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In-Depth Characterization of the Phaseolin Protein Diversity of Common Bean (Phaseolus vulgaris L.) Based on Two-Dimensional Electrophoresis and Mass Spectrometry

María De La Fuente1*, María López-Pedrouso2, Jana Alonso3, Marta Santalla1, Antonio M. De Ron1, Gonzalo Álvarez2 and Carlos Zapata2


1Department of Plant Genetic Resources, Misión Biológica de Galicia-CSIC, ES-36080 Pontevedra, Spain

2Department of Genetics, Santiago de Compostela University, ES-15782 Santiago de Compostela, Spain
3Proteomics Laboratory, University Hospital Complex of Santiago de Compostela-CHUS, ES-15706 Santiago de Compostela, Spain

Article history:

Received January 31, 2012
Accepted March 8, 2012

Key words:

common bean, glycoproteins, mass spectrometry, phaseolin protein, Phaseolus vulgaris, seed proteome, two-dimensional electrophoresis

Summary:
Phaseolin is the major seed storage protein of common bean. It comprises a complex set of glycoproteins heterogeneous in their polypeptide composition that is encoded by a gene family. Analyses of phaseolin banding patterns by one-dimensional electrophoresis (SDS-PAGE) have been central to the current understanding of the diversity of wild and cultivated common beans. In this work, we have carried out a detailed description and interpretation of phaseolin diversity in cultivated common beans of different geographic origins (Mesoamerican and Andean gene pools) based on the current two-dimensional electrophoresis (2-DE) technology and mass spectrometry (MS). High-quality 2-DE gel images revealed very complex phaseolin patterns across the studied cultivars. Specifically, patterns of phaseolin within cultivars were organized in a horizontal string of multiple isospot pairs varying in isoelectric point and molecular mass. The degree of similarity among phaseolin patterns was estimated from the percentage of spots shared between pairs of cultivars. Analyses of proteomic distances between phaseolin types by non-metrical multidimensional scaling revealed that 2-DE phaseolin profiles are more similar among cultivars belonging to the same gene pool. However, higher differentiation was found among cultivars of the Andean gene pool. Analysis of genetic variations of the PCR-based SCAR marker of phaseolin seed protein was in general agreement with 2-DE phaseolin patterns, but provided supplementary information regarding diversity among cultivars. Furthermore, the molecular basis responsible for the complexity of 2-DE phaseolin patterns was investigated. Thus, identification of phaseolin spots from 2-DE gels by MALDI-TOF and MALDI-TOF/TOF MS showed that each single isospot pair contained only one type (α or β) of phaseolin polypeptide, but pairs with higher and lower molecular mass corresponded to α- and β-type polypeptides, respectively. In addition, partial and total deglycosylation of seed protein extracts with the enzyme PNGase F indicated that differences between isospots of each pair are exclusively due to a different extent of glycosylation of the same type of phaseolin polypeptide. Taken together, our observations provide new insights into the study of genetic differentiation of common bean populations based on phaseolin diversity.

 


*Corresponding author:          mfuente@mbg.csic.es
                                               ++34 986 854 800
                                               ++34 986 841 362

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