Transcriptomic and proteomic data provide new insights into cold-treated potato tubers with T- and D-type cytoplasm.

MAIN CONCLUSION: Tuber-omics in potato with the T- and D-types of cytoplasm showed different sets of differentially expressed genes and proteins in response to cold storage. For the first time, we report differences in gene and protein expression in potato (Solanum tuberosum L.) tubers possessing the T- or D-type cytoplasm. Two F1 diploid reciprocal populations, referred to as T and D, were used. The pooling strategy was applied for detection of differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) in tubers consisting of extreme chip colour after cold storage. RNA and protein bulks were constructed from contrasting phenotypes. We recognized 48 and 15 DEGs for the T and D progenies, respectively. DEPs were identified in the amyloplast and mitochondrial fractions. In the T-type cytoplasm, only 2 amyloplast-associated and 5 mitochondria-associated DEPs were detected. Of 37 mitochondria-associated DEPs in the D-type cytoplasm, there were 36 downregulated DEPs in the dark chip colour bulks. These findings suggest that T- and D-type of cytoplasm might influence sugar accumulation in cold-stored potato tubers in different ways. We showed that the mt/nucDNA ratio was higher in D-possessing tubers after cold storage than in T progeny. For the D-type cytoplasm, the pt/nucDNA ratio was higher for tubers characterized by dark chip colour than for those with light chip colour. Our findings suggest that T- and D-type cytoplasm might influence sugar accumulation in cold-stored potato tubers in different ways.

FTSH4 and OMA1 mitochondrial proteases reduce moderate heat stress-induced protein aggregation.

The threat of global warming makes uncovering mechanisms of plant tolerance to long-term moderate heat stress particularly important. We previously reported that Arabidopsis (Arabidopsis thaliana) plants lacking mitochondrial proteases FTSH4 or OMA1 suffer phenotypic changes under long-term stress of 30°C, while their growth at 22°C is not affected. Here we found that these morphological and developmental changes are associated with increased accumulation of insoluble mitochondrial protein aggregates that consist mainly of small heat-shock proteins (sHSPs). Greater accumulation of sHSPs in ftsh4 than oma1 corresponds with more severe phenotypic abnormalities. We showed that the proteolytic activity of FTSH4, and to a lesser extent of OMA1, as well as the chaperone function of FTSH4, is crucial for protecting mitochondrial proteins against aggregation. We demonstrated that HSP23.6 and NADH dehydrogenase subunit 9 present in aggregates are proteolytic substrates of FTSH4, and this form of HSP23.6 is also a substrate of OMA1 protease. In addition, we found that the activity of FTSH4 plays an important role during recovery from elevated to optimal temperatures. Isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic analyses, along with identification of aggregation-prone proteins, implicated mitochondrial pathways affected by protein aggregation (e.g. assembly of complex I) and revealed that the mitochondrial proteomes of ftsh4 and oma1 plants are similarly adapted to long-term moderate heat stress. Overall, our data indicate that both FTSH4 and OMA1 increase the tolerance of plants to long-term moderate heat stress by reducing detergent-tolerant mitochondrial protein aggregation.

Chronic Activation of AMPK Induces Mitochondrial Biogenesis through Differential Phosphorylation and Abundance of Mitochondrial Proteins in Dictyostelium discoideum.

Mitochondrial biogenesis is a highly controlled process that depends on diverse signalling pathways responding to cellular and environmental signals. AMP-activated protein kinase (AMPK) is a critical metabolic enzyme that acts at a central control point in cellular energy homeostasis. Numerous studies have revealed the crucial roles of AMPK in the regulation of mitochondrial biogenesis; however, molecular mechanisms underlying this process are still largely unknown. Previously, we have shown that, in cellular slime mould Dictyostelium discoideum, the overexpression of the catalytic α subunit of AMPK led to enhanced mitochondrial biogenesis, which was accompanied by reduced cell growth and aberrant development. Here, we applied mass spectrometry-based proteomics of Dictyostelium mitochondria to determine the impact of chronically active AMPKα on the phosphorylation state and abundance of mitochondrial proteins and to identify potential protein targets leading to the biogenesis of mitochondria. Our results demonstrate that enhanced mitochondrial biogenesis is associated with variations in the phosphorylation levels and abundance of proteins related to energy metabolism, protein synthesis, transport, inner membrane biogenesis, and cellular signalling. The observed changes are accompanied by elevated mitochondrial respiratory activity in the AMPK overexpression strain. Our work is the first study reporting on the global phosphoproteome profiling of D. discoideum mitochondria and its changes as a response to constitutively active AMPK. We also propose an interplay between the AMPK and mTORC1 signalling pathways in controlling the cellular growth and biogenesis of mitochondria in Dictyostelium as a model organism.

Protein Processing in Plant Mitochondria Compared to Yeast and Mammals.

Limited proteolysis, called protein processing, is an essential post-translational mechanism that controls protein localization, activity, and in consequence, function. This process is prevalent for mitochondrial proteins, mainly synthesized as precursor proteins with N-terminal sequences (presequences) that act as targeting signals and are removed upon import into the organelle. Mitochondria have a distinct and highly conserved proteolytic system that includes proteases with sole function in presequence processing and proteases, which show diverse mitochondrial functions with limited proteolysis as an additional one. In virtually all mitochondria, the primary processing of N-terminal signals is catalyzed by the well-characterized mitochondrial processing peptidase (MPP). Subsequently, a second proteolytic cleavage occurs, leading to more stabilized residues at the newly formed N-terminus. Lately, mitochondrial proteases, intermediate cleavage peptidase 55 (ICP55) and octapeptidyl protease 1 (OCT1), involved in proteolytic cleavage after MPP and their substrates have been described in the plant, yeast, and mammalian mitochondria. Mitochondrial proteins can also be processed by removing a peptide from their N- or C-terminus as a maturation step during insertion into the membrane or as a regulatory mechanism in maintaining their function. This type of limited proteolysis is characteristic for processing proteases, such as IMP and rhomboid proteases, or the general mitochondrial quality control proteases ATP23, m-AAA, i-AAA, and OMA1. Identification of processing protease substrates and defining their consensus cleavage motifs is now possible with the help of large-scale quantitative mass spectrometry-based N-terminomics, such as combined fractional diagonal chromatography (COFRADIC), charge-based fractional diagonal chromatography (ChaFRADIC), or terminal amine isotopic labeling of substrates (TAILS). This review summarizes the current knowledge on the characterization of mitochondrial processing peptidases and selected N-terminomics techniques used to uncover protease substrates in the plant, yeast, and mammalian mitochondria.

Targeted Proteomics Approach Toward Understanding the Role of the Mitochondrial Protease FTSH4 in the Biogenesis of OXPHOS During Arabidopsis Seed Germination.

Seed germination provides an excellent model to study the process of mitochondrial biogenesis. It is a complex and strictly regulated process which requires a proper biogenesis of fully active organelles from existing promitochondrial structures. We have previously reported that the lack of the inner mitochondrial membrane protease FTSH4 delayed Arabidopsis seed germination. Here, we implemented a targeted mass spectrometry-based approach, Multiple Reaction Monitoring (MRM), with stable-isotope-labeled standard peptides for increased sensitivity, to quantify mitochondrial proteins in dry and germinating wild-type and ftsh4 mutant seeds, lacking the FTSH4 protease. Using total seed protein extracts we measured the abundance of the peptide targets belonging to the OXPHOS complexes, AOX1A, transport, and inner membrane scaffold as well as mitochondrial proteins that are highly specific to dry and germinating seeds. The MRM assay showed that the abundance of these proteins in ftsh4 did not differ substantially from that observed in wild-type at the level of dry seed and after stratification, but we observed a reduction in protein abundance in most of the examined OXPHOS subunits in the later stages of germination. These changes in OXPHOS protein levels in ftsh4 mutants were accompanied by a lower cytochrome pathway activity as well as an increased AOX1A amount at the transcript and protein level and alternative pathway activity. The analyses of the steady-state transcript levels of mitochondrial and nuclear genes encoding OXPHOS subunits did not show significant difference in their amount, indicating that the observed changes in the OXPHOS occurred at the post-transcriptional level. At the time when ftsh4 seeds were fully germinated, the abundance of the OXPHOS proteins in the mutant was either slightly lowered or comparable to these amounts in wild-type seeds at the similar developmental stage. By the implementation of an integrative approach combining targeted proteomics, quantitative transcriptomics, and physiological studies we have shown that the FTSH4 protease has an important role in the biogenesis of OXPHOS and thus biogenesis of mitochondria during germination of Arabidopsis seeds.

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.