Cloning and expression analysis of mevalonate kinase and phosphomevalonate kinase genes associated with the MVA pathway in Santalum album

Sandalwood (Santalum album L.) is highly valued for its fragrant heartwood and extracted oil. Santalols, which are the main components of that oil, are terpenoids, and these are biosynthesized via the mevalonic acid (MVA) pathway. Mevalonate kinase (MK) and phosphomevalonate kinase (PMK) are key enzymes in the MVA pathway. Little is known about the genes that encode MK and PMK in S. album or the mechanism that regulates their expression. To isolate and identify the functional genes involved in santalol biosynthesis in S. album, an MK gene designated as SaMK, and a PMK gene designated as SaPMK, were cloned from S. album. The sequences of these genes were analyzed. A bioinformatics analysis was conducted to assess the homology of SaMK and SaPMK with MK and PMK genes from other plants. The subcellular localization of SaMK and SaPMK proteins was also investigated, as was the functional complementation of SaMK and SaPMK in yeast. Our results show that the full-length cDNA sequences of SaMK and SaPMK were 1409 bp and 1679 bp long, respectively. SaMK contained a 1381 bp open reading frame (ORF) encoding a polypeptide of 460 amino acids and SaPMK contained a 1527 bp ORF encoding a polypeptide of 508 amino acids. SaMK and SaPMK showed high homology with MK and PMK genes of other plant species. Functional complementation of SaMK in a MK-deficient mutant yeast strain YMR208W and SaPMK in a PMK-deficient mutant yeast strain YMR220W confirmed that cloned SaMK and SaPMK cDNA encode a functional MK and PMK, respectively, mediating MVA biosynthesis in yeast. An analysis of tissue expression patterns revealed that SaMK and SaPMK were constitutively expressed in all the tested tissues. SaMK was highly expressed in young leaves but weakly expressed in sapwood. SaPMK was highly expressed in roots and mature leaves, but weakly expressed in young leaves. Induction experiments with several elicitors showed that SaMK and SaPMK expression was upregulated by methyl jasmonate. These results will help to further study the role of MK and PMK genes during santalol biosynthesis in S. album.

Bioinformatics analysis of the deduced SaMK and SaPMK proteins. The ExPASy online tool was used to calculate the physicochemical properties of the deduced SaMK and SaPMK proteins. The results are shown in Table 2. The predicted relative molecular weight (MW) of the SaMK protein is 41.3 kDa and the relative MW of the SaPMK protein is 54.6 kDa. The theoretical isoelectric points of SaMK and SaPMK are 5.23 and 5.92, respectively. The instability index of SaMK is 33 and that of SaPMK is 46, indicating that they are both stable proteins ( Table 2). The total average hydropathicity of SaMK is 0.113, indicating that it is a hydrophobic protein, while that of SaPMK is − 0.073, indicating that it is a hydrophilic protein (Table 2). Transmembrane domain and signal peptides were predicted by the TMHMM Server and SignalP, respectively. SaMK and SaPMK have no transmembrane domain or signal peptide (Fig. 1), indicating that they are non-secretory proteins.

Molecular evolution of the deduced SaMK and SaPMK proteins.
To investigate the evolutionary relationships among deduced SaMK and SaPMK proteins with other MKs and PMKs from angiosperms, gymnosperms, fungi, and bacteria, phylogenetic trees were constructed using the NJ method with MEGA 7. As shown in Fig. 4a, MKs from different species appeared to evolve into four groups, with bacteria as an ancient group. SaMK belonged to the angiosperms group and was clustered into one group with Siraitia grosvenorii and H. brasiliensis. As shown in Fig. 4b, PMKs from different species also evolved into four groups with bacteria as the ancient group. SaPMK was in the same group with dicotyledons and was clustered into one group with H. brasiliensis and Tripterygium wilfordii. These results suggest that SaMK and SaPMK shared a common evolutionary origin with MK and PMK proteins of other plants.

Subcellular localization of SaMK and SaPMK proteins.
To further verify the subcellular localization of SaMK and SaPMK, subcellular localization of SaMK-YFP and SaPMK-YFP (yellow fluorescent protein) were  www.nature.com/scientificreports/ studied using a modified polyethylene glycol method to transform SaMK-YFP and SaPMK-YFP constructs into A. thaliana protoplasts. It was found that both SaMK and SaPMK were located in the cytosol (Fig. 5).

Tissue-specific expression of SaMK and SaPMK.
To determine the tissue-specific expression patterns of SaMK and SaPMK genes in S. album, total RNA was extracted from roots, heartwood, sapwood, young leaves, mature leaves and shoots, and qRT-PCR was performed. The results of qRT-PCR are shown in Fig. 7. SaMK and SaPMK were constitutively expressed in all tissues of S. album. As shown in Fig. 7a, the lowest level of SaMK transcript was observed in sapwood, and the highest expression level in young leaves followed by mature leaves and shoots, approximately 7.77-, 6.59-and 2.72-fold higher than in sapwood. The expression level of SaPMK ( Fig. 7b) was lowest in young leaves but was highest in roots followed by mature leaves and sapwood, approximately 5.84-, 5.38-and 3.93-fold higher than in young leaves.
Expression of SaMK and SaPMK in response to MeJA. MeJA is a plant-specific signaling molecule that is involved in the regulation of various biological processes 53 . In the present study, we measured the expression level of SaMK and SaPMK in S. album roots, shoots and leaves after treatment with 100 μM MeJA (Fig. 8).
The expression of both genes was significantly induced by MeJA. The change in transcript level of SaMK and SaPMK in S. album roots, shoots and leaves after MeJA treatment was consistent, all increasing gradually and peaking at 12 h and then gradually decreasing compared with control seedlings. However, the level of increase in different tissues differed.

Discussion
Terpenoids, including monoterpenes, sesquiterpenes and diterpenes, play an important role in plant physiology and ecology 54 . In recent years, many studies have documented the molecular regulation of sesquiterpene biosynthesis 55 . Santalol, a sesquiterpenoid, is the most dominant aromatic and active ingredient in sandalwood essential oil 7 . Santalol is mainly synthesized via the MVA pathway. The functions of MK and PMK proteins in the MVA pathway have been studied in many plant species 36,44 . MK expression level is related to the precursors of terpenoid biosynthesis, IPP and DMAPP, which can indirectly regulate the biosynthesis of terpenoids, and    Sandalwood is considered to be one of the most valuable trees in the world 67 . Its value lies mainly in its heartwood and the essential oil extracted from heartwood 3 . Santalol is responsible for the pleasant fragrance www.nature.com/scientificreports/ of sandalwood 13 and most of the oil's pharmacological activity 68 . Thus, it is important to investigate whether or not SaMK and SaPMK transcripts may be related to the accumulation of santalol in different S. album tissues.
qRT-PCR showed that SaMK and SaPMK genes were constitutively expressed in all the tested tissues, but at different levels. The SaMK transcript level in young leaves was significantly higher than in other tissues, and its expression level was lowest in sapwood. A similar expression pattern was observed in Ginkgo biloba, in which GbMK genes were highly expressed in roots and leaves 46 . In H. brasiliensis, the HbMK gene was highly expressed in latex, followed by xylem and mature leaves 48 . The level of the SaPMK transcript in roots was significantly higher than in other tissues, and its expression level was lowest in young leaves. In H. brasiliensis, the HbPMK gene was highly expressed in xylem, followed by latex 48 . In Panax ginseng, the PgPMK gene was highly expressed in fine roots, followed by lateral roots 68 . These studies revealed that MK and PMK genes may have distinct spatial and temporal expression patterns in different plant species. MeJA is an important plant growth regulator involved in diverse developmental processes, such as seed germination, root growth, fertility, fruit ripening, and senescence 69 . Previous studies showed that there is a relationship between MeJA and terpene metabolism 70,71 . MeJA promoted the production of monoterpenoids and sesquiterpenoids in Ocimum basilicum 72 , Sarcophyton glaucum 69 , Salvia miltiorrhiza and G. biloba 73,74 . In S. album, MeJA induced the expression of SaTPS1 and SaTPS2 in leaves 24 . Thus, studying the expression profiles of SaMK and SaPMK following treatment with MeJA is important because it may provide insight into the regulation of these genes in santalol biosynthesis. In the present study, the expression levels of SaMK and SaPMK increased significantly in S. album roots, shoots and leaves after treatment with 100 μM MeJA, peaking at 12 h after treatment, then gradually decreasing, indicating that these inducible genes might be involved in signal molecule-related responses to environmental stimuli. The MK gene transcript was induced by 1 mM MeJA in G. biloba 46 and the PMK gene transcript was induced by 100 μM MeJA in P. ginseng 68 .
The characterization and expression profiles of SaMK and SaPMK genes may contribute to an understanding of the biosynthesis of sesquiterpenes in S. album at the molecular level and the regulatory mechanisms involved in the MVA pathway.

Materials and methods
Plant material. Five-year-old sandalwood trees growing in South China Botanical Garden (SCBG), Guangzhou, China, were used. Permission and guidance was obtained from SCBG and the local government for using this plant material for this study. Young (light green) and mature (dark green) leaves, heartwood, sapwood, roots and shoots were collected and wrapped in tin foil, frozen immediately in liquid nitrogen, and stored at − 80 °C for subsequent analyses. Two-month-old young seedlings (6-8 leaves) of S. album were sprayed with 100 μM MeJA (dissolved in 2% ethanol) until the leaf surfaces were wet, and 2% ethanol served as the control for each treatment. Samples (leaves, shoots and roots) were collected at 0, 2, 6, 12, 24, 48 and 72 h after treatment and stored at − 70 °C for further analyses. Each treatment was repeated three times.  (Table 3). The sequence information of 5′ and 3′ RACE PCR product clones were used to design primers from the start and stop codon to obtain the internal fragments. The amplified PCR products were purified by a gel DNA purification kit (Tiangen, Beijing, China) and ligated into the pMD18-T vector (Takara Bio Inc.). The recombined plasmids were transformed into Escherichia coli DH5α competent cells (Takara Bio Inc.) and sequenced at the Beijing Genomics Institution (BGI, Shenzhen, China).

Bioinformatics analysis and molecular evolution analysis of SaMK and SaPMK. SaMK and
SaPMK gene sequences were assembled and translated into amino acid sequences using DNAMAN software.

Subcellular localization of SaMK and SaPMK proteins.
A vector pSAT6-EYFP containing the enhanced yellow fluorescent protein (EYFP) ORF was used in this study. The cDNAs encoding SaMK and SaPMK were amplified with two pairs of primers, YFP-MK-F and YFP-MK-R, and YFP-PMK-F and YFP-PMK-R, respectively ( Table 1). The PCR product of MK was digested with EcoRI and SamI, the PCR product of PMK was digested with EcoRI and BamHI, and the pSAT6-EYFP vectors were digested with corresponding restriction endonucleases. The digested fragments were ligated into the linearized pSAT6-EYFP vector to generate pSAT6-  www.nature.com/scientificreports/ EYFP-SaMK and pSAT6-EYFP-SaPMK fusion constructs. The fusion expression vectors and the pSAT6-EYFP vector were transformed into A. thaliana mesophyll protoplasts through PEG-mediated transformation following a previously described method 77 . A confocal laser-scanning microscope (Leica TCS SP8 STED 3X, Wetzlar, Germany) was used to observe YFP fluorescence in transformed protoplasts after overnight incubation in a constant temperature incubator (SPH-2102C, Shanghai, China) at 22 °C. Fluorescence was excited for YFP at 514 nm, for Chl at 543 nm and for m-Cherry at 587 nm.

Tissue-specific analysis and expression profiles of SaMK and SaPMK induced by MeJA.
To investigate the expression levels of SaMK and SaPMK genes in different tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) and their expression profiles after MeJA treatment, qRT-PCR was carried out according to the manufacturer's instructions. About 1.0 μg of total RNA was reverse transcribed into first-strand cDNA using the PrimeScript RT reagent kit (Takara Bio Inc.) according to the manufacturer's protocols. The reactions were performed by ABI7500 fluorescence quantitative PCR (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) using iTaq Universal SYBR Green supermix as the buffer (Applied Biosystems). The housekeeping gene, β-actin, was selected as the internal control 75 for the normalization of all reactions. All experiments were performed in triplicate and mean values were analyzed. Significant differences (p < 0.05) between means were tested with Duncan's multiple range test. The 2 −ΔΔCT method was used to analyze the relative expression level of genes 80 .

Data availability
All data generated or analyzed during this study are included in this published article.