Assessment of Mitochondrial Protein Composition and Purity by Mass Spectroscopy.

While the routine mitochondrial extraction and isolation protocols have not fundamentally changed since the introduction of density gradients, the approaches we use to examine the proteome have. The initial characterisation of mitochondrial proteomes was carried out using two-dimensional gel electrophoresis in 2001 and gel spot mass spectrometry have now largely been superseded as the throughput and sensitivity of commercial mass spectrometers increases. Whist many of these early studies established the components of the mitochondrial proteome, as gels were replaced by gel free approaches the numbers of confirmed components rapidly increased. In this chapter we present gel-based approaches for the separation and concentration of mitochondrial proteins for their characterization by mass spectrometry. We also describe two gel-free approaches which can be used to quantity the degree of contamination arising during the isolation of mitochondria. These approaches are equally suitable for studies comparing one treatment to another.

Energy costs of salt tolerance in crop plants.

Agriculture is expanding into regions that are affected by salinity. This review considers the energetic costs of salinity tolerance in crop plants and provides a framework for a quantitative assessment of costs. Different sources of energy, and modifications of root system architecture that would maximize water vs ion uptake are addressed. Energy requirements for transport of salt (NaCl) to leaf vacuoles for osmotic adjustment could be small if there are no substantial leaks back across plasma membrane and tonoplast in root and leaf. The coupling ratio of the H+ -ATPase also is a critical component. One proposed leak, that of Na+ influx across the plasma membrane through certain aquaporin channels, might be coupled to water flow, thus conserving energy. For the tonoplast, control of two types of cation channels is required for energy efficiency. Transporters controlling the Na+ and Cl- concentrations in mitochondria and chloroplasts are largely unknown and could be a major energy cost. The complexity of the system will require a sophisticated modelling approach to identify critical transporters, apoplastic barriers and root structures. This modelling approach will inform experimentation and allow a quantitative assessment of the energy costs of NaCl tolerance to guide breeding and engineering of molecular components.

Assessment of respiration in isolated plant mitochondria using Clark-type electrodes.

Mitochondrial respiration involves two key gas exchanges, the consumption of oxygen and the release of carbon dioxide. The ability to measure the consumption of oxygen via Clark-type electrodes has been one of the key techniques for advancing our knowledge of mitochondrial function in whole organisms, tissue samples, cells, and isolated subcellular fractions. In plants, oxygen electrode analyses provided the first evidence for some of the unique respiratory properties of plant mitochondria. This chapter briefs the principles of respiration and oxidative phosphorylation, how oxygen consumption measurements can be used to assess the quality of isolated mitochondrial preparations, and how these measurements can answer important questions in plant biochemistry and physiology. Finally, it presents instructions on assembling the oxygen electrode apparatus and how to conduct various assays.