Reduced cytochrome c levels in Arabidopsis disrupt stability of complex IV without affecting complexes I and III.

WELCHEN, ELINA; HILDEBRANDT, T; LEWEJOHANN, DAGMAR; GONZALEZ, DANIEL H.; BRAUN, HANS PETER.

Congreso; International Congress on Plant Mitochondrial Biology – ICPMB (2011).; 2011

Arabidopsis contains two cytochrome c genes, CYTC-1(At1g22840) and CYTC-2 (At4g10040). For CYTC-1, we have analyzed two mutants with T-DNA insertions in the first intron (a1) and downstream of the stop codon (a2). Insertional mutant a1 is a knock-out line while a2 homozygous plants have low transcript levels as measured by Northern analysis. The CYTC-2 mutant lines contain insertions within exons 1 and 3 (b1 and b2 respectively) and it was not possible to detect expression of this gene by qRT-PCR in mutant plants. We obtained double CYT-C mutants by reciprocal crossing. Knock-out of both genes was lethal and produced an arrest of embryo development, showing that cytochrome c function is essential at early stages of plant development. In double homozygous a2b mutants, Western blot analysis indicated that cytochrome c levels were less than 5% respective to wild type. These mutants showed smaller rosettes with a pronounced decrease in the size of parenchymatic cells, reduced flowering stem elongation and root growth, and a delay in senescence. Mitochondria prepared from a2b1 cell cultures showed lower levels of respiration rates but higher alternative respiration compared to wild type. Furthermore, the reduced levels of cytochrome c in plant mitochondria severely affect the activity and the amount of complex IV, without affecting complexes I and III. One possibility is that cytochrome c is required for the stability of Complex IV, as observed in other systems. It is surprising that, even if cytochrome c is essential, a significant decrease in its levels produces only mild phenotypic alterations. A small amount of this protein may be necessary for its role as an electron carrier, remaining in close contact with the inner membrane as part of supercomplexes. The excess protein could remain in a soluble pool in the intermembrane space, participating as electron carrier in other processes like ascorbate metabolism, mitochondrial import mediated by MIA40 and -oxidation pathways.