Hsp101-1 Orchestrates Thermotolerance in Rice via Pre-Activated Transcriptional Networks and Modular Cross-Tissue Coordination

<b>Background/Objectives:</b> Rice production faces threats from rising temperatures, demanding thermotolerant varieties. This study characterizes transcriptomic dynamics and identifies <i>Hsp101-1</i> (<i>heat shock protein 101-1</i>)-associated gene regulatory modules in rice under reproductive-stage heat stress. <b>Methods:</b> Transcriptomics and WGCNA (weighted gene co-expression network analysis) were conducted in flag leaves and spikelets for wild-type (WT) and <i>Hsp101-1</i>-overexpressing (OE) lines under 40 °C stress at six time points (0-24 h) to reveal the change in gene expressions. <b>Results:</b> The number of DEGs (differentially expressed genes) revealed substantial pre-existing differences in WT and OE lines. Pre treatment, OE flag leaves showed 545 upregulated and 676 downregulated DEGs versus WT leaves. Post heat shock, the number of DEGs in flag leaves and spikelets was significantly reduced by 70-80%. KEGG enrichment of common DEGs across time points showed both WT and OE flag leaves enriched for ribosome biogenesis, ribosomes, and chaperones/folding catalysts. WGCNA identified that the MEdarkslateblue module correlated negatively with WT and positively with OE flag leaves. The MEturquoise module was suppressed at 1 h but activated by 8 h. Spikelet analysis identified the MElightpink4 module (negative correlation with WT, positive with OE) and a similarly dynamic MEturquoise module. Venn analysis identified 76 shared module genes, 71 of which were upregulated in the OE line, indicating that Hsp101-1 activates common protective targets. Hsp101-1's expression in the WT line was low basally, significantly upregulated at 1-8 h post shock, and returned to low levels by 24 h. <b>Conclusions:</b><i>Hsp101-1</i> enhances thermotolerance by (1) constitutively pre-stabilizing transcriptomic networks and reducing transcriptional fluctuations under heat stress and (2) modularly coordinating tissue-specific responses, providing a climate resilience framework.