Chapter 17
Elucidating Carotenoid Biosynthetic Enzyme Localization
and Interactions Using Fluorescent Microscopy
Maria Shumskaya, Rena F. Quinlan, and Eleanore T. Wurtzel
Abstract
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.
Key words Carotenoid enzymes, Biosynthetic pathway, Plants, Maize, Bimolecular fluorescence,
Plastid localization, Protein interaction, Microscopy
1 Introduction
Carotenoids constitute a large class of lipophilic pigments that are
biosynthesized by plants and other organisms [1]. In plants, carotenoids and their bioactive derivatives have manifold roles, ranging from photosynthetic light capture and photoprotection to
signaling needed to promote proper growth and development.
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_17,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
223
Elucidating Carotenoid Biosynthetic Enzyme Localization
and Interactions Using Fluorescent Microscopy
Maria Shumskaya, Rena F. Quinlan, and Eleanore T. Wurtzel
Abstract
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.
Key words Carotenoid enzymes, Biosynthetic pathway, Plants, Maize, Bimolecular fluorescence,
Plastid localization, Protein interaction, Microscopy
1 Introduction
Carotenoids constitute a large class of lipophilic pigments that are
biosynthesized by plants and other organisms [1]. In plants, carotenoids and their bioactive derivatives have manifold roles, ranging from photosynthetic light capture and photoprotection to
signaling needed to promote proper growth and development.
Manuel Rodrı ´guez-Concepcio ´ n and Ralf Welsch (eds.), Plant and Food Carotenoids: Methods and Protocols,
Methods in Molecular Biology, vol. 2083, https://doi.org/10.1007/978-1-4939-9952-1_17,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
223