Combinatorial Evolution of a Terpene Synthase Gene Cluster Explains Terpene Variations in Oryza

Abstract

Terpenes are specialized metabolites ubiquitously produced by plants via the action of terpene synthases (TPSs). There are enormous variations in the types and amounts of terpenes produced by individual species. To understand the mechanisms responsible for such vast diversity, here we investigated the origin and evolution of a cluster of tandemly arrayed TPS genes in Oryza In the Oryza species analyzed, TPS genes occur as a three-TPS cluster, a two-TPS cluster, and a single TPS gene in five, one, and one species, respectively. Phylogenetic analysis revealed the origins of the two-TPS and three-TPS clusters and the role of species-specific losses of TPS genes. Within the three-TPS clusters, one orthologous group exhibited conserved catalytic activities. The other two groups, both of which contained pseudogenes and/or nonfunctional genes, exhibited distinct profiles of terpene products. Sequence and structural analyses combined with functional validation identified several amino acids in the active site that are critical for catalytic activity divergence of the three orthologous groups. In the five Oryza species containing the three-TPS cluster, their functional TPS genes showed both conserved and species-specific expression patterns in insect-damaged and untreated plants. Emission patterns of volatile terpenes from each species were largely consistent with the expression of their respective TPS genes and the catalytic activities of the encoded enzymes. This study indicates the importance of combinatorial evolution of TPS genes in determining terpene variations among individual species, which includes gene duplication, retention/loss/degradation of duplicated genes, varying selection pressure, retention/divergence in catalytic activities, and divergence in expression regulation.

Figure 1.

A TPS gene cluster in seven species of Oryza. The phylogeny of the seven Oryza species was adopted from Stein et al. (2018). The blocks depict the arrangement of tandemly arrayed TPS genes and their neighboring genes. Blocks of the same color represent apparent orthologous genes.

Figure 2.

Evolutionary analysis of the TPS gene cluster. A, Phylogenetic tree of the Oryza TPSs based on DNA sequences of complete coding regions with ZmTPS10 from maize as outgroup. B, Dot-plot analysis covering genomic DNAs for Obr080/100 in O. brachyantha, Op100+Op120 in O. punctata, and Os080+Os100+Os120 in O. sativa. C and D, Two inferred models for the evolutionary trajectory of the TPS cluster in the seven species of Oryza. The blocks depict TPS genes. The same color indicates shared catalytic activities. Blocks with an X depict pseudogenes.

Figure 3.

Catalytic activities of TPSs. Os080, Or080, Oba120, and Op120 are indicated as pseudogenes and were not analyzed. NE indicates that the respective gene is nonexistent. All other genes were expressed in E. coli, and partially purified recombinant proteins were assayed with the substrate FPP. The produced terpenes were separated and analyzed by gas chromatography-mass spectrometry (GC-MS). The traces of the MS detector are shown for the active enzymes. Products were identified as follows: 1, 7-epi-sesquithujene; 2, (E)-α-bergamotene; 3, sesquisabinene A; 4, (E)-β-farnesene; 5, zingiberene; 6, β-bisabolene; 7, β-sesquiphellandrene; 8, (E)-γ-bisabolene; C, nonterpene contamination.

Figure 4.

Activity restoration of Og080 and Os080. A, Comparative sequence analysis for residue 527 (referring to Ob080 and Og080) among the active enzymes characterized in this study and some known sesquiterpene TPS from other species. B, Activity of Og080-Y527D. C, Comparison of sequences of functional On080 and two nonfunctional Os080 and Or080 to indicate a deletion of TAGC in the two pseudogenes. D, Activity of Os080-TAGC.

Figure 5.

Analysis of selection pressure on three lineages leading to “100”TPSs, “080”TPSs, and “120”TPSs. These TPSs are intact TPSs from five Oryza species containing a three-TPS cluster.

Figure 6.

The active site cavity of On080. The model was created using the crystal structure of δ-cadinene synthase (Protein Data Bank identifier 3g4dA) from Gossypium arboreum (Gennadios et al., 2009). The image shows a view from the opening (illustrated as a blue circle) to the bottom of the active site cavity. Amino acids in the active site differing between On080 and Oba080 are shown in red.

Figure 7.

Activity analysis of On080 mutants. A, Clustering analysis of 16 On080 mutants together with On080 wild-type enzyme and Oba080 wild-type enzyme based on the proportions of their sesquiterpene products. B, Product profiles of three representative mutants On080-I294T, On080-I294T+F375S+G519S, and On080-I294T+F375S+E406Q+G519S. Peak numbers correspond to those in Figure 3: 1, 7-epi-sesquithujene; 2, (E)-α-bergamotene; 3, sesquisabinene A; 4, (E)-β-farnesene; 6, β-bisabolene; 7, β-sesquiphellandrene. TIC,Total ion chromatogram; ND, not detected or 0%.

Figure 8.

Activity analysis of Or100 mutants. A, Product profiles of four Or100 mutants (Or100-Y373F, Or100-T292I, Or100-T292I+Y373F, and Or100-Q404E) together with Or100 wild-type enzyme. Peak numbers correspond to those in Figure 3: 4, (E)-β-farnesene; 5, zingiberene; 6, β-bisabolene; 7, β-sesquiphellandrene. B, Clustering analysis of four Or100 mutants together with On080, Or100, and Or120 wild-type enzymes and one artificial mutant Or100-Q404E′ based on the proportions of their sesquiterpene products. Or100-Q404E′ was based on Or100-Q404E with zingiberene removed from the product profile. TIC, Total ion chromatogram; ND, not detected or 0%.

Figure 9.

Expression analysis of functional TPS genes of the three-TPS cluster in the five species of Oryza. Genes that are underlined indicate nonfunctional genes, and the expression for such genes was not measured. The expression of individual functional TPS genes in each species was quantified as fold change in insect-damaged plants versus undamaged control plants using RT-qPCR. Data are presented as means ± sd (n = 3). Asterisks indicate statistical significance at P ≤ 0.05 (*) and P ≤ 0.01 (**).

Figure 10.

Scheme for the biochemical reaction mechanisms involving the functional divergence of three orthologous groups of TPSs (“080”TPSs, “100”TPSs, and “120”TPSs). The four labeled numbers (#4, #5, #6, and #7) correspond to the peak numbers in Figure 3.