SUMMARYMitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein-protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot-gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re-annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource (https://complexomemap.de/at_mito_lea ves) to deposit the data and to allow straightforward access and custom data analyses.

The respiratory electron transport chain (ETC) couples electron transfer from organic substrates onto molecular oxygen with proton translocation across the inner mitochondrial membrane. The resulting proton gradient is used by the ATP synthase complex for ATP formation. In plants, the ETC is especially intricate. Besides the “classical” oxidoreductase complexes (complex I–IV) and the mobile electron transporters cytochrome c and ubiquinone, it comprises numerous “alternative oxidoreductases.” Furthermore, several dehydrogenases localized in the mitochondrial matrix and the mitochondrial intermembrane space directly or indirectly provide electrons for the ETC. Entry of electrons into the system occurs via numerous pathways which are dynamically regulated in response to the metabolic state of a plant cell as well as environmental factors. This mini review aims to summarize recent findings on respiratory electron transfer pathways in plants and on the involved components and supramolecular assemblies.

Background: L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the final step of the L-ascorbate biosynthesis pathway and at the same time is essential for complex I accumulation. Results: The active GLDH is localized within three different subcomplexes of complex I. Conclusion: Evidence is increasing that GLDH represents a complex I assembly factor. Significance: New insights into mitochondrial complex I assembly in Arabidopsis thaliana.

Proline accumulates in many plant species in response to environmental stresses. Upon relief from stress, proline is rapidly oxidized in mitochondria by proline dehydrogenase (ProDH) and then by pyrroline-5-carboxylate dehydrogenase (P5CDH). Two ProDH genes have been identified in the genome of the model plant Arabidopsis thaliana To gain a better understanding of ProDH1 functions in mitochondria, proteomic analysis was performed. ProDH1 polypeptides were identified in Arabidopsis mitochondria by immunoblotting gels after 2D blue native (BN)-SDS/PAGE, probing them with an anti-ProDH antibody and analysing protein spots by MS. The 2D gels showed that ProDH1 forms part of a low-molecular-mass (70-140 kDa) complex in the mitochondrial membrane. To evaluate the contribution of each isoform to proline oxidation, mitochondria were isolated from wild-type (WT) and prodh1, prodh2, prodh1prodh2 and p5cdh mutants. ProDH activity was high for genotypes in which ProDH, most likely ProDH1, was strongly induced by proline. Respiratory measurements indicate that ProDH1 has a role in oxidizing excess proline and transferring electrons to the respiratory chain.

Proline has multiple functions in plants. Besides being a building block for protein biosynthesis proline plays a central role in the plant stress response and in further cellular processes. Here, we report an analysis on the integration of proline dehydrogenase (ProDH) into mitochondrial metabolism in Arabidopsis thaliana. An experimental system to induce ProDH activity was established using cell cultures. Induction of ProDH was measured by novel photometric activity assays and by a ProDH in gel activity assay. Effects of increased ProDH activity on other mitochondrial enzymes were systematically investigated. Activities of the protein complexes of the respiratory chain were not significantly altered. In contrast, some mitochondrial dehydrogenases had markedly changed activities. Activity of glutamate dehydrogenase substantially increased, indicating upregulation of the entire proline catabolic pathway, which was confirmed by co-expression analyses of the corresponding genes. Furthermore, activity of D-lactate dehydrogenase was increased. D-lactate was identified to be a competitive inhibitor of ProDH in plants. We suggest that induction of D-lactate dehydrogenase activity allows rapid upregulation of ProDH activity during the short-term stress response in plants.