An in vivo plant platform to assess genes encoding native and synthetic enzymes for carotenoid biosynthesis

This chapter provides an overview of how to test enzyme function in plants using candidate genes encoding carotenoid biosynthetic enzymes and/or engineered enzyme versions for synthetic biology applications. A step-by-step guide explains transformation and screening of Agrobacterium tumefaciens containing a binary vector, preparation of inoculum, and transient expression of candidate or retooled enzymes in Nicotiana benthamiana leaf tissue. In planta expression is especially useful for screening candidate carotenoid biosynthetic enzymes because many of the substrates that these enzymes would use are already present in the leaf. In this assay pigments are extracted from leaf discs expressing transgenes and changes in pigment profiles resulting from the activity of these introduced enzymes are scored using High Performance Liquid Chromatography (HPLC). If a transgene product modifies a carotenoid substrate, differences are easily revealed by comparison against wildtype pigment profiles. Additionally, this in planta method presents the researcher with an opportunity to evaluate candidate carotenoid biosynthetic enzyme function in the existing context of N. benthamiana photosynthetic chloroplasts. Photosynthetic phenotypes that arise from changes in pigment composition can be determined by Pulse Amplitude Modulated (PAM) fluorescence measurements, if desired.