|CARVALHO, LUIZ - Embrapa Genetic Resources|
|CHEN, SONGBI - Chinese Academy Of Agricultural Sciences|
|DE SOUZA, CLAUDIA - Federal University Of Para|
|VIERIA, EDUARDO - Embrapa Genetic Resources|
Submitted to: The Open Biochemistry Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/12/2012
Publication Date: 11/26/2012
Citation: Carvalho, L.J., Lippolis, J.D., Chen, S., De Souza, C.R., Vieria, E.A., Anderson, J.V. 2012. Characterization of carotenoid-protein complexes and gene expression analysis associated with carotenoid sequestration in pigmented cassava (Manihot esculenta Crantz) storage root. The Open Biochemistry Journal. 6:116-130.
Interpretive Summary: Separation of proteins fractions obtained from intense yellow pigmented cassava roots identified a unique carotinoid-protein complex. Proteins associated with the carotenoid-protein complex were isolated and further characterized. Peptide sequences obtained from these proteins were identified using public sequence databases. A total of 83 peptides were identified, which matched to 26 proteins names in the public databases. Small Heat Shock Proteins (sHSPs) were the most abundant proteins present in the carotenoid-protein complex, which included three families of HSPs with a variety of putative functionality. Further analysis to identify transcript abundance of six proteins isolated from roots of intense yellow cassava root indicated that all are highly expressed. Transcript for a novel allele gene coding for a sHSP (HSP21) identified four single point mutations in intense yellow compared to white phenotypes of cassava. Cumulatively, these results indicate that sHSP play an important role in sequestration mechanism of carotenoids in cassava storage root, support the correlation between total carotenoid and protein content in the root tissue of pigmented cassava, and may provide new approaches for increasing nutritional value of domesticated cassava.
Technical Abstract: Carotenoid-protein complex separation by size exclusion chromatography, protein fractionation by SDS-PAGE, and shotgun PROTEOMICS technology were used to identify and characterize carotenoid associated proteins (CAPs) of chromoplast-enriched suspensions from cassava intense yellow storage root. A non-denatured carotenoid-protein complex was separated in fractions 33-49 (peak 1) and a non-carotenoid-associated proteins in fractions 108-122 (peak 2). Proteins from each peak were separated by SDS-PAGE and in gel tryptic digested. Peptide sequences were directly obtained from the LC_MS/MS spectrum and peptide mass analyzed using MASCOT search engine. Protein identification was established using public sequence databases, and enhancement of confidence using four cassava EST databases from CIAT, EMBRAPA, ESTIMA, and RIKEN that were translated in all six reading frames and annotated to the Arabidopsis and Manihot database. Sequences from peak 1 and 2 identified 83 and 106 peptides, respectively, which matched to 26 and 39 known proteins names. Small Heat Shock Proteins (sHSPs) were the most abundant proteins present in the carotenoid-protein complex. Pfam domain analysis indicated three families of HSPs present in the carotenoid-protein complex with a variety of putative functionality. Western blot analysis recognized the presence of Fibrillin and Or_proteins in chromoplast enriched suspension and peak 2 of intense yellow root but not in peak 1 and white root. Performed qRT_PCR identified transcript abundance of four proteins identified in peaks 1 and 2 together with Fibrillin and Or_proteins indicating that all of them are highly expressed in intense yellow root. Transcript for CAP4 protein indicated the presence of a heteroduplex DNA strand distinct in the white and intense yellow phenotype, as judged by their dissociation curve from the qRT_PCR result and PCR product sequence. Cumulatively, these results indicate that sHSP play a important role in sequestration mechanism of carotenoids in cassava storage root, support the correlation between total carotenoid and protein content in the root tissue of pigmented cassava, and may provide new approaches for increasing nutritional value of domesticated cassava.