Biology subjects

Schwarzländer, M.

Publications and source records attributed to Schwarzländer, M..

14 recordsLinked to original sources

bioRxiv2025

Advanced illumination-imaging reveals photosynthesis-triggered pH, ATP and NAD redox signatures across plant cell compartments

Photosynthesis provides energy and organic substrates to most life. In plants, photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects energy physiology within and beyond the chloroplast. We developed a new standard for the live-monitoring of subcellular energy physiology by combining confocal imaging of genetically encoded fluorescent protein biosensors with advanced on-stage illumination technology to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions in Arabidopsis mesophyll cells. Our findings reveal a stromal alkalinization signature induced by photosynthetic proton pumping, extending to the cytosol and mitochondria as an alkalinization wave. Photosynthesis leads to increased MgATP2- levels in both the stroma and cytosol. Additionally, we observed reduction of the NAD pool driven by photosynthesis-derived electron export. Arabidopsis lines defective in chloroplast NADP- and mitochondrial NAD-dependent malate dehydrogenases show more reduced cytosolic NAD redox status even in darkness, highlighting the involvement of chloroplasts and mitochondria in shaping cytosolic redox metabolism via malate metabolism. Our study sets a novel methodological standard for precision live-monitoring of photosynthetic cell physiology. Applying this technology reveals signatures of photosynthetic physiology within and beyond the chloroplast with unprecedented resolution. Those signatures link photosynthetic activity and the fundamental biochemical functions of phototrophic cells. Significance statementBy applying novel live microscopy monitoring using fluorescent protein biosensors in plant cells, we reveal that dark-light transitions trigger profound re-orchestration of subcellular pH, ATP and NAD redox physiology not limited to chloroplasts but extending into the cytosol and the mitochondria.

plant biology↗

bioRxiv2025

Hydrogen Sulfide modulates Flagellin-Induced Stomatal Immunity

Stomata are natural pores through which plants exchange gases with the environment, mainly carbon dioxide and oxygen required for photosynthesis and respiration, as well as water vapor through evapotranspiration. However, they also serve as entry points for microbial pathogens such as Pseudomonas syringae pv. tomato (Pst) bacteria. To prevent microbe invasion, guard cells detect pathogens-associated molecular patterns (PAMPs), including the bacterial peptide flagellin (flg22), triggering stomatal closure. This study identifies hydrogen sulfide (H2S) and its cytosolic source L-CYSTEINE DESULFHIDRASE 1 (DES1), as key players in stomatal immunity. We demonstrate that H2S and DES1 are involved in flg22- and bacterial-induced responses, including stomatal closure and modulation of reactive oxygen species (ROS) production. We have found that knock out mutants in DES1 gene exhibits reduced susceptibility to Pst spray-inoculation and lower apoplastic and cytosolic H2O2 levels in response to flg22. Additionally, H2S independently induces cytosolic H2O2 levels in guard cells without requiring RBOHD activity. All together, these findings establish H2S and its source, DES1, as critical components of the stomatal immune response. One Sentence SummaryH2S and DES1 actively participate in flg22-induced stomatal closure modulating apoplastic and cytosolic ROS production.

plant biology↗

bioRxiv2024

The unusual orf117Sha locus causes desynchronization of pollen development in the Arabidopsis gametophytic Sha CMS

Cytoplasmic male sterility (CMS) is of major agronomical relevance in hybrid breeding. In gametophytic CMS, abortion of pollen is determined by the grain genotype, while in sporophytic CMS, it is determined by the mother plant genotype. While several CMS mechanisms have been dissected at the molecular level, gametophytic CMS has not been straightforwardly accessible. We used the gametophytic Sha-CMS in Arabidopsis to characterize the cause and process of pollen abortion by implementing in vivo biosensing in single pollen and mitoTALEN mutagenesis. We obtained conclusive evidence that orf117Sha is the CMS-causing gene, despite distinct characteristics from other CMS-genes. We measured the in vivo cytosolic ATP content in single pollen, followed pollen development and analyzed pollen mitochondrial volume in two genotypes that differed only by the presence of the orf117Sha locus. Our results show that the Sha-CMS is not triggered by ATP deficiency. Instead, we observed desynchronization of a pollen developmental program. Pollen death occurred independently in pollen grains at diverse stages and was preceded by mitochondrial swelling. We conclude that pollen death is grain-autonomous in Sha-CMS and propose that mitochondrial permeability transition, which was previously described as a hallmark of developmental and environmental-triggered cell death programs, precedes pollen death in Sha-CMS. HighlightThe Arabidopsis CMS-causing gene orf117Sha does not limit pollen ATP supply. Pollen-centered approaches show desynchronization of development and mitochondrial swelling before pollen death, which occurred at diverse stages.

plant biology↗

bioRxiv2023

Chloroplasts lacking class I glutaredoxins are functional but show a delayed recovery of protein cysteinyl redox state after oxidative challenge

Redox status of protein cysteinyl residues is mediated via glutathione (GSH)/glutaredoxin (GRX) and thioredoxin (TRX)-dependent redox cascades. An oxidative challenge can induce post-translational protein modifications on thiols, such as protein S-glutathionylation. Class I GRX are small thiol-disulfide oxidoreductases that reversibly catalyse S-glutathionylation and protein disulfide formation. TRX and GSH/GRX redox systems can provide partial backup for each other in several subcellular compartments, but not in the plastid stroma where TRX/light-dependent redox regulation of primary metabolism takes place. While the stromal TRX system has been studied at detail, the role of class I GRX on plastid redox processes in vivo is still unknown. We generate knockout lines of GRXC5 as the only chloroplast class I GRX of the moss Physcomitrium patens. While we find that class I PpGRXC5 has high activities in glutathione-dependent oxidoreductase assays using hydroxyethyl disulfide or redox-sensitive GFP2 (roGFP2) as substrates in vitro, {Delta}grxc5 plants show no detectable growth defect or stress sensitivity, in contrast to mutants with a less negative stromal EGSH ({Delta}gr1). Using stroma-targeted roGFP2, we show increased protein Cys oxidation and decreased reduction rates after oxidative challenge in {Delta}grxc5 plants in vivo, indicating kinetic uncoupling of the protein Cys redox state from glutathione redox potential. Protein Cys disulfide and S-glutathionylation formation rates after H2O2 treatment remained unchanged. Lack of class I GRX function in the stroma did not result in impaired carbon fixation. Our observations suggest specific roles for class I GRX in the efficient redox equilibration between EGSH and protein Cys in the plastid stroma as well as negligible cross-talk with metabolic regulation via the TRX system. We propose a model for stromal class I GRX function as efficient kinetic couplers of protein Cys redox state to the dynamic stromal EGSH and highlight the importance of identifying in vivo target proteins of GRXC5. One sentence summaryRemoval of class I GRX activity in the chloroplast stroma of P. patens kinetically uncouples GRX-dependent cysteine redox changes from the local glutathione redox potential, without an effect on NPQ or photosynthetic carbon reactions.

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

High robustness of cytosolic glutathione redox potential under combined salt and osmotic stress in barley as revealed by the biosensor Grx1-roGFP2

O_LIBarley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. C_LIO_LIWe generated and characterized stable homozygous barley lines (cv. Golden Promise Fast) expressing the genetically-encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. C_LIO_LIOur results demonstrate functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) that caused growth retardation and showed only a minor oxidation after 96 h of treatment. C_LIO_LIWe conclude that the generated reporter lines are a novel resource to study stress resilience in barley. C_LI One sentence summaryGeneration and characterization of barley plants stably expressing Grx1-roGFP2 reveal high robustness of cytosolic glutathione redox potential (EGSH) under combined salt and osmotic stress.

plant biology↗

bioRxiv2020

Photosynthetic activity triggers pH and NAD redox signatures across different plant cell compartments

A characteristic feature of most plants is their ability to perform photosynthesis, which ultimately provides energy and organic substrates to most life. Photosynthesis dominates chloroplast physiology but represents only a fraction of the tightly interconnected metabolic network that spans the entire cell. Here, we explore how photosynthetic activity affects the energy physiological status in cell compartments beyond the chloroplast. We develop precision live monitoring of subcellular energy physiology under illumination to investigate pH, MgATP2- and NADH/NAD+ dynamics at dark-light transitions by confocal imaging of genetically encoded fluorescent protein biosensors in Arabidopsis leaf mesophyll. We resolve the in vivo signature of stromal alkalinisation resulting from photosynthetic proton pumping and observe a similar pH signature also in the cytosol and the mitochondria suggesting that photosynthesis triggers an alkalinisation wave that affects the pH landscape of large parts of the cell. MgATP2- increases in the stroma at illumination, but no major effects on MgATP2- concentrations in the cytosol were resolved. Photosynthetic activity triggers a signature of substantial NAD reduction in the cytosol that is driven by photosynthesis-derived electron export. Strikingly, cytosolic NAD redox status was deregulated in mutants of chloroplastic NADP- and mitochondrial NAD-dependent malate dehydrogenases even at darkness, pinpointing the participation of the chloroplasts and mitochondria in shaping cytosolic redox metabolism in vivo with a dominant function of malate metabolism. Our data illustrate how profoundly and rapidly changes in photosynthetic activity affect the physiological and metabolic landscape throughout green plant cells. One-sentence summaryDark-light transitions trigger profound re-orchestration of subcellular pH and NAD redox physiology not only in the chloroplast but also beyond, in the cytosol and the mitochondria, as revealed by precision live-monitoring using fluorescent protein biosensors.

plant biology↗

bioRxiv2020

An evolutionary conserved lysine acetylation hotspot regulates plant mitochondrial malate dehydrogenase activity

Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a posttranslational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 highly activated it. Comparative homology modelling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity. Significance statementWe explore the role of lysine acetylation as a mechanism to directly modulate mitochondrial metabolism in land plants by generating the lysine acetylome of the moss Physcomitrium patens and comparing with available angiosperm data. We found acetylation of evolutionarily conserved lysines as a strategy to fine-tune the activity of mitochondrial malate dehydrogenase in a species-dependent molecular context.

plant biology↗

bioRxiv2020

Chloroplast-derived photo-oxidative stress causes changes in H2O2 and EGSH in other subcellular compartments

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers such as the highly reduced glutathione pool serve as reservoirs of reducing power for several ROS scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide (GSSG) and a shift of the glutathione redox potential (EGSH) towards less negative values is considered a hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically-encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol or mitochondria. Treatment of seedlings with MV caused a rapid oxidation in chloroplasts and subsequently also in the cytosol and mitochondria. The MV-induced oxidation was significantly boosted by illumination with actinic light and largely abolished by inhibitors of photosynthetic electron transport. In addition, MV also induced an autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress acclimation in plants. One sentence summaryMethyl viologen-induced photooxidative stress causes an increase of H2O2 and oxidation of glutathione in chloroplasts, cytosol and mitochondria as well as autonomous oxidation in mitochondria.

plant biology↗

bioRxiv2020

The Arabidopsis mitochondrial dicarboxylate carrier 2 maintains leaf metabolic homeostasis by uniting malate import and citrate export

Malate is the major substrate for respiratory oxidative phosphorylation in illuminated leaves. In the mitochondria malate is converted to citrate either for replenishing tricarboxylic acid (TCA) cycle with carbon, or to be exported as substrate for cytosolic biosynthetic pathways or for storage in the vacuole. In this study, we show that DIC2 functions as a mitochondrial malate/citrate carrier in vivo in Arabidopsis. DIC2 knockout (dic2-1) results in growth retardation that can only be restored by expressing DIC2 but not its closest homologs DIC1 or DIC3, indicating that their substrate preferences are not identical. Malate uptake by non-energised dic2-1 mitochondria is reduced but can be restored in fully energised mitochondria by altering fumarate and pyruvate/oxaloacetate transport. A reduced citrate export but an increased citrate accumulation in substrate-fed, energised dic2-1 mitochondria suggest that DIC2 facilitates the export of citrate from the matrix. Consistent with this, metabolic defects in response to a sudden dark shift or prolonged darkness could be observed in dic2-1 leaves, including altered malate, citrate and 2-oxoglutarate utilisation. There was no alteration in TCA cycle metabolite pools and NAD redox state at night; however, isotopic glucose tracing reveals a reduction in citrate labelling in dic2-1 which resulted in a diversion of flux towards glutamine, as well as the removal of excess malate via asparagine and threonine synthesis. Overall, these observations indicate that DIC2 is responsible in vivo for mitochondrial malate import and citrate export which coordinate carbon metabolism between the mitochondrial matrix and the other cell compartments. SIGNIFICANCE STATEMENTMitochondria are pivotal for plant metabolism. One of their central functions is to provide carbon intermediates for the synthesis of critical building blocks, such as amino acids. Malate import and citrate export are two of the most recognised and specialised features of the mitochondrial role in the plant cellular metabolic network, yet the possibility that a single carrier would unite both functions has not been considered. Here, we have demonstrated that DIC2 preferentially fulfils these two functions in Arabidopsis thaliana in vivo, making it a bifunctional gateway for two major metabolite fluxes into and out of the mitochondrial matrix in the plant cell. Our results highlight the significance of DIC2 in cooperation with other mitochondrial carriers in maintaining metabolic balance even under challenging environmental conditions.

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

Low-glutathione mutants are impaired in growth but do not show an increased sensitivity to moderate water deficit

Glutathione is considered a key metabolite for stress defense and elevated levels have frequently been proposed to positively influence stress tolerance. To investigate whether glutathione affects plant performance and the drought tolerance of plants, wild-type Arabidopsis plants and an allelic series of five mutants (rax1, pad2, cad2, nrc1, and zir1) with reduced glutathione contents between 21 and 63 % compared to wild-type glutathione content were phenotypically characterized for their shoot growth under control and water-limiting conditions using a shoot phenotyping platform. Under non-stress conditions the zir1 mutant with only 21 % glutathione showed a pronounced dwarf phenotype. All other mutants with intermediate glutathione contents up to 62 % in contrast showed consistently slightly smaller shoots than the wild-type. Moderate drought stress imposed through water withdrawal until shoot growth ceased showed that wild-type plants and all mutants responded similarly in terms of chlorophyll fluorescence and growth retardation. These results lead to the conclusion that glutathione is important for general plant performance but that the glutathione content does not affect tolerance to moderate drought conditions typically experienced by crops in the field.

plant biology↗

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

Chloroplasts Require Glutathione Reductase to Balance Reactive Oxygen Species and Maintain Efficient Photosynthesis

O_LIThiol-based redox-regulation is vital to coordinate chloroplast functions depending on illumination. Yet, how the redox-cascades of the thioredoxin and glutathione redox machineries integrate metabolic regulation and reactive oxygen species (ROS) detoxification remains largely unresolved. We investigate if maintaining a highly reducing stromal glutathione redox potential (EGSH) via glutathione reductase (GR) is necessary for functional photosynthesis and plant growth.\nC_LIO_LISince absence of the plastid/mitochondrial GR is embryo-lethal in Arabidopsis thaliana, we used the model moss Physcomitrella patens to create knock-out lines. We dissect the role of GR in chloroplasts by in vivo monitoring stromal EGSH dynamics, and reveal changes in protein abundances by metabolic labelling.\nC_LIO_LIWhereas stromal EGSH is highly reducing in wildtype and clearly responsive to light, the absence of GR leads to a partial oxidation, which is not rescued by light. Photosynthetic performance and plant growth are decreased with increasing light intensities, while ascorbate and zeaxanthin levels are elevated. An adjustment of chloroplast proteostasis is pinpointed by the induction of plastid protein repair and degradation machineries.\nC_LIO_LIOur results indicate that the plastid thioredoxin and glutathione redox systems operate largely independently. They reveal a critical role of GR in maintaining efficient photosynthesis.\nC_LI

plant biology↗