An efficient system composed of maize protoplast transfection and HPLC–MS for studying the biosynthesis and regulation of maize benzoxazinoids

Insect herbivory poses a major threat to maize. Benzoxazinoids are important anti-insect secondary metabolites in maize, whose biosynthetic pathway has been extensively studied. However, yet little is known about how benzoxazinoids are regulated in maize, partly due to lack of mutant resources and recalcitrance to genetic transformation. Transient systems based on mesophyll- or cultured cell-derived protoplasts have been exploited in several plant species and have become a powerful tool for rapid or high-throughput assays of gene functions. Nevertheless, these systems have not been exploited to study the regulation of secondary metabolites. A protocol for isolation of protoplasts from etiolated maize seedlings and efficient transfection was optimized. Furthermore, a 10-min-run-time and highly sensitive HPLC–MS method was established to rapidly detect and quantify maize benzoxazinoids. Coupling maize protoplast transfection and HPLC–MS, we screened a few genes potentially regulating benzoxazinoid biosynthesis using overexpression or silencing by artificial microRNA technology. Combining the power of maize protoplast transfection and HPLC–MS analysis, this method allows rapid screening for the regulatory and biosynthetic genes of maize benzoxazinoids in protoplasts, before the candidates are selected for in planta functional analyses. This method can also be applied to study the biosynthesis and regulation of other secondary metabolites in maize and secondary metabolites in other plant species, including those not amenable to transformation.