Molecular and Biochemical Mechanisms Underlying Carotenoid Biosynthesis in Capsicum annuum

Research output: ThesisDoctoral Thesis

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Colour is a commercially important quality trait for many fruit crops, including the sweet bell pepper (Capsicum annuum). Due to the significant financial value and nutritional benefits conferred on crop products by colour, it is essential that we understand the mechanisms which govern its development. Mature fruit colour in Capsicum is controlled by three loci: c1, c2, and y. The c2 locus has been associated with the gene encoding the fruit specific enzyme catalysing the first step of carotenoid biosynthesis, phytoene synthase (Psy-1), the y locus with the gene encoding the enzyme capsanthin-capsorubin synthase (Ccs), and the identity of the c1 locus remains undetermined. The present work aims to investigate fruit colour from two different perspectives: orange and yellow fruit phenotypes in relation to the ripening-fruit copy of the enzyme catalysing the first step in carotenoid biosynthesis (Psy-1), and yellow and white fruit phenotypes in relation to the c1 locus.
Firstly, a discovery panel of sixteen Capscium annuum accessions ranging in colour from red to white was used to further investigate the role of the enzyme PSY-1 in determining pepper mature fruit colour. Reverse-phased chromatography and quantitative real-time PCR (qRT-PCR) were used to investigate the molecular mechanisms governing colour in this panel of lines. It was demonstrated that Psy-1 expression correlated with colour, and that expression levels of most other genes in the carotenoid biosynthesis pathway were unaffected by a lack of Psy-1 expression; validating that this is the most important regulatory gene in the pathway. The structure of the plastids in which carotenoids accumulate and the specialisations of their sub-compartments were investigated with electron microscopy, sub-chromoplast fractionation and enzyme activity assays. These studies showed that PSY-1 protein was absent from accessions lacking Psy-1 transcript; that the enzyme is located in the same plastid sub-structure as most of the carotenoids it synthesises; and that this is also the region of the plastid where the enzyme is active. Gas Chromatography-Mass Spectrometry (GC-MS) was also employed to elucidate the impact of a lack of Psy-1 on the wider metabolism of the fruit, revealing that the largest metabolic changes were in fatty acids capable of esterifying carotenoids. The multiple lines of investigation validate the contribution of Psy-1 to fruit colour.
A second diversity panel consisted of twelve lines, none of which possessed a functional copy of either the y or c2 loci. Six were positive for the c1 locus and produced yellow ripe fruit, and six were negative for all three loci and produced cream coloured or white ripe fruit. Carotenoids were profiled, and broader fruit metabolism was investigated with GC-MS. This demonstrated that carotenoids were only detectable at trace levels in ripe fruit of lines lacking the c1 locus, and that these lines possessed elevated levels of Very Long Chain Fatty Acids (VLCFAs), which are too long to esterify carotenoids. This could have implications for their storage during the chloroplast to chromoplast transition. Chloroplast and chromoplast structures were studied with electron microscopy, revealing that accessions lacking functional copies of the c1 locus possessed aberrant chloroplast structures at the mature green stage, which could alter their ability to retain carotenoids throughout ripening. Furthermore, the transcriptomes of c1 positive and negative accessions were generated with RNA-seq, and interrogated for potential candidate genes for association with the c1 locus. Candidate genes for further analysis were generated; these include a NAC transcription factor, an acyl transferase, and a plastid-specific kinase, among others.
Original languageEnglish
Awarding Institution
  • Royal Holloway, University of London
  • Fraser, Paul, Supervisor
Thesis sponsors
Award date1 Mar 2018
Publication statusUnpublished - 2018

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