**1. Introduction**

Plants emit an astonishing number of volatile metabolites during growth and development, and these have various roles, some with biological effects, that are considered beneficial to plants and humans [1]. For ornamental plants, floral volatiles have a dual function, to attract pollinators, and in defense against pests, herbivores, and pathogens [2–4]. Orchids, economically important floricultural crops, possess an abundance of floral volatile terpenes. Among them, monoterpenes, especially geraniol, linalool, and their oxygenated derivatives, are predominant components of floral scents [4,5]. Geraniol is an acyclic monoterpene alcohol released from several ornamental plants, such as citronella, geranium, herbs, roses, and orchids (*Phalaenopsis bellina* and *Dendrobium o*ffi*cinale*) [5–8], and is extensively used in fragrance and cosmetics industries because of its pleasant rose-like scent.

Geraniol is synthesized from geranyl pyrophosphate (GPP), the universal five-carbon precursor for the biosynthesis of all monoterpenes, and is catalyzed by a terpene synthase (TPS), which has been designated as geraniol synthase (GES, EC 3.1.7.11) [6,9]. GPP, as an immediate precursor of monoterpenes, is proceeded by a condensation reaction of two C5-isoprene building units, namely isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [9]. Recent studies have thoroughly characterized two well-established pathways, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway, that generate IPP and DMAPP, [7–9]. Several enzymes, including 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS), and GPP synthase (GPPS), contribute to GPP biosynthesis [10], providing the GPP substrate for GES to generate geraniol. Taken together, GES is a mono-TPS that specifically catalyzes the formation of geraniol from GPP in the MEP pathway. for the biosynthesis of all monoterpenes, and is catalyzed by a terpene synthase (TPS), which has been designated as geraniol synthase (GES, EC 3.1.7.11) [6,9]. GPP, as an immediate precursor of monoterpenes, is proceeded by a condensation reaction of two C5-isoprene building units, namely isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) [9]. Recent studies have thoroughly characterized two well-established pathways, the cytosolic mevalonic acid (MVA) pathway and the plastidic methylerythritol phosphate (MEP) pathway, that generate IPP and DMAPP, [7–9]. Several enzymes, including 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1 deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (HDS), and GPP synthase (GPPS), contribute to GPP biosynthesis [10], providing the GPP substrate for GES to generate geraniol. Taken together, GES is a mono-TPS that specifically catalyzes the formation of geraniol from GPP in the MEP pathway. In plants, two kinds of enzymatic reactions can produce geraniol from GPP, either a TPS-based

Geraniol is synthesized from geranyl pyrophosphate (GPP), the universal five-carbon precursor

In plants, two kinds of enzymatic reactions can produce geraniol from GPP, either a TPS-based canonical pathway, which is catalyzed by GES in chloroplasts/plastids [6], or a phosphatase-based non-canonical pathway, which is catalyzed by nudix hydrolase (NUDX) in the cytoplasm (Figure 1) [9,11]. Thus far, the *GES* gene has already been identified and functionally characterized in multiple horticultural plants, including *CitTPS16* in *Citrus sinensis* [12], *LoTPS3* in *Lathyrus odoratus* [13], *GES* in *Ocimum basilicum* [6], and *PbGDPS* in *P. bellina* [14], all of which can produce geraniol from GPP in vitro. However, no TPS with GES activity has been identified in *Rosa rugosa* to date. Only one *NUDX* gene, *RhNUDX1*, converts GPP into geranyl monophosphate (GP), which is then hydrolyzed to geraniol by a petal-derived phosphatase [11]. In orchids, *PbGDPS*, which encodes GPP synthase, may play a key role in regulating the biosynthesis of monoterpenes (geraniol and linalool) in *P. bellina* [14]. In addition, the transcript levels of two TPS genes (*PbTPS5* and *PbTPS10*) are consistent with the production of geraniol and linalool in *P. bellina* [15], although their functionality has not yet been verified. Although geraniol is an important floral volatile compound in *D. o*ffi*cinale*, a medicinal orchid [7], the *GES* gene responsible for geraniol biosynthesis in *D. o*ffi*cinale* has not yet been characterized. canonical pathway, which is catalyzed by GES in chloroplasts/plastids [6], or a phosphatase-based non-canonical pathway, which is catalyzed by nudix hydrolase (NUDX) in the cytoplasm (Figure 1) [9,11]. Thus far, the *GES* gene has already been identified and functionally characterized in multiple horticultural plants, including *CitTPS16* in *Citrus sinensis* [12], *LoTPS3* in *Lathyrus odoratus* [13], *GES* in *Ocimum basilicum* [6], and *PbGDPS* in *P. bellina* [14], all of which can produce geraniol from GPP in vitro. However, no TPS with GES activity has been identified in *Rosa rugosa* to date. Only one *NUDX* gene, *RhNUDX1*, converts GPP into geranyl monophosphate (GP), which is then hydrolyzed to geraniol by a petal-derived phosphatase [11]. In orchids, *PbGDPS*, which encodes GPP synthase, may play a key role in regulating the biosynthesis of monoterpenes (geraniol and linalool) in *P. bellina* [14]. In addition, the transcript levels of two TPS genes (*PbTPS5* and *PbTPS10*) are consistent with the production of geraniol and linalool in *P. bellina* [15], although their functionality has not yet been verified. Although geraniol is an important floral volatile compound in *D. officinale*, a medicinal orchid [7], the *GES* gene responsible for geraniol biosynthesis in *D. officinale* has not yet been characterized.

**Figure 1.** The pathway of the *GES*/*NUDX* genes responsible for the formation of geraniol in planta [9–11]. C5 precursors DMAPP and IPP are generated by the cytosol mevalonic acid (MVA) and the plastid methylerythritol phosphate (MEP) pathways. DMAPP, dimethylallyl pyrophosphate; GES, geraniol synthase; GPP, geranyl pyrophosphate; GPPS, GPP synthase; IPP, isopentenyl **Figure 1.** The pathway of the *GES*/*NUDX* genes responsible for the formation of geraniol in planta [9–11]. C5 precursors DMAPP and IPP are generated by the cytosol mevalonic acid (MVA) and the plastid methylerythritol phosphate (MEP) pathways. DMAPP, dimethylallyl pyrophosphate; GES, geraniol synthase; GPP, geranyl pyrophosphate; GPPS, GPP synthase; IPP, isopentenyl pyrophosphate; NUDX,

Phosphatase-based non-canonical pathway

pyrophosphate; NUDX, nudix hydrolase; TPS, terpene synthase.

nudix hydrolase; TPS, terpene synthase.

Herein, using the *D. officinale* genome database [16,17], and according to phylogenetic analysis and sequence homology, three *GES* genes (named *DoGES1–3*), with putative roles in the production of geraniol, were screened. The transcriptional regulatory functions of *DoGES1*, a member of the TPS family, in response to the accumulation of geraniol in *D. officinale* was investigated in different plant tissues (roots, stems, leaves, and flowers), harvest times (8:00, 11:00, 14:00, and 17:00), flower organs (petals, sepals, and gynostemium), and flowering periods (budding, semi-open flowers, fully open flowers). An in vitro assay of recombinant protein in *Escherichia coli* BL21 star (DE3) as well as in vivo Herein, using the *D. o*ffi*cinale* genome database [16,17], and according to phylogenetic analysis and sequence homology, three *GES* genes (named *DoGES1–3*), with putative roles in the production of geraniol, were screened. The transcriptional regulatory functions of *DoGES1*, a member of the TPS family, in response to the accumulation of geraniol in *D. o*ffi*cinale* was investigated in different plant tissues (roots, stems, leaves, and flowers), harvest times (8:00, 11:00, 14:00, and 17:00), flower organs (petals, sepals, and gynostemium), and flowering periods (budding, semi-open flowers, fully open flowers). An in vitro assay of recombinant protein in *Escherichia coli* BL21 star (DE3) as well as in vivo

transient overexpression in *Nicotiana benthamiana* indicated that *DoGES1* was responsible for geraniol biosynthesis, advancing our understanding of geraniol biosynthesis in *D. o*ffi*cinale*. transient overexpression in *Nicotiana benthamiana* indicated that *DoGES1* was responsible for geraniol biosynthesis, advancing our understanding of geraniol biosynthesis in *D. officinale*.

#### **2. Results 2. Results**

#### *2.1. Identification of Candidate GES Genes from the D. o*ffi*cinale Genome 2.1. Identification of Candidate GES Genes from the D. officinale Genome*

From *D. o*ffi*cinale* genomic annotation data, three candidate GES sequences with best matches to known GES proteins [6,12,13,15] were retrieved by BLASTN, and named DoGES1, DoGES2, and DoGES3 (Table S1). Multiple sequence alignment demonstrated that three DoGES proteins had highly conserved aspartate-rich motifs (DDxxD) and NSE/DTE motifs at the *C*-terminal, and an RRX8W domain at the *N*-terminal (Figure 2), suggesting that DoGES1-3 were all TPSs. Among them, DDxxD and NSE/DTE were essential for the cofactor Mg2<sup>+</sup> or Mn2<sup>+</sup> to catalyze the synthesis of monoterpenes [18,19], and the RRX8W domain was also involved in the cyclization of monoterpene synthase [20]. From *D. officinale* genomic annotation data, three candidate GES sequences with best matches to known GES proteins [6,12,13,15] were retrieved by BLASTN, and named DoGES1, DoGES2, and DoGES3 (Table S1). Multiple sequence alignment demonstrated that three DoGES proteins had highly conserved aspartate-rich motifs (DDxxD) and NSE/DTE motifs at the *C*-terminal, and an RRX8W domain at the *N*-terminal (Figure 2), suggesting that DoGES1-3 were all TPSs. Among them, DDxxD and NSE/DTE were essential for the cofactor Mg2+ or Mn2+ to catalyze the synthesis of monoterpenes [18,19], and the RRX8W domain was also involved in the cyclization of monoterpene synthase [20].

**Figure 2.** Comparison of deduced amino acid sequences of DoGES proteins in *Dendrobium officinale*. The Asp-rich domain DDXXD, the RRX8W motif, and the NSE/DTE motif, which are highly conserved in plant TPS proteins and required for TPS activity, are indicated. Completely conserved sequences **Figure 2.** Comparison of deduced amino acid sequences of DoGES proteins in *Dendrobium o*ffi*cinale*. The Asp-rich domain DDXXD, the RRX8W motif, and the NSE/DTE motif, which are highly conserved in plant TPS proteins and required for TPS activity, are indicated. Completely conserved sequences are shaded in black, identical sequences in dark grey, and similar sequences in light grey.

are shaded in black, identical sequences in dark grey, and similar sequences in light grey.

#### *2.2. Phylogenetic Analysis of DoGES Proteins in the D. o*ffi*cinale Genome*

gene related to floral scent formation.

*2.2. Phylogenetic Analysis of DoGES Proteins in the D. officinale Genome*  To investigate the evolutionary relationship of DoGES proteins with other reported GES proteins, a phylogenetic tree was generated by the neighbor-joining method (Figure 3; Table S2). All three DoGES proteins clustered in the TPS-b subfamily, which is specific to angiosperms and is responsible To investigate the evolutionary relationship of DoGES proteins with other reported GES proteins, a phylogenetic tree was generated by the neighbor-joining method (Figure 3; Table S2). All three DoGES proteins clustered in the TPS-b subfamily, which is specific to angiosperms and is responsible for encoding monoterpene synthases [21].

for encoding monoterpene synthases [21]. Based on the transcription levels in different tissues (roots, stems, leaves, and flowers), *DoGES1* exhibited high expression in flowers, while *DoGES2* and *DoGES3* were mainly expressed in roots and leaves, respectively (Figure S1). Consequently, DoGES1 was selected for our candidate study Based on the transcription levels in different tissues (roots, stems, leaves, and flowers), *DoGES1* exhibited high expression in flowers, while *DoGES2* and *DoGES3* were mainly expressed in roots and leaves, respectively (Figure S1). Consequently, DoGES1 was selected for our candidate study gene related to floral scent formation.

*Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 15

**Figure 3.** Phylogenetic positioning of GES proteins within representative samples of known plant TPS proteins. The neighbor-joining tree was generated using MEGA 7.0 software after the alignment of full-length DoGES proteins in *D. officinale* with other plant TPS proteins. The seven subfamilies TPSa-g are delimited based on the taxonomic distribution of the TPS families [19]. All sequences that were used can be retrieved from Supplementary Table S2. syn, synthase. **Figure 3.** Phylogenetic positioning of GES proteins within representative samples of known plant TPSproteins. The neighbor-joining tree was generated using MEGA 7.0 software after the alignment of full-length DoGES proteins in *D. o*ffi*cinale* with other plant TPS proteins. The seven subfamilies TPS-a-g are delimited based on the taxonomic distribution of the TPS families [19]. All sequences that were used can be retrieved from Supplementary Table S2. syn, synthase.

#### *2.3. Molecular Cloning and Analysis of DoGES1 from D. officinale Flowers 2.3. Molecular Cloning and Analysis of DoGES1 from D. o*ffi*cinale Flowers*

*2.4. Subcellular Localization of DoGES1* 

RNA isolated from *D. officinale* flowers during the blossoming period were used as template and amplified via nested PCR. Full-length cDNA sequences of *DoGES1* have a 1749-bp long open reading frame (ORF) that encodes 582 amino acids with a theoretical isoelectric point of 5.34 and a molecular weight of 67.99 kDa (Figure S2). The DoGES1 sequence was submitted to GenBank Data Libraries RNA isolated from *D. o*ffi*cinale* flowers during the blossoming period were used as template and amplified via nested PCR. Full-length cDNA sequences of *DoGES1* have a 1749-bp long open reading frame (ORF) that encodes 582 amino acids with a theoretical isoelectric point of 5.34 and a molecular weight of 67.99 kDa (Figure S2). The DoGES1 sequence was submitted to GenBank Data Libraries under accession number MT875214.

under accession number MT875214. The DoGES1 secondary structure, which was determined using the SOPMA program (http://npsa-pbil.ibcp.fr/), shows that it harbors 69.24% α-helixes, 23.37% random coils, 3.78% β-turns, and 3.61% extended strands. The Chlorop 1.1 tool predicted that DoGES1 contains a 34 amino acid long *N*-terminal chloroplast transit peptide. To determine the subcellular localization of DoGES1, three subcellular localization tools (AtSubP [22], Plant-mPLoc [23], and pLoc-mPlant [24]) were used. All of them demonstrated that DoGES1 was located in chloroplasts, and was thus likely a mono-TPS in the MEP-pathway, but not in the cytosolic MVA pathway. The DoGES1 secondary structure, which was determined using the SOPMA program (http: //npsa-pbil.ibcp.fr/), shows that it harbors 69.24% α-helixes, 23.37% random coils, 3.78% β-turns, and 3.61% extended strands. The Chlorop 1.1 tool predicted that DoGES1 contains a 34 amino acid long *N*-terminal chloroplast transit peptide. To determine the subcellular localization of DoGES1, three subcellular localization tools (AtSubP [22], Plant-mPLoc [23], and pLoc-mPlant [24]) were used. All of them demonstrated that DoGES1 was located in chloroplasts, and was thus likely a mono-TPS in the MEP-pathway, but not in the cytosolic MVA pathway.

#### *2.4. Subcellular Localization of DoGES1 Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 5 of 15

To confirm the intracellular localization of DoGES1, pSAT6-EYFP-DoGES1 was transformed to the mesophyll protoplasts of 4-week-old *Arabidopsis thaliana* leaves. Yellow fluorescent signals were visualized by confocal laser scanning microscopy. The images indicate that DoGES1 was located in chloroplasts (Figure 4), similar to *LiTPS2*, which encodes a mono-TPS in lily (*Lilium longiflorum* 'Siberia') [25], indicating that DoGES1 may be responsible for monoterpene synthesis. To confirm the intracellular localization of DoGES1, pSAT6-EYFP-DoGES1 was transformed to the mesophyll protoplasts of 4-week-old *Arabidopsis thaliana* leaves. Yellow fluorescent signals were visualized by confocal laser scanning microscopy. The images indicate that DoGES1 was located in chloroplasts (Figure 4), similar to *LiTPS2*, which encodes a mono-TPS in lily (*Lilium longiflorum* 'Siberia') [25], indicating that DoGES1 may be responsible for monoterpene synthesis.

**Figure 4.** Subcellular localization of DoGES1 in *Dendrobium officinale*. Yellow fluorescence indicates the DoGES1-YFP fusion protein signal. Red fluorescence is chloroplast autofluorescence. The merged images indicate a combination of chloroplast autofluorescence and YFP fluorescence. **Figure 4.** Subcellular localization of DoGES1 in *Dendrobium o*ffi*cinale*. Yellow fluorescence indicates the DoGES1-YFP fusion protein signal. Red fluorescence is chloroplast autofluorescence. The merged images indicate a combination of chloroplast autofluorescence and YFP fluorescence.
