Proteomics of Araucaria angustifolia seed development

Author(s)

Tiago Santana Balbuena

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB)
• Defense date: 2009-05-28

Examining board members

Eny Iochevet Segal Floh; Marcia Regina Braga; Gilberto Barbosa Domont; Carlos Alberto Labate; Maria Magdalena Rossi

• Advisor: Eny Iochevet Segal Floh

Abstract

The aim of the present work was to characterize the seed development of Araucaria angustifolia through proteomics in order to understand the physiological and biochemical changes during this process. For that, initially, three different protein extraction methods were evaluated. The extraction based on protein solubilization in 7 M urea, 2 M thiourea, 1% dithiothreitol, 2% Triton-100, 1 mM phenylmethylsulphonyl fluoride, 5 µM pepstatin, followed by 20% trichloroacetic acid precipitation showed the highest gel resolution and reprodutivity and, thus, was chosen to be used in the analysis of the proteome of A. angustifolia seeds. One aspect that hampers the proteome study of unsequenced species is the low protein representativity in databases. So, protein identification is usually carried out through homology. Strategies based on 2-DE result in high keratin contamination. In the present work a spectra filtering software was developed and evaluated for use in homology driven proteomics. The software reduced the time of search, improved alignment quality and did not result in lost of positive identifications. Finally, using the described strategies, the changes in the proteome of A. angustifolia seeds were studied. Ninety six proteins were identified and classified according to their biological functions and expression profiles during seed development. The identified proteins may be used as protein markers of early and late embryogenesis. Proteins involved in the control of oxidative metabolism were highly expressed during the early stages of seed development; while, carbon metabolism and storage proteins were highly expressed in late stages. Considerations on the improvement and control of somatic embryogenesis through medium manipulation and protein markers screening using data generated are also discussed. (AU)

• FAPESP's process: 05/58747-8 - Comparative proteomic analysis and biochemical variations during the zygotic and somatic embryogenesis of Araucaria angustifolia (Bert.) O. Kuntze.
• Grantee: Tiago Santana Balbuena
• Support Opportunities: Scholarships in Brazil - Doctorate