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

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.

The foxtail millet (Setaria italica) terpene synthase gene family

“…rice, switchgrass) or (+)-CPP (e.g. maize, wheat) in addition to the ubiquitously occurring ent-CPP (Schmelz et al, 2014;Murphy et al, 2018;Pelot et al, 2018). Although syntenic orthologs for SiTPS6 and SiTPS9 were present in all analyzed genomes (except for B. distachyon), the respective maize, rice and switchgrass orthologs did not show functional conservation.…”

“…Although syntenic orthologs for SiTPS6 and SiTPS9 were present in all analyzed genomes (except for B. distachyon), the respective maize, rice and switchgrass orthologs did not show functional conservation. For example, the SiTPS6 orthologs of rice (OsCPS2) and maize (ZmCPS4) encode for an ent-CPP synthase and 8,13-CPP synthase, respectively, rather than syn-CPP synthases (Prisic et al, 2004;Murphy et al, 2018). Likewise, of the identified maize and rice orthologs to the (+)-CPP synthase SiTPS9, only ZmCPS3 encodes for a (+)-CPP synthase, whereas rice OsCPS4 encodes a syn-CPP synthase and maize ZmCPS4 forms 8,13-CPP (Xu et al, 2004;Otomo et al, 2004;Murphy et al, 2018).…”

“…For example, the SiTPS6 orthologs of rice (OsCPS2) and maize (ZmCPS4) encode for an ent-CPP synthase and 8,13-CPP synthase, respectively, rather than syn-CPP synthases (Prisic et al, 2004;Murphy et al, 2018). Likewise, of the identified maize and rice orthologs to the (+)-CPP synthase SiTPS9, only ZmCPS3 encodes for a (+)-CPP synthase, whereas rice OsCPS4 encodes a syn-CPP synthase and maize ZmCPS4 forms 8,13-CPP (Xu et al, 2004;Otomo et al, 2004;Murphy et al, 2018). Close phylogenetic interrelations of SiTPS6 with ZmCPS4 and of SiTPS9 with ZmCPS3 and OsCPS4 are consistent with this functional divergence.…”

“…© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd, The Plant Journal, (2020), 103, 781-800 wheat (+)-CPP synthase TaCPS2 (Wu et al, 2012;Murphy et al, 2018) (Figures 1a and S1). The remaining 28 TPS candidates were designated as putative class I TPSs on account of the presence of only the conserved C-terminal DDxxD motif ( Figure S2).…”

“…Similarly, the predicted ent-kaurene synthases SiTPS28/29 (chromosome 7) clustered in a syntenic network with ent-kaurene synthases from rice (OsKS1, chromosome 4), maize (ZmKSL3/5, chromosome 2) and switchgrass (PvKSL13/15, chromosome 7). Additionally, S. italica class II diTPS candidates potentially involved in specialized metabolism, such as SiTPS6, were in syntenic networks with the ent-CPP synthase of specialized metabolism in rice (OsCPS2, chromosome 2) and 8,13-CPP synthases from maize (ZmCPS4, chromosome 4) and switchgrass (PvCPS3, chromosome 1) (Prisic et al, 2004;Murphy et al, 2018;Pelot et al, 2018). Likewise, SiTPS9 was placed in a syntenic network with ZmCPS4 and the maize (+)-CPP synthase ZmCPS3 (chromosome 10) (Murphy et al, 2018), PvCPS3 (Pelot et al, 2018) and the rice syn-CPP synthase OsCPS4 (chromosome4) (Xu et al, 2004;Otomo et al, 2004).…”