**1. Introduction**

Plants can tolerate considerable biotic and abiotic stresses in their complex and changing environments, including drought, high salt, extreme temperatures, and oxidation [1,2]. To mitigate stresses, plants have developed several protective mechanisms. Heat shock proteins (HSPs) can maintain protein homeostasis and prevent or repair the misfolding of proteins in abiotic stresses response. Moreover, HSPs are evolutionarily conserved molecular chaperones widely found among various plant taxa [3–5]. Plant HSPs also play critical roles in the folding, transport, degradation, and assembly of proteins under normal and stress conditions [6]. In response to high temperatures, plant cells dramatically increase the concentrations of HSPs to prevent heat-related damage and increase plant thermotolerance [7]. In addition, HSPs are also involved in plant growth and development under normal conditions, including the growth of flowers and seeds as well as fruit set, development [8], tuberization [9], and nutrient uptake [10]. HSPs are present in the cell membrane

**Citation:** Liu, Y.-L.; Liu, S.; Xiao, J.-J.; Cheng, G.-X.; ul, H.S.; Gong, Z.-H. CaHSP18.1a, a Small Heat Shock Protein from Pepper (*Capsicum annuum* L.), Positively Responds to Heat, Drought, and Salt Tolerance. *Horticulturae* **2021**, *7*, 117. https:// doi.org/10.3390/horticulturae 7050117

Academic Editor: Yuyang Zhang

Received: 1 April 2021 Accepted: 11 May 2021 Published: 18 May 2021

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and cytoplasm, nucleus, and cell organelles such as the mitochondria, chloroplasts, and endoplasmic reticulum [11,12].

HSPs, based on their sequence homology and molecular weight, are generally grouped into the following different families: HSP20s, HSP60s, HSP70s, HSP90s, and HSP100s [13,14]. Of the five conserved families, HSP20s, are also called small heat shock proteins (s HSPs). The molecular weights of HSP20s range between 15 and 42 kDa [13,15,16]. Furthermore, one of the distinctive characteristics of HSP20s is their ability to bind to substrate proteins without ATP, and they also have a strong ability to bind to denatured substrates [15–18]. Thus, s HSPs are highly able to maintain the stability of foreign proteins in cells to prevent them from aggregating. Although there are many types of substrate proteins, s HSPs have a flexible N-terminus and α-crystallin domain (ACD) hydrophobic surface that can adapt to bind these different protein substrates. In addition, s HSPs can be combined with different substrates in different ways, which makes s HSPs able to bind to a wider variety of proteins and to provide more complicated mechanisms of action among HSPs [19].

Korotaeva et al. [20] and Nieto-Sotelo et al. [21] showed that different HSPs are differentially expressed in different species, and even among different genotypes of the same species. It has been reported that the overexpression of AtHSP17. 6A increased the penetration resistance of *Arabidopsis* [22]. *AtHSP21* improved the heat resistance of transgenic *Arabidopsis* and extended the memory time of plants subjected to heat resistance, such that *Arabidopsis* was more heat resistant when subjected to heat stress again [23]. Some studies have also reported that HSP gene expression positively regulated protective enzyme activities. For example, in *Arabidopsis*, overexpression of *AtHSP17.8* enhanced SOD activity [24]. Similarly, overexpression of *HSP16.9* in tobacco increased the activities of POD, CAT, and SOD [25].

Pepper (*Capsicum annuum* L.) is one of the most important economical and medicinal vegetable crops worldwide [26].Pepper is usually cultivated in warm regions under temperatures of 15–34 ◦C [27]. Salt, drought, and heat stress can limit pepper growth and development and severely damage pepper pollination and seed set, which can lead to flower and fruit abscission and thus lower pepper fruit yield and quality [28,29].HSP20s in pepper play a major role in environmental stress responses, and a total of 35 pepper HSP20s were identified by Guo et al. [30]. All HSP20s were named based on their molecular weights, and stress-related cis-elements were detected in the promoter regions, including heat shock elements (HSEs), TATA boxes, CCAAT motifs, and TC-rich repeats [26]. Many CaHSP20 genes are not expressed across different pepper tissues (i.e., root, stem, leaf, and flower tissues). In recent years, the functions of CaHSP22.4, CaHSP25.9, CaHSP16.4, CaHSP24.2, and CaHSP26 have been identified. CaHSP16.4 is localized to the cytoplasm and nucleus, while in *Arabidopsis* lines with *CaHSP16.4* overexpression, increased tolerance to heat stress has been observed [31]. Guo et al. [26] also found that overexpression of CaHSP22.4, which is located in the mitochondria and cytoplasm, increased heat tolerance in *Arabidopsis*, with the expression increasing when pepper plants were subjected to high temperature. Similarly, the CaHSP25.9 protein was localized to the cell membrane and cytoplasm, and positively regulates heat, salt, and drought stress tolerance in pepper (*Capsicum annuum* L.) [32]. Pepper CaHSP24.2 is localized to mitochondria, the cytoplasm, and chloroplasts, where *CaHSP24.2* enhances the thermo-tolerance of transgenic *Arabidopsis* plants and regulates the expression of heat stress-related genes [30]. He et al. [33] overexpressed *CaHSP26*, which enhanced the tolerance of heat stress in *Arabidopsis*. Interestingly, heat-tolerance and salt-tolerance decreased in *CaHSP22.0*-silenced pepper [34]. All these studies suggest that sHSP20s may participate in responses to heat stress [35] and contribute to the acquisition of pepper thermo-tolerance [30].

Among the 35 *CaHSP20s* examined, the expression level of *CaHSP18.1a* was increased in both the B6 and R9 lines under heat stress [30]. Moreover, sequence analysis showed that *CaHSP18.1a* contained an HSE, and some other stress-related elements were also identified [30]. Based on the above findings, we analyzed the subcellular localization and expression pattern of *CaHSP18.1a* in different pepper tissues, as well as its response to salt, drought, and heat stresses. Virus-induced gene silencing (VIGS) was preliminarily used to analyze the functions of *CaHSP8.1a* in response to stress in pepper plants. In addition, overexpression (OE) in transgenic *Arabidopsis thaliana* indicated that *CaHSP18.1a* plays a positive regulatory role in the responses to heat, salt, and drought stress. Our results provide a basis for further functional studies of *CaHSP18.1a* in other important crop species and in its role in stress tolerance.
