Identification and Molecular Characterization of Geranyl Diphosphate Synthase (GPPS) Genes in Wintersweet Flower

Geranyl diphosphate synthase (GPPS) is a plastid localized enzyme that catalyzes the biosynthesis of Geranyl diphosphate (GPP), which is a universal precursor of monoterpenes. Wintersweet (Chimonanthus praecox L.), a famous deciduous flowering shrub with a strong floral scent character, could have GPPS-like homologs that are involved in monoterpenes biosynthesis, but it remains unclear. In the present study, five full-length GPPS and geranylgeranyl diphosphate synthases (GGPPS) genes were identified in the wintersweet transcriptome database. The isolated cDNAs showed high protein sequence similarity with the other plants GPPS and GGPPS. The phylogenetic analysis further classified these cDNAs into four distinct clades, representing heterodimeric GPPS small subunits (SSU1 and SSU2), homodimeric GPPS, and GGPPS. Analysis of temporal expression revealed that all genes have the highest transcript level at the full-open flower stage. From tissue-specific expression analysis, CpGPPS.SSU1 and CpGGPPS1 were predominantly expressed in petal and flower, whereas CpGPPS.SSU2, GPPS, and GGPPS2 showed a constitutive expression. Additionally, the subcellular localization assay identified the chloroplast localization of SSUs and GGPPSs proteins, and the yeast two-hybrid assay showed that both CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS proteins. Taken together, these preliminary results suggest that the heterodimeric GPPS can regulate floral scent biosynthesis in wintersweet flower.


Introduction
The floral scent is one of the main features that define the aesthetic value of cut flowers and ornamental plants [1]. It is made up of the specialized metabolites of plants, which enable them to interact with their environment by attracting pollinators and repelling pests, such as herbivores, pathogens, and parasites [2]. The floral scent is the composite of different volatile organic compounds (VOCs), which mainly include terpenoids, phenylpropanoids, benzenoid compounds, and fatty acid derivatives [3]. Moreover, the content and composition of VOCs vary among species and contribute to their distinct fragrance [4]. Overall, terpenoids account for a range of secondary metabolites with numerous volatile constituents, which are derived from two interconvertible five-carbon (C5) precursors (isopentenyl diphosphate (IPP) and its allylic isomer, dimethylallyl diphosphate (DMAPP)) [5]. These C5-isoprene building units are produced from two independent pathways: (1) the mevalonic

Sequence Comparison, Gene Structure, and Phylogenetic Analysis
Multiple protein sequence alignment of the GPPS/GGPPS-like genes was performed with the MEGA 7 software. The results showed that the amino acid sequence of c90672, c80517, and c51170 contain the two highly conserved aspartate-rich regions [(FARM (DDX [2][3][4]

Spatiotemporal Expression Pattern of CpGPPS/GGPPS Genes
In plants, the formation of VOCs is a spatially and developmentally regulated phenomenon [4,24,25]. The transcript level of CpGPPS/GGPPS genes was examined using real-time quantitative polymerase chain reaction (qRT-PCR) in various plant tissues, including the stamen, pistil, petal, flower, and leaf. Relative expression levels of all the CpGPPS/GGPPS genes were compared with reference to the relative expression of CpGGPPS2 in the stamen. All the CpGPPS/GGPPS genes showed variation in the spatial expression intensities. Among the five genes, CpGPPS.SSU1 and CpGGPPS1 showed the highest transcript, followed by CpGPPS, CpGPPS.SSU2, and CpGGPPS2. In detail, CpGPPS.SSU1 and CpGGPPS1 genes were highly expressed in the petal and flower but had low expression levels in the leaf, pistil, and stamen. Moreover, CpGPPS.SSU2, CpGPPS, and CpGGPPS2 were expressed in all the examined tissues, with high transcript levels in the petal ( Figure 2).
The temporal expression analysis of CpGPPS/GGPPS genes in flowers revealed that all genes had the lowest expression levels in stage 1 ( Figure 3). Moreover, the expression levels of all CpGPPS/GGPPS genes, except CpGGPPS2 genes, increased gradually and reached a peak in stage 4, whereas their expression levels were significantly decreased in stage 5 ( Figure 3). Interestingly, a prominent change in the temporal expression profile was observed for CpGPPS.SSU1 and CpGGPPS1 genes. Their level of expression was significantly increased in stage 4, which was approximately 245-and 35-time higher than that of stage 1, respectively ( Figure 3).    highest transcript, followed by CpGPPS, CpGPPS.SSU2, and CpGGPPS2. In detail, CpGPPS.SSU1 and CpGGPPS1 genes were highly expressed in the petal and flower but had low expression levels in the leaf, pistil, and stamen. Moreover, CpGPPS.SSU2, CpGPPS, and CpGGPPS2 were expressed in all the examined tissues, with high transcript levels in the petal ( Figure 2). The temporal expression analysis of CpGPPS/GGPPS genes in flowers revealed that all genes had the lowest expression levels in stage 1 ( Figure 3). Moreover, the expression levels of all CpGPPS/GGPPS genes, except CpGGPPS2 genes, increased gradually and reached a peak in stage 4, whereas their expression levels were significantly decreased in stage 5 ( Figure 3). Interestingly, a prominent change in the temporal expression profile was observed for CpGPPS.SSU1 and CpGGPPS1 genes. Their level of expression was significantly increased in stage 4, which was approximately 245and 35-time higher than that of stage 1, respectively ( Figure 3).

Subcellular Localization of CpGPPS/GGPPS Genes
In silico study indicated the presence of putative transit peptide in CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, and CpGGPPS2 proteins with the chloroplast localization, while the CpGPPS protein was found to be localized in the mitochondria (Table 1). To validate, protein sequences of CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, CpGGPPS2, and CpGPPS were fused to the N-terminus of the PRI101-EGFP vector, and these recombinant vectors were transiently expressed into the Nicotiana benthamiana leaves. Through microscopy, the green fluorescent protein (GFP) fluorescence signals of CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, and CpGGPPS2 were detected in the chloroplasts, whereas CpGPPS-GFP fusion protein fluorescence signals were detected in the mitochondria ( Figure 4).

Subcellular Localization of CpGPPS/GGPPS Genes
In silico study indicated the presence of putative transit peptide in CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, and CpGGPPS2 proteins with the chloroplast localization, while the CpGPPS protein was found to be localized in the mitochondria (Table 1). To validate, protein sequences of CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, CpGGPPS2, and CpGPPS were fused to the N-terminus of the PRI101-EGFP vector, and these recombinant vectors were transiently expressed into the Nicotiana benthamiana leaves. Through microscopy, the green fluorescent protein (GFP) fluorescence signals of CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, and CpGGPPS2 were detected in the chloroplasts, whereas CpGPPS-GFP fusion protein fluorescence signals were detected in the mitochondria ( Figure 4).

CpGPPS.SSU1 and CpGPPS.SSU2 Interaction with GGPPS
To validate if the CpGPPS.SSU1 and CpGPPS.SSU2 can interact with the GGPPS/GPPS.LSU to form a heterodimer, we performed the yeast 2 hybrid (Y2H) assay. The result revealed that yeast cells carrying the plasmid pairs BD-CpGPPS.SSU1+AD-CpGGPPS2, BD-CpGPPS.SSU2+AD-CpGGPPS2, BD-CpGPPS.SSU2+AD-AtGGPPS11 and the positive control plasmid pair grew and turned blue on the QDO/X medium, indicating that the testing plasmid pair could interact and activate the reporter genes in the GAL4 system. On the other hand, yeast cells harboring the plasmid pairs, namely BD-
The scent of the wintersweet flower is composed of different volatile compounds, of which αlinalool is the most abundant monoterpene volatile compound, accounting for about 36% of the wintersweet floral scent [28]. Moreover, the formation of monoterpenes possesses tissue specificity and is often related to the maturity of a given tissue [29,30]. The wintersweet flower develops a strong and specific fragrance during flower opening [31]. Further, wintersweet flowers contain the maximum number of scent-emitting nectaries at the fully opened flower stage. Dominantly, the maximum number of scent-emitting nectaries resides on petals during flower development [32]. In the present study, CpGPPS/GGPPS genes were expressed in all tested tissues, with varying levels (Figure 2). Namely, the CpGPPS.SSU1 and CpGGPPS1 were highly expressed in the petal and flower, suggesting that these genes could have an important role in scent-producing tissues. Temporal

Discussion
Short-chain prenyl diphosphate synthases are enzymes of the isoprenoid pathway that use IPP and DMAPP to produce central intermediates in the isoprenoid metabolism. Short-chain prenyl diphosphate synthases are represented by three enzymes: GPPS, FPPS, and GGPPS [26]. Isolation and functional characterization of GPPSs from different plant species have confirmed the existence of homodimeric and heterodimeric forms, which are comprised of two similar subunits and one GPPS.SSU with one GPPS.LSU, respectively [15]. From the wintersweet transcriptome database [23], we isolated five transcripts that were annotated as GPPS/GGPPS-like genes: one homodimeric GPPS (CpGPPS), two GPPS small subunits (CpGPPS.SSU1, CpGPPS.SSU2), and two GGPPS (CpGGPPS1, and CpGGPPS2) sequences (Table 1). Subsequent sequence analysis indicated that homodimeric CpGPPS and both CpGGPPSs have two conserved aspartate-rich motifs, containing FARM (DD(X) 2-4 D) and SARM (DDXXD) (Figure S1C,D; Table 1), which are essential for catalysis and substrate binding [27]. However, CpGPPS.SSUs lack one or both of these motifs ( Figure S1A,B; Table 1). Besides, the CxxxC motif is needed for physical interaction between both subunits of heterodimeric GPPS [13][14][15]. In the present study, two CxxxC motifs were found in CpGPPS.SSUs and one motif in CpGGPPSs ( Figure S1A,B,D; Table 1), which is consistent with the previous findings from other plant species.
The scent of the wintersweet flower is composed of different volatile compounds, of which α-linalool is the most abundant monoterpene volatile compound, accounting for about 36% of the wintersweet floral scent [28]. Moreover, the formation of monoterpenes possesses tissue specificity and is often related to the maturity of a given tissue [29,30]. The wintersweet flower develops a strong and specific fragrance during flower opening [31]. Further, wintersweet flowers contain the maximum number of scent-emitting nectaries at the fully opened flower stage. Dominantly, the maximum number of scent-emitting nectaries resides on petals during flower development [32]. In the present study, CpGPPS/GGPPS genes were expressed in all tested tissues, with varying levels (Figure 2). Namely, the CpGPPS.SSU1 and CpGGPPS1 were highly expressed in the petal and flower, suggesting that these genes could have an important role in scent-producing tissues. Temporal expression analysis revealed that all CpGPPS/GGPPS genes, except CpGGPPS2, showed the maximum expression at fully opened flower stage, followed by reduction at the senescence stage ( Figure 3). These expression profiles are consistent with the finding of Xiang et al. [21], who reported that the C. praecox L. 'H29' flowers had the maximum monoterpene contents at the partially opened (58.06%) and fully opened stage flowers (57.89%), followed by a sudden decline at senescence (9.44%). Overall, these results suggest that the spatiotemporal expression of CpGPPS.SSU1 and CpGGPPS1 is strictly confined to the scent-emitting tissues with a good association to monoterpene contents. Moreover, Chen et al. (2015) reported the constitutive expression of AtGPPS.SSU2 and found its involvement in monoterpene biosynthesis [16]. This finding indicates that we cannot neglect the role of CpGPPS.SSU2 in scent biosynthesis in the wintersweet flower and suggest further investigations.
All genes/enzymes involved in the MEP pathway are known to have a transit peptide for their plastid targeting. The subcellular localization experiments in Catharanthus roseus and Litsea cubeba displayed the plastidial localization of GPPS/GGPPS [12,18,33]. On the other hand, there are few reports which support the mitochondrial, cytosolic, and endoplasmic reticulum localization of this pathway's genes [26]. In the present study, the subcellular localization assay revealed that CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, and CpGGPPS2 proteins have strong GFP signals in the chloroplast region similar to the previous findings of GPPS.SSU1 from A. majus, GPPS.SSU2 from C. annuum and GGPPSs from A. thaliana [14,26]. However, the GFP fluorescence of CpGPPS protein was not observed in the chloroplast, likely because it is localized in the mitochondria (Figure 4), consistent with the previous result of homodimeric GPPS in C. roseus [12]. These results support the chloroplast localization of CpGPPS.SSU1 and CpGPPS.SSU2 genes, further suggesting the functional involvement of these genes in monoterpene biosynthesis and metabolism.
In the heterodimeric GPPS gene, LSU is active and functional itself, while SSU is inactive and needs to interact with the LSU for functioning [9,12]. Through Y2H assay, both the CpGPPS.SSU1 and CpGPPS.SSU2 showed interaction with CpGGPPS2. Moreover, CpGPPS.SSU2 also interacted with the AtGGPPS11 (Figure 5). These results support the possibility of heterodimeric GPPS existence in the wintersweet plant and warrant speculation that the SSU of heterodimeric GPPS could have a role in modifying or accelerating the product specificity of LSU or GGPPS. These assertions are consistent with previous suggestions. For instance, following the bacterial genetic complementation assay, when LiGPPS.SSU1 and LiGPPS.SSU2 were individually co-transformed with catalytically active LiGGPPS, both SSU interacted and reduced the carotenoid contents [34]. Moreover, when AmGPPS.SSU1 was transformed in tobacco, it act as a modifier and increases the GPP, as well as monoterpene biosynthesis, at the expense of GGPP-derived compounds, i.e., gibberellins, chlorophyll, and carotenoids [14]. On the other hand, MpGPPS.SSU1 act like an accelerator when overexpressed in tobacco, as it promotes shoot branching and early flowering by an elevated content of cytokinin and gibberellin. Wang et al. [18] reported that the virus-induced gene silencing of CaSSU2 led to a reduction in carotenoid content in red pepper fruit. However, the knowledge about this phenomenon remains unknown in wintersweet. In the future, all CpGPPS/GGPPS candidate genes need to be further investigated by exploiting techniques such as in vitro enzymatic assays and genetic transformation to obtain deeper insights.

Plant Material and Sampling
A healthy and vigorous wintersweet plant (C. praecox L. 'H29') was selected as the study material. The experimental plant was grown under natural field conditions in the campus of Huazhong Agricultural University (Wuhan, China). Flower samples were collected at five developmental stages (stage 1 to stage 5), from bud development to senescence ( Figure 6). In detail, flower developmental stages were characterized with the following distinct morphological characters: Closed bud with green petals (stage 1), closed bud with yellow petals (stage 2), partial open flower (stage 3), open flower but not pollinated (stage 4), and senescent flower (stage 5). For spatial expression analysis, the leaf, open flower, stamen, pistil, and petal samples were collected. All samples were immediately frozen in liquid nitrogen and stored at −80 • C for further use.

Database Mining and Sequencing of CpGPPS cDNAs
The GPPS/GGPPS sequences of A. thaliana and L. cubeba were used as a query to blast against the C. praecox genome database (unpublished), and the floral transcriptome database [23] to retrieve all the putative CpGPPS/GGPPS protein sequences from wintersweet. A blastp analysis of the retrieved CpGPPS/GGPPS protein sequences was carried out by searching against the NCBI (https://www.ncbi.nlm.nih.gov/), Sol genomics (https://solgenomics.net/), and TAIR (https://www.arabidopsis.org/index.jsp) databases to find their homologs in other plant species. The largest ORF of CpGPPS/GGPPSs were amplified using the high-fidelity DNA polymerases (Vazyme, China) with gene-specific primers (Table S2). PCR was performed in a 50-µL reaction mixture comprising phanta max superfidelity DNA polymerases (11.5 µL, 1 unit/µL), forward and reverse primer (each 2.5 µL, 10 µM/µL), cDNA template (2 µL, 1 µg/µL) and ddH2O (31.5 µL) was subjected to the following PCR instructions: 95 °C for 5 min, and 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and a final extension of 72 °C for 7 min. The amplicons were purified using the Tiangel Midi Purification Kit (Tiangen, China) and cloned into a pEASY-T1 cloning vector (Transgen, China) for sequencing. The consistent sequencing results of each CpGPPS/GGPPSs were used for subsequent analysis.

RNA Extraction and qRT-PCR
Total RNA was extracted from different samples using the Hipure plant RNA purification kit (Magen Biotech, China), following the manufacturer's instructions. One microgram of high-quality RNA was used to synthesize the first strand of cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China). qRT-PCR was performed in the 10-µL reaction mixture comprising 5 µL of SYBR ® Premix Ex Taq™ II mix (Takara, Dalian, China), 0.3 µL of primer (forward and reverse each; the final concentration of 10 µM), 3.9 µL of ddH2O, and 0.5 µL of cDNA template with a final concentration of 300 ng/µL. The reactions were carried out using a Quant studio 7 flex real-time system (Applied Biosystems Life Technologies, New York), following the

Database Mining and Sequencing of CpGPPS cDNAs
The GPPS/GGPPS sequences of A. thaliana and L. cubeba were used as a query to blast against the C. praecox genome database (unpublished), and the floral transcriptome database [23] to retrieve all the putative CpGPPS/GGPPS protein sequences from wintersweet. A blastp analysis of the retrieved CpGPPS/GGPPS protein sequences was carried out by searching against the NCBI (https://www.ncbi. nlm.nih.gov/), Sol genomics (https://solgenomics.net/), and TAIR (https://www.arabidopsis.org/index. jsp) databases to find their homologs in other plant species. The largest ORF of CpGPPS/GGPPSs were amplified using the high-fidelity DNA polymerases (Vazyme, China) with gene-specific primers (Table S2). PCR was performed in a 50-µL reaction mixture comprising phanta max superfidelity DNA polymerases (11.5 µL, 1 unit/µL), forward and reverse primer (each 2.5 µL, 10 µM/µL), cDNA template (2 µL, 1 µg/µL) and ddH 2 O (31.5 µL) was subjected to the following PCR instructions: 95 • C for 5 min, and 35 cycles of 95 • C for 30 s, 60 • C for 30 s, 72 • C for 45 s, and a final extension of 72 • C for 7 min. The amplicons were purified using the Tiangel Midi Purification Kit (Tiangen, China) and cloned into a pEASY-T1 cloning vector (Transgen, China) for sequencing. The consistent sequencing results of each CpGPPS/GGPPSs were used for subsequent analysis.

RNA Extraction and qRT-PCR
Total RNA was extracted from different samples using the Hipure plant RNA purification kit (Magen Biotech, China), following the manufacturer's instructions. One microgram of high-quality RNA was used to synthesize the first strand of cDNA using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, China). qRT-PCR was performed in the 10-µL reaction mixture comprising 5 µL of SYBR ® Premix Ex Taq™ II mix (Takara, Dalian, China), 0.3 µL of primer (forward and reverse each; the final concentration of 10 µM), 3.9 µL of ddH 2 O, and 0.5 µL of cDNA template with a final concentration of 300 ng/µL. The reactions were carried out using a Quant studio 7 flex real-time system (Applied Biosystems Life Technologies, New York), following the manufacturer's protocol. The reaction was started with an initial incubation at 50 • C for 2 min and 95 • C for 5 min, then subjected to 40 cycles of 95 • C for 10 s, 60 • C for 20 s, and 72 • C for 20 s. qRT-PCR was performed in three biological replicates, and each biological replicate consisted of three technical replicates. Moreover, β-actin was used as a housekeeping gene to normalize the relative expression of target genes. Relative gene expression was analyzed following the 2 -∆∆CT method [37].

Subcellular Localization
For protein localization, we generated the green fluorescent fusion protein in frame with Sma1/EcoR1 cloning sites of the PRI101-EGFP vector driven by CaMV 35S promoter. The coding sequence of CpGPPS/GGPPSs were amplified (without stop codon) from C. praecox cDNA using gene-specific primers (Table S2). Amplified fragments were fused with the 5 end of the GFP sequence to generate a cassette comprising of 35S-CpGPPSs-EGFP. The resulting constructs of CpGPPSs-EGFP fusion were sequenced to confirm the protein fusions. All the constructs were then transferred into A. tumefaciens strain (GV3101+p19) by a freeze-thaw method. Transformed Agrobacterium clone was grown in LB medium until the OD 600 value reached 0.4-0.6 and was then used to infiltrate the leaves of 3-5-week-old N. benthamiana plants. The suspension solution was infiltrated on the lower surface of leaves using a 1-mL sterile needleless syringe, followed by a dark period for two days [38]. After 2-3 days of infiltration, the fluorescence signal of the GFP was examined at 488 nm under a confocal microscope (TCS-SP8, Leica, Germany).

Yeast 2-Hybrid Assay (Y2H)
The truncated versions of CpGPPS.SSU1, CpGPPS.SSU2, CpGGPPS1, CpGGPPS2, and AtGGPPS11 without the predicted plastid target peptide were amplified and sequenced using the gene-specific primers (Table S2). The amplified fragments of CpGPPS.SSU1 and CpGPPS.SSU2 were cloned in frame with the binding domain (GAL4) of the pGBKT7 (bait) vector, while CpGGPPS1, CpGGPPS2, and AtGGPPS11 were introduced in frame with the activation domain (GAL4) of the PGADT7 (prey) vector to use in protein interaction evaluation. The respective pairs of bait and prey vectors were co-transformed into the yeast strain (AH109) by the LiAc/DNA/PEG method, following the Yeast Protocols Handbook from Clontech (http://www.clontech.com). Co-transformed AH109 cells were cultured on the synthetic dropout (SD) media without Leu and Trp, and the interaction/activation of two proteins was studied on the SD media lacking Leu, Trp, His, and Ade.

Statistical Analysis
The data were evaluated by the HSD post-hoc test in the ANOVA program of Statistix 8.1 (Florida, USA). Different lowercase letters on bars indicate significant differences at p < 0.05.

Conclusions
Five GPPS/GGPPS genes (CpGPPS.SSU1, CpGPPS.SSU2, CpGPPS, CpGGPPS1, and CpGGPPS2) were isolated in wintersweet flower. qRT-PCR results showed that the expression of CpGPPS.SSU1 was predominantly confined to the fragrance-producing tissues and was upregulated during flower development, indicating important roles in floral volatile emission. In addition, the present data confirm that both CpGPPS.SSUs can interact with GGPPS-like proteins, and localization experiments further support the presence of chloroplast localized heterodimeric GPPS in the wintersweet flower. Collectively, the present data warrant consideration of heterodimeric GPPS.SSU as a target for the manipulation of floral scent production. Further studies will better define the modes of regulation and confirm the roles of heterodimeric GPPS in wintersweet flower scent production.