Phytochemistry 172 (2020) 112289

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Phytochemistry

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Review Specialized diterpenoid metabolism in monocot crops: Biosynthesis and ☆ chemical diversity T

∗ Katherine M. Murphya, Philipp Zerbea, a Department of Plant Biology, University of California-Davis, One Shields Avenue, Davis, CA, 95616, USA

ARTICLE INFO ABSTRACT

Keywords: Among the myriad specialized metabolites that plants employ to mediate interactions with their environment, Diterpene biosynthesis diterpenoids form a chemically diverse group with vital biological functions. A few broadly abundant diterpe- Plant specialized metabolism noids serve as core pathway intermediates in plant general metabolism. The majority of plant diterpenoids, Crop defense however, function in specialized metabolism as often species-specific chemical defenses against herbivores and Plant-environment interactions microbial diseases, in below-ground allelopathic interactions, as well as abiotic stress responses. Dynamic net- Plant natural products works of anti-microbial diterpenoids were first demonstrated in rice (Oryza sativa) over four decades ago, and more recently, unique diterpenoid blends with demonstrated antibiotic bioactivities were also discovered in maize (Zea mays). Enabled by advances in -omics and biochemical approaches, species-specific diterpenoid- diversifying enzymes have been identified in these and other Poaceous species, including wheat (Triticum aestivum) and switchgrass (Panicum virgatum), and are discussed in this article with an emphasis on the critical diterpene synthase and cytochrome P450 monooxygenase families and their products. The continued investigation of the biosynthesis, diversity, and function of terpenoid-mediated crop defenses provides founda- tional knowledge to enable the development of strategies for improving crop resistance traits in the face of impeding pest, pathogen, and climate pressures impacting global agricultural production.

1. Introduction 2013). However, despite the extensive advances in agricultural prac- tices in the past 50 years, impeding climate shifts and associated pest Poaceous, monocot grain crops are the most important source of and disease pressures cause substantial economic losses across small- global food security. Grain consumption accounts for more than 50% of and large-scale agrosystems (Chakraborty and Newton, 2011; de Sassi world daily caloric intake, the majority of which is provided by only and Tylianakis, 2012), and threaten our ability to meet the required three crops, maize (Zea mays), wheat (Triticum aestivum), and rice agricultural outputs (Liang et al., 2017; Ray et al., 2012, 2015). In 2015 (Oryza sativa)(Food and Agriculture Organization of the United alone, fungal diseases caused a nearly 14% loss in U.S. maize produc- Nations, 2009; Tilman et al., 2011). Beyond their importance for food tion, not including harvest loss and human health risks caused by ac- and livestock feed production, monocot crops, such as maize, sorghum companying mycotoxin contamination (Crop Protection Network, (Sorghum bicolor), and switchgrass (Panicum virgatum), are of economic 2017). Similarly, insect and other herbivore damage is a major con- value as feedstock for lignocellulosic biofuel production (Mullet et al., tributor to crop losses worldwide (Oerke, 2006). At a global level, pest- 2014; Tubeileh et al., 2016). For example, 36% of maize produced in and disease-related crop losses are estimated to constitute 22–30% in the U.S. was used for ethanol production in 2016 (U.S. Department of the major cereal crops, rice, maize, and wheat, highlighting the need to Energy, 2016). develop crops that can provide nutritious food while being able to To meet the needs of a growing global population, the United withstand increasingly detrimental environmental conditions (Savary Nations has forecasted a required doubling of crop production (Food et al., 2019; Wurtzel and Kutchan, 2016). To address this challenge, and Agriculture Organization of the United Nations, 2009; Ray et al., research eﬀorts have focused on elucidating the natural defenses

Abbreviations: diTPS, diterpene synthase; GGDP, geranylgeranyl diphosphate; P450, cytochrome P450-dependent monooxygenase; CDP, copalyl diphosphate; MS, mass spectrometry; CPS, copalyl diphosphate synthase; KSL, kaurene synthase-like ☆ This invited review is associate with the award to Dr. Philipp Zerbe of the 2018 Elsevier/Phytochemical Society of North America Young Investigator Award in recognition of his outstanding contribution to studies of the biosynthesis, diversity and funcion of plant terpenoid metabolism. ∗ Corresponding author. E-mail address: [email protected] (P. Zerbe). https://doi.org/10.1016/j.phytochem.2020.112289 Received 13 December 2019; Received in revised form 24 January 2020; Accepted 28 January 2020 0031-9422/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289 mechanisms underlying crop resilience, thus providing essential re- Unlike other terpenoids, the biosynthesis of labdane-related struc- sources for improving crop traits (Schmelz et al., 2014; Wurtzel and tures proceeds via dual biosynthetic cyclization and/or rearrangement Kutchan, 2016). reactions catalyzed by the activity of pairs of class II and class I diTPS A major mechanism by which plants interact with and adapt to their enzymes (Peters, 2010; Zerbe and Bohlmann, 2015). After initial pro- environments is through the production of elaborate cocktails of spe- tonation-initiated cyclization of GGDP into distinct bicyclic copalyl cialized metabolites. Constitutive and inducible metabolite groups ei- diphosphate (CDP) intermediates by class II diTPSs, class I diTPSs cat- ther proven or predicted to contribute to the defense against biotic and alyze the ionization of the diphosphate ester and downstream sec- abiotic stressors in monocot crops include terpenoids, phenylpropa- ondary cyclization and/or rearrangements (Peters, 2010; Karunanithi noids, oxylipins, benzoxazinoids, and carotenoids (Christensen et al., and Zerbe, 2019). Functionalization of the resulting diterpene scaffolds 2015; de Bruijn et al., 2018; Schmelz et al., 2014). Among these me- is then facilitated by P450s and select other enzyme classes such as tabolites, diterpenoids (20-carbon terpenoids) comprise a vast group of reductases, dehydrogenases, and transferases. Oxygenation reactions more than 10,000 chemically and functionally diverse natural products. catalyzed by often promiscuous P450s is essential toward forming dis- A few ubiquitously distributed diterpenoids play key roles in develop- tinct bioactive diterpenoids. Presently known P450s with functions in mental processes as intermediates of general metabolism, such as the monocot diterpenoid biosynthesis belong to the CYP71, CYP76, CYP99, biosynthesis of chlorophyll, plastoquinones, and gibberellin (GA) phy- and CYP701 families (Banerjee and Hamberger, 2018; Bathe and tohormones (Liu and Lu, 2016; Salazar-Cerezo et al., 2018). However, Tissier, 2019; Nelson and Werck-Reichhart, 2011; Schmelz et al., 2014). most diterpenoids are specialized metabolites that often represent sig- Class II and class I diTPS pairs involved in GA metabolism represent nature molecules of individual plant species or families, and biosynth- the ancestral form of these pairwise reactions and have served as a esis of which is commonly tissue- or organ-specific and underlies a strict genetic archetype to the diversification of plant specialized diterpenoid regulation by environmental stimuli (Celedon and Bohlmann, 2019; metabolism (Zi et al., 2014). Through repeated events of gene and Gershenzon and Dudareva, 2007; Schmelz et al., 2014; Tholl, 2015). genome duplication, accompanied by expansive sub- or neo-functio- Often occurring in combination with volatile mono- and sesqui-terpe- nalization, species-specific multi-enzyme diTPS and P450 families have noid defenses, higher molecular weight and typically extensively evolved (Zi et al., 2014; Bathe and Tissier, 2019; Chen et al., 2011). functionalized diterpenoids have been established as local anti- These protein families can form dynamic, modular pathway networks, microbial phytoalexins in monocot crops such as maize and rice (Block where functionally distinct and often substrate- or product-promiscuous et al., 2019; Hammerbacher et al., 2019; Peters, 2006; Schmelz et al., enzyme modules can act in different combinations to fine-tune the 2014). formation of specific diterpenoids in response to different environ- During the past decades a broader spectrum of diterpenoid functions mental stimuli (Karunanithi and Zerbe, 2019; Banerjee and Hamberger, has been discovered, including defenses against insect pests, bacterial 2018). Such modular pathway networks appear to be the predominant and fungal diseases, and competing weedy plants, as well as abiotic organizational structure of diterpenoid metabolism in Poaceous crops, stressors (Schmelz et al., 2014). The diversity of specialized diterpe- leading to species-specific diterpenoid blends (Schmelz et al., 2014). noids is realized by diverse families of diterpene synthase (diTPS) and Genome-wide discovery of diterpenoid pathways in rice, maize, cytochrome P450 monooxygenase (P450) enzymes that form modular wheat and switchgrass revealed diTPS families of 10–31 members (Ding pathway networks to convert a central diterpenoid precursor, ger- et al., 2019; Mafu et al., 2018; Murphy et al., 2018; Pelot et al., 2018; anylgeranyl diphosphate (GGDP), into an array of compounds with Prisic et al., 2004; Wu et al., 2012; Xu et al., 2004, 2007a; Zhou et al., distinct scaffold arrangements and secondary functional decorations 2012), and mining of the publicly available genomes of the related that define the range of diterpenoid bioactivities (Banerjee and Poaceous grasses Brachypodium distachyon, foxtail millet (Setaria ita- Hamberger, 2018; Karunanithi and Zerbe, 2019). Advances in the in- lica), barley (Hordeum vulgare), and sorghum (Sorghum bicolor) suggest tegration of genome sequencing, DNA synthesis, and biochemical and similar family sizes of 4–10 members (Chen et al., 2011). Notably, with genetic approaches have accelerated the analysis of diterpenoid meta- the exception of the allotetraploid switchgrass, diTPS gene family sizes bolism beyond the well-established diterpenoid pathway networks in appear independent of the genome size and ploidy across these crops rice and maize (Schmelz et al., 2014), and multi-enzyme diTPS and (Fig. 1). Similarly, in all presently investigated Poaceous species, the P450 families along with species-specific diterpenoid products have class I diTPS families have undergone a more extensive expansion and recently been described in wheat and switchgrass (Pelot et al., 2018; functional divergence, whereas class II diTPSs feature a narrower cat- Wu et al., 2012; Zhou et al., 2012). Building on prior reviews on spe- alytic range with several products commonly occurring across monocot cialized diterpenoid metabolism (Celedon and Bohlmann, 2019; Chen crops and the plant kingdom as a whole (Fig. 1). et al., 2011; Karunanithi and Zerbe, 2019 ; Peters, 2006; Schmelz et al., In addition to ent-CPS genes critical for GA biosynthesis, biochem- 2014), this review provides an overview on recent research progress ical diTPS characterization showed that, to current knowledge, all regarding the diverse biosynthesis, biochemistry, and physiological Poaceous crops contain CPS enzymes producing a CDP compound of importance of monocot diterpenoid metabolism. either syn- or normal/(+)-stereochemistry (Bensen et al., 1995; Harris et al., 2005; Murphy et al., 2018; Pelot et al., 2018; Prisic et al., 2004; 2. Monocot crops employ species-specific diterpenoid networks Wu et al., 2012)(Fig. 1). Additional enzymes producing CDP isomers, such as 8,13-CDP and terpentedienyl diphosphate (TDP), and the hy- 2.1. Biosynthesis of specialized diterpenoids in monocot crops droxylated CDP compound 8-hydroxy labdadienyl diphosphate (LDP), have further been reported in maize and switchgrass (Murphy et al., Monocot diterpenoids almost invariably belong to the large group of 2018; Pelot et al., 2018). Due to their importance for crop yield and labdane-type terpenoids, comprising bi-, tri-, and tetra-cyclic structures stress resilience, intensive research efforts have focused on a deeper with a core decalin ring structure (Peters, 2010). As with all terpenoids, understanding of these dynamic metabolic networks and the biological diterpenoids derive from the universal five-carbon precursor iso- function of specialized diterpenoids in monocot crops, recent advances pentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate on which are highlighted below. (DMADP) that are formed via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways (Davis and 2.2. Biosynthesis and function of diterpenoids in rice Croteau, 2000; Peters, 2010). The MEP pathway is the primary route toward producing the central GGDP diterpenoid precursor, although Rice has served as a long-standing monocot model system to in- some MVA-MEP crosstalk has been demonstrated (Hemmerlin et al., vestigate the biosynthesis, evolution, and function of specialized di- 2003; Laule et al., 2003). terpenoids (Peters, 2006; Schmelz et al., 2014). Pioneered by early

2 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289

appear common among grasses with similar functional ent-CPS pairs shown or predicted in maize, wheat, and switchgrass (Fig. 1). Downstream of ent- and syn-CDP, a family of nine kaurene-synthase- like (KSL) class I diTPSs have been characterized in rice (Alamgir et al., 2016; Schmelz et al., 2014; Xu et al., 2007a)(Fig. 2). Functional diversity of these class I diTPSs enables the formation of an array of diterpene scaffolds, among which syn-stemarene formed by OsKSL8, syn- stemodene formed by OsKSL11, and cassadiene produced by OsKLS7 are uniquely found in rice (Jia et al., 2016; Morrone et al., 2006, 2011; Nemoto et al., 2004; Xu et al., 2007a). Beyond the functional variation within the rice class I diTPS family, at least OsKSL10 shows substrate promiscuity, converting ent-CPP into ent-sandaracopimaradiene en route to bioactive oryzalexins and forming syn-labda-8 (14),15-diene from syn-CDP as a precursor to yet unidentified diterpenoids (Kanno et al., 2006; Xu et al., 2007a). This expansive functional diversity of the rice diTPS family exemplifies the ease with which new diterpenoid structures can evolve through gene duplication and subsequent neo- functionalization of enzyme functions and their use in combinatorial pathway branches. For example, a single Ile to Thr substitution in the active site of the rice ent-kaurene synthase, OsKS1 of general metabolism, was shown to be sufficient to alter product specificity from the GA precursor ent-kaurene to the specialized diterpene ent-pimaradiene (Xu et al., 2007b). This evolutionary plasticity is reflected in the natural variation of diterpenoid profiles and diTPS functions across rice varieties. For example, the wild rice species O. rufipogon has been shown to form momilactones and phytocassanes, but lacks oryzalexins A-F and oryzalexin S due to a functional difference in the OsKSL10 homolog of O. rufipogon that forms ent-miltaradiene instead of ent-sandaracopimaradiene (Miyamoto et al., 2016; Toyomasu et al., 2016). Similar functional differences have also been described for OsKSL5 that forms ent-isokaurene in O. sativa var. indica, while the homolog in O. sativa var. Fig. 1. Size and functional diversity of diterpene synthase families in Poaceous japonica carries a single point mutation resulting in the production of crops. (A) Genome size (grey) and number of demonstrated or predicted di- ent-pimaradiene instead (Xu et al., 2007b). Likewise, O. sativa var. ja- terpene synthases (diTPSs; black) in selected Poaceous crop species. Genome ponica OsKSL8 generates syn-stemarene, while the O. sativa var. indica sizes were retrieved from public repositories (https://phytozome.jgi.doe.gov/; homolog forms syn-stemodene (Morrone et al., 2006; Nemoto et al., https://www.maizegdb.org/; http://www.gramene.org/). (B) Presence of 2004; Toyomasu et al., 2016). These findings highlight not only the functionally characterized Poaceous class II diTPSs involved in general (black rapid divergence of plant diterpenoid metabolism, but underscore the circles), specialized (white circles), and general and/or specialized (grey cir- importance of understanding metabolic variation beyond established cles) metabolism. Triangles represent class I diTPSs shown to function in model cultivars and inbred lines in order to capture the extent of che- combination with individual class II diTPSs in in vitro or in vivo activity assays. mical diversity of such natural defenses. In addition to the rice diTPS family, several P450 enzymes of the work demonstrating the inducible formation of diterpenoids in rice cell CYP701A, CYP71Z, CYP76M, and CYP99A subfamilies with roles in suspension cultures (Wickham and West, 1987), several decades of re- functionally decorating the different diterpene scaffolds have been search have demonstrated that rice produces a unique repertoire of identified (Ko et al., 2008; Swaminathan et al., 2009; Wang et al., 2011, specialized labdane diterpenoids, comprised of oryzalides, phyto- 2012a, 2012b; Wu et al., 2011; Ye et al., 2018). Alike the presence of cassanes, momilactones and oryzalexins derived from kaurene, cassa- duplicated ent-CPSs, the rice genome contains two paralogous ent- diene, and pimaradiene/stemarene scaffolds, respectively (Peters, kaurene oxidase genes, OsKO2 (CYP701A6) and OsKOL4 (CYP701A8). 2006; Schmelz et al., 2014). OsKO2 is required for GA metabolism, whereas OsKOL4 shows different Numerous diTPSs, P450s, and select other enzymes that govern substrate- and regio-specificity of the catalyzed hydroxylation reaction diterpenoid chemical diversity in rice have been identified (Fig. 2). and yields intermediates of oryzalexin and phytocassane biosynthesis Three class II diTPSs that produce CDP of ent- (OsCPS1 and OsCPS2) or (Ko et al., 2008; Wang et al., 2012b). Recent studies further identified syn- (OsCPS4) stereochemistry define the stereochemical range of rice the role of short-chain alcohol dehydrogenase/reductases (SDRs) in rice diterpenoids (Otomo et al., 2004; Prisic et al., 2004; Sakamoto et al., diterpenoid metabolism. This currently includes OsSDR110C-MS2 and 2004; Xu et al., 2004). Interestingly, class II diTPSs producing a CDP of OsSDR110C-MS1 (OsMAS) capable of catalyzing the final oxidation (+)-stereochemistry, abundant in many angiosperms (Zerbe and reaction in the biosynthesis of the major rice diterpenoid, momilactone Bohlmann, 2015), is absent in rice, with the exception of the wild rice A, as well as OsSDR110C-MI3 and OsSDR110C-MS3 that oxidize or- species, Oryza brachyantha, where a (+)-CPS but not a syn-CPS has yzalexin D en route to the formation of oryzalexins A-C (Atawong et al., been reported (Toyomasu et al., 2018). The two rice ent-CPS are cata- 2002; Kitaoka et al., 2016; Shimura et al., 2007). Importantly, con- lytically redundant, yet were demonstrated to have distinct metabolic trasting diterpenoid-metabolic genes in other investigated Poaceous roles in planta, where OsCPS1 is required for GA biosynthesis, and crops (see below), some diterpenoid pathways in rice are organized in OsCPS2 is a stress-elicited gene dedicated to specialized diterpenoid form of biosynthetic gene clusters (Boutanaev et al., 2015; Miyamoto metabolism, as demonstrated with knock-out plant mutants and tran- et al., 2016; Shimura et al., 2007). This includes the formation of mo- script localization (Otomo et al., 2004; Prisic et al., 2004; Toyomasu milactone A through the functions of the chromosomally clustered syn- et al., 2008, 2015). Duplicated, catalytically redundant ent-CPS genes CPS OsCPS4, the syn-pimara-7,15-diene synthase OsKSL4, CYP99A3, with distinct roles in GA and specialized diterpenoid biosynthesis and the SDR OsMAS (Shimura et al., 2007; Wang et al., 2011). Likewise,

3 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289

Fig. 2. Diterpenoid metabolism in rice. Overview of the established modular diterpenoid-metabolic network in rice (Oryza sativa), involving the conversion of the central precursor geranylgeranyl diphosphate (GGDP) through the combined activity of class II diterpene synthases (green boxes), class I diterpene synthases (blue boxes), cytochrome P450 monooxygenases (orange) and select other enzyme classes (pink). Representative pathway intermediates and end products are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) phytocassane biosynthesis relies on a biosynthetic cluster comprised of bacterial leaf blight (Xanthomonas oryzae pv. Oryzae), another de- OsCPS2, the ent-cassadiene synthase OsKSL7 and two P450s, namely structive rice pathogen (Kono et al., 2004; Watanabe et al., 1996; CYP76M7 and CYP71Z7, that catalyze oxygenation of the olefin scaf- Watanabe et al., 1992). In addition, in rice roots, phytocassanes A-E fold at C11 and C3 (Swaminathan et al., 2009; Wu et al., 2011). and, to lesser degree, oryzalexins A-D provide pathogen resistance by The chemical diversity of rice diterpenoids echoes their broad range suppressing spore germination, as shown, for example, for M. oryzae of physiological functions (for detailed review articles see Schmelz (Koga et al., 1995, 1997; Sesma and Osbourn, 2004). et al., 2014; Kato-Noguchi and Peters, 2013; Peters, 2006). Several rice Substantial genetic evidence for the defensive importance of di- diterpenoids show both overlapping and distinct stress-elicited accu- terpenoids was provided by genetic studies using rice knock-out mutant mulation and activation patterns and bioactivities (Hasegawa et al., lines. For example, loss of function in the ent-CPS gene, Oscps2, that is 2010; Horie et al., 2015; Ren and West, 1992; Toyomasu et al., 2008, critical for forming phytocassanes, oryzalides, and oryzalexins A-F, re- 2014; Umemura et al., 2003). For example, in above-ground tissues sulted in an increased susceptibility to the pathogens M. oryzae and X. momilactones, oryzalexins, and phytocassanes exhibit strong, elicited oryzae (Lu et al., 2018). Surprisingly, a mutant line deficient in mo- production at the site of attack after inoculation with selected patho- milactone and oryzalexin S formation through knock-out of the syn-CPS gens, including economically impactful fungal diseases such as Mag- gene, Oscps4, showed enhanced resistance, not susceptibility, to X. or- naporthe oryzae, M. grisea, and Rhizoctonia solani (Koga et al., 1997; yzae and M. oryzae, despite the induced diterpenoid accumulation in Umemura et al., 2003; Lu et al., 2018). In vitro bioactivity studies fur- response to this pathogen (Lu et al., 2018). Conversely, susceptibility to ther showed that rice oryzalides exert antibiotic activity against the wheat pathogen M. poae was increased in this mutant. Earlier crop

4 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289 resistance studies using other Oscps4 knock-out lines also showed dif- Formation of ent-CDP is facilitated by one of two class II diTPS fering impacts of gene function loss on the susceptibility to M. oryzae enzymes, ANTHER EAR 1 and ANTHER EAR 2 (ZmAn1, ZmAn2) that (Xu et al., 2012; Toyomasu et al., 2014). These differing findings be- are catalytically redundant, yet have clearly distinct physiological roles. tween in vitro assays, inducibility, and mutant susceptibilities highlight Genetic studies showed that ZmAn1 supplies GA metabolism, based on that the interrelations of pathogen-elicited production and antibiotic the dwarf phenotype and formation of anthers in the ears due to GA efficacy of distinct diterpenoids and complex blends thereof are not deficiency in the corresponding Zman1 maize mutant (Bensen et al., clear cut and require further detailed investigation of the underlying 1995). Conversely, knock-out of Zman2 results in normal GA levels, but mechanisms. adeficiency in kauralexins and dolabralexins, and shows a higher stress Insight into relevant modes of action is further complicated by the susceptibility while otherwise a normal phenotype under healthy con- multi-functional nature of several rice diterpenoids beyond the well- ditions, thus demonstrating a role of ZmAn2 in specialized metabolism established defensive role as antimicrobial phytoalexins. For example, (Harris et al., 2005; Mafu et al., 2018; Vaughan et al., 2015). momilactone A and B and phytocassanes A-E were identified in root In addition to ZmAn1 and ZmAn2, the maize genome (B73 version exudates, supporting their role in below-ground plant-environment in- 4.0) contains two other class II diTPSs, recently shown to produce the teractions (Kato-Noguchi and Ino, 2003; Toyomasu et al., 2008). In- specialized diterpenoid intermediates (+)-CDP (ZmCPS3), and 8,13- deed, momilactones A and B were shown to have potent allelopathic CDP with LDP as a minor byproduct (ZmCPS4) in in vivo biochemical properties, inhibiting the growth of selected rice weeds such as barn- assays (Murphy et al., 2018)(Fig. 3). Presence of a (+)-CPS but lack of yard grass (Echinochloa crus-galli), which impacts commercial rice cul- a syn-CPS enzyme sets maize apart from rice, where syn-CDP derived tivation (Kato-Noguchi et al., 2010; Kato-Noguchi and Peters, 2013; momilactones represent core bioactive products (Fig. 2). While Toyomasu et al., 2014). Interestingly, Xuan and coworkers showed that pathway products derived from these class II diTPS activities are yet to despite this activity, momilactones A and B were not correlated with be demonstrated in planta and their biological function remains un- weed resistance in a study of 30 rice cultivars (Xuan et al., 2016). known, root-specific inducible gene expression of at least ZmCPS4 in In addition to functions in biotic plant-microbe and plant-plant in- response to oxidative stress suggests a possible role in abiotic stress teractions, increased momilactone A and B production in rice leaves in tolerance. response to crude honeydew or chewing activity of the rice brown plant Next to these four class II diTPSs, the maize B73 and Mo17 genomes hopper (Nilaparvata lugens) suggested roles in the defense against insect contain a family of seven class I diTPSs (ZmTPS1 and ZmKSL1-6) (Ding pests (Alamgir et al., 2016; Wari et al., 2019). In vitro assays demon- et al., 2019; Mafu et al., 2018). Among these, ZmTPS1 [previously strating activity of momilactone A and B as strong α-amylase and α- shown to also function as a sesquiterpene synthase (Schnee et al., glucosidase inhibitors further substantiated such a function (Quan 2002)], ZmKSL3, and ZmKSL5 form a tandem array of ent-kaurene et al., 2019). However, lack of metabolite accumulation following ex- synthases, of which ZmKSL3 seemingly feeds into GA biosynthesis ex- posure to other insect pests, such as rice skipper (Parnara guttata)or clusively, whereas the other two genes show patterns of stress-inducible lawn armyworm (Spodoptera mauritia), may indicate species-specific expression and have possible functions in specialized terpenoid meta- antifeedant activities (Alamgir et al., 2016). Furthermore, several stu- bolism (Fu et al., 2016). Among the remaining class I diTPSs, activities dies revealed that combined herbivory and pathogen stress can result in of ZmKSL1 and ZmKSL6 are presently unresolved, whereas a recent increased pathogen resistance after insect pest attack, suggesting a functional genomics approach demonstrated that ZmKSL2 provides the possible priming role of rice diterpenoids to fend off microbial patho- central ent-isokaurene intermediate en route to kauralexin diterpenoid gens following insect-derived wounding (Gomi et al., 2010; Kanno acids (Ding et al., 2019)(Fig. 3). In addition, ZmKSL2 produces ent- et al., 2012). kaurene as a minor byproduct that can serve as an alternate substrate in Beyond their functions in biotic interactions, some rice diterpenoids kauralexin biosynthesis and a critical GA precursor, supporting the have been suggested to also contribute to the abiotic stress responses. origin of ZmKSL2 from a GA-metabolic ent-kaurene synthase pro- Transcript abundance of oryzalexin and phytocassane pathway genes genitor. Neo-functionalization of ZmKSL2 toward a positional isomer of was increased in rice leaves following UV irradiation of rice leaves ent-kaurene, combined with the highly stress-elicited expression pattern (Horie et al., 2015; Park et al., 2013). UV stress further induced mo- of the corresponding gene, illustrate how catalytic as well as regulatory milactone accumulation in the rhizosphere (Kato-Noguchi et al., 2007), divergence of duplicated diTPS genes may lead to the evolution of and momilactones have been correlated with drought tolerance (Xuan specialized metabolites, while minimizing interference with parallel et al., 2016). These latter results may point toward a possible role of hormone-metabolic pathways sharing a common precursor pool (Ding momilactones in mediating interactions with beneficial root micro- et al., 2019). biota. However, analysis of the Oscps2 and Oscps4 rice mutants for al- In addition to ZmKSL2, a gene-to-metabolite approach identified terations of the rhizosphere microbiome composition showed no sig- ZmKSL4 as a dolabradiene synthase and led to the discovery of the nificant impact in support of this hypothesis (Lu et al., 2018). previously hidden, and perhaps unique, group of maize dolabralexin diterpenoids (Mafu et al., 2018). Thus, ZmKSL2 and ZmKSL4 mark 2.3. The maize diterpenoid network critical bifurcation points immediately downstream of ZmAn2, enabling the production of chemically diverse diterpenoid scaffolds (Fig. 3). In Similar to rice, maize produces a complex array of bioactive ter- vitro biochemical assays further showed that both ZmKSL2 and ZmKSL4 penoids that differ considerably in their abundance and composition are promiscuous with respect to their CDP substrates, also converting across different cultivars, as well as between developmental stages, (+)-CDP and syn -CDP, but not 8,13-CDP, into a range of pimarane- and tissues, and environmental conditions (Block et al., 2019; Huffaker abietane-type scaffolds (Ding et al., 2019; Mafu et al., 2018; Murphy et al., 2011; Mafu et al., 2018; Schmelz et al., 2011). Early maize stu- et al., 2018). While syn-CDP is absent in maize, conversion of (+)-CDP dies revealed numerous volatile terpenoid pathways with functions in occurs in maize, but the resulting products yet await discovery in planta. pest and pathogen defense (Ding et al., 2017; Huffaker et al., 2011; Similar to rice diterpenoid metabolism, several P450s of the Köllner et al., 2004, 2008a, 2008b; Lenk et al., 2012; Schnee et al., CYP701A and the CYP71Z subfamilies have been characterized in 2002, 2006). Bioactive diterpenoids were discovered only more re- maize that show substantial substrate and product promiscuity to en- cently and, to current knowledge, comprise two groups, namely kaur- able the production of an array of different compounds from only a alexins and dolabralexins (Mafu et al., 2018; Schmelz et al., 2011). handful of hydrocarbon precursors. In a similar fashion as shown for Similar to rice, these diterpenoids are formed through a modular, P450 of the CYP701A family in rice and Arabidopsis (Morrone et al., branching, pathway network originating from the same ent-CDP pre- 2010; Wang et al., 2012b), the promiscuous P450, CYP701A43, cata- cursor that is also shared with GA biosynthesis (Fig. 3). lyzes the carboxylation of both ent-kaurene and ent-isokaurene toward

5 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289

OPP

GGDP

ZmAN1 ZmAN2 ZmCPS3 ZmCPS4

OPP OPP OPP OPP OPP

OH LDP ent-CDP ent-CDP (+)-CDP 8,13-CDP minor product major product ZmTPS1 ZmKSL3 ZmKSL2 ZmKSL4 ZmKSL4 ZmKSL4 ZmKSL5

minor product unidentiﬁed pimaranes

ent-kaurene ent-isokaurene dolabradiene pimara-8(14),15-diene

CYP701A26 CYP701A43 CYP71Z16/18 CYP701A43 CYP71Z16/18

GAs KB1-4 Dolabralexins CYP71Z16/18 ZmKR2

KA1-4

KA1 KA2 KA3 KA4 COOH COOH COOH COOH

COOH CHO OH

KB1 KB2 KB3 KB4 COOH COOH COOH COOH Kauralexins

COOH CHO OH

epoxydolabrene epoxydolabrenol trihydroxydolabrene O O OH OH

Dolabralexins HO HO

Fig. 3. Diterpenoid metabolism in maize. Overview of the established modular diterpenoid-metabolic network in maize (Zea mays), involving the conversion of the central precursor geranylgeranyl diphosphate (GGDP) through the combined activity of class II diterpene synthases (green boxes), class I diterpene synthases (blue boxes), cytochrome P450 monooxygenases (orange) and select other enzyme classes (pink). Representative pathway intermediates and end products are highlighted. KA, A-series kauralexins 1-4; KB, B-series kauralexins 1-4 . (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

GA and kauralexin biosynthesis (Ding et al., 2019). Additionally, a dolabradiene through hydroxylation at C3 and addition of an unusual steroid 5α reductase, ZmKR2, has been characterized to catalyze the extracyclic epoxide group to yield dolabralexins (Ding et al., 2019; desaturation of these P450 products as an additional pathway route to Mafu et al., 2018). Moreover, both CYP71Z16 and CYP71Z18 were kauralexin production (Ding et al., 2019). Furthermore, two closely shown to convert the sesquiterpene (S)-β-macrocarpene to form zeal- related P450 enzymes, CYP71Z16 and CYP71Z18, are capable of con- exins as a group of acidic maize sesquiterpenoids with critical defensive verting ent-kaurene, ent-isokaurene, and dolabradiene into diﬀerent functions (Mafu et al., 2018; Mao et al., 2016), thus highlighting the downstream products (Ding et al., 2019; Mafu et al., 2018; Mao et al., functional plasticity of these P450 enzymes. Notably, diterpenoid-me- 2016). While ent-kaurene and ent-isokaurene undergo carboxylation at tabolic diTPSs and P450s do not form biosynthetic clusters in maize, as C16 toward formation of kauralexins, CYP71Z16/18 converts has been shown for at least some pathways in rice, suggesting an

6 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289

Fig. 4. Diterpenoid metabolism in wheat. Overview of the established modular diterpenoid-metabolic network in wheat (Triticum aestivum), involving the conversion of the central precursor geranylgeranyl diphosphate (GGDP) through the combined activity of class II diterpene synthases (green boxes) and class I diterpene synthases (blue boxes). Representative pathway intermediates are highlighted. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) independent evolution consistent with the fact that rice does not form momilactones have not yet been described for any maize terpenoid. kauralexins despite the presence of an ent-isokaurene synthase However, similar to the accumulation of rice diterpenoids in response (OsKSL6) (Xu et al., 2007a). to abiotic stress (Horie et al., 2015; Kato-Noguchi et al., 2007; Park Alike to rice chemical defenses, maize diterpenoids exhibit diverse et al., 2013; Xuan et al., 2016), kauralexins and dolabralexins accu- biological activities (for comprehensive reviews see Schmelz et al., mulate in roots in response to oxidative, drought, and elevated CO2 2014; Block et al., 2019). As the major bioactive diterpenoid groups stress, and Zman2 plants are more susceptible to these abiotic pertur- currently known in maize, kauralexins and dolabralexins exhibit both bations, suggesting broader below-ground biological functions (Mafu overlapping and separate biological activities, inducibility, and tissue- et al., 2018; Vaughan et al., 2014, 2015). specificity. Both kauralexins and dolabralexins were shown to accu- Drawing on the potent bioactivities of maize and rice diterpenoids, a mulate at high levels in response to major maize fungal pathogens, recent study demonstrated that overexpression of the functionally including F. verticillioides and F. graminearum, as well as Rhizopus mi- promiscuous maize CYP71Z18 enzyme in rice plants resulted in the crosporus, Cochliobolus heterostrophus, and Colletotrichum graminicola as formation of new diterpenoid products (including those with the shown for kauralexins (Christensen et al., 2018; Mafu et al., 2018; characteristic epoxide group of dolabralexins). The resulting mutants Schmelz et al., 2011). Their critical role in mitigating biotic stress was further showed enhanced resistance to the rice pathogen M. oryzae further supported by the dramatically decreased pathogen resistance of (Shen et al., 2019). These findings highlight how the modularity of the Zman2 mutant deficient in kauralexins and dolabralexins plant diterpenoid-metabolic networks can be harnessed through com- (Christensen et al., 2018). A similar pathogen susceptibility phenotype binatorial pathway engineering to develop new crop protection strate- was also observed in the Zmksl2 mutant (which lacks kauralexins but gies for increasing crop productivity with reduced chemical inputs. contains normal dolabralexins levels) after stem inoculation with F. graminearum, thus demonstrating that kauralexin and dolabralexin 2.4. Diterpenoid-metabolic networks in other monocot crops functions are not redundant (Ding et al., 2019). Complementary physiological roles are also supported by the dif- While the biosynthesis and function of specialized diterpenoids in fi ferent tissue-speci city of kauralexins and dolabralexins. Pathogen- maize and rice have been extensively studied, knowledge of species- elicited accumulation of kauralexins along with the expression of the specific diterpenoid metabolism in related Poaceous crops has long corresponding pathway genes was show to predominantly occur in lagged behind. Increasing availability of genomic resources for above-ground tissues with limited root production, whereas dolabra- monocot crops enabled the discovery of similar mid-size diTPS families lexins appear to primarily occur in maize roots, albeit with substantial in several species, including barley, sorghum, and wheat (Fig. 1)(Chen variation among genotypes (Ding et al., 2019; Mafu et al., 2018; et al., 2011). Although gene mapping revealed a more expansive diTPS Schmelz et al., 2011). Kauralexins have further been shown to have an family in barley, biochemical analysis, to date, characterized only a pair even broader role in plant stress responses, including functions as anti- of ent-CPS and ent-kaurene synthase enzymes likely functioning in GA feedant agents in the defense against insect pests such as the European metabolism (Spielmeyer et al., 2004; Wu et al., 2012; Zhou et al., corn borer (Ostrinia nubilalis)(Schmelz et al., 2011; Vaughan et al., 2012). 2015). Transcript accumulation of relevant biosynthetic genes in re- More extensive in vitro diTPS functional studies have been under- sponse to O. nubilalis and subsequent R. microporus treatment further taken in wheat and revealed a diTPS family of four class II (TaCPS1-4) indicated possible priming functions similar to those hypothesized for and six class I (TaKSL1-6) enzymes (Wu et al., 2012; Zhou et al., 2012) rice diterpenoids (Dafoe et al., 2011; Schmelz et al., 2011). (Fig. 4). The identified class II diTPSs encode for three catalytically Anti-herbivory activity has not yet been demonstrated for dolabra- redundant ent-CPSs (TaCPS1,3 and 4) and a (+)-CPS enzyme (TaCPS2). lexins. Likewise, allelopathic functions as demonstrated for rice The latter function is also present in maize, but not rice, while maize

7 K.M. Murphy and P. Zerbe Phytochemistry 172 (2020) 112289 and wheat both lack a syn-CPS (Fig. 1). TaCPS2 is most closely related enzyme functions (Fig. 5). to the rice syn-CPS, OsCPS4, which may suggest a more recent evolu- So far characterized class II diTPSs include two ent-CPS enzymes tionary divergence (Wu et al., 2012). (PvCPS14 and PvCPS15) with likely separate functions in specialized Consistent with diterpenoid metabolism in maize and rice, bio- and GA metabolism as shown for rice and maize, a syn-CPS (PvCPS8), chemical characterization of the six wheat class I diTPSs demonstrated and an 8,13-CPS (PvCPS3), all representing CDP products present in expansive substrate promiscuity of several enzymes that likely function one or more other Poaceous species (Fig. 1)(Pelot et al., 2018). In as part of a dynamic pathway network. In addition, to the GA precursor addition, switchgrass contains class II diTPSs producing the ent-ste- ent-kaurene formed by TaKSL5 and TaKSL6, coupled class II and class I reoisomer of LDP (PbCPS11) and terpentedienyl diphosphate (TDP, assays with ent-CDP and (+)-CDP as substrates, revealed several pi- PvCPS1). These class II diTPS products have not been observed in maradiene, isopimaradiene, abietadiene, and ent-beyerene products, monocot species, but are common precursors of specialized diterpe- the latter formed by TaKSL5 represents a perhaps unique wheat diTPS noids in many dicot species (Peters, 2010; Zerbe and Bohlmann, 2015). product (Zhou et al., 2012). However, none of these metabolites have Structure-guided mutagenesis of the TDP synthase PvCPS1 revealed been identified in wheat plants. Interestingly, the catalytic capacity of active site residues relevant for controlling the enzyme's product spe- TaKSL1 and TaKSL4 expanded to the conversion of also syn-CDP into cificity toward clerodane rather than other labdane scaffolds, sug- corresponding pimarane scaffolds (Morrone et al., 2011), highlighting gesting their emergence after the split of the switchgrass lineage (Pelot that the capacity for conversion is maintained in the KSL family and et al., 2018). Several highly substrate-promiscuous class I diTPSs supporting that loss of the syn-CPS functionality may have been a more (PvKSL2-5 and PvKSL8) were shown to convert the various class II recent event. Given the metabolic potential of the wheat diTPS family, diTPS products into a range of different diterpene scaffolds (Pelot et al., it appears plausible that yet unidentified downstream pathway products 2018)(Fig. 5). Surprisingly, this excluded 8,13-CDP, which was not fulfill similar physiological functions as those shown for diterpenoids in converted by any tested KSL enzyme (Pelot et al., 2018). This result is rice and maize. This hypothesis is also supported by the increased ex- consistent with biochemical analyses of the maize biosynthetic network pression of selected wheat diTPS genes (TaKSL1-3 and TaKSL5) in UV- (Murphy et al., 2018), which also contains an 8,13-CPS, and leave the stressed leave tissue (Zhou et al., 2012). However, the presence and metabolic fate of this pathway intermediate elusive. The full chemical identity of the corresponding pathway products in wheat are still un- diversity of diterpenoid metabolism in switchgrass is yet to be unknown. covered with several enzyme functions still unknown. Moreover, de- Diterpenoid metabolism in allotetraploid switchgrass differs sub- spite the expansive diversity of switchgrass diterpenoids, their physio- stantially from other investigated crop species, featuring a far larger logical relevance has not been comprehensively investigated. Gene and more diversified diTPS family comprised of 15 class II diTPSs and expression studies showed inducible transcript accumulation for all so 15 class I enzymes (Pelot et al., 2018). This diTPS family expansion far characterized specialized diTPSs in roots and leaves in response to might be attributed to a higher tolerance for diTPS neo-functionaliza- oxidative and UV irradiation stress, respectively (Pelot et al., 2018). tion due to the presence of diTPS genes on both subgenomes, many of Consistent with increased gene expression, two pathway products, 9β- which with predictably redundant catalytic activities. This is further hydroxy-syn-pimar-15-ene and manoyl oxide, accumulated, albeit at supported by the fact that the closely related diploid species P. hallii moderate levels, in roots and/or leaves (Pelot et al., 2018). Considering contains nine predicted diTPS gene models, similar to the diTPS fa- the common functionalization of diTPS products by P450s and other milies of rice, maize, and wheat. The larger number of diTPSs in the enzymes, it appears likely, however, that these compounds do not re- switchgrass genome indeed is reflected in a broader spectrum of present the bioactive pathway end products, and more detailed studies

Fig. 5. Diterpenoid metabolism in switchgrass. Overview of the established modular diterpenoid-metabolic network in switchgrass (Panicum virgatum), involving the conversion of the central precursor geranylgeranyl diphosphate (GGDP) through the combined activity of class II diterpene synthases (green boxes) and class I diterpene synthases (blue boxes). Representative pathway products are highlighted. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the Web version of this article.)

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