1. Introduction
Heat shock proteins (HSPs) are evolutionarily ancient, highly conserved, and ubiquitously present across various organisms, including archaea, prokaryotes, and eukaryotes [1]. These proteins enhance stress tolerance under adverse environmental conditions such as salinity, elevated temperatures, heavy metal exposure, and drought. HSPs achieve this by stabilizing cellular structures, facilitating the transport and proper folding of proteins, and maintaining essential cellular functions [2,3,4,5].
HSPs are categorized into six classes based on their molecular weights: HSP110, HSP90, HSP70, HSP60, HSP40, and HSP20 [6]. Among these, HSP20, also called small heat shock proteins (sHSPs), have a molecular weight predominantly ranging from 15 to 25 kDa [7]. Subcellular localization studies have further classified HSP20 into five subfamilies. The cytoplasmic subfamily (CI-CVI) is found in the cytoplasm or nucleus, while the mitochondrial subfamily (MTI and MTII) resides in the mitochondria. Additionally, the ER, CP, and PX subfamilies are localized in the endoplasmic reticulum, chloroplasts, and peroxisomes, respectively [8]. All members of the HSP20 family have a highly conserved α-crystallin structural domain (ACD) located in the C-terminal region, which is encoded by approximately ninety amino acids and contains multiple β-strand structures, an opposite sequence of 80–100 amino acids and two conserved regions: the CRI (N-terminal consensus region I) and the CRII (C-terminal consensus region II) [9,10]. In recent years, with the improvement and development of genome-sequencing technology, the HSP20 gene family has gradually been comprehensively identified and analyzed in different species, such as grapes [11], tomato [12], watermelon [13], African bermudagrass [14], and Prunus mume [15]. HSP20, abundant in plants, is the most commonly produced protein under heat stress and is widely involved in plants’ response to biotic and abiotic stresses [16]. Overexpression of the small heat shock protein gene (OSHSP18.6) in rice results in reduced malondialdehyde levels and increased catalase and superoxide dismutase activity [17]. Overexpression of sHSP in cucurbit crops inhibits viral RNA accumulation and delays disease progression [18]. Overexpression of a small heat shock protein gene (RcHSP17.8) from the rose of China in tobacco increased the tolerance of recombinant strains and transgenic tobacco to abiotic stresses [19]. In Arabidopsis, overexpression of AtHSP17.6A and AtHSP17.6C resulted in rapid accumulation under high-temperature stress and showed enhanced heat tolerance [20]. Furthermore, transgenic plants (MsHSP16.9 and TaHSP23.9) demonstrated robust growth at elevated temperatures and conferred a protective effect on Arabidopsis [21,22]. Thus, HSP20 plays an important positive role in ameliorating abiotic stress in plants.
Chickpeas (Cicer arietinum L.), the world’s third most-produced legume, are cultivated in over fifty countries worldwide and are renowned for their high nutritional value, including proteins, carbohydrates, and minerals [23]. In recent years, the effects of high temperatures on chickpea growth and development have become increasingly evident as a consequence of global warming [24,25,26]. High temperatures reduce chickpea yields by reducing pollen viability [27] and stigma receptivity [28] during flowering and pod development, leading to fertilization failure. High temperatures also affect the grain filling period, leading to reduced yield and quality [28]. During the reproductive growth stage, high temperatures may affect the development of floral organs and the viability of pollen, which can lead to poor pollination and fertilization and subsequently reduce the rate of fructification. However, although the HSP20 gene family has been reported in many plants, the mechanism of action of the HSP20 gene family under heat stress in chickpeas (Cicer arietinum L.) is unclear. Therefore, an in-depth study on the whole-genome identification of the HSP20 gene family in chickpeas under high-temperature stress has important theoretical and practical significance for revealing the response mechanism of chickpeas under heat stress and breeding varieties with high-temperature tolerance. In this study, we identified chickpea HSP20 family genes and systematically analyzed the phylogenetic relationships, gene structures, structural domains, chromosomal locations, and cis-acting elements, which are expected to provide a reference for unraveling the functions of the HSP20 family members in the regulation of chickpea development and stress response.
2. Materials and Methods
2.1. Identification of the HSP20 Gene Family Members in Chickpeas
Chickpea genome and protein sequences were downloaded from the National Center for Biotechnology Information (NCBI) database. The Hidden Markov Model (HMM) profile of Hsp20 (PF00011) downloaded from the Pfam protein family database (
2.2. Chromosomal Distribution, Gene Duplication Event, Synteny Analyses, Gene Structure, and the Conserved Domain of CaHSP20s
Chromosome information was obtained from Phytozome, and a chromosomal location map of the genes was generated using TBtools (v.1.120) [32]. The gene structures of CaHSP20 were created using the website Gene Structure Display Server (GSDS 2.0,
2.3. Protein–Protein Interaction Network of CaHSP20s
Based on the protein sequences of the twenty-one identified CaHSP20 genes, the complete set of corresponding representative transcript protein sequences was searched on the STRING database (
2.4. Phylogenetic Analysis of HSP20s
The HSP20 amino acid sequences of Arabidopsis [36,37], soybeans [38], and peanuts [39] were downloaded and combined with the identified CaHSP20 protein sequences for multiple alignments, which were performed using ClustalW (v.2.0.11). First, all protein sequences were aligned using MAFFT (version 7). Second, a rootless neighbor-joining (NJ) phylogenetic tree was constructed using MEGA 11 with default parameters and 1000 bootstrap tests. Finally, CaHSP20 genes were divided into subgroups based on the classification of HSP20 in other species and the topology of the phylogenetic tree. The tree was later colored using iTOL (
2.5. Prediction of Promoter Cis-Elements of CaHSP20s
The promoter sequence upstream of the transcription start site of each CaHSP20s gene, which is 1.5 kb in length, was extracted from the chickpea genome database and analyzed using PlantCARE online software (
2.6. The Analysis of Tissue Expression Profile for CaHSP20s Genes
To determine the expression profiles of CaHSP20s in the different tissues of chickpeas, a comparative analysis of published RNA-sequencing data (series accession number GSE147831) [41] was conducted (
2.7. Plant Materials and Growth Conditions
The chickpea seeds were sterilized in 3% sodium hypochlorite and then washed five times using distilled water. Seeds were pre-germinated on moist filter paper in a Petri dish for 48 h at 25 °C in the dark, and pre-germinated seeds were planted into each pot containing soil and vermiculite (2:1 ratio). The plants were grown at 25 ± 1 °C with a 16 h/8 h (light/dark), as described by Pandey et al. [44]. The chickpea seedlings at twenty-one days old were used for the high-temperature stress experiments. The chickpea seedlings were treated with heat at temperatures of 28 °C, 37 °C, and 42 °C. Samples of leaves and stems were collected immediately before heat treatments (0 h), as well as at 24 and 48 h after the high-temperature stress, and immediately frozen in liquid nitrogen and stored frozen at −80 °C until use.
2.8. RNA Isolation and Real-Time PCR Analysis
The total RNA was extracted from leaves and stems of chickpeas using a HiPure Universal RNA Mini Kit (MGBio, Shanghai, China), and reverse transcription was performed using the MonScript™ RTIII Super Mix with dsDNase (Monad Biotech Co., Ltd., Shanghai, China). The quantitative PCR of the target genes was carried out on a CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA) using MonAmp™ ChemoHS qPCR Mix (Monad Biotech Co., Ltd., Shanghai, China) according to the manufacturer’s instructions. The relative mRNA expression levels were determined using a comparative threshold cycle (CT) method [45], and the data were from three independent biological replicates. The primers for quantitative real-time PCR assays are listed in Supplementary File S3.
2.9. Statistical Analysis
All of the experiments were conducted in triplicate, and the results are presented as the means ± standard deviations (SDs). Statistical significance was determined using Student’s t-test, with a p-value of less than 0.05 considered statistically significant.
3. Results
3.1. Identification and Characterization of CaHSP20s Gene Family
In this study, the HSP20 gene family of chickpeas was identified using the HSP20 protein sequences of Arabidopsis thaliana and rice (Supplementary File S1) to construct a hidden Markov model (HMM). As a result, twenty-one HSP20 genes were identified in chickpeas and named CaHSP20-1 to CaHSP20-21 according to their positions in the genome (Table 1). The genomic mRNA of the identified HSP20S ranged from 396 to 1191 bp. The amino acid residue numbers of the putative HSP20 proteins varied from 131 to 242, the coding sequence of the CaHSP20 proteins varied from 396 to 729 bp, and their isoelectric point (pI) and molecular weight (MW) ranged from 5.24 to 9.35 and 15.15 to 27.46 kDa, respectively (Table 1). The instability index and hydropathy index of the CaHSP20 proteins varied from 30.45 (CaHSP20-5) to 63.28 (CaHSP20-16) and from −0.84 (CaHSP20-11) to −0.25 (CaHSP20-13), respectively (Table 1).
3.2. Chromosome Location, Gene Duplication Event, and Synteny Analyses of CaHSP20s Family
The CaHSP20 genes were found to be unevenly distributed across seven chickpea chromosomes (Figure 1 and Figure 2). Chromosome 2 contained four CaHSP20s, chromosome 5 contained five CaHSP20s, and chromosome 6 contained eight CaHSP20s. The remaining four chromosomes each harbored only one CaHSP20 gene (Figure 1 and Figure 2). The locations of the CaHSP20 repeat sequences in the chickpea genome were analyzed, revealing that five (23.8%) of the CaHSP20 genes exhibited tandem repeats. As shown in Figure 1, four pairs of segmental duplications were identified on chromosomes 5 and 6: CaHSP20-10/CaHSP20-11, CaHSP20-17/CaHSP20-18, CaHSP20-17/CaHSP20-19, and CaHSP20-18/CaHSP20-19 (Figure 1 and Figure 2). There are no segmental duplicate gene pairs on the same and different chromosomes of CaHSP20, as shown in Figure 2. We also compared homologous blocks of the chickpea, soybean, peanut, and Arabidopsis genomes and found that many HSP20 genes are homologous (Figure 3). Twenty-seven CaHSP20 genes were homologous to soybean genes, twenty-three were homologous to peanut genes, and seven were homologous to Arabidopsis genes (Figure 3). The number of collinear gene pairs between chickpeas and other members of the legume family (soybeans or peanuts) was found to be greater than that between distantly related Arabidopsis thaliana, with the HSP20 gene family of chickpeas (Cicer arietinum L.) and soybeans (Glycine max) having the most homologous gene pairs. This finding suggests that chickpeas and soybeans are more closely related, which is consistent with the phylogenetic analysis results. The divergence times of these repeats were determined by calculating the Ka/Ks ratios. Tandem repeat events were found to have occurred within the last fifty-three million years (MYA), with all CaHSP20 paralogs undergoing purifying selection (Ka/Ks < 1) (Table 2). These results indicate that tandem duplication events have played a significant role in the expansion of the CaHSP20 gene family in chickpeas. The observation of purifying selection suggests that these genes have conserved their functions over time, likely contributing to the plant’s adaptation to various environmental stresses.
3.3. Construction of the Protein Interaction Networks of the CaHSP20s Family
Protein–protein interaction (PPI) analysis is an important method for studying the relationship between proteins. It can help us to understand the interactions between proteins and their role in cellular processes. By analyzing the connections in the network, interactions between different proteins can be revealed [46,47]. In the present study, the STRING database was used to construct the PPI network of the chickpea CaHSP20 family, with a total of 13 members participating in network construction (Figure 4). It is clear from the network that CaHSP20-7, CaHSP20-8, CaHSP20-15, and CaHSP20-12 interact most closely with other members, indicating that they may be key members involved in responding to heat stress and maintaining cell homeostasis. In particular, CaHSP20-8, which is in the core region, interacts with multiple genes (CaHSP20-1, CaHSP20-7, CaHSP20-9, CaHSP20-10, CaHSP20-5, CaHSP20-12, CaHSP20-15, CaHSP20-11, CaHSP20-13). These interacting proteins are of great significance for further study of the role of CaHSP20s in chickpeas under various stress conditions.
3.4. Phylogenetic Analysis of the CaHSP20s Gene Family in Chickpeas
To investigate the evolutionary relationships and classification of HSP20 family genes, a neighbor-joining phylogenetic tree was constructed with full-length protein sequences of twenty-one CaHSP20 proteins, nineteen AtHSP20 proteins, thirty-three GmHSP20, and twenty-seven AdHSP20 proteins (Figure 5). The CaHSP20 proteins were divided into ten subgroups, including nine CI, three P, two CII, and one each of PO, CVI, MI, ER, CV, CIV, and CIII based on previously reported results and phylogeny analysis [48] (Figure 5). The concentration of HSP20s was evident in chickpeas, Arabidopsis, soybeans, and peanuts. From an evolutionary perspective, chickpeas and soybeans are the most closely genetically related, followed by peanuts and Arabidopsis. Roughly half of the CaHSP20s were localized in the cytoplasm or nucleus, meaning that they mainly play roles in the cytosol.
3.5. Gene Structure, Conserved Motifs, and Sequence Analysis of CaHSP20s Family
To fully understand the diverse functions of these CaHSP20 proteins, their conserved motifs were analyzed using the Multiple Em for Motif Elicitation (MEME) suite (
3.6. Promoter Analysis of CaHSP20s Genes
To explore the potential function and regulatory mechanisms of the CaHSP20 genes, the cis-acting elements within the 2000 bp promoter sequences of CaHSP20s were analyzed using the PlantCARE database. The analysis revealed several phytohormone and stress response-related cis-acting elements, including the MeJA Response Element (MeJARE), Anaerobic Response Element (ARE), ABA Response Element (ABRE), Drought Response Element (DRE), Low-Temperature Responsive Element (LTRE), Gibberellin Response Element (GARE), Defense and Stress Response Element (DSRE), SA Response Element (SARE), and Auxin Response Element (AuxRE) (Figure 8). Among the twenty-one CaHSP20, fourteen contain ARE elements, nine contain DRE elements, seventeen contain ABA elements, twelve contain MeJA elements, eight contain GARE and LTRE elements, five contain SA elements, ten contain AuxRE elements, and seven contain DSRE elements (Figure 8). These results suggest that the CaHSP20 genes may play roles in various plant hormone signaling pathways and stress responses, indicating their potential involvement in the plant’s mechanisms of adaptation to environmental stimuli.
3.7. Tissue-Specific Expression Patterns of CaHSP20s Genes
To further investigate the expression patterns of CaHSP20s in chickpeas, their expression profiles across various tissues, including the roots, stems, leaves, buds, capsules, apices, calli, and seedlings, were analyzed using publicly available RNA sequencing data (
3.8. Expression Profiling of CaHSP20s Genes under Heat Stress Treatment
To explore the potential function of CaHSP20s in response to heat stress, the expression of CaHSP20s in the leaves and stems of heat-treated chickpeas was analyzed using qRT-PCR. The observations revealed that the expression levels of 10 CaHSP20 genes (CaHSP20-17, CaHSP20-13, CaHSP20-9, CaHSP20-20, CaHSP20-11, CaHSP20-2, CaHSP20-7, CaHSP20-3, CaHSP20-12, and CaHSP20-5) were sensitive to heat stress, with their relative expression levels gradually increasing as the temperature rose from 25 °C to 42 °C. In the leaves, the expression levels of most CaHSP20s were highest within a short period (Figure 10A). Among these genes, CaHSP20-17, CaHSP20-20, CaHSP20-3, and CaHSP20-5 from the CI group exhibited the greatest increases in expression. This suggests that CI group genes play a significant role in responding to heat stress (Figure 10A). In addition, the relative expression heat maps of these 10 CaHSP20 genes were constructed using relative expression values to assess their expression levels under heat stress (Figure 10B,D). Compared with the 0 h treatment, all CaHSP20s were upregulated to varying degrees after the 42 °C heat stress treatment. As shown in Figure 10C, the relative expression levels of CaHSP20-9, CaHSP20-13, CaHSP20-7, CaHSP20-3, CaHSP20-12, and CaHSP20-5 genes in the stems reached their peak after the time increased to 24 h under the condition of 37 °C. When the temperature was 42 °C, the relative expression levels of CaHSP20-3 and CaHSP20-5 showed an increasing trend with the increase in time, differing from the trend that the expression levels of other genes first increased and then slowly decreased. The relative expression level of CaHSP20-3 increased by about 10,000 times at 24 h and 48 h (compared with 0 h), and the relative expression level of CaHSP20-5 increased by about 8000 times at 24 h and 9000 times at 48 h. In the leaves (Figure 10A,B), the relative expression content of most genes did not change much when treated at 25 °C for 24 h and 48 h compared with 0 h, while the relative expression content of CaHSP20-9 increased significantly when treated at 25 °C for 48 h. The expression levels of CaHSP20-13, CaHSP20-11, CaHSP20-2, and CaHSP20-7 peaked after treatment at 48 °C for 24 h. In the entire process of heat stress, the expression levels of six genes, including CaHSP20-17, CaHSP20-7, CaHSP20-20, CaHSP20-3, CaHSP20-12, and CaHSP20-5, were always high in the leaves and stems. CaHSP20-3, CaHSP20-5, CaHSP20-17, and CaHSP20-20 were clustered into the CI group; in comparison, the other three genes were scattered in the ER and P groups, indicating that these genes may have great significance in the response of chickpeas to heat stress. Overall, our findings indicate that most CaHSP20 genes are responsive to heat stress, highlighting their potential importance in heat stress tolerance in chickpeas.
4. Discussion
HSP20, as a class of molecular chaperones, is ubiquitous and simultaneously plays a central role in biotic and abiotic stress resistance [49,50]. Recent studies have reported the genome-wide identification and systemic functional analysis of the HSP20 gene family in various plant species, including grapes [11], tomatoes [12], watermelons [13], African bermudagrass [14], apples [48], and other species [50,51]. In this study, we systematically identified twenty-one HSP20 genes in chickpeas and grouped them into 10 subfamilies based on their phylogenetic clades and amino acid sequences (Figure 2). This classification is consistent with previous reports on HSP20 in other plant species, confirming the conserved nature of these genes across different species [11,15,16,50,51]. Instability is considered to be the common feature of stress protein [52], which may be associated with the CaHSP20 gene and can be found quickly during induction.
Furthermore, this classification of CaHSP20s was also supported by conserved domain, motif, and gene structure analyses showing that each subfamily shares similar domains, motifs, and exon–intron structures (Figure 6 and Figure 7). Conserved domain and motif analyses showed that all the identified CaHSP20s had a typical ACD domain, and most CaHSP20 family members in the same category had similar motif types and numbers (Figure 6); previous research has shown that the HSP20 genes may exhibit different biological functions, which could be dependent on the types and numbers of motifs [12,51]. When plants respond to various stresses, genes with few or no introns are rapidly activated to respond to external stimuli promptly [53]. Analysis of the exon–intron structure of CaHSP20s revealed that all members of the CaHSP20 gene family contain only one or two exons. Among them, thirteen genes were found to contain only one exon, eight genes contained two exons and different CaHSP20 genes in each subgroup showed similar exon–intron structures. In soybean species, most GmHsp20 gene members contain one or two exons (sixteen genes contain one exon, sixteen genes contain two exons, and one gene contains three exons). Different GmHsp20 genes in each subfamily exhibit similar exon–intron structures. In peanuts, six members of the AdHSP20 gene contain one exon, fifteen members of the AdHSP20 gene contain two exons, and six members of the AdHSP20 gene possess between 3 and 11 exons. Through analysis, the exon–intron structures of chickpeas, soybeans, and peanuts showed similarities and close evolutionary relationships (Figure 7). This finding suggests the conservation of the gene structures of the HSP20 family in plants. Transcription factors (TFs) play a crucial role in regulating plant growth, development, and stress resistance through the control of gene expression [54]. To obtain additional information about the regulation of CaHSP20s, we explored the cis-acting elements in their 2.0 kb promoter regions. Several cis-acting elements with phytohormone and/or stress response were identified, namely, MeJARE, ARE, ABRE, DRE, LTRE, GARE, DSRE, SARE, and AuxRE, which indicates that the CaHSP20 family members may widely be involved in responses to various phytohormone and/or stress in plants to exert their biological function (Figure 8). This wide range of cis-acting elements is consistent with the range in previous studies on the stress-responsive functions of HSP20 genes in plants [10,52]. Among the nine common plant hormones in plants, five of them, including ABA, auxin, BR, GA, and MeJA, play significant roles in the response to heat stress [55]. Among them, MeJA represents the highest number, with forty-four in total. MeJA transcription factors, as one of the most widely distributed transcription factor families in plants, have been proven to play a key role in plant stress responses, especially under abiotic stresses such as high temperatures [56]. When plants are subjected to high-temperature stress, MeJA can protect the cell membrane by enhancing the activity of some antioxidant enzymes and enhancing the osmotic regulation ability of cells. MeJA may increase the expression of genes responding to high-temperature stress, and it is speculated that the CaHsp20 gene family is involved in the response to high-temperature stress by regulating the activity of antioxidant enzymes in cells. Therefore, we suggest that CaHsp20 plays an important role in chickpeas’ response to heat stress.
It has been shown that gene duplication events, which include segmental and tandem duplications, are essential in genomic rearrangement and frequently cause the expansion of a gene family [57,58]. In this study, four tandem duplication pairs were involved in gene duplications (Table 2), indicating that segmental duplication may be the predominant gene-duplication event in the expansion of the HSP20 gene family in chickpeas. This finding is also consistent with a previous study showing that tandem duplication contributes to HSP20 gene family expansion during evolution [57]. In our study, Ka/Ks ratios of both segmentally and tandemly duplicated gene pairs were significantly less than 1, suggesting that purifying selection plays a predominant role in the evolution of the HSP20 gene family in chickpeas.
Gene expression patterns frequently provide crucial information regarding gene functions [59,60]. Tissue expression of the Hsp20 gene in plants has been detected and analyzed in several species, including soybeans, peanuts, apples, and pumpkins [3,38,39,48]. Therefore, the expression levels of CaHSP20 in the root, stem, leaf, bud, capsule, apice, calli, and seedling tissues were determined using public RNA-Seq data [41]. The results indicated that most CaHSP20 genes were expressed in various tissues and are involved in the development of various plant tissues (Figure 10), suggesting their essential roles in chickpea growth and development. This result corroborates previous studies in which HSP20 was shown to have important role functions in plant growth and development [11,12,13,48,50,51]. However, most CaHsp20 genes are underexpressed at all stages of development, and only a few genes are highly expressed in specific organs or during development. The same results have also been reported in cotton, and it is speculated that the CaHsp20 gene in chickpeas has a similar function to GhHsp20 in cotton and is part of a complex transcriptional network that regulates stamen development [61]. Five genes (CaHSP20-2, CaHSP20-7, CaHSP20-12, CaHSP20-13, and CaHSP20-17) were found to be highly expressed in all tissues. This finding is similar to the results for SlHsp17.7A, SlHsp17.6B, SlHsp17.6C, and SlHsp24.54 genes in tomatoes, meaning that these five genes may have specific housekeeping functions in chickpea cells under normal growth conditions [30]. However, the expression of some CaHsp20 genes in different tissues varies. For example, the expression levels of CaHSP20-4 and CaHSP20-6 were found to be relatively high in seeds but low in leaves, at almost 0, indicating that these CaHsp20 genes are mainly involved in the growth and development of chickpea seeds. As an important reproductive organ of chickpeas, these CaHsp20 genes still accumulate specifically in seeds without stress, thus further indicating that CaHSP20 plays a crucial role in maintaining cell homeostasis during meiosis, fertilization, and seed formation [62]. There is no uniform expression pattern of the CaHSP20 gene in chickpeas, indicating that different CaHSP20 proteins in chickpeas may have diversified functions in plant growth, development, and stress response. This finding supports the idea that there is no uniform expression pattern for all HSP20 genes in plants, consistent with results obtained in Arabidopsis, rice, and capsicum [49].
The results of a study on the potato HSP20 gene showed that the relative expression levels of fourteen StHsp20 genes were significantly upregulated by more than a hundred-fold in response to heat stress [50]. Analysis of soybean HSP20 gene expression also indicated that most CmHsp20 genes are highly induced in roots and leaves, and the expression level was found to be much higher than that of the control group [38]. Similarly, the gene expression analysis conducted in the present study showed that six genes, including CaHSP20-17, CaHSP20-20, CaHSP20-7, CaHSP20-3, CaHSP20-12, and CaHSP20-5, possessed higher expression levels in the leaves and stems under high-temperature stress. Compared with the 0 h control group, their content increased by a hundred or even a thousand times. The expression levels of most of the above genes were significantly upregulated between 25 °C and 42 °C with the increase in temperature and treatment time, indicating that these six genes play an important role in the heat stress response of chickpeas. Studies on the HSP20 gene in pumpkin showed that the relative expression levels of six Hsp20 genes (CmoHsp20-7, CmoHsp20-13, CmoHsp20-18, CmoHsp20-22, CmoHsp20-26, and CmoHsp20-32) were extremely upregulated after 3 h of heat stress [3]. In the present study, CaHSP20-17, CaHSP20-5, CaHSP20-7, and CaHSP20-20 genes that significantly responded to high-temperature stress were clustered into the CI group; in comparison, CaHSP20-3 and CaHSP20-12 were distributed in the ER and P groups.
Moreover, certain CaHSP20 genes, such as CaHSP20-17, CaHSP20-13, CaHSP20-2, and CaHSP20-7, exhibited significantly higher expression levels in leaves; similarly, CaHSP20-17, CaHSP20-13, CaHSP20-2, CaHSP20-7, and CaHSP20-12 showed elevated expression in flowering tissues (Figure 9). These tissue-specific expression patterns suggest potential functional specializations of these genes, indicating their significant roles in these particular tissues. In the results of the analysis of relative gene expression levels, these ten CaHSP20 genes were found to contain ABA, DRE, LTRE, and other abiotic stress response elements, and most of the genes were found to contain cis-regulatory elements related to abscisic acid (ABA), gibberellin (GARE), AuxRE, and other hormones. Among them, CaHSP20-17, CaHSP20-7, CaHSP20-20, CaHSP20-3, CaHSP20-12, and CaHSP20-5 genes from CI, ER, and P families possessed high expression levels in the leaves and stems in response to high-temperature stress. Moreover, almost all contain plant hormones, such as abscisic acid (ABA). Plant hormones are key signaling compounds that regulate plant growth, development, and stress responses. The expression levels of these genes were significantly upregulated when the leaves and stems were subjected to high temperatures, indicating that the response to high-temperature stress may be related to the regulation of hormones. This finding is consistent with the conclusion of previous studies in that auxin, abscisic acid, gibberellin, brassinosterol, and jasmonic acid play important roles in plant heat stress response [55]. CaHSP20-3 also contains DSRE elements related to defense and stress. When treated at 42 °C, the expression level of CaHSP20-3 is upregulated with the increase in treatment time. It is speculated that CaHSP20-3’s positive response to high-temperature stress may be related to DSRE and other elements.
Recent projections indicate that the Earth’s global average temperature will rise by 1.5 °C within two decades. Studies have demonstrated that for each 1 °C increase in the average annual temperature, yields of key crops such as rice, wheat, and maize decrease by 3–8 percent [63]. Consequently, investigating plants’ response mechanisms to high-temperature stress and enhancing heat tolerance in crops is imperative for ensuring global food security and fostering human development. Heat shock proteins, particularly HSP20, play a vital role in coping with extreme temperatures [59,64,65]. To determine whether CaHSP20 genes are involved in chickpea heat tolerance, we analyzed their expression profiles in leaves using RNA-Seq data with FPKM values of ≥0.3. The qRT-PCR results indicated that the expression of CaHSP20-17, CaHSP20-13, CaHSP20-9, CaHSP20-20, CaHSP20-11, CaHSP20-2, CaHSP20-7, CaHSP20-3, CaHSP20-12, and CaHSP20-5 was significantly upregulated under high-temperature stress in chickpeas. Additionally, their relative expression levels increased progressively with rising temperatures (25–42 °C), showing a consistent trend in both leaf and stem tissues (Figure 10). These findings suggest a role for these CaHSP20 genes in responding to high-temperature stress. The rapid upregulation observed in these CaHSP20 genes may be attributed to their compact gene structures, which have fewer introns. Previous research has shown that genes with such compact structures can facilitate rapid and timely responses to various plant stresses [66]. Remarkably, the relative expression levels of CaHSP20-17, CaHSP20-20, CaHSP20-7, CaHSP20-3, CaHSP20-12, and CaHSP20-5 increased by hundreds or even thousands of times as temperatures rose from 25 °C to 42 °C. These six CsHSP20 genes appear primarily involved in the heat stress response pathway and represent potential candidate genes for breeding heat-tolerant chickpea varieties. CaHSP20s contribute not only to the formation of heat tolerance but also play significant roles in the broader process of plant adaptations to environmental changes.
In the present study, the expression pattern of the CaHSP20 gene family provides genetic options for solving the issue of poor growth due to high-temperature stress in chickpeas using molecular breeding methods. For example, CaHSP20-17, CaHSP20-20, CaHSP20-3, CaHSP20-12, and CaHSP20-5 showed consistently high expression levels throughout the heat stress process, suggesting that these five genes may be important in the response of chickpeas to heat stress. The analysis of cis-acting elements in the promoter region provides a theoretical basis for further analysis of the regulatory mechanisms of other factors on CaHSP20s; anaerobic-induced cis-acting elements, drought-induced elements, and other stress-related cis-acting elements may be transcription factor binding sites. The presence of many hormone-related cis-acting elements suggests that CaHSP20s may be closely related to hormone regulation or signal transduction. However, these speculations require further investigation.
5. Conclusions
In the study presented herein, a comprehensive analysis of the HSP20 gene family in chickpeas was conducted at a genome-wide level, with a total of twenty-one CaHSP20 genes being identified. These genes were found to be distributed unevenly across seven chromosomes and were categorized into ten subfamilies based on their evolutionary relationships. To further investigate the evolutionary connections among HSP20 gene family members, the structure, conserved motifs, cis-acting elements, protein interaction networks, and homology of these genes were investigated. Additionally, the expression patterns of CaHSP20s in various organs and under different stress conditions were studied using qRT-PCR, offering insights for future research on the roles of HSP20 genes. The results of the present study provide a theoretical basis for analyzing the molecular regulatory mechanisms under heat stress and provide a basis for identifying important candidate HSP20 genes involved in chickpeas’ response to heat stress.
All authors contributed to the study’s conception and design. Material preparation, data collection, and data analyses were performed by S.L., Y.W., Y.L. (Yang Li), J.Y., Z.Z., H.Z., D.H., and Y.L. (Yanmin Liu) The first draft of the manuscript was written by S.L., and all authors commented on previous versions of the manuscript. All authors have read and agreed to the published version of the manuscript.
The original contributions presented in the study are included in the article/
The authors would like to thank the experimental materials provided by bread of life farm.
Author Jianguo Yan was employed by the company Huzhou Lvteng Ecological Agriculture Company Limited. The remaining 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.
Footnotes
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Enlarge this image.Figure 1. Chromosomal locations of CaHSP20 genes. Tandemly duplicated genes are indicated in red. Chromosome numbers are shown at the top of each bar. The scale presented on the left indicates chromosome sizes in megabases (Mb).
Enlarge this image.Figure 2. Homology of the CaHSP20 gene. Gray lines indicate all homology blocks in the chickpea genome.
Enlarge this image.Figure 3. Synchronization analysis of the HSP20 gene in chickpeas with three other representative plants (soybean, peanut, and Arabidopsis). (A) Chickpeas and soybeans. (B) Chickpeas and peanuts. (C) Chickpeas and Arabidopsis. Gray lines indicate significant blocks of co-linearity within and between plant genomes; red lines indicate homologous HSP20 gene pairs. Chromosome numbers are indicated at the top of each chromosome.
Enlarge this image.Figure 4. Schematic depicting the protein interaction network of caHSP20s. Each node represents a protein, and each edge represents an interaction. Purple lines represent experimentally validated protein interactions, green lines represent protein interactions predicted based on gene neighborhoods, blue lines represent protein interactions predicted based on gene co-occurrence, and black lines represent protein co-expression.
Enlarge this image.Figure 5. Phylogenetic analysis of HSP20 proteins from chickpeas, Arabidopsis, soybeans, and peanuts. The full length of the amino acid sequences of twenty-one CaHSP20 proteins, nineteen AtHSP20 proteins, thirty-three GmHSP20 proteins, and twenty-seven AdHSP20 proteins were aligned using the MUSCLE program in MEGA 11.0, and the phylogenetic tree was constructed with MEGA 11.0 using the maximum likelihood (ML) method with 1000 bootstrap replicates.
Enlarge this image.Figure 6. A schematic diagram of phylogenetic relationships and conserved motifs of the HSP20 gene family in chickpeas. The phylogenetic tree was constructed using the maximum-likelihood method with 1000 bootstrap replicates for each branch using MEGA 11.0. Conserved motifs were identified using Multiple Em for Motif Elicitation (MEME). A model exhibition of 10 motif compositions in CaHSP20 complete amino acid sequences was prepared using MEME.XML (v.5.5.5) through TBtools (v.1.120). Each conserved motif is represented by a specific color box and corresponds one-to-one in the structural diagram. The locations of different motifs are proportional to their sequence lengths.
Enlarge this image.Figure 7. A schematic diagram of phylogenetic relationships and gene structure of the HSP20 gene family in chickpeas. The phylogenetic tree was constructed using the maximum likelihood method with 1000 bootstrap replicates for each branch using MEGA 11.0. The gene structure of the CaHSP20s diagrammatic was produced using TBtools (v.1.120), with the coding sequence (CDS) represented by a green box and the intron shown as a black line.
Enlarge this image.Figure 8. A schematic representation of molecular phylogenetic relationships and 2000 bp promoters of HSP20 in chickpeas. The phylogenetic tree was constructed using the maximum-likelihood method with 1000 bootstrap replicates for each branch using MEGA 11.0. The 2000 bp promoter sequences of CaHSP20s were analyzed using PlantCARE and visualized using TBtools (v.1.098661). Differently colored boxes represent different cis-acting elements.
Enlarge this image.Figure 9. Expression profile of CaHSP20 genes in eight different tissues: roots, stems, leaves, buds, capsules, apices, calli, and seedlings of chickpeas. Transcriptome data for CaHSP20s gene tissue expression analysis were downloaded from the National Center for Biotechnology (NCBI) Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra/, accessed on 5 May 2024) under the accession number GSE147831. The heatmap was constructed using each gene Log2 (FPKM + 1) values via TBtools (v.1.120), ranging from a low expression level (blue) to a high expression level (red).
![View Image - Figure 10. The relative expression levels of chickpea HSP20 genes. (A) The relative expression level of the HSP20 gene in the leaves at 25 °C, 37 °C, and 42 °C, and (C) The relative expression level of the HSP20 gene in the stems at 25 °C, 37 °C, and 42 °C. All data shown are the means and SDs of three biological replicates. The asterisks indicate the significance level (* p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001) based on Duncan’s multiple range test. (B,D) Heat maps of CaHSP20 gene expression in the leaves and stems at different times under heat stress (25 °C, 37 °C, and 42 °C), respectively. The graph size and color represent the expression level of each gene in each sample.](https://media.proquest.com/media/hms/THUM/20/sObeZ?_a=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%3D%3D&_s=BTP%2BMcf5q09AvoZfMLTItMA7xFI%3D "View Image - Figure 10. The relative expression levels of chickpea HSP20 genes. (A) The relative expression level of the HSP20 gene in the leaves at 25 °C, 37 °C, and 42 °C, and (C) The relative expression level of the HSP20 gene in the stems at 25 °C, 37 °C, and 42 °C. All data shown are the means and SDs of three biological replicates. The asterisks indicate the significance level (* p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001) based on Duncan’s multiple range test. (B,D) Heat maps of CaHSP20 gene expression in the leaves and stems at different times under heat stress (25 °C, 37 °C, and 42 °C), respectively. The graph size and color represent the expression level of each gene in each sample.")
Enlarge this image.Figure 10. The relative expression levels of chickpea HSP20 genes. (A) The relative expression level of the HSP20 gene in the leaves at 25 °C, 37 °C, and 42 °C, and (C) The relative expression level of the HSP20 gene in the stems at 25 °C, 37 °C, and 42 °C. All data shown are the means and SDs of three biological replicates. The asterisks indicate the significance level (* p [less than] 0.05, ** p [less than] 0.01, and *** p [less than] 0.001) based on Duncan’s multiple range test. (B,D) Heat maps of CaHSP20 gene expression in the leaves and stems at different times under heat stress (25 °C, 37 °C, and 42 °C), respectively. The graph size and color represent the expression level of each gene in each sample.
Structural and biochemical features of the HSP20 gene family in chickpeas.
| Name | Gene ID | Chr | Gene Start | Gene End | Strand | mRNA | CDS | Protein | pI | MW | Instability Index | Hydropathy Index |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CaHSP20-1 | Ca_06952 | Chr01 | 15999417 | 16000315 | reverse | 899 | 582 | 193 | 5.73 | 22.43 | 34.09 | −0.69 |
| CaHSP20-2 | Ca_10257 | Chr02 | 32641982 | 32642736 | forward | 755 | 633 | 210 | 9.16 | 24.44 | 37.68 | −0.83 |
| CaHSP20-3 | Ca_12525 | Chr02 | 30242080 | 30242541 | reverse | 462 | 462 | 153 | 6.85 | 17.44 | 41.85 | −0.66 |
| CaHSP20-4 | Ca_14336 | Chr02 | 29023413 | 29024198 | reverse | 786 | 420 | 139 | 7.03 | 15.48 | 46.12 | −0.34 |
| CaHSP20-5 | Ca_14692 | Chr02 | 5192005 | 5192424 | forward | 420 | 420 | 139 | 5.96 | 15.86 | 30.45 | −0.55 |
| CaHSP20-6 | Ca_19633 | Chr03 | 18515549 | 18516052 | reverse | 504 | 504 | 167 | 7.27 | 19.44 | 48.30 | −0.81 |
| CaHSP20-7 | Ca_12168 | Chr04 | 3702122 | 3702712 | reverse | 591 | 591 | 196 | 6.45 | 22.71 | 41.12 | −0.57 |
| CaHSP20-8 | Ca_04038 | Chr05 | 45931029 | 45931899 | forward | 871 | 690 | 229 | 7.67 | 26.03 | 39.09 | −0.66 |
| CaHSP20-9 | Ca_11311 | Chr05 | 42969085 | 42970157 | forward | 1073 | 729 | 242 | 9.35 | 27.46 | 57.14 | −0.72 |
| CaHSP20-10 | Ca_01653 | Chr05 | 35740491 | 35740964 | forward | 474 | 474 | 157 | 5.79 | 17.63 | 42.36 | −0.45 |
| CaHSP20-11 | Ca_01654 | Chr05 | 35738083 | 35738649 | forward | 567 | 567 | 188 | 5.46 | 21.37 | 40.02 | −0.84 |
| CaHSP20-12 | Ca_20062 | Chr05 | 22777083 | 22778273 | forward | 1191 | 576 | 191 | 6.32 | 22.08 | 43.37 | −0.77 |
| CaHSP20-13 | Ca_16264 | Chr06 | 54104152 | 54104622 | forward | 471 | 471 | 156 | 5.90 | 17.62 | 43.35 | −0.25 |
| CaHSP20-14 | Ca_13815 | Chr06 | 45062577 | 45062972 | forward | 396 | 396 | 131 | 5.49 | 15.15 | 41.72 | −0.75 |
| CaHSP20-15 | Ca_15814 | Chr06 | 34622371 | 34623153 | forward | 783 | 624 | 207 | 5.67 | 23.30 | 61.30 | −0.60 |
| CaHSP20-16 | Ca_14594 | Chr06 | 27635845 | 27636312 | forward | 468 | 468 | 155 | 5.56 | 17.87 | 63.28 | −0.73 |
| CaHSP20-17 | Ca_06336 | Chr06 | 17172499 | 17172921 | forward | 423 | 423 | 140 | 5.24 | 15.69 | 42.04 | −0.73 |
| CaHSP20-18 | Ca_06335 | Chr06 | 17171634 | 17172116 | forward | 483 | 483 | 160 | 5.57 | 18.41 | 62.45 | −0.71 |
| CaHSP20-19 | Ca_06334 | Chr06 | 17163907 | 17164389 | reverse | 483 | 483 | 160 | 5.82 | 18.38 | 58.18 | −0.68 |
| CaHSP20-20 | Ca_05301 | Chr06 | 15188993 | 15189463 | reverse | 471 | 471 | 156 | 6.19 | 17.86 | 52.39 | −0.71 |
| CaHSP20-21 | Ca_16176 | Chr07 | 33565963 | 33566633 | forward | 671 | 450 | 149 | 7.79 | 16.97 | 47.51 | −0.62 |
Bp: base pair. aa: amino acid. CDS: coding sequence. pI: isoelectric point. MW: molecular weight.
Ka, Ks, and Ka/Ks values for the duplication gene pairs from.
| Seq1 | Seq2 | Ka | Ks | Ka/Ks Ratio | Date (MY) | Duplication Type |
|---|---|---|---|---|---|---|
| CaHSP10 | CaHSP11 | 0.327 | 1.005 | 0.325 | 33.500 | Tandem |
| CaHSP19 | CaHSP18 | 0.008 | 0.117 | 0.068 | 3.898 | Tandem |
| CaHSP19 | CaHSP17 | 0.253 | 1.230 | 0.206 | 40.985 | Tandem |
| CaHSP18 | CaHSP17 | 0.247 | 1.576 | 0.157 | 52.539 | Tandem |
Supplementary Materials
The following supporting information can be downloaded at
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