Experimental approaches to studying translation in plant semi-autonomous organelles.

Plant mitochondria and chloroplasts are semi-autonomous organelles originated from free-living bacteria and retaining respective reduced genomes during evolution. As a consequence, relatively few of the mitochondrial and chloroplast proteins are encoded in the organellar genomes and synthesized by the organellar ribosomes. Since the both organellar genomes encode mainly components of the energy transduction systems, oxidative phosphorylation in mitochondria and photosynthetic apparatus in chloroplasts, understanding the organellar translation is critical to a thorough comprehension of the key aspects of mitochondrial and chloroplast activity affecting plant growth and development. Recent studies have clearly shown that translation is a key regulatory node in the expression of plant organellar genes, underscoring the need for an adequate methodology to study this unique stage of gene expression. The organellar translatome can be analysed by studying newly synthesized proteins or the mRNA pool recruited to the organellar ribosomes. In this review, we present in some detail the experimental approaches used to date for studying translation in the plant bioenergetic organelles. Their benefits and limitations, as well as the critical steps are discussed. Additionally, we briefly mention several recently developed strategies to study organellar translation that have not yet been applied to plants.

Assessment of Protein Synthesis in Mitochondria Isolated from Rosette Leaves and Liquid Culture Seedlings of Arabidopsis.

Mitochondria are subcellular organelles with their own genome and expression system, including translation machinery to make proteins. Several independent studies have shown that translation is an essential regulatory step in expression of the plant mitochondrial genome. Thus, the study of mitochondrial translation seems to be crucial for the comprehension of plant mitochondrial biogenesis and maintenance. In organello protein synthesis in isolated mitochondria is a direct method to visualize the translational products of this organellar genetic system. In this method, highly purified, functional mitochondria synthesize proteins in the presence of radiolabeled amino acids, such as methionine, and an energy regeneration system. The labeled, newly synthesized polypeptides are separated by SDS-polyacrylamide gel electrophoresis and are detected by autoradiography. Here we describe the detailed protocol for in organello labeling of translation products that was optimized for mitochondria isolated from rosette leaves and liquid culture seedlings of Arabidopsis thaliana plants.

Joint inhibition of mitochondrial complex IV and alternative oxidase by genetic or chemical means represses chloroplast transcription in Arabidopsis.

Changes in the functional state of mitochondria have profound effects on other cellular compartments. Genome-wide expression analysis of Arabidopsisrps10 mutants with an RNAi-silenced expression of mitoribosomal S10 protein has revealed extensive transcriptional reprogramming. A meta-analysis comparing expression datasets of 25 mitochondrial perturbations showed a high similarity of the aox1a:rpoTmp mutant, which is defective in the alternative oxidase (AOX1a) and dual-targeted mitochondrial and plastid RNA polymerase (RPOTmp), to rps10. Both rps10 and aox1a:rpoTmp showed a significantly decreased electron flux through both the cytochrome and the alternative respiratory pathways, and a markedly decreased the expression of nuclear-encoded components of the chloroplast transcription machinery. In line with this, a decreased level of plastid transcripts was observed in rps10 and aox1a:rpoTmp, which was reflected in a reduced rate of chloroplast transcription. Chemical treatment of wild-type seedlings with respiratory inhibitors showed that only simultaneous and direct inhibition of complex IV and AOX activity decreased the level of plastid transcripts. Taken together, both chemical and genetic studies show that the limitation of the activity of two mitochondrial terminal oxidases, complex IV and AOX, negatively impacts chloroplast transcription. Salicylic acid and oxygen are discussed as putative mediators of the signalling pathway between mitochondria, nucleus and chloroplasts. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

An Update on Mitochondrial Ribosome Biology: The Plant Mitoribosome in the Spotlight.

Contrary to the widely held belief that mitochondrial ribosomes (mitoribosomes) are highly similar to bacterial ones, recent experimental evidence reveals that mitoribosomes do differ significantly from their bacterial counterparts. This review is focused on plant mitoribosomes, but we also highlight the most striking similarities and differences between the plant and non-plant mitoribosomes. An analysis of the composition and structure of mitoribosomes in trypanosomes, yeast, mammals and plants uncovers numerous organism-specific features. For the plant mitoribosome, the most striking feature is the enormous size of the small subunit compared to the large one. Apart from the new structural information, possible functional peculiarities of different types of mitoribosomes are also discussed. Studies suggest that the protein composition of mitoribosomes is dynamic, especially during development, giving rise to a heterogeneous populations of ribosomes fulfilling specific functions. Moreover, convincing data shows that mitoribosomes interact with components involved in diverse mitochondrial gene expression steps, forming large expressosome-like structures.

Deficiency of mitoribosomal S10 protein affects translation and splicing in Arabidopsis mitochondria.

The ribosome is not only a protein-making machine, but also a regulatory element in protein synthesis. This view is supported by our earlier data showing that Arabidopsis mitoribosomes altered due to the silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 differentially translate mitochondrial transcripts compared with the wild-type. Here, we used ribosome profiling to determine the contribution of transcriptional and translational control in the regulation of protein synthesis in rps10 mitochondria compared with the wild-type ones. Oxidative phosphorylation system proteins are preferentially synthesized in wild-type mitochondria but this feature is lost in the mutant. The rps10 mitoribosomes show slightly reduced translation efficiency of most respiration-related proteins and at the same time markedly more efficiently synthesize ribosomal proteins and MatR and TatC proteins. The mitoribosomes deficient in S10 protein protect shorter transcript fragments which exhibit a weaker 3-nt periodicity compared with the wild-type. The decrease in the triplet periodicity is particularly drastic for genes containing introns. Notably, splicing is considerably less effective in the mutant, indicating an unexpected link between the deficiency of S10 and mitochondrial splicing. Thus, a shortage of the mitoribosomal S10 protein has wide-ranging consequences on mitochondrial gene expression.

[Ribosome profiling as an innovative tool to study the process of protein synthesis]. / Profilowanie rybosomów jako innowacyjne narzedzie do badania procesu syntezy bialek.

The ribosome profiling approach (Ribo-seq) is currently the most effective method to study the protein synthesis in vivo. This technique relies on sequencing of ribosome protected mRNA fragments (so-called ribosomal footprints) allowing to indicate the exact positions of ribosomes on transcripts. Advanced bioinformatic analysis of Ribo-seq data enables selection of ribosomal footprints originated from translating ribosomes, providing information about authentic translational status of mRNA. Here, authors present principles of ribosome profiling method with special attention to basic experimental and bioinformatics strategies important for obtaining satisfactory results. Review includes examples of applications of ribosome profiling technique in various biological systems, giving the significant insight into the translation process and its regulation.

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.

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.