Chapter 5
Determination of In Vitro and In Vivo Activities of Plant
Carotenoid Cleavage Oxygenases
Lourdes Go ´ mez-Go ´ mez, Gianfranco Diretto, Oussama Ahrazem,
and Salim Al-Babili
Abstract
Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important
functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are
generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of
enzymes. The activity of plant CCDs was unraveled more than 20 years ago, with the characterization of the
maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme
in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed
during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific
carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of
detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting
from well-established methods.
Key words Apocarotenoids, Carotenoids, CCD, Enzymatic activity
1 Introduction
In all clades of life, enzymatic breakage of carotenoids gives rise to
biologically active molecules, known as apocarotenoids. In plants,
apocarotenoids play important functions, which include their role
as hormones, such as abscisic acid (ABA) and strigolactones
(SL) [1–3] and as signals regulating different developmental processes [4–6] and involved in thermotolerance and adaptation to
environmental stress [7, 8]. In addition, apocarotenoids contribute
plant-to-plant communication [9, 10], and to herbivore and pathogen defense [11–14], and attract animals for pollination and seed
dispersal [15–18].
Carotenoid cleavage, the initial step in the biosynthesis of
apocarotenoids, is generally mediated by nonheme iron
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_5,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
63
Determination of In Vitro and In Vivo Activities of Plant
Carotenoid Cleavage Oxygenases
Lourdes Go ´ mez-Go ´ mez, Gianfranco Diretto, Oussama Ahrazem,
and Salim Al-Babili
Abstract
Carotenoid cleavage products, apocarotenoids, are biologically active compounds exerting important
functions as chromophore, hormones, signaling molecules, volatiles, and pigments. Apocarotenoids are
generally synthesized by the carotenoid cleavage dioxygenases (CCDs) that comprise a ubiquitous family of
enzymes. The activity of plant CCDs was unraveled more than 20 years ago, with the characterization of the
maize VP14, the first identified CCD. The protocol developed to determine the activity of this enzyme
in vitro is still being used, with minor modifications. In addition, in vivo procedures have been developed
during these years, mainly based on the exploitation of Escherichia coli cells engineered to produce specific
carotenoid substrates. Further, technological developments have led to significant improvements, contributing to a more efficient detection of the reaction products. This chapter provides an updated set of
detailed protocols suitable for the in vitro and in vivo characterization of the activities of CCDs, starting
from well-established methods.
Key words Apocarotenoids, Carotenoids, CCD, Enzymatic activity
1 Introduction
In all clades of life, enzymatic breakage of carotenoids gives rise to
biologically active molecules, known as apocarotenoids. In plants,
apocarotenoids play important functions, which include their role
as hormones, such as abscisic acid (ABA) and strigolactones
(SL) [1–3] and as signals regulating different developmental processes [4–6] and involved in thermotolerance and adaptation to
environmental stress [7, 8]. In addition, apocarotenoids contribute
plant-to-plant communication [9, 10], and to herbivore and pathogen defense [11–14], and attract animals for pollination and seed
dispersal [15–18].
Carotenoid cleavage, the initial step in the biosynthesis of
apocarotenoids, is generally mediated by nonheme iron
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_5,
© Springer Science+Business Media, LLC, part of Springer Nature 2020
63