Sulfate availability critically influences plant growth, yet the role of small RNAs, particularly microRNAs (miRNAs), in regulating responses to sulfate deficiency remains poorly understood. Here, we conducted a temporal analysis of sulfate deficiency-responsive miRNAs in the roots and leaves of Solanum lycopersicum (tomato), using an updated miRNA annotation in the SL4.0 genome. We found 40 differentially expressed miRNAs, including 2 novel, tomato-specific miRNAs. Tomato miRNAs showed an important time- and organ-specific regulation, similar to the described response of the mRNA transcriptome. Integration with transcriptomic data and Degradome-seq analysis highlighted both canonical and non-canonical targets for sulfate-responsive miRNAs. miR395, the most extensively studied miRNA, was found to control not only its conserved targets involved in sulfate transport and assimilation, but also genes involved in redox homeostasis, photosynthesis and chloride transport. Notably, most targets were repressed in leaves, suggesting miRNA-mediated downregulation of energy-intensive processes, while root targets were predominantly upregulated, including genes related to protein remodeling and antioxidant defense. Comparative analysis with Arabidopsis thaliana revealed a broader functional repertoire in tomato, suggesting species-specific adaptations to sulfate deficiency. Overall, our results underscore the critical role of miRNAs in fine-tuning organ-specific metabolic reprogramming during nutrient stress, expanding the current understanding of the regulatory landscape underlying sulfate deficiency in plants.

Sulfur is an essential element for biological molecules, with sulfate as the primary sulfur source for plants. Under sulfate-deficiency, plants undergo physiological, metabolic, transcriptional, and post-transcriptional changes. Although Solanum lycopersicum (tomato) exhibits extensive transcriptome reprogramming during sulfate deficiency, the role of microRNAs (miRNAs) in this response remains poorly explored. In this study, we analyzed miRNAs expression in tomato roots and leaves at 3- and 4-weeks post-sowing under sulfate-sufficient and sulfate-deficient conditions, leveraging a de novo annotation of miRNA genes in the SL4.0 genome. We identified several miRNAs showing tissue- and time-specific expression changes in response to sulfate deficiency, with a potential role in processes such as lignin, chlorophyll, and polysaccharides metabolism. Consistent with other plants, miR395 emerged as a key sulfur metabolism and transport regulator. Notably, we identified a novel miR395 target, SlERF2a, an Ethylene-Responsive Factor predicted to regulate numerous sulfate deficiency-responsive genes, including genes involved in plastid organization, detoxification, sulfur assimilation and starvation response. These results suggest a broader role for miR395 in sulfate-deficiency responses in tomato compared to other plants. This work provides new insights into the post-transcriptional regulation of sulfate-deficiency in tomato, providing an updated miRNA annotation for future studies on miRNA-mediated regulation in this relevant crop.