Upstream ORFs regulate translation of the Arabidopsis ZIF2 transporter and affect ER stress tolerance

Translational regulation mediated by upstream open reading frames (uORFs) is an important mechanism controlling gene expression in eukaryotes, yet the functional characterization of uORFs in plant transporter genes remains limited. ZINC-INDUCED FACILITATOR 2 (ZIF2) is an Arabidopsis thaliana tonoplast-localized zinc transporter whose expression is regulated at the translational level via alternative splicing of its 5 untranslated region (UTR), but the role of additional cis-regulatory elements within this region has remained unexplored. Here, we characterize the functional significance of three uORFs present in the ZIF2 5UTR and elucidate their contribution to translational control and stress responses. We demonstrate that two of the three uORFs (uORF2 and uORF3) act as negative regulators of ZIF2 translation. uORF2 is constitutively translated and plays the primary role in uORF-mediated translational inhibition, whereas uORF3 functions as a fail-safe mechanism that represses translation only when uORF2 is inactive. We further show that uORF2-mediated repression depends on the amino acid sequence of the encoded peptide and identify a short motif critical for its function. Importantly, uORF-mediated regulation of ZIF2 is physiologically relevant in planta: plants carrying mutations that abolish uORF2 translation accumulate higher levels of ZIF2 protein and display increased tolerance to endoplasmic reticulum stress. Together, our findings reveal a uORF-based fail-safe regulatory mechanism that modulates ZIF2 expression, contributes to plant stress adaptation, and is consistent with translational inhibition mediated by peptide-dependent ribosome stalling.