Additional byproducts present in the ZmCPS4 product profile represent manoyl oxide as based on comparison to a manoyl oxide standard produced from the coupled reaction of G. robusta GrTPS1 and MvELS from M. vulgare , and a closely related unidentified diterpenoid . These products are likely resulting from rearrangement of the ZmCPS4 products 8,13-CPP and LPP after dephosphorylation by endogenous E. coli phosphatases, as has been described for various related class II diTPSs . To test for possible downstream products of 8,13-CPP and LPP, ZmCPS4 was co-expressed with the currently known maize class I diTPS functions, including the ent-kaurene synthase ZmKSL3 and the dolabradiene synthase ZmKSL4 . No new products were detected when co-expressing ZmCPS4 and ZmKSL3 as compared to the expression of ZmCPS4 alone . When combining ZmCPS4 with ZmKSL4, compound identified as manoyl oxide was significantly increased and, albeit at low abundance, an additional product was formed that predictably represents a labdane diterpene olefin as based on characteristic mass fragments of m/z 272 and 257 . To further investigate the functions of ZmCPS3 and ZmCPS4, we next examined their gene expression patterns using publicly available transcriptome and proteome data across various organs, tissues, and developmental stages of unstressed maize plants . With the exception of 5-days-old primary root and 2 cm tassels, 1–2 mm anthers, and mature pollen , all samples were from the B73 genotype . Detectable transcripts of ZmCPS3 were distributed across all organs and tissues with highest abundance included germinating kernels . Notably,hydroponic indoor growing system when specifically analyzing gene expression in roots, transcript of ZmCPS3 was observed to be significantly more abundant with expression levels 5–481 times higher as compared to ZmAN1, ZmAN2, and ZmCPS4 in the same tissue .

Transcript of ZmCPS4 was detected at only trace levels and present exclusively in primary and secondary root and root cortex tissues . Consistent with observed transcript abundance, query of public proteome data showed that protein levels of ZmCPS3 were highest in root tissues .Previous studies demonstrated that ZmAN2 gene expression is induced under both pathogen and oxidative stress in above- and below-ground tissues . In the context of these findings, the relatively higher transcript abundance of ZmCPS3 in roots and the low but possibly root-specific expression of ZmCPS4 suggested a role of these enzymes in below ground stress responses. To investigate this hypothesis, we analyzed transcript abundance of ZmCPS3 and ZmCPS4 using a previously reported set of samples of 53-days-old maize Mo17 roots incision inoculated with fungal spores of F.v. and F.g. and harvested 7 days later . Plants treated by incision-wounding only were used as controls. QPCR analysis showed no significant fold change in ZmCPS3 transcript abundance in roots exposed to F.v. or F.g. as compared to wounded controls . On average, gene expression of ZmCPS4 was significantly decreased in response to F.v. and F.g. elicitation as compared to control plants. To examine ZmCPS3 and ZmCPS4 gene expression in response to abiotic stress, qPCR analyses were performed on two week-old roots of maize plants that were exposed to 1 mM CuSO4 treatment , previously shown to induce diter penoid biosynthesis . All treatments were compared to a water treated control at each time point using the 2−11 Ct method in which the control has a fold change of 1. Transcript levels of ZmCPS3 in CuSO4-treated roots did not differ significantly from those observed in the roots of water-treated control plants at 2 and 4 h post treatment, but were significantly reduced at the 24 h post treatment time point Conversely, ZmCPS4 showed increased transcript abundance in CuSO4-treated roots as early as 2 h post treatment and with an up to sixfold change at 4 h, before decreasing again after 12 h, with a peak of sixfold increase in transcript abundance .

Rapidly increasing biotic and abiotic pressures can overcome the natural defense systems of plants, leading to substantial harvest losses in major food crops . Given the proven protective properties of crop-specific diterpenoid arsenals, a deeper knowledge of their biosynthesis and biological functions may aid new solutions to optimize crop stress resilience traits and mitigate yield loss . Elucidation of the enzyme activities of ZmCPS3 and ZmCPS4 completes the functional range of the maize class II diTPS family, which controls the early committed reactions responsible for the diversity of maize diterpenoid pathways. Advances in the discovery and mechanistic analysis of diTPSs across the plant kingdom increasingly enable the prediction of diTPS functions , as exemplified here for ZmCPS3 and ZmCPS4. However, accurate computational annotation of diTPS activities remains to be constrained by the vast sequence and functional space of the enzyme family, thus necessitating biochemical characterization. Modular co-expression assays with an expanding catalog of diTPSs of known substrate/product-specificity can be leveraged for efficient diTPS functional analysis, and were applied in this study for the identification of ZmCPS3 as a -CPP synthase and ZmCPS4 as an 8,13-CPP synthase . While absent in rice , a -CPP synthase has been identified in wheat, where -CPP can be further converted by class I diTPSs to form labdane structures, such as pimara- 8,15-diene, abietadiene, and isopimara-7,15-diene . While corresponding end-products and physiological functions have yet to be discovered in maize and wheat, the wide distribution of -CPP-derived diterpenoids in both angiosperm and gymnosperm species suggests roles in stress defense . Similar to the recently demonstrated accumulation and predicted defensive functions of dolabralexins in maize roots , a possible function of ZmCPS3 in below ground stress responses could be hypothesized based on the higher transcript abundance in roots as compared to ZmCPS4, ZmAN1, and ZmAN2. However, the observed lack of elicited ZmCPS3 expression in maize roots in response to Fusarium infection or oxidative stress did not support an inducible defense role for ZmCPS3-derived pathways and metabolites.

Although ZmCPS3 and its protein product were more highly expressed in roots as compared to other healthy tissues, its broad tissue- and development-wide presence may suggest a possibly more constitutive function. Tissue-wide expression levels of ZmCPS3 suggest a conceptual parallel to the high constitutive levels of benzoxazinoid pathway enzymes and metabolites in maize seedlings, which have been shown to be important for the maize biotic and abiotic stress responses . The largely constitutive and moderately inducible role is supported by a separate yet related study, where ZmCPS3 transcript levels displayed an approximately twofold accumulation in the leaves of a resistant maize recombinant inbred line two weeks after challenge with gray leaf spot . In addition to the -CPP synthase ZmCPS3, characterization of ZmCPS4 as an 8,13-CPP synthase adds an uncommon scaffold to the diterpenoid network of maize . Mechanistically, the proposed ZmCPS4-catalyzed reaction will proceed through terminal deprotonation of the common -labda-13E-en-8-yl+ diphosphate intermediate at C-9 to yield 8,13-CPP, as opposed to the more typical deprotonation of the carbocation at the exocyclic C-17 methyl to form the -CPP or ent-CPP isomers . ZmCPS4-catalyzed formation of LPP as a minor product will require hydroxylation at C-8 of the -labda-13E-en-8-yl+ diphosphate intermediate . Dual product activity is rarely observed in class II diTPSs . Lacking available ZmCPS4 maize mutants to enable in planta gene function studies, it can only be speculated if LPP represents a native ZmCPS4 product or results from a possibly reduced enzyme activity in vitro that could cause a slower conversion of the intermediary carbocation and thus facilitate water quenching toward formation of LPP. Notably, an 8,13-CPP synthase was also recently discovered in switch grass,vertical rack system where the enzyme was characterized as a single product class II diTPS . By contrast, an 8,13-CPP synthase function is absent in rice and wheat . Although a geneloss in these species cannot be excluded, this selective presence of 8,13-CPP synthases indicates the independent evolution of this function in maize and switch grass. Similar to ZmCPS3, no pathogen-elicited gene expression was observed for ZmCPS4; in fact, transcript abundance was significantly decreased in response to Fusarium infection . This contrasts the well-established pathogen-elicited gene expression of ZmAN2 , and may suggest that ZmAN2-derived pathways are the predominant inducible mediators of pathogen defense responses .

Conversely, CuSO4-induced expression of ZmCPS4 in roots points to a possible role in below ground plant-environment interactions as also proposed for kauralexins and dolabralexins . The identity of predominant pathway end-products derived from -CPP, 8,13-CPP, and possibly LPP, as well as their corresponding roles in plant-environmental adaptation will require further investigation and ultimately the generation and analysis of defined genetic mutants in future studies. Currently, the biochemical characterization of ZmCPS3 and ZmCPS4 expands the known chemical landscape surrounding maize diterpenoid metabolism and completes the characterization of predicted class II diTPSs in the maize genome. Modular pathway networks composed of class II and class I diTPSs are likely operating in maize to convert the ZmCPS3 and/or ZmCPS4 products into a broader spectrum of specialized diterpenoids. In vitro formation of several labdane-type diterpenoids through the sequential activity of ZmKSL4, but not the ent-kaurene synthase ZmKSL3, with ZmCPS3 and ZmCPS4 support this hypothesis. However, no -CPP, 8,13-CPP or LPP derivatives have yet been demonstrated in maize and the biological role of ZmCPS3/4 pathway branches has to be demonstrated in planta. Nevertheless, functional knowledge of all maize class II diTPSswill now enable a detailed investigation of modular diterpenoidmetabolic pathway branches in maize that are formed by class II diTPSs and known or thus far uncharacterized class I diTPSs as the biochemical foundation for maize diterpenoid diversity. Such insight can be of substantial value to elucidate and ultimately harness the genetic basis of crop stress resilience . The use of reclaimed wastewater for irrigation and biosolids or animal wastes as fertilizers in agriculture is on the rise worldwide . There are many benefits from reuse of these waste materials, such as augmenting water supply, increasing soil nutrient content and improving crop yields . However, concerns remain about the safety of such practices , as they introduce a multitude of trace contaminants, including pharmaceuticals and personal care products , into the agroeco systems . In recent years, the fate of PPCPs in the soil plant continuum has been extensively studied . Furthermore, several studies have considered uptake and accumulation of subsets of PPCPs by different crop species . Although many PPCPs are inherently bioactive substances, their toxicity to plants is comparatively less understood. A few studies showed that exposure to PPCPs affected plant development and physiological functions . For example, root growth and development were markedly reduced when pinto beans were exposed to chlortetracycline antibiotics . Tetracyclines and sulfonamides were shown to negatively affect seed germination , and the influence varied among plant species and the different PPCPs considered in the study . Although such studies have shown various phytotoxic effects from PPCPs, the corresponding physiological and molecular mechanisms contributing to the toxicity were not adequately explored. Once taken up by the plant root, PPCPs may be metabolized, leading to their detoxification, inactivation and sequestration . Another nodal point in the response of plant cells to xenobiotics is reactive oxygen species generation, ultimately imposing oxidative stress to fundamental plant bio-molecules . Recent studies suggested that ROS overproduction-triggered oxidative damage may be the cause of the longer-term visual phytotoxic responses , such as root growth inhibition and seed germination reduction. For instance, phenanthrene-induced oxidative stress in Arabidopsis was responsible for the observed reductions in germination and root growth and the damage to organelle structures . On the other hand, plants have developed sophisticated antioxidant mechanisms to protect their cellular components from oxidative damage . However, to our knowledge, so far little information is available on the potential impacts of PPCPs on ROS metabolism in higher plants, such as ROS production, oxidative damage and antioxidant system responses. When exposed to single compounds of PPCPs, the observed toxic effects to plants were generally low . However, PPCPs always enter agroecosystems as mixtures of many compounds. Comparison between individual and mixtures of PPCPs in aquatic organisms suggested that exposure to single PPCPs underestimated the actual environmental effect, and did not allow prediction of the risk of mixtures at environmentally relevant doses . This study was designed to evaluate PPCP accumulation and potential effects on ROS production and oxidative damage. Cucumber seedlings were exposed hydroponically to a mixture of 17 PPCPs at incremental levels covering environmentally relevant occurrence .