Identification of Putative Candidate Genes from Galphimia spp. Encoding Enzymes of the Galphimines Triterpenoids Synthesis Pathway with Anxiolytic and Sedative Effects

Abstract

Galphimia spp. is popularly used in Mexican traditional medicine. Some populations of Galphimia exert anxiolytic and sedative effects due to the presence of the modified triterpenoids galphimines. However, the galphimine synthesis pathway has not yet been elucidated. Hence, in this study, a comparative transcriptome analysis between two contrasting populations of Galphimia spp., a galphimine-producer, and a non-galphimine-producer, is performed using RNA-Seq in the Illumina Next Seq 550 platform to identify putative candidates genes that encode enzymes of this metabolic pathway. Transcriptome functional annotation was performed using the Blast2GO in levels of gene ontology. For differential expression analysis, edgeR, pheatmap, and Genie3 library were used. To validate transcriptome data, qPCR was conducted. In producer and non-producer plants of both populations of Galphimia spp., most of the transcripts were grouped in the Molecular Function level of gene ontology. A total of 680 differentially expressed transcripts between producer and non-producer plants were detected. In galphimine-producer plants, a larger number of highly expressed transcripts related to acyclic and polycyclic terpene synthesis were identified. As putative candidate genes involved in the galphimine synthesis pathway, P450 family members and enzymes with kinase activity were identified.

Keywords: Galphimia spp.; functional annotation; transcriptome.

Figure 1

Percentage of annotated sequences of galphimine-producer plants (leaves samples, non-galphimine producer plants, (leaves samples) and control (roots samples) in gene ontology levels Biological Process (BP), Molecular Function (MF), and Cellular Component (CC).

Figure 2

Enzyme distribution code of annotated sequences (percentage) in galphimine producer plants (leaves samples), non-galphimine producer plants (leaves samples), and control (roots samples).

Figure 3

Volcano plot of differentially expressed genes (a) galphimine producer plants (leaves samples) versus non-galphimine producer plants (leaves samples) (b) galphimine producer plants (leaves samples) versus control (roots samples). X-axis and y-axis represent log2 fold-change differences between the compared samples and statistical significance as the negative log of differentially expressed genes p-values, respectively. The significantly up-regulated and down-regulated genes are indicated with red and green dots, respectively, while non-significant genes are shown as black dots.

Figure 4

Comparison of transcripts with higher differential expression values between two populations of Galphimia spp. GP1, GP2 (leaves of galphimine producer plants). NGP1, NPG2 (leaves of non-galphimine producer plants). High values are represented in red color, the lowest values are represented in blue.

Figure 5

Comparison of transcripts with higher differential expression values between leaves and roots of galphimine producer plants. C1, C2 (roots). GP1, GP2 (leaves). High values are represented in red color, the lowest values are represented in blue.

Figure 6

Co-expression network of transcripts related to the synthesis pathway of triterpenes and members of the cytochrome P450 family in leaves of the galphimine producer plants and non-galphimine producer plants. Transcripts are represented by spheres and dotted lines are their interaction. HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase), DXS (1-deoxy-D-xylulose-5-phosphate synthase), CDP-ME (4-diphosphocytidyl-2-C-methyl-D-erythritol kinase), HMB-PPS (4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase), IPP isomerase (isopentenyl diphosphate isomerase), GPPS (geranyl diphosphate synthase), FT (farnesyl transferase), TS (terpene synthase), SS (squalene synthase), SE (squalene epoxidase), BAS (beta-amyrin synthase), LS (lanosterol synthase).

Figure 7

Relative expression of putative candidate genes in leaves of galphimine producer plants (GP) and non-galphimine producer plants (NGP) was calculated by the 2^−ΔΔCt method in quantitative PCR analysis. Y-axis represents the fold change value between GP and NGP. AMPA (5’-AMP-activated protein kinase), Ser/Thr (serine/threonine-protein kinase AtPK2/AtPK19).

Figure 8

CT values of leaves of galphimine producer plants (GP) and non-galphimine producer plants (NGP) obtained by quantitative PCR analysis. AMPA (5’-AMP-activated protein kinase), Ser/Thr (serine/threonine-protein kinase AtPK2/AtPK19).

Figure 9

Proposed galphimines synthesis pathway mediated by cytochrome P450 in Galphimia spp. plants. Solid arrows indicate pathway reactions identified in previous work [18,28]. Dotted arrows indicate reactions proposed in this study.