m-AAA Complexes Are Not Crucial for the Survival of Arabidopsis Under Optimal Growth Conditions Despite Their Importance for Mitochondrial Translation.

For optimal mitochondrial activity, the mitochondrial proteome must be properly maintained or altered in response to developmental and environmental stimuli. Based on studies of yeast and humans, one of the key players in this control are m-AAA proteases, mitochondrial inner membrane-bound ATP-dependent metalloenzymes. This study focuses on the importance of m-AAA proteases in plant mitochondria, providing their first experimentally proven physiological substrate. We found that the Arabidopsis m- AAA complexes composed of AtFTSH3 and/or AtFTSH10 are involved in the proteolytic maturation of ribosomal subunit L32. Consequently, in the double Arabidopsis ftsh3/10 mutant, mitoribosome biogenesis, mitochondrial translation and functionality of OXPHOS (oxidative phosphorylation) complexes are impaired. However, in contrast to their mammalian or yeast counterparts, plant m-AAA complexes are not critical for the survival of Arabidopsis under optimal conditions; ftsh3/10 plants are only slightly smaller in size at the early developmental stage compared with plants containing m-AAA complexes. Our data suggest that a lack of significant visible morphological alterations under optimal growth conditions involves mechanisms which rely on existing functional redundancy and induced functional compensation in Arabidopsis mitochondria.

AtOMA1 Affects the OXPHOS System and Plant Growth in Contrast to Other Newly Identified ATP-Independent Proteases in Arabidopsis Mitochondria.

Compared with yeast, our knowledge on members of the ATP-independent plant mitochondrial proteolytic machinery is rather poor. In the present study, using confocal microscopy and immunoblotting, we proved that homologs of yeast Oma1, Atp23, Imp1, Imp2, and Oct1 proteases are localized in Arabidopsis mitochondria. We characterized these components of the ATP-independent proteolytic system as well as the earlier identified protease, AtICP55, with an emphasis on their significance in plant growth and functionality in the OXPHOS system. A functional complementation assay demonstrated that out of all the analyzed proteases, only AtOMA1 and AtICP55 could substitute for a lack of their yeast counterparts. We did not observe any significant developmental or morphological changes in plants lacking the studied proteases, either under optimal growth conditions or after exposure to stress, with the only exception being retarded root growth in oma1-1, thus implying that the absence of a single mitochondrial ATP-independent protease is not critical for Arabidopsis growth and development. We did not find any evidence indicating a clear functional complementation of the missing protease by any other protease at the transcript or protein level. Studies on the impact of the analyzed proteases on mitochondrial bioenergetic function revealed that out of all the studied mutants, only oma1-1 showed differences in activities and amounts of OXPHOS proteins. Among all the OXPHOS disorders found in oma1-1, the complex V deficiency is distinctive because it is mainly associated with decreased catalytic activity and not correlated with complex abundance, which has been observed in the case of supercomplex I + III2 and complex I deficiencies. Altogether, our study indicates that despite the presence of highly conservative homologs, the mitochondrial ATP-independent proteolytic system is not functionally conserved in plants as compared with yeast. Our findings also highlight the importance of AtOMA1 in maintenance of proper function of the OXPHOS system as well as in growth and development of Arabidopsis thaliana.

Lack of FTSH4 Protease Affects Protein Carbonylation, Mitochondrial Morphology, and Phospholipid Content in Mitochondria of Arabidopsis: New Insights into a Complex Interplay.

FTSH4 is one of the inner membrane-embedded ATP-dependent metalloproteases in mitochondria of Arabidopsis (Arabidopsis thaliana). In mutants impaired to express FTSH4, carbonylated proteins accumulated and leaf morphology was altered when grown under a short-day photoperiod, at 22°C, and a long-day photoperiod, at 30°C. To provide better insight into the function of FTSH4, we compared the mitochondrial proteomes and oxyproteomes of two ftsh4 mutants and wild-type plants grown under conditions inducing the phenotypic alterations. Numerous proteins from various submitochondrial compartments were observed to be carbonylated in the ftsh4 mutants, indicating a widespread oxidative stress. One of the reasons for the accumulation of carbonylated proteins in ftsh4 was the limited ATP-dependent proteolytic capacity of ftsh4 mitochondria, arising from insufficient ATP amount, probably as a result of an impaired oxidative phosphorylation (OXPHOS), especially complex V. In ftsh4, we further observed giant, spherical mitochondria coexisting among normal ones. Both effects, the increased number of abnormal mitochondria and the decreased stability/activity of the OXPHOS complexes, were probably caused by the lower amount of the mitochondrial membrane phospholipid cardiolipin. We postulate that the reduced cardiolipin content in ftsh4 mitochondria leads to perturbations within the OXPHOS complexes, generating more reactive oxygen species and less ATP, and to the deregulation of mitochondrial dynamics, causing in consequence the accumulation of oxidative damage.