A maize protoplast transfection system for studying the biosynthesis of volatile terpenoids

Volatile terpenoids are produced by almost all plants. They often function as important defensive secondary metabolites. Currently, research aimed at elucidating the biosynthesis of maize terpenes is limited. Thus, a rapid and efficient platform for identifying genes involved in the synthesis/regulation of maize volatile terpenes is highly desirable. Through optimization of various conditions, including light, substrate accumulation, nutrient and ionic composition of the culture medium, culture vessel size and conditions, organic solvent extraction, and promoter selection, we established a maize protoplast transfection system for analyzing the biochemical functions of terpene synthases (TPSs) and the functions of regulatory factors, such as transcription factors. Specifically, dim light-cultured maize seedlings are first treated with methyl jasmonate, followed by preparation of protoplasts and transfection of target genes driven by the ubiquitin promoter. The transfected protoplasts were cultured overnight in glass headspace tubes. Maize protoplasts were extracted by n-pentane and concentrated, and after being heated, the volatile terpenes were absorbed by solid-phase microextraction and analyzed on a gas chromatograph. This system was successfully used to detect the products of ZmTPS10, (E)-β-farnesene and (E)-α-bergamotene and to analyze the function of the transcription factor ZmMYC2a in regulating the production of (E)-β-farnesene and (E)-α-bergamotene. Our system provides a rapid and efficient platform for studying the synthesis and regulation of maize terpene volatiles and can also be adopted for other plant species.