Biology subjects

Wirtz, M.

Publications and source records attributed to Wirtz, M..

8 recordsLinked to original sources

bioRxiv2025

Impaired methyl recycling induces substantial shifts in sulfur utilization in Arabidopsis

The tight coordination of sulfur metabolism and growth regulation is predicated upon nutrient availability. Central to this balancing act is the utilization of cysteine (Cys) for the formation of methionine (Met) and S-adenosylmethionine (SAM). Plants that are severely deficient in regenerating Met due to reduced methylthioadenosine (MTA) nucleosidase activity experience numerous developmental abnormalities. Here, we assess the developmental, metabolic, and regulatory effects associated with decreased MTA recycling. We show that MTA over-accumulation predominantly occurs in reproductive tissues and leads to reduced levels of Cys, Met, and SAM, as well as elevated S-adenosylhomocysteine. These disruptions of primary sulfur utilization also lead to the misregulation of energy metabolism and altered cell cycle progression. RNA-seq experiments show a general down-regulation of many developmental and reproductive genes. Targeted metabolite analyses demonstrate clear impacts on the methyl index which are reflected in the results of bisulfite-sequencing experiments including global alterations in CG gene-body methylation levels and decreases of CHG and CHH methylation in transposable elements. Our findings demonstrate the broad impacts of MTA metabolism on plant development, sulfur utilization and the maintenance of the methyl index.

plant biology↗

bioRxiv2023

Altered iron-sulfur cluster transfer in Arabidopsis mitochondria reveals lipoyl synthase as a Janus-faced enzyme that generates toxic sulfide

Iron-sulfur (Fe-S) cluster are vital cofactors in all domains of life. Mitochondrial Fe-S cluster assembly occurs in two major steps to first build [2Fe-2S] clusters and subsequently assemble these into [4Fe-4S] clusters. The two assembly machineries are interconnected by glutaredoxin S15 (GRXS15) that transfers [2Fe-2S] clusters to the second machinery. Diminished cluster transfer activity of GRXS15 in Arabidopsis mitochondria causes specific defects associated with lipoyl synthase (LIP1) activity. Conversely, overexpression of LIP1 in wild-type plants causes the release of toxic amounts of sulfide that can be detoxified by increasing the capacity for sulfide fixation through overexpression of O-acetylserine-(thiol)-lyase. The release of sulfide by lipoyl synthase causes a disturbance of mitochondrial sulfide homeostasis resulting in distinct and readily observable macroscopic phenotypes. These phenotypes enable a direct readout of consequences resulting from defects in Fe-S cluster assembly or targeted modulation of Fe-S cluster flux in mitochondria.

plant biology↗

bioRxiv2020

Branched-chain amino acid catabolism depends on GRXS15 through mitochondrial lipoyl cofactor homeostasis

Iron-sulfur (Fe-S) clusters are ubiquitous cofactors in all life and are used in a wide array of diverse biological processes, including electron transfer chains and several metabolic pathways. Biosynthesis machineries for Fe-S clusters exist in plastids, the cytosol and mitochondria. A single monothiol glutaredoxin (GRX) has been shown to be involved in Fe-S cluster assembly in mitochondria of yeast and mammals. In plants, the role of the mitochondrial homologue GRXS15 has only partially been characterized. Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype. In an in-depth metabolic analysis, we show that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis and the electron transport chain. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, 2-oxoglutarate, glycine and branched-chain amino acids (BCAAs). The most pronounced accumulation occurred in branched-chain -keto acids (BCKAs), the first degradation products resulting from deamination of BCAAs. In wild-type plants, pyruvate, 2-oxoglutarate, glycine and BCKAs are all metabolized through decarboxylation by four mitochondrial lipoyl cofactor-dependent dehydrogenase complexes. Because these enzyme complexes are very abundant and the biosynthesis of the lipoyl cofactor depends on continuous Fe-S cluster supply to lipoyl synthase, this could explain why lipoyl cofactor-dependent processes are most sensitive to restricted Fe-S supply in GRXS15 K83A mutants. One-sentence summaryDeficiency in GRXS15 restricts protein lipoylation and causes metabolic defects in lipoyl cofactor-dependent dehydrogenase complexes, with branched-chain amino acid catabolism as dominant bottleneck.

plant biology↗

bioRxiv2019

The proteomic inventory reveals the chloroplast ribosome as nexus within a diverse protein network

Chloroplast gene expression is tightly regulated and majorly controlled on the level of protein synthesis. Fine-tuning of translation is vital for plant development, acclimation to environmental challenges and for the assembly of major protein complexes such as the photosynthesis machinery. However, many regulatory mediators and the interaction network of chloroplast ribosomes are not known to date. We report here on a deep proteomic analysis of the plastidic ribosome interaction network in Chlamydomonas reinhardtii cells. Affinity-purification of ribosomes was achieved via endogenous affinity tagging of the chloroplast-encoded protein Rpl5, yielding a specific enrichment of >650 chloroplast-localized proteins. The ribosome interaction network was validated for several proteins and provides a new source of mainly conserved factors directly linking translation with central processes such as protein folding, photosystem biogenesis, redox control, RNA maturation, energy and metabolite homeostasis. Our approach provided the first evidence for the existence of a plastidic co-translational acting N-acetyltransferase (cpNAT1). Expression of tagged cpNAT1 confirmed its ribosome-association, and we demonstrated the ability of cpNAT1 to acetylate substrate proteins at their N-terminus. Our dataset establishes that the chloroplast protein synthesis machinery acts as nexus in a highly choreographed, spatially interconnected protein network and underscores its wide-ranging regulatory potential during gene expression. ONE-SENTENCE SUMMARYAffinity purification of Chlamydomonas reinhardtii chloroplast ribosomes and subsequent proteomic analysis revealed a broad spectrum of interactors ranging from global translation control to specific pathways.

biochemistry↗

bioRxiv2019

Redox-mediated Kick-Start of Mitochondrial Energy Metabolism drives Resource-efficient Seed Germination

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is re-activated to drive germination when the external conditions are favorable. Since the switchover from quiescence to re-activation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag-based (iodoTMT) thiol redox proteomics. The redox state across all Cys-peptides was shifted towards reduction from 27.1 % to 13.0 %. A large number of Cys-peptides (412) were redox-switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid cycle (TCA). Active site Cys-peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.

plant biology↗

bioRxiv2019

Staphylococcus aureus uses the bacilliredoxin (BrxAB)/bacillithiol disulfide reductase (YpdA) redox pathway to defend against oxidative stress under infections

Staphylococcus aureus is a major human pathogen and has to cope with reactive oxygen and chlorine species (ROS, RCS) during infections. The low molecular weight thiol bacillithiol (BSH) is an important defense mechanism of S. aureus for detoxification of ROS and HOCl stress to maintain the reduced state of the cytoplasm. Under HOCl stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolations, which are reduced by bacilliredoxins (BrxA and BrxB). The NADPH-dependent flavin disulfide reductase YpdA is phylogenetically associated with the BSH synthesis and BrxA/B enzymes and was proposed to function as BSSB reductase. Here, we investigated the role of the bacilliredoxin BrxAB/BSH/YpdA pathway in S. aureus COL under oxidative stress and macrophage infection conditions in vivo and in biochemical assays in vitro. Using HPLC thiol metabolomics, a strongly enhanced BSSB level and a decreased BSH/BSSB ratio were measured in the S. aureus COL ypdA deletion mutant under control and NaOCl stress. Monitoring the BSH redox potential (EBSH) using the Brx-roGFP2 biosensor revealed that YpdA is required for regeneration of the reduced EBSH upon recovery from oxidative stress. In addition, the ypdA mutant was impaired in H2O2 detoxification as measured with the novel H2O2-specific Tpx-roGFP2 biosensor. Phenotype analyses further showed that BrxA and YpdA are required for survival under NaOCl and H2O2 stress in vitro and inside murine J-774A.1 macrophages in infection assays in vivo. Finally, NADPH-coupled electron transfer assays provide evidence for the function of YpdA in BSSB reduction, which depends on the conserved Cys14 residue. YpdA acts together with BrxA and BSH in de-bacillithiolation of S-bacilithiolated GapDH. In conclusion, our results point to a major role of the BrxA/BSH/YpdA pathway in BSH redox homeostasis in S. aureus during recovery from oxidative stress and under infections.

microbiology↗

bioRxiv2019

Arabidopsis glutathione reductase 2 is indispensable in plastids, while mitochondrial glutathione is safeguarded by additional reduction and transport systems

O_LIA highly negative glutathione redox potential (EGSH) is maintained in the cytosol, plastids and mitochondria of plant cells to support fundamental processes, including antioxidant defence, redox regulation and iron-sulfur cluster biogenesis. Out of two glutathione reductase (GR) proteins in Arabidopsis, GR2 is predicted to be dual-targeted to plastids and mitochondria, but its differential roles in these organelles remain unclear.\nC_LIO_LIWe dissected the role of GR2 in organelle glutathione redox homeostasis and plant development using a combination of genetic complementation and stacked mutants, biochemical activity studies, immunogold labelling and in vivo biosensing.\nC_LIO_LIOur data demonstrate that GR2 is dual-targeted to plastids and mitochondria, but embryo lethality of gr2 null mutants is caused specifically in plastids. Whereas lack of mitochondrial GR2 leads to a partially oxidised glutathione pool in the matrix, the ABC transporter ATM3 and the mitochondrial thioredoxin system provide functional backup and maintain plant viability.\nC_LIO_LIWe identify GR2 as essential in the plastid stroma, where it counters GSSG accumulation and developmental arrest. By contrast a functional triad of GR2, ATM3 and the thioredoxin system in the mitochondria provides resilience to excessive glutathione oxidation.\nC_LI

plant biology↗

bioRxiv2019

Differential N-end rule degradation of RIN4/NOI fragments generated by the AvrRpt2 effector protease

The protein RPM1-INTERACTING PROTEIN4 (RIN4) is a central regulator of both layers of plant immunity systems, the so-called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). RIN4 is targeted by several effectors, including the Pseudomonas syringae protease effector AvrRpt2. Cleavage of RIN4 by AvrRpt2 generates unstable RIN4 fragments, whose degradation leads to the activation of the resistance protein RPS2 (RESISTANT TO P. SYRINGAE2). Hence, identifying the determinants of RIN4 degradation is key to understanding RPS2-mediated ETI, as well as virulence functions of AvrRpt2. In addition to RIN4, AvrRpt2 cleaves host proteins from the nitrate-induced (NOI) domain family. Although cleavage of NOI-domain proteins by AvrRpt2 may contribute to PTI regulation, the (in)stability of these proteolytic fragments and the determinants that regulate their stability have not been examined. Notably, a common feature of RIN4 and of many NOI-domain protein fragments generated by AvrRpt2 cleavage is the exposure of a new N-terminal residue that is destabilizing according to the N-end rule. Using antibodies raised against endogenous RIN4, we show that the destabilization of AvrRpt2-cleaved RIN4 fragments is independent of the N-end rule pathway (recently renamed N-degron pathway). By contrast, several NOI-domain protein fragments are bona fide substrates of the N-degron pathway. The discovery of this novel set of substrates considerably expands the number of proteins targeted for degradation by this ubiquitin-dependent pathway, for which very few physiological substrates are known in plants. Our results also open new avenues of research to understand the role of AvrRpt2 in promoting bacterial virulence.\n\nOne sentence summaryAnalysis of RIN4/NOI fragments released after cleavage by the bacterial effector protease AvrRpt2 reveals a novel role of the N-end rule in the degradation of NOI-domain proteins, but not of RIN4.

plant biology↗