**4. Discussion**

Plants are inevitably subjected to various extreme environmental conditions, such as heat, drought, oxidation and salt damage [36]. Under such adverse conditions, sHsP20s make a valuable protective contribution [19]. Plant HSPs are linked to heat tolerance and have been confirmed in many species [31,49]. Guo et al. [30] identified sHsP20s in pepper, and showed that *CaHSP18.1a* was induced in different tissues of pepper plants under heat stress, but the function of *CaHSP18.1a* under heat, salt, and drought stress has not been further studied. In this study, we identified that *CaHSP18.1a* is positively involved in plant tolerance to heat and salt, drought stress.

*CaHSP18.1a* was responsive to heat stress in both R9 and B6 plants and strongly induced [30]. In accordance with the results, we also found that the expression level of *CaHSP18.1a* was strongly induced in both the R9 and B6 lines after heat stress treatment (Figure 1B–D). However, under heat stress, the expression of *CaHSP18.1a* in R9 plants was higher than that in B6 plants (Figure 1B–D). This may be because R9 is a thermo-tolerant cultivar, it has better thermo-tolerance and adaptability than B6 under heat stress. The heat tolerance of plants is related to the dynamic expression patterns of heat stress-related genes [50]. Under heat stress, other HSP20s or HSPs in R9 are also strongly and rapidly induced in the early stage of heat stress (0.5–1 h). It had also been reported that the expression level of *CaHSP25.8* and *CaHSP30.1* in R9 was higher than B6, but with the extension of heat stress treatment time, the expression level of these two genes in B6 were higher than R9 [30]. However, these results also showed that the expression of *CaHSP18.1a* was lower at V in R9 than in B6. The expression of pepper HSP20s is regulated by many transcription factors, such as HSFs [51]. Under heat stress, it is because HSFs that regulate the expression of *CaHSP18.1a* in pepper variety R9 and B6 are different, or the expression of the same HSFs that regulate the expression of *CaHSP18.1a* is different in R9 and B6, causing the differential expression of *CaHSP18.1a* in B6 and R9 [51,52]. Therefore, the difference in the expression of *CaHSP18.1a* between heat-resistant and heat-sensitive varieties is due to the above reasons. However, the relationship between the function of this gene and the heat-resistance mechanism of pepper still needs further research.

In addition, *CaHSP18.1a* was induced under salt and drought stress (Figure 1E–H). The expression of *CaHSP18.1a* in R9 leaves and roots was highest under the 150 mM, 100 mM NaCl treatments, respectively (Figure 1E,F). The expression of *CaHSP18.1a* in R9

leaves was the highest after the 150 mM mannitol treatment; the highest expression of *CaHSP18.1a* was observed in R9 roots subjected to the 50 mM mannitol treatment (Figure 1G,H). However, the expression of the *CaHSP18.1a* decreased at higher NaCl and mannitol concentrations. The response pattern of *CaHSP18.1a* that rapidly and sharply responded to salt and drought stress in a short time, and then had slight variations, was similar to quite a few HSP20s such as *TaHSP23.9* [53] and *ClHSP22.8* [54]. Thus, *CaHSP18.1a* may play a role in pepper which rapidly adapts to drought and salt stress.

VIGS technology is an important method used to study gene function under adverse environments [55]. In the R9 line, silencing of *CaHSP16.4* reduces heat tolerance and drought resistance of pepper plants [31]; *CaHSP22.0*-silenced peppers showed more sensitivity to salt and heat stress, which was mainly reflected in decreased antioxidant enzyme activity, increased leaf conductivity, and increased superoxide anion and MDA contents [34]. MDA content and REL are products of cell membrane lipid peroxidation, which damages the integrity of plasma membranes under salt or heat stress [35] and may sensitize plants to subsequent stress [56]. MDA content, total chlorophyll content, and REL are widely used to determine the degree to which plants have been damaged by abiotic stress [4]. It has also been reported that proline content, MDA content, and POD and SOD activity of pepper were significantly related to the variation in heat tolerance and temperature stress time, which can be used as an index for heat resistance identification [57].

In this study, after treatments with high temperature, salt, and drought stress, the content of MDA and REL in peppers that had been silenced for *CaHSP18.1a* was higher than that of the controls, indicating that the damage to cell membranes increased in *CaHSP18.1a*-silenced plants (Figure 3A–C). It was also found that the *CaHSP18.1a*-silenced plants had lower total chlorophyll content when exposed to heat stress (Figure 3B and Supplementary Figure S1C). These results demonstrated that silencing of *CaHSP18.1a* reduced pepper stress tolerance (Figure 3). In contrast, overexpression of *CaHSP18.1a* in *Arabidopsis thaliana* transgenic lines was associated with minimal injury symptoms, increased REL, and decreased MDA content compared with WT plants (Figures 4 and 6). These results showed that *CaHSP18.1a* increases plant tolerance to heat, salt, and drought stresses.

HSP20s are widely distributed in plants, and their location may be related to their function, as exemplified by *AtHsP21* being localized to chloroplasts [23]. *CaHSP18.1a* was predicted to have cytoplasm localization [30]. Subcellular localization of *CaHSP18.1a* is shown in Figure 2, which confirmed that it is localized to the cell membrane.

Studies have shown that HSP20 is a molecular chaperone that can also participate in antioxidant mechanisms of plants [6,58]. HSP20scan cooperate with the plant's antioxidant scavenging system to protect plants from secondary damage [59,60]. For example, overexpression of *AtHSP17.6* can increase CAT enzyme activity and further regulate abiotic stress responses [61]. Furthermore, the over-expression of *ZmHSP16.9* in tobacco can increase the activities of POD, CAT and SOD, and enhance oxidative stress tolerance [62]. In this study, SOD, CAT, POD, and APX enzyme activities in overexpression of *CaHSP18.1a* plants were significantly enhanced under heat and drought stress. This is similar to the results obtained with *Arabidopsis* transformed with *CaHSP25.9*; that is, by increasing the activities of ROSscavenging related antioxidant enzymes, the heat, salt, and drought tolerance of plants can be increased [31,63]. It has been reported that plants have built defense mechanisms that scavenge excess reactive oxygen species (ROS) throughout their long evolutionary histories [64–66], such as ROS-scavenging non-enzymatic antioxidants (e.g., ascorbic acid (AsA), glutathione, and proline) [67] and antioxidant enzymes (e.g., peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX)) that prevent secondary oxidative stress caused by abiotic stress [68–70]. Moreover, the expression levels of *AtSOD1, AtAPX1, AtAPX3*, and *AtCAT1* were also increased by heat and drought stress, and were higher in the *CaHSP18.1a* transgenic *Arabidopsis* than in the WT. This indicated that *CaHSP18.1a* may improve stress resistance through the ROS-scavenging system, but the specific mechanism needs further study.

Many stress-related genes are involved in plant responses to heat, salt, and drought stresses. It has been reported that *AtHsfA2* is a heat shock transcription factor that enables prolonged acquired thermo-tolerance, and it can enhance tolerance to salt and osmotic stresses [71–73]. s HSPs are downstream target genes of HsfA2 [74]. Burke [75,76] have also shown that *AtHSA32* and *AtHSP101* expression can be induced by high temperature and participate in the acquired thermo-tolerance of plants. *AtMYB44* can be induced by salt, drought, and other stresses to participate in the abscisic acid (ABA) signaling pathway; Refs. [77,78] found that the ABA signaling response gene *AtDREB2A* can be induced by low temperature stress. The drought responsive gene *AtRD29A* was up-regulated under heat, salt, and drought stresses [79], while the molecular chaperone HSP70 participates in drought and heat stress responses [28]. *NCED3* is related to biological metabolism and also participates in defense responses to drought stress [80]. Notably, HSP20s can regulate many of these stress-related genes [31,36]. For example, *CaHSP16.4* and *OsMSR-4* can increase the expression of these genes in transgenic seedlings, thereby enhancing stress resistance [31,81]. In this study, we assessed the expression levels of 13 stress-related genes in WT and transgenic plants. *CaHSP18.1a* enhanced heat tolerance in transgenic *Arabidopsis*, which may be closely related to its regulation of the expression of many heatstress-related genes in *Arabidopsis*. Overexpressed genotypes compared with wild type under normal conditions also showed higher values of stress-related genes expression, higher expression of *AtP5CS,* higher expression of *AtNCED*, higher expression of *AtMYB*, and higher expression of *AtRD29, AtHsfA2, AtRab1* and *AtHSP30*. These results showed that *CaHSP18.1a* may play an important role in regulation of these genes. Under heat stress, the expressions levels of *AtHsfB4, AtHSFA8, AtHSFA2, AtHSFA7a, AtHSPC30, AtHSFA3, AtHSP70,* and *AtHSP101* in transgenic plants were significantly higher than WT plants (Figure 5). In particular, the *AtHSPC30* and *AtHSP70* transcripts were present at levels nearly 3-fold higher in transgenic seedings than in WT plants; the transcript level of *AtHsfA2* was also up-regulated in transgenic plants. The expression levels of *AtHSP70, AtHSP101, AtDREB2A, AtMYB124, AtNCED3, AtRD29A*, and *AtRAB1* were higher (Figure 7) in the *CaHSP18.1a* transgenic *Arabidopsis* than in WT plants under drought stress. Similar results were also reported by Feng and Huang [31,32]. Thus, *CaHSP18.1a* may respond to heat and drought stress through its complex regulatory network.
