Abstract
Diterpenoids are the major group of antimicrobial phytoalexins in rice1,2. Here, we report the discovery of a rice diterpenoid gene cluster on chromosome 7 (DGC7) encoding the entire biosynthetic pathway to 5,10-diketo-casbene, a member of the monocyclic casbene-derived diterpenoids. We revealed that DGC7 is regulated directly by JMJ705 through methyl jasmonate-mediated epigenetic control3. Functional characterization of pathway genes revealed OsCYP71Z21 to encode a casbene C10 oxidase, sought after for the biosynthesis of an array of medicinally important diterpenoids. We further show that DGC7 arose relatively recently in the Oryza genus, and that it was partly formed in Oryza rufipogon and positively selected for in japonica during domestication. Casbene-synthesizing enzymes that are functionally equivalent to OsTPS28 are present in several species of Euphorbiaceae but gene tree analysis shows that these and other casbene-modifying enzymes have evolved independently. As such, combining casbene-modifying enzymes from these different families of plants may prove effective in producing a diverse array of bioactive diterpenoid natural products.
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Data availability
The sequences data of 424 O. sativa accessions are available in the National Center for Biotechnology Information (NCBI) under the BioProject PRJNA171289 (ref. 17). The single nucleotide polymorphisms information of 424 O. sativa accessions is available in RiceVarMap (http://ricevarmap.ncpgr.cn/v1). The pangenome data were acquired from the pangenome dataset (https://figshare.com/collections/Novel_sequences_structural_variations_and_gene_presence_variations_of_Asian_cultivated_rice/3876022/1 and http://cgm.sjtu.edu.cn/3kricedb/)18,19,24. Thirteen accessions of O. rufipogon were selected from 446 diverse O. rufipogon accessions from Asia and Oceania, and represented all the major genetically distinct clusters in O. rufipogon; the other ten wild rice are from EnsemblPlants (http://plants.ensembl.org/index.html) and National Genomics Data Center (https://bigd.big.ac.cn/search?dbId=gwh&q=Oryza), including O. barthii (AA), O. glumipatula (AA), O. glaberrima (AA), O. meridionalis (AA), O. longistaminata (AA), O. nivara (AA), O. brachyantha (FF), O. punctata (BB) and O. brachyantha (GG)24. Genes reported in the study are deposited in the NCBI. The genes can be found in GenBank or Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/analyses_search_locus.shtml) under the following accession numbers: OsTPS28, MN833254; OsCYP71Z21, LOC_Os07g11870; OsCYP71Z2, LOC_Os07g11739; OsCYP71Z22, LOC_Os07g11970; OsCYP71Z30, LOC_Os07g11890. Source data are provided with this paper.
Change history
16 December 2020
A Correction to this paper has been published: https://doi.org/10.1038/s41477-020-00838-1.
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Acknowledgements
We thank J. D. Keasling, G. P. Lomonossoff and Z. Zhao for their advice and their gift of the expression vectors and strains. We also thank D. R. Nelson, University of Tennessee, for help in naming the OsCYP71Z30. This work was supported by the National Science Fund for Distinguished Young Scholars (grant no. 31625021), the State Key Programme of National Natural Science Foundation of China (grant no. 31530052) and the Hainan University Startup Fund KYQD(ZR)1866 to J.L.
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J.L. designed the research. J.L., L.-L.C., L.Q., M.Y. and X.L. supervised this study. C.Z., L. Lei, S.Z., Z.L., F.Z., M.Z., Y. Sun, Y. Shi, K.L., T.C., M.H., I.G., Z.Y. and T.T. participated in the material preparation. C.Z., C.Y., Yi Li, X.W. and J.S. carried out the metabolite analyses. C.Z., Z.L., S.Z., C.Y., X.Z., H.G., M.P., M.Z., Yufei Li, Z.Y., L. Liu, S.S., J.S., X.J., Yi Li, T.T. and Z.W. performed the data analyses. C.Z., L. Lei, Z.L., S.Z. and C.Y. performed most of the experiments. J.L., C.Z., I.G. and A.R.F. wrote the manuscript.
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Extended data
Extended Data Fig. 1 The distribution of the worldwide collection of rice accessions in this study.
The core collection of 424 cultivated rice accessions in this study has a wide geographic distribution. Colour dots indicate different subspecies/type of cultivated rice.
Extended Data Fig. 2 Functional analyses of OsTPS28, OsCYP71Z2 and OsCYP71Z21.
a, Gas chromatography of the reaction products of OsTPS28 with GGDP. GGDP, geranylgeranyl diphosphate. Casbene and neocembrene reference compounds were purified from infiltrated N. benthamiana leaves by the method described previously26. Compound 1, casbene; Compound 2, neocembrene. b, Gas chromatography of in vitro enzyme assays showing the 10-keto-casbene C5 oxidase activity of yeast-expressed CYP71Z2 in the present of NADPH. Microsomes prepared from yeast containing PESC-URA empty vector were used as a negative control. 10-keto-casbene reference compound was purified from rice leaves by the method described previously13,14. Compound 3, 10-keto- casbene; Compound 4, 5,10-diketo-casbene. c, Gas chromatography of the extracts prepared from the leaves of N. benthamiana infiltrated with OsTPS28 overexpressing vector.
Extended Data Fig. 3 Mass spectrum and structure of 5,10- diketo-casbene.
a, Mass spectrum and structure of the product in N. benthamiana leaves simultaneously overexpressing OsTPS28, OsCYP71Z2 and OsCYP71Z21. b, Mass spectrum of 5,10-diketo-casbene reference. LC-MS, liquid chromatography–mass spectrometry. c, 1H NMR (left) and 13C NMR (right) results of 5,10-diketo-casbene.
Extended Data Fig. 4 The expression profiles of genes from DGC7.
The genes from DGC7 are indicated in bold. The transcript abundances of indicated genes in different organs at different stages were shown: expression levels of OsTPS28, OsCYP17Z2 and OsCYP71Z21 is correlated at different developmental stages. The numerical values for blue-to-red gradient represent normalized expression levels from quantitative real-time PCR analysis.
Extended Data Fig. 5 The casbene-type diterpene biosynthesis via distinct biosynthetic routes in rice and castor.
The casbene-type diterpene biosynthetic pathways in rice and castor. Chr.7, chromosome 7; GGDP, geranylgeranyl diphosphate.
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Zhan, C., Lei, L., Liu, Z. et al. Selection of a subspecies-specific diterpene gene cluster implicated in rice disease resistance. Nat. Plants 6, 1447–1454 (2020). https://doi.org/10.1038/s41477-020-00816-7
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DOI: https://doi.org/10.1038/s41477-020-00816-7
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