Evolution of Terpene Synthases in Orchidaceae

Abstract

Terpenoids are the largest class of plant secondary metabolites and are one of the major emitted volatile compounds released to the atmosphere. They have functions of attracting pollinators or defense function, insecticidal properties, and are even used as pharmaceutical agents. Because of the importance of terpenoids, an increasing number of plants are required to investigate the function and evolution of terpene synthases (TPSs) that are the key enzymes in terpenoids biosynthesis. Orchidacea, containing more than 800 genera and 28,000 species, is one of the largest and most diverse families of flowering plants, and is widely distributed. Here, the diversification of the TPSs evolution in Orchidaceae is revealed. A characterization and phylogeny of TPSs from four different species with whole genome sequences is available. Phylogenetic analysis of orchid TPSs indicates these genes are divided into TPS-a, -b, -e/f, and g subfamilies, and their duplicated copies are increased in derived orchid species compared to that in the early divergence orchid, A. shenzhenica. The large increase of both TPS-a and TPS-b copies can probably be attributed to the pro-duction of different volatile compounds for attracting pollinators or generating chemical defenses in derived orchid lineages; while the duplications of TPS-g and TPS-e/f copies occurred in a species-dependent manner.

Keywords: Orchidaceae; evolution; phylogenetic tree; terpene synthase.

Figure 1

The MVA (left) and MEP (right) pathways responsible for IPP and DMAPP biosynthesis and monoterpene biosynthesis in plants. AACT, acetoacetyl-CoA thiolase; CMK, 4-(cytidine 5′ -diphospho)-2-C-methyl-d-erythritol kinase; DMAPP, dimethylallyl diphosphate; DXR, 1-deoxy-d-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxyd- xylulose 5-phosphate synthase; FDP, farnesyl diphosphate; FPPS, farnesyl diphosphate synthase; G3P, d-glyceraldehyde 3-phosphate; GDPS, geranyl diphosphate synthase; GDP, geranyl diphosphate; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl- CoA synthase; IDI, isopentenyl diphosphate isomerase; IPP, isopentenyl diphosphate; MCT, 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase; MDD, mevalonate diphosphate decarboxylase; MDS, 2-C-methyld-erythritol 2,4-cyclodiphosphate synthase; MVK, mevalonate kinase; MVAP, mevalonate 5-phosphate; MVAPP, mevalonate diphosphate; PMK, phosphomevalonate kinase; TPS, terpene synthase.

Figure 2

Phylogenetic analysis of terpene synthases. TPSs in Orchidaceae, including A. shenzhenica; V. planifolia; D. catenatuml P. equestris Phalaenopsis aphrodite; P. bellina, Arabidopsis thaliana, and Abies grandis; and S. moellendorffii were used. Sequence analysis was performed using MEGA 7.0 to create a tree using the nearest neighbor-joining method. The coding sequence was used for analysis. The numbers at each node represent the bootstrap values. Various colors mean distinct subfamilies and special symbols represent different plant species, with solid circles, tangle, diamond, and triangle illustrating Orchidaceae, Arabidopsis thaliana, A. grandis, and S. moellendorffii, respectively.

Figure 3

The amino acid sequences of the predicted motifs in TPS proteins. (A) Twenty-five classical motifs in TPS proteins were analyzed using the MEME tool. The width of each motif ranges from 6 to 50 amino acids. The font size represents the strength of conservation. (B) The amino acid sequences of five highly conserved motifs in TPS proteins.

Figure 4

Motif structures of TPS proteins. (A–D) are TPS-a, -b, -e/f, and -g subfamilies, respectively. Twenty-five classical motifs in TPS proteins were analyzed by using the MEME tool. The width of each motif ranged from 6 to 50 amino acids. Different color blocks represent distinct motifs. Star indicates TPSs of A. shenzhenica, and the red solid circle indicates the out group of Apostasia TPSs. The red and blue rectangle squares reveal orthologous and paralogous gene pairs, respectively.

Figure 4

Motif structures of TPS proteins. (A–D) are TPS-a, -b, -e/f, and -g subfamilies, respectively. Twenty-five classical motifs in TPS proteins were analyzed by using the MEME tool. The width of each motif ranged from 6 to 50 amino acids. Different color blocks represent distinct motifs. Star indicates TPSs of A. shenzhenica, and the red solid circle indicates the out group of Apostasia TPSs. The red and blue rectangle squares reveal orthologous and paralogous gene pairs, respectively.

Figure 5

Gene clusters in Orchidaceae genome. Clustered genes in the genomic scaffolds of A. shenzhenica (A), V. planifolia (B), D. catenatum (C), and P. equestris (D), respectively. The TPS genes located on the scaffolds are identified from the assembled whole genome sequences of A. shenzhenica, V. planifolia, D. catenatum, and P. equestris. The direction of arrows illustrates the forward translation of genes in the scaffolds. Various colors indicate the distinct TPS subfamilies. Blue, green, purple, and bisque colors represent TPS genes in TPS-a, -b, -e/f, and -g subfamilies, respectively. Break lines indicate the shrink length of genes.