Tomato (Solanum lycopersicum L.) is an economically valuable fruit worldwide. Its yield and development are acutely affected by harsh environmental conditions such as drought, salinity, and extreme temperatures. Salinity and drought stress conditions lead to harmful physiological effects—for instance, stunted plant growth, failure in seed germination, and reduction in fruit yield. Moreover, drought causes osmotic stress that results in the decrease of photosynthetic rate and plant development (Diouf et al., 2018; Zhou et al., 2019). In the FAO 2019 report, the data stated a substantial increase in tomato production to 182 million tons from 37 million tons that was in 2017.

The dehydration-responsive element-binding (DREB) gene family is one of the most prominent transcription regulators that bind with DRE/cis regulatory elements (CREs) to induce tolerance under biotic and abiotic stresses in plants (Du et al., 2018). It is also known as cis-binding factor, belongs to the ethylene-responsive element-binding factor (ERF) family, and contains conserved AP2 domain (Khan, 2011). The presence of valine (V) and glutamic acid (E) at the 14th and 19th positions in the AP2 domain adds more clarity to the DREB family from its other sub-family members. The AP2 domain contains 60 to 70 amino acid sequences that are responsible for plant defense mechanisms to combat stressful circumstances (Wu et al., 2015). YRG (19–22 aa long) and RAYDS are two conserved elements that are characteristic features of the AP2 domain. The core region of RAYD is supposed to be involved in the existence of an amphipathic α-helix (Chai et al., 2020). DREB is a sub-family of ERF transcription factors with conserved AP2 domain. The three-dimensional structure of AP2 domain was revealed in AtERF1 gene through nuclear magnetic resonance imaging that demonstrated the presence of an α-helix and three antiparallel β-sheets in its structural organization (Lata and Prasad, 2011; Li Z. et al., 2021). DREB genes attach to drought-responsive (DRE/CRT) elements with the assistance of a V residue, two W residues, and four R residues (Mushtaq et al., 2021). The phosphorylation of DREB genes is essential for their activation, supported by a threonine- or serine-rich sequence present next to the AP2 domain (Lata and Prasad, 2011). The DRE/CRT elements have a core motif consisting of ACCGAC/GCCGAC that directly interacts with DREB genes under abiotic stresses (Vazquez-Hernandez et al., 2017). The DREB gene family further splits into six classes, i.e. A1–A6, based on their fundamental structural and functional characteristics (Chai et al., 2020).

Subsequently, the DREB gene family has been identified in a number of plants including Arabidopsis (Hwang et al., 2012), pearl millet, strawberry, tobacco (Cheng et al., 2013), rice, lettuce (Park et al., 2020), bananas, wheat, bell pepper, maize (Liu et al., 2013), soybean (Hou et al., 2022), and potato (Mushtaq et al., 2021). Previous studies have identified DREB1, DREB2, and DREB3 in tomatoes. SlDREB1 has a remarkable role in response to drought stress. It enhances the concentration of soluble sugars and accumulates osmolytes that further balance the osmotic pressure and increase the tolerance response under water-deficient conditions (Jiang et al., 2017). SlDREB2 is involved in the regulation of stress signaling pathways. Studies have shown its contribution in the development of early vegetative and reproductive stages. It regulates the synthesis of proline that further assists plants in response to salt and hormonal changes (Hichri et al., 2016), whereas SlDREB3 is a negative regulator of the abscisic acid (ABA) pathway by altering ABA signaling. It is also known as the ripening gene by controlling the ethylene-dependent and ethylene-independent pathways (Gupta et al., 2022).

Despite this, limited studies have been conducted on the characterization of the DREB gene family in S. lycopersicum. The present research data extensively covers phylogenetic evaluation, gene structure analysis, conserved motifs, chromosomal localization, gene duplication events, synteny analysis for the detection of orthologues in interspecies, gene ontology, characterization of cis-regulatory elements, prediction of subcellular localization, protein modeling, prediction of pocket binding sites, disorder region analysis, and interactive networks with other proteins. Overall, our data has the potential information that serves as a strong infrastructure for unravelling the extensive structural and functional relationships of intricate genes for genetic engineering to enhance tolerance in tomato genotypes under environmental stresses.

The present study covers extensive data on 58 SlDREB gene sequences. These 58 SlDREB genes were classified into A1–A6 classes based on the classification of AtDREB genes. The conserved amino acid analysis revealed that valine (V) is replaced by isoleucine (I) at the 14th position due to their same chemical structure, polarity, and characteristics in three SlDREB genes. Both are aliphatic, non-polar, and hydrophobic, whereas glutamic acid (E) is being replaced by V, Q, D, H, L, and A at the 19th position (Zhang et al., 2022). The genome-wide analysis discovered the presence of 58 SlDREB genes, which can vary in numbers in different plants due to the variance in genome sizes and genes present in their genome. The physiochemical properties, variation in amino acid structure, and protein binding affinity contribute multiplicity in protein function to withstand harsh environmental conditions (Salih et al., 2019; Panja et al., 2020). Thus, the variations in amino acids at conserved positions in SlDREB genes proved that these genes regulate many biological and molecular mechanisms associated with plant growth and interact with other stress-regulatory proteins. The total length of SlDREB proteins varied between 113 to 419 amino acids. Other properties like isoelectric point and molecular weight fluctuated between 4.42–9.41 and 12.35–46.94 kDa ( Supplementary Table S1 ).

The phylogenetic analysis of SlDREB and AtDREB genes revealed that the variation in gene distribution patterns among A1–A6 classes, respectively, is due to genetic diversity. The study discovered the presence of seven members in A1 class and seven in A2 class, 16 members related to A4 class, seven members involved in A5, and 21 members present in A6 subgroup, respectively. It also demonstrated the absence of A3 class in the SlDREB gene family because no SlDREB member showed homology with AT2G40220.1 (member of A3 class) ( Figure 2 ). Similarly, another related study reported the absence of A3 class in Ananas comosus (L.) Merr. (Chai et al., 2020). The research data illustrates that SlDREB24 (Solyc06g050520, accession no: AF500011 or DREB1) has a significant role under drought stress (Jiang et al., 2017). SlDREB22 (Solyc05g052410, GenBank accession: ADZ15315 or DREB2), known as SlDREB2, is likewise a potential regulator of stress-responsive genes and is expressed in early-seedling stage (Hichri et al., 2016). SlDREB18 (Solyc04g072900) (GenBank accession: AF506825.1), named LeDREB3, showed enhanced tolerance against many abiotic stresses like drought, salinity, and osmotic pressure (Islam and Wang, 2009, Wang G. et al., 2019), and SlDREB2 (Solyc06g066540) was recently characterized in response to heat stress (Mao et al., 2020). Furthermore, the gene structure analysis discovered the presence of introns in S. lycopersicum, unlike in Arabidopsis. The AtDREB genes showed the lack of intronic regions in its coding sequence, but studies revealed the occurrence of introns in various plants like S. tuberosum (Mushtaq et al., 2021), soybean (Zhou et al., 2020), Ananas comosus (L.) Merr., and Saccharum spontaneum (Zhen et al., 2021). Studies revealed that 12 SlDREB genes contain introns, out of which nine SlDREBs had one intron and the remaining three SlDREB genes entailed two introns. Thus, S. lycopersicum shows a similarity with Ananas comosus, Solanum tuberosum, Saccharum spontaneum, and soybean in terms of gene structure due to the presence of introns in the coding region. According to studies, gene structures with no or few introns are more active in response towards stress conditions (Jeffares et al., 2008). The difference in intron and exon organization clarifies the functional diversity of DREB genes that occurred due to variations in climatic conditions and stresses in a variety of plant species (Zhen et al., 2021).

To gain insight into the conserved motifs that have an important role in protein functionality and interactions, a motif analysis was accomplished. The results demonstrated the presence of conserved motifs in TFs on the same chromosome as in Ananas comosus (L.) Merr. and Solanum melongena L. (Li et al., 2021). In SlDREB proteins, 20 conserved motifs were discovered, out of which two motifs were present throughout all SlDREB sequences. It was observed that A1, A4, and A5 classes are more closely related, while A2 and A6 classes shared a combined clade due to the same conserved motifs. The AP2 domain consists of one alpha helix made up of 18 amino acids that assist in the interactions of DREB genes with other proteins, followed by three antiparallel beta sheets on the major groove of the DNA (Song et al., 2020). The identified 20 motifs in the SlDREB sequences have a variable composition that is the possible reason for the classification of DREB genes into different classes with high structural resemblance with the AP2 domain. Motifs 1, 2, and 3 are specifically present in the AP2 domain, which is the characteristic domain of DREB genes, whereas a difference in motif composition evaluated the formation of two clades in the phylogenetic tree. The difference in motif composition is responsible for the molecular interactions with different proteins and assists in DNA binding (Alberts et al., 2002; Defoort et al., 2018). This study proved that a similar pattern of intron–exon organization and conserved motifs was observed in the members belonging to same groups, thus elucidating the reliability of the phylogenetic relationship represented in Figure 3 .

The random distribution of SlDREB genes provides an insight for their stability in a changing environment. Gene duplication events occurred in genes for adaptive evolution that is a major cause of plant diversity and adaptive specialization in genes (Flagel and Wendel, 2009). Polyploidy was observed in Arabidopsis, soybean, cotton, and potato (Nakano et al., 2006). Whole-genome duplication events occur in two ways: tandem duplication and segmental duplication. A tandem duplication event is responsible for the addition of a copied gene into the array, while segmental duplication is accountable for duplicating and spreading the fragments or the entire array. S. spontaneum, Z. mays, and P. trichocarpa go through both mechanisms of polyploidy. Whole-genome duplication was also examined in S. lycopersicum. Our results confirmed the occurrence of duplication events in 15 gene pairs, out of which eight gene pairs (32–33, 34–39, and 35–40) present on chromosome 08 (8–15, 27–29, and 50–51) exist on chromosomes 03, 06, and 11, and 43–44 and 45–47 belong to chromosome 10, undergoing tandem duplication events which were present on the same chromosomes ( Figure 4 ).

In contrast, segmental duplication events were observed in seven gene pairs (36–52, 26–53, 11–54, 41–48, 17–58, 14–28, and 22–24) ( Figure 5 ). In the tandem duplication method, gene pairs with the same cis regulatory elements, due to their presence on the same chromosome, were observed and involved in the regulation of similar functions (Flagel and Wendel, 2009). The present study’s data has revealed that the last gene duplication event occurred 4 million years ago in the 43–44 gene pair, which belongs to the A2 class and exhibits tandem duplication ( Supplementary Table S5 ). Genes that are mostly involved in biological processes such as biotic and abiotic stress signaling undergo tandem duplications (Huang et al., 2020). To evaluate the presence of orthologous genes in interspecies, synteny analysis was employed. In total, 32 orthologs of SlDREB genes were found in Arabidopsis, whereas 47 orthologs were observed in S. tuberosum due to the close evolutionary relationship with S. lycopersicum, with both belonging to the same family ( Figure 6 ). For the analysis of functional genomics and molecular processes conducted by each SlDREB gene, gene ontology annotation was employed. The results revealed that the DNA binding ability of SlDREB genes is uniform, based on studies conducted in P. vulgaris. The GO annotation analysis also discovered that the SlDREB genes primarily localized in the nucleus. Furthermore, the subcellular localization prediction showed the presence of SlDREBs in 13 other organelles as illustrated in the heat map ( Figure 8 ).

In this study, 103 CREs were found in SlDREB sequences through promoter analysis. These 103 CREs have been classified into seven distinct categories depending on the functions and activation of the biological, cellular, and molecular pathways associated with them. Seven functional groups include light-responsive elements, hormone-related cis elements, development-related cis elements, biotic stress response elements, abiotic stress-responsive cis elements, promoter-related cis elements, and unidentified CREs (Ain-Ali et al., 2021). The promoter-related cis regulatory elements frequently occurred in all genes and secured about 69%, followed by light-responsive at 8%, abiotic stress-responsive at 8%, development-related at 6%, hormone-related at 3%, and biotic stress-responsive elements at 1% ( Figure 9 ). The following cis elements are promoter-related: TATA-box, CAAT-box, AT~TATA box, A-box, and TATA elements. Among these elements, TATA-box and CAAT-box frequently occurred in every sequence and accommodated transcription factors by acting as their binding sites. The TATA element assists in the binding of TATA-box and activates the initiation site for transcription (Bae et al., 2015). The TATA box binds with RNA polymerase II for the initiation of transcription. AT~TATA box, and A-box are promoter binding sites. Moreover, A-box is an α-amylase promoter that is also identified in WRKY genes (Zhou et al., 2010). According to studies in rice, A-box assists in the regulation of the flowering stage (Mongkolsiriwatana et al., 2009). Thus, the analysis proved that SlDREB genes have a strong role in transcriptional regulation ( Figure 10A ).

The cis regulatory elements involved in light response are G-box or G-Box, TCT-motif, AE-box, GATA-motif, Sp1, GT1-motif, Box 4, ACE, LAMP-element, GA-motif, Gap-box, ATCT-motif, 3-AF1, chs-CMA1a, chs-CMA2a, AT1-motif, ATC-motif, TCCC-motif, I-box, chs-Unit 1 m1, AAAC-motif, Box II, CAG-motif, MRE, LS7, chs-CMA2b, ACA-motif, 3-AF3 binding site, and L-box. G-box and Box-4 abundantly appeared and regulated the transcription of light-responsive genes (Gilmartin et al., 1990). Moreover, G-box, ACE, Sp1, and GATA-motif also regulate the responses against abiotic stresses (Shariatipour and Heidari, 2020). The TCT motif controls photoperiodism, which is crucial for circadian rhythms for the proper development of plants and for the flowering stage (Zhao et al., 2018). Studies have evaluated the involvement of G-box in the phosphorylation of light-responsive genes through the calcium pathway upon receiving light signal and upregulated rd29 in response to drought as well (Kurt and Filiz, 2018). It is also involved in ethylene and ABA signaling pathway by acting as a binding site for bZIP proteins and responds to many abiotic stresses such as UV light and hormonal changes (Ma et al., 2013; Saidi and Hajibarat, 2018). Box-4 is a highly conserved DNA module in genes that are lightly sensitive. WRKY genes are also highly concentrated with box-4 element (Yin et al., 2013). The GT-1 motif is involved in the upregulation of light responses (Wang C. et al., 2019). The same pattern of cis elements was observed in S. tuberosum, with the exception of 3-AF1 binding site, 3-AF3 binding site, chs-CMA2b, and ACA motif elements that are only specified in S. lycopersicum ( Figure 10B ). The light-responsive elements are highly associated with photoperiods that regulate circadian rhythms and many other processes like flowering stage (Ain-Ali et al., 2021).

Abiotic stress conditions severely affect the growth and development of tomato crop. AT-rich sequence, ARE, CCAAT-box, DRE core, DRE 1, GC-motif, LTR, MBS, MBS 1, STRE, TC-rich repeats, MYB, MYC, MYB recognition site, MYB binding site, MYB like site, and AT-rich element are associated with abiotic stress-responsive elements ( Figure 11A ). MYB and MYC are dehydration-responsive motifs mostly observed in SlDREB gene promoters, whereas MBS and MBS1 act as binding sites for MYB that are engaged in drought response, biotic stress, and gene regulation of flavonoid biosynthesis (Li et al., 2015; Kaur et al., 2017). MYC is involved in the regulation of many hormone-related pathways. JA is one of the hormones mainly regulated through the MYC element in response to stresses (Yanfang et al., 2018). AT-rich elements act as binding site for AT-rich binding proteins and functions as activation mediator upon signal perception. Moreover, ARE is responsible for anaerobic induction and consists of two elements: GT and GC motif. GT motif has a high resemblance with MYB binding site and induces a response against anaerobic and water-deficient conditions (Dolferus et al., 2001). CCAAT-box acts as MYBHv1 binding site for heat shock factors against heat stress (Chaudhary et al., 2019). DRE core and DRE 1 are dehydration-responsive elements, whereas GC-motif is involved in anoxic-specific inducibility (Brown et al., 2003), LTR and STRE are low temperature stress-responsive elements, and TC-rich repeats are involved in defense signaling pathway (Lu et al., 2019; Niu et al., 2020). The cis elements associated with abiotic stress response appeared third in terms of occurrence and found to be consistent with prior studies conducted on S. tuberosum except the MYB like site.

Furthermore, among the results discovered were ABRE, AuxRE, AuxRR-Core, CGTCA-Motif, ERE, GARE-motif, P-Box, TATC-box, TCA-element, TGACG-motif, TGA-element, ABRE4, ABRE2, ABRE3a, AT~ABRE, and TCA motifs in the hormone-related cis elements category. Abscisic acid (ABA)-responsive element (ABRE) is associated with hormonal regulation and abiotic stress signaling. ABRE4, ABRE2, ABRE3a, and AT~ABRE act as binding sites (Verma et al., 2016). Another related study demonstrates that AuxRE, TGA, and AuxRR-Core cis elements are auxin-responsive and have a regulatory role in the expression of DREB genes under drought stress (Kolachevskaya et al., 2019). Moreover, auxin plays a significant role in plant development and defense signaling mechanisms against biotic and abiotic stresses, particularly in response to drought stress. Auxin, as a key growth hormone, assures the effective working of the plant physiology under stressful environments (Sharif et al., 2022), whereas CGTCA and TGACG-motif act as methyl jasmonate-responsive elements (Concha et al., 2013). Similar hormone-related elements were described in Saccharum spontaneum and Solanum tuberosum except ABRE4, ABRE2, ABRE3a, AT~ABRE, and TCA elements (Kaur et al., 2017). These hormone-related elements may have a direct or indirect interaction with signaling pathways in response to various stresses ( Figure 11B ).

In development-related cis elements, several motifs were discovered, such as AAGAA-motif, CCGTCC-box, AP-1, AC-1, AC-II, as-I, CARE, circadian, CAT-box, dOCT, E2Fb, F-box, GCN4-motif, HD-zip 1, HD-zip 3, MSA like, NON, O2 Site, re2f1, RY element, AACA-motif, and CCGTCC motif ( Figure 12A ). The AAGAA-motif and as-1 element, available abundantly, are pathogenesis-related promoters. The AAGAA-motif is responsible for the development of xylem and assists in water transportation. The As-1 element acts as a promoter binding site for many hormone-responsive genes, especially under auxin and methyl jasmonate stress signaling. The CCGTCC-box, CARE, and CAT-box act as cis elements in meristem-specific stimulation (Laloum et al., 2013). Moreover, AC-1 and AC-II are engaged in xylem-specific activation. HD-zip 1 and HD-zip 3 participate in palisade mesophyll cell differentiation and vascular development and regulate biological processes associated with macromolecules (Vulavala et al., 2017). AACA-motif and GCN4-motif act as endosperm expression elements and regulate the circadian rhythms (Kaur et al., 2017). O2 site is involved in the regulation of zein metabolism, and RY element assists in seed -specific regulation. However, E2Fb and F-box are engaged in cell cycle regulation, and MSA like is a mitosis-specific activator (Li L. et al., 2021). The present work demonstrates that S-box, W-box, WRE 3, and WUN-motif cis regulatory elements were recognized to function in response to biotic stresses and, furthermore, to interact with WRKY transcription factor which acts as a wound-responsive element as depicted in Figure 12B (Yin et al., 2013; Liu et al., 2016). According to another related study, W-box regulates seed dormancy and defense mechanisms against biotic stresses (Rinerson et al., 2015). These elements were also reported in Saccharum spontaneum and Solanum tuberosum. However, among 103 cis elements, 10 motifs which cover almost 5% occurrence were found with unidentified functions. Parallel studies revealed the existence of conserved motifs that might have a crucial role in many biological processes. Thus, the presence of these motifs in the promoter regions of SlDREB genes indicates their potentialities in plant defense signaling and processes associated with growth and development in tomato.

Moreover, structural variations were detected in five SlDREB representatives from each class through secondary and tertiary structure analysis, which gave insight to the development of stable protein structures. Secondary structures were created by SOPMA server that exhibited different percentages of α-helices, β turn, random coils, and extended strands. The spatial arrangement of amino acids present in the random coils of DREB proteins can vary depending on the intrinsic nature of protein. These factors contributed to the structural and functional versatility and molecular interactions of proteins (Vatansever et al., 2017). Furthermore, Phyre2 server assisted in the prediction of tertiary structures. Models that covered more than 30% coverage were further subjected for refinement. For structural refinement and validation, Galaxy Refine server and PROCHECK server were employed to complete the gaps between protein sequences and conformations to create stable protein structures and to check the stability through the generation of Ramachandran plots. Validation is considered as a core step in structural determination (Pražnikar et al., 2019). Studies reported that the residues lying from 85% to more than 90% in favored region is considered as a good model. Hence, this study indicated the presence of 91.2% for SlDREB1, 88.8% for SlDREB2, 89.4% for SlDREB51, 90.3% for SlDREB7, and 92.8% for SlDREB54 residues present in the favored region. The percentages were represented through a Ramachandran plot that further verified the models having good stereo-chemical properties ( Supplementary Table S10 ). Stabilized models were visualized through ChimeraX that constructed high-quality models with their respective α-helix and β turns ( Supplementary Table S11 ).

There are specific regions on proteins that are responsible to interact with other proteins and molecules in response to different cellular and biological processes, and these are known as ligand binding sites (Kawabata, 2010). The present study data has revealed the presence of active catalytic sites on five SlDREB protein structures that are crucial for binding with different proteins having variable binding capacities as indicated in orange region ( Supplementary Table S12A ). Each catalytic site is comprised of specific amino acids that contribute to the binding capacity, assortment in protein functions, interactions with other molecules, and structural versatility (Coleman and Sharp, 2010).

Furthermore, proteins consist of disorder regions that allow them to change their functions, expand their interactions, and induce structural versatility in response to the changing environment. These disorder regions are flexible parts that allow proteins to bind with greater affinity to different molecules and thus contribute to various defense signaling pathways and cell cycle processes. The disorder region analysis provides thorough understanding of the spatial regulations and interactions of signaling pathways associated with proteins. The presence of these flexible regions in transcription factors allow their binding with signaling cascades and other stress-responsive proteins (Pazos et al., 2013). Disorder regions were predicted in five representative SlDREB proteins ( Supplementary Table S12B ). The binding plasticity of disorder regions assists in the attachment to other gene families like NAC, GRAS, and dehydrins. These interactions generate a signaling cascade in developmental processes like seed development, response to hormonal changes, and drought stress (Hsiao, 2022).

Protein–protein interactions are necessary for the regulation of different developmental and adaptive processes for normal plant growth (Braun et al., 2013). Transcription factors stimulate other proteins and co-factors for the activation of distinct regulatory pathways that control transcription in response to various stresses (Francois et al., 2020). In response to both biotic and abiotic stresses, thousands of molecules like reactive oxygen species (ROS), hormones, and proteins interact at the cellular and molecular levels for the activation of kinases and other signaling cascades. For the complete integration of protein function, its cellular abundance, and biochemical activities, it is crucial to gain insight to interactive pathways (Struk et al., 2019). The protein–protein interactions of five SlDREB proteins were detected through STRING that revealed its physical and functional associations. In the present study ( Figure 13 ), nodes indicate spliced isoforms and the interaction of query protein (red node) with different interactors due to post-translational modifications. Each node represents all the proteins produced by a single protein-coding gene locus. Moreover, colored edges represent the type of evidence in protein associations, blue and pink edges represent known interactions, and green, red, and dark blue colors are known for predicted interactions, whereas other colors indicate the evidence recovered from text mining or co-occurrence.

Moreover, our results demonstrate that SlDREB1 interacts with 10 interactors, namely: Solyc10g084370.1.1, Solyc02g077970.2.1, Solyc03g044300.2.1, Solyc02g082990.2.1, Solyc02g089570.2.1, Solyc02g089580.2.1, Solyc09g010820.2.1, Solyc02g077710.1.1, HSFA3, and Solyc02g065640.2.1. Out of 10 interactors, two members (Solyc10g084370.1.1 and Solyc09g010820.2.1) showed the presence of SANT domain which belongs to homeobox superfamily and a MYB-related protein that acts as a DNA binding domain and regulates histone acetylation (Boyer et al., 2004). Histone acetylation is essential in the cell cycle and in response to abiotic stress (light, heat, UVB, sugar, salt, and wound) and hormonal change (salicylic, ethylene, abscisic acid and auxin) (Jiang et al., 2020). Histone acetyltransferase genes previously discovered in rice (Liu et al., 2012), Triticum aestivum (Gao et al., 2021), and Arabidopsis thaliana (Pandey et al., 2002) (Solyc02g077970.2.1) belong to the LEA protein family which are connected with salinity and water stress signaling and have been reported in many plants like Vigna glabrescens (Singh et al., 2022), cotton (Magwanga et al., 2018), Salvia miltiorrhiza (Chen et al., 2021), and Triticum aestivum (Liu et al., 2019). The presence of AP2 domain was detected in one member (Solyc03g044300.2.1) that contributed to transcriptional regulation and responded to stresses as found in wheat (Magar et al., 2022) and castor bean (Xu et al., 2013). However, four members of SlDREB1 interactors—namely, Solyc02g082990.2.1, Solyc02g089570.2.1, Solyc02g089580.2.1, and Solyc02g065640.2.1—include the KIN1 and KIN2 domains, also known as cor6.6. in Arabidopsis, which is engaged in low temperature and responses to disturbance in abscisic acid levels, as abscisic acid is a vital hormone that activates in retort to stress stimuli (Wang et al., 1995). Solyc02g077710.1.1 is an E-6 like protein which is necessary for fiber development (John, 1996). Solyc09g009100.2.1 is the last interactor that is a heat shock transcription factor A3 (HSFA3) involved in response to heat stress, DNA binding, and regulation of transcription as illustrated in Figure 13A .

Several proteins were likewise associated with SlDREB24, which belongs to the A2 subgroup, i.e., HSFA3, Solyc08g005270.2.1, PRO2, Solyc06g019170.2.1, Solyc05g009710.2.1, MED25, ICE1a, Solyc05g010300.2.1, TCP10, and SIZ1 ( Figure 13B ). Solyc08g005270.2.1 contains the RST domain which is present in PARP and TAF4s factors and belongs to the SRO gene family. It is engaged in transcriptional regulation by interacting with transcription factors and maintains ribosyl transferase activity. Ribosyl transferase activity is linked with oxidative stress and responsible for ADP-ribosyl polymerases (Jaspers et al., 2010). Extensive analysis of RST domain has been carried out in wheat (Liu et al., 2014), tomato (Zhen et al., 2021), banana (Zhang et al., 2019), Sesamum indicum (Liu et al., 2021), and rice (You et al., 2014), whereas two members [Solyc08g043170.2.1 (PRO2) and Solyc06g019170.2.1) are known as delta-1-pyrroline-5-carboxylate synthase. These members are associated with proline biosynthesis and osmoregulation. Proline synthesis is important in plants as it works as an antioxidant and signaling molecule under stress conditions, which further activates the defense signaling cascades. Furthermore, it controls osmotic pressure and ROS levels, thus making the plant tolerant to stresses (Hayat et al., 2012). In Solyc05g009710.2.1, MED25 (Solyc12g070100.1.1) mediator complex subunit25_von Willebrand factor type A was detected, which performs as a scaffold protein for transcription factors and RNA polymerase II and regulates hormonal cascades under biotic and abiotic stresses (Kazan, 2017). MED25 has been reported in Arabidopsis, where it is discovered to deal with drought stress (Elfving et al., 2011). Additionally, ICE1a (Solyc06g068870.2.1), also known as inducer of CBI expression1, is a DNA binding protein present in the nucleus and confers tolerance in plants against cold stress (Chinnusamy et al., 2003). The RPOLD domain in Solyc05g010300.2.1 contributes to protein dimerization and cellular response to elevated temperatures. Moreover, TCP10 and SIZ1 (SP-RING-type zinc finger domain), detected in Solyc07053410.2.1 and Solyc11g069160.1.1, are responsible for protein–protein interactions and DNA binding TF activity and play a crucial role in zinc ion binding activity and protein SUMOylating. Studies in rice revealed that protein SUMOylation is a process in which local proteins act as signal transducers to activate signal cascades under stress (Raorane et al., 2013).

SlDREB51 likewise belongs to the A4 class. It shows interactions with Solyc07g008700.2.1, Solyc07g043270.2.1, Solyc00g007190.2.1, Solyc01g094460.2.1, Solyc02g082490.2.1, Solyc09g009250.2.1, Solyc03g046570.2.1, Solyc01g009840.1.1, Solyc08g080400.1.1, and Solyc08g007840.1.1 ( Figure 13C ). FAR1-related sequence9 (Solyc07g008700.2.1) protein and Solyc07g043270.2.1 are a far-red elongated hypocotyl 3 isoform x involved in transcription regulation, response to hormonal change, and light stress. Other functions that are revealed in other plants are the biosynthesis of chlorophyll and starch and circadian rhythms (Ma and Li, 2018). The sigma factor PP2C-like phosphatases in Solyc00g007190.2.1 and Solyc02g082490.2.1 regulate protein dephosphorylation and serine/threonine phosphatase activity in the signaling pathway. Additionally, Solyc01g094460.2.1 contains AT-hook and DUF 296 domain which is a domain of unknown function, and Solyc03g046570.2.1 (BES1-interacting MYC like protein) acts as a DNA binding protein and controls protein dimerization activity. SlDREB51 also interacts with Solyc08g007840.1.1 (SlDREB34), which demonstrated that SlDREB members are associated with each other for the regulation of DNA binding activity and response to biotic and abiotic stresses. The Solyc01g009840.1.1 and Solyc08g080400.1.1 proteins belong to the GRAS family, which is engaged in gibberellin signaling pathway and functions in plant growth and development. The GRAS family has been analyzed in various plants like rice, Populus and Arabidopsis (Liu and Widmer, 2014), Glycine max (Wang et al., 2020), Prunus mume (Lu et al., 2015), and tomato (Huang et al., 2015).

The present study has established that SlDREB7 is A5 class member and associated with the following proteins: Solyc06g053340.2.1, HSFA3, Solyc12g021360.1.1, Solyc05g032660.2.1, Solyc02g093280.2.1, SSR2, Solyc04g071310.2.1, Solyc09g072630.2.1, Solyc09g031610.2.1, and IAA36 ( Figure 13D ). One member (Solyc06g053340.2.1) belongs to the bZIP (basic-leucine zipper domain) family which is a prominent TF family associated with stress signaling pathways and developmental processes. In addition, studies on Arabidopsis and potato have revealed its functions in senescence, light and biotic stress response, and seed growth (Wang et al., 2021). Solyc06g053340.2.1 is a UVR domain-containing protein, and Solyc05g032660.2.1 is SDR, which shows oxidoreductase activity. SDR is of a heterogenous protein family which is involved in secondary metabolic and developmental pathways such as catabolism and flower growth (Moummou et al., 2012). However, Solyc02g093280.2.1 is recognized as bHLH DNA-binding protein that controls circadian rhythm, response to cold and light stress, carpel development, and protein dimerization. bHLH is a significant transcription factor family which is discovered in several plant species, i.e., Capsicum annuum L. (Zhang et al., 2020), tomato (Sun et al., 2015), and maize (Li et al., 2018). Moreover, succinic semialdehyde reductase isofom2 SSR2, recognized in Solyc03g121720, is an RNA recognition motif domain, and Solyc04g071310.2.1 is a CDK-activating kinase assembly factor-related protein that is associated with kinase activity and nucleotide excision repair, whereas ROP-interactive CRIB motif-containing protein 7 (Solyc09g072630.2.1) belongs to the Rho family that reacts to oxygen deficiency, cellular development, and hormone-mediated pathways (Yang and Fu, 2007). Finally, damaged DNA-binding 2 protein (Solyc09g031610.2.1) and auxin-responsive protein (IAA36) (Solyc06g066020.2.1) control the DNA repair mechanism, UV damage excision repair, protein poly-ubiquitination, response to UV-A and B, transcription, and auxin-activated signaling pathway (Reed, 2001).

STRING interactions revealed the network of SlDREB54 (member of the A6 subgroup) with Solyc01g109180.2.1 (long-chain acyl-CoA synthetase 2) which regulates and controls beta-oxidation for lipid metabolism. The second member is Solyc12g027870.1.1, a cyclin-dependent kinase E1 (CDKE1) that acts as a catalytic domain, mediating phosphorylation and the kinase pathways. In a study, CDKs revealed their functions in cell cycle regulations (Malumbres, 2014). Solyc05g054490.2.1 belongs to the 3-oxo-5-alpha-steroid 4-dehydrogenase family protein that functions in fatty acid biosynthesis. However, Solyc02g093640.2.1 is a fourth interactor (SDR protein) engaged in oxidoreductase activity. Solyc01g066050.2.1 (fifth interactor) is a transmembrane protein that belongs to the TPR-like superfamily present in the plasma membrane. The TPR family is involved in phytohormone signaling like abscisic acid, cytokinins, and gibberellins and also regulates ethylene signaling pathways. Furthermore, it regulates biological processes under osmotic stress conditions in plants (Schapire et al., 2006), and Solyc02g088190.2.1 consists of a SANT domain.

Moreover, Solyc05g054200.2.1 and Solyc07g047610.2.1 (transcriptional adapter ada2b isoform x1), associated with cold stress signaling, are involved in histone acetylation and chromatin remodeling. SlGPAT6 (Solyc09g014350.2.1) is a glycerol-3-phosphate 2-O-acyltransferase 6 protein detected in cutin biosynthesis in tomato (Petit et al., 2021). Additionally, SlDREB54 revealed its interaction with Solyc01g090140.1.1 protein [belongs to 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase superfamily] that functions in the biosynthesis of plant pigments, flavonoids, and gibberellins and regulates oxidoreductase activity (Kawai et al., 2014) ( Figure 13E ). The transcriptome analysis of tomato predicted that about 29 SlDREB genes are actively involved in regulating the defense pathways against abiotic stresses such as drought and heat stress. Only four DREB genes have already been characterized through an expression analysis that revealed their significant role against abiotic stresses. Recent studies conducted on SlDREB24 (Solyc06g050520, accession no: AF500011 or DREB1) showed its remarkable role in response to drought stress. It enhances the sugar concentration and accumulates osmolytes that further normalize the osmotic pressure and increase the response to drought stress (Jiang et al., 2017). SlDREB22 (Solyc05g052410, GenBank accession: ADZ15315 or DREB2), known as SlDREB2, is also a potential regulator of stress-responsive genes. According to spatiotemporal expression, this gene is also induced during early-seedling stage and is present in the vegetative parts of a plant. It is highly expressed in the leaves in defense against drought and salt stress (Hichri et al., 2016). Consequently, SlDREB18 (Solyc04g072900; GenBank accession: AF506825.1), named LeDREB3, has been reported to enhance the tolerance against many abiotic stresses like drought, salinity, and osmotic pressure. It is also known as the negative regulator of the ABA pathway, thus affecting many developmental processes like germination and senescence. It is also engaged in many physiological processes like root architecture development (Islam and Wang, 2009; Wang G. et al., 2019). Similar studies also revealed the expression of SlDREB28 (Solyc06g066540) under heat stress. It functions in response to many biotic and abiotic stresses such as salinity, drought by a fluctuating hormone level that contributes to increasing tolerance against stress. It assists in the biosynthesis of phytohormones (jasmonic acid, salicylic acid, and ethylene) and binds to DRE element under stress conditions (Mao et al., 2020). Other genes that were downregulated or not expressed under heat and drought stress might be involved in other abiotic stresses, developmental mechanisms, and the regulation of several stress-resistant genes involved in biotic responses. Previous studies on expression analysis exhibit the role of DREB genes in fruit development and ripening (Zhang et al., 2022). Overall, the present study data illustrates the comprehensive interactive networks of SlDREBs with various other stress-responsive proteins associated with the regulation of development, growth, and stress tolerance processes in tomato.

In conclusion, our results revealed a total of 58 SlDREB genes in tomato, with the presence of a conserved AP2 domain. Only three DREB genes indicated the replacement of valine with isoleucine due to the same polarity, structure, and chemical characteristics. Multiple sequence alignment and phylogenetic evaluation demonstrated the classification of SlDREB genes into five subgroups, excluding the A3 class, unlike Arabidopsis, which comprised A1–A6 classes. Gene duplication events proved the occurrence of polyploidy by tandem and segmental duplication methods. The evaluation of (Ka/Ks) values confirmed the existence of strong purification selection during the evolution of plants by calculating the divergence time. Moreover, synteny analysis revealed 32 and 47 orthologs in Arabidopsis and Solanum tuberosum, respectively. The cis-regulatory element analysis discovered 103 motifs that were further divided into seven categories. These elements have been implicated in light response, hormonal modifications, development, abiotic stress-responsive, biotic stress, and promoter binding sites. Gene ontology data illustrated that the SlDREB genes are involved in various biological and molecular processes. A subcellular localization assessment confirmed that the SlDREB genes primarily resided in the nucleus and were observed in 13 other organelles in variable concentrations. The secondary structure analysis assisted in determining essential points, whereas the tertiary structures of SlDREB proteins and validation of protein models provide a thorough understanding of homology modeling. The results of active ligand binding sites and disordered regions provide a deep insight into the protein’s structural and functional plasticity and versatility. Moreover, interactive network analysis demonstrated the connections of SlDREB proteins with other potential interactors. These interactions result in extensive signaling pathways and the functional diversity of SlDREB proteins. Transcriptome analysis revealed that 29 SlDREB genes regulate the defense signaling mechanisms against drought and heat stress. Overall, our data has potential information that serves as a robust infrastructure for unravelling the extensive structural and functional relationships of intricate genes requisite for genetic engineering, advance breeding, and biogenetics to develop stress tolerant/over-expressive tomato genotypes.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

FM designed the project and scheme of work. HM and FM conducted the research, analyzed the data, and wrote the manuscript. FM, RA, and AG reviewed the manuscript. All authors contributed to the article and approved the submitted version.

We acknowledge the Higher Education Commission, Pakistan, and the National University of Sciences and Technology (NUST), Pakistan, for providing research facilities.

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.

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.