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Article

Genome-Wide Identification and Expression Analysis of ADK Gene Family Members in Cotton under Abiotic Stress

by
Peijun Huang
1,2,
Ziwei Lin
2,
Yuzhi Zhang
2,
Yu Gao
2,
Songjuan Tan
2,
Shuai Wang
2,
Xiaoyu Cao
2,
Hongyan Shi
2,
Chao Sun
1,
Jiangping Bai
1,* and
Xiongfeng Ma
1,2,3,4,*
1
College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China
2
State Key Laboratory of Cotton Bio-Breeding and Integrated Utilization, Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, China
3
Western Agricultural Research Center, Chinese Academy of Agricultural Sciences, Changji 831100, China
4
College of Mechanical and Electrical Engineering, Shihezi University, Shihezi 518000, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(14), 7821; https://doi.org/10.3390/ijms25147821
Submission received: 4 June 2024 / Revised: 11 July 2024 / Accepted: 12 July 2024 / Published: 17 July 2024
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

:
Adenosine kinase (ADK) is a key enzyme widely distributed in plants, playing an important role in maintaining cellular energy homeostasis and regulating plant growth, development, and responses to environmental stresses. However, research on ADK genes in cotton (Gossypium hirsutum), an economically significant crop, has been limited. This study identified 92 ADK genes from four cotton species (G. arboreum, G. raimondii, G. hirsutum, and G. barbadense) using HMMER and Local BLASTP methods and classified them into six groups. Chromosomal localization revealed a random distribution of ADK genes in G. hirsutum, with 13 genes located on the At subgenome and 14 genes on the Dt subgenome. Gene structure analysis showed consistency in exon–intron organization within subgroups, while conserved motif analysis identified subgroup-specific motifs, indicating functional diversity. Synteny and collinearity mapping analysis revealed that the primary expansion mechanisms of the ADK gene family in cotton are polyploidy and segmental duplication. Cis-regulatory elements in GhADK promoters were classified into light response, hormone response, developmental regulation, and stress response. We also analyzed the expression patterns of GhADK genes under a low temperature (4 °C) and drought conditions. Most GhADK genes responded to cold stress with different expression patterns, indicating their roles in rapid response and long-term cold adaptation. Under drought stress, expression patterns varied, with some genes showing sustained high expression levels. The qRT-PCR validation of transcriptomic data confirmed the stress-induced expression patterns of selected GhADK genes. Functional analysis through the VIGS silencing of GhADK25 demonstrated its importance in cold and drought stress responses, with silencing resulting in poor growth under stress, highlighting its significance in stress tolerance. This study provides a basis for further understanding the evolutionary relationships and functions of the cotton ADK gene family.

1. Introduction

Adenosine monophosphate (AMP), a precursor molecule in the nucleotide metabolism pool, is one of the main mononucleotides that constitute cellular RNA. The formation of AMP is usually accompanied by the release of energy within the organism [1]. The proportions of AMP, adenosine diphosphate (ADP), and adenosine triphosphate (ATP)—the energy molecule and precursor in the carbohydrate metabolism pool—determine the energy charge ratio and twice carbohydrate metabolism, which directly affects plant growth, development, and adaptation to adverse environmental conditions [2,3]. Adenylate kinase (ADK, EC 2.7.4.3) is a universally present and abundant monophosphate transferase found in almost all living organisms [4]. It catalyzes the reversible phosphorylation reaction between ATP and AMP (ATP + AMP ↔ 2ADP) and is considered a key enzyme in maintaining the balance of energy metabolism and various adenylate pools [5,6]. ADK usually consists of an AMP domain, an ATP domain, and a relatively conserved core domain (CORE) [7,8].
Studies have shown that ADK is highly conserved in both animals and plants, and its activity has been confirmed in plants such as Arabidopsis thaliana, Oryza sativa (rice), Zea mays (maize), Pisum sativum (pea), and Solanum tuberosum (potato). The subcellular localization of ADK is distributed in different organelles within cells, being reported in the cytoplasm, mitochondria, nucleus, and plastids [9,10,11]. For example, inhibiting StADK expression in potato plastids significantly increased the adenylate content and starch yield [12]. In Arabidopsis, T-DNA insertion mutants disrupting the ADK gene At2g37250 exhibited an increased amino acid content and enhanced root growth [13]. Another study indicated that the disruption of the ADK gene At5g47840 led to a loss of chloroplast integrity, causing albino seedlings from the early embryonic to seedling development stages [2]. Additionally, ADK3 interacts with the chloroplast glyceraldehyde-3-phosphate dehydrogenase, forming a stable complex in green Arabidopsis chloroplasts, which may be a potential mechanism for regulating the crucial ATP-NADPH ratio in the Calvin–Benson cycle [14].
ADK plays a regulatory role in plant growth and development and participates extensively in plant responses to abiotic stresses. For instance, when maize roots and stems were treated with solutions of different Ca2+/Na+ ratios, a significant relationship was found between the ADK content and salt stress response [15]. In tomato, gene microarray analysis showed that the expression of an ADK homolog (SGN-U214214) was suppressed in salt-treated tissues [16]. Furthermore, other microarray data indicated that the expression of the ADK gene (SGN-U232826) was induced by drought stress in drought-tolerant tomatoes [17]. Using pea seeds as a model, a study on the adenylate balance during seed dehydration and rehydration revealed that ADK activity plays a critical role in maintaining the adenylate balance during seed desiccation and maturation [18].
Upland cotton (Gossypium hirsutum), a species within the Malvaceae family, originated from South America. Archaeological evidence indicates that it was first discovered in ancient cultures of northern Chile around 3500 to 2500 BCE. In the 15th century, Europeans introduced it to Asia and Africa. Today, it stands as one of the world’s most significant crops and a critical economic crop in China [19]. Abiotic and biotic stresses are the significant challenges in crop production worldwide, and climate change will likely lead to more severe abiotic and biotic stress conditions [20]. Despite the recognized importance of ADK genes in regulating plant growth and stress resistance, research on the crucial fiber crop cotton (Gossypium hirsutum) is still insufficient [21]. This study identified ADK genes from the cotton genome using bioinformatics methods and analyzed their phylogenetic relationships, sequence characteristics, gene localization, chromosomal localization, evolutionary relationships, and cis-elements in promoters. Additionally, using existing transcriptome data, we analyzed the transcriptome of cotton under different abiotic stresses at various time points. We further studied the dynamic expression patterns of the ADK family in response to abiotic stresses (such as drought and cold) using qRT-PCR. The function of GhADK25 under drought stress was validated through virus-induced gene-silencing technology. These findings provide valuable information for the further exploration of the functions and regulatory mechanisms of the ADK family in cotton.
This study aims to deepen our understanding of the ADK gene family in cotton, including their evolutionary relationships, regulatory mechanisms, and responses under abiotic stress conditions. By elucidating these aspects, we aim to provide valuable insights for enhancing the stress tolerance of cotton crops, thus contributing to sustainable agriculture in the face of climate change.

2. Results

2.1. Identification and Sequence Analysis of Cotton ADK Genes

Combining the HMMER search and Local BLASTP methods, we totally identified 92 ADK genes in four cotton genomes. G. arboreum, G. raimondii, G. hirsutum, and G. barbadense were found to have 16, 14, 30, and 32 ADK proteins, respectively (Additional File S1: Table S1). G. barbadense and G. hirsutum harbored twice as many ADK proteins as G. arboretum and G. raimondii. We named the 30 G. hirsutum ADK proteins GhADK1–GhADK30 according to their locations on the chromosome. The physicochemical properties of all ADK members in G. hirsutum were summarized in Table 1, including the names and IDs of genes, the chromosomal and strand locations, the length of the CDS and amino acid sequence, the molecular weight (MW) and the isoelectric point (pI), and the prediction of subcellular localization. In detail, the GhADKs proteins ranged from 214 aa to 746 aa in length, and the average length was 301 aa, with the predicted molecular weights (MW) being from 24.07 to 83.78 kDa and the theoretical isoelectric points (pI) being from 6.15 to 9.68. The subcellular localization prediction results showed that the localization predictions of 11 ADK proteins were displayed in the cytoplasm, 8 ADK proteins were located in the mitochondria, 7 ADK proteins were located in chloroplast, and the rest were located in the extracellular (2) and membrane-bound chloroplast (2).

2.2. Phylogenetic Analysis and Classification of the GhADK Gene Family

To better understand the evolutionary relationship of GhADKs, we built a phylogenetic tree of the full-length sequences of 92 cotton ADK proteins, 7 Arabidopsis ADK proteins, 7 rice ADK proteins, 11 tomato ADK proteins, and 12 potato ADK proteins (Figure 1).
The phylogenetic analysis revealed that all the ADK proteins were clustered into six groups (Group I–Group VI). The species’ ADK genes were present in almost every clade. Therefore, the subgroups classification was considered more reliable. Group V contained the most ADK members, including ten, six, ten, and four ADK genes of G. hirsutum, G. arboreum, G. barbadense, and G. raimondii, respectively. As the second largest branch, Group II and Group III contained the same number of family members, with 24 members, including six GhADKs, three GaADKs, six GbADKs, and three GrADKs. Group I has 19 members, representing 14.7% of the total ADK genes, including four, two, four, and two ADK genes belonging to G. hirsutum, G. arboreum, G. barbadense, and G. raimondii, respectively. Group VI and Group IV occupied ten and eleven ADK members, including two GhADKs, one GaADKs, two GbADKs, and one GrADK. The increase rate of groups II, III, and V in GhADKs is relatively large compared with that in Arabidopsis and other selected species; the expansion of the three groups may have resulted from gene duplications. The phylogenetic analysis results indicated genetic differentiation between ADK genes in other species and upland cotton.

2.3. Chromosomal Mapping and Gene Duplication of the ADK Gene Family in G. hirsutum

To further explore the molecular mechanisms underlying the expansion in G. hirsutum, we first mapped all identified GhADKs onto the chromosomes (Figure 2).
As a result of the analysis, we found that 27 of the 30 ADK genes were distributed on the chromosomes randomly, with the remaining 3 ADK members located on the scaffolds, which are not shown in Figure 2. Moreover, 13 GhADKs were located at the At-subgenome and 14 GhADKs were located at the Dt-subgenome. Among them, ten chromosomes contained only one member, while four chromosomes contained two genes, and three chromosomes contained three genes. Interestingly, most ADKs were distributed on both ends of the chromosomes. We also found that the numbers of genes on some chromosomes were not the same, such as for A01 and D01, A04 and D04, A05 and D05, A08 and D08, and A10 and D10, which may be caused by the incomplete genome-sequencing or the gene being lost during evolution. In particular, we did not identify tandem duplication events (TEDs) in GhADKs, which also occurred in G. arboretum, G. raimondii, and G. barbadense.
To better analyze the evolutionary relationship of the ADK gene family in G. hirsutum, we chose G. hirsutum as the main species and other cotton species as the control. The syntenic and collinearity maps were conducted on ADK genes of four cotton species (Figure 3).
As the result shows, both allotetraploid cotton species, G. hirsutum and G. barbadense (AD genome), contained 24 pairs of segmental duplication genes, respectively. In the diploid ancestral cotton species, G. arboretum only contained five pairs of segmental duplication genes, while G. raimondii contained the fewest, with two pairs. The results of the collinearity analysis revealed that the main expansion mechanisms of the cotton species ADK gene family are polyploidy and segmental duplication events.
In addition, homology analyses of the ADKs between G. hirsutum, G. arboretum, G. raimondii, G. barbadense, Arabidopsis, Rice, Populus trichocarpa, and Glycine max were conducted. As shown in Figure 4 and Figure 5, the ADK genes in upland cotton had the most homologous gene pairs in G. barbadense (53 orthologous gene pairs), followed by G. arboretum (26 orthologous gene pairs) and G. raimondii (26 orthologous gene pairs). However, fewer homologous genepairs were observed between G. hirsutum and Arabidopsis (12 orthologous gene pairs), Rice (no orthologous gene pairs), Glycine max (19 orthologous gene pairs), and Populus trichocarpa (14 orthologous gene pairs).

2.4. Gene Structure Analysis and Conserved Motif Detection of Upland Cotton ADK Genes

The gene structure can provide valuable information regarding possible evolutionary relationships and gene functions. Therefore, the organization of exons and introns in all GhADK genes was investigated through phylogenetic analysis (Figure 6).
This is consistent with the result in Figure 1. In the GhADK gene family, the number of exons varied from 4 to 19. The result showed that the members were clustered in the same subgroups and generally shared highly similar exon–intron structures, including intron numbers and exon/intron lengths. We also found that the length of exon s in the same subgroup was identical, while the intron length varied a lot. Our results also showed that GhADK members shared a similar genetic structure with some differences; for example, in Group V, most members contained six exons, but GhADK30 contained eight exons, while GhADK8 contained five exons. This raises the possibility of a certain degree of functional diversity among the genes in this family.
To further characterize the structural diversity and determine the functions of GhADKs, the MEME suite software was used to identify 10 motifs among 30 members. As shown in Figure 5, motifs 1, 3, 5, and 7 were widely distributed in all GhADKs, which were annotated to encode the ADK domain based on a Pfamscan and SMART data search. However, some motifs were recognized to be specific to certain subgroups; for example, motif 6 was unique to Group V, motif 9 was unique to Group V and Group III, and motif 8 was only found in Groups I and II. Moreover, in Group V, GhADK8 and GhADK30 only contained five motifs, which were different from those of other members. Thus, we supported the idea that changes in gene function or errors in genome annotation may cause genes in the same family.
Substantially, genes in the same group shared a similar gene structure and motif organization, suggesting that these proteins may all have similar functions. However, differentiating group-specific gene structures and motifs would allow for functional specialization.

2.5. Analysis of Cis-Acting Elements of the G. hirsutum ADK Gene Promoter

Cis-acting regulatory elements play a key role in the regulation of gene transcription initiation through interacting with their corresponding trans-regulatory factors, especially encountering environmental biotic and abiotic factors. To further study the transcriptional regulation mechanism of the GhADK gene family, the cis-acting elements in the upstream 2000 bp promoter sequences of 30 GhADK genes were analyzed (Figure 7).
Generally, these identified cis-elements were divided into four functional categories: light response, hormone response, development regulation, and stress response. The detailed information of putative cis-acting elements in each GhADK is listed in Additional File S5 (in this study, we only selected the + chain for the statistic). Among them, the promoters of 30 ADK genes contained at least one light-responsive element (e.g., Box 4, G-box, and GATA-motif), especially the two most abundant type elements, Box 4 and G-box. The upstream regions of most ADK genes contained a phytohormone-related component, such as ABRE, ERE, and P-box. Furthermore, we also identified cis-elements involved in plant growth and development, such as CAT-box, dOCT, and GCN4_motif. In addition, the promoters of 30 ADK genes contained one or more stress-related elements; ARE involved in the regulation of gene expression in the absence of oxygen was the most abundant, followed by MBS (MYB binding site involved in drought-inducibility), LTR (involved in low-temperature responsiveness), and DRE involved in dehydration, low temperatures, salt stresses, and so on. These findings suggested that GhADKs may be in various stress responses, which can provide more helpful information in exploring the regulatory mechanisms of the G. hirsutum ADK gene family.

2.6. Expression Profiles of GhADK Genes under Cold Treatment and Drought Stress

Previous studies have shown that ADK genes regulate plant responses to abiotic stressors (drought and cold). To investigate the possible functions of the ADK gene family in G. hirsutum, the expression profiles of ADK genes exposed to low-temperature treatment (4 °C) and drought treatment were also examined based on transcriptome data. We analyzed the expression patterns of the upland cotton GhADKs gene after 0, 1, 3, 6, 12, and 24h of cold or drought treatment, respectively. GhADK6/22/23/24/29 could not be detected in the two stresses. We deducted these genes as pseudogenes, or they may be expressed only under particular conditions.
The results indicated that, under cold stress, the majority of the GhADK genes (25 out of 30) were consistently induced during the treatment (Figure 8a).
Among them, six genes were upregulated and nineteen genes were downregulated at 1 h, suggesting that these genes were regulated for a rapid response to cold stress. GhADK17/18/19/21/25/28 showed a down–up–downregulation trend under cold treatment. Two genes (GhADK18/28) reached their peak expression at 3 h, and GhADK1/GhADK20 reached their peak at 6 h, indicating that these genes may be cold acclimation genes. After 12 h of treatment, GhADK4 and GhADK19 were significantly down-regulated and GhADK13/16/25 were upregulated considerably, suggesting that these genes may be involved in a cascade of downstream signal pathways by interacting with other proteins. Also, GhADK4/8/21/30 were significantly downregulated and GhADK16/25 were significantly upregulated after 24 h of cold treatment; these genes could be crucial for long-term cold acclimation. In these cold-induced genes, GhADK19, GhADK20, and GhADK25 were highly expressed at every treatment point; contrarily, most genes had low expression at all times. These fundings suggest that this gene family may play a negative regulatory role in cold stress responses.
During drought stress, GhADK3 was significantly upregulated at the 1 h point and dramatically downregulated at 3/6/12/24h. GhADK8/15/21/27 were down-regulated at 6 h, while GhADK28 was significantly upregulated. Among the GhADKs, GhADK1/9/12/18/21/28/30 reached their peak expression at 12 h, and GhADK8/20/26 reached their peak expression at the 24 h time point. Four genes (GhADK19/20/25/28) were highly expressed at every moment; contrarily, most genes have been at relatively low expression levels. Combining the transcriptomic data under cold and drought conditions, we found that GhADK7/13 were upregulated and 12 GhADKs were downregulated at 1 h. Seven genes were upregulated and eighteen were downregulated at 3 h under cold and drought conditions. These results suggested that each ADK member had its unique inducible expression pattern and thus may have its own functional specificity in plant stress responses.

2.7. qRT-PCR Verification of Upland Cotton ADK Family Members

Transcriptome data analysis showed that most GhADK members exhibited different expression patterns under various stress treatments. To verify the reliability of the above results, several candidate genes with altered expression levels were randomly selected for qRT-PCR experiments. The treated cotton leaves were harvested at the indicated time points. Primers specific to GhADK were used, with the details listed in Supplementary Materials Table S3. The expression patterns of eight genes (GhADK1, GhADK4, GhADK11, GhADK17, GhADK20, GhADK21, GhADK25, and GhADK28) were examined after 0, 1, 3, 6, 12, and 24 h of cold or drought treatment (Figure 9).
GhADK1, GhADK4, GhADK17, GhADK21, GhADK25, and GhADK28 showed an upregulation trend under cold stress, with elevated expression levels in the later stages. GhADK1 was significantly upregulated at 3 h, indicating a rapid response to cold stress. GhADK20 and GhADK17 were significantly upregulated at 6 h. GhADK1 was significantly downregulated at 12 h. GhADK4, GhADK17, GhADK25, and GhADK28 had higher expression levels at 12 and 24 h, suggesting these genes may be crucial for long-term cold adaptation. Under drought stress, GhADK11 and GhADK20 were upregulated at 1 h and downregulated at 6, 12, and 24 h. Among the GhADKs, GhADK1, GhADK4, GhADK17, GhADK21, GhADK28, and GhADK25 reached their peak expression at 12 h.
Combining the transcriptome data under cold and drought conditions, we found that GhADK21 was upregulated at 1 h, while 5 GhADKs were downregulated at 1 h. At 12 h, four genes were upregulated and four genes were downregulated. These results suggest that each ADK member has its unique inducible expression pattern and may therefore have its own functional specificity in plant stress responses.

2.8. Functional Analysis of GhADK25 Silencing in Cotton under Drought and Cold Stress

Based on the results of qRT-PCR, a candidate gene GhADK25 was selected for VIGS gene silencing. Injection was performed on two cotyledons of the test variety TM-1 and after 7 days of treatment, as shown in cold (Figure 10a) and drought (Figure 11a).
The appearance of bleaching in the cotyledons indicated successful PDS silencing. Subsequently, samples were taken from the remaining silenced plants, with each sample subjected to qRT-PCR validation in triplicate and compared with control groups and untreated materials. The results revealed that the control group TRV:00 (referred to hereafter as the control group) exhibited significantly better leaf and plant growth under cold and drought stress compared to silenced plants (Figure 10c and Figure 11c). The qRT-PCR silencing efficiency detection results are shown in Figure 10d and Figure 11d. Compared to the control group, the expression level of GhADK25 in the silenced plants was significantly reduced, indicating that the viral injection caused the differential expression of GhADK25 in the silenced plants. In addition, the physiological and chemical characteristics of MDA (malondialdehyde) and T-AOC (total antioxidant capacity) showed significant differences between GhADK25-silenced plants and controls. Compared to the controls, the T-AOC in the silenced seedlings decreased, while the MDA increased. These results indicate that silencing GhADK25 compromises the plant’s ability to tolerate drought and cold stress (Figure 10b and Figure 11b).

3. Discussion

Cotton (Gossypium spp.) is one of the most important economic crops worldwide as a source of natural fibers, edible oil, and protein. With the continuous development of sequencing technology, the genomes of cotton species have been sequenced, already making the genome-wide identification and analysis of gene families in plants common. ADK proteins were reported to be involved in the regulation of plant growth and development and in responses to various environmental stresses in plants [13]. However, systematic studies of ADK genes in cotton species are lacking. Hence, our research conducted a comprehensive exploration of the ADK family in cotton, mainly focusing on the allotetraploid cotton G. hirsutum, to understand its roles, their evolutionary relationships, and its expression in response to abiotic stresses.

3.1. Systematical and Comprehensive Analyses of ADK Proteins

In the present study, we identified 16, 14, 30, and 32 ADK proteins in G. arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. Our results showed that the number of ADK proteins in tetraploid cotton is almost twice as much as that in diploid cotton, which indicates that allotetraploid cotton is produced via the natural hybridization of diploid cotton seeds containing the A genome and D genome [22]. The phylogenetic tree suggested that ADK proteins in cotton could be divided into six groups, similar to Arabidopsis and other plants. Each subgroup contains the ADK genes of four cotton species; the number of ADK proteins in some branches differed from that of different species. For example, group V included 10, 6, 10, and 4 ADK genes of G. hirsutum, G. arboreum, G. barbadense, and G. raimondii, respectively, but only 2 in arabidopsis, 3 in rice, 2 in potato, and 2 in tomato. This situation is also seen in other groups. ADK proteins were unevenly distributed on 16 chromosomes in upland cotton, but we also found that the number and location of genes on each chromosome were almost one to one. The gene’s structure and motif analyses indicated that the ADK genes in the same subgroup shared similar arrangements, increasing the gene diversity among different subgroups. Notably, in the same subgroup, GhADK8 and GhADK30 lacked motifs 2, 4, and 9, and GhADK4 and GhADK19 have a long intron, which made them different from other ADK gene members because of their large size, these gains or losses suggesting that these motifs and different structures might be involved in the functional divergence of GhADKs. The analysis of cis-acting elements of the G. hirsutum ADK gene promoter showed that the G. hirsutum ADK family may exert its biological functions mainly through different pathways.

3.2. Evolution and Expansion of the ADK Gene Family in Cotton

To date, ADK genes have been identified in various plants. The number of these ADKs in Gossypium was more significant than that in other species such as arabidopsis (7), rice (7), potato (12), and tomato (11) [10,13,23], indicating that more ADK proteins in cotton participate in the stress-resistant signaling pathway. Generally, the size of the gene number may be affected by two reasons: the genome sizes and the duplication event. For example, the upland cotton has a greater than 2.27 GB genome [24], while Arabidopsis only contains a 125 Mb genome, rice only contains a 386 Mb genome, tomato contains a less than 1.2 Gb genome, and so on [25,26,27]. Next, homologous exchanges were present in the upland cotton genome, and G. raimondii underwent a whole-genome duplication event about 13–20 million years ago [28].
Tandem duplication and segmental duplication events are the main driving forces of gene family expansion, which can help plants adapt to varying environmental conditions [29]. A total of 24 putative segmental gene pairs with 22 upland cotton ADK genes were identified in the whole genome. This finding suggests that more than half of the ADK genes in G. hirsutum may not originate from the same ancestor. We did not find a tandem duplication event in four cotton genomes. A similar phenomenon was observed in the tomato ADK gene family: it contained only three pairs of duplicated genes among a total of 11 tomato ADK genes [23]. According to these results, we supposed that the expansion of the ADK gene family may be mainly through the segmental supplication event. On the other hand, the number of ADK genes in the two tetraploid species was almost the sum of those in the two ancestral diploid progenitors, which provided evidence that polyploidy is another important factor. At the same time, we found that the ratio of ADKs in allotetraploid cotton and diploid cotton was less than 2:1, which may be the result of evolutionary selection in the process of hybridizing two diploid cotton plants to form allotetraploid cotton.
All the duplicated genes in cotton underwent the purifying selection, as indicated by the Ka/Ks ratio being <1, suggesting that all of those duplicated genes experienced a solid purifying selective pressure and subfunctionalized during evolution.

3.3. Gene Expression Profiles and Functional Divergence of ADKs in G. hirsutum

Expression analyses could provide insight into the potential functions of genes. In many plants, the ADK genes responded rapidly to biotic and abiotic stresses [15,18]. Hence, the expression pattern of GhADK genes under cold and drought conditions was performed using publicly available RNA-seq data. Interestingly, most duplicated genes showed different expressions between the duplicates. For example, GhADK11 and its paralog GhADK20, GhADK11 and its paralog GhADK9, and GhADK11 and its paralog GhADK25 displayed distinct expression patterns under cold stress. These findings indicate different roles of these genes under various stress. However, some duplicated genes still showed a similar expression between the duplicates. For example, GhADK9 and its paralog GhADK25, GhADK28 and its paralog GhADK14, and GhADK1 and its paralog GhADK17 showed the same expression patterns under cold stress. This is also the case under the drought treatment; for example, GhADK9 and its paralog GhADK25 and GhADK13 and its paralog GhADK27 showed the same expression patterns. These results suggest that they might have redundant roles. All these findings suggest that the duplicated genes might undergo different evolutionary pressures, which helped in analyzing the functional genes in upland cotton.
Also, we found that most GhADKs presented opposite expression patterns under cold and drought stress. For example, GhADK8, GhADK21, and GhADK26 were downregulated during cold stress but were upregulated under drought stress. GhADK27 and GhADK3 were upregulated during cold stress but were downregulated under drought stress. This phenomenon is similar to the expression pattern of ADK members in Medicago sativa. In tomato studies, it was shown that most SlADK genes were upregulated by drought induction [23]. In upland cotton, the situation was similar. So, the expression patterns of ADK genes show that there are differences between different species under abiotic stress, suggesting that positive and negative regulatory mechanisms may exist.

3.4. The Potential Roles of ADK Genes in Response to Cold and Drought Stress

In plants, adapting to abiotic stresses such as cold and drought is crucial for survival and productivity. Adenylate kinase (ADK) genes have been implicated in regulating plant responses to these stressors, particularly energy transfer and metabolic homeostasis. Under 4 °C treatment, GhADK25 was induced between 3 and 24 h (Figure 9b). Further functional analysis of GhADK25 using the VIGS method showed that under cold conditions, plants with silenced GhADK25 suffered more damage compared to control plants (Figure 10c). In our study, GhADK25 was also induced between 3 and 24 h after PEG 6000 treatment (Figure 9a). Further functional analysis using VIGS revealed that under drought conditions, plants with silenced GhADK25 suffered more damage than the control plants (Figure 11c). Understanding the potential roles of ADK genes in cold and drought stress tolerance can provide valuable insights into the molecular mechanisms underlying plant stress adaptation.
Previous studies, including those referenced in the material text, have demonstrated that ADK genes play a pivotal role in regulating the energy transfer rate and effectiveness from mitochondria to hexokinase in vitro [1]. This function is critical for maintaining metabolic homeostasis under stressful conditions, where energy production and utilization are often compromised. ADK enzymes catalyze the reversible phosphorylation of AMP and ADP, which is a crucial step in maintaining the adenylate pool and energy charge [30]. By modulating the levels of AMP, ADP, and ATP, ADK genes can influence the cell’s metabolic state and its ability to respond to stress.

4. Materials and Methods

4.1. Identification and Bioinformatics Analysis of the ADK Gene Family in Cotton

The genome datasets of Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense were downloaded from the COTTONGEN (https://www.cottongen.org/ (accessed on 3 April 2024)) [31] database. Two methods were used to identify cotton ADK family genes comprehensively. First, the ADK protein sequences of Arabidopsis, rice, and soybean were obtained from the Uniprot (https://www.uniprot.org/ (accessed on 3 April 2024)) [32] website and were used as query sequences to conduct a local BLASTP analysis against the cotton genomic database, with an e-value of 1 × 10−50 as the threshold. Second, the hidden Markov model (HMM) file corresponding to the ADK domain (PF00406) was downloaded from the Pfamscan database (https://www.ebi.ac.uk/Tools/pfa/pfamscan/ (accessed on 3 April 2024)) [33]; then, the HMM model was used as a probe to perform a BLASTP against the cotton genome database under the bio-Linux operating system, with the threshold expectation value set to 1 × 10−20. Then, the standard IDs of the genes obtained by the two methods were selected as the candidate genes. The conserved domains of the candidate sequences were annotated using the SMART (http://smart.embl-heidelberg.de/ (accessed on 3 April 2024)) [34], Pfamscan, and CDD Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 3 April 2024)) [35] for detecting the ADK domain. The biophysical properties of potential GhADKs, including the number of amino acids, molecular weights (MW), and isoelectric points (pI), were generated using ExPASy (http://web.expasy.org/protparam/ (accessed on 4 April 2024)) [36]. The subcellular localization of the ADK proteins were predicted via the online website Softberry (http://www.softberry.com/ (accessed on 3 April 2024)) [37].

4.2. Multiple Alignments and Phylogenetic Analysis

The ADK protein sequences of Arabidopsis, rice, soybean, Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense were used for multiple sequence alignment using MAFFT version 7 (https://mafft.cbrc.jp/alignment/software/ (accessed on 7 April 2024)) [38] with auto strategy parameters. An unrooted phylogenetic tree was generated through IQ-TREE version 1.6.2 [39] with the Maximum Likelihood (ML) method. Branch support for the tree topology was estimated by using a bootstrap analysis with 1000 replicates. The resulting phylogenetic tree was visualized using the iTOL (http://itol.embl.de/help.cgi (accessed on 10 April 2024)) [40] online tool.

4.3. Chromosomal Localization and Gene Duplication Analysis

The chromosomal distribution of GhADKs genes was visualized by the MapInspect version 1.0 (https://mapinspect.software.informer.com/ (accessed on 3 April 2024)) graphical tool according to the genomic coordinates retrieved from the Gossypium hirsutum genome database. As previously studies described, gene duplication events included tandem and segmental duplication. Two or more genes located on the same chromosome were arranged at a 200 kb distance and shared more than 70% of their identities, as analyzed with BLASTP, which can be defined as tandem duplications. The Multiple collinear scanning toolkits (MCScanX) [41] with default parameters were used to detect the segmental duplication patterns of the GhADKs. The Circos program was used to draw collinearity maps of the duplicated gene pairs between four cotton species as well as the synteny blocks of the orthologous ADK genes between Gossypium hirsutum and Arabidopsis, soybean, poplar, and rice. To further analyze the divergence of duplicated genes, the synonymous substitution rate (Ks) and non-synonymous substitution rate (Ka) were calculated using the TBTOOLS [42] software (V2.097). Based on a rate of λ (6.1 × 10−9) substitutions per site per year, the divergence time (T) was calculated using the Ks value and the following formula: T = Ks/(2 × 6.1 × 10−9)10−6 Mya.

4.4. Gene Structure Analysis and Conserved Motif Detection of the ADK Gene Family in Gossypium hirsutum

To further study the gene structures of GhADKs, the coding sequence (CDS) and gff3 format files of Gossypium hirsutum were downloaded from the cotton genome database. Subsequently, the diagrammatic sketch containing CDS, UTR, and Intron was utilized by TBtools software. To investigate the conserved motifs of ADK proteins, the complete amino acid sequences were analyzed using MEME (Multiple Expectation Maximization for Motif Elicitation) (http://meme.nbcr.net/meme/cgibin/meme.cg (accessed on 12 April 2024)) [43]. The parameters were shown follows: the number of repetitions was set to zero or one; the maximum number of motifs was set to 15; and other optional parameters were set to default.

4.5. Promoter Cis-Regulatory Analysis of the ADK Gene Family in Gossypium hirsutum

To further investigate the cis-elements in the promoter, we obtained the 2 kbp promoter sequences of GhADK genes from the genome annotation files. Then, the obtained sequences were subjected to analysis using the PlantCARE Database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 14 April 2024)) [44]. In this study, we only selected thosethat may be typical and functional cis-elements; the ubiquitous cis-acting elements in most genes were filtered out, such as CAAT-box, TATA-box, TATC-box, and so on. Also, Promoter sequences with three members (GhADK28, GhADK29, GhADK30) were not extracted and were therefore not analyzed.

4.6. Expression Analysis of GhADKs

The gene expression data of GhADKs under stress conditions such as cold, heat, drought, and salinity were obtained from unpublic transcriptome data. The transcript abundance was represented by fragments per kilobase of exon per million mapped reads (FPKM) values calculated based on RNA-Seq reads. To clearly see the difference in the expression level and more convenient statistics, the RPKM values were log2-transformed. The results were presented as heatmaps using TBTOOLS software (V2.097).

4.7. Plant Materials and Treatments, RNA Isolation, and Quantitative Real-Time PCR Analysis

Quantitative real-time PCR (qPCR) analysis: cDNA was synthesized from the isolated RNA using a reverse transcription kit. qPCR was performed using specific primers targeting the genes of interest and a SYBR Green-based master mix. The reaction mixtures were subjected to thermal cycling conditions, including an initial denaturation step, followed by a series of amplification cycles. The fluorescence signals were monitored during each cycle to quantify the accumulation of PCR products. The relative gene expression levels were calculated using the comparative Ct method, with normalization to a reference gene. Statistical analysis was performed to determine significant differences in gene expression between the treatment groups.

4.8. Virus-Induced Gene Silencing (VIGS) Treatment of ADK25 in Cotton Seedlings

Based on the gene silencing sequence design website (https://vigs.solgenomics.net/ (accessed on 3 April 2024) [22], a 300 bp specific coding sequence of ADK25 was designed for virus-induced gene silencing (VIGS) treatment. The primers used are listed in Additional File S7. The fragment was amplified and cloned into a tobacco rattle virus vector (pTRV2) using a ClonExpress® Ultra One Step Cloning Kit (C115, Vazyme, Nanjing, China). TRV2::ADK25 was transferred into the Agrobacterium tumefaciens strain LGV3101. TRV2::00 was used as a negative control. The Agrobacterium tumefaciens strains of the vector were mixed with the Agrobacterium tumefaciens strains containing pTRV1 at a ratio of 1:1. The mixture was injected into the cotyledons of cotton seedlings using a 1 mL needleless syringe. After 24 h in the dark, the plants were cultivated in a constant temperature light room (28 °C, 16 h/8 h, day/night).

4.9. Measurement of Physio-Biochemical Attributes

Ion permeability was determined using 10 rosette leaves of identical sizes. Before and after treatment, the malondialdehyde (MDA) and total antioxidant capacity (T-AOC) were measured according to the manufacturer’s instructions (Solarbio, Beijing, China).

5. Conclusions

This study systematically analyzed the ADK gene family in cotton (Gossypium hirsutum) using bioinformatics methods. The results revealed significant variations in the length and molecular weight of GhADKs proteins, with a diverse range of theoretical isoelectric points, highlighting their structural diversity and potential functional differences. Subcellular localization predictions indicated that ADK proteins predominantly localize to the cytoplasm, mitochondria, and chloroplasts, suggesting specific functions and regulatory roles in different cellular compartments. Further transcriptome analysis unveiled dynamic expression patterns of the ADK gene family in response to abiotic stresses such as drought and cold in cotton. Particularly, the functional validation of GhADK25 under drought stress using VIGS technology emphasized the crucial role of ADK genes in cotton’s response to environmental stresses. In summary, this study provides important foundational data and insights into the molecular mechanisms of ADK genes in cotton growth, development, and stress responses. Future research could explore the regulatory networks of the ADK gene family under different growth stages and environmental conditions, as well as their potential applications in breeding and improvement efforts.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25147821/s1.

Author Contributions

X.M., C.S. and J.B.: conceived and designed the experiments. P.H., Z.L., Y.Z. and Y.G.: performed the experiments and analyzed the data. P.H.: drafted the manuscript. S.T., S.W., X.C., H.S. and X.M.: revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Biological Breeding of Stress-Tolerant and High-Yield Cotton Varieties (2023ZD04040).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Material.

Acknowledgments

We sincerely thank Xiongfeng Ma (Cotton Research Institute) for his valuable advice and financial support of this research. To the entire research group, friends, and any other person who contributed, we are grateful for your help.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis of cotton species ADK proteins with A. thaliana, O. sativa, S. lycopersicum, and S. tuberosum. IQTREE was used to construct the phylogenetic tree using the maximum likelihood method with 1000 bootstrap replications. Each color indicates an individual group (I–VI).
Figure 1. Phylogenetic analysis of cotton species ADK proteins with A. thaliana, O. sativa, S. lycopersicum, and S. tuberosum. IQTREE was used to construct the phylogenetic tree using the maximum likelihood method with 1000 bootstrap replications. Each color indicates an individual group (I–VI).
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Figure 2. The chromosomal distribution of ADK genes in upland cotton (Gossypium hirsutum).
Figure 2. The chromosomal distribution of ADK genes in upland cotton (Gossypium hirsutum).
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Figure 3. Schematic representation of the duplication of ADK genes in the cotton genome. The gene duplication events from (a) G. arboretum, (b) G. barbadense, (c) G. hirsutum, and (d) G. raimondii were exhibited on their respective chromosomes. The number of each chromosome is indicated inside each bar. The scale on the box above is in megabases (Mb). ADK gene pairs with a syntenic relationship are linked by red lines.
Figure 3. Schematic representation of the duplication of ADK genes in the cotton genome. The gene duplication events from (a) G. arboretum, (b) G. barbadense, (c) G. hirsutum, and (d) G. raimondii were exhibited on their respective chromosomes. The number of each chromosome is indicated inside each bar. The scale on the box above is in megabases (Mb). ADK gene pairs with a syntenic relationship are linked by red lines.
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Figure 4. Synteny analyses of ADK genes between G. hirsutum and (a) A. thaliana; (b) O. sativa; (c) Glycine max; and (d) Populus trichocarpa.
Figure 4. Synteny analyses of ADK genes between G. hirsutum and (a) A. thaliana; (b) O. sativa; (c) Glycine max; and (d) Populus trichocarpa.
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Figure 5. Synteny analysis of ADK family genes in cotton. Orthologous relationships of ADK genes were investigated between (a) G. hirsutum and G. arboretum; (b) G. hirsutum and G. barbadense; and (c) G. hirsutum and G. raimondii.
Figure 5. Synteny analysis of ADK family genes in cotton. Orthologous relationships of ADK genes were investigated between (a) G. hirsutum and G. arboretum; (b) G. hirsutum and G. barbadense; and (c) G. hirsutum and G. raimondii.
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Figure 6. Conserved motifs and structure of ADK genes in upland cotton. Maximum likelihood phylogenetic tree of the GhADKs. The Conserved motifs in ADK genes were predicted, and the different colors represent different motifs. The intron–exon structures of the ADK genes were analyzed. The exons and introns were represented by yellow boxes and gray lines, respectively.
Figure 6. Conserved motifs and structure of ADK genes in upland cotton. Maximum likelihood phylogenetic tree of the GhADKs. The Conserved motifs in ADK genes were predicted, and the different colors represent different motifs. The intron–exon structures of the ADK genes were analyzed. The exons and introns were represented by yellow boxes and gray lines, respectively.
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Figure 7. The distribution of cis-acting elements in promoters of GhADKs. The corresponding number of them was indicated by the color scale.
Figure 7. The distribution of cis-acting elements in promoters of GhADKs. The corresponding number of them was indicated by the color scale.
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Figure 8. Expression analysis of GhADK genes in G. hirsutum under cold and drought stresses. The RNA-Seq expression profiles of G. hirsutum were used to identify the relative expression levels of GhADK genes. Levels of gene expression are depicted in different colors on the scale. cold (a) and drought (b) treatments, the red color represents a high expression and the green color represents a low expression. The detailed FPKM values are present in Additional File S6.
Figure 8. Expression analysis of GhADK genes in G. hirsutum under cold and drought stresses. The RNA-Seq expression profiles of G. hirsutum were used to identify the relative expression levels of GhADK genes. Levels of gene expression are depicted in different colors on the scale. cold (a) and drought (b) treatments, the red color represents a high expression and the green color represents a low expression. The detailed FPKM values are present in Additional File S6.
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Figure 9. Through qRT-PCR analysis, the expression profiles of GhADKs under various stress treatments were studied. Treated cotton leaves were harvested at the specified time points. To ensure that the expression of the same gene under different treatments as well as the expression of different genes under the same treatment can both be clearly displayed and compared for cold (a) and drought (b) treatments, the color scale represents the log2 mean value of relative expression levels from three independent biological replicates (n = 3).
Figure 9. Through qRT-PCR analysis, the expression profiles of GhADKs under various stress treatments were studied. Treated cotton leaves were harvested at the specified time points. To ensure that the expression of the same gene under different treatments as well as the expression of different genes under the same treatment can both be clearly displayed and compared for cold (a) and drought (b) treatments, the color scale represents the log2 mean value of relative expression levels from three independent biological replicates (n = 3).
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Figure 10. Regulation of ADK25 under cold stress. (a) Albino phenotype. (b) Effects of silencing ADK25 on the MDA content and T-AOC in cotton under cold stress. (c) Phenotype of plants; negative control (left) and silenced plant (right). (d) Silencing efficiency of ADK25 was detected by qRT-PCR. Asterisks represent differences by Student’s t-test: **, p < 0.01 (n = 3).
Figure 10. Regulation of ADK25 under cold stress. (a) Albino phenotype. (b) Effects of silencing ADK25 on the MDA content and T-AOC in cotton under cold stress. (c) Phenotype of plants; negative control (left) and silenced plant (right). (d) Silencing efficiency of ADK25 was detected by qRT-PCR. Asterisks represent differences by Student’s t-test: **, p < 0.01 (n = 3).
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Figure 11. Regulation of ADK25 under drought stress. (a) Albino phenotype. (b) Effects of silencing ADK25 on the MDA content and T-AOC in cotton under drought stress. (c) Phenotype of plants; negative control (left) and silenced plant (right). (d) Silencing efficiency of ADK25 was detected by qRT-PCR. Asterisks represent differences by Student’s t-test:; **, p < 0.01 (n = 3).
Figure 11. Regulation of ADK25 under drought stress. (a) Albino phenotype. (b) Effects of silencing ADK25 on the MDA content and T-AOC in cotton under drought stress. (c) Phenotype of plants; negative control (left) and silenced plant (right). (d) Silencing efficiency of ADK25 was detected by qRT-PCR. Asterisks represent differences by Student’s t-test:; **, p < 0.01 (n = 3).
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Table 1. The information of ADK gene family in G. hirsutum.
Table 1. The information of ADK gene family in G. hirsutum.
Gene IDGene nameChrStartEndPIMW (kDa)AA
GhADK1Gh_A05G2360A0528948631289532257.1432528.07296
GhADK2Gh_A05G2963A0573057726730590346.1529723.98265
GhADK3Gh_A05G3557A0591270041912713056.3725040.75222
GhADK4Gh_A06G0410A06688633769006418.5665724.32590
GhADK5Gh_A07G0462A07597259559749067.1130968.44273
GhADK6Gh_A07G0465A07599564159988946.9726899.23245
GhADK7Gh_A07G2086A0777433461774351106.1530273.43272
GhADK8Gh_A08G1415A0889538235895407468.524830.9230
GhADK9Gh_A09G2184A0974822698748256727.6426854.17244
GhADK10Gh_A10G1601A1086412249864155066.3732856.6303
GhADK11Gh_A12G1706A1278779245787815767.6926803.97244
GhADK12Gh_A12G1810A1280690562806936568.4926316.29236
GhADK13Gh_A13G0108A13126841912705127.0326556.53237
GhADK14Gh_D01G1585D0149745145497467506.9126725.94245
GhADK15Gh_D04G0048D047745227756767.624074.71214
GhADK16Gh_D04G0753D0415612652156139616.1529856.14267
GhADK17Gh_D05G2627D0527184500271891018.4132934.66299
GhADK18Gh_D06G0443D06637084463801208.9466355.03594
GhADK19Gh_D07G0526D07594836059592389.0833159.95290
GhADK20Gh_D07G0529D07597876059820186.5526957.26245
GhADK21Gh_D07G2304D0754335943543375966.5130197.38272
GhADK22Gh_D09G2390D0950793345507963398.2226720.04244
GhADK23Gh_D10G1856D1051921697519249676.2432658.46301
GhADK24Gh_D10G2125D1058219589582231128.3732771.56301
GhADK25Gh_D12G1868D1251286089512884258.2626892.19245
GhADK26Gh_D12G1980D1252858886528644098.5283784.67746
GhADK27Gh_D13G0125D13124613912482318.426423.47237
GhADK28Gh_A01G2126scaffold147_A011999052015126.9626852.05245
GhADK29Gh_A10G2351scaffold2716_A10591494258.3732710.48301
GhADK30Gh_A12G2555scaffold3185_A12118085711863239.6834073.49317
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MDPI and ACS Style

Huang, P.; Lin, Z.; Zhang, Y.; Gao, Y.; Tan, S.; Wang, S.; Cao, X.; Shi, H.; Sun, C.; Bai, J.; et al. Genome-Wide Identification and Expression Analysis of ADK Gene Family Members in Cotton under Abiotic Stress. Int. J. Mol. Sci. 2024, 25, 7821. https://doi.org/10.3390/ijms25147821

AMA Style

Huang P, Lin Z, Zhang Y, Gao Y, Tan S, Wang S, Cao X, Shi H, Sun C, Bai J, et al. Genome-Wide Identification and Expression Analysis of ADK Gene Family Members in Cotton under Abiotic Stress. International Journal of Molecular Sciences. 2024; 25(14):7821. https://doi.org/10.3390/ijms25147821

Chicago/Turabian Style

Huang, Peijun, Ziwei Lin, Yuzhi Zhang, Yu Gao, Songjuan Tan, Shuai Wang, Xiaoyu Cao, Hongyan Shi, Chao Sun, Jiangping Bai, and et al. 2024. "Genome-Wide Identification and Expression Analysis of ADK Gene Family Members in Cotton under Abiotic Stress" International Journal of Molecular Sciences 25, no. 14: 7821. https://doi.org/10.3390/ijms25147821

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