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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 chlorophyteChlamydomonas reinhardtii. Surprisingly, unlike other studied MCAs and similar to caspases, CrMCA-II dimerizes bothin vitroandin vivo. Furthermore, activation of CrMCA-IIin vivocorrelates with the dimerization. Most of CrMCA-II in the cell is present as a zymogen attached to the plasma membrane (PM). Deletion ofCrMCA-IIby 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.

The imaging of plant seedlings, fungal mycelia and bacterial colonies grown on Petri plates is common in phenotyping assays, and is typically done manually despite the procedures being time-consuming and laborious. The main reason for this is the still limited availability of existing automated phenotyping tools and facilities. Additionally, constructing a custom-made automated solution is a daunting task for most research groups specializing in biology.Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photos of up to four vertically oriented Petri plates in a single experiment. SPIRO was designed for biologists by biologists; thus, its assembly does not require experience in engineering or programming and its operation is sufficiently intuitive to be carried out without training. SPIRO has a small footprint optimal for fitting into standard incubators for plants and microbes, and is equipped with an LED light source for imaging in the dark, thus allowing acquisition of photos under optimal growth conditions. SPIRO’s web-based user interface allows setting up experiments and downloading data remotely, without interfering with samples growth. SPIRO’s excellent image quality is optimal for automated image processing, which we demonstrate with two semi-automated assays for analysis of commonly used phenotypic traits: seed germination and root growth.Moreover, the robot can be easily customized for a specific use, as all information about SPIRO, including the models for 3D-printed structural components, control software, and scripts for image analysis, are released under permissive open-source licenses.

SUMMARYAdaptation to stress depends on the modulation of gene expression. Regulation of mRNA stability and degradation in stress granules (SGs), - cytoplasmic membraneless organelles composed of messenger ribonucleoprotein (mRNP) complexes, - plays an important role in fine-tuning of gene expression. In addition, SG formation can modulate stress signaling pathways by protein sequestration. Molecular composition, structure, and function of SGs in plants remain obscure. Recently, we established Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) as integral component of SGs in Arabidopsis thaliana. Here, we combined purification of TSN interactome with cell biology, reverse genetics and bioinformatics to study composition and function of SGs in plants. We found that under both normal (in the absence of stress) and stress conditions TSN interactome is enriched in the homologues of known mammalian and yeast SG proteins, in addition to novel or plant-specific SG components. We estimate that upon stress perception, approximately half of TSN interactors are recruited to SGs de novo, in a stress-dependent manner, while another half represent a dense protein-protein interaction network pre-formed before onset of stress. Almost all TSN-interacting proteins are moderately or highly disordered and approximately 20% of them are predisposed for liquid-liquid phase separation (LLPS). This suggests that plant SGs, similarly to mammalian and yeast counterparts, are multicomponent viscous liquid droplets. Finally, we have discovered that evolutionary conserved SNF1-related protein kinase 1 (SnRK1) interacts with TSN in heat-induced SGs and that SnRK1 activation critically depends on the presence of TSN and formation of SGs. Altogether, our results establish TSN as a docking platform for SG-associated proteins and important stress signal mediator in plants.

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.