*3.1. Phenotypic and Physiological Responses of 17-03 and H1023 under HS*

To accurately evaluate the heat resistance of pepper in the seedling stage under HS, 17-03 (heat-tolerant variety) and H1023 (heat-sensitive variety) were treated at 42 ◦C for 3 d and then recovered for 1 d at 25 ◦C. Compared with the leaves of seedlings of 17-03 and H1023 that did not undergo HS (Figure 1a,d), the leaves of seedlings 17-03 after heat treatment were slightly bent (Figure 1b), while the leaves of H1023 were severely sagged, and the lower leaves were severely wilted. Additionally, the growth points of the plants were necrotic after treatment at 42 ◦C for 3 d (Figure 1e). After recovering at 25 ◦C for 1 d, the down-bent leaves of 17-03 were completely extended (Figure 1c), while those of H1023 only partially recovered and did not stretch out completely. Additionally, the edges of the leaves had different degrees of withering (Figure 1f).

**Figure 1.** Phenotypic and physiological responses of 17-03 and H1023 under heat stress (HS). Seedlings of 17-03 and H1023 in control (**a**,**d**), treated at 42 ◦C for 3 d (**b**,**e**), and recovered for 1 d after 3 d of heat treatment (**c**,**f**). Relative electrolyte leakage (**g**) and proline content (**h**) in leaves treated at 42 ◦C for 0 d and 3 d and recovered for 1 d. Three independent biological replicates were used in each treatment, with 9 plants (6–8 true leaves) per replicate. Data are presented as mean ±SEM of three independent biological replicates. Asterisks indicate statistically significant differences between tolerant and sensitive genotypes. \*\*, *p* < 0.01, Student's *t* test.

In this study, relative electrolyte leakage and proline content were measured in the treated plants to evaluate heat tolerance. Under normal growth conditions, there was no significant difference in the relative electrolyte leakage and proline content between the two varieties (Figure 1g,h). However, after 3 d of heat treatment at 42 ◦C and 1 d recovery at 25 ◦C, there was a remarkable increase in relative electrolyte leakage in the two varieties, with levels being significantly lower in 17-03 than in H1023 (Figure 1g). The proline content in plants increased after HS and recovery in both varieties (Figure 1h). After 3 d of heat treatment, the proline content increased, but there was no significant difference between the two varieties. However, the proline content further increased significantly in the recovery stage, and the proline content in 17-03 was obviously higher than that in H1023. These results show that 17-03 was more heat-tolerant than H1023, as the cell membranes were protected from damage, and osmotic stress was alleviated by increasing the levels of proline, an important osmotic protectant.
