Experimental Analysis of the Rice Mitochondrial Proteome, Its Biogenesis, and Heterogeneity    

Abstract

Mitochondria in rice (Oryza sativa) are vital in expanding our understanding of the cellular response to reoxygenation of tissues after anaerobiosis, the crossroads of carbon and nitrogen metabolism, and the role of respiratory energy generation in cytoplasmic male sterility. We have combined density gradient and surface charge purification techniques with proteomics to provide an in-depth proteome of rice shoot mitochondria covering both soluble and integral membrane proteins. Quantitative comparisons of mitochondria purified by density gradients and after further surface charge purification have been used to ensure that the proteins identified copurify with mitochondria and to remove contaminants from the analysis. This rigorous approach to defining a subcellular proteome has yielded 322 nonredundant rice proteins and highlighted contaminants in previously reported rice mitochondrial proteomes. Comparative analysis with the Arabidopsis (Arabidopsis thaliana) mitochondrial proteome reveals conservation of a broad range of known and unknown function proteins in plant mitochondria, with only approximately 20% not having a clear homolog in the Arabidopsis mitochondrial proteome. As in Arabidopsis, only approximately 60% of the rice mitochondrial proteome is predictable using current organelle-targeting prediction tools. Use of the rice protein data set to explore rice transcript data provided insights into rice mitochondrial biogenesis during seed germination, leaf development, and heterogeneity in the expression of nucleus-encoded mitochondrial components in different rice tissues. Highlights include the identification of components involved in thiamine synthesis, evidence for coexpressed and unregulated expression of specific components of protein complexes, a selective anther-enhanced subclass of the decarboxylating segment of the tricarboxylic acid cycle, the differential expression of DNA and RNA replication components, and enhanced expression of specific metabolic components in photosynthetic tissues.

Nucleoid-Enriched Proteomes in Developing Plastids and Chloroplasts from Maize Leaves: A New Conceptual Framework for Nucleoid Functions

“…Conversely, coexpression in many cases implies the presence of functional linkages between genes or proteins, allowing for the identification of new components of processes or protein complexes. Cluster analysis has been used extensively for transcripts (Eisen et al, 1998;Belacel et al, 2006;Long et al, 2008) and more recently for proteomics (Dong et al, 2008;Huang et al, 2009;Pontén et al, 2009;Quintana et al, 2009;Majeran et al, 2010;Olinares et al, 2010). Cluster analysis is based on the notion of unsupervised learning in which data objects within the same cluster are similar to one another and dissimilar to the objects in other clusters.…”

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

“…Samples were then dried in a vacuum centrifuge, resuspended in 5% (v/v) acetonitrile and 0.1% (v/v) formic acid, and analyzed on an Agilent XCT Ultra Ion Trap mass spectrometer (Agilent Technologies) according to Meyer et al (2007). Resulting MS/MS-derived spectra were analyzed against our in-house rice mitochondrial protein database mainly extracted from our recently generated rice mitochondrial protein list (Huang et al, 2009) and our in-house Arabidopsis mitochondrial protein database extracted from SUBA ; http:// www.plantenergy.uwa.edu.au/applications/suba/index.php). The database was searched using the Mascot search engine version 2.2.03 with selection nonenzyme digestion and utilizing error tolerances of 61.2 D for MS and 60.6 D for MS/MS, Max Missed Cleavages set to 1, with variable modification of Oxidation (M), Carbamidomethyl (C), and Acetylation (N-term), instrument set to ESI-TRAP, and peptide charge set at 2 + and 3 + .…”

“…Such analysis will not identify spectra for the N-terminal peptides of mature mitochondrial proteins that are derived from the processing carried out by MPP. In this study, we have exploited our lists of the Arabidopsis and rice mitochondrial proteomes (Heazlewood et al, 2004Huang et al, 2009) to build smaller databases in order to carry out searches of archived MS/MS spectra with the digestion parameter set to "no enzyme." This allows matching of any peptides after trypsin digestion, even those derived from digestion with another enzyme such as MPP.…”

Alterations of Mitochondrial Protein Assembly and Jasmonic Acid Biosynthesis Pathway in Honglian (HL)-type Cytoplasmic Male Sterility Rice

“…Among these 522 proteins, 245 proteins were confirmed as mitochondrial components through comparison with a list of 322 nonredundant rice mitochondrial proteins previously compiled ( Fig. 2A; a list of these proteins is shown in supplemental Table S1A) (16).…”

“…Therefore, it is difficult to attribute a precise function to any specific CMS protein in mitochondria, especially when the CMS protein shares limited homology to any known protein. In searching for clues to the mechanism of CMS, past studies have used proteomics to obtain a global view of the composition and spatial distribution of mitochondrial proteins (8,15,16). In this study, quantitative proteomics was used to compare the mitochondrial proteome between YtA and its maintainer line YtB.…”