Characterization of Geraniol Synthase from the Peltate Glands of Sweet Basil<sup><a href="#fn1" rid="fn1" class=" fn">1</a></sup>
Abstract
The monoterpene fraction of the lemon-scented sweet basil (Ocimum basilicum) cv Sweet Dani consists mostly of citral (a mixture of geranial and neral), with lower levels of geraniol and nerol. These compounds are stored in the peltate glands found on the leaf epidermis. Younger leaves, which have a higher density of such glands, also have a higher content of monoterpenes than older leaves. Geraniol synthase (GES) activity, generating geraniol from geranyl diphosphate, was shown to be localized exclusively or almost exclusively to glands. GES activity resides in a homodimeric protein that was purified to near homogeneity. Basil GES requires Mn as a divalent metal cofactor for activity and produces only geraniol from geranyl diphosphate. Km values of 21 and 51 μm were obtained for geranyl diphosphate and Mn, respectively. In the presence of O-labeled water, GES catalyzed the formation of O-geraniol from geranyl diphosphate, indicating that the reaction mechanism of GES is similar to that of other monoterpene synthases and is different from the action of phosphatases. A GES cDNA was isolated based on analysis of a glandular trichome expressed sequence tag database, and the sequence of the protein encoded by this cDNA shows some similarity to sequences of other terpene synthases. The expression of the GES cDNA in Escherichia coli resulted in a protein with enzymatic activity essentially identical to that of plant-purified GES. RNA gel-blot analysis indicated that GES is expressed in glands but not in leaves of basil cv Sweet Dani, whose glands contain geraniol and citral, and not in glands or leaves of another basil variety that makes other monoterpenes but not geraniol or citral.
Geraniol is an acyclic monoterpene alcohol emitted from the flowers of many species, notably roses (Rosa hybrida; Bayrak, 1994; Antonelli et al., 1997; Rao et al., 2000). It is also present in vegetative tissues of many herbs (Charles and Simon, 1992; Mallavarapu et al., 1998; Mockute and Bernotiene, 1999; Vieira et al., 2001) and is often found together with geranial and neral, which are the oxidation products of geraniol (Miyazawa and Kameoka, 1988). The mixture of geranial and neral, also called citral, imparts a “lemon” flavor, and lemongrass (Cymbopogon citratus Stapf.; Singh-Sangwan et al., 1993), ginger (Zingiber officinale Rosc.; Miyazawa and Kameoka, 1988), and some varieties of sweet basil (Ocimum basilicum; Grayer et al., 1996; Simon et al., 1999) such as basil cv Sweet Dani are particularly rich in citral (Morales and Simon, 1997). However, at present, there is no definitive proof of whether citral is synthesized from geraniol by an alcohol dehydrogenase (Sangwan et al., 1993; Singh-Sangwan et al., 1993; Hallahan et al., 1995; Sekiwa-Iijima et al., 2001) or by an oxidase (Potty and Bruemmer, 1970; Banthorpe et al., 1976), nor is it known if geraniol is the only substrate whose oxidation leads to the formation of citral or whether nerol, the cis-isomer of geraniol, can also serve as a precursor (Corbier and Ehret, 1988; Ikeda et al., 1991; Hallahan et al., 1995).
Geraniol itself is likely to be synthesized from geranyl diphosphate, the universal precursor of all monoterpenes (Croteau, 1987; Gershenzon and Croteau, 1993; McGarvey and Croteau, 1995; Wise and Croteau, 1999), although no report to date has identified a specific geraniol synthase (GES). Two types of enzymatic reactions have been hypothesized to lead to geraniol synthesis from geranyl diphosphate, either a phosphatase- or monoterpene synthase-based catalysis. However, in the absence of purified and characterized GES, the question of whether GES employs a similar mechanism to the one used by other monoterpene synthases (and by sesquiterpene and diterpene synthases, acting on farnesyl diphospate and geranylgeranyl diphosphate, respectively) has remained unanswered. The general mechanism of terpene synthases involves the removal of the diphosphate group and the generation of an intermediate with a carbocation as the first step. In the various terpene synthases, such intermediates further rearrange to generate the high number of terpene skele- tons observed in nature (Gershenzon and Croteau, 1993; McGarvey and Croteau, 1995). However, the structure of geraniol, whose carbon skeleton is identical to that of its precursor geranyl diphosphate, hypothetically allows for an alternative mechanism of simply breaking the phosphoester bond by a phosphatase to generate geraniol.
In this paper, we report the purification and characterization of GES from basil cv Sweet Dani and GES cDNA isolation and expression in Escherichia coli. Furthermore, the reaction mechanism of GES was investigated using [O]-labeled water. We show that GES is a member of the terpene synthase family based on sequence analysis, and its reaction mechanism is similar to that of other terpene synthases.
Acknowledgments
We thank Drs. Rod Wing (Arizona Genomics Institute, University of Arizona, Tucson) and Cari Soderlund (Arizona Genomics Computational Laboratory, University of Arizona, Tucson) for their assistance in production and maintenance of the basil EST databases, Dr. George Tsaprailis (Proteomics Core Facility of the Southwest Environmental Health Sciences Center, University of Arizona, Tucson) for assistance with peptide sequencing and analysis, Dr. Jihong Wang (University of Michigan, Ann Arbor) for help with protein purification, and Ms. Yue Yang (University of Michigan, Ann Arbor) for help with the RNA gel blots.
Notes
This work was supported by the U.S. Department of Agriculture-Binational Agricultural Research and Development Fund (grant no. IS-3332-02C), by the National Research Initiative Competitive Grants Program-U.S. Department of Agriculture (grant no. 2001-35318-10006), and by the National Science Foundation (grant no. 0210170).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032946.