Identification of Volatile Compounds and Terpene Synthase (TPS) Genes Reveals ZcTPS02 Involved in β-Ocimene Biosynthesis in Zephyranthes candida

Abstract

Zephyranthes candida is a frequently cultivated ornamental plant containing several secondary metabolites, including alkaloids, flavonoids, and volatile organic compounds (VOCs). However, extensive research has been conducted only on non-VOCs found in the plant, whereas the production of VOCs and the molecular mechanisms underlying the biosynthesis of terpenes remain poorly understood. In this study, 17 volatile compounds were identified from Z. candida flowers using gas chromatography-mass spectrometry (GC-MS), with 16 of them being terpenoids. Transcriptome sequencing resulted in the identification of 17 terpene synthase (TPS) genes; two TPS genes, ZcTPS01 and ZcTPS02, had high expression levels. Biochemical characterization of two enzymes encoded by both genes revealed that ZcTPS02 can catalyze geranyl diphosphate (GPP) into diverse products, among which is β-ocimene, which is the second most abundant compound found in Z. candida flowers. These results suggest that ZcTPS02 plays a vital role in β-ocimene biosynthesis, providing valuable insights into terpene biosynthesis pathways in Z. candida. Furthermore, the expression of ZcTPS02 was upregulated after 2 h of methyl jasmonate (MeJA) treatment and downregulated after 4 h of the same treatment.

Keywords: VOCs; monoterpenes; terpene synthase.

Figure 1

GC-MS analysis of volatiles from Z. candida flowers, showing the gas chromatographs of volatile compounds emitted from Z. candida flowers. The numbers (ordered by retention time) represent the peaks of volatiles. 1, α-thujene; 2, β-thujene; 3, β-myrcene; 4, D-limonene; 5, β-terpinyl acetate; 6, α-pinene; 7, β-ocimene; 8, (+)-4-carenel; 9, allo-ocimene; 10, limonene oxide; 11, (+)-(E)-limonene oxide; 12, myroxide; 13, (E)-9-octadecene; 14, linalool; 15, humulene; 16, α-terpineol; 17, α-farnesene; 18, (E)-β-ocimene. *, impurities.

Figure 2

Phylogenetic tree analysis. The phylogenetic tree was constructed using the 1000 repeated neighborhood connection method. Bootstrap values exceeding 40% are indicated for the respective nodes. The scale quantifies the evolutionary distance in substitutions per site. Color classifications represent the TPS-a to TPS-g subfamilies. The TPS-b subfamily is represented by green, TPS-a by blue, TPS-g by orange, TPS-d by purple, TPS-c by red, and TPS-e/f by pink. The red dots indicate the two sequences studied in this paper. At, Arabidopsis thaliana; Os, Oryza sativa.

Figure 3

Sequence alignment analysis. Alignment of Z. candida TPS amino acid sequences with functionally characterized TPSs from other species. The numbers in the figure represent the amino acid sequences. 1, (+)-limonene synthase 1 (Citrus limon) (accession no. AAM53944); 2, (+)-limonene synthase 2 (Citrus limon) (accession no. AAM53946); 3, E-β-ocimene synthase, partial (Arabidopsis thaliana) (accession no. AAN65379); 4, β-ocimene synthase (Phaseolus lunatus) (accession no. ABY65110); 5, ZcTPS01; 6, ZcTPS02. The sequences were aligned by Genomenet and analyzed with ESPript 3.0. The red background shading represents 100% identity and light red represents 70% identity. The blue frame shows the similarity across groups. The terpene synthases motifs RRx₈W, RxR, DDxxD, and NSE/DTE are indicated by the black box.

Figure 4

Biochemical characterization of enzymes encoded by ZcTPS. In vitro enzymatic assays of ZcTPSs were performed and the resulting enzyme products were collected by SPME and subsequently analyzed through GC-MS. (A) Chromatogram of terpenes generated by the ZcTPS01 protein purified from BL21(DE3) pLysS cells in a reaction with GPP. (B) Chromatogram of terpenes generated by the ZcTPS02 protein from BL21(DE3) pLysS cells in a reaction with GPP. (C) Chemical structure and mass spectra of β-ocimene. 7, β-ocimene; 9, allo-ocimene; 18, (E)-β-ocimene. *, impurities.

Figure 5

Subcellular localization of ZcTPS02. After infiltration for 36 h with GV3101, pictures were obtained using a confocal laser microscope. The upper panel shows pCAMBIA 1300-35S-sGFP transformed tobacco leaf cells and the lower panel shows ZcTPS02-GFP transformed cells. The GFP emission signal in the left panel is green, the middle is chlorophyll red fluorescence and bright-field, and the two signals on the right overlap. Bars, 20 µm.

Figure 6

Relative expression of ZcTPS02 after MeJA treatment. The expression of ZcTPS02 after MeJA treatment. Error bars represent the SD of three independent experiments. CK2 and CK4 were treated with 0.1% ethanol for 2 and 4 h. T2 and T4 were treated with MeJA for 2 and 4 h. The * indicates a significant difference (p < 0.05).