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.