**4. Discussion**

High temperature is one of the key climatic parameters affecting plant growth and development, resulting in crop yield losses [47]. HS can restrict photosynthesis, increase photorespiration and transpiration rate through stomatal regulation, and reduce plant biomass [7]. Pepper is a highly temperature-sensitive crop [3]. Although the physiological effects of HS on pepper have been widely studied, changes in pepper at the molecular level in response to HS and subsequent recovery are poorly understood. Therefore, to better understand the HS response in pepper, it is necessary to uncover the mechanisms underlying it. In the present study, we investigated the phenotypic and physiological changes in pepper seedlings of two varieties during and after recovery from HS. Furthermore, we comparatively analyzed heat-induced transcriptomic changes to obtain a global view of HS responses in pepper.

Under high temperature stress, the structure and function of the cell protoplasmic membrane are initially damaged, resulting in an increase in cell membrane permeability and intracellular electrolyte leakage and finally leading to an increase in electrolyte leakage of tissue leachate [48]. Therefore, the degree of electrolyte extravasation and high temperature injury can be determined by measuring the relative electrical conductivity of the tissue extract. In this study, 17-03 and H1023 were verified as high temperature-resistant and high

temperature-sensitive varieties, respectively (Figure 1b,c,f,g), and were used to explore the responses and recovery patterns of pepper to HS and the possible mechanisms of the different heat resistances. There was a remarkable increase in relative electrolyte leakage in both 17-03 and H1023 after HS, but the levels were significantly lower in 17-03 than in H1023 (Figure 1h), indicating that 17-03 could alleviate damage to cellular membranes during HS. However, there was little change in the two varieties from HS to recovery (Figure 1h), indicating that the damage to the cell membrane caused by HS is irreversible. Proline, an amino acid and a compatible solute, has been widely reported to accumulate in response to various abiotic stresses, such as high temperatures [37]. After 3 d of heat treatment and 1 d of recovery, proline levels were significantly increased in both 17-03 and H1023. While there was no significant difference in proline content between 17-03 and H1023 during heat treatment (Figure 1h), the proline content increase in 17-03 during recovery was higher than that in H1023 (Figure 1h), indicating that the self-repairing ability of 17-03 after HS was stronger than that of H1023. Based on these data, we conclude that 17-03 is more heat-tolerant, as it protects cell membranes from damage and alleviates osmotic stress by increasing the proline levels.

Moreover, we obtained accurate data from transcriptome analyses based on RNAseq and analyzed the genes of metabolic pathways that were significantly affected by HS and participated in the process of plant restoration. In 17-03 and H1023, there were more DEGs in the HS stage than in the recovery stage (Figure 2a,b), indicating that the regulatory mechanism of HS response was more active at a transcriptional level. There were significantly more upregulated DEGs than downregulated DEGs in CK1\_vs\_M1 (Figure 2b). However, in the other groups, the number of up- and downregulated DEGs was almost the same (Figure 2b). Moreover, the DEGs after heat treatment were mostly different from those during recovery in 17-03 and H1023 (Figure 2a). These results indicate that the defense and recovery mechanisms of pepper may have common regulatory pathways, and that there are different pathways for response, resistance, and repair.

Out of a total of 11,633 DEGs (Table S3), 5133 were assigned a GO classification (Table S3). GO enrichment analysis showed that DEGs after heat treatment were commonly enriched in "photosynthesis, light harvesting" (GO:0009765), "cellular glucan metabolic process" (GO:0006073), and "nucleosome assembly" (GO:0006334) in 17-03 and H1023 (Table S4). These findings are similar to the HS response in sweet maize (*Zea mays* L.) [49]. DEGs were also significantly enriched in "DNA replication" (GO:0006260), "DNA replication initiation" (GO:0006270), "protein folding" (GO:0006457), "protein-DNA complex assembly" (GO:0065004), "protein-DNA complex" (GO:0032993), and "nucleosomal DNA binding" (GO:0031492) in 17-03 after heat treatment (Figure 4; Table S4), which may confer increased resistance to high temperatures. The DEGs of H1023 and 17-03 during recovery were enriched with different GO terms and KEGG pathways (Figures 4 and 5, Tables S4 and S5), indicating that the repair pathways were different, which is likely due to the different degrees of high-temperature damage. In the KEGG pathway analysis, DEGs involved in HS response were predicted to function in metabolic pathways and the biosynthesis of secondary metabolites in 17-03 and H1023, which is similar to the results of previous studies [50,51].

Hsps, which are involved in multiple biological processes, such as signal transduction during HS, and have deduced functions, such as being chaperones, the folding and unfolding of cellular proteins, and the protection of functional sites from the adverse effects of high temperature, range in molecular mass from 10 to 200 kDa [52]. Hsps have functions as molecular chaperones that affect protein quality and were initially identified as proteins that were upregulated during heat treatment [51]. Many Hsps have been detected as heat response factors in tomato [53] and grape [54] plants. In this study, a total of 47 Hsps were significantly differentially expressed in the four groups. Among them, 45 Hsps were common differentially expressed in 17-03 and H1023 after heat treatment (Figure 6; Table S6). Hsps can accumulate rapidly in sensitive organs and play important roles in protecting the metabolic apparatus of cells, thus acting as a key factor in the adaptation of plants to high

temperatures [55]. In this study, almost all DEGs encoding Hsps were upregulated in the four groups, the expression of which was the highest after heat treatment (Figure 6), which may play a role in protection under and after HS in 17-03 and H1023.

In addition to Hsps, various other TF genes, such as genes of Hsfs, NAC, and WRKY TFs, were also affected by HS [56]. Hsfs combine with cis-acting Hsps to play important roles in both basal and acquired thermotolerance [26,51]. Here, 17 DEGs encoding Hsfs were identified and most were upregulated in the four groups (Figure 6; Table S6). Moreover, some Hsfs were significantly highly expressed in H1023 after recovery from HS; such as CA02g11030, CA03g06850, CA05g00840, CA06g08710, CA07g15920, and CA10g20440 (Figure 6). These significantly expressed Hsfs could play important roles in the long-term HS response of pepper, by combining with the cis-acting regulatory elements, called heat shock elements, in the promoter regions of Hsps.

Plant NAC TFs have been reported to play an important role in modulating HS responses. For example, overexpression of Arabidopsis ANAC042 leads to significant thermotolerance in transgenic plants [57]. Our data indicated that the expression of 38 TFs encoded by NAC domain-containing genes was also heat-regulated (Figure 7; Table S7). Interestingly, most upregulated NAC TFs were more highly expressed in H1023 than in 17-03, during and after recovery from HS (Figure 7). WRKY TFs are one of the largest TF families in plants and have also been reported to participate in regulating plant HS response [58]. In this study, 35 WRKY TFs responded to HS in the four groups, and almost half of them positively regulated thermotolerance (Figure 7). Moreover, some WRKY TFs negatively regulated thermotolerance, such as CA01g01920 and CA01g23300 (Figure 7).
