Discovery, Biosynthesis and Stress-Related Accumulation of Dolabradiene-Derived Defenses in Maize

Abstract

Terpenoids are a major component of maize (<i>Zea mays</i>) chemical defenses that mediate responses to herbivores, pathogens, and other environmental challenges. Here, we describe the biosynthesis and elicited production of a class of maize diterpenoids, named dolabralexins. Dolabralexin biosynthesis involves the sequential activity of two diterpene synthases, <i>ENT</i>-COPALYL DIPHOSPHATE SYNTHASE (ZmAN2) and KAURENE SYNTHASE-LIKE4 (ZmKSL4). Together, ZmAN2 and ZmKSL4 form the diterpene hydrocarbon dolabradiene. In addition, we biochemically characterized a cytochrome P450 monooxygenase, ZmCYP71Z16, which catalyzes the oxygenation of dolabradiene to yield the epoxides 15,16-epoxydolabrene (epoxydolabrene) and 3β-hydroxy-15,16-epoxydolabrene (epoxydolabranol). The absence of dolabradiene and epoxydolabranol in <i>Zman2</i> mutants under elicited conditions confirmed the in vivo biosynthetic requirement of ZmAN2. Combined mass spectrometry and NMR experiments demonstrated that much of the epoxydolabranol is further converted into 3β,15,16-trihydroxydolabrene (trihydroxydolabrene). Metabolite profiling of field-grown maize root tissues indicated that dolabralexin biosynthesis is widespread across common maize cultivars, with trihydroxydolabrene as the predominant diterpenoid. Oxidative stress induced dolabralexin accumulation and transcript expression of <i>ZmAN2</i> and <i>ZmKSL4</i> in root tissues, and metabolite and transcript accumulation were up-regulated in response to elicitation with the fungal pathogens <i>Fusarium verticillioides</i> and <i>Fusarium graminearum</i> Consistently, epoxydolabranol significantly inhibited the growth of both pathogens in vitro at 10 µg mL^-1, while trihydroxydolabrene-mediated inhibition was specific to <i>F</i><i>verticillioides</i> These findings suggest that dolabralexins have defense-related roles in maize stress interactions and expand the known chemical space of diterpenoid defenses as genetic targets for understanding and ultimately improving maize resilience.