Recurrent evolutionary switches of mitochondrial cytochrome c maturation systems in Archaeplastida.

Mitochondrial cytochrome c maturation (CCM) requires heme attachment via distinct pathways termed systems I and III. The mosaic distribution of these systems in Archaeplastida raises questions about the genetic mechanisms and evolutionary forces promoting repeated evolution. Here, we show a recurrent shift from ancestral system I to the eukaryotic-specific holocytochrome c synthase (HCCS) of system III in 11 archaeplastid lineages. Archaeplastid HCCS is sufficient to rescue mutants of yeast system III and Arabidopsis system I. Algal HCCS mutants exhibit impaired growth and respiration, and altered biochemical and metabolic profiles, likely resulting from deficient CCM and reduced cytochrome c-dependent respiratory activity. Our findings demonstrate that archaeplastid HCCS homologs function as system III components in the absence of system I. These results elucidate the evolutionary trajectory and functional divergence of CCM pathways in Archaeplastida, providing insight into the causes, mechanisms, and consequences of repeated cooption of an entire biological pathway.

Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells.

Plastids are complex organelles that vary in size and function depending on the cell type. Accordingly, they can be referred to as amyloplasts, chloroplasts, chromoplasts, etioplasts, or proplasts, to only cite a few. Over the past decades, methods based on density gradients and differential centrifugation have been extensively used for the purification of plastids. However, these methods need large amounts of starting material, and hardly provide a tissue-specific resolution. Here, we applied our IPTACT (Isolation of Plastids TAgged in specific Cell Types) method, which involves the biotinylation of plastids in vivo using one-shot transgenic lines expressing the Translocon of the Outer Membrane 64 (TOC64) gene coupled with a biotin ligase receptor particle and the BirA biotin ligase, to isolate plastids from mesophyll and companion cells of Arabidopsis using tissue specific pCAB3 and pSUC2 promoters, respectively. Subsequently, a proteome profiling was performed, which allowed the identification of 1672 proteins, among which 1342 were predicted to be plastidial, and 705 were fully confirmed according to the SUBA5 database. Interestingly, although 92% of plastidial proteins were equally distributed between the two tissues, we observed an accumulation of proteins associated with jasmonic acid biosynthesis, plastoglobuli (e.g. NAD(P)H dehydrogenase C1, vitamin E deficient 1, plastoglobulin of 34 kDa, ABC1-like kinase 1) and cyclic electron flow in plastids originating from vascular tissue. Besides demonstrating the technical feasibility of isolating plastids in a tissue-specific manner, our work provides strong evidence that plastids from vascular tissue have a higher redox turnover to ensure optimal functioning, notably under high solute strength as encountered in vascular cells.

Two wrongs make a right: heat stress reversion of a male-sterile Brassica napus line.

Male-sterile lines play important roles in plant breeding to obtain hybrid vigour. The male sterility Lembke (MSL) system is a thermosensitive genic male sterility system of Brassica napus and is one of the main systems used in European rapeseed breeding. Interestingly, the MSL system shows high similarity to the 9012AB breeding system from China, including the ability to revert to fertile in high temperature conditions. Here we demonstrate that the MSL system is regulated by the same restorer of fertility gene BnaC9-Tic40 as the 9012AB system, which is related to the translocon at the inner envelope membrane of chloroplasts 40 (TIC40) from Arabidopsis. The male sterility gene of the MSL system was also identified to encode a chloroplast-localized protein which we call BnChimera; this gene shows high sequence similarity to the sterility gene previously described for the 9012AB system. For the first time, a direct protein interaction between BnaC9-Tic40 and the BnChimera could be demonstrated. In addition, we identify the corresponding amino acids that mediate this interaction and suggest how BnaC9-Tic40 acts as the restorer of fertility. Using an RNA-seq approach, the effects of heat treatment on the male fertility restoration of the C545 MSL system line were investigated. These data demonstrate that many pollen developmental pathways are affected by higher temperatures. It is hypothesized that heat stress reverses the male sterility via a combination of slower production of cell wall precursors in plastids and a slower flower development, which ultimately results in fertile pollen. The potential breeding applications of these results are discussed regarding the use of the MSL system in producing thermotolerant fertile plants.

Assessment of Mitochondrial Protein Topology and Membrane Insertion.

Analyzing the membrane integrity and topology of a mitochondrial protein is essential for truly understanding its function. In this chapter, we demonstrate how to analyze mitochondrial membrane proteins using both an immunological-based assay and an in vivo self-assembling GFP approach. First, immunological approaches to investigate the solubility or membrane association of a protein within mitochondria are described. With this method, we demonstrate how the topology of soluble domains of a membrane-integrated protein can be determined. Using protein-specific antibodies, the localization of the soluble domains of a protein are analyzed by a proteolytic-cleavage approach using proteinase K in mitochondria, outer membrane-ruptured mitochondria, and solubilized mitochondrial membranes. In a second approach, we determine the topology of plant mitochondrial proteins using an in vivo self-assembling GFP localization approach.

The OXA2a Insertase of Arabidopsis Is Required for Cytochrome c Maturation.

In yeast (Saccharomyces cerevisiae) and human (Homo sapiens) mitochondria, Oxidase assembly protein1 (Oxa1) is the general insertase for protein insertion from the matrix side into the inner membrane while Cytochrome c oxidase assembly protein18 (Cox18/Oxa2) is specifically involved in the topogenesis of the complex IV subunit, Cox2. Arabidopsis (Arabidopsis thaliana) mitochondria contain four OXA homologs: OXA1a, OXA1b, OXA2a, and OXA2b. OXA2a and OXA2b are unique members of the Oxa1 superfamily, in that they possess a tetratricopeptide repeat (TPR) domain at their C termini. Here, we determined the role of OXA2a by studying viable mutant plants generated by partial complementation of homozygous lethal OXA2a transfer-DNA insertional mutants using the developmentally regulated ABSCISIC ACID INSENSITIVE3 (ABI3) promoter. The ABI3p:OXA2a plants displayed growth retardation due to a reduction in the steady-state abundances of both c-type cytochromes, cytochrome c 1 and cytochrome c The observed reduction in the steady-state abundance of complex III could be attributed to cytochrome c 1 being one of its subunits. Expression of a soluble heme lyase from an organism with cytochrome c maturation system III could functionally complement the lack of OXA2a. This implies that OXA2a is required for the system I cytochrome c maturation of Arabidopsis. Due to the interaction of OXA2a with Cytochrome c maturation protein CcmF C-terminal-like protein (CCMFC) in a yeast split-ubiquitin based interaction assay, we propose that OXA2a aids in the membrane insertion of CCMFC, which is presumed to form the heme lyase component of the cytochrome c maturation pathway. In contrast with the crucial role played by the TPR domain of OXA2b, the TPR domain of OXA2a is not essential for its functionality.

The Plant Mitochondrial TAT Pathway Is Essential for Complex III Biogenesis.

Twin arginine translocation (TAT) pathways have been extensively studied in bacteria and chloroplasts for their role in membrane translocation of folded proteins. However, an increasing number of organisms have been found to contain mitochondria-located TAT subunits, including plant mitochondria, which contain TAT subunits, though in an unusual arrangement with only TatB and TatC subunits. To date, no confirmed function has been attributed to mitochondrial TAT pathways in any organism. Using a truncation mutant approach, we demonstrate that the plant mitochondrial TatB (MTTATB) is required for complex III biogenesis. More specifically, MTTATB performs at a late stage in complex III biogenesis, conveying the translocation of the C terminus of the Rieske FeS subunit back across the inner membrane. This work confirms that plant mitochondria retained a functional TAT pathway for the Rieske FeS translocation, most likely from the original mitochondrial ancestor. It is hypothesized that the original mitochondria contained a bacteria-derived TAT pathway required for at least the Rieske FeS translocation. In several eukaryotic lineages, this mitochondrial TAT pathway was lost and replaced by BCS1. Interestingly, plant mitochondria appear to assemble complex III in the same subunit order as yeast and mammals but in contrast use bacteria-like assembly factors for this process.

Tissue-specific isolation of Arabidopsis/plant mitochondria - IMTACT (isolation of mitochondria tagged in specific cell types).

Plant cells contain numerous subcompartments with clearly delineated metabolic functions. Mitochondria represent a very small fraction of the total cell volume and yet are the site of respiration and thus crucial for cells throughout all developmental stages of a plant's life. As such, their isolation from the rest of the cellular components is a basic requirement for numerous biochemical and physiological experiments. Although procedures exist to isolate plant mitochondria from different organs (i.e. leaves, roots, tubers, etc.), they are often tedious and do not provide resolution at the tissue level (i.e. phloem, mesophyll or pollen). Here, we present a novel method called IMTACT (isolation of mitochondria tagged in specific cell types), developed in Arabidopsis thaliana (Arabidopsis) that involves biotinylation of mitochondria in a tissue-specific manner using transgenic lines expressing a synthetic version of the OM64 (Outer Membrane 64) gene combined with BLRP and the BirA biotin ligase gene. Tissue specificity is achieved with cell-specific promoters (e.g. CAB3 and SUC2). Labeled mitochondria from crude extracts are retained by magnetic beads, allowing the simple and rapid isolation of highly pure and intact organelles from organs or specific tissues. For example, we could show that the mitochondrial population from mesophyll cells was significantly larger in size than the mitochondrial population isolated from leaf companion cells. To facilitate the applicability of this method in both wild-type and mutant Arabidopsis plants we generated a set of OM64-BLRP one-shot constructs with different selection markers and tissue-specific promoters.

Single organelle function and organization as estimated from Arabidopsis mitochondrial proteomics.

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.

Arabidopsis DGD1 SUPPRESSOR1 Is a Subunit of the Mitochondrial Contact Site and Cristae Organizing System and Affects Mitochondrial Biogenesis.

Mitochondrial and plastid biogenesis requires the biosynthesis and assembly of proteins, nucleic acids, and lipids. In Arabidopsis (Arabidopsis thaliana), the mitochondrial outer membrane protein DGD1 SUPPRESSOR1 (DGS1) is part of a large multi-subunit protein complex that contains the mitochondrial contact site and cristae organizing system 60-kD subunit, the translocase of outer mitochondrial membrane 40-kD subunit (TOM40), the TOM20s, and the Rieske FeS protein. A point mutation in DGS1, dgs1-1, altered the stability and protease accessibility of this complex. This altered mitochondrial biogenesis, mitochondrial size, lipid content and composition, protein import, and respiratory capacity. Whole plant physiology was affected in the dgs1-1 mutant as evidenced by tolerance to imposed drought stress and altered transcriptional responses of markers of mitochondrial retrograde signaling. Putative orthologs of Arabidopsis DGS1 are conserved in eukaryotes, including the Nuclear Control of ATP Synthase2 (NCA2) protein in yeast (Saccharomyces cerevisiae), but lost in Metazoa. The genes encoding DGS1 and NCA2 are part of a similar coexpression network including genes encoding proteins involved in mitochondrial fission, morphology, and lipid homeostasis. Thus, DGS1 links mitochondrial protein and lipid import with cellular lipid homeostasis and whole plant stress responses.

Assembly of the Complexes of the Oxidative Phosphorylation System in Land Plant Mitochondria.

Plant mitochondria play a major role during respiration by producing the ATP required for metabolism and growth. ATP is produced during oxidative phosphorylation (OXPHOS), a metabolic pathway coupling electron transfer with ADP phosphorylation via the formation and release of a proton gradient across the inner mitochondrial membrane. The OXPHOS system is composed of large, multiprotein complexes coordinating metal-containing cofactors for the transfer of electrons. In this review, we summarize the current state of knowledge about assembly of the OXPHOS complexes in land plants. We present the different steps involved in the formation of functional complexes and the regulatory mechanisms controlling the assembly pathways. Because several assembly steps have been found to be ancestral in plants-compared with those described in fungal and animal models-we discuss the evolutionary dynamics that lead to the conservation of ancestral pathways in land plant mitochondria.

OXA2b is Crucial for Proper Membrane Insertion of COX2 during Biogenesis of Complex IV in Plant Mitochondria.

The evolutionarily conserved YidC/Oxa1/Alb3 proteins are involved in the insertion of membrane proteins in all domains of life. In plant mitochondria, individual knockouts of OXA1a, OXA2a, and OXA2b are embryo-lethal. In contrast to other members of the protein family, OXA2a and OXA2b contain a tetratricopeptide repeat (TPR) domain at the C-terminus. Here, the role of Arabidopsis (Arabidopsis thaliana) OXA2b was determined by using viable mutant plants that were generated by complementing homozygous lethal OXA2b T-DNA insertional mutants with a C-terminally truncated OXA2b lacking the TPR domain. The truncated-OXA2b-complemented plants displayed severe growth retardation due to a strong reduction in the steady-state abundance and enzyme activity of the mitochondrial respiratory chain complex IV. The TPR domain of OXA2b directly interacts with cytochrome c oxidase subunit 2, aiding in efficient membrane insertion and translocation of its C-terminus. Thus, OXA2b is crucial for the biogenesis of complex IV in plant mitochondria.

Plant Mitochondrial Inner Membrane Protein Insertion.

During the biogenesis of the mitochondrial inner membrane, most nuclear-encoded inner membrane proteins are laterally released into the membrane by the TIM23 and the TIM22 machinery during their import into mitochondria. A subset of nuclear-encoded mitochondrial inner membrane proteins and all the mitochondrial-encoded inner membrane proteins use the Oxa machinery-which is evolutionarily conserved from the endosymbiotic bacterial ancestor of mitochondria-for membrane insertion. Compared to the mitochondria from other eukaryotes, plant mitochondria have several unique features, such as a larger genome and a branched electron transport pathway, and are also involved in additional cellular functions such as photorespiration and stress perception. This review focuses on the unique aspects of plant mitochondrial inner membrane protein insertion machinery, which differs from that in yeast and humans, and includes a case study on the biogenesis of Cox2 in yeast, humans, two plant species, and an algal species to highlight lineage-specific similarities and differences. Interestingly, unlike mitochondria of other eukaryotes but similar to bacteria and chloroplasts, plant mitochondria appear to use the Tat machinery for membrane insertion of the Rieske Fe/S protein.

To Mia or not to Mia: stepwise evolution of the mitochondrial intermembrane space disulfide relay.

The disulfide relay system found in the intermembrane space (IMS) of mitochondria is an essential pathway for the import and oxidative folding of IMS proteins. Erv1, an essential member of this pathway, has been previously found to be ubiquitously present in mitochondria-containing eukaryotes. However, the other essential protein, Mia40, was found to be absent or not required in some organisms, raising questions about how the disulfide relay functions in these organisms. A recent study published in BMC Biology demonstrates for the first time that some Erv1 proteins can function in oxidative folding independently of a Mia40 protein, providing for the first time strong evidence that the IMS disulfide relay evolved in a stepwise manner.See research article: 10.1186/s12915-017-0445-8.

TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana.

The translocon on the outer membrane of mitochondria (TOM) facilitates the import of nuclear-encoded proteins. The principal machinery of mitochondrial protein transport seems conserved in eukaryotes; however, divergence in the composition and structure of TOM components has been observed between mammals, yeast, and plants. TOM9, the plant homolog of yeast Tom22, is significantly smaller due to a truncation in the cytosolic receptor domain, and its precise function is not understood. Here we provide evidence showing that TOM9.2 from Arabidopsis thaliana is involved in the formation of mature TOM complex, most likely by influencing the assembly of the pore-forming subunit TOM40. Dexamethasone-induced RNAi gene silencing of TOM9.2 results in a severe reduction in the mature TOM complex, and the assembly of newly imported TOM40 into the complex is impaired. Nevertheless, mutant plants are fully viable and no obvious downstream effects of the loss of TOM complex, i.e., on mitochondrial import capacity, were observed. Furthermore, we found that TOM9.2 can bind calmodulin (CaM) in vitro and that CaM impairs the assembly of TOM complex in the isolated wild-type mitochondria, suggesting a regulatory role of TOM9.2 and a possible integration of TOM assembly into the cellular calcium signaling network.

The PPR protein SLOW GROWTH 4 is involved in editing of nad4 and affects the splicing of nad2 intron 1.

KEY MESSAGE: SLO4 is a mitochondrial PPR protein that is involved in editing nad4, possibly required for the efficient splicing of nad2 intron1. Pentatricopeptide repeat (PPR) proteins constitute a large protein family in flowering plants and are thought to be mostly involved in organellar RNA metabolism. The subgroup of PLS-type PPR proteins were found to be the main specificity factors of cytidine to uridine RNA editing. Identifying the targets of PLS-type PPR proteins can help in elucidating the molecular function of proteins encoded in the organellar genomes. In this study, plants lacking the SLOW GROWTH 4 PPR protein were characterized. Slo4 mutants were characterized as having restricted root growth, being late flowering and displaying an overall delayed growth phenotype. Protein levels and activity of mitochondrial complex I were decreased and putative complex I assembly intermediates accumulated in the mutant plants. An editing defect, leading to an amino acid change, in the mitochondrial nad4 transcript, encoding for a complex I subunit, was identified. Furthermore, the splicing efficiency of the first intron of nad2, encoding for another complex I subunit, was also decreased. The change in splicing efficiency could however not be linked to any editing defects in the nad2 transcript.

Plant mitochondria contain the protein translocase subunits TatB and TatC.

Twin-arginine translocation (Tat) pathways have been well-characterized in bacteria and chloroplasts. Genes encoding a TatC protein are found in almost all plant mitochondrial genomes but to date these have not been extensively investigated. For the first time it could be demonstrated that this mitochondrial-encoded TatC is a functional gene that is translated into a protein in the model plant Arabidopsis thaliana A TatB--like subunit localized to the inner membrane was also identified that is nuclear-encoded and is essential for plant growth and development, indicating that plants potentially require a Tat pathway for mitochondrial biogenesis.

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function.

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

Glutaredoxin S15 Is Involved in Fe-S Cluster Transfer in Mitochondria Influencing Lipoic Acid-Dependent Enzymes, Plant Growth, and Arsenic Tolerance in Arabidopsis.

Glutaredoxins (Grxs) are small proteins that function as oxidoreductases with roles in deglutathionylation of proteins, reduction of antioxidants, and assembly of iron-sulfur (Fe-S) cluster-containing enzymes. Which of the 33 Grxs in Arabidopsis (Arabidopsis thaliana) perform roles in Fe-S assembly in mitochondria is unknown. We have examined in detail the function of the monothiol GrxS15 in plants. Our results show its exclusive mitochondrial localization, and we are concluding it is the major or only Grx in this subcellular location. Recombinant GrxS15 has a very low deglutathionylation and dehydroascorbate reductase activity, but it binds a Fe-S cluster. Partially removing GrxS15 from mitochondria slowed whole plant growth and respiration. Native GrxS15 is shown to be especially important for lipoic acid-dependent enzymes in mitochondria, highlighting a putative role in the transfer of Fe-S clusters in this process. The enhanced effect of the toxin arsenic on the growth of GrxS15 knockdown plants compared to wild type highlights the role of mitochondrial glutaredoxin Fe-S-binding in whole plant growth and toxin tolerance.

In vitro and in vivo protein uptake studies in plant mitochondria.

The study of protein uptake into mitochondria is an important tool for investigating the subcellular distribution of proteins and the molecular mechanisms that determine location. Here we describe five techniques that allow the quantitative or qualitative monitoring of protein uptake into mitochondria using both in vitro and in vivo approaches.