Genome-wide detection of terpene synthase genes in holy basil (Ocimum sanctum L.)

Abstract

Holy basil (Ocimum sanctum L.) and sweet basil (Ocimum basilicum L.) are the most commonly grown basil species in India for essential oil production and biosynthesis of potentially volatile and non-volatile phytomolecules with commercial significance. The aroma, flavor and pharmaceutical value of Ocimum species is a significance of its essential oil, which contains most of the monoterpenes and sesquiterpenes. A large number of plants have been studied for characterization and identification of terpene synthase genes, involved in terpenoids biosynthesis. The goal of this study is to discover and identify the putative functional terpene synthase genes in O. sanctum. HMMER search was performed by using a set of 13 well sequenced and annotated plant genomes including the newly sequenced genome of O. sanctum with Pfam-A database locally, using HMMER 3.0 hmmsearch for the two Pfam domains (PF01397 and PF03936). Using this search method 81 putative terpene synthases genes (OsaTPS) were identified in O. sanctum; the study further reveals 47 OsaTPS were putatively functional genes, 19 partial OsaTPS, and 15 OsaTPS as probably pseudogenes. All these identified OsaTPS genes were compared with other plant species, and phylogenetic analysis reveals the subfamily classification of OsaTPS in TPS-a, -b, -c, -e, -f and TPS-g subfamilies clusters. This genome-wide identification of OsaTPS genes, their phylogenetic analysis and secondary metabolite pathway mapping predictions together provide a comprehensive understanding of the TPS gene family in Ocimum sanctum and offer opportunities for the characterization and functional validation of numbers of terpene synthase genes.

Fig 1. Representation of class I and class II domain within terpene synthase protein structure; class I consists of conserved DDXXD motif in its α-domain, whereas class II contains DXDD conserved motif.

Fig 2. Representation of the gene structure of putative functional TPS genes from Ocimum sanctum.

Genes were predicted by FGENESH tool and Augustus but also determined manually by mapping to previously TPS characterized genes. The black line indicates the introns; yellow rectangles indicate exons, red lines show conserved DDXXD motif and the green line shows DxDD motif. Arrow indicated in the figure shows the incomplete gene sequence or loss of sequence. Color boxes indicate the TPS subfamily TPS-a (from OsaTPS1 to OsaTPS19), TPS-b from (OsaTPS-19 to OsaTPS34), TPS-c from (OsaTPS35 to OsaTPS39), TPS-e (OsaTPS-40) and OsaTPS41, TPS-f (OsaTPS42) and TPS-g from (OsaTPS43 to OsaTPS47).

Fig 3. Discovered 47 putative functional TPS genes from O. sanctum: Neighbor-joining phylogenetic tree was constructed using 1,000 bootstrap value and classified into terpene synthase gene subfamily as TPS-a, -b, -c, -e, -f and TPS-g.

Bootstrap values assigned in the tree, which was higher than 80%, values below 80% were not shown in the figure.

Fig 4. Representation of phylogenetic tree of O. sanctum compared to other species of TPS-a subfamily.

18 OsaTPS represented in the tree were compared with the neighboring species. The Neighbor-joining method was used with 1,000 replicates for bootstrap values. The values = 80% and = 95% were designated in S2 Fig. OsaTPS35 and Pt0092s00200, two TPS gene were used as an out-group.

Fig 5. Phylogenetic tree of putative functional TPS genes from O. sanctum were represented, here TPS-b and TPS-g were grouped in a single tree.

TPS-b was sub-classified into TPS-b1 and TPS-b2, where TPS-b1 shows only S. lycopersicum species TPS genes and TPS-b2 shows mixed species in which 16 OsaTPS were shown. In TPS-g subfamily 5 OsaTPS were represented. The phylogenetic tree was constructed with 1,000 replicates for bootstrap values using the neighbor-joining method.

Fig 6. TPS-c, -e, and TPS-f, combined phylogenetic tree were represented from O. sanctum putatively functional TPS genes compared with other species.

The phylogenetic tree was constructed with 1,000 replicates for bootstrap values using the neighbor-joining method. Here in TPS-c clade five OsaTPS were shown and compared, TPS-e and TPS-f show two OsaTPS and single OsaTPS42. Whereas OsaTPS1 was shown as outgroup.

Fig 7. Conserved motifs of the TPS gene family presented in the O. sanctum putative functional TPS genes were shown.

All the conserved amino acids were highlighted in the grey shades. 47 OsaTPS were shown and classified into TPS gene subfamily. Here RR(X)₈W conserved motif is conserved in OsaTPS-b subfamily except for OsaTPS19 (angiosperm monoterpene synthase). Variation of the RR(X)₈W motif is found in OsaTPS-a subfamily of sesquiterpene and diterpene synthase that have putative N-terminal plastid peptides, OsaTPS-c and–e lack twin-arginine residues but consist only tryptophan residue. NSE/DTE motifs were continuously seen conserved except OsaTPS-c subfamily. Motif RXR in all OsaTPS are shown genes either in same or modified form except some OsaTPS. Motif DDXXD and DXDD were shown highly conserved in all putative functional O. sanctum TPS genes, but in two genes, i.e., OsaTPS6 and OsaTPS25 DDXXD motif is absent, which might be due to sequence assembly error.

Fig 8. Annotation result of 47OsaTPS protein sequences, show higher sequence homology with Ocimum basilicum followed by Nicotiana tabacum, Pogostemon cablinsesamum and Salvia officinalis species.

The pie chart represents the sequence similarity matching of OsaTPS with different plant species.

Fig 9. Proposed secondary metabolite synthesis pathway was constructed for O. sanctum.

Monoterpenoid, Diterpenoid and Sesquiterpenoid pathways show the presence of putatively functional OsaTPS genes.