Biology subjects

Keech, O.

Publications and source records attributed to Keech, O..

6 recordsLinked to original sources

bioRxiv2024

GCS-H2 is essential for growth as it acts as the main relay for mitochondrial lipoylation in heterotrophic tissues of Arabidopsis thaliana

H-protein is part of the multi-protein glycine cleavage system found in both prokaryotes and eukaryotes. The Arabidopsis thaliana genome contains three loci for genes encoding H-protein. We show that in land plants, there are two clades of mitochondrial H-proteins; one that clusters with H1 and H3, and another that clusters with H2, an isoform mostly expressed in heterotrophic tissue and for which a null mutation results in a >99% impeded growth. After showing that all three isoforms fulfill the same metabolic function, we evidenced that the impaired root growth in h2 results from an altered cell cycle progression concomitant to a lower Target Of Rapamycin kinase activity. Subsequent metabolomic approaches revealed an accumulation of storage sugars and a significant decrease of the TCA intermediates and several vitamins in the h2 root cells as compared to the wild-type ones. Additional investigations evidenced that H2 acts as the main relay for lipoylation in root mitochondria, which diverges from the current lipoylation model proposed for photosynthetic tissues. Together, this work provides new insight on the control of the cell cycle by mitochondrial metabolism, and also challenges the current dogma for lipoylation in mitochondria of plant cells. Once sentence summaryThe absence of GCS-H2, an isoform of H-protein found in heterotrophic tissue, impedes the cell cycle, and drastically affects plant growth as it acts as the main relay for mitochondrial lipoylation.

plant biology↗

bioRxiv2023

PLANT UNCOUPLING MITOCHONDRIAL PROTEIN 2 localizes to the Golgi

Mitochondria act as cellular hubs of energy transformation and metabolite conversion in most eukaryotes. Plant mitochondrial electron transport chains are particularly flexible, featuring alternative components, such as ALTERNATIVE NAD(P)H DEHYDROGENASES and ALTERNATIVE OXIDASES (AOXs), that can bypass proton translocation steps. PLANT UNCOUPLING MITOCHONDRIAL PROTEINS (named PUMPs or plant UCPs) have been identified in plants as homologues of mammalian Uncoupling Proteins (UCPs), and their biochemical and physiological roles have been investigated in the context of mitochondrial energy metabolism. To dissect UCP function in Arabidopsis, the two most conserved (UCP1 and UCP2) have been targeted in recent work by combining mutant lines to circumvent potential functional redundancy in vivo. Such approaches rely on the assumption that both proteins reside in the inner mitochondrial membrane as a prerequisite for functional redundancy. Yet, contradicting results have been reported on UCP2 localization in plants. Here we provide evidence that, conversely to UCP1, which is an abundant inner mitochondrial membrane protein, UCP2 localizes to the Golgi rather than to mitochondria. Based on multiple lines of new and prior evidence, we summarize the consensus view that we have reached and provide an example of how open, critical exchange within the research community is able to constructively address ambiguities. Our observations and considerations provide direction to the ongoing discussion about the functions of UCP proteins. They further offer new perspectives for the study of Golgi membrane transport and subcellular targeting principles of membrane proteins. Since 20 to 30 % of genes in plant genomes are predicted to encode transmembrane proteins and the function of most of those proteins has not been experimentally investigated, we highlight the importance of using independent evidence for localization as a prerequisite for understanding physiological function of membrane proteins.

plant biology↗

bioRxiv2022

The mitochondrial ferredoxin-like is essential for the formation of complex I-containing respiratory supercomplexes in Arabidopsis thaliana

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of five multiprotein complexes (complexes I to V). Analyses of the OXPHOS system by native gel electrophoresis revealed an organization of OXPHOS complexes in supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mFDX-like). This protein was previously found associated with complex I, being part of the bridge domain linking the matrix and membrane arms of the complex. A phylogenetic analysis suggests that the Arabidopsis thaliana mFDX-like evolved from classical mitochondrial ferredoxin, but it lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster essential for the electron transfer function of ferredoxins. Accordingly, our biochemical study shows that AtmFDX-like does not bind an Fe-S cluster, and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9 approach. These lines do not show any growth phenotype under standard growth condition. However, the characterization of the OXPHOS system demonstrates that mFDX-like is important for the assembly of complex I, and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III to form supercomplexes.

plant biology↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗

bioRxiv2019

Enhancing the biological relevance of Gene Co-expression Networks: A plant mitochondrial case study

Gene Co-expression Networks (GCNs) are obtained by a variety of mathematical of models commonly derived on data sampled from diverse developmental processes, tissue types, pathologies, mutant backgrounds, and stress conditions. These networks aim to identify genes with similar expression dynamics, but are prone to introduce false-positive and -negative relations, especially in the instance of large and highly complex datasets. With the aim of optimizing the relevance of edges in GCNs and enhancing global biological insight, we propose a novel approach that involves a data-centering step performed simultaneously per gene and per sub-experiment, called centralisation within sub-experiments (CSE).\n\nUsing a gene set encoding for the plant mitochondrial proteome as a case study, our results show that CSE-based GCNs had significantly more edges within the majority of the considered functional sub-networks, such as the mitochondrial electron transport chain and its sub-complexes, than GCNs not using CSE; thus demonstrating that the CSE-based GCNs are efficient at predicting those canonical functions and associated pathways, also referred to as the \"core network\". Furthermore, we show that CSE, in conjunction with conventional correlation analyses can be used to fine-tune the prediction of the function for uncharacterised genes; while in combination with analyses based on non-centralised data can augment those conventional stress analyses with the innate connections underpinning the dynamic system examined.\n\nTherefore, CSE appears as an alternative method to conventional batch correction approaches. The method is easy to implement into a pre-existing GCN analysis pipeline and can provide accentuated biological relevance to conventional GCNs by allowing users to delineate a \"core\" gene network.\n\nAuthor SummaryGene Co-expression networks (GCNs) are the product of a variety of mathematical models that identify causal relationships in gene expression dynamics, but are prone to the misdiagnoses of false-positives and -negatives, especially in the instance of large and highly complex datasets. In light of the burgeoning output of next generation sequencing projects performed on any species, under different developmental or clinical conditions, the statistical power and complexity of these networks will undoubtedly increase, while their biological relevance will be fiercely challenged. Here, we propose a novel approach to primarily generate a \"core\" GCN with augmented biological relevance. Our method, which involves data-centering steps and thus effectively removes all primary treatment / tissue /patient effects, is simple to employ and can be easily implemented into pre-existing GCN analysis pipelines. The gained biological relevance of such an approach was validated using a subcellular gene set encoding for the plant mitochondrial proteome, and by applying numerous steps to challenge its application.

bioinformatics↗

bioRxiv2019

The mitogenome of Norway spruce and a reappraisal of mitochondrial recombination in plants

Plant mitogenomes can be difficult to assemble because they are structurally dynamic and prone to intergenomic DNA transfers, leading to the unusual situation where an organelle genome is far outnumbered by its nuclear counterparts. As a result, comparative mitogenome studies are in their infancy and some key aspects of genome evolution are still known mainly from pre-genome, qualitative methods. To help address these limitations, we combined machine learning and in silico enrichment of mitochondrial-like long reads to assemble the bacterial-sized mitogenome of Norway spruce (Pinaceae: Picea abies). We conducted comparative analyses of repeat abundance, intergenomic transfers, substitution and rearrangement rates, and estimated repeat-by-repeat homologous recombination rates. Prompted by our discovery of highly recombinogenic small repeats in P. abies, we assessed the genomic support for the prevailing hypothesis that intramolecular recombination is predominantly driven by repeat length, with larger repeats facilitating DNA exchange more readily. Overall, we found mixed support for this view: recombination dynamics were heterogeneous across vascular plants and highly active small repeats (ca. 200 bp) were present in about a third of studied mitogenomes. As in previous studies, we did not observe any robust relationships among commonly-studied genome attributes, but we identify variation in recombination rates as a underinvestigated source of plant mitogenome diversity.

genomics↗