More recently, both kauralexins and dolabralexins have been shown to accumulate under drought and below-ground oxidative stress, consistent with a possible protective role of diterpenoids in abiotic stress responses . Furthermore, a kauralexin- and dolabralexindeficient maize mutant an2showed increased susceptibility to both drought stress and pathogen attack . These findings highlight the biological importance of diterpenoids in conferring stress resilience, and underscore the need to better understand the biosynthesis and chemical ecology of diterpenoids in cereal crops. In angiosperms, the structural diversity of labdane-type diterpenoids is determined by the activity of class II and class I diTPS enzymes that act sequentially to transform the central precursor GGPP into different scaffolds . Class II diTPSs control the first committed reaction, catalyzing the protonation-initiated cyclization of GGPP into bicyclic labdadienyl/CPP intermediates of distinct stereochemistry and/or regio-specific oxygenations. Class I diTPSs then convert these intermediates via ionization of the diphosphate group and a variety of possible downstream cyclization and rearrangement reactions of the intermediary carbocation . Multi-gene diTPS families of 9–31 members were identified in the genomes of maize, rice, wheat , and switch grass . Functional characterization of class II diTPSs in these species demonstrated the formation of both common and distinct CPP stereoisomers. All species produce ent-CPP as a precursor for gibberellin phytohormones, as well as specialized diterpenoids at least in rice and maize . In addition, wheat forms the enantiomeric -CPP en route to pimarane and abietane diterpenoids, whereas rice and switch grass form syn-CPP en route to, for example, rice oryzalexins and momilactones . The switch grass class II diTPS family appears to have functionally diverged more extensively to also produce 8,13- CPP and the clerodane diterpenoid precursor clerodienyl diphosphate . In maize, only two of the four class II diTPSs present in the genome , namely, ANTHER EAR 1 and ANTHER EAR 2 ,hydroponic grow kit have been functionally analyzed and demonstrated to produce ent-CPP.

Despite their catalytic redundancy, ZmAN1 and ZmAN2 serve different physiological functions in maize. Knock-out mutants of Zman1 display characteristic gibberell indeficient phenotypes including dwarfism and anther formation in ears that demonstrate a role of ZmAN1 in general metabolism . By contrast, ZmAN2 is stress-inducible and an2 mutants exhibit normal growth and reproductive phenotypes, but display enhanced susceptibility to fungal disease and environmental stress associated with the lack of kauralexins and/or dolabralexins . While these genetic studies clarified the dependency of kauralexin and dolabralexin metabolism on ZmAN2 activity, knowledge of the downstream class I diTPS reactions remains incomplete. Only KAURENE SYNTHASE-LIKE 4 and the P450s CYP71Z16/18 have been recently identified to catalyze the conversion of ent-CPP into dolabradiene and downstream dolabralexins . These enzymes expand our knowledge of previously reported maize ent-kaurene synthases that convert ent-CPP into ent-kaurene en route to gibberellin biosynthesis and possibly specialized diterpenoid metabolism . Herein reported is the biochemical characterization of the two remaining class II diTPSs present in the maize genome, COPALYL DIPHOSPHATE SYNTHASE 3 and COPALYL DIPHOSPHATESYNTHASE 4 , to delineate the scope of yet unknown specialized diterpenoid pathways in maize. Unlike ZmAN2, interrogation of transcriptomic and proteomic datasets did not support a role of ZmCPS3 and ZmCPS4 in highly inducible pathogen defenses. A pattern of largely constitutive gene expression of ZmCPS3 and moderately inducible expression of ZmCPS4 under root exposure to abiotic stress may suggest more constitutive functions in maize ecological adaptation.Gas chromatography mass spectrometry analysis of enzyme products was performed on an Agilent 7890B GC interfaced with a 5977 Extractor XL MS Detector at 70 eV and 1.2 mL min−1 He flow, using an Agilent HP5-MS column with a sample volume of 1 µL and the following GC parameters: pulsed splitless injection at 250 and 50◦C oven temperature; hold at 50◦C for 3 min, 20◦C min−1 to 300◦C, hold 3 min. MS data from 90 to 600 mass-to-charge ratio were collected after a 8 min solvent delay.

Products were identified using comparison to authentic standards or, where these were not available, comparison to published mass spectra and the National Institute of Standards and Technology mass spectral library . Diterpenoids were produced via large-scale enzyme coexpression cultures as described above. Hexane extracts were dried using rotary evaporation, resuspended in hexane, and purified by silica column chromatography using a hexane:ethyl acetate gradient as the mobile phase. Fractions were further purified on an Agilent 1100 series HPLC with diode array UV detector and an Agilent ZORBAX Eclipse Plus-C8 column at a 0.5 mL min−1 flow rate and H2O/acetonitrile gradient as mobile phase. Product purity was verified using GC-MS analysis as outlined above. Purified products were dissolved in 0.6 mL deuterated chloroform containing tetramethylsilane . NMR spectra were acquired at room temperature on a Bruker Avance III 800 spectrometer equipped with a 5 mm CPTCI.All spectra were acquired using standard experiments on a Bruker TopSpin 3.2 software, including 1D 1H and 1D 13C spectra .For analysis of class II diTPS gene and protein abundance, publicly available transcriptome and proteome inventories were investigated that represent a range of organs and tissues at different developmental stages of healthy maize plants . All samples derive from B73, with the exception of 2 cm tassels, 1–2 mm anthers, and mature pollen , and 5-days-old primary root . Transcript abundance and protein expression levels were retrieved directly from this public resource , and were scaled by color either individually by gene or absolute across all four genes of interest. Plant samples used for gene expression analysis were prepared previously . Briefly, maize seed was germinated in the dark on wetted paper for 4 days at 23◦C. Seedlings were transferred and grown hydroponically for 12 days under 16/8 h light/darkness at 28◦C, light intensity of 180 µmol photons m−2 s −1 , and ∼60% relative humidity. A total of 1 mM CuSO4 or the corresponding water control was added to the hydroponic medium. Root samples were collected at the time points indicated, with three biological replicates per time point, and immediately frozen in liquid nitrogen for further analysis.

Plants samples used for gene expression analysis were derived from a previous study . Maize plants were grown in the greenhouse for 53 days in individual, 10 L pots and supplemented with 14-14-14 Scotts Miracle Grow fertilizer. Large nodal roots were punctured with a 0.6 mm dia steel pin at 1 cm intervals and inoculated with 10 µL of 1 × 107 conidia mL−1 of Fusarium verticillioides , Fusarium graminearum , or water control at each wound site. To avoid damage to other tissues not undergoing treatment, sampling was limited to roots on the outer edge of the soil, in contact with the vertical plastic pot wall. Root samples were collected after 7 days and immediately frozen in liquid nitrogen before further processing. The maize genome contains four class II diTPSs , which comprise the known entCPP synthases ZmAN1 and ZmAN2 located on chromosome 1 , and the previously uncharacterized ZmCPS3 and ZmCPS4 positioned on chromosomes 4 and 10, respectively. ZmCPS3 and ZmCPS4 share a protein sequence identity of 55% with each other and 46–56% with ZmAN1 and ZmAN2. Phylogenetic analysis placed ZmCPS3 and ZmCPS4 separate from most ent-CPP synthases on a branch primarily consisting of class II diTPSs that produce prenyl diphosphates of specialized metabolism . Although this suggested a role of both enzymes in specialized metabolism,vertical farming racks the functional diversity of diTPSs in this group did not allow inference of possible ZmCPS3 and ZmCPS4 functions. Therefore, to inform biochemical analyses, we interrogated key active site residues with known impact on class II diTPS product specificity . Previous studies identified a His-Asn catalytic dyad that is widely conserved among ent-CPP synthases, including ZmAN1 and ZmAN2, and was shown to direct class II diTPS catalysis toward ent-CPP formation . Neither ZmCPS3 nor ZmCPS4 possess a His-Asn catalytic dyad, but instead feature a Leu-Phe residue pair . These residues are consistent with a recently characterized 8,13-CPP synthase from switch grass . In addition, presence of a Tyr residue in position 497 and 458 of ZmCPS3 and ZmCPS4, respectively, is consistent with known monocot ent-CPP and -CPP synthases, but contrasts known syn-CPP synthases from rice and switchgrass that feature a His residue in this position . Although insufficient to allow an unambiguous functional annotation, these active site characteristics disfavored an ent-CPP or syn-CPP synthase activity, and supported a possible function of ZmCPS3 and ZmCPS4 as 8,13-CPP or -CPP synthases or related specialized class II diTPSs. To test the predicted enzyme activity of ZmCPS3, a synthetic full-length gene was co-expressed with a GGPP synthase from A. grandis using an in vivo E. coli expression platform engineered for diterpenoid production . In vivo expression of class II diTPS enzymes using this system readily yields dephosphorylated products, presumably due to the activity 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. of E. coli endogenous phosphatases and thus enables direct hexane extraction and analysis of the corresponding diterpene alcohols . For clarity, structures depicted below represent the native prenyl diphosphate products, but were detected via GC-MS and NMR analysis as the corresponding alcohols. Expression of ZmCPS3 resulted in a major product with a retention time of 11.25 min and a fragmentation pattern showing dominant mass ions of m/z 137, 257, and 275 that closely matched the mass spectrum of copalol produced by ZmAN2 . Two additional byproducts detected in the ZmCPS3 product profile represented unconverted GGPP substrate and an unidentified non-diterpenoid contaminant . Next we defined the stereochemistry of the ZmCPS3 product by co-expressing ZmCPS3 with characterized maize class I diTPSs that display catalytic specificity toward CPP substrates of different stereochemistries. ZmKSL3 converts entCPP into ent-kaurene , whereas ZmKSL4 forms dolabradiene from ent-CPP and pimarane-type diterpene olefins with -CPP as a substrate . As controls, we co-expressed ZmAN2 with either ZmKSL3 or ZmKSL4 to generate ent-kaurene and dolabradiene , respectively . Co-expression of ZmCPS3 and ZmKSL3 did not result in any detectable class I diTPS product . The combined activity of ZmCPS3 with ZmKSL4 did not yield dolabradiene, but resulted in several pimarane-related products , the most abundant of which was identified as pimara-8,14-diene by comparison to reference mass spectra . These products are consistent with the previously reported activity of ZmKSL4 with the established -CPP synthase, A. grandis abietadiene synthase variant D621A . On the basis of these results, ZmCPS3 was designated as a -CPP synthase. Escherichia coli co-expression of the full-length, synthetic gene encoding ZmCPS4 with the A. grandis GGPP synthase yielded a major product with a retention time of 11.26 min, indicating a related but distinct compound as compared to – CPP and ent-CPP formed by ZmCPS3 and ZmAN2 in vitro, respectively . Presence of signature mass ions of m/z 275 and 257 in the fragmentation pattern of this product indicated the expected labdane structure, but additional major mass ions of m/z 205 and 149 suggested a structure distinct from the common ent-CPP, -CPP, or syn-CPP products observed in monocot crops . Indeed, the fragmentation pattern of compound 12 matched the product of a recently identified class II diTPS from switch grass that forms 8,13-CPP . To further verify the identity of this ZmCPS4 product, nuclear magnetic resonance spectroscopy was performed. For this purpose, large-scale E. coli co-expression cultures were used to produce an excess of 1 mg of the product, which was then purified using silica column chromatography and semi-preparative high-pressure liquid chromatography . For the purified product, 1D 1H and 13C NMR spectra were acquired and validated the ZmCPS4 product as 8,13-CPP in comparison to published spectra . In addition to the primary 8,13-CPP product, a minor, more polar, ZmCPS4 product with a retention time of 11.79 min was also observed . This product was identified as labda-13-en-8-ol diphosphate based on comparison to the retention time and mass spectrum of LPP produced by a known LPP synthase from G. robusta . Low abundance of this product prevented further stereochemical analysis via NMR.