As sessile organisms, plants are more prone to environmental stresses that could cause an adverse impact on growth and development, such as drought, salinity, cold, and high temperature (Filomena et al., 2009; Herman et al., 2017). For instance, to cope with the heat stress, plants regulate the gene expression patterns, resulting in a nearly exclusive synthesis of stress proteins, particularly heat shock proteins (HSPs; Sun et al., 2002). HSPs as molecular chaperones were distributed in diverse organisms and played crucial roles in assisting protein folding and preventing protein aggregation (Becker and Craig, 1994; Tyedmers et al., 2010). They are now also known to function in developmental stages and to respond to other abiotic stresses such as low temperature, drought, salinity, and stress-induced oxidative stress (Smirnoff, 1998; Yadav et al., 2021).

Generally, HSPs could be categorized into five protein families according to the molecular weight and sequence homology: HSP100s/ClpB, HSP90s, HSP70s/DnaK, HSP60s, and HSP20s (Wang et al., 2004; Waters, 2013). HSP20s are also called small HSP (sHSP), and its molecular weight is approximately between 12 and 42 kDa (Wang et al., 2004). Commonly, HSP20s capture the substrate proteins in an ATP-independent manner and prevent the irreversible aggregation of stress-denaturing proteins (Cashikar et al., 2005). The release and folding of the HSP20–substrate complexes are not spontaneous, and the ATP-dependent chaperones, including HSP70s and HSP100s, could cooperate in the processes (Lee et al., 1997; Haslbeck and Vierling, 2015). The HSP20 sequences contain the central conserved domain, the α-crystallin domain (ACD), which is flanked by a variable N-terminal region and a short C-terminal extension (Caspers et al., 1995; Kriehuber et al., 2010). The ACD is the signature domain of HSP20s containing 80–100 amino acids, and its structure in plants is a β-sandwich including three and four strands in an antiparallel direction and an extended strand (β6; Scharf et al., 2001; Basha et al., 2012). Unlike other HSP families, the HSP20 family is more variable and diverged in plant kingdom (Basha et al., 2012). On the basis of subcellular locations, seven subfamilies (CI, CII, CIII, M, P, ER, and Px) were primarily defined in Arabidopsis (Scharf et al., 2001). Another five subfamilies, including four cytoplasmic subfamilies (CIV, CV, CVI, and CVII) and one mitochondrial subfamily MII (Siddique et al., 2008), were added into the former classification. Totally, HSP20 proteins could be classified into 12 subfamilies (CI, CII, CIII, CIV, CV, CVI, CVII, MI, MII, ER, P, and Po; Scharf et al., 2001; Ma et al., 2006; Siddique et al., 2008). Among the subfamilies, CI to CVII are localized to the cytoplasmic/nuclear, M (MI and MII) are localized to the mitochondria, and ER, P, and Po are localized to the endoplasmic reticulum (ER), plastids (Ps), and peroxisomes (Pos), respectively.

In recent years, as more plant genomes have been assembled, the HSP20 gene family was identified across many plant species including Arabidopsis (Arabidopsis thaliana; Siddique et al., 2008), rice (Oryza sativa; Ouyang et al., 2009), soybean (Glycine max; Lopes-Caitar et al., 2013), wheat (Triticum aestivum; Muthusamy et al., 2017), potato (Solanum tuberosum; Zhao et al., 2018), and apple (Malus domestica; Yao et al., 2020). Of the species, there are 19 HSP20 genes in Arabidopsis, 39 in rice, 51 in soybean, 163 in wheat, 48 in potato, and 41 in apple. Cynodon transvaalensis Burtt–Davy, commonly called African bermudagrass, is a perennial warm-season turfgrass species. Recently, the genome of African bermudagrass was assembled (Cui et al., 2021), which makes it possible to characterize the HSP20 gene family in African bermudagrass genome-widely. African bermudagrass is primarily endemic to damp and uncultivated areas including Orange Free State, southern Transvaal and northern Cape Province of South Africa, and was further introduced to other countries such as Greece, Iran, the United States, Madagascar, and Australia (Beard, 2013). Although the commercial use of African bermudagrass for turf was relatively limited, African bermudagrass exhibits a great value as a parent in hybridization with tetraploid C. dactylon and has been utilized to breed a lot of leading triploid bermudagrass cultivars.

Here we utilized bioinformatics methods to identify HSP20 genes of African bermudagrass and uncovered their chromosomal positions, gene duplication events, and phylogenetic relationships. Moreover, the expression patterns of HSP20 genes were analyzed with RNA-seq data to determine their responses to abiotic stresses including drought, salinity, and extreme temperatures. The protein three-dimensional (3D) structures and their protein–protein interaction (PPI) networks were all predicted to resolve the possible regulation mechanisms of HSP20s. The findings in the study would provide valuable information for further investigations of the functions and regulatory mechanisms of potentially important HSP20 genes in modulating African bermudagrass tolerance to abiotic stresses, including high temperature.

Heat shock protein 20s, as molecular chaperones, are a ubiquitous protein family found in both prokaryotes and eukaryotes, and they are the most abundant HSP family in plants (Waters, 2013). With the ever-increasing availability of plant genomes and transcriptomes, the HSP20 genes have been identified from some monocots and dicots plants, such as Arabidopsis (Scharf et al., 2001; Siddique et al., 2008), soybean (Lopes-Caitar et al., 2013), potato (Zhao et al., 2018), apple (Yao et al., 2020), rice (Ouyang et al., 2009), bread wheat (Muthusamy et al., 2017), and switchgrass (Yan et al., 2017). However, no studies have been conducted on an overall identification and characterization of the HSP20 genes from African bermudagrass, an important warm-season turfgrass species. The completion of high-quality African bermudagrass genome assembly has just provided an opportunity to identify and characterize HSP20s at the whole-genome level (Cui et al., 2021).

Here, a total of 41 HSP20 genes were identified in African bermudagrass genome. The HSP20 gene number of African bermudagrass was higher than those of Arabidopsis (19; Siddique et al., 2008) and rice (39; Ouyang et al., 2009), similar to those of apple (41; Yao et al., 2020) and watermelon (44; He et al., 2018), and less than those of potato (48; Zhao et al., 2018), soybean (51; Lopes-Caitar et al., 2013), and wheat (117; Wang et al., 2017). The differences in HSP20 gene numbers are most likely due to the genome size differences and the fact of gene duplications during plant evolution. Among the species, the number of HSP20 genes in Arabidopsis is the least, which was thought to be related to its smaller genome size (Zhao et al., 2018). Gene duplication plays important roles in the expansion of the number of gene families in plants (Blanc and Wolfe, 2004). The sHSPs are known to undergo a lineage-specific gene expansion, diversifying early in land plant evolution, potentially in response to stress in the terrestrial environment, and expand again in seed plants and again in angiosperms (Wang et al., 2017; Waters and Vierling, 2020). The expansion of HSP20 genes in African bermudagrass genome was possibly owing to the whole genome duplication events (WGD) during evolution (Cui et al., 2021). The gene duplications were then investigated, and the captured paralogs included six pairs of WGD/segmental duplicates and nine pairs of tandem duplicates. Compared with WGD/segmental duplicates, tandem duplicates accounted for a larger proportion, and they were likely to play more important roles in HSP20 evolution. Additionally, the HSP20 syntenic genes were also predicted in monocots (maize and rice) and dicots (Arabidopsis and soybean). As expected, maize and rice contained more syntenic genes with HSP20s of African bermudagrass than Arabidopsis and soybean did, which provided the evidence that African bermudagrass had a closer evolutionary relationship with monocot plants than dicot plants (Cui et al., 2021), which indicated that CtHSP20s might have experienced species-specific duplications after the monocot/dicot divergence event, and it was also reported that African bermudagrass had experienced two WGD events after the divergence between monocots and dicots (Cui et al., 2021). Only three CtHSP20s (CtHSP20-3, CtHSP20-35, and CtHSP20-41) had syntenic genes among four species, and they belonged to the conserved subfamilies CI, Po, and CVI, respectively. Combining the inferred orthologs in Supplementary Table 2, we deducted that these three genes could originate from a common ancestor before the divergence between monocots and dicots, and their functions might be conserved and important in angiosperms.

Furthermore, the phylogenetic tree was utilized to uncover the HSP20s evolutionary relationships. In the current study, 172 HSP20s containing CtHSP20s together with HSP20s from other four species were categorized into 12 distinct subfamilies (CI to CVII, MI, MII, ER, P, and Po). Among the sequences, 117 HSP20s accounting for 68.02% were clustered into the cytoplasm subfamilies (CI to CVII), which was consistent with previous findings in other plants (Siddique et al., 2008; Zhao et al., 2018), and the cytoplasm was suggested to be the primary cellular site for HSP20s accumulation and function (Lopes-Caitar et al., 2013; Yao et al., 2020). Interestingly, some HSP20 members clustered in the same subfamily from various species were more related than those in different subfamily belonging to the same species, indicating that there was synteny among different plant species, and these HSP20s were conserved across multiple species (Lopes-Caitar et al., 2013). The colored collinear links in Figure 3A represent the syntenic relationships of HSP20s, and the syntenic genes could evolve from a common ancestor of the HSP20 family. We also noted that M subfamily members were adjacent to P subfamily members, which implied that they might undergo a closer divergence time (Waters and Vierling, 2020). Among CtHSP20s, the phylogenetic tree exhibited similar patterns with their motif composition and distribution. There was no CtHSP20s in CIV and CVII subfamilies, which might be caused by gene loss during evolution. As for motifs, Motif 1, Motif 2, and Motif 4 were conservative and widespread, and they could be more responsible for HSP20 functions. Overall, the diversity of HSP20 family could be driven by environmental selection pressures and continuous evolution of plants.

HSP20 genes could be responsible for different stresses (Waters et al., 1996; Yao et al., 2020). In our research, the expression patterns of CtHSP20s were investigated on the basis of their transcriptome data from various abiotic stresses. As a result, 41 CtHSP20s genes could be classified into three groups (Groups I to III; Figure 6). The unified expression patterns were observed in Group I, and almost all genes were up-regulated under HTS and down-regulated under other treatments. Similarly, many of the HSP20s in various plant species were also up-regulated in response to heat stress (Guo et al., 2015; Zhao et al., 2018; Yao et al., 2020). Considering numerous studies have reported the positive role of sHSPs in plant thermotolerance (Waters, 2013; Haslbeck and Vierling, 2015; Waters and Vierling, 2020), the Group I genes could be the major ones contributing to the heat-stress tolerance, and the functions of these genes needed to be validated in further studies. In Group II, three genes (CtHSP20-14, CtHSP20-35, and CtHSP20-36) maintained higher expression levels under RTS and LTS instead of HTS, and they could be low temperature responsive. Particularly, CtHSP20-14 showed more obvious down-regulation under HTS, and it was one of the 10 CtHSP20 genes that had orthologs in the two monocot plants (rice and maize), but not in the two dicot plants (Arabidopsis and soybean; Supplementary Table 2). It would be interested to further study the role of CtHSP20-14 in response to temperature stresses since it could be monocot specific, and its function has not been characterized. The remaining two genes (CtHSP20-9 and CtHSP20-10) only expressed under HTS and LTS treatments. Intriguingly, CtHSP20-9 and CtHSP20-10 belonged to the same pair of WGD/segmental duplicates, and experienced positive selection (Ka/Ks > 1) as well. In addition, they were among the most conserved CtHSP20s across various plant species according to the orthologous analysis (Supplementary Table 2). Thus, we speculated that CtHSP20-9 and CtHSP20-10 were possibly selected in response to extreme temperatures during evolution. In Group III, almost all the genes were down-regulated among DSS, SSS, HTS and LTS compared with the control, which suggested that these genes could be negatively regulated to adapt to abiotic stresses. Two studies from a research group reported negative effects on growth and stress tolerance in A. thaliana plants expressing either an Agrostis stolonifera cytosolic AsHSP17 or chloroplast AsHSP26.8a constitutively (Sun et al., 2016, 2020). Due to the importance of cis-elements in gene promoters for plant responses to environmental stresses (Yamaguchi-Shinozaki and Shinozaki, 2005), we further identified them in the putative promoter regions of African bermudagrass HSP20 genes. Based on the results, stress-responsive and hormone-related elements were closely related to abiotic stresses. As reported (Sarkar et al., 2009; Lopes-Caitar et al., 2013), under heat stress, heat shock transcription factors (HSFs) could bind to HSEs to regulate the expression levels of HSP20 genes. In this study, we further verified that the correlation relationship between HSEs and up-regulated genes was extremely significant (Fisher’s exact test, ^∗∗p < 0.01), which suggested that HSFs could upregulate HSP20 genes for African bermudagrass to cope with high temperature. Additionally, the light-responsive elements were also widespread among all CtHSP20s, which was consistent with the results in apple, grape, and pepper (Guo et al., 2015; Ji et al., 2019; Yao et al., 2020). Light is essential to plant growth and development, and the finding confirmed that HSP20s could be not only important for environmental stresses but also for normal growth and development of plants (Waters and Vierling, 2020; Yao et al., 2020).

Although HSP20s were relatively small among HSP families, HSP20 oligomers often contained 12 to 40 subunits (Basha et al., 2012; Haslbeck and Vierling, 2015), and the ability to assemble into large oligomers of 12 or more subunits is the key to the function of many HSP20s (van Montfort et al., 2001; Hanazono et al., 2013). In our analysis, 16 proteins were homo-12-mer and successful modeling with identities from 34.82 to 83.33%. The structures of CtHSP20-9, CtHSP20-10, and CtHSP20-30 were slightly different from the others (Figure 9) with relatively lower homology modeling identities of 34.82, 37.86, and 37.86%. Combined with the prior analyses, gene duplication and selection events might both contribute to the evolution of CtHSP20-9 and CtHSP20-10. Notably, three HSP20s (CtHSP20-17, CtHSP20-22, and CtHSP20-27) were homodimers based on modeling with identities of 75, 62.37, and 30.77% (Supplementary Figure 1). A few HSP20s were found to be present as small oligomers of two to four subunits (Kokke et al., 1998; Basha et al., 2013). These small oligomers (e.g., tetramers) were thought to be the building blocks of larger HSP20 complexes, and that higher multimer formation was a prerequisite to fulfill their chaperone-like activity (Kokke et al., 1998). A striking feature of HSP20 oligomers is their dynamic behavior. Some HSP20s can readily exchange subunits and form hetero-oligomeric complex in a temperature-dependent manner, which is very likely crucial to their functions (Stengel et al., 2010; Chen et al., 2014). As expected, the integrated PPI networks of CtHSP20s found that the majority of the HSP20 proteins were enriched. The interaction degrees of the proteins on the inner circular layout were generally higher than those on the outer ones. In addition to the HSP20 proteins, some ATP-dependent chaperones containing HSP100 (ClpB1), HSP70 (OsJ_039939), and HSP90 (Os04T0107900-02) were also enriched in the networks. The current HSP20/sHSP function model proposes that HSP20s act as ATP-independent molecular chaperones to capture stress-denatured proteins as substrates. The HSP20-bound substrates are then prevented from irreversible denaturation and can be reactivated by ATP-dependent chaperones (HSP70 and co-chaperones, along with the protein disaggregase HSP100) for refolding (Haslbeck and Vierling, 2015). HSP70 and HSP100 could be helpful in releasing and refolding substrate proteins (Haslbeck and Vierling, 2015). In tomato, HSP70 and HSP90 were reported to directly interact with HSFs to regulate downstream gene expressions (Hahn et al., 2011). The PPI networks provided the evidence that different kinds of chaperones could cooperate together in response to environmental stresses, and further researches would be warranted to explore the mechanisms in more detail.

In this study, a total of 41 CtHSP20s were identified and confirmed in African bermudagrass genome. The CtHSP20s were randomly localized on different chromosomes, and they were classified into 12 subfamilies based on the phylogenetic tree and cellular locations. The gene duplications, expression profiles, cis-elements, 3D structure, and PPI networks were conducted to resolve the characteristics of CtHSP20s. The PPI network revealed that different kinds of chaperones could cooperate together in response to environmental stresses. Additionally, the HSP20 genes was clustered into three groups (Groups I, II, and III) based on the expression profiles, and most CtHSP20s were up-regulated under HTS as expected, especially those in Group I. Interestingly, in Group II, a monocot-specific HSP20, CtHSP20-14 maintained higher expression levels under both RTS and LTS, but not HTS. Moreover, a pair of WGD/segmental duplicates CtHSP20-9 and CtHSP20-10 were among the most conserved HSP20s across different plant species, and they seemed to be positively selected in response to extreme temperatures during evolution. This work would aid in elucidating further functional characterizations of CtHSP20s in the future.

Publicly available datasets were analyzed in this study. This data can be found here: https://ngdc.cncb.ac.cn/gsub/submit/bioproject/PRJCA003581.

FC collected the public dataset, performed the data analysis, and wrote the manuscript. GT helped collect the plant materials. XW and KW conceived this study and reviewed the manuscript. All of the authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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