*2.5. DEGs in Response to Di*ff*erent Heat Treatment Exposure*

The 3559 DEGs were further compared in all four heat treatments; 210, 542, and 1308 common DEGs were found between WD\_1 and WD\_2, WD\_2 and WD\_6, and WD\_6 and WD\_12, respectively (Figure 4a).

To further investigate the dynamic expression of the 3559 DEGs at different time points of heat treatment, heatmap was constructed using *R* package firstly (Figure S2), and then genes with similar expression pattern were plotted using *R* language (Figure 6). As shown in Figure 6, the 3559 DEGs were mainly classified into nine clusters according to their expression level changes in the four heat treatments as compared to the control. Cluster 1 contained 129 DEGs that were shared across all treatments, accounting for less than 4% of the total DEGs. Further analysis showed that 33 and 96 of these genes were consistently up- or down-regulated, respectively, with different fold changes from 1 to 12 h of heat treatment. Cluster 2 contained 20 genes that were regulated by short and intermediate heat treatments but not by long-term treatments, while Cluster 3 contained 279 genes that were not regulated by 1 h heat shock (WD\_1), but by longer time of heat treatments after that (WD\_2, WD\_6, and WD\_12). Cluster 4 contained 30 genes that were regulated by the two short-term heat treatments, but not by the intermediate and long-term heat treatment. Cluster 5 contained 819 genes that were regulated by the intermediate and long-term heat treatments, but not by the short-term heat treatments. Clusters 6 to 9 contained 126, 166, 872, and 732 DEGs were identified to be specifically regulated by 1, 2, 6, and 12 h of heat treatment, respectively (Figure 6, Table S2). Expressions of the remaining 386 DEGs were fluctuant with the time of treatments increase (other cluster), e.g., LOC\_Os04g59440 was induced by WD\_1 and WD\_6, but not by WD\_2 and WD\_12.

**Figure 6.** Expression of genes in different clusters. (**a**) cluster 1, genes regulated by all treatments; (**b**) cluster 2, genes regulated by short and intermediate heat treatments but not by long-term treatments; (**c**) cluster 3, genes not regulated by 1 hour heat shock, but by longer time of heat treatments after that; (**d**) cluster 4, genes regulated by the two short-term heat treatments, but not by the intermediate and long-term heat treatment; (**e**) cluster 5, genes regulated by intermediate and long-term heat treatments, but not by short-term heat treatments; (**f–i**) clusters 6–9, genes specifically regulated by 1, 2, 6, and 12 h of heat treatment, respectively. Different color lines indicated different genes.
