Elucidating Carotenoid Biosynthetic Enzyme Localization and Interactions Using Fluorescent Microscopy.

Carotenoids are essential for survival of all plants, where these colorful pigments and derivatives are biosynthesized, as well as for humans and other species that obtain plant-derived carotenoids in their diets and rely upon them for vitamin biosynthesis or antioxidant actions. The plant carotenoid biosynthetic pathway consists of nuclear encoded enzymes that are imported into chloroplasts and other plastids. The pathway structural genes are known and have been targeted for metabolic engineering to improve carotenoid profiles or content. However, results are not always as expected because there remain fundamental gaps in understanding how the pathway is physically organized. Many of the enzymes have been found in high molecular weight complexes which are poorly described. Elucidation of enzyme localization as well as enzyme interactions in vivo are needed for advancing the carotenoid field and facilitating our understanding of the three-dimensional organization of this important pathway. Fluorescent protein fusions with carotenoid enzymes can provide in vivo information when these fusions are introduced and transiently expressed in plant cells. Current advances in fluorescent microscopy, especially confocal microscopy, provide the resolution needed to localize fluorescently tagged carotenoid enzymes within suborganellar locations of plastids. Interactions between carotenoid biosynthetic enzymes can be determined using bimolecular fluorescence complementation (BiFC), a method whereby genes of interest are fused with sequences encoding nonfluorescent N- and C-terminal halves of YFP (yellow fluorescent protein), and then introduced into plant protoplasts to allow expression and visualization by fluorescence microscopy. The YFP fluorescence is restored only if the N and C-terminal regions are brought together by interacting fusion partners. Here we describe the methodology, with extensive tips and notes, for determining in vivo carotenoid enzyme localization and enzyme interactions by transient expression of enzyme-fluorescent protein fusions.