L. x intermedia, a high EO yielding natural hybrid of L. angustifolia and L. latifolia, produces an EO constituted of monoterpenes, mainly linalool, linalyl acetate, borneol, camphor and 1,8-cineole. Due to the relatively high levels of borneol and camphor, L. x intermedia EOs typically are used in low-value products such as air fresheners and soaps. The cloning of genes responsible for borneol and camphor production opens several avenues for reducing the production of these compounds, and hence increasing the commercial value of the EO in L. x intermedia plants. In this context, the cloning of BPPS, an enzyme that catalyzes the committed step (conversion of GPP to BPP) in borneol (and camphor) production is of key importance.
To date, five BPPS genes have been reported from different plants (Wise et al. 1998; Despinasse et al. 2017; Hurd et al. 2017; Wang et al 2018; Ma et al 2021). Most produce BPP and multiple monoterpenes from GPP, with BPP (measured as a derivative alcohol borneol) proportion of 30% (L. angustifoila), 65.9% (A. villosum), 57–75% (S. officinialis), 88.7% (C. burmanni) and 90% (P. dulicis).
Phylogenetic analysis and genome organization of (+)-LiBPPS
In this study, we identified (+)-LiBPPS, which encoded 593 amino acids consisting of all a single copy of conserved motifs present in typical plant TPSs, such as RR(x8)W, DDxxD and (N,D)D(L,I,V)x(S,T)x3E (Wise et al. 1998). The first motif, RR(x8)W, is located at the very beginning of the N-terminus of the protein and is required in mono-TPSs for cyclization (Williams et al. 1998), or protein stabilization (Hyatt et al. 2007). The DDxxD and ((N,D)D(L,I,V)x(S,T)x3E) motifs presented at C-terminus are responsible for substrate binding and coordination of divalent metal ion cofactors (Degenhardt et al. 2009). Uniquely, a double copy of RR(x8)W motif was detected from P. dulicis BPPS, in which the first copy inhibited the in vitro protein activity (Hurd et al. 2017). However, alterations of the common residues in these conserved motifs are often observed through deletion and/or substitution without compromising the functional activities. For example, a deletion of an arginine (R) at RR(x8)W motif of cadinol and germacrene D synthases, and substitution of an aspartate (D) by glutamate (E) at DDxxD motif of germacrene D synthases were identified in L. angustifolia (Jullien et al. 2014).
Based on analysis of deduced amino acid sequences, (+)-LiBPPS is highly homologous to the L. angustifolia limonene synthase (LaLimS), L. x intermedia 3-carene synthase (Li3CarS), and Lavandula viridis α-pinene synthase (LvPinS) and fenchol synthase (LvFencS) (Landmann et al. 2007; Benabdelkader et al. 2015; Adal et al. 2017). All these TPSs are clustered together under the angiosperm TPS-b subfamily, a monoterpene synthase subfamily (Bohlmann et al. 1998). Intriguingly, (+)-LiBPPS is distantly related to most of the known BPPSs from different plants, including that from L. angustifolia, and closes to SoBPPS, with which it shares 65.48% identity at the amino acid level. This indicates that plant mono-TPSs originated from the same ancestor or one has evolved from the other through gene duplication, leading to divergent functional evolution through subfunctionalization and / or neofunctionalization of TPSs. It is not also surprising that (+)-LiBPPS is more homologous to other Lavandula TPSs, as most TPS genes with different functions have more similarity to each other within a given species than TPSs with the same function in a different species. For example, Li3CarS shares 75% amino acid identity with LaLimS (from half-parent species) compared to 29–50% sequence homology with 3-carene synthases of P. abies and S. stenophylla (Adal et al. 2017). While, in Sitka spruce, 3-carene synthase gene had 89–92% nucleotides identity with another mTPS, (-)-sabinene synthase (Roach et al. 2014).
The genomic structure of (+)-LiBPPS has typical features of the Class III TPSs, which are known by seven exons and six introns (Chen et al. 2011). The common conserved motifs are also located at the same exon positions of most TPSs in several angiosperms.
In vitro activity of the recombinant (+)-LiBPPS
The genes and encoding enzymes for most major Lavandula monoterpene synthases have been identified and functionally characterized in vitro (Landmann et al. 2007; Demissie et al. 2011; Demissie et al. 2012; Adal et al. 2017; Adal et al. 2019). The (+)-LiBPPS cloned in this study was found to encode a monoterpene synthase that catalyzes the conversion of GPP to (+)-bornyl diphosphate (BPP), and a few other monoterpenes. The generation of BPP and multiple monoterpenes by (+)-LiBPPS in vitro was not surprising, as most of the known BPPSs and some other mono-TPSs are multiproduct monoterpenes (Despinasse et al. 2017; Hurd et al. 2017; Wang et al 2018; Ma et al 2021, Wise et al. 1998; Fäldt et al. 2003; Demissie et al. 2012, Adal et al 2017). The product profile of LiBPPS (which includes 61% BPP, measured as nearest prenyl alcohol, (+)-borneol) is closer to A. villosum BPPS (65.9% BPP) (Wang et al. 2018)d officinalis BPPS (57–75% BPP) (Wise et al. 1998; Despinasse et al. 2017), than L. angustifolia BPPS (30% BPP) (Despinasse et al. 2017), C. burmanni BPPS (88.7%) (Ma et al. 2021)d dulicus BPPS (90%) (Hurd et al. 2017). A few minor monoterpenes with a total proportion of 39% were also detected from L. x intermedia BPPS activity. Production of a few minor monoterpene products by the (+)-LiBPPS was not surprising, as most other reported BPPS proteins are multiproduct enzymes.
(+)-LiBPPS and LaBPPS are differentially expressed in lavenders
Both (+)-LiBPPS and LaBPPS are constitutively expressed in L. x intermedia leaf and flower tissues, with a strong transcriptional expression observed in leaves. Given that high levels of monoterpenes derived from BPP (i.e., borneol and camphor) are produced in leaves, compared to flowers, in L. x intermedia (Boeckelmann 2008; Hassiotis et al. 2014; Seidler- Lozykowska et al. 2014), this finding was not surprising.
Expression of (+)-LiBPPS impacts the biosynthesis of monoterpenes in the transgenic L. latifolia
Metabolic engineering of monoterpenes has been reported in Lamiaceaces, particularly in lavender and mint, (Mahmoud and Croteau 2001; Muñoz-Bertomeu et al. 2006; Muñoz-Bertomeu et al. 2008; Lange et al. 2011; Mendoza-Poudereux et al. 2014; Wang et al. 2016; Tsuro et al. 2019; Li et al. 2020). These studies targeted mainly the DXP pathway genes or terpene synthase genes. For example, expression of the Arabidopsis DXS gene increases the concentration of monoterpenes and essential oil yield without impacting primary metabolites, such as chlorophyll and carotenoids in L. latifolia transgenic plant leaves and inflorescences (Muñoz-Bertomeu et al. 2006). A high level of linalool was also produced through the expression of a full-length clarkia linalool synthase in L. latifolia leaves (Mendoza-Poudereux et al. 2014).
In this study, we expressed the full-length (+)-LiBPPS cDNA in sense or antisense orientation under the control of the CaMV 35S promoter in L. latifolia plants. Plants that most strongly expressed the transgene in sense did not survive to full maturity. The exact nature of this effect is unclear and requires further investigation. However, most likely the unregulated production of large amounts of BPP and monoterpenes derived from BPP (in particular camphor (Fig. 1), a known agent of allelopathy) is lethal to the plant. Given that normally BPPS and other monoterpene synthase genes are expressed in the secretory cells of glandular trichomes, which are specialized to produce and store large quantities of monoterpenes, the above scenario makes sense. To avoid this issue, BPPS should be expressed under the control of a trichome-specific promoter.
Reduced BPPS transcript levels in transgenic plants expressing (+)-LiBPPS in antisense correlate well with the accumulation of borneol/camphor in these plants. This suggests that (+)-LiBPPS controls the synthesis of these monoterpenes through the supply of the primary precursor bornyl diphosphate. This outcome was not unexpected as the production of numerous monoterpenes is controlled through the transcriptional regulation of the corresponding terpene synthase gene. For example, the production of 1,8 cineole was recently shown to be controlled via the regulation of the cineol synthase gene in lavender (Demissie et al., 2012; Tsuro et al. 2019).