*3.6. Analysis of HS-Responsive Heat Shock Proteins and Heat Transcription Factors*

Hsps and Hsfs are sensitive to HS, indicating that they play important roles in the HS response. Based on the results of transcriptome sequencing, 47 Hsp members were identified as differentially expressed in at least one of the four comparison groups (Figure 6; Table S6). Most of these were dramatically upregulated in pepper leaves after 3 d of heat treatment and were more highly expressed in H1023 than in 17-03 (Figure 6). Moreover, seven Hsps were expressed at higher levels after recovery from HS. CA01g13220, CA02g11030, CA09g06120, and CA10g10840 were highly expressed in H1023 cells (Figure 6), and CA01g31330 was highly expressed in 17-03 and H1023 cells (Figure 6). Furthermore, the expression levels of five Hsps (CA04g02800, CA09g03220, CA09g06120, CA11g13160, and CA11g13170) markedly decreased after 3 d of heat treatment (Figure 6). Similarly, 17 significantly differentially expressed Hsfs were identified, most of which were upregulated during and after recovery from HS (Figure 6). Among them, six (CA02g11030, CA03g06850, CA05g00840, CA06g08710, CA07g15920, and CA10g20440) were significantly highly expressed in H1023 after recovery from HS (Figure 6). These results indicate that these significantly differentially expressed Hsps and Hsfs might play an important role in plant protection in the long-term HS response of pepper.

**Figure 6.** Heatmap of differentially expressed genes (DEGs) encoding Hsps and Hsfs in the comparison groups CK1\_vs\_T1, CK2\_vs\_T2, CK1\_vs\_M1, and CK2\_vs\_M2. The color gradient represents the normalized fragments per kilobase of transcript per million mapped reads (FPKM) value (Z-score) of DEGs. The redder the bars, the higher the gene expression level.

## *3.7. Analysis of HS-Responsive Transcription Factors*

Transcription factors (TFs) play an important role in plant growth and development, as well as in biotic and abiotic stress response networks. Transcriptome analysis showed that many TFs in pepper were regulated by high temperatures and participated in plant recovery. A total of 49 TF families of 635 TFs were differentially expressed during heat treatment and recovery in 17-03 and H1023, including HSF, NAC, WRKY, ERF, bHLH, MYB, C2H2, B3, GRAS, bZIP, and HD-ZIP (Table S7). In this study, 38 DEGs encoding NAC proteins were identified. Among them, most were upregulated during and after recovery from HS. Moreover, the expression levels of most NAC TFs were higher in H1023 than in 17-03 during and after recovery from HS (Figure 7). WRKY proteins have also been reported to play important roles in heat response. Here, 35 DEGs encoding WRKY proteins were identified, with almost half of them upregulated in both H1023 and 17-03 during and after recovery from HS (Figure 7). Moreover, five WRKY genes (CA01g01280, CA01g34460, CA09g05110, CA09g11940, and CA12g09290) were upregulated in 17-03 but downregulated in H1023 after recovery from HS (Figure 7). Furthermore, some WRKY genes, such as CA11g05370, CA02g18540, CA09g08120, CA11g03750, and CA01g22410,

were significantly highly expressed in H1023 after recovery from HS, but not at other times in H1023 or in 17-03 (Figure 7).

**Figure 7.** Heatmap of differentially expressed genes (DEGs) encoding NAC and WRKY proteins in the comparison groups CK1\_vs\_T1, CK2\_vs\_T2, CK1\_vs\_M1, and CK2\_vs\_M2. The color gradient represents the normalized fragments per kilobase of transcript per million mapped reads (FPKM) value (Z-score) of DEGs; the redder the bars, the higher the gene expression level.
