Ceramides play a significant role in the response of Pogostemon cablin to bacterial wilt by regulating the ABA pathway

Abstract

As a strategic resource for both medicine and essential oil, the healthy development of the Pogostemon cablin industry is crucial for the traditional medicine and fragrance sectors. Bacterial wilt represents one of the most significant threats to patchouli cultivation; however, the molecular mechanisms underlying P. cablin's response to bacterial wilt remain unexplored. Here, we conducted transcriptome and metabolome analyses, revealing an increase in the expression of genes associated with lipid pathways and a corresponding rise in the concentration of lipid metabolites in P. cablin following infection by the bacterial wilt pathogen SY1. Further lipidomics analysis demonstrated a significant upregulation of ceramide levels due to SY1 infection. Additionally, hormone analysis indicated that SY1 significantly induced an increase in abscisic acid (ABA) concentration, accompanied by the upregulation of genes involved in the ABA synthesis pathway and its downstream signaling pathways. Furthermore, we treated P. cablin seedlings with the ceramide synthase inhibitor FB1, which significantly reduced ceramide concentration in P. cablin. FB1 treatment also inhibited the expression of ABA-synthesizing genes, leading to a notable decrease in ABA concentration and downstream pathway genes. These data indicate that ceramides and ABA may participate in P. cablin's response to SY1.

Keywords: Pogostemon cablin; ABA; Bacterial wilt; Plant defense response.

Fig. 1

Transcriptomic analysis of P. cablin plants before and after inoculation with the patchouli bacterial wilt pathogen SY1. A Phenotype of patchouli inoculated with pathogen SY1 for 7 days. B Volcano plot analysis of the gene expression in the samples with or without SY1 inoculation. The x-axis indicates log2 (fold change), while the y-axis represents -log10 (adjusted P-value). C KEGG pathway clustering analysis for differentially expressed genes induced by SY1

Fig. 2

Metabolomic analysis of P. cablin plants before and after inoculation with the patchouli bacterial wilt pathogen SY1. A Volcano plot analysis of the metabolite abundance in the samples with or without SY1 inoculation. The x-axis indicates log2 (fold change), while the y-axis represents -log10 (adjusted P-value). B The pathway heatmap analysis of untargeted metabolomics in samples with and without SY1 inoculation. This heatmap visualizes the log2 fold changes of key metabolic pathways, where color intensity indicates the degree of upregulation (positive values) or downregulation (negative values)

Fig. 3

The expression of the lipid transfer protein gene in patchouli was found to be up-regulated following SY1 infection. A The expression levels of the lipid transfer protein genes (PatLTPs) are presented based on transcriptome data. B The expression level of these PatLTPs was analyzed using quantitative PCR. C The structural analysis of the protein encoded by these PatLTPs

Fig. 4

Ceramides accumulated following SY1 infectionin P. cablin. Sphingolipids were extracted from P. cablin before and after inoculation with SY1. The long chain base (LCB), ceramide (Cer), hydroxyceramide (hCer), and glucosylceramide (GlcCer) were separated and identified using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS). The experiment was conducted twice, yielding similar results from independent samples. Significant differences from the untreated sample are indicated by asterisks, based on Student’s t-test (*P<0.05; ** P<0.01; *** P<0.001). A The analysis of sphingolipid levels in P. cablin following SY1 inoculation. B A comparison of ceramide concentration in P. cablin following SY1 inoculation

Fig. 5

ABA pathway was activated following SY1 infection in P. cablin. A The level of ABA increased after SY1 infection in P. cablin. OPDA: cis-(+)-12-oxo-phytodienoic acid; JA-Ile: (+)-7-iso-jasmonoyl-l-isoleucine; SAG: salicylate 2-O-β-D-glucoside. B The expression levels of ABA pathway genes were up-regulated in response to SY1 infection in P. cablin. PatNCED1: 9-cis-epoxy carotenoid dioxygenase; PatPP2C:protein phosphatase 2C; PatABF:ABA responsive element binding factor. Significant differences were indicated by different letters, as determined by Student’s t-test (P < 0.05). These experiments were conducted at least twice, yielding consistent results

Fig. 6

The expression of the PatWRKY was found to be up-regulated following SY1 infection in P. cablin. A The expression levels of the PatWRKY are presented based on transcriptome data. B The expression level of these PatWRKY was analyzed using quantitative PCR. Significant differences were indicated by different letters, as determined by Student’s t-test (P < 0.05). These experiments were conducted at least twice, yielding consistent results

Fig. 7

Ceramides reduced after FB1 treatment in P. cablin. A The gene expression of PatLTPs was down-regulated after FB1 treatment in P. cablin. B The level of total ceramide was reduced after FB1 treatment in P. cablin. C A comparison of ceramide concentration after FB1 treatment in P. cablin. Significant differences from the untreated sample are indicated by asterisks, based on Student’s t-test (*P<0.05; ** P<0.01; *** P<0.001)

Fig. 8

The expression of the PatWRKY was found to be down-regulated after FB1 treatment in P. cablin. The expression of PatWRKY genes was quantified using quantitative PCR. Significant differences were indicated by different letters, as determined by Student’s t-test (P < 0.05). These experiments were conducted at least twice, yielding consistent results

Fig. 9

ABA pathway was inhibited after FB1 treatment in P. cablin. A The expression level of ABA pathway genes was down-regulated in response to FB1 treatment in P. cablin. B The level of ABA reduced after FB1 treatment in P. cablin. Significant differences were indicated by different letters, as determined by Student’s t-test (P < 0.05). These experiments were conducted at least twice, yielding consistent results