Characterization under quasi-native conditions of the capsanthin/capsorubin synthase from Capsicum annuum L.

Chromoplasts are typical plastids of fruits and flowers, 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, finding a significant 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-purified with a non-proteinaceous fraction of carotenoid aggregates carrying a high molecular weight and separable only by Size Exclusion Chromatography. This last finding 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, confirming 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.

Piano D ，Cocco E ，Guadalupi G ，Kalaji HM ，Kirkpatrick J ，Farci D ... -  《-》

Correlations of carotenoid content and transcript abundances for fibrillin and carotenogenic enzymes in Capsicum annum fruit pericarp.

The fruits of Capsicum spp. are especially rich sites for carotenoid synthesis and accumulation, with cultivar-specific carotenoid accumulation profiles. Differences in chromoplast structure as well as carotenoid biosynthesis are correlated with distinct carotenoid accumulations and fruit color. In the present study, the inheritance of chromoplast shape, carotenoid accumulation profiles, and transcript levels of four genes were measured. Comparisons of these traits were conducted using fruit from contrasting variants, Costeño Amarillo versus Costeño Red, and from F1 hybrids; crosses between parental lines with novel versions of these traits. Intermediate chromoplast shapes were observed in the F1, but no association between specific carotenoid accumulation and chromoplast shape was detected. Increased total carotenoid content was associated with increased β-carotene and violaxanthin content. Transcript levels for phytoene synthase (Psy) and β-carotene hydroxylase (CrtZ-2) were positively correlated with increased levels of specific carotenoids. No correlation was detected between transcript levels of capsanthin/capsorubin synthase (Ccs) and carotenoid composition or chromoplast shape. Transcript levels of fibrillin, were differentially correlated with specific carotenoids, negatively correlated with accumulation of capsanthin, and positively correlated with violaxanthin. The regulation of carotenoid accumulation in chromoplasts in Capsicum fruit continues to be a complex process with multiple steps for control.

Kilcrease J ，Rodriguez-Uribe L ，Richins RD ，Arcos JM ，Victorino J ，O'Connell MA ... -  《-》

Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts.

We report a comprehensive proteome analysis of chromoplasts from bell pepper (Capsicum annuum L.). The combination of a novel strategy for database-independent detection of proteins from tandem mass spectrometry (MS/MS) data with standard database searches allowed us to identify 151 proteins with a high level of confidence. These include several well-known plastid proteins but also novel proteins that were not previously reported from other plastid proteome studies. The majority of the identified proteins are active in plastid carbohydrate and amino acid metabolism. Among the most abundant individual proteins are capsanthin/capsorubin synthase and fibrillin, which are involved in the synthesis and storage of carotenoids that accumulate to high levels in chromoplasts. The relative abundances of the identified chromoplast proteins differ remarkably compared with their abundances in other plastid types, suggesting a chromoplast-specific metabolic network. Our results provide an overview of the major metabolic pathways active in chromoplasts and extend existing knowledge about prevalent metabolic activities of different plastid types.

Siddique MA ，Grossmann J ，Gruissem W ，Baginsky S ... -  《PLANT AND CELL PHYSIOLOGY》

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid.

The late steps of carotenoid biosynthesis in plants involve the formation of xanthophylls. Little is known about the enzymology of these steps. This paper reports the purification to homogeneity of a xanthophyll biosynthetic enzyme from Capsicum annuum chromoplasts, which catalyzes the conversion of the ubiquitous 5,6-epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. Owing to its bifunctionality, the name capsanthin-capsorubin synthase is proposed for this new enzyme. The purified enzyme is a monomer with a molecular mass of 50 kDa. Antibodies raised against this enzyme allowed the isolation of a full-length cDNA clone encoding a capsanthin capsorubin synthase high molecular weight precursor. The primary deduced structure reveals the presence of a consensus nucleotide binding site. The capsanthin-capsorubin synthase gene is specifically expressed during chromoplast development in fruits accumulating ketocarotenoids, but not in mutants impaired in this biosynthetic step.

Bouvier F ，Hugueney P ，d'Harlingue A ，Kuntz M ，Camara B ... -  《PLANT JOURNAL》

Manipulation of Carotenoid Metabolic Flux by Lycopene Cyclization in Ripening Red Pepper ( Capsicum annuum var. conoides) Fruits.

Carotenoids are essential phytonutrients for the human body. Higher plants usually synthesize and accumulate carotenoids in their leaves, flowers, and fruits. Most carotenoids have either two β-rings on both ends or β- and ε-rings separately on two ends of their molecules and are synthesized from the acyclic lycopene as the precursor. Lycopene β- and ε-cyclases (LCYB and LCYE, respectively) catalyze the β- and ε-cyclization of lycopene, respectively, and regulate the metabolic flux from lycopene to its downstream β,β-branches (by LCYB alone) and β,ε-branches (by LCYE and LCYB). In this study, we identified and characterized genes for two LCYBs (CaLCYB1 and CaLCYB2), one LCYE (CaLCYE1), and a capsanthin/capsorubin synthase (CaCCS1) which is also able to β-cyclize lycopene from the red pepper ( Capsicum annuum var. conoides) genome. By quantifying transcript abundances of these genes and contents of different carotenoid components in ripening fruits, we observed a correlation between the induction of both CaLCYBs and the accumulation of carotenoids of the β,β-branch during ripening. Although capsanthin was accumulated in ripened fruits, our quantification demonstrated a strong induction of CaCCS1 at the breaker stage, together with the simultaneous repression of CaLCYE1 and the decrease of lutein content, suggesting the involvement of CaCCS1 in competing against CaLCYE1 for synthesizing carotenoids of the β,β-branch. Our results provide important information for future metabolic engineering studies to manipulate carotenoid biosynthesis and accumulation in fruits.

Wang Q ，Cao TJ ，Zheng H ，Zhou CF ，Wang Z ，Wang R ，Lu S ... -  《-》