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Article

Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance

by
Yating Su
1,†,
Jieyun Fang
1,†,
Muhammad Zeeshan Ul Haq
1,
Wanli Yang
1,
Jing Yu
1,
Dongmei Yang
1,
Ya Liu
1,2,* and
Yougen Wu
1,*
1
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), School of Tropical Agriculture and Forestry, Hainan University, Sanya 572025, China
2
Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, Hainan University, Haikou 570228, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2024, 13(15), 2077; https://doi.org/10.3390/plants13152077
Submission received: 28 June 2024 / Revised: 20 July 2024 / Accepted: 24 July 2024 / Published: 26 July 2024
(This article belongs to the Special Issue Physiological and Genetic Mechanisms of Abiotic Stress Tolerance in Crops II)
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Abstract

:
Casparian strip membrane domain proteins (CASPs), regulating the formation of Casparian strips in plants, serve crucial functions in facilitating plant growth, development, and resilience to abiotic stress. However, little research has focused on the characteristics and functions of AhCASPs in cultivated peanuts. In this study, the genome-wide identification and expression analysis of the AhCASPs gene family was performed using bioinformatics and transcriptome data. Results showed that a total of 80 AhCASPs members on 20 chromosomes were identified and divided into three subclusters, which mainly localized to the cell membrane. Ka/Ks analysis revealed that most of the genes underwent purifying selection. Analysis of cis elements suggested the possible involvement of AhCASPs in hormonal and stress responses, including GA, MeJA, IAA, ABA, drought, and low temperature. Moreover, 20 different miRNAs for 37 different AhCASPs genes were identified by the psRNATarget service. Likewise, transcriptional analysis revealed key AhCASPs responding to various stresses, hormonal processing, and tissue types, including 33 genes in low temperature and drought stress and 41 genes in tissue-specific expression. These results provide an important theoretical basis for the functions of AhCASPs in growth, development, and multiple stress resistance in cultivated peanuts.

1. Introduction

Cultivated peanut (Arachis hypogaea L.), also known as the peanut or long-lived fruit, is an annual herbaceous plant of the legume Arachis. Peanuts are native to South America, including Brazil, Peru, and other countries. They are mainly distributed in Asia, Africa, and America, as well as in tropical and subtropical agricultural climate zones [1]. Peanuts are an important oil crop with economic value worldwide [2]. Peanuts are renowned for their nutritional richness, containing oil (40–60%), protein (10–20%), carbohydrates, minerals, essential vitamins, antioxidants, and unsaturated fatty acids [3]. In addition, peanuts are also the main source of important medicinal compounds such as sterols, arginine, resveratrol, and flavonoids [4]. These compounds effectively prevent or treat chronic diseases, including cardiovascular disease, diabetes, and cancer, and have high medicinal value [5]. Peanuts have been introduced and cultivated in over 100 countries or regions worldwide, including China, the US, and India [6]. China is a significant producer and consumer of peanuts in the global market, with an area of about 5 million hectares under cultivation and an annual yield of more than 18 million tons [7,8]. However, peanuts often suffer from abiotic stresses (including drought, salt, extreme temperature, and heavy metal stress) during their growth and development, seriously affecting peanut germination, development, flowering, and fruiting processes. These issues contribute to reduced yield and quality of peanuts and thus limit the peanut industry’s growth in terms of sustainability and high-quality production [9]. Therefore, an in-depth study of the peanut stress response and regulation of molecular mechanisms is urgently needed to ensure high-quality cultivation and production of peanuts and create new stress-tolerant germplasm.
Abiotic stress is a general term for various environmental factors detrimental to plant growth, such as drought, salt, extreme temperature, and heavy metals [10]. When a peanut plant senses abiotic stress, it initiates a series of early stress-responsive genes that activate or repress the expression of downstream-associated genes/transcription factors via different signaling pathways [11]. These stress-responsive and regulatory genes regulate various physiological and biochemical responses in plants, such as osmotic pressure regulation, stomatal opening and closing, antioxidant system activation, etc. For drought stress, drought signaling induces the expression of transcription factors such as DREB [12], WRKY [13], NAC [14], bHLH [15], and bZIP [16], which, respectively, regulates the expression of downstream drought resistance genes, and then improves the drought resistance ability of crops. Meanwhile, it is also reported that functional genes related to water transport (PIP, TIP), E3 ligase SIZ 1, and dehydrating protein DHN are induced under drought conditions and resist drought stress by regulating the water potential, osmotic potential and ROS accumulation [17]. Currently, known anti-drought signaling pathways include the ABA signaling pathway, Ca2+ signaling pathway, and mitogen-activated protein kinase (MAPKs) cascade signaling pathway [18]. In resisting salt stress, the SOS pathway is crucial, with SOS1 helping plants to resist salt stress by reducing intracellular Na+ toxicity through the transport of Na+ outside the cell [19]. It has been found that transcription factors such as NAC72 [20], ERF1 [21], MYB42 [22], WRKY81 [23], etc., can respond to salt stress and activate the expression of stress-related genes, thus improving the salt-tolerant ability of crops [24]. CBFS/EREBs constitute the central component of the regulatory network that functions under low-temperature stress, and their activity is modulated by ICE1 [25]. Furthermore, LEA and HSPs proteins safeguard cells from denaturation and preserve membrane fluidity under low temperatures [26]. Previous studies have demonstrated conclusively that the formation and integrity of Casparian strips, such as CASP1, CIF1/2, GSO1/2, and MYB36 [27,28], play an important role in stress. What kind of genes are involved in forming and regulating peanut Casparian strip? Do these genes have stress tolerance functions? Which signaling pathways are involved? These questions need to be further answered.
The Casparian strip is a special wall structure surrounded by endothelial cells’ radial and transverse walls. It has the characteristics of bolted and lignified band thickening [29]. The main function of this structure is to screen, block, and cut off unwanted ions or macromolecules from entering the vascular column [28,30]. Current investigations have revealed that the formation and regulation of the Casparian strip encompass the intricate interplay of numerous genes and signaling pathways [31,32], such as Casparian strip domain proteins (CASPs) [33], leucine receptor kinase (GSO1/SGN3) [34], enhanced suberin 1 (ESB1) mutant [35], MYB domain protein 36 (MYB36) [36] and endodermis Casparian strip integrity factor (CIF1/2) [37]. As membrane proteins specific to the Casparian strip-forming region, CASPs feature a distinctive transmembrane topology, including the amino and carboxyl termini in the cytoplasm and conserved extracellular ring structures. These properties are critical for forming Casparian strips, ensuring their core function in the plant cell wall architecture [38]. In the Arabidopsis genome, 39 members belong to the CASPs family, while in the rice genome, 19 members have been identified [39]. In addition, there are 48 in cotton [40], 61 in banana [41], and 156 in patchouli [42]. These results indicate differences in the number of CASPs family members among different species. Further studies have found that CASPs family genes play an important role in salt tolerance [43], cold tolerance [44], and other abiotic stresses. However, the research on the biological function of plant CASPs genes mainly focuses on model plants, such as Arabidopsis thaliana and rice. The related research on peanut AhCASPs genes has not been reported. Regarding the peanut AhCASPs gene family, the number of members remains unknown. Furthermore, it is necessary to determine which specific members can respond to adversity stress. The stress regulation signaling pathways implicated in peanut AhCASPs also require thorough examination and investigation.
In this study, we conducted a rigorous bioinformatics analysis of the whole genome data of cultivated peanuts, resulting in the screening and identification of 80 AhCASPs family members. Subsequently, a comprehensive study was performed to elucidate the physicochemical properties of these proteins, the distribution across chromosomes, the promoter cis-acting elements, and their evolutionary expression characteristics. In addition, through the analysis of peanut transcriptome data of different treatments, plant growth regulators, and tissues, the key AhCASPs candidate genes involved in peanut stress (33 genes), hormone stress (40 genes), and tissue specificity (41 genes) were screened and identified. The findings of this study furnish a theoretical foundation for examining the AhCASPs gene family’s functionality and its significance in peanut’s response to stress conditions.

2. Results

2.1. Identification of AhCASPs Genes in Peanut

We used a two-step method to ascertain the members of the AhCASPs gene family in the peanut genome. The first Blast obtained 185 candidate AhCASPs genes based on the Pfam (PF04535) file. Subsequently, using the known AtCASPs protein sequence from A. thaliana as a query and blast alignment of the peanut proteome database, 91 candidate AhCASPs genes in peanuts were retrieved. The genes initially identified as potential candidates were subjected to a comparative analysis with those subsequently identified on the second occasion, and the repeated transcripts were deleted. Finally, 80 members of the AhCASPs gene family have been successfully identified and obtained. To understand the physical and chemical properties of 80 peanut AhCASPs proteins, we used the online analysis tool ExPASy (https://www.expasy.org/ accessed on 27 April 2024) to analyze their protein sequences comprehensively. We predicted the isoelectric point and molecular weight of peanut AhCASPs proteins (Table 1). The results indicated that the amino acid length of peanut AhCASPs protein was between 72 and 810 amino acids). The gene with the least number of amino acids was AhCASP65 (containing 72 amino acids), and the gene with the most significant number of amino acids was AhCASP16 (containing 810 amino acids). The molecular weight of AhCASPs was between approximately 7734.14 and 89,355.45 Da, including 17 acidic proteins (pI ≤ 7) and 63 basic proteins (pI ≥ 7). In the AhCASPs gene family, 51 AhCASPs genes had an instability coefficient of less than 40, which were stable proteins. The instability coefficient of 29 AhCASPs genes was greater than 40, which belonged to unstable proteins. The average hydrophilic coefficient of AhCASP33, AhCASP40, AhCASP72, AhCASP30, AhCASP25, AhCASP66, AhCASP61, AhCASP9, AhCASP11, AhCASP67, and AhCASP24 is less than 0, indicating that these proteins exhibit hydrophilic properties. Furthermore, the average hydrophilic coefficient of the 69 AhCASPs genes exceeded 0, suggesting that they are classified as non-hydrophilic proteins.
The results of in silico analysis showed that the localization of different AhCASPs family members was different: 69.5% of the AhCASPs family proteins were located in the cell membrane, 19.5% of the AhCASPs proteins were located in the chloroplast, and 14.6% of the AhCASPs proteins were situated within the nucleus. It is worth noting that AhCASP6 and AhCASP52 are located on peroxisomes, AhCASP22 is located on chloroplasts and Golgi apparatus, and AhCASP65 is located on cell membranes and Golgi apparatus, indicating that they may play different biological functions. In summary, the results showed that the peanut AhCASPs family proteins mainly play biological functions in the cell membrane and adhere to the structural characteristics consistent with membrane proteins.

2.2. Chromosome Distribution, Collinearity, and Ka/Ks Analysis

According to the chromosomal localization analysis of 80 AhCASPs family genes in peanuts, the distribution map of AhCASPs family genes on peanut chromosomes was drawn (Figure 1). The results indicated that AhCASPs genes were distributed on each peanut chromosome and distributed on 20 chromosomes. According to the physical location on the chromosome, all AhCASPs genes were renamed uniformly. Only one gene was distributed on chromosomes 4, 7, 10, 17, 18, and 20; the remaining genes were mostly distributed on chromosome 3, with eleven genes. A rigorous analysis of gene density reveals that the genes within the three subclusters exhibit a primary concentration at both extremities of the chromosome. Specifically, the gene density is notably higher at the termini, whereas it is comparatively lower in the central region of the chromosome. AhCASP6, AhCASP22, AhCASP27, AhCASP52, and the other 23 genes were distributed in the low-density region, with the remaining genes distributed in medium or high-density areas, respectively.
In addition, the collinear analysis of the genomes between A. thaliana, rice, and peanut (Figure 2) revealed that there were 47 linear relationships between 26 genes in A. thaliana and 35 genes in peanut; there were 15 linear relationships between 9 genes in rice and 9 genes in peanut. Moreover, 46 peanut AhCASPs genes were more conservative, neither collinear with CASPs genes in A. thaliana nor collinear with CASPs genes in rice. Some CASPs genes are related to at least two pairs of homologous genes, especially AhCASP43, AhCASP64, AhCASP7, AhCASP8, AhCASP21, AhCASP18, AhCASP56, and AhCASP2, which are closely related to other genomes and may play an important role in the evolution of CASPs gene family. The comparative analysis of CASPs genes between peanuts and other plants holds significant importance in establishing interspecific genetic relationships and forecasting gene functionality, thereby contributing to a deeper understanding of genetic diversity and potential applications in plant biology.
The Ka/Ks value is an important parameter for evaluating the evolution of coding sequences and determining the type of selection pressure after repetition [45]. Generally, a Ka/Ks ratio greater than 1, equal to 1, and less than 1 indicates that the gene underwent positive, neutral, and negative or stable selection, respectively [46,47]. In order to understand the selection pattern of AhCASPs, we revealed the Ka, Ks, and Ka/Ks ratios of all gene pairs (Table 2). Among the 32 pairs of homologous gene pairs identified, except for AhCASP2/43, AhCASP29/71, and AhCASP30/72, the Ka/Ks values of other 93.75% (30/32) were less than 1, indicating that the AhCASPs genes in these groups were subjected to purification selection pressure. The results showed that most AhCASPs genes experienced robust purification and selection during evolution, while only a few genes diverged and produced new biological functions. The difference time range of 32 pairs of genome-wide repetitive gene pairs was 0.41~151.82 Mya. Among them, the differentiation time of AhCASP29/71 in the CASP-b subcluster was the shortest, only 0.41 Mya.

2.3. Conserved Motifs, Conserved Domains, and Gene Structure Analysis

The MEME online analysis tool predicted and identified the peanut AhCASPs protein sequence motifs. The statistical results (Figure 3a) showed that 10 motifs were identified from 80 AhCASPs family members, namely motifs 1–10. The variation in conserved motifs within each cluster is notable and significant. Although AhCASP65 contains only one conserved motif, AhCASP78, AhCASP23, and AhCASP68 contain only two motifs, and the number of motifs of the remaining genes is 3–7. It is worth noting that, as shown in Figure 3a, motifs 2, 3 and 10 are widely distributed in the AhCASPs family, occupying 80.4%, 95.1%, and 97.6%, respectively, and they are all situated in proximity to the c-terminus of the respective proteins. These findings indicate a high level of conservation among these three motifs within the AhCASPs gene family, potentially indicating their significant roles in various standard biological functions. Upon integration of the phylogenetic tree and motif analysis, it is evident that the motif distribution of AhCASPs exhibits distinct patterns across different branches. For example, motif 10 is only present in subcluster C; subcluster A lacks motif 6 and motif 9; motif 5 only exists in subcluster B. These results indicate that AhCASPs genes with close evolutionary relationships have similar motif compositions.
The analysis results of 80 identified AhCASPs protein domains are shown in Figure 3b. AhCASP16 protein contained the PLN03081 superfamily domain and the MARVEL structure. AhCASP58 and AhCASP61 contained the DUF588 structure and TCR domain. The remaining genes contained at least one conserved domain, mainly DUF588 and MARVEL domains. Studies have shown that some scholars have broadened the scope of phylogenetic analysis beyond the plant realm and have uncovered a noteworthy degree of conservation among the CASPs and MARVEL protein families. Notably, these conserved residues are positioned in the transmembrane domains, implying that these specific domains are crucial for the localization of CASPs [33].
The intron/exon structure, intron type, and number of genes are typical evolutionary marks of gene families [48]. Therefore, further construction of a protein phylogenetic tree and intron/exon structure map of the peanut AhCASPs gene family can further evaluate the structural characteristics of related genes. As shown in Figure 3c, 78 AhCASPs members contain 1–8 introns and 2–9 exons, of which AhCASP65 and AhCASP78 have no introns.

2.4. Phylogenetic Relationship of Peanut AhCASPs

To comprehensively understand the evolutionary relationships among peanut AhCASPs genes, a rigorous multiple sequence alignment analysis was conducted encompassing 80 peanut AhCASPs genes and 39 AtCASPs genes derived from the model plant A. thaliana. Subsequently, phylogenetic trees were generated with the alignment results (Figure 4). According to the genetic relationship, 119 CASPs genes were divided into CASP-a, CASP-b, and CASP-c sub-clusters. Among them, the CASP-c subcluster was the most distributed, with a total of 23 AtCASPs genes and 44 AhCASPs genes. A total of 6 AtCASPs genes and 18 AhCASPs genes were distributed in the CASP-a subcluster. There were 10 AtCASPs genes and 18 AhCASPs genes distributed in the CASP-b subcluster. Prior investigations have revealed a definitive association between the AT2G36100 gene (AtCASP1) and salt tolerance in rice [49]. Furthermore, the AT3G55390 gene (AtCASPL4C1) has been documented to be triggered by low-temperature conditions and exerts a negative regulatory effect on plant growth [50]. Additionally, the AT4G03540 gene (AtCASPL1C1) constructs an extracellular barrier, thereby effectively augmenting the salt tolerance capability of sweet sorghum [43]. Therefore, it is speculated that the highly homologous AhCASPs genes may possess analogous functionalities. So far, the specific biological function of the peanut AhCASPs genes remains unclarified, but the function of some CASPs genes in the model plant A. thaliana has been verified. A class of CASPs proteins with similar evolutionary relationships have identical structures and often play similar biological functions. Therefore, the biological function of the peanut AhCASPs gene can be speculated and verified according to the clustering analysis results of peanut and model plant A. thaliana CASPs protein.

2.5. Prediction of Cis-Acting Elements of AhCASPs Promoter

Cis-acting elements constitute a specific category of DNA sequences located in the initiation region of gene transcription, which play a key role in regulating gene transcription. They promote or inhibit gene transcription by binding to transcription factors [51,52]. The promoter sequences of 2000 bp upstream of ATG of the 80 AhCASPs CDS was extracted and analyzed. After filtering out unknown and untrustworthy elements, a total of three types of cis-acting elements were identified, including abiotic stress response, plant hormone response, and growth and development response elements (Figure 5 and Figure 6). Four abiotic stress response elements have been identified: light, anaerobic, drought, and low temperature. Among these elements, the following motifs have been detected: I-box, ATCT, Box 4, GT1, GA (accounting for 79.7% in light response), ARE (13%), MBS (4.4%), and LTR (2.8%). Among them, most are light response elements, suggesting that the AhCASPs gene family may play a role in regulating plant photomorphogenesis. There were five types of response elements related to hormone regulation (gibberellin (GA), methyl jasmonate (MeJA), SA, auxin (IAA), and ABA, including ABRE (34.5%), CGTCA-motif/TGACG-motif (36.8%), AuxRE/AuxRR-core/TGA-element (10.6%), P-box/TATC-box/GARE-motif (9.9%) and TCA-element (8.3%)). Among them, ABRE and CGTCA-motif/TGACG-motif are the most prominent types, accounting for 34.5% and 36.8% of all identified cis-acting hormone elements. In addition, after in-depth research, we identified seven growth and developmental response elements, including endosperm expression, meristem expression, circadian control, zein metabolism, cell cycle regulation, seed specificity, and MYBHv1 binding sites. These crucial components encompass CAT-box (24.6%), O2-site (21.7%), CCAAT-box (18.8%), GCN4_motif (14.5%), circadian (11.6%), RY-element (5.1%), and MSA-like (3.6%). It is surmised that the AhCASPs family is potentially implicated in diverse developmental processes and has a significant role in hormone regulation and stress response mechanisms.

2.6. miRNA Target Gene Prediction and GO Enrichment Analysis

The psRNATarget service has been utilized to examine the regulatory mechanisms of miRNAs with the expression of the AhCASPs gene. In order to better study how miRNAs regulate AhCASPs genes, we identified 20 different miRNAs for 37 different AhCASPs genes (Figure 7). The results showed that Ath-miR172d-5p targeted the most genes. Twelve miRNAs, including ath-miR2933a, ath-miR2933b, ath-miR861-3p, ath-miR163, ath-miR419, ath-miR426, ath-miR5012, ath-miR5021, ath-miR5027, ath-miR5641, ath-miR8184, ath-miR838, each targeted two different genes, respectively. Seven miRNAs including ath-miR418, ath-miR447c-3p, ath-miR5630a, and ath-miR5630b only regulate one gene, AhCASP52, AhCASP56, AhCASP32, AhCASP32, AhCASP6, AhCASP63, and AhCASP15. Seven genes, including AhCASP29, AhCASP56, and AhCASP32, were targeted by more than one miRNA.
In order to gain a deeper understanding of the molecular-level function of AhCASPs genes, we conducted a GO enrichment analysis encompassing 80 AhCASPs (Figure 8). GO enrichment analysis can be divided into three categories: molecular function (MF), cellular component (CC) and biological process (BP). At the molecular function level, obsolete cofactor binding (GO: 0048037) as well as iron–sulfur cluster binding (GO: 0051536), metal cluster binding (GO: 0051540), binding (GO: 0005488), small molecule binding (GO: 0036094) and other processes were significantly enriched. At the cellular component level, the highly enriched items were the cell periphery (GO: 0071944), plasma membrane (GO: 0005886), and membrane (GO: 0016020). At the level of biological processes, the highly enriched terms were cellular component organization (GO: 0016043), multicellular organismal process (GO: 0032501), cellular component organization or biogenesis (GO: 0071840), anatomical structure development (GO: 0048856), developmental process (GO: 0032502), etc.

2.7. Differential Expression Analysis of AhCASPs Genes

According to rigorous studies, it has been established that numerous plant hormones play a pivotal role in the response to abiotic stress. They serve as intermediaries in the plant’s adaptive response to such stress conditions, thereby safeguarding the plants from the adverse impacts of abiotic stress [53]. In the analysis of cis-acting elements, we found that the AhCASPs promoter region has many elements that respond to stress and various plant hormone signals, indicating that the AhCASPs gene is postulated to contribute significantly to plants’ stress regulation response mechanisms. In order to delve deeper into the functional role of AhCASPs family genes in adverse stress conditions, we conducted a comprehensive analysis of the transcriptional expression patterns of the AhCASPs gene family, utilizing transcriptome data derived from cultivated peanuts under low-temperature and drought stress, as well as exposure to various hormone treatments. In order to understand the expression changes of AhCASPs under different hormones, the FPKM value of transcriptome data was used to screen and construct the response heat map of 40 AhCASPs genes to five stress-related plant growth regulators (SA, paclobutrazol, ABA, brassinolide, and ethylene) (Figure 9a). After SA treatment, compared with the control group and other hormone treatments, the expression levels of AhCASP3, AhCASP25, AhCASP1, AhCASP42, AhCASP6, and AhCASP61 genes were significantly increased, and the expression levels of AhCASP66, AhCASP5, and AhCASP44 genes were down-regulated. The response of AhCASPs genes to paclobutrazol was divided into two types. In the first type, the expression levels of 16 genes were up-regulated, while in the second type, except for AhCASP28 and AhCASP10, the expression levels of other genes were down-regulated. Following ABA treatment, a comparative analysis was conducted against the control group and alternative hormone treatments, revealing distinct variations in the gene expression levels of the target subjects. AhCASP21, AhCASP64, AhCASP75, AhCASP26, AhCASP5, AhCASP44, and AhCASP15 were significantly up-regulated, and the expression levels of the other 33 genes were down-regulated. After brassinolide treatment, the expression levels of AhCASP15, AhCASP53, AhCASP12, and AhCASP49 genes were significantly up-regulated, and the expression levels of 36 genes such as AhCASP62, AhCASP75, AhCASP10, and AhCASP26 were down-regulated. Following ethylene treatment, in comparison to the control group and alternative hormonal interventions, the transcriptional levels of AhCASP73 and AhCASP7 genes were observed to increase, while the expression levels of the remaining 38 genes underwent a decrease.
To investigate the different expression patterns and tissue expression specificity of AhCASPs family genes, we used the FPKM value of transcriptome data to screen and analyze the expression levels of 63 AhCASPs genes in 13 organs of cultivated peanut (including pericarp, embryo, testa, florescence, gynophore, root, root tip, root nodule, root and stem, stem, stem tip, leaf, and cotyledon). We used TBtools-II software to draw an expression heat map of these organs (Figure 9b). The findings indicate that distinct expression patterns of various genes exist across different organs. Among them, AhCASP13 and AhCASP50 were highly expressed in the seed coat; the expression levels of AhCASP25, AhCASP39, and AhCASP53 were the highest in embryos. AhCASP73, AhCASP17, and AhCASP31 had higher expression levels in leaves. The expression levels of AhCASP6, AhCASP41, and AhCASP4 were higher in roots, flowering, and cotyledons, and the expression levels of these genes were lower in other organs. It is hypothesized that various AhCASPs genes may solely exhibit biological functions in designated organs. In addition, the response of different AhCASPs to low temperature and drought treatment was not the same. After low temperature treatment (Figure 9c), the expression levels of 21 genes, such as AhCASP15, were up-regulated, and 12 genes, such as AhCASP26, were down-regulated. The expression trend of 11 genes, such as AhCASP12, AhCASP49, AhCASP5, and AhCASP44, under drought treatment was notably elevated compared to the control group (normal irrigation conditions). The expression levels of AhCASP15, AhCASP53, AhCASP21, AhCASP64, AhCASP31, AhCASP3, and AhCASP42 were significantly down-regulated. Notably, the expression levels of AhCASP11, AhCASP55, AhCASP70, and AhCASP62 were significantly diminished under conditions of low temperature and drought, respectively. This observation suggests these four genes are likely unrelated to the adaptive response to this environmental stress but may play a key role in other biological processes.

3. Discussion

CASPs are membrane proteins that are exclusively found in the Casparian strip. This gene is important in regulating plant growth and development and stress response mechanisms, as it is a critical component in forming and controlling the Casparian strip [33,38]. Several plant species, including A. thaliana (39 members) [33], rice and cotton (19 and 48 members, respectively) [39,40], and patchouli (156 members) [42], have been identified as containing this gene family. However, as an important oil crop, the identification and analysis of the peanut AhCASPs gene family have not been reported. Moreover, they exhibit vulnerability to diverse abiotic stresses, encompassing phenomena such as hypothermic conditions and drought. Therefore, identifying and analyzing the response of peanut AhCASPs genes to various abiotic stresses is of great significance. In this study, a total of 80 AhCASPs genes were identified in the peanut genome and divided into 3 sub-clusters along with Arabidopsis CASPs genes (Figure 4). Compared with Arabidopsis and rice, the number of AhCASPs genes in peanuts increased significantly, which may be because the cultivated peanut is an allotetraploid (AABB type genome; 2n = 4x = 40) [54,55,56]. Several variables determine the number of gene family members in different species, including natural selection, genome doubling time, and tandem duplication [57]. Previous research has demonstrated that genes frequently endure tandem repeat events during evolution to enhance the number of gene family members [46]. Simultaneously, gene replication can induce functional differentiation among genes and expedite the emergence of new genes [58]. This study showed that 32 gene pairs in the peanut AhCASPs gene family had a Ka/Ks ratio < 1, suggesting that the AhCASPs gene may be retained to promote the amplification of CASPs genes in peanuts and may be subject to purification selection pressure. Unsurprisingly, the two genes in each pair are from the same subfamily, which strongly implies that CASPs genes are more conserved within the same subfamily.
Transcriptional regulation is the primary mechanism of gene expression regulation in eukaryotes. Cis elements are critical components in regulating gene transcription, and they play a critical role in various biological processes, including hormonal responses, abiotic stress reactions, and developmental trajectories [59]. The auxin-induced promoters are typically the source of the cis-acting elements that contain the AuxRE and DR5-motif [60]. Light-induced promoters act as an important regulatory sequence, usually containing core elements such as G-box, rich in AT, GT1-motif, and I-box [61]. Promoters that harbor CATGTG and CACG cis-acting elements are responsive to drought stress conditions [62]. In plants, IAA (indole-3-acetic acid), GA (gibberellic acid), SA (salicylic acid), ABA (abscisic acid), and MeJA (methyl jasmonate) are essential for the regulation of plant growth, ensuring the proper development and functioning of the plant’s various biological development, processes, and stress adaptation mechanisms [63,64]. The peanut genome was used to extract the promoter sequences of 2000 bp upstream of ATG of the 80 AhCASPs CDS, and their cis-acting elements were identified in this study. The cis-acting elements associated with stress and hormones were the primary focus of our analysis (Figure 5 and Figure 6). The findings indicated that the cis-acting elements associated with hormone response were ABA, GA, IAA, MeJA, and SA. Transcriptome data analysis showed that ABA and SA treatments had different induction effects on AhCASPs genes. Various hormones play significant roles in the regulatory mechanism of CASPs, especially ABA and MeJA hormones, which occupy more central and critical positions in the hormonal regulatory system [65]. Additionally, we have identified cis-acting elements that are associated with the stress response. These elements primarily respond to environmental stresses like drought and low temperature. The close proportion of cis-acting elements in different species and hypoxia suggests that families of CASPs may have a similar regulation pattern in peanuts. The CASPs family may be involved, collectively, in the control of several hormonal and stress responses to cis-acting elements that improve plant tolerance to the external environment, allow plants to react to biotic and abiotic stresses, and help to understand the complex mechanisms underlying cis-acting regulatory components in the stress response process.
It has previously been reported that miRNAs are responsive to non-biological stress [66], suggesting that these miRNA families hold significant potential in regulating biological processes associated with stress-related responses. Particular miRNA families are well-established in their roles in the developmental progression of plants and their adaptive responses to abiotic stresses [67,68]. Previous studies have documented the induction of miR861 in A. thaliana under high light conditions [69]. Furthermore, miRNAs such as ath-miR2933a, ath-miR2933b, and ath-miR5021 have been identified as key regulators in the calcium signaling pathway, mediating the regulation of calcium response genes during drought stress [70]. miR408 and ath-miR5632-5p are implicated in diverse abiotic stress responses, including drought [71,72]. Additionally, miRNA172, which inhibits the AP2 gene, has been reported to regulate flower development and flowering time across various plant species, including A. thaliana, barley, soybean, and rice [73,74,75,76]. In turmeric, miR5021 participates in the biosynthesis of terpenoids and isoquinoline alkaloids [77]. Our research has identified a total of 20 distinct miRNAs targeting 37 different AhCASPs genes (Figure 7). The identified differential expression patterns of AhCASPs genes have been observed to exhibit a correlation with stress-responsive miRNAs that specifically target these genes. This correlation underscores the significance of AhCASPs in the adaptation to stress conditions and plant developmental processes.
The stress and hormone transcriptional expression analysis indicated that the transcriptional expression patterns of AhCASPs under stress could be divided into high and medium–low expressions. Notably, the expression levels of AhCASP55, AhCASP73, AhCASP48, AhCASP31, AhCASP42, AhCASP3, and AhCASP41 under low-temperature stress and the expression levels of AhCASP3, AhCASP31, AhCASP73, AhCASP44, AhCASP62, AhCASP5, AhCASP49, and AhCASP12 under drought stress were high. These genes contained low-temperature- and drought-response elements (Figure 5). This indicates that these genes may respond to low temperature or drought stress. Previous studies have found that Arabidopsis AT3G55390 (AtCASPL4C1) is crucial in resisting low-temperature stress conditions [50]. Some members of banana MaCASPs are specifically expressed under low-temperature induction [41]. In addition, AhCASP76, AhCASP50, AhCASP54, and AhCASP76, AhCASP71, AhCASP50, AhCASP79, AhCASP1, AhCASP39, AhCASP69, AhCASP11, AhCASP53, and other genes were down-regulated under cold and drought stress, respectively. Therefore, how and what role different AhCASPs play in different stress conditions remains to be further verified.

4. Material and Methods

4.1. Identification of AhCASPs Genes

The Arabidopsis protein sequence and genome files were obtained from the TAIR website (https://www.arabidopsis.org/ accessed on 2 March 2024), and the peanut genome and gene annotation files were downloaded from the peanut website (http://peanutgr.fafu.edu.cn/ accessed on 2 March 2024). Two distinct methodologies were employed to search for and identify members of the peanut AhCASPs family. Firstly, 39 protein sequences downloaded from the Arabidopsis website were used as reference sequences for blast alignment [78] to obtain candidate AhCASPs family members. Subsequently, a search was conducted on the Pfam website [79] for the Pfam identifier (PF04535) on the AhCASPs family domain, and the respective Hidden Markov Model (HMM) was then procured for further analysis. The Simple HMM search online program [80] was utilized to initially screen protein sequences analogous to the hidden Markov model domain, applying a stringent e-value threshold of less than 10−5 to identify potential candidate members. They were submitted to CDD (https://www.ncbi.nlm.nih.gov/CDD accessed on 13 March 2024) and SMART (http://smart.embl.de/ accessed on 8 March 2024); to ensure the accuracy of the domain, it is necessary to verify it and subsequently eliminate any sequences that do not include the specified domain. The ProtParam (https://web.expasy.org/protparam/ accessed on 22 March 2024) online program [81] was employed to forecast its physicochemical properties, including relative molecular weight (Mw), amino acid number, hydrophilic large average (GRAVY) and isoelectric point (pI). The Plant-mPLoc 2.0 online software (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 11 April 2024) was used to predict the in silico analysis of AhCASPs [82].

4.2. Chromosome Distribution, Collinearity, and Ka/Ks Analysis

The Ka/Ks values for both repetitive genes and tandem repeats were precisely computed using the KaKs_Calculator 2.0 online program [83]. The peanut’s differentiation time (Mya) was calculated using the formula T = Ks/2r. Based on prior research, the value of r, representing the neutral substitution rate, was established at 8.5 × 10−9 [55].

4.3. Conserved Motifs, Domains, and Gene Structure Analysis

The positional data on exons, introns, and untranslated regions (UTRs) of the AhCASPs family genes were retrieved from the peanut genome annotation file (gff), and the structural representation of AhCASPs genes was subsequently generated utilizing TBtools-II software (version 2.085). The conserved motifs of AhCASPs were analyzed using MEME online (http://meme-suite.org/, accessed on 12 April 2024). The maximal number of motifs is 10, and the length ranges from 6 to 50 amino acids [84]. The conserved domains were inputted into the AhCASPs protein sequence using the online tool Batch CD-Search to generate the corresponding domain files [85]. Visual analysis was conducted using TBtools-II [86].

4.4. Phylogenetic Relationship of Peanut AhCASPs

MEGA 11 software was employed to conduct multiple sequence alignments on the full-length Arabidopsis and peanut CASPs sequences to investigate the phylogenetic relationship of peanut AhCASPs family members [87]. The NJ method was employed to generate the phylogenetic tree, which was then used to create the comparison results. The phylogenetic tree’s bootstrap method was configured to 1000, and all other parameters were set to their default values. Ultimately, the tree was colored using iTOL (https://itol.embl.de/, accessed on 24 April 2024) [88].

4.5. Prediction of Cis-Acting Elements of AhCASPs Promoter

The 2000 base pair region upstream of the AhCASPs start codon (ATG) was successfully extracted, and the cis-acting elements in this promoter were predicted and analyzed using PlantCARE software (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 14 April 2024) [89]. In addition, cis-acting elements were visualized using the TBtools-II software.

4.6. miRNA Target Gene Prediction and GO Enrichment Analysis

We used the eggNOG-mapper online tool (http://eggnog-mapper.embl.de/, accessed on 8 April 2024) to perform a detailed functional annotation of the peanut genome file [90]. This analysis method can effectively reveal the molecular function of genes, biological processes, and their complex association with cellular components. In addition, we also used GO enrichment analysis to accurately label related metabolic pathways, thus providing a crucial reference for deepening our comprehension of these genes’ role in specific metabolic processes. By combining the comprehensive analysis of eggNOG-mapper and TBtools-II, we can more fully understand the functional characteristics of the identified genes and their specific roles in the metabolic process.
In order to identify miRNAs that target the transcripts of AhCASPs family members, the coding sequence (CDS) was submitted to psRNATarget (https://www.zhaolab.org/psRNATarget/, accessed on 24 April 2024) to set the following parameters: (1) the expected value was 3; (2) Max UPE was 25 and flank length upstream was 17 NT; and the downstream was 13 NT to find the target of miRNA and analyze the targeting relationship of miRNA-AhCASPs [91]. Finally, the Cytoscape software (version 3.10.2) was used to visualize the target relationship network [92].

4.7. Differential Expression Analysis of AhCASPs Genes

Transcriptome data (http://peanutgr.fafu.edu.cn accessed on 10 May 2024) were used to extract the expression data of all AhCASPs in different stress conditions (blank, drought, and low temperature), different plant growth regulators (ABA, brassinolide, ethephon, paclobutrazol, and SA) and different tissues and organs (root, stem, leaf, peel, embryo, and other 13 organs) [93]. Transcriptome data of AhCASPs genes were screened from the above data, and preliminary screening of these data was performed using Excel 2019. Then, TBtools-II was used to reprocess the data and make heat maps for visualization.

5. Conclusions

Peanuts are an important economic and oil crop, providing oil and protein for human nutrition. During the growth phase, peanut production is subject to various adverse stress conditions, including extreme temperatures, elevated salinity, and drought. Many peanut gene families have been reported with the establishment of the peanut genome database, but our understanding of the peanut AhCASPs gene family is minimal. Based on their phylogenetic tree connection, the 80 peanut AhCASPs genes were identified in this research and grouped into three subclusters. Sequence analysis showed that most AhCASPs proteins contained conserved MAVEL and DUF588 domains. Apart from AhCASP2/43, AhCASP29/71, and AhCASP30/72, of the 32 pairings of homologous gene pairs found, 93.75% (30/32) of the other pairs had Ka/Ks values < 1, suggesting that purification selection pressure was acting on the AhCASPs genes in these groups. The analysis of cis-acting elements revealed that 80 AhCASPs genes harbored many hormone-regulatory, light-responsive, and environmental-stress-associated elements. Thus, to explore the many functions of the members of the AhCASPs family in peanuts, future studies should concentrate on the functional identification of AhCASPs genes. The findings of this work provide significant new information on the evolution of the AhCASPs gene family in peanuts and provide the theoretical groundwork for further research on the stress-responsive function of AhCASPs and molecular processes of CASPs genes in other species.

Author Contributions

Conceptualization, Y.S., J.F. and Y.L.; methodology, Y.S., J.F. and M.Z.U.H.; software, Y.S. and J.F.; validation, Y.S., J.F., Y.W. and D.Y.; formal analysis, Y.S., J.F., Y.L. and J.Y.; investigation, Y.S., J.F., M.Z.U.H., J.Y. and Y.L.; resources, Y.S., J.F., Y.L., M.Z.U.H. and Y.W.; data curation, Y.S., J.F. and M.Z.U.H.; visualization, Y.S., Y.W. and J.Y.; writing—original draft preparation, Y.S., J.F. and Y.L.; writing—review and editing, Y.S., J.F., Y.L., D.Y., M.Z.U.H., W.Y., J.Y. and Y.W.; supervision, Y.L.; project administration, Y.L. and Y.W.; funding acquisition, Y.L. and Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by Hainan Provincial Natural Science Foundation of China (No. 824RC502); Startup Funding from Hainan University (No. KYQD(ZR)23018); the Foundation of Key Laboratory of Tropical Crops Nutrition of Hainan province, Zhanjiang, China (No. 23KLTCN01); and the Technology Service Agreement of Hainan University (No. HD-KYH-2024020).

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosomal localization of peanut AhCASPs family genes. The scale provided represents the chromosome size (Mbp); Chr01–Chr20 represents the name of 20 chromosomes of the peanut genome. The blue and red colors represent low and high gene densities, respectively.
Figure 1. Chromosomal localization of peanut AhCASPs family genes. The scale provided represents the chromosome size (Mbp); Chr01–Chr20 represents the name of 20 chromosomes of the peanut genome. The blue and red colors represent low and high gene densities, respectively.
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Figure 2. Interspecific collinearity analysis of peanut, Arabidopsis, and rice. A grey line represents the collinear block, while a red line highlights the CASPs homologous gene pairs.
Figure 2. Interspecific collinearity analysis of peanut, Arabidopsis, and rice. A grey line represents the collinear block, while a red line highlights the CASPs homologous gene pairs.
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Figure 3. Analysis of conserved motifs, gene domains, and gene structure of peanut AhCASPs. Conserved motifs of AhCASPs (a); the color boxes represent different conserved motifs, as shown in the scheme on the right side of the figure. Conserved domain of AhCASPs (b): The exon–intron structure of AhCASPs (c), UTR, and CDS represent untranslated regions and coding sequences, respectively.
Figure 3. Analysis of conserved motifs, gene domains, and gene structure of peanut AhCASPs. Conserved motifs of AhCASPs (a); the color boxes represent different conserved motifs, as shown in the scheme on the right side of the figure. Conserved domain of AhCASPs (b): The exon–intron structure of AhCASPs (c), UTR, and CDS represent untranslated regions and coding sequences, respectively.
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Figure 4. Phylogenetic tree analysis of CASPs genes in peanut and Arabidopsis. The red dotted line was 39 CASPs proteins in A. thaliana; different color branches represent different subclusters.
Figure 4. Phylogenetic tree analysis of CASPs genes in peanut and Arabidopsis. The red dotted line was 39 CASPs proteins in A. thaliana; different color branches represent different subclusters.
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Figure 5. Cis-acting elements of peanut AhCASPs family members. The upstream sequence of AhCASPs gene promoter is 2000 bp and encompasses a diverse array of cis-acting elements, encompassing elements responsive to light, hormones, drought conditions, low temperatures, anaerobic environments, and wounding, in addition to specific elements associated with meristems, seeds, and endosperms.
Figure 5. Cis-acting elements of peanut AhCASPs family members. The upstream sequence of AhCASPs gene promoter is 2000 bp and encompasses a diverse array of cis-acting elements, encompassing elements responsive to light, hormones, drought conditions, low temperatures, anaerobic environments, and wounding, in addition to specific elements associated with meristems, seeds, and endosperms.
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Figure 6. Regarding the cis-regulatory elements in the AhCASPs promoter region, detailed statistical and analytical results are as follows: (a,c,e) the total number of AhCASPs genes involved in abiotic stresses, phytohormones, and cis-element growth and developmental categories has been counted. Specifically, the (b,d,f) pie charts show the percentage proportion of several cis-elements in each category, including (b) abiotic stress response, (d) phytohormone response, and (f) plant growth and development response. Different colors represent different cis-acting elements and their proportions in the AhCASPs gene.
Figure 6. Regarding the cis-regulatory elements in the AhCASPs promoter region, detailed statistical and analytical results are as follows: (a,c,e) the total number of AhCASPs genes involved in abiotic stresses, phytohormones, and cis-element growth and developmental categories has been counted. Specifically, the (b,d,f) pie charts show the percentage proportion of several cis-elements in each category, including (b) abiotic stress response, (d) phytohormone response, and (f) plant growth and development response. Different colors represent different cis-acting elements and their proportions in the AhCASPs gene.
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Figure 7. Predicted miRNAs that may target AhCASPs. The pink shape corresponds to the AhCASPs gene, and the blue represents the indicated miRNA.
Figure 7. Predicted miRNAs that may target AhCASPs. The pink shape corresponds to the AhCASPs gene, and the blue represents the indicated miRNA.
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Figure 8. Highly enriched GO annotations in MF, CC, and BP classifications of AhCASPs genes.
Figure 8. Highly enriched GO annotations in MF, CC, and BP classifications of AhCASPs genes.
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Figure 9. Heatmap of expression patterns of peanut AhCASPs based on FPKM values for different hormones (a), different organs (b), and different treatments (c). TBtools was used to perform log2 transformation and visualization of FPKM values. The upper right scale represents the level of gene expression.
Figure 9. Heatmap of expression patterns of peanut AhCASPs based on FPKM values for different hormones (a), different organs (b), and different treatments (c). TBtools was used to perform log2 transformation and visualization of FPKM values. The upper right scale represents the level of gene expression.
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Table 1. Physicochemical properties and in silico analysis of peanut AhCASPs.
Table 1. Physicochemical properties and in silico analysis of peanut AhCASPs.
Gene IDGene NameLengthMolecular Weight (Da)pIInstability IndexAliphatic IndexGRAVYSubcellular
AH01G00870.1AhCASP115817,410.6405.03032.870108.0400.946Cell membrane
AH01G22790.1AhCASP220022,455.8809.51034.390101.0000.495Chloroplast
AH01G27590.1AhCASP318419,521.2109.86030.550125.0000.726Chloroplast
AH01G27600.1AhCASP419220,653.2509.55029.280114.7400.617Cell membrane
AH02G02150.1AhCASP517018,992.3108.24033.890110.1200.680Cell membrane
AH02G14400.1AhCASP612914,159.91010.53032.350123.8000.561Peroxisome
AH02G19940.1AhCASP715517,076.3609.25042.09099.4800.751Cell membrane
AH02G23610.1AhCASP817419,302.0906.72025.71092.5900.217Cell membrane/Chloroplast/Peroxisome
AH03G00710.1AhCASP912813,763.5206.69053.33089.920−0.098Cell membrane
AH03G01440.1AhCASP1019920,893.6805.80045.11089.7500.261Cell membrane
AH03G01480.1AhCASP1112813,763.5206.69053.33089.920−0.098Cell membrane
AH03G04600.1AhCASP1215216,577.6606.68029.900120.5900.968Cell membrane
AH03G14800.1AhCASP1316618,174.9009.61021.420127.5300.887Cell membrane/Nucleus
AH03G17620.1AhCASP1422223,645.6409.02033.690101.5800.539Cell membrane
AH03G30890.1AhCASP1520522,831.8808.09028.81088.5400.477Cell membrane
AH03G36880.1AhCASP1681089,355.4508.83042.510100.3000.272Chloroplast
AH03G39480.1AhCASP1720922,590.1607.57048.11092.0600.412Cell membrane
AH03G42570.1AhCASP1818720,528.56010.15043.820115.2400.811Chloroplast
AH03G44420.1AhCASP1919120,647.4809.61024.700111.3600.536Chloroplast/Nucleus/Peroxisome
AH04G05340.1AhCASP2020121,647.6609.24026.370112.4400.674Cell membrane/Chloroplast/Golgi apparatus/Peroxisome
AH05G06060.1AhCASP2115316,646.6604.46029.570110.3301.061Cell membrane
AH05G18110.1AhCASP2218419,606.3209.37026.220129.4600.930Chloroplast/Golgi apparatus
AH05G29740.1AhCASP2317518,952.2409.55028.090115.2600.704Chloroplast
AH05G30620.1AhCASP2427329,920.4808.79047.58089.300−0.052Chloroplast/Nucleus
AH05G31520.1AhCASP2525828,998.7807.71063.51066.940−0.307Nucleus
AH05G37160.1AhCASP2618019,143.0407.71046.22089.0600.441Cell membrane
AH06G18360.1AhCASP2723426,676.0709.91028.460111.2000.496Chloroplast
AH06G24120.1AhCASP2819421,650.2308.35045.760100.0500.537Cell membrane
AH06G25820.1AhCASP2920022,063.6609.39045.660100.6000.149Nucleus
AH06G26150.1AhCASP3037040,467.5507.75061.55072.190−0.324Nucleus
AH07G11350.1AhCASP3121122,581.9105.88024.94087.0100.363Cell membrane
AH08G15760.1AhCASP3222223,721.7608.91034.200100.2700.586Cell membrane/Nucleus
AH08G17980.1AhCASP3320522,704.0308.98057.46075.120−0.439Nucleus
AH09G03020.1AhCASP3420522,248.6909.58049.21093.8000.290Nucleus
AH09G11490.1AhCASP3514515,417.4109.87026.260126.9700.806Cell membrane/Chloroplast/Golgi apparatus/Peroxisome
AH09G21610.1AhCASP3615216,448.4607.75032.140115.6600.892Cell membrane
AH09G31200.1AhCASP3723425,999.4606.78040.50084.6200.213Cell membrane
AH10G25080.1AhCASP3819220,478.1909.28021.540123.9100.748Cell membrane
AH11G08640.1AhCASP3915817,376.6205.03034.090110.5100.953Cell membrane
AH11G21400.1AhCASP4028931,997.8607.16060.85064.500−0.383Nucleus
AH11G30050.1AhCASP4119220,653.2509.55029.280114.7400.617Cell membrane
AH11G30070.1AhCASP4218319,441.1209.86033.650124.1000.745Cell membrane/Chloroplast/Golgi apparatus/Nucleus/Peroxisome
AH11G35090.1AhCASP4318520,578.6509.49030.560106.0000.487Chloroplast/Nucleus
AH12G02290.1AhCASP4417018,992.3108.24033.890110.1200.680Cell membrane
AH12G22250.1AhCASP4515517,076.3609.25042.09099.4800.751Cell membrane
AH12G35210.1AhCASP4619721,076.5306.39020.650113.4000.674Cell membrane
AH12G35220.1AhCASP4719720,858.5308.62032.210114.0100.827Cell membrane
AH13G03220.1AhCASP4819520,666.4605.40043.30091.5900.274Cell membrane
AH13G06630.1AhCASP4915616,936.8205.61037.610110.5800.855Cell membrane
AH13G17400.1AhCASP5016618,128.8109.61020.910128.6700.887Cell membrane
AH13G20190.1AhCASP5122223,717.8009.04037.090103.7400.584Cell membrane
AH13G29210.1AhCASP5220822,435.4909.60030.120113.5600.447Peroxisome
AH13G34990.1AhCASP5322224,705.1008.09034.41090.5400.441Cell membrane
AH13G40110.1AhCASP5416117,258.6709.64033.040138.1400.988Cell membrane/Nucleus
AH13G42370.1AhCASP5520922,589.1307.57046.28091.5800.401Cell membrane
AH13G45190.1AhCASP5618720,521.58010.15038.000114.1700.831Chloroplast
AH13G47090.1AhCASP5719120,643.4809.61022.840113.9300.548Chloroplast/Nucleus/Peroxisome
AH14G06350.1AhCASP5823326,029.9409.63033.82085.2800.194Cell membrane/Cell wall/Chloroplast/Mitochondrion/Nucleus/Peroxisome
AH14G06890.1AhCASP5920121,680.6509.24025.410109.5500.644Cell membrane/Chloroplast/Golgi apparatus/Peroxisome
AH14G18560.1AhCASP6012413,547.1709.37022.580128.7900.841Cell membrane
AH14G22800.1AhCASP6129232,598.0409.51048.55073.420−0.216Cell membrane/Mitochondrion/Nucleus
AH14G32740.1AhCASP6212813,859.6409.23014.940137.7300.930Cell membrane/Chloroplast
AH14G40830.1AhCASP6317118,650.6306.26025.350105.5000.645Cell membrane
AH15G02130.1AhCASP6415316,630.6604.46029.570110.9801.078Cell membrane
AH15G14530.1AhCASP65727734.1408.34036.500105.8300.590Cell membrane/Golgi apparatus
AH15G21900.1AhCASP6625828,924.6807.71062.47067.710−0.304Nucleus
AH15G23250.1AhCASP6727530,269.9108.90046.84087.240−0.077Chloroplast
AH15G24290.1AhCASP6817518,998.3309.55031.870114.1700.699Chloroplast
AH16G06190.1AhCASP6920222,036.0109.27035.040108.5600.648Chloroplast
AH16G29800.1AhCASP7019421,654.2408.35045.61097.5300.512Cell membrane
AH16G32140.1AhCASP7120022,098.5108.52046.37098.1500.111Nucleus
AH16G32610.1AhCASP7237340,707.7307.13060.93071.610−0.351Nucleus
AH17G10700.1AhCASP7321122,581.9105.88024.94087.0100.363Cell membrane
AH18G06070.1AhCASP7422223,731.8008.91033.460100.7200.586Nucleus
AH19G00710.1AhCASP7518019,143.0407.71046.22089.0600.441Cell membrane
AH19G04550.1AhCASP7620522,248.6909.58049.21093.8000.290Nucleus
AH19G27380.1AhCASP7715216,383.3406.80031.650113.0900.912Cell membrane
AH19G31300.1AhCASP78758172.8009.70025.450123.3300.681Cell membrane/Chloroplast/Peroxisome
AH19G36900.1AhCASP7919521,762.8508.93042.00090.0500.251Cell membrane
AH20G32430.1AhCASP8019120,292.9209.30027.180122.5100.721Cell membrane
Note. Length: length of amino acid (aa); pI: isoelectric point; GRAVY: grand average of hydropathicity.
Table 2. Non-synonymous (Ka) and synonymous (Ks) substitution rates of homologous AhCASPs gene pairs.
Table 2. Non-synonymous (Ka) and synonymous (Ks) substitution rates of homologous AhCASPs gene pairs.
Gene 1Gene 2KaKsKa/KsDivergence Time (Mya)
AhCASP1AhCASP390.00280.04450.06282.62
AhCASP2AhCASP430.00950.00761.25810.45
AhCASP3AhCASP420.00740.04360.16952.56
AhCASP1AhCASP640.11171.08430.103063.78
AhCASP3AhCASP690.49462.58090.1917151.82
AhCASP17AhCASP310.26800.94840.282655.79
AhCASP16AhCASP540.07940.17200.461610.12
AhCASP17AhCASP550.00870.04450.19432.62
AhCASP18AhCASP560.01670.04510.36992.65
AhCASP13AhCASP500.00530.03370.15811.98
AhCASP14AhCASP510.03260.04380.74522.58
AhCASP12AhCASP490.10790.18750.575311.03
AhCASP10AhCASP480.00230.06680.03383.93
AhCASP17AhCASP730.26790.90530.295953.25
AhCASP20AhCASP590.00660.02840.23051.67
AhCASP21AhCASP390.11171.08430.103063.78
AhCASP25AhCASP400.28721.12720.254866.31
AhCASP21AhCASP640.00290.02690.10801.58
AhCASP22AhCASP650.01860.07990.23274.70
AhCASP23AhCASP680.01030.04680.21932.75
AhCASP24AhCASP670.02770.07000.39614.12
AhCASP25AhCASP660.00510.03940.12902.32
AhCASP29AhCASP710.01100.00701.58320.41
AhCASP30AhCASP720.00840.00751.11920.44
AhCASP31AhCASP550.26290.92120.285454.19
AhCASP32AhCASP740.01000.01890.52821.11
AhCASP36AhCASP770.00580.03700.15742.18
AhCASP37AhCASP790.00460.04870.09442.86
AhCASP38AhCASP800.01910.03420.56052.01
AhCASP40AhCASP660.28431.10550.257165.03
AhCASP39AhCASP640.11511.01310.113659.59
AhCASP55AhCASP730.26280.87940.298851.73
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MDPI and ACS Style

Su, Y.; Fang, J.; Zeeshan Ul Haq, M.; Yang, W.; Yu, J.; Yang, D.; Liu, Y.; Wu, Y. Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance. Plants 2024, 13, 2077. https://doi.org/10.3390/plants13152077

AMA Style

Su Y, Fang J, Zeeshan Ul Haq M, Yang W, Yu J, Yang D, Liu Y, Wu Y. Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance. Plants. 2024; 13(15):2077. https://doi.org/10.3390/plants13152077

Chicago/Turabian Style

Su, Yating, Jieyun Fang, Muhammad Zeeshan Ul Haq, Wanli Yang, Jing Yu, Dongmei Yang, Ya Liu, and Yougen Wu. 2024. "Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance" Plants 13, no. 15: 2077. https://doi.org/10.3390/plants13152077

APA Style

Su, Y., Fang, J., Zeeshan Ul Haq, M., Yang, W., Yu, J., Yang, D., Liu, Y., & Wu, Y. (2024). Genome-Wide Identification and Expression Analysis of the Casparian Strip Membrane Domain Protein-like Gene Family in Peanut (Arachis hypogea L.) Revealed Its Crucial Role in Growth and Multiple Stress Tolerance. Plants, 13(15), 2077. https://doi.org/10.3390/plants13152077

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