Abstract
The Two-component system (TCS) consists of Histidine kinases (HKs), Phosphotransfers (HPs), and response regulator (RR) proteins. It has an important role in signal transduction to respond to a wide variety of abiotic stresses and hence in plant development. Brassica oleracea (cabbage) is a leafy vegetable, which is used for food and medicinal purposes. Although this system was identified in several plants, it had not been identified in Brassica oleracea yet. This genome-wide study identified 80 BoTCS genes consisting of 21 HKs, 8 HPs, 39 RRs, and 12 PRRs. This classification was done based on conserved domains and motif structure. Phylogenetic relationships of BoTCS genes with Arabidopsis thaliana, Oryza sativa, Glycine max, and Cicer arietinum showed conservation in TCS genes. Gene structure analysis revealed that each subfamily had conserved introns and exons. Both tandem and segmental duplication led to the expansion of this gene family. Almost all of the HPs and RRs were expanded through segmental duplication. Chromosomal analysis showed that BoTCS genes were dispersed across all nine chromosomes. The promoter regions of these genes were found to contain a variety of cis-regulatory elements. The 3D structure prediction of proteins also confirmed the conservation of structure within subfamilies. MicroRNAs (miRNAs) involved in the regulation of BoTCSs were also predicted and their regulatory roles were also evaluated. Moreover, BoTCSs were docked with abscisic acid to evaluate their binding. RNA-seq-based expression analysis and validation by qRT-PCR showed significant variation of expression for BoPHYs, BoERS1.1, BoERS2.1, BoERS2.2, BoRR10.2, and BoRR7.1 suggesting their importance in stress response. These genes showing unique expression can be further used in manipulating the plant’s genome to make the plant more resistant the environmental stresses which will ultimately help in the increase of plant’s yield. More specifically, these genes have altered expression in shade stress which clearly indicates their importance in biological functions. These findings are important for future functional characterization of TCS genes in generating stress-responsive cultivars.
1 Introduction
Two-component system (TCS) is a major signal transduction system that is involved in several biological processes by regulating the signaling of histidine-aspartate. Thus, it responses to environmental stimuli such as cell growth and division, oxygen sensing, osmotic sensing, host recognition, and chemotaxis in both prokaryotes and eukaryotes. In bacteria, the two-component system relies on the phosphorylation of aspartic acid and histidine residues. Moreover, this system has also been identified in slime molds and fungi (; ). Protein phosphorylation is one of the most common methods for intracellular signaling. Protein kinases use ATP as the phosphate donor to phosphorylate their substrates. They are divided into five classes based on the receptor amino acid: histidine kinases (HK), serine-threonine kinases (STK), cysteine kinases (CK), tyrosine kinases (TK), and aspartyl kinases (AK) ().
Plants use two-component signaling elements in a variety of signaling pathways, the best known of which is cytokinin signaling (; ). A His-to-Asp phosphorelay is involved in the signaling of the plant hormone cytokinin. The HKs associated sensory extracellular extracytoplasmic hormone-binding domain (CHASE), as well as the receiver and cytoplasmic HK, make up the classic cytokinin receptor (). The functionality of HKs is based on TCS. This system consists of a His-kinase that recognizes the environmental stimuli and autophosphorylates its conserved histidine (His) residue, which is subsequently transferred to a conserved aspartic acid (Asp) residue in another group of signal transducers known as response regulators (RRs). Phosphorylation of RRs provides the ability to control the downstream signaling, allowing the external stimulus to be converted into an internal signal (). In bacteria, fungi (slime molds, yeast), and plants a His-to-Asp phosphorelay is formed which is more complex and uses a hybrid kinase including both a receiver domain (Rec) and an HK domain in a single protein. Another domain called His-containing phosphotransfer (HP) domain is also present in both prokaryotes and in eukaryotes which is a signaling component. The phosphate is transported from HK to RR through a multistep His-to-Asp phosphorelay ().
In plants, three distinct families of HKs are present: cytokinin receptors, phytochromes, and ethylene receptors which play an important role in phytochrome signaling. Although TCS elements have all the necessary conserved residues, phytochromes, and ethylene receptors are divergent signaling elements that do not have conserved residues for phosphotransfer activity in their HK motif (). In Arabidopsis, there are three kinases (AHK1, AHK5, and CKl1) that do not belong to the cytokinin receptors. Cytokinin receptors’ basic structure consists of an input domain, a Rec domain, a transmitter domain (having a conserved histidine/H residue that provides the site of autophosphorylation), and many trans-membrane domains. Another domain called cyclase/CHASE domain is also present in three cytokinin receptors (AHK2/3/4) which is a possible cytokinin recognition site. A C2H4/ethylene binding domain is shared by five ethylene receptors (ETR1/2, ERS1/2, and EIN4). Five phytochromes (PHYA, B, C, D, and E) share a chromophore binding (PHY) domain and two PAS (Per/Arndt/Sim) folds. In Arabidopsis, a conserved motif “XHQXKGSSXS” is present in AHPs that regulates the transfer of phosphate group HK’s receiver to the RR’s Rec domain. AHP6 lacks the conserved His residue, therefore, is called a pseudo-His-containing phosphotransfer (HP) protein (Z. ).
Response regulators (RRs) are classified into four groups based on domain structure and phylogenetic analysis: type-A, type-B, type-C, and pseudo-response regulators (PRRs). Type-A RRs are the key transcriptional targets for cytokinin signaling that contain a Rec domain. Type-B RRs function as the positive transcriptional regulator of cytokinin signaling. These RRs contain a Rec as well as Myb-like DNA-binding domain at C-terminus. Type-C RRs also contain the Rec domain and lack the Myb domain. Therefore, these are phylogenetically closer to Type-A RRs (). A multistep phosphorelay comprising cytokinin receptors, type-B RRs, and phosphotransfer proteins mediates the initial steps of cytokinin signaling. Type-B RRs regulate gene expression, including transcriptional activation of Type-A RRs by relaying the cytokinin signal from the membrane to the nucleus. Type-A RRs are negative regulators of the initial signal transduction pathway mediating downstream responses to cytokinin (). Finally, PRRs have a receiver domain in which the D residue is replaced by E, as well as the C-terminus CCT domain has a function associated with protein-protein interactions ().
Abscisic acid (ABA) and stress responses are significantly mediated by the cytokinin-responsive TCS. Different stresses impose a negative impact on plant development and crop yield. Under osmotic stress, ABA is generated and plays a crucial role in plant stress response as well as in tolerance. During the growth and germination of the seed, it mediates gene expression. Furthermore, it is also found that the ABA and other signaling components interact during seed development and stress response. Controlling the expression of ABA signaling factors may help to cope better with stresses (; ). Various abiotic stresses like drought and excessive salinity have a negative impact on plant growth and yield. To survive in this variety of conditions, plants have evolved various complex signaling networks at the system, cellular, and molecular level. In plant cells, the TCS genes mediate phosphorylation, which is a major method of signal transduction regarding stress (Z. ). TCS genes have previously been identified in Arabidopsis (), rice (), banana (), melon (Cucumis melo L.) (P. ), cucumber (), C. arietinum (), tomato (), soybean () and wheat (). Different TCS genes have different expression patterns. TCS responses to environmental factors vary greatly depending on individual TCS members, conditions, and species. In C. arietinum, Arabidopsis, soybean, and cucumber, members of the TCS gene family are found to be involved in the regulation of abiotic stresses including dehydration, drought, cold, and salt stress [19, 21, 22]. Similarly, TCS genes in rice are involved in ABA-induced increased expression of antioxidant enzymes and gene activity [12, 46].
Brassica oleracea L. var. Capitata f. Rubra (Red cabbage) (2 n = 18) is a Mediterranean and southwestern European native herbaceous leafy vegetable that currently grows worldwide. It is a member of the Brassicaceae family, with an annual global production of 58 million tons of fresh heads from 3.1 million ha in over 130 countries. Since Brassica crops are mostly grown in an arid or semiarid environment, they are subjected to a variety of abiotic stresses. In crops, two strategies can be employed to cope with abiotic stresses. These include biotechnological approaches and crop management. Agronomic methods like irrigation with high-quality water, and changing sowing times can all contribute to reducing the effects of abiotic stresses. For vegetable cultivation, this technique is neither sustainable nor cost-effective. Therefore, a long-term solution for stress-resistant vegetable cultivation is required. Plants contain various stress-related genes and their expression helps to regulate stress tolerance in plants. These stress-related genes are required to produce stress-tolerant crop varieties. This genetic mechanism can be utilized to produce high yields during stressful conditions. The active genes have been exploited to generate stress-tolerant cops (; ). Similarly, TCS genes have been found in a variety of plants including Brassica oleracea, and they may help to improve food production by resisting a variety of environmental factors. Since, B. oleracea is economically important food, getting higher yield by making plant more resistant to stress would be helpful. Therefore, the goal is to identify certain genes important in stress resistance and manipulating them to make plant more resistant to environmental stresses. For this purpose, the functional diversity and expression profiles of BoTCS genes were evaluated in this work. In addition, the expression profiling of these genes under shade stress will help to study the stress effect on plant physiology. Our study aimed to discover TCS genes that are responsive to changes in shade, which could be useful in the breeding of B. oleracea. As TCS genes have various roles in biological processes, it is crucial to conduct a comprehensive investigation of these genes in B. oleracea. By identifying these shade-responsive TCS genes, we hope to contribute to the understanding of the molecular mechanisms underlying the shade response in B. oleracea and provide valuable information for future breeding efforts. Our research underscores the importance of studying specific gene families in different crops to uncover their potential roles in important biological processes.
2 Materials and methods
2.1 Identification of Brassica oleracea TCS genes and their subcellular localization
For the identification of TCS proteins in B. oleracea, amino acid sequences from Arabidopsis TCS proteins were retrieved from the Ensembl plants database (http://plants.ensembl.org/index.htm) and were used as queries in BLASTP searches against NCBI (https://www.ncbi.nlm.nih.gov/) () database of B. oleracea. The default parameters of the BLASTP tool were used for this search. All the retrieved non-redundant protein sequences were subjected to domain analysis using online servers: Pfam (http://pfam.xfam.org/) (), CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART (http://smart.embl-heidelberg.de/) (). Protein sequences containing specific domains including histidine-kinases receptor domain, response regulator domain, and other specific domains were kept as candidate TCS proteins, while those without conserved domains were excluded. Details such as exon number, chromosome number, and location were obtained from NCBI.
Physiochemical characteristics of all BoTCS proteins were determined using the ExPASy ProtParam online tool (https://web.expasy.org/protparam/) (), including molecular weight (in Da Da), grand average of hydropathicity (GRAVY), isoelectric point (pI, point at which molecule is electrically neutral), instability index (II) and aliphatic index (AI). The subcellular localizations were predicted with the online CELLO v.2.5 tool (http://cello.life.nctu.edu.tw/) ().
2.2 Phylogenetic, gene structure, and conserved motif analysis
To determine the evolutionary relationship of TCS proteins from B. oleracea and diverse plant species, protein sequences of B. oleracea, Arabidopsis thaliana, C. arietinum, Glycine max, and Oryza sativa were used. Multiple sequence alignment of these proteins was generated using the ClustalW tool and a Maximum-likelihood (ML) tree was constructed by using IQ-Tree () with a bootstrap value of 1,000 and was edited online using iTOL: Interactive Tree of Life (https://itol.embl.de/) ().
The Gene Structure Display Server 2.0 (GSDS) (http://gsds.gao-lab.org/) () was used to predict gene exon/intron structure using coding (CDS) and genomic sequences of B. oleracea and A. thaliana as input data. The CDS and genomic sequences were retrieved through NCBI. The MEME (Multiple EM for Motif Elicitation) tools (https://meme-suite.org/meme/) () were used to determine the conserved motifs in the BoTCS sequence. The maximum motif number was set to 20, all other parameters were left at their default values and the meme.xml file was obtained. This file was used to analyze conserved motifs using TBtools.
2.3 Identification of cis-regulatory elements and chromosomal mapping, gene duplication events, and syntenic analysis of BoTCS genes
The 2 kb upstream sequences from the translation start site of BoTCS genes were retrieved from the NCBI-nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/) (). To examine the cis-elements in these promoter sequences, the PlantCARE online server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) () was used and TBtools simple bioSequence viewer was used to visualize results. A genetic relational map of B. oleracea chromosomes was created by using TBtools advanced Circos. The position of each B. oleracea TCS gene on its chromosomes was predicted using the NCBI Gene database (https://www.ncbi.nlm.nih.gov/gene) (; ). Similarly, TBtools was used to perform syntenic analysis (). The gene duplication events were analyzed by using DnaSP v.6 tool (). Further, their synonymous as well as non-synonymous substitution rates were also calculated. To analyze their evolutionary events, the divergence time was calculated. The following formula was used to calculate the time of duplication: T = Ks/2x (x = 6.56 * 10−9) (; ).
2.4 miRNA target prediction
The Plant microRNA Encyclopedia database (PmiREN; https://pmiren.com) () was utilized to acquire mature miRNA sequences of B. oleracea. For putative miRNA target prediction CDS sequences of the potential target, BoTCSs were utilized and were submitted at the psRNATarget server (https://www.zhaolab.org/psRNATarget/home) () along with the respective mature miRNA sequences of B. oleracea with default considerations. The regulatory association between target BoTCSs and predicted miRNAs was visualized using Cytoscape software ().
2.5 Three-dimensional structure of BoTCSs and molecular docking analysis
The three-dimensional (3D) structure of the protein is essential for a proper understanding of its functions. Therefore, the I-TASSER web server was used to predict the 3D structures of BoTCS proteins (https://zhanggroup.org/I-TASSER/) (). The UCSF Chimera tool was used to visualize the 3D structures (). In addition, models were verified using the SAVES v6.0 web server (https://saves.mbi.ucla.edu/) (). The 3D structure of ABA was retrieved using PubChem (https://pubchem.ncbi.nlm.nih.gov/) ID (5,280,896). Further, to check the binding pattern of these proteins with specific ligand, molecular docking analysis was performed which showed the interactions as well as interacting residues among the proteins and ligand. The molecular docking analysis of BoTCSs and ABA was done using PyRx virtual screening software (https://pyrx.sourceforge.io/downloads). Interaction study of protein-ligand complexes was also done using UCSF Chimera () and Discovery Studio after the successful docking of ABA ligand against all 80 BoTCSs.
2.6 Differential expression profiling of BoTCS genes under different stress conditions
RNA-seq data for light/shade [BioProject: SRR14774565] and tissue-specific responses [BioProject: SRR13759392] was retrieved from the NCBI-SRA database (https://www.ncbi.nlm.nih.gov/sra) to investigate the differential expression pattern of TCS genes in B. oleracea. This data was used to compare the expression pattern of BoTCS genes in shade and light at specific times (1h and 6 h after stress application). For tissue-specific responses, SRA data for leaf, bud, stem, root, silique, flower, and the leafy head was used. The genome and GFF files were downloaded from the Phytozome database (https://phytozome-next.jgi.doe.gov/). Bowtie2 tool () was used to build genomic sequence indexes for B. oleracea and further the genome was mapped using pair-end clean reads. The alignment file was then used in the next step of abundance estimation which provided the differential expression of genes. The expression level of genes was estimated using the cufflinks tool, which provided FPKM values. FPKM values were used to generate a heatmap (; ). Heatmap was generated using TBtools ().
2.7 Plant growth, treatment, and quantitative real-time PCR validation
Seedlings of cabbage (B. oleracea var. capitata) were grown at 23°C ± 2°C for 25 days under natural light. For shade treatment-seedlings were kept in shade for specific periods. First, the duration for keeping seedlings in shade was 1 h and then the second time the duration was increased to 6 h. For RNA extraction leaves/roots of plants were collected and sampled as a biological replicate. These samples were immediately frozen in liquid nitrogen and kept at −80°C until needed.
TRIzol reagent was used to extract RNA from leaf samples as directed by the manufacturer and quantified using a NanoDrop spectrophotometer (). One microgram of total RNA was reverse transcribed with dsDNase using the Maxima H Minus First-strand cDNA synthesis kit. 10X dsDNase buffer, total RNA, dsDNase, and nuclease-free H2O were added to an RNase-free tube on ice to obtain a total volume of 10 mL. The mixture was gently mixed and spun before being incubated at 37°C for 2 min in a hot water bath and then stored on ice. To synthesize the strands, chemicals (5X RT buffer, 10 mM dNTP mix, oligoT primer, and maxima H minus reverse transcriptase enzyme) were then added and gently mixed before being centrifuged briefly. The mixture was then incubated for 30 min at 50°C and 5 min at 85°C. Finally, the mixture was kept at a temperature of around −80°C until it was used. iTaq universal SYBR Green Super Mix and Real-time PCR detection system (CFX96 Touch™ Real-TimePCR Detection System were used to carry out qRT-PCR. Primers were designed using the online NCBI Primer-BLAST Program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) () and their specificity was verified by using an online tool Oligo Calculator (http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html) (). The overall workflow of this study is presented in Supplementary Figure S1.
2.8 Data analysis
RNA-seq analysis provided FPKM values which were further analyzed to find differentially expressed genes. In tissue-specific expression analysis, genes with zero FPKM values were considered as having no change in expression and higher values were supposed to have an increasing expression. While in shade stress, genes having FPKM values greater than one were considered as upregulated. Whereas, genes having FPKM values less than one were considered as downregulated ones. qRT-PCR results were analyzed by applying Student’s t-test (p < 0.05 = > *, p < 0.01 = >**) to compare control and shade stress and calculated the significance level.
3 Results
3.1 Identification of TCS proteins in Brassica oleracea
A total of 80 TCS genes; 21 HKs, 8 HPs, 39 RRs, and 12 PRRs, were identified in B. oleracea after checking for specific domains and removal of redundancy. The identified TCS members were named according to the proposed nomenclature in previous studies on this gene family on other plants. The tree shows the evolutionary relationships among the members of this gene family.
3.1.1 HK protein family in Brassica oleracea
The genome of B. oleracea has 21 members of the HK family, which is comparable to the other species including Soybean (21), M. acuminata, and M. balbisiana (25) (Supplementary Table S2). The HK family is further classified into three subgroups CHK/HK, PHY, and ETR. Out of 21 HK genes identified in B. oleracea, 9 members were classified as CHK/HK. Both PHY and ETR subgroups contained six members each. All of the HKs possess a conserved HK domain that contains five conserved signature motifs, namely, H, N, G1, F, and G2.
Histidine Kinases subgroup members either possess a simple histidine kinase domain or a CHASE domain associated with the HK domain, and the CHASE domain is crucial for proteins to recognize and bind cytokinin (). Brassica oleracea has four CHKs (BoHK2.1/2.2/3/4) and 5 HK (BoHK1/5.1/5.2, BoCKI1.1, and BoCKI1.2) proteins contain HK domain followed by HATPase_c domain (Figure 1; Supplementary Table S3). The CHKs have a CHASE domain followed by HK HATPase_c and Rec domains. Additional Transmembrane (TM) domains present in the CHK proteins have been shown to play an important role in membrane-associated signal transduction in plants (). A similar pattern for motif conservation was observed in these HKs. HKs contained six conserved motifs and CHKs also contained the same motifs with one additional motif (Figure 2C). Gene structure showed that these proteins contain six to thirteen introns. This conservation of gene structure and motif patterns suggests that they are structurally similar and potentially involved in similar biological functions such as signal transduction.
FIGURE 1
FIGURE 2
Ethylene receptors have a common domain structure containing GAF, HK, HATPase_c, and Rec domains. In B. oleracea six ethylene receptors were identified. BoETRs and BoERSs are representative of the ethylene receptor family with or without the Rec domain and are involved in ethylene response. The ETR1 proteins (BoETR1 and BoETR2) had all the standard domains present in ethylene receptors. The ERS proteins (BoERS2.1 and BoERS2.2) show quite a difference in the presence of domains. The general domain organization includes the GAF domain and lacks the Rec domain (Figure 1; Supplementary Table S3). Motif analysis showed that these proteins have at least five motifs conserved in them. Gene structure revealed that BoETRs contain two to five introns.
Phytochrome/photoreceptors are characterized by the presence of a chromophore binding group and two PAS domains that enable plants to respond to light stimuli and regulate the processes of growth and development. There are six members of the PHY subgroup in B. oleracea. Domain analysis showed that phytochromes contain a GAF domain, an N-terminal PHY domain (involved in light absorption), two PAS domains, and one His Kinase domain involved in signal transduction. The domain PHY, GAF, and PAS play important roles in responding to red light and far-red light signals during plant growth and development in Arabidopsis (). Like other HKs this subgroup also contained highly conserved motifs as well as gene structure. Gene structure showed that these contain two to five introns (Figure 2B).
3.1.2 HP protein family in Brassica oleracea
Histidine-containing phosphotransmitters (HPs/Hpts) are the signal mediators in multistep His-to-Asp phosphorelay and originated in plants (). Eight BoHP genes were identified: seven authentic and one pseudo-Hpt. All BoTCS are small proteins with 145–156 amino acids. Each member contains a conserved phosphorylation motif (XHQXKGSSXS) with a conserved Histidine phosphorylation site (H residue). All the BoHPs contained this residue except BoHP6 in which H residue is replaced by N residue. Similarly, in AHP6 and APHP1, which are Arabidopsis pseudo-Hpts, their conserved His residue was replaced by N. These pseudo-Hpts are also sharing high similarity which suggests that they may share similar functionality. Same pattern was observed in cucumber and rice (Supplementary Table S2) (; ). Gene structure revealed that each of the eight BoHP genes contains six to seven exons with five to six introns, respectively, except BoHP6. BoHP6 contains five exons and four introns (Figure 2E). Motif analysis suggested that all members had almost four motifs (motifs 1, 2, 3, and 16) conserved in them. While the rest of the motifs had random distribution across the members (Figure 2F).
3.1.3 RR protein family in Brassica oleracea
A total of 51 RR genes were identified in B. oleracea of which 18 are RR-Type-A, 16 RR-Type-B, 5 RR-Type-C, and 12 PRRs (Supplementary Table S4). The final response to the environment is provided by RRs and the RR family in Arabidopsis has been shown to contain conserved aspartic acid (D) and lysine (K) residue. All the sequences containing the Rec domain only are identified as RR-Type-A and contain conserved D-D-K motifs which is the final receiver of the phosphate group with highly conserved D-residue at N-terminal ().
RR-Type-B family members are different from RR-Type-A family members because they contain an additional DNA binding domain (MYB). It has been shown that RR-Type-B functions as a transcription factor. The 16 RR-Type-B family members were found in S. bicolor (Supplementary Table S2). They also contain a conserved N-terminal D residue which plays a key role in receiving the phosphate group to initiate downstream processes. Different from RR-Type-A, five identified RR-Type-C contained a Rec domain but lacked C-terminal extension. Arabidopsis has two members of the RR-Type-C family (ARR22 and ARR24). These are divergent from RR-Type-A and do not express during cytokinin response. Brassica oleracea was found to contain five members of this family (BoRR22.1, BoRR22.2, BoRR24.1, BoRR24.2, and BoRR24.3) having significant homologous relationships with their Arabidopsis counterparts. All these five proteins had three motifs conserved.
Brassica oleracea had 12 BoPRRs a conserved Rec domain and CCT domain, which play an important role in regulating circadian rhythms. These are further divided into type-B PRRs and Cloak-associated PRRs. Type-B PRRs have a Myb domain instead of a CCT motif and include BoPRR2.1/2.2 and BoPRR6.1. Cloak-associated PRRs have the conserved arginine and lysine in the CCT motif and include BoPRR1, BoPRR3, BoPRR5, BoPRR7.1, BoPRR7.2, and BoPRR9.1. All these BoPRR genes also have conserved gene structures and motifs (Figure 3).
FIGURE 3
3.2 Physiochemical characteristics of BoTCS proteins
The sequence characteristics of 80 BoTCS proteins were analyzed (Supplementary Table S4). The BoTCSs are present on nine chromosomes (Chr), Chr 1-9. The exon number ranged from 3 to 19. The amino acid length varied from 134 (BoRR24.3) to 1,327 (BoRR24.3) aa. Accordingly, the molecular weight (MW) ranged from 15,030.16 to 146,733.58. The ExPasy ProtParam tool showed that BoTCS proteins had a very different isoelectric point (pI) with a value ranging from 4.65 to 9.2. This suggests the acidic and basic nature of proteins. Instability index (II) ranged from 22.62 (stable) to 81.76 (unstable), aliphatic index (AI) value ranging from 61.38 to 116.92, and grand average of hydropathicity index (GRAVY) values from −0.741 to −0.08 which showed protein’s hydrophobic as well as hydrophilic nature. BoTCS proteins localize mostly in the plasma membrane, nucleus, cytoplasm, and mitochondria (Supplementary Table S4).
3.3 Comparative phylogenetic analysis of two-component system proteins in diverse plant species
TCS genes in B. oleracea, A. thaliana, C. arietinum, Oryza sativa, and Glycine max have closer phylogenetic relationships. Based on these closer relationships, a phylogenetic and evolutionary analysis of five different plant species (mentioned above) was carried out (Figure 4). TCS gene family members were divided into three main families HKs, HPs, and RRs. HK family is further divided into five subgroups: CKI1 and AHK1, Cytokinin receptors, CKI2, ethylene receptors, and phytochromes. BoCKI1.1, BoCKI1.2, and BoHK1 are the members of the CKI1 and AHK1 subgroups which are orthologues of AtCKI1 and AHK1. AHK1 protein is involved in osmosensation and primarily expresses in the A. thaliana roots during salt stress (). Cytokinin receptor subfamily has four members in B. oleracea while three members (BoHK2.1, BoHK2.2, BoHK3, and BoHK4) were present in A. thaliana (AHK2, AHK3, and AHK4). In A. thaliana this subfamily controls various cytokinin-regulated responses like seed size, cell division, leaf senescence, and other stress responses . Similarly, other plant species also have similar phylogenetic relationships with plants. These closer relationships among B. oleracea, their A. thaliana orthologues, and other plant members suggest that they may have a functional similarity. BoHK5.1 and BoHK5.2 belong to the CKI2 subfamily. Six members of the ethylene receptor subfamily are present in B. oleracea having homologous relationships with ETR1, ASR1, and EIN4. Since the phylogenetic analysis has shown homologous relationships among the TCS members, it suggests that they have similar functions in different plant species as they emerged from the same ancestral sequence (Figure 4).
FIGURE 4
Phytochromes or photoreceptors control growth and developmental process during light stress. Five members of this subfamily are present in A. thaliana while the current study has found six members in S. bicolor. Direct homologous relationships are found among all the members belonging to five plants. All the BoHPs are having close relationships with A. thaliana. This subfamily has six and eight members in A. thaliana and B. oleracea, respectively.
RRs from five plants were used to perform phylogenetic analysis and are classified as true RRs and pseudo-RRs (PRRs). True RRs are divided into further subgroups: RR-Type-A, RR-Type-B, and RR-Type-C. RR-Type-B is further classified as Type-B1, Type-B2, and Type-B3 RRs. All these BoRRs are closely related to their A. thaliana homologs. RRs give final responses to the environmental factors. RR-Type-B and RR-Type-C are mostly involved in cytokinin activities whereas RR-Type-A is found exclusively in land plants and is considered new members because of their absence in unicellular alga. PRRs are further classified into two subgroups: Clock-associated PRRs and Type-b PRRs based on their domain organization. Both subgroups contain seven and 5 members respectively.
3.4 Promoter analysis of BoTCS genes
The promoter sequences of the BoTCS genes were analyzed to predict the cis-regulatory elements to understand their functional as well as transcriptional roles. Several abiotic stress and hormone-related cis-regulatory elements have been found.
Major classes of cis-elements found include Stress-responsive (low temperature, light), MYB-binding sites, and hormone-responsive elements. The element involved in low-temperature stress (LTR: CCGAAA), light-responsive element (GATA motif: GATAGGG, Box II: TGGTAATAA, LAMP element: CTTTATCA), gibberellin-responsive element (GARE-motif: TCTGTTG), abscisic acid-responsive element (ABRE: ACGTG), element involved in salicylic acid response (TCA-element: CCATCTTTTT), MYB binding site (MRE: AACCTAA), cis-regulatory element involved in circadian control (circadian: CAAAGATATC), MeJa-responsive element (CGTCA-motif: CGTCA) and auxin-responsive element (TGA-element: AACGAC) were common in almost all BoTCS genes (Figure 5; Supplementary Table S5). All these elements induce a response to abiotic stress and growth hormone regulation. An element for anaerobic induction (ARE) is also found. These results imply that TCS family members of B. oleracea have cis-acting elements in their promoter sequence and hence can play pivotal roles in abiotic stress tolerance. Most of these cis-elements were associated with shade stress which further led to the involvement and regulation of these genes in shade stress. Most importantly, BoPHYs, BoERS1.1, BoERS2.1, BoERS2.2, BoRR10.2, and BoRR7.1 contained shade-stress related elements which can be used for shade stress tolerant breedings.
FIGURE 5
3.5 Chromosomal localization of BoTCS genes
To better understand the genomic distribution of BoTCS genes, their location on chromosomes and duplication events were predicted by performing syntenic analysis. The nine chromosomes had a different number of genes distributed on them (Supplementary Figure S2). The distribution of genes is extremely random, like chromosome 2 contains only six genes while chromosome 4 contains 12 genes. All BoRRs were distributed on every chromosome. Similarly, every gene family has its member on every chromosome.
Gene duplication events were also identified for this family. Gene duplication event is an important event in evolution; therefore, it led to analyze the evolution of these genes. Both tandem and segmental duplications were analyzed. The potential gene duplication events identified were 27. Among duplication types, tandem duplication was found in three pairs (BoCKI1.1/BoCKl1.2, BoRR8.1/BoRR9.2, and BoRR15.1/BoRR15.2). Rests of the pairs were segmentally duplicated. Synonymous rate (Ks), non-synonymous (Ka), and their ratio (Ka/Ks) were calculated (Table 1). Using these values, duplication time was also calculated. The Ks value for these duplications ranged from 0.18 to 0.8922. Therefore, the time of divergence ranged from 2.41 to 40.236 million years ago (mya).
TABLE 1
| Duplicated gene pairs | Ka | Ks | Ka/Ks | Time (MYAa) | Duplication type |
|---|---|---|---|---|---|
| BoCKI1.1/BoCKl1.2 | 0.1064 | 0.124 | 0.858065 | 4.133333333 | Tandem |
| BoERS1/BoETR1 | 0.6122 | 1.4943 | 0.40969 | 49.81 | Segmental |
| BoERS2.1/BoERS2.2 | 0.1048 | 0.1308 | 0.801223 | 4.36 | Segmental |
| BoPHYAa/BoPHYAb | 0.0704 | 0.4248 | 0.165725 | 14.16 | Segmental |
| BoPHYB/BoPHYD | 0.0767 | 0.0917 | 0.836423 | 3.056666667 | Segmental |
| BoHP2.1/BoHP2.2 | 0.107 | 0.2665 | 0.401501 | 8.883333333 | Segmental |
| BoHP2.1/BoHP3 | 0.1064 | 0.2726 | 0.390315 | 9.086666667 | Segmental |
| BoHP4.1/BoHP4.2 | 0.1051 | 0.2858 | 0.36774 | 9.526666667 | Segmental |
| BoRR1.1/BoRR1.2 | 0.1571 | 0.4618 | 0.340191 | 15.39333333 | Segmental |
| BoRR1.2/BoRR2.1 | 0.5566 | 1.7376 | 0.320327 | 57.92 | Segmental |
| BoRR2.1/BoRR2.2 | 0.576 | 0.6956 | 0.828062 | 23.18666667 | Segmental |
| BoRR3/BoRR4.2 | 0.2012 | 0.1001 | 2.00999 | 3.336666667 | Segmental |
| BoRR6.1/BoRR6.2 | 0.1163 | 0.0812 | 1.432266 | 2.706666667 | Segmental |
| BoRR7.1/BoRR7.2 | 0.0675 | 0.1367 | 0.493782 | 4.556666667 | Segmental |
| BoRR7.2/BoRR15.2 | 0.1805 | 0.1399 | 1.290207 | 4.663333333 | Segmental |
| BoRR8.1/BoRR9.1 | 0.1531 | 0.0812 | 1.885468 | 2.706666667 | Segmental |
| BoRR8.1/BoRR9.2 | 0.2305 | 0.0846 | 2.724586 | 2.82 | Tandem |
| BoRR8.2/BoRR9.1 | 0.197 | 0.0723 | 2.724758 | 2.41 | Segmental |
| BoRR10.1/BoRR10.2 | 0.159 | 0.7166 | 0.221881 | 23.88666667 | Segmental |
| BoRR10.1/BoRR12 | 0.3779 | 0.824 | 0.458617 | 27.46666667 | Segmental |
| BoRR10.1/BoRR14 | 0.8922 | 1.2071 | 0.739127 | 40.23666667 | Segmental |
| BoRR10.2/BoRR12 | 0.2627 | 0.3342 | 0.786056 | 11.14 | Segmental |
| BoRR13/BoRR21 | 0.1424 | 0.1337 | 1.065071 | 4.456666667 | Segmental |
| BoRR15.1/BoRR15.2 | 0.18 | 0.1416 | 1.271186 | 4.72 | Tandem |
| BoRR22.1/BoRR22.2 | 0.0629 | 0.4582 | 0.137276 | 15.27333333 | Segmental |
| BoPRR2.1/BoPRR2.2 | 0.1844 | 0.4836 | 0.381307 | 16.12 | Segmental |
| BoPRR5/BoPRR9.1 | 0.6968 | 0.8073 | 0.863124 | 26.91 | Segmental |
Calculation of Ka, Ks, and Ka/Ks values and divergence time of the duplicated BoTCS gene pairs.
aMYA: million years ago.
3.6 MiRNA target prediction of BoTCSs
A total of 186 miRNAs were identified that targeted 80 BoTCSs, with expectation values ranging from 3 to 5 (Figure 6; Supplementary Table S6). 13 miRNAs were targeting BoCKI1.2, 12 miRNAs were targeting BoERS2.2, while some other BoTCSs were targeted by less than 10 miRNAs. Among these miRNAs, most of them were responsible for inhibiting the cleavage of target transcript while only a few were involved in inhibiting the translation of target genes.
FIGURE 6
3.7 BoTCS protein’s structure prediction
A protein’s structural features indicate its potential interactions or binding with the other molecules as well as its functional correlations. In this study, the three-dimensional structures of 6 BoTCS proteins were predicted by using the I-TASSER server. These models had higher stereochemical characteristics locally as well as globally. In these models, the major structural component is found in the helix. The structural symmetry of proteins belonging to a particular family is similar. From the HK family, models of two proteins BoHK5.1 and BoETR2 are predicted. Different helices are shown in blue, green, yellow, and orange colors. Helices are the main structural component present in these proteins with fewer loops and sheets. From the HP family, BoHP1 and BoHP6 were modeled and helices mainly constitute these proteins. From the RR family, BoRR6.1 and BoRR9.2 had their structures modeled. These proteins have helices as well as loops (Supplementary Figure S3). This conservation of secondary structures among proteins suggests their similarity in functions as well.
3.8 Molecular docking analysis of BoTCS proteins with abscisic acid (ABA)
ABA was docked against 6 BoTCSs to check their binding because TCSs regulate a variety of biological activities and responses to environmental stimuli (Figure 7). The cytokinin-responsive TCS controls ABA and osmotic stress responses ().
FIGURE 7
Docking complexes have −6.1 Kcal/Mol (BoRR9.2) to −5 Kcal/Mol (BoHP6) global energy. RMSD value for BoHK5.1 is 2.659 Å. Its conserved residues are HIS A:641, HIS A:643, ARG A:669, GLU A:673, and LYS A: 674 BoETR2 is having conserved residues, ARG A:4, ALA A:7, SER A:8, LEU A:11, ILE A:12, TYR A:61, and PHE A:62 with their RMSD value of 2.644 Å BoHP1 conserved residues are GLN A:34, PRO A:39, VAL A:141, ILE A:147, and ALA A:149 having an RMSD value of 2.216 Å BoHP6’s conserved residues include PHE A:21, HIS A:22, GLU A:28, LEU A:31, and ARG A:95 with an RMSD value of 2.82 Å BoRR6.1 has conserved residues including LEU A:24, LYS A:79, ASN A:81, and PHE A:160 RMSD value of 0.498 Å BoRR9.2 conserved residues include VAL A:40, LEU A: 41, LEU A: 42, LYS A:80, ARG A:452, PRO A:453, and ARG A:454 with RMSD value of 0.465 Å (Supplementary Table S4). All these binding affinities of the BoTCS proteins imply that they can bind with the ligand to perform specific functions.
3.9 Analysis of differently expressed genes in the TCS family of Brassica oleracea
According to previous studies and studies of cis-regulatory elements, it is found that TCS genes can play a key role in abiotic stress response. In the current study, the expression of BoTCS genes was investigated using RNA-seq data to understand the tissue-specific and stress-responsive (light/shade) expression of these genes. The expression of BoTCS genes was estimated in different tissues including leaf, bud, stem, root, flower, and leafy head. The majority of TCS genes were not expressed or weakly expressed in all tissues. However, members of the PHY subfamily were mostly expressed in all tissues. Similarly, BoETR2, BoEIN4, and BoRR10.1 were highly expressed in roots (Figure 8A). In stress response (shade stress for 1 and 6 h) majority of TCS genes were expressed (Figure 8B). Members of the PHY family were mostly highly upregulated in response to shade. In response to shade stress of 6 h, BoPHYAa, BoPHYAb, BoPHYB, BoPHYC, BoPHYD, BoPHYE, BoERS2.1, BoERS2.2, BoRR8.2, BoRR10.2, BoPRR5, BoPRR7.1, BoPRR7.2, BoPRR9.1, and BoPRR9.2 were upregulated and BoRR3, BoRR4.1, BoRR4.2, BoRR5.1, BoRR5.2, BoRR6.1, BoRR6.2, BoRR7.1, BoRR7.2, BoRR8.1, BoRR9.1 were downregulated (Figure 7B). To validate the expression level of stress-responsive BoTCS genes, real-time RT-qPCR was performed. It was observed that most of the genes demonstrated expression patterns similar to RNA-seq data except BoPHYAb, BoERS2.2, BoRR8.2, BoRR10.2, BoRR5.2, and BoRR6.1 (Figure 8C).
FIGURE 8
4 Discussion
Cytokinins are the key plant hormones that regulate various processes in plants including germination, apical dominance, development of flower and fruit, pathogen interaction of plants, and cell division of several tissues. Signal transduction of cytokinin occurs using a phosphor-relay which is derived from TCS (L. ). Phosphorylation of proteins is one of the most common methods for intracellular signaling where an outside stimuli is received and processed to generate the required response. Protein kinases use ATP as the phosphate donor to phosphorylate their substrates ().
In this study, the main objective was to find a Two-Component System (TCS) in B. oleracea, its involvement in light/shade stress, and how they express in multiple tissues. To accomplish this objective, a comprehensive genomic analysis was carried out in B. oleracea which identified 21 HKs, 8 HPs, and 51 RRs. In Arabidopsis a number of these genes identified were as follows: 17HKs, 6 HPs, and 33 RRs (). Similarly in S. bicolor, 13HKs,5 HPs, and 19RRs were identified () In melon 51 members of this family were identified with no identified Type-C RR. All these differences in gene number across species is attributed to the duplication events. Type-C RR is thought to be the earliest RR and changes in its promoters may have led to the emergence of Type-A RRs ().
The phylogenetic study showed that B. oleracea TCS proteins are divided into three Groups: BoHKs, BoHPs, and BoRRs. These groups were further divided into subfamilies. A similar grouping was found in Arabidopsis and rice (). On the phylogenetic tree, these subfamilies were dispersed and proteins belonging to the same subfamilies were clustered together. HKs were further grouped into CKI1 and CKI2 according to their domains. CKI1 contained AHK1 and CKI2 group contained true histidine kinase BoHK5 (). The presence of the CHASE domain makes identification of the clade regarding cytokinin receptors. RRs subfamilies cluster as a larger clade in the phylogenetic tree which further subdivides the RRs according to their specific clades ().
The numbers of genes and phylogenetic analysis show that different plants have a different number of genes belonging to the same superfamily. Various events like gene duplication as well as whole genome duplication can lead to the difference in the number of genes in multiple plants. Duplication studies revealed that mainly segmental duplication led to the expansion of this gene family in S. bicolor. Segmental duplication was the main mechanism of duplication in A. thaliana, Brassica rapa (Chinese cabbage), and C. arietinum. Although tandem duplication is also found but mainly the genes are segmentally duplicated. In tomato, both events have been identified. This shows the role of duplication in the expansion of this gene family in plants.
In the promoter region of BoTCS genes, several cis-acting elements for the regulation of abiotic stress responses are found. These include low-temperature responsive, light responsive, drought-inducibility, and hormone-responsive elements (auxin, gibberellins, salicylic acid, and MeJa responsive elements). Similar results were observed for S. bicolor genes which contained elements for light, drought, and hormone responsiveness (), Similarly, in watermelon and cucumber, drought-responsive elements, abscisic acid-responsive and elements for anaerobic induction were found (). In banana, several hormone-responsive elements were found abundantly. This shows that TCS-mediated signaling is induced by hormones, developmental conditions, and various stresses. In plants, complementary miRNAs and the target genes control various developmental, metabolic, and stress-related responses. Similarly, for BoTCSs, predicted associated miRNAs were mostly responsible for inhibiting the cleavage of target transcripts. Only a few of these predicted miRNAs were involved in the inhibition of the target gene’s translation.
Expression analysis demonstrated that several genes express in the root, stem, bud, leaves, and leafy head and under shade/light treatment (Figure 7). In tomatoes, 12 genes from HK and RR families were found to express highly in roots which is the main site for the synthesis of cytokinins (). These genes are likely to be involved in cytokinin-mediated signal transduction in Arabidopsis as well its homologs (). In S. bicolor, the expression was showed by ethylene receptors and RRs mainly. In tomatoes, similar proteins HKl especially ethylene receptors and PRRs were expressed during the ripening of the fruit. It is been found that these genes are involved in the development and ripening of fruits (; ). Most of the genes in Chinese cabbage and soybean responded to drought and salt stresses. Most of the tomato genes were expressed under drought stress while Arabidopsis genes show a decline in expression under drought stress ().
This expression profiling of TCS is important for understanding the function of these genes under different abiotic stresses. Cis-regulatory elements for abiotic stresses are found in all subfamilies of BoTCS, especially RRs. Similarly, most of the genes from the RR subfamily are found to have higher expression in multiple tissues as well as in shade stress. This reveals the involvement of BoTCSs especially BoRRs in abiotic stress tolerance. In Medicago truncatula few TCS genes were highly expressed in different organs suggesting their tissue-specific role (). In chickpea, RRs were localized in the nucleus and interacted with the promoters of nodulation genes which indicate that they act as a transcription factor and regulate the early stages of nodulation (). In Arabidopsis, HKs and the type-A RRs that induce cold stress had a negative role in cold stress through inhibition of ABA response (). In B. rapa, gene expression regarding high salinity and drought stress was found. Ethylene receptors are involved in pollen thermotolerance in tomatoes. Similarly, TCS genes express under dehydration stress in G. max. GATA-motif and Protein binding element in the promoter region of TCS genes were identified which is consistent with the result of previous studies (; ). These studies show the evolution of the TCS gene family in B. oleracea and how this evolution led to the expansion of the genes as well as their functionality. These studies require further biotechnological analyses, however, all the information gained through this study is valuable for the future studies on the functional insights of the B. oleracea under environmental stresses. The genes showing distinct expression pattern can be used in the development of genetically modified crops which will not only be resistant to the stresses but also provide an increase in crop yield.
5 Conclusion
In B. oleracea, 80 genes have been identified as potential members of the TCS gene family. HKs accounted for 21 of the 80 identified genes, whereas HPs and RRs accounted for 8 and 51 genes respectively. Moreover, further information regarding conserved domains, motifs, cis-regulatory elements, phylogenetic analysis, and protein structure prediction showed the conservation of the TCS gene family in diverse plant species. Gene duplication events for maximum genes were identified and mainly the segmental duplication caused the expansion of these genes. MiRNAs also showed associations with certain BoTCS genes. The genes showing unique expression (higher or lower) in light/shade stress are ideal candidates for gene manipulation and characterization in breeding for shade tolerance. The qRT-PCR results verified the expression variation of genes including BoERS1, BoERS2.1, BoERS2.2, BoRR10.2, BoRR7.1, and BoPHYs. This would help to acquire a better understanding of TCS and further genomic research may help to increase plant resistance to abiotic stresses.
Statements
Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
Author contributions
MJ conceived the idea, supervised, and provided funding for lab experiments; MS and BU collected the literature review for the design of the work, performed the analysis, and wrote first draft; KF, RZ, and MN provided the technical expertise to strengthen the basic concept, data acquisition and analysis; FA and SS helped in experimental setup and qPCR data analysis; MT and MAN helped in analyzing data, drawing figures and data interpretation; FA, MJ, KA, AA, and SE helped in manuscript preparation, lab experiment and discussion; MAN, KA, and AA were involved in the final development and scientific proofreading of the manuscript. All authors contributed to the article and approved the submitted version.
Acknowledgments
The authors extend their appreciation to the Researchers Supporting Project Number (RSP-2023R369), King Saud University, Riyadh, Saudi Arabia.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fgene.2023.1142544/full#supplementary-material
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Summary
Keywords
cabbage, two-component system, cytokinin, cell signaling, abiotic stress, genome-wide, expression analysis, phylogeny
Citation
Sadaqat M, Umer B, Attia KA, Abdelkhalik AF, Azeem F, Javed MR, Fatima K, Zameer R, Nadeem M, Tanveer MH, Sun S, Ercisli S and Nawaz MA (2023) Genome-wide identification and expression profiling of two-component system (TCS) genes in Brassica oleracea in response to shade stress. Front. Genet. 14:1142544. doi: 10.3389/fgene.2023.1142544
Received
11 January 2023
Accepted
26 April 2023
Published
30 May 2023
Volume
14 - 2023
Edited by
Sajid Shokat, Nuclear Institute for Agriculture and Biology (NIAB), Pakistan
Reviewed by
Anuj Kumar, Dalhousie University, Canada
Bindu Yadav, Jawaharlal Nehru University, India
Syed Adeel Zafar, University of California, Riverside, United States
Updates
Copyright
© 2023 Sadaqat, Umer, Attia, Abdelkhalik, Azeem, Javed, Fatima, Zameer, Nadeem, Tanveer, Sun, Ercisli and Nawaz.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Muhammad Rizwan Javed, rizwan@gcuf.edu.pk; Muhammad Amjad Nawaz, amjad_ucauos@yahoo.com
† These authors have contributed equally to this work and share first authorship
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