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Genome-wide analysis of the terpene synthase gene family in physic nut (Jatropha curcas L.) and functional identification of six terpene synthases

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Abstract

Plant terpenes constitute a large class of compounds and have numerous biological roles as either primary or secondary metabolites. Terpene synthases (TPSs) play key roles on catalyzing the formation of different terpenes; they are divided into seven subfamilies based on sequence relatedness (TPSa–h). TPS-a proteins catalyze the formation of sesquiterpenes and diterpenes in plants. Physic nut (Jatropha curcas L.) is an attractive biofuel tree, but its seeds contain diterpene derivatives, which make them inedible for animals. In this study, 59 putative TPS genes (JcTPS01 to JcTPS59) were identified in the physic nut genome, and 26 belong to the TPS-a subfamily. Eight among the 26 TPS-a genes showed expression in developing seeds of physic nut in the present study. After heterologous expression of these eight genes in Escherichia coli and in vitro enzyme assays, six were shown to have TPS activities. Two (JcTPS09 and JcTPS11) catalyzed the production of diterpene casbene, which was consistent with earlier findings. The other four (JcTPS02, 23, 55, and 56) catalyzed the production of sesquiterpenes. These results may facilitate the efforts for identifying TPS genes involving the physic nut terpene synthesize.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31270705). Thanks to Dr. Feng Chen from the University of Tennessee, Knoxville for giving some helpful suggestions and language editing. We also thank Dr. Feng Chen for kindly providing the plasmid of SmMTPSL17-TOPO.

Data archiving statement

All identified amino acid of TPS gene sequences were deposited into the NCBI database (http://www.ncbi.nlm.nih.gov/). The accession numbers are listed in Fig. S1.

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Authors and Affiliations

  1. Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, 510650, China

    Wangdan Xiong, Pingzhi Wu, Yongxia Jia, Xiaoyi Wei, Liangxiong Xu, Yaping Chen, Meiru Li, Huawu Jiang & Guojiang Wu

  2. University of Chinese Academy of Sciences, Beijing, 100049, China

    Wangdan Xiong

  3. State Key Laboratory of Tree Genetics and Breeding, the Research Institute of Forestry, Chinese Academy of Forestry, Beijing, 100091, China

    Yanfang Yang & Deyou Qiu

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  1. Wangdan Xiong
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  2. Pingzhi Wu
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Correspondence to Guojiang Wu.

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Communicated by A. Brunner

Wangdan Xiong and Pingzhi Wu contributed equally to this work.

Electronic supplementary material

Fig. S1

The information of putative TPS genes in physic nut. (DOCX 28 kb)

Fig. S2

Sequence alignments of JcTPS proteins. (DOCX 93 kb)

Fig. S3

Gene components of physic nut cluster region on scaffold 123_27 and castor bean cluster region on scaffold 30169. The orthologous genes based on the phylogenetic analysis were indicated by lines. ADH, alcohol dehydrogenase; AT, acyl-transferase; CS, casbene synthase; CYP, cytochrome P450; NCS, neo-cembrene synthase. (GIF 76 kb)

High resolution image (TIFF 1249 kb)

Fig. S4

Proteins of recombinant synthases and analysis by SDS–PAGE (7.5 %). The gel was stained with Coomassie Brilliant Blue R-250. Lane 1, protein standard ladder; lanes 2 and 3, soluble and insoluble fractions, respectively, from lysate of E. coli BL21 transformed with native plasmid pET-32a; lanes 4 and 5, soluble and insoluble fractions, respectively, from lysate of E. coli BL21 expressing recombinant protein; lane 6, purified recombinant protein. (GIF 110 kb)

High resolution image (TIFF 3459 kb)

Fig. S5

Gas chromatography-mass spectrometry (GC-MS) analysis of the diterpene products formed by the recombinant proteins in vitro assays. a Gas chromatograms showing terpene yielded by recombinant JcTPS09, JcTPS11, and RcCS3, using GGPP as substrate. Species: Jatropha curcas (Jc), Ricinus communis (Rc). b Mass spectrum of the substances in the indicated peaks of the chromatograms. (GIF 50 kb)

High resolution image (TIFF 700 kb)

Fig. S6

Unrooted phylogenetic tree of TPS-a proteins. The amino acid sequences of TPS proteins were aligned using Clustal X 1.83 and the compressed phylogenetic tree was constructed by MEGA 5.0 using the Neighbor-Joining method. Species: Euphorbia esula (Ee), E. fischeriana (Ef), E. peplus (Ep), Homalanthus nutans (Hn), Jatropha curcas (Jc), Ricinus communis (Rc), Triadica sebifera (Ts). (GIF 13 kb)

High resolution image (TIFF 841 kb)

Fig. S7

Gas chromatography-mass spectrometry (GC-MS) analysis of germacrene D, bicyclogermacrene and beta-elemene. a Gas chromatograms showing terpenes yielded by recombinant JcTPS02 and SmMTPSL17, using FPP as substrate. Species: Jatropha curcas (Jc), Selaginella moellendorffii (Sm). b Mass spectrum of germacrene D, bicyclogermacrene, peak 1 and peak 2. c Gas chromatograms showing terpenes yielded by recombinant JcTPS56 and (±)-beta-elemene. d Mass spectrum of components from the standard (±)-beta-elemene and peak 7. (GIF 78 kb)

High resolution image (TIFF 650 kb)

Table S1

Primer sequences used in this study. (XLSX 10 kb)

Table S2

Overview of the JcTPS genes. (XLSX 16 kb)

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Xiong, W., Wu, P., Jia, Y. et al. Genome-wide analysis of the terpene synthase gene family in physic nut (Jatropha curcas L.) and functional identification of six terpene synthases. Tree Genetics & Genomes 12, 97 (2016). https://doi.org/10.1007/s11295-016-1054-3

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