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Article

Genome-Wide Identification of B3 DNA-Binding Superfamily Members (ABI, HIS, ARF, RVL, REM) and Their Involvement in Stress Responses and Development in Camelina sativa

by
Mahmoud Kandeel
1,2,*,
Mohamed A. Morsy
3,4,
Hany M. Abd El-Lateef
5,6,
Mohamed Marzok
7,8,
Hossam S. El-Beltagi
9,10,
Khalid M. Al Khodair
11,
Ibrahim Albokhadaim
1 and
Katharigatta N. Venugopala
3,12
1
Department of Biomedical Sciences, College of Veterinary Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia
2
Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
3
Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia
4
Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia 61511, Egypt
5
Department of Chemistry, College of Science, King Faisal University, Al-Ahsa 31982, Saudi Arabia
6
Department of Chemistry, Faculty of Science, Sohag University, Sohag 82524, Egypt
7
Department of Clinical Sciences, College of Veterinary Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia
8
Department of Surgery, Faculty of Veterinary Medicine, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
9
Agricultural Biotechnology Department, College of Agriculture and Food Sciences, King Faisal University, Al-Ahsa 31982, Saudi Arabia
10
Biochemistry Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
11
Department of Anatomy, College of Veterinary Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia
12
Department of Biotechnology and Food Science, Faculty of Applied Sciences, Durban University of Technology, Durban 4000, South Africa
 
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Author to whom correspondence should be addressed.
Agronomy 2023, 13(3), 648; https://doi.org/10.3390/agronomy13030648
Submission received: 4 January 2023 / Revised: 13 February 2023 / Accepted: 21 February 2023 / Published: 23 February 2023
(This article belongs to the Special Issue GMO and New Breeding Techniques for Abiotic Stress Tolerance in Crops)
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Abstract

:
The B3 DNA-binding superfamily is a group of gene families that contain a B3 domain in their proteins. Members of this superfamily are involved in responses to stresses including salt, drought, and cold stress. The B3 DNA-binding superfamily has not been fully studied in Camelina sativa. A total of eighty-seven members of this superfamily were identified in C. sativa. The identified genes were placed into five groups based on a phylogenetic analysis. All the proteins were predicted to be nuclear. The RAV and ARF gene family had the most conserved motifs, with nine out of ten motifs being preserved, while the REM gene family was discovered to have the fewest, with just one conserved motif being present. The RAV and REM gene families showed the least protein–protein interactions. The CsARF5 and CsARF7 genes showed the highest potent interaction score with multiple auxin-responsive proteins. A qPCR analysis was carried out on six genes that showed stress-induced expression changes. CsREM17, CsREM5, and CsRAVL5 were discovered to be considerably increased in response to drought stress, while CsARF10, CsARF4, and CsREM34 were found to be downregulated to a large extent. The B3 DNA-binding superfamily regulates abscisic acid signaling, which in turn influences plant growth and stress resistance.

1. Introduction

Camelina sativa is an oil-yielding and flowering plant with distinguished four-petaled, pale yellow-colored, and cross-shaped flowers of the family Brassicaceae that is commonly known as the “Gold of Pleasure”, “False Flax”, “Wild Flax”, “German Sesame”, and sometimes “Siberian Oilseed”. C. sativa is natively cultivated as an oilseed crop in North America, Europe, and some areas of Central Asia [1]. Oil from C. sativa is typically used in cooking or as fuel or burnt in lamps [2]. C. sativa is being researched due to its unusually high contents of omega-3 fatty acids (up to 45%) [3]. Its seeds contain up to 43% oil that is rich in natural antioxidants, up to 32% protein [4], and up to 3% erucic acid. These seeds contain up to 110 mg of vitamin E per 100 g, and they are well suited for cooking due to their unique aroma and flavor resembling that of almond [5]. Its oil is registered under the brand name “Olej rydzowy tradycyjny” in the European Union as a traditional specialty guaranteed product [6]. The cultivation period of C. sativa is short, and it can only be cultivated in temperate climate zones in light or medium soil. It is seeded in the spring season from March to May, and in favorable conditions the yield of seeds can reach up to 2700 kg/ha [7]. This all makes C. sativa a very important plant to work on. Previously, little work has been conducted on this plant, especially in the context of the B3 DNA-binding super family (ABI3, HIS, ARF, RAV, and REM) [8].
Abiotic stresses on plants are caused by salinity [9], drought, and cold [10], as well as several other factors that are caused by global climate change. In this study, we will discuss the role of the B3 DNA-binding superfamily in the fight against drought, salt, and cold stress, as well as its role in the development of different parts of the plant body. The B3 DNA-binding superfamily plays a significant role in the response to stresses in plants, as well as in the development of different parts of the plant body [11]. Here, we studied the role of this gene family in the development of the roots, leaf, stem, and flower of C. sativa together with its involvement in drought, salt, and cold stresses in the same plant [12].
The B3 DNA-binding superfamily is a group of families consisting of ABI3 (abscisic acid insensitive 3) [13], HIS (high-level expression of sugar-inducible genes) [14], ARF (auxin response factors) [15], RAV (related to ABI3/VP1) [16], and REM (reproductive meristem) [17]. All of these gene families have relatively different functions. Proteins with the B3 domain are found to be involved in many plant processes. The ABI3 gene family is a transcriptional activator family, and the HIS gene family is a repressor, which is also involved in the development and maturation of plant seeds [18]. The RAV gene family is not well characterized, but the members of this family are thought to be involved in development, growth, and flowering time in various plants [19]. The ARF gene family is the only one among the five that has been studied extensively, and it has been identified to have functional importance in response to auxins as well as a role in the regulation of plant systems [20]. The REM gene family is not well studied, and thus we have less information about its function and involvement in plant processes [21]. The B3 DNA-binding superfamily is predominantly involved in hormone and signaling pathways such as auxins, gibberellins, and abscisic acid. The main reason for grouping all these families together as one superfamily is that all the proteins of these families share a common domain, namely the B3 domain [22]. In this study, the B3 DNA-binding superfamily was assessed using a number of bioinformatics analyses to identify the genes in C. sativa and the effects of salt, drought, and cold stresses on the expression of these genes.

2. Materials and Methods

2.1. Plant Materials, Treatments, and Growth Conditions

C. sativa plants were grown in a growth chamber for three weeks at a humidity level of 60% and a temperature of 20 °C ± 5 °C. Camelina is much more resilient to cold temperatures than most oil seeds and can also sustain itself through drought conditions. Sixteen hours were spent exposing the plants to light, followed by an eight-hour period in which the plants were exposed to darkness each day. Upon the germination of the seedlings, they were treated with a 1 M NaCl solution for the treatment of salt stress after two weeks of growing. The preliminary treatment of the plants was conducted using a Hoagland solution (a mixture of many essential nutrients for the growth and development of plants) [23,24]. The plants were divided into two groups: “treatment” and “control.” They were all given water every day before the plants were stressed (treatment). After that, the “treatment” plants were still given water every day, but the “control” plants were only given water with a special solution called Hoagland solution. No water or treatment was given to the plants that were under drought stress. A week after the plants had been subjected to drought stress, the first batch of samples was collected, and the samples were quickly frozen using liquid nitrogen and then stored at −80 °C until extracted for qPCR. During plant growth, no changes in plant phenotype were observed after the 3rd day of stresses application. The plants started to wilt after the sixth day.

2.2. Genome-Wide Identification of B3 DNA-Binding Superfamily in C. sativa

Members of the B3 DNA-binding superfamily were found on the TAIR database [25], and the sequences of the members of the family along with their accession numbers were retrieved from the NCBI database, which were annotated for further analysis using a comprehensive text editor Notepad++ [26]. Homologs of C. sativa were identified using NCBI BLAST, and the results were manually verified using NCBI and UniProt [27].

2.3. Phylogenetic Analysis

The B3 DNA-binding superfamily genes in Arabidopsis thaliana were retrieved from the TAIR database [28]. These sequences were used to retrieve the corresponding genes from C. sativa in the NCBI gene repository. The identified sequences were then subjected to SIAS tools (developed by the Immunomedicine Group) to check for redundancy. After analyzing the protein sequences from different species, an analysis known as phylogenetic analysis was carried out to check their evolutionary importance and the relationships between the proteins from different species [29]. A maximum likelihood tree was constructed using IQ-Tree based on 251 protein sequences from A. thaliana, S. alba, and C. sativa (Figure 1). The derived tree was then colored and decorated using the online server-based tool iTOL [30].

2.4. Characteristics of B3 Domain-Containing Proteins

The physicochemical properties of the B3 DNA-binding superfamily proteins were predicted using the online server-based tool ProtParam, which is hosted by the Swiss Institute of Bioinformatics [31], and the sub-cellular localization of the proteins was predicted using another online server-based tool, ProtComp 9.0, which is hosted by the famous bioinformatics tool website Softberry [32].

2.5. Gene Structure and Motif Analysis of B3 Proteins

The exon and intron structures of the B3 DNA-binding superfamily genes were predicated using GSDS (Gene Structure Display Server Tool v2.0), which takes the CDS and genomic sequences of the genes to predict the structure of the genes of the family [33]. The motifs of the SBP-box gene family were identified using MEME (Multiple Em for Motif Elicitation), in which the site distribution was kept at zero or one sequence per occurrence, and the number of motifs was set to 10 (could be adjusted to any value within an acceptable range). This took only the protein sequence as the input and gave the motifs of the protein as the result, and the motif logos were also designed by MEME [34].

2.6. Promoter Region and Regulatory Elements Analysis

A 2000 bp upstream region (promoter and UTR regions) of every gene of the superfamily (B3 DNA-binding superfamily) was retrieved from the NCBI nucleotide database [35], and the sequences were stored in a file. The cis-regulatory elements were predicted by the online server-based tool Plantcare, which utilizes the promoter and UTR region sequences of the genes [36]. The Plantcare database takes only a single sequence at a time and took roughly an hour, sometimes more, to process the task, but once we had the results for all of the genes, we compiled and visualized the results using tbTools, which is an open-source program hosted on GitHub [37].

2.7. Protein–Protein Interaction and Domain Analysis

To study the protein–protein interactions, a web-based tool called STRINGdb was used. All the sequences of the B3 DNA-binding superfamily proteins were pasted into the search box, and the tool carried out all the work automatically [24]. Manual intervention was required to rectify certain aspects, such as clustering (5 clusters using K-mean clustering), the arrangement of protein nodes, etc. The lines of interaction were set to be dotted and straight to represent the weak and strong interactions, respectively [38].
Protein domains were identified by utilizing the NCBI conserved domain database, which is commonly known as the Web CD-Search Tool. It takes a protein batch as an input and returns the domains present in the proteins as results in the form of tabular data, which were used to visualize the domains using tbTools [39].

2.8. Gene Expression Analysis Using NGS Data

To study the expression of the genes of the B3 DNA-binding superfamily, the transcriptomic data on root (SRR935368), leaf (SRR935362), stem (SRR935365), and flower (SRR935369), as well as on the abiotic stresses of drought (SRR935373), NaCl (SRR935377), and cold (SRR935370), were retrieved from an NCBI database called the SRA database [40]. All the available reads were mapped to the C. sativa genome using a tool of the online web-based toolkit Galaxy, BOWTIE2 [41]. Cufflinks, which is another tool of Galaxy, was used to calculate the expression levels of the genes of the gene family in the form of the FPKM values, which were utilized to make the head map of the gene expression using tbTools, which makes it easy to understand visually, and the results were clustered based on expression [42].

2.9. RNA Extraction, cDNA Synthesis, and qRT PCR

Thermo Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the amount of total RNA that was isolated from C. sativa leaf samples using the Trizol technique. A First-Strand Synthesis kit was used to synthesize cDNA from 1 µg of RNA. To prepare it for future analysis, the cDNA was kept at −20 °C. A qRT PCR analysis was carried out using an iTaq Universal SYBR Green Super-Mix and a qRT PCR detection system (CFX96 TouchTM RT PCR Detection System, Bio_Rad Labs, Hercules, CA, USA). Gene-specific primers were designed using the internet tool Oligo Calculator (http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html/ (accessed on 13 July 2022)), which were then confirmed by the NCBI-primer BLAST algorithm (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (accessed on 13 July 2022)) (Supplementary Table S1). Each gene’s expression was analyzed in triplicate, and the CsGAPDH gene served as the housekeeping gene.

3. Results

3.1. Identification of B3 Genes

A total of 86 B3 genes were found to be present in A. thalian, of which 86 were identified in C. sativa. All of these genes were part of a superfamily that comprises five families.
The ABI (abscisic acid insensitive) gene family comprises three members, ABI3, LEC2, and FUS3. All three members of this family play important roles in plant development. The ABI3 gene is important for seed maturation and functions as a regulator for the transition between embryo development and early seedling maturation. It also interacts with other members of the family to regulate the accumulation of chlorophyll and anthocyanin. LEC2 and FUS3 regulate and ensure the abundance of ABI3 proteins in seeds [43].
The HSI (high-level expression of sugar-inducible) gene family is a B3 domain containing a family redundantly responsible for the events after the germination of seeds [44]. Genes of this family have been found to be overexpressed when subjected to drought stress [45].
The ARF (auxin response factors) family comprises 23 members in A. thaliana and the same number in C. sativa, of which all possess a B3 domain in addition to the AUX_IAA superfamily domain and Auxin_resp domain. The members of this family are involved in the hormone regulation and development of plants [46], including the elongation of shoots, formation of lateral roots, differentiation of vascular tissues, apical margin patterning, and response to environmental stimuli [47].
RAV (related to ABI3/VP1) is a transcription factor gene family that comprises two domains, one B3 domain at the N-terminus and an AP2 domain at the C-terminus. In A. thaliana, the family contains 13 members, and the same number was identified in C. sativa [48]. Members of this gene family are involved in hormone signaling, along with the growth of organs and tissues in various plants, and it plays a crucial role in response to biotic and abiotic stresses in plants [17].
The REM (reproductive meristem) gene family is part of the B3 DNA-binding superfamily. Over the years, a number of researchers and institutes have studied this gene family, but we have very little knowledge about its function in plants [17]. Here, we studied the involvement of the REM gene family in the development and growth of different parts of C. sativa, as well as in responses to abiotic stresses such as drought, salt, and cold stress in the same plant. There were 45 members of the REM gene family in A. thaliana, of which 44 were identified in C. sativa.

3.2. Phylogenetic Analysis of B3 Genes

The maximum likelihood tree that was constructed based on 251 protein sequences from A. thaliana, S. alba, and C. sativa showed that all the members of the gene family are present in a total of five groups based on the families of the superfamily [49]. Members such as AtABI3, SaABI3, and CaABI3 belong to the ABI3 gene family; AtHSI2, SaHSI2, and CsHSI2 belong to the HIS gene family; AtNGA4, SaNGA4, and CsNGA4 belong to the RAV gene family; AtARF1, SaARF1, and CsARF1 belong to the ARF gene family; AtREM1, SaREM1, and CsREM1 belong to the REM gene family; and so on. Each group had approximately the same number of members from different species. Almost all their homologs were available in the NCBI database and were retrieved for phylogenetic analysis [50]. Members of the ABI group, such as CsLEC2 and AtLEC2, were found to be orthologs, and members of the HIS group, such as CsHSI and AtHSI, CsHSI2L2 and AtHSI2L1, and CsHSI2L2 and AtHSI2L2 were orthologs. Members of the RAV group, such as CsRAVL2 and AtRAVL2, CsRAVL3 and AtRAVL3, CsRAV1.2 and AtRAV1.2, CsRAV2 and AtRAV2, CsNGA1 and AtNGA1, CsNGA3 and AtNGA3, and CsTEM1 and AtTEM1 were found to be orthologs. Members of the ARF group, such as CsARF1 and AtARF1, CsARF2 and AtARF2, CsARF3 and AtARF3, CsARF4 and AtARF4, CsARF5 and AtARF5, CsARF7 and AtARF7, CsARF8 and AtARF8, CsARF9 and AtARF9, CsARF10 and AtARF10, CsARF11 and AtARF11, CsARF16 and AtARF16, and CsARF19 and AtARF19 were found to be orthologs. Members such as CsARF12 and CsARF20, CsARF22 and CsARF14, and CsARF21 and CsARF15 were found to be paralogs. Members of the REM group, such as CsREM2 and AtREM2, CsREM3 and AtREM3, CsREM4 and AtREM4, CsREM5 and AtREM5, CsREM6 and AtREM6, CsREM9 and AtREM9, CsREM10 and AtREM10, CsREM11 and AtREM11, CsREM12 and AtREM12, CsREM13 and AtREM13, CsREM14 and AtREM14, CsREM15 and AtREM15, CsREM16 and AtREM16, CsREM17 and AtREM17, CsREM20 and AtREM20, CsREM21 and AtREM21, CsREM22 and AtREM22, CsREM23 and AtREM23, CsREM24 and AtREM24, CsREM25 and AtREM25, CsREM26 and AtREM26, CsREM27 and AtREM27, CsREM28 and AtREM28, CsREM29 and AtREM29, CsREM30 and AtREM30, CsREM31 and AtREM31, CsREM32 and AtREM32, CsREM33 and AtREM33, CsREM34 and AtREM34, and CsREM40 and AtREM40 were found to be orthologs. Members such as CsREM7 and CsREM8, CsREM18 and CsREM19, CsREM35, CsREM36 and CsREM37, CsREM38 and CsREM39, and CsREM41 and CsREM42 were found to be paralogs.

3.3. Characteristics of B3 Domain-Containing Proteins

The physiochemical properties of the 3 ABI3, 3 HSI, 23 ARF, 13 RAV, and 44 REM proteins are represented in the form of a table (Table 1). After analyzing the protein sequences, it was found that the members of the HIS and ARF gene families were relatively higher in terms of their amino acid count and were found to have higher molecular weights as well. The isoelectric points (the value of pH at which the net charge on a protein molecule is zero) of the proteins were also predicted theoretically and found to be about 7.05 on average for all proteins. All the proteins were predicted to be nuclear, which means that their role is likely associated with the nucleus of the cell. In addition, several other properties of the proteins were found and are provided in Table 1.

3.4. Gene Structure and Conserved Motif Analysis of B3 Proteins

The exon and intron structures of B3 genes are shown in Figure 2, and the gene structures are grouped based on the phylogenetic results. The number of introns differs greatly between the genes of the B3 DNA-binding superfamily, as well as within individual families [51]. Some members of the RAV family were found to have genes consisting of only a single whole exon with no intron present in them, and some members of the ARF family were found to have much higher exon counts. A motif analysis revealed that the motifs are conserved among families of the superfamily but vary greatly in their level of conservation [52]. The motif “LFEKTLTASDTSTHGGLSVPKRHAEKCFP” was highly conserved in the ABI3, HIS, RAV, and ARF gene families, and is labeled number 2 in Figure 2. Motif 1, with the sequence “AKDLHGNEWRFRHSYRGSPRRHLLTTGWSRFVKTKKLVAGD”, was also conserved in the RAV gene family. The ARF gene family had the highest number of the conserved motifs in their sequences, and nine of the ten motifs were found to be conserved in almost all the members of this gene family. The REM gene family was found to have the smallest number of conserved motifs, and there was only a single motif, “GWKEFVEDHDLRDGDFLVFRHDGDMVFH”, which is labeled motif 7, that was found to be conserved in all the sequences of this gene family.

3.5. Promoter Region and Regulatory Elements Analysis

Cis-regulatory sequences or enhancers and promoters (cis-regulatory elements) are critical for controlling the expression of genes, thereby determining how an organism develops and how it functions. In some species, some mutations have the potential to contribute to the variation of phenotypic expression by causing changes to these sequences (regulatory elements). In the present study, the 2000 bp promoter sequences of eighty-six C. sativa sequences were analyzed to identify sixty-three types of cis-regulatory elements (Figure 3). Each gene promoter had certain regulatory elements, and the most common was the TATA-box. This element helps determine the direction of gene transcription and the strand of DNA that should be read by the transcription machinery [53]. ARE regulates gene expression under low-oxygen conditions, and ABRE is a target for the bZIP protein, which is a basic helix–loop–helix transcription factor. Box-4, G-box, and motifs such as TGACG, CGTCA, TCT, and TG1 are also involved in gene regulation. Lastly, LTR regulates gene expression in response to light [54]. MBS (MYB-binding site involved in drought response) regulates gene expression in response to drought stress through interaction with MYB proteins. Similarly, the MRE (MYB-binding site involved in light responsiveness) regulates gene expression in response to light. Another important regulatory element is the O2 site, which helps regulate the expression of transcription factors involved in carbon and amino acid metabolism and abiotic stress resistance. The CAT-box is also a cis-acting regulatory element that is specifically involved in the regulation of gene expression related to meristem development. The AT-rich element plays a role in the initiation of DNA replication and synthesis. Lastly, I-box, ACE, and A-box are additional cis-acting regulatory elements that contribute to gene expression regulation in plants [55]. Additionally, there are several other cis-acting DNA regulatory elements that have been found to be present in smaller numbers in plants. These elements include the CAG-motif, AuxRE, L-box, LS7, and HD-Zip 1. These elements play a role in gene expression regulation, but their exact function and significance are still being studied.

3.6. Protein–Protein Interaction and Domain Analysis

The domain analysis of C. sativa showed that all the members of the B3 DNA-binding superfamily had a conserved domain, which was the B3 domain. The proteins of the ABI and HSI gene families had only a single domain (B3 domain). Members of the ARF gene family had an Auxin_resp domain and AUX_IAA domains in addition to the B3 domain, some members of the RAV gene family had an AP2 domain in addition to a B3 domain, and members of the REM gene family also had only a single domain (B3 domain) (Figure 4).
A protein–protein interaction analysis showed that members of the superfamily were interacting with one another and with other proteins in the cell, while some were not interacting with any other proteins. Most of the proteins that did not show any interaction with other proteins were members of the RAV and REM gene families. Only one member of the RAV gene family (CsRAVL1) showed a very strong interaction with XP_010452952.1 (succinate dehydrogenase subunit 3-1, mitochondrial) and several related but uncharacterized proteins. Members of the ARF gene family showed strong interactions overall with other proteins of the family, some members of the RAV gene family, and other proteins in the cell. The strongest interaction score was found between CsARF5 and CsARF7 and XP_010457463.1, XP_010475075.1, XP_010457463.1, and XP_010483317.1. All these are auxin-responsive proteins, indicating multiple and strong interactions between single CsARF and several proteins. The complete interaction profile is provided in Supplementary Table S2.

3.7. Gene Expression Analysis Using NGS

The current study involves the transcriptomic sequencing data analysis of the B3 DNA-binding superfamily in the development of the flowers, stem, root, and leaf and in cold, salt, and drought stress [56]. It was seen that members of the superfamily, such as CsARF8, CsREM8, CsREM12, CsREM34, CsRAVL3, CsREM26, CsREM33, CsRAVL1, CsREM3, CsREM7, CsREM13, CsREM18, CsREM20, CsREM24, CsREM11, CsREM15, CsREM25, CsNGA1, CsNGA2, CsREM28, CsNGA3, and CsRAVL2 were highly upregulated in the development of flowers, while some other members showed relatively less expression than those mentioned above in the expression profile. Some members of a gene superfamily showed upregulation in response to cold stress (CsREM1, CsARF18, CsRAV1.2, etc.) (Figure 5). A few members were upregulated for drought stress (CsHSI2, CsREM7, etc.) and some were downregulated (CsARF10, CsARF4). Some members showed no expression for any of the observed stresses (CsREM38, CsABI3, etc.).

3.8. RNA Extraction, Gene Expression, and qRT-PCR Analysis

Using real-time amplification (qRT-PCR) to calculate the transcript abundance of six B3 genes from C. sativa leaves, the transcript abundance of all six genes was calculated (Figure 6). Abiotic stressors such as drought were applied to C. sativa. The genes expressed differently in response to drought stress. The qPCR-based quantification of six genes was conducted in response to drought, including CsREM17, CsREM5, CsRAVL5, CsARF10, CsARF4, and CsREM34. The differential regulation of all B3 genes was observed after three days and seven days of drought stress. When exposed to drought stress, CsREM17, CsREM5, and CsRAVL5 were found to be strongly elevated, and genes such as CsARF10, CsARF4, and CsREM34 were found to be downregulated to a very high degree.

4. Discussion

As the B3 DNA-binding superfamily contributes to the development of plants as well as their adaptation to abiotic stresses [48], genes of this superfamily have been identified and studied in several plant species. However, the B3 genes in oil-yielding plants such as C. sativa have yet to be studied thoroughly [57]. In the present study, we conducted extensive research on these crucial genes in C. sativa that involved the identification of these B3 genes; the prediction of their physiochemical properties; the classification of these genes into distinct groups via a phylogenetic analysis; an analysis of the gene structure and the protein conserve motif [58]; a promoter region and cis-regulatory element analysis; the identification of the expression patterns of these genes in various tissues of C. sativa and under salt, drought, and cold stresses; and a qPCR analysis of the differentially expressed genes under drought stress [59].
A total of 87 B3 genes were found to be present in A. thaliana, of which 86 were identified in C. sativa and 83 were identified in S. alba. The genome sizes of A. thaliana, C. sativa, and S. alba were 119.75 Mb, 453.11 Mb, and 201.60 Mb, respectively. The B3 genes were clustered into five distinct groups that were the families within the superfamily. The characteristics of the B3 domain-containing proteins showed that practically all of these proteins were present inside the nucleus, and the GRAVY (grand average of hydropathy) was less than zero for all the proteins in the superfamily [60]. The domain analysis of these genes showed that all of the proteins had a common conserved domain, namely the B3 domain, because all of these genes belong to the B3 DNA-binding superfamily. In addition to this, the domain members of the ARF family had two more domains, namely the AUXIN_resp and AUX_IAA domains [17], and some of the members of the RAV gene family had another domain, the AP2 domain. More than 60 stress and hormone-related types of cis-regulatory element were also identified from the promoter regions of the genes. To understand the involvement of these genes in the plant functions of C. sativa, the expression pattern of 86 B3 genes in the development of the root, shoot, leaf, and flower, as well as the expression pattern of these genes when the plant was under salt, drought, and cold stresses, was observed [61]. When exposed to drought stress, CsREM17, CsREM5, and CsRAVL5 were found to be strongly elevated/upregulated, and genes such as CsARF10, CsARF4, and CsREM34 were found to be downregulated to a very high degree. It has been shown that both CsARF4 and CsARF10 play important roles in the auxin response during plant development and growth. Meristem development is supported by CsREM17, CsREM5, and CsREM34. Abscisic acid intolerance is caused by CsRAVL5. Therefore, these genes share in the regulation of plant growth and stress response through their modulation of abscisic acid’s function.
In the citrus B3 superfamily, CsREM17 and CsREM5 play key roles in various plant physiological development processes such as somatic embryogenesis. During differentiation, CsREM17 expression in particular showed higher levels in embryogenic calluses, indicating its potential association with EC initiation during cold or drought stress, which was revealed in our study [62]. As for the role of CsRAVL5 during cold or drought stress, it seems to be novel as it has not been reported previously. Moreover, this corroborates findings showing that the majority of genes associated with the B3 family showed differential expression in root tissues under stress conditions when compared to control tissue [63].
CsARF10a, CsARF10b, and CsARF10c auxin response factor (RF10) genes revealed that CsARF10a has a closer evolutionary relationship with SlARF10, but CsARF10b and CsARF10c had a stronger resemblance with AtARF10. The quantitative qRT-PCR analysis showed that the CsARF10 gene subfamily had high transcriptional levels in leaves, stems, root, and flowers. Furthermore, the qPCR findings revealed that, despite having a comparable transcriptional pattern, CsARF10a had a high expression level, but CsARF10b and CsARF10c mRNA levels were comparatively low [64]. The CsARF4 gene is known as a key potential gene family with a role in reproductive growth [65]. The downregulation of CsARF10, CsARF4, and CsREM34 genes appears to be important during drought stress. Drought control, for example, has been found to be a possible factor in drought stress that involves downregulating the ethylene and methionine biosynthesis pathways by Medicago [66].

5. Conclusions

The B3 domain and its associated proteins are involved in many of plants’ vital functions such as their responses to a number of stresses, including salt, drought, and cold stresses, as well as in the development of plants. However, its significance has not been well studied, and this domain has been given little attention by researchers or cultivators. The B3 DNA-binding superfamily and individual gene families within this superfamily have been studied in different plant species, but their role has not been fully disclosed in C. sativa (false flax). Through this study, it was determined that 86 members of this superfamily were identified in C. sativa. The identified genes were placed into five groups (families) based on a phylogenetic analysis. RNA seq data was used for the gene expression analysis of the B3 genes, which revealed the expression pattern of the genes under different stresses and in different plant tissues. The strongest interaction score was identified between CsARF5 and CsARF7 and XP_010457463.1, XP_010475075.1, XP_010457463.1, and XP_010483317.1. These were all auxin-responsive proteins, demonstrating the multiple and robust interactions of one CsARF with various proteins. Finally, the gene expression pattern of this set of genes affects plants’ growth and resistance to stress through the modulation of abscisic acid signaling. This study paves the way for the better cultivation of C. sativa all over the globe.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13030648/s1, Table S1: The primers used in the qPCR studies and their properties. Table S2: The outputs of the protein–protein interaction profiles.

Author Contributions

Conceptualization, M.K. and M.A.M.; methodology, M.K.; software, M.K.; validation, M.K., H.M.A.E.-L., and M.M.; formal analysis, M.K.; investigation, K.N.V. and I.A.; resources, M.K.; data curation, M.K. and H.S.E.-B.; writing—original draft preparation, M.K.; writing—review and editing, K.M.A.K.; funding acquisition, M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This project was funded by the Deputyship for Research and Innovation at the Ministry of Education of Saudi Arabia through the project number INSTR004.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are available in the manuscript and Supplementary Materials. The sequences and further details can be requested from the corresponding author.

Acknowledgments

The authors extend their appreciation to the Deputyship for Research and Innovation at the Ministry of Education of Saudi Arabia for funding this research work through the project number INSTR004.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Maximum likelihood tree of C. sativa B3 DNA-binding superfamily with two other species, A. thaliana and S. alba.
Figure 1. Maximum likelihood tree of C. sativa B3 DNA-binding superfamily with two other species, A. thaliana and S. alba.
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Figure 2. Gene structure prediction and protein motif analysis. On the left panel are the results of the gene structure analysis using GSDS, displaying the organization of exons and introns in the B3 genes. Yellow boxes represent exons, and introns are represented by lines. The right panel shows the motif structure of the B3 proteins.
Figure 2. Gene structure prediction and protein motif analysis. On the left panel are the results of the gene structure analysis using GSDS, displaying the organization of exons and introns in the B3 genes. Yellow boxes represent exons, and introns are represented by lines. The right panel shows the motif structure of the B3 proteins.
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Figure 3. On the left panel are the results for the cis-acting regulatory element analysis of B3 genes in C. sativa, and on the right panel is the graph for the most abundant cis-acting elements.
Figure 3. On the left panel are the results for the cis-acting regulatory element analysis of B3 genes in C. sativa, and on the right panel is the graph for the most abundant cis-acting elements.
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Figure 4. A conserved domain analysis of the B3 genes is presented on the left panel of the picture, and the protein–protein interaction is shown on the right panel.
Figure 4. A conserved domain analysis of the B3 genes is presented on the left panel of the picture, and the protein–protein interaction is shown on the right panel.
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Figure 5. This diagram represents all the B3 genes in C. sativa and their respective in silico expression. On the left panel is the expression profile for the development of tissues such as flower, stem, leaf, and root, and on the right panel is the expression profile for different stresses such as cold, salt, and drought stress.
Figure 5. This diagram represents all the B3 genes in C. sativa and their respective in silico expression. On the left panel is the expression profile for the development of tissues such as flower, stem, leaf, and root, and on the right panel is the expression profile for different stresses such as cold, salt, and drought stress.
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Figure 6. The B3 superfamily’s relative qRT-PCR test in response to drought. To produce an objective average value, the experiment was conducted in triplicate. In untreated plants, each gene had a default expression value of 1. Bars have been placed in each column. * displays the significance of the PCR results, and ** shows the most significant results. (A) The gene expression of CsREM17. (B) The gene expression of CsARF10. (C) The gene expression of CsRAVL5. (D) The gene expression of CsARF4. (E) The gene expression of CsREM5. (F) The gene expression of CsREM34.
Figure 6. The B3 superfamily’s relative qRT-PCR test in response to drought. To produce an objective average value, the experiment was conducted in triplicate. In untreated plants, each gene had a default expression value of 1. Bars have been placed in each column. * displays the significance of the PCR results, and ** shows the most significant results. (A) The gene expression of CsREM17. (B) The gene expression of CsARF10. (C) The gene expression of CsRAVL5. (D) The gene expression of CsARF4. (E) The gene expression of CsREM5. (F) The gene expression of CsREM34.
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Table
Table 1. Characteristics of B3 domain-containing proteins.
Table 1. Characteristics of B3 domain-containing proteins.
Sr. NoProtein NameGene IDProtein IDNo of Amino
Acids		Molecular WeightTheoretical PIExonGravySubcellular LocalizationChromosomal Presence
1CsABI3LOC104789694XP_010513655.173480,974.185.46−0.72Nuclear1
2CsLEC2LOC104741439XP_010460616.136341,214.215.66−0.654Nuclear14
3CstFUS3LOC104710489XP_019084789.131135,112.25.766−0.596Nuclear9
4CsHSI2LOC104700440XP_010414264.180287,796.917.867−0.706Nuclear12
5CsHSI2 L1LOC104721632XP_010437965.178086,067.37.9217−0.568Nuclear11
6CsHSI2 L2LOC104722899XP_010439457.174282,894.297.8711−0.691Nuclear11
7CsARF1LOC104787179XP_010511014.166273,817.85.7515−0.578Nuclear5
8CsARF2LOC104762323XP_010483894.185695,433.416.1113−0.724Nuclear3
9CsARF3LOC104700045XP_010413793.160766,706.766.5910−0.408Nuclear7
10CsARF4LOC104737879XP_010456443.179087,759.615.7312−0.446Nuclear2
11CsARF5LOC104776101XP_010498407.190399,769.395.7313−0.418Nuclear3
12CsARF6LOC104757377XP_010478419.1922101,737.485.9514−0.525Nuclear17
13CsARF7LOC104706424XP_010420922.11160128,463.736.4715−0.706Nuclear8
14CsARF8LOC104732603XP_010450468.182090,929.955.9118−0.5Nuclear12
15CsARF9LOC104717887XP_010433831.163271,383.586.1916−0.639Nuclear10
16CsARF10LOC104700695XP_010414540.170577,675.847.94−0.378Nuclear7
17CsARF11LOC104784659XP_010508005.160967,855.636.1313−0.443Nuclear5
18CsARF12LOC104757783XP_010478873.161269,178.486.9914−0.478Nuclear17
19CsARF13LOC104756528XP_019094802.153160,278.526.7714−0.468Nuclear17
20CsARF14LOC104756528XP_010477432.159767,719.726.814−0.504Nuclear17
21CsARF15LOC104743784XP_019091439.161369,565.258.414−0.482Nuclear14
22CsARF16LOC104730315XP_010447772.167574,647.426.514−0.426Nuclear12
23CsARF17LOC104751681XP_010471984.157963,193.655.553−0.435Nuclear16
24CsARF18LOC104788487XP_010512556.159166,668.736.9112−0.522Nuclear5
25CsARF19LOC104756263XP_010477125.11080119,499.136.2214−0.616Nuclear17
26CsARF20LOC104757783XP_019095192.159867,756.857.2514−0.499Nuclear17
27CsARF21LOC104743784XP_019091440.159968,090.578.414−0.491Nuclear14
28CsARF22LOC104756528XP_019094801.158366,245.046.7714−0.513Nuclear17
29CsARF23LOC104777428XP_019095962.153760,694.936.5114−0.437Nuclear3
30CsNGA3LOC104738690XP_010457183.135539,887.085.72−0.772Nuclear14
31CsRAVL1LOC104782023XP_010505139.124628,619.436.423−0.945Nuclear4
32CsNGA1LOC104783271XP_010506701.131936,000.836.282−0.784Nuclear4
33CsRAVL2LOC104764914XP_010486825.126930,297.476.973−0.644Nuclear19
34CsNGA2LOC104788506XP_010512577.130634,732.767.251−0.76Nuclear5
35CsNGA4LOC104737629XP_010456151.131035,336.326.561−0.787Nuclear13
36CsRAVL3LOC104708489XP_010423372.129132,641.66.423−0.768Nuclear8
37CsRAVL4LOC104744026XP_010463359.132437,718.486.51−0.76Nuclear14
38CsRAVL5LOC104742629XP_010461945.135540,225.026.051−0.717Nuclear14
39CsRAV1.1LOC104755609XP_010476321.134838,913.399.221−0.656Nuclear17
40CsTEM1LOC104756941XP_010477910.136340,376.439.221−0.605Nuclear17
41CsRAV1.2LOC104779763XP_010502448.135239,352.359.371−0.655Nuclear4
42CsRAV2LOC104701513XP_019083256.135439,772.069.561−0.516Nuclear7
43CsREM1LOC104746303XP_010466051.126229,984.727.797−0.874Nuclear15
44CsREM2LOC104760694XP_010481961.123126,284.149.816−0.531Nuclear18
45CsREM3LOC104735367XP_010453458.127331,233.057.637−0.763Nuclear2
46CsREM4LOC104742474XP_010461785.122725,875.239.495−0.742Nuclear14
47CsREM5LOC104765761XP_010487822.134139,281.669.169−0.473Nuclear19
48CsREM6LOC104729928XP_019089716.141647,681.488.035−0.453Nuclear12
49CsREM7LOC104746283XP_010466029.120924,011.276.253−0.502Nuclear15
50CsREM8LOC104765764XP_010487826.120823,804.035.993−0.494Nuclear19
51CsREM9LOC104777862XP_010500489.119822,706.825.593−0.498Nuclear3
52CsREM10LOC104729094XP_010446288.131936,980.785.793−0.696Nuclear11
53CsREM11LOC104737509XP_010456016.127631,073.664.74−0.648Nuclear2
54CsREM12LOC104737493XP_010455999.130434,281.974.464−0.648Nuclear2
55CsREM13LOC109124983XP_019082402.127031,433.696.684−0.717Nuclear6
56CsREM14LOC104755982XP_010476776.113615,995.974.522−0.493Nuclear17
57CsREM15LOC104761860XP_010483307.131236,060.475.184−0.67Nuclear18
58CsREM16LOC104781186XP_010504088.127731,509.144.714−0.511Nuclear4
59CsREM17LOC104757181XP_010478207.139044,619.764.957−0.844Nuclear17
60CsREM18LOC104764308XP_010486112.118320,644.824.453−0.504Nuclear19
61CsREM19LOC104769721XP_010492301.113415,123.964.843−0.222Nuclear20
62CsREM20LOC104735855XP_010454021.130835,573.799.644−0.705Nuclear13
63CsREM21LOC104770134XP_010492802.128933,338.388.534−0.427Nuclear20
64CsREM22LOC104781821XP_010504892.133138,736.049.754−0.718Nuclear1
65CsREM23LOC104747383XP_010467314.130335,083.259.984−0.683Nuclear15
66CsREM24LOC104766732XP_010488975.129833,918.049.594−0.482Nuclear19
67CsREM25LOC104734977XP_010452977.129934,293.549.844−0.477Nuclear13
68CsREM26LOC104736573XP_010454877.128132,344.636.95−0.619Nuclear13
69CsREM27LOC104706867XP_010421397.128532,181.325.256−0.464Nuclear8
70CsREM28LOC104741183XP_010460280.182393,431.219.439−0.681Nuclear14
71CsREM29LOC104752106XP_010472478.181392,247.718.5610−0.576Nuclear16
72CsREM30LOC104752107XP_019094010.170578,020.729.427−0.567Nuclear16
73CsREM31LOC104702990XP_010417225.181591,497.378.8512−0.669Nuclear7
74CsREM32LOC104752112XP_010472483.155063,046.049.038−0.627Nuclear16
75CsREM33LOC104702985XP_010417221.153361,426.549.587−0.707Nuclear7
76CsREM34LOC104721678XP_010438016.152559,246.635.437−0.809Nuclear11
77CsREM35LOC104717033XP_010432841.153960,045.556.886−0.604Nuclear10
78CsREM36LOC104717033XP_010432842.152658,614.926.876−0.617Nuclear10
79CsREM37LOC104730115XP_010447524.152858,966.16.26−0.634Nuclear12
80CsREM38LOC104727641XP_019087943.134740,051.439.028−0.682Nuclear11
81CsREM39LOC104727641XP_019087942.156564,747.079.498−0.657Nuclear11
82CsREM40LOC104730118XP_010447527.126229,896.886.115−0.526Nuclear12
83CsREM41LOC104730120XP_019089533.149255,514.56.277−0.654Nuclear12
84CsREM42LOC104730120XP_010447530.146352,334.836.197−0.744Nuclear12
85CsREM43LOC104782484XP_010505731.123127,013.514.71−0.592Nuclear4
86CsREM45LOC104743162XP_010462576.132537,293.417.981−0.631Nuclear14
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MDPI and ACS Style

Kandeel, M.; Morsy, M.A.; Abd El-Lateef, H.M.; Marzok, M.; El-Beltagi, H.S.; Al Khodair, K.M.; Albokhadaim, I.; Venugopala, K.N. Genome-Wide Identification of B3 DNA-Binding Superfamily Members (ABI, HIS, ARF, RVL, REM) and Their Involvement in Stress Responses and Development in Camelina sativa. Agronomy 2023, 13, 648. https://doi.org/10.3390/agronomy13030648

AMA Style

Kandeel M, Morsy MA, Abd El-Lateef HM, Marzok M, El-Beltagi HS, Al Khodair KM, Albokhadaim I, Venugopala KN. Genome-Wide Identification of B3 DNA-Binding Superfamily Members (ABI, HIS, ARF, RVL, REM) and Their Involvement in Stress Responses and Development in Camelina sativa. Agronomy. 2023; 13(3):648. https://doi.org/10.3390/agronomy13030648

Chicago/Turabian Style

Kandeel, Mahmoud, Mohamed A. Morsy, Hany M. Abd El-Lateef, Mohamed Marzok, Hossam S. El-Beltagi, Khalid M. Al Khodair, Ibrahim Albokhadaim, and Katharigatta N. Venugopala. 2023. "Genome-Wide Identification of B3 DNA-Binding Superfamily Members (ABI, HIS, ARF, RVL, REM) and Their Involvement in Stress Responses and Development in Camelina sativa" Agronomy 13, no. 3: 648. https://doi.org/10.3390/agronomy13030648

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