Abstract
Rice growth under aerobic and anaerobic conditions allowed aspects of mitochondrial biogenesis to be identified as dependent on or independent of an oxygen signal. Analysis of transcripts encoding mitochondrial components found that a subset of these genes respond to oxygen (defined as aerobic), whereas others are relatively unaffected by oxygen availability. Mitochondria formed during growth in anaerobic conditions had reduced protein levels of tricarboxylic acid cycle components and cytochrome-containing complexes of the respiratory chain and repressed respiratory functionality. In general, the capacity of the general import pathway was found to be significantly lower in mitochondria isolated from tissue grown under anaerobic conditions, whereas the carrier import pathway capacity was not affected by changes in oxygen availability. Transcript levels of genes encoding components of the protein import apparatus were generally not affected by the absence of oxygen, and their protein abundance was severalfold higher in mitochondria isolated from anaerobically grown tissue. However, both transcript and protein abundances of the subunits of the mitochondrial processing peptidase, which in plants is integrated into the cytochrome bc(1) complex, were repressed under anaerobic conditions. Therefore, in this system, an increase in import capacity is correlated with an increase in the abundance of the cytochrome bc(1) complex, which is ultimately dependent on the presence of oxygen, providing a link between the respiratory chain and protein import apparatus.
Highlights
EXPERIMENTAL PROCEDURESPlant Material—Dehulled, sterilized rice seeds (Oryza sativa cv. Amaroo) were germinated in the dark, submerged in rice growth media (0.5 mM MES,3 0.5 mM CaCl2, pH 6.5) with carbenicillin (6 mg/liter), and incubated at 30 °C
Eukaryotic responses to oxygen deficit have been performed using the facultative anaerobe, Saccharomyces cerevisiae, where growth under aerobic and anaerobic conditions is associated with changes in mitochondrial ultrastructure and respiratory activity [1, 2]
We have previously established a system for examining mitochondrial biogenesis during rice germination and early seedling development under normal growth conditions [25], where we have found that the maturation of mitochondria is facilitated by high levels of protein import components already present in promitochondria within the dry seed and which is driven by oxidation of external NADH allowing for the rapid resumption of respiratory and metabolic functions to support early seedling establishment
Summary
Plant Material—Dehulled, sterilized rice seeds (Oryza sativa cv. Amaroo) were germinated in the dark, submerged in rice growth media (0.5 mM MES,3 0.5 mM CaCl2, pH 6.5) with carbenicillin (6 mg/liter), and incubated at 30 °C. Western Blotting and Immunodetection—Mitochondrial protein samples were separated by SDS-PAGE (10 g/lane), transferred to a nitrocellulose membrane, and analyzed using antibodies raised to mitochondrial proteins. Primary MS/MS data were analyzed using the Mascot server (available on the World Wide Web at www.matrixscience.com/), searching against an in house data base comprising TIGR and NCBI rice protein sets with error tolerances of MS of Ϯ1.2 Da and MS/MS of Ϯ0.6 Da. In Vitro Import Assays—Precursor proteins were generated from the soybean alternative oxidase [31], soybean FAd subunit of ATP synthase [32], maize phosphate carrier Imports were performed with 20 g of mitochondrial protein as previously described [33], and substrates were included to final concentrations as follows: ATP (0.75 mM), NADH (10 mM), and succinate (5 mM).
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