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Abstract Aldoximes are amino acid derivatives that serve as intermediates for numerous specialized metabolites including cyanogenic glycosides, glucosinolates, and auxins. Aldoxime formation is mainly catalyzed by cytochrome P450 monooxygenases of the 79 family (CYP79s) that can have broad or narrow substrate specificity. Except for SbCYP79A1, aldoxime biosynthetic enzymes in the cereal sorghum (Sorghum bicolor) have not been characterized. This study identified nine CYP79-encoding genes in the genome of sorghum. A phylogenetic analysis of CYP79 showed that SbCYP79A61 formed a subclade with maize ZmCYP79A61, previously characterized to be involved in aldoxime biosynthesis. Functional characterization of this sorghum enzyme using transient expression in Nicotiana benthamiana and stable overexpression in Arabidopsis thaliana revealed that SbCYP79A61 catalyzes the production of phenylacetaldoxime (PAOx) from phenylalanine but, unlike the maize enzyme, displays no detectable activity against tryptophan. Additionally, targeted metabolite analysis after stable isotope feeding assays revealed that PAOx can serve as a precursor of phenylacetic acid (PAA) in sorghum and identified benzyl cyanide as an intermediate of PAOx-derived PAA biosynthesis in both sorghum and maize. Taken together, our results demonstrate that SbCYP79A61 produces PAOx in sorghum and may serve in the biosynthesis of other nitrogen-containing phenylalanine-derived metabolites involved in mediating biotic and abiotic stresses. 

SUMMARY Zea mays(maize) makes phytoalexins such as sesquiterpenoid zealexins, to combat invading pathogens. Zealexins are produced from farnesyl diphosphate in microgram per gram fresh weight quantities. As farnesyl diphosphate is also a precursor for many compounds essential for plant growth, the question arises as to howZ. maysproduces high levels of zealexins without negatively affecting vital plant systems. To examine if specific pools of farnesyl diphosphate are made for zealexin synthesis we made CRISPR/Cas9 knockouts of each of the three farnesyl diphosphate synthases (FPS) inZ. maysand examined the resultant impacts on different farnesyl diphosphate‐derived metabolites. We found that FPS3 (GRMZM2G098569) produced most of the farnesyl diphosphate for zealexins, while FPS1 (GRMZM2G168681) made most of the farnesyl diphosphate for the vital respiratory co‐factor ubiquinone. Indeed,fps1mutants had strong developmental phenotypes such as reduced stature and development of chlorosis. The replication and evolution of thefpsgene family inZ. maysenabled it to produce dedicated FPSs for developmentally related ubiquinone production (FPS1) or defense‐related zealexin production (FPS3). This partitioning of farnesyl diphosphate production between growth and defense could contribute to the ability ofZ. maysto produce high levels of phytoalexins without negatively impacting its growth.