Biology subjects

Ugalde, J. M.

Publications and source records attributed to Ugalde, J. M..

9 recordsLinked to original sources

bioRxiv2026

Nanoscale regulation of ROS signaling at the plasma membrane tunes the plant response to osmotic stress

The spatiotemporal organization of proteins and lipids within membranes is crucial for ensuring proper cellular signaling. While the segregation of proteins and lipids into membrane nanodomains is well established, it remains unclear whether nanodomains can generate gradients of small diffusible molecules. In plants, reactive oxygen species (ROS), especially hydrogen peroxide (H2O2), act as a key signaling molecules in response to environmental stimuli such as osmotic stress. However, how extracellular H2O2 affects intracellular signaling has remained unknown. Here, we show that osmotic stimulation induces the formation of localized, H2O2-rich nano-environments at the cytoplasmic face of the plasma membrane (PM) in Arabidopsis root cells. Using a PM-tethered H2O2 biosensor, we found that these oxidized nanodomains arise from the clustering of RESPIRATORY BURST OXIDASE HOMOLOGs (RBOHs) and RHO OF PLANTS 6 (ROP6), in coordination with aquaporin-mediated H2O2 transport via the PLASMA MEMBRANE INTRINSIC PROTEIN2;7 (PIP2;7). These local redox hotspots at the PM create a feedforward loop in which H2O2 enhances ROP6 nanoclustering thereby amplifying ROS signaling. Disruption of H2O2 production or transport dampens both ROP6 clustering and anisotropic cell expansion, indicating a crucial role for spatially-confined redox signaling in regulating plant growth under osmotic stress. Our findings propose a model in which ROP6/RBOHD-F/PIP2;7 nanodomains function as discrete redox signaling units, redefining ROS signaling at the PM as a structured, signal-specific, and compartmentalized process.

cell biology↗

bioRxiv2025

Root growth in Arabidopsis depends on the amount of glutathione and not the glutathione redox potential

Cell cycle activity and hence plant growth strictly depends on glutathione homeostasis. Despite compelling evidence for this dependency based on glutathione depletion, the cause for the cell cycle arrest had remained unclear. Cell cycle control may depend on either the absolute amount of glutathione or on the glutathione redox potential (EGSH). To unambiguously distinguish those two options, we characterized an allelic series of six Arabidopsis mutants affected in glutamate-cysteine ligase, which catalyses the first step for the biosynthesis of reduced glutathione (GSH). When grown under the same conditions, even mutants with 20% and 50% of wild-type GSH amounts are slightly stunted. The most severely compromised mutants, zir1 and rml1, were crossed with either gr1, which lacks cyto-nuclear glutathione disulfide reductase and was used to induce a pronounced shift in EGSH, or with bir6, which has a diminished glutathione consumption and thus exhibits slightly increased levels of GSH. Based on theoretical considerations, these levels are not expected to shift the EGSH to any significant extent. Our study shows that deleting GR1 in the zir1 or rml1 background does not result in an obvious phenotypic change. By contrast, deleting BIR6 was sufficient to suppress the growth arrest in rml1 and to attenuate the growth in zir1. These findings demonstrate that root growth is dependent on the availability of sufficient amounts of GSH, and not affected by pronounced changes in EGSH. This insight provides a decisive step towards understanding the mechanisms underpinning the proposed role of glutathione in cell cycle and growth control. HighlightCrosses of GSH-deficient Arabidopsis mutants with mutants affected in glutathione reduction or GSH consumption pinpoint the GSH amount rather than the glutathione redox potential as causative for changes in root growth.

plant biology↗

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↗

bioRxiv2023

Localization of four class I glutaredoxins in the cytosol and the secretory pathway and characterization of their biochemical diversification

Class I glutaredoxins (GRXs) are catalytically active oxidoreductases and considered key proteins mediating reversible glutathionylation and deglutathionylation of protein thiols during development and stress responses. To narrow in on putative target proteins, it is mandatory to know the subcellular localization of the respective GRXs and to understand their catalytic activities and putative redundancy between isoforms in the same compartment. We show that GRXC1 and GRXC2 are cytosolic proteins with GRXC1 being attached to membranes through myristoylation. GRXC3 and GRXC4 are identified as type II membrane proteins along the early secretory pathway with their enzymatic function on the luminal side. Comparison of all four studied GRXs for their oxidoreductase function highlights biochemical diversification with GRXC1 and GRXC2 being better reductants than GRXC3 and GRXC4 with bis(2-hydroxyethyl) disulfide and oxidized roGFP2 as substrates. Vice versa, GRXC3 and GRXC4 are better oxidants of reduced roGFP2 in the reverse reaction. Analysis of electrostatic surface potentials mirrors the phylogenetic classification of class I GRXs but cannot fully account for the observed kinetic differences in their interaction with roGPF2. Despite localization of two class I GRXs each in the cytosol and the endomembrane system, the respective double null mutants are viable without obvious phenotypes. Summary statementWe identify Arabidopsis glutaredoxins GRXC3 and GRXC4 as type II membrane proteins in the secretory pathway and GRXC1 as attached to membranes through N-terminal myristoylation. Cytosolic GRXC1 and GRXC2 and luminal GRXC3 and GRXC4 display distinct biochemical properties in their redox activities.

plant biology↗

bioRxiv2021

Endoplasmic reticulum oxidoreductin (ERO) provides resilience against reductive stress and hypoxic conditions by mediating luminal redox dynamics

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. While many key players involved in oxidative protein folding are known, our understanding of how redox homeostasis in the ER is maintained and how EROs, the Cys residues of nascent proteins, and the luminal glutathione redox buffer interact is limited. Here, we isolated viable ero1 ero2 double mutants largely deficient in ERO activity, which rendered the mutants highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER lumen in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress, as induced by acute exposure to dithiothreitol, was delayed in ero1 ero2 mutants. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in the ero1 ero2 mutant reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis. One sentence summaryDynamic monitoring the ER luminal glutathione redox potential highlights the role of EROs in defining redox conditions and the interplay between different redox inputs during hypoxia and reductive stress.

plant biology↗

bioRxiv2020

Live monitoring of ROS-induced cytosolic redox changes with roGFP2-based sensors in plants

Plant cells produce reactive oxygen species (ROS) as by-products of oxygen metabolism and for signal transduction. Depending on their concentration and their site of production, ROS can cause oxidative damage within the cell, and must be effectively scavenged. Detoxification of the most stable ROS, hydrogen peroxide (H2O2), via the glutathione-ascorbate pathway may transiently alter the glutathione redox potential (EGSH). Changes in EGSH can thus be considered as a proxy of the oxidative load in the cell. Genetically encoded probes based on roGFP2 enable extended opportunities for in vivo monitoring of H2O2 and EGSH dynamics. Here, we report detailed protocols for live monitoring of both parameters in the cytosol with the probes Grx1-roGFP2 for EGSH and roGFP2-Orp1 for H2O2, respectively. The protocols have been adapted for live cell imaging with high lateral resolution on a confocal microscope and for multiparallel measurements in whole organs or intact seedlings in a fluorescence microplate reader. Elicitor-induced ROS generation is used as an example for illustration of the opportunities for dynamic ROS measurements that can easily be transferred to other scientific questions and model systems.

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

GSTU7 affects growth performance and acts as an antagonist of oxidative stress induced by methyl viologen

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis genome codes for 53 GSTs, divided into seven subclasses, however understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5 and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-class GSTUs. Specifically, TGA2 controls the expression of several GSTUs under UV-B light and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutively expressed GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared to WT plants. GSTU7 was found to act as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus protecting cells from oxidative stress.

plant biology↗

bioRxiv2020

Transcription factor TGA2 is essential for UV-B stress tolerance controlling oxidative stress in Arabidopsis

Plants possess a diversity of Reactive Oxygen Species (ROS)-processing enzymes involved in sensing and controlling ROS levels under basal and stressful conditions. There is little information on the transcriptional regulators that control the expression of these ROS-processing enzymes, particularly at the onset of the defense response to abiotic stress. Filling this gap, this paper reports a critical role for Arabidopsis TGA class II factors (TGA2, TGA5, and TGA6) in the tolerance response to UV-B light and photooxidative stress, by activating the expression of genes with antioxidative roles. We identified two clusters of genes responsive to UV-B and activated by TGA2/5/6 were identified using RNAseq and clustering analysis. The GSTU gene family, which encodes glutathione transferase enzymes from the Tau subclass, was overrepresented in these clusters. We corroborated the TGA2-mediated activation in response to UV-B for three model genes (GSTU7, GSTU8, and GSTU25) using RT-qPCR and ChIP analyses. Interestingly, using tga256 mutant and TGA2- and GSTU7-complemented mutant plants, we demonstrated that TGA2-mediated induction of GSTU genes is essential to control ROS levels and oxidative damage after UV-B and MeV treatments. This evidence positions TGA class II factors, particularly TGA2, as a key players in the redox signaling network of Arabidopsis plants. HighlightArabidopsis TGA2 transcription factor is part of the redox-signaling network controlling ROS levels and oxidative damage in tolerance response to UV-B and photooxidative stress, via activation of antioxidant GSTU genes.

plant biology↗