Genome-wide detection and classification of terpene synthase genes in Aquilaria agallochum

Abstract

Agarwood, one of the precious woods in the globe, is produced by Aquilaria plant species during an upshot of wounding and infection. Produced as a defence response, the dark, fragrant resin gets secreted in the plant's duramen, which is impregnated with fragrant molecules with the due course. Agarwood has gained worldwide popularity due to its high aromatic oil, fragrance, and pharmaceutical value, which makes it highly solicited by numerous industries. Predominant chemical constituents of agarwood, sesquiterpenoids, and 2-(2-phenylethyl) chromones have been scrutinized to comprehend the scientific nature of the fragrant wood and develop novel products. However, the genes involved in the biosynthesis of these aromatic compounds are still not comprehensively studied in Aquilaria. In this study, publicly available genomic and transcriptomics data of Aquilaria agallochum were integrated to identify putative functional terpene synthase genes (TPSs). The in silico study enabled us to identify ninety-six TPSs, of which thirty-nine full-length genes were systematically classified into TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g subfamilies based on their gene structure, conserve motif, and phylogenetic comparison with TPSs from other plant species. Analysis of the cis-regulatory elements present upstream of AaTPSs revealed their association with hormone, stress and light responses. In silico expression studies detected their up-regulation in stress induced tissue. This study provides a basic understanding of terpene synthase gene repertoire in Aquilaria agallochum and unlatches opportunities for the biochemical characterization and biotechnological exploration of these genes.

Supplementary information: The online version contains supplementary material available at 10.1007/s12298-021-01040-z.

Keywords: Aquilaria agallochum; Classification; Phylogenetic analysis and functional prediction; Terpene synthase.

Fig. 1

Conserved motifs in the putative terpene synthases identified in the genome of Aquilaria agallochum. In the TPS-a subfamily, RDR and DDXXD motifs were conserved, while R(R)X₈W and DTE were not conserved. In the TPS-b subfamily, the DDXXD motif was conserved, and R(R)X₈W motif was conserved only in AaTPS28 and AaTPS29. In the TPS-c subfamily, the DXDD motif was present, and in TPS-e and f, R(R)X₈W was not seen but, DDXXD was found to be conserved

Fig. 2

Overview of the gene structure of 39 putative terpene synthase genes in Aquilaria agallochum. Intron–exon boundaries are presented where the black rectangular box depicts the exon, and the thin line represents the intron. Genes are presented in the different boxes based on the subfamily

Fig. 3

Neighbor-joining tree constructed between 39 AaTPS using 1000 bootstrap replications with substitution and p-distance model. Subfamily based classification is shown with labelling and different branch shade

Fig. 4

Neighbor-joining tree constructed between other plants terpene synthases and 39 AaTPS using 1000 bootstrap replications with substitution and p-distance model

Fig. 5

Functional classification of AaTPS based on Gene Ontology (GO). a. Percentage of AaTPS genes predicted to be involved in various Biological processes (most genes involved in terpenoid, isoprenoid, lipid biosynthetic and metabolic process). b. Percentage of AaTPS genes predicted to be involved in various molecular functions (highest number of genes were involved in ion binding, terpene synthase, and lyase activity, and fewer genes in isomerase and cyclase activity)

Fig. 6

Overview of predicted secondary metabolic pathway of putative AaTPSs based on mapping with the KEGG database and characterized TPS genes in Aquilaria plant species a. Representation of the possible involvement of predicted terpene synthases in the biosynthesis of terpenes in A. agallochum. b. Characterized terpene synthases in Aquilaria plant species to date. AaTPSa3 showed the highest sequence similarity of 98.30% to delta-guaiene synthase characterized from cultured cells of Aquilaria crassna

Fig. 7

Overview of the expression level of AaTPSs in stress induced tissue and their possible CREs in the upstream region. a. Predicted CREs in upstream regions of AaTPS genes. Stress-responsive elements include (W box: WRKY transcription factors in stress responses, LTR: Low-temperature-responsive element, WUN-motif: wound-responsive element, MBS: MYB binding site involved in drought-inducibility, ARE: regulatory elements essential for anaerobic induction, DRE dehydration-responsive elements, TC-rich repeats: stress-responsive elements); Hormone-responsive elements include (TCA-element salicylic acid responsiveness, TGA-element auxin-responsive element, TATC-box: gibberellin-responsiveness, ABRE: abscisic acid-responsive elements, TGACG-motif: MeJA-responsiveness); and light responsive elements (TCT-motif, L-box, G-box, GATA-motif, Box-4, 3-AF1, GT-1 motif, chs-CMA1a, MRE, ACE, TCCC-motif, ATCT-motif, ATC-motif, I-box, GA-motif, AE-box, LAMP-element, AAAC-motif, CAG-motif, Gap-box, Box II, Sp1, and sbp-CMA1c. b. Log fold change of the differentially regulated (p < 0.05) AaTPS genes in stress-induced tissues based on RNA-seq data retrieved from NCBI. Subfamily members are shown in a different coloured bar: TPS-a (green), TPS-b (red), TPS-g (violet). c. Differentially expressed genes (Log fold change > 1 or < − 1) in tissues exposed to 0.5 mM Methyl jasmonate (MeJ) when compared to control tissues (no treatment). Subfamily members are shown in a different coloured bar: TPS-a (green), TPS-b (red), TPS-f (blue), and TPS-g (violet). d. Differentially expressed AaTPS genes (*marked) (Log fold change > 1 or < 1) in tissues exposed to Far Red light compared to normal white light (FR/W), and Red light compared to white (R/W). Members are coloured coded based on subfamily

Fig. 8

Homology model of A. agallochum AaTPSa19. a. Modelled structure of AaTPSa19 with substrate FPP along with Mg²⁺ trio. The N-terminal region is shown in cyan colour, and the C terminal region in pale blue with Mg²⁺ trio and the ligand FPP. The two conserved motif DTE (violet colour) and metal binding DDXXD motif (green colour) was found to be in close proximity to FPP and metal ions. b. Alignment of 5-epi-aristolochene synthase (red colour) from Nicotiana tabacum and AaTPSa19 (pale green) of A. agallochum (RMSD = 0.172). c. Probable polar (red stick) and non-polar (black stick) interaction (yellow colour dashes) with FPP (cyan deep teal colour) and Mg²⁺ trio in the pocket region. The residue ASP-306 and ASP-310 (green coloured stick) of metal binding motif are found to form polar interaction with two Mg²⁺. d. Surface view of the modelled protein where N terminal (pale cyan), C terminal (blue) and pocket region are shown. Residues involving in polar and non-polar interactions are shown in red and black colour respectively