Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy Research article Characterization under quasi-native conditions of the capsanthin/ capsorubin synthase from Capsicum annuum L Dario Piano a,b , Emma Cocco a , Giulia Guadalupi a , Hazem M. Kalaji b,c , Joanna Kirkpatrick d , Domenica Farci c,∗ a Department of Life and Environmental Sciences, Laboratory of Photobiology and Plant Physiology, University of Cagliari, V.le S. Ignazio da Laconi 13, 09123, Cagliari, Italy b Department of Plant Physiology, Warsaw University of Life Sciences - SGGW, Nowoursynowska Str. 159, 02776, Warsaw, Poland c White Hill Company, Ciolkowskiego 161, 15-545, Bialystok, Poland d Leibniz Institute on Aging – Fritz Lipmann Institute, Beutenbergstraβe 11, 07745, Jena, Germany ARTICLEINFO Keywords: Capsanthin/capsorubin synthase Carotenoids Chromoplast Fruit ripening Membrane protein complex Oxidative stress Red bell pepper ABSTRACT Chromoplasts are typical plastids of fruits and fowers, deriving from chloroplasts through complex processes of re-organization and recycling. Since this transition leads to the production of reactive species, chromoplasts are characteristic sites for biosynthesis and accumulation of carotenoids and other antioxidants. Here, we have analysed the chromoplast membranes from Capsicum annuum L. fruits, fnding a signifcant expression of the capsanthin/capsorubin synthase. This enzyme was isolated by a very mild procedure allowing its analyses under quasi-native conditions. The isolated complex appeared as a red coloured homo-trimer, suggesting the retention of at least one of the typical carotenoids from C. annuum. Moreover, the protein complex was co-purifed with a non-proteinaceous fraction of carotenoid aggregates carrying a high molecular weight and separable only by Size Exclusion Chromatography. This last fnding suggested a relationship between the carotenoids synthesis on chromoplast membranes, the presence, and storage of organised carotenoids aggregates typical for chromoplasts. Further MS analyses also provided important hints on the interactome network associated to the capsanthin/ capsorubin synthase, confrming its functional relevance during ripening. Results are discussed in the frame of the primary role played by carotenoids in quenching the growing oxidative stress during fruits ripening. 1. Introduction Fruit ripening is an ensemble of complex physiological processes guided by hormonal and environmental factors (White, 2002; Ozga and Reinecke, 2003; Prasanna et al., 2007). During development, fruits' cells undergo extreme morphologic and metabolic changes, among which there is the chloroplast-to-chromoplast transition (Summer and Cline, 1999; Li and Hui, 2013; Sun et al., 2018). This process sees the dismantling of thylakoid membranes and the recycling of their com- ponents, which are mainly exported and used in other compartments of the plant. At the same time important processes associated to the bio- synthesis of antioxidants, antibiotic molecules, and favouring compo- nents also take place (Spurr and Harris 1968; Howard et al., 2000; Sun et al., 2018). During ripening, the fruit passes from an auto- to a het- erotrophic metabolism, becoming a “passive” organ subject to respira- tion of organic acids and accumulation of sugars (Hall, 1977; Seymour et al., 1993; Fisher et al., 2012; Batista-Silva et al., 2018). This exposes ripening fruits to oxidation events, with concomitantly increased levels of Reactive Species (RS). Therefore, during ripening, RS quenching assumes a pivotal role and must be fnely controlled in order to reach a precise species-specifc synchrony between fruits and seeds maturity (Bouvier et al., 1998; Prasanna et al., 2007; Osorio et al., 2013; Airaki et al., 2015; Kumar et al., 2016; Corpas et al., 2018). In this context, the complex mechanism of RS scavenging, fnely regulated by hormones, makes use of carotenoids, favonoids, and derivatives of phenylpropa- noids (White, 2002; Blokhina et al., 2003; Prasanna et al., 2007; Wang et al., 2013). Among these groups of molecules, carotenoids have the strongest scavenging efciency and they are the main class of anti- oxidants in ripened fruits. This is particularly true in the case of the Capsicum annuum fruits, for which the development and physiology has been extensively studied (Kilcrease et al., 2013; Chaki et al., 2015; Palma et al., 2015), especially in light of their nutritional, medical, and economic importance worldwide (Palevitch and Craker, 1996; Palma et al., 2015; Arimboor et al., 2015; Fernández-Bedmar and Alonso- https://doi.org/10.1016/j.plaphy.2019.09.007 Received 8 August 2019; Received in revised form 2 September 2019; Accepted 4 September 2019 ∗ Corresponding author. E-mail address: farcidomenica@live.it (D. Farci). Plant Physiology and Biochemistry 143 (2019) 165–175 Available online 05 September 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved. T