Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.
Subscribe to Journal
Get full journal access for 1 year
only $5.42 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Dixon, R. A. Natural products and plant disease resistance. Nature 411, 843–847 (2001).
Gershenzon, J. & Dudareva, N. The function of terpene natural products in the natural world. Nat. Chem. Biol. 3, 408–414 (2007).
Pichersky, E. & Lewinsohn, E. Convergent evolution in plant specialized metabolism. Ann. Rev. Plant Biol. 62, 549–566 (2011).
Turlings, T. C. J. & Erb, M. Tritrophic interactions mediated by herbivore-induced plant volatiles: mechanisms, ecological relevance, and application potential. Annu. Rev. Entomol. 63, 433–452 (2018).
Lopez-Nieves, S. et al. Relaxation of tyrosine pathway regulation underlies the evolution of betalain pigmentation in Caryophyllales. New Phytol. 217, 896–908 (2018).
Moghe, G. D. & Last, R. L. Something old, something new: conserved enzymes and the evolution of novelty in plant specialized metabolism. Plant Physiol. 169, 1512–1523 (2015).
Chae, L., Kim, T., Nilo-Poyanco, R. & Rhee, S. Y. Genomic signatures of specialized metabolism in plants. Science 344, 510–513 (2014).
Chen, F., Tholl, D., Bohlmann, J. & Pichersky, E. The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 66, 212–229 (2011).
Peters, R. J. Two rings in them all: the labdane-related diterpenoids. Nat. Prod. Rep. 27, 1521–1530 (2010).
Ma, K. W. & Ma, W. Phytohormone pathways as targets of pathogens to facilitate infection. Plant Mol. Biol. 91, 713–725 (2016).
Robert-Seilaniantz, A., Navarro, L., Bari, R. & Jones, J. D. Pathological hormone imbalances. Curr. Opin. Plant Biol. 10, 372–379 (2007).
Robert-Seilaniantz, A., Grant, M. & Jones, J. D. G. Hormone crosstalk in plant disease and defense: more than just jasmonate–salicylate antagonism. Annu. Rev. Phytopathol. 49, 317–343 (2011).
Hedden, P. & Thomas, S. G. Gibberellin biosynthesis and its regulation. Biochem. J. 444, 11–25 (2012).
Mitchell, J. W., Skaggs, D. P. & Anderson, W. P. Plant growth-stimulating hormones in immature bean seeds. Science 114, 159–161 (1951).
Phinney, B. O., West, C. A., Ritzel, M. & Neely, P. M. Evidence for ‘gibberellin-like’ substances from flowering plants. Proc. Natl Acad. Sci. USA 43, 398–404 (1957).
Yabuta, T. Biochemistry of the ‘bakanae’ fungus of rice. Agric. Hort. (Tokyo) 10, 17–22 (1935).
Zi, J., Mafu, S. & Peters, R. J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism. Annu. Rev. Plant Biol. 65, 259–286 (2014).
Gao, Y., Honzatko, R. B. & Peters, R. J. Terpenoid synthase structures: a so far incomplete view of complex catalysis. Nat. Prod. Rep. 29, 1153–1175 (2012).
Schmelz, E. A. et al. Biosynthesis, elicitation and roles of monocot terpenoid phytoalexins. Plant J. 79, 659–678 (2014).
Lu, X. et al. Inferring roles in defense from metabolic allocation of rice diterpenoids. Plant Cell 30, 1119–1131 (2018).
Christensen, S. A. et al. Commercial hybrids and mutant genotypes reveal complex protective roles for inducible terpenoid defenses in maize. J. Exp. Bot. 69, 1693–1705 (2018).
Fu, J. et al. A tandem array of ent-kaurene synthases in maize with roles in gibberellin and more specialized metabolism. Plant Physiol. 170, 742–751 (2016).
Mafu, S. et al. Discovery, biosynthesis and stress-related accumulation of dolabradiene-derived defenses in maize. Plant Physiol. 176, 2677–2690 (2018).
Schmelz, E. A. et al. Identity, regulation, and activity of inducible diterpenoid phytoalexins in maize. Proc. Natl Acad. Sci. USA 108, 5455–5460 (2011).
Vaughan, M. M. et al. Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance. Plant Cell Environ. 38, 2195–2207 (2015).
Bensen, R. J. et al. Cloning and characterization of the maize An1 gene. Plant Cell 7, 75–84 (1995).
Mao, H., Shen, Q. & Wang, Q. CYP701A26 is characterized as an ent-kaurene oxidase with putative involvement in maize gibberellin biosynthesis. Biotechnol. Lett. 39, 1709–1716 (2017).
Mafu, S. et al. Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis. Proc. Natl Acad. Sci. USA 113, 2526–2531 (2016).
Harris, L. J. et al. The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase. Plant Mol. Biol. 59, 881–894 (2005).
Wisecaver, J. H. et al. A global coexpression network approach for connecting genes to specialized metabolic pathways in plants. Plant Cell, 29, 944–959 (2017).
Murphy, K. M., Ma, L. T., Ding, Y. Z., Schmelz, E. A. & Zerbe, P. Functional characterization of two class II diterpene synthases indicates additional specialized diterpenoid pathways in maize (Zea mays). Front. Plant Sci. 9, 1542 (2018).
Morrone, D. et al. Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering. Appl. Microbiol. Biotechnol. 85, 1893–1906 (2010).
Morrone, D. et al. Evident and latent plasticity across the rice diterpene synthase family with potential implications for the evolution of diterpenoid metabolism in the cereals. Biochem. J. 435, 589–595 (2011).
Zerbe, P. & Bohlmann, J. Plant diterpene synthases: exploring modularity and metabolic diversity for bioengineering. Trends Biotechnol. 33, 419–428 (2015).
Bak, S. et al. Cytochromes P450. Arabidopsis Book 9, e0144 (2011).
Wu, Y. S., Hillwig, M. L., Wang, Q. & Peters, R. J. Parsing a multifunctional biosynthetic gene cluster from rice: biochemical characterization of CYP71Z6 & 7. FEBS Lett. 585, 3446–3451 (2011).
Wang, X. Y. et al. Genome alignment spanning major poaceae lineages reveals heterogeneous evolutionary rates and alters inferred dates for key evolutionary events. Mol. Plant 8, 885–898 (2015).
Flint-Garcia, S. A. et al. Maize association population: a high-resolution platform for quantitative trait locus dissection. Plant J. 44, 1054–1064 (2005).
Hedden, P. & Sponsel, V. A century of gibberellin research. J. Plant Growth Regul. 34, 740–760 (2015).
Kono, T. J. Y., Brohammer, A. B., McGaugh, S. E. & Hirsch, C. N. Tandem duplicate genes in maize are abundant and date to two distinct periods of time. G3 8, 3049–3058 (2018).
Helliwell, C. A., Poole, A., James Peacock, W. & Dennis, E. S. Arabidopsis ent-kaurene oxidase catalyzes three steps of gibberellin biosynthesis. Plant Physiol. 119, 507–510 (1999).
Meyer, J., Berger, D. K., Christensen, S. A. & Murray, S. L. RNA-Seq analysis of resistant and susceptible sub-tropical maize lines reveals a role for kauralexins in resistance to grey leaf spot disease, caused by Cercospora zeina. BMC Plant Biol. 17, 197 (2017).
Yang, D. L. et al. Altered disease development in the eui mutants and Eui overexpressors indicates that gibberellins negatively regulate rice basal disease resistance. Mol. Plant 1, 528–537 (2008).
Langlois, V. S., Zhang, D., Cooke, G. M. & Trudeau, V. L. Evolution of steroid-5alpha-reductases and comparison of their function with 5beta-reductase. Gen. Comp. Endocrinol. 166, 489–497 (2010).
Li, J. M., Biswas, M. G., Chao, A., Russell, D. W. & Chory, J. Conservation of function between mammalian and plant steroid 5 alpha-reductases. Proc. Natl Acad. Sci. USA 94, 3554–3559 (1997).
Uemura, M. et al. Novel 5 alpha-steroid reductase (SRD5A3, type-3) is overexpressed in hormone-refractory prostate cancer. Cancer Sci. 99, 81–86 (2008).
Garcia, N., Li, Y., Dooner, H. K. & Messing, J. Maize defective kernel mutant generated by insertion of a Ds element in a gene encoding a highly conserved TTI2 cochaperone. Proc. Natl Acad. Sci. USA 114, 5165–5170 (2017).
McMullen, M. D., Frey, M. & Degenhardt, J. in Handbook of Maize: Its Biology (eds Bennetzen, J. L. & Hake, S. C.) 271–289 (Springer, 2009).
Wouters, F. C., Blanchette, B., Gershenzon, J. & Vassão, D. G. Plant defense and herbivore counter-defense: benzoxazinoids and insect herbivores. Phytochem. Rev. 15, 1127–1151 (2016).
Bagnaresi, P. et al. Comparative transcriptome profiling of the early response to Magnaporthe oryzae in durable resistant vs susceptible rice (Oryza sativa L.) genotypes. PLoS ONE 7, e51609 (2012).
Toyomasu, T. et al. Reverse-genetic approach to verify physiological roles of rice phytoalexins: characterization of a knockdown mutant of OsCPS4 phytoalexin biosynthetic gene in rice. Physiol. Plant. 150, 55–62 (2014).
Brohammer, A. B., Kono, T. J. Y., Springer, N. M., McGaugh, S. E. & Hirsch, C. N. The limited role of differential fractionation in genome content variation and function in maize (Zea mays L.) inbred lines. Plant J. 93, 131–141 (2018).
Schnable, J. C., Springer, N. M. & Freeling, M. Differentiation of the maize subgenomes by genome dominance and both ancient and ongoing gene loss. Proc. Natl Acad. Sci. USA 108, 4069–4074 (2011).
Pelot, K. A. et al. Functional diversity of diterpene synthases in the biofuel crop switchgrass. Plant Physiol. 178, 54–71 (2018).
Miyazaki, S., Katsumata, T., Natsume, M. & Kawaide, H. The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P. FEBS Lett. 585, 1879–1883 (2011).
Morrone, D., Chen, X. M., Coates, R. M. & Peters, R. J. Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis. Biochem. J. 431, 337–344 (2010).
Vrabka, J. et al. Production and role of hormones during interaction of Fusarium species with maize (Zea mays L.) seedlings. Front. Plant Sci. 9, 1936–1936 (2019).
Hedden, P. & Phinney, B. O. Comparison of ent-kaurene and ent-isokaurene synthesis in cell-free systems from etiolated shoots of normal and dwarf-5 maize seedlings. Phytochemistry 18, 1475–1479 (1979).
Bohlmann, J., Meyer-Gauen, G. & Croteau, R. Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc. Natl Acad. Sci. USA 95, 4126–4133 (1998).
Laursen, T. et al. Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum. Science 354, 890–893 (2016).
Mehrshahi, P., Johnny, C. & DellaPenna, D. Redefining the metabolic continuity of chloroplasts and ER. Trends Plant Sci. 19, 501–507 (2014).
Wiemann, P. et al. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog. 9, e1003475 (2013).
Lu, X., Hershey, D. M., Wang, L., Bogdanove, A. J. & Peters, R. J. An ent-kaurene-derived diterpenoid virulence factor from Xanthomonas oryzae pv. oryzicola. New Phytol. 206, 295–302 (2015).
Williams-Carrier, R. et al. Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize. Plant J. 63, 167–177 (2010).
Ding, Y. Z. et al. Selinene volatiles are essential precursors for maize defense promoting fungal pathogen resistance. Plant Physiol. 175, 1455–1468 (2017).
Huffaker, A. et al. Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Physiol. 156, 2082–2097 (2011).
Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).
Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010).
Kremling, K. A. G. et al. Dysregulation of expression correlates with rare-allele burden and fitness loss in maize. Nature 555, 520 (2018).
Obayashi, T. & Kinoshita, K. Rank of correlation coefficient as a comparable measure for biological significance of gene coexpression. DNA Res. 16, 249–260 (2009).
Horevaj, P., Milus, E. A. & Bluhm, B. H. A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels. J. Appl. Microbiol. 111, 396–406 (2011).
Zhang, Z. W. et al. Mixed linear model approach adapted for genome-wide association studies. Nat. Genet. 42, 355–U118 (2010).
Yu, J. M. et al. A unified mixed-model method for association mapping that accounts for multiple levels of relatedness. Nat. Genet. 38, 203–208 (2006).
Lipka, A. E. et al. GAPIT: genome association and prediction integrated tool. Bioinformatics 28, 2397–2399 (2012).
Bradbury, P. J. et al. TASSEL: software for association mapping of complex traits in diverse samples. Bioinformatics 23, 2633–2635 (2007).
Cook, J. P. et al. Genetic architecture of maize kernel composition in the nested association mapping and inbred association panels. Plant Physiol. 158, 824–834 (2012).
Elshire, R. J. et al. A robust, simple genotyping-by-sequencing (GBS) approach for high diversity species. PLoS ONE 6, e19379 (2011).
Samayoa, L. F., Malvar, R. A., Olukolu, B. A., Holland, J. B. & Butron, A. genome-wide association study reveals a set of genes associated with resistance to the Mediterranean corn borer (Sesamia nonagrioides L.) in a maize diversity panel. BMC Plant Biol. 15, 35 (2015).
VanRaden, P. M. Efficient methods to compute genomic predictions. J. Dairy Sci. 91, 4414–4423 (2008).
Turner, S. D. qqman: an R package for visualizing GWAS results using Q–Q and Manhattan plots. Preprint at https://doi.org/10.1101/005165 (2014).
Schmelz, E. A., Engelberth, J., Tumlinson, J. H., Block, A. & Alborn, H. T. The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites. Plant J. 39, 790–808 (2004).
Bach, S. S. et al. High-throughput testing of terpenoid biosynthesis candidate genes using transient expression in Nicotiana benthamiana. Methods Mol. Biol. 1153, 245–255 (2014).
Kitaoka, N., Lu, X., Yang, B. & Peters, R. J. The application of synthetic biology to elucidation of plant mono-, sesqui-, and diterpenoid metabolism. Mol. Plant 8, 6–16 (2015).
Zerbe, P. et al. Gene discovery of modular diterpene metabolism in nonmodel systems. Plant Physiol. 162, 1073–1091 (2013).
Brazelton, V. A. et al. A quick guide to CRISPR sgRNA design tools. GM Crops Food 6, 266–276 (2015).
Char, S. N. et al. An agrobacterium‐delivered CRISPR/Cas9 system for high‐frequency targeted mutagenesis in maize. Plant Biotechnol. J. 15, 257–268 (2017).
We thank A. Steinbrenner, P. Weckwerth, K. Dressano, J. Chan, K. O’Leary, M. Broemmer, H. Riggleman, S. Reyes, S. Delgado and J. Tran for help in planting, treatments and sampling (UCSD). Thanks also to L. Smith (UCSD) for shared UCSD Biology Field Station management. This work was supported by a grant from the National Science Foundation Plant-Biotic Interactions Program (grant no. 1758976 to E.S. and P.Z.), by a Department of Energy Joint Genome Institute Community Science Program grant (no. CSP2568 to P.Z., E.S. and A.H.) and by a fellowship provided by the National Science Foundation Graduate Research Fellowship Program (to K.M.M.).
The authors declare no competing interests.
Peer Review Information: Nature Plants thanks Hugues Renault and other, anonymous, reviewers for their contribution to the peer review of this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–22 and Supplementary Table 6.
Southern leaf blight (SLB; Cochliobolous heterostrophus) induced gene expression in maize leaves.
Summary of SLB-induced expression levels of diterpene synthases and related pathway genes in maize leaves.
Summary of SLB-induced expression levels of the maize cytochrome P450 gene family.
Co-expression analyses of ZmKSL2 with maize P450 gene family and all maize genes.
GWAS mapping interval identified using the ratio of highly oxidized kauralexins to total kauralexins.
Using the ratio of kauralexin A-/B-series metabolites as a trait, GWAS identifies an interval containing steriod 5a reductase candidates.
Protein fold changes of DiTPS pathway enzymes in the stem tissues (Zea mays var. W22) elicited with heat-killed Fusarium venenatum.
Maize mapping lines used for replicated genome wide association studies (GWAS) in the Goodman diversity panel and quantitative trait loci mapping in the nested association mapping subpopulation B73 × M162W.
Primers used for qrtPCR analysis.
Primers used for mutant identification.
Primers used for gene cloning into the pLIFE33 expression vector from cDNA.
Abbreviations and accession identification numbers for diterpene synthases, kaurene oxidases, steriod 5α-reductases and CYP71 family proteins used for phylogenetic analysis in this study.
Gene sequences used in enzyme co-expression studies including native sequences, synthetic sequences, and codon-optimized sequences for expression in E. coli.