Unfolding of RNA secondary structure impairs RNA stability to fine-tune phosphate starvation responses in rice roots

The availability of essential macronutrients, such as inorganic phosphate (Pi) and nitrogen, limits global crop production. In vivo RNA secondary structure (RSS) regulates nearly all steps of the RNA life cycle and is dynamic under stress conditions. However, the roles of RSS in plant responses to nutrient deficiency remain unclear. Here, we used dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) to generate high-quality, deep-coverage in vivo RSS profiles of rice (Oryza sativa) roots in response to Pi deficiency (-P) or nitrogen deficiency (-N) stress. -P increased global RSS diversity and triggered RSS unfolding in thousands of transcripts. By comparing -P and -N RSS profiles, we identified -P-specific RSS-unfolding regions in rice roots. These regions, characterized by low GC content, were enriched within the coding sequences of many Pi starvation response transcripts. Ribosome profiling suggested that -P-specific RSS unfolding was not associated with translational regulation. In contrast, transcriptome-wide RNA decay assays under normal, -P, and Pi-refeeding conditions revealed global regulation of RNA stability in response to -P in rice roots; decreased RNA half-life was linked to RSS unfolding. Transcriptome analysis and analyses of transgenic rice plants with altered RSS demonstrated that -P-specific RSS unfolding lowers RNA stability, thus fine-tuning the accumulation of Pi starvation response transcripts and Pi homeostasis. This study systemically elucidates the dynamic roles and regulatory functions of RSS in the Pi starvation response in rice roots. Our findings underscore the importance of RSS in modulating nutrient-deficient stress responses.