Overexpression of Panax ginseng sesquiterpene synthase gene confers tolerance against Pseudomonas syringae pv. tomato in Arabidopsis thaliana

Abstract

Sesquiterpenes are an abundant group belonging to the terpenoid family, with a C15 structure comprise of three isoprene units. Many sesquiterpenes are volatile compounds and it act as chemical messenger in plant signalling, particularly in the defense mechanism against biotic and abiotic stresses. Panax ginseng Meyer is important medicinal herbs with various reported pharmacological efficacies in which its triterpenoid saponins, called ginsenosides, were mostly studied. However, there have been few studies on volatile sesquiterpenes compounds regulation on P. ginseng. As slow-growing perennial plant, P. ginseng received many kind of stresses during its cultivation. The pathogen attack is one of the most devastated perturbation for ginseng yield. Thus, we aimed to analyze P. ginseng STS gene (PgSTS) expressions in ginseng organs as well as mono-, sesquiterpenes contents from ginseng seedlings treated with elicitors. qRT-PCR and GC-MS analysis showed that two elicitors- salicylic acid (SA) and methyl jasmonate (MeJA) triggered PgSTS expression at different time points and significantly induced mono-, sesquiterpene yield. Overexpression of PgSTS in Arabidopsis also induced high terpene content and conferred tolerance against Pseudomonas syringae pv. tomato infection. These results suggested that PgSTS transcripts are involved in terpenoid biosynthesis in response to environmental stress mediated by MeJA and SA elicitors; thus, generate tolerance against pathogen attack.

Keywords: Gene expression; Ginseng; Methyl jasmonate; Salicylic acid; Sesquiterpene synthase; Terpene content.

Fig. 1

Terpene contents of 4-week-old control and SA or MeJA treated ginseng seedlings. The data represent the mean ± SE of three independent replicates

Fig. 2

Phylogenetic tree of PgSTS proteins and close homologs. The phylogenetic tree was constructed using the ClustalX program (neighbor-joining method). The bar represents 0.1 substitutions per amino acid position

Fig. 3

Alignment of PgSTSs and close homologs. Boxed domains represent conserved motifs RRx₈W and DDxxD, which are known STS functional domains. Residues conserved in all sequences are marked with asterisks. Colons and dots indicate the positions of amino acid residues with strong and weak similarity, respectively. Residues that are identical in at least half of the sequences are printed with a black background, residues that are similar in at least half of the sequences are printed with a gray background, and residues with <50 % similarity are printed with a white background. EtACS (ADK94034), CaSTC (ABK63808), PgSTS (AGS16741), TgSTS2 (AFV09099), and TgSTS1 (AFV09098)

Fig. 4

3-D structural view of STS. a Stereo view of a cartoon diagram with alpha and beta helices. b PgSTS was superimposed with the X-RAY structure of (+)-δ-cadinene synthase from Gossypium arboreum (GaDCS) (Gennadios et al. 2009). Structurally conserved regions (SCRs) and structurally variable regions (SVRs) are shown in green and blue, respectively (color figure online)

Fig. 5

Organ-specific expression of PgSTS. Four-year-old field samples were collected and used to quantify the expression pattern of STS. Relative gene expression was determined by qRT-PCR. The data represent the mean ± SE of three independent replicates

Fig. 6

Expression levels of PgSTS in SA- (a) and MeJA- (b) treated seedlings of P. ginseng. A quantitative expression profile of STS was determined at various time intervals. The Ct value of the actin gene was obtained and used as the relative calibrator according to the formula 2^–ΔΔCt. The data represent the mean ± SE of three independent replicates. Average values of treated samples are significantly different compared to the control at *P < 0.001 according to Student’s t test

Fig. 7

Plant height (a) and relative mRNA expression levels (b) of PgSTSox lines. The data represent the mean ± SE of three independent replicates

Fig. 8

Sesquiterpene content of PgSTS-overexpressing transgenic Arabidopsis plants. The data represent the mean ± SE of three independent replicates

Fig. 9

In vitro assay of PgSTSox lines against bacterial pathogens. Healthy leaves of wild-type and transgenic plants were infected with Pseudomonas syringe a photographs were taken after 2 and 4 days of treatment, b bacterial populations per leaf disc (1 cm²) were counted after 2 and 4 days of syringe infiltration containing P. syringe, and the corresponding leaves were compared with the empty vector control (Pro35S: YFP) and two independent transgenic lines. The data represent the mean ± SE of three independent replicates. Averages of treated samples were significantly different compared to the control, *P < 0.05