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First published online August 6, 2004; 10.1104/pp.104.044388 Plant Physiology 135:1956-1966 (2004) © 2004 American Society of Plant Biologists
Characterization of a Root-Specific Arabidopsis Terpene Synthase Responsible for the Formation of the Volatile Monoterpene 1,8-Cineole1Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, Michigan 48109–1048 (F.C., E.P., D.T.); Max Planck Institute for Chemical Ecology, D–07745 Jena, Germany (J.P., J.G., D.T.); and Biotechnology Laboratory, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 (D.K.R., J.B.)
Arabidopsis is emerging as a model system to study the biochemistry, biological functions, and evolution of plant terpene secondary metabolism. It was previously shown that the Arabidopsis genome contains over 30 genes potentially encoding terpene synthases (TPSs). Here we report the characterization of a monoterpene synthase encoded by two identical, closely linked genes, At3g25820 and At3g25830. Transcripts of these genes were detected almost exclusively in roots. An At3g25820/At3g25830 cDNA was expressed in Escherichia coli, and the protein thus produced was shown to catalyze the formation of 10 volatile monoterpenes from geranyl diphosphate, with 1,8-cineole predominating. This protein was therefore designated AtTPS-Cin. The purified recombinant AtTPS-Cin displayed similar biochemical properties to other known monoterpene synthases, except for a relatively low Km value for geranyl diphosphate of 0.2 µM. At3g25820/At3g25830 promoter activity, measured with a  -glucuronidase (GUS) reporter gene, was primarily found in the epidermis, cortex, and stele of mature primary and lateral roots, but not in the root meristem or the elongation zone. Although the products of AtTPS-Cin were not detected by direct extraction of plant tissue, the recent report of 1,8-cineole as an Arabidopsis root volatile (Steeghs M, Bais HP, de Gouw J, Goldan P, Kuster W, Northway M, Fall R, Vivanco JM [2004] Plant Physiol 135: 47–58) suggests that the enzyme products may be released into the rhizosphere rather than accumulated. Among Arabidopsis TPSs, AtTPS-Cin is most similar to the TPS encoded by At3g25810, a closely linked gene previously shown to be exclusively expressed in flowers. At3g25810 TPS catalyzes the formation of a set of monoterpenes that is very similar to those produced by AtTPS-Cin, but its major products are myrcene and (E)--ocimene, and it does not form 1,8-cineole. These data demonstrate that divergence of organ expression pattern and product specificity are ongoing processes within the Arabidopsis TPS family.
Terpenes comprise a very large class of plant secondary metabolites that serve a variety of different functions in basic and specialized metabolism. The majority of these compounds are likely to operate as direct defense agents because they often exhibit toxic and deterrent effects against plant pathogens and herbivores (Langenheim, 1994  ; Gershenzon and Kreis, 1999). However, since all monoterpenes, many sesquiterpenes, and a few diterpenes have high vapor pressure at ambient temperature and volatilize easily, these compounds could also act at a distance from the plant, serving as attractants or deterrents for pollinators and herbivores (Dobson, 1994; Dudareva and Pichersky, 2000; Pichersky and Gershenzon, 2002), or as signals released after damage by herbivores to attract their predators and parasitoids (Pare and Tumlinson, 1999; Dicke and Van Loon, 2000; Kessler and Baldwin, 2001). In fact, volatile monoterpenes and sesquiterpenes have frequently been shown to be emitted from flowers and other aerial parts of the plant, and their release is often developmentally regulated or induced by damage (Chen et al., 2003b; Dudareva et al., 2003; Arimura et al., 2004). Curiously, volatile monoterpenes and sesquiterpenes have also been reported to be synthesized and accumulated in roots and rhizomes of various plant species (Wichtmann and Stahl-Biskup, 1987; Bos et al., 2002; Kovacevic et al., 2002). As a consequence of their antimicrobial and antiherbivore activity, these substances may serve as direct defenses of below-ground tissue when they accumulate. However, if volatile terpenes were released from roots, they could also play interesting roles in interactions with other rhizosphere organisms analogous to roles of terpenes emitted from aerial parts.
Monoterpenes contain a 10-carbon backbone and are all synthesized from the common precursor geranyl diphosphate (GPP) by the action of various monoterpene synthases (Wise and Croteau, 1999
The Arabidopsis genome contains more than 30 genes that encode proteins with sequence similarity to the TPS family (Aubourg et al., 2002
The Arabidopsis cDNAs of At3g25820 and At3g25830 Are Identical and Have High Sequence Similarity to Other Monoterpene Synthases
The Arabidopsis genome database shows three tandemly linked TPS genes, At3g25810, At3g25820, and At3g25830 on chromosome 3 (Fig. 1). Gene At3g25810 was previously found to be expressed exclusively in flowers and to encode a TPS that catalyzes the formation mostly of myrcene and (E)-
The At3g25820/At3g25830-Encoded Protein Expressed in E. coli Converts GPP to 1,8-Cineole as the Principal Product
Because sequence analysis indicated that the protein encoded by At3g25820 and At3g25830 contains a transit peptide (Aubourg et al., 2002
The crude protein extracts from bacterial cultures expressing the truncated At3g25820/At3g25830-encoded protein were incubated with GPP or FPP in assay buffer as previously described for other TPSs (Bohlmann et al., 2000
Biochemical Properties of At3g25820/At3g25830-Encoded AtTPS-Cin The E. coli-expressed mature AtTPS-Cin was purified and its biochemical properties determined. The crude enzyme preparation was subjected to a combination of DEAE and Mono-Q ion-exchange chromatography to yield a nearly homogeneous protein with a molecular mass of approximately 60 kD (Fig. 4). The enzyme showed a pH optimum at 8.0. In the absence of a divalent metal ion, AtTPS-Cin was inactive, but activity was restored by provision of either Mg2+ or Mn2+. The maximum activity levels with either ion were nearly identical, but with Mg2+, 40 mM were required to achieve these levels, while with Mn2+, the enzyme was fully active at a concentration of 1 mM. The specific activity of Arabidopsis AtTPS-Cin with GPP was 10.9 ± 0.4 pkat mg–1 protein, giving a kcat of 0.001 s–1. The enzyme had an apparent Km value for GPP of 0.2 ± 0.03 µM.
At3g25820/At3g25830 Are Expressed in Specific Tissues of the Roots
In a preliminary RT-PCR characterization of the entire family of TPS genes in Arabidopsis (ecotype Col), we reported that At3g25820/At3g25830 transcripts were found in roots and at much lower levels in siliques also, but not in leaves, flowers, or stems under normal growing conditions (Chen et al., 2003b
We looked at the expression of At3g25820/At3g25830 in roots and seedlings in more detail by examining the staining patterns of transgenic Arabidopsis Col plants carrying an At3g25820/At3g25830 promoter:: -glucuronidase (GUS) fusion construct. Five to 10 seedlings of four independent T2 lines showed consistently strong GUS staining in the mature primary and lateral roots. However, GUS activity was never observed in a 0.2- to 0.5-cm-long zone situated at the tip of the root, which includes the apical meristem and elongation zone (Fig. 6, A and B). The staining pattern of intact roots in comparison to that of transverse and lateral root sections suggested a developmental change in the location of At3g25820/At3g25830 promoter activity. Whereas younger root sections often showed GUS staining primarily in the vascular system, in older root zones, strong GUS activity was exclusively or additionally observed in the cortex and epidermal cell layer (Fig. 6, B–E). Staining was found with less consistency in root hairs (Fig. 6, B and C). In addition to the roots, GUS staining also occurred in the hypocotyl and, to a lesser degree, in the petioles of the seedling, but the rest of the aerial parts were not stained (Fig. 6A). GUS activity was not affected by the addition of Suc or kanamycin to the medium. In mature plants, GUS staining was also observed in roots, but not in stems or flowers (data not shown). Control lines carrying the promoterless GUS insertion cassette did not show any GUS activity.
The products of AtTPS-Cin were sought in planta by solvent extraction and solid-phase microextraction of ground roots of Arabidopsis plants grown in soil or under hydroponic conditions. However, neither 1,8-cineole nor any of the other enzyme products were detectable by GC-MS analysis above the level of 10 to 20 ng g–1 fresh weight. Two AtTPS-Cin products, myrcene and (–)-limonene, have previously been found in headspace collections of aerial parts of Arabidopsis (ecotype Col; Chen et al., 2003b ). However, these are unlikely to represent the activity of AtTPS-Cin since At3g25820/At3g25830 expression is almost completely absent in aerial parts (Fig. 5), and myrcene and (–)-limonene are also known to be products of other Arabidopsis TPSs, including the enzyme encoded by At3g25810 (Bohlmann et al., 2000; Chen et al., 2003b; Fäldt et al., 2003).
At3g25820 and At3g25830 Encode a Root-Specific AtTPS-Cin
While much research has focused on terpenoid biosynthesis in aerial parts of plants, little is known about the formation of terpenes in below-ground organs. Here we show that the tandem Arabidopsis genes At3g25820 and At3g25830, with identical open reading frames and promoter regions, encode a root-localized monoterpene synthase that produces the cyclic ether 1,8-cineole as its principal product when expressed in E. coli. The enzyme also catalyzes the formation of nine other monoterpenes (eight olefins and one alcohol) as minor products, and has properties generally similar to those of other monoterpene synthases. An exception is the kcat value, which is approximately one order of magnitude lower than the turnover rates usually reported for plant monoterpene and sesquiterpene synthases (Cane, 1999
The nearly exclusive root-specific expression of At3g25820 and At3g25830 was revealed by RT-PCR and promoter::GUS fusion analysis. To our knowledge, this is the first report of a functionally characterized root-specific monoterpene synthase gene. Among sesquiterpene synthases, a gene encoding an epi-aristolochene synthase from Nicotiana attenuata was shown to be expressed in roots (Bohlmann et al., 2002 ), while a number of the uncharacterized root-expressed TPS genes of Arabidopsis are likely to encode diterpene synthases (Aubourg et al., 2002; Chen et al., 2003b).
Our inability to detect 1,8-cineole and other products of AtTPS-Cin in Arabidopsis roots is puzzling given the high apparent level of At3g25820/At3g25830 gene expression in this organ. Due to the presence of a potential plastidic transit peptide, AtTPS-Cin is most likely located in root plastids that have been shown, in other plant species, to contain an active methylerythritol phosphate pathway for formation of the C5 building blocks of terpenoids (Hans et al., 2004
On the other hand, our findings are consistent with previous studies on Arabidopsis aerial parts where, despite high expression of several TPS genes (Chen et al., 2003b
The volatilization of 1,8-cineole and other AtTPS-Cin products from Arabidopsis roots would be consistent with the overall staining pattern observed in the At3g25820/At3g25830 promoter::GUS fusion transformants. Expression is strongest in the outer cell layers, the cortex, and the epidermis of mature roots, from which these lipophilic compounds could readily diffuse or be released directly into the atmosphere. In younger roots, GUS staining was weaker and localized in the vascular system, while staining was completely absent from the root tip. These observations are supported by recent cell type-specific microarray expression profiling of Arabidopsis roots, which showed that transcripts of At3g25820/At3g25830 were detected primarily in fully elongated epidermal and lateral root cap cells (localized expression domain group 6; Birnbaum et al., 2003
The lack of accumulation of 1,8-cineole and other AtTPS-Cin products in the plant may also be attributed to their rapid conversion to other metabolites. However, except for limonene and sabinene, bona fide metabolites of AtTPS-Cin monoterpenes have only rarely been reported from plant species (Wise and Croteau, 1999
1,8-Cineole and several other AtTPS-Cin products have long been known to be active against plant pests. For example, 1,8-cineole is toxic and deterrent to certain insect and mammalian herbivores (Tripathi et al., 2001
In evaluating the biological significance of AtTPS-Cin products, it will be important to keep in mind that these compounds do not appear to accumulate in plant tissue, but are instead released from the roots. A large variety of primary and secondary metabolites are known to be exuded from the roots of many plant species (Walker et al., 2003a
Besides being of direct benefit to plants, exudates could also serve as signals for a diverse group of rhizosphere organisms. For example, a group of sesquiterpene lactones that are released in low amounts from roots of several different plant species, including sorghum, maize, and rice, trigger the germination of the parasitic weeds Orobanche and Striga spp. (Bouwmeester et al., 2003
The close chromosomal proximity of At3g25810 to At3g25820 and At3g25830 and their high levels of coding sequence similarity (78% identity on the protein level), as well as intron number, position, and sequence, suggest that a relatively recent tandem duplication gave rise to a pair of genes composed of At3g25810 and the At3g25820/At3g25830 progenitor, followed by an even more recent tandem duplication that gave rise to the two genes At3g25820 and At3g25830, which are presently still identical (Aubourg et al., 2002
In addition to divergence in the mode of expression, genes At3g25810 and At3g25820/At3g25830 have also diverged with respect to the enzymatic reaction catalyzed by the proteins they encode. The major product of the reaction catalyzed by AtTPS-Cin is 1,8-cineole, which is not produced by the At3g25810-encoded protein. However, the nine other enzyme products of AtTPS-Cin (Fig. 3) are also produced by the At3g25810 protein (Fig. 7). The TPS encoded by gene At3g25810 was originally reported to catalyze the formation of myrcene and (E)-
Peters and Croteau (2003) In conclusion, the observed differences in spatial expression and product specificity of the two closely related enzymes AtTPS-Cin and At3g25810 TPS demonstrate the ongoing evolutionary process of duplication and divergence in gene families involved in plant secondary metabolism.
Plant Material and Growth Conditions Arabidopsis (L.) Heynh (ecotype Columbia) plants were grown from seed on potting soil in a climate-controlled growth chamber (22°C, 55% relative humidity, 150 µmol m–2 s–1 photosynthetically active radiation, 16-h/8-h photoperiod), if not stated otherwise. Roots were collected from 6-week-old bolted mature plants for RNA extraction.
GPP and FPP were purchased from Echelon Biosciences (Salt Lake City). [1-3H]GPP and [1-3H]FPP were products of American Radiolabeled Chemicals (St. Louis).
Total RNA was isolated from roots of mature Arabidopsis plants with an RNeasy plant mini kit (Qiagen, Valencia, CA). DNA contamination was removed with deoxyribonuclease (Qiagen) treatment. Five micrograms of the purified RNA sample were used to synthesize cDNA with an oligo(dT) primer and a First-Strand cDNA Synthesis kit (Amersham Biosciences, Uppsala) in a 15-µL reaction. Two primers, 5'-ATGGCAACTTTACGTATAAG-3' and 5'-TCAACCAAGACGATTTACATC-3', which correspond to the start and the end of the coding region of At3g25820 and At3g25830, respectively, were subsequently used for PCR amplification of At3g25820/At3g25830 cDNA. The PCR reaction volume was 25 µL and contained 100 ng of each primer, 0.2 mM of each deoxynucleoside triphosphate, 1 µL cDNA, and 0.75 units of Taq DNA Polymerase (Fisher, Pittsburgh). A programmable thermal controller (PTC-100; MJ Research, Waltham, MA) was used with an initial denaturation step of 96°C for 1 min, followed by 30 cycles of 94°C for 30 s, 54°C for 30 s, 72°C for 60 s, and a final elongation step of 72°C for 10 min. The resulting cDNA fragment was cloned into the expression vector pCRT7/CT-TOPO (Invitrogen, Carlsbad, CA) for sequencing.
A truncated version of the At3g25820/At3g25830 cDNA was prepared in which the first 46 codons were removed and an ATG codon was added immediately upstream of the two Arg codons 47 and 48. This was accomplished by PCR using a primer pair with the forward primer 5'-ATGCGACGCTCCGGAAACTATCAAC-3' and the reverse primer 5'-TCAACCAAGACGATTTACATC-3' and a second primer pair with the forward primer 5'-CACCATGCGACGCTCCGGAAACTATCAACCT-3' and the reverse primer 5'-TCAACCAAGACGATTTACATCTAA-3', respectively. The product of the reaction with the first primer pair was inserted into the pCRT7/CT-TOPO expression vector (Invitrogen), while the product amplified in the reaction with the second primer pair was inserted into the expression vector pET101/D-TOPO (Invitrogen). The resulting plasmids were transformed into the E. coli BL21 Codon Plus strain (Stratagene, La Jolla, CA). Bacterial growth conditions, induction of expression of the introduced gene, and protein extraction procedures were as previously described (Chen et al., 2003b
TPS assays and volatile terpene product separation and analysis were carried out essentially as previously described (Chen et al., 2003b
Protein purification protocols essentially followed the procedures previously described (Chen et al., 2003a
Purified AtTPS-Cin enzyme was used for biochemical characterization. A standard assay for determining biochemical properties was carried out in a final volume of 50 µL with 0.1 µg purified enzyme, and 10 µM of [1-3H]GPP (2 or 9 MBq µmol–1) or [1-3H]FPP (9 MBq µmol–1). Buffer, salt, and incubation conditions were the same as previously described (Chen et al., 2003b For pH optimum determination, assays with saturated substrate were carried out in 100 mM Bis-Tris propane buffers with pH 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. For determination of the metal ion requirement, assays were carried out in buffer A in the presence of MgCl2 (concentrations from 0 to 50 mM) or MnCl2 (concentrations from to 0 to 8 mM). For kinetic assays, appropriate enzyme concentrations and incubation times were determined so that the reaction velocity was linear during the reaction time period. To determine the Km value for GPP, different concentrations of GPP were applied in standard assays in three replications. Calculation of the apparent Km value was obtained by Lineweaver-Burk plot analysis.
Procedures for total RNA isolation from mature plants and cDNA synthesis were the same as those described for cDNA cloning. For RT-PCR analysis of At3g25820/At3g25830 expression in different organs, primers designed to amplify a fragment of approximately 0.9 kb were as follows: forward primer 5'-TATTTGATGTGATCATCGACC-3' and reverse primer 5'-GGAACACTTAAGATATAAAAGGT-3'. The two primers used for PCR amplification of 18S rRNA were: forward 5'-GACGGAGAATTAGGGTTCGATTC-3' and reverse 5'-CCAACTAAGAACGGCCATGCAC-3'. Initially, PCR reactions were performed with At3g25820/At3g25830-specific primers with 0.1, 0.2, 0.5, and 1.0 µL cDNA for 30 cycles with an annealing temperature of 54°C. Each PCR reaction of 25 µL contained 0.4 µM forward primer, 0.4 µM reverse primer, and 0.2 mM of dATP, dTTP, dGTP, and dCTP, respectively. Amplified products were separated on a 1.0% agarose gel and quantified using the Bio-Rad Quantity One program (Hercules, CA). Analysis showed that the amounts of amplified products increased linearly with increasing amounts of cDNA. 0.2 µL cDNA was chosen as the optimal amount of template for further PCR reactions. For 18S rRNA, PCR reactions were carried out in separate tubes under conditions similar to those described for At3g25820/At3g25830, except that the reactions were performed for 20 cycles.
Genomic DNA was isolated from Arabidopsis (ecotype Col) leaves as described by Rogers and Bendich (1985)
For sectioning of GUS-stained roots, root tissue was embedded in a glycol methacrylate resin as described by Beeckman and Viane (2000)
Soil-grown roots were harvested from 4-week-old prebolting Arabidopsis plants (ecotype Col). Soil particles were removed from roots by repeated submersion and rinsing in tap water and the tissue was immediately analyzed. Alternatively, roots were harvested from plants in the rosette stage grown hydroponically under short-day conditions for 4 to 5 weeks in Hoagland solution (Gibeaut et al., 1997 Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number AY691947.
We thank Katrin Heisse for technical assistance, Aleksandra Skirycz for help with embedding and sectioning of Arabidopsis roots, and Jorge Vivanco for stimulating discussion and unpublished data. Received April 8, 2004; returned for revision June 2, 2004; accepted June 2, 2004.
1 This work was supported by the National Science Foundation (grant no. IBN–0211697 to E.P.), by the Max Planck Society (to J.P., J.G., and D.T.), and by the Natural Sciences and Engineering Research Council of Canada (to J.B.).  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.044388. * Corresponding author; e-mail tholl{at}ice.mpg.de; fax 49–3641–571302.
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TOP
ABSTRACT
RESULTS
-thujene (0.6%), (–)-(1S)-
-selinene synthase and 
-humulene synthase. J Biol Chem 273: 2078–2089
