Molecular Cloning and Expression Analysis of Geranyllinalool Synthase Gene (SgGES) from Salvia guaranitica Plants

Abstract

Salvia guaranitica is considered one of the most significant medicinal and aromatic herbs in terms of nutritional and medical benefits due to its wealth of important active components. Among these compounds, terpenoids are the most prominent and abundant, particularly monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20). They are biologically advantageous to plants and perform a multitude of functions. The current study aimed to clone the S. guaranitica gene that encodes for geranyllinalool synthases (SgGES, EC: 4.2.3.144), with consideration for these features. The open reading frame of the 867-amino-acid protein encoded by SgGES consists of 2.721 base pairs. In addition, the SgGES protein has five domains that belong to the terpene synthase family, which are related to the terpene and terpenoid synthase domains. We manipulated and overexpressed the SgGES gene in Nicotiana tabacum to explore its function. When compared to the GUS control, the transgenic N. tabacum plants displayed an increase in leaf production and diameter when compared with the wild-type plants. Finally, analysis of transgenic plants using gas chromatography/mass spectrometry (GC-MS) showed that SgGES is responsible for producing various terpene species, especially diterpenes.

1. Introduction

The genus Salvia (Lamiaceae) has more than 1000 species of woody, fragrant shrubs, some of which, like S. przewalskii, S. miltiorrhiza, S. allagospadonopsis, S. aegyptiaca, S. officinalis, S. africana, S. splendens, and S. japonica, are economically significant and are globally cultivated for their numerous medicinal uses and to produce essential oils (EOs). Although a plethora of Salvia species can be found on Earth, the majority of them are concentrated in three distinct geographical regions: East Asia (100 species), West Asia (200 species), and Central and South America (500 species). In contrast, the remaining species of Salvia are dispersed throughout the remainder of the world [1,2,3,4].

In recent years, EOs from Salvia species have emerged as a critical resource in pharmacological and aromatic research, focusing on the discovery and characterization of bioactive compounds. The essential oils derived from Salvia display significant bioactivities, including choleretic effects, antioxidant properties, antibacterial activity, anticancer properties, antimutagenic effects, and anti-inflammatory capabilities [5,6,7]. Terpenoids have been classified as the largest class of secondary metabolites and natural products. They were detected in both plants and other species, with over 40,000 unique structural variations present [2,4,8,9,10]. Isopentenyl diphosphate (IPP), which consists of five carbon atoms (C5), serves as the structural basis for terpenoid compounds; see Figure 1 [11,12]. The original names of these compounds were derived from the Pistacia terebinthus tree, which was the source from which all these unique terpene chemicals were first identified. Ruzicka and Wallach altered and depicted the structure of the terpene unit [13,14,15,16]. Certain terpenes play a vital role in the fundamental metabolic processes of plants and are indispensable for plant growth, flowering, and development. Examples of such terpenes include carotenoid pigments, the phytol side chains of chlorophyll, phytohormones, and certain constituents of cellular membranes [17,18]. Nonetheless, most terpenes that have been discovered are classified as secondary metabolites and are crucial to the way plants interact with their natural environments. Terpenes, both volatile and nonvolatile, are known to play a significant role in the defense against insects and microbes, as well as in the attraction of pollinators and the management of photo-oxidative stress [19]. To gain a comprehensive understanding of the functions of terpenes and terpenoid compounds, extensive research is currently being conducted [2,4,9,10,20,21]. Salvia species are widely recognized for their significant concentration of EOs, with mono- and sesquiterpenes constituting most of the constituents of their fragrant essential oils [2,22,23,24,25,26,27,28]. According to [4], the primary sesquiterpenes in the Chinese cultivar of S. guaranitica are germacrene-A, isocaryophyllene, germacrene-C, humulene, caryophyllene, germacrene-D, pi-a-murolene, caryophyllene oxide, and d-cadinene. The pharmacological and physiological roles of these sesquiterpenes are diverse, and many of the genes responsible for synthesizing these compounds in Salvia are yet to be identified. In this study, the gene encoding the geranyllinalool synthase (SgGES, EC: 4.2.3.144) enzyme from S. guaranitica was cloned and subsequently subjected to functional characterization. Additionally, the recombinant SgGES catalyzed the conversion of geranylgeranyl diphosphate (GGPP) into a range of terpene products, including diterpenes in various forms.

2. Materials and Methods

2.1. Plant Materials

S. guaranitica plantlets were collected from the Wuhan Botanical Garden (WBG), Chinese Academy of Sciences, located in Hubei, Wuhan, China. Following that, the plantlets were cultivated in the greenhouse of the National Research Centre in Cairo, Egypt. Young leaves from five-year-old plants were collected and rapidly frozen in liquid nitrogen for 15 min and stored at −80 °C to isolate RNA and prepare for gene cloning.

2.2. Sequence Characterization of SgGES

The sequence of the SgGES gene was selected based on the highest sequence similarity it displayed when compared with genes responsible for plant diterpene synthesis that are already known. The PROTPARAM server (http://web.expasy.org/protparam/, accessed on 15 January 2024) was utilized to evaluate the physical and chemical characteristics of SgGES. We employed the iPSORT prediction tool (http://ipsort.hgc.jp/, accessed on 16 June 2024) to analyze the putative transit peptide for open reading frames (ORFs) of SgGES. To compare and analyze the sequence of SgGES with known proteins, we used the NCBI BLASTX tool (http://blast.ncbi.nlm.nih.gov/, accessed on 5 June 2023). The PhyML Server aided the understanding of the evolutionary relationships of the SgGES protein and other plant TPS proteins between the SgGES protein and other plant TPS proteins, without requiring any adjustments to the parameters of the tool (http://www.phylogeny.fr/, accessed on 16 June 2024) [2,4,29,30]. Moreover, PROTPARAM software (http://web.expasy.org/protparam, accessed on 15 May 2023) facilitated the determination of the physiochemical properties of SgGES.

2.3. Putative Tissue Expression Pattern and Subcellular Localization of SgGES

Based on the Arabidopsis eFP browsers (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi, accessed on 16 June 2024), tissue-specific expression data from seventy-two (e.g., Dry seed, Imbibed seed, 24 h, 1st Node, Flower Stage 12, Stamens, Cauline Leaf, Cotyledon, Root, Entire Rosette After Transition to Flowering, Flower Stage 9, Flower Stage 10/11, Flower Stage 12, Flower Stage 15, Flower Stage 12, Carpels, Flower Stage 12, Petals, Flower Stage 12, Sepals, Flower Stage 15, Carpels, Flower Stage 15, Petals, Flower Stage 15, Sepals, Flower Stage 15, Stamen, Flowers Stage 15, Pedicels, Leaf 1 + 2, Leaf 7, Petiole, Leaf 7, Distal Half, Leaf 7, Proximal Half, Hypocotyl, Root, Rosette Leaf 2, Rosette Leaf 4, Rosette Leaf 6, Rosette Leaf 8, Rosette Leaf 10, Rosette Leaf 12, Senescing Leaf, Shoot Apex, Inflorescence, Shoot Apex, Transition, Shoot Apex, Vegetative, Stem, 2nd Internode, Mature Pollen, Seeds Stage 3 w/Siliques, Seeds Stage 4 w/Siliques, Seeds Stage 5 w/Siliques, Seeds Stage 6 w/o Siliques, Seeds Stage 7 w/o Siliques, Seeds Stage 8 w/o Siliques, Seeds Stage 9 w/o Siliques, Seeds Stage 10 w/o Siliques, Vegetative Rosette, Globular—apical, Globular—basal, Heart—cotyledons, Heart—root, Torpedo—cotyledons, Torpedo—apical, Torpedo—root, Torpedo—basal, Torpedo—meristem, cross-section of leaf/mesophyll cells no ABA, cross-section of leaf/mesophyll cells with 100 μM ABA, surface view of leaf/guard cells no ABA, surface view of leaf/guard cells with 100 μM ABA, mesophyll cells without ABA/cordycepin and actinomycin added during protoplasting, mesophyll cells without ABA/no cordycepin nor actinomycin, mesophyll cells with 100 μM ABA/cordycepin and actinomycin added during protoplasting, mesophyll cells with 100 μM ABA/no cordycepin nor actinomycin, guard cells without ABA/cordycepin and actinomycin added during protoplasting, guard cells without ABA/no cordycepin nor actinomycin, guard cells with 100 μM ABA/cordycepin and actinomycin added during protoplasting, guard cells with 100 μM ABA/no cordycepin nor actinomycin, stem epidermis/top of stem, whole stem/top of stem, stem epidermis/bottom of stem, and whole stem/bottom of stem; see Supplementary Materials Table S1) tissues were analyzed to generate expression profiles. The putative sub-cellular localizations of the SgGES gene was analyzed using the Cell-eFP browsers (http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi, accessed on 16 June 2024) [31,32]. The color boxes represent the expression scale (e.g., red color—high expression and yellow color—low expression).

2.4. RNA Extraction and cDNA Synthesis

Six tissues from five-year-old S. guaranitica were used to extract RNA using TransZol Reagent (Invitrogen, Carlsbad, CA, USA). Also, RNA was extracted from leaves of wild-type and transgenic N. tabacum for semi-quantitative RT-PCR (sqRT-PCR). For cDNA synthesis, 1 μg of RNAs was used to synthesize the first-strand cDNA using TransScript^® First-Strand cDNA Synthesis Super Mix kit (Invitrogen, Carlsbad, CA, USA), as described by the authors of [2,33].

2.5. Isolating the Full-Length SgGES Gene

The complete SgGES cDNA was used as a template to amplify the full-length gene using short gene-specific forward (5′-ATGATAAACATTATGTATACAAGCTAGCC-3′) and reverse (5′-CTAATTAAGGAAA AGTGAGAAACAAACAC-3′) primers and TaKaRa Taq DNA Polymerase enzyme (TaKaRa, Beijing, China). The amplification conditions were 4 min at 96 °C; 33 cycles for 13 s at 96 °C; 30 s at 58 °C; 96 s at 68 °C; and then 14 min at 68 °C. The first PCR product was used as a template for the second PCR using long gene-specific forward (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGATAAACATTATGTAT-3′) and reverse (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTAAAGTGAGAAACAAACAC-3′) primers. We adopted the same polymerase enzyme and PCR program. The amplicon was cloned into a pDONR-221 vector (Invitrogen, Carlsbad, CA, USA) and then pB2GW7 vector with the cauliflower mosaic virus (CaMV) 35S promoter as a strong constitutive promoter (Invitrogen, Carlsbad, CA, USA) and sequenced as described by the authors of [2,4].

2.6. Nicotiana Tabacum Leaf Transformation Procedure Using Plant Tissue Culture Method and Preparation of Agrobacterium Cultures for Infection

To ensure that SgGES is consistently expressed in tobacco plants, we utilized the Agrobacterium tumefaciens strain GV3101 to infect N. tabacum leaves. The pB2GW7-SgGES vector was introduced into the A. tumefaciens strain GV3101 by the direct electroporation method. This strain carried the construct plasmid pB2GW7-SgGES, and the expression of our target gene in this plasmid was driven by the 35S promoter (see Supplementary Materials Figure S1). Utilizing leaf discs from tobacco, a slightly modified protocol was implemented that was originally described by the authors of [2,34,35,36]. Transgenic tobacco plants were regenerated, and then the positive transgenic plantlets were selected with 55 mg/L hygromycin. More than 10 individual transgenic tobacco lines were generated and examined with RT-PCR for positive transgenic lines. Transgenic plants with good and healthy roots were transferred to the greenhouse for acclimatization. To facilitate this, the transgenic plants were provided with regular watering and fertilization using Miracle-Gro fertilizer (Scott’s Company, Marysville, OH, USA). Plants were grown in growth chambers with a temperature of 22 °C during the day and 20 °C at night and a humidity of 60–70%. The photoperiod was set at 16 h during the day and 8 h at night, and the light density was 100–150 μ moles m⁻² s⁻¹, with fluorescent bulbs used for flowering and vegetative growth. Finally, the transgenic tobacco and control plants were analyzed for growth, leaf morphology, and terpenoid profiling.

2.7. Semi-Quantitative RT-PCR (sqRT-PCR) Analysis

To confirm the gene transfer process, sqRT-PCR was conducted using a PCR system from Biometra (Biometra, Jena, Germany). The NtEF-1α gene was utilized as a housekeeping gene (forward primer 5′-TGGTTGTGACTTTTGGTCCCA-3′; reverse primer 5′-ACAAACCCACGCTTGAGATCC-3′) with a length of 155 base pairs, while the SgGES gene was amplified using the forward primer 5′-CATGGTGTGTACCCTGTTAG-3′ and reverse primer 5′-GATCTTTGAGGCGGTAGATG-3′, with a length of 159 base pairs. The primers for our study were designed using the online platform provided by IDTdna (http://www.idtdna.com/scitools/Applications/RealTimePCR/, accessed on 16 June 2024). The sqRT-PCR reaction was carried out utilizing the Biometra Thermocycler T-Gradient Thermo Block system, with the program executed as follows: an initial denaturation phase at 94 °C for 5 min, followed by 33 cycles of denaturation at 96 °C for 34 s, annealing at 59 °C for 30 s, and extension at 72 °C for 1.3 min. The final extension phase was carried out at 72 °C for 10 min. The PCR products were then analyzed on a 1.35% agarose gel to assess gene expression levels.

2.8. Terpenoid Extraction and GC-MS Analysis

Intact leaves from both transgenic and non-transgenic N. tabacum plant lines were frozen in liquid nitrogen (LN2) and subsequently crushed using a ceramic mortar and pestle before being immersed in the solvent n-hexane for 73 h. This process ensures the complete extraction of terpenoids from the leaves, as previously described [2,9,10,20,21]. An approximately 1 µL aliquot of each extract from a 1.5 mL crimp vial was analyzed using the Shimadzu GC-MS system (Tokyo, Japan) with three technical replicates. The analysis was performed by the Wiley GC/MS Library, the Volatile Organic Compounds (VOCs) Analysis S/W software https://www.intertek.com/chemicals/voc-testing/, accessed on 16 June 2024, and the NIST Library, which was utilized as a reference to identify terpenoids according to previously described methods [2,4].

2.9. Quantitative Real-Time PCR (qRT-PCR) Analyses

qRT-PCR was employed to determine the quantity of SgGES transcripts responsible for the synthesis of geranyllinalool synthases. For this purpose, tissue samples were obtained from various plant parts, including flowers, flowering buds, roots, stems, young leaves, and old leaves. A total of three biological replicates were collected for each sample type. The housekeeping gene used in this reaction was SgACTIN. Its forward primer was 5′-CTGGATTTGCGGGAGATG-3′, and its reverse primer was 5′ CCGTGCTCAATTGGATACTT-3′. For SgGES, the forward primer used was 5′-GTAACTCGCATCGCACAT-3′, and the reverse primer used was 5-GAAGCAAGAGACGCTATCAG-3. The qRT-PCR reaction was carried out using an IQTM5 System, SYBR Green. The program was modified to include the following settings: 94 °C for 12 s, 59 °C for 30 s, and 72 °C for 22 s, followed by 65 °C for 6 s and 93 °C for 6 s. The expression levels were calculated by comparing our target gene cycle thresholds (CTs) with the housekeeping gene SgACTIN using the 2^−ΔΔCt method [33,37,38,39]. Values are reported as the means ± SE from three independent RNA pool replicates.

3. Results

3.1. Full-Length Geranyllinalool Synthase (SgGES) Clone and Sequence Analysis

The open reading frame (ORF) of the SgGES gene spans a length of 2721 base pairs and is responsible for encoding a protein consisting of 867 amino acids. This protein has a predicted instability index of approximately 45.48, a theoretical isoelectric point (pI) of 9.54, an aliphatic index of 94.94, a grand average of hydropathicity (GRAVY) of −0.037, a molecular mass of 100.172 kilodaltons (kDa), and a total of 56 negatively charged residues (Asp + Glu) and 101 positively charged residues (Arg + Lys). The N-terminal region of SgGES is composed of a thirty-amino-acid peptide (MINIMYTSPTLGDFHESKRWQNNISNYTYI), which forms a lengthy signal peptide, comparable to the monoterpene synthases (600–650 aa) and sesquiterpene synthases (550–580 aa). Moreover, this signal peptide in N-terminus was used as an intracellular postal code for the targeting of the protein to secretory pathways, such as cell membranes or chloroplasts. The ‘iPSORT’ program has revealed that SgGES is in either the chloroplast or the mitochondria, where the biosynthesis of farnesyl diphosphate (FPP) occurs.

NCBI-BLASTx analysis, depicted in

Table 1. BLASTX analysis SgGES compared with the NCBI protein database for gene identification purposes.

NCBI Accession	a Descriptiona	Organism	E Value	Max Score	Total Score	Query Cover	Identity (%)	Accession Length
XP_042043873.1	(E,E)-geranyllinalool synthase-like	Salvia splendens	0.0	1582	1582	90%	98.54%	837
XP_041991789.1	(E,E)-geranyllinalool synthase-like	Salvia splendens	0.0	1575	1575	90%	98.18%	837
XP_047940334.1	(E,E)-geranyllinalool synthase-like	Salvia hispanica	0.0	1526	1526	90%	94.66%	834
XP_041994219.1	(E,E)-geranyllinalool synthase-like	Salvia splendens	0.0	1435	1435	90%	87.90%	851
XP_047964745.1	(E,E)-geranyllinalool synthase-like	Salvia hispanica	0.0	1414	1414	90%	87.44%	850
XP_057764549.1	S-linalool synthase	Salvia miltiorrhiza	0.0	1184	1184	90%	74.76%	820
QEV81852.1	Terpene synthase 13	Prunella vulgaris	0.0	1067	1067	90%	66.07%	881
PIN19742.1	Alpha-farnesene synthase	Handroanthus impetiginosus	0.0	993	993	90%	60.83%	846
UVE15964.1	Geranyllinalool synthase	Leonurus japonicus	0.0	989	989	84%	64.99%	827
QIQ55998.1	Putative terpene synthase 7	Eremophila drummondii	0.0	872	872	83%	59.04%	792

^a Description—homology search using BLASTX.

, revealed that SgGES exhibited varying levels of identity with its homologous diterpene synthase proteins from various plants, including Salvia splendens (98.54%), Salvia hispanica (87.44%), Salvia miltiorrhiza (74.76%), and others. The SgGES gene sequence alignment with known and putative TPS genes from Lamiaceae and other plants helped to predict its potential function. This prediction indicates that the SgGES protein contains multiple domains, as reported by the Superfamily, Pfam, and InterPro databases. As a result, the SgGES protein comprises five terpene synthase family (TSF) domains, which include two terpenoid cyclases/protein prenyltransferase alpha-alpha toroid domains (IPR008930/SSF48239, spanning from 35 to 133 and 235 to 440), an isoprenoid synthase domain (IPR008949/SSF48576, from 463 to 783), a terpene synthase N-terminal domain (IPR001906/PF01397, from 226 to 425), and a terpene synthase metal-binding domain (IPR005630/PF03936, from 466 to 727) (as depicted in Figure 2). The previous domains have been widely recognized in various mono-, sesqui-, and diterpene synthases [2,4,9,10,12,40,41]. SgGES was classified into the TPS-a subfamily of angiosperm sesquiterpene synthases, as determined by phylogenetic analysis results (Figure 3). This subfamily is primarily responsible for synthesizing mono-, sesqui-, and diterpene synthases [2,4,42,43].

3.2. Putative Tissue Expression and Subcellular Localization of SgGES Gene

We performed a BlastP search against A. thaliana genomics with the Phytozome database (https://phytozome-next.jgi.doe.gov/, accessed on 16 June 2024) to monitor the putative tissue expression pattern of SgGES in A. thaliana with the nucleotide sequence of SgGES as a query. As a result, several nucleotides closely related to the SgGES sequences, especially (AT1G61120), with high BLAST identities % and e-values (73% and 5.81^e-4) were identified. The tissue expression of the SgGES gene in Arabidopsis discovered by our data was analyzed across seventy-two tissues using the BAR database (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi, accessed on 16 June 2024) and the Arabidopsis Electronic Fluorescent Pictograph browsers (eFP browsers (http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi, accessed on 16 June 2024) (Figure 4A–E). As demonstrated by the Arabidopsis eFP browsers, the SgGES gene (AT1G61120) was found in the majority of the tissues and was highly expressed in Flower Stage 15, Sepals (303.35), Flower Stage 15 (128.03), Rosette Leaf 4 (85.63), Flower Stage 12, Sepals (85.28), Leaf 7, Distal Half (58.53), Flower Stage 12 (49.55), and Rosette Leaf 6 (38.42).

Moreover, the expression of the SgGES (AT1G61120) gene displayed an elevated expression specifically at the embryo development stage; for example, our gene was highly expressed in Heart—cotyledons (46.13), followed by Torpedo—cotyledons (45.97), Torpedo—root (21.47), and Torpedo—basal (21.38) (Figure 4B and Supplementary Materials Table S1). On the other hand, the application of 100 µM Abscisic acid (ABA) led to an increase in the expression of the SgGES gene in both guard and mesophyll cells, regardless of the presence of cordycepin and actinomycin during protoplasting. In mesophyll cells treated with 100 µM ABA, cordycepin and actinomycin were added during protoplasting, resulting in a value of 154.49. This was followed by the cross-section of mesophyll cells in the presence of 100 µM ABA, which had a value of 80.39. In comparison, mesophyll cells without ABA, cordycepin, and actinomycin during protoplasting had a value of 74.61, while the cross-section of mesophyll cells without ABA had a value of 43.29 (as shown in Figure 4C and the Supplementary Materials Table S1). In addition, the SgGES gene was highly expressed in the stem epidermis/top of stem (9.76), followed by stem epidermis/bottom of stem (8.03), whole stem/bottom of stem (3.15), and whole stem/top of stem (2.8); see Figure 4D. Furthermore, the subcellular localization of the (SgGES gene: AT1G61120) gene was predicted using ePlant and cell eFP (http://bar.utoronto.ca/cell_efp/cgi-bin/cell_efp.cgi, accessed on 16 June 2024) and displayed various expression levels for 14 cell organelles, as depicted in Figure 4E.

3.3. Monitoring the Expression of SgGES Using qRT-PCR

The transcripts of SgGES at various tissues of S. guaranitica (e.g., flowering buds, flowers, roots, stems, young and old leaves) were measured using qRT-PCR. The results revealed that flowers displayed the highest expression levels of all the tissues analyzed, followed by bud flowers, young leaves, roots, old leaves, and stems (Figure 5).

3.4. Functional Expression of SgGES Gene N. tabacum Plants

We utilized N. tabacum plants as a transgenic expression system for conducting a further investigation on the function of the SgGES gene (Figure 6A). Subsequently, we confirmed the presence of positive transgenic tobacco plants using semi-quantitative RT-PCR (Figure 6B).

3.5. Phenotypic Evaluation

Comparisons of the phenotype between N. tabacum plants transformed with SgGES and their wild-type counterparts were conducted. The assessment included evaluations of leaf morphology, growth, and terpene metabolism. The transformed plants have large green oval leaves compared with the control plants (non-transformant plants), which have small green oval leaves. Moreover, the transformed plants are growing fast and have larger numbers of leaves than the control plants.

3.6. Overexpression of the SgGES Gene Altered the Terpene Profiles in Transgenic and Non-Transgenic Tobacco Plants

To explore the impact of overexpressing the SgGES gene on the terpene profiles of N. tabacum leaves, GC-MS was employed to compare the changes in metabolic profiles between transgenic plants and non-transgenic controls. The results revealed a significant increase in numerous terpenes in transgenic N. tabacum leaves that showed expression of the SgGES gene compared to the non-transgenic plants (Figure 7 and

Table 2. The major terpenoid compositions in transgenic N. tabacum leaves over-expressing SgGES.

N	Compound Name	R.T (min.)	Formula	Molecular Mass (g mol−1)	Terpene of Type	% Peak Area
						AtW.T	SgTPS-V
1	3,4-Dimethylcyclohexanol	16.114	C8H16O	128.212		0.04
2	L-(-)-Nicotine	25.9	C10H14N2	162.232		23.06
3	Cycloheptasiloxane, tetradecamethyl-	29.69	C14H42O7Si7	519.0776			0.02
4	Trans-β-Ionone	30.644	C13H20O	192.2973			0.05
5	Topanol	31.134	C15H24O	220.3505	Sesqui	0.06
6	Stavox	31.345	C15H24O	220.3505	Sesqui		0.15
7	Cyclooctasiloxane, hexadecamethyl-	34.574	C16H48O8Si8	593.2315			0.16
8	Carbamic acid, methyl-, 3-methylphenyl ester	36.029	C9H11NO2	165.1891			0.06
9	Tetradecanal	37.76	C14H28O	212.3715	Fatty acids		0.09
10	Octadeamethyl-cyclononasiloxane	38.761	C18H54O9Si9	667.3855			0.32
11	Pentadecanoic acid	39.636	C15H30O2	242.3975	Fatty acids		0.14
12	N-Pentadecanal	40.583	C15H30O	226.3981	Fatty acids	0.17
13	Oxirane, tetradecyl-	40.777	C16H32O	240.4247			1.29
14	Linolenic acid;Methyl linolenate	42.253	C19H32O2	292.4562	Fatty acids	0.86
15	Alpha.-Linolenic acid, trimethylsilyl ester	42.458	C21H38O2Si	350.6107	Fatty acids		0.8
16	Palmitic acid, methyl ester	43.187	C17H34O2	270.4507	Fatty acids		0.1
17	Methyl α-linolenate	44.012	C19H32O2	292.4562	Fatty acids		3.48
18	N-Hexadecanoic acid	44.285	C16H32O2	256.4241	Fatty acids	4.36
19	Palmitic acid	44.686	C16H32O2	256.4241	Fatty acids		11.74
20	Cycloartanyl acetate	45.616	C32H54O2	470.77		0.63
21	Hexadecamethyl-cyclooctasioxane	45.865	C16H48O8Si8	593.2315			0.3
22	Labda-8,13-(E)-dien-15-ol	45.95	C20H34O	290.4834	Di		0.2
23	Stearin, 2-mono	46.238	C21H42O4	358.5558			0.11
24	Geranylgeraniol	46.367	C22H36O2	332.52	Di	0.35
25	Guaia-1(10),11-diene;	46.392	C15H24	204.3511	Sesqui		0.65
26	All-trans-Retinol acetate	46.593	C22H32O2	328.4883		0.55
27	(+)-Ledol; d-Ledol	46.691	C15H26O	222.3663	Sesqui		0.63
28	Linoleic acid, methyl ester	46.888	C19H34O2	294.4721	Fatty acids	0.11
29	Retinol, acetate, all-trans	46.926	C22H32O2	328.4883			0.87
30	Linolenic acid	47.041	C19H32O2	292.4562	Fatty acids	0.47
31	Methyl linoleate	47.192	C19H34O2	294.4721	Di		0.45
32	Cis-Phytol	47.267	C20H40O	296.531	Di	4.71
33	Farnesyl butyrate	47.341	C19H32O2	292.4562	Di		0.74
34	Phytol	47.603	C20H40O	296.531	Di		4.82
35	Citronellyl geranylate	47.702	C20H34O2	306.4828	Di	1.32
36	(Z)-β-Elemene;	48.037	C15H24	204.3511	Di	0.27
37	(2E,6E)-Farnesyl pentanoate	48.064	C20H34O2	306.4828	Di		1.22
38	Phytol, trimethylsilyl ether	48.303	C23H48OSi	368.7121	Di		0.21
39	geranylgeraniol	48.418	C22H36O2	332.52	Di		0.28
40	Linolenic acid	48.532	C18H30O2	278.4296	Fatty acids	8.02
41	α-Linolenic acid	48.907	C18H30O2	278.4296	Fatty acids		23.73
42	Geranylgeraniol	48.948	C22H36O2	332.52	Di	1.17
43	4,8,13-Duvatriene-1,3-Diol	49.156	C20H34O2	306.4828		0.68
44	Stearic acid	49.281	C18H36O2	284.4772	Fatty acid		2.86
45	(+)-Ledol	49.552	C15H26O	222.3663	Sesqui		1.15
46	4,8,13-Duvatriene-1,3-Diol	50.049	C20H34O2	306.4828		43.79
47	α-Limonene diepoxide	50.216	C10H16O2	168.2328	Mono	0.84
48	(E,E)-Geranyllinalool	50.461	C20H34O	290.4834	Di		33.8
49	Behenyl alcohol	51.405	C22H46O	326.6		0.23
50	d-Ledol	51.493	C15H26O	222.3663	Sesqui		0.07
51	Octadeamethyl-cyclononasiloxane	52.037	C18H54O9Si9	667.3855			0.65
52	Methyl 11,14,17-icosatrienoate	52.978	C21H36O2	320.5093		0.24
53	Cyclononasiloxane, octadecamethyl-	53.436	C18H54O9Si9	667.3855			0.05
54	7,10-Hexadecadienoic acid, methyl ester	53.481	C17H30O2	266.4189		0.7
55	Squalene	54.046	C30H50	410.718	Tri		0.43
56	n-Heneicosane	54.964	C21H44	296.5741			0.05
57	Octadeamethyl-cyclononasiloxane	56.355	C18H54O9Si9	667.3855			0.79
58	Methyl linolenate, α	57.393	C19H32O2	292.4562	Fatty acids		0.09
59	n-Pentatriacontane	58.253	C35H72	492.9462		0.52
60	n-Pentatriacotane	58.992	C35H72	492.9462			0.79
61	Phthalic acid dioctyl ester	59.688	C24H38O4	390.5561	Ester	0.49
62	phthalic acid	60.497	C24H38O4	390.5561	Ester		0.15
63	Methyl 11,14,17-icosatrienoate	60.973	C21H36O2	320.5093		0.4
64	Homomyrtenol	61.574	C11H18O	166.26	Mono		0.13
65	Octadeamethyl-cyclononasiloxane	62.682	C18H54O9Si9	667.3855			1.06
66	n-Heneicosane	63.222	C21H44	296.5741	Alkane	0.18
67	n-Tetracontane	64.117	C40H82	563.0791	Alkane		0.14
68	n-Nonacosane	66.567	C29H60	408.7867	Alkane	0.09
69	Isovaleric acid, allyl ester	68.645	C8H14O2	142.1956			0.23
70	henicosane	68.682	C21H44	296.5741	Alkane	1.55
71	Tetracontane	69.7	C40H82	563.0791	Alkane		1.61
72	Octadeamethyl-cyclononasiloxane	70.054	C18H54O9Si9	667.3855			1.15
73	Tetrapentacontane	71.42	C54H110	759.4512			0.03
74	9,12-Octadecadien-1-ol, (Z,Z)-	71.563	C18H34O	266.462		0.07
75	(-)-Myrtenol	72.087	C10H16O	152.2334	Mono	0.15
76	n-Pentatriacontane	72.462	C35H72	492.9462			0.19
77	Montanyl alcohol	72.767	C28H58O	410.7595		0.33
78	α-Curcumene	73.038	C15H22	202.3352	Sesqui		0.15
79	3-Methyloctadecane	73.842	C19H40	268.5209			0.03
80	Octadecyl chloride	74.477	C18H37Cl	288.939		0.63
81	Nonacosane	75.547	C29H60	408.7867	Alkane		0.38
82	n-Octadecyl chloride;	76.096	C18H37Cl	288.939		0.04
83	Octadeamethyl-cyclononasiloxane	77.732	C18H54O9Si9	667.3855			1.17
84	n-Nonacosane	78.098	C29H60	408.7867	Alkane	2.84
85	n-Octacosane	78.668	C28H58	394.7601	Alkane	0.08
86	n-Nonacosane	79.245	C29H60	408.7867	Alkane		0.27
	Total % monoterpene	0.99	0.13	667.3855			0.65
	Total % sesquiterpene	0.33	2.8	320.5093		0.24
	Total % diterpene	7.55	41.72	667.3855			0.05
	Total % triterpene		0.43	266.4189		0.7
	Total % ester	0.49	0.15	410.718	Tri		0.43
	Total % of Alkane	4.74	2.4	296.5741			0.05
	Total % of fatty acid.	13.99	40.17	667.3855			0.79

). In leaves of N. tabacum plants overexpressing SgGES, (E,E)-geranyllinalool appeared as the main diterpene compound (33.8%), followed by α-Linolenic acid (23.73%) and Palmitic acid (11.74%) as fatty acids, whereas, (+)-Ledol compounds (1.15%) and Squalene compounds (0.43%) showed up as the main sesquiterpene and triterpene compounds, respectively.

4. Discussion

4.1. Cloning and Sequence Analysis of SgGES Gene from S. guaranitica

The SgGES gene was identified, and its full-length cDNA was obtained from the leaves of S. guaranitica by utilizing a conserved sequence that is remarkably similar to the sequences found in other plant species, including those identified in our query. The full-length cDNA of the SgGES gene was acknowledged and isolated from the leaves of S. guaranitica based on the conserved sequence, which is highly similar to our query sequence and the other sequences identified in different plant species, including Salvia splendens, Salvia hispanica, Salvia miltiorrhiza, Prunella vulgaris, Handroanthus impetiginosus, Leonurus japonicus, and Eremophila drummondii.

When contrasted with diterpene and other terpene synthases, the SgGES protein comprises five domains, which have been identified and designated by the Superfamily, Pfam, and InterPro databases. The first two domains are the terpenoid cyclases/protein prenyltransferase alpha-alpha toroid (IPR008930/SSF48239, from 35 to 133 and IPR008930/SSF48239, from 235 to 440), and the third is the isoprenoid synthase domain (IPR008949/SSF48576, from 463 to 783), while the other domains are terpene synthase N-terminal domain (IPR001906/PF01397, from 226 to 425) and terpene synthase metal-binding domain (IPR005630/PF03936, from 466 to 727) (Figure 2). The putative SgGES protein sequence showed various highly conserved domains that are responsible for coordinating the binding between divalent metal ion co-factors and substrate [2,4,12,40,41,44,45,46], (Figure 2). Furthermore, they are well known for their capacity to bind a tri-nuclear-magnesium cluster, two magnesium ions, and one magnesium ion [12,45,47]. This magnesium cluster binds and interacts with the diphosphate moiety of Geranylgeranyl diphosphate (GFPP) and FPP to catalyze the C20-substrate GGPP and C15-substrate FPP formation [12,40,41,46,48,49,50]. Finally, the terpene synthase family of proteins consistently includes one or two conserved domains [2,4,9,51]. To further uncover the evolutionary relations between SgGES and other plant terpene synthase genes, we used the neighbor-joining method parameters, which generated an evolutionary tree. The classification results pointed to the placement of the SgGES protein in the TPS-a subfamily, which is known to encode mono-, sesqui-, and diterpenes. The capacity of SgGES to generate diverse types of terpenes has been previously highlighted by [2,4] (Figure 3).

4.2. Putative Tissue Expression Pattern and Subcellular Localizations of SgGES Gene

To uncover the physiological functions of SgGES, we screened its putative expression patterns in 72 tissues. The high similarity between SgGES and the AT1G61120 gene from A. thaliana facilitated this process. The presence of SgGES in the tested tissues aligns with the findings of Ali et al. [2,4,9,29], who reported that most TPS genes (e.g., SoFLDH, SgTPSV, SoTPS3, SgGERIS, SoLINS2, GmTPS21, SoTPS4, SgFARD, SoNEOD, and SoHUMS) from S. officinalis, Glycine max, and S. guaranitica exhibited high expression levels in various tissues and developmental stages, particularly in flowers, roots, leaves, stems, and seeds. Moreover, the putative subcellular localization for the SgGES protein revealed that our target gene is predominantly present in the cytosol, followed by the plastids, mitochondria, and nuclei. These results align with the findings of Wang et al., Ali et al., Chen et al., and Makhadmeh et al. [2,4,9,31,32,52,53], who reported that numerous TPS genes exhibited clear localization in the plastids, mitochondria, and nuclei (Figure 4E).

4.3. Analysis of SgGES Gene Expression via qRT-PCR

Our study involved the use of qRT-PCR to assess the expression levels of the SgGES gene in different tissues. The qPCR data showed that it is most abundant in flowers, followed by flowering buds, young leaves, roots, old leaves, and stems. These findings are congruent with previous studies, which have reported high expression of TPS synthesis genes in flowers, young leaves, bud flowers, roots, old leaves, and stems [2,4,54,55]. The expression of the SgGES gene in stems is believed to be influenced by several factors. These factors may include gene-regulatory mechanisms that control post-transcriptionally and/or post-translationally (Figure 5). Additionally, the expression levels of SgGES might be related to tissue developmental stages, which greatly influence the expression of terpene synthase in Salvia [2,4,54,55] (Figure 5).

4.4. N. tabacum Plant Phenotypes and Terpene Profile Were Altered by Overexpression of the SgGES Gene

The role of SgGES in N. tabacum plants was investigated by introducing an overexpression vector pB2GW7-SgGES into Nicotiana plants using Agrobacterium. We validated the expression of our target gene in the positive transgenic N. tabacum plants via sqRT-PCR (Figure 6B). The transgenic lines exhibited a higher expression level of the SgGES gene expression when compared to the wild type, thus confirming the presence of the target gene. In the following step, some transgenic plants were chosen (named OE-SgGES -7, OE-SgGES-9, and OE-SgGES-10) for terpene analysis. The results of the morphological analysis indicated that the transgenic plants displayed an accelerated rate of leaf growth in comparison to the wild-type plants (Figure 6A). These results are consistent with the research conducted by Ali et al. [2,4,35], which demonstrated that in A. thaliana and N. tabacum, overexpression of genes involved in TPS synthesis and terpenoid biosynthesis (SgGPS, SgLINS, SgFPPS, SoTPS3, SoTPS6, SoCINS, SoLINS, SoFLDH, SoSABS, SoTPA4, SgGPS, and SoNEOD) from S. guaranitica and S. officinalis resulted in enhanced growth and augmented flower development compared to wild-type plants. The reason behind the superiority of transgenic plants with our terpene gene at the levels of growth and development compared with control plants is because of the roles of various terpenes and terpenoids in various metabolic pathways, such as the biosynthesis of terpenoids and steroids; KEGG:map01062, plant secondary metabolites; KEGG:map01060, ubiquinone and other terpenoid–quinone biosynthesis; KEGG:map00130 and biosynthesis of plant hormones; and KEGG:map01070 [56]. Several genes belonging to the TPS family were found to be important in various cell-specific processes, including the synthesis of thalianol as a mono-, sesqui-, di-, and triterpenes; 1,8-cineole; Z-γ-bisabolene; Rhizathalene; and β-amyrin [57,58,59,60,61,62,63,64]. This means that these genes can co-express initially in diverse cells, tissues, and organs to produce a different plant phenotype, hence confirming the role of TPS genes in plant growth, flowering, and development [57,58,59,60,61,62,63,64].

It was essential to analyze the metabolic profiles of tobacco plants after introducing the SgGES gene to identify the specific terpenes, and for this purpose, a GC-MS system was employed. The mono-, sesqui-, di-, and triterpene peaks were easily visible; the amount of each component was represented by the percentage of peak area (% peak area). To recognize those terpenes in our transgenic tobacco plants, the libraries of mass spectra from wild-type tobacco extracts were used as a reference. The data presented in Figure 7 and Table 2 unequivocally demonstrate a significant modification in the transgenic plants as a novel peak at the retention time of 50.461 was detected. This peak was identified as (E,E)-geranyllinalool based on the matched mass with the Wiley GC/MS Library, NIST Library, and VOC Analysis S/W software. On the other hand, the production of terpenes by the overexpression of terpene synthase genes in tobacco was described formerly by [2,41,51]. Diverse terpene synthase genes have been identified as capable of producing multiple compounds simultaneously, such as carene, (±)-linalool, cineole, myrcene, β-amyrin, and terpinolene synthases [11,12,65,66,67,68,69]. Based on our perspective, SgGES plays a pivotal part in the synthesis of (E,E)-geranyllinalool via the diterpenoid biosynthesis pathway, which is a prominent feature of diterpene biosynthesis processes.

5. Conclusions

S. guaranitica is a valuable Chinese medicinal herb with distinctive pharmacological effects. As a result, cloning and characterizing several genes involved in secondary metabolic pathways will help metabolic engineering of S. guaranitica and other therapeutic plants. In this investigation, we cloned the SgGES gene as a plant geranyllinalool biosynthetic from S. guaranitica. SgGES overexpression in N. tabacum enhanced leaf formation in the OE-SgGES-7 and OE-SgGES-9 transgenic lines. In comparison to control plants, these previous lines produced a high amount of (E,E)-geranyllinalool. The presence of (E,E)-geranyllinalool in these transgenic lines demonstrates that N. tabacum plants may synthesize the same product via the common mevalonate pathway (MVK) of diterpene biosynthesis, emphasizing the potential role of this gene in producing various types of terpenes, particularly the diterpene (E,E)-geranyllinalool. This study showed that the diterpene gene could be studied using the N. tabacum plant as a model plant, which can be used to improve the essential oil composition in S. guaranitica and other species via metabolic engineering.