| Abstract: | Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.Although extensive monoterpenol (especially linalool) oxidative metabolism has been described in many plant species, leading to fragrant and bioactive compounds as diverse as alcohols, aldehydes, acids, and epoxides (Williams et al., 1982; Matich et al., 2003, 2011; Luan et al., 2005, 2006; Ginglinger et al., 2013), pyranoid or furanoid linalool derivatives (Pichersky et al., 1994; Raguso and Pichersky, 1999), and geraniol-derived iridoids and secoiridoids (Dinda et al., 2007a, 2007b, 2011; Tundis et al., 2008), limited information is available on the enzymes generating these oxygenated compounds. Involvement of a cytochrome P450 (P450) enzyme extracted from Vinca rosea (now renamed Catharanthus roseus) in the hydroxylation of geraniol and nerol was suggested as early as 1976 (Madyastha et al., 1976). The first plant P450 gene to be isolated, CYP71A1 from avocado (Persea americana) fruit, was later shown to encode an enzyme with geraniol/nerol epoxidase activity (Hallahan et al., 1992, 1994). To our knowledge, a connection with compounds formed in the fruit has not yet been established. The geraniol 8-hydroxylase (often named geraniol 10-hydroxylase) CYP76B6, involved in the biosynthesis of secoiridoids and monoterpene indole alkaloid anticancer drugs in C. roseus, was found to belong to the CYP76 family in 2001 (Collu et al., 2001). The catalytic function of this enzyme was recently revised, and was shown to include a second oxidation activity, the conversion of 8-hydroxygeraniol into 8-oxogeraniol (Höfer et al., 2013). The same work also revealed a geraniol 8- and 9-hydroxylase activity of CYP76C4 from Arabidopsis thaliana. More recently, another CYP76 enzyme (CYP76A226) from C. roseus was found to metabolize oxidized geraniol derivatives and to have an iridoid oxidase activity, catalyzing the triple oxygenation of cis-trans-nepetalactol into 7-deoxyloganetic acid for the biosynthesis of secoiridoids and terpene indole alkaloids (Miettinen et al., 2014; Salim et al., 2014). Not all CYP76 enzymes seem to be devoted to the metabolism of monoterpenols. In most cases, however, CYP76s seem to be involved in terpenoid metabolism. CYP76Ms from monocots were found to metabolize diterpenoids for the synthesis of antifungal phytocassanes (Swaminathan et al., 2009; Wang et al., 2012; Wu et al., 2013), CYP76AH1 from Salvia miltiorhizza and its ortholog CYP76AH4 from rosemary (Rosmarinus officinalis) were shown to hydroxylate the norditerpene abietatriene in the pathway to labdane-related compounds (Zi and Peters, 2013), whereas CYP76Fs from sandalwood (Santalum album) were found to hydroxylate the sesquiterpenes santalene and bergamotene (Diaz-Chavez et al., 2013). CYP76B1 from Helianthus tuberosus was, however, found to metabolize herbicides belonging to the class of phenylurea (Robineau et al., 1998; Didierjean et al., 2002), but its physiological function was not reported. Other P450s from soybean (Glycine max; CYP71A10; Siminszky et al., 1999) or tobacco (Nicotiana tabacum; CYP71A11 and CYP81B1; Yamada et al., 2000) were also reported to metabolize phenylurea, but their physiological function was not investigated.A. thaliana ecotype Columbia-0 (Col-0) emits no geraniol and only tiny amounts of linalool, and extensive volatile profiling of different tissues detected only minor amounts of lilac aldehydes (oxygenated linalool derivatives; Rohloff and Bones, 2005). However, ectopic expression of a linalool/nerolidol synthase of strawberry (Fragaria × anannasa cv Elsanta) revealed a potentially efficient oxidative linalool metabolism in A. thaliana rosette leaves (Aharoni et al., 2003). Only recent work started to explore linalool metabolism in A. thaliana, which was found mainly localized in the flowers (Ginglinger et al., 2013). This work demonstrated the existence of two linalool synthases producing different enantiomers, and the concomitant involvement of two P450 enzymes, CYP76C3 and CYP71B31, with predominance of CYP76C3, in linalool oxidation. It also suggested the presence of partially redundant enzymes that may contribute to floral linalool metabolism.A family of eight CYP76 genes is detected in the A. thaliana genome. We report here an evolutionary and functional analysis of this family. We show that members of the CYP76C subfamily, when successfully expressed in yeast (Saccharomyces cerevisiae), all metabolize monoterpenols with different substrate specificities. Although CYP76Cs seem specific to Brassicaceae, they share common functions with CYP76s from other plants, such as CYP76B1 from H. tuberosus and CYP76B6 from C. roseus. These functions include not only monoterpenol oxidation, but also metabolism and detoxification of herbicides belonging to the class of phenylurea. Because of this property, CYP76Cs can be used simultaneously for monoterpenol oxidation and as selectable markers for plant transformation. |
