Structural and regulatory features associated with elevated accumulation of the rice ERFVII transcription factor SUB1A-1.

Abstract

Flooding-induced hypoxia severely limits crop productivity, and Group VII Ethylene Response Factors (ERFVIIs) function as central transcriptional regulators of physiological and transcriptional acclimation to hypoxia. In rice, the ERFVII transcription factor SUB1A-1 confers strong submergence tolerance. It displays unusually high steady-state protein levels and reduced sensitivity to oxygen-dependent turnover despite retaining a canonical N-terminal degron motif that promotes rapid turnover of other ERFVIIs under normoxia. The basis of this property remains incompletely resolved. Here, we investigate structural and regulatory features associated with SUB1A-1 accumulation using ex vivo protein accumulation assays, domain-truncation analyses, biophysical characterization, and promoter-activity assays. We show that the C-terminal region, especially Gly256 to Ala281, of SUB1A-1 is associated with increased steady-state protein levels, and that its removal is associated with reduced accumulation and effects consistent with increased turnover in this assay. Full-length SUB1A-1 forms higher-order assemblies, and removal of the C-terminal region yields a smaller species by size-exclusion chromatography. In addition, SUB1A-1-dependent transcriptional activation requires clustered GCC-box motifs within target promoters, indicating that promoter context strongly influences regulatory output. Notably, the GCC1-box motifs most relevant to SUB1A-1 activity coincide with HRPE-like sequence motifs, suggesting that the local promoter sequence context may further shape SUB1A-1-dependent activation. Together, these observations indicate that elevated accumulation of SUB1A-1 correlates with C-terminal-associated assembly behavior and promoter-associated regulatory features. We propose that these properties may reduce functional accessibility to N-degron pathway components, providing a framework for understanding how SUB1A-1 is structurally and functionally configured to support transcriptional regulation under submergence and other energy-limiting conditions.