Introduction
Rosemary (Salvia rosmarinus Schleid.) is a well-known Mediterranean perennial shrub that belongs to the mint family (Lamiaceae). It has been cultivated worldwide for its culinary, aromatic, ornamental, and therapeutic properties (Allegra, Tonacci, Pioggia, Musolino, & Gangemi, 2020; Degner, Papoutsis, & Romagnolo, 2009; Freedman, 2019; Neves, Neves, & Oliveira, 2018), with a global market size of rosemary products estimated 2,224 million USD in 2021. The use of rosemary dates back to ancient times, with evidence of its use for embalming in Egyptian tombs 3000 B.C. and as a herbal medicine in ancient Greece and Rome 500 B.C. Currently, rosemary extracts are widely used in cooking, food preservation, cosmetics, and herbal medicine due to their high antimicrobial and antioxidant activities (Degner et al., 2009). Rosemary is considered one of the most effective herbs for treating headaches, poor circulation, inflammatory diseases, and physical and mental fatigue (Nematolahi, Mehrabani, Karami-Mohajeri, & Dabaghzadeh, 2018; Ojeda-Sana, van Baren, Elechosa, Juarez, & Moreno, 2013; Rašković et al., 2014).
Essential oils of rosemary contain more than 30 components, including flavones (genkwanin, isoscutellarein 7- O-glucoside), caffeoyl derivatives (rosmarinic acid), phenolic monoterpenes (1,8-cineole (eucalyptol), α-pinene, camphene, limonene) (al-Sereiti, Abu-Amer, & Sen, 1999; Angioni et al., 2004; Mena et al., 2016; Sharma, Velamuri, Fagan, & Schaefer, 2020), and diterpenes (carnosic acid, carnosol), which are considered as the major bioactive components. Monoterpenes in rosemary are the main source of the aromatic properties of the fragrance and essential oil (Christopoulou et al., 2021; Micić et al., 2021), which have been shown to possess olfactory properties that influence cognitive performance including memory (Moss, Cook, Wesnes, & Duckett, 2003). The diterpenoids in rosemary leaves are reported to be responsible for their antioxidant, antibacterial, and anticancer properties (Alsamri et al., 2021; Bao et al., 2020; Ngo, Williams, & Head, 2011; Veenstra & Johnson, 2021; M. H. Yu et al., 2013). Despite the commercial interest and increasing demand for rosemary, improvements through breeding have been very limited (Maurizio, Francesconi, Perinu, & Vais, 2002). The lack of high-quality genome information has hindered the understanding of how its terpenoid bioactives are made and any improvements in productivity possible through genetic selection. Therefore, understanding the genes responsible for biosynthesis of the various terpenoids made in rosemary and their regulation will lay a foundation for molecular breeding for improved and sustainable production. Rosemary essential oil is characterized by a high content of monoterpenes, including 1,8-cineole, α-pinene, limonene (Flamini et al., 2022; Rašković et al., 2014). In fact, 1,8-cineole is the major constituent of rosemary, accounting for 23%-49% of the oil (Christopoulou et al., 2021; Flamini et al., 2022; Rašković et al., 2014). Compared to other aromatic plants in the mint family, such as mentha, lavender and ocimum, which contain lower levels of 1,8-cineole, ranging from 0.5% and 8% (Pokajewicz, Białoń, Svydenko, Fedin, & Hudz, 2021; Senthoorraja et al., 2021; Singh & Pandey, 2018; Yang, Jeon, Lee, Shim, & Lee, 2010), rosemary is exceptional in its ability to synthesize large amounts of 1,8-cineole (Raal, Orav, & Arak, 2007).
Monoterpenes, including 1,8-cineole, are derived from the precursor geranyl diphosphate (GPP) via the mevalonate pathway (MVA) in the cytosol (Mendoza-Poudereux et al., 2015; Wu et al., 2020). The phosphate bond of GPP is broken by monoterpene synthase, generating the geranyl cation, which is then isomerized and cyclized to form a terpinyl cation intermediate (N. Srividya, Davis, Croteau, & Lange, 2015; J. Xu et al., 2017). Limonene synthase directly catalyzes the deprotonation of terpinyl cation to synthesize limonene. The product profile of any monoterpene synthase is determined by the conformation of its substrate or intermediate in the active site pocket of the enzyme. The terpinyl cation can be further deprotonated to form a more stable intermediate and generate a variety of monoterpene profiles (Gao, Honzatko, & Peters, 2012). Specifically, 1,8-cineole synthase catalyzes, the cyclization of the terpinyl cation and traps water to generate 1,8-cineole (Piechulla et al., 2016; N. Srividya et al., 2015; Wedler, Pemberton, & Tantillo, 2015). Monoterpene synthases share a common tertiary structure, with similar polar pockets that include conserved active site motifs, such as RR(X)8W, which is responsible for substrate isomerization (Williams, McGarvey, Katahira, & Croteau, 1998), an RXR motif that protects the carbocation intermediate against nucleophilic attack (Starks, Back, Chappell, & Noel, 1997), and a DDXXD motif that provides the main divalent metal binding site (Starks et al., 1997). A NALV motif is necessary to produce 1,8-cineole but not alpha-terpineol (Piechulla et al., 2016). Despite the structural elucidation of 1,8-cineole synthase in Salvia fruticosa , the molecular and structural basis of 1,8-cineole synthase activity in rosemary remains unclear.
Carnosic acid and carnosol, which are the primary active diterpenes found in S. rosmarinus extracts, exhibit significant antioxidant properties (Veenstra & Johnson, 2021). The biosynthesis of these compounds begins with geranylgeranyl diphosphate (GGPP) supplied by the plastidial methylerythritol phosphate pathway (MEP) (Bergman, Davis, & Phillips, 2019; Forestier, Brown, Harvey, Larson, & Graham, 2021). Diterpene synthases initiated diterpenoid biosynthesis, by cyclizing GGPP to form various hydrocarbon backbone structures. Ent-copalyl diphosphate synthase (CPS) and kaurene synthases (KSL) catalyze the cyclization GGPP to form miltiradiene (Su et al., 2016), which can be spontaneously oxidized to ferruginol. The oxidation network of abietane diterpenes is complex in the genus Salvia , with cytochromes P450 enzymes of the subfamily CYP76AK serving as C-20 oxidases, contributing to oxygenations at position C-20 (Bathe, Frolov, Porzel, & Tissier, 2019). In S. rosmarinus , the genes CYP76AK7 andCYP76AK8 encode enzymes that can catalyze three sequential C-20 oxidations, converting 11-hydroxy ferruginol to carnosic acid (Ignea et al., 2016). However, in S. miltiorrhiza , a congeneric medicinal plant in East Asia, the CYP76AK1 gene was found to catalyze a single hydroxylation at position C-20, resulting in the production of 11,20-hydroxy ferruginol, which is the precursor of tanshinone biosynthesis (Ignea et al., 2016; Scheler et al., 2016). Notably,CYP76AK6–8 and CYP76AK1 accept the same substrate, leading to the diversity of diterpenes present in S. rosmarinusand S. miltiorrhiza (Bathe et al., 2019; Scheler et al., 2016).
Belonging to genus Salvia , S. rosmarinus is native to the west coast of the Mediterranean Sea and is a typical European species ofSalvia , S. miltiorrhiza is mainly distributed in Eastern Asia, it has been derived into a separate lineage during the long history of evolution, while S. splendens is native to South America and it is a common garden ornamental plant. Rosemary has a long history and a solid position in the spice industry, which stems from the ability of producing essential oils in leaves. Most species of the genusSalvia do not possess this ability, including S. miltiorrhiza and S. splendens . In addition, the antioxidant property of rosemary attributed to the diterpenoids in leaves, including carnosic acid and carnosol. S. miltiorrhiza , a traditional medicinal plant, has antioxidant property as well. It was mainly attributed to diterpenoids in the hairy root with different structures, such as tanshinone IIA. The cultivation of S. splendens was mainly for ornamental purposes, and few secondary metabolites extracted from the plant. Rosemary is aromatic and its ability to produce essential oils is unique within species of genus Salvia. In addition, the high levels of carnosic acid and carnosol in rosemary leaves had not been found in other species of the genus Salviaexcept for Salvia officinalis , and the reasons behind the secondary metabolites of rosemary are worth of further exploring.
In this study, we present a reference genome sequence of S. rosmarinus that was generated by combining Illumina and PacBio data and assembled using Hi-C technologies. The genome was assembled into twelve pseudochromosomes with a super-N50 of 107.45 Mb, totaling 1.24 Gb. Though the previous article of rosemary genome assembly had revealed the biosynthesis of carnosic acid (Han et al., 2023), essential oil was considered as important metabolite of rosemary. We performed comparative genomic analysis with the published genomes of S. miltiorrhizaand S. splendens , and identified tandem gene duplications encoding 1,8-cineole synthase and limonene synthase, which are highly expressed in leaves and contribute to the large accumulation of monoterpenes, particularly 1,8-cineole. Additionally, we identified theCYP76AK6–8 genes responsible for carnosol synthesis and used molecular docking to reveal the differential diterpenoid synthesis mechanism between S. rosmarinus and S. miltiorrhiza.