Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Oct 23:9:1542.
doi: 10.3389/fpls.2018.01542. eCollection 2018.

Functional Characterization of Two Class II Diterpene Synthases Indicates Additional Specialized Diterpenoid Pathways in Maize (Zea mays)

Katherine M Murphy  1 , Li-Ting Ma  1   2 , Yezhang Ding  3 , Eric A Schmelz  3 , Philipp Zerbe  1
Affiliations

Functional Characterization of Two Class II Diterpene Synthases Indicates Additional Specialized Diterpenoid Pathways in Maize (Zea mays)

Katherine M Murphy et al. Front Plant Sci. .

Abstract

As a major staple food, maize (Zea mays) is critical to food security. Shifting environmental pressures increasingly hamper crop defense capacities, causing expanded harvest loss. Specialized labdane-type diterpenoids are key components of maize chemical defense and ecological adaptation. Labdane diterpenoid biosynthesis most commonly requires the pairwise activity of class II and class I diterpene synthases (diTPSs) that convert the central precursor geranylgeranyl diphosphate into distinct diterpenoid scaffolds. Two maize class II diTPSs, ANTHER EAR 1 and 2 (ZmAN1/2), have been previously identified as catalytically redundant ent-copalyl diphosphate (CPP) synthases. ZmAN1 is essential for gibberellin phytohormone biosynthesis, whereas ZmAN2 is stress-inducible and governs the formation of defensive kauralexin and dolabralexin diterpenoids. Here, we report the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 (ZmCPS3) and COPALYL DIPHOSPHATE SYNTHASE 4 (ZmCPS4). Functional analysis via microbial co-expression assays identified ZmCPS3 as a (+)-CPP synthase, with functionally conserved orthologs occurring in wheat (Triticum aestivum) and numerous dicot species. ZmCPS4 formed the unusual prenyl diphosphate, 8,13-CPP (labda-8,13-dien-15-yl diphosphate), as verified by mass spectrometry and nuclear magnetic resonance. As a minor product, ZmCPS4 also produced labda-13-en-8-ol diphosphate (LPP). Root gene expression profiles did not indicate an inducible role of ZmCPS3 in maize stress responses. By contrast, ZmCPS4 showed a pattern of inducible gene expression in roots exposed to oxidative stress, supporting a possible role in abiotic stress responses. Identification of the catalytic activities of ZmCPS3 and ZmCPS4 clarifies the first committed reactions controlling the diversity of defensive diterpenoids in maize, and suggests the existence of additional yet undiscovered diterpenoid pathways.

Keywords: Zea mays; diterpene synthase; diterpenoid biosynthesis; plant specialized metabolism; plant stress response.

PubMed Disclaimer

Figures

FIGURE 1
FIGURE 1
Class II diterpene synthase functions in maize diterpenoid metabolism. Maize deploys functionally diverse families of class II and class I diterpene synthases (diTPSs) that act sequentially to form the core scaffolds of diverse biologically active diterpenoids that mediate plant growth and responses to both biotic and abiotic stress. Among the four class II diTPSs present in the maize genome, previous studies showed that ZmAN1 functions in gibberellin phytohormone biosynthesis, whereas ZmAN2 controls the formation of kauralexin and dolabralexin diterpenoids as major components of the maize chemical defense system (Schmelz et al., 2011; Mafu et al., 2018). ZmCPS3 and ZmCPS4 represent additional specialized class II diTPSs that form (+)-CPP and 8,13-CPP as major products, respectively, representing previously hidden precursors of maize specialized diterpenoid metabolism. GGPP, geranylgeranyl diphosphate; CPP, copalyl diphosphate; LPP, labda-13-en-8-ol diphosphate.
FIGURE 2
FIGURE 2
Active site determinants of monocot class II diterpene synthases. Illustrated is a protein sequence alignment highlighting key active site residues with demonstrated impact on product specificity of known monocot class II diTPSs. ZmCPS3 and ZmCPS4 feature distinct residues in select active site positions defining enzyme product specificity, thus suggesting a (+)-CPP synthase, 8,13-CPP synthase, or other specialized class II diTPS function for ZmCPS3 and ZmCPS4. Residue positions are numbered in reference to ZmAN2. Zm, Zea mays; Os, Oryza sativa; Ta, Triticum aestivum; Hv, Hordeum vulgare; Pv, Panicum virgatum.
FIGURE 3
FIGURE 3
Functional characterization of the maize diterpene synthase ZmCPS3. Extracted ion chromatograms (EIC, m/z 257) and mass spectra of reaction products resulting from E. coli co-expression assays of ZmAN2 and ZmCPS3 alone, as well as in combination with the ent-kaurene synthase ZmKSL3 (Fu et al., 2016) or the dolabradiene synthase ZmKSL4 (Mafu et al., 2018). (+)-copalol (i.e., dephosphorylated copalyl diphosphate, CPP) 1, geranylgeraniol (i.e., dephosphorylated geranylgeranyl diphosphate, GGPP) 2, plasticizer contaminant 3, pimara-8,14-diene 5, ent-copalol (i.e., dephosphorylated ent-CPP) 8, ent-CPP derivative 9, ent-kaurene 10, dolabradiene 11, unidentified pimarane-type products 4, 6, and 7. Where applicable, depicted structures represent the native prenyl diphosphate class II diTPS products, whereas spectra are derived from the corresponding dephosphorylated compounds that are formed during enzyme co-expression analyses by the activity of endogenous E. coli phosphatases.
FIGURE 4
FIGURE 4
Functional characterization of the maize diterpene synthase ZmCPS4. (A) Extracted ion chromatograms (EIC, m/z 257) of reaction products resulting from E. coli co-expression assays of ZmCPS4 alone or in combination with the ent-kaurene synthase ZmKSL3 (Fu et al., 2016) or the dolabradiene synthase ZmKSL4 (Mafu et al., 2018). Expression of the LPP synthase GrTPS1 (Zerbe et al., 2013) was used to produce a labda-13-en-8-ol (i.e., dephosphorylated labda-13-en-8-ol diphosphate, LPP) standard 13, and co-expression of GrTPS1 and the multi-functional class I diTPS MvELS (Zerbe et al., 2014) enabled the formation of a manoyl oxide 15 standard. (B) Mass spectrum of the ZmCPS4 product, 8,13-copalol (i.e., dephosphorylated 8,13-CPP) 12. (C) Structural identification of the ZmCPS4 product as 8,13-CPP as verified by NMR analysis. Compounds 14, 16, and 17 represent unidentified LPP and 8,13-CPP derivatives. (D) Comparison of mass spectra of the ZmCPS4 products, compounds 13 and 15 to those of authentic standards of labda-13-en-8-ol (i.e., dephosphorylated labda-13-en-8-ol diphosphate, LPP) produced by GrTPS1 and manoyl oxide produced by combining GrTS1 and MvELS. Where applicable, depicted structures represent the native prenyl diphosphate class II diTPS products, whereas spectra are derived from the corresponding dephosphorylated compounds that are formed during enzyme co-expression analyses by the activity of endogenous E. coli phosphatases.
FIGURE 5
FIGURE 5
Gene expression of maize class II diterpene synthases across maize developmental stages and tissues. (A) Heat map showing the individually scaled mRNA expression (FPKM) of class II diTPS transcripts as based on a publically available transcriptome data panel comprising various maize tissue types and developmental stages (Walley et al., 2016). (B) Heat map showing only root samples, scaled to an absolute (not individual) scale. All samples are derived from maize genotype B73 with the exception of 5-days-old primary root (Mo17 inbred) and 2 cm tassels, 1–2 mm anthers, and mature pollen (W23 inbred).
FIGURE 6
FIGURE 6
Transcript abundance of ZmCPS3 and ZmCPS4 under biotic and abiotic stress. Transcript abundance was measured by qPCR and is depicted as average fold change (2-ΔΔCt) of the transcript abundance of ZmCPS3 (A) and ZmCPS4 (B) in Mo17 roots incision-inoculated with live conidia, separately with Fusarium verticillioides (F.v.) and F. graminearum (F.g.). Plants treated by incision and application of water only were used as controls. Treatments occurred in 53-days-old plants with tissue harvests 7 days later (n = 4). Average fold change (2-ΔΔCt) of the transcript abundance of ZmCPS3 (C) and ZmCPS4 (D) in Golden Queen roots treated with 1 mM CuSO4 and sampled over 24 h as compared to a water-treated control (n = 3). A fold change of one is equal to no change from the control. Transcript abundance was normalized to the maize internal reference gene EF1α. Error bars represent propagated standard error of the mean fold change of the biological replicates. Asterisks indicate significant change compared to control with P-value < 0.05 as measured with two-tailed Student’s t-tests. A Dunnett’s test was used for fungal treated samples to compare to the single wounded control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahmad S., Veyrat N., Gordon-Weeks R., Zhang Y., Martin J., Smart L., et al. (2011). Benzoxazinoid metabolites regulate innate immunity against aphids and fungi in maize. Plant Physiol. 157 317–327. 10.1104/pp.111.180224 - DOI - PMC - PubMed
    1. Bensen R. J., Johal G. S., Crane V. C., Tossberg J. T., Schnable P. S., Meeley R. B., et al. (1995). Cloning and characterization of the maize An1 gene. Plant Cell 7 75–84. 10.1105/tpc.7.1.75 - DOI - PMC - PubMed
    1. Caniard A., Zerbe P., Legrand S., Cohade A., Valot N., Magnard J. L., et al. (2012). Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture. BMC Plant Biol. 12:119. 10.1186/1471-2229-12-119 - DOI - PMC - PubMed
    1. Chakraborty S., Newton A. C. (2011). Climate change, plant diseases and food security: an overview. Plant Pathol. 60 2–14. 10.1111/j.1365-3059.2010.02411.x - DOI - PubMed
    1. Chaturvedi R., Venables B., Petros R. A., Nalam V., Li M., Wang X., et al. (2012). An abietane diterpenoid is a potent activator of systemic acquired resistance. Plant J. 71 161–172. - PubMed