer, Boston, 16.1 Ci/mmol), or [3H]GGPP (Perkin-Elmer, Boston, 23 Ci/mmol) using standard reaction conditions (Rising et al., 2000
The product profile for the Ni2+ affinity column-purified RT-110 enzyme was determined by incubating approximately 100 nM enzyme with 46 µM FPP in a 2.5-mL reaction. Reactions were incubated 30 min, then extracted twice with 2 mL of pentane, which was concentrated under nitrogen to approximately 50 µL for GC-MS analysis. GC-MS analysis was performed with an HP-GCDplus equipped with a DB-5ms capillary column (30 mx0.25 mm, 0.25-µm phase thickness) and run with He as the carrier gas at 1 mL/min. Sample injections were splitless with an injection port temperature of 250°C. The oven was programmed to hold at 100°C for 1 min and then increased to 270°C with an 8°C/min ramp. The dominant product was compared to an authentic sample of 5-epi-aristolochene (Zhao et al., 2004
The reaction product profile for the anion-exchange purified At5g44630 enzyme was determined similarly. Partially purified enzyme (4 mg) was incubated with 11.5 µM FPP in a total volume of 10 mL of reaction buffer (20 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.2 mM MnCl2) for 30 min before sequential extractions with 10 mL of hexane. The hexane extracts were combined, concentrated to 1 mL, passed over a 1-mL silica gel column, and the hexane eluate was reconcentrated to 30 µL. Reaction products were identified by MS using a Thermo Finnigan DSQ GC-MS system equipped with a Restec Rtx-5 capillary column (30 mx0.32 mm, 0.25-µm phase thickness). One-microliter samples were injected in the splitless mode at 250°C with an initial oven temperature of 70°C for 1 min followed by a 4°C per min gradient to 230°C. Mass spectra were recorded at 70 eV, scanning from 35 to 300 atomic mass units, and compared to NIST and MassFinder library standards for verification. Tentative compound identifications are based on standard NIST and MassFinder computer algorithms matching unknowns to library standards with similarity values in excess of 30%, visual comparison of the mass spectral patterns, and retention time comparisons to those reported in Chen et al. (2003)
Upon request, all materials described in this publication will be made available in a timely manner for noncommercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining all such permission will be the responsibility of the requestor. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY313939 (g110) and AY876386 (RT-At5g44630). Received January 6, 2005; returned for revision February 23, 2005; accepted February 24, 2005.
1 This work was supported by the Kentucky Tobacco Research and Development Center and the National Science Foundation (to J.C.), by the National Institutes of Health (R.M.C.), and by the Kentucky Agricultural Experiment Station. 
2 These authors contributed equally to the paper.
3 Present address: Biology Department, University of Nebraska-Omaha, Omaha, NE, 68182.
4 Present address: Allylix Inc., A165 ASTeCC, University of Kentucky, Lexington, KY 40546–0286.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.059386. * Corresponding author; e-mail chappell{at}uky.edu; fax 859–257–7125.
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ABSTRACT
RESULTS
-cyclization onto the 10, 11 double bond creates the 5-membered B ring and results in centering of the secondary carbocation at C10 of the B ring. A 1,4-hydride shift from the A ring repositions the reactive carbocation back onto the A ring, which creates a key branch point for the diversion of reaction intermediates to either