3.4.1. Overexpression of CaHSP18.1a Enhances Plant Tolerance of Heat Stress

First, we transformed pVBG2307:*CaHSP18.1a* into *Agrobacterium tumefaciens* strain GV3101, which was used to transfect *Arabidopsis thaliana* using the dipping method; successful transformants were identified through resistance gene screening and molecular level detection until homozygous T3 lines were obtained (Supplementary Figure S2A). The wild-type (WT) line and five transgenic lines were cultured on Murashige and Skoog (MS) medium for 10 days, and the lengths of their roots were measured. The survival rate of WT plants was lower than those of the OE1, OE2, and OE3 seedlings (Supplementary Figure S2C,D). Transgenic plants and WT plants were cultured under normal growth conditions for 48 days, and the growth rates of the OE1, OE2, and OE3 lines exceeded those of WT plants (Supplementary Figure S2D). Thus, the OE3, OE2, and OE1 lines were selected for follow-up experiments.

The obtained transgenic lines and WT plants were heat treated (42 ◦C for 24 h) at the 3-week stage. After heat-stress treatment, the WT plants showed wilting symptoms. Notably, restorable wilt or indistinct-symptoms were observed among the *CaHSP18.1a*-OE seedlings (Figure 4A), indicating that *CaHSP18.1a* plays an active role in increasing the thermotolerance of transgenic *Arabidopsis*. In addition, REL and MDA content increased significantly in both the OE and WT lines after heat treatment, while the MDA content was notably lower in the transgenic lines relative to the WT plants (Figure 4B). In addition, the SOD and POD activities of *CaHSP18.1a*-OE seedlings were clearly higher than those of the WT plants (Figure 4C,D). However, the catalase (CAT) activity did not significantly differ between the WT and transgenic lines (Figure 4E). *CaHSP18.1a* played a role in transgenic *Arabidopsis*, probably by regulating the expression of endogenous genes.

In the present study, among 18 stress-related genes, 12 were up-regulated in transgenic lines, while the other 6 did not change much (Figure 5). Among the up-regulated genes, *AtHSPC30, AtAPX3, AtCAT, AtHSP70*, and *AtRab1* were more prominently expressed in the transgenic OE3 line. Moreover, the expression of the 18 stress-related genes was markedly increased in both transgenic *Arabidopsis* and WT plants under heat stress. However, the expression of these genes in WT seedlings was lower than that in transgenic seedlings (Figure 5).

**Figure 4.** Heat resistance of transgenic *CaHSP18.1a*-OE *Arabidopsis* plants. (**A**) Phenotypes of 42 ◦C-treated wild-type (WT) and transgenic *Arabidopsis*; (**B**–**C**) Malonaldehyde (MDA) and relative electrolyte leakage (REL) of WT and transgenic *Arabidopsis*; (**D**–**G**) Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbic acid peroxidase (APX) activity of WT and transgenic *Arabidopsis*. Data are means with standard deviations of three biological replicates. Different letters denote statistical significance (*p* ≤ 0.05).

**Figure 5.** Expression patterns of stress–response genes in wild-type (WT), OE1, OE2, and OE3 lines before and after 42 ◦C heat treatment for 24 h. Data are means with standard deviations of three biological replicates. Different letters denote statistical significance (*p* ≤ 0.05).
