Characterization of a novel levopimaradiene synthase gene responsible for the biosynthesis of terpene trilactones in Ginkgo biloba

ABSTRACT

Terpene trilactones (TTLs) are the main medicinal compounds of Ginkgo biloba. Levopimaradiene synthase (LPS) is the crucial enzyme that catalyzes TTLs biosynthesis in G. biloba. In this study, a novel LPS gene (designated as GbLPS2) was cloned from G. biloba leaves. The open reading frame of GbLPS2 gene was 2520 bp in length, encoding a predicted polypeptide of 840 amino acids. Phylogenetic analysis revealed that the GbLPS2 was highly homologous with reported LPS proteins in other plants. On the basis of the genomic DNA (gDNA) template, a 4308 bp gDNA sequence of GbLPS2 and a 913 bp promoter sequence were amplified. Cis-acting elements in promoter analysis indicated that GbLPS2 could be regulated by methyl jasmonate (MeJA) and abscisic acid (ABA). Tissue-specific expression analysis revealed that GbLPS2 was mainly expressed in roots and ovulate strobilus. MeJA treatment could significantly induce the expression level of GbLPS2 and increase the content of TTLs. This study illustrates the structure and the tissue-specific expression pattern of GbLPS2 and demonstrates that exogenous hormones regulated the expression of GbLPS2 and TTL content in G. biloba. Our results provide a target gene for the enhancement of TTL content in G. biloba via genetic engineering.

Introduction

Ginkgo biloba L. is an ancient plant with great economic and medicinal value.^1–3 The leaf extracts of G. biloba contain a variety of medicinal compounds that have been widely used in the clinical treatment of cardiovascular and cerebrovascular diseases all over the world.⁴ Terpene trilactones (TTLs), including ginkgolide (diterpenoids) and bilobalide (sesquiterpenoids), were considered as the main effective components in leaf extracts of G. biloba.^5–8 Pharmacological studies have revealed that TTLs could improve the symptoms of cardiocerebrovascular diseases for their specific platelet-activating factor (PAF) antagonist,⁹ and they are widely used to treat age-related chronic neurological disorders, such as memory reduction and dementia.¹⁰ The standardized G. biloba extract (EGb761) stipulated that the TTL content should not be less than 6%.^11,12 However, due to the low TTLs content in G. biloba, the biological extraction processes are inefficient and expensive. Moreover, due to the unique chemical structure of TTLs, which make it difficult to synthesis via chemical method. Therefore, increasing the TTL content through genetic engineering is a viable option to make the biological extraction process economically sustainable.

Plants synthesize highly diverse types of terpenoids and an estimated 25,000 terpenoids have already been identified.¹³ Terpenoids have various functions in plants such as regulating growth and development and improving biotic and abiotic stress tolerance of plants.^14–16 In higher plants, terpenoids, including TTLs, can be synthesized via the mevalonate (MVA) pathway and the 2-C-methyl-D-erythriver-4-phosphate (MEP) pathway. The MVA pathway utilizes acetyl coenzyme A as a precursor and catalyzes the synthesis of IPP and DMAPP successively through multiple-enzyme pathways.¹⁷ IPP and DMAPP are condensed by geranylgeranyl diphosphate synthase (GGPPS) to synthesize 20-carbon geranylgeranyl diphosphate (GGPP), which serves as a linear skeleton for TTLs biosynthesis. GGPP is cyclized by the enzyme levopimaradiene synthase (LPS) to form levopimaradiene (LP) and subsequently translocated from plastids to cytoplasm.¹⁸ The cyclization of GGPP by terpenoid synthase followed by the oxidation process by cytochrome P450 (CYP450) results in the synthesis of ginkgolide^19-21 (Figure 1).

Figure 1.

Terpene trilactones biosynthesis pathway in Ginkgo biloba.

Studies have shown that the TTLs in plants were mainly synthesized in actively growing tissues. For instance, ginkgolides and bilobalides were first synthesized in roots before being transported to the other organs and tissues through the phloem of G. biloba.^22,23 Many studies have revealed that overexpression of the key genes involves in TTL biosynthesis pathways could increase the TTL content in plants.^24,25 In addition, our previous studies revealed that the transcript of genes that participated in TTL biosynthesis is positively responsive to hormones,^26,27 including methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), and ethylene (Eth).

In recent years, the presence of genes encoding homologous of the terpene synthase has been noted in G. biloba. Such as the reported of three 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) genes and two 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) genes in G. biloba.^27–30 In this study, we reported a novel levopimaradiene synthase gene GbLPS2, which encoded a putative protein consisting of 840 amino acids. Multiple sequence alignment showed that GbLPS2 contains the conserved motifs of LPS protein. Phylogenetic analysis indicated GbLPS2 is paralogous of the reported LPS proteins in other plants. Tissue-specific analysis indicated that GbLPS2 was mainly expressed in roots. Hormone treatment results revealed that GbLPS2 was induced by MeJA. Besides, the TTL content was increased after treatment with MeJA and ABA. These findings could help further insights into the molecular mechanisms of TTLs biosynthesis. The GbLPS2 gene could serve as an important genetic engineering target gene for increasing TTL content in G. biloba.

Materials and methods

Plant materials and hormone treatments

A 32-year-old cultivar “Jiafoshou” of G. biloba was grown in the Ginkgo Botanical Garden of Yangtze University in China. For tissue-specific pattern analysis, the ovulate strobilus and staminate strobilus were collected in March 2018, and the leaves, roots, stems, and fruits were collected in mid-May 2018. The 2-year-old seedlings of G. biloba were grown in a greenhouse at 25°C under 14 h of photoperiod, and the leaves were sprayed with salicylic acid (SA), Eth, MeJA, and ABA that dissolved in 0.01% (v/v) Tween-20 with a final concentration of 10 mM, 10 mM, 100 μM, and 100 μM, respectively. Leaves treated with deionized water at 0 h were used as the control, and leaf samples were subsequently harvested for RNA extraction and TTL content determination at 0, 1, 2, 3, 4, and 5 d. Each treatment was conducted in triplicate. All the samples were quickly frozen in liquid nitrogen and stored in a refrigerator at −80°C.

DNA and RNA extraction

Genomic DNA was extracted from the leaves of G. biloba by using a modified CTAB method in accordance with Clarke and Moran.³¹ Total RNA was extracted from various tissues of G. biloba by using a TaKaRa MiniBEST Plant RNA Extraction Kit (Takara, China) following the manufacturer’s instructions. The concentration and quality of DNA and RNA were detected using a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and agarose gel electrophoresis.

Isolation of GbLPS2 cDNA and gDNA sequence

The specific cDNA was synthesized following the instruction in the PrimeScript first-strand cDNA synthesis kit(Takara, China). Primers GbLPS2-F and GbLPS2-R were designed based on the sequence of the G. biloba transcriptome database, while gDNA clone primers GbLPS2-TF and GbLPS2-TR were designed in accordance with the results of GbLPS2 cDNA sequencing. The amplified products were purified using a TaKaRa MiniBEST Agarose Gel DNA Extraction Kit (Takara, China), subcloned into the pMD19-T vector (Takara, China), and then sequenced (Sangon, China). All the primers used in this study are listed in Table S1.

Cloning of GbLPS2 promoter

Promoter-specific primers GbLPS2-pU1 and GbLPS2-pU2 were designed in accordance with the ORF sequence of GbLPS2. Adaptor primers AP1 and AP2 were synthesized by Sangon Biotech (Shanghai, China). All the primers used in this study are listed in Table S1. The GbLPS2 promoter was amplified using the Genome Walker Universal Kit following the manufacturer’s instructions. The DNA library was constructed using Stu I, Pvu II, Dra I, EcoR V, and Ssp I to digest the DNA at 37°C for 3, 6, 1, 2, and 1 h, respectively. The first step of nested PCR was carried out with GbLPS2-pU1 and AP1 following the instructions for Advantage 2 Polymerase Mix (Clontch, USA). The first PCR product was diluted 50 times with deionized water and used as the template of the second PCR, which was performed using GbLPS2-pU2 and AP2. The second PCR product was subcloned into the pMD19-T vector and sequenced.

Bioinformatics and Phylogenetic analyses

The sequencing data were assembled and analyzed using Vector NTI 11.5.1. The deduced GbLPS2 protein sequence was blasted using NCBI and Expasy online blast engine (https://blast.ncbi.nlm.nih.gov/Blast.cgi and https://www.expasy.org/resources/uniprot-blast). Multiple sequence alignments were performed on DNAMAN 8.0. The conserved domains were predicted using InterPro Scan (http://www.ebi.ac.uk/interpro/). Multiple sequence alignments were conducted by MEGA 7.0. The phylogenetic tree of the LPS protein in plants was constructed via the neighbor-joining method by using Clustalx 2.0 and MEGA 7.0. The promoter sequence was analyzed using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict the cis-acting elements.

Quantification of GbLPS2 in different tissues or hormone treatment

The total RNA of various tissues and the leaves treated with hormones were extracted using the MiniBEST Plant RNA Extraction Kit (Takara, China). Then, 600 ng of total RNA was reverse transcribed using PrimeScript RT reagent Kit (Takara, China) following the instructions. The forward and reverse primers LPS-qrtF and LPS-qrtR were designed with Primer Premier 5.0 in accordance with the cDNA sequence of GbLPS2. qRT-PCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) and run on a 9600 Real-Time PCR machine (Bioer, China) following the manual’s recommendations. Glyceraldehyde-3-phosphate dehydrogenase housekeeping gene (GAPDH) (forward primer GAPDH-F and reverse primer GAPDH-R) was selected as the reference gene for the normalization of all reactions³² (Table. S1). GbLPS2 and GAPDH were analyzed simultaneously for each sample. For post-treatment analysis, the transcription levels of GbLPS2 with different hormone treatments were compared with the controls. For tissue-specific analysis, the GbLPS2 transcription level of other tissues was compared with the fold change relative to fruits. The relative expression level of GbLPS2 was calculated using the 2^−ΔΔCt method.³³ Each sample was biologically and technically repeated three times.

Determination of TTL content

The TTL content in G. biloba was evaluated using a high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) in accordance with Kaur’s method.³⁴ It was calculated as the sum of ginkgolide and bilobalide contents and expressed by fresh weight percentages. The standards were purchased from Yuanye Bio-Technology Co., Ltd (Shanghai, China). 3 g of samples were ground with liquid nitrogen, the powders were well dispersed with 20 mL ethyl acetate, and the mixture was sonicated using the Scientz-650E ultrasonic crusher (Scientz, China) for 30 min. The supernatant was redispersed to 5 mL by methanol and filtered through a 0.45 μm membrane. The samples were injected and analyzed using HPLC-ELSD (UltiMate 3000, Thermo, USA) with Agilent C-18 column (5 μm, 4.6 nm × 250 mm). The mobile phase was methanol:tetrahydrofuran:water = 20:8:72 with a 1 mL/min flow rate. All measurements were performed in triplicate, and all data were represented as means ± SE (n = 3).

Isolation of cDNA and gDNA from GbLPS2

The GbLPS2 cDNA (GenBank Accession No. KX904943.1) was isolated via RT-PCR by using gene-specific primers based on the sequence deposited in the G. biloba transcriptome database. Sequence analysis revealed that GbLPS2 contained a complete ORF of 2520 bp encoding a protein of 840 amino acid residues. The deduced GbLPS2 protein shared 68.4% identity with the reported GbLPS protein (GenBank Accession No. AAL09965.1). On the basis of the sequencing result, the specific primers were designed, and a 4308 bp fragment of GbLPS2 gDNA sequence was isolated (Fig. S1). Alignment analysis of cDNA and gDNA sequences revealed that the GbLPS2 genome consisted of 14 introns and 15 exons (Figure 2).

Figure 2.

Genomic structure of GbLPS2 gene

Promoter sequence analysis of GbLPS2

A 913 bp promoter fragment of GbLPS2 was isolated via nested PCR (Fig. S1). The Promoter 2.0 Prediction Server predicted that the transcriptional start site (TSS) was located in the −83 bp upstream of the codon ATG site. The cis-acting elements of the GbLPS2 promoter are displayed in Table 1. The TATA-box was located at the position of −33 bp. The cis-acting elements in the GbLPS2 promoter including LTR, CAAT-box, SP1 CATT-motif, CGTCA motif, G-box, MBS, CCAAT-box, Opaque-2(O2) site, and ABRE (Table 1). These findings indicated that GbLPS2 could be regulated by exogenous and endogenous signals, such as light, low temperature, MeJA, ABA, MYC, and MYB transcription factor.

Table 1.

List of putative cis-acting elements in the GbLPS2 promoter in G. biloba.

Element	Signal Sequence	Position	Expected function
TATA-box	TATATATA	−33	core promoter elements around −30 of transcription start site
LTR	CCGAAA	−59	low-temperature-responsive element
CAAT-box	CAAAT	−175	common cis-acting element in promoter and enhancer regions
Spl	CCACCCAACG	−239	light responsive element
CGTCA-motif	TGACGTCA	−451	methyl jasmonate (MeJA)-responsiveness element
G-box	GACATGTGTT	−489	MYC2 binding cis-elements
MBS	CAACTG	−638	MYB binding site involved in drought-inducibility
CCAAT-box	CAACGG	−711	MYBHv1 binding site
CATT-motif	ATTCCC	−767	light-responsive element
O2-site	GATGGCATGG	−826	zein metabolism regulation element
ABRE	AACCCGG	−906	cis-acting element involved in the abscisic acid responsiveness

Bioinformatic analysis of the deduced GbLPS2 protein

GbLPS2 contained 840 amino acids, the molecular weight and isoelectric point were 95 kDa and 5.31, respectively. Structural analysis showed that the first 53 amino acid residues in the N-terminal of GbLPS2 had lower similarity with LPS in other plants, whereas the residue similarity after the 53^rd reached approximately 78% (Figure 3). GbLPS2 was composed of two domains contains the N-terminal (residues 264–476) and C-terminal (residues 519–839), and three aspartic acid-rich motifs (Figure 3). A DDxIx motif (residues 60–64) was reported to stabilize carbocations and direct deprotonation. The N-terminal domain contained a highly conserved D (I/V) xxTA aspartic acid-rich motif (residues 369–374), which initiated the cyclization of GGPP. The third aspartic acid-rich motif DDxxD (residues 593–597) coordinated Mg²⁺ to bind with the substrate and contained a feature of class I diterpene synthesis. Therefore, the deduced GbLPS2 protein shared high sequence similarity with LPS.

Figure 3.

Multi-sequence alignments of deduced GbLPS2 with LPS of other plants. The aspartic acid-rich motifs DDxIx, D (I/V) xxTA and DDxxD are underlined. Additional sequences include Picea sitchensis (PsLPS, ADZ45517.1), Pinus contorta (PcLPS, M4HYC6.1), Abies grandis (AgAS, Q38710.1), Pseudolarix amabilis (PaLPS, AGN70885.1), Ginkgo biloba (GbLPS, Q947C4.1; GbLPS2, KX904943.1)

Phylogenetic analysis of GbLPS2

A phylogenetic tree was constructed using ClustalX 2.0 and MEGA 7.0 to investigate the phylogenetic relationships between GbLPS2 and other plant LPS proteins. Phylogenetic analysis revealed that LPS was divided into four branches (bryophyta, pteridophyte, gymnosperms, and angiosperms; Figure 4). The GbLPS2 branch was clustered with Abies grandis, G. biloba, and Taiwania cryptomerioides in gymnosperm class. The above results indicated that GbLPS2 is a potential LPS involved in TTL biosynthesis.

Figure 4.

The phylogenetic analysis of GbLPS2 with the other terpene synthases. The GenBank Accession numbers of LPS proteins are as follows: Ginkgo biloba (GbLPS, Q947C4.1; GbLPS2, KX904943.1), Abies grandis (AgAS, Q38710.1), Taiwania cryptomerioides (TcDTS, AOG18234.1), Amborella trichopoda (AtLPS, XP_020527156.1), Phoenix dactylifera (PdLPS, XP_008795586.1), Dryopteris fragrans (DfKS, AVB77349.1), Lygodium japonicum (LjKS, BAQ20600.1), Marchantia polymorpha (MpDTS, APP91795.1), Zostera marina (ZmDPS, KMZ61416.1), Castanea mollissima (CmDPS, AEF32082.1)

Expression profile of GbLPS2 and TTLs content in various tissues

Tissue-specific expression analysis indicated that the expression of GbLPS2 was varied among different tissues. The expression of GbLPS2 in ovulate strobilus and roots were approximately 20 and 2 times higher than that in the fruits, respectively. Meanwhile, the expression of GbLPS2 in stems, leaves, and staminate strobilus was similar to that in the level of fruits (Figure 5a). As showed in Figure 5b, TTLs were detected in all studied tissues. The highest content was observed in roots at 457.8 μg g⁻¹, followed by stems, leaves, and fruits at 89.1 μg g⁻¹, 72.9 μg g⁻¹, and 44.3 μg g⁻¹, respectively. The lowest content was found in ovulate strobilus at 24.7 μg g⁻¹ (Table. S2).

Figure 5.

The expression of GbLPS2 (a) and TTLs content (b) in various tissues of G. biloba. The gene expression level of GbLPS2 in fruits was set to 1, and those of GbLPS2 in other tissues were accordingly accounted and presented as the relative fold changes. R: roots, S: stems, L: leaves, SS: staminate strobilus, OS: ovulate strobilus, F: fruits. Data are shown as the mean ± SD of triplicate assays. Means with different letters are significantly different at ρ < 0.05 by one-way ANOVA, with Tukey’s honestly significant difference test

Analysis of GbLPS2 transcript level and TTLs content under different hormone treatments

Two-year-old seedlings were treated with MeJA, SA, ABA, and Eth to investigate the effect of hormones on the expression of GbLPS2 and the biosynthesis of TTLs in G. biloba. The results showed that the expression of GbLPS2 in the CK treatment sharply decreased at day 1, and returned to the initial level at day 2, followed by a slight decrease from 2 to 5d (Figure 6a). Under MeJA treatment, the GbLPS2 expression level increased significantly after 1d of treatment, followed by a slight decrease and subsequently increased after 2 to 5d treatment (Figure 6b). In the SA treatment group, there are no obvious changes in GbLPS2 expression level (Figure 6c). In response to ABA treatment, GbLPS2 showed a continuous downregulation of its transcripts from 0 to 2d, and it increased by 13.3-fold relative to the control group at day 5 (Figure 6d). The expression of GbLPS2 rapidly decreased in response to Eth treatment, reached a minimum level by 94.1% relative to the control group within 1 day, and then increased from day 1 to day 3 (Figure 6e).

Figure 6.

GbLPS2 relative expression level and TTL content of different hormones treatment. (a): CK, (b): MeJA, (c): SA, (d): ABA, (e): Eth. The gene expression level of GbLPS2 at day 0 of CK were set to 1, and those of GbLPS2 under hormones treatment were accordingly accounted and presented as the relative fold changes. Data is shown as the mean ± SD of triplicate assays. Means with different letters are significantly different at ρ < 0.05 by one-way ANOVA, with Tukey’s honestly significant difference test

The TTL content increased from day 1 to day 5 in the control group (Figure 6a) and gradually increased from day 0 to day 2 in the MeJA treatment group (Figure 6b). Under SA treatment, a constant decrease of TTL content was observed from day 0 to day 3, subsequently reached a peak at day 4 by 1.17-fold relative to day 0 (Figure 6c). In the ABA treatment group, the TTL content continuously increased from day 1 to day 4 and reached a peak at day 4 by 1.66-fold relative to day 0 (Figure 6d). Under Eth treatment, there were no significant changes in the content of TTL from day 0 to day 4, except for a decrease at day 2 (Figure 6e).

Isolation and identification of a novel LPS from G. biloba

LPS catalyzes GGPP for cyclization to produce levopimaradiene.¹⁸ This is the key step in the TTLs biosynthesis pathway of G. biloba. After a series of complex reactions, levopimaradiene eventually produces ginkgolides, but how these ginkgolides are biosynthesized is still unclear. In this study, a novel LPS from G. biloba named GbLPS2 was cloned and characterized. Multiple alignments indicated that GbLPS2 and LPS proteins are different before the 53^rd residue, and the amino acid sequences are conserved to a large extent (about 78%) after 53^rd residue, thus we speculate that the function of GbLPS2 protein is similar to LPS protein in other plants. The N-terminal of GbLPS2 contains the conserved domain of terpenoid synthase while the C-terminal contains the conserved domain of terpenoid cyclase.³⁵ Besides, we found that the three aspartate motifs of the GbLPS2 protein were critical for terpene trilactones synthesis and this is consistent with previous reports.^18,35 It was reported that the aspartic acid-rich motif D (I/V) xxTA was able to initiate the cyclization of GGPP, and the aspartic acid-rich motif DDxxD was believed to combine complex divalent metal ions for metal-dependent ionization of pyrophosphate.³⁶ The catalysis of terpene synthase requires the binding of Mg²⁺ combination,^37,38 and these motifs founding indicate LPS may require Mg²⁺ binding to activate catalysis.

Sequence analysis of GbLPS2 promoter fragment

Analysis of cis-acting elements revealed that GbLPS2 was regulated by light, low temperature, hormones, and other factors. The first cis-acting elements TATA-box combines with RNA polymerase II to promote gene transcription, thus affecting the transcriptional rate.³⁹ This cis-acting element is usually positioned at 32 ± 7 bp upstream of the transcription start site,⁴⁰ and its sequence format is TATAWAW (W represents A or T). Another conserved eukaryotic cis-element, CAAT-box, is the binding site of transcription factor CBF on the DNA sequence that controls the frequency of transcription initiation to affect the level of transcription of the target gene.⁴¹ Otherwise, we identified some cis-acting elements involved in light responsiveness, such as CATT-motif, G-box, and Sp1.^42–44 Furthermore, G-box is also a classic MYC2-binding sequence,⁴⁵ CCAAT-box belongs to stress-related cis-elements that could bind to MYBHv1 transcription factor,⁴⁶ O2-site is reported a basic Leu zipper transcription factor responsible for the production of protein bodies in maize endosperm.^47–49 The MBS cis-acting element belongs to the MYB transcription factor binding site under drought stress.⁵⁰ These results indicate that GbLPS2 could be regulated by various transcription factors. CGTCA-motifs and TGACG-motifs were identified as cis-acting elements in response to MeJA.⁵¹ Besides, MeJA could regulate the response of plants to abiotic stress, and it is an elicitor of the secondary metabolic pathway.^27,52 These signal response elements suggested that the expression of GbLPS2 may be regulated by various abiotic signals, and the expression of GbLPS2 regulated by the promoter could be improved to a certain extent by exogenous hormone treatment.

The tissue-specific of GbLPS2 expression and TTLs content

Among different tissues of G. biloba, the highest TTL content was observed in roots, followed by stems, leaves, and the lowest was observed in ovulate strobilus. This result was consistent with the previous report that TTLs are synthesized in roots, further translocated to shoots, and eventually accumulated in leaves.^28,53 Tissue-specific expression analysis revealed that GbLPS2 was highly expressed in ovulate strobilus, followed by roots, staminate strobilus, fruits, stems, and leaves. A previous study has shown that GbLPS was expressed in roots and staminate strobilus.²⁴ Whereas, this study revealed that GbLPS2 is expressed throughout all studied tissues. Meanwhile, there was one unexpected result that the highest transcription level of GbLPS2 was observed in ovulate strobilus with a 10.66-fold change relative to roots. The possibility is that GbLPS2 in ovulate strobilus is involved in the biosynthesis of yet to be identified TTLs. Overall, these findings suggested that GbLPS2 participated in the biosynthesis of TTLs in G. biloba.

Multiple hormones influence the expression of GbLPS2 and TTLs content

Phytohormones, such as MeJA, SA, ABA, and Eth, have been proved to regulate the secondary metabolism of plants, including terpenoids.^52,54 It is reported that Eth/JA can induce plant defense response to pathogens.⁵⁵ In this study, the TTL content showed a tendency to gradually increase after 1–2d of MeJA treatment, which was consistent with the report of MeJA treatment increased the total amount of terpenoids in sweet basil.^56,57 Meanwhile, the expression of GbLPS2 showed an increasing tendency under MeJA treatment, which was consistent with the reported genes GbDXS2, GbCMK2, and GbHDR2 were positively regulated by MeJA treatment.^25,58

SA plays an important role in plant immunity and mediates the basic defense of biotrophic pathogens.⁵⁹ Transcriptome analysis of G. biloba indicated SA could induce the expression of a series of genes involved in TTLs biosynthesis.⁶⁰ In this study, the TTL content decreased from 0–3d after treated with SA. In parallel, the expression of GbLPS2 showed a similar tendency. The results were consistent with the previous characterization of GbHMGR2 and GbHMGR3²⁸.

In this study, we found that ABA treatment continuously promoted the TTL content in G. biloba, while the expression of GbLPS2 was continuously inhibited under ABA treatment. Similar results have been reported that exogenous ABA application significantly increased sesquiterpene nerolidol production, and significantly repressed terpene biosynthesis genes in Salvia hispanica.^61,62 Moreover, it was reported that exogenous ABA treatment triggers endogenous MeJA accumulation to regulate the accumulation of TTLs.⁶³ In this study, the ABRE element was present in the promoter region of GbLPS2. Therefore, ABA signaling may directly or through MeJA signaling pathway indirectly regulate the expression of GbLPS2 to regulate the accumulation of TTLs.

Eth signal transduction pathway involved in the regulation of defense in response to various pathgens.⁶⁴Numerous studies suggest that the phenylpropanoid pathway regulated by Eth signaling could lead to the synthesis of terpenoids, flavonoids, and stilbenes.^65–67 In this research, the relative expression of GbLPS2 under Eth treatment continuously at a low level from day 1 to day 5. The TTL content showed no significant change after Eth treatment except for day 2. Rao et al.²⁸ reported that G. biloba showed a continuously decreased tendency of TTL content under SA treatment. Besides, Rajasekaran et al.⁶⁸ discovered that terpenes could be part of an ethylene-independent abscission pathway in Balsam fir, this founding was similar to our results in this study. Besides, further studies are needed to investigate the effect of exogenous ethylene on TTL biosynthesis.

Conclusion

In this study, a novel levopimaradiene synthesis gene named GbLPS2 was cloned and characterized. GbLPS2 contains LPS conserved domain DDXIX motif, D (I/V) XXTA motif, and DDXXD motif, and has a close relationship with the reported GbLPS. The GbLPS2 promoter fragments contain O2-site, CATT-motif, CCAAT-box, MBS, G-box, CGTCA-motif, SP1, CAAT-box, LTR, ABRE, and TATA-box cis-acting elements. Tissue-specific pattern analysis revealed that GbLPS2 was mainly expressed in roots and ovulate strobilus. The TTLs mainly accumulated in the root of G. biloba. Exogenous hormones could alter the expression of GbLPS2 and influence the TTL content through a complex signaling network. These results are helpful for us to deeply understand the biosynthesis mechanism of TTLs, and provide the candidate gene for improving TTL content through genetic engineering of G. biloba.

Funding Statement

This work was supported by the National Natural Science Foundation of China (No. 31971693)

Availability of data and materials

The datasets used and analyzed in this study are available from the corresponding author upon reasonable request.

Author’s contribution

Feng Xu and Shuiyuan Cheng designed the whole experiment and drafted the manuscript. Qiling Song performed the exogenous hormone treatment experiment and determined the TTLs contents. Qiangwen Chen collated and analyzed the data, and wrote the manuscript. Huan Han and Xian Zhang contributed to cDNA cloning and qRT-PCR analysis. Xiaoyan Yang, Jiabao Ye, WeiWei Zhang, and Yongling Liao provide technical guidance for experiments. All authors have reviewed and approved the manuscript.

Declaration of interest statement

The authors declare that they have no competing interests.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website.