Background/Objective: The midday depression of photosynthesis, a physiological phenomenon driven by environmental stress, impacts plant productivity. This study aims to elucidate the molecular and physiological responses underlying midday depression in two mulberry species, Ewu No. 1 (Ew1) and Husan No. 32 (H32), to better understand their species-specific stress adaptation mechanisms. Methods: RNA-seq analysis was conducted on leaf samples collected at three time points (10:00 a.m., 12:00 p.m., and 4:00 p.m.), identifying 22,630 differentially expressed genes (DEGs). A comparative Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to reveal the involvement of key metabolic and signaling pathways in stress responses. Results: Ew1 displayed enhanced stress tolerance by upregulating genes involved in energy management, water conservation, and photosynthetic processes, maintaining higher photosynthetic rates under midday stress. In contrast, H32 adopted a more conservative response, downregulating genes related to photosynthesis and metabolism, favoring survival at the expense of productivity. The KEGG analysis highlighted starch and sucrose metabolism and plant hormone signaling as critical pathways contributing to these species-specific responses. Conclusions: Ew1’s adaptive molecular strategies make it more suitable for environments with variable light and temperature conditions, while H32’s conservative approach may limit its productivity. These findings provide valuable insights for breeding programs aimed at improving stress tolerance and photosynthetic efficiency in mulberry and other crops, particularly under fluctuating environmental conditions.

Grafting profoundly influences fruit tree performance, yet the molecular mechanisms underlying rootstock–scion interactions in mulberry (Morus multicaulis) remain poorly understood. To investigate the regulatory networks linking grafting to scion physiology, we performed an integrated analysis combining small RNA sequencing, transcriptome-based KEGG pathway assessment, and qRT-PCR validation of key target genes across five rootstock–scion combinations. High-throughput miRNA profiling of phloem tissues identified 71 conserved and 156 novel miRNAs, which exhibited distinct, genotype-dependent expression patterns. Recurrent activation of nutrient- and stress-responsive families (e.g., miR399, miR397, and miR395) was observed. Target prediction and functional enrichment analyses revealed that these miRNAs likely regulate pathways related to metabolic processes, hormone signaling, cell wall modification, and stress responses. The expression trends of their predicted target genes—including TCP4, Laccase-3, and ATP sulfurylase 1—were subsequently confirmed by qRT-PCR, revealing significant rootstock-specific regulation. Collectively, this study establishes a reference framework for elucidating the molecular mechanisms underlying scion biological processes in Morus species, which is of great significance for improving fruit quality, enhancing abiotic and biotic stress resistance, and boosting long-term planting productivity.