https://doi.org/10.1093/plcell/koad172 THE PLANT CELL 2023: 35: 3325–3344 Arabidopsis metacaspase MC1 localizes in stress granules, clears protein aggregates, and delays senescence Research Article Nerea Ruiz-Solaní ,1,2 Jose Salguero-Linares ,1,‡ Laia Armengot ,1,2 Jaime Santos ,3 Irantzu Pallarès ,3 Katarina P. van Midden ,4 Ujjal J. Phukkan ,1,† Seda Koyuncu ,5 Júlia Borràs-Bisa ,1 Liang Li ,1 Crina Popa ,1 Frederik Eisele ,6 Anna Maria Eisele-Bürger ,6 Sandra Malgrem Hill ,6 Emilio Gutiérrez-Beltrán ,7,8 Thomas Nyström ,6 Marc Valls ,1,2 Ernesto Llamas ,9 David Vilchez ,5,10,11 Marina Klemenčič ,4 Salvador Ventura 3 and Nuria S. Coll 1,12,* 1 Centre for Research in Agricultural Genomics (CRAG), CSIC-IRTA-UAB-UB, Bellaterra 08193, Spain 2 Department of Genetics, Microbiology and Statistics, Universitat de Barcelona, Barcelona 08028, Spain 3 Institut de Biotecnologia i de Biomedicina, Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Barcelona 08193, Spain 4 Department of Chemistry and Biochemistry, Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana 1000, Slovenia 5 Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne 50931, Germany 6 Department of Microbiology and Immunology, The Sahlgrenska Academy at the University of Gothenburg, Gothenburg 41390, Sweden 7 Instituto de Bioquímica Vegetal y Fotosíntesis (Universidad de Sevilla and Consejo Superior de Investigaciones Científicas), 41092 Seville, Spain 8 Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Biología, Universidad de Sevilla, Sevilla 41012, Spain 9 Cluster of Excellence on Plant Sciences (CEPLAS), Institute for Plant Sciences, University of Cologne, Cologne D-50674, Germany 10 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne 50931, Germany 11 Faculty of Medicine, University Hospital Cologne, Cologne 50931, Germany 12 Consejo Superior de Investigaciones Científicas (CSIC), Barcelona 08001, Spain *Author for correspondence: nuria.sanchez-coll@cragenomica.es ‡ Present address: The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, UK. † Present address: School of Plant Sciences, University of Arizona, Tucson, AZ, USA. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Nuria S. Coll (nuria.sanchez-coll@cragenomica.es). Abstract Stress granules (SGs) are highly conserved cytoplasmic condensates that assemble in response to stress and contribute to main­ taining protein homeostasis. These membraneless organelles are dynamic, disassembling once the stress is no longer present. Persistence of SGs due to mutations or chronic stress has been often related to age-dependent protein-misfolding diseases in animals. Here, we find that the metacaspase MC1 is dynamically recruited into SGs upon proteotoxic stress in Arabidopsis (Arabidopsis thaliana). Two predicted disordered regions, the prodomain and the 360 loop, mediate MC1 recruitment to and release from SGs. Importantly, we show that MC1 has the capacity to clear toxic protein aggregates in vivo and in vitro, acting as a disaggregase. Finally, we demonstrate that overexpressing MC1 delays senescence and this phenotype is dependent on the presence of the 360 loop and an intact catalytic domain. Together, our data indicate that MC1 regulates senescence through its recruitment into SGs and this function could potentially be linked to its remarkable protein aggregate-clearing activity. Received January 4, 2023. Accepted June 21, 2023. Advance access publication July 1, 2023 © The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. Open Access This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 3326 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. Introduction An essential property of SGs is their dynamism: to be func­ tional, they must be inducible and reversible. In mammals, To cope with stress, eukaryotic cells are equipped with multiple cumulative evidence links altered SG dynamics with patholo­ sophisticated mechanisms that ultimately confer robustness gies featuring aberrant protein coalescence leading to aggre­ against various perturbations. As part of their stress responses, gation (Baradaran-Heravi et al. 2020; Marcelo et al. 2021). In cells must readjust proteostasis (protein homeostasis), which is several neurodegenerative diseases, mutations in LCRs/IDRs achieved through an arrest in protein synthesis and activation of certain proteins disrupt their biophysical properties, lead­ of protein quality control (PQC) mechanisms to prevent accu­ ing to enhanced LLPS and formation of pathological protein mulation of misfolded proteins in the cytoplasm, potentially aggregates (Baradaran-Heravi et al. 2020). Pathological SGs causing proteotoxicity (Alberti and Carra 2018). The proteo­ undergo a liquid-to-solid transition and persist even after static capacity of cells declines with age, which may reduce the stress has passed, acting as undissolvable protein traps. their capacity to dispose of potentially harmful protein aggre­ This is the case of polyglutamine (polyQ) pathologies, such gates (Vilchez et al. 2014; Hipp et al. 2019). as Huntington’s disease, caused by abnormal polyQ exten­ An important stress response mechanism in fungi, animals, sions, that make them more aggregation prone (Sanchez and plants is the formation of stress granules (SGs). SGs are et al. 2021). biomolecular condensates assembled in the cytosol under Plants may have evolved extremely efficient mechanisms stress conditions with a highly dynamic behavior, containing to deal with toxic protein aggregation. It has been recently a combination of mRNA and proteins, many of which have demonstrated that overexpression of synthetic extended RNA-binding ability (Jain et al. 2016; Markmiller et al. 2018; polyQ proteins, which normally aggregate and cause cell Youn et al. 2019). These membraneless compartments death in animal models, does not cause deleterious defects were originally viewed as sites of accumulation and disposal in plants (Llamas et al. 2022). In fact, plants overexpressing of stalled mRNAs but are currently emerging as major or­ synthetic protein variants that constitutively aggregate do chestrators of stress responses (Buchan and Parker 2009; not show major defects (Jung et al. 2020; Llamas et al. Maruri-López et al. 2021). 2022). This may indicate that plants have evolved extremely Current models predict that SG formation is mediated by efficient mechanisms to deal with protein aggregation. liquid–liquid phase separation (LLPS) promoted by multiva­ Selective autophagy has been previously implicated in deg­ lent molecules, such as proteins featuring low complexity re­ radation of protein aggregates or aggrephagy during proteo­ gions (LCRs)/intrinsically disordered regions (IDRs) (Protter toxic stress (Jung et al. 2020). However, whether formation of and Parker 2016). Assembly and clearance of SGs are finely these protein aggregates is related to molecular condensa­ regulated, with initial formation of a dense core by LLPS fol­ tion including SG formation and dynamics has not been lowed by recruitment of peripheral proteins (Jain et al. 2016; addressed. Markmiller et al. 2018). Core components are proteins con­ Metacaspases are cysteine proteases present in plants, taining IDRs and RNA-binding domains together with pro­ yeast, and protozoa (Uren et al. 2000). Plant metacaspases teins involved in translation, whereas the shell is composed are divided into Type I if they bear an N-terminal prodo­ of an array of mRNA, proteins, and small molecules that main and Type II, if no prodomain is present but instead vary depending on the species, cell type, and developmental a long linker between the catalytic subunits exists (Uren stage (González-García et al. 2011; Jain et al. 2016; Niewidok et al. 2000; Klemenčič and Funk 2019). Several metacaspases et al. 2018; Kosmacz et al. 2019; Guillén-Boixet et al. 2020). have been shown to play important roles in stress responses Compared to yeast and mammals, little is known about (Coll et al. 2010; Hander et al. 2019; Minina et al. 2020; Luan plant SGs, despite their important role in stress responses, in­ et al. 2021; Pitsili et al. 2023), although in most cases the cluding heat, hypoxia, salt, and drought (Sorenson and mode of action of these proteases remains obscure. The Bailey-Serres 2014; Yan et al. 2014; Gutierrez-Beltran et al. model plant Arabidopsis thaliana (hereafter Arabidopsis) 2015; Marondedze et al. 2020). Among these stress responses, encodes 9 metacaspases in its genome. MC1-3 (AtMC1-3/ heat is the best characterized so far, as it presents an arche­ AtMCA-Ia-c) is a Type I metacaspase, while MC4-9 typal form of acute stress resulting in proteotoxicity that (AtMC4-9/AtMCA-IIa-f) is a Type II metacaspase (Minina must be handled by various mechanisms, including SG for­ et al. 2020). We previously showed that plants lacking mation (Maruri-López et al. 2021). SGs provide efficient regu­ MC1 exhibit accelerated senescence and accumulate aggre­ latory platforms under stress conditions (Maruri-López et al. gated proteins, indicating a potential role of MC1 in pro­ 2021), serving as (i) mRNA reorganization centers, wherein teostasis (Coll et al. 2014). In addition, a portion of MC1 their fate is determined (reinitiation, decay, and storage), relocalizes to insoluble protein deposits (IPODs) under pro­ (ii) temporary protein storage centers, to protect them teotoxic stress conditions. Data from our lab and others in­ from unfolding, and (iii) enzyme recruitment centers, to fa­ dicate that MC1 may help stabilizing various proteins cilitate rapid activation of certain metabolic pathways. (Roberts et al. 2013; Lema Asqui et al. 2018; Wang et al. Plant SG component catalogs and molecular markers have 2021). However, the specific mechanisms whereby MC1 started to become available in recent years opening new ave­ contributes to protein stabilization and aggregate clearance nues of research. remain unknown. Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3327 Here, we demonstrate that MC1 is dynamically recruited Schmit et al. 2021). IDRs have been proposed to act as one to SGs upon proteotoxic stress. This SG localization is of the main driving forces of condensate assembly, although mediated by a C-terminal IDR, the 360 loop. We show that the exact mechanism by which this occurs remains to be fully MC1 participates in the clearance of pathological aggregates elucidated (Posey et al. 2018; Alberti et al. 2019). We used a in evolutionarily distant organisms ranging from yeast and combination of 2 predictive algorithms (D2P2, https://d2p2. animals to plants. In vitro, recombinant MC1 (rMC1) alone pro, and DISOPRED3, http://bioinf.cs.ucl.uk/psipred; Oates acts as a highly efficient disaggregase. In plants, this function et al. 2013; Jones and Cozzetto 2015) to pinpoint potential can be harnessed to delay senescence, as observed in MC1 IDRs within MC1 amino acid sequence. Based on these pre­ overexpressing lines. dictions, MC1 encompasses 2 major IDRs (Fig. 2A), 1 at the N-terminal prodomain and another at a region of the predicted C-terminal p10 catalytic domain known as Results the 360 loop (van Midden et al. 2021). Since aggregation pro­ pensity is also considered an intrinsic determinant of phase MC1 dynamically localizes to cytoplasmic SGs upon separation (Babinchak and Surewicz 2020), we used acute proteotoxic stress AGGRESCAN3D (A3D) to predict the structural aggregation To gain a deeper understanding of MC1 function, we generated propensity of MC1 on top of its AlphaFold predicted struc­ transgenic lines expressing MC1 tagged with a GFP under the ture (http://biocomp.chem.uw.edu.pl/A3D/; Zambrano control of the 35S promoter in the mc1 mutant background et al. 2015). Interestingly, MC1 displays a strong propensity (mc1 Pro35S:MC1-GFP) and evaluated its subcellular localiza­ to aggregate at the predicted IDRs (Fig. 2B). In particular, tion. Under basal conditions, MC1-GFP showed a diffuse pat­ the 360 loop shows the longest stretch of amino acids with tern in both cytoplasm and nucleus (Fig. 1A). Heat high aggregation propensity scores. stress (HS) treatment (39 °C for 40 min; Gutierrez-Beltran The 360 loop of MC1 is a highly hydrophobic sequence et al. 2015) resulted in rapid formation of dynamic cytoplasmic only present in plant Type I metacaspases (van Midden puncta that grew in size over time and disappeared shortly et al. 2021). In fungi, protozoa, and red algae, Type I metacas­ after returning the plants to nonstress conditions (Figs. 1, A pases do not contain the 360 loop and, interestingly, these and B, and S1). The same heat-responsive relocalization pattern proteins are soluble when full length is produced recombi­ was observed when MC1-GFP was expressed under the control nantly in vitro (McLuskey et al. 2012; Wong et al. 2012). of its native promoter (Supplemental Fig. S2, A and B). To In contrast, previous efforts to produce recombinant plant evaluate if such puncta correspond to SGs, we used cyclohex­ Type I metacaspases proved unsuccessful due to the fact imide (CHX), an inhibitor of translational elongation that pre­ that their full-length versions are highly insoluble (van vents SG assembly and forces the disassembly of existing SGs Midden et al. 2021). Removal of the 360 loop and the prodo­ (Weber et al. 2008). Application of CHX blocked the appear­ main was necessary to express soluble MC1 in Escherichia coli ance of the observed heat stress-induced puncta (Fig. 1, C (Fig. 3, A and B), similar to what was previously shown with the and D), indicating that they may indeed correspond to SGs. single Type I metacaspase of the green algae Chlamydomonas To further examine if MC1 colocalized with SGs, we used the reinhardtii CrMCA-I (van Midden et al. 2021). Removal of the plant SG markers translationally controlled tumor protein 360 loop alone was not sufficient to solubilize MC1 (Fig. 3B). (TCTP) and Tudor staphylococcal nuclease 2 (TSN2), which lo­ The soluble MC1 variant devoid of the prodomain and the cate into SGs specifically under HS conditions (Gutierrez- 360 loop carrying an N-terminal hexahistidine (6xHis) tag (re­ Beltran et al. 2021). We observed that transgenic plants stably ferred to as rMC1) was purified to homogeneity by coexpressing MC1-RFP (Pro35S:MC1-RFP) and GFP-TCTP nickel-affinity chromatography (Supplemental Fig. S4A) and (Pro35S:GFP-TCTP) or TSN2-GFP (Pro35S:TSN2-GFP) show further isolated by size-exclusion chromatography removing cytoplasmic colocalization in SGs under heat stress conditions minor impurities (Fig. 3C). Importantly, rMC1 was catalytically (Fig. 1E). Furthermore, MC1 immunoprecipitated with both active as shown by its ability to cleave Arabidopsis SERPIN1, an TCTP and TSN2 in transgenic plants subjected to HS inhibitor and previously reported in planta substrate of MC1 (Fig. 1F). Similarly, MC1 colocalized with other well-known (Lema Asqui et al. 2018) (Supplemental Fig. S5A). rMC1 be­ SG markers RBP47 and TSN2 (Lorković et al. 2000; haved as a canonical Type I metacaspase, showing dependency Gutierrez-Beltran et al. 2021) in protoplasts from transgenic on calcium ions at low millimolar concentrations (1 to 10 mM) MC1-GFP lines transiently expressing RFP-RBP47 or RFP-TSN2 and a neutral pH (pH 7) for maximum cleavage of the fluoro­ (Supplemental Fig. S3, A and B). Together, these data demon­ genic substrate Z-FR-7-amino-4-methylcoumarin (AMC) strate that MC1 dynamically relocalizes to SGs upon heat (Supplemental Fig. S5, B and C, respectively), similar to treatment, disappearing upon stress removal. CrMCA-I (van Midden et al. 2021). In agreement with the ob­ served trypsin-like activity of metacaspases, rMC1 cleaved the The IDRs of MC1 are aggregation prone and confer trypsin substrate β-casein (rβ-casein) (Supplemental Fig. S5D) insolubility in vitro (Lee et al. 2007). We also purified rMC1 carrying a point mu­ SGs are enriched in proteins containing predicted IDRs tation in the catalytic cysteine (C220) to alanine (rMC1CA) (Guillén-Boixet et al. 2020; Gutierrez-Beltran et al. 2021; (Supplemental Fig. S4, B and C). Importantly, rMC1CA was 3328 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. A B C D E F Figure 1. MC1 is recruited to SGs upon heat stress. A) Five-day-old mc1 Arabidopsis seedlings expressing Pro35S:MC1-GFP were heat stressed at 39 °C for 40 min (HS), followed by incubation at 22 °C for up to 120 min (recovery). Images of root tips were taken at indicated time points. Bars = 5 μm. B) Kinetics of the assembly and disassembly of MC1 cytoplasmic foci. Graph shows means ± SD of 3 independent experiments, each including 5 seedlings. Eight to 10 cells for each seedling were analyzed for SG quantification. “Stop HS” corresponds to the time point when plants were trans­ ferred from 39 °C to 22 °C. C) Treatment with CHX inhibits the formation of MC1 foci in root tip cells. For CHX treatment, 5-d-old seedlings ex­ pressing Pro35S:MC1-GFP were incubated with 200 ng/μl CHX for 30 min at 22 °C before HS. Images show localization of MC1-GFP in heat-stressed (39 °C for 40 min) root tip cells of 5-d-old seedlings previously treated with CHX or DMSO (control). D) Quantification of MC1-GFP foci in the experiment shown in C). Upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median and whiskers mark the highest and lowest values. Three independent experiments, each containing at least 5 individual measurements, were per­ formed. Means with different letters are significantly different at P < 0.05 (1-way ANOVA). E) Colocalization of GFP-TCTP or TSN2-GFP with MC1-RFP in heat-stressed (39 °C for 40 min) root tip cells of 5-d-old seedlings expressing both Pro35S:GFP-TCTP or Pro35S:TSN2-GFP together with Pro35S:MC1-RFP. Inset show enlarged boxed areas. Pearson coefficients (rP) of colocalization of GFP-TCTP, TSN2-GFP, and MC1-RFP represent the mean of 5 replicate measurements from 3 independent experiments. Scale bars = 5 μm. F) Immunoprecipitation (IP) of GFP-TCTP or TSN2-GFP and MC1-RFP in protein extracts prepared from leaves of 3-wk-old transgenic Arabidopsis seedlings expressing either Pro35S:GFP-TCTP or Pro35S: TSN2-GFP with Pro35S:MC1-RFP. Samples were kept in control (NS) conditions or heat stressed (HS, 39 °C for 40 min). Input and IP fractions were analyzed by immunoblotting using α-GFP or α-RFP. Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3329 A distinct IDRs that are aggregation prone and confer high in­ solubility for protein overexpression and isolation in vitro. When removed, proteolytically active rMC1 can be expressed and isolated. The IDRs of MC1 regulate its dynamic recruitment into SGs To determine whether the prodomain and 360 loop IDRs of MC1 are important for its translocation into SGs, we gener­ ated transgenic plants stably expressing GFP-tagged trun­ cated versions lacking the prodomain (ΔNMC1) or the 360 loop (MC1Δ360) under the control of the 35S promoter in the mc1 mutant background (mc1 Pro35S:ΔNMC1-GFP and mc1 Pro35S:MC1Δ360-GFP) (Supplemental Fig. S6). We also included transgenic plants carrying a full-length version of MC1 with the catalytic cysteine in position 220 mutated to an alanine (Supplemental Fig. S6), which renders the protease inactive (mc1 ProMC1:MC1CA-GFP) (Supplemental Fig. S5, A B and D) (Coll et al. 2010; Lema Asqui et al. 2018) to determine whether the catalytic activity of MC1 was required for its SG targeting. Under basal conditions, all MC1 versions showed a diffused cytoplasmic localization (Fig. 4A). Except for ΔNMC1, all other MC1 variants also localized in the nucleus (Fig. 4A). As shown above (Fig. 1), heat stress (39 °C for 40 min) resulted in the rapid recruitment of MC1 into cyto­ plasmic SGs. Removal of the prodomain resulted in the for­ mation of significantly larger SGs without altering their number per cell, while removal of the 360 loop resulted in smaller and less abundant SGs (Fig. 4, A to C). The catalytic­ ally inactive version of MC1 displayed wild-type (WT)–like SGs in response to heat stress (Fig. 4, A to C). Together, these data indicate that the 360 loop may be important for the cor­ rect recruitment of MC1 into SGs during stress, while the prodomain may help with the clearance of MC1-containing SGs. To address the association dynamics of MC1 with SGs, we used fluorescence recovery after photobleaching (FRAP) ana­ lysis. MC1-GFP fluorescence partially recovered after photo­ bleaching and mutating the catalytic site of MC1 Figure 2. MC1 contains intrinsically disordered and aggregation-prone regions. A) Prediction of IDRs of MC1. Top, scheme of MC1 protein struc­ (MC1CA-GFP) did not alter its recovery rate (Fig. 4, C and ture. Zf: LSD1-Zinc finger domain within the prodomain (amino acids D). However, mutating the IDRs of MC1 significantly altered 1-77); H164 and C220 correspond to the amino acids of the catalytic the recovery capacity of the protein. On one hand, the few dyad within the large p20 catalytic subunit; 360 loop (amino acids MC1Δ360-GFP-containing SGs showed a higher recovery 318-346): hydrophobic loop within the small p10 catalytic subunit. rate, compared to WT, which suggests that the 360 loop is Middle and bottom, prediction of the disordered regions by D2P2 important for the stable association of MC1 with SGs. (https://d2p2.pro) and DISOPRED3 (http://bioinf.cs.ucl.ac.UK/psipred), re­ (Fig. 4, C and D). In contrast, ΔNMC1-GFP-containing SGs spectively. Precision above the dotted line show disordered scores higher showed slower recovery rate, indicating a potential role of than 0.5 and precision dots below the dotted line show disordered scores this IDR in the dynamic association of MC1 with SGs. lower than 0.5. B) A3D structure of MC1. The prodomain and the 360 Altogether, these results show that MC1 dynamically associ­ loop are highlighted. The amino acid sequences of the prodomain and ates with SGs. The 360 loop of MC1 might mediate its stable 360 loop are shown and the amino acids with high A3D scores (aggrega­ tion-prone) are highlighted. recruitment into SGs while the prodomain might be neces­ sary for its disassembly from SGs. To further address the role of MC1 in SGs, we measured unable to cleave SERPIN1 or rβ-casein (Supplemental Fig. S5, A the dynamics of Rbp47 in WT and MC1-deficient plants. and D). Together, these data show that MC1 contains 2 Five-day-old seedlings of WT and mc1 expressing RFP-Rbp47 3330 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. A B C Figure 3. The prodomain and the 360 loop confer insolubility to MC1. A) Schematic representation of MC1 full length, MC1Δ360 (without 360 loop) and ΔNMC1Δ360 (without prodomain and 360 loop) domain architecture. B) SDS–PAGE Coomassie-stained gels (upper panels) and western blot analysis (lower panels) of lysates from total, soluble, or insoluble fractions of E. coli cells expressing either MC1Δ360 or ΔNMC1Δ360 (rMC1) carrying an N-terminal 6xHis tag. Arrow denotes expected molecular weight of each of the 2 MC1 variants. C) Size-exclusion chromatography of concentrated eluates obtained by nickel-affinity chromatography (Supplemental Fig. S3A). The inlet shows an SDS–PAGE Coomassie-stained gel of fractions 14 to 16 ml of the eluted volume from a Superdex 75 column. were exposed to heat stress (39 °C for 40 min), and the asso­ detect and quantify protein aggregates, in 5-d-old WT and ciation of Rbp47 with SGs was assessed by FRAP. MC1 defi­ mc1 seedlings after heat stress. Seedlings were subjected to ciency resulted in a reduced exchange rate of RFP-Rbp47 90′ of a moderate HS at 37 °C, followed by 90′ of recovery between SGs and the cytoplasm (Supplemental Fig. S7, A at 22 °C and a severe HS at 45 °C for 90′. Samples were col­ and B). This result suggests that MC1 might modulate the as­ lected after 1 d of recovery at 22 °C, using nonstressed seed­ sociation of other SG proteins to SGs. lings as control. We analyzed accumulation of aggregated forms of actin, Hsp90, and proteins containing polyQ MC1 can specifically degrade aggregated proteins stretches, all of them had been shown to aggregate in Sustained stress or certain pathological conditions lead to Arabidopsis after heat stress (Llamas et al. 2022). Under basal the formation of protein associations or aggregates that, in conditions, protein aggregates (actin, Hsp90-tagged, or contrast to SGs, are nonregulated and nondynamic, having polyQ-containing proteins) are barely detectable both in detrimental consequences for the cell, tissue, and even at WT and mc1 mutants, as they can be efficiently cleared by the organismal level (Morimoto 2008). Indeed, SGs may PQC mechanisms (Figs. 5A and S8, A to D). Stresses such have an important role in the pathogenesis of proteotoxicity- as HS result in a sudden overaccumulation of misfolded pro­ derived conditions, although their exact function remains to teins that often surpasses the PQC capacity of the cell and be elucidated (Marcelo et al. 2021). Because MC1 is recruited results in protein aggregation detectable by filter trap to SGs and we previously observed that mc1 knock-out mu­ (Fig. 5A). Plants lacking MC1 accumulated higher quantities tant plants have increased accumulation of aggregated pro­ of aggregated proteins than WT after proteotoxic stress, in­ teins (Coll et al. 2014), we sought to understand whether dicating a reduced capacity to manage protein aggregation its function may be linked to clearance of protein aggregates and proteotoxic stress in the mutant. The overaccumulation under stressful/pathological conditions. of protein aggregates in mc1 mutants did not affect their First, we investigated whether Arabidopsis plants lacking thermotolerance (Supplemental Fig. S8A), possibly owing MC1 show defects in protein aggregate clearance and sur­ to the fact that the experiment was performed on very young vival after proteotoxic stress. To monitor changes in protein plants to avoid age as an additive effect and these may be ex­ aggregation, we used filter trap analysis, a robust method to tremely proficient at dealing with protein aggregation. Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3331 A B C D E Figure 4. The 360 loop and the prodomain of MC1 are involved, respectively, in recruitment to and clearance from SGs. A) Five-day-old mc1 seed­ lings expressing Pro35S:MC1-GFP, Pro35S:MC1C220A-GFP, Pro35S:ΔNMC1-GFP, or Pro35S:MC1Δ360loop-GFP were heat stressed at 39 °C for 40 min (HS), followed by incubation at 22 °C for up to 120 min (recovery). Images of root tips were taken at indicated time points. Bars = 5 μm. B) Quantification of the number of condensates in the experiment shown in A). C) Quantification of the area (µm2) of condensates in the experiment shown in B). D) Selected time frames (prebleach and 0, 3, and 15 s after bleaching) from FRAP analysis of MC1-GFP, MC1C220A-GFP, ΔNMC1-GFP, and MC1Δ360loop-GFP foci formed upon heat stress (40 min at 39 °C) in root tip cells of mc1 seedlings expressing Pro35S:MC1-GFP, Pro35S: MC1C220A-GFP, Pro35S:ΔNMC1-GFP, or Pro35S:MC1Δ360loop-GFP, respectively. Bars = 2 μm. E) Initial signal recovery (%) of the experiment shown in C). In B), C), and E), upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median and whiskers mark the highest and lowest values. In B) and C), 3 independent experiments, each including 3 to 5 seedlings. For each seedling, ∼30 cells from the root meristem were analyzed. In E), 3 independent experiments, each containing at least 8 measurements of different SGs, were performed. Means with different letters are significantly different at P < 0.05 (1-way ANOVA). Second, we assessed the capacity of MC1 to disassemble rMC1 treatment caused a 90% reduction of TTR aggregates pathological protein aggregates in vitro. We coincubated (Fig. 5B). This protein aggregate clearance activity was de­ equimolar concentrations of rMC1 with aggregates of human pendent on MC1 catalytic activity, as evidenced by the ab­ transthyretin (TTR). TTR is a homotetrameric thyroxine sence of disaggregation in the catalytically dead mutant transport protein in which tetramer dissociation events rMC1CA. Indeed, a 3-fold higher turbidity signal was ob­ lead to aggregation (Westermark et al. 1990; Quintas et al. served in the rMC1CA-treated samples suggesting that in­ 2001). Extracellular insoluble deposits of TTR in several hu­ active MC1 becomes aggregated when TTR insoluble man organs give rise to distinct progressive and fatal clinical assemblies are present in the reaction. Visual inspection of syndromes known as TTR amyloidosis (Goren et al. 1980; TTR samples by transmission electron microscopy (TEM) Hou et al. 2007; Rapezzi et al. 2010). Using turbidity measure­ (Fig. 5C) confirmed the disaggregation activity of MC1. The ments to monitor protein aggregation, we observed that need of catalytic activity for disaggregation is consistent 3332 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. A B C D E Figure 5. MC1 can specifically clear protein aggregates in vitro and in vivo and the lack of MC1 results in protein aggregate accumulation. A) Filter trap analysis of protein extracts from 5-d-old Arabidopsis mc1 or WT seedlings in control conditions (NS) or subjected to a severe heat stress (HS, 90 min at 37 °C, 90 min at 22 °C and 90 min at 45 °C). SDS-resistant aggregates were detected using antibodies against actin, HSP90-1 or polyQ proteins. rHS/NS represents the ratio between HS and NS protein levels. Signal intensity of the bands was quantified using ImageJ. Two independent experiments were performed with similar results. B) Turbidity assays of end-point disaggregation reactions using light scattering at 360 nm. rMC1 and the catalytic inactive form rMC1C220A (rMC1CA) were coincubated with TTR aggregates or native tetrameric TTR for 24 h at 37 °C. rMC1 proteins and TTR aggregates, incubated for the same period, were also measured as controls. Data represent 3 individual measurements. Upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median and whiskers mark the highest and lowest values. Means with different letters are significantly different at P < 0.05 (1-way ANOVA). C) Electron microscopy images of end-point disaggregation reactions of TTR aggregates incubated with or without purified rMC1 for 24 h at 37 °C. D) SDS–PAGE analysis of the end- point samples shown in panel C). E) Filter trap analysis showing mRFP-Q74 aggregation levels in HEK293 cells. HEK293 cells were transfected with mRFP-Q74 and GFP-MC1 or mRFP-Q74 and GFP as a control. mCherry antibody was used to detect Q74 SDS-resistant aggregates. with the observation that aggregated TTR becomes signifi­ of the protease by its rapid autoprocessing in the presence of cantly degraded in presence of rMC1 (Lane 3), as demon­ calcium (Fig. 5D, Lane 10), as previously described (Zhu et al. strated by SDS–PAGE (Fig. 5D). Notably, rMC1 acts as a 2020). specific disaggregase, clearing protein aggregates but not Finally, we tested the capacity of MC1 to degrade protein the functional form of proteins, since it is unable to degrade aggregates in vivo. To this end, we used 2 well-established soluble TTR in its native tetrameric state (nTTR) (Fig. 5D, model systems: (i) human embryonic kidney (HEK) cells ex­ Lane 3 and 4, respectively). The obtained data indicate that pressing a polyQ-expanded Huntingtin form (Q74) that causes rMC1 targets and disassembles specifically the aggregated aggregation and proteotoxicity used as a proxy for the neuro­ and pathogenic form of TTR. Notably, this disaggregase activ­ degenerative Huntington’s disease (Jimenez-Sanchez et al. ity toward TTR aggregates was not observed in samples incu­ 2015) and (ii) yeast expressing a constitutively misfolded car­ bated with MC4 (Fig. 5, B and D). We confirmed the activity boxypeptidase (ΔssCPY∗) that forms insoluble protein Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3333 aggregates upon stress (Park et al. 2007). Coexpression of full- formation of cytoplasmic condensates containing the SG length MC1 with Q74 fused to mCherry in HEK cells resulted marker TSN2 and MC1 (Supplemental Fig. S11). This may in­ in a reduction of protein aggregates in comparison to expres­ dicate that SGs may also form during developmental processes sion of Q74 fused to mCherry alone, as demonstrated by filter leading to proteotoxicity such as senescence, although further trap analysis using anti-mCherry antibody (Figs. 5E and S6E). research will be needed to determine the nature and dynamics In yeast, we expressed ΔssCPY∗ fused to the prototrophic of these compartments. marker Leu2 and a C-terminal myc tag (ΔssCL∗) in WT, a mu­ tant lacking the single metacaspase gene MCA1 in yeast (ymca1Δ) and ymca1Δ complemented with a WT copy of Discussion the Arabidopsis MC1. All strains grew normally on control SGs are membraneless organelles formed by LLPS under media (Supplemental Fig. S6A, left panel), while on selective stress conditions and act both as storage compartments media, lacking leucine WT yeast had reduced growth capacity and microreactors where signaling takes place (Alberti and due to degradation of misfolded ΔssCL∗ by PQC systems Carra 2018). Their functionality inside healthy cells is linked (Supplemental Figs. S7A, right panel, and S7B). In contrast to their ability to assemble and disassemble dynamically in and as previously shown, ymca1Δ was not able to degrade response to changing environments (i.e. assembling upon ΔssCL∗ and therefore could grow normally on a leucine- stress perception and disassembling when the causative selective media (Hill et al. 2014). This phenotype could be fully stress subdues). SGs contain a high proportion of proteins complemented by AtMC1 that due to its ability to degrade, bearing IDRs/LCRs, which, together with RNA, drive forma­ ΔssCL∗ restored yeast WT growth levels (Supplemental Figs. tion of the condensate but at the same time are extremely S7A, right panel, and S7B). Altogether, these data demonstrate misfolding/aggregation prone. Therefore, they are closely sur­ that MC1 can degrade protein aggregates in vitro and in vivo veilled by the PQC machinery. Reduced proteostatic capacity and a lack of MC1 leads to abnormal protein aggregate accu­ derived from aging and mutations affecting the phase separ­ mulation under proteotoxic stress. ation behavior of these proteins lead to chronic activation of integrated stress responses. This will eventually surpass the MC1 delays senescence PQC capacity of the cell, resulting in the accumulation of Based on all the evidence presented above demonstrating misfolded and aggregated proteins and leading to the forma­ the recruitment of MC1 into SGs and its aggregate clearing tion of persistent SGs, which are linked to disease (Wolozin function, we hypothesized that overproduction of the pro­ 2012; Wang et al. 2022). tein in plants may minimize the effects of proteotoxic stress Sessile organisms such as plants cannot flee from extreme occurring during plant aging and contribute to fitness. and prolonged stress situations, such as heat or drought. Previously, we reported that the lack of MC1 led to early sen­ Therefore, they must be equipped with extremely efficient escence in Arabidopsis (Coll et al. 2014). Here, we confirmed PQC mechanisms to deal with massive protein misfolding these results using a dark-induced senescence assay and and aggregation. In fact, proteins containing aggregation- tested the effects of stably overexpressing WT full-length prone polyQ proteins are enriched in plants, but no polyQ MC1 and its mutant variants (MC1, MC1CA, ΔNMC1, and pathologies have been reported, in contrast to animals MC1Δ360) in a mc1 mutant background. Individual leaves (Llamas et al. 2022). In this context, it has been recently shown from 3-wk-old plants were covered with aluminum foil, that the chloroplasts could act as important protein degrad­ and 8 d later, they were uncovered to evaluate senescence ation machines to maintain proteostasis of polyQ-containing visually and by means of chlorophyll quantification and proteins (Llamas et al. 2022). Plants may also respond to pro­ photosynthetic efficiency. In uncovered leaves (basal condi­ teotoxic stress by actively regulating protein solubility and tions), all lines showed similar total chlorophyll levels and phase behavior, similar to yeast that can tolerate high levels photosynthetic efficiency (Fig. 6, B and D). Leaf senescence of insoluble proteins and form solid-like condensates resulted in a drop in chlorophyll levels and photosynthetic (Franzmann and Alberti 2019). Insoluble proteins within these activity in WT plants. In contrast, plants overexpressing condensates, including SGs, may have evolved to become MC1 displayed a clear delay in senescence (Fig. 6A), accom­ stress sensors serving an adaptive function (Franzmann and panied by higher chlorophyll levels (Fig. 6C) and higher Alberti 2019). photosynthetic activity than WT (Fig. 6E). Importantly, the protease catalytic activity of MC1, the pro­ MC1 as a SG component domain, and the 360 loop were all required for the observed In this work, we focus on the characterization of MC1, an antiaging phenotype, since mc1 plants stably expressing Arabidopsis Type I metacaspase. MC1 was previously shown MC1CA, ΔNMC1 and MC1Δ360 behaved like WT (Fig. 6, A to participate in immunogenic cell death and aging, although to E). Reverse transcription quantitative PCR (RT-qPCR) ana­ its mode of action remained obscure (Coll et al. 2010, 2014). lysis using the senescence marker gene STAY GREEN1 (SGR1; Aging caused remobilization of MC1 from the soluble frac­ Sakuraba et al. 2012) confirmed the delayed senescence tion to insoluble protein aggregates. Further, absence of phenotype in plants overexpressing MC1 (Supplemental Fig. MC1 caused overaccumulation of insoluble protein aggre­ S10). Importantly, dark-induced senescence resulted in the gates in aging cells, potentially leading to the observed 3334 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. A B C D E Figure 6. Overexpression of MC1 delays dark-induced senescence. A) Representative leaf images of Arabidopsis WT, mc1 mutants and Pro35S: MC1-GFP mc1, ProMC1:MC1C220A-GFP mc1, Pro35S:ΔNMC1-GFP mc1, and Pro35S:MC1Δ360loop-GFP mc1 grown for 3 wk under controlled growth conditions (16-h light/8-h dark photoperiod) and covered for 8 d to induce senescence. For each plant, only Leaves 5 and 6 were either dark ac­ climated or used as controls. Bars = 0.5 cm. B) and C) Total chlorophyll concentration (µg/mg FW) of uncovered (control, B) or covered (senes­ cence, C) leaves of 3-wk-old Arabidopsis WT, mc1 mutants, and Pro35S:MC1-GFP mc1, ProMC1:MC1C220A-GFP mc1, Pro35S:ΔNMC1-GFP mc1, and Pro35S:MC1Δ360loop-GFP mc1. Means with different letters are significantly different at P < 0.05 (1-way ANOVA). D) and E) PSII maximum effi­ ciency (Fv/Fm) quantifications capacity of uncovered (control, D) or covered (senescence, E) leaves of 3-wk-old Arabidopsis WT, mc1 mutants, and Pro35S:MC1-GFP mc1, ProMC1:MC1C220A-GFP mc1, Pro35S:ΔNMC1-GFP mc1, and Pro35S:MC1Δ360loop-GFP mc1. Means with different letters are significantly different at P < 0.05 (Kruskal–Wallis test). In B to D) and E), upper and lower box boundaries represent the first and third quantiles, respectively; horizontal lines mark the median and whiskers mark the highest and lowest values. Four independent experiments, each containing at least 10 leaves for each phenotype, were performed. accelerated senescence phenotype in mc1 mutant plants. stress on young seedlings, which lack persistent or aberrant Aging, with its overall loss of proteostatic capacity, unveils condensates that accumulate as a result of aging mutant phenotypes linked to PQC failure that remain hidden (Gutierrez-Beltran et al. 2015; Kosmacz et al. 2018, 2019). in young cells due to their very efficient control of protein We showed that upon heat stress, MC1 relocalizes into misfolding/aggregation. Thus, to investigate the role of distinct cytoplasmic puncta that disappear during the MC1 in acute proteotoxic stress in cells with full proteostatic recovery phase after stress removal (Figs. 1, A and B, and S2). capacity, we used a simple, well-characterized system, heat These puncta correspond to SGs based on their dynamics Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3335 (Figs. 1, A and B, and 4C), sensibility to CHX (Fig. 1, C and D), 2014). Here, we demonstrate that in plants, MC1 is recruited and colocalization/coimmunoprecipitation with SG markers to SGs upon heat stress, but what is its function and how is in planta (Figs. 1, E and F, and S3) (Kosmacz et al. 2019; it connected to aggregate clearance? First, we showed that Gutierrez-Beltran et al. 2021). How much of the total MC1 mutants lacking MC1 accumulated higher levels of aggregate- protein pool localizes to SGs remains to be established. prone aggregated proteins, such as polyQ-containing, HSP90, Phase separation often drives the formation of SGs (Alberti or actin, than WT plants under basal condition, a phenotype and Carra 2018; Maruri-López et al. 2021; Allen and Strader exacerbated after applying heat stress (Fig. 5A). Second, 2022). The main features of proteins that form membraneless MC1 exhibited a strong and evolutionarily conserved capacity compartments such as SGs through LLPS are (i) presence of to degrade protein aggregates, as shown in various well- IDRs, (ii) complex domain organization, and (iii) their mar­ established systems in vitro (human protein) and in vivo (yeast ginal solubility in the cell (Alberti and Carra 2018). MC1 is and human cells) (Sant’Anna et al. 2016; Koyuncu et al. 2018; predicted to encompass 2 main IDR regions in its amino Llamas et al. 2022) (Figs. 5, S8, and S9). In particular, rMC1 acid sequence, 1 coinciding with the N-terminal prodomain showed an extraordinary capacity to clear aggregated TTR, a and the second near the C-terminus, known as the 360 loop pathological form of the protein that causes a diversity of life- (van Midden et al. 2021) (Fig. 2A). Further, these 2 regions are threatening pathologies (Saelices et al. 2015). MC1 proteolysis predicted to be highly insoluble and aggregation prone, in of TTR was prodomain and 360 loop independent but de­ particular the 360 loop (Fig. 2B). In agreement with this pre­ pendent on its catalytic activity. In fact, mutation of the cata­ diction, MC1 became soluble only when both the prodomain lytic cysteine in MC1 resulted in increased insolubility and and the 360 loop were simultaneously removed (Fig. 3). self-aggregation of the protein, indicating a marked change Interestingly, the highly hydrophobic 360 loop is only present in its biophysical properties. Still, it remains to be defined in plant Type I metacaspases (van Midden et al. 2021). Type I whether MC1-containing condensates are the physiological metacaspases from protozoa and fungi do not possess this sites of protein aggregate clearance and what is the exact domain and, accordingly, they are soluble in vitro and they MC1 mode of action therein. can be readily purified without removal of any domain MC1 proteolytic activity was specifically directed toward (Lee et al. 2010; McLuskey et al. 2012). aggregated forms of the protein, since monomeric TTR was In this regard, our data show that the 360 loop mediates not processed by MC1 (Fig. 5D). The protein aggregate- MC1 recruitment into SGs during heat stress, as demon­ targeted behavior of MC1 could potentially be due to its strated by the drastic reduction in MC1-containing SGs slow kinetics. MC1 is an active protease, as shown by its abil­ formed in the 360 loopless mutant (Fig. 4). In contrast, in mu­ ity to self-cleave and cleave its inhibitor serpin in vivo (Lema tants lacking the prodomain, SGs still formed upon heat Asqui et al. 2018) and in vitro (Supplemental Fig. S4A). Here, stress. However, these granules are less dynamic, showing re­ we show that in addition to that, rMC1 can cleave duced recovery after photobleaching (Fig. 4). This may indi­ metacaspase-specific synthetic substrates in vitro in the pres­ cate that the prodomain is involved in SG clearance. ence of calcium and neutral pH (Supplemental Fig. S5, B Considering that the prodomain has been proposed to act and C). However, compared to Arabidopsis Type II metacas­ as a negative regulator of Type I metacaspase activity based pases, MC1 displays slower and/or less efficient protease activ­ on structural, as well as genetic data (Lee et al. 2010; ity toward typical metacaspase substrates (Vercammen et al. McLuskey et al. 2012), it is tempting to speculate that 2004; Hander et al. 2019; Zhu et al. 2020). We hypothesize the prodomain contains certain amino acids or motifs that precisely this slower kinetics may favor its disaggregase ac­ that may mediate recognition and degradation of MC1 by tivity, rather than a quicker protein-processing activity that granulostasis, which has been shown to involve chaperone- could cleave monomeric forms of TTR. Notwithstanding, mediated PQC, autophagy, or the ubiquitin–proteasome this newly discovered function of MC1 may inspire further re­ system (Alberti and Carra 2018). Interestingly, maize (Zea search on protein disaggregases as an avenue for therapeutic mays) MC1 has been shown to form puncta and colocalize intervention in age-related protein-misfolding diseases. with autophagosomes after heat stress when transiently ex­ pressed in Nicotiana benthamiana (Luan et al. 2021), indicat­ Antiaging role of MC1: physiological implications of ing that autophagy may be a possible degradation route for the role of MC1 in protein aggregate clearance MC1-containing SGs as shown for other aggregation-prone In previous work, we showed that plants lacking MC1 dis­ proteins (Munch et al. 2014; Jung et al. 2020). played accelerated senescence, which has been confirmed here using a different senescence-inducing system (Fig. 6A; MC1 can specifically clear protein aggregates Coll et al. 2014). Beyond that, here we present data demon­ From previous work in yeast (Lee et al. 2010; Hill et al. 2014) strating that overexpression of MC1 delays leaf senescence and plants (Coll et al. 2014), it was unclear how MC1 contri­ (Fig. 6A). The onset of senescence triggers the formation of butes to aggregate clearance. Work with the yeast metacas­ cytoplasmic MC1-containing puncta that could also corres­ pase yMCA1 showed that upon heat stress and aging, the pond to SGs, as they also contain TSN2 (Supplemental Fig. protein relocalizes to PQC condensates known as juxtanuclear S11). Considering that in our system, we use dark as a trigger quality control compartment (JUNQ) and IPOD (Hill et al. for senescence, 1 may speculate that the observed 3336 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. condensates are processing bodies (PBs) rather than SGs, since sterile dH2O. Sterile seeds were sown in solid ½ MS medium PBs have been shown to act as mRNA reservoirs under dark with vitamins and stratified 48 h at 4 °C. Plants were grown conditions (Jang et al. 2019). In contrast to SGs, PBs are consti­ vertically under long day (LD) conditions (16-h light/8-h tutively present in the cell, but they have been shown to in­ dark) at 22 °C. For dark-induced senescence studies, 1-wk-old crease in number and size during stress conditions and seedlings were transferred into soil and grown for an add­ exchange proteins and RNAs with SGs (Chicois et al. 2018; itional 2 wk under LD conditions. Gutierrez-Beltran et al. 2021; Solis-Miranda et al. 2023). The following transgenic lines in mc1 background were used A recent metaproteomic analysis of stress-induced conden­ for SG visualization and dark-induced senescence studies: sates showing distinct protein signatures for SGs and PBs Pro35S:MC1-GFP, Pro35S:MC1C220A-GFP, Pro35S:ΔNMC1-GFP, has revealed that MC1 is only present in SGs across various Pro35S:MC1Δ360loop-GFP, ProMC1:MC1-GFP, and ProMC1: stress conditions (Solis-Miranda et al. 2023). Still, it will be in­ MC1C220A-GFP (Supplemental Data Set 1). Transgenic Col-0 teresting to determine the composition of MC1-containing lines expressing Pro35S:GFP-TCTP (Gutierrez-Beltran condensates under natural and dark-induced senescence et al. 2021) and Pro35S:MC1-RFP or ProTSN2:TSN2-GFP to define the degree of resemblance to stress-induced (Gutierrez-Beltran et al. 2021) and Pro35S:MC1-RFP were condensates. used for SG colocalization experiments. Additionally, Col-0 Mutation of the conserved MC1 catalytic cysteine or re­ WT and mc1 mutant plants expressing 35S:RFP-Rbp47 were moval of the 360 loop abolishes the observed senescence de­ used for FRAP analysis. lay caused by overexpression of the protein. This indicates that MC1 proteolytic activity as well as its recruitment into Plasmid construction SGs may be involved in this antiaging function of the protein. All constructs and primers used in this study are described in This prolife function of MC1 is evolutionarily conserved, as Supplemental Data Sets S2 and S3, respectively. To generate overexpression of yeast MC1 can also extend replicative life­ Pro35S:MC1-GFP, Pro35S:MC1-RFP, Pro35S:ΔNMC1-GFP, span, a function partly dependent on the presence of an in­ Pro35S:MC1Δ360loop-GFP, ProMC1:MC1-GFP, and ProMC1: tact catalytic cysteine and attributed to a role in protein MC1C220A-GFP constructs, the coding sequence and native aggregate management as part of PQC (Hill et al. 2014). promoter (∼1 kb) of Arabidopsis MC1 (AT1G02170) were An interesting question is why altering the levels of MC1 in amplified from Col-0 cDNA and genomic DNA, respectively. knock-out mutants did not result in an obvious phenotypic Plasmids were assembled through GreenGate cloning effect compared to WT in response to heat (Supplemental (Lampropoulos et al. 2013). To generate Pro35S:RFP-Rbp47 Fig. S8), such as the 1 observed during senescence. A plausible and Pro35S:RFP-TSN2, the coding sequences of Rbp47 explanation coming from the animal field is that young indi­ (AT3G19130) and TSN2 (AT5G61780) were amplified from viduals/tissues/cells have multiple and very active misfolded Col-0 cDNA and cloned into pGWB655 following the protein clearance mechanisms, which can efficiently manage Gateway strategy (Karimi Inzé and Depicker 2002). proteotoxicity ensued from stress situations, such as heat For purification of rMC1, the MC1 coding sequences lack­ stress, even in the presence of mutations affecting PQC. ing the 360 loop and with or without the prodomain In contrast, old individuals/tissues/cells experience a global (MC1Δ360 or ΔNMC1Δ360/rMC1) (Fig. 2) were synthesized decrease in proteostasis, uncovering the effect of mutations (Twist Bioscience) with codon optimization for expression in affecting PQC, which is the causative ground of many E. coli. Synthetic sequences contained NdeI and XhoI restric­ age-associated protein-misfolding diseases that appear late tion sites at the 5´ and 3´ ends of the sequence, respectively. in life (Alberti and Hyman 2021). Both synthetic genes and destination vector pET28 b(+) were All considered, a plausible hypothesis is the following cut with NdeI and XhoI and subsequently ligated so that an (Fig. 7): (i) proteotoxic stress, such as heat stress, triggers N-terminal 6xHis tag precedes the start site of the MC1 var­ the formation of SGs; (ii) MC1 is recruited to SGs via the iants. QuickChange Site-Directed Mutagenesis (Agilent 360 loop; and (iii) once there, MC1 participates in protein Technologies) was used to cause point mutations in the cata­ clearance via its proteolytic activity to help dissolving the lytic site of rMC1. granules. In favor of this hypothesis, removal of the catalytic To complement the yeast metacaspase mutant strain cysteine of MC1 does not affect recruitment into SGs, but it ymca1Δ with Arabidopsis MC1 (AT1G02170), we con­ alters aggregate clearance. structed a gene replacement cassette by PCR-directed hom­ ologous recombination (Gardner and Jaspersen 2014). The cassette consisted of 3 fragments: (i) yeast 5′ flanked Materials and methods with N-acetyltransferase (NAT) resistance gene and a Materials used and growth conditions glyceraldehyde-3-phosphate dehydrogenase (GPD) constitu­ All experiments were performed using A. thaliana Col-0 eco­ tive promoter, (ii) Arabidopsis MC1 gene fused to a type. Lines used for this work are listed in Supplemental Data C-terminal HA tag, and (iii) 3′ flanking of the yeast yMCA1 Set 1. The single mutant mc1 has been previously described gene. These 3 fragments were PCR amplified and fused into (GK-096A10; Coll et al. 2010). All seeds were surface sterilized the final recombinant DNA by using the double-joint PCR with 35% NaClO for 5 min and washed 5 times for 5 min with method as previously described (Yu et al. 2004). The resulting Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3337 A B Figure 7. Working model on the role of MC1 in SGs. A) Upper panel (WT plants): Under basal conditions, no SGs are detectable and MC1 presents a diffuse nucleo-cytoplasmic localization pattern. Upon perception of an acute, reversible stress, MC1 is recruited to SGs where it hypothetically clears misfolded/aggregated proteins. Under chronic or irreversible stress, the proteostatic capacity of the cell is surpassed and toxic protein aggregates that cannot be cleared start accumulating in the cytoplasm. Lower panel (mc1 mutant plants): Plants devoid of MC1 cannot cope as WT with pro­ teotoxic stress. Any stress may result in accumulation of protein aggregates that over time manifest as the observed accelerated senescence pheno­ type. B) MC1 is recruited to SGs via its 360 loop. Once in SGs, it clears aggregated proteins via its disaggregase activity. Release of MC1 from SGs is dependent on the prodomain. Nuclear schemes in A) and B) were obtained from Bioicons/Servier/license CC BY 3.0. C) MC1 is recruited to SGs via its 360 loop. Once in SGs, it clears aggregated proteins via its disaggregase activity. Release of MC1 from SGs is dependent on the prodomain. DNA product (NATNT2:pGPD:AtMC1-HA) was transformed plants were collected and digested in an enzyme solution (1% into the ymca1Δ mutant strain KanMX4. cellulose R10, 0.25% macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 0.1% BSA, and 20 mM MES at pH 5.7) for 1 Protoplasts and plant transformation or 2 h. Protoplasts were collected through a 70-micron Arabidopsis protoplasts were obtained as previously de­ strainer, washed twice with ice-cold W5 solution (154 mM scribed (Truskina et al. 2020). In short, leaves from 3-wk-old NaCl, 125 mM CaCl2, 5 mM KCl, 5 mM glucose, and 2 mM 3338 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. MES at pH 5.7), and incubated on ice for 30 min. The proto­ For detection of fluorescent signals, GFP was excited at plasts were then resuspended in mannitol-magnesium solu­ 488 nm and mRFP at 543 nm. tion (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES at pH Root meristem cells from 5-d-old seedlings vertically 5.7) at a final concentration of 2.5 × 105 cells/ml. To trans­ grown under LD conditions (16-h light/8-h dark) at 22 °C form the protoplasts, 30 μg of the appropriate plasmid were used to determine protein subcellular localization. For were mixed with 200 μl of protoplast solution. heat stress treatment, 5-d-old seedlings were transferred to Immediately, 210 μl of PEG solution (40% PEG 4000, 0.2 M a hot air incubator at 39 °C and incubated for 40 min. For mannitol, and 0.1 M CaCl2) was added and the protoplasts CHX treatment, 5-d-old seedlings were incubated in ½ MS li­ were incubated for 5 min at room temperature and then quid medium with 200 ng/µl CHX for 30 min. A number of washed twice in W5 solution. The protoplasts were resus­ granules were quantified with ImageJ using the SiCE spot de­ pended in 500 μl of the W5 solution and incubated for tector macro for automatic quantification (Bayle et al. 2017). 24 h in 16-h light/8-h dark growth chamber. Before imaging, the protoplasts were incubated at 39 °C for 40 min in a hot FRAP air incubator. The assay was performed as described previously (Moschou Arabidopsis plants were transformed as described previ­ et al. 2013). Five-day-old seedlings grown vertically in LD con­ ously through the Agrobacterium tumefaciens–mediated ditions were incubated for 40 min at 39 °C in a hot air incu­ floral-dip method (Clough and Bent 1998). bator. During analyses, the Olympus FV1000 software was set up for the acquisition of 2 prebleach images, 1 bleach scan, Yeast strains and spot dilution assays and 30 postbleach scans. A region 2 µm in diameter was Yeast media preparation and molecular biology techniques bleached using a laser intensity of 100% at 488 nm. were carried out using standard methods (Lázaro-Silva Prebleach and postbleach scans were at the minimum pos­ et al. 2015). All experiments were done using the genetic sible laser power. A zoom factor of 5 was used. background of Saccharomyces cerevisiae strain BY4741 Analyses of fluorescence intensities during FRAP were per­ (Supplemental Data Set 4). To test the capacity of each yeast formed in the bleached regions. One region of interest out­ strain to remove misfolded proteins, yeast cells were trans­ side of the bleached area was also measured to serve as the formed with the plasmid pFE15 encoding the fusion con­ background. The background values were subtracted from struct ΔssCL∗myc (Supplemental Data Set 2; Eisele and the fluorescence recovery values, and the resulting values Wolf 2008). Growth phenotypes were assessed with spot di­ were normalized by the first postbleach time point. Initial sig­ lution assays. Ten-fold serial dilutions were made, ranging nal recovery (%) = 100 × (Ifinal, postbleach − Iinitial, post­ from undiluted to a 104 dilution. Five microliters of each di­ bleach)/(Iprebleach − Iinitial, postbleach), where I is the lution were spotted onto the corresponding selective media normalized signal intensity (relative to the background (-ura or -ura -leu plates), and plates were incubated for at intensity). least 3 d before images were taken. Protein purification HEK293T cells transfection and protein extraction E. coli OverExpress C41 (DE3) Chemically Competent Cells The MC1 (AT1G02170) gene was codon optimized for expres­ from BioCat GmbH (Heidelberg, Germany) or E. coli BL21 sion in animal cells and synthetized (Twist Bioscience). To strain containing the pBB542 vector (de Marco et al. 2007) generate ProCMV:GFP-MC1, the synthetic gene was cloned were transformed with expression plasmids and grown in ei­ in the pDEST-CMV-N-GFP vector by Gateway technology ther autoinduction media or LB, respectively. Cells were grown (Addgene). ProCMV:mRFP-Q74 (Balaji et al. 2022), ProCMV: first at 37 °C with continuous shaking until OD600 reached 0.6 GFP-MC1, and ProCMV:GFP (Llamas et al. 2022) were used and then transferred to 25 °C for overnight growth. In the case for transfection of HEK cells (CRL-1573) (Supplemental of expression in E. coli Chaperone Competent Cells BL21, iso­ Data Set 5) following the protocol described in (Llamas propyl β-D-1-thiogalactopyranoside (IPTG) at 1 mM concen­ et al. 2022). After 72 h of incubation, cells were lysed in non­ tration was added to 400-ml cell cultures when transferred denaturing native lysis (300 mM NaCl, 100 mM HEPES pH 7.4, to 25 °C to induce protein expression. The pellet from over­ 2 mM EDTA, and 2% Triton X-100) supplemented with 1× night cultures was resuspended in 20 mM HEPES, pH 7.5, plant protease inhibitor (Merck), scraped from the tissue cul­ and 500 mM NaCl and sonicated on ice. A centrifugation of ly­ ture plates, and homogenized through a syringe needle sates at 25,000 × g for 20 min was performed to remove cell (27G). Samples were centrifuged at 10,000 × g for 10 min at debris and insoluble proteins. Soluble lysate was filtered 4 °C and supernatant was collected. Protein concentration through 0.45 µM sterile filters and loaded into a 5-ml nickel was determined with the Pierce BCA Protein Assay Kit ion HisTrap purification column (Cytiva, Marlborough, (Thermo Fisher). MA, USA). Washes of the columns were performed with 20 mM HEPES, pH 7.5, 500 mM NaCl, and 20 mM imidazole. Microscopy analysis Elution of proteins was performed by increasing imidazole Microscopy images were acquired with an Olympus FV1000 concentrations up to 250 mM. The cleanest elutions were con­ inverted confocal microscope with a 60×/water objective. centrated using Amicon filters and loaded onto a Superdex 75 Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3339 size-exclusion chromatography column (GE Healthcare Life NaCl, 0.5% Nonidet P-40, 10% glycerol, 1 mM EDTA pH 8, Sciences, Chicago, IL, USA) connected to an AKTA FPLC sys­ 5 mM DTT, 1% PVPP, and 1× Protease Inhibitor Cocktail tem. The Superdex 75 column was equilibrated in 20 mM [Sigma, P599]) and centrifuged for 10 min at 14,000 × g at HEPES, pH 7.5, and 500 mM NaCl. A flow rate of 0.75 ml per 4 °C; 5× Laemmli sample buffer was added to 100-µl super­ minute was used to separate proteins. Samples belonging to natant and boiled for 5 min. Equal amounts of supernatant the most prominent peaks were kept and loaded onto an were loaded on 12% SDS–PAGE gels. Antibodies used for im­ SDS–PAGE gel to verify the purity of the samples. MC4 was munoblotting were as follows: α-GFP-HRP (1:5,000 Miltenyi provided by F. van Breusegem and it is described in Biotec), α-RFP-HRP (1:5,000 Abcam), α-myc (1:10,000, Vercammen et al. (2004). Sigma-Aldrich), α-actin (dilution 1:5,000, Agrisera), α-Hsp90-1 (1:2,000 Abcam), and α-polyQ (1:1,000 Merck). Enzymatic activity assays Protease activity was measured by quantification of the fluores­ Filter trap assay cence intensity released from the AMC group of the fluorogenic Protein extracts were obtained with native lysis buffer substrate Z-FR-AMC (PeptaNova, Sandhausen, Germany) (300 mM NaCl, 100 mM HEPES pH 7.4, 2 mM EDTA, and 2% at 383- and 455-nm excitation and emission wavelengths, Triton X-100) supplemented with EDTA-free protease inhibi­ respectively, in a Tecan Infinite M200 Microplate Reader tor cocktail. When processing plant protein extracts, 1× System (Männedorf, Switzerland). All proteolytic assays were plant protease inhibitor (Merck) was added to native lysis performed in 20 mM HEPES (pH 7.0) containing 150 mM buffer. In experiments with HEK cells, cells were homoge­ NaCl, varying CaCl2 concentrations and 5 mM DTT. For estima­ nized by passing 7 times through syringe needle (27 G). tion of pH optima, buffers containing 100 mM acetate (pH 4 Cellular debris was removed by several centrifugation steps to 5.5), 100 mM MES (pH 6 to 6.5), 100 mM HEPES (pH 7.0 at 8,000 × g for 10 min at 4 °C. Supernatant was recollected to 8.0), 100 mM Tris (pH 8.5 to 9), and 100 mM CAPS (pH 9.5 and protein concentration determined with the Pierce BCA to 11) were used. 0.2 μg of recombinant protease was used Protein Assay Kit (Thermo Fisher). A cellulose acetate mem­ and the concentration of fluorogenic substrates was 5 μM. brane filter (GE Healthcare Life Sciences) was placed in a slot blot apparatus (Bio-Rad) coupled to a vacuum system. The Preparation of TTR aggregates membrane was equilibrated with 3 washes with equilibration TTR was expressed and purified following previously de­ buffer (native buffer supplemented with 0.5% SDS). scribed procedures (Pinheiro et al. 2021). Briefly, TTR aggre­ Approximately 150 µg of protein extract was supplemented gation was induced by mixing 7 μM purified TTR with an with SDS at a final concentration of 0.5% and loaded and fil­ equal volume of 400 mM sodium acetate, 200 mM KCl, and tered through the membrane. Then, the membrane was pH 4.4, obtaining a final TTR concentration of 3.5 μM. washed 3 times with 0.2% SDS. The membrane was blocked Samples were incubated for 72 h at 37 °C in quiescent condi­ in 3% BSA in TBST for 30 min followed by 3 washes with tions. Aggregated samples were centrifuged at 20,000 × g for TBST. The membrane was incubated with indicated antibody 1 h to recover the insoluble material that was subsequently and then washed 3 times for 5 min and incubated with sec­ resuspended in 20 mM HEPES, 150 mM NaCl, and pH 7.5 to ondary antibodies in TBST 3% BSA for 30 min. The mem­ a concentration of 100 μM. brane was developed using an Odyssey DLx (Licor). Extracts were also analyzed by SDS–PAGE and western blot­ In vitro disaggregation assay ting to determine loading controls. End-point disaggregation reactions were performed by coin­ cubating TTR aggregates at a concentration of 7 μM with 0.25 mg/ml of protease at 37 °C in presence of 5 mM DTT Heat treatments and 5 mM CaCl2. Protease disaggregation was monitored Thermotolerance assays were performed using 5-d-old using sample turbidity, SDS–PAGE, and TEM. seedlings. Seedlings were grown at 22 °C for 5 d, put in a hot-air incubator set at 37 °C for 90 min, put in a growth Turbidity assay chamber set at 37 °C for 90 min, incubated at 45 °C for Sample turbidity was monitored as an indicator of the amount 90 min, and allowed to recover at 22 °C for 8 d. The percen­ of aggregated material using synchronous light scattering. The tages of seedlings in different phenotypic classes were calcu­ spectra were recorded in a JASCO Spectrofluorometer lated based on results from 3 biological replicates. In each FP-8200 with an excitation wavelength of 360 nm and emis­ biological replicate, at least 50 seedlings were used for sion range from 340 to 380 nm. Excitation and emission band­ each genotype. width were set to 5 nm. The light scattered at 360 nm was used as a measure of turbidity. Dark-induced senescence assay Leaves 5 and 6 of 3-wk-old plants grown in LD conditions Protein extraction and immunoblotting were covered with aluminum foil for 8 d (Li et al. 2016). Five hundred milligrams of leaf material were mixed with Control plants kept without covered leaves were grown in 2 ml of extraction buffer (50 mM HEPES pH 7.3, 150 mM parallel. 3340 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. Chlorophyll analysis Accession numbers Covered and uncovered leaves from 3 different plants were Sequence data for the genes described in this study can be snap frozen in liquid nitrogen and ground with TissueLyser II found in the TAIR database (https://www.arabidopsis.org) (QIAGEN). A 50-mg aliquot of crushed leaf material was mixed and NCBI under the following accession numbers: MC1 with 1.5 ml of 80% prechilled acetone and thoroughly mixed (AT1G02170), RBP47 (AT3G19130), TCTP (AT3G16640), and for 5 min. Samples were centrifuged at 20,000 × g for 1 min, yMCA1 (Q08601). and the supernatant was transferred to spectrophotometer cuvettes. Chlorophyll was then quantified at 663 and 646 nm with a spectrophotometer (UV-2600, Shimadzu) as Acknowledgments previously described (Lichtenthaler and Wellburn 1983). Thanks to F. van Breusegem for providing MC4 recombinant protein and I. Rubio-Somoza for providing us with plasmids Pulse amplitude modulation (PAM) fluorometric pGGD-RBCSt (D-F) and pGGD-UBQt (D-F) and the members measurements of the Microscopy Services of the UAB for their assistance. After 30 min of dark adaptation, the kinetics of chlorophyll fluorescence in whole rosettes were monitored by measuring F0 in the dark and Fm with initial saturation pulse using Author Contributions Imaging PAM M-series, MAXI version device (Walz). Fv/Fm N.R.-S. and N.S.C. conceived the study; N.R.-S., J.S.-L., L.A., and Fv′/Fm′ (PSII efficiency) ratio for the maximum quan­ and N.S.C. designed experiments; N.R.-S., J.S.-L., L.A., I.P., tum efficiency upon dark and light conditions was calculated J.S., K.P.v.M., U.J.P., S.K., J.B.-B., L.L., C.P., F.E., A.M.B.-E., E.L., according to the manufacturer’s instructions. and S.M.H. performed the experiments; E.G.-B. provided es­ sential material; N.R.-S., J.S.-L., L.A., I.P., J.S., and K.P.v.M., ana­ RT-qPCR analysis lyzed the data; T.N., M.V., D.V., M.K., S.V., and N.S.C. The Maxwell RSC Plant RNA kit (Promega) was used to isolate supervised the work; and N.R.-S. and N.S.C. wrote the manu­ RNA from covered and uncovered leaves from 3 different script with comments from J.S.-L., L.A., I. P., K.P.v.M., U.J.P., plants. Two micrograms of RNA were reverse transcribed into S.K., F.E., E.G.-B., M.V., E.L., D.V., and S.V. All authors re­ cDNA with the High-Capacity cDNA Reverse Transcription viewed the manuscript. Kit with RNase inhibitor (Applied Biosystems). RT-qPCRs were performed with LightCycler SYBRgreen I master (Roche) in a LightCycler 480 System (Roche). SGR1 (AT4G22920) expres­ Supplemental data sion was normalized to expression of ACT8 (AT1G49240), and The following materials are available in the online version of data were analyzed using the ΔΔCT method. Primers for this article. RT-qPCR used in this study were previously described and are Supplemental Figure S1. The size of MC1-containing SGs listed in Supplemental Data Set 3 (Ueda et al. 2020). changes over time. Supplemental Figure S2. MC1-GFP expressed under the Bioinformatic analyses control of its own promoter relocalizes to cytoplasmic con­ IDRs of MC1 were predicted using the D2P2 database (D2P2, densates upon heat stress. http://d2p2.pro/; Oates et al. 2013) and DISOPRED3 (http:// Supplemental Figure S3. MC1 colocalizes with SG mar­ bioinf.cs.ucl.uk/psipred; Jones and Cozzetto 2015). LLPS pre­ kers in protoplasts upon heat stress. disposition was evaluated using the PSPredictor tool (http:// Supplemental Figure S4. Production and purification of www.pkumdl.cn:8000/PSPredictor/; Chu et al. 2022). Analysis rMC1 in E. coli cells. of the aggregation propensity of MC1 amino acids was per­ Supplemental Figure S5. rMC1 is a proteolytically active formed with A3D (http://biocomp.chem.uw.edu.pl/A3D/; enzyme and behaves as a canonical Type I metacaspase. Zambrano et al. 2015) using as input file the AlphaFold2 Supplemental Figure S6. Immunoblot analysis of trans­ (https://alphafold.ebi.ac.uk/; Jumper et al. 2021) predicted genic plants expressing the different MC1 variants. MC1 structure. Supplemental Figure S7. MC1 mediates the association of Rbp47b with SGs. Statistical analysis Supplemental Figure S8. mc1 knock-out mutants do not All quantification analyses and statistical tests were per­ display thermotolerance or total protein accumulation dif­ formed with R software. T-test was used to compare the sig­ ferences compared to WT. nificance of differences between 2 experimental groups. For Supplemental Figure S9S. MC1 participates in the clear­ comparing the significance of differences between multiple ance of terminally misfolded proteins. experimental groups, 1-way ANOVA or Kruskal–Wallis Supplemental Figure S10. Relative expression of the sen­ 1-way analysis were performed as indicated in each experi­ escence marker SGR1 (AT4G22920). ment. Different letters show statistically significant differ­ Supplemental Figure S11. Dark-induced senescence re­ ences between samples. Statistical analysis results are listed sults in the formation of MC1-containing cytoplasmic in Supplemental Data Set 6. condensates. Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3341 Supplemental Data Set 1. Arabidopsis lines used in this Alberti S, Gladfelter A, Mittag T. Considerations and challenges in study. studying liquid–liquid phase separation and biomolecular conden­ sates. Cell 2019:176(3):419–434. https://doi.org/10.1016/j.cell.2018. Supplemental Data Set 2. List of plasmids used in this 12.035 study. Alberti S, Hyman AA. Biomolecular condensates at the nexus of cellu­ Supplemental Data Set 3. List of primers and synthetic se­ lar stress, protein aggregation disease and ageing. Nat Rev Mol Cell quences used in this study for genotyping and cloning. Biol. 2021:22(3):196–213. https://doi.org/10.1038/s41580-020- Supplemental Data Set 4. Yeast strains used in this study. 00326-6 Allen JR, Strader LC. Beating the heat: phase separation in plant stress Supplemental Data Set 5. Other materials used in this granules. Dev Cell. 2022:57(5):563–565. https://doi.org/10.1016/J. study. DEVCEL.2022.02.012 Supplemental Data Set 6. Table for statistical analysis. Babinchak WM, Surewicz WK. Liquid–liquid phase separation and its mechanistic role in pathological protein aggregation. J Mol Biol. 2020:432(7):1910–1925. https://doi.org/10.1016/J.JMB.2020.03.004 Funding Balaji V, Müller L, Lorenz R, Kevei É, Zhang WH, Santiago U, Gebauer J, Llamas E, Vilchez D, Camacho CJ, et al. A dimer–mono­ Research at CRAG was supported by grants mer switch controls CHIP-dependent substrate ubiquitylation and PID2019-108595RB-I00 funded by MCIN/AEI/10.13039/ processing. Mol Cell. 2022:82(17):3239–3254.e11. https://doi.org/10. 501100011033 and AGL2016-78002-R funded by MCIN/AEI/ 1016/J.MOLCEL.2022.08.003 10.13039/501100011033 and by “ERDF A way of making Baradaran-Heravi Y, Van Broeckhoven C, van der Zee J. Stress gran­ Europe” (to N.S.C. and M.V.); fellowship BES-2017-080210 ule mediated protein aggregation and underlying gene defects in the FTD-ALS spectrum. Neurobiol Dis. 2020:134:104639. https://doi.org/ funded by MCIN/AEI/10.13039/501100011033 and by “ESF 10.1016/J.NBD.2019.104639 Investing in your future” (to J.S.-L.); FPU19/03778 funded by Bayle V, Platre M, Jaillais Y. Automatic quantification of the number MU (o Ministerio de Universidades) (to N.R.-S.); by of intracellular compartments in Arabidopsis thaliana root cells. Bio the “Severo Ochoa Programme for Centres of Excellence Protoc. 2017:7(4):e2145. https://doi.org/10.21769/BIOPROTOC.2145 in R&D” (SEV-2015-0533 and CEX2019-000902-S funded Buchan JR, Parker R. Eukaryotic stress granules: the ins and outs of translation. Mol Cell. 2009:36(6):932–941. https://doi.org/10.1016/J. by MCIN/AEI/10.13039/501100011033); by the grant MOLCEL.2009.11.020 PID2020-119737GA-I00 funded by the Ministerio de Ciencia e Chicois C, Scheer H, Garcia S, Zuber H, Mutterer J, Chicher J, Innovacion (MCIN/AEI/10.13039/501100011033) (to E.G.-B); Hammann P, Gagliardi D, Garcia D. The UPF1 interactome reveals by the Spanish Ministry of Science and Innovation (MICINN) interaction networks between RNA degradation and translation re­ grant PID2019-105017RB-I00 (to S.V.), by ICREA, pression factors in Arabidopsis. Plant J. 2018:96(1):119–132. https:// doi.org/10.1111/TPJ.14022 ICREA-Academia 2020 (to S.V); by the MICINN fellowship Chu X, Sun T, Li Q, Xu Y, Zhang Z, Lai L, Pei J. Prediction of liquid– (FPU17/01157) (to J.S.); and by the CERCA Programme/ liquid phase separating proteins using machine learning. BMC Generalitat de Catalunya. L.A. is supported by a Maria Bioinformatics 2022:23(1):72. https://doi.org/10.1186/S12859-022- Zambrano postdoctoral fellowship by de Ministerio de 04599-W/FIGURES/5 Universidades and the European Union—NextGenerationEU. Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 1998:16(6): This work was also funded by the Deutsche 735–743. https://doi.org/10.1046/J.1365-313X.1998.00343.X Forschungsgemeinschaft (DFG) Germany’s Excellence Strategy Coll NS, Smidler A, Puigvert M, Popa C, Valls M, Dangl JL. The plant (CECAD, EXC 2030-390661388 to D.V. and CEPAS EXC-2048/ metacaspase AtMC1 in pathogen-triggered programmed cell death 1–390686111 to E.L.) and the Else Kröner-Fresenius-Stiftung and aging: functional linkage with autophagy. Cell Death Differ. (2021-EKSE.95) (to D.V.). We also acknowledge support from 2014:21(9):1399. https://doi.org/10.1038/CDD.2014.50 Coll NS, Vercammen D, Smidler A, Clover C, Van Breusegem F, COST Actions PROTEOSTASIS (BM1307) and ProteoCure Dangl JL, Epple P. Arabidopsis type I metacaspases control cell death. (CA20113), supported by COST (European Cooperation in Science 2010:330(6009):1393–1397. https://doi.org/10.1126/SCIENCE. Science and Technology) (N.R.-S., U.P., L.L., and N.S.C). We ac­ 1194980/SUPPL_FILE/COLL.SOM.PDF knowledge support of the publication fee by the CSIC Open de Marco A, Deuerling E, Mogk A, Tomoyasu T, Bukau B. Access Publication Support Initiative through its Unit of Chaperone-based procedure to increase yields of soluble recombin­ ant proteins produced in E. coli. BMC Biotechnol. 2007:7(1):32. Information Resources for Research (URICI). https://doi.org/10.1186/1472-6750-7-32 Conflict of interest statement. None declared. Eisele F, Wolf DH. Degradation of misfolded protein in the cytoplasm is mediated by the ubiquitin ligase Ubr1. FEBS Lett. 2008:582(30): 4143–4146. https://doi.org/10.1016/J.FEBSLET.2008.11.015 Franzmann TM, Alberti S. Protein phase separation as a stress survival Data availability strategy. Cold Spring Harb Perspect Biol. 2019:11(6):a034058. https:// The data that support the findings of this study are available doi.org/10.1101/CSHPERSPECT.A034058 from the corresponding author upon reasonable request. Gardner JM, Jaspersen SL. Manipulating the yeast genome: deletion, mutation, and tagging by PCR. Methods Mol Biol. 2014:1205: 45–78. https://doi.org/10.1007/978-1-4939-1363-3_5 References González-García MP, Vilarrasa-Blasi J, Zhiponova M, Divol F, Mora-García S, Russinova E, Caño-Delgado AI. Brassinosteroids Alberti S, Carra S. Quality control of membraneless organelles. J Mol control meristem size by promoting cell cycle progression in Biol. 2018:430(23):4711–4729. https://doi.org/10.1016/j.jmb.2018.05. Arabidopsis roots. Development 2011:138(5):849–859. https://doi. 013 org/10.1242/dev.057331 3342 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. Goren H, Steinberg MC, Farboody GH. Familial oculoleptomeningeal et al. Protein and metabolite composition of Arabidopsis stress gran­ amyloidosis. Brain: J Neurol. 1980:103(3):473–495. https://doi.org/10. ules. New Phytol. 2019:222(3):1420–1433. https://doi.org/10.1111/ 1093/BRAIN/103.3.473 NPH.15690 Guillén-Boixet J, Kopach A, Holehouse AS, Wittmann S, Jahnel M, Kosmacz M, Luzarowski M, Kerber O, Leniak E, Gutiérrez-Beltrán E, Schlüßler R, Kim K, Trussina IREA, Wang J, Mateju D, et al. Moreno JC, Gorka M, Szlachetko J, Veyel D, Graf A, et al. RNA-induced conformational switching and clustering of G3BP drive Interaction of 2′,3′-cAMP with Rbp47b plays a role in stress granule stress granule assembly by condensation. Cell 2020:181(2):346– formation. Plant Physiol. 2018:177(1):411–421. https://doi.org/10. 361.e17. https://doi.org/10.1016/J.CELL.2020.03.049/ATTACHMENT 1104/pp.18.00285 /4A66A2D6-41BF-46CF-BE60-045CAC185392/MMC2.ZIP Koyuncu S, Saez I, Lee HJ, Gutierrez-Garcia R, Pokrzywa W, Fatima Gutierrez-Beltran E, Elander PH, Dalman K, Dayhoff GW 2nd, A, Hoppe T, Vilchez D. The ubiquitin ligase UBR5 suppresses pro­ Moschou PN, Uversky VN, Crespo JL, Bozhkov PV. Tudor staphylo­ teostasis collapse in pluripotent stem cells from Huntington’s disease coccal nuclease is a docking platform for stress granule components patients. Nat Commun. 2018:9(1):2886. https://doi.org/10.1038/ and is essential for SnRK1 activation in Arabidopsis. EMBO J. S41467-018-05320-3 2021:40(17):e105043. https://doi.org/10.15252/embj.2020105043 Lampropoulos A, Sutikovic Z, Wenzl C, Maegele I, Lohmann JU, Gutierrez-Beltran E, Moschou PN, Smertenko AP, Bozhkov PV. Forner J. Greengate—a novel, versatile, and efficient cloning system Tudor staphylococcal nuclease links formation of stress granules for plant transgenesis. PLoS One 2013:8(12):83043. https://doi.org/10. and processing bodies with mRNA catabolism in Arabidopsis. 1371/JOURNAL.PONE.0083043 Plant Cell 2015:27(3):926–943. https://doi.org/10.1105/TPC.114. Lázaro-Silva DN, De Mattos JC, Castro HC, Alves GG, Amorim LM. 134494 The use of DNA extraction for molecular biology and biotechnology Hander T, Fernández-Fernández ÁD, Kumpf RP, Willems P, training: a practical and alternative approach. Creat Educ. 2015:6(08): Schatowitz H, Rombaut D, Staes A, Nolf J, Pottie R, Yao P, et al. 762–772. https://doi.org/10.4236/CE.2015.68079 ‘Damage on plants activates Ca2+-dependent metacaspases for re­ Lee RE, Brunette S, Puente LG, Megeney LA. Metacaspase Yca1 is re­ lease of immunomodulatory peptides’. Science 2019:363(6433): quired for clearance of insoluble protein aggregates. Proc Natl Acad eaar7486. https://doi.org/10.1126/SCIENCE.AAR7486 Sci U S A. 2010:107(30):13348–13353. https://doi.org/10.1073/PNAS. Hill SM, Hao X, Liu B, Nyström T. Life-span extension by a metacas­ 1006610107/SUPPL_FILE/PNAS.201006610SI.PDF pase in the yeast Saccharomyces cerevisiae. Science 2014:344(6190): Lee N, Gannavaram S, Selvapandiyan A, Debrabant A. 1389–1392. https://doi.org/10.1126/science.1252634 Characterization of metacaspases with trypsin-like activity and their Hipp MS, Kasturi P, Hartl FU. The proteostasis network and its decline putative role in programmed cell death in the protozoan parasite in ageing. Nat Rev Mol Cell Biol. 2019:20(7):421–435. https://doi.org/ leishmania. Eukaryot Cell. 2007:6(10):1745. https://doi.org/10.1128/ 10.1038/S41580-019-0101-Y EC.00123-07 Hou X, Aguilar MI, Small DH. Transthyretin and familial amyloidotic Lema Asqui S, Vercammen D, Serrano I, Valls M, Rivas S, Van polyneuropathy. FEBS J. 2007:274(7):1637–1650. https://doi.org/10. Breusegem F, Conlon FL, Dangl JL, Coll NS. AtSERPIN1 is an inhibi­ 1111/J.1742-4658.2007.05712.X tor of the metacaspase AtMC1-mediated cell death and autocatalyt­ Jain S, Wheeler JR, Walters RW, Agrawal A, Barsic A, Parker R. ic processing in planta. New Phytol. 2018:218(3):1156–1166. https:// ATPase-modulated stress granules contain a diverse proteome and doi.org/10.1111/NPH.14446 substructure. Cell 2016:164(3):487–498. https://doi.org/10.1016/J. Li L, Kubiszewski-Jakubiak S, Radomiljac J, Wang Y, Law SR, Keech CELL.2015.12.038 O, Narsai R, Berkowitz O, Duncan O, Murcha MW, et al. Jang GJ, Yang JY, Hsieh HL, Wu SH. Processing bodies control the select­ Characterization of a novel β-barrel protein (AtOM47) from the ive translation for optimal development of Arabidopsis young seed­ mitochondrial outer membrane of Arabidopsis thaliana. J Exp Bot. lings. Proc Natl Acad Sci U S A. 2019:116(13):6451–6456. https: 2016:67(21):6061–6075. https://doi.org/10.1093/JXB/ERW366 //doi.org/10.1073/PNAS.1900084116/SUPPL_FILE/PNAS.1900084116.­ Lichtenthaler HK, Wellburn AR. Determinations of total carotenoids SD01.XLSX and chlorophylls a and b of leaf extracts in different solvents. Jimenez-Sanchez M, Lam W, Hannus M, Sönnichsen B, Imarisio S, Biochem Soc Trans. 1983:11(5):591–592. https://doi.org/10.1042/ Fleming A, Tarditi A, Menzies F, Dami TE, Xu C, et al. siRNA screen BST0110591 identifies QPCT as a druggable target for Huntington’s disease. Llamas E, Koyuncu S, Lee HJ, Gutierrez-Garcia R, Dunken N, Nat Chem Biol. 2015:11(5):347–354. https://doi.org/10.1038/ Charura N, Torres-Montilla S, Schlimgen E, Pulido P, NCHEMBIO.1790 Rodriguez-Concepcion M, et al. Chloroplast protein import deter­ Jones DT, Cozzetto D. DISOPRED3: precise disordered region predic­ mines plant proteostasis and retrograde signaling. bioRxiv, 484971. tions with annotated protein-binding activity. Bioinformatics https://www.biorxiv.org/content/10.1101/2022.03.19.484971v1% 2015:31(6):857. https://doi.org/10.1093/BIOINFORMATICS/BTU744 0Ahttps://www.biorxiv.org/content/10.1101/2022.03.19.484971v1. Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, abstract, 20 March 2022, preprint: not peer reviewed. Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, et al. Highly Lorković ZJ, Wieczorek Kirk DA, Klahre U, Hemmings-Mieszczak M, accurate protein structure prediction with AlphaFold. Nature Filipowicz W. RBP45 and RBP47, two oligouridylate-specific 2021:596(7873):583–589. https://doi.org/10.1038/s41586-021- hnRNP-like proteins interacting with poly(A)+ RNA in nuclei of 03819-2 plant cells. RNA 2000:6(11):1610–1624. https://doi.org/10.1017/ Jung H, Lee HN, Marshall RS, Lomax AW, Yoon MJ, Kim J, Kim JH, S1355838200001163 Vierstra RD, Chung T. Arabidopsis cargo receptor NBR1 mediates Luan QL, Zhu YX, Ma S, Sun Y, Liu XY, Liu M, Balint-Kurti PJ, Wang selective autophagy of defective proteins. J Exp Bot. 2020:71(1): GF. Maize metacaspases modulate the defense response mediated by 73–89. https://doi.org/10.1093/JXB/ERZ404 the NLR protein Rp1-D21 likely by affecting its subcellular localiza­ Karimi M, Inzé D, Depicker A. GATEWAY vectors for tion. Plant J. 2021:105(1):151–166. https://doi.org/10.1111/tpj.15047 Agrobacterium-mediated plant transformation. Trends Plant Sci. Marcelo A, Koppenol R, de Almeida LP, Matos CA, Nóbrega C. Stress 2002:7(5):193–195. https://doi.org/10.1016/S1360-1385(02)02251-3 granules, RNA-binding proteins and polyglutamine diseases: too Klemenčič M, Funk C. Evolution and structural diversity of metacas­ much aggregation? Cell Death Dis. 2021:12(6):592. https://doi.org/ pases. J Exp Bot. 2019:70(7):2039–2047. https://doi.org/10.1093/ 10.1038/s41419-021-03873-8 JXB/ERZ082 Markmiller S, Soltanieh S, Server KL, Mak R, Jin W, Fang MY, Luo Kosmacz M, Gorka M, Schmidt S, Luzarowski M, Moreno JC, EC, Krach F, Yang D, Sen A, et al. Context-dependent and disease- Szlachetko J, Leniak E, Sokolowska EM, Sofroni K, Schnittger A, specific diversity in protein interactions within stress granules. Cell Proteostatic role of the metacaspase MC1 THE PLANT CELL 2023: 35; 3325–3344 | 3343 2018:172(3):590–604.e13. https://doi.org/10.1016/J.CELL.2017.12. amyloidoses and the heart: a clinical overview. Nat Rev Cardiol. 032 2010:7(7):398–408. https://doi.org/10.1038/NRCARDIO.2010.67 Marondedze C, Thomas L, Lilley KS, Gehring C. Drought stress causes Roberts M, Tang S, Stallmann A, Dangl JL, Bonardi V. Genetic re­ specific changes to the spliceosome and stress granule components. quirements for signaling from an autoactive plant NB-LRR intracellu­ Front Mol Biosci. 2020:6:163. https://doi.org/10.3389/FMOLB.2019. lar innate immune receptor. PLoS Genet. 2013:9(4):e1003465. https:// 00163/BIBTEX doi.org/10.1371/JOURNAL.PGEN.1003465 Maruri-López I, Figueroa NE, Hernández-Sánchez IE, Chodasiewicz Saelices L, Johnson LM, Liang WY, Sawaya MR, Cascio D, Ruchala P, M. Plant stress granules: trends and beyond. Front Plant Sci. Whitelegge J, Jiang L, Riek R, Eisenberg DS. Uncovering the mech­ 2021:12:722643. https://doi.org/10.3389/fpls.2021.722643 anism of aggregation of human transthyretin. J Biol Chem. McLuskey K, Rudolf J, Proto WR, Isaacs NW, Coombs GH, Moss CX, 2015:290(48):28932. https://doi.org/10.1074/JBC.M115.659912 Mottram JC. Crystal structure of a Trypanosoma brucei metacaspase. Sakuraba Y, Schelbert S, Park SY, Han SH, Lee BD, Andrès CB, Proc Natl Acad Sci U S A. 2012:109(19):7469–7474. https://doi.org/ Kessler F, Hörtensteiner S, Paek NC. STAY-GREEN and chlorophyll 10.1073/PNAS.1200885109 catabolic enzymes interact at light-harvesting complex II for chloro­ Minina EA, Staal J, Alvarez VE, Berges JA, Berman-Frank I, Beyaert R, phyll detoxification during leaf senescence in Arabidopsis. Plant cell Bidle KD, Bornancin F, Casanova M, Cazzulo JJ, et al. Classification 2012:24(2):507–518. https://doi.org/10.1105/TPC.111.089474 and nomenclature of metacaspases and paracaspases: no more con­ Sanchez II, Nguyen TB, England WE, Lim RG, Vu AQ, Miramontes R, fusion with caspases. Mol Cell. 2020:77(5):927–929. https://doi.org/ Byrne LM, Markmiller S, Lau AL, Orellana I, et al. Huntington’s dis­ 10.1016/J.MOLCEL.2019.12.020 ease mice and human brain tissue exhibit increased G3BP1 granules Morimoto RI. Proteotoxic stress and inducible chaperone networks in and TDP43 mislocalization. J Clin Invest. 2021:131(12):e140723. neurodegenerative disease and aging. Genes Dev. 2008:22(11): https://doi.org/10.1172/JCI140723 1427–1438. https://doi.org/10.1101/GAD.1657108 Sant’Anna R, Gallego P, Robinson LZ, Pereira-Henriques A, Ferreira Moschou PN, Smertenko AP, Minina EA, Fukada K, Savenkov EI, N, Pinheiro F, Esperante S, Pallares I, Huertas O, Almeida MR, Robert S, Hussey PJ, Bozhkov PV. The caspase-related protease se­ et al. Repositioning tolcapone as a potent inhibitor of transthyretin parase (EXTRA SPINDLE POLES) regulates cell polarity and cytokin­ amyloidogenesis and associated cellular toxicity. Nat Commun. esis in Arabidopsis. Plant Cell 2013:25(6):2171. https://doi.org/10. 2016:7(1):10787. https://doi.org/10.1038/NCOMMS10787 1105/TPC.113.113043 Schmit JD, Feric M, Dundr M. How hierarchical interactions make Munch D, Rodriguez E, Bressendorff S, Park OK, Hofius D, Petersen membraneless organelles tick like clockwork. Trends Biochem Sci. M. Autophagy deficiency leads to accumulation of ubiquitinated 2021:46(7):525–534. https://doi.org/10.1016/j.tibs.2020.12.011 proteins, ER stress, and cell death in Arabidopsis. Autophagy Solis-Miranda J, Chodasiewicz M, Skirycz A, Fernie AR, Moschou PN, 2014:10(9):1579–1587. https://doi.org/10.4161/AUTO.29406 Bozhkov PV, Gutierrez-Beltran E. Stress-related biomolecular con­ Niewidok B, Igaev M, Pereira da Graca A, Strassner A, Lenzen C, densates in plants. Plant Cell 2023:35(9):3187–3204. https://doi.org/ Richter CP, Piehler J, Kurre R, Brandt R. Single-molecule imaging 10.1093/PLCELL/KOAD127 reveals dynamic biphasic partition of RNA-binding proteins in stress Sorenson R, Bailey-Serres J. Selective mRNA sequestration by granules. J Cell Biol. 2018:217(4):1303–1318. https://doi.org/10.1083/ OLIGOURIDYLATEBINDING PROTEIN 1 contributes to translational JCB.201709007 control during hypoxia in Arabidopsis. Proc Natl Acad Sci U S A. Oates ME, Romero P, Ishida T, Ghalwash M, Mizianty MJ, Xue B, 2014:111(6):2373–2378. https://doi.org/10.1073/PNAS.1314851111/ Dosztányi Z, Uversky VN, Obradovic Z, Kurgan L, et al. D2p2: SUPPL_FILE/SM04.MP4 database of disordered protein predictions. Nucleic Acids Truskina J, Han J, Chrysanthou E, Galvan-Ampudia CS, Lainé S, Resarch 2013:41(D1):D508–D516. https://doi.org/10.1093/NAR/ Brunoud G, Macé J, Bellows S, Legrand J, Bågman AM, et al. A net­ GKS1226 work of transcriptional repressors modulates auxin responses. Nature Park SH, Bolender N, Eisele F, Kostova Z, Takeuchi J, Coffino P, Wolf 2020:589(7840):116–119. https://doi.org/10.1038/s41586-020-2940-2 DH. The cytoplasmic Hsp70 chaperone machinery subjects mis­ Ueda H, Ito T, Inoue R, Masuda Y, Nagashima Y, Kozuka T, Kusaba folded and endoplasmic reticulum import-incompetent proteins to M. Genetic interaction among phytochrome, ethylene and abscisic degradation via the ubiquitin–proteasome system. Mol Biol Cell. acid signaling during dark-induced senescence in Arabidopsis thali­ 2007:18(1):153–165. https://doi.org/10.1091/MBC.E06-04-0338/ ana. Front Plant Sci. 2020:11:564. https://doi.org/10.3389/FPLS.2020. ASSET/IMAGES/LARGE/ZMK0010779040010.JPEG 00564 Pinheiro F, Varejão N, Esperante S, Santos J, Velázquez-Campoy A, Uren AG, O’Rourke K, Aravind LA, Pisabarro MT, Seshagiri S, Reverter D, Pallarès I, Ventura S. Tolcapone, a potent aggregation Koonin EV, Dixit VM. Identification of paracaspases and metacas­ inhibitor for the treatment of familial leptomeningeal amyloidosis. pases two ancient families of caspase-like proteins, one of which plays FEBS J. 2021:288(1):310–324. https://doi.org/10.1111/FEBS.15339 a key role in MALT lymphoma. Mol Cell. 2000:6(4):961–967. https:// Pitsili E, Rodriguez-Trevino R, Ruiz-Solani N, Demir F, Kastanaki E, doi.org/10.1016/s1097-2765(00)00094-0 Dambire C, de Pedro R, Vercammen D, Salguero-Linares J, Hall van Midden KP, Peric T, Klemenčič M. Plant type I metacaspases are H, et al. A phloem-localized Arabidopsis metacaspase (AtMC3) im­ proteolytically active proteases despite their hydrophobic nature. proves drought tolerance. New Phytologist. https://doi.org/10. FEBS Lett. 2021:595(17):2237–2247. https://doi.org/10.1002/1873- 1111/nph.19022, 15 June 2023, preprint: not peer reviewed. 3468.14165 Posey AE, Holehouse AS, Pappu RV. Phase separation of intrinsically Vercammen D, van de Cotte B, De Jaeger G, Eeckhout D, Casteels P, disordered proteins. Methods Enzymol. 2018:611:1–30. https://doi. Vandepoele K, Vandenberghe I, Van Beeumen J, Inzé D, Van org/10.1016/BS.MIE.2018.09.035 Breusegem F. Type II metacaspases Atmc4 and Atmc9 of Protter DSW, Parker R. Principles and properties of stress granules. Arabidopsis thaliana cleave substrates after arginine and lysine. J Trends Cell Biol. 2016:26(9):668. https://doi.org/10.1016/J.TCB.2016. Biol Chem. 2004:279(44):45329–45336. https://doi.org/10.1074/JBC. 05.004 M406329200 Quintas A, Vaz DC, Cardoso I, Saraiva MJ, Brito RM. Tetramer dissoci­ Vilchez D, Saez I, Dillin A. The role of protein clearance mechanisms in ation and monomer partial unfolding precedes protofibril formation organismal ageing and age-related diseases. Nat Commun. 2014:5(1): in amyloidogenic transthyretin variants. J Biol Chem. 2001:276(29): 1–13. https://doi.org/10.1038/ncomms6659 27207–27213. https://doi.org/10.1074/JBC.M101024200 Wang J, Gan Y, Cao J, Dong X, Ouyang W. Pathophysiology of stress Rapezzi C, Quarta CC, Riva L, Longhi S, Gallelli I, Lorenzini M, granules: an emerging link to diseases (review). Int J Mol Med. Ciliberti P, Biagini E, Salvi F, Branzi A. Transthyretin-related 2022:49(4):44. https://doi.org/10.3892/IJMM.2022.5099 3344 | THE PLANT CELL 2023: 35; 3325–3344 Ruiz-Solaní et al. Wang S, Xue M, He C, Shen D, Jiang C, Zhao H, Niu D. AtMC1 associ­ mRNA levels in Arabidopsis. J Exp Bot. 2014:65(20):5933–5944. ates with LSM4 to regulate plant immunity through modulating https://doi.org/10.1093/JXB/ERU334 pre-mRNA splicing. Mol Plant-Microbe Interact. 2021:34(12): Youn JY, Dyakov BJA, Zhang J, Knight JDR, Vernon RM, Forman-Kay 1423–1432. https://doi.org/10.1094/MPMI-07-21-0197-R JD, Gingras AC. Properties of stress granule and P-body proteomes. Weber C, Nover L, Fauth M. Plant stress granules and mRNA process­ Mol Cell. 2019:76(2):286–294. https://doi.org/10.1016/J.MOLCEL. ing bodies are distinct from heat stress granules. Plant J. 2008:56(4): 2019.09.014 517–530. https://doi.org/10.1111/J.1365-313X.2008.03623.X Yu JH, Hamari Z, Han KH, Seo JA, Reyes-Domínguez Y, Westermark P, Sletten K, Johansson B, Cornwell GG 3rd. Fibril in se­ Scazzocchio C. Double-joint PCR: a PCR-based molecular tool nile systemic amyloidosis is derived from normal transthyretin. Proc for gene manipulations in filamentous fungi. Fungal Genet Natl Acad Sci U S A. 1990:87(7):2843–2845. https://doi.org/10.1073/ Biol. 2004:41(11):973–981. https://doi.org/10.1016/J.FGB.2004.08. PNAS.87.7.2843 001 Wolozin B. Regulated protein aggregation: stress granules and neuro­ Zambrano R, Jamroz M, Szczasiuk A, Pujols J, Kmiecik S, Ventura S. degeneration. Mol Neurodegener. 2012:7(1):56. https://doi.org/10. AGGRESCAN3D (A3D): server for prediction of aggregation proper­ 1186/1750-1326-7-56 ties of protein structures. Nucleic Acids Res. 2015:43(W1): Wong AHH, Yan C, Shi Y. Crystal structure of the yeast metacaspase W306–W313. https://doi.org/10.1093/NAR/GKV359 Yca1. J Biol Chem. 2012:287(35):29251. https://doi.org/10.1074/JBC. Zhu P, Yu XH, Wang C, Zhang Q, Liu W, McSweeney S, Shanklin J, M112.381806 Lam E, Liu Q. Structural basis for Ca2+-dependent activation of a Yan C, Yan Z, Wang Y, Yan X, Han Y. Tudor-SN, a component of stress plant metacaspase. Nat Commun. 2020:11(1):2249. https://doi.org/ granules, regulates growth under salt stress by modulating GA20ox3 10.1038/S41467-020-15830-8