| Description: | Purpose: We will identify nuclear genes that exhibit altered expression from inhibition of the cytochrome respiratory pathway (CRP), which is one branch of the mitochondrial electron transport chain (mtETC). Rationale: One way to alter flux through metabolic pathways is to alter the levels of proteins in the pathways. Plant mitochondria contain many proteins involved in various metabolic processes, including the TCA cycle, respiration, and photorespiration. Most mtETC complexes consist of nuclearly-encoded mitochondrial proteins (NEMPs) and mitochondrially-encoded mitochondrial proteins (MEMPs). NEMPs are produced in coordination with MEMPs of the same complex and pathway and with other proteins in multi- organelle pathways. Enzymes involved in photorespiration, for example, are located in chloroplasts, mitochondria, and peroxisomes and many of the proteins are nuclearly-encoded. We know little about how plants control the expression of NEMPs, especially in coordination with expression of the other enzymes in multi-organelle pathways. We are identifying the signal transduction pathways through which plant cells control expression of NEMPs. We are beginning by exploring changes in expression of NEMP genes following inhibition of the CRP while preparing to study NEMP gene regulation more broadly. Inhibition of the CRP by antimycin A (AA) causes an accumulation of the transcript of nuclearly-encoded, mitochondrial alternative oxidase (specifically the aox1a gene), which is in the second branch of the mtETC. We are isolating Arabidopsis mutants that no longer increase AOX expression in response to AA. It will be interesting to determine if our mutants are also mutants in altered expression of other genes induced by AA treatment. We hypothesize there will be increases in expression of nuclearly-encoded subunits of complex I of the mtETC and decreases in expression of other CRP proteins. Tissue and treatment: We will use 21 day old Arabidopsis plants (Columbia, C24, stock CS906). The tissues from which we will isolate mRNA are: 1) untreated leaves; and 2) leaves treated with 10 mM antimycin A and incubated in the light for 4 hr. This AA treatment causes an accumulation of aox1a transcript, which is a positive control for testing the RNA from our treatments, and possibly, in the microarray experiment. We hope aox1a is on the microarrays because it is found only in higher plants, some fungi, Chlamydomonas and trypanosomes but not in other eukaryotes. |
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