Mitochondrial CLPP2 Assists Coordination and Homeostasis of Respiratory Complexes.

Protein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear- and mitochondrial-encoded subunits for complex assembly in mitochondria await discovery. We generated knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis (Arabidopsis thaliana). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes.

Fewer genes than organelles: extremely low and variable gene copy numbers in mitochondria of somatic plant cells.

Plant mitochondrial genomes are split into sub-genomes, i.e. genes are distributed across various sub-genomic molecules. To investigate whether copy numbers vary between individual mitochondrial genes, we used quantitative real-time PCR in combination with flow cytometric determination of nuclear DNA quantities to determine absolute per-cell-copy numbers of four mitochondrial genes in various Arabidopsis organs and the leaves of tobacco (Nicotiana tabacum) and barley (Hordeum vulgare). The copy numbers of the investigated mitochondrial genes (atp1, rps4, nad6 and cox1) not only differed from each other, but also varied between organs and changed during the development of cotyledons and leaves in Arabidopsis. We found no correlation between altered gene copy numbers, transcript levels and O(2) consumption. However, per cell, both the number of mitochondria and the number of gene copies increased with growing cell size. Gene copy numbers varied from approximately 40 (cox1 in young leaves) to approximately 280 (atp1 in mature leaves), and the mean number of mitochondria was approximately 300 in young leaves and 450 in mature leaves. Thus, cells are polyploid with respect to their mitochondrial genomes, but individual mitochondria may contain only part of the genome or even no DNA at all. Our data supports structural models of the mitochondrial genome in non-dividing cells of angiosperms that predict localization of the genes on sub-genomic molecules rather than master chromosomes. The data indicate control of the number of individual genes according to the genotype and developmental program(s) via amplification and/or degradation of sub-genomic molecules.