Clustering and high expression of 1,8-cineole synthase genes in S.
rosmarinus leaf tissue
The co-expression results showed that limonene synthases in S.
rosmarinus were highly correlated with the synthesis of monoterpenes
(p-value = 0.0102, Table S21). A total of three limonene synthases and
three 1,8-cineole synthases were identified in S. rosmarinus . All
genes encoding limonene synthases (SrLS-1 , SrLS-2 ,SrLS-3 ) and 1,8-cineole synthases (SrCinS-3 ,SrCinS-4 ) are clustered on pseudochromosome 3, within a 1.1 Mb
region (Figure 5b, Table S25). Transcriptome analysis revealed that the
expression levels of the three limonene synthases (SrLS-1 ,SrLS-2 , SrLS-3 ) were 8.44, 9.80 and 6.67 times higher in
leaves than in roots (Figure 5c), respectively, while the expression
levels of the two 1,8-cineole synthases (SrCinS-3 ,SrCinS-4 ) were 5.57 and 8.81 times higher in leaves than in roots
(Figure 5c). Additionally, covariance analysis was performed on S.
rosmarinus , S. miltiorrhiza and S. splendens , revealing
that the genes encoding 1,8-cineole synthase on pseudochromosome 2 did
not have homologues in S. miltiorrhiza and S. splendens.
We constructed an evolutionary tree of 24 species to analyze limonene
synthases and 1,8-cineole synthases, which revealed that SrCinSsand SrLSs formed two clades with 40.47%–99.16% sequence
identity ((Figure S19, Table S29). 3D models of SrCinSs andSrLSs (pdbid: 2ong) were generated and validated using
Ramachandran plots (Figure S21). The models of both enzymes were highly
similar in stereospecificity, as indicated by the average root means
square displacements (RMSDs) (0.38Å–1.03Å) between the predicted models
(Figure S22, S21a). Docking studies showed that. intermediate terpinol
cations are located near the active pockets of SrCinS-3 andSrCinS-4 (Figure 5d). And eight amino acid residues of the active
pocket (Cys-250, Trp-253, Asn-274, Thr-278, Met-458, His-502, Tyr-496
and Ser-454) that all lie within 10 Å distance of the docking site and
have a direct effect on biosynthesis of 1,8-cineole were examined
(Kampranis et al., 2007; Piechulla et al., 2016; N. Srividya et al.,
2015; J. Xu et al., 2017). Cys-379, Trp-382, Tyr-626, and Thr-278
maintained their original polarity in SrCinS-3 andSrCinS-4. Mutations S512G and A278T in silico analysis indicated
that these residues retained their original polarity in SrCinS-3and SrCinS-4 . However, A278T became more hydrophilic and M622I
lost its original polarity (Figure S24, Table S27). Based on these
findings, we hypothesize that SrCinS-3 and SrCinS-4 are responsible for
1,8-cineole biosynthesis. Furthermore, we found that S512G and A278T,
which promote the accumulation of 1,8-cineole in tobacco terpene
synthases (Piechulla et al., 2016), were retained in SrCinS-3 and
SrCinS-4 in rosemary, their association with high 1,8-cineole
accumulation in rosemary.
The limonene synthases in S. rosmarinus were observed to dock
with terpinyl cations near the active pocket (Figure S23), indicating
that the crystallographic structures of limonene synthase could accept
terpinyl cation intermediate. Mutations in the key sites of the active
pocket can affect the product diversity of terpene synthases as the
product profile is determined by the conformation of the substrate or
intermediate. Analysis of the active pockets of SrLSs showed that
they deviate from the ancestral limonene synthase pattern (N. Srividya
et al., 2015; Narayanan Srividya, Lange, & Lange, 2020), with changes
in polarity observed for several important sites (Thr-278, His-502,
Tyr-496 and Ser-454, see Table S27). M519I and T279V mutations directly
led the loss of polarity of original residues, but compensatory
mutations were observed for Asn-274 (to Phe-274) and Cys-250 (to
Asn-250), which maintained the polarity of the active site (J. Xu et
al., 2017) (Figure S23, Table S27). The polarity changes in the active
sites resulted in a larger active pocket, potentially enhancing the
production of more abundant terpenoids by attenuating the stability of
the carbon positive ion.
The transcriptional expression of genes started from the specific
binding of the promoter region upstream of the gene to RNA polymerase;
therefore, we extracted the promoter sequences of SrCinSs andSrLSs and analyzed the promoter elements using Promoter 2.0
(Knudsen, 1999). TATA-boxes are essential for binding to RNA polymerase
and activating transcription among the various elements (Orphanides,
Lagrange, & Reinberg, 1996). The results showed the number of
TATA-boxes nearly doubled in the promoter sequence of SrCinS-3(Table S28), greatly enhancing its expression and promoting the
accumulation of 1,8-cineole. Additionally, we examined G-box elements in
the promoter region of SrCinSs, and five G-boxes were targeted onSrCinS-3 , significantly increasing its binding probability with
the bHLH gene family. Furthermore, we discovered twoSrbHLH143 near the gene cluster (Figure 5b), and their expression
was 2.24-fold and 1.63-fold higher in leaves than in roots (Figure 5c),
leading to a speculation that SrbHLH143 may play a regulatory
role in the biosynthesis of 1,8-cineole. The expression of the bHLH
family was reported to be significantly correlated with changes in
1,8-cineole content in Artemisia absinthium (Yi et al., 2021),
and G-box was cis-acting DNA regulatory element which could bind with
bHLH transcription factor(Qian et al., 2007). Conversely, the promoter
region of other 1,8-cineole synthases had incomplete G-box elements,
resulting in low expression.