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

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.