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Article

Genome-Wide Survey and Expression Analysis of B-Box Family Genes in Cucumber Reveal Their Potential Roles in Response to Diverse Abiotic and Biotic Stresses

by
Chuxia Zhu
1,
Lingdi Xiao
1,
Yaqi Hu
1,
Liu Liu
1,
Haoju Liu
1,
Zhaoyang Hu
1,
Shiqiang Liu
1 and
Yong Zhou
1,2,*
1
College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China
2
Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education, Jiangxi Agricultural University, Nanchang 330045, China
*
Author to whom correspondence should be addressed.
Agriculture 2022, 12(6), 827; https://doi.org/10.3390/agriculture12060827
Submission received: 20 May 2022 / Revised: 2 June 2022 / Accepted: 7 June 2022 / Published: 9 June 2022
(This article belongs to the Special Issue Effects of Abiotic Stress on Horticultural Crops)
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Abstract

:
As a class of zinc finger transcription factors, B-box (BBX) proteins play diverse roles in numerous biological processes, and they have been identified in a series of plant species in recent years. However, the roles of BBX genes in regulating cucumber growth regulation and stress response have not yet been established. Here, a total of 22 BBX family genes were identified via an analysis of the latest cucumber genome data, which were classified into five groups (I–V) on the basis of their phylogenetic features and number of B-box domains and CCT domains. The CsBBX genes were unevenly distributed across the seven cucumber chromosomes, and segmental duplication was found to play a significant role in the expansion of the cucumber BBX gene family. Gene structure and motif composition analysis suggested that the evolutionarily close CsBBXs have similar conserved motif composition and gene structure. Most CsBBX genes possessed 1–3 introns, and intron gain rather than intron loss could contribute to the different structures of CsBBX genes across different groups during their evolution. Promoter analysis revealed the presence of 13 kinds of hormone-related and nine kinds of stress-related cis-regulatory elements in the promoter regions of these CsBBX genes. Expression analysis via RNA-seq and qRT-PCR suggested that the CsBBX genes exhibit differential expression in different tissues and in response to various abiotic and biotic stresses. This work constitutes a starting point for further revealing the function of the CsBBX genes and sheds light on the potential molecular mechanism of stress resistance in cucumber.

1. Introduction

Zinc finger transcription factors (TFs), which are widely present in all eukaryotes, are vital regulators playing important regulatory roles in various essential biological processes of plants. B-box (BBX) proteins are a class of zinc finger TFs characterized by the presence of one or two specific B-box domains with approximately 40 amino-acid residues at the N-terminus [1,2]. On the basis of the consensus sequence and specificity of the zinc ion binding site, the B-box domains can be divided into two types, namely, B-box 1 and B-box 2 [2,3], both of which are highly conserved across species and have certain functions in the protein–protein interaction between BBXs and other proteins, such as TOPLESS [4], early flowering 3 (ELF3) [5], ABF [6], and LONG HYPOCOTYL 5 (HY5) [7,8,9]. In addition, some BBX proteins contain a conserved CCT (CONSTANS, CO-like, and TOC1) domain at the C-terminus, which is involved in nuclear protein transport, transcriptional regulation, and protein–protein interaction. For instance, the CCT domains of CO and HEADING DATE1 (HD1) can form a heterotrimer with NF-YB/YC (NUCLEAR FACTOR-YB/YC) dimers, and then specifically bind to a conserved ‘CCACA’ motif upstream of the promoter regions of their target genes [10,11]. Members of the BBX family in various plant species can be classified into five groups (I–V) on the basis of their numbers of B-box and CCT domains, with group I and group II having one CCT domain and two B-box domains, group III comprising one CCT domain and one B-box domain, group IV including two B-box domains, and group V having one B-box domain [2,12].
BBX proteins are known to participate in a wide range of physiological processes in plants. For example, four members of group IV in Arabidopsis (BBX18, BBX19, BBX24, and BBX25) negatively regulate photomorphogenesis, while three other members in this group (BBX20, BBX21, and BBX22) positively regulate photomorphogenesis [13,14]. In addition, some BBXs are associated with light-induced proanthocyanidin biosynthesis and anthocyanin accumulation. For example, PpBBX16 and PpBBX18 can positively regulate anthocyanin synthesis by interacting with PpHY5, while PpBBX21 interferes with the formation of the PpBBX18–PpHY5 heterodimer, thereby repressing anthocyanin biosynthesis [15,16]. In apple, MdBBX21 can also interact with MdHY5 and observably enhance the promoter activity of MdMYB1 under light, thereby regulating anthocyanin accumulation [17]. Under light stress, MdBBX22 can bind to the promoter of mdm-miR858, inducing its transcription to inhibit proanthocyanidin accumulation and subsequently indirectly promoting anthocyanin synthesis in the peel [18]. Furthermore, UV-B can promote the transcription of MdBBX22 via both transcriptional and post-translational regulations, thereby acting as a cofactor of MdHY5 to activate UV-B-induced anthocyanin biosynthesis [19]. Moreover, numerous reports have documented that BBXs also play crucial roles in diverse developmental processes, such as seed germination [20], shade avoidance [21], leaf senescence [22,23], and circadian regulation [24], as well as in the responses of plants to various biotic and abiotic stresses. For example, BBX18 and BBX23 can repress the protein levels of ELF3 and, therefore, positively regulate thermomorphogenesis in Arabidopsis [5]. Additionally, BBX18 promotes thermomorphogenesis by interfering with PSEUDO-RESPONSE REGULATOR 5 (PRR5)-mediated inhibition of PIF4 in response to high temperature [25]. A tomato BBX member SlBBX17 acts as a negative regulator of plant growth but positively regulates heat tolerance [26]. Overexpression of a chrysanthemum BBX gene CmBBX22 in Arabidopsis delayed leaf senescence and improved drought tolerance [22]. Similarly, transgenic Arabidopsis plants overexpressing apple MdBBX1 and MdBBX10 exhibited enhanced tolerance to multiple abiotic stresses [27,28].
Cucumber, a popular and economically important crop of the Cucurbitaceae family, is widely cultivated in many countries. It produces tender fruits rich in nutrients and provides people with various health-promoting properties. However, its growth and development are always negatively affected by a number of abiotic stresses, such as extreme temperature and salt. Additionally, a variety of infective agents, such as downy mildew (DM), powdery mildew (PM), and root-knot nematodes (RKNs), can destroy the production of cucumber, as well as negatively affect other Cucurbitaceae species [29,30]. Therefore, it is quite necessary to excavate more potential resistance genes and define their functional roles in the stress resistance of cucumber. BBXs have been identified and functionally characterized in many plants, where they are generally encoded by a multigene family, such as Arabidopsis thaliana [3], Oryza sativa [31], Solanum tuberosum [32], Malus domestica [33], Dendrobium officinale [34], Phyllostachys edulis [35], Vitis vinifera [36,37,38], and Capsicum annuum [39,40]. In addition, a recent study identified 26 cucumber BBX genes and found that some of them are associated with fruit development and carotenoid biosynthesis [41]. To date, little information is available on the cucumber BBX genes in responses to various environmental stimuli. In this work, we carried out a genome-wide analysis of BBX genes in cucumber according to the cucumber genome v3 (an updated version in the cucumber ‘Chinese long 9930’ genome), including their gene and protein sequences, conserved motifs, gene duplication events, and cis-acting elements. Additionally, the expression profiles of the cucumber BBX genes under abiotic stresses (heat, cold, and salt) and biotic stresses (DM, PM, and RKN) were also investigated. The results provide a biological basis for illustrating the functions of the BBX genes in the abiotic and biotic stress response of cucumber.

2. Materials and Methods

2.1. Identification and Characterization of BBX Family Members in Cucumber

To identify and annotate the BBX family members in cucumber, a search was firstly conducted against the cucumber genomic database v3.0 (with “9930” as the reference genome, http://cucurbitgenomics.org/, accessed on 20 May 2022) using the hidden Markov model (HMM) profile of the B-box domain (PF00643) with the HMMER software. Moreover, the Arabidopsis BBX proteins from the TAIR database (https://www.arabidopsis.org/, accessed on 20 May 2022) were used as the query sequences to search against the cucumber genomic database with blastp program (the cutoff value was 1 × 10−5). Then, all putative BBX protein sequences were sent to PFAM (http://pfam.xfam.org/, accessed on 20 May 2022), SMART (http://smart.emblheidelberg.de/, accessed on 20 May 2022), as well as InterproScan (https://www.ebi.ac.uk/interpro/search/, accessed on 20 May 2022) online tools to guarantee the presence of the complete BBX domain. Identified CsBBX proteins were first subjected to the ProtParam website (http://www.expasy.org/, accessed on 20 May 2022) to calculate the molecular weight (MW), isoelectric point (pI), and grand average of hydropathicity (GRAVY) values, and their subcellular localizations were subsequently predicted with the Plant-mPLoc server (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 20 May 2022).

2.2. Multiple Sequence Alignment and Phylogenetic Analysis

Multiple sequence alignment of cucumber BBX proteins was carried out by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 20 May 2022) with default settings, and the results were displayed with the GeneDoc software. To study the evolutionary relationships, full-length BBX protein sequences from cucumber, grape, pear, Arabidopsis, and rice were first aligned using MAFFT (https://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 20 May 2022) with default parameters, and then the comparison results were uploaded to Molecular Evolutionary Genetics Analysis (MEGA) 7.0 software to construct the phylogenetic tree with the following parameters: Poisson correction, pairwise deletion, and a bootstrap test with 1000 replicates.

2.3. Gene Structure and Conserved Motif Analysis

The coding region sequence (CDS) and genomic DNA (gDNA) sequences were downloaded from the cucumber genomic database and submitted to GSDS online software (http://gsds.gao-lab.org/, accessed on 20 May 2022) to analyze the gene structure of each CsBBX gene. The Multiple Expectation Maximization for Motif Elicitation (MEME) (http://meme-suite.org/tools/meme, accessed on 20 May 2022) was used to examine the conserved motif arrangements of CsBBX proteins. The maximum number and minimum motif width of different motifs was set to 10 and 6, respectively.

2.4. Chromosomal Distribution, Gene Duplication, and Cis-Acting Elements Analysis of CsBBX Genes

The positional information of CsBBX genes was retrieved from the cucumber genomic database, and the MG2C (http://mg2c.iask.in/mg2c_v2.1/, accessed on 20 May 2022) online tool was employed to determine the chromosomal distributions of CsBBX genes. The Multiple Collinearity Scan toolkit (MCScanX) software was used to analyze the tandem and segment duplication events of CsBBX genes within the cucumber genome. To investigate the distribution of cis-acting elements within the promoter regions of CsBBX genes, the 2.0 kb upstream promoter sequence of the start codon (ATG) of each CsBBX gene was extracted and submitted to the PlantCARE server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 May 2022).

2.5. Expression Profiling of the CsBBX Genes via RNA-Seq Data

To determine the tissue-specific expression of CsBBX genes, the RNA-seq data of various tissues at different developmental stages were retrieved from the Sequence Read Archive (SRA) database in NCBI (accession number: PRJNA80169). For the determination of the expression patterns of CsBBX genes in response to diverse abiotic stresses, the RNA-seq data were downloaded using the published RNA-seq dataset under heat stress (0, 3, and 6 h under 42 °C treatment; accession number: PRJNA634519) [42] and salt stress (leaves and roots of the seedlings after salt treatment under the accession numbers of PRJNA477930 and PRJNA511946; CK-L and Na-L, and CK-R and Na-R were the control and salt-stressed samples of leaves and roots, respectively) [43,44]. The RNA-seq datasets under diverse biotic stresses were also downloaded, including powdery mildew (PM, the PM susceptible cultivar D8 and PM resistant cultivar SSL508-28 at 0 h and 48 h after inoculation with the PM pathogen, accession number: PRJNA321023) [45], downy mildew (DM, the DM-susceptible cultivar Vlaspik and DM-resistant cultivar PI 197088 mock inoculated or infected with Pseudoperonospora cubensis at 1, 2, 3, 4, and 6 dpi, accession number: PRJNA285071) [46], and root-knot nematode (RKN, the RKN-susceptible line CC3 and RKN-resistant line IL10-1 at 0, 1, 2, and 3 dpi after infection with Meloidogyne incognita, accession number: SRP125669) [47], to analyze the expression patterns of the CsBBX genes under biotic stresses. The relative expression levels of CsBBX genes were calculated and normalized as transcripts per kilobase million (TPM) values for further analysis. These results were represented as heat maps using TBtools [30]. The CsBBX genes with expression values at a threshold of |log2 (fold change)| ≥ 1 and p < 0.05 were defined as differentially expressed.

2.6. Plant Materials, Abiotic Stress Treatment, and qRT-PCR Analysis

Two week old seedlings of cucumber variety ‘9930’ were treated with cold and salt stress treatments following the methods described in our previous report [30]. Briefly, 2 week old seedlings were incubated with 1/2 Hoagland nutrient solution containing 200 mM NaCl or transferred to 4 °C for salt stress or cold stress, respectively. The leaves were collected respectively for three biological replicates at 0, 6, 12, and 24 h after the application of each treatment. Total RNA was extracted with the Trizol reagent, and then cDNA was reverse-transcribed using random primers. The quantitative real-time PCR (qRT-PCR) experiment was conducted with TB Green Premix Ex TaqII Kit (TaKaRa Biotechnology, Dalian, China) on a Roche Lightcyler 480II PCR System in triplicate according to the previously described method [48]. The 2−ΔΔCt method was used to analyze the qRT-PCR results, and the primer sequences are provided in Table S1.

3. Results

3.1. Identification and Characterization of CsBBX Genes in Cucumber

To identify the CsBBX genes in cucumber, sequence homology analysis and protein domain validation were performed. As a result, only 22 genes encoding the corresponding conserved BBX domain were obtained, which were designated as CsBBX1–CsBBX22 on the basis of their position on chromosomes 1–7 (Table 1). A comparison of the BBX genes with the published cucumber genomes is presented in Table S2. The CDS length of the CsBBX genes ranged from 399 bp (CsBBX22) to 1476 bp (CsBBX10). The CsBBX proteins contained 132 to 491 amino acids, and their molecular weight (MW) ranged from 14.63 kDa (CsBBX22) to 54.84 kDa (CsBBX15). The theoretical isoelectric point (pI) of the CsBBX proteins ranged from 4.31 (CsBBX9) to 8.40 (CsBBX8). The GRAVY scores of all CsBBX proteins were negative (ranged from −0.949 to −0.041), indicating that all CsBBX proteins are hydrophilic. According to the predicted subcellular localization results, all CsBBX proteins are located in the nucleus.

3.2. Phylogenetic Analysis of BBX Family Members

To reveal the evolutionary relationship of the BBX proteins in cucumber and other plants, a phylogenetic tree involving the full-length amino-acid sequences of BBX proteins from cucumber, grape [37], pear [49], Arabidopsis [50], and rice [31] was constructed by MEGA 7.0. Phylogenetic analysis revealed that these BBX family members can be divided into five clades (I–V) (Figure 1), which was in accordance with the results in previous studies [12,51]. For the 22 CsBBX proteins, clade IV had the largest number of members (seven), followed by clades V, I, II, and III, which had five, four, three, and three members, respectively (Figure 1). Nearly all BBX proteins fell into the five clades on the basis of the number of B-box and CCT domains, except for CsBBX13 and CsBBX15, which were not categorized as expected. CsBBX13 was located in clade I but had one B-box and one CCT domain, while CsBBX15 was located in clade V but had two B-box domains and one CCT domain (Figure 1 and Figure S1).

3.3. Protein Motif and Gene Structure Analysis of BBX Members in Cucumber

To understand the structural features of CsBBX proteins, we first created a phylogenetic tree of the CsBBX proteins (Figure 2A and Figure S1). The CsBBX proteins were clustered into five groups (I–V) on the basis of the diversity of their conserved domains following the previous study [3]. All CsBBX proteins had the conserved B-box signature sequence CX2CX8CX7X2CDX3H with conserved cysteine (Cys), histidine (His), and aspartic acid (Asp) (Figure 2A and Figure S1), among which the Asp residue is considered to be involved in transcriptional activation and DNA binding for BBX proteins [14,34].
Subsequently, we analyzed the conserved motifs of CsBBX proteins by MEME and predicted a total of 10 distinct motifs (designated as motifs 1–10). Among them, motif 9 and motif 5 constituted the BBX domain; motif 1 and motif 3 were also annotated as the BBX domain, while motif 2 was associated with the CCT domain (Figure 2B). All CsBBXs had motif 1 and motif 4, with the exception of CsBBX4. Motif 2 was exclusively present in the CsBBXs of group I–III, while motif 6, motif 7, and motif 10 were only found in the CsBBXs of group III, II, and IV, respectively. In addition, all the CsBBXs in group I and CsBBX13 from another group contained motif 8, while motif 5 and motif 9 were exclusively present in the CsBBXs of group II (Figure 2B).
To explore the structural diversity of CsBBX genes, we compared the CDS and corresponding gDNA sequence to illustrate a gene structural diagram (Figure 2C). The results showed that the CsBBX genes had 0–4 introns, and most of them contained 1–3 introns. Amongst them, CsBBX15 had the largest number of introns, whereas three CsBBX genes (CsBBX5, CsBBX8, and CsBBX22) were found to be intronless. Notably, all CsBBX genes in group III possessed only one intron, while the CsBBX genes in group II and group IV had 2–4 introns (except for CsBBX5) (Figure 2C).

3.4. Chromosomal Location and Gene Duplication of the CsBBX Genes

To visualize the location of CsBBX genes on chromosomes, their annotations and positions were downloaded and visualized by MG2C online software. As shown in Figure 3, all CsBBX genes were unevenly located at seven chromosomes of cucumber. Chromosome 3 and chromosome 2 had the smallest (one) and largest (five) number of CsBBX genes, respectively. Chromosomes 4 and 6 contained four CsBBX genes; chromosomes 1 and 7 were observed to each have only three CsBBX genes, followed by chromosome 5, which contained two CsBBX genes. In addition, analysis of gene duplication events indicated that a total of nine pairs of CsBBX genes underwent segmental duplication (Figure 3).

3.5. Cis-Acting Elements in the Promoter Regions of the CsBBX Genes

To determine the potential biological function of CsBBX genes, the PlantCARE server was used to analyze their promoter regions. By screening the function of cis-acting elements, we found 13 and nine types of cis-acting elements associated with hormone and stress responses, respectively (Figure 4). For the hormone-related cis-acting elements, ABRE (involved in abscisic acid (ABA) response) was the most abundant cis-element in CsBBX genes, followed by gibberellin-related elements (TATC-box, P-box, and GARE-motif), and then auxin-responsive elements (AuxRE, TGA-box, AuxRR-core, and TGA-element). Notably, the CsBBX9 gene had 11 ABRE elements in its promoter region, implying that CsBBX9 may play a key role in ABA response. In terms of elements associated with MeJA (CGTCA-motif and TGACG-motif) and salicylic acid (TCA-element), they were widely distributed in 17 and 12 CsBBX genes, respectively. In addition, the CsBBX4 gene had more cis-elements related to MeJA than other CsBBX genes, indicating that it may function in MeJA response. As for stress-related cis-elements, all the CsBBX genes had one to six AREs (related to anaerobic induction) with the exception of CsBBX10 and CsBBX22. Moreover, the GC-motif (related to anoxic specific inducibility) was only present in CsBBX19, and there were three MBS elements (involved in drought response) in CsBBX14. As for the STRE element (involved in stress response), it was widely present in most CsBBX genes (18 out of 22) (Figure 4), suggesting that the CsBBX genes may participate in multiple stress responses.

3.6. Expression Patterns of CsBBX Genes in Different Tissues of Cucumber

To investigate the potential roles of CsBBX genes in plant growth and development, the tissue expression patterns of 22 CsBBX genes were analyzed according to the available RNA-seq data. As shown in Figure 5, all CsBBX genes had expression in at least one tested tissue, with CsBBX3, CsBBX9, CsBBX10, CsBBX12, CsBBX18, CsBBX20, and CsBBX21 having high expression in all tested tissues. Two CsBBX genes (CsBBX2 and CsBBX22) and one CsBBX gene (CsBBX13) were exclusively expressed in the root and tendril, respectively, implying that they may play a critical role in the corresponding tissues. Furthermore, CsBBX6, CsBBX7, CsBBX16, and CsBBX21 were more abundantly expressed in the root, stem, leaf, and flower, respectively, while four CsBBX genes (CsBBX8, CsBBX11, CsBBX12, and CsBBX19) showed the highest expression in the tendril. Interestingly, 10 CsBBX genes (CsBBX1, CsBBX3, CsBBX4, CsBBX6, CsBBX10, CsBBX13, CsBBX15, CsBBX16, CsBBX17, and CsBBX22) exhibited relatively higher expression levels in unfertilized ovaries than in unexpanded ovaries and fertilized ovaries, indicating that they might play a role in ovary development (Figure 5).

3.7. Expression Patterns of CsBBX Genes under Multiple Abiotic Stresses

To investigate the functions of CsBBX genes in response to environmental stimuli, the expression patterns of CsBBX genes under heat and salt stresses were determined according to RNA-seq data from the public available transcriptional database. Upon heat stress, the expression of CsBBX1, CsBBX4, CsBBX14, CsBBX16, CsBBX19, and CsBBX20 decreased significantly compared with the control (0 h), while that of CsBBX7, CsBBX17, CsBBX18, and CsBBX22 was significantly upregulated. Furthermore, the expression levels of CsBBX9, CsBBX11, and CsBBX13 declined at 3 h, while they were induced at 6 h under heat stress (Figure 6A). The transcriptional levels of CsBBX genes in leaf and root samples of cucumber under salt stress are shown in Figure 6B. In leaves, the expression levels of CsBBX8 and CsBBX22 were significantly increased, while those of CsBBX13 and CsBBX18 were significantly inhibited under salt stress. In addition, salt stress obviously downregulated the expression levels of CsBBX2, CsBBX5, and CsBBX13 in roots. These findings suggest that the CsBBX genes might play different roles in response to multiple abiotic stresses.
The transcriptional levels of six selected CsBBX genes under cold and salt stress were further determined by qRT-PCR. Under cold stress, the transcription of CsBBX1 showed no significant change at all test time points, while other CsBBX genes displayed significant decreases in expression at certain time points. Amongst them, CsBBX7 and CsBBX10 were downregulated at the early time points (6 h or 12 h), followed by an increase at the late timepoint (24 h). However, CsBBX8 showed quite different changes in transcription level, as its transcription was significantly increased at 6 h, and then decreased later (Figure 7). Under salt stress, the expression level of CsBBX1 showed no significant change, while that of other CsBBX genes was significantly induced at certain time points (Figure 8).

3.8. Expression Patterns of CsBBX Genes in Response to Biotic Stress

To unravel the role of CsBBX genes in response to biotic stress, the expression data of CsBBX genes under the inoculation with PM, DM, and RKN were downloaded. Under PM inoculation, a total of six and six CsBBX genes were differentially expressed in the D8 (PM-susceptible) and SSL508-28 (PM-resistant) cucumber lines, respectively (Figure 9A). In the PM-susceptible line, CsBBX4, CsBBX7, CsBBX13, and CsBBX16 displayed downregulated expression, while CsBBX22 was upregulated after PM treatment compared with the control. In the PM-resistant line, CsBBX4, CsBBX7, CsBBX12, and CsBBX22 were obviously upregulated, whereas CsBBX15 and CsBBX16 were significantly downregulated after PM treatment (Figure 9A). Under DM inoculation, a total of 13 and 15 CsBBX genes were differentially expressed in the Vlaspik (DM-susceptible) and PI 197088 (DM-resistant) cucumber lines, respectively. Amongst them, CsBBX6, CsBBX11, CsBBX13, and CsBBX15 were downregulated over time in both sensitive and resistant cucumber plants, while two other CsBBX genes (CsBBX4 and CsBBX16) were downregulated at the earlier timepoint (1 dpi), but their expression levels subsequently increased in both sensitive and resistant cucumber plants (Figure 9B). Compared with those in the control, CsBBX7, CsBBX17, and CsBBX19 exhibited upregulated expression after DM treatment at certain time points in the DM-susceptible line, while CsBBX4, CsBBX14, CsBBX18, and CsBBX19 showed upregulated expression at certain time points in the DM-resistant line (Figure 9B). Under RKN inoculation, a total of 10 genes (CsBBX4, CsBBX7, CsBBX8, CsBBX9, CsBBX11, CsBBX13, CsBBX16, CsBBX17, CsBBX19, and CsBBX22) were upregulated, and three genes (CsBBX2, CsBBX5, and CsBBX14) were downregulated in both sensitive and resistant cucumber plants (Figure 9C). However, the expression of CsBBX3 was obviously increased after RKN treatment in CC3 (RKN-susceptible), whereas its expression showed no change in IL10-1 (RKN-resistant) cucumber plants (Figure 9C).

4. Discussion

Plant-specific TFs such as BBXs play crucial roles in different growth and development processes, as well as responses to diverse stresses [1]. In the current study, a comprehensive analysis of the cucumber BBX gene family was carried out on the basis of the updated version cucumber genome (v3), and a total of 22 BBX genes were identified (Table 1). The number of cucumber BBX genes is comparable to that in sorghum (24) [52], grape (25) [36,37], petunia (28) [53], rice (30) [31], tomato (31) [51], and Arabidopsis (32) [3], while the genome size varies greatly among these species. A recent study demonstrated that genome and segmental duplication play a significant role in the expansion of the BBX gene family [12]. In this study, only nine segmental duplication gene pairs were identified, and no tandem duplication was detected (Figure 3). Similar results were also reported in many other plants [12,49,52], suggesting that the BBX gene family in cucumber has been expanded, mainly attributed to segmental duplication.
According to the phylogenetic results of the BBX proteins from cucumber and four other plant species, the CsBBX proteins can also be divided into five clades, and each CsBBX has at least one homolog in these plant species (Figure 1), implying that their functions may be evolutionarily conserved. Protein motif and gene structure analysis revealed that the evolutionarily close CsBBXs exhibit strong conservation in gene architecture and domain organization, but CsBBX genes in group II and group IV tend to have more introns than those in other three groups (no more than two introns) (Figure 2). Both gain and loss of introns may occur during the evolution of genes within families, which may be responsible for their functional divergence. According to the hypothetical model of B-box domain evolution [12,54], it can be speculated that intron gain rather than intron loss has contributed to the different structures of CsBBX genes across different groups (particularly group II and group IV) during their evolution.
Promoters can provide directional support for the expression and possible role of genes in the interaction between plants and environment stimuli [29]. CsBBX promoters include a series of hormone- and stress-related cis-elements (Figure 4), which have also been observed in the promoters of the BBX genes in other plant species, such as Solanum lycopersicum [55], Vitis vinifera [38], Gossypium hirsutum [56], and Brassica rapa [57], and the BBX genes in these plants are regulated by diverse stress treatments. In the present study, six CsBBX genes displayed variations in expression under salt stress according to RNA-seq and qRT-PCR results (Figure 6B and Figure 8), indicating that CsBBX genes might play crucial roles in response to salt stress. Wintersweet (Chimonanthus praecox) CpBBX19 was upregulated by four types of abiotic stress (heat, cold, salt, and drought), and overexpression of CpBBX19 promoted salt and drought stress tolerance in transgenic Arabidopsis plants [58]. In addition, qRT-PCR results showed that five out of the six selected CsBBX genes were differentially expressed under cold stress (Figure 7), suggesting that CsBBX genes might also participate in response to cold stress. A previous report showed that SlBBX7, SlBBX9, and SlBBX20 positively participate in cold stress tolerance in tomato plants [51]. Apple MdBBX37 can fine-tune jasmonic acid (JA)-mediated cold stress tolerance through the MIEL1–BBX37–ICE1–CBF and JAZ–BBX37–ICE1–CBF pathways [59]. In particular, a total of 13 CsBBX genes were found to be responsive to heat stress, and seven CsBBX genes (CsBBX7, CsBBX9, CsBBX11, CsBBX13, CsBBX17, CsBBX18, and CsBBX22) were significantly upregulated after heat stress treatment (Figure 6A), implying that they may be the key regulators of heat tolerance in cucumber. In a recent study, SlBBX17 was found to be induced by heat stress, and its overexpression increased the heat tolerance of tomato by modulating the expression of heat stress-responsive genes [26]. In Arabidopsis, BBX18 plays a vital role in high-temperature-induced hypocotyl growth by not only preventing PRR5 from suppressing PIF4 but also promoting the degradation of ELF3 [5,25]. CsBBX11 and CsBBX19 are close homologs of BBX18 (Figure 1), both of which showed great variations in expression level in response to heat stress (Figure 6A), implying their possible roles in thermomorphogenesis.
Accumulating evidence has demonstrated that BBXs also play essential roles in regulating plant defense responses. For instance, the expression level of two group II BBX genes, OsCOL9 and MaCOL1, was significantly increased after the infection of Magnaporthe oryza and Colletotrichum musae, respectively [60]. In Ipomoea trifida, the expression of 14 and 10 ItfBBX genes was significantly upregulated under β-aminobutyric acid (BABA) and benzothiadiazole S-methyl ester (BTHT) treatments, respectively [61]. In the present study, a total of eight, 15, and 15 CsBBX genes displayed significant variations in expression level after PM, DM, and RKN inoculation, respectively (Figure 9). In addition, many cis-acting elements related to hormone, including JA and SA, were identified in most CsBBX genes (Figure 4), indicating that the CsBBX genes are involved in response to biotic stress and hormonal signal transduction pathways. In a previous study, the expression of 10 VviBBX genes was upregulated, while that of seven other VviBBX genes was downregulated under PM treatment in grape, and the VviBBX genes were also found to be regulated by multiple phytohormones to various degrees [37]. Notably, CsBBX4 was differentially expressed upon the infection of all three phytopathogens in both susceptible and resistant varieties, suggesting that it may be a key regulator of the defense response to PM, DM, and RKN inoculation.

5. Conclusions

In conclusion, genome-wide identification and characterization of the BBX gene family were conducted in cucumber, and the phylogenetic relationship, protein and gene structures, and tissue expression patterns of the CsBBX genes were systematically analyzed. A total of 22 CsBBX genes were identified, and they were distributed across the seven cucumber chromosomes. Amongst them, 11 CsBBX genes made up to nine segmental duplication events, while no tandem duplication events were found. Simultaneously, our analysis found that the CsBBX proteins were clustered into five structural groups (I–V) according to the diversity of their conserved domains, and evolutionarily close CsBBXs exhibited strong conservation in gene structure and conserved motif organization. Furthermore, promoter analysis indicated that CsBBX genes contained many cis-elements that are responsive to various hormones and stresses, and the expression patterns of stress-responsive CsBBX genes were elucidated on the basis of RNA-seq and qRT-PCR results. These findings provide useful information for further functional characterization of CsBBX genes in cucumber growth regulation and stress response, further laying a foundation for cucumber crop improvement.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture12060827/s1: Figure S1. Amino-acid sequence conservation within the B-box domains of cucumber BBX proteins; Table S1. The gene-specific primers used for qRT-PCR; Table S2. List of the cucumber BBX genes identified in this study.

Author Contributions

Data curation, C.Z., L.X., Y.H., L.L. and Y.Z.; funding acquisition, H.L., S.L. and Y.Z.; investigation, C.Z., L.X., L.L., Z.H. and Y.Z.; methodology, C.Z., L.X., Y.H. and S.L.; resources, C.Z., Y.H. and Y.Z.; software, C.Z., L.X., H.L. and Z.H.; validation, L.X.; writing—original draft, C.Z. and Y.Z.; writing—review and editing, S.L. and Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Academic and Technical Leader Plan of Jiangxi Provincial Main Disciplines (20204BCJ22023), the Science and Technology Project of Jiangxi Provincial Department of Education (GJJ200436), and the National Natural Science Foundation of China (31860566).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree analysis of BBX protein sequences. The full-length BBX protein sequences from Arabidopsis, rice, grape, pear, and cucumber were used to construct the phylogenetic tree with the MAFFT and MEGA 7.0 software. AtBBX, OsBBX, VviBBX, PbBBX, and CsBBX represent the BBX family members of Arabidopsis, rice, grape, pear, and cucumber, respectively. The BBX proteins in the phylogenetic tree were divided into five clades (I–V) with different colors.
Figure 1. Phylogenetic tree analysis of BBX protein sequences. The full-length BBX protein sequences from Arabidopsis, rice, grape, pear, and cucumber were used to construct the phylogenetic tree with the MAFFT and MEGA 7.0 software. AtBBX, OsBBX, VviBBX, PbBBX, and CsBBX represent the BBX family members of Arabidopsis, rice, grape, pear, and cucumber, respectively. The BBX proteins in the phylogenetic tree were divided into five clades (I–V) with different colors.
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Figure 2. Phylogenetic tree (A), conserved motif arrangement (B), and gene structure diagram (C) of BBX gene family in cucumber. (A) The NJ phylogenetic tree was created using the MEGA 7.0 program with 1000 bootstrap repeats, and CsBBX proteins in the phylogenetic tree were divided into five groups (I–V) with different colors. (B) Distribution of conserved motifs in CsBBX proteins identified by MEME. The length and name of conserved motifs are represented by boxes with different colors. (C) Gene architecture of the CsBBX genes. The yellow and blue boxes represent the CDSs and UTRs, respectively, while the black lines show the introns.
Figure 2. Phylogenetic tree (A), conserved motif arrangement (B), and gene structure diagram (C) of BBX gene family in cucumber. (A) The NJ phylogenetic tree was created using the MEGA 7.0 program with 1000 bootstrap repeats, and CsBBX proteins in the phylogenetic tree were divided into five groups (I–V) with different colors. (B) Distribution of conserved motifs in CsBBX proteins identified by MEME. The length and name of conserved motifs are represented by boxes with different colors. (C) Gene architecture of the CsBBX genes. The yellow and blue boxes represent the CDSs and UTRs, respectively, while the black lines show the introns.
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Figure 3. Distribution of the CsBBX genes on seven cucumber chromosomes. The scale on the left represents megabases (Mb). The segmental duplication genes are marked with blue lines.
Figure 3. Distribution of the CsBBX genes on seven cucumber chromosomes. The scale on the left represents megabases (Mb). The segmental duplication genes are marked with blue lines.
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Figure 4. Types and numbers of hormone- and stress-related cis-elements in CsBBX promoter regions.
Figure 4. Types and numbers of hormone- and stress-related cis-elements in CsBBX promoter regions.
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Figure 5. Expression levels of CsBBX genes in various cucumber tissues. The expression data were displayed and clustered with TBtools using log2-transformed TPM values (log2 (TPM + 1)).
Figure 5. Expression levels of CsBBX genes in various cucumber tissues. The expression data were displayed and clustered with TBtools using log2-transformed TPM values (log2 (TPM + 1)).
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Figure 6. Expression patterns of the CsBBX genes in cucumber exposed to various abiotic stresses based on data retrieved from available RNA-seq data. (A) The expression heatmap of the CsBBX genes under heat stress at 0, 3, and 6 h. The data in the boxes indicate log2 (TPM + 1) values. (B) The expression heatmap of the CsBBX genes under salt stress. CK-L and Na-L, and CK-R and Na-R are the control and salt-stressed samples of leaves and roots, respectively. The data in the boxes indicate log2 (TPM + 1) values.
Figure 6. Expression patterns of the CsBBX genes in cucumber exposed to various abiotic stresses based on data retrieved from available RNA-seq data. (A) The expression heatmap of the CsBBX genes under heat stress at 0, 3, and 6 h. The data in the boxes indicate log2 (TPM + 1) values. (B) The expression heatmap of the CsBBX genes under salt stress. CK-L and Na-L, and CK-R and Na-R are the control and salt-stressed samples of leaves and roots, respectively. The data in the boxes indicate log2 (TPM + 1) values.
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Figure 7. qRT-PCR analysis of six selected CsBBX genes under cold stress. The seedlings were kept at 4 °C for cold stress, and leaf samples were collected at 0, 6, 12, and 24 h. Data were normalized to the CsAct3 gene expression level, and the value of each CsBBX gene at 0 h was normalized as “1.0”. Different lowercase letters indicate significant differences among all the test data.
Figure 7. qRT-PCR analysis of six selected CsBBX genes under cold stress. The seedlings were kept at 4 °C for cold stress, and leaf samples were collected at 0, 6, 12, and 24 h. Data were normalized to the CsAct3 gene expression level, and the value of each CsBBX gene at 0 h was normalized as “1.0”. Different lowercase letters indicate significant differences among all the test data.
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Figure 8. qRT-PCR analysis of six selected CsBBX genes under salt stress. The seedlings were treated with 200 mM NaCl for salt stress, and leaf samples were collected at 0, 6, 12, and 24 h. Data were normalized to the CsAct3 gene expression level, and the value of each CsBBX gene at 0 h was normalized as “1.0”. Different lowercase letters indicate significant differences among all the test data.
Figure 8. qRT-PCR analysis of six selected CsBBX genes under salt stress. The seedlings were treated with 200 mM NaCl for salt stress, and leaf samples were collected at 0, 6, 12, and 24 h. Data were normalized to the CsAct3 gene expression level, and the value of each CsBBX gene at 0 h was normalized as “1.0”. Different lowercase letters indicate significant differences among all the test data.
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Figure 9. Expression profiles of CsBBX genes in cucumber inoculated with PM (A), DM (B), and RKN (C). (A) Expression of CsBBX genes in response to PM treatment in cucumber cultivars D8 (PM-susceptible) and SSL508-28 (PM-resistant). (B) Expression of CsBBX genes in Vlaspik (DM-susceptible) and PI 197088 (DM-resistant) cultivars of cucumber after DM inoculation. Mock, control sample. (C) Expression of CsBBX genes at different time points after infection with M. incognita in a RKN-susceptible line (CC3) and RKN-resistant line (IL10-1). The expression data were displayed and clustered with TBtools using log2 (TPM + 1) values; dpi, days after inoculation.
Figure 9. Expression profiles of CsBBX genes in cucumber inoculated with PM (A), DM (B), and RKN (C). (A) Expression of CsBBX genes in response to PM treatment in cucumber cultivars D8 (PM-susceptible) and SSL508-28 (PM-resistant). (B) Expression of CsBBX genes in Vlaspik (DM-susceptible) and PI 197088 (DM-resistant) cultivars of cucumber after DM inoculation. Mock, control sample. (C) Expression of CsBBX genes at different time points after infection with M. incognita in a RKN-susceptible line (CC3) and RKN-resistant line (IL10-1). The expression data were displayed and clustered with TBtools using log2 (TPM + 1) values; dpi, days after inoculation.
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Table
Table 1. Identification and characterization of BBX gene family in cucumber.
Table 1. Identification and characterization of BBX gene family in cucumber.
GeneAccession No. (v3)Chromosome: LocationProteinGroup
CDS/bpAApIMW/kDaGRAVYSubcellular Localization
CsBBX1CsaV3_1G003800chr1: 2,364,830–2,367,23310833605.25 41.63 −0.949 NucleusIII
CsBBX2CsaV3_1G011690chr1: 7,244,708–7,246,4267952645.29 29.49 −0.316 NucleusV
CsBBX3CsaV3_1G031230chr1: 18,437,778–18,448,74811343775.85 41.24 −0.308 NucleusI
CsBBX4CsaV3_2G008220chr2: 4,503,501–4,505,2289603198.20 35.16 −0.395 NucleusI
CsBBX5CsaV3_2G015160chr2: 12,644,521–12,645,3745431806.64 19.76 −0.301 NucleusIV
CsBBX6CsaV3_2G029230chr2: 19,152,113–19,154,4099213066.65 32.67 −0.221 NucleusIV
CsBBX7CsaV3_2G032640chr2: 21,557,067–21,559,99610143376.08 36.93 −0.505 NucleusI
CsBBX8CsaV3_2G035230chr2: 23,569,718–23,570,6604021338.40 14.66 −0.041 NucleusV
CsBBX9CsaV3_3G044220chr3: 36,085,222–36,088,6418252744.31 30.15 −0.829 NucleusV
CsBBX10CsaV3_4G002870chr4: 1,790,472–1,794,05914764915.50 54.36 −0.545 NucleusII
CsBBX11CsaV3_4G005810chr4: 3,819,528–3,822,9145611866.44 20.76 −0.605 NucleusIV
CsBBX12CsaV3_4G008210chr4: 5,753,161–5,758,5568942975.31 32.30 −0.384 NucleusIV
CsBBX13CsaV3_4G009980chr4: 7,751,656–7,755,20210353445.24 38.47 −0.772 NucleusIII
CsBBX14CsaV3_5G034320chr5: 27,321,989–27,323,30612124035.55 45.47 −0.710 NucleusIII
CsBBX15CsaV3_5G034710chr5: 27,519,572–27,522,23614644876.81 54.84 −0.614 NucleusII
CsBBX16CsaV3_6G003540chr6: 2,826,773–2,829,26810053348.33 38.40 −0.823 NucleusIII
CsBBX17CsaV3_6G006790chr6: 5,555,910–5,558,0077142374.89 26.09 −0.285 NucleusIV
CsBBX18CsaV3_6G009750chr6: 7,889,202–7,896,87012484155.14 45.47 −0.481 NucleusII
CsBBX19CsaV3_6G046900chr6: 27,677,337–27,679,7975071686.59 18.86 −0.567 NucleusIV
CsBBX20CsaV3_7G000270chr7: 395,757–397,68910503495.91 39.33 −0.359 NucleusIV
CsBBX21CsaV3_7G002350chr7: 1,863,318–1,866,69112244075.42 44.56 −0.520 NucleusII
CsBBX22CsaV3_7G003780chr7: 2,810,583–2,811,4463991326.40 14.63 −0.242 NucleusV
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Zhu, C.; Xiao, L.; Hu, Y.; Liu, L.; Liu, H.; Hu, Z.; Liu, S.; Zhou, Y. Genome-Wide Survey and Expression Analysis of B-Box Family Genes in Cucumber Reveal Their Potential Roles in Response to Diverse Abiotic and Biotic Stresses. Agriculture 2022, 12, 827. https://doi.org/10.3390/agriculture12060827

AMA Style

Zhu C, Xiao L, Hu Y, Liu L, Liu H, Hu Z, Liu S, Zhou Y. Genome-Wide Survey and Expression Analysis of B-Box Family Genes in Cucumber Reveal Their Potential Roles in Response to Diverse Abiotic and Biotic Stresses. Agriculture. 2022; 12(6):827. https://doi.org/10.3390/agriculture12060827

Chicago/Turabian Style

Zhu, Chuxia, Lingdi Xiao, Yaqi Hu, Liu Liu, Haoju Liu, Zhaoyang Hu, Shiqiang Liu, and Yong Zhou. 2022. "Genome-Wide Survey and Expression Analysis of B-Box Family Genes in Cucumber Reveal Their Potential Roles in Response to Diverse Abiotic and Biotic Stresses" Agriculture 12, no. 6: 827. https://doi.org/10.3390/agriculture12060827

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