*4.5. Cis-Acting Elements Analysis of TPS Genes in D. o*ffi*cinale*

The promoter sequences, 2000 bp upstream of the translational start site (ATG), of TPS genes in *D. o*ffi*cinale* were obtained from the *D. o*ffi*cinale* genome [23]. Afterwards, the online software PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was employed to investigate putative *cis*-regulatory elements in the promoter region of *DoTPS* genes in *D. o*ffi*cinale*.

#### *4.6. Gene Expression Analysis Based on Transcriptome Data*

To gain insight into the tissue-specific transcription levels of *DoTPS* family genes, raw data from the RNA-sequencing of 10 different tissues (i.e., flower buds, green root tips, gynostemium (column), labellum (lip), leaves, pollinia, sepals, stems, roots, and the white part of roots) in two-year-old *D. o*ffi*cinale* adult plants was downloaded from NCBI under BioProject PRJNA262478 [25]. To study the

effects of cold acclimation (0 ◦C for 20 h, CA) and non-acclimation (20 ◦C for 20 h, CK) on *DoTPS* gene expression, the raw RNA-sequencing reads were retrieved from a reported transcriptome database [29]. Fragments per kilobase of transcript per million mapped reads (FPKM) values of *DoTPS* genes in tested samples were used to evaluate transcription abundance. *DoEF-1*α was selected as the internal reference gene for normalizing each expression value. The heat maps of the *DoTPS* genes' expression patterns were illustrated using the TBtools software with default settings (https://github.com/CJ-Chen/TBtools), and the gradient color from green to red is expressed as the log2-transformed expression levels of each *DoTPS* gene.

#### *4.7. Gas Chromatography–Mass Spectrometry Analysis of Geraniol and Linalool in Flowers of D. o*ffi*cinale*

The frozen flowers of *D. o*ffi*cinale* (500 mg) were ground to a fine powder in liquid nitrogen, and then blended with precooled dichloromethane (3 mL) by vortexing for 2 min, followed by shaking at 25 ◦C for 8 h in the dark. The supernatant was collected by 13,000× *g* centrifugation, and concentrated to 200 µL using a stream of N<sup>2</sup> before analysis by gas chromatography–mass spectrometry (GC-MS, Shimadzu Co., Kyoto, Japan) equipped with a 30-m Supelcowax-10 column (0.25 mm diameter × 0.25 µm film thickness). The temperature program was isothermal at 60 ◦C for 3 min, then increased at a rate of 4 ◦C min−<sup>1</sup> to 240 ◦C, and maintained at 240 ◦C for 20 min. MS analyses were performed in full-scan mode with a mass range from *m*/*z* 40 to 200. Geraniol and linalool were identified against the NIST 2008 mass spectra library (https://chemdata.nist.gov/) as described previously [44].

## *4.8. Prokaryotic Expression and DoTPS10 Enzyme Assay in Escherichia coli*

The full-length *DoTPS10* was amplified from first-strand cDNA, as published previously [41]. The obtained PCR product was purified and inserted into the pMD-18T vector (TaKaRa) for sequencing. The gene-specific primers used for *DoTPS10* are indicated in Table S14.

A 1797-bp ORF without a stop codon (TAA) of *DoTPS10* was cloned into the pET32a vector with *Sal*I and *Xho*I restriction sites. *DoTPS10* expression in *E. coli* BL21 (DE3) cells and purification using a His-trap Ni-sepharose high performance column (GE Healthcare, Fairfield, CT, USA) were described in our previous study [45]. The purified pET32a-DoTPS10 protein was fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

In vitro DoTPS10 enzyme assays were performed in screw-cap 5-mL glass vials containing 1 mL of 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer (10 mM, pH 7.0, containing 5 mM dithiothreitol, 10 mM MgCl2, and 10 mM GPP as substrate) and 100 µg of DoTPS10 protein. The reactions were overlaid with 200 µL of *n*-hexane and incubated at 30 ◦C for 1 h. The mixtures were mixed vigorously for 1 min to obtain the enzymatic products. The organic phase was removed, and 1 µL was detected using GC-MS as described above. For comparison, His-tagged protein (empty pET32a) was used as the blank control.

## *4.9. Subcellular Localization of DoTPS10 in A. thaliana Mesophyll Protoplasts*

To determine the localization of DoTPS10, a 1797-bp ORF without a stop codon (TAA) of *DoTPS10* was introduced into the pSAT6-EYFP-N1 vector with an *Nco*I restriction site, and was transiently transformed into mesophyll protoplasts from four-week-old *A. thaliana* leaves. After transformation in darkness at 22 ◦C for 20 h, YFP signals were evaluated using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany) with an excitation wavelength of 514 nm.

#### *4.10. Statistical Analysis*

IBM SPSS statistics software version 22.0 for Windows (IBM Corp., Armonk, NY, USA) was used to carry out one-way analysis of variance (ANOVA) among different samples using three replications. Duncan's multiple range test (DMRT) was used to determine significant differences (*p* < 0.05).
