Biology subjects

Stael, S.

Publications and source records attributed to Stael, S..

11 recordsLinked to original sources

bioRxiv2026

OEF18 is a membrane-anchored organellar Ca{superscript 2}⁺ sensor linking calcium signaling to jasmonate-mediated defense and stress acclimation

Changes in intracellular calcium ion (Ca{superscript 2}) concentrations generate characteristic signatures that are decoded by specialized Ca{superscript 2}-binding proteins (CaBP). Although substantial progress has been made in understanding cytosolic calcium signaling pathways, calcium signaling within organelles, particularly chloroplasts, remains poorly understood, partly because only a few EF-hand CaBP have been identified in organelles. Here, we describe a novel EF-hand protein of 18 kDa, that was found to be associated with the chloroplast envelope and peroxisomal membrane and was therefore named OEF18 (ORGANELLAR EF-HAND PROTEIN OF 18 kDa). OEF18 has a very unusual structure, containing an N-terminal myristoylation site, followed by one EF-hand in the N-terminus facing to the cytosol, and a transmembrane domain in the C-terminus. OEF18 membrane-targeting was found to be mediated by ANKYRIN REPEAT-CONTAINING PROTEIN 2A (AKR2A) via the C-terminal transmembrane domain of OEF18. Furthermore, the EF-hand in OEF18 bound Ca{superscript 2} at a physiological concentration that led to a large protein conformational change, inducing oligomerization of the N-terminal part. We found that oef18 mutants accumulated less jasmonic acid (JA) and its bioactive conjugate JA-Ile, likely causing a defect in the insect herbivore response. Wild-type OEF18 complemented the herbivory phenotype of oef18 mutants, whereas an EF-hand point mutant lacking Ca{superscript 2}-binding capacity failed to restore the wild-type response. Furthermore, OEF18 was required for resistance to salt stress in combination with dark-induced senescence. Together, these results establish OEF18 as a previously unrecognized organellar Ca{superscript 2} sensor that couples Ca{superscript 2} perception to JA-mediated defense and abiotic stress responses in plants.

plant biology↗

bioRxiv2026

Starvation-induced autophagy occurs independently of the ATG1 complex in Chlamydomonas

The survival of eukaryotes during starvation depends on effective nutrient recycling via autophagy. Accordingly, loss of autophagy-related (ATG) proteins, including the nutrient-sensing ATG1 kinase complex, typically results in reduced fitness or lethality under nutrient limitation. The green alga Chlamydomonas reinhardtii provides a tractable model for autophagy studies, as its ATG repertoire is encoded by single-copy genes. Here, we generated a comprehensive library of ATG deletion mutants and examined their growth and autophagy during starvation. Surprisingly, starvation-induced autophagy occurred in the absence of ATG1 complex components (ATG1, ATG11, ATG13, and ATG101), revealing ATG1-independent autophagy and challenging the canonical model for autophagy initiation.

molecular biology↗

bioRxiv2025

Processing and release of the maize phytocytokine Zip1

Phytocytokines are endogenous peptides that modulate plant immunity outcomes, yet how their maturation and spatial deployment are controlled remains unclear. Here we show that the maize phytocytokine precursor PROZIP1 is controlled by a spatially separated, two-stage proteolytic pathway that mechanistically uncouples signal activation from extracellular attenuation. PROZIP1 associates with the endoplasmic reticulum and undergoes intracellular, arginine-dependent processing by type II metacaspases, generating a C-terminal PROZIP1 fragment (Ct-PROZIP1). This processing licenses PROZIP1 for export to the apoplast via an ER-Golgi-independent route. Proteomic mapping and mutational analyses identify arginine residues flanking the Zip1 peptide as critical for efficient processing and secretion. The calcium-dependent metacaspase ZmMC9 specifically processes PROZIP1, thereby efficiently generating the bioactive Ct-PROZIP1 fragment. In the apoplast, Ct-PROZIP1 is further processed by papain-like cysteine proteases and additional extracellular proteases, contributing to Zip1 turnover and signal clearance. While the free Zip1 peptide is detected at later stages, Ct-PROZIP appears to be the primary signaling entity in modulating pathogen-induced immune responses. Together, these findings demonstrate a previously unknown complexity in peptide signaling, suggesting a multilayered control of phytocytokine activity that provides spatial and temporal precision to disease modulation in maize.

plant biology↗

bioRxiv2025

Lack of AtMC1 catalytic activity triggers autoimmunity dependent on NLR stability

Plants utilize cell surface-localized pattern recognition receptors (PRRs) and intracellular nucleotide-binding leucine-rich repeat (NLR) receptors to detect non-self and elicit robust immune responses. Fine-tuning the homeostasis of these receptors is critical to prevent their hyperactivation. Here, we show that Arabidopsis plants lacking metacaspase 1 (AtMC1) display autoimmunity dependent on immune signalling components downstream of NLR and PRR activation. Overexpression of a catalytically inactive AtMC1 in an atmc1 background triggers severe autoimmunity partially dependent on the same immune signalling components. Overexpression of the E3 ligase SNIPER1, a master regulator of NLR homeostasis, fully reverts the AtMC1-dependent autoimmunity phenotype, inferring that a broad defect in NLR turnover may underlie the severe phenotype observed. Catalytically inactive AtMC1 localizes to punctate structures that are degraded through autophagy. Altogether and considering previous evidence on the proteostatic functions of AtMC1, we speculate that Wt AtMC1 may either directly or indirectly control NLR protein levels, thereby preventing autoimmunity.

plant biology↗

bioRxiv2024

Burning glass effect of water droplets triggers an ER-derived calcium response in the chloroplast stroma of Arabidopsis thaliana leaves

Plants require water and light for photosynthesis, but light, when focused by water droplets on leaves, can create high light intensity spots that are harmful to plants. As excessive light intensity can reduce growth or even induce cell death, it is vital for plants to detect and react to changes in light exposure and acclimate to high light stress. Ca2+ signaling was previously implicated in high light acclimation. However, the dynamics of free Ca2+ concentration in the chloroplast, the primary site of photosynthesis, or in the nucleus and in the cytoplasm, where transcription and translation for long-term acclimation occurs, remain unknown. Here we studied the dynamics and mechanism of the Ca2+ response to high light exposure. Focusing light through a glass bead to mimic water droplets triggered an increase of the free Ca2+ concentration in the chloroplast stroma of Arabidopsis thaliana. This finding was corroborated using established and newly developed genetically encoded calcium indicators, which revealed a biphasic increase in the stromal free Ca2+ concentration when exposed to varying intensities and qualities of light. Among photosynthetic by-products, reactive oxygen and lipophilic species in particular, have been implicated in high light stress acclimation. A H2O2 signature was induced, albeit with different dynamics than the Ca2+ response, while chemical inhibition of the photosynthetic electron transport points towards singlet oxygen as a potential culprit of the high light-induced increase in stromal free Ca2+ concentration. The observed dynamics differed from those of a heat-shock induced Ca2+ signature, although temperature had a positive effect on the Ca2+ response. Based on Ca2+ inhibitor treatments and the free Ca2+ concentration dynamics, we suggest that the high light-induced stromal Ca2+ is derived from the endoplasmic reticulum rather than from the cytoplasm. In conclusion, inspired by the burning glass effect of water droplets on leaves, we uncovered a Ca2+ response that implicates a novel mechanism for plants to acclimate to high light stress--a process that will become increasingly relevant in a changing climate.

plant biology↗

bioRxiv2024

Shedding light on plant proteolysis: genetically encoded fluorescent sensors as tools for profiling protease activities

Proteolysis, a ubiquitous process in living organisms, is driven by proteases that regulate numerous signaling pathways through the hydrolysis of peptide bonds in protein substrates. Understanding the temporal and spatial dynamics of proteolysis and the activation of proteases is crucial for elucidating their roles in biological pathways. Here, we introduce a suite of genetically encoded FRET reporters designed to detect various proteolytic activities in plants. These sensors effectively reported in planta the specific activity of both Tobacco Etch Virus protease and caspase-3. Furthermore, we developed sensors for detecting plant metacaspase activity, validated through both in vitro and in planta experiments. These experiments revealed the spatial dynamics of proteolysis triggered by metacaspase activation following wounding and programmed cell death in roots. The implementation of these tools in plant biology research opens new avenues for investigating proteolytic mechanisms, significantly enhancing the potential for in-depth studies. Our work demonstrates the feasibility of using these sensors to detect diverse protease activities in vivo with high spatiotemporal resolution. These plant proteolytic biosensors hence represent a valuable toolbox for understanding protease functions within their natural context, paving the way for future advancements in plant biology research.

plant biology↗

bioRxiv2023

Thermoprotection by a cell membrane-localized metacaspase in a green alga

Caspases are restricted to animals, while other organisms, including plants possess metacaspases (MCAs), a more ancient and broader class of structurally-related yet biochemically distinct proteases. Our current understanding of plant MCAs is derived from studies in streptophytes, and mostly in Arabidopsis expressing nine MCAs with partly redundant activities. In contrast to streptophytes, most chlorophytes contain only one or two hitherto uncharacterized MCAs, providing an excellent platform for MCA research. Here we investigate CrMCA-II, a single type II MCA from a model chlorophyte Chlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes both in vitro and in vivo. Furthermore, activation of CrMCA-II in vivo correlates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion of CrMCA-II by CRISPR/Cas9 compromises thermotolerance leading to increased cell death under heat stress. Adding back either wild-type or catalytically dead CrMCA-II restores thermoprotection, suggesting that its proteolytic activity is dispensable for this effect. Finally, we link the non-proteolytic role of CrMCA-II in thermotolerance to the ability to modulate PM fluidity. Our study reveals an ancient, MCA-dependent thermotolerance mechanism retained by Chlamydomonas and probably lost during the evolution of multicellularity.

plant biology↗

bioRxiv2023

Seed Longevity is Controlled by Metacaspases

To survive extreme desiccation, seeds enter dormancy that can last millennia. This dormancy involves the accumulation of protective but structurally disordered storage proteins through unknown adjustments of proteolytic surveillance mechanisms. Mutation of all six types II metacaspases (MCAs)-II in the model plant Arabidopsis revealed their essential role in modulating these proteolytic mechanisms. MCA-II mutant seeds fail to properly target at the endoplasmic reticulum (ER) the AAA ATPase Cell Division Cycle 48 (CDC48) to dispose of misfolded proteins. MCA-IIs cleave a CDC48 adaptor, the ubiquitination regulatory X domain-containing (PUX) responsible for localizing CDC48 to the lipid droplets. When cleaved, CDC48-PUX is inactivated and allows a lipid droplet-to-ER shuttling of CDC48, an important step in the regulation of seeds lifespan. In sum, we uncover antagonism between proteolytic pathways bestowing longevity. One-Sentence SummaryMetacaspase proteases confer seed longevity by antagonizing CDC48 activity.

plant biology↗

bioRxiv2023

Structure-function analysis of a calcium-independent metacaspase reveals a novel proteolytic pathway for lateral root emergence

Metacaspases are part of an evolutionarily broad family of multifunctional cysteine proteases, involved in disease and normal development. Despite the extensive study of metacaspases in the two decades since their discovery, the structure-function relationship of metacaspases remains poorly understood. Furthermore, previous studies on their function have been thwarted by the redundancy in gene copy number and potential phenotypic suppression of genetic mutations, especially in plants. Here, we have solved the X-ray crystal structure of an Arabidopsis thaliana type II metacaspase (AtMCA-IIf) that belongs to a particular sub-group that does not require calcium ions for activation. Compared to crystal structures of other metacaspases and caspases, the AtMCA-IIf active site is structurally similar and poses a conundrum for the catalytic mechanism of the cysteine-histidine dyad. To study metacaspase activity in plants, we developed an in vitro chemical screen to identify small molecule metacaspase inhibitors. Several hits with a minimal thioxodihydropyrimidine-dione (TDP) structure were identified, some being specific inhibitors of AtMCA-IIf. We provide a mechanistic basis for inhibition by the TDP-containing compounds through molecular docking onto the AtMCA-IIf crystal structure. Finally, a TDP-containing compound (TDP6) was effective at inhibiting lateral root emergence in vivo, likely through the inhibition of metacaspases that are specifically expressed in the endodermal cells overlaying developing lateral root primordia. In the future, the small compound inhibitors and crystal structure of AtMCA-IIf can be used to study metacaspases in various other species, such as important human pathogens including those causing neglected diseases.

plant biology↗

bioRxiv2022

Proximity labelling allows to study novel factors in chloroplast development

Chloroplast development is initiated by light-signals triggering the expression of nuclear encoded chloroplast genes in a first phase, followed by massive structural changes in the transition from proplastids to mature chloroplasts in the second phase. While the molecular players involved in the first phase are currently emerging, regulatory components of the second phase, demanding high plastid translational capacity and RNA processing, are still enigmatic. This is mostly due to the very limited amount of plant material at the early phases of development that makes biochemical studies such as identifying protein interaction networks very difficult. To overcome this problem, we developed a TurboID-based proximity labelling workflow that requires only very limited sample amounts to obtain mechanistic insights into protein interaction networks present in the early stages of plastid development. We used the CGL20a protein, a novel factor involved in chloroplast development, as bait for in vivo proximity labelling in developing seedlings 7 days after germination. We found that CGL20a resides in a nexus of RNA binding proteins mainly associated to ribosomal RNA (rRNA) including different ribosome-associated proteins. One-sentence summaryThe use of plastid-specific in vivo proximity labelling in Arabidopsis seedlings allows to identify novel components in chloroplast development in higher plants.

plant biology↗

bioRxiv2017

The Proteolytic Landscape Of An Arabidopsis Separase-Deficient Mutant Reveals Novel Substrates Associated With Plant Development

Digestive proteolysis executed by the proteasome plays an important role in plant development. Yet, the role of limited proteolysis in this process is still obscured due to the absence of studies. Previously, we showed that limited proteolysis by the caspase-related protease separase (EXTRA SPINDLE POLES [ESP]) modulates development in plants through the cleavage of unknown substrates. Here we used a modified version of the positional proteomics method COmbined FRActional DIagonal Chromatography (COFRADIC) to survey the proteolytic landscape of wild-type and separase mutant RADIALLY SWOLLEN 4 (rsw4) root tip cells, as an attempt to identify targets of separase. We have discovered that proteins involved in the establishment of pH homeostasis and sensing, and lipid signalling in wild-type cells, suggesting novel potential roles for separase. We also observed significant accumulation of the protease PRX34 in rsw4 which negatively impacts growth. Furthermore, we observed an increased acetylation of N-termini of rsw4 proteins which usually comprise degrons identified by the ubiquitin-proteasome system, suggesting that separase intersects with additional proteolytic networks. Our results hint to potential pathways by which separase could regulate development suggesting also novel proteolytic functions.

plant biology↗