Biology Reference
In-Depth Information
Chapter 34
Plant Mitochondrial Proteomics
Shaobai Huang , Richard P. Jacoby , A. Harvey Millar , and Nicolas L. Taylor
Abstract
Mitochondria are responsible for a number of major biochemical processes in plant cells including
oxidative phosphorylation and photorespiration. Traditionally their primary role has been viewed as
the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the
transfer of electrons to O
2
. More recently its role in the synthesis of many metabolites such as amino
acids, lipids, and vitamins has been revealed. They also contain large number of transporters including
members of the mitochondrial carrier substrate family (MCSF) that allow the exchange of metabolites
with the cytosol. Mitochondria also contain their own genome and actively transcribe and translate a
set of proteins that are coordinated with proteins encoded by the nuclear genome to produce large
multisubunit enzymes. To reveal the full diversity of metabolism carried out by mitochondria signifi -
cant efforts have sought to uncover the protein profi le of mitochondria from both crops and model
plants. Successful proteomic analysis depends on the preparation of high-quality isolated mitochondria,
coupled to high-resolution proteomic techniques for identifi cation, quantitation, and assessment of the
degree of contamination by other organelles and cellular compartments. Here we outline a mitochon-
drial isolation protocol that can be applied to a range of plant tissues, and detail methods of assessing
the quality and purity of the resultant sample, including calculations of respiratory control ratio, marker
enzyme assays, differential in-gel electrophoresis, and quantitative gel-free mass spectrometry.
Key words
Proteomics, Mitochondria, Mass spectrometry, Gradient purifi cation, 2D-Polyacrylamide
gel electrophoresis, 2D-Differential in-gel electrophoresis, Spectral counting
1
Introduction
Plant mitochondria are well known as the site of production of the
mobile energy currency ATP, for their role in the recycling of phos-
phoglycolate in photorespiration and the oxidation of organic acids
by the TCA cycle. In addition they also have well-studied roles in
plant development, fertility, abiotic stress tolerance, disease suscep-
tibility and defense, senescence, and programmed cell death.
Recent advances in peptide mass spectrometry coupled with the
complete sequencing and extensive annotation of the nuclear and
organelle genomes of a variety of plant species have been impor-
