A survey of the plant mitochondrial proteome in relation to development

Abstract

To expand the functional analysis of plant mitochondria, we have undertaken the building of the proteome of pea mitochondria purified from leaves (green and etiolated), roots and seeds. In the first stage, we focused our proteomic exploration on the soluble protein complement of the green leaf mitochondria. We used traditional two-dimensional polyacrylamide gel electrophoresis, in combination with size exclusion chromatography as a third dimension, to identify the major proteins and further resolve their macromolecular complexity. The two-dimensional map of soluble proteins of green leaf mitochondria revealed 433 spots (with Coomassie blue staining) and around 73% of the proteins (in mass) were identified using three different approaches: Edman degradation, matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry. Quite a lot of the polypeptides were present in multiforms which indicated the presence of isoforms or the occurrence of post-translational modifications. Among these proteins, we uncovered an abundant family that was identified as aldehyde dehydrogenases, representing approximately 7.5% of the soluble proteins. The comparative analysis of soluble mitochondrial proteomes led to the identification of a number of proteins which were specifically present in root or in seed mitochondria, thus revealing the impact of tissue differentiation at the mitochondrial level.

Towards an Analysis of the Rice Mitochondrial Proteome

“…Numbers on the horizontal axis are pI, and numbers on the vertical axis are apparent molecular mass (in kilodaltons). Asterisks in final columns indicate the presence of orthologs in the IEF/SDS-PAGE proteome reports from Arabidopsis (At; Kruft et al, 2001;Millar et al, 2001) and pea (Ps; Bardel et al, 2002). Several other Arabidopsis proteins identified elsewhere by a variety of different electrophoresis techniques have also been included for comparison: a, Sweetlove et al (2002);b, Millar and Heazlewood (2003);and c, Werhahn and Braun (2002).…”

“…Extensive discussions of many of the components identified here and their biological roles can be found in the earlier reports on the proteomes of Arabidopsis and pea (Kruft et al, 2001;Millar et al, 2001;Bardel et al, 2002). Here, we have largely restricted any extensive comment to components that were not identified in these earlier studies (Table II).…”

“…These carriers were identified mainly through LC-MS/MS, which is in agreement with the difficulty of identifying these proteins in two-dimensional gel separations from Arabidopsis (Millar and Heazlewood, 2003). None of these carrier proteins were identified in the IEF/SDS-PAGE approaches in Arabidopsis or pea (Millar et al, 2001;Bardel et al, 2002).…”

“…Several reports have recently attempted to identify the protein contents of plant mitochondria from Arabidopsis (Kruft et al, 2001; Millar et al, 2001) and pea (Pisum sativum;Bardel et al, 2002) using isoelectric focusing (IEF)/SDS-PAGE-based separation of proteins. These three reports each identified 40 to 90 proteins using this arraying technique coupled with identification using antibodies, N-terminal sequencing, matrix-assisted laser-desorption/ionization-timeof-flight (TOF) peptide mass fingerprinting, and collision-induced tandem mass spectrometry (MS/ MS) spectra of single peptides.…”

Refining the Definition of Plant Mitochondrial Presequences through Analysis of Sorting Signals, N-Terminal Modifications, and Cleavage Motifs

“…To adequately address these questions, a large number of presequences need to be assembled to define motifs that differentiate presequence classes. Traditionally, the N-terminal sequences of plant mitochondrial proteins have been obtained by Edman degradation either from purified mitochondrial protein complexes or in proteome studies (Braun and Schmitz, 1995;Jänsch et al, 1996;Millar et al, 1998Millar et al, , 1999Kruft et al, 2001;Bardel et al, 2002). The presequences could only be obtained by comparison of these N-terminal sequences with the preprotein sequence deduced from full-length cDNA sequences, which were only available in a small number of cases.…”