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. 2022 Jan 18:9:uhab020.
doi: 10.1093/hr/uhab020. Online ahead of print.

Characterization of Terpene synthase variation in flowers of wild aquilegia species from Northeastern Asia

Song Yang  1 , Ning Wang  1 , Shadrack Kimani  1   2 , Yueqing Li  1 , Tingting Bao  1 , Guogui Ning  3 , Linfeng Li  4 , Bao Liu  1 , Li Wang  1 , Xiang Gao  1
Affiliations

Characterization of Terpene synthase variation in flowers of wild aquilegia species from Northeastern Asia

Song Yang et al. Hortic Res. .

Abstract

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and β-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545 L affected the ratio of β-sesquiphellandrene to β-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

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Figures

Figure 1
Figure 1
Phylogenetic analysis, gene expression detection and subcellular localization of Aquilegia TPS genes. a Phylogenetic analysis of Aquilegia coerulea TPS sequences. The TPS sequences were processed by Clustal Omega and input into MEGA-X to construct the neighbor-joining tree. Bootstrap values represent 1000 replicates. The TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g clades are highlighted with colored lines. b The relative expression levels of 42 TPSs in Aquilegia flowers collected from Zuojia, Jilin Province, China. All the transcripts were normalized to β-actin and compared with A. oxysepala TPS1. c Subcellular localization of the highly expressed TPS7, TPS8, TPS9 and TPS27 genes in Arabidopsis protoplasts. Green, GFP fluorescence detected in the green channel; red, chlorophyll autofluorescence detected in the red channel; merged, merged green and red channel images; and bright light, bright field image. Scale bar =25 μm. d Representative flowers of A. oxysepala, A. japonica and their hybrid. Scale bar =1 cm.
Figure 2
Figure 2
Volatile terpene emissions and TPS expression of Aquilegia individuals from the Zuojia population. a Floral volatile terpene profiles of representative Aquilegia individuals. b Volatile terpenes emitted from fully blooming flowers of Aquilegia individuals. Data represent the relative peak area in specific individuals. c Volatile terpenes released from flowers at different developmental stages of representative A. oxysepala 7#, A. japonica 7#, and their hybrid. Data represent the relative content calculated by the standard curve of (+)-limonene. d Relative expression levels of TPS7, TPS8, TPS9 and TPS27 in flowers of Aquilegia individuals. e Relative expression levels of TPS7, TPS8, TPS9 and TPS27 in flowers at different developmental stages of representative A. oxysepala 7#, A. japonica 7#, and their hybrid. The transcripts were normalized by AqIPP2 (GenBank KC854337) and compared with the lowest expression level of TPS in specific species. All the data were calculated as log2. Red and blue boxes indicate high and low expression levels, respectively. The detailed terpene contents are listed in Table S1 and Table S2. Here, 1 to 29 represent different volatiles: 1, (1R)-(+)-α-pinene; 2, (1S)-(−)-β-pinene; 3, α-phellandrene; 4, β-phellandrene; 5, β-pinene; 6, myrcene; 7, (+)-limonene; 8, 3-carene; 9, (E)-β-ocimene; 10, linalool; 11, α-terpineol; 12, an unidentified sesquiterpene; 13, (−)-α-copaene; 14, α-bergamotene; 15, an unidentified sesquiterpene; 16, (Z)-β-farnesene; 17, an unidentified sesquiterpene; 18, α-curcumene; 19, (E)-β-farnesene; 20, himachalene; 21, β-caryophyllene; 22, zingiberene; 23, β-bisabolene; 24, β-sesquiphellandrene; 25, α-caryophyllene; 26, an unidentified sesquiterpene; 27, cubebene; 28, α-muurolene; and 29, an unidentified sesquiterpene.
Figure 3
Figure 3
In vitro enzymatic analysis of AoTPS7, AjTPS8 and AoTPS9 using four acyclic prenyl diphosphate substrates. a SDS–PAGE and western blotting analysis of the purified Aquilegia TPS proteins. The recombinant proteins were indicated by anti-His-Tag antibody. b Enzymatic products of AoTPS7. c Enzymatic products of AjTPS8. d Enzymatic products of AoTPS9. The x-axis represents the retention time, and the y-axis represents the abundance of each compound. The enzymatic products are detailed in Table S4. Numbers above the peaks represent the following: 1, (1R)-(+)-α-pinene; 2, (1S)-(−)-β-pinene; 3, (1S)-(−)-β-pinene derivative; 4, α-phellandrene; 5, β-phellandrene; 6, β-pinene; 7, myrcene; 8, (+)-limonene; 9, (3R)-(+)-isosylvestrene; 10, 3-carene; 11, (Z)-β-ocimene; 12, γ-terpinene; 13, terpinolene; 14, linalool; 15, α-terpineol; 16, geraniol; 17, α-bergamotene; 18, an unidentified sesquiterpene; 19, an unidentified sesquiterpene; 20, an unidentified sesquiterpene; 21, an unidentified sesquiterpene; 22, β-chamigrene; 23, α-farnesene; 24, (E)-β-farnesene; 25, β-himachalene; 26, α-cedrene; 27, (Z,E)-α-farnesene; 28, zingiberene; 29, β-bisabolene; 30, (+)-α-longipinene; 31, α-patchoulene; 32, α-himachalene; 33, β-sesquiphellandrene; 34, α-caryophyllene; 35, (E)-nerolidol; and 36, farnesol methyl ether.
Figure 4
Figure 4
Possible pivotal amino acid residues of TPS7 for producing (+)-limonene. a Amino acid sequence alignment of AjTPS7 (from A. japonica 7#), AoTPS7 (from A. oxysepala 7#) and HTPS7. The conserved motifs are indicated by lines. The polymorphic residues between AjTPS7 and AoTPS7 are marked with red boxes, while polymorphic residues between HTPS7 and AoTPS7 are indicated by black boxes. The numbers in each line indicate the positions of the last residue. b Volatile terpenes detected in control and transgenic tobacco overexpressing TPS7. TPS7 was stably transformed into Nicotiana tabacum (cv. K326). The seedlings in tissue culture bottles were directly used for volatile terpene detection. The lower panel indicates the typical mass spectrum of (+)-limonene. c Enzymatic assays of AoTPS7, AjTPS7, and a series of mutant TPS7 proteins based on the AjTPS7 backbone. GPP was employed as the substrate in these assays. d Tabulated contents of (+)-limonene catalyzed by AoTPS7, AjTPS7 and mutant TPS proteins. Data represent the mean ± SD of three replicates. The (+)-limonene content was calculated by a standard curve. One-way ANOVA was carried out to compare significant differences (Duncan, P < 0.05).
Figure 5
Figure 5
Possible amino acid residues responsible for the product plasticity in Aquilegia TPS8. a Amino acid sequence alignment between AoTPS8 (from A. oxysepala 7#) and AjTPS8 (from A. japonica 7#). The conserved TPS motifs are indicated by lines, and polymorphic amino acids are marked with red boxes. The numbers in each line indicate the positions of the last residue. b Volatile compounds detected in the control and transgenic Nicotiana benthamiana transiently overexpressing TPS8. AtFPS2, which encodes Arabidopsis thaliana farnesyl diphosphate synthase in FPP synthesis, was coinfiltrated into tobacco leaves. The tobacco leaves were sampled and analyzed by GC–MS analysis. Tobacco leaves infiltrated with AtFPS2 alone were used as controls. c Enzymatic assays of AoTPS8, AjTPS8, and a series of mutant TPS8 proteins based on the AoTPS8 backbone. (E, E)-FPP was employed as the substrate in these assays. The enzymatic products are detailed in Table S5. d Relative contents of β-sesquiphellandrene and β-bisabolene catalyzed by AoTPS8, AjTPS8 and mutant TPS proteins. Data represent the mean ± SD of two replicates calculated from relative peak areas. One-way ANOVA was carried out to compare significant differences (Duncan, P < 0.05).
Figure 6
Figure 6
Volatile terpene release, main TPS expression and key residue polymorphisms of A. oxysepala and A. japonica from Changbai Mountain. a Volatile terpenes emitted from flowers of A. oxysepala and A. japonica individuals from the Changbai Mountain population. Data represent the relative content calculated from peak areas and then log2 transformed. The detailed terpene contents are listed in Table S6. Here, 1 to 29 represent different volatiles: 1, (1R)-(+)-α-pinene; 2, (1S)-(−)-β-pinene; 3, α-phellandrene; 4, β-phellandrene; 5, β-pinene; 6, myrcene; 7, (+)-limonene; 8, 3-carene; 9, (E)-β-ocimene; 10, linalool; 11, α-terpineol; 12, an unidentified sesquiterpene; 13, (−)-α-copaene; 14, α-bergamotene; 15, an unidentified sesquiterpene; 16, (Z)-β-farnesene; 17, an unidentified sesquiterpene; 18, α-curcumene; 19, (E)-β-farnesene; 20, himachalene; 21, β-caryophyllene; 22, zingiberene; 23, β-bisabolene; 24, β-sesquiphellandrene; 25, α-caryophyllene; 26, an unidentified sesquiterpene; 27, cubebene; 28, α-muurolene; and 29, an unidentified sesquiterpene. b Relative expression levels of TPS7 and TPS8 in Aquilegia individuals from the Changbai Mountain population. Data represent changes relative to the lowest expression level of TPS in specific species. Data are the mean of three replicates and calculated as log2. c Key amino acid residue polymorphisms of TPSs among Aquilegia individuals from both Zuojia and Changbai Mountain. A. oxysepala 1#-7#, A. japonica 1#-7# and the hybrid are from Zuojia. A. oxysepala 8#-12# and A. japonica 8#-12# are from Changbai Mountain. The number in front of each line indicates the key residue position.
Figure 7
Figure 7
Proposed model for volatile terpene biosynthesis in flowers of Aquilegia populations endemic to Northeastern Asia. The parent TPS7 was highly expressed in A. oxysepala (Zuojia and the Changbai mountain population), the hybrid and A. japonica (the Changbai mountain population). The active parent TPS7 generated a high amount of (+)-limonene, whereas the mTPS7 mutated protein was hardly expressed in A. japonica (the Zuojia population), although it produced a low amount of (+)-limonene. The parent TPS8 was highly expressed in A. japonica (the Zuojia population) when compared with the less expressed mTPS8 in the other populations. Moreover, the parent TPS8 yielded a high amount of β-sesquiphellandrenes, while the mutant mTPS8 produced more β-bisabolenes. The arrows represent high expression. The squares and circles under TPSs indicate A. oxysepala and A. japonica, respectively. The different background colors of the same shape indicate the same species from different populations. The hexagon indicates the natural hybrid from the Zuojia population. The three-dimensional models of the TPSs were created according to the crystal structure of 5-epi-aristolochene synthase (PDB ID: 5IL3) from tobacco61. The active cavity is illustrated in dotted circles. Polymorphic amino acids in or adjacent to the active cavity are marked. The different background colors of (+)-limonene, β-sesquiphellandrenes and β-bisabolenes represent different contents produced by the enzymes.
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