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Biochemical and Molecular Mechanisms Underlying Colour Retention in Capsicum annuum. Alexandra Holden1, Harriet Berry1, Daniel Rickett2, Paul Fraser1. 1Department of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, UK | email: [email protected] 2Syngenta, Jealott’s Hill International Research Centre, Bracknell, Berkshire, RG42 6EY. Background Capsicum annuum • Chilli pepper (Capsicum annuum) is the most widely grown spice product. • Colour retention is a key quality trait. • Colour is lost over time during storage, leading to less valuable crop. Carotenoids • Carotenoids are coloured lipophilic tetraterpenes, synthesised by photosynthetic plants (Fig. 1). • Carotenoid quantity and composition, and sub-chromoplast sequestration mechanisms, are directly linked to red colour phenotype. • Capsanthin (Fig. 2), and its esters, are primarily responsible for red colour of C. annuum. Objective 1: Metabolic profiling of a C. annuum population x Spec Assay HPLC Metabolic profiling of a C. annuum population displaying variation in colour retention phenotype will identify differences in carotenoid profile, along with other metabolites, in high and low retention lines. GC-MS This data will then be used in QTL analysis for locating genomic regions containing candidate genes responsible for carotenoid degradation. Fig 3. Carotenoid profile HPLC chromatograms. Fig 1. Carotenoid modification pathway, from phytoene1. Fig 2. Capsanthin structure. Objective 2: Identification of candidate genes involved in carotenoid degradation Using QTL data, loci in the C. annuum genome containing candidate genes involved in colour loss mechanisms will be identified. Candidate genes will be identified through bioinformatic data mining using the Capsicum genome sequence. We hypothesise that candidate genes may include carotenoid cleavage dioxygenases, or genes involved in carotenoid sequestration and storage. Gene expression techniques, such as RNA-seq and qPCR, will determine the gene expression profile in high retention lines. Protein expression will determine function. Objective 3: Subchromoplast structural study Objective 4: Characterisation of lipid composition and peroxidation Carotenoids are sequestered in organelles, known as chromoplasts; plastoglobules, within chromoplasts, are lipoproteins associated with carotenoids2. Plastoglobule structure is dependent on the carotenoids present. Fibrillin is a protein commonly found associated with carotenoids in plastoglobules in C. annuum3. Lipid peroxidation is thought to cause carotenoid degradation. Carotenoids protect lipids from oxidation by scavenging ROS5. The antioxidative role of carotenoids is a mechanism for carotenoid degradation. Lipid peroxidation rate will be assessed in different lines to determine whether a correlation exists. Variation in lipid composition in sub- Fig 4. Chromoplast4. Understanding of the storage mechanisms will highlight the role that such structures play in carotenoid degradation, and hence, colour loss. Electron microscopy will be used to determine sub-chromoplast structure, and enzyme assays will determine enzyme localisation. Fig 5. Waxy cuticle structure. chromoplast fractions of different lines will be characterised by GC-MS. Fruit waxy cuticle structure will be observed by electron microscopy to determine whether cuticle ultrastructure correlates with colour retention. Expected outcomes and impact Understanding colour loss and carotenoid degradation mechanisms in C. annuum will: • • • • Result in the breeding of high colour retention chilli pepper varieties leading to greater economic return for growers Increase understanding of colour loss mechanisms in a wide array of species Allow the nutritional benefit of chilli peppers to be exploited, as carotenoids may be degraded slower Provide a basis on which genome editing may be carried out, following identification of genes involved in colour loss Acknowledgements and References I would like to thank members of the Fraser Group at Royal Holloway for help and support with this project. This PhD studentship is funded by BBSRC CASE studentships in collaboration with Syngenta. 1. 2. 3. 4. 5. Fraser PD, Bramley PM (2004). Progress in Lipid Research 43 (3):228-265. Austin JR, Frost E, Vidi P-A, Kessler F, Staehelin LA (2006). The Plant Cell 18 (7):1693-1703. Deruère J, Römer S, d'Harlingue A, Backhaus RA, Kuntz M, Camara B (1994). The Plant Cell 6 (1):119-133. Botte CY, Marechal E (2013). Trends in Plant Science 19 (2):71-78. Sharma P, Jha AB, Dubey RS, Pessarakli M (2012). Journal of Botany 2012:26.