A conserved clade of ER-membrane tethered SBP/SPL transcription factors regulate cell death mediated by ER stress in plants

A conserved clade of ER-membrane tethered SBP/SPL transcription factors regulate cell death mediated by ER stress in plants | 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 A conserved clade of ER-membrane tethered SBP/SPL transcription factors regulate cell death mediated by ER stress in plants Mehdi Kabbage, Austin VanDenTop, Davis Macnabb, Zhengshu Ma, Ryan Kessens, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7794624/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract SQUAMOSA promoter-binding proteins (SBPs) are key regulators of plant growth, development, and stress responses, with their roles in programmed cell death (PCD) gaining increasing attention. In this study, we investigate the role of a tomato SBP/SPL transcription factor, and its Arabidopsis thaliana homologs, AtSPL1 and AtSPL12, in ER stress-induced PCD. All three homologs share the conserved SBP-Box domain, and C-terminal Ankyrin and transmembrane domains (TMDs). We show that the TMD anchors these transcription factors to the ER membrane, and their transient expression in Nicotiana benthamiana induced spontaneous cell death, mediated by the C-terminal TMDs. Upon ER stress induction, specifically with tunicamycin, we observed a striking shift of these transcription factors from the ER to the nucleus, marking a crucial step in their activation of PCD. This nuclear translocation underscores their role as ER stress sensors. RNA sequencing following expression of these transcription factors revealed the upregulation of several protease classes that may be responsible for the execution of PCD. Overexpression of individual proteases induced cell death, suggesting that a coordinated protease response is necessary for full PCD induction. We also identify the interacting protein SINAT2, a RING-type E3 ubiquitin ligase, as a key regulator of SBP/SPL stability. SINAT2 physically interacts with these transcription factors, promoting their proteasomal degradation as evidenced by protein accumulation assays, and mitigates the PCD phenotype. The Arabidopsis thaliana atspl1/12 double mutants were insensitive to tunicamycin-induced ER stress, and failed to exhibit the typical growth suppression seen in wild-type and single mutants upon tunicamycin treatment. This suggests that AtSPL1 and AtSPL12 are essential for stress perception and response under ER stress conditions. These findings shed light on the roles of ER membrane-tethered SBP/SPL transcription factors in stress signaling and the execution of cell death, emphasizing their potential as targets for enhancing stress resilience in plants through genetic engineering. Biological sciences/Molecular biology Biological sciences/Cell biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Proper regulation of programmed cell death (PCD) is essential to plant growth and survival. In plants, PCD participates in various aspects of development, including xylem maturation, embryogenesis, and senescence ( 1 , 2 ). Beyond development, PCD also plays a critical role in plant responses to abiotic stresses such as drought, salinity, and extreme temperatures, helping to eliminate damaged cells and maintain overall plant health ( 3 , 4 ). Additionally, PCD is a key factor in defense responses against pathogens, and its (mis)regulation dictates disease outcomes to a range of plant pathogens ( 5 – 7 ). However, the specific biochemical networks that govern plant PCD remain largely unknown. While PCD mechanisms are poorly understood in plants, comparison to well-characterized systems in animals has revealed useful insights. Plant and animal cells undergoing PCD exhibit several conserved morphological hallmarks ( 8 – 10 ). However, despite these morphological similarities, the molecular machinery that drives plant PCD appears to have diverged significantly ( 11 ). Plant genomes lack direct homologs to the canonical elements of apoptosis as they are understood in animals. For example, caspases, cysteine proteases that orchestrate apoptosis in animals, have no direct homologs in plants, but plant proteases with caspase-like activity have been identified based on their ability to cleave caspase substrates ( 12 – 14 ). Remarkably, while sequence conservation of a core apoptotic pathway does not appear to extend to plants, ectopic expression of animal apoptotic regulators in plants can direct cell death outcomes ( 15 – 18 ). Notably, we previously showed that the expression of the insect Inhibitor of Apoptosis, SfIAP, inhibits cell death in tobacco and tomato to protect against biotic and abiotic stresses ( 19 , 20 ). This implies a certain level of functional similarity in apoptotic regulators between plants and animals. We have previously used ectopic expression of SfIAP to elucidate how this IAP suppresses PCD in plants ( 21 ). Plant endogenous proteins physically interacting with SfIAP or plant genes required for its function are likely to have roles in plant PCD. We identified several candidate SfIAP interactors in tomato, notably members of the SQUAMOSA promoter-binding protein (SBP) family, a group of transcription factors largely linked to developmental processes, such as flowering, phase transition and fruit ripening ( 22 , 23 ). Two of our candidates, SlySBP8b and SlySBP12a, induced spontaneous cell death upon expression in Nicotiana benthamiana ( 21 ). Interestingly, other studies showed that silencing the SBP gene, Colorless non-ripening (Cnr), in tomato delays ripening ( 24 ), and the deletion of the Arabidopsis thaliana homolog AtSPL14 conferred enhanced tolerance to the cell death inducing mycotoxin FB1 ( 25 ), both phenotypes were also observed in SfIAP-overexpressing plants ( 19 , 20 ). Furthermore, the SBP homolog, NbSPL6 is required for HR cell death and R mediated resistance in Nicotiana benthamiana ( 26 , 27 ). These SBPs can also be targeted by pathogen effectors to establish disease, as shown in the soybean rust pathosystem ( 28 ). Overall, these findings indicate that SBP transcription factors may be involved in cell death regulation in response to developmental cues or environmental insults. Herein, we focus this study on the two Arabidopsis thaliana ER localized AtSPL 1/12, homologs of the tomato SlySBP12a identified in our initial yeast two-hybrid screen ( 21 ). We show that AtSPL 1/12 translocate to the nucleus upon ER stress to induce cell death, by upregulating the expression of cell death inducing proteases. AtSPL1/12 are themselves regulated by the RING-type E3 ubiquitin ligase, SINAT2. Indeed, SINAT2 physically binds AtSPL 1/12 and suppresses their accumulation, thereby preventing the induction of cell death. This requires the RING domain, which facilitates the ubiquitination and subsequent proteasomal degradation of AtSPL1/12. We propose that AtSPL1/12 act as novel ER stress sensors which dissociate from the ER membrane upon ER stress to execute cell death. Given the evolutionary conservation of this transcription factor family, these findings may uncover fundamental regulatory mechanisms of PCD conserved across diverse plant species. Methods and Materials Plant Growth Conditions Nicotiana benthamiana plants were grown under a 16 h light/8 h dark cycle at 26°C and 60% relative humidity. Arabidopsis thaliana mutant and wild-type lines were maintained under a 16 h light/8 h dark cycle at 22°C. For protoplast isolation, Arabidopsis thaliana was grown under a 12 h light/12 h dark cycle under similar conditions. Plasmid Construction For yeast two-hybrid (Y2H), SlySBP8b and SlySBP12a(ΔTMD) cDNAs were amplified with NdeI and PstI sites and cloned into the bait vector pGBKT7. For plant overexpression, Arabidopsis thaliana cDNA was amplified with attB adapters and recombined into pDONR™/Zeo using BP Clonase II (Invitrogen). LR reactions with pEarleyGate104 (YFP-tag) or pEarleyGate201 (HA-tag), both driven by the CaMV 35S promoter, were used for destination cloning. Truncated versions of AtSPL1(ΔTMD), AtSPL12(ΔTMD), and SINAT2(ΔRING) were generated using primers listed in Supplemental Table 1. All constructs were sequence-verified and transformed into Agrobacterium tumefaciens strain GV3101. Yeast-Two Hybrid Y2H assays were conducted using the Matchmaker Gold system (Takara Bio USA, San Jose, CA). Bait constructs (pGBK:SlySBP8b, pGBK:SlySBP12a(ΔTMD)) were transformed into Saccharomyces cerevisiae strain AH109 and screened against a normalized Arabidopsis thaliana cDNA prey library in pGADT7. Mating and selection followed the manufacturer’s protocol. Positive interactions were identified by growth on QDO medium supplemented with X-α-Gal and Aureobasidin A, and prey plasmids were sequenced to identify interactors. Transient Expression in Nicotiana benthamiana Agrobacterium tumefaciens (GV3101 strain) harboring the vectors of interest were grown overnight on LB agar with kanamycin (50 µg/ml), gentamycin (25 µg/ml) and Rifampicin (15 µg/ml) at 28°C. Colonies were scraped and resuspended in infiltration buffer (10 mM MgSO₄, 9 mM MES, 10 mM MgCl₂, 300 µM acetosyringone, pH 5.7) to an OD600 of 0.9, incubated at room temperature for 2–4 hours before infiltration. 4–5 week old Nicotiana benthamiana were infiltrated on the two youngest leaves using a needleless 1 mL syringe. For protein extraction, infiltrated leaf tissue was flash-frozen, ground in extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Triton X-100) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Lysates were clarified by centrifugation and mixed with 2× Laemmli buffer containing 10% β-mercaptoethanol, then boiled for 5 min. Proteins were resolved on 12% SDS-PAGE gels, transferred to PVDF membranes, and stained with Ponceau S for total protein. HA-tagged proteins were detected using anti-HA primary antibody (Cell Signaling Technology, Danvers, MA3724S) and HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology 7074P2). Signal was visualized with SuperSignal™ West Dura substrate (Thermo Fisher Scientific). Electrolyte Leakage Assay Leaf disks were collected 24 h post-infiltration (8 per replicate) and rinsed in deionized water. Wash water was replaced with 4 ml fresh deionized water, and conductivity was measured using an ECTestr 11 + MultiRange meter (Oakton) to assess ion leakage. Co-Immunoprecipitation Agrobacterium tumefaciens GV3101 harboring 35S:YFP or 35S:YFP-SINAT2 were co-infiltrated with cultures expressing HA-tagged SlySBP12a(ΔTMD), AtSPL1(ΔTMD), or AtSPL12(ΔTMD). At 48 h post-infiltration, leaves were harvested, flash-frozen, and ground to a fine powder in liquid nitrogen. Proteins were extracted using buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.2% NP-40, with protease inhibitors, at 2 mL per gram of tissue. Lysates were clarified and incubated overnight at 4°C with anti-GFP magnetic agarose beads (GFP-Trap MA; Proteintech, Rosemont, IL). Beads were washed three times with extraction buffer lacking detergent and eluted by boiling in 2× SDS loading buffer. Proteins were separated on 12% SDS-PAGE gels, transferred to PVDF membranes, and probed with anti-GFP (Cell Signaling 2955S) and anti-HA (Cell Signaling 3724S) antibodies. Secondary antibodies were HRP-conjugated goat anti-mouse (Cell Signaling 7076P2) and goat anti-rabbit (Cell Signaling 7074P2). Signal was detected using SuperSignal™ West Dura substrate. Protoplast Isolation and Expression Protoplasts were isolated from 3-week-old Arabidopsis thaliana rosettes using the Tape Sandwich method ( 29 ). Transfections were performed with 30µg total plasmid DNA (equal ratios in co-expression), using PEG-mediated transformation ( 30 ). Imaging was performed ~ 18–24 h post-transfection. Confocal Microscopy Microscopy was performed using a Zeiss LSM 980 confocal microscope with a 40× water-immersion objective. YFP and DHE were excited at 514 nm; emissions were detected at 525–550 nm and 606–659 nm, respectively. Chlorophyll and mCherry signals were excited at 561 nm, with emission detection at 657–724 nm (chlorophyll) and 606–651 nm (mCherry). Protoplasts were stained with 5 µM dihydroethidium (DHE) and imaged 10 min after staining. Abiotic Stress Assay Arabidopsis thaliana mutants atspl1 (SALK_134584) and atspl12 (SALK_142295) were obtained from the Arabidopsis Biological Resource Center (ABRC) and crossed to generate the double mutant atspl1/12 . All mutants were in the background ecotype Col-0. Seeds were surface sterilized with 50% bleach as described previously ( 31 ) and stratified in darkness for three days. Seeds were sown on ½ MS plates and grown at 22°C with 16hr/8hr photoperiod for seven days. Seedlings were then submerged in ½ MS liquid containing 0.5µg/mL Tm for 6 h, washed twice in ½ MS, and grown for a further 5 days on 1/2 MS agar. Seedlings were photographed and root lengths measured using ImageJ measurement tools. Results Cell death induction by the tomato SlySBP12a is conserved in Arabidopsis homologs AtSPL1 and AtSPL12 Given that members of the SQUAMOSA promoter binding protein (SBP) family are conserved across all land plants, we investigated whether this cell death-associated function is conserved in the Arabidopsis thaliana homologs. SBP-box transcription factors are defined by a conserved zinc finger DNA-binding domain, the SBP-box. Sequence homology analysis revealed that AtSPL1 and AtSPL12 are the closest Arabidopsis thaliana homologs of SlySBP12a, sharing 52.6 and 53.4% peptide sequence homology, respectively (Suppl. Figure 1). The protein domain structure in SlySBP12a, AtSPL1 and AtSPL12 are highly similar, with all sharing the family-characteristic SBP-box domain, as well as multiple Ankyrin repeat domains and a C-terminal transmembrane domain (Fig. 1 a). To assess functional conservation, we transiently expressed AtSPL1 and AtSPL12 in Nicotiana benthamiana leaves. Both AtSPL1 and AtSPL12 induced a spontaneous cell death phenotype comparable to that triggered by SlySBP12a (Fig. 1 b). We previously showed that a C-terminal transmembrane domain (TMD) in SlySBP12a regulates the cell death response upon overexpression ( 21 ). Consistent with this, the deletion of this domain in either homolog enhanced the intensity of the cell death phenotype upon expression in Nicotiana benthamiana (Fig. 1 b). These results demonstrate that the cell death-inducing function mitigated by the C-terminal transmembrane domain of SlySBP12a in AtSPL1 and AtSPL12. C-terminal transmembrane domain controls the subcellular localization in AtSPL1 and 12 We demonstrated that SlySBP12a localizes to both the nucleus and the endoplasmic reticulum (ER) membrane in tomato protoplasts, with this dual localization being dependent on the C-terminal TMD. Truncation of the TMD abolished ER localization, resulting in exclusive nuclear localization ( 21 ). To determine whether this localization pattern is conserved in the Arabidopsis thaliana homologs, full-length AtSPL1 and AtSPL12 were expressed in Arabidopsis thaliana protoplasts and visualized using fluorescent compartment-specific markers. Both full-length transcription factors co-localized predominantly with the ER marker HDEL-mCherry (Fig. 2 a). Upon truncation of their respective TMDs (AtSPL1ΔTMD and AtSPL12ΔTMD), localization shifted entirely to the nucleus, as indicated by co-localization with the nuclear stain dihydroethidium (DHE) (Fig. 2 b). This shift coincides with enhanced cell death (Fig. 1 b), suggesting that these transcription factors likely translocate from to the ER to nucleus under specific conditions to initiate cell death. ER stress triggers nuclear translocation of AtSPL1 and AtSPL12 We next sought to determine the mechanism driving translocation from the ER to the nucleus. We hypothesized that ER stress could act as a signal promoting this shift. To test this, we included SlySBP12a alongside AtSPL1 and AtSPL12 in an ER stress induction assay, as the regulatory mechanism of SlySBP12a localization had not been previously established. Protoplasts expressing each transcription factor were treated with either tunicamycin, a potent inducer of the unfolded protein response (UPR), or with DMSO as a mock control. After incubation, localization was assessed using co-staining with HDEL-mCherry or DHE. In mock-treated samples, SlySBP12a, AtSPL1, and AtSPL12 maintained their ER membrane localization (Fig. 3 a). In contrast, tunicamycin-treated protoplasts exhibited a marked shift in localization. Fluorescence from YFP-tagged transcription factors no longer overlapped with the ER marker, and co-localization with the nuclear stain DHE was clearly observed (Fig. 3 b). These results indicate that ER stress induces translocation of SlySBP12a, AtSPL1, and AtSPL12 from the ER membrane to the nucleus. Nuclear localization activates the transcription of proteases associated with cell death Having established the nuclear translocation of our SBP/SPL transcription factors, we next investigated its downstream effects on gene expression, reasoning that the transcription factor likely regulates nuclear targets involved in the observed cell death phenotype. To identify candidate genes under regulation by SBP/SPL transcription factors, we conducted RNA sequencing following transient overexpression of SlySBP12aΔTMD in Nicotiana benthamiana leaves (Suppl. Figure 2). Among the differentially expressed genes, proteases emerged as a notable functional category, consistent with their observed involvement in plant cell death activation. We prioritized these candidates for functional validation, selecting a subset of protease-encoding genes with a Log2 Fold Change above 2 compared to the empty vector control (Suppl. Table 2). To test whether these upregulated proteases could independently induce cell death, each protease was transiently expressed in Nicotiana benthamiana leaves. While most induced mild chlorosis, the phenotype was more pronounced than the empty vector control (Fig. 4 a). To quantitatively assess membrane disruption, we performed ion leakage assays, which revealed that all tested proteases, except serine carboxypeptidases 3 and 31, significantly increased conductivity compared to control infiltrations (Fig. 4 b). To assess whether these proteases are likely direct transcriptional targets of these transcription factors, we analyzed the upstream promoter regions of the induced genes. Notably, all tested proteases contained a predicted SBP-binding site within 2 kb of the transcriptional start site, and with ≥ 90% identity to the SlySBP12a consensus motif (Fig. 4 c). These results suggest that specific proteases are directly activated upon SBP/SPL nuclear translocation, and may contribute to the execution of cell death. AtSPL1 and AtSPL12 interact with the E3 Ligase protein Seven in Absentia 2 (SINAT2) To uncover protein interactors that might modulate SBP/SPL transcription factor activity, we performed a yeast two-hybrid (Y2H) screen using SlySBP12a as bait against a tomato cDNA library. Sequencing of positive colonies yielded a list of candidate interactors, and identified the candidate interactor Seven in Absentia 2 (SINAT2), a RING-type E3 ubiquitin ligase from the SINAT family as a potential regulator of SlySBP12a activity. This family is involved in the ubiquitination and degradation of regulatory proteins, including nuclear transcription factors, that mediate plant responses to both abiotic and biotic stress ( 32 – 34 ). Given this role and the importance of post-translational regulation in stress-responsive signaling, SINAT2 emerged as a compelling candidate for modulating the stability and activity SlySBP12a and its Arabidopsis thaliana homologs AtSPL1 and AtSPL12. To confirm physical interaction between SINAT2 and the transcription factors we performed co-immunoprecipitation using a YFP-tagged SINAT2 construct transiently expressed in Nicotiana benthamiana . HA-tagged AtSPL1, AtSPL12, and SlySBP12a were successfully co-immunoprecipitated with YFP–SINAT2, confirming their interaction in planta (Fig. 5 , Suppl. Figure 6), demonstrating conserved protein–protein interactions across species. No HA signal was detected when SBP/SPL constructs were co-expressed with free YFP, indicating that the observed interactions are specific to SINAT2. SINAT2 attenuates cell death imposed by SBP/SPL transcription factors Having confirmed the physical interaction between SINAT2 and AtSPL1/AtSPL12/SlySBP12a, we next sought to determine its functional relevance in modulating the cell death phenotype imposed by these transcription factors. Transient expression of SINAT2 with all three homologs in Nicotiana benthamiana led to a marked visual reduction in the cell death phenotype compared to co-expression with an empty vector control (Fig. 6 a). Cell death suppression by SINAT2 was quantified using ion leakage assays. Leaves co-infiltrated with SINAT2 and SBP/SPL transcription factors exhibited significantly lower conductivity than those infiltrated with SBP/SPLs and empty vector, thus confirming the ability of SINAT2 to inhibit the cell death phenotype displayed by these transcription factors (Fig. 6 b). When co-expressed with the animal cell death inducing protein Bax, SINAT2 did not inhibit Bax-induced cell death, indicating SINAT2 inhibition is specific to SBP/SPL induced death (Suppl. Figure 3). These results identify SINAT2 as a conserved specific negative regulator of SBP/SPL-induced cell death. SINAT2 reduces SBP/SPL accumulation via the Ubiquitin–Proteasome System Given that SINAT2 directly interacts with these SBP/SPL transcription factors, we next examined its functional role, specifically the mechanism by which it suppresses cell death induced by SBP/SPL in Nicotiana benthamiana . As an E3 ubiquitin ligase, we reasoned that SINAT2 may promote the ubiquitination and subsequent proteasomal degradation of its target proteins. We therefore hypothesized that SINAT2 mitigates SBP/SPL-induced cell death by limiting the accumulation of these transcription factors. Immunoblot analysis revealed that HA-tagged SBP/SPL proteins accumulated to high levels when co-infiltrated with empty vector control. In contrast, co-infiltration with SINAT2 led to a marked reduction in protein levels in a time course experiment, consistent with targeted degradation (Fig. 6 c, Suppl. Figure 6). To determine whether this reduction is mediated by the 26S proteasome, we treated infiltrated leaves with the proteasome inhibitor MG132. Upon MG132 treatment, SBP/SPL protein levels were restored to those observed in the empty vector control, suggesting that SINAT2 promotes SBP/SPL degradation via the ubiquitin–proteasome pathway (Fig. 6 c, Suppl. Figure 6). The RING domain of SINAT2 is required for SBP/SPL destabilization and cell death suppression To determine whether SINAT2's E3 ligase activity is responsible for SBP/SPL degradation and suppression of cell death, we generated a mutant construct lacking the RING domain (SINAT2ΔRING), which is required for ubiquitin ligase activity (Suppl. Figure 4). Co-infiltration of SlySBP12aΔTMD, AtSPL1ΔTMD, or AtSPL12ΔTMD with SINAT2ΔRING failed to suppress the cell death phenotype associated with SBP/SPL expression, in contrast to co-infiltration with wild-type SINAT2, which strongly attenuated cell death (Fig. 7 a). This visual phenotype was confirmed by ion leakage assays, which indicates a significant decrease in conductivity when co-infiltrated with wild-type SINAT2 compared to the empty vector, but no significant change when co-infiltrated with SINAT2ΔRING (Fig. 7 b). This assay confirms that SINAT2ΔRING lacks the ability to attenuate cell death induced by SlySBP12aΔTMD, AtSPL1ΔTMD, or AtSPL12ΔTMD. Furthermore, we monitored SBP/SPL protein accumulation in the presence of SINAT2 or SINAT2ΔRING in Nicotiana benthamiana in a time course experiment. Immunoblot analysis revealed that SINAT2ΔRING failed to reduce SBP/SPL protein accumulation to the level of the wild-type SINAT2 construct (Fig. 7 c, Suppl. Figure 6). These results demonstrate that SINAT2 requires its RING domain to promote proteasomal degradation of SBP/SPL transcription factors and to suppress their cell death activity. Loss of AtSPL1/12 Confers Partial Insensitivity to Tunicamycin To further investigate the role of SBP/SPL transcription factors in ER stress signaling, we leveraged Arabidopsis thaliana genetic resources, including T-DNA insertion mutants for AtSPL1 and AtSPL12, and generated an atspl1/12 double mutant by crossing AtSPL1 and AtSPL12 single mutant lines (Suppl. Figure 5). We focused on ER stress because these SBP/SPL proteins translocate to the nucleus upon ER perturbation, and we assayed responsiveness by treating Col-0 and atspl lines with Tunicamycin, a well-characterized ER-stress inducer. Comparing growth on Mock versus Tunicamycin plates, Col-0 roots on Tunicamycin retained 18% of their Mock growth, whereas the atspl1/12 double mutant retained 45% of its growth compared to Mock treatment; the atspl1 and atspl12 single mutants resembled Col-0 (Fig. 8 ). These data indicate that loss of AtSPL1 and AtSPL12 confers partial insensitivity to Tunicamycin-induced growth suppression and may be considered as potential targets for enhanced stress tolerance. Discussion SBP-box transcription factors are conserved across land plants and are best known for regulating key developmental processes, including vegetative phase change, floral induction, and leaf morphology ( 22 , 23 ). However, mounting evidence suggests that certain members of this family may also play roles in environmental stress responses, both abiotic and biotic ( 25 , 26 ). Our findings demonstrate a novel function for a clade of membrane tethered SBP-box transcription factors, linked by the presence of a C-terminal transmembrane domain, that function to transmit ER stress signals and induce transcriptional changes that result in activation of programmed cell death. We previously demonstrated that overexpression of SlySBP12a from tomato triggers spontaneous cell death in Nicotiana benthamiana ( 21 ). Here, we show that this cell death-inducing activity is conserved in its Arabidopsis thaliana homologs, AtSPL1 and AtSPL12. Both genes were identified based on sequence similarity and the presence of C-terminal TMDs. Transient expression of AtSPL1 and AtSPL12 in Nicotiana benthamiana caused strong lesion mimic phenotypes, phenotypically indistinguishable from SlySBP12a. These results suggest that the ability to induce PCD is conserved within this TMD-containing SBP/SPL subgroup and is likely linked to their membrane localization. Like SlySBP12a, AtSPL1 and AtSPL12 localize to the ER membrane via C-terminal transmembrane domains (TMDs), which constrain activity under basal conditions. Removing the TMD causes full nuclear accumulation and a stronger cell death phenotype, indicating that the TMD acts as a negative regulatory gatekeeper. This architecture parallels other ER-tethered transcription factors in plants. bZIP28 is released by proteolytic cleavage, and bZIP60 is activated through IRE1-dependent splicing during ER stress ( 35 – 38 ). A similar framework applies to the NTM1-like NAC subgroup, which carries an N-terminal DNA-binding domain and a C-terminal TMD, responds to ER, oxidative, or mitochondrial stress, and can be activated by diverse mechanisms: ANAC017 via rhomboid protease cleavage, GmNTL1 via oxidative cysteine modification, and others via alternative splicing that removes the TMD ( 39 – 41 ). The shared architecture and stress-responsive mobility suggest SBP/SPLs may constitute a previously unrecognized class of membrane-tethered transcription factors. Unlike the well-characterized bZIPs and NACs, however, the molecular switch that controls SBP/SPL release remains unresolved. Defining the trigger and the timing relative to ER-stress signaling will be essential to explain how stress cues are converted into a transcriptional program for cell death. Nuclear-localized SlySBP12a lacking the TMD domain strongly upregulates multiple proteases linked to plant PCD, some of which cause chlorosis, and ion leakage when expressed individually, but none replicate the full cell death phenotype. This suggests that robust PCD requires coordinated activity of multiple proteolytic pathways, as seen in hypersensitive responses and other PCD models ( 42 ). The breadth of proteases induced also points to a potentially layered proteolytic network that integrates signals from different subcellular compartments. Interestingly, among the induced genes were a cathepsin-like protease and a subtilisin-like serine protease, both members of families previously shown to cleave caspase substrates and were implicated in plant PCD ( 12 , 43 ). Cathepsins can display caspase-like activity and contribute to vacuole-mediated cell collapse during stress, while subtilases, such as phytaspases, promote immune-associated PCD by relocalizing during stress ( 44 – 46 ). Their induction downstream of SlySBP12a suggests that this transcription factor directly engages established proteolytic effectors, activating a number of proteases with complementary roles in cell death execution. Future work should test co-expression of these proteases to determine whether their effects are redundant, additive, or synergistic, and confirm direct promoter binding by SlySBP12a through assays such as Yeast-1 Hybrid. If validated, these proteases could serve as biomarkers and tools for engineering tunable cell death pathways in crops. In addition to transcriptional regulation, we show that SBP/SPL protein levels are post-translationally regulated by SINAT2, a RING-type E3 ubiquitin ligase. In Arabidopsis thaliana , SINA/SINAT ligases, including SINAT2, indirectly modulates ABA-linked drought responses by controlling ESCRT components such as FREE1 and VPS23A, and SINA2 directly promotes ABA-mediated drought tolerance ( 33 , 47 ). In cotton, SINA orthologs are induced by Verticillium dahliae and enhance resistance when overexpressed, underscoring broader roles in stress ( 48 ). Through the E3 ligase mutants and inhibition by MG132, we demonstrate that SINAT2 regulates SBP/SPLs by ubiquitination and subsequent degradation via the 26S proteasome. These results align with prior work showing that SINAT-family E3 ligases regulate stress-responsive transcription factors ( 34 , 49 – 51 ). The 26S proteasome adds an important layer of control to SBP/SPL turnover, acting as the primary degradation machinery for ubiquitinated proteins and tuning transcription factor activity in response to cellular cues. Similar proteasome-dependent regulation of membrane-tethered or stress-responsive transcription factors, such as DREB2A, ABI5, and NAC proteins, is known to maintain proper stress thresholds ( 51 – 53 ). Given the activity of SBP/SPLs, proteasomal degradation may buffer against excessive cell death, allowing fine-tuned responses. Mapping how SBP/SPL stability is shaped by proteasomal activity will be critical, and targeted experiments such as proteasome reporter lines or real-time degradation assays offer a direct path to uncovering how this turnover integrates into the plant’s broader stress-response and developmental networks. Functionally, the ER-stress insensitivity of atspl1/12 places AtSPL1 and AtSPL12 within the UPR machinery that couples ER stress cues to growth restraint. This assignment aligns with executor-type precedents such as NAC089, whose nucleus-localized, TMD-deleted form drives ER-stress–induced PCD and whose knockdown confers tunicamycin tolerance, while the cytoprotective cochaperone AtBAG7 exemplifies the inhibitor arm, with atbag7 mutants hypersensitive to tunicamycin, heat, and cold ( 54 , 55 ). Together, these examples underscore that the plant UPR is a balance of pro-survival buffering and pro-death outputs. Within that balance, AtSPL1/12 acts as redundant executors that impose growth restraint once proteotoxic burden crosses a threshold; with mutants showing continued growth despite ER stress. Interestingly, Chao et al. (2017) reported that atspl1/12 mutants show no overt phenotypes under standard conditions but are impaired in basal thermotolerance of developing flowers due to reduced induction of heat-responsive genes ( 56 ). This may seem at odds with our findings, however the discrepancy likely reflects differences in tissue, developmental stage, and stress context. Chao et al. examined floral tissues under acute heat stress, whereas we assayed root growth in 1-week-old seedlings under ER stress, conditions that recruit distinct signaling pathways. That AtSPL1/12 function in both settings suggests they act as versatile, context-dependent regulators integrating environmental cues in a tissue- and input-specific manner, a hallmark of membrane-tethered transcription factors such as bZIPs and NACs ( 57 – 59 ). Our findings support a model where a specific clade of SBP/SPL transcription factors are ER-tethered under basal conditions and translocate to the nucleus during ER stress, where they activate programmed cell death genes, including proteases. Their accumulation is counterbalanced by SINAT2, which targets SBP/SPLs for ubiquitin–proteasome degradation (Fig. 8 b). This dual control, ER release and regulated turnover, fine-tunes the timing and magnitude of SBP/SPL activity during stress. Our findings extend SBP/SPL roles beyond development to stress signaling and cell fate, defining an ER-anchored/SINAT2 module as a new layer of control and a potential lever for engineering more resilient, precisely tuned cell-death responses in crops. Declarations Acknowledgements We thank Dr. Sarah Swanson, director of the Newcomb Imaging Center for technical support with confocal microscopy. We also thank the lab of Dr. Andrew Bent (University of Wisconsin-Madison) for providing the Bax control plasmid. This project was partially supported by USDA-HATCH (Award # WIS04031) and the Department of Plant Pathology at the University of Wisconsin-Madison. Conflict of Interest The authors declare no competing interests. Author Contributions A.V. and M.K. contributed to study conception and design. A.V., D.M. and Z.M. executed the experiments. A.V., M.K., and B.W. analyzed and interpreted the data. A.V. prepared the figures and drafted the manuscript. 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10:40:50","extension":"png","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":190918,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/4b11440f7819ba153d1c5340.png"},{"id":93127013,"identity":"070d7dfb-d237-4ffd-86d6-b5bf2143e87f","added_by":"auto","created_at":"2025-10-09 10:40:50","extension":"xml","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":134596,"visible":true,"origin":"","legend":"","description":"","filename":"CDD2536240structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/53911c98d5c5f6499bb4659e.xml"},{"id":93127015,"identity":"f3d8f955-a483-4390-8333-fc3de2493a9f","added_by":"auto","created_at":"2025-10-09 10:40:50","extension":"html","order_by":30,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":148496,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/b853192335867ace44306b1d.html"},{"id":93127117,"identity":"d6733ca5-4b4e-40ea-b4cf-586a8a04c954","added_by":"auto","created_at":"2025-10-09 10:48:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4707316,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification and characterization of SlySBP12a homologs in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e(a) Domain structure of SlySBP12a, AtSPL1, and AtSPL12. Sequences were retrieved from Plant Transcription Factor Database and domains predicted with SMART. The amino acid sequence of the SBP box is depicted, with the residues of the Zinc Fingers and NLS sequence highlighted. (b) \u003cem\u003eNicotiana benthamiana\u003c/em\u003eleaves infiltrated with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. The left half of each leaf was transformed with empty vector as a negative control while the right half was transformed with the corresponding AtSPL gene containing an N‐terminal HA tag and driven by a 35S promoter. Images were taken 5 days post‐transformation.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/8491e48d905d4cc6e655e664.png"},{"id":93127116,"identity":"b9e18548-6148-43c4-aa7f-d7010179f549","added_by":"auto","created_at":"2025-10-09 10:48:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2145429,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization of AtSPL1 and AtSPL12.\u003c/strong\u003e (a) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts were transfected with plasmids encoding YFP-AtSPL1 or YFP-AtSPL12 and imaged by CLSM. HDEL-mCherry (red) was used as an Endoplasmic Reticulum counterstain while the magenta signal represents chloroplast autofluorescence. (b) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts were transfected with plasmids encoding YFP-AtSPL1(ΔTMD) or YFP-AtSPL12(ΔTMD) and imaged by CLSM. Dihydroethidium (red) was used as a nuclear counterstain while the magenta signal represents chloroplast autofluorescence.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/68103655fc5240fb6b0389aa.png"},{"id":93128563,"identity":"e3ab6b86-ef85-47d0-951f-b0cb212409fa","added_by":"auto","created_at":"2025-10-09 11:04:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1838664,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eER stress induces translocation of SBP/SPLs from the ER to the nucleus.\u003c/strong\u003e(a) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts were transfected with plasmids encoding YFP-AtSPL1, YFP-AtSPL12, or YFP-SlySBP12a and incubated with DMSO (Mock) for 2 hours before imaging by CLSM. HDEL-mCherry (red) was used as an Endoplasmic Reticulum counterstain while the magenta signal represents chloroplast autofluorescence. (b) \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts were transfected with plasmids encoding YFP-AtSPL1, YFP-AtSPL12, or YFP-SlySBP12a and incubated with 5ug/mL Tunicamycin for 2 hours before imaging by CLSM. Dihydroethidium (red) was used as a nuclear counterstain while the magenta signal represents chloroplast autofluorescence.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/ce6df48e4669cd9b574f60a2.png"},{"id":93128282,"identity":"57924dcd-095c-4c6d-9b25-f3d07bcfde0e","added_by":"auto","created_at":"2025-10-09 10:56:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5403523,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransient expression of upregulated proteases induces cell death\u003c/strong\u003e. (a) \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves infiltrated with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. The left half of each leaf was transformed with empty vector as a negative control while the right half was transformed with the corresponding protease gene. Images were taken 5 days post‐transformation. (b) Electrolyte leakage assay used to quantify cell death. Significance for each protease is in reference to the EV sample. Data is presented as mean ± SEM (n=12). (c) Promoter sequences 2kb upstream of starting codon with SlySBP12a binding sites marked, indicating the matching sequence in the promoter and the matching strand direction.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/dfabbd07f0e9e7a8464a01a8.png"},{"id":93126982,"identity":"c362f1ee-f874-428b-a055-b4120d3d267e","added_by":"auto","created_at":"2025-10-09 10:40:49","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1122828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCoimmunoprecipitation of SINAT2 with AtSPL1, AtSPL12 and SlySBP12a in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eNicotiana benthamiana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003eYFP-SINAT2 or free YFP was transiently co-expressed with HA-AtSPL1(ΔTMD), HA-AtSPL12(ΔTMD) or HA-SlySBP12a(ΔTMD) in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Proteins were immunoprecipitated with α‐YFP magnetic agarose beads. A portion of each sample was taken before immunoprecipitation to serve as the input control. An immunoblot was performed on input and elution fractions using the indicated antibodies to detect the epitope‐tagged proteins.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/a875454bda1aabb4394952e0.png"},{"id":93127125,"identity":"56891a95-01ee-435e-9e0a-e9c17d4cb6ed","added_by":"auto","created_at":"2025-10-09 10:48:49","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7112050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCo-expression with interacting protein SINAT2 reduces cell death by SBP/SPLs.\u003c/strong\u003e(a) \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves infiltrated with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. The left half of each leaf was co-infiltrated with the corresponding SBP/SPL and empty vector at a 1:1 ratio while the right half was transformed with the corresponding SBP/SPL and SINAT2 at a 1:1 ratio. Images were taken 5 days post‐transformation. (b) Electrolyte leakage assay used to quantify cell death. Data is presented as mean ± SEM (n=18). (c) HA-tagged SBP/SPL constructs were transiently co-expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003eleaves with either an empty vector (top row), SINAT2 (middle row), or SINAT2 with 50 µM MG132 proteasome inhibitor applied 24 hours post-infiltration (bottom row). Total protein was extracted at 36, 48, 60, and 72 hours post-infiltration and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA antibody to detect SBP/SPL protein levels.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/d875ff3e3f0b6dd9584e575c.png"},{"id":93126996,"identity":"27cfb469-6726-47f7-8e16-7273a05228f1","added_by":"auto","created_at":"2025-10-09 10:40:49","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4281129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSINAT2 inhibits cell death by SBP/SPLs using its RING domain.\u003c/strong\u003e (a) \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves infiltrated with \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e. Whole leaves were co-infiltrated with the corresponding SBP/SPL and either empty vector, wild type SINAT2, or SINAT2ΔRING at a 1:1 ratio. Images were taken 5 days post‐transformation. (b) Electrolyte leakage assay used to quantify cell death. Data is presented as mean ± SEM (n=12). (c) HA-tagged SBP/SP L constructs were transiently co-expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves with the corresponding SBP/SPL and either wild type SINAT2 or SINAT2ΔRING at a 1:1 ratio. Total protein was extracted at 24, 36, 48, 60, and 72 hours post-infiltration and analyzed by SDS-PAGE followed by immunoblotting with an anti-HA antibody to detect SBP/SPL protein levels\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/427173ffecef3b582b3bc5ef.png"},{"id":93127121,"identity":"eeabb6b5-6f77-4ff5-b209-22194343e10c","added_by":"auto","created_at":"2025-10-09 10:48:49","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1720674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eatspl1/12\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edouble mutants are less sensitive to ER stress.\u003c/strong\u003e(a) Root growth of wildtype Col-0, \u003cem\u003eatspl1\u003c/em\u003e and \u003cem\u003eatspl12\u003c/em\u003e single mutants, and \u003cem\u003eatspl1/12\u003c/em\u003e over a 5-day period following Tunicamycin or Mock DMSO treatment. Data is presented as mean ± SEM (n=21). (b) Summary model of cell death induction by SBP/SPL transcription factors and its attenuation by SINAT2 through ubiquitin–proteasome–mediated degradation.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/bb696dfbe020dcb6fd361a9e.png"},{"id":93129506,"identity":"a62c549d-3c57-4284-a452-4fe11b4e5af0","added_by":"auto","created_at":"2025-10-09 11:13:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":32432705,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/8fdba309-671f-4efa-92b9-7707f03ed3a1.pdf"},{"id":93126976,"identity":"d68600f8-d634-467d-aa70-1d2a5e8ab595","added_by":"auto","created_at":"2025-10-09 10:40:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2834579,"visible":true,"origin":"","legend":"Supplementary Figure 6","description":"","filename":"SuppFig6OriginalWesternBlots.docx","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/796104a9c5248225ceb28833.docx"},{"id":93128280,"identity":"0ccdbcdd-bfac-47dc-90d4-f14d6750e124","added_by":"auto","created_at":"2025-10-09 10:56:49","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":975206,"visible":true,"origin":"","legend":"Supplementary Figures and Tables","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7794624/v1/774f3f50018359289ca2a3cb.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"A conserved clade of ER-membrane tethered SBP/SPL transcription factors regulate cell death mediated by ER stress in plants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eProper regulation of programmed cell death (PCD) is essential to plant growth and survival. In plants, PCD participates in various aspects of development, including xylem maturation, embryogenesis, and senescence (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Beyond development, PCD also plays a critical role in plant responses to abiotic stresses such as drought, salinity, and extreme temperatures, helping to eliminate damaged cells and maintain overall plant health (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Additionally, PCD is a key factor in defense responses against pathogens, and its (mis)regulation dictates disease outcomes to a range of plant pathogens (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). However, the specific biochemical networks that govern plant PCD remain largely unknown.\u003c/p\u003e\u003cp\u003eWhile PCD mechanisms are poorly understood in plants, comparison to well-characterized systems in animals has revealed useful insights. Plant and animal cells undergoing PCD exhibit several conserved morphological hallmarks (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). However, despite these morphological similarities, the molecular machinery that drives plant PCD appears to have diverged significantly (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Plant genomes lack direct homologs to the canonical elements of apoptosis as they are understood in animals. For example, caspases, cysteine proteases that orchestrate apoptosis in animals, have no direct homologs in plants, but plant proteases with caspase-like activity have been identified based on their ability to cleave caspase substrates (\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Remarkably, while sequence conservation of a core apoptotic pathway does not appear to extend to plants, ectopic expression of animal apoptotic regulators in plants can direct cell death outcomes (\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Notably, we previously showed that the expression of the insect Inhibitor of Apoptosis, SfIAP, inhibits cell death in tobacco and tomato to protect against biotic and abiotic stresses (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This implies a certain level of functional similarity in apoptotic regulators between plants and animals.\u003c/p\u003e\u003cp\u003eWe have previously used ectopic expression of SfIAP to elucidate how this IAP suppresses PCD in plants (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Plant endogenous proteins physically interacting with SfIAP or plant genes required for its function are likely to have roles in plant PCD. We identified several candidate SfIAP interactors in tomato, notably members of the SQUAMOSA promoter-binding protein (SBP) family, a group of transcription factors largely linked to developmental processes, such as flowering, phase transition and fruit ripening (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Two of our candidates, SlySBP8b and SlySBP12a, induced spontaneous cell death upon expression in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Interestingly, other studies showed that silencing the SBP gene, Colorless non-ripening (Cnr), in tomato delays ripening (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), and the deletion of the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homolog AtSPL14 conferred enhanced tolerance to the cell death inducing mycotoxin FB1 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), both phenotypes were also observed in SfIAP-overexpressing plants (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Furthermore, the SBP homolog, NbSPL6 is required for HR cell death and R mediated resistance in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). These SBPs can also be targeted by pathogen effectors to establish disease, as shown in the soybean rust pathosystem (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Overall, these findings indicate that SBP transcription factors may be involved in cell death regulation in response to developmental cues or environmental insults.\u003c/p\u003e\u003cp\u003eHerein, we focus this study on the two \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ER localized AtSPL 1/12, homologs of the tomato SlySBP12a identified in our initial yeast two-hybrid screen (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). We show that AtSPL 1/12 translocate to the nucleus upon ER stress to induce cell death, by upregulating the expression of cell death inducing proteases. AtSPL1/12 are themselves regulated by the RING-type E3 ubiquitin ligase, SINAT2. Indeed, SINAT2 physically binds AtSPL 1/12 and suppresses their accumulation, thereby preventing the induction of cell death. This requires the RING domain, which facilitates the ubiquitination and subsequent proteasomal degradation of AtSPL1/12. We propose that AtSPL1/12 act as novel ER stress sensors which dissociate from the ER membrane upon ER stress to execute cell death. Given the evolutionary conservation of this transcription factor family, these findings may uncover fundamental regulatory mechanisms of PCD conserved across diverse plant species.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant Growth Conditions\u003c/h2\u003e\u003cp\u003e\u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants were grown under a 16 h light/8 h dark cycle at 26\u0026deg;C and 60% relative humidity. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutant and wild-type lines were maintained under a 16 h light/8 h dark cycle at 22\u0026deg;C. For protoplast isolation, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was grown under a 12 h light/12 h dark cycle under similar conditions.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePlasmid Construction\u003c/h3\u003e\n\u003cp\u003eFor yeast two-hybrid (Y2H), SlySBP8b and SlySBP12a(ΔTMD) cDNAs were amplified with NdeI and PstI sites and cloned into the bait vector pGBKT7. For plant overexpression, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e cDNA was amplified with attB adapters and recombined into pDONR\u0026trade;/Zeo using BP Clonase II (Invitrogen). LR reactions with pEarleyGate104 (YFP-tag) or pEarleyGate201 (HA-tag), both driven by the CaMV 35S promoter, were used for destination cloning. Truncated versions of AtSPL1(ΔTMD), AtSPL12(ΔTMD), and SINAT2(ΔRING) were generated using primers listed in Supplemental Table\u0026nbsp;1. All constructs were sequence-verified and transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101.\u003c/p\u003e\n\u003ch3\u003eYeast-Two Hybrid\u003c/h3\u003e\n\u003cp\u003eY2H assays were conducted using the Matchmaker Gold system (Takara Bio USA, San Jose, CA). Bait constructs (pGBK:SlySBP8b, pGBK:SlySBP12a(ΔTMD)) were transformed into \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain AH109 and screened against a normalized \u003cem\u003eArabidopsis thaliana\u003c/em\u003e cDNA prey library in pGADT7. Mating and selection followed the manufacturer\u0026rsquo;s protocol. Positive interactions were identified by growth on QDO medium supplemented with X-α-Gal and Aureobasidin A, and prey plasmids were sequenced to identify interactors.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransient Expression in\u003c/b\u003e \u003cb\u003eNicotiana benthamiana\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e (GV3101 strain) harboring the vectors of interest were grown overnight on LB agar with kanamycin (50 \u0026micro;g/ml), gentamycin (25 \u0026micro;g/ml) and Rifampicin (15 \u0026micro;g/ml) at 28\u0026deg;C. Colonies were scraped and resuspended in infiltration buffer (10 mM MgSO₄, 9 mM MES, 10 mM MgCl₂, 300 \u0026micro;M acetosyringone, pH 5.7) to an OD600 of 0.9, incubated at room temperature for 2\u0026ndash;4 hours before infiltration. 4\u0026ndash;5 week old \u003cem\u003eNicotiana benthamiana\u003c/em\u003e were infiltrated on the two youngest leaves using a needleless 1 mL syringe.\u003c/p\u003e\u003cp\u003eFor protein extraction, infiltrated leaf tissue was flash-frozen, ground in extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% Triton X-100) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). Lysates were clarified by centrifugation and mixed with 2\u0026times; Laemmli buffer containing 10% β-mercaptoethanol, then boiled for 5 min. Proteins were resolved on 12% SDS-PAGE gels, transferred to PVDF membranes, and stained with Ponceau S for total protein. HA-tagged proteins were detected using anti-HA primary antibody (Cell Signaling Technology, Danvers, MA3724S) and HRP-conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology 7074P2). Signal was visualized with SuperSignal\u0026trade; West Dura substrate (Thermo Fisher Scientific).\u003c/p\u003e\n\u003ch3\u003eElectrolyte Leakage Assay\u003c/h3\u003e\n\u003cp\u003eLeaf disks were collected 24 h post-infiltration (8 per replicate) and rinsed in deionized water. Wash water was replaced with 4 ml fresh deionized water, and conductivity was measured using an ECTestr 11\u0026thinsp;+\u0026thinsp;MultiRange meter (Oakton) to assess ion leakage.\u003c/p\u003e\n\u003ch3\u003eCo-Immunoprecipitation\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e GV3101 harboring 35S:YFP or 35S:YFP-SINAT2 were co-infiltrated with cultures expressing HA-tagged SlySBP12a(ΔTMD), AtSPL1(ΔTMD), or AtSPL12(ΔTMD). At 48 h post-infiltration, leaves were harvested, flash-frozen, and ground to a fine powder in liquid nitrogen. Proteins were extracted using buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.2% NP-40, with protease inhibitors, at 2 mL per gram of tissue. Lysates were clarified and incubated overnight at 4\u0026deg;C with anti-GFP magnetic agarose beads (GFP-Trap MA; Proteintech, Rosemont, IL). Beads were washed three times with extraction buffer lacking detergent and eluted by boiling in 2\u0026times; SDS loading buffer. Proteins were separated on 12% SDS-PAGE gels, transferred to PVDF membranes, and probed with anti-GFP (Cell Signaling 2955S) and anti-HA (Cell Signaling 3724S) antibodies. Secondary antibodies were HRP-conjugated goat anti-mouse (Cell Signaling 7076P2) and goat anti-rabbit (Cell Signaling 7074P2). Signal was detected using SuperSignal\u0026trade; West Dura substrate.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eProtoplast Isolation and Expression\u003c/h2\u003e\u003cp\u003eProtoplasts were isolated from 3-week-old \u003cem\u003eArabidopsis thaliana\u003c/em\u003e rosettes using the Tape Sandwich method (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Transfections were performed with 30\u0026micro;g total plasmid DNA (equal ratios in co-expression), using PEG-mediated transformation (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Imaging was performed\u0026thinsp;~\u0026thinsp;18\u0026ndash;24 h post-transfection.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eConfocal Microscopy\u003c/h3\u003e\n\u003cp\u003eMicroscopy was performed using a Zeiss LSM 980 confocal microscope with a 40\u0026times; water-immersion objective. YFP and DHE were excited at 514 nm; emissions were detected at 525\u0026ndash;550 nm and 606\u0026ndash;659 nm, respectively. Chlorophyll and mCherry signals were excited at 561 nm, with emission detection at 657\u0026ndash;724 nm (chlorophyll) and 606\u0026ndash;651 nm (mCherry). Protoplasts were stained with 5 \u0026micro;M dihydroethidium (DHE) and imaged 10 min after staining.\u003c/p\u003e\n\u003ch3\u003eAbiotic Stress Assay\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eArabidopsis thaliana\u003c/em\u003e mutants \u003cem\u003eatspl1\u003c/em\u003e (SALK_134584) and \u003cem\u003eatspl12\u003c/em\u003e (SALK_142295) were obtained from the Arabidopsis Biological Resource Center (ABRC) and crossed to generate the double mutant \u003cem\u003eatspl1/12\u003c/em\u003e. All mutants were in the background ecotype Col-0. Seeds were surface sterilized with 50% bleach as described previously (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e) and stratified in darkness for three days. Seeds were sown on \u0026frac12; MS plates and grown at 22\u0026deg;C with 16hr/8hr photoperiod for seven days. Seedlings were then submerged in \u0026frac12; MS liquid containing 0.5\u0026micro;g/mL Tm for 6 h, washed twice in \u0026frac12; MS, and grown for a further 5 days on 1/2 MS agar. Seedlings were photographed and root lengths measured using ImageJ measurement tools.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eCell death induction by the tomato SlySBP12a is conserved in\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e \u003cb\u003ehomologs AtSPL1 and AtSPL12\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven that members of the SQUAMOSA promoter binding protein (SBP) family are conserved across all land plants, we investigated whether this cell death-associated function is conserved in the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs. SBP-box transcription factors are defined by a conserved zinc finger DNA-binding domain, the SBP-box. Sequence homology analysis revealed that AtSPL1 and AtSPL12 are the closest \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs of SlySBP12a, sharing 52.6 and 53.4% peptide sequence homology, respectively (Suppl. Figure\u0026nbsp;1). The protein domain structure in SlySBP12a, AtSPL1 and AtSPL12 are highly similar, with all sharing the family-characteristic SBP-box domain, as well as multiple Ankyrin repeat domains and a C-terminal transmembrane domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess functional conservation, we transiently expressed AtSPL1 and AtSPL12 in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. Both AtSPL1 and AtSPL12 induced a spontaneous cell death phenotype comparable to that triggered by SlySBP12a (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We previously showed that a C-terminal transmembrane domain (TMD) in SlySBP12a regulates the cell death response upon overexpression (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Consistent with this, the deletion of this domain in either homolog enhanced the intensity of the cell death phenotype upon expression in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). These results demonstrate that the cell death-inducing function mitigated by the C-terminal transmembrane domain of SlySBP12a in AtSPL1 and AtSPL12.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eC-terminal transmembrane domain controls the subcellular localization in AtSPL1 and 12\u003c/h2\u003e\u003cp\u003eWe demonstrated that SlySBP12a localizes to both the nucleus and the endoplasmic reticulum (ER) membrane in tomato protoplasts, with this dual localization being dependent on the C-terminal TMD. Truncation of the TMD abolished ER localization, resulting in exclusive nuclear localization (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). To determine whether this localization pattern is conserved in the \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs, full-length AtSPL1 and AtSPL12 were expressed in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e protoplasts and visualized using fluorescent compartment-specific markers. Both full-length transcription factors co-localized predominantly with the ER marker HDEL-mCherry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Upon truncation of their respective TMDs (AtSPL1ΔTMD and AtSPL12ΔTMD), localization shifted entirely to the nucleus, as indicated by co-localization with the nuclear stain dihydroethidium (DHE) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). This shift coincides with enhanced cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb), suggesting that these transcription factors likely translocate from to the ER to nucleus under specific conditions to initiate cell death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eER stress triggers nuclear translocation of AtSPL1 and AtSPL12\u003c/h2\u003e\u003cp\u003eWe next sought to determine the mechanism driving translocation from the ER to the nucleus. We hypothesized that ER stress could act as a signal promoting this shift. To test this, we included SlySBP12a alongside AtSPL1 and AtSPL12 in an ER stress induction assay, as the regulatory mechanism of SlySBP12a localization had not been previously established.\u003c/p\u003e\u003cp\u003eProtoplasts expressing each transcription factor were treated with either tunicamycin, a potent inducer of the unfolded protein response (UPR), or with DMSO as a mock control. After incubation, localization was assessed using co-staining with HDEL-mCherry or DHE. In mock-treated samples, SlySBP12a, AtSPL1, and AtSPL12 maintained their ER membrane localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, tunicamycin-treated protoplasts exhibited a marked shift in localization. Fluorescence from YFP-tagged transcription factors no longer overlapped with the ER marker, and co-localization with the nuclear stain DHE was clearly observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). These results indicate that ER stress induces translocation of SlySBP12a, AtSPL1, and AtSPL12 from the ER membrane to the nucleus.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eNuclear localization activates the transcription of proteases associated with cell death\u003c/h2\u003e\u003cp\u003eHaving established the nuclear translocation of our SBP/SPL transcription factors, we next investigated its downstream effects on gene expression, reasoning that the transcription factor likely regulates nuclear targets involved in the observed cell death phenotype. To identify candidate genes under regulation by SBP/SPL transcription factors, we conducted RNA sequencing following transient overexpression of SlySBP12aΔTMD in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves (Suppl. Figure\u0026nbsp;2). Among the differentially expressed genes, proteases emerged as a notable functional category, consistent with their observed involvement in plant cell death activation. We prioritized these candidates for functional validation, selecting a subset of protease-encoding genes with a Log2 Fold Change above 2 compared to the empty vector control (Suppl. Table\u0026nbsp;2).\u003c/p\u003e\u003cp\u003eTo test whether these upregulated proteases could independently induce cell death, each protease was transiently expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. While most induced mild chlorosis, the phenotype was more pronounced than the empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). To quantitatively assess membrane disruption, we performed ion leakage assays, which revealed that all tested proteases, except serine carboxypeptidases 3 and 31, significantly increased conductivity compared to control infiltrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). To assess whether these proteases are likely direct transcriptional targets of these transcription factors, we analyzed the upstream promoter regions of the induced genes. Notably, all tested proteases contained a predicted SBP-binding site within 2 kb of the transcriptional start site, and with \u0026ge;\u0026thinsp;90% identity to the SlySBP12a consensus motif (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). These results suggest that specific proteases are directly activated upon SBP/SPL nuclear translocation, and may contribute to the execution of cell death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAtSPL1 and AtSPL12 interact with the E3 Ligase protein Seven in Absentia 2 (SINAT2)\u003c/h2\u003e\u003cp\u003eTo uncover protein interactors that might modulate SBP/SPL transcription factor activity, we performed a yeast two-hybrid (Y2H) screen using SlySBP12a as bait against a tomato cDNA library. Sequencing of positive colonies yielded a list of candidate interactors, and identified the candidate interactor Seven in Absentia 2 (SINAT2), a RING-type E3 ubiquitin ligase from the SINAT family as a potential regulator of SlySBP12a activity. This family is involved in the ubiquitination and degradation of regulatory proteins, including nuclear transcription factors, that mediate plant responses to both abiotic and biotic stress (\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Given this role and the importance of post-translational regulation in stress-responsive signaling, SINAT2 emerged as a compelling candidate for modulating the stability and activity SlySBP12a and its \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs AtSPL1 and AtSPL12.\u003c/p\u003e\u003cp\u003eTo confirm physical interaction between SINAT2 and the transcription factors we performed co-immunoprecipitation using a YFP-tagged SINAT2 construct transiently expressed in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. HA-tagged AtSPL1, AtSPL12, and SlySBP12a were successfully co-immunoprecipitated with YFP\u0026ndash;SINAT2, confirming their interaction \u003cem\u003ein planta\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, Suppl. Figure\u0026nbsp;6), demonstrating conserved protein\u0026ndash;protein interactions across species. No HA signal was detected when SBP/SPL constructs were co-expressed with free YFP, indicating that the observed interactions are specific to SINAT2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSINAT2 attenuates cell death imposed by SBP/SPL transcription factors\u003c/h2\u003e\u003cp\u003eHaving confirmed the physical interaction between SINAT2 and AtSPL1/AtSPL12/SlySBP12a, we next sought to determine its functional relevance in modulating the cell death phenotype imposed by these transcription factors. Transient expression of SINAT2 with all three homologs in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e led to a marked visual reduction in the cell death phenotype compared to co-expression with an empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). Cell death suppression by SINAT2 was quantified using ion leakage assays. Leaves co-infiltrated with SINAT2 and SBP/SPL transcription factors exhibited significantly lower conductivity than those infiltrated with SBP/SPLs and empty vector, thus confirming the ability of SINAT2 to inhibit the cell death phenotype displayed by these transcription factors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). When co-expressed with the animal cell death inducing protein Bax, SINAT2 did not inhibit Bax-induced cell death, indicating SINAT2 inhibition is specific to SBP/SPL induced death (Suppl. Figure\u0026nbsp;3). These results identify SINAT2 as a conserved specific negative regulator of SBP/SPL-induced cell death.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eSINAT2 reduces SBP/SPL accumulation via the Ubiquitin\u0026ndash;Proteasome System\u003c/h2\u003e\u003cp\u003eGiven that SINAT2 directly interacts with these SBP/SPL transcription factors, we next examined its functional role, specifically the mechanism by which it suppresses cell death induced by SBP/SPL in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. As an E3 ubiquitin ligase, we reasoned that SINAT2 may promote the ubiquitination and subsequent proteasomal degradation of its target proteins. We therefore hypothesized that SINAT2 mitigates SBP/SPL-induced cell death by limiting the accumulation of these transcription factors.\u003c/p\u003e\u003cp\u003eImmunoblot analysis revealed that HA-tagged SBP/SPL proteins accumulated to high levels when co-infiltrated with empty vector control. In contrast, co-infiltration with SINAT2 led to a marked reduction in protein levels in a time course experiment, consistent with targeted degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, Suppl. Figure\u0026nbsp;6). To determine whether this reduction is mediated by the 26S proteasome, we treated infiltrated leaves with the proteasome inhibitor MG132. Upon MG132 treatment, SBP/SPL protein levels were restored to those observed in the empty vector control, suggesting that SINAT2 promotes SBP/SPL degradation via the ubiquitin\u0026ndash;proteasome pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, Suppl. Figure\u0026nbsp;6).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eThe RING domain of SINAT2 is required for SBP/SPL destabilization and cell death suppression\u003c/h2\u003e\u003cp\u003eTo determine whether SINAT2's E3 ligase activity is responsible for SBP/SPL degradation and suppression of cell death, we generated a mutant construct lacking the RING domain (SINAT2ΔRING), which is required for ubiquitin ligase activity (Suppl. Figure\u0026nbsp;4). Co-infiltration of SlySBP12aΔTMD, AtSPL1ΔTMD, or AtSPL12ΔTMD with SINAT2ΔRING failed to suppress the cell death phenotype associated with SBP/SPL expression, in contrast to co-infiltration with wild-type SINAT2, which strongly attenuated cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). This visual phenotype was confirmed by ion leakage assays, which indicates a significant decrease in conductivity when co-infiltrated with wild-type SINAT2 compared to the empty vector, but no significant change when co-infiltrated with SINAT2ΔRING (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). This assay confirms that SINAT2ΔRING lacks the ability to attenuate cell death induced by SlySBP12aΔTMD, AtSPL1ΔTMD, or AtSPL12ΔTMD. Furthermore, we monitored SBP/SPL protein accumulation in the presence of SINAT2 or SINAT2ΔRING in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e in a time course experiment. Immunoblot analysis revealed that SINAT2ΔRING failed to reduce SBP/SPL protein accumulation to the level of the wild-type SINAT2 construct (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, Suppl. Figure\u0026nbsp;6). These results demonstrate that SINAT2 requires its RING domain to promote proteasomal degradation of SBP/SPL transcription factors and to suppress their cell death activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eLoss of AtSPL1/12 Confers Partial Insensitivity to Tunicamycin\u003c/h2\u003e\u003cp\u003eTo further investigate the role of SBP/SPL transcription factors in ER stress signaling, we leveraged \u003cem\u003eArabidopsis thaliana\u003c/em\u003e genetic resources, including T-DNA insertion mutants for AtSPL1 and AtSPL12, and generated an \u003cem\u003eatspl1/12\u003c/em\u003e double mutant by crossing AtSPL1 and AtSPL12 single mutant lines (Suppl. Figure\u0026nbsp;5). We focused on ER stress because these SBP/SPL proteins translocate to the nucleus upon ER perturbation, and we assayed responsiveness by treating Col-0 and \u003cem\u003eatspl\u003c/em\u003e lines with Tunicamycin, a well-characterized ER-stress inducer. Comparing growth on Mock versus Tunicamycin plates, Col-0 roots on Tunicamycin retained 18% of their Mock growth, whereas the atspl1/12 double mutant retained 45% of its growth compared to Mock treatment; the atspl1 and atspl12 single mutants resembled Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These data indicate that loss of AtSPL1 and AtSPL12 confers partial insensitivity to Tunicamycin-induced growth suppression and may be considered as potential targets for enhanced stress tolerance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSBP-box transcription factors are conserved across land plants and are best known for regulating key developmental processes, including vegetative phase change, floral induction, and leaf morphology (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). However, mounting evidence suggests that certain members of this family may also play roles in environmental stress responses, both abiotic and biotic (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Our findings demonstrate a novel function for a clade of membrane tethered SBP-box transcription factors, linked by the presence of a C-terminal transmembrane domain, that function to transmit ER stress signals and induce transcriptional changes that result in activation of programmed cell death.\u003c/p\u003e\u003cp\u003eWe previously demonstrated that overexpression of SlySBP12a from tomato triggers spontaneous cell death in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Here, we show that this cell death-inducing activity is conserved in its \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs, AtSPL1 and AtSPL12. Both genes were identified based on sequence similarity and the presence of C-terminal TMDs. Transient expression of AtSPL1 and AtSPL12 in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e caused strong lesion mimic phenotypes, phenotypically indistinguishable from SlySBP12a. These results suggest that the ability to induce PCD is conserved within this TMD-containing SBP/SPL subgroup and is likely linked to their membrane localization.\u003c/p\u003e\u003cp\u003eLike SlySBP12a, AtSPL1 and AtSPL12 localize to the ER membrane via C-terminal transmembrane domains (TMDs), which constrain activity under basal conditions. Removing the TMD causes full nuclear accumulation and a stronger cell death phenotype, indicating that the TMD acts as a negative regulatory gatekeeper. This architecture parallels other ER-tethered transcription factors in plants. bZIP28 is released by proteolytic cleavage, and bZIP60 is activated through IRE1-dependent splicing during ER stress (\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). A similar framework applies to the NTM1-like NAC subgroup, which carries an N-terminal DNA-binding domain and a C-terminal TMD, responds to ER, oxidative, or mitochondrial stress, and can be activated by diverse mechanisms: ANAC017 via rhomboid protease cleavage, GmNTL1 via oxidative cysteine modification, and others via alternative splicing that removes the TMD (\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). The shared architecture and stress-responsive mobility suggest SBP/SPLs may constitute a previously unrecognized class of membrane-tethered transcription factors. Unlike the well-characterized bZIPs and NACs, however, the molecular switch that controls SBP/SPL release remains unresolved. Defining the trigger and the timing relative to ER-stress signaling will be essential to explain how stress cues are converted into a transcriptional program for cell death.\u003c/p\u003e\u003cp\u003eNuclear-localized SlySBP12a lacking the TMD domain strongly upregulates multiple proteases linked to plant PCD, some of which cause chlorosis, and ion leakage when expressed individually, but none replicate the full cell death phenotype. This suggests that robust PCD requires coordinated activity of multiple proteolytic pathways, as seen in hypersensitive responses and other PCD models (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). The breadth of proteases induced also points to a potentially layered proteolytic network that integrates signals from different subcellular compartments. Interestingly, among the induced genes were a cathepsin-like protease and a subtilisin-like serine protease, both members of families previously shown to cleave caspase substrates and were implicated in plant PCD (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Cathepsins can display caspase-like activity and contribute to vacuole-mediated cell collapse during stress, while subtilases, such as phytaspases, promote immune-associated PCD by relocalizing during stress (\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Their induction downstream of SlySBP12a suggests that this transcription factor directly engages established proteolytic effectors, activating a number of proteases with complementary roles in cell death execution.\u003c/p\u003e\u003cp\u003eFuture work should test co-expression of these proteases to determine whether their effects are redundant, additive, or synergistic, and confirm direct promoter binding by SlySBP12a through assays such as Yeast-1 Hybrid. If validated, these proteases could serve as biomarkers and tools for engineering tunable cell death pathways in crops.\u003c/p\u003e\u003cp\u003eIn addition to transcriptional regulation, we show that SBP/SPL protein levels are post-translationally regulated by SINAT2, a RING-type E3 ubiquitin ligase. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, SINA/SINAT ligases, including SINAT2, indirectly modulates ABA-linked drought responses by controlling ESCRT components such as FREE1 and VPS23A, and SINA2 directly promotes ABA-mediated drought tolerance (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). In cotton, SINA orthologs are induced by Verticillium dahliae and enhance resistance when overexpressed, underscoring broader roles in stress (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Through the E3 ligase mutants and inhibition by MG132, we demonstrate that SINAT2 regulates SBP/SPLs by ubiquitination and subsequent degradation via the 26S proteasome. These results align with prior work showing that SINAT-family E3 ligases regulate stress-responsive transcription factors (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe 26S proteasome adds an important layer of control to SBP/SPL turnover, acting as the primary degradation machinery for ubiquitinated proteins and tuning transcription factor activity in response to cellular cues. Similar proteasome-dependent regulation of membrane-tethered or stress-responsive transcription factors, such as DREB2A, ABI5, and NAC proteins, is known to maintain proper stress thresholds (\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Given the activity of SBP/SPLs, proteasomal degradation may buffer against excessive cell death, allowing fine-tuned responses. Mapping how SBP/SPL stability is shaped by proteasomal activity will be critical, and targeted experiments such as proteasome reporter lines or real-time degradation assays offer a direct path to uncovering how this turnover integrates into the plant\u0026rsquo;s broader stress-response and developmental networks.\u003c/p\u003e\u003cp\u003eFunctionally, the ER-stress insensitivity of \u003cem\u003eatspl1/12\u003c/em\u003e places AtSPL1 and AtSPL12 within the UPR machinery that couples ER stress cues to growth restraint. This assignment aligns with executor-type precedents such as NAC089, whose nucleus-localized, TMD-deleted form drives ER-stress\u0026ndash;induced PCD and whose knockdown confers tunicamycin tolerance, while the cytoprotective cochaperone AtBAG7 exemplifies the inhibitor arm, with \u003cem\u003eatbag7\u003c/em\u003e mutants hypersensitive to tunicamycin, heat, and cold (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Together, these examples underscore that the plant UPR is a balance of pro-survival buffering and pro-death outputs. Within that balance, AtSPL1/12 acts as redundant executors that impose growth restraint once proteotoxic burden crosses a threshold; with mutants showing continued growth despite ER stress.\u003c/p\u003e\u003cp\u003eInterestingly, Chao et al. (2017) reported that \u003cem\u003eatspl1/12\u003c/em\u003e mutants show no overt phenotypes under standard conditions but are impaired in basal thermotolerance of developing flowers due to reduced induction of heat-responsive genes (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). This may seem at odds with our findings, however the discrepancy likely reflects differences in tissue, developmental stage, and stress context. Chao et al. examined floral tissues under acute heat stress, whereas we assayed root growth in 1-week-old seedlings under ER stress, conditions that recruit distinct signaling pathways. That AtSPL1/12 function in both settings suggests they act as versatile, context-dependent regulators integrating environmental cues in a tissue- and input-specific manner, a hallmark of membrane-tethered transcription factors such as bZIPs and NACs (\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur findings support a model where a specific clade of SBP/SPL transcription factors are ER-tethered under basal conditions and translocate to the nucleus during ER stress, where they activate programmed cell death genes, including proteases. Their accumulation is counterbalanced by SINAT2, which targets SBP/SPLs for ubiquitin\u0026ndash;proteasome degradation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). This dual control, ER release and regulated turnover, fine-tunes the timing and magnitude of SBP/SPL activity during stress. Our findings extend SBP/SPL roles beyond development to stress signaling and cell fate, defining an ER-anchored/SINAT2 module as a new layer of control and a potential lever for engineering more resilient, precisely tuned cell-death responses in crops.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Sarah Swanson, director of the Newcomb Imaging Center for technical support with confocal microscopy. We also thank the lab of Dr. Andrew Bent (University of Wisconsin-Madison) for providing the Bax control plasmid. This project was partially supported by USDA-HATCH (Award # WIS04031) and the Department of Plant Pathology at the University of Wisconsin-Madison.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.V. and M.K. contributed to study conception and design. A.V., D.M. and Z.M. executed the experiments. A.V., M.K., and B.W. analyzed and interpreted the data. A.V. prepared the figures and drafted the manuscript. M.K., B.W., and R.K. reviewed and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequencing read files available at NCBI Sequence Read Archives (SRA) BioProject PRJNA1337887. \u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eBeers EP. Programmed cell death during plant growth and development. Cell Death Differ. 1997 Dec;4(8):649\u0026ndash;61.\u003c/li\u003e\n \u003cli\u003eXie F, Vahldick H, Lin Z, Nowack M. Killing me softly - programmed cell death in plant reproduction from sporogenesis to fertilization. Curr Opin Plant Biol. 2022 Aug 9;69:102271.\u003c/li\u003e\n \u003cli\u003evan Doorn WG, Beers EP, Dangl JL, Franklin-Tong VE, Gallois P, Hara-Nishimura I, et al. Morphological classification of plant cell deaths. 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Plant J. 2014;79(6):1033\u0026ndash;43.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\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":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7794624/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7794624/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSQUAMOSA promoter-binding proteins (SBPs) are key regulators of plant growth, development, and stress responses, with their roles in programmed cell death (PCD) gaining increasing attention. In this study, we investigate the role of a tomato SBP/SPL transcription factor, and its \u003cem\u003eArabidopsis thaliana\u003c/em\u003e homologs, AtSPL1 and AtSPL12, in ER stress-induced PCD. All three homologs share the conserved SBP-Box domain, and C-terminal Ankyrin and transmembrane domains (TMDs). We show that the TMD anchors these transcription factors to the ER membrane, and their transient expression in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e induced spontaneous cell death, mediated by the C-terminal TMDs. Upon ER stress induction, specifically with tunicamycin, we observed a striking shift of these transcription factors from the ER to the nucleus, marking a crucial step in their activation of PCD. This nuclear translocation underscores their role as ER stress sensors. RNA sequencing following expression of these transcription factors revealed the upregulation of several protease classes that may be responsible for the execution of PCD. Overexpression of individual proteases induced cell death, suggesting that a coordinated protease response is necessary for full PCD induction. We also identify the interacting protein SINAT2, a RING-type E3 ubiquitin ligase, as a key regulator of SBP/SPL stability. SINAT2 physically interacts with these transcription factors, promoting their proteasomal degradation as evidenced by protein accumulation assays, and mitigates the PCD phenotype. The \u003cem\u003eArabidopsis thaliana atspl1/12\u003c/em\u003e double mutants were insensitive to tunicamycin-induced ER stress, and failed to exhibit the typical growth suppression seen in wild-type and single mutants upon tunicamycin treatment. This suggests that AtSPL1 and AtSPL12 are essential for stress perception and response under ER stress conditions. These findings shed light on the roles of ER membrane-tethered SBP/SPL transcription factors in stress signaling and the execution of cell death, emphasizing their potential as targets for enhancing stress resilience in plants through genetic engineering.\u003c/p\u003e","manuscriptTitle":"A conserved clade of ER-membrane tethered SBP/SPL transcription factors regulate cell death mediated by ER stress in plants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-09 10:40:44","doi":"10.21203/rs.3.rs-7794624/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-11-10T15:45:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-11-05T05:54:57+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-31T09:08:38+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-10-22T01:07:05+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-10-09T10:23:15+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-10-09T09:34:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T15:44:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-06T23:12:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2025-10-06T23:12:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6dd8f14e-c8d6-492d-a368-cc04b5a03901","owner":[],"postedDate":"October 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":56015115,"name":"Biological sciences/Molecular biology"},{"id":56015116,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2026-04-13T21:25:55+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-09 10:40:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7794624","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7794624","identity":"rs-7794624","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}