*3.1. Expression of the CaHSP18.1a in Pepper Plants under Abiotic Stress*

To confirm whether heat, drought, and salt have an effect on the expression of *CaHSP18.1a*, R9 and B6 pepper lines were used to analyze the expression of *CaHSP18.1a* under heat, drought, and salt stress. Under heat stress (Figure 1A,B), the expression levels of *CaHSP18.1a* were significantly upregulated (samples IV, V, and VI) at 3 h in the roots, stems, and leaves of R9 plants and peaked at 6 h (sample Point V) in B6 plants. However, during the 22 ◦C recovery stage, the recovery times of *CaHSP18.1a* in the R9 and B6 strains differed. In roots, the expression levels of *CaHSP18.1a* in R9 and B6 plants returned to baseline after a 6 h recovery at the normal temperature (sample VII point) (Figure 1B). In stems, the expression level of *CaHSP18.1a* returned to a normal level after 3 h under the 22 ◦C recovery conditions for both the R9 and B6 plants (point VI in samples) (Figure 1C). In leaves, the expression level of *CaHSP18.1a* returned to normal levels at 24 h (sample VIII point) (Figure 1D). We also analyzed the expression pattern of *CaHSP18.1a* under salt and drought stresses (Figure 1E–H). After 6-h NaCl treatments at different concentrations, the expression of *CaHSP18.1a* in R9 and B6 leaves and roots was highest under the 150 mM NaCl, 100 mM NaCl treatments, respectively. The transcription of *CaHSP18.1a* was higher in R9 under different concentrations of NaCl treatment (Figure 1E, F). After 6-h treatments with different concentrations of mannitol, the expression of *CaHSP18.1a* in R9 and B6 leaves was the highest after the 150 mM mannitol treatment; the highest expression of *CaHSP18.1a* was observed in R9 and B6 roots subjected to the 50mM mannitol treatment (Figure 1G, H). In addition, the transcription of *CaHSP18.1a* was higher in R9 under different concentrations of mannitol. This analysis showed that the expression of *CaHSP18.1a* in pepper was induced by heat, salt, and drought. The response times of *CaHSP18.1a* in different organs of different pepper lines differed, and the response was more intense and more early in the R9 line, which suggests that *CaHSP18.1a* plays a substantial role in plant responses to heat stress.

**Figure 1.** The expression characteristics of *CaHSP18.1a* in peppers in response to heat stress. (**A**) Time course of heat stress treatment and normal temperature recovery; the sampling time points are represented by triangles (pepper sample points I–VIII); (**B**–**D**) The expression levels of *CaHSP18.1a* in roots, stems, and leaves of R9 and B6 plants at each sampling time point; the expression levels of B6 and R9 plants were based on the reference level of their samples, and *CaUBI-3* was selected as the reference gene.(**E**–**H**) The expression levels of *CaHSP18.1a* following salt and drought treatment in R9 and B6 leaves and roots. The data presented are means with standard deviations of three biological replicates. Different letters denote statistical significance (*p* ≤ 0.05).
