A possible approach for gel-based proteomic studies in recalcitrant woody plants.

Mónica Sebastiana,Joana Martins,Sílvia Ferreira,Ana Varela Coelho,Fátima Vaz,Tânia Simões,Filipa Monteiro,Andreia Figueiredo,Maria Salomé Pais,Catarina Franco,Deborah Penque

doi:10.1186/2193-1801-2-210

Abstract

Woody plants are particularly difficult to investigate due to high phenolic, resin, and tannin contents and laborious sample preparation. In particular, protein isolation from woody plants for two-dimensional gel electrophoresis (2-DE) is challenging as secondary metabolites negatively interfere with protein extraction and separation. In this study, three protein extraction protocols, using TCA, phenol and ethanol as precipitation or extraction agents, were tested in order to select the more efficient for woody recalcitrant plant gel-based proteomics. Grapevine leaves, pine needles and cork oak ectomycorrhizal roots were used to represent woody plant species and tissues. The phenol protocol produced higher quality 2-DE gels, with increased number of resolved spots, better spot focusing and representation of all molecular mass and isoelectric point ranges tested. In order to test the compatibility of the phenol extracted proteomes with protein identification several spots were excised from the phenol gels and analyzed by mass spectrometry (MALDI-TOF/TOF). Regardless the incomplete genome/protein databases for the plant species under analysis, 49 proteins were identified by Peptide Mass Fingerprint (PMF). Proteomic data have been deposited to the ProteomeXchange with identifier PXD000224. Our results demonstrate the complexity of protein extraction from woody plant tissues and the suitability of the phenol protocol for obtaining high quality protein extracts for efficient 2-DE separation and downstream applications such as protein identification by mass spectrometry.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-2-210) contains supplementary material, which is available to authorized users.

Highlights

Nowadays, proteomics constitutes one of the priority research areas in biological sciences
Both qualitative and quantitative differences were found among 2-DE patterns for the three protein extraction protocols
All three extraction protocols resulted in good quality well-resolved gels (Figure 1D,E,F)

Summary

Introduction

Proteomics constitutes one of the priority research areas in biological sciences. Knowledge generated from these and other model plants need to be applied to other plant species. On the Gymnosperm group, much research has been conducted on the genus Pinus (Wu et al 2008; Valledor et al 2008, 2010; Wang et al 2013), with Maritime pine (Pinus pinaster Ait.) being one of the most representative species used for reforestation in South-western Europe. Grapevine (Vitis vinifera) is considered the most important fruit plant throughout the world, much proteomic research has been conducted in the last decade on this species (reviewed in Giribaldi and Giuffrida 2010). Cork production from cork-oak supports an industry of economic and social relevance in Mediterranean countries, but few proteomic studies have been conducted (Gómez et al 2009; Ricardo et al 2011)

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