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Article

Genome-Wide Identification and Expression Analysis under Abiotic Stress of BrAHL Genes in Brassica rapa

by
Xiaoyu Zhang
†,
Jiali Li
†,
Yunyun Cao
,
Jiabao Huang
* and
Qiaohong Duan
*
College of Horticulture Science and Engineering, Shandong Agricultural University, Tai’an 271018, China
*
Authors to whom correspondence should be addressed.
†
These authors contribute equally to this work.
Int. J. Mol. Sci. 2023, 24(15), 12447; https://doi.org/10.3390/ijms241512447
Submission received: 12 July 2023 / Revised: 31 July 2023 / Accepted: 1 August 2023 / Published: 4 August 2023
(This article belongs to the Section Molecular Genetics and Genomics)
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Abstract

:
The AT-hook motif nuclear localized (AHL) gene family is a highly conserved transcription factor critical for the growth, development, and stress tolerance of plants. However, the function of the AHL gene family in Brassica rapa (B. rapa) remains unclear. In this study, 42 AHL family members were identified from the B. rapa genome and mapped to nine B. rapa chromosomes. Two clades have formed in the evolution of the AHL gene family. The results showed that most products encoded by AHL family genes are located in the nucleus. Gene duplication was common and expanded the BrAHL gene family. According to the analysis of cis-regulatory elements, the genes interact with stress responses (osmotic, cold, and heavy metal stress), major hormones (abscisic acid), and light responses. In addition, the expression profiles revealed that BrAHL genes are widely expressed in different tissues. BrAHL16 was upregulated at 4 h under drought stress, highly expressed under cadmium conditions, and downregulated in response to cold conditions. BrAHL02 and BrAHL24 were upregulated at the initial time point and peaked at 12 h under cold and cadmium stress, respectively. Notably, the interactions between AHL genes and proteins under drought, cold, and heavy metal stresses were observed when predicting the protein-protein interaction network.

1. Introduction

The AT-Hook motif nuclear localized (AHL) gene family is a highly conserved transcription factor critical for the growth, development, and stress tolerance of plants [1,2,3,4]. AHL proteins consist of the AT-hook motif and the Plant and Prokaryote Conserved (PPC/DUF296) domain [5]. The AT-hook motif has a conserved palindromic core sequence (i.e., Arg-Gly-Arg) that binds to AT-rich DNA [6,7]. In terrestrial plants, the PPC/DUF296 domain is localized at the carboxyl end relative to the AT-hook motif [8]. The AHL gene interacts with other proteins through PPC/DUF296 to participate in plant biological activities [5]. It has been identified in terrestrial plant species such as Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa) [3,4].
AHL genes are implicated in the process in response to abiotic and biotic stresses. The drought avoidance and tolerance of O. sativa are positively affected by OsAHL1 containing an AT-hook motif. Specifically, the overexpression of OsAHL1 improves stress tolerance in rice [4]. In Populus trichocarpa, PtrAHL34 induced by drought stress in roots and leaves can enhance drought tolerance [9]. PtrAHL14 and PtrAHL17 have a positive effect under cold conditions by modulating PtrA/NINV7-mediated Suc catabolism [10]. In A. thaliana, AtAHL10 phosphorylation regulated by abscisic acid (ABA) induced 1 (HAI1) is involved in drought stress and defense processes [11]. The phosphorylation modifies the AtAHL13 protein in response to pathogen-associated molecular pattern-triggered immunity [12]. The overexpression of AtAHL20 is highly susceptible to Pseudomonas syringae and negatively affects plant immunity [13]. In Cicer arietinum L., CaAHL18 exhibits a high expression level against the severe foliar disease Ascochyta blight [14]. In Vernicia fordii, AHL genes modulate V. fordii seed development and respond to Fusarium wilt, which exerts a negative effect on seed oil accumulation [15]. Based on previous studies, AHL genes are also critical in the physiological and developmental processes of plants. In A. thaliana, AtAHL22 is involved in FLOWERING LOCUS T chromatin modification and delays the flowering time [16,17]. AtAHL15 can form the secondary xylem. On the other hand, mutant AHL15 reduces the development of secondary xylem [18]. AtAHL18 modulates the architecture of the root system, and the mutant AHL18 is shown to possess reduced primary root elongation and a lateral root number [19]. In Zea mays, barren stalk fastigiate-1 is an AT-hook protein forming the seed-bearing inflorescences and is also annotated as the maize ear [20].
Brassica rapa (B. rapa, Chinese cabbage) is a leafy vegetable that belongs to the cruciferous family. Notably, B. rapa experiences yield reduction under adverse conditions, including drought, extreme temperatures, and heavy metal stress [21]. Previous studies have demonstrated that AHL genes can mediate stress tolerance in different plants [8,9,10,11]. However, few studies elucidated the function of AHL genes in B. rapa. In this study, BrAHL genes were identified from the B. rapa genome. Their physicochemical properties, structures, and expression profiles were analyzed. It is hypothesized that the BrAHL genes in plants can respond to abiotic stress.

2. Results

2.1. Identification and Physicochemical Properties of the BrAHL Gene Family

In order to analyze the basic characteristics of the BrAHL gene family, 42 conserved AHL family members were identified from the whole genome and labeled as BrAHL1 to BrAHL42. Their physical and chemical properties were analyzed by the ExPASy tool. The measured relative molecular weights ranged between 27,045.57 and 45,472.21 D, the protein isoelectric points ranged between 5.04 and 10.42, and the length of the amino acids ranged between 261 and 429 aa. Furthermore, more than 60% of the proteins had isoelectric points greater than 7, indicating that most BrAHL proteins were enriched in basic amino acids. Subcellular localization showed that over 90% of AHL proteins were located in the nucleus, suggesting the possibility of the BrAHL gene family being involved in intranuclear regulation, consistent with a previous finding that AHLs are nuclear-localized proteins [3]. BrAHL10 is located in chloroplasts, and only BrAHL27 is distributed in both the nucleus and cytosol. In addition, BrAHL31, BrAHL32, BrAHL33, BrAHL34, and BrAHL35 can be detected in both the nucleus and chloroplasts (Table S1).

2.2. Phylogenetic Analysis of the BrAHL Gene Family

A phylogenetic analysis was performed on the identified AHL family members to further investigate the evolutionary relationship of the BrAHL gene family. In Figure 1, branches indicate different evolutionary clades, with different colors representing different species. The number in each branch denotes the percentage of reliability. The selected species include B. rapa, A. thaliana, and Solanum lycopersicum, and the results indicate that two clades (A and B) are formed during the evolution of this gene family. All family members are unevenly distributed in two clades. Clade A consists of 13 BrAHLs, 15 AtAHLs, and 13 SIAHLs, and Clade B includes 29 BrAHLs, 14 AtAHLs, and 19 SlAHLs. The close phylogenetic relationship between B. rapa and the rest of the AHL families reveals the consistent evolution of AHL genes among different species, suggesting that the homologous genes may have similar functions.

2.3. Gene Structure and Conserved Motifs of the BrAHL Gene Family

The evolutionary relationship of gene families can be represented by the gene structure and motifs. In this study, the gene structure and motifs were explored by comparing coding sequences with corresponding genomic DNA sequences. In Figure 2B, the green boxes represent exons, the yellow boxes represent the conserved domain of AHL genes, and the black lines connecting exons denote introns. Most BrAHL genes in the same clade have similar exon intron lengths and numbers, especially the paralogous pairs in the same clade. It can be seen from Figure 2A that the number of exons in the BrAHL gene family is consistent (ranging from one to six), with most members containing five exons. All the members in Clade A have introns, while 12 members in Clade B have no intron. In addition, BrAHL12 and BrAHL16 have four introns, while BrAHL42 has only one intron in Clade B (Figure 2B). The conserved motifs of 42 BrAHL members were analyzed, revealing the similarity of conserved motifs in the same clade. All the gene family members contain Motif 1 and Motif 5. Motif 6 only appears in Clade A, and Motif 9 appears only in Clade B. Most gene family members in Clade B have motif 7, while only five gene family members in Clade A have motif 7 (Figure 2C). Motif 5 and motif 8 consist of highly conserved R-G-R-P-R-K-Y and R-G-R-P amino acid sequences, respectively. Among them, R-G-R-P is the core sequence shared by the two motifs. It can prevent changes in DNA conformation by combining with the minor groove of DNA, resulting in the binding between transcription factors and the major groove [7] (Figure 2D). This result is consistent with the previous study on Brassica napus [22].

2.4. Collinear and Homologous Gene Pairs in the BrAHL Gene Family

To further investigate the evolutionary relationships, a homology map of the AHL genes was constructed using the genes of A. thaliana, a model plant belonging to Brassicaceae. The red lines in Figure 3 represent the collinear gene pairs, and the green blocks represent chromosomes. All the BrAHL family members share homologs with A. thaliana, indicating a good homology between the two species and the similar functions of the genes. According to the location of the AHL genes throughout the chromosomes, forty-two BrAHL genes are unevenly distributed on nine chromosomes, with one–seven genes on each chromosome. Chromosome A 09 contains the most genes with seven family members. Chromosomes A 01 and A 04 have six family members. Chromosomes A 03, A 06, and A 07 have five members. Chromosome A 05 has the least genes, with only one family member (Figure 3).
The collinearity of the BrAHL gene family was analyzed to select duplicate genes based on two criteria (comparison rate of two genes > 75% and comparative similarity > 75%). Groups of gene pairs located on different chromosomes were obtained. Five or fewer genes positioned within 100 kb on the same chromosome are considered tandem duplication [23]. Ka and Ks represent the substitutions at each synonymous and nonsynonymous site, respectively. The Ka/Ks values of most gene pairs are lower than 1, indicating that purifying selection affects the evolution of most gene pairs and suppresses the differentiation of duplicate genes. Only the BrAHL27-BrAHL26 obtained from tandem duplication have Ka/Ks greater than 1, belonging to positive selection. In addition, most of the genes in the BrAHL gene family are characterized by segmental duplications, indicating a high degree of homology in this gene family (Table S2).

2.5. Cis-Regulatory Element Analyses of the BrAHL Gene Family

Cis-regulatory elements affect the initiation and efficiency of gene transcription by binding to transcription factors [24]. In this study, cis-regulatory elements of the BrAHL gene family were analyzed to determine the potential functions of BrAHL genes, and a 2000-bp sequence upstream of the gene start codon was downloaded. Based on the promoter sequences of BrAHL genes, 20 types of cis-regulatory elements were predicted. Most of these elements were associated with abiotic stress responses, plant growth and development, and hormonal responses. BrAHL01, BrAHL04, BrAHL06, BrAHL07, BrAHL08, BrAHL16, BrAHL18, BrAHL25, and BrAHL40 have drought-inducible cis-regulatory elements. BrAHL02, BrAHL07, BrAHL08, BrAHL10, BrAHL13, BrAHL15, BrAHL16, BrAHL19, BrAHL21, BrAHL24, BrAHL34, and BrAHL39 have low-temperature responsiveness cis-regulatory elements. The number and distribution of promoter cis-regulatory elements vary significantly (Figure 4A). All 42 BrAHL genes contain considerable light-responsive elements, suggesting that the BrAHL genes may function in counteracting heavy metal stress. More than 90% of genes have anaerobic-responsive elements and five plant hormone-responsive elements. In addition, some members have elements related to stress responses, including drought and low temperatures (Figure 4B). Based on the above results, it can be speculated that the BrAHL genes may be involved in the regulation of stress, light, and hormone responses.

2.6. Expression Profile of BrAHL Genes in Different Tissues

In order to investigate the expression of BrAHL genes, the tissue expression profiles of this gene family in B. rapa root, stem, leaf, flower, silique, and callus were explored. The results revealed that each member differed in tissue expression. Therefore, it is speculated that the expression of BrAHL genes has different effects on plant development at different stages. As shown in Figure 5, BrAHL02, BrAHL16, BrAHL17, and BrAHL20 are widely expressed in tissues, and BrAHL04, BrAHL05, BrAHL08, BrAHL12, BrAHL19, and BrAHL29 exhibit a relatively low expression in different tissues. In contrast, BrAHL13, BrAHL23, BrAHL25, BrAHL26, BrAHL27, and BrAHL35 are less expressed in organs. The high expressions of BrAHL01, BrAHL02, BrAHL04, BrAHL16, BrAHL17, BrAHL18, BrAHL24, BrAHL28, BrAHL29, and BrAHL41 in roots suggest that they might participate in the regulation of drought stress. Notably, BrAHL18 shows a higher expression in the leaf than in other tissues, and BrAHL36 exhibits a higher expression level in silique than in other organs.

2.7. Expression Profile of BrAHL Genes under Osmotic Treatment

Drought stress negatively affects plant growth and development, decreasing yield and vegetable quality [25]. Under drought conditions, the root system is the primary organ that responds to stress [26]. To further investigate the effects of BrAHL genes under drought conditions, the RNA of BrAHL genes was extracted at 2, 4, 6, and 12 h of osmotic treatment, and the qRT-PCR was used to analyze the expression level of the ten family genes abundantly expressed in the roots. Half of the BrAHL family members were upregulated under osmotic treatment. As shown in Figure 6, BrAHL01 is upregulated at all time points and peaks at 4 h. BrAHL28 shows a similar trend and reaches the maximum at 6 h. Notably, BrAHL01, BrAHL16, BrAHL17, and BrAHL41 are induced at 4 h of drought stress and are significantly suppressed at the other three time points, suggesting their potential effects on stress tolerance. BrAHL02 shows moderate upregulations at all time points. BrAHL18 is upregulated at 2 h and downregulated at 4 h, while BrAHL04 exhibits the opposite trend.

2.8. Expression Profile of BrAHLs under Cold Treatment

Low temperatures can suppress plant growth and food quality, potentially generating reactive oxygen species (ROS) [27,28]. To have insights into the expression of BrAHL genes under such stress, RNA samples of the family genes under cold treatment at 2, 4, 6, and 12 h were extracted, and the expression levels of ten genes in the BrAHL gene family were analyzed using qRT-PCR. The presence of four cold stress-induced genes indicated their involvement in regulating biological processes under low temperatures. BrAHL02 was upregulated at the first two time points, was significantly downregulated at 6 h, and peaked at 12 h. BrAHL18 was elevated gradually at all durations and reached the climax at 12 h. BrAHL24 was downregulated at 2 h and 6 h and dramatically upregulated at 4 h and 12 h. The expression trend of BrAHL28 was the opposite. It is worth noting that BrAHL01, BrAHL04, BrAHL16, BrAHL17, BrAHL29 and BrAHL41 were all inhibited at tested periods while coping with cold stress (Figure 7).

2.9. Expression Profile of BrAHL Genes under Cadmium (Cd) Treatment

Cd is a highly toxic heavy metal that substantially risks plant growth [29]. To better explore the expression of BrAHL genes under Cd stress, the RNA of the gene family under Cd treatment at 2, 4, 6, and 12 h was extracted, and the expression level of ten genes in the BrAHL gene family was analyzed using qRT-PCR. The results showed that most family genes were upregulated, indicating their positive response to Cd exposure. BrAHL01, BrAHL04, BrAHL16, BrAHL17, and BrAHL41 showed a consistent trend and peaked at 6 h under Cd stress. BrAHL02, BrAHL28, and BrAHL29 were suppressed at all time points. BrAHL18 and BrAHL24 were slightly upregulated at the first time point and suppressed more significantly than the control at the next three time points (Figure 8).

2.10. Protein-Protein Interaction Networks of the AHL Genes

Protein-protein interaction networks are composed of proteins involved in basic life processes through mutual interactions, such as gene expression regulation, signal transduction, and metabolism control. The protein-protein interaction networks were predicted, whereby circles represent the different gene members, and lines denote the interactions. In addition, the degree centrality of nodes exhibits a positive correlation with the circle sizes and shades of color. Given that BrAHL genes are closely related to AtAHL genes, protein interactions were predicted to reveal the potential functions of these proteins. AT2G33620 (homologous gene of BrAHL16) was observed to interact with HAI1, which functions as a negative regulator in response to osmotic stress and drought [11]. It is inferred that BrAHL16 is probably involved in regulation under drought stress (Figure 9A). AT4G22770 (homologous gene of BrAHL02) interacted with AT-HSFA5, a heat shock protein (HSP). HSPs are induced by unfavorable environments, suggesting that BrAHL02 potentially affects the process under cold conditions (Figure 9B). AT3G04590 (homologous gene of BrAHL24) was found to have interactions with far-red elongated hypocotyls (FRSs) in regulating the light control of plant development. Notably, photosynthesis is the primary process affected by stress conditions, e.g., heavy metal stress [30,31,32]. It can be speculated that BrAHL24 participates in heavy metal stress tolerance (Figure 9C).

3. Discussion

AHL genes are implicated in plant growth, physiological processes, and stress tolerance. The role of AHL genes was not recognized in B. rapa. In this study, 42 BrAHL genes were identified in B. rapa, and their genomic features were analyzed, such as evolutionary relationships, gene structures, conserved motifs, duplicate gene pairs, cis-regulatory elements, and expression profiles.
Gene duplication serves as a main driving force in the evolution of genomes and genetic systems [33,34], facilitating the formation of novel gene functions and species evolution [35,36]. Duplicate genes provide raw materials for new genes, which in turn promotes the generation of new functions. Four major patterns of evolution involve fragmental, tandem, whole genome, and gene transposition duplications [37,38]. Among them, segmental and tandem duplications are considered primary contributors to the expansion of plant gene families [39]. Tandem duplication derives from unequal crossing-over events [40], and multiple episodes of these crossovers may increase or decrease the copy number in different gene families. Tandem replication mainly occurs in the region of chromosome recombination and forms a gene cluster with identical sequences and functions. Segmental duplication results in the duplicated blocks of genomic DNA typically within 200 kb in size, and these segments contain high-copy repeats and gene sequences characterized by an intron exon structure [41]. Brassicaceae genomes were estimated to undergo three rounds of whole genome duplication [42,43]. In the BrAHL gene family, most Ka/Ks values of studied gene pairs were lower than 1, indicating that purifying the selection influences the evolution of most gene pairs and suppresses the differentiation of duplicate genes. Remarkably, BrAHL27 and BrAHL26 had Ka/Ks values greater than 1, indicative of positive selection. In the case of BrAHL genes, segmental duplication was primarily responsible for the gene family expansion, and one instance of tandem duplication was also seen. The amplification of genes by tandem duplication was more closely related to abiotic and biotic stress than non-tandem duplications [25,26,44]. In this sense, it is inferred that gene duplication is involved in BrAHL gene family expansion and plant stress tolerance.
Drought stress often negatively influences plant growth and development, potentially decreasing yield and vegetable quality [45]. Under drought conditions, roots are the primary organs coping with stress [46]. Given this scenario, ABA can be employed to regulate the physiological activities of plants under drought stress [27,47]. Notably, the MYB binding site (MBS) cis-regulatory element is involved in drought-inducibility elements. An ABA-responsive element is involved in ABA responsiveness and plays an important role in dealing with drought stress. In the BrAHL gene family, BrAHL16 expressed abundantly in roots and possessed MBS and ABA-responsive elements. This gene was upregulated at 4 h under osmotic stress and downregulated at the following time points. In O. sativa, OsAHL1 was induced under drought stress. In OsAHL1 over-expressing plants, water loss was slower, and the volume of the total root, upper root, and lower root in plants was higher than in wild-type plants under drought stress. OsAHL1 regulated its target genes (HSP101, OsCDPK7, OsRNS4, Rab16b, and AP2-EREBP) to improve drought tolerance. It is indicated that drought resistance is enhanced by the over-expression of OsAHL1 [4]. In A. thaliana, some AtAHL10 phosphorylation sites were de-phosphorylated by HAI1, ultimately suppressing plant growth under drought stress. Notably, AHL10 had more seedling weight and root elongation than the wild type under moderate soil drying. HAI1-regulated phosphorylation of AtAHL10 could affect plant growth under drought stress [11]. In Populus trichocarpa, PtrAHL34 with an MBS cis-element exhibited a high expression level in roots and was induced by drought stress at 6 h and declined to a normal level at 24 h [9]. It can be elucidated that BrAHL16 is involved in the regulation of drought stress.
Cold stress hampers plant growth and food quality and may produce ROS [28,48]. The response of plants to extreme temperatures is mediated by plant hormones [49,50]. One fundamental mechanism is to induce ROS production and activate nicotinamide adenine dinucleotide phosphate oxidases in response to temperature changes [51,52]. A ong-terminal repeat cis-regulatory element is vital in low-temperature response. In the BrAHL gene family, BrAHL02 had low-temperature cis-regulatory elements and was gradually expressed at 2 h and 4 h, was downregulated at 6 h, and peaked at 12 h. From the protein-protein interaction networks, the homologous gene in A. thaliana (AT4G22770) interacted with AT-HSFA5. HSFA4 and HSFA5 belong to distinct subgroups within the class A HSF, characterized by identical DNA-binding domains and conserved C-terminal motifs [53]. Heat shock factors (HSFs) are transcriptional regulators that mediate adverse conditions such as extreme temperatures [54,55]. Some HSFs act as molecular peroxide sensors that respond to alterations in ROS levels under stress [56]. HSFA4A transcription is stimulated by stress conditions (e.g., cold stress) and produces ROS [29]. Based on the findings, BrAHL02 probably plays an important role under cold conditions.
Cd exposure is considered a potential threat to plant growth due to its toxic effects. [30]. Notably, photosynthesis is the primary process affected by adverse conditions such as heavy metal stress [31,32,57]. Photosynthetic response to stress conditions is extremely complicated and occurs at different sites of cells or leaves at various time scales [58]. In the BrAHL gene family, BrAHL24 with light-responsive cis-regulatory elements was induced at the initial two periods, was downregulated at 6 h, and peaked at 12 h. Considering protein-protein interaction networks, the homologous gene AT3G04590 interacted with far-red elongated hypocotyls (FRSs). FRSs are crucial in the light control process that regulates the development of roots and flowers [59]. It is concluded that BrAHL24 probably has potential functions under Cd stress.

4. Materials and Methods

4.1. Identification of AHL Members and Their Physical and Chemical Properties

A total of 29 A. thaliana AHL protein sequences were downloaded from the TAIR database (http://www.arabidopsis.org/, accessed on 1 July 2023). The possible members of the BrAHL family were searched through the two-way BLAST in the B. rapa genome version 1.5 (http://brassicadb.cn/, accessed on 1 July 2023) [60]. The gene family was further identified using conserved domain verification [61]. The physicochemical properties, such as the isoelectric point and molecular weight, were analyzed on the ExPASy server (https://www.expasy.org/, accessed on 1 July 2023) [62]. The data were acquired in batches using TBtools (version 1.120) and sorted out [63]. The subcellular localization was predicted using WoLF PSORT (http://www.genscript.com/wolf-psort.html, accessed on 1 July 2023) [64].

4.2. Phylogenetic Analysis of the Family Genes

According to the ID information of the identified family genes, the protein sequences were combined with other species utilizing ClustalW [65] multiple sequence alignment. The reliability of the phylogenetic tree was evaluated using 1000 bootstrap duplications. At the same time, based on Jones Taylor Thornton (JTT) model and gamma distribution, we also constructed the phylogenetic tree using the maximum-likelihood (ML) method in MEGA-11 [45]. The evolutionary tree was beautified on the website (https://itol.embl.de/itol.cgi, accessed on 1 July 2023) [66].

4.3. Analysis of Gene Structure and Cis-regulatory Elements

MEME tool (http://meme-suite.org/, accessed on 1 July 2023) and Batch CD-Search on the NCBI website were adopted to predict conserved motifs and domains, respectively [67]. TBtools software (version 1.120) was employed to combine the conserved motif and domain with the phylogenetic tree for mapping [63]. The 2000-bp sequence upstream of the BrAHL gene initiation codon was downloaded through the Ensembl Plants website (http://plants.ensembl.org/index.html, accessed on 1 July 2023) [60].

4.4. Analysis of Collinearity and Gene Pairs

The MCScanX [23] was utilized to identify the collinear relationships between the chromosomes within the genome. Sequentially, duplication analysis was performed on the identified duplicate gene pairs between family genes. The results were illustrated using the Circos software [68]. Synonymous and nonsynonymous substitutions were selected to calculate the Ka/Ks values for all paralogous genes [69].

4.5. Analysis of the Expression Profiles

RNA-seq data (accession number: GSE43245) were collected for the transcriptome sequencing of B. rapa tissues (http://brassicadb.cn, accessed on 1 July 2023). All transcriptomic expression data were homogenized using log2. A cluster heatmap of the gene family expression was constructed to show the differential expression of the family members in different tissues [70].

4.6. Total RNA Extraction and qRT-PCR

Chinese cabbage seedlings were grown to six leaves in Hoagland nutrient solution (Coolaber, Beijing, China) and treated with PEG 6000 (15%), 4 °C, and 100 µM CdCl2 to simulate osmotic, cold, and Cd stress in a hydroponic system for 2, 4, 6 and 12 h. Normal Chinese cabbage seedlings were used as a control. Following RNA extraction, RNA molecules in the sample were verified with 1% agarose gel electrophoresis, and retrotranscription was performed to prepare the cDNA. The qRT-PCR primers were obtained from the qPrimerDB-qPCR primer database (https://biodb.swu.edu.cn/qprimerdb/, accessed on 1 July 2023). BrActin2 was used as the reference gene and synthesized by Qingdao Qingke Zixi Biotechnology Co., Ltd. (Qingdao, China). The total RNA sample was obtained using an RNA extraction kit (Black, Beijing, China) using HiScript II QRT SuperMix for qPCR. The resulting RNA was then reverse transcribed using a total RNA extraction kit (Novizan, Nanjing, China) to obtain the cDNA. Ten-fold dilution with dd H2O was added to test the expression of BrAHL family genes. The 20 L reaction system was configured according to the following instructions of the ChamQ Universal SYBR qPCR Master Mix kit (Novizan, Nanjing, China): 10 μL of SYBR green master mix, 2 μL of cDNA, 7.2 μL of dd H2O, and 0.4 μL each of the upstream and downstream primers. The PCR reaction was performed with a qTOWER3 (Analytik Jena AG, Jena, Germany) at 40 cycles of 95 °C for 30 s, 95 °C for 10 s, and 60 °C for 22 s. The relative expression of the Chinese cabbage genes was calculated using the 2−∆∆CT method [71] and presented by TBtools (version 1.120) [63]. The primer sequences are shown in Table S3.

4.7. Protein-Protein Interaction Network Analysis

STRING (http://cn.string-db.org/, accessed on 1 July 2023) [27] is a comprehensive protein-protein interaction database that includes known and predicted interactions. The protein sequences of the studied family genes were submitted to this database to query the relationships among the genes and subsequently map the protein-protein interaction networks utilizing Cytoscape (version 3.9.1) [72].

5. Conclusions

In this study, 42 AHL family members were identified from the B. rapa genome and mapped to nine B. rapa chromosomes. The expression profile revealed that BrAHLs were widely expressed in different tissues. Specifically, BrAHL16 was probably involved in drought stress response. BrAHL02 was induced under cold stress. BrAHL24 may undergo regulation under Cd conditions. Notably, the protein-protein interaction network prediction demonstrated that AT2G33620 (homologous gene of BrAHL16) interacted with HAI1. It is speculated that BrAHL16 participates in the regulation under drought stress. AT4G22770 (homologous gene of BrAHL02) was revealed to interact with AT-HSFA5, suggesting that BrAHL02 functions under cold conditions. AT3G04590 (homologous gene of BrAHL24) had interactions with far-red elongated hypocotyls (FRSs), indicating BrAHL24's involvement in response to heavy metal stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms241512447/s1.

Author Contributions

Q.D. conceived the project; Q.D. and J.H. conceptualized and designed the experiments; X.Z. and J.L. performed the bioinformatic analysis and experiments with the help from Y.C.; X.Z. wrote the manuscript; J.H. and Q.D. revised the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

The funder was Q.D. and J.H. This research was funded by the Key Program of Shandong Province Science Foundation (ZR2020KC017) and Shandong Provincial Natural Science Foundation (ZR2022MC021).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

Data are contained within the article/Supplementary Materials.

Conflicts of Interest

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

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Figure 1. Phylogenetic analysis of the AHL gene family between B. rapa and A. thaliana, and S. lycopersicum. Branches indicate different evolutionary clades, with different colors representing different species. The number in each branch denotes the percentage of reliability.
Figure 1. Phylogenetic analysis of the AHL gene family between B. rapa and A. thaliana, and S. lycopersicum. Branches indicate different evolutionary clades, with different colors representing different species. The number in each branch denotes the percentage of reliability.
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Figure 2. Analysis of the gene structure and protein conserved domains of BrAHL genes. (A). Phylogenetic analysis of BrAHL genes. (B). Gene structure of BrAHL genes. (C). Conserved protein motifs of BrAHL genes. (D). Motifs of BrAHL genes. The black boxes represent two amino acid sequences, respectively.
Figure 2. Analysis of the gene structure and protein conserved domains of BrAHL genes. (A). Phylogenetic analysis of BrAHL genes. (B). Gene structure of BrAHL genes. (C). Conserved protein motifs of BrAHL genes. (D). Motifs of BrAHL genes. The black boxes represent two amino acid sequences, respectively.
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Figure 3. Collinearity between B. rapa and A. thaliana. The red lines represent the collinear gene pairs, and the green blocks represent chromosomes.
Figure 3. Collinearity between B. rapa and A. thaliana. The red lines represent the collinear gene pairs, and the green blocks represent chromosomes.
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Figure 4. Cis-regulatory elements analysis in the promoter region of BrAHL genes. (A). Cis-regulatory elements of BrAHL genes. (B). Proportion of cis-regulatory elements.
Figure 4. Cis-regulatory elements analysis in the promoter region of BrAHL genes. (A). Cis-regulatory elements of BrAHL genes. (B). Proportion of cis-regulatory elements.
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Figure 5. Expression of BrAHL genes in different tissues. Red color represents increased expression, and blue denotes decreased expression.
Figure 5. Expression of BrAHL genes in different tissues. Red color represents increased expression, and blue denotes decreased expression.
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Figure 6. Expression of BrAHL genes under drought stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
Figure 6. Expression of BrAHL genes under drought stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
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Figure 7. Expression of BrAHL genes under cold stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
Figure 7. Expression of BrAHL genes under cold stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
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Figure 8. Expression of BrAHL genes under Cd stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
Figure 8. Expression of BrAHL genes under Cd stress. Data are presented as means (±SD) of three biological replicates. Asterisks or n.s. above the data bars indicate a significant difference (two-tailed t-test * p < 0.05 ** p < 0.01) or no significant difference, respectively.
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Figure 9. Protein-protein interaction network prediction of AHL genes. Circles represent the proteins. Lines represent the interaction. The degree centrality of nodes exhibits a positive correlation with the circle sizes and shades of color. (A). AT2G33620 (homologous gene of BrAHL16) PPIs. (B). AT4G22770 (homologous gene of BrAHL02) PPIs. (C). AT3G04590 (homologous gene of BrAHL24) PPIs.
Figure 9. Protein-protein interaction network prediction of AHL genes. Circles represent the proteins. Lines represent the interaction. The degree centrality of nodes exhibits a positive correlation with the circle sizes and shades of color. (A). AT2G33620 (homologous gene of BrAHL16) PPIs. (B). AT4G22770 (homologous gene of BrAHL02) PPIs. (C). AT3G04590 (homologous gene of BrAHL24) PPIs.
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Zhang, X.; Li, J.; Cao, Y.; Huang, J.; Duan, Q. Genome-Wide Identification and Expression Analysis under Abiotic Stress of BrAHL Genes in Brassica rapa. Int. J. Mol. Sci. 2023, 24, 12447. https://doi.org/10.3390/ijms241512447

AMA Style

Zhang X, Li J, Cao Y, Huang J, Duan Q. Genome-Wide Identification and Expression Analysis under Abiotic Stress of BrAHL Genes in Brassica rapa. International Journal of Molecular Sciences. 2023; 24(15):12447. https://doi.org/10.3390/ijms241512447

Chicago/Turabian Style

Zhang, Xiaoyu, Jiali Li, Yunyun Cao, Jiabao Huang, and Qiaohong Duan. 2023. "Genome-Wide Identification and Expression Analysis under Abiotic Stress of BrAHL Genes in Brassica rapa" International Journal of Molecular Sciences 24, no. 15: 12447. https://doi.org/10.3390/ijms241512447

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