Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature

Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature Ive De Smet, Shao-Li Yang, Xiangyu Xu, Hongyan Liu, Anton Gorkovskiy, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7157930/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Protein evolution is shaped by sequence variation that modulates protein properties, e.g., through gain or loss of post-translational modifications. Among these, reversible phosphorylation alters a protein’s overall electrical charge and enables organisms to dynamically respond to environmental fluctuations. In plants, the hydro-active opening of stomata, microscopic pores that regulate gas exchange and leaf temperature, is governed by phosphorylation-dependent signalling. Here, we identify a mechanism involving the deubiquitylase UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24) that promotes stomatal opening in Arabidopsis thaliana under heat. UBP24 is phosphorylated at serine 360 by the kinase OPEN STOMATA 1 (OST1), which is activated by B4 RAF kinases in response to heat stress. This phosphorylation stabilizes UBP24, enabling deubiquitylation of a plasma membrane H⁺-ATPase to promote stomatal opening. This reveals a novel heat-responsive signalling pathway that evolved in vascular plants to regulate stomatal function. Strikingly, a similar evolutionary feature exists in Saccharomyces cerevisiae, where the UBP24 homolog Ubp3 requires a constitutively negatively charged residue at the homologous site to support growth after heat shock. Our findings uncover a conserved molecular mechanism in which negative charge, via phosphorylation or acidic residues, modulates deubiquitylase function, supporting adaptive thermal responses across eukaryotes and highlighting how charge-based regulation promotes cellular resilience under stress. Biological sciences/Plant sciences/Plant stress responses/Heat Biological sciences/Plant sciences/Plant signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Main Protein evolution relies on variation within protein sequences, impacting activity, interactions and stability through – for example – gaining or losing regulatory post-translational modifications 1 . Rapid and reversible protein phosphorylation enables organisms to respond efficiently to fluctuating environments and enables phenotypic plasticity 2 . Homologous proteins display a high conversion probability from negatively charged (aspartic and glutamic acid) to phosphorylatable residues (serine and threonine) to generate tuneable electrostatic interactions 3 . The successful colonisation of the green lineage, also known as Viridiplantae, to terrestrial environments required several adaptations, including the evolution of microscopic pores on the body surface, called stomata 4 . The tight regulation of gas and water vapor exchange to control photosynthesis and water loss through stomata, enabled plants to grow and survive under climatically changing environments 4,5 . Liverworts and some mosses have static pores 6,7 , while in vascular plants, stomatal dynamics are achieved by the action of a pair of guard cells that allow or restrict the aperture. Stomatal closure is triggered by, for example, drought and involves ABA-activated Sucrose Nonfermenting 1-Related Protein Kinases (SnRK2s) 8 . In contrast, high temperature promotes stomatal opening to contribute to evaporative cooling through TARGET OF TEMPERATURE 3/MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4) 9–11 . A major driving force behind the dynamics of guard cells is the ion channels on the plasma membrane and the tonoplast, which control solute concentration in the cytosol, thereby driving water flow resulting in changing turgor pressure 12–16 . Protein levels and the activities of these ion channels and transporters are, to a large extent, regulated by phosphorylation, also in response to high temperature 17–20 . For example, this occurs in Arabidopsis plasma membrane H + -ATPASEs 1/2 (AHA1/2) 11 , which play a crucial role in high temperature-mediated stomatal aperture dynamics 19,21 . High temperature-activated TOT3/MAP4K4 phosphorylates the C-terminal regulatory domain of AHA1, thereby activating AHA1 and promoting stomatal opening under high temperature 11 . Here, we identified and characterized a mechanism involving the deubiquitylase UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24) that promotes stomatal opening in Arabidopsis thaliana under high temperature. We demonstrate that UBP24 is phosphorylated at serine 360 by the kinase OPEN STOMATA 1 (OST1), which is activated by B4 RAF kinases in response to high temperature stress. This phosphorylation event stabilizes UBP24, enabling deubiquitylation of a plasma membrane H⁺-ATPase to promote stomatal opening. This reveals a novel signalling mechanism that regulates the hydro-active opening of plant stomata under high temperature that evolved in vascular plants. Strikingly, we find that such a heat-responsive protein feature is conserved in the distantly related eukaryote Saccharomyces cerevisiae , where the UBP24 homolog Ubp3 requires a constitutively negatively charged residue at the equivalent position to support growth during heat shock. Our findings uncover an evolutionarily conserved molecular mechanism in which negative charge, conferred either via phosphorylation or by acidic residues, fine-tunes deubiquitylase function to support adaptive thermoresponsive signaling in eukaryotes. This work highlights how modular charge-based regulation has evolved to maintain cellular performance under thermal stress. To identify conserved proteins and regulated phosphosites that may mediate high temperature responses, including stomatal dynamics, we conducted a comparative phosphoproteome analysis between Triticum aestivum (wheat, Cadenza variety) and Arabidopsis thaliana seedlings, that were exposed to high temperature (34/35°C), which triggers significant stomatal opening and increased stomatal conductance 11 (Extended Data Fig. 1a and 3b and Supplementary Data 1) 22 . By overlapping significantly high temperature-regulated phosphorylation events from Arabidopsis and wheat datasets, we found 8 proteins with increased and 15 proteins with decreased phosphorylation levels in both species (Extended Data Fig. 1b,c). Three of the 8 co-increasing proteins have a conserved phosphorylation site between Arabidopsis and wheat. One of these candidates, TOT3, has been shown to regulate hypocotyl elongation and stomatal opening in response to high temperatures 11,23 . We then focused on another candidate, UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24), which undergoes high temperature-mediated phosphorylation at S360 (Fig. 1a). To validate phosphorylation at S360, we immunoprecipitated UBP24 protein from GFP-UBP24 -overexpressing Arabidopsis seedlings exposed to 35°C for one hour, and subsequent mass spectrometry analysis pinpointed UBP24 S360 as a reproducible high temperature-upregulated phosphosite (Fig. 1a and Supplementary Data 2). To assess a deeper conservation of S360 in UBP24, we performed an alignment of UBP24 putative orthologs from 55 species within the green lineage (Fig. 1b and Extended Data Fig. 2). In vascular plants, the relevant phosphorylatable serine (S360 in Arabidopsis ) is deeply conserved within the RxxS/T motif. Mapping crucial stomatal evolutionary events in the green lineage, such as presence and dynamics of stomata 4 , revealed that the presence of the S360 phosphosite coincides with the evolution of hydroactive stomatal opening (Fig. 1b). This suggests that UBP24 S360 phosphorylation might be associated with regulation of stomatal dynamics in evolution. Indeed, UBP24 was previously shown to play a role in abscisic acid (ABA)-mediated stomatal closure 24 . To test whether UBP24 is involved in the regulation of stomatal opening under high temperature, we conducted a stomatal bioassay (Extended Data Fig. 3a) 25 . Wild-type (Col-0) stomata exhibited a notable increase in aperture at temperatures of 35°C and 42°C, but not at 28°C, compared to control temperature (21°C), and stomatal aperture was greater at 42°C than at 35°C (Extended Data Fig. 3b). We therefore conducted subsequent stomatal bioassays at 42°C. Both the T-DNA mutant ubp24-1 and the CRISPR-Cas9 edited mutant ubp24-crispr showed impaired stomatal opening in the stomatal bioassay at 42°C (Fig. 1c,d and Extended Data Fig. 3c,d), and leaf imprinting followed by scanning electron microscopy corroborated these results (Extended Data Fig. 3e,f). Furthermore, the ubp24-1 stomatal aperture phenotype at high temperature could be complemented by a UBP24:YFP-UBP24 construct (Fig. 1e and Extended Data Fig. 3j). In addition, an inactive UBP24(C206S) variant 24 does not rescue the ubp24-1 high temperature stomatal aperture phenotype, indicating that activity of the deubiquitylating enzyme UBP24 plays a crucial role in the regulation of stomatal opening under high temperature (Extended Data Fig. 3g,h). Taken together, these results support that UBP24 plays an important role in stomatal dynamics under high temperature. To test for a role of UBP24 phosphorylation at S360, we introduced a phosphodead UBP24(S360A) or a phosphomimetic UBP24(S360D) variant, driven by the native UBP24 promoter, into the ubp24-1 mutant. The stomatal bioassay demonstrated that, like the UBP24(WT), the phosphomimetic UBP24(S360D) variant rescued the ubp24-1 stomatal opening phenotype (Fig. 1e and Extended Data Fig. 3j). However, the phosphodead UBP24(S360A) variant was unable to rescue this phenotype, regardless of the UBP24 expression level (Fig. 1e and Extended Data Fig. 3i,j). The results indicate that phosphorylation of UBP24 at S360 plays a crucial role in regulating stomatal opening under high temperatures. To understand how phosphorylation at S360 affects UBP24 under high temperature, we first used AlphaFold2 and molecular dynamics simulation to model different UBP24 variants under high temperature. Given that the predicted N-terminal region of UBP24 is highly disordered, we elected to input solely the UBP24 ubiquitin-specific protease (USP) domain, which contains the S360 phosphorylation site and accounts for 65% of the protein, for molecular dynamics simulation. The simulation results, the root mean square deviation (RMSD) of protein atomic positions and native contacts between alpha carbons, demonstrated that the phosphorylated UBP24 USP domain (pS360) is less sensitive to the high temperatures environment than the unphosphorylated variant (Fig. 1f,g and Extended Data Fig. 4c,d). Regardless of the temperature, 21°C or 42°C, the pS360 variant exhibited a lower energy than the unphosphorylated variant (Fig. 1h,i), further supporting that phosphorylated UBP24 is more stable. The results of the RMSD and native contact simulation analyses demonstrate that the phosphomimetic UBP24(S360D) USP domain exhibits increased stability in comparison with the phosphodead UBP24(S360A) variant (Extended Data Fig. 4a,b,e,f). To confirm if protein stability is regulated by phosphorylation at S360, we exposed ubp24-1 transgenic seedlings expressing WT, S360A, or S360D YFP-tagged UBP24 to 35°C for one hour. Here, we used 35°C, because the solubility of UBP24 decreases after extreme high temperature treatment (Extended Data Fig. 4g). The accumulation of UBP24(S360A) was limited under both 21°C and 35°C incubation conditions, while the UBP24(S360D) variant demonstrated a marked increase in protein level at 35°C (Fig. 1j,k). We hypothesised that the increase in UBP24(S360D) protein level may be attributable to slower degradation. Indeed, treating seedlings expressing UBP24(WT) or UBP24(S360D) with cycloheximide (CHX), demonstrated a higher protein stability of the UBP24(S360D) variant compared to UBP24(WT) under control temperature (Fig. 1l,m). These findings suggest that the phosphorylation of S360 in UBP24 stabilises the protein, thereby facilitating stomatal opening under high temperature. Since UBP24 S360 was previously listed as an OST1/SnRK2.6 target 26 , we explored if OST1 phosphorylates UBP24 at S360 in response to high temperature. We showed a high temperature-enhanced interaction between OST1 and UBP24 that was not influenced by ABA (Fig. 2a–c). To confirm that OST1 phosphorylates UBP24, we performed kinase assays. To circumvent the issue of insoluble UBP24 under high temperature (Extended Data Fig. 4g), we enhanced OST1 activity with ABA as an alternative to high temperature in the Nicotiana benthamiana leaf epidermis. The in planta kinase assay demonstrated that the phosphorylation level of UBP24, specifically at S360, increased in the presence of ABA-activated OST1 (Fig. 2d,e and Supplementary Data 4). An in vitro kinase assay further indicated that recombinant OST1 directly phosphorylates UBP24 S360 (Fig. 2f and Extended Data Fig. 5a). In addition, using transgenic OST1 isolated from Arabidopsis seedlings exposed to high temperature for a maximum of one hour, displayed increased kinase activity with respect to the recombinant UBP24 fragment substrate (Fig. 2i, Col-0). To explore this further, we used the ost1-3 mutant, which showed closed stomata under dark conditions 27 and an inability to open stomata after high temperature treatment in our stomatal bioassay (Fig. 2g). We introduced the phosphomimetic UBP24(S360D) variant into ost1-3 , which rescued the stomatal opening defect under high temperature (Fig. 2g and Extended Data Fig. 5b). Taken together, our results show that OST1 directly phosphorylates UBP24 at S360 under high temperature to facilitate stomatal opening. We observed that in our experimental conditions, specifically under darkness, no increase in ABA level, as visualised through the Arabidopsis ABA biosensor lines (nlsABACUS2s 28 ), occurred in guard cells following high temperature treatment (Extended Data Fig. 5c). This suggested that the activation of OST1 under high temperature conditions might not require ABA. Indeed, the high temperature-triggered opening of stomata in our stomatal bioassay still occurred in the presence of the ABA biosynthesis inhibitor abamine SG 29 and in the ABA biosynthesis mutant aba2-11 30 (Extended Data Fig. 5d,e). Taken together, these results suggest that high temperature-mediated activation of OST1 does not require ABA. Previously, it was shown that B4 RAF kinases can activate SnRK2s under osmotic stress in an ABA-independent manner 31 , and RAF24 phosphorylates OST1 under osmotic stress 32 . We noticed that RAF20 and RAF24, which belong to the B4 clade, are more phosphorylated at high temperature (Extended Data Fig. 5f and Supplementary Data 1). We thus hypothesised that B4 RAF kinases phosphorylate and activate OST1 at high temperatures to facilitate stomatal opening. Indeed, a septuple null mutant for the entire B4 family of RAF kinases ( raf ) 32,33 impairs stomatal opening and this phenotype can be complemented by either RAF20 or RAF24 (Fig. 2h). RAF20 (Extended Data Fig. 5g) and RAF24 32 , can directly phosphorylate OST1 in the activation loop at S175. With respect to its substrate UBP24, mCherry-OST1 isolated from raf seedlings exhibited lower kinase activity than the one isolated from WT seedlings and failed to be activated after high temperature treatment (Fig. 2i,j). These results support that B4 RAF kinases activate OST1 under high temperature in an ABA-independent manner, leading to OST1-mediated phosphorylation of UBP24. To identify interactors and possible deubiquitylation targets of UBP24, we performed immunoprecipitation followed by mass spectrometry detection (IP-MS/MS) on an Arabidopsis line overexpressing GFP-tagged UBP24 under control temperature (Fig. 3a and Supplementary Data 2). One of the putative interactors, the proton pump AHA1, is a major regulator of blue light and high temperature-mediated stomatal opening 11,15 , making this a promising candidate. Indeed, AHA1 protein levels in Col-0 Arabidopsis seedlings decreased under high temperature (Fig. 3k,l). Furthermore, AHA1 interacted with UBP24 (Fig. 3b,c), and a decrease in proton pump activity in ubp24-1 roots associated with reduced primary root growth were observed compared to Col-0 (Extended Data Fig. 6a–d). To test if UBP24 stabilises the AHA1 protein level via deubiquitylation, we co-expressed RFP-AHA1 with UBP24(WT) or UBP24(C206S) in N. benthamiana leaves. After treatment with concanamycin A (concA) to block vacuolar degradation, a decrease in AHA1 protein levels, an increase in AHA1 ubiquitylation and an increase in AHA1 internalisation were observed for UBP24(C206S) compared to UBP24(WT) (Fig. 3d–g). In line with these results, ubp24-1 seedlings expressing RFP-AHA1 displayed a substantial decrease in RFP-AHA1 protein expression, which was not caused by transcriptional regulation (Fig. 3j‒l and Extended Data Fig. 6h). Together, these results support that UBP24 deubiquitylates AHA1 under high temperature to prevent its degradation. A number of ubiquitinome analyses have demonstrated that AHA1 and AHA2 can be ubiquitin-modified at several lysine sites 34,35 . The distribution of these sites is principally confined to the N-terminal catalytic A domain, the nucleotide binding P-N domain, and the C-terminal regulatory R domain (Extended Data Fig. 6e) 36 . Given that the activities of AHA1 and AHA2 are primarily regulated by the R domain, we replaced five lysine residues located within the AHA1 R domain with arginine, thereby generating a ubiquitylation-deficient AHA1(5KR) variant (Extended Data Fig. 6e). To ascertain the potential impact of the ubiquitylation on AHA1, we expressed RFP-AHA1 and single lysine K48 or K63 ubiquitin in N. benthamiana leaves. Following the selective enrichment of K48- or K63-linkage ubiquitylated proteins, the AHA1(5KR) resulted in a decrease in ubiquitylation for K63-linkage (Extended Data Fig. 6f,g). We thus hypothesised that the K63 ubiquitylation of the R domain of AHA1 regulates AHA1 internalisation and stability 37,38 . By expressing AHA1(WT) and AHA1(5KR) in N. benthamiana leaves followed by concA treatment, we found that AHA1 internalisation was reduced, which may prevent the subsequent degradation of AHA1 (Fig. 3h,i). Additionally, the AHA1(5KR) mutation partially restored the AHA1 protein levels in ubp24-1 (Fig. 3j,k). Furthermore, the stomatal opening defect in ubp24-1 was rescued by AHA1(5KR) under high temperatures, but not by AHA1(WT) (Fig. 3m and Extended Data Fig. 6i). These findings show that UBP24 stabilises AHA1 at high temperatures through deubiquitylation, thereby contributing to stomatal opening. Protein phosphorylation, which introduces a negative charge on proteins, can impact protein-protein interactions and protein conformations, ultimately affecting their function 39,40 . Our results demonstrate that OST1-mediated phosphorylation of UBP24 at S360, impacting UBP24 molecular charge and stability, is a key component of stomatal dynamics in Arabidopsis . Of note, Ubp3, a S. cerevisiae ortholog of UBP24, is required for growth after a high temperature shock, since S. cerevisiae without Ubp3 grew slower after high temperature shock (Extended Data Fig. 7) 41 . Interestingly, at the position corresponding to UBP24 S360, animal (Usp10) and S. cerevisiae (Ubp3) orthologs of UBP24 contained the negatively charged amino acids aspartic or glutamic acid, corresponding to phosphomimetic amino acids (Fig. 4a and Extended Data Fig. 2 and Supplementary Data 3). We furthermore observed that 8 Saccharomyces species with distinct thermal preferences did not display any variation at the E691 (in S. cerevisiae ) position (Fig. 4b, lower part). Moreover, in an adaptive evolution experiment in which the temperature was gradually increased from 25°C to 40°C every 50 generations, for a total of 600 generations or until strains went extinct, there was no variation at the E691 position (Fig. 4b, upper part) 42 . This supports that retaining E691 in Ubp3 is important for thermoresponsive yeast growth. To test if the presence of the negatively charged amino acid is indeed important for thermoresponsive growth, we generated a S. cerevisiae yeast line replacing E691 of Ubp3 with alanine. Growth of the E691A yeast was slower at all temperatures tested compared to the WT, with or without a high temperature shock, but this was most obvious at the suboptimal temperature of 20°C (Fig. 4c,d). Molecular dynamics simulations of the Ubp3(WT) and the Ubp3(E691A) USP domain showed that Ubp3(E691A) increased energy regardless of temperature, suggesting instability of the E691A variant (Fig. 4e,f). Taken together, these results demonstrate that the charge of UBP24 at S360, which is mediated through phosphorylation, or Ubp3 at E691, in Arabidopsis or yeast, respectively, is crucial to respond to or grow at higher temperatures. Our study reveals that the evolutionary tuning of molecular charge state of UBP24 through protein phosphorylation shapes eukaryotic responses to high temperature. The majority of evolutionary studies focus on the presence or absence of specific genes 4 . However, this ignores the evolutionary effect on finetuning of protein activities through specific amino acid exchanges. While putative UBP24 orthologs are present in green lineage organisms and while the origin of the UBP24 regulatory kinase OST1 can be traced back to streptophyte algae 43 , we showed that the presence of a charge-tuneable site coincides with the evolution of hydro-active opening of stomata in vascular plants. OST1-mediated phosphorylation and subsequent stabilisation of UBP24 leads to a stabilisation of AHA1 on the plasma membrane by deubiquitylation, facilitating stomatal opening. A role for OST1 in promoting stomatal opening is at odds with the current understanding of the positive role of OST1 in ABA-induced stomatal closure 14,44,45 ; but, our results indicate that the role of OST1 in stomatal opening under high temperature is ABA-independent and relies on activation by B4 RAF kinases. In addition, we found that UBP24 orthologs outside the green lineage contain an aspartic or glutamic acid instead of a phosphorylation-mediated tuneable site and we demonstrated that this conserved Ubp3 charge is necessary for optimal thermoresponsive growth of yeast. Taken together, our results pinpoint that a role in thermoresponsiveness of UPB24 protein orthologs is deeply conserved and that the charge on these proteins, either being tuneable through kinases and phosphatases in vascular plants or being fixed in the genome to encode an aspartic or glutamic acid, is a key component for thermoresponsiveness of these proteins. Methods Plant materials and growing conditions All the Arabidopsis mutants and transgenic lines were in Columbia-0 (Col-0) ecotype. The ubp24-1 24 , ost1-3 46 , nlsABACUS2-100n 28 , nlsABACUS2-400n 28 , aba2-11 47 , ost2-2D 48 , raf 32,33 , RAF20:RAF20-sYFP 33 , RAF24:RAF24-sYFP 33 were described previously. Arabidopsis seeds were surface sterilised by 70% ethanol followed by 1% sodium hypochlorite solution. After washing with sterilised water, seeds were incubated at 4°C under darkness for 2 days. Sterilised seeds were sown on ½ Murashige and Skoog (MS) medium containing 1% sucrose, 0.5% MES and 0.8% (w/v) plant agar, pH adjusted to 5.7 with KOH. Seeds were grown horizontally under long-day photoperiod (16 h light/8 h dark, intensity 70 to100 μmol m −2 s −1 ) at 21°C. For stomatal bioassays, seedlings after 10-day growth on plates were transfered to soil and grown under long-day photoperiod (16 h light/8 h dark, intensity 70 to100 μmol m −2 s −1 ) at 21°C before analysis. For phosphoproteome analysis, seeds were grown vertically under continuous light (intensity 100 μmol m −2 s −1 ) at 21°C before harvesting. For high temperature treatment, Arabidopsis seedlings grown as described above were incubated at 35°C for 1 hour before analysis. Arabidopsis mutants and constructs Details on generating the CRISPR-edited ubp24 mutant ( ubp24-crispr ) and on generating and expressing constructs can be found in the Supplementary Information. Yeast strain construction and growth measurement The prototrophic diploid version of Saccharomyces cerevisiae strain S288C 49 was used for genome editing, and details on constructing the strains can be found in the Supplementary Information. For growth measurements, yeast cells were initially inoculated at a high dilution into YP5%GLU liquid medium, composed of 1% (w/v) yeast extract, 2% (w/v) peptone, and 5% (w/v) glucose, and cultured in 96-well microplates (655180, Greiner Bio-One) at 30°C with continuous agitation at 900 rpm until cultures reached the early exponential phase, as determined by an optical density at 600 nm (OD 600 ) of approximately 0.1. Cells were then further diluted in fresh YP5%GLU medium and regrown to an OD 600 of 0.1 to ensure uniform physiological state prior to subsequent treatments. Following growth, cell suspensions were washed three times with YP medium (1% yeast extract, 2% peptone) to remove residual glucose and metabolites, and resuspended in 150 μL of YP medium. Each sample was divided equally, with 75 μL subjected to high temperature shock at 45°C for 30 minutes in PCMT Thermoshaker (Grant-bio) with continuous agitation at 400 rpm, while the remaining aliquot was maintained at room temperature as a control. For growth kinetics assessment, yeast suspensions were diluted 1:50 in 150 μL of YP5%GLU medium and transferred to the Bioscreen C automated growth analysis system (Oy Growth Curves Ab). Growth experiments were conducted at 20°C, 25°C, and 30°C, with each condition including triplicate biological replicates and blank controls (medium only). Optical density at 600 nm was measured every 15 minutes for up to 5 days. Continuous shaking with the medium amplitude was used. Data were processed using BioScreener software, with blank OD 600 values subtracted to normalise growth curves. Stomatal bioassay The assay was adapted from a previously published method 25 with some modifications. The fourth to sixth true leaves were detached from 4-week-old Arabidopsis plants grown on soil. The abaxial epidermal peels, prepared by the sandwich method with label tape and Scotch tape, were floated on the stomatal closing buffer (50 mM KCl, 0.1 mM CaCl 2 and 10 mM MES, pH adjusted to 6.5 with KOH). After incubation for 2 hours at 21°C in the dark, the peels were transferred to dark incubators at 21°C, 28°C, 35°C or 42°C for 2 hours. For abamine SG treatment, 100 µM abamine SG in dimethyl sulfoxide (DMSO), or the same volume of DMSO for mock, was added to the stomatal closing buffer prior to the first incubation at 21°C in the dark. The peels were then mounted with stomatal closure buffer and observed using an Olympus BX51 upright microscope with 40x objective lens and DIC modules. The stomatal apertures (stomatal width divided by length) were measured by Fiji (ImageJ) software. Protein expression and cycloheximide assay To determine protein expression levels in Arabidopsis transgenic lines, three to five 10-day-old seedlings were harvested, frozen in liquid nitrogen and then ground with stainless steel beads. The material powder was incubated with 1x Laemmli sample buffer (62.5 mM Tris base, 2% SDS and 10% glycerol, pH adjusted to 6.8 with HCl) for 5 minutes at room temperature. For UBP24 protein variants, samples were incubated at 70°C for 10 minutes prior to loading on the gel. For AHA1 protein variants, samples were loaded directly onto the gel without high temperature incubation. SDS-PAGE was performed on 4‒20% mini-PROTEAN TGX stain-free precast gels at 150 V for 50 minutes. Before transfer to PVDF membrane, the gels were visualised using the ChemiDoc Imaging System (Biorad) for loading controls. Western blotting was performed as follows. Transfer was performed using Trans-Blot Turbo mini PVDF transfer packs on the Trans-Blot Turbo Transfer System (Biorad). The PVDF membranes were then blocked with 5% skim milk in 1x PBST or TBST buffer. The membranes were incubated with antibodies, 1:5000 anti-GFP HRP, 1:2000 anti-RFP or 1:5000 anti-mouse HRP. The signal was detected using SuperSignal West Femto maximum sensitivity substrate and visualised on the ChemiDoc Imaging System (Biorad). The signal intensity was measured by Fiji (ImageJ) software. For cycloheximide assays, the 10-day-old Arabidopsis seedlings were transferred to 12-well plates containing ½ Murashige and Skoog (MS) medium containing 1% sucrose, 0.5% MES and 200 µM cycloheximide, and pH adjusted to 5.7 with KOH. At the indicated time point, seedlings were harvested and used for protein level detection. Detection procedures were described above. Co-immunoprecipitation (co-IP) Final expression vectors for co-IP were transformed into Agrobacterium tumefaciens strain C58C1 by heat shock. Agrobacteria containing the indicated vectors or P19 were suspended in the infiltration buffer (10 mM MgCl 2 , 10 mM MES, pH adjusted to 5.7 with KOH) and then infiltrated into Nicotiana benthamiana leaves with the combination described in figures. After 60 hours, the leaves of N. benthamiana plants were harvested and ground into fine powder using liquid nitrogen. Proteins were extracted with extraction buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 1x cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack protease inhibitor cocktail, pH adjusted to 7.5 with HCl). After centrifugation to remove debris, the supernatant was transferred to new tubes containing GFP-Trap magnetic agarose. The mixture was incubated for 1 hour at 4°C with top-bottom rotating. The GFP-Trap magnetic agarose beads were collected using DynaMag-2 magnet (Invitrogen). After washing three times with the extraction buffer, the beads were resuspended in 1x NuPAGE LDS sample buffer and then incubate at 70°C for 10 minutes. The beads were trapped, and the supernatant were loaded on the gel for SDS-PAGE and western blotting. The procedure for protein band detection was described above. For HA-tagged OST1, 1:10000 anti-HA HRP antibody was used. In vitro kinase assay Final expression vectors for recombinant protein expression (see Supplementary Information for details) were transformed into Escherichia coli strain Rosetta(DE3) by electroporation. The positive colonies were inoculated in Luria-Bertani medium. After reaching the log phase, the cultures were treated with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) and incubated under 18°C for 16 hours. The cells were harvested, sonicated and centrifuged. The soluble fractions were incubated with Glutathione Sepharose 4B or PureCube 100 Ni-INDIGO agarose for purification of target proteins. To enrich OST1 from Arabidopsis transgenic lines, the total protein from wild-type or raf mutant containing OST1:mCherry-OST1 was extracted with the extraction buffer extraction buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1x cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack protease inhibitor cocktail and PhosSTOP, pH adjusted to 7.5 with HCl). After removing the debris by centrifugation, the supernatant was incubated with RFP-Trap magnetic agarose at 4°C for 1 hour with top-bottom rotating. The RFP-Trap magnetic agarose beads were collected using DynaMag-2 magnet (Invitrogen). After three washes with extraction buffer, the protein on the beads was eluted with 200 mM glycine-HCl (pH 2.5), and then neutralised with 1 M Tris-HCl (pH 10.4). The in vitro kinase assay was performed on either (γ-32P)ATP-radioactive or adenosine-5-o-(3-thio)-triphosphate (ATPγS)-based method. The (γ-32P)ATP-radioactive method was described previously 11 . Briefly, the 3:1 (w/w) ratio of substrate and kinase protein were incubated at 30°C for 1 hour in kinase assay buffer (10 mM Tris base, 2 mM MnCl 2 , 10 mM MgCl 2 , 0.5 mM DTT, 5 µM ATP and 5 µCi [γ-32P]ATP, pH adjusted to 7.5 with HCl). The reaction was stopped by adding 4x NuPAGE LDS sample buffer and 10x NuPAGE sample reducing agent to a final concentration 1x and then incubated at 70°C for 10 minutes. SDS-PAGE was performed. Protein bands were visualised by autoradiography for phosphorylation and InstantBlue Coomassie protein stain for loading control. In the ATPγS-based method, the kinase assay buffer was replaced with another buffer (20 mM Tris base, 100 mM NaCl, 2 mM MnCl 2 , 10 mM MgCl 2 , 1 mM DTT and 330 µM ATPγS,, pH adjusted to 7.5 with HCl). After the incubation at 30°C for half (for OST1 from Arabidopsis seedlings) or 1 (for recombinant OST1 and RAF20) hour, the reaction was stopped with 24 mM EDTA and alkylated with 100 mM p-Nitrobenzyl mesylate (PNBM) at 25°C for 1 hour. NuPAGE LDS sample buffer and NuPAGE sample reducing agent were added and then incubated at 70°C for 10 minutes before SDS-PAGE. Protein bands were visualised by a 1:5000 anti-thiophosphate ester antibody and a 1:10000 anti-rabbit HRP antibody for phosphorylation, and the signal of stain-free gel for loading control. In planta deubiquitylation assay The method for enrichment of ubiquitylated proteins was similar to the one for co-IP described above. Briefly, the agroinfiltrated N. benthamiana leaves were harvested after 60 hours. The ground materials were incubated with extraction buffer. The soluble protein extract was incubated with Pierce anti-HA magnetic beads at 4°C for 1 hour. After washing three times with the extraction buffer, the beads were resuspended in 1x NuPAGE LDS sample buffer and then incubate at 70°C for 10 minutes. The supernatant was loaded on the gel for SDS-PAGE and western blotting. The procedure for protein band detection was described above. Confocal microscopy For imaging with a Leica SP8 inverted confocal laser scanning microscope, objective lens HC PL APO CS2 40x/1.10 water and HC PL APO CS2 63x/1.20 water, white light laser (1.5 mW, range from 470 to 670 nm), detectors HyD and HyD SMD2, and software LASX were used. For imaging with a Leica Stellaris 5 LiAchroic upright confocal laser scanning microscope, objective lens HC PL APO 40x/1.10 W CORR CS2 water and HC PL APO 63x/1.20 W CORR CS2 water, diode lasers 488 (20 mW) and 561 (20 mW) nm, detectors HyD S, and software LASX were used. For imaging with a Zeiss inverted LSM710 confocal laser scanning microscope, objective lens C-Apochromat 40x/NA 1.20 W Korr M27 water and C-Apochromat 63x/NA 1.20 W Korr M27 water, argon laser 515 nm and diode lasers 559 nm, detectors PMT, and software Zen black were used. EYFP was excited by a 514 (white light) or 515 (argon) nm laser, and the emission was collected from 520 to 555 nm (with lifetime gating from 1 to 6 ns when using the Leica SP8). mRFP1 was excited by a 561 (white light) or 559 (argon) nm laser, and the emission was collected from 566 to 620 nm (with lifetime gating from 1 to 6 ns when using the Leica SP8). The laser power and detector gain were optimised and maintained constant throughout the experiment. Bimolecular fluorescence complementation (BiFC) Final expression vectors for BiFC were transformed into Agrobacterium tumefaciens strain C58C1 by heat shock. Agrobacteria containing the BiFC vector or P19 were suspended in the infiltration buffer (10 mM MgCl 2 , 10 mM MES, pH adjusted to 5.7 with KOH) and then co-infiltrated into N. benthamiana leaves. After 60 hours, the N. benthamiana plants were treated 1 hour at 40°C for UBP24-OST1 interaction detection. The florescence signal was imaged from leaf abaxial epidermis with the confocal setting described above. Imaging of RFP-AHA1 For observing AHA1 endocytosis, 35S:GFP-UBP24 , 35S:GFP-UBP24(C206S) , 35S:RFP-AHA1 or 35S :RFP-AHA1(5KR) was infiltrated into N. benthamiana leaves with the combinations showed in the figure. After 1 µM concanamycin A treatment overnight, the abaxial leaf epidermis was observed under a confocal microscope with the setting described above. For observing AHA1 protein expression, the 10-day-old Arabidopsis transgenic lines expressing RFP-AHA1 variants in wild-type or ubp24-1 background were used. Protein extraction and sample preparation for phosphoproteomics The procedure has been described previously 23 with some modifications. For the high temperature-treated Arabidopsis thaliana , seedlings from four biological replicates were harvested and ground to a fine powder using in liquid nitrogen. The samples were homogenised in the buffer (50 mM Tris base, 0.1 M KCl, 30% sucrose, 5 mM EDTA, 1 mM DTT, 1x cOmplete™ ULTRA Tablets, EDTA-free, protease inhibitor cocktail and PhosSTOP, pH adjusted to 8 by HCl), sonicated on ice for 30 seconds with 2-second on/1-second off cycles, and centrifuged at 4°C for 15 min at 3220 g. The soluble proteins were precipitated using methanol/chloroform method and resuspended in 8 M urea dissolved in 50 mM triethylammonium bicarbonate buffer (TEAB, pH 8). Cysteine alkylation was performed using 15 mM tris(carboxyethyl)phosphine (TCEP) and 30 mM iodoacetamide for 2 hours at 30°C. The protein solutions were diluted with 50 mM TEAB (pH 8) to 1 M urea. 200 µg proteins determined by the Pierce 660 nm protein assay kit were firstly pred-igestedpredigested with EndoLysC (SignalChem) at an enzyme-to-substrate ratio of 1:5 (w:w) for 2 hours and then digested with sequencing grade modified trypsin overnight at an enzyme-to-substrate ratio of 1:10 (w:w). Peptides were desalted using C18 ODS SampliQ solid phase extraction (SPE) columns according to the manufacturer’s guidelines, eluted to 70% (v/v) acetonitrile (ACN) containing 0.1% (v/v) trifluoroacetic acid (TFA), and dried by vacuum. Dried peptide pellets were dissolved in 80% (v/v) ACN containing 6% (v/v) TFA and incubated with 1 mg MagReSyn Ti-IMAC magnetic microspheres for 20 minutes with continuous agitation. After washing the microspheres once with 60% ACN, 1% TFA, 200 mM NaCl and twice with 60% ACN, 1% TFA, the phosphopeptides were eluted with elution buffer (40% ACN, 5% NH 4 OH) followed by acidification to pH 3 with 100% formic acid. The peptides were dried by vacuum for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analysis, database searching and data analysis The procedure has been described previously 23 with some modifications. Samples were analysed by LC-MS/MS using an Ultimate 3000 RSLC nano LC (Thermo Fisher Scientific) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The samples were loaded on an analytical (made in-house, 25 cm column, 1.9 µm beads, 75 μm internal diameter, Dr. Maisch, Ammerbuch-Entringen, Germany) with solvent 0.1% TFA in water. The samples were separated by solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in water/ACN, 20/80 [v/v]) with a linear gradient from solvent A/solvent B, 98/2 (v/v) to solvent A/solvent B, 45/55 (v/v) over 2.5 hour at 300 nL min −1 flow rate followed by a 5-minutes wash till 99% of solvent B. The MS setting was identical as previously described. The phosphoproteomics data of high temperature-treated Arabidopsis seedlings produced in this study have been deposited to the ProteomeXchange Consortium via the PRIDE 50 partner repository with the dataset identifier PXD066011. The spectra were searched using MaxQuant (version 2.1.4.0) on a high performance computer (HPC-UGent) against a proteome database Araport11 51 for Arabidopsis thaliana or Nicotiana tabacum protein sequences from UniProt 52 (Proteome ID: UP000084051) combining with particular protein sequences from Arabidopsis thaliana . The “Phospho(STY).txt” output filewasimputed by PhosR, analyzed by Perseus (version 2.0.10.0). In planta kinase assay and immunoprecipitation followed by LC-MS/MS (IP-MS) The protein enrichment method is similar to the method used in co-IP. For the in planta kinase assay, the agroinfiltrated N. benthamiana leaves were treated with DMSO (mock) or 50 µM abscisic acid in infiltration buffer for 2 hours before harvested. The extraction buffer was replaced to 150 mM Tris base, 150 mM NaCl, 10% glycerol, 10 mM EDTA, 1% NP-40, 10 mM DTT, 1x cOmplete™ ULTRA Tablets, EDTA-free, protease inhibitor cocktail and PhosSTOP, pH adjusted by HCl to 7.5. The wash buffer was replaced to 20 mM Tris base, 150 mM NaCl and 0.5% NP-40, pH adjusted by HCl to 7.5. For immunoprecipitation GFP-UBP24 from Arabidopsis transgenic lines, the extraction buffer was the same as described in CoIP. The wash steps were performed with the extraction buffer. After the washing steps, both experiments were followed by washing GFP-Trap magnetic beads once with 50 mM TEAB (pH 8). The on-bead digestion was performed by 0.5 µg trypsin in 50 mM TEAB. The peptides in the supernatant were alkylated on cysteine residues using 10 mM TCEP and 30 mM iodoacetamide. After reduction with 5 mM DTT, the peptide solution was treated with an additional 0.5 µg of trypsin and digested overnight. Digestion was stopped by adding TFA to a final concentration of 1%. Desalting was performed by OMIX C18 pipette tips according to the manufacturer’s guidelines, and elution in 60% ACN, 40% water, 0.1% TFA. The samples were vacuum dried and analysed by LC-MS/MS as described above. Protein sequence alignment and phylogenetic analysis The proteome databases of selected species were obtained from Araport 11 51 , NCBI 53 , UniProt 52 , PLAZA 5.0 54 and ORCAE 55 . The Arabidopsis thaliana UBP24 protein sequence from Araport 11 was used for BLASTp analysis. When multiple UBP24 orthologs were found, the protein with the lowest E-score was selected. The entire protein sequences of the UBP24 orthologs were used for multiple sequence alignment and neighbour-joining phylogenetic relationships analysis using Clustal Omega. The results were visualised using Interactive Tree Of Life (iTOL) version 6. Saccharomyces Ubp3 sequences and alignment To investigate amino acid variation in the Ubp3 proteins across eight species of the Saccharomyces genus with different thermal profiles ( S. eubayanus, S. uvarum, S. arboricola, S. kudriavzevii, S. mikatae, S. jurei, S. paradoxus, and S. cerevisiae ), we analysed both ancestral and experimentally evolved strains from a previously published study 56 . Ancestral strains correspond to natural isolates and were used as starting points for thermal evolution adaptation. Evolved strains were derived from these ancestors exposed to gradually increasing temperatures from 25ºC to 40ºC every 50 generations for a total of 600 generations, or until they were extinct 56 . We reconstructed the UBP3 coding sequences using whole-genome resequencing data. Reads from each strain were previously mapped to species-specific reference genomes, and variant calling was performed using GATK version 4.3.0.0 57 . Using BCFtools consensus 58 , we generated consensus genome sequences for each strain by applying the variant calls to the reference sequence. Genomic coordinates of the UBP3 gene were retrieved from the reference genome annotations, and the corresponding nucleotide sequences were extracted using BEDtools getfasta 59 . Protein sequences were inferred from the nucleotide sequences using NCBI’s ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) tool, selecting the longest open reading frame consistent with the annotated UBP3 ORF in S. cerevisiae S288C reference strain. Multiple sequence alignment of the predicted protein sequences was performed using MUSCLE (https://www.ebi.ac.uk/jdispatcher/msa/muscle?stype=protein) with default parameters. Amino acid differences between strains were identified and visualised across the alignment. The reconstructed UBP3 nucleotide and protein sequences used in this study are available through GenBank accession numbers PV855420-PV855652. Protein structure prediction and molecular dynamics (MD) simulation The structure of the wild-type UBP24 USP domain was predicted by AlphaFold2 with default settings. The predicted structure underwent mutagenesis, resulting in three different variants: S360A, S360D, and S360-phosphorylation (pS360). This mutagenesis and solvate with TIP3P water molecules 60 and 150 mM NaCl was facilitated by the CHARMM-GUI. All-atom MD simulations were performed using NAMD 3.0b6 with the CHARMM36m force field. The solvated protein models were minimised for 1000 steps followed by simulation under the NVT (constant number of atoms, volume and temperature) system at 295 K or 337 K using a Langevin thermostat. Four independent 100 ns simulations of all variant UBP24 USP domain were performed with structure saving per 200 ps. The simulation results were analysed by Python package MDAnalysis. The RMSD, free energy and native contact was calculated by C-alpha. Diagram plotting, statistics and reproducibility All the figures were generated by Python package matplotlib and seaborn and the layout composition was done in Inkscape. All the statistical analysis were performed by Microsoft Excel 2019 or R package stats, emmean and multcomp. All calculated p values and associated statistical outputs are provided in the Source data. No sample size predetermination was used. During the experiments and outcome assessment, investigators were not blinded to the allocation. Each experiment was repeated at least twice with similar results. Declarations Data availability Data are available in the Article, Supplementary information and Source data. Mass spectrometry phosphoproteomics data of Arabidopsis thaliana are available via ProteomeXchange with identifier PXD066011. The nucleotide and protein sequences of Saccharomyces species used in this study are available through GenBank accession numbers PV855420-PV855652. Acknowledgements We thank A. M. Jones (University of Cambridge) for sharing nlsABACUS2 lines, D. Weijers (Wageningen University) for sharing raf and RAF complementing Arabidopsis seeds and E. Farmer (University of Lausanne) for sharing ost2-2D seeds. We acknowledge the VIB Proteomics Core for mass spectrometry analysis. We thank Carina Braeckman (VIB) for helping with Arabidopsis transformation. We are grateful to Bert De Rybel and Daniel Van Damme (VIB) for their comments on the manuscript. We acknowledge funding from the Research Foundation - Flanders (FWO.OPR.2019.0009.01 to I.D.S.), VIB (to S.L.Y and I.D.S.), the Knut and Alice Wallenberg Foundation (to R.S.), Taiwanese Government Scholarship to Study Abroad (to S.L.Y.), UGent BOF postdoctoral mandate no. 01P12219 (to L.D.V.) and no. 01P11322 (to T.Z), UGent BOF doctoral mandate no. 01CD7122 (to X.X.), and China Scholarship Council grant no. 202204910025 (to H.L.), grant no. 201706910095 (to T.Z.) and grant no. 201706350153 (to X.X.). Author information S.L.Y., I.D.S., K.G., K.J.V, and L.D.V designed experiments. S.L.Y. performed most of the experiments. X.X. supported the radioactive in vitro kinase assay. 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Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79 , 926–935 (1983). Additional Declarations There is NO Competing Interest. Supplementary Files YangSupplementaryData1.xlsx Extanded Data Table 1 YangSupplementaryData2.xlsx Extanded Data Table 2 YangSupplementaryData3.xlsx Extanded Data Table 3 YangSupplementaryData4.xlsx Extanded Data Table 4 YangSupplementaryInformation.pdf Supplementary Information Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Gorkovskiy","suffix":""},{"id":542699769,"identity":"b454da46-2f49-42b9-908d-b38fe5dc0a3e","order_by":5,"name":"Tingting Zhu","email":"","orcid":"","institution":"VIB-UGent Center for Plant Systems Biology","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Zhu","suffix":""},{"id":542699770,"identity":"dc415910-9a59-44a9-95ac-cbafbe462fe0","order_by":6,"name":"Cássio Flávio Fonseca de Lima","email":"","orcid":"","institution":"VIB-UGent Center for Plant Systems Biology","correspondingAuthor":false,"prefix":"","firstName":"Cássio","middleName":"Flávio Fonseca","lastName":"de Lima","suffix":""},{"id":542699771,"identity":"37b4c8af-983d-44a6-af7b-26003b91ab5e","order_by":7,"name":"Brigitte Van De Cotte","email":"","orcid":"","institution":"VIB-UGent Center for Plant Systems Biology","correspondingAuthor":false,"prefix":"","firstName":"Brigitte","middleName":"Van","lastName":"De Cotte","suffix":""},{"id":542699772,"identity":"244da085-779e-40d9-b303-7d04b1304d90","order_by":8,"name":"Jennifer Molinet","email":"","orcid":"","institution":"Department of Zoology, Stockholm University","correspondingAuthor":false,"prefix":"","firstName":"Jennifer","middleName":"","lastName":"Molinet","suffix":""},{"id":542699773,"identity":"f4930c36-eb49-4daa-9a82-4744a35348c7","order_by":9,"name":"Karin Voordeckers ","email":"","orcid":"https://orcid.org/0000-0001-6397-840X","institution":"VIB-KU Leuven Center for Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Karin","middleName":"","lastName":"Voordeckers ","suffix":""},{"id":542699774,"identity":"e3f21ff0-657d-491e-a087-41ff0d93390e","order_by":10,"name":"Kevin Verstrepen ","email":"","orcid":"","institution":"VIB-KU Leuven Center for Microbiology","correspondingAuthor":false,"prefix":"","firstName":"Kevin","middleName":"","lastName":"Verstrepen ","suffix":""},{"id":542699775,"identity":"35515f72-3335-4931-8f06-57bd37ae0a87","order_by":11,"name":"Rike Stelkens","email":"","orcid":"","institution":"Department of Zoology, Stockholm University","correspondingAuthor":false,"prefix":"","firstName":"Rike","middleName":"","lastName":"Stelkens","suffix":""},{"id":542699776,"identity":"12b6d2b7-aff0-4658-b87b-d125d3f1c619","order_by":12,"name":"Kris Gevaert","email":"","orcid":"https://orcid.org/0000-0002-4237-0283","institution":"VIB-UGent Center for Medical Biotechnology and UGent Department of Biomolecular Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kris","middleName":"","lastName":"Gevaert","suffix":""},{"id":542699777,"identity":"8cd5ffa9-a5bd-4772-ae73-4b6ae5cea44b","order_by":13,"name":"Lam Dai Vu","email":"","orcid":"","institution":"VIB","correspondingAuthor":false,"prefix":"","firstName":"Lam","middleName":"Dai","lastName":"Vu","suffix":""}],"badges":[],"createdAt":"2025-07-18 13:05:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7157930/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7157930/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95631887,"identity":"6f1c9def-7c2b-4b50-97bb-14e4641e3ab1","added_by":"auto","created_at":"2025-11-11 11:39:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":465835,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh temperature-triggered phosphorylation of UBP24 promotes protein stability and stomatal opening.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea, \u003c/strong\u003eDiagram of the UBP24 USP domain and detected phosphosites from two independent datasets. The orange, blue, and grey lollipops represent phosphosites that were significantly high temperature-upregulated, significantly high temperature-downregulated, and no significant changed upon high temperature, respectively. The full list and statistical analysis are provided in Supplementary Data 1 and 2. \u003cstrong\u003eb\u003c/strong\u003e, Simplified phylogenic tree of representative green lineage species (branch lengths not scaled). The critical evolutionary events of single origin for stomata hypothesis are mapped. Within the amino acid sequence alignment of putative UBP24 orthologs, the grey shading represents the phosphorylated motif RxxS/T; the green shading represents the vascular plant-specific phosphosite. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e, Stomatal morphologies (\u003cstrong\u003ec\u003c/strong\u003e) and quantification of apertures (\u003cstrong\u003ed\u003c/strong\u003e, pore width divided by pore length) of wild-type (Col-0) and \u003cem\u003eubp24\u003c/em\u003e mutants after 21°C or 42°C treatment under darkness. In \u003cstrong\u003ed\u003c/strong\u003e, Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cem\u003en\u003c/em\u003e = 50 stomata per condition. \u003cstrong\u003ee\u003c/strong\u003e, Quantification of stomatal apertures of \u003cem\u003eubp24-1 \u003c/em\u003eexpressing UBP24 variants at 21°C or 42°C under darkness. The other independent lines are provided in Extended Data Fig. 3j. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cem\u003en\u003c/em\u003e = 50 stomata per condition. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e, The root-mean-square-deviation (RMSD) of WT (\u003cstrong\u003ef\u003c/strong\u003e) and phosphorylated (\u003cstrong\u003eg\u003c/strong\u003e) UBP24 USP domain from MD simulation trajectories. Three of four simulations showed similar results. \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e, The total energy of UBP24 USP domain at MD simulation trajectories (\u003cstrong\u003eh\u003c/strong\u003e) and steady states (\u003cstrong\u003ei\u003c/strong\u003e). Three of four simulations showed similar results. In \u003cstrong\u003ei\u003c/strong\u003e, two-way ANOVA, \u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003cem\u003en\u003c/em\u003e = 355 datapoints.\u003cstrong\u003e j\u003c/strong\u003e,\u003cstrong\u003ek\u003c/strong\u003e, Representative Western blot (\u003cstrong\u003ej\u003c/strong\u003e) and semi-quantification (\u003cstrong\u003ek\u003c/strong\u003e) using GFP antibody for UBP24 protein detection in \u003cem\u003eArabidopsis\u003c/em\u003e after 1 hour treatment at 21°C or 35°C in the dark. In \u003cstrong\u003ek\u003c/strong\u003e, UBP24 protein intensities in 3replicates. Two-way ANOVA, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cstrong\u003el\u003c/strong\u003e,\u003cstrong\u003em\u003c/strong\u003e, Representative Western blot (\u003cstrong\u003el\u003c/strong\u003e) and semi-quantification (\u003cstrong\u003em\u003c/strong\u003e) using GFP antibody for UBP24 protein detection in \u003cem\u003eArabidopsis\u003c/em\u003e after 200 µM cycloheximide (CHX) treatment at indicated time. In\u003cstrong\u003e m\u003c/strong\u003e, UBP24 protein intensities in 3 replicates. One-way ANOVA, \u003cem\u003ep\u003c/em\u003e ≤ 0.05, statistical analysis was applied separately for each group. ns indicates no significant difference. For the box plots in \u003cstrong\u003ed\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003ek\u003c/strong\u003e and \u003cstrong\u003em\u003c/strong\u003e, the centre lines represent medians, the box limits represent the first and the third quartiles, and the whiskers represent 1.5× the interquartile ranges.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/5282dfd5124ad1e214c161cc.png"},{"id":95631896,"identity":"f5b2aac6-9d44-4d79-961b-cfa1011ea7a2","added_by":"auto","created_at":"2025-11-11 11:39:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":355950,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRAF-activated OST1 phosphorylates UBP24 at S360 under high temperature.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e,\u003cstrong\u003eb\u003c/strong\u003e, Representative confocal images (\u003cstrong\u003ea\u003c/strong\u003e) and quantification of YFP signal particle sizes (\u003cstrong\u003eb\u003c/strong\u003e) of co-expressing split cYFP-tagged GST or OST1 with nYFP-UBP24 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Free RFP used as expression control. In \u003cstrong\u003eb\u003c/strong\u003e, two-tailed Student’s t-test. ND indicates no signal detected. The numbers of particles measured (\u003cem\u003en\u003c/em\u003e) in 5 comparable images with the same area for each condition is shown below the plot. \u003cstrong\u003ec\u003c/strong\u003e, Representative Western blot after GFP-trap co-immunoprecipitation from \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ed\u003c/strong\u003e, Schema of detected UBP24 phosphosites by co-expressing OST1 with or without activation by ABA in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The orange, and grey lollipops represent phosphosites that were significantly upregulated, or did not significant change, respectively. The full list and statistical analysis are provided in Supplementary Data 4. \u003cstrong\u003ee\u003c/strong\u003e, Phosphopeptide intensities of UBP24 S360 across different treatments. The numbers within the brackets in the detected phosphopeptide indicate the localization probabilities. One-way ANOVA, \u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003cem\u003en\u003c/em\u003e = 4 replicates. \u003cstrong\u003ef\u003c/strong\u003e, Representative radioactive blot of UBP24 S360 phosphorylation by recombinant OST1 using \u003cem\u003ein vitro\u003c/em\u003e kinase assay. UBP24\u003csup\u003e311–411\u003c/sup\u003e represents a fragment of 100 amino acids used for the substrate. \u003cstrong\u003eg\u003c/strong\u003e, Quantification of stomatal apertures of \u003cem\u003eost1-3\u003c/em\u003e complemented with UBP24(S360D) at 21°C or 42°C under darkness. The other independent lines are provided in Extended Data Fig. 5b. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cem\u003en\u003c/em\u003e = 50 stomata per condition. \u003cstrong\u003eh\u003c/strong\u003e, Quantification of stomatal apertures of the septuple null mutant of the entire B4 clade RAF kinases (\u003cem\u003eraf\u003c/em\u003e) and RAF20-YFP or RAF24-YFP expressed in \u003cem\u003eraf\u003c/em\u003e at 21°C or 42°C under darkness. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. \u003cem\u003en\u003c/em\u003e = 50 stomata per condition. \u003cstrong\u003ei\u003c/strong\u003e,\u003cstrong\u003ej\u003c/strong\u003e, Representative Western blot (\u003cstrong\u003ei\u003c/strong\u003e) and semi-quantification for the intensities (\u003cstrong\u003ej\u003c/strong\u003e) of UBP24 phosphorylation by high temperature-activated OST1 using an ATPγS-based \u003cem\u003ein vitro \u003c/em\u003ekinase assay. OST1 was enriched by RFP-trap immunoprecipitation from high temperature-treated transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines \u003cem\u003eOST1:mCherry-OST1\u003c/em\u003e in WT or \u003cem\u003eraf\u003c/em\u003e background. The substrate used is identical to that in \u003cstrong\u003ef\u003c/strong\u003e. In \u003cstrong\u003ej\u003c/strong\u003e, intensities of UBP24 100 amino acid fragment in 3 replicates. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05. The asterisks in \u003cstrong\u003ef\u003c/strong\u003e and \u003cstrong\u003ei\u003c/strong\u003e indicate non-specific protein bands. For the box plots in \u003cstrong\u003eb\u003c/strong\u003e, \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003eh\u003c/strong\u003e and\u003cstrong\u003e j\u003c/strong\u003e, the centre lines represent medians, the box limits represent the first and the third quartiles, and the whiskers represent 1.5× the interquartile ranges.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/8944772d50be41997edf67ce.png"},{"id":95657151,"identity":"b3892ad8-03d5-4850-92d5-df141d8307e8","added_by":"auto","created_at":"2025-11-11 16:20:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUBP24 deubiquitylates and stabilises AHA1 to promote stomatal opening.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Volcano plot of enriched proteins after UBP24 pull-down from a UBP24-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e transgenic line. Yellow dots represent all significant candidates compared to pull-down product from WT seedlings. Red dots represent proteins involved in stomatal movement regulation based on Gene Ontology terms. \u003cstrong\u003eb\u003c/strong\u003e, Representative confocal images of co-expressing split cYFP-tagged GST or AHA1 with nYFP-UBP24 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves at 21°C. Free RFP used as expression control. \u003cstrong\u003ec\u003c/strong\u003e, Representative Western blot after GFP-trap co-immunoprecipitation from \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. \u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003ee\u003c/strong\u003e, Representative Western blot (\u003cstrong\u003ed\u003c/strong\u003e) and semi-quantification for RFP-AHA1 protein intensities (\u003cstrong\u003ee\u003c/strong\u003e) after HA-trap immunoprecipitation to enrich ubiquitylated proteins from \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The 3×HA-UBQ1 was used as the ubiquitin source. In \u003cstrong\u003ee\u003c/strong\u003e, RFP-AHA1 protein intensities in 3 replicates. Two-tailed Mann-Whitney U test. \u003cstrong\u003ef\u003c/strong\u003e,\u003cstrong\u003eg\u003c/strong\u003e, Representative confocal images (\u003cstrong\u003ef\u003c/strong\u003e) and quantification for AHA1 vesicles (\u003cstrong\u003eg\u003c/strong\u003e) of co-expressing enzyme-dead UBP24(C206S) or wild-type UBP24(WT) with AHA1 using the \u003cem\u003eN. benthamiana\u003c/em\u003eleaves. In \u003cstrong\u003eg\u003c/strong\u003e, AHA1 vesicle density (vesicle number per cell/cell area) was log-transformed. Two-tailed Mann-Whitney U test. \u003cem\u003en\u003c/em\u003e = 66 cells. \u003cstrong\u003eh\u003c/strong\u003e,\u003cstrong\u003ei\u003c/strong\u003e, Representative confocal images (\u003cstrong\u003eh\u003c/strong\u003e) and quantification for AHA1 vesicles (\u003cstrong\u003ei\u003c/strong\u003e) of wild-type AHA1(WT) and ubiquitylation-deficient AHA1(5KR) in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. In \u003cstrong\u003ei\u003c/strong\u003e, AHA1 vesicle density (vesicle number per cell/cell area) was log-transformed. Two-tailed Mann-Whitney U test. \u003cem\u003en\u003c/em\u003e = 60 cells. \u003cstrong\u003ej\u003c/strong\u003e, Representative confocal images of \u003cem\u003eArabidopsis\u003c/em\u003e transgenic lines at 21°C. The rainbow color gradient of the images represents RFP-AHA1 fluorescence intensity from low to high from black and blue to red and white. \u003cstrong\u003ek\u003c/strong\u003e,\u003cstrong\u003el\u003c/strong\u003e, Representative Western blot (\u003cstrong\u003ek\u003c/strong\u003e) and semi-quantification (\u003cstrong\u003el\u003c/strong\u003e) using RFP antibody for AHA1 protein detection in \u003cem\u003eArabidopsis\u003c/em\u003e after 1 hour treatment at 21°C or 35°C in the dark. In \u003cstrong\u003el\u003c/strong\u003e, AHA1 protein intensities in 3replicates. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05, statistical analysis was applied separately for each group. ns indicates no significant difference. \u003cstrong\u003em\u003c/strong\u003e, Quantification of stomatal apertures of \u003cem\u003eubp24-1\u003c/em\u003e expressing AHA1(WT) or AHA1(5KR) at 21°C or 42°C under darkness. The other independent lines are provided in Extended Data Fig. 6g. Kruskal-Wallis test, \u003cem\u003ep\u003c/em\u003e ≤ 0.05.For the box plots in \u003cstrong\u003ee\u003c/strong\u003e, \u003cstrong\u003eg\u003c/strong\u003e, \u003cstrong\u003ei\u003c/strong\u003e, \u003cstrong\u003el\u003c/strong\u003e and\u003cstrong\u003e m\u003c/strong\u003e, the centre lines represent medians, the box limits represent the first and the third quartiles, and the whiskers represent 1.5× the interquartile ranges.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/e22f2096ee50a79baa22da8c.png"},{"id":95631891,"identity":"256f2efd-e88f-4e19-a900-c0c408642fa4","added_by":"auto","created_at":"2025-11-11 11:39:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":400219,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvolutionary tuning of negative charge on Arabidopsis UBP24 orthologs regulates high temperature responses in yeast.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e, Amino acid alignment of \u003cem\u003eArabidopsis\u003c/em\u003e UBP24 orthologs from representative species in Extended Data Fig. 2. The green shading represents the higher plant-specific phosphosites; the orange shading represents the comparative aspartic acid or glutamic acid in fungi and animals. \u003cstrong\u003eb\u003c/strong\u003e, Diagram of evolutionary comparison of Ubp3 between \u003cem\u003eSaccharomyces\u003c/em\u003e species. The orange dots represent mutations that occurred during directed evolution experiments. The bar plot below shows the identity of each amino acid between ancestors. \u003cstrong\u003ec\u003c/strong\u003e,\u003cstrong\u003ed\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eGrowth curves (\u003cstrong\u003ec\u003c/strong\u003e) and time to reach maximum growth rate (\u003cstrong\u003ed\u003c/strong\u003e) of WT and substitute-mutated Ubp3 E691A in \u003cem\u003eS. cerevisiae\u003c/em\u003e S288C under different temperatures after 42°C high temperature-shock treatment. The shading in \u003cstrong\u003ec\u003c/strong\u003e and the whiskers/caps in \u003cstrong\u003ed\u003c/strong\u003e represent the standard error (SE). Two-tailed Student's t-test. \u003cem\u003en\u003c/em\u003e = 3, 3, 5 for 30°C, 25°C, 20°C, respectively. ns indicates no significant difference. \u003cstrong\u003ee\u003c/strong\u003e,\u003cstrong\u003ef\u003c/strong\u003e, The total energy of Ubp3 USP domain at MD simulation trajectories (\u003cstrong\u003ee\u003c/strong\u003e) and steady states (\u003cstrong\u003ef\u003c/strong\u003e). Three simulations showed similar results. In \u003cstrong\u003ef\u003c/strong\u003e, two-way ANOVA, \u003cem\u003ep\u003c/em\u003e ≤ 0.05, \u003cem\u003en\u003c/em\u003e = 701 datapoints. For the box plots in \u003cstrong\u003ef\u003c/strong\u003e, the centre lines represent medians, the box limits represent the first and the third quartiles, and the whiskers represent 1.5× the interquartile ranges.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/7eec3aecbaa56d7e7b3a2733.png"},{"id":95797410,"identity":"7cc350ad-4316-4c19-ae7f-62ed98656b38","added_by":"auto","created_at":"2025-11-13 08:04:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2764887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/41413f1f-4f4e-4a8e-b88e-58de87cae45e.pdf"},{"id":95631890,"identity":"a8dd1ebf-44b6-4f3a-b4f8-720a88464536","added_by":"auto","created_at":"2025-11-11 11:39:12","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":620307,"visible":true,"origin":"","legend":"Extanded Data Table 1","description":"","filename":"YangSupplementaryData1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/0e57febe65eec72b66f6a4ef.xlsx"},{"id":95631895,"identity":"d57f75e7-ad60-422e-819d-581c9295d80f","added_by":"auto","created_at":"2025-11-11 11:39:15","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":634156,"visible":true,"origin":"","legend":"Extanded Data Table 2","description":"","filename":"YangSupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/aa93bc2e5a33c20a6a3fe319.xlsx"},{"id":95631897,"identity":"f2cbe9ef-98f6-4445-bd27-366bcb2ea412","added_by":"auto","created_at":"2025-11-11 11:39:15","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":54671,"visible":true,"origin":"","legend":"Extanded Data Table 3","description":"","filename":"YangSupplementaryData3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/0b2e9fb4d4f736affed3163d.xlsx"},{"id":95631888,"identity":"f79a6d4b-ab4a-4a1f-b4ec-368013369546","added_by":"auto","created_at":"2025-11-11 11:39:12","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":715911,"visible":true,"origin":"","legend":"Extanded Data Table 4","description":"","filename":"YangSupplementaryData4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/81ea8c3392a539ce0f62ba98.xlsx"},{"id":95631898,"identity":"f73f234e-4c8f-4664-87d8-2a75e43adba2","added_by":"auto","created_at":"2025-11-11 11:39:15","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":434189,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"YangSupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7157930/v1/de3ecf9975a3050e7c9cb819.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature","fulltext":[{"header":"Main","content":"\u003cp\u003eProtein evolution relies on variation within protein sequences, impacting activity, interactions and stability through – for example – gaining or losing regulatory post-translational modifications\u003csup\u003e1\u003c/sup\u003e. Rapid and reversible protein phosphorylation enables organisms to respond efficiently to fluctuating environments and enables phenotypic plasticity\u003csup\u003e2\u003c/sup\u003e. Homologous proteins display a high conversion probability from negatively charged (aspartic and glutamic acid) to phosphorylatable residues (serine and threonine) to generate tuneable electrostatic interactions\u003csup\u003e3\u003c/sup\u003e. The successful colonisation of the green lineage, also known as Viridiplantae, to terrestrial environments required several adaptations, including the evolution of microscopic pores on the body surface, called stomata\u003csup\u003e4\u003c/sup\u003e. The tight regulation of gas and water vapor exchange to control photosynthesis and water loss through stomata, enabled plants to grow and survive under climatically changing environments\u003csup\u003e4,5\u003c/sup\u003e. Liverworts and some mosses have static pores\u003csup\u003e6,7\u003c/sup\u003e,\u0026nbsp;while in vascular plants, stomatal dynamics are achieved by the action of a pair of guard cells that allow or restrict the aperture. Stomatal closure is triggered by, for example, drought and involves ABA-activated Sucrose Nonfermenting 1-Related Protein Kinases (SnRK2s)\u003csup\u003e8\u003c/sup\u003e.\u0026nbsp;In contrast, high temperature promotes stomatal opening to contribute to evaporative cooling through TARGET OF TEMPERATURE 3/MITOGEN-ACTIVATED PROTEIN KINASE KINASE KINASE KINASE4 (MAP4K4)\u003csup\u003e9–11\u003c/sup\u003e.\u0026nbsp;A major driving force behind the dynamics of guard cells is the ion channels on the plasma membrane and the tonoplast, which control solute concentration in the cytosol, thereby driving water flow resulting in changing turgor\u0026nbsp;pressure\u003csup\u003e12–16\u003c/sup\u003e.\u0026nbsp;Protein levels and the activities of these ion channels and transporters are, to a large extent, regulated by phosphorylation, also in response to high temperature\u003csup\u003e17–20\u003c/sup\u003e.\u0026nbsp;For example,\u0026nbsp;this occurs in\u0026nbsp;\u003cem\u003eArabidopsis\u003c/em\u003e plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPASEs 1/2 (AHA1/2)\u003csup\u003e11\u003c/sup\u003e, which play a crucial role in high temperature-mediated stomatal aperture\u0026nbsp;dynamics\u003csup\u003e19,21\u003c/sup\u003e.\u0026nbsp;High temperature-activated TOT3/MAP4K4 phosphorylates the C-terminal regulatory domain of AHA1, thereby activating AHA1 and promoting stomatal opening under high\u0026nbsp;temperature\u003csup\u003e11\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eHere, we identified and characterized a mechanism involving the deubiquitylase UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24) that promotes stomatal opening in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e under high temperature. We demonstrate that UBP24 is phosphorylated at serine 360 by the kinase OPEN STOMATA 1 (OST1), which is activated by B4 RAF kinases in response to high temperature stress. This phosphorylation event stabilizes UBP24, enabling deubiquitylation of a plasma membrane H⁺-ATPase to promote stomatal opening. This reveals a novel signalling mechanism that regulates the hydro-active opening of plant stomata under high temperature that evolved in vascular plants. Strikingly, we find that such a heat-responsive protein feature is conserved in the distantly related eukaryote \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, where the UBP24 homolog Ubp3 requires a constitutively negatively charged residue at the equivalent position to support growth during heat shock. Our findings uncover an evolutionarily conserved molecular mechanism in which negative charge, conferred either via phosphorylation or by acidic residues, fine-tunes deubiquitylase function to support adaptive thermoresponsive signaling in eukaryotes. This work highlights how modular charge-based regulation has evolved to maintain cellular performance under thermal stress.\u003c/p\u003e\n\u003cp\u003eTo identify conserved proteins and regulated phosphosites that may mediate high temperature responses, including stomatal dynamics, we conducted a comparative phosphoproteome analysis between \u003cem\u003eTriticum aestivum\u003c/em\u003e (wheat, Cadenza variety) and \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003eseedlings, that were exposed to high temperature (34/35°C), which triggers significant stomatal opening and increased stomatal conductance\u003csup\u003e11\u003c/sup\u003e (Extended Data Fig. 1a and 3b and Supplementary Data 1)\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;By overlapping significantly high temperature-regulated phosphorylation events from \u003cem\u003eArabidopsis\u003c/em\u003e and wheat datasets, we found 8 proteins with increased and 15 proteins with decreased phosphorylation levels in both species (Extended Data Fig. 1b,c). Three of the 8 co-increasing proteins have a conserved phosphorylation site between \u003cem\u003eArabidopsis\u003c/em\u003e and wheat. One of these candidates, TOT3, has been shown to regulate hypocotyl elongation and stomatal opening in response to high\u0026nbsp;temperatures\u003csup\u003e11,23\u003c/sup\u003e.\u0026nbsp;We then focused on another candidate, UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24), which undergoes high temperature-mediated phosphorylation at S360 (Fig. 1a). To validate phosphorylation at S360, we immunoprecipitated UBP24 protein from \u003cem\u003eGFP-UBP24\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e seedlings exposed to 35°C for one hour, and subsequent mass spectrometry analysis pinpointed UBP24 S360 as a reproducible high temperature-upregulated phosphosite (Fig. 1a and Supplementary Data 2). To assess a deeper conservation of S360 in UBP24, we performed an alignment of UBP24 putative orthologs from 55 species within the green lineage (Fig. 1b and Extended Data Fig. 2). In vascular plants, the relevant phosphorylatable serine (S360 in \u003cem\u003eArabidopsis\u003c/em\u003e) is deeply conserved within the RxxS/T motif. Mapping crucial stomatal evolutionary events in the green lineage, such as presence and dynamics of\u0026nbsp;stomata\u003csup\u003e4\u003c/sup\u003e,\u0026nbsp;revealed that the presence of the S360 phosphosite coincides with the evolution of hydroactive stomatal opening (Fig. 1b). This suggests that UBP24 S360 phosphorylation might be associated with regulation of stomatal dynamics in evolution. Indeed, UBP24 was previously shown to play a role in abscisic acid (ABA)-mediated stomatal\u0026nbsp;closure\u003csup\u003e24\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eTo test whether UBP24 is involved in the regulation of stomatal opening under high temperature, we conducted a stomatal bioassay (Extended Data Fig. 3a)\u003csup\u003e25\u003c/sup\u003e.\u0026nbsp;Wild-type (Col-0) stomata exhibited a notable increase in aperture at temperatures of 35°C and 42°C, but not at 28°C, compared to control temperature (21°C), and stomatal aperture was greater at 42°C than at 35°C (Extended Data Fig. 3b). We therefore conducted subsequent stomatal bioassays at 42°C. Both the T-DNA mutant \u003cem\u003eubp24-1\u003c/em\u003e and the CRISPR-Cas9 edited mutant \u003cem\u003eubp24-crispr\u003c/em\u003e showed impaired stomatal opening in the stomatal bioassay at 42°C (Fig. 1c,d and Extended Data Fig. 3c,d), and leaf imprinting followed by scanning electron microscopy corroborated these results (Extended Data Fig. 3e,f). Furthermore, the \u003cem\u003eubp24-1\u003c/em\u003e stomatal aperture phenotype at high temperature could be complemented by a \u003cem\u003eUBP24:YFP-UBP24\u003c/em\u003e construct (Fig. 1e and Extended Data Fig. 3j). In addition, an inactive UBP24(C206S)\u0026nbsp;variant\u003csup\u003e24\u003c/sup\u003e does not rescue the \u003cem\u003eubp24-1\u003c/em\u003e high temperature stomatal aperture phenotype, indicating that activity of the deubiquitylating enzyme UBP24 plays a crucial role in the regulation of stomatal opening under high temperature (Extended Data Fig. 3g,h). Taken together, these results support that UBP24 plays an important role in stomatal dynamics under high temperature.\u003c/p\u003e\n\u003cp\u003eTo test for a role of UBP24 phosphorylation at S360, we introduced a phosphodead UBP24(S360A) or a phosphomimetic UBP24(S360D) variant, driven by the native \u003cem\u003eUBP24\u003c/em\u003e promoter, into the \u003cem\u003eubp24-1\u003c/em\u003e mutant. The stomatal bioassay demonstrated that, like the UBP24(WT), the phosphomimetic UBP24(S360D) variant rescued the \u003cem\u003eubp24-1\u0026nbsp;\u003c/em\u003estomatal opening phenotype (Fig. 1e and Extended Data Fig. 3j). However, the phosphodead UBP24(S360A) variant was unable to rescue this phenotype, regardless of the \u003cem\u003eUBP24\u003c/em\u003e expression level (Fig. 1e and Extended Data Fig. 3i,j). The results indicate that phosphorylation of UBP24 at S360 plays a crucial role in regulating stomatal opening under high temperatures.\u003c/p\u003e\n\u003cp\u003eTo understand how phosphorylation at S360 affects UBP24 under high temperature, we first used AlphaFold2 and molecular dynamics simulation to model different UBP24 variants under high temperature. Given that the predicted N-terminal region of UBP24 is highly disordered, we elected to input solely the UBP24 ubiquitin-specific protease (USP) domain, which contains the S360 phosphorylation site and accounts for 65% of the protein, for molecular dynamics simulation. The simulation results, the root mean square deviation (RMSD) of protein atomic positions and native contacts between alpha carbons, demonstrated that the phosphorylated UBP24 USP domain (pS360) is less sensitive to the high temperatures environment than the unphosphorylated variant (Fig. 1f,g and Extended Data Fig. 4c,d). Regardless of the temperature, 21°C or 42°C, the pS360 variant exhibited a lower energy than the unphosphorylated variant (Fig. 1h,i), further supporting that phosphorylated UBP24 is more stable. The results of the RMSD and native contact simulation analyses demonstrate that the phosphomimetic UBP24(S360D) USP domain exhibits increased stability in comparison with the phosphodead UBP24(S360A) variant (Extended Data Fig. 4a,b,e,f). To confirm if protein stability is regulated by phosphorylation at S360, we exposed \u003cem\u003eubp24-1\u003c/em\u003e transgenic seedlings expressing WT, S360A, or S360D YFP-tagged UBP24 to 35°C for one hour. Here, we used 35°C, because the solubility of UBP24 decreases after extreme high temperature treatment (Extended Data Fig. 4g). The accumulation of UBP24(S360A) was limited under both 21°C and 35°C incubation conditions, while the UBP24(S360D) variant demonstrated a marked increase in protein level at 35°C (Fig. 1j,k). We hypothesised that the increase in UBP24(S360D) protein level may be attributable to slower degradation. Indeed, treating seedlings expressing UBP24(WT) or UBP24(S360D) with cycloheximide (CHX), demonstrated a higher protein stability of the UBP24(S360D) variant compared to UBP24(WT) under control temperature (Fig. 1l,m). These findings suggest that the phosphorylation of S360 in UBP24 stabilises the protein, thereby facilitating stomatal opening under high temperature.\u003c/p\u003e\n\u003cp\u003eSince UBP24 S360 was previously listed as an OST1/SnRK2.6 target\u003csup\u003e26\u003c/sup\u003e,\u0026nbsp;we explored if OST1 phosphorylates UBP24 at S360 in response to high temperature. We showed a high temperature-enhanced interaction between OST1 and UBP24 that was not influenced by ABA (Fig. 2a–c). To confirm that OST1 phosphorylates UBP24, we performed kinase assays. To circumvent the issue of insoluble UBP24 under high temperature (Extended Data Fig. 4g), we enhanced OST1 activity with ABA as an alternative to high temperature in the \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaf epidermis. The \u003cem\u003ein planta\u003c/em\u003e kinase assay demonstrated that the phosphorylation level of UBP24, specifically at S360, increased in the presence of ABA-activated OST1 (Fig. 2d,e and Supplementary Data 4). An \u003cem\u003ein vitro\u003c/em\u003e kinase assay further indicated that recombinant OST1 directly phosphorylates UBP24 S360 (Fig. 2f and Extended Data Fig. 5a). In addition, using transgenic OST1 isolated from \u003cem\u003eArabidopsis\u003c/em\u003e seedlings exposed to high temperature for a maximum of one hour, displayed increased\u0026nbsp;kinase activity with respect to the recombinant UBP24 fragment substrate (Fig. 2i, Col-0). To explore this further, we used the \u003cem\u003eost1-3\u003c/em\u003e mutant, which showed closed stomata under dark\u0026nbsp;conditions\u003csup\u003e27\u003c/sup\u003e and an inability to open stomata after high temperature treatment in our stomatal bioassay (Fig. 2g). We introduced the phosphomimetic UBP24(S360D) variant into \u003cem\u003eost1-3\u003c/em\u003e, which rescued the stomatal opening defect under high temperature (Fig. 2g and Extended Data Fig. 5b). Taken together, our results show that OST1 directly phosphorylates UBP24 at S360 under high temperature to facilitate stomatal opening.\u003c/p\u003e\n\u003cp\u003eWe observed that in our experimental conditions, specifically under darkness, no increase in ABA level, as visualised through the \u003cem\u003eArabidopsis\u003c/em\u003e ABA biosensor lines (nlsABACUS2s\u003csup\u003e28\u003c/sup\u003e),\u0026nbsp; occurred in guard cells following high temperature treatment (Extended Data Fig. 5c). This suggested that the activation of OST1 under high temperature conditions might not require ABA. Indeed, the high temperature-triggered opening of stomata in our stomatal bioassay still occurred in the presence of the ABA biosynthesis inhibitor abamine\u0026nbsp;SG\u003csup\u003e29\u003c/sup\u003e and in the ABA biosynthesis mutant \u003cem\u003eaba2-11\u003c/em\u003e\u003csup\u003e30\u003c/sup\u003e (Extended Data Fig. 5d,e). Taken together, these results suggest that high temperature-mediated activation of OST1 does not require ABA. Previously, it was shown that B4 RAF kinases can activate SnRK2s under osmotic stress in an ABA-independent\u0026nbsp;manner\u003csup\u003e31\u003c/sup\u003e,\u0026nbsp;and RAF24 phosphorylates OST1 under osmotic\u0026nbsp;stress\u003csup\u003e32\u003c/sup\u003e.\u0026nbsp;We noticed that RAF20 and RAF24, which belong to the B4 clade, are more phosphorylated at high temperature (Extended Data Fig. 5f and Supplementary Data 1). We thus hypothesised that B4 RAF kinases phosphorylate and activate OST1 at high temperatures to facilitate stomatal opening. Indeed, a septuple null mutant for the entire B4 family of RAF kinases (\u003cem\u003eraf\u003c/em\u003e)\u003csup\u003e32,33\u003c/sup\u003e impairs stomatal opening and this phenotype can be complemented by either RAF20 or RAF24 (Fig. 2h). RAF20 (Extended Data Fig. 5g) and\u0026nbsp;RAF24\u003csup\u003e32\u003c/sup\u003e,\u0026nbsp;can directly phosphorylate OST1 in the activation loop at S175. With respect to its substrate UBP24, mCherry-OST1 isolated from \u003cem\u003eraf\u003c/em\u003e seedlings exhibited lower kinase activity than the one isolated from WT seedlings and failed to be activated after high temperature treatment (Fig. 2i,j). These results support that B4 RAF kinases activate OST1 under high temperature in an ABA-independent manner, leading to OST1-mediated phosphorylation of UBP24.\u003c/p\u003e\n\u003cp\u003eTo identify interactors and possible deubiquitylation targets of UBP24, we performed immunoprecipitation followed by mass spectrometry detection (IP-MS/MS) on an \u003cem\u003eArabidopsis\u003c/em\u003e line overexpressing GFP-tagged UBP24 under control temperature (Fig. 3a and Supplementary Data 2). One of the putative interactors, the proton pump AHA1, is a major regulator of blue light and high temperature-mediated stomatal opening\u003csup\u003e11,15\u003c/sup\u003e, making this a promising candidate. Indeed, AHA1 protein levels in Col-0 \u003cem\u003eArabidopsis\u003c/em\u003e seedlings decreased under high temperature (Fig. 3k,l). Furthermore, AHA1 interacted with UBP24 (Fig. 3b,c), and a decrease in proton pump activity in \u003cem\u003eubp24-1\u003c/em\u003e roots associated with reduced primary root growth were observed compared to Col-0 (Extended Data Fig. 6a–d). To test if UBP24 stabilises the AHA1 protein level via deubiquitylation, we co-expressed RFP-AHA1 with UBP24(WT) or UBP24(C206S) in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. After treatment with concanamycin A (concA) to block vacuolar degradation, a decrease in AHA1 protein levels, an increase in AHA1 ubiquitylation and an increase in AHA1 internalisation were observed for UBP24(C206S) compared to UBP24(WT) (Fig. 3d–g). In line with these results, \u003cem\u003eubp24-1\u003c/em\u003e seedlings expressing \u003cem\u003eRFP-AHA1\u003c/em\u003e displayed a substantial decrease in RFP-AHA1 protein expression, which was not caused by transcriptional regulation (Fig. 3j‒l and Extended Data Fig. 6h). Together, these results support that UBP24 deubiquitylates AHA1 under high temperature to prevent its degradation.\u003c/p\u003e\n\u003cp\u003eA number of ubiquitinome analyses have demonstrated that AHA1 and AHA2 can be ubiquitin-modified at several lysine sites\u003csup\u003e34,35\u003c/sup\u003e. The distribution of these sites is principally confined to the N-terminal catalytic A domain, the nucleotide binding P-N domain, and the C-terminal regulatory R domain (Extended Data Fig. 6e)\u003csup\u003e36\u003c/sup\u003e.\u0026nbsp;Given that the activities of AHA1 and AHA2 are primarily regulated by the R domain, we replaced five lysine residues located within the AHA1 R domain with arginine, thereby generating a ubiquitylation-deficient AHA1(5KR) variant (Extended Data Fig. 6e). To ascertain the potential impact of the ubiquitylation on AHA1, we expressed \u003cem\u003eRFP-AHA1\u003c/em\u003e and single lysine K48 or K63 ubiquitin in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Following the selective enrichment of K48- or K63-linkage ubiquitylated proteins, the AHA1(5KR) resulted in a decrease in ubiquitylation for K63-linkage (Extended Data Fig. 6f,g). We thus hypothesised that the K63 ubiquitylation of the R domain of AHA1 regulates AHA1 internalisation and\u0026nbsp;stability\u003csup\u003e37,38\u003c/sup\u003e.\u0026nbsp;By expressing AHA1(WT) and AHA1(5KR) in \u003cem\u003eN. benthamiana\u0026nbsp;\u003c/em\u003eleaves followed by concA treatment, we found that AHA1 internalisation was reduced, which may prevent the subsequent degradation of AHA1 (Fig. 3h,i). Additionally, the AHA1(5KR) mutation partially restored the AHA1 protein levels in \u003cem\u003eubp24-1\u0026nbsp;\u003c/em\u003e(Fig. 3j,k). Furthermore, the stomatal opening defect in \u003cem\u003eubp24-1\u003c/em\u003e was rescued by AHA1(5KR) under high temperatures, but not by AHA1(WT) (Fig. 3m and Extended Data Fig. 6i). These findings show that UBP24 stabilises AHA1 at high temperatures through deubiquitylation, thereby contributing to stomatal opening.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eProtein phosphorylation, which introduces a negative charge on proteins, can impact protein-protein interactions and protein conformations, ultimately affecting their function\u003csup\u003e39,40\u003c/sup\u003e.\u0026nbsp;Our results demonstrate that OST1-mediated phosphorylation of UBP24 at S360, impacting UBP24 molecular charge and stability, is a key component of stomatal dynamics in \u003cem\u003eArabidopsis\u003c/em\u003e. Of note, Ubp3, a \u003cem\u003eS. cerevisiae\u003c/em\u003e ortholog of UBP24, is required for growth after a high temperature shock, since \u003cem\u003eS. cerevisiae\u003c/em\u003e without Ubp3 grew slower after high temperature shock (Extended Data Fig. 7)\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;Interestingly, at the position corresponding to UBP24 S360, animal (Usp10) and \u003cem\u003eS. cerevisiae\u003c/em\u003e (Ubp3) orthologs of UBP24 contained the negatively charged amino acids aspartic or glutamic acid, corresponding to phosphomimetic amino acids (Fig. 4a and Extended Data Fig. 2 and Supplementary Data 3). We furthermore observed that 8 \u003cem\u003eSaccharomyces\u003c/em\u003e species with distinct thermal preferences did not display any variation at the E691 (in \u003cem\u003eS.\u003c/em\u003e \u003cem\u003ecerevisiae\u003c/em\u003e) position (Fig. 4b, lower part). Moreover, in an adaptive evolution experiment in which the temperature was gradually increased from 25°C to 40°C every 50 generations, for a total of 600 generations or until strains went extinct, there was no variation at the E691 position (Fig. 4b, upper part)\u003csup\u003e42\u003c/sup\u003e.\u0026nbsp;This supports that retaining E691 in Ubp3 is important for thermoresponsive yeast growth. To test if the presence of the negatively charged amino acid is indeed important for thermoresponsive growth, we generated a \u003cem\u003eS. cerevisiae\u0026nbsp;\u003c/em\u003eyeast line replacing E691 of Ubp3 with alanine. Growth of the E691A yeast was slower at all temperatures tested compared to the WT, with or without a high temperature shock, but this was most obvious at the suboptimal temperature of 20°C (Fig. 4c,d). Molecular dynamics simulations of the Ubp3(WT) and the Ubp3(E691A) USP domain showed that Ubp3(E691A) increased energy regardless of temperature, suggesting instability of the E691A variant (Fig. 4e,f). Taken together, these results demonstrate that the charge of UBP24 at S360, which is mediated through phosphorylation, or Ubp3 at E691, in \u003cem\u003eArabidopsis\u003c/em\u003e or yeast, respectively, is crucial to respond to or grow at higher temperatures.\u003c/p\u003e\n\u003cp\u003eOur study reveals that the evolutionary tuning of molecular charge state of UBP24 through protein phosphorylation shapes eukaryotic responses to high temperature. The majority of evolutionary studies focus on the presence or absence of specific genes\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;However, this ignores the evolutionary effect on finetuning of protein activities through specific amino acid exchanges. While putative UBP24 orthologs are present in green lineage organisms and while the origin of the UBP24 regulatory kinase OST1 can be traced back to streptophyte\u0026nbsp;algae\u003csup\u003e43\u003c/sup\u003e, we showed that the presence of a charge-tuneable site coincides with the evolution of hydro-active opening of stomata in vascular plants. OST1-mediated phosphorylation and subsequent stabilisation of UBP24 leads to a stabilisation of AHA1 on the plasma membrane by deubiquitylation, facilitating stomatal opening. A role for OST1 in promoting stomatal opening is at odds with the current understanding of the positive role of OST1 in ABA-induced stomatal\u0026nbsp;closure\u003csup\u003e14,44,45\u003c/sup\u003e; but, our results indicate that the role of OST1 in stomatal opening under high temperature is ABA-independent and relies on activation by B4 RAF kinases. In addition, we found that UBP24 orthologs outside the green lineage contain an aspartic or glutamic acid instead of a phosphorylation-mediated tuneable site and we demonstrated that this conserved Ubp3 charge is necessary for optimal thermoresponsive growth of yeast. Taken together, our results pinpoint that a role in thermoresponsiveness of UPB24 protein orthologs is deeply conserved and that the charge on these proteins, either being tuneable through kinases and phosphatases in vascular plants or being fixed in the genome to encode an aspartic or glutamic acid, is a key component for thermoresponsiveness of these proteins.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and growing conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the \u003cem\u003eArabidopsis\u003c/em\u003e mutants and transgenic lines were in Columbia-0 (Col-0) ecotype. The \u003cem\u003eubp24-1\u003c/em\u003e\u003csup\u003e24\u003c/sup\u003e, \u003cem\u003eost1-3\u003c/em\u003e\u003csup\u003e46\u003c/sup\u003e, nlsABACUS2-100n\u003csup\u003e28\u003c/sup\u003e, nlsABACUS2-400n\u003csup\u003e28\u003c/sup\u003e, \u003cem\u003eaba2-11\u003c/em\u003e\u003csup\u003e47\u003c/sup\u003e, \u003cem\u003eost2-2D\u003c/em\u003e\u003csup\u003e48\u003c/sup\u003e, \u003cem\u003eraf\u003c/em\u003e\u003csup\u003e32,33\u003c/sup\u003e,\u0026nbsp;\u003cem\u003eRAF20:RAF20-sYFP\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e,\u003cem\u003e\u0026nbsp;RAF24:RAF24-sYFP\u003c/em\u003e\u003csup\u003e33\u003c/sup\u003e were described previously. \u003cem\u003eArabidopsis\u003c/em\u003e seeds were surface\u0026nbsp;sterilised\u0026nbsp;by 70% ethanol followed by 1% sodium hypochlorite solution.\u0026nbsp;After washing with\u0026nbsp;sterilised\u0026nbsp;water, seeds were incubated at 4\u0026deg;C under darkness for 2 days.\u0026nbsp;Sterilised\u0026nbsp;seeds were sown on \u0026frac12; Murashige and Skoog (MS) medium containing 1% sucrose, 0.5% MES and 0.8% (w/v) plant agar, pH adjusted to 5.7 with KOH. Seeds were grown horizontally under long-day photoperiod (16\u0026thinsp;h light/8\u0026thinsp;h dark, intensity 70 to100 \u0026mu;mol\u0026thinsp;m\u003csup\u003e\u0026minus;2\u003c/sup\u003e\u0026thinsp;s\u003csup\u003e\u0026minus;1\u003c/sup\u003e) at 21\u0026deg;C. For stomatal bioassays, seedlings after 10-day growth on plates were transfered to soil and grown under long-day photoperiod (16\u0026thinsp;h light/8\u0026thinsp;h dark, intensity 70 to100 \u0026mu;mol\u0026thinsp;m\u003csup\u003e\u0026minus;2\u003c/sup\u003e\u0026thinsp;s\u003csup\u003e\u0026minus;1\u003c/sup\u003e) at 21\u0026deg;C before analysis. For phosphoproteome analysis, seeds were grown vertically under continuous light (intensity 100 \u0026mu;mol\u0026thinsp;m\u003csup\u003e\u0026minus;2\u003c/sup\u003e\u0026thinsp;s\u003csup\u003e\u0026minus;1\u003c/sup\u003e) at 21\u0026deg;C before harvesting. For high temperature treatment, \u003cem\u003eArabidopsis\u003c/em\u003e seedlings grown as described above were incubated at 35\u0026deg;C for 1 hour before analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;mutants and constructs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails on generating the CRISPR-edited \u003cem\u003eubp24\u003c/em\u003e mutant (\u003cem\u003eubp24-crispr\u003c/em\u003e) and on generating and expressing constructs can be found in the Supplementary Information.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYeast strain construction\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and growth measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe prototrophic diploid version of\u003cem\u003e\u0026nbsp;Saccharomyces cerevisiae\u003c/em\u003e strain S288C\u003csup\u003e49\u003c/sup\u003e was used for \u0026nbsp; genome editing, and details on constructing the strains can be found in the Supplementary Information. For growth measurements, yeast cells were initially inoculated at a high dilution into YP5%GLU liquid medium, composed of 1% (w/v) yeast extract, 2% (w/v) peptone, and 5% (w/v) glucose, and cultured in 96-well microplates (655180, Greiner Bio-One) at 30\u0026deg;C with continuous agitation at 900 rpm until cultures reached the early exponential phase, as determined by an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of approximately 0.1. Cells were then further diluted in fresh YP5%GLU medium and regrown to an OD\u003csub\u003e600\u003c/sub\u003e of 0.1 to ensure uniform physiological state prior to subsequent treatments. Following growth, cell suspensions were washed three times with YP medium (1% yeast extract, 2% peptone) to remove residual glucose and metabolites, and resuspended in 150 \u0026mu;L of YP medium. Each sample was divided equally, with 75 \u0026mu;L subjected to high temperature shock at 45\u0026deg;C for 30 minutes in PCMT Thermoshaker (Grant-bio) with continuous agitation at 400 rpm, while the remaining aliquot was maintained at room temperature as a control. For growth kinetics assessment, yeast suspensions were diluted 1:50 in 150 \u0026mu;L of YP5%GLU medium and transferred to the Bioscreen C automated growth analysis system (Oy Growth Curves Ab). Growth experiments were conducted at 20\u0026deg;C, 25\u0026deg;C, and 30\u0026deg;C, with each condition including triplicate biological replicates and blank controls (medium only). Optical density at 600 nm was measured every 15 minutes for up to 5 days. Continuous shaking with the medium amplitude was used. Data were processed using BioScreener software, with blank OD\u003csub\u003e600\u003c/sub\u003e values subtracted to normalise growth curves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStomatal bioassay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe assay was adapted from a previously published method\u003csup\u003e25\u003c/sup\u003e with some modifications. The fourth to sixth true leaves were detached from 4-week-old \u003cem\u003eArabidopsis\u003c/em\u003e plants grown on soil. The abaxial epidermal peels, prepared by the sandwich method with label tape and Scotch tape, were floated on the stomatal closing buffer (50 mM KCl, 0.1 mM CaCl\u003csub\u003e2\u003c/sub\u003e and 10 mM MES, pH adjusted to 6.5 with KOH). After incubation for 2 hours at 21\u0026deg;C in the dark, the peels were transferred to dark incubators at 21\u0026deg;C, 28\u0026deg;C, 35\u0026deg;C or 42\u0026deg;C for 2 hours. For abamine SG treatment, 100 \u0026micro;M abamine SG in dimethyl sulfoxide (DMSO), or the same volume of DMSO for mock, was added to the stomatal closing buffer prior to the first incubation at 21\u0026deg;C in the dark. The peels were then mounted with stomatal closure buffer and observed using an Olympus BX51 upright microscope with 40x objective lens and DIC modules. The stomatal apertures (stomatal width divided by length) were measured by Fiji (ImageJ) software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and cycloheximide assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine protein expression levels in \u003cem\u003eArabidopsis\u003c/em\u003e transgenic lines, three to five 10-day-old seedlings were harvested, frozen in liquid nitrogen and then ground with stainless steel beads. The material powder was incubated with 1x Laemmli sample buffer (62.5 mM Tris base, 2% SDS and 10% glycerol, pH adjusted to 6.8 with HCl) for 5 minutes at room temperature. For UBP24 protein variants, samples were incubated at 70\u0026deg;C for 10 minutes prior to loading on the gel. For AHA1 protein variants, samples were loaded directly onto the gel without high temperature incubation. SDS-PAGE was performed on 4‒20% mini-PROTEAN TGX stain-free precast gels at 150 V for 50 minutes. Before transfer to PVDF membrane, the gels were visualised using the ChemiDoc Imaging System (Biorad) for loading controls. Western blotting was performed as follows.\u0026nbsp;Transfer was performed using Trans-Blot Turbo mini PVDF transfer packs on the Trans-Blot Turbo Transfer System (Biorad). The PVDF membranes were then blocked with 5% skim milk in 1x PBST or TBST buffer. The membranes were incubated with antibodies, 1:5000 anti-GFP HRP, 1:2000 anti-RFP or 1:5000 anti-mouse HRP. The signal was detected using SuperSignal West Femto maximum sensitivity substrate and visualised on the ChemiDoc Imaging System (Biorad). The signal intensity was measured by Fiji (ImageJ) software. For cycloheximide assays, the 10-day-old \u003cem\u003eArabidopsis\u003c/em\u003e seedlings were transferred to 12-well plates containing \u0026frac12; Murashige and Skoog (MS) medium containing 1% sucrose, 0.5% MES and 200 \u0026micro;M cycloheximide, and pH adjusted to 5.7 with KOH. At the indicated time point, seedlings were harvested and used for protein level detection. Detection procedures were described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation (co-IP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinal expression vectors for co-IP were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain C58C1 by heat shock. \u003cem\u003eAgrobacteria\u003c/em\u003e containing the indicated vectors or P19 were suspended in the infiltration buffer (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES, pH adjusted to 5.7 with KOH) and then infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves with the combination described in figures. After 60 hours, the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e plants were harvested and ground into fine powder using liquid nitrogen. Proteins were extracted with extraction buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 1x cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack protease inhibitor cocktail, pH adjusted to 7.5 with HCl). After centrifugation to remove debris, the supernatant was transferred to new tubes containing GFP-Trap magnetic agarose. The mixture was incubated for 1 hour at 4\u0026deg;C with top-bottom rotating. The GFP-Trap magnetic agarose beads were collected using DynaMag-2 magnet (Invitrogen). After washing three times with the extraction buffer, the beads were resuspended in 1x NuPAGE LDS sample buffer and then incubate at 70\u0026deg;C for 10 minutes. The beads were trapped, and the supernatant were loaded on the gel for SDS-PAGE and western blotting. The procedure for protein band detection was described above. For HA-tagged OST1, 1:10000 anti-HA HRP antibody was used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro kinase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinal expression vectors for recombinant protein expression (see Supplementary Information for details) were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e strain Rosetta(DE3) by electroporation. The positive colonies were inoculated in Luria-Bertani medium. After reaching the log phase, the cultures were treated with 0.5 mM isopropyl \u0026beta;-D-thiogalactopyranoside (IPTG) and incubated under 18\u0026deg;C for 16 hours. The cells were harvested, sonicated and centrifuged. The soluble fractions were incubated with Glutathione Sepharose 4B or PureCube 100 Ni-INDIGO agarose for purification of target proteins. To enrich OST1 from \u003cem\u003eArabidopsis\u003c/em\u003e transgenic lines, the total protein from wild-type or \u003cem\u003eraf\u003c/em\u003e mutant containing \u003cem\u003eOST1:mCherry-OST1\u003c/em\u003e was extracted with the extraction buffer extraction buffer (50 mM Tris base, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1x cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack protease inhibitor cocktail and PhosSTOP, pH adjusted to 7.5 with HCl). After removing the debris by centrifugation, the supernatant was incubated with RFP-Trap magnetic agarose at 4\u0026deg;C for 1 hour with top-bottom rotating. The RFP-Trap magnetic agarose beads were collected using DynaMag-2 magnet (Invitrogen). After three washes with extraction buffer, the protein on the beads was eluted with 200 mM glycine-HCl (pH 2.5), and then neutralised with 1 M Tris-HCl (pH 10.4). The \u003cem\u003ein vitro\u003c/em\u003e kinase assay was performed on either (\u0026gamma;-32P)ATP-radioactive or adenosine-5-o-(3-thio)-triphosphate (ATP\u0026gamma;S)-based method. The (\u0026gamma;-32P)ATP-radioactive method was described previously\u003csup\u003e11\u003c/sup\u003e. Briefly, the 3:1 (w/w) ratio of substrate and kinase protein were incubated at 30\u0026deg;C for 1 hour in kinase assay buffer (10 mM Tris base, 2 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.5 mM DTT, 5 \u0026micro;M ATP and 5 \u0026micro;Ci [\u0026gamma;-32P]ATP, pH adjusted to 7.5 with HCl). The reaction was stopped by adding 4x NuPAGE LDS sample buffer and 10x NuPAGE sample reducing agent to a final concentration 1x and then incubated at 70\u0026deg;C for 10 minutes. SDS-PAGE was performed. Protein bands were visualised by autoradiography for phosphorylation and InstantBlue Coomassie protein stain for loading control. In the ATP\u0026gamma;S-based method, the kinase assay buffer was replaced with another buffer (20 mM Tris base, 100 mM NaCl, 2 mM MnCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT and 330 \u0026micro;M ATP\u0026gamma;S,, pH adjusted to 7.5 with HCl). After the incubation at 30\u0026deg;C for half (for OST1 from \u003cem\u003eArabidopsis\u003c/em\u003e seedlings) or 1 (for recombinant OST1 and RAF20) hour, the reaction was stopped with 24 mM EDTA and alkylated with 100 mM p-Nitrobenzyl mesylate (PNBM) at 25\u0026deg;C for 1 hour. NuPAGE LDS sample buffer and NuPAGE sample reducing agent were added and then incubated at 70\u0026deg;C for 10 minutes before SDS-PAGE. Protein bands were visualised by a 1:5000 anti-thiophosphate ester antibody and a 1:10000 anti-rabbit HRP antibody for phosphorylation, and the signal of stain-free gel for loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn planta deubiquitylation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe method for enrichment of ubiquitylated proteins was similar to the one for co-IP described above. Briefly, the agroinfiltrated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were harvested after 60 hours. The ground materials were incubated with extraction buffer. The soluble protein extract was incubated with Pierce anti-HA magnetic beads at 4\u0026deg;C for 1 hour. After washing three times with the extraction buffer, the beads were resuspended in 1x NuPAGE LDS sample buffer and then incubate at 70\u0026deg;C for 10 minutes. The supernatant was loaded on the gel for SDS-PAGE and western blotting. The procedure for protein band detection was described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfocal microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor imaging with a Leica SP8 inverted confocal laser scanning microscope, objective lens HC PL APO CS2 40x/1.10 water and HC PL APO CS2 63x/1.20 water, white light laser (1.5 mW, range from 470 to 670 nm), detectors HyD and HyD SMD2, and software LASX were used. For imaging with a Leica Stellaris 5 LiAchroic upright confocal laser scanning microscope, objective lens HC PL APO 40x/1.10 W CORR CS2 water and HC PL APO 63x/1.20 W CORR CS2 water, diode lasers 488 (20 mW) and 561 (20 mW) nm, detectors HyD S, and software LASX were used. For imaging with a Zeiss inverted LSM710 confocal laser scanning microscope, objective lens C-Apochromat 40x/NA 1.20 W Korr M27 water and C-Apochromat 63x/NA 1.20 W Korr M27 water, argon laser 515 nm and diode lasers 559 nm, detectors PMT, and software Zen black were used.\u003c/p\u003e\n\u003cp\u003eEYFP was excited by a 514 (white light) or 515 (argon) nm laser, and the emission was collected from 520 to 555 nm (with lifetime gating from 1 to 6 ns when using the Leica SP8). mRFP1 was excited by a 561 (white light) or 559 (argon) nm laser, and the emission was collected from 566 to 620 nm (with lifetime gating from 1 to 6 ns when using the Leica SP8). The laser power and detector gain were optimised and maintained constant throughout the experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBimolecular fluorescence complementation (BiFC)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinal expression vectors for BiFC were transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain C58C1 by heat shock. Agrobacteria containing the BiFC vector or P19 were suspended in the infiltration buffer (10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM MES, pH adjusted to 5.7 with KOH) and then co-infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. After 60 hours, the \u003cem\u003eN. benthamiana\u003c/em\u003e plants were treated 1 hour at 40\u0026deg;C for UBP24-OST1 interaction detection. The florescence signal was imaged from leaf abaxial epidermis with the confocal setting described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImaging of RFP-AHA1\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor observing AHA1 endocytosis, \u003cem\u003e35S:GFP-UBP24\u003c/em\u003e, \u003cem\u003e35S:GFP-UBP24(C206S)\u003c/em\u003e, \u003cem\u003e35S:RFP-AHA1\u003c/em\u003e or 35S\u003cem\u003e:RFP-AHA1(5KR)\u003c/em\u003e was infiltrated into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves\u0026nbsp;with the combinations showed in the figure. After 1 \u0026micro;M concanamycin A treatment overnight, the abaxial leaf epidermis was observed under a confocal microscope with the setting described above. For observing AHA1 protein expression, the 10-day-old \u003cem\u003eArabidopsis\u003c/em\u003e transgenic lines expressing RFP-AHA1 variants in wild-type or \u003cem\u003eubp24-1\u003c/em\u003e background were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction and sample preparation for phosphoproteomics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedure has been described previously\u003csup\u003e23\u003c/sup\u003e with some modifications. For the high temperature-treated \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, seedlings from four biological replicates were harvested and ground to a fine powder using in liquid nitrogen. The samples were homogenised in the buffer (50\u0026thinsp;mM Tris base, 0.1\u0026thinsp;M KCl, 30% sucrose, 5\u0026thinsp;mM EDTA, 1\u0026thinsp;mM DTT, 1x cOmplete\u0026trade; ULTRA Tablets, EDTA-free, protease inhibitor cocktail and PhosSTOP, pH adjusted to 8 by HCl), sonicated on ice for 30 seconds with 2-second on/1-second off cycles, and centrifuged at 4\u0026deg;C for 15\u0026thinsp;min at 3220 g. The soluble proteins were precipitated using methanol/chloroform method and resuspended in 8 M urea dissolved in 50 mM triethylammonium bicarbonate buffer (TEAB, pH 8). Cysteine alkylation was performed using 15\u0026thinsp;mM tris(carboxyethyl)phosphine (TCEP) and 30\u0026thinsp;mM iodoacetamide for 2 hours at 30\u0026deg;C. The protein solutions were diluted with 50 mM TEAB (pH 8) to 1 M urea. 200 \u0026micro;g proteins determined by the Pierce 660 nm protein assay kit were firstly pred-igestedpredigested with EndoLysC (SignalChem) at an enzyme-to-substrate ratio of 1:5 (w:w) for 2\u0026thinsp;hours and then digested with sequencing grade modified trypsin overnight at an enzyme-to-substrate ratio of 1:10 (w:w). Peptides were desalted using C18 ODS\u0026nbsp;SampliQ solid phase extraction (SPE) columns according to the manufacturer\u0026rsquo;s guidelines, eluted to 70% (v/v) acetonitrile (ACN) containing 0.1% (v/v) trifluoroacetic acid (TFA), and dried by vacuum. Dried peptide pellets were dissolved in 80% (v/v) ACN containing 6% (v/v) TFA and incubated with 1\u0026thinsp;mg MagReSyn Ti-IMAC magnetic microspheres for 20 minutes with continuous agitation. After washing the microspheres once with 60% ACN, 1% TFA, 200\u0026thinsp;mM NaCl and twice with 60% ACN, 1% TFA, the phosphopeptides were eluted with elution buffer (40% ACN, 5% NH\u003csub\u003e4\u003c/sub\u003eOH) followed by acidification to pH 3 with 100% formic acid. The peptides were dried by vacuum for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS analysis, database searching and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedure has been described previously\u003csup\u003e23\u003c/sup\u003e with some modifications. Samples were analysed by LC-MS/MS using an Ultimate 3000 RSLC nano LC (Thermo Fisher Scientific) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The samples were loaded on an analytical (made in-house, 25 cm column, 1.9 \u0026micro;m beads, 75\u0026thinsp;\u0026mu;m internal diameter, Dr. Maisch, Ammerbuch-Entringen, Germany) with solvent 0.1% TFA in water. The samples were separated by solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in water/ACN, 20/80 [v/v]) with a linear gradient from solvent A/solvent B, 98/2 (v/v) to solvent A/solvent B, 45/55 (v/v) over 2.5 hour at 300\u0026thinsp;nL\u0026thinsp;min\u003csup\u003e\u0026minus;1\u003c/sup\u003e flow rate followed by a 5-minutes wash till 99% of solvent B. The MS setting was identical as previously described. The phosphoproteomics data of high temperature-treated \u003cem\u003eArabidopsis\u003c/em\u003e seedlings produced in this study have been deposited to the ProteomeXchange Consortium via the PRIDE\u003csup\u003e50\u003c/sup\u003e partner repository with the dataset identifier PXD066011. The spectra were searched using \u0026nbsp;MaxQuant (version 2.1.4.0) on a high performance computer (HPC-UGent) against a proteome database Araport11\u003csup\u003e51\u003c/sup\u003e for \u003cem\u003eArabidopsis thaliana\u003c/em\u003e or \u003cem\u003eNicotiana tabacum\u0026nbsp;\u003c/em\u003eprotein sequences from UniProt\u003csup\u003e52\u003c/sup\u003e (Proteome ID: UP000084051) combining with particular protein sequences from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. The \u0026ldquo;Phospho(STY).txt\u0026rdquo; output filewasimputed by PhosR, analyzed by Perseus (version 2.0.10.0).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn planta kinase assay and immunoprecipitation followed by LC-MS/MS (IP-MS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein enrichment method is similar to the method used in co-IP. For the \u003cem\u003ein planta\u003c/em\u003e kinase assay, the agroinfiltrated \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were treated with DMSO (mock) or 50 \u0026micro;M abscisic acid in infiltration buffer for 2 hours before harvested. The extraction buffer was replaced to 150 mM Tris base, 150 mM NaCl, 10% glycerol, 10 mM EDTA, 1% NP-40, 10 mM DTT, 1x cOmplete\u0026trade; ULTRA Tablets, EDTA-free, protease inhibitor cocktail and PhosSTOP, pH adjusted by HCl to 7.5. The wash buffer was replaced to 20 mM Tris base, 150 mM NaCl and 0.5% NP-40, pH adjusted by HCl to 7.5. For immunoprecipitation GFP-UBP24 from \u003cem\u003eArabidopsis\u003c/em\u003e transgenic lines, the extraction buffer was the same as described in CoIP. The wash steps were performed with the extraction buffer. After the washing steps, both experiments were followed by washing GFP-Trap magnetic beads once with 50 mM TEAB (pH 8). The on-bead digestion was performed by 0.5 \u0026micro;g trypsin in 50 mM TEAB. The peptides in the supernatant were alkylated on cysteine residues using 10\u0026thinsp;mM TCEP and 30\u0026thinsp;mM iodoacetamide. After reduction with 5 mM DTT, the peptide solution was treated with an additional 0.5 \u0026micro;g of trypsin and digested overnight. Digestion was stopped by adding TFA to a final concentration of 1%. Desalting was performed by OMIX C18 pipette tips according to the manufacturer\u0026rsquo;s guidelines, and elution in 60% ACN, 40% water, 0.1% TFA. The samples were vacuum dried and analysed by LC-MS/MS as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein sequence alignment and phylogenetic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proteome databases of selected species were obtained from Araport 11\u003csup\u003e51\u003c/sup\u003e, NCBI\u003csup\u003e53\u003c/sup\u003e, UniProt\u003csup\u003e52\u003c/sup\u003e, PLAZA 5.0\u003csup\u003e54\u003c/sup\u003e and ORCAE\u003csup\u003e55\u003c/sup\u003e.\u0026nbsp;The \u003cem\u003eArabidopsis thaliana\u003c/em\u003e UBP24 protein sequence from Araport 11 was used for BLASTp analysis.\u0026nbsp;When multiple UBP24 orthologs were found, the protein with the lowest E-score was selected. \u0026nbsp;The entire protein sequences of the UBP24 orthologs were used for multiple sequence alignment and neighbour-joining phylogenetic relationships analysis using Clustal Omega. The results were visualised\u0026nbsp;using\u0026nbsp;Interactive Tree Of Life (iTOL) version 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSaccharomyces\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;Ubp3 sequences and alignment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate amino acid variation in the Ubp3 proteins across eight species of the \u003cem\u003eSaccharomyces\u003c/em\u003e genus with different thermal profiles (\u003cem\u003eS. eubayanus, S. uvarum, S. arboricola, S. kudriavzevii, S. mikatae, S. jurei, S. paradoxus, and S. cerevisiae\u003c/em\u003e), we analysed both ancestral and experimentally evolved strains from a previously published study\u003csup\u003e56\u003c/sup\u003e. Ancestral strains correspond to natural isolates and were used as starting points for thermal evolution adaptation. Evolved strains were derived from these ancestors exposed to gradually increasing temperatures from 25\u0026ordm;C to 40\u0026ordm;C every 50 generations for a total of 600 generations, or until they were extinct\u003csup\u003e56\u003c/sup\u003e. We reconstructed the UBP3 coding sequences using whole-genome resequencing data. Reads from each strain were previously mapped to species-specific reference genomes, and variant calling was performed using GATK version 4.3.0.0\u003csup\u003e57\u003c/sup\u003e. Using BCFtools consensus\u003csup\u003e58\u003c/sup\u003e, we generated consensus genome sequences for each strain by applying the variant calls to the reference sequence. Genomic coordinates of the \u003cem\u003eUBP3\u003c/em\u003e gene were retrieved from the reference genome annotations, and the corresponding nucleotide sequences were extracted using BEDtools getfasta\u003csup\u003e59\u003c/sup\u003e. Protein sequences were inferred from the nucleotide sequences using NCBI\u0026rsquo;s ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) tool, selecting the longest open reading frame consistent with the annotated \u003cem\u003eUBP3\u003c/em\u003e ORF in \u003cem\u003eS. cerevisiae\u003c/em\u003e S288C reference strain. Multiple sequence alignment of the predicted protein sequences was performed using MUSCLE \u0026nbsp;(https://www.ebi.ac.uk/jdispatcher/msa/muscle?stype=protein) with default parameters. Amino acid differences between strains were identified and visualised across the alignment. The reconstructed UBP3 nucleotide and protein sequences used in this study are available through GenBank accession numbers PV855420-PV855652.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein structure prediction and molecular dynamics (MD) simulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe structure of the wild-type UBP24 USP domain was predicted by AlphaFold2 with default settings. The predicted structure underwent mutagenesis, resulting in three different variants: S360A, S360D, and S360-phosphorylation (pS360). This mutagenesis and solvate with TIP3P water molecules\u003csup\u003e60\u003c/sup\u003e and 150 mM NaCl was facilitated by the CHARMM-GUI. All-atom MD simulations were performed using NAMD 3.0b6 with the CHARMM36m force field. The solvated protein models were minimised for 1000 steps followed by simulation under the NVT (constant number of atoms, volume and temperature) system at 295 K or 337 K using a Langevin thermostat. Four independent 100 ns simulations of all variant UBP24 USP domain were performed with structure saving per 200 ps. The simulation results were analysed by Python package MDAnalysis. The RMSD, free energy and native contact was calculated by C-alpha.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiagram plotting, statistics and reproducibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the figures were generated by Python package matplotlib and seaborn and the layout composition was done in Inkscape. All the statistical analysis were performed by Microsoft Excel 2019 or R package stats, emmean and multcomp. All calculated \u003cem\u003ep\u003c/em\u003e values and associated statistical outputs are provided in the Source data. No sample size predetermination was used. During the experiments and outcome assessment, investigators were not blinded to the allocation. Each experiment was repeated at least twice with similar results.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are available in the Article, Supplementary information and Source data. Mass spectrometry phosphoproteomics data of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e are available via ProteomeXchange with identifier\u0026nbsp;PXD066011. The nucleotide and protein sequences of \u003cem\u003eSaccharomyces\u003c/em\u003e species used in this study are available through GenBank accession numbers PV855420-PV855652.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank A. M. Jones (University of Cambridge) for sharing nlsABACUS2 lines, D. Weijers (Wageningen University) for sharing \u003cem\u003eraf\u003c/em\u003e and RAF complementing Arabidopsis seeds and E. Farmer (University of Lausanne) for sharing \u003cem\u003eost2-2D\u003c/em\u003e seeds. We acknowledge the VIB Proteomics Core for mass spectrometry analysis. We thank Carina Braeckman (VIB) for helping with \u003cem\u003eArabidopsis\u003c/em\u003e transformation. We are grateful to Bert De Rybel and Daniel Van Damme (VIB) for their comments on the manuscript. We acknowledge funding from\u0026nbsp;the Research Foundation - Flanders (FWO.OPR.2019.0009.01 to I.D.S.),\u0026nbsp;VIB (to S.L.Y and I.D.S.), the Knut and Alice Wallenberg Foundation (to R.S.), Taiwanese Government Scholarship to Study Abroad (to S.L.Y.), UGent BOF postdoctoral mandate no. 01P12219 (to L.D.V.) and no. 01P11322 (to T.Z), UGent BOF doctoral mandate no. 01CD7122 (to X.X.), and China Scholarship Council grant no. 202204910025 (to H.L.), grant no.\u0026nbsp;201706910095 (to T.Z.) and\u0026nbsp;grant no. 201706350153 (to X.X.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.L.Y., I.D.S., K.G., K.J.V, and L.D.V designed experiments. S.L.Y. performed most of the experiments. X.X. supported the radioactive \u003cem\u003ein vitro\u003c/em\u003e kinase assay. H.L. performed stomatal bioassay for wild-type plants across gradient temperature treatment. B.V.D.C. prepared mass spectrometry samples and assisted with immunoprecipitation and selection of Arabidopsis lines. J.M. and R.S. performed amino acid sequence alignments across yeast species via discussions with R.S. A.G. and K.V. performed yeast growth experiments. S.L.Y. and I.D.S. wrote the manuscript and all other authors edited or commented.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVila, J. A. Proteins\u0026rsquo; evolution upon point mutations. \u003cem\u003eACS Omega\u003c/em\u003e \u003cstrong\u003e7\u003c/strong\u003e, 14371\u0026ndash;14376 (2022).\u003c/li\u003e\n\u003cli\u003eStuder, R. 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Phys.\u003c/em\u003e \u003cstrong\u003e79\u003c/strong\u003e, 926\u0026ndash;935 (1983).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7157930/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7157930/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Protein evolution is shaped by sequence variation that modulates protein properties, e.g., through gain or loss of post-translational modifications. Among these, reversible phosphorylation alters a protein’s overall electrical charge and enables organisms to dynamically respond to environmental fluctuations. In plants, the hydro-active opening of stomata, microscopic pores that regulate gas exchange and leaf temperature, is governed by phosphorylation-dependent signalling. Here, we identify a mechanism involving the deubiquitylase UBIQUITIN-SPECIFIC PROTEASE 24 (UBP24) that promotes stomatal opening in Arabidopsis thaliana under heat. UBP24 is phosphorylated at serine 360 by the kinase OPEN STOMATA 1 (OST1), which is activated by B4 RAF kinases in response to heat stress. This phosphorylation stabilizes UBP24, enabling deubiquitylation of a plasma membrane H⁺-ATPase to promote stomatal opening. This reveals a novel heat-responsive signalling pathway that evolved in vascular plants to regulate stomatal function. Strikingly, a similar evolutionary feature exists in Saccharomyces cerevisiae, where the UBP24 homolog Ubp3 requires a constitutively negatively charged residue at the homologous site to support growth after heat shock. Our findings uncover a conserved molecular mechanism in which negative charge, via phosphorylation or acidic residues, modulates deubiquitylase function, supporting adaptive thermal responses across eukaryotes and highlighting how charge-based regulation promotes cellular resilience under stress.","manuscriptTitle":"Evolutionary tuning of molecular charge state of UBP24 shapes eukaryotic responses to high temperature","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 11:39:06","doi":"10.21203/rs.3.rs-7157930/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-plants","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nplants","sideBox":"Learn more about [Nature Plants](http://www.nature.com/nplants/)","snPcode":"","submissionUrl":"","title":"Nature Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99dbc7ee-64d3-48ea-a47b-3b90cbcd7a8b","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57732561,"name":"Biological sciences/Plant sciences/Plant stress responses/Heat"},{"id":57732562,"name":"Biological sciences/Plant sciences/Plant signalling"}],"tags":[],"updatedAt":"2026-05-05T12:11:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 11:39:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7157930","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7157930","identity":"rs-7157930","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}