Firstly, the A. thaliana full-length protein sequences of TCS were retrieved from Ensemble plants database (http://plants.ensembl.org/index.html). These sequences were used as query sequence to execute a BLASTp program search to identify the TCS gene family members in S. bicolor. All putative sequences were further evaluated to check the presence of a specific domain using different domain databases including, CDD (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) (Marchler-Bauer et al., 2011), SMART (http://smart.embl-heidelberg.de/) (Letunic et al., 2012) and Pfam (https://pfam.xfam.org/) (Punta et al., 2012). This step was taken to eliminate the sequences that lacked specific conserved domains required for TCS protein function. The sequences were manually sorted to remove the redundancy, and the remaining proteins were considered as identified TCS proteins. Molecular weight (MW), theoretical isoelectric point (pI), aliphatic index, instability index and the grand average of hydrophobicity (GRAVY) values were calculated by using the online tool ProtParam from ExPASY server (https://web.expasy.org/protparam/) (Gasteiger et al., 2003). In addition, the subcellular localization of SbTCS genes was determined by using online CELLO v.2.5 (http://cello.life.nctu.edu.tw/) (Yu et al., 2006).

To comprehend the SbTCS genes’ evolutionary relationship, multiple sequence alignment of the identified TCS proteins of S. bicolor, and already reported sequences of A. thaliana, C. arietinum O. sativa, and G. max was performed using ClustalW tool, and the neighbor-joining (NJ) tree was created using MEGA7 (https://www.megasoftware.net/) (Kumar et al., 2016) with a bootstrap value of 1,000. The exon and intron structures were visualized using the online software Gene Structure Display Server (GSDS) (http://gsds.gao-lab.org/) through matching the genomic sequences and coding sequences (CDS) of identified TCS genes, which were retrieved from NCBI (Hu B. et al., 2015). Furthermore, the MEME (Multiple EM for Motif Elicitation) tool (https://meme-suite.org/meme/tools/meme) was used to predict the specific conserved motifs of each TCS protein sequence (Bailey et al., 2015). The maximum number of motifs was set to 20, and other parameters were at the default setting.

By using TBtool advance Circos, a genetic relation map of chromosomes was constructed. Moreover, NCBI-gene database was used to predict the position of each TCS gene on the chromosomes of S. bicolor (Yu et al., 2006; Brown et al., 2015). The syntenic analysis was conducted using TBtool (Chen et al., 2018). DnaSP v.6 tool was utilized to analyze the gene duplication events. Synonymous and non-synonymous substitution rates were calculated to determine the selective pressure on the duplicated genes (Rozas et al., 2017). Divergence time was also calculated to perceive the evolutionary events. The following formula calculated the duplication time: T = Ks/2x (x = 6.56 × 10^–9) (He et al., 2016b).

To understand the expression pattern of these identified SbTCSs under saline/alkali and drought stress, RNA-seq data (BioProject: PRJNA319738 for drought stress tolerance and BioProject: PRJNA591555 for saline/alkali stress) was downloaded from NCBI Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra). The genome annotation in .fna and .gtf extension were downloaded from (https://www.ncbi.nlm.nih.gov/assembly/GCF_000003195.3/). Indexes of S. bicolor genome sequence were built using bowtie2, and paired-end clean reads with high quality were mapped to the S. bicolor genome. The expression level of the annotated genes in the reference genome was then calculated by the cufflinks program. The normalized FPKM (fragment per kilobase of transcript per million fragments mapped reads) values of each SbTCS were calculated, and differentially expressed genes were identified. The heatmap was generated to envision the expression through TBtool (Chen et al., 2018).

Plants (S. bicolor, JS2002) were grown for 28 days in a growth chamber under controlled conditions: 25–27°C day-night temperature with 12-h light and 65% humidity. Plants were exposed to drought stress (well-watered and limited water supply), 10 mM or moderate saline-alkali soil stress (6 and 24 h), and 50 mM or severe saline-alkali soil stress (6 and 24 h). The Saline-alkali solution was used to apply saline-alkali soil stress; this solution was made of Na₂CO₃ and NaHCO₃ (1:9, v/v) with half-strength Hoagland’s nutrient solution including Na⁺ at 150 mM and pH 9.5 (Hu G. et al., 2015). 3% salt content were used for moderate and 5% salt content were used for severe condition. For RNA extraction purposes, leaf samples were collected from all the pots (Control, Drought, moderate saline-alkali, and severe saline-alkali) with three biological replicates and then speedily frozen in liquid nitrogen and kept at −80°C until further use.

In the presence of liquid nitrogen, leaf samples were grounded into fine powder by using sterile pestles and mortar. By using the Fastlane cell cDNA kit the complementary DNA (cDNA) was synthesized (Qiagen, Switzerland). A Nanodrop spectrophotometer (NanoDrop 2000 spectrophotometer, Thermo Fisher Scientific) was used to quantify RNA. The qPCR reactions were executed in Applied Biosystem Real-Time PCR Detection System using SYBR Green Master kit (Applied Biosystems, United Kingdom). Gene-specific primers were designed through the online tool “Oligo Calculator” (http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html), and specificity of these primers was then confirmed by NCBI Primer-BLAST program (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) (Supplemental Table S1). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene has been used as the reference gene to normalize gene expression (Sudhakar Reddy et al., 2016).

A BLASTp search was performed to identify the putative members of the TCS gene family in S. bicolor by employing 47 A. thaliana TCS query protein sequences. A total of 37 TCS genes were identified in the genome of S. bicolor, which were further divided into 13 HKs, 5 HPs, and 19 RRs.

The genome of S. bicolor contains 13 HKs (SbHKs/SbHKLs). This number is comparatively more significant than those present in Oryza sativa L. (5), Zea mays (11), Triticum aestivum (7), and Populus trichocarpa (12). However, other legumes such as A. thaliana (17), Lotus japonicas (14), Cucumis melo L. (17), and Physcomitrella patens (18) have a similar number of HKs, which reveals their significance among plants (Table 1). We identified six genes encoding members of the cytokinin receptor family in S. bicolor, i.e., SbHK1, SbHK2, SbHK3, SbHK4, SbHK5, and SbCKl1. The conserved residues required for HK activity were present in all members. Domain analysis of these HKs confirmed that five members (except for SbCKl1) contained a conserved HisKa domain, having a conserved His phosphorylation site. Moreover, all six members had a conserved RR (Rec) domain in which a highly conserved Asp, which acts as the photoreceptor, is present (Figure 1; Supplementary Table S2, ). Genetic and molecular analyses of A. thaliana have shown that five ethylene receptors are involved in ethylene response. These receptors have a GAF protein-protein interactive domain, a HisKa domain, and a HATPase_c domain. Similarly, four genes encoding ethylene receptors were predicted in S. bicolor (SbERS1, SbETR1, SbEIN4.1, and SbEIN4.2). Of these, SbERS1 and SbETR1 encoded almost the same domains as those of A.thaliana. SbEIN4.1 was found to contain GAF, HisKa, and a conserved Rec domain, whereas SbEIN4.2 contained a GAF and a conserved Rec domain (Figure 1; Supplementary Table S2).

Members of the phytochrome family/photoreceptors allow plants to regulate their growth and developmental process and respond to light stimuli. PHYA, PHYB, PHYC, PHYD, and PHYE are the members of this family that have been reported in A. thaliana. Phytochromes contain a PHY domain at the N-terminal (involved in the absorption of light), a GAF domain, one HisKa, and two PAS domains involved in the transduction of signals. Sensor H proteins have a similar structure to that of phytochromes, which are soluble proteins. They have a signal transduction HisKa domain at the C-terminus and a sensor domain at the N-terminus. Since the phytochrome family lacks all five conserved motifs, they are referred to as divergent HKs. Instead of having HK activity, they have another Ser/Thr kinase activity (Ahmad et al., 2020). Three members of the phytochrome family were found in S. bicolor (SbPHYA, SbPHYB, and SbPHYC). All the domains (PAS, PHY, GAF, and HisKa domains) necessary for the regulation of light and signal transduction were present in their protein products, making them actual photoreceptors.

Five out of six members of the A. thaliana HP (AHP1–AHP6) family are true HPs; one member, AHP6, is considered a pseudo-HP due to the absence of a conserved residue (H) required to obtain phosphate from the donor proteins (Bleecker 1999). Except for AHP6/APHP1, all the other members encode a conserved phosphorylation motif (XHQXKGSSXS). In AHP6/APHP1, the N residue has replaced the H residue of the phosphorylation motif. In S. bicolor, five members of the HP family were discovered to have a conserved domain, “Hpt” (Figure 1; Supplementary Table S2). Three of these five members (SbHP1, SbHP2, and SbHP5) were classified as true HPs since they have the phosphorylation motif, while the other two members (SbHP3 and SbHP4) were considered pseudo-HPs because the H residue is missing from their phosphorylation motif.

RRs modulate the final responses to different environmental stimuli. In this study, 19 RR family members were identified in S. bicolor, including both standard and pseudo-RRs. Previous studies have reported that A. thaliana has 32 members of the RR family (Mochida et al., 2010) and that O. sativa has 22 (Du et al., 2007). RRs act as terminal components in the TCS signaling pathway, acting as phosphorylation-activated switches. This RR functioning catalyzes the transport of phosphoryl groups to the Asp residue of its conserved domain. A significant feature of RRs is that they contain conserved K (lysine) and D (aspartic acid) residues, which are found in the Rec domain of A. thaliana RR family members. On the basis of the domains that are conserved, RR family members are further classified into three subfamilies: type-A RRs, type-B RRs, and type-C RRs. Type-A RRs have a Rec domain with a conserved D residue and a C-terminal extension. The Rec and DNA binding domains (Myb) are present in type-B RRs. Type-C RRs have a similar structure to that of type-A RRs but do not have a C-terminal extension. Another type of RR is known as the pseudo-response regulator (PRR). These RRs have a conserved Rec domain with an E residue (in place of the D residue) and a C-terminal CCT motif (Ahmad et al., 2020). Among the 19 S. bicolor RR family members, 2, 3, 7, and 7 are type-C RRs, type-A RRs, PRRs, and type-B RRs, respectively.

Three members of the type-A RR family in S. bicolor were identified, namely SbRR4, SbRR9, and SbRR10. These putative members of the type-A RR family were similar to their A. thaliana counterparts, with all three possessing a conserved Rec domain. Nuclear proteins are the most common members of the type-B RR family. These are different from the members of the type-A RR family since they contain a Myb-DNA binding domain. It has been reported that type-B RRs act as transcription factors (Ahmad et al., 2020). Seven members of this family were identified in S. bicolor (SbRR16, SbRR17, SbRR18, SbRR19, SbRR20, SbRR24.1, and SbRR24.2). This number is higher than that in T. aestivum (2) and lower than that in many other plant members, including A. thaliana and O. sativa. All seven members were found to have conserved Rec and Myb domains, except for SbRR24.1 and SbRR24.2, from which the Myb domain is missing. In A. thaliana, there are two members of the type-C RR family (ARR22 and ARR24). Like type-A RRs, they also have a conserved Rec domain, but possess a very small C-terminal. They are not very closely related to type-A RRs, according to phylogenetic analysis. It has also been shown that they are not expressed during cytokinin response. S. bicolor was found to contain two members of the type-C RR family (SbRR13 and SbRR14). These members exhibited significant homologous relationships with their A. thaliana counterparts and possessed a conserved Rec domain.

Divergent RRs, also known as PRRs, are another type of RRs found in a variety of plant species. In A. thaliana, there are nine divergent RR family members (ARR1–9). They have the entire Rec domain but lack a conserved DDK motif. Seven PRRs were found in the S. bicolor genome: SbPRR1, SbPRR2.1, SbPRR2.2, SbPRR3.1, SbPRR3.2, SbPRR5, and SbPRR9. PRRs have been further divided into two groups based on the C-terminal extension: type-B PRRs and clock-associated PRRs. In type-B PRRs (SbPRR2.1 and SbPRR2.2), instead of the CCT motif, a Myb domain is present. While clock-associated PRRs (SbPRR1, SbPRR3.2, SbPRR5, and SbPRR9) have the conserved amino acids arginine and lysine in the CCT motif, in SbPRR3.1, only a pseudo-rec domain is present.

The detailed physio-chemical characteristics of 37 SbTCS proteins are shown in Table 2 and Supplementary Table S4. The SbTCSs are present in 10 chromosomes (Chr), Chr 1–10. The exon numbers ranged from 2 to 14. The protein length ranged from 132 (SbRR13) to 1,178 (SbPHYB) amino acids (aa). ExPASy analysis revealed that the SbTCS proteins exhibited very different isoelectric point (pI) values (ranging from 4.55 to 9.69), molecular weight values (ranging from 14,169.76 to 122,065.35), aliphatic index values (ranging from 57.62 to 106.71), and grand average of hydropathicity index (GRAVY) values (ranging from −0.827 to 0.224). SbTCS proteins were shown to be located in the cytoplasmic, mitochondrial, and nuclear membranes.

This study aimed to look at the evolutionary pattern and phylogenetic relationship of TCS proteins in S. bicolor; a neighbor-joining tree was constructed using the full-length protein sequence alignment of the identified TCS proteins from S. bicolor, A. thaliana, G. max, O. sativa, and C. aritenum (Figure 2). These proteins were divided into three groups: RRs, phosphotransfer proteins (HPs), and HKs. HKs were divided into five subgroups (CKl2, HK1 and CKl1, ethylene receptor, cytokinin receptor, and phytochrome families) according to the phylogenetic tree. Three cytokinin receptors were present in A. thaliana (AHK2, AHK3, and AHK4), as well as in S. bicolor (SbHK2, SbHK3, and SbHK4). Phylogenetic analysis revealed that SbHK3 and SbHK4 were orthologues of AHKs. Cytokinin receptors in A. thaliana have been functionally characterized and shown to control a variety of cytokinin-regulated processes, such as leaf senescence, stress responses, seed size, vascular differentiation, and cell division. Based on phylogenetic analysis, it can be suggested that the predicted cytokinin receptors of S. bicolor have similar functions as their A. thaliana counterparts.

AHK1 is a transmembrane protein which plays a role in osmosensing. It is largely expressed in the roots of A. thaliana in salt stress conditions. In S. bicolor, SbHK1 and SbCKl1 are the two orthologues of AHK1. In the CKl2 group, phylogenetic analysis indicated that SbHK5 is an orthologue of AHK5/ACKl2. The ethylene receptors SbETR1 and SbERS1 are present as direct homologs of ERS1 and ETR1. Similarly, SbEIN4.1 and SbEIN4.2 proteins are present on another clade, making a direct orthologue relationship with A. thaliana ETR2 and ERS2.

Phytochromes or photoreceptors are found to be involved in growth and development in light stress conditions. A. thaliana contains five photoreceptors. These contain a C-terminal, a GAF domain, an N-terminal PHY domain, one HisKa, and two PAS domains, which are involved in signal transduction pathways. Three photoreceptors (SbPHYA, SbPHYA, and SbPHYC) were identified in S. bicolor, with the same domains as those present in A. thaliana. Five HPs were found in S. bicolor, showing close phylogenetic relationships to the true HPs of A. thaliana and O. sativa. These HPs were grouped according to their phylogenetic relationships with their A. thaliana counterparts. SbHP1 and SbHP2 were grouped as being AHP1-like. SbHP3, SbHP4, and SbHP5 were grouped as being AHP4-like HPs.

Protein sequences of S. bicolor, O. sativa, A. thaliana, G. max, and C. Arietinum were used for the phylogenetic analysis of RRs. RRs are classified as PRRs (divergent RRs), type-A RRs, type-B RRs, or type-C RRs. This third family of TCS genes regulates the final responses to environmental stresses. PRRs are not considered to be true RRs due to the absence of a DDK conserved motif. It was confirmed that SbRRs are true RRs, since they have close phylogenetic relationships with their A. thaliana and O. Sativa counterparts. The evolution pattern of this family in S. bicolor revealed that this is segmentally duplicated. The same results have been found in O. sativa and A. thaliana in which it is also segmentally duplicated. Because type-A RRs are not found in unicellular algae and are observed only in land plants, they are considered relatively new members of the RR family and have been suggested to perform some novel functions in those organisms. Their structure contains a Rec domain and a conserved DDK motif that is important for receiving the phosphate group. Mainly, cytokinin activates type-A RRs, and this cytokinin induction partially depends on type-B RRs.

A crucial evolutionary feature of a gene is its exon-intron structure and it provides information about its functional diversity. Therefore, the exon-intron organization of the AtTCS and SbTCS genes was further analyzed (Figure 3). The results showed that the members of the cytokinin receptor (HK) family had exon numbers ranging from 5 in SbHK1 to 14 in SbHK5 with introns 4–13. The A. thaliana HK family has exons 11–14 and introns 10–13. SbCKl1 was found to have 8 exons and 7 introns, whereas AtCKl1 contains 9 exons and 9 introns. Ethylene receptor family members SbEIN4.1 and SbEIN4.2 were shown to contain 3 exons and 2 introns, whereas SbERS1 and SbETR1 contained 6 exons and 5 introns. A. thaliana ERS1 contains 6 exons and 5 introns, ETR1 contains 7 exons and 6 introns, and AtEIN4, ETR2, and ERS2 have 2–3 exons and 1–2 introns. In the phytochrome family members, SbPHYA was found to have 6 exons and 5 introns. SbPHYB and SbPHYC were both shown to have 4 exons. The HP family members have 5–7 exons and 4–6 introns. A. thaliana HP family members have 3–5 exons and 2–4 introns.

The S. bicolor RR family genes had a number of exons varying from 2 to 12 and an intron number varying from 1 to 11. The maximum number of exons and introns in SbPRR3.2 was found to be 12 and 11, respectively. The same number of exons and introns were found in AtRRs. These results showed that the groups and members with closer phylogenetic relationships contain a similar exon-intron structure. Subsequently, we used the MEME software to predict the conserved motifs of these TCS genes (Figure 5). The overall number of identified motifs was 20. Motifs 2, 8, 15, 16, 17, and 18 were conserved in the whole cytokinin receptor family of S. bicolor and A. thaliana . The same motifs were present in SbCKl1 and CKl1. The members of the ethylene receptor sub-family also contain similar motifs to those of the cytokinin receptor family but with the additional conserved motif 13; SbERS1 and SbETR1 lacked motifs 2 and 18. The S. bicolor and A. thaliana phytochrome family members contain an average of 10 conserved motifs (1, 4, 9, 11, 12, 14, 19, 15, 16, and 17). Only motif 20 is conserved in the members of the HP family.

Motifs 1, 2, 3, and 5 were conserved in all members of the type-A RR family. Type-B RRs contain the same motifs as type-A RRs and contain two more conserved motifs: motifs 6 and 7. The same motifs were present in two type-C RR members (SbRR24.1 and SbRR24.2). SbRR13 and SbRR14 encoded only two conserved motifs (motifs 2 and 5). The members of the PRRs family contain the same set of motifs as type-A RRs, except for two members, which contain only two motifs (motifs 6 and 7). The members of the same gene families share the same motifs, indicating that there is no significant functional and sequence divergence between them. Collectively evolutionary analysis revealed that TCS is conserved.

To examine the genomic distribution of the SbTCS genes, their chromosome gene location and duplication events were identified using syntenic analysis. All the identified S. bicolor TCS family members were found to be distributed on 10 chromosomes (Figure 4). These genes are unevenly distributed, since chr1 contains 9 (maximum number) genes, while chromosome 7 contains only one gene. The HK(L)s are randomly distributed on all S. bicolor chromosomes, except for chr2, chr5, chr7, and chr8. The members of the HP family are located on chr2, chr3, chr7, and chr9. RR family members are distributed on all chromosomes, except for chr7 and chr9.

The duplication events were analyzed for the SbTCS gene family. Since gene duplication provides raw material for development, the evolution of new genes in the genome was also analyzed. The number of tandem and segmental duplication events was observed to increase in a number of plant genes. When identifying the potential genomic duplication events, five pairs of TCS syntenic paralogs were found in the S. bicolor genome. This indicated that the SbTCSs have a high gene family expansion (Figure 6). In this study, the duplicated pairs resulting from segmental duplication include SbPRR3.1/SbPRR3.2, SbPRR5/SbPRR9, SbEIN4.1/SbEIN4.2, SbRR18/SbRR20, and SbRR9/SbRR10. In S. bicolor, multiple pairs exhibited segmental duplication, implying that the expansion of SbTCS genes is mainly due to segmental duplication. A similar expansion pattern exists in other plants, such as in G. max (Mochida et al., 2010), A. thaliana (Hutchison and Kieber, 2002), and Chinese cabbage (Liu et al., 2014). The synonymous rate (Ks), non-synonymous rate (Ka), and the Ka/Ks ratio of these duplications were calculated, and the Ks values were used to speculate on the duplication time (Table 3). The Ks of five segment duplicates ranged from 0.143 to 0.632. Therefore, the divergent time ranged from 10.89939024 to 48.17073171 Mya.

For a better understanding of the transcriptional regulation and functional role of the SbTCS genes, their promoter sequences were investigated to predict the cis-regulatory elements. Several hormone-related and abiotic stress-related cis-regulatory elements were identified, of which TATA- and CAAT-box were present in almost all of the 37 SbTCSs (Figure 5A; Supplementary Table S3). Among them, the methyl jasmonate (meJa)-responsiveness elements were found in 27 SbTCSs. The ABA-responsive element (ABRE) involved in the abscisic acid response was present in 23 SbTCSs. A large number of cis-regulatory elements were associated with light signaling, including the GATA motif, ACE, box 4, G-box, and the TCCC and TCT motifs. Gibberellin-responsive elements (GARE motif, TATC-box, and P-box) were present in almost 10 SbTCSs. Auxin-responsive elements, such as AuxRR-core and TGA-element, and salicylic acid-responsive elements (TCA element and SARE) were also found. In addition, low temperature-responsiveness (LTR) elements were present in 11 SbTCSs, and drought-inducibility element (MBS) was found in 14 SbTCSs. These results demonstrate that the TCS genes are potentially involved in growth and developmental processes related to hormone metabolism and signal transduction networks. The presence of LTRs, TC-rich repeats (defense-responsive element), and MBS (MYB binding site), which are associated with drought-inducibility, suggest that TCS plays a vital role in the development of plant and multiple abiotic stress responses.

Gene ontology enrichment analysis of all the SbTCS genes was performed. For this purpose, a well-known open-source DAVID bioinformatics resource 6.7 was used. All the identified genes were subjected to DAVID gene ontology analysis by using the entrez_gene_id. The results revealed the identified genes were classified into three main functional biological categories annotated by GO, that including molecular function, cellular component and biological process (Figure 5B). In the category of biological processes 23 out of 37 genes were found to be involved and the highest proportion (14 out of 23) was found in the phosphorelay signal transduction system. Moreover 9 out of 23 genes were found to be involved in transcription, DNA-templated. In the category of molecular function 21 out of 37 genes were found to be involved in different functions, including phosphorelay sensor kinase activity (10), protein histidine kinase binding (4), histidine phosphotransfer kinase activity (4), photoreceptor activity (3), transcription factor activity; sequence-specific DNA binding (6), and DNA binding (7). Lastly in the category of cellular component, 25 out of 37 genes were found to be involved in two components including intracellular and nuclear.

To examine the expression level of the 37 SbTCS genes under different abiotic stress conditions, the publicly available RNA-seq data of S. bicolor was obtained from the SRA-NCBI database. The results showed that SbHK3, SbPHYA, SbHP3, and SbHP5 were upregulated under drought stress in leaves. Most members of the RR family were upregulated under drought stress, whereas the expression levels of SbETR1, SbRR4, SbRR10, SbRR9, SbRR3.1, SbPRR5, SbPRR1, and SbPRR9 were decreased (Figure 6A).

In the salt treatment, many HK family members were showed to be upregulated, including SbPHYA, SbEIN4.1, and SbEIN4.1. Almost all members of the HP family were upregulated during salt stress conditions. The RR family members, including SbRR20, SbRR16, SbRR18, SbRR19, and SbPRR1, were also upregulated. There were no expression changes observed in SbHP3, SbPRR2.2, SbRR10, SbRR9, and SbPRR3.2 (Figure 6B). These results indicate that the S. bicolor TCS genes potentially play a crucial role in diverse abiotic responses.

To further endorse the expression of SbTCS genes, 15 differentially expressed SbTCS genes based on RNA-seq analysis (belonging to different groups) were selected. The findings revealed that the overall expression trend of these genes obtained through qRT-PCR analysis was highly consistent with the RNA-seq data (Figure 7). Furthermore, compared with the control, the results revealed that drought stress treatment enhanced the expression of the SbHK3, SbPHYA, SbHP1, SbHP2, SbHP3, SbPR2.2, SbRR16, and SbRR18 genes up to several folds higher. Meanwhile, compared with the control, drought stress decreased the expression of the SbHK4, SbPRR1, SbPRR3.1, SbRR9, and SbRR10 genes. Drought stress seems to not have affected the expression of SbPRR2.1 and SbPRR3.2.

Additionally, SbHK3, SbHP2, SbPRR1, SbRR16, and SbRR18 were shown to be significantly highly expressed and SbHP3, SbPRR2.2, SbPRR3.1, SbRR9, and SbRR10 were shown to not be significantly expressed in both mild and severe salt stress conditions. No expression changes were observed for SbPRR2.1 and SbPRR3.2 under salt stress conditions. SbPHYA and SbPRR1 were found to be significantly overexpressed, by up to three-fold, under salt stress conditions. All of these findings show the potential functional importance of TCS genes in the growth and development of S. bicolor under different abiotic stresses.