TPS and CYP clustered in phylogenetic tree
Two TPS genes having the sizes of 1803 and 1800 bp were obtained from the same primer, and were named LaTPS7 and LaTPS8 , respectively; LaTPS7 and LaTPS8 displayed an identical nucleotide sequence in the first 33 bp of N and in the last 23 bp of C (Figure S2). Phylogenetic trees based on deduced amino-acid sequences of each gene showed that these two genes belonged to the TPS-b subfamily, which consists of monoterpene synthases (Chen et al. 2011) (Figure 1a).LaTPS7showed the highest identity withL. angustifolia limonene synthase when searched via BLAST in NCBI, andLaTPS8 showed highest identity with L. peduncuiatapinene synthase in a phylogenetic tree. Both of these proteins contained the conserved arginine-tryptophan (RRX8W) motif in the N terminal, and an aspartate-rich (DDXXD) and (NSE/DTE) motif (which chelates divalent metal ions such as Mg2+ or Mn2+) in the C terminal. Synthases need these motifs to cyclise terpene precursors such as GPP, NPP, or/and FPP (Figure S3, S4).
The CYP sequence was submitted to CYP450 nomenclature committee and designated as LaCYP71D582 . The protein showing highest identity with La CYP71D582 was P. barbatus Pb CYP71D378 (Figure 1b), which functions in forskolin (Pateraki et al. 2017) with conserved motifs (A/G)GX(D/E)T(T/S), EXXR, and FXXGXRXCXG (Figure S5). Secondary protein structure with four membrane-spanning domains and active site is shown in supplementary figure S5. CYP clans, used to build the phylogenetic tree, were related to terpene metabolism (Nelson and Werck-Reichhart, 2011), and the candidate CYP was clustered into the CYP71 clan, which is the largest clan among the CYPs (Figure 1b). This result indicates that it is highly possible to catalyze terpenes (Nelson et al. 2004). These results indicate that La CYP71D582 may participate in terpene metabolism
Quantitative RT-PCR analysis of candidate-gene expression during budding stage
The expression level of LaTPS7 , LaTPS8 andLaCYP71D582 were accessed via quantitative real-time PCR (qRT-PCR) during different periods of budding, blossoming and fading stages of glandular trichomes in leaves and flowers (Figure 2). LaTPS7 , LaTPS8 , andLaCYP71D582 were highly expressed in the glandular trichomes of flower during budding stage, but showed low expression in other organs. Meanwhile, the expression levels of LaTPS7 , LaTPS8 , andLaCYP71D582 decreased during blossoming and fading stage, showing a similar expression pattern, and indicating that the three genes perform similar functions during plant development (Figure 2). This expression pattern may provide a temporal basis for consecutive catalysis: products from TPSs may be further catalyzed by CYP.