**Table 1.** Detailed annotations of the *TaTPPs* in wheat.

112

Subcellular Localization.

We further investigated the duplication events in the *TaTPP* gene family in the context of wheat being hexapolyploid and having big genomes. Genes are usually considered duplicated when the query cover and identity value of gene sequences are more than 80% [71]. It has also been reported that genes are considered duplicated when protein sequence similarity and identity are more than 70% and 75%, respectively [72]. By analyzing the sequences, we found 27 pairs of *TaTPPs* with a sequence identity ranges from 82.14% to 95.25% and 100% query cover within all gene pairs (Tables S4 and S5) and identified in the same phylogenetic tree clade (Figure 3). We further computed the non-synonymous (Ka) and synonymous (Ks) substitutions, as well as the Ka/Ks ratios, for the 27 *TaTPP* gene pairs to determine the selection pressure on the duplicated *TaTPPs* (Table S5). These gene pairs Ka/Ks ratios were smaller than one, indicating that they developed under functional restriction with negative or purifying selection. The divergence period ranged from 2.93 to 13.3 million years ago (MYA), showing that these gene pairs were duplicated recently (Table S5).

**Figure 2.** Graphical presentations of *TaTPPs* chromosomal distribution of on wheat chromosomes. The name of the gene on the right side and the location of the *TaTPPs* is indicated by the colored circular circle on the chromosomes. The three subgenomes chromosomal numbers are shown at the top of each bar.

**Figure 3.** Phylogenetic analysis of TaTPP proteins. The tree was generated using MEGA X by the maximum likelihood method with 1000 bootstrap values. All the species and protein ID used for constructing tree were presented in Table S6.

#### *3.3. Phylogenetic and Conserved Domain Analyses of TaTPP Proteins*

A phylogenetic tree containing full length TPP protein sequences from twelve plant species was constructed by the maximum likelihood method to better understand the evolutionary relations among the TaTPP proteins with other species (Figure 3, Table S6), including five species from monocot: *Hordeum vulgare*, *Brachypodium distachyon*, *Oryza sativa*, *Zea maize*, *Sorghum bicolor*; and 6 species from dicot: *Arabidopsis thaliana*, *Glycine max*, *Populus trichocarpa*, *Solanum tuberosum*, *Solanum lycopersicum*, *Vitis vinifera*. The results indicated that TPP proteins were divided into eleven clades, where clade I was the largest with 30 members. Clades II to XI (total 10 clades in order) included 21, 24, 10, 6, 13, 10, 1, 10, 8, and 2 members, respectively (Figure 3).

Plants classified as dicots and monocots were divided into distinct clades. Proteins from monocot plants were grouped into clade I, clade II, clade V, clade VI, and clade VII, whereas proteins from dicot plants were grouped into clade III, clade IV, clade VIII, clade IX, clade X, and clade XI. The highest number of TaTPP proteins were grouped into clade I and clade II, which had nine proteins in each clade. In addition, clades V, VI, VII contained four, six, and three TaTPP proteins, respectively (Figure 3). Most of the wheat TPP proteins were closely related to *H. vulgare*, *B. distachyon*, and *O. sativa*, suggesting their conserved function with those plant species and offering information that can be used to conduct a more in-depth functional analysis. All the *TaTPPs* were assembled into 11 groups, as sequences from A, B, and D subgenome of 11 groups clustered together in the phylogenetic

tree (Figure 3) and protein sequence identity was more the 88% between A, B, and D subgenome of each group (Table S4). Thus, we considered the protein sequences from A, B, and D subgenome of each group are copies of separate *TaTPP* genes and named them according to the ascending order of the chromosomal location (Table 1).

Further, the Pfam database was utilized to find the important component domains of TaTPP proteins [59]. All the TaTPP proteins contain a specific Trehalose PPase domain (PF02358). In addition, a stress antifungal domain was found in TaTPP-5A, TaTPP7-A and TaTPP7-D (Figure 4a). We used MEME suite 5.1.1 to evaluate motif sequences for 31 TaTPPs and found six significant motifs (motifs 1–6) (Figure 4b). All the motifs were found to be conserved in all TaTPP proteins except for TaTPP5-A, which lacks lacks motif 3 (Figure 4b).

**Figure 4.** Conserved domain and motif of TaTPPs. (**a**) The conserved domain of TaTPP members was identified from Pfam and SMART databases and presented using TBtools. (**b**) The conserved motifs of TaTPP members. Six motifs were identified using MEME program and presented with different colored boxes.

#### *3.4. Gene Structure and Evolution Analyses of TaTPPs*

The exon-intron structures of *TaTPPs* were studied to better understand their structural features (Figure 5). The *TaTPP* gene family had a lot of variation in terms of gene structure, according to gene structure analyses as introns ranged from 4 to 13. Most of the *TaTPPs* contain eight or nine introns. A maximum of 13 introns was found in *TaTPP7-D* and a minimum of four introns were observed in *TaTPP8-B* (Figure 5). Moreover, different *TaTPPs* showed different intron phase patterns. *TaTPP1-A*, *TaTPP1-D*, *TaTPP5-A*, *TaTPP6*, *TaTPP8*, *TaTPP9* showed phase 0 and *TaTPP2*, *TaTPP3*, *TaTPP4, TaTPP5-B*, *TaTPP7-A*, *TaTPP10*, *11* showed phase 2 patterns, whereas *TaTPP1-B* and *TaTPP7-D* exhibited all phases (Phase 0,1,2) (Figure 5).

**Figure 5.** Structural organizations of *TaTPPs*. The introns are shown by black lines, whereas the exons are represented by pink boxes and untranslated regions (UTRs) are represented with green boxes. Intron phase, 0: phase 0, 1: phase 1 and 2: phase 2 denotes that a codon is not disrupted by introns, a codon between the first and second bases is disrupted by an intron and a codon between the second and third bases is disrupted by an intron, respectively.

Further, the Multiple Collinearity Scan toolkit was used to investigate the synteny networks between *TaTPPs* and other wheat relatives and model plants. The results showed that 27, 26, 13, 33, 26, and 22 orthologous gene pairs were identified between *TaTPPs* and other *TPPs* in *B. distachyon*, *O. sativa*, *A. thaliana*, *H. vulgare, Z. mays*, and *S. bicolor*, respectively (Figure 6 and Table S7). A collinear relation was observed for 19, 18, 9, 22, 17 and 19 *TaTPPs* with other *TPPs* in *B. distachyon*, *O. sativa*, *A. thaliana*, *H. vulgare*, *Z. mays*, and *S. bicolor*, respectively. *TaTPP6*, *TaTPP9*, *TaTPP10* and *TaTPP11* were shown to have more than one pair of orthologs. Thus, these *TaTPPs* might have a crucial role in the evolution of *TPPs*. These findings imply that *TaTPPs* in wheat may have evolved from other plant species orthologous genes.

**Figure 6.** Syntenic relationship between *TaTPPs* with rice, *Arabidopsis*, *Brachypodium,* sorghum, and maize. The collinear blocks within wheat and other plant genomes are shown by gray lines in the background, while the syntenic *TaTPP* gene pairs are highlighted by red lines.

#### *3.5. 3-D Protein Structure Analysis*

The 3-D structure reveals a few key residues linked to biological processes or intended outcomes [73]. Thus, we used SWISS-MODEL to identify the 3-D model of TaTPP proteins (Figure S1a). For all TaTPP proteins, the 3-D structures were analyzed using template "5gvx.1.A." and predicted 3-D structures covering the N-terminus and C-terminus regions of 31 TaTPP proteins (Figure S1a). Within 4 A◦ , three conserved residues that worked as ligands were identified. The interaction of those ligands with chain A and the magnesium ion (Mg2+) indicates that TaTPP proteins have distinct catalytic activities, which have also been reported for AtTPP, ZmTPP, ScTPP, CaTPP, and EcTPP that have a catalytic function and they are all similar to one other by 80% [39,74]. Further, we used SOPMA to calculate the secondary structure elements of protein sequences (Table S8). TaTPP proteins were found to contain a range of 35.70% to 47.99% α helix, 13.41% to 18.18% extended strand, 6.62% to 9.93% β turn and 8.38% to 41.21% random coil (Table S8). All TaTPPs except TaTPP5, TaTPP7, TaTPP9-A, and TaTPP10-B had a coiled coil-like structure in the C-terminus and one Mg2+ ligand each was observed in all the TaTPPs (Figure S1a).

To validate TaTPP protein structures, we employed SWISS-MODEL analysis and the MolProbity server (Figure S1b and Table S9). The produced Ramachandran plot has an average favored region of 94.07%, an average allowed region of 99.08%, and an average outer region of 0.91% (Table S9). The average sequence identity was 34.95%, with a similarity of 37%, covering 68% of the query sequences obtained by the X-ray Method in 2.6 A◦ (Table S9). The ligand interaction between chain A and Mg2+ was confirmed with the Protein–Ligand Interaction Pipeline (PLIP), and the residue site was noticed to be highly conserved. We investigated these conserved residues further in all TaTPP protein sequence alignments and found that they include aspartic acid (D/Asp), which is conserved in motif 3 and motif 6. (Figure S2, Table S9). For improved visual clarity, the side chains of the catalytic triads were expanded with the TaTPP1-A residues (Figure S3).

#### *3.6. Analysis of Cis-Regulatory Elements*

To examine the responses of *TaTPPs* members to various stimuli, the 2 kb promoter sequences upstream of the start codon of these genes were submitted to the PlantCARE service to predict their *Cis*-regulatory elements (CREs). A total of 90 CREs with a frequency of 1985 were identified in all *TaTPP* promoters (Figure 7, Table S10). Among them, 72 CREs were related to phytohormones, stress, growth, and development (Figure 7, Table S10). All of the identified CREs were divided into five groups according to their known functions (Tables S10 and S11). Group I contained four core *Cis*-elements, including AT~TATA-box, CAAT-box, TATA, TATA-box. TATA-box (which comprises TATA and AT TATA-box) is a critical promoter element found in approximately 30% of transcription start sites and the CAAT-box is a kind of promoter that may influence the choice of transcription start location [75]. TATA-box and CAAT-box are generally present 25–30 bp and ~75 bp upstream of the transcription start site, respectively, and both of them are found in a wide range across all the promoters.

**Figure 7.** Putative *Cis*-acting regulatory elements (CREs) of *TaTPPs*. The CREs were identified with the 2 kb upstream sequences of the start codon using the PlantCARE online server and presented using TBtools. Red color indicates the CREs with high frequency, while black color indicates CREs with zero frequency.

Group II contained 44 stress-related CREs, among them 20 were light-responsive *Cis*-elements such as 3-AF1 binding site, 3-AF1 binding site, ABRE4, ACE, ATCT-motif, Box 4, AE-box, Box II, LAMP-element. The stress-responsive CREs consist of one anaerobicresponsive element (ARE), one low-temperature-responsive element (LRT), one droughtresponsive element (MBS), two wound-responsive elements (WUN-motif, box S), one coldand dehydration-responsive (DRE core) and 17 defense- and stress-responsive elements (as-1, TC-rich repeats, W box, CCAAT-box, MYB, MYB recognition site, Myb, Myb-binding site, MYB-like sequence, MYC, Myc, STRE, WRE3, Unnamed\_1, Unnamed\_8, GC-motif, AT-rich sequence). There were 12 CREs in group III, involved in cell development including seed specific expression (AAGAA-motif, RY-element), cellular development and cell cycle regulation (AC-I, MSA-like), meristem expression (CAT-box, CCGTCC-box, CCGTCC-box), circadian control (circadian), differentiation of palisade mesophyll cells (HD-Zip I), cell cycle regulation (MSA-like), and endosperm expression (GCN4\_motif).

Additionally, the hormone-responsive CREs in group IV included 16 CREs such as abscisic-acid-responsive element (ABRE, ABRE2), auxin-responsive elements (AuxRR-core and TGA-element), salicylic-acid-responsive element (TCA-element, TCA, SARE), methyljasmonate-responsive elements (TGACG and CGTCA motifs), ethylene-responsive element (ERE) and gibberellin-responsive elements (GARE-motif, P-box, and TATC-box). There were also 14 CREs in group V with unknown functions. CTAG-motif and A-box might act as a CRE, Unnamed\_2 might act as an antisense transcript, BOX III might function as a protein binding site and Unnamed\_16 was found to be involved in sugar transporter family genes. Most *TaTPPs* possessed one or more CREs associated with hormone and stressrelated activities, suggesting that *TaTPPs* may be engaged in a variety of physiological processes as a result of diverse environmental adaptations.

#### *3.7. Transcriptional Patterns of TaTPPs in Different Organs and Developmental Stages of Wheat*

To investigate the transcription level of *TaTPP* genes in different wheat organs and development stages, mRNA transcripts data was collected from Genevestigator and visualized with a heatmap in Figures 8 and S4. The transcript data were divided into six groups. Group I included callus, Group II included primary cells (cell culture, spike cell, spikelet cell, floret cell, stamen cell, anther cell, meiocyte, microspore), Group III included seedlings (seedling, coleoptile, root, radicle, radicle tip), Group IV included inflorescence (inflorescence, spike, rachis, spikelet, floret, stamen, anther, pistil, ovary, lemma, awn, glume, caryopsis, embryo, endosperm, aleurone layer, starchy endosperm, endosperm transfer layer, pericarp, outer pericarp), Group V included shoot (shoot, culm (stem), internode, peduncle, leaf, blade (lamina), sheath, flag leaf, blade (lamina), ligule, sheath, crown, shoot apex, shoot apical meristem, axillary bud) and Group VI included rhizome (rhizome, roots, nodal root, unspecified root type, root tip, root, apical meristem). Our results showed that *TaTPP1*, *TaTPP8*, *TaTPP9-A*, *TaTPP9-D* and *TaTPP10-B* had the highest transcriptions in most of the organs compared to other *TaTPPs* (Figure 8). In addition, high expression was observed for *TaTPP2-D*, *TaTPP5-B*, *TaTPP6-A* and *TaTPP6-B* only in the rhizome group. In contrast, other *TaTPPs* had no expression in most of the organs (Figure 8).

Further, we observed the mRNA transcripts level of *TaTPPs* during different developmental stages of wheat, such as germination, seedling growth, tillering, stem elongation, booting, inflorescence emergence, anthesis, milk development, dough development, and ripening (Figure S4). A number of *TaTPPs* were expressed differently at various stages of wheat development. For example, *TaTPP8* and *TaTPP4-A* were found to be expressed in all stages, whereas *TaTPP1* and *TaTPP9* were induced in all except the ripening stage. *TaTPP4-D* was expressed in all except stem elongation and *TaTPP3-D* was expressed in all except tillering and ripening stages. *TaTPP5* and *TaTPP7* showed very low expression in all wheat developmental stages and other *TaTPPs* were either slightly or highly expressed in one or more developmental stages (Figure S4). These findings suggest that various *TaTPPs* may have a role in the development of various tissues at different development stages.

**Figure 8.** Transcription profiles of *TaTPPs* in different wheat organs. mRNA transcription data of *TaTPPs* in different wheat organs were retrieved from Genevestigator and presented using MeV software.

#### *3.8. Transcriptions of TaTPPs Were Induced in Response to ABA, Abiotic Stresses and Leaf Senescence*

Wheat seedlings treated with ABA or abiotic stress (drought and salinity) were used to analyze the transcriptional pattern of the *TaTPPs* in wheat. Under ABA treatment, *TaTPP1* and *TaTPP4* exhibited upregulated transcriptions at most of the time points and significant upregulation was observed from 3 to 12 hpt (hours post treatment). Moreover, three *TaTPPs* (*TaTPP7*, *TaTPP8* and *TaTPP9*) were upregulated immediately after ABA treatment and transcriptions decreased with an increase in ABA treatment time points. Transcriptions were significantly downregulated for *TaTPP2*, *TaTPP3* and *TaTPP11* at most of the time points after ABA treatments compared to control (0 hpt) (Figure 9a). The transcriptional patterns of *TaTPP* members were examined following drought stress in wheat to provide insight into the underlying functional roles of wheat *TPPs* in response to drought stress. During the drought stress treatment, only *TaTPP1* and *TaTPP4* showed significant upregulations at a later time post treatment. A slight upregulation or significant downregulation was observed for all other *TaTPPs* after drought stress in wheat (Figure 9b). The transcriptional levels of *TaTPP* members were examined to elucidate the mechanism of gene responses to leaf senescence in wheat. Most of the *TaTPP* members were slightly or highly induced during leaf senescence, *TaTPP1* showed obvious upregulated transcriptions at 19 and 22 days after anthesis compared to the control (0 days after anthesis) (Figure 9c).

Further, we analyzed the transcriptions of *TaTPP* members under salt stress by qRT-PCR to observe the involvement of *TaTPPs* in wheat salt tolerance (Figure 10). Significant upregulation of the transcripts was observed for *TaTPP1*, *TaTPP2*, *TaTPP4* and *TaTPP9* at an early stage of salt treatment and downregulations were observed at the later stage of salt treatment. Moreover, *TaTPP7* showed a significant upregulation only at 12 h post treatment (hpt) compared to the control (0 hpt). In contrast, either no changes or significant downregulations were observed for other *TaTPP* members compared to the control (Figure 10). However, no expression was observed for *TaTPP5* and *TaTPP6* by qRT-PCR in all aspects. Overall, these findings suggest that *TaTPPs* act as an important regulator of wheat abiotic stress and leaf senescence responses.

**Figure 9.** Relative transcript profiles of *TaTPPs* in response to (**a**) abscisic acid (ABA), (**b**) drought stress and (**c**) leaf senescence. The relative transcripts of all genes were analyzed using qRT-PCR. The relative transcript levels of *TaTPPs* were measured using the comparative threshold (2−∆∆CT) method. Data normalized with the transcripts of wheat elongation factor, *TaEF-1α*. The 0 h post treatment (**a,b**) or 0 days after anthesis (**c**) was used as a control and standardized with 1. Red and green colors denote strong and weak transcription of *TaTPPs*, respectively. The heat map was generated with TBtools and tree was constructed with the average linkage clustering method.

**Figure 10.** Relative transcript profiles of *TaTPPs* in response to salt stress. The relative transcripts of all genes were analyzed using qRT-PCR. The relative transcript levels of *TaTPPs* were measured using the comparative threshold (2−∆∆CT) method. Data normalized with the transcripts of wheat elongation factor, *TaEF-1α*. The 0 h post treatment was used as a control and standardized with 1. Values represent the mean ± SD from three independent biological samples. Asterisks (*p* < 0.05) or double asterisks (*p* < 0.01) designate significant differences from 0 hpt by the Student's *t*-test.

#### *3.9. Protein–Protein Interaction Analysis of TaTPPs*

The STRING database was used to build a network to study protein–protein interactions between TaTPPs and other wheat proteins (Figure S5 and Table S12). From

prediction results, it was found that TaTPPs can interact with five other wheat proteins. Traes\_1AL\_7531AC097.1, Traes\_1BL\_2AE952A77.1 and Traes\_1DL\_50B29C62B.2 have encoded an enzyme called TRE, which is hydrolyzed Trehalose to synthesize two molecules of glucose. Moreover, Traes\_6DL\_33F8A5EF4.1 has encoded TPS enzyme which produces T6P, a phosphorylated intermediate, from UDPG and G6P and Traes\_4AS\_4B8E78B13.1 was an unknown protein. Thus, our results suggesting that TaTPPs might interact with other enzymes that are involved trehalose biosynthesis pathway to accelerate the trehalose biosynthesis process.

#### **4. Discussion**

The *TPP* gene family has been characterized as catalytic enzymes that mainly function in trehalose biosynthesis [18,76,77]. Despite their catalytic function, a portion of *TPP* genes has been identified to be involved in growth and development, response in abiotic and biotic stress and senescence [27–29,31–33,35,42,78,79]. Although wheat is one of the most economically important cereal crops, systemic studies on *TPP* homologs in wheat have not been reported yet.

In the present study, we analyzed wheat *TPPs* with other species and identified 31 *TPPs* in wheat based on the Chinese Spring genome sequence (Table 1). The highest number of *TPPs* were found in wheat and these genes were distributed over 17 chromosomes (Figure 2). In comparison to previously described *TPPs* in *Arabidopsis*, rice, maize, and purple false brome, the wheat *TPP* gene family has been significantly extended with relatively more *TPPs* [38,74,80–82]. The major driving forces for extending the gene family in various plant species are gene duplication mechanisms, which include segmental, tandem, and whole-genome duplications [83,84]. All the *TaTPPs* are distributed unevenly on the wheat chromosome and the number ranges from 1 to 5 on each chromosome (Figure 2). Gene duplication analysis revealed that 27 pairs of *TaTPPs* duplicated within the wheat genome (Tables S4 and S5). The gap between genes on the chromosomal map of common wheat was higher than 200 kb (Figure 2), indicating that these genes were not formed via tandem duplication [85]. In addition, Ka/Ka ratio was less than one for all pairs of duplicated genes, suggesting that *TaTPPs* were subjected to a rigorous purifying selection (Table S5) and a comparable segmental duplication event was also observed for *TPPs* in rice [74]. Natural whole-genome duplicating processes might have led to the expansion of the *TaTPP* gene family. Thus, these findings suggest that whole-genome and segmental duplications might be vital in the expansion and evolution of *TaTPPs*.

Phylogenetic analysis of 31 TaTPP proteins and 11 other plant species showed that these proteins clustered into 11 groups, where TPPs from monocots and dicots species were grouped into separate clades (Figure 3). TaTPP proteins were grouped into clade I, clade II, clade V, clade VI, and clade VII and closely related to *Brachypodium*, rice, and barley TPPs, suggesting that TaTPP proteins might originate from a common ancestor. TaTPP5 and TaTPP7 have moved far away from the cluster of all other TPPs in the radiation tree (Figure S6) that was similar to OsTPP11 and OsTPP12 as previously reported [74]. The *TaTPP* gene structure study demonstrated that the majority of *TaTPPs* had highly conserved gene structures. The size of an intron has a significant impact on the size of a gene. The number of introns in *TaTPPs* ranged from 4 to 13 and most of the *TaTPPs* had 8 or 9 introns (Table 1). The difference in total intron length between the largest gene *TaTPP7-A* (32 kb) and the shortest gene *TaTPP-8B* (2.3 kb), resulted in a significant variation in gene size. Further, multiple alignments of TaTPP protein sequences revealed that the Trehalose\_PPase domain and conserved motif are conserved within the *TaTPPs* (Figure S2). Among the identified six motifs, all the motifs were highly conserved in all *TaTPPs* except *TaTPP5-A*, which lacks motif 3. All the *TaTPPs* had a complete Trehalose\_PPase domain, suggesting the various proteins' functional equivalence and evolutionary relationships. In addition, *TaTPP5-B* and *TaTPP7* had a stress-antifungal domain which has been reported to be involved in disulphide bridges and response to salt stress [86,87]. Subcellular localization prediction showed that most of the TaTPPs are localized in the chloroplast, whereas some

of them are found in the mitochondrion or secreted protein (Table 1). In *Arabidopsis* or rice, different localizations were also detected. For instance, AtTPPD and AtTPPE were localized in the chloroplast whereas AtTPPA, AtTPPB, AtTPPC, AtTPPF, and AtTPPH were found in the cytosol and AtTPPG, AtTPPI, and AtTPPJ showed localization in the nucleus [88]. This variation in the localization of TaTPPs might be due to a lack of conserved N-terminus (Figure S2). According to the 3-D structure analysis, all TaTPPs were highly conserved and showed Mg2+ ligand-binding sites in SWISSMODEL (Figure S1a), which are shown to have a role in catalysis by activating or inhibiting a variety of enzymes [89,90]. To investigate the *TPP* gene synteny relationship in wheat and other plant species, we identified 27, 26, 13, 33, 26 and 22 orthologous gene pairs between *TaTPPs* and other *TPPs* in *B. distachyon*, *O. sativa*, *A. thaliana*, *H. vulgare*, *Z. mays*, and *S. bicolor*, respectively (Figure 6 and Table S7). These findings imply that *TaTPPs* in wheat might have evolved from other plant species orthologous genes.

A non-coding DNA sequence found in the promoter region of a gene is known as a CREs. Different CREs distribution in promoter regions may indicate variations in gene regulation and function [91]. To identify the CREs, we used 2kb promoter regions of all *TaTPPs* and classified into five groups according to their known functions (Figure 7, Table S10). Stress related CREs were identified in high frequency compared to cellular development and hormone related CREs, suggesting the involvement of *TaTPPs* in response to stress. ABRE, At~ABRE, ABRE3a, and ABRE4 are ABA-responsive CREs that play important roles in seed dormancy, stomatal closure, leaf senescence, and plant biotic and abiotic stress responses. Multiple ABREs or their combinations have been reported to act as CEs (Coupling Elements) in the formation of ABA-responsive complex (ABRC) [92–97]. The ABRE CREs were predicted in all *TaTPPs* promotor with high frequency and ABRE3a and ABRE4 CREs were found in most of the *TaTPPs* except *TaTPP5*, *TaTPP7*, *TaTPP9* and *TaTPP11*. Following that, we also discovered TGA-element, and AuxRR-core (auxinresponsive element), TCA-element (salicylic acid responsiveness), CGTCA-motif (MeJA responsiveness) and *p*-box and TATC-box (gibberellin responsive element), among other hormone-related CREs [98], that might potentially induce possible signal transduction pathways for wheat *TPPs* during stress response. Furthermore, other CREs linked to a variety of development and stress were also predicted in *TaTPP* promoters with high frequency, including MBS (drought inducibility), MYC (drought-responsive CRE) MYB and STRE (stress response element), as-1(Defense response), Unnamed\_1 (ABRE-like CRE, responsible for biotic and abiotic stress responses), ARE (anaerobic induction CRE), Unnamed\_4 (might responsible for tissue specific expression) and AAGAA-motif (involved in seed specific expression) [91]. These findings suggest that *TPP* gene family members in wheat may be controlled by a variety of developmental events, hormones, and stress; however, additional experimental investigations will be required to validate this.

Higher transcriptional levels of *TaTPPs* were observed in different wheat organs and developmental stages. *TaTPP1*, *TaTPP8* and *TaTPP9* were expressed in most organs and developmental stages but predominantly expressed in the roots, suggesting that they could be important for root physiology. (Figures 8 and S4). Previous evidence showed that *AtTPPE* modulates ABA-mediated root growth and a rice *TPP*, *OsTPP7*, enhanced the anaerobic germination [41,99]. Wang et. al. [27] reported that seed germination was regulated by *OsTPP1* via crosstalk with the ABA catabolism pathway. In addition, high expression was also observed for *TaTPP1*, *TaTPP8* and *TaTPP9* in all developmental stages except dough development and ripening, suggesting that these genes might have a significant association with wheat developmental processes. Plants have developed sensory and response systems that enable them to adjust physiologically to environmental stress conditions such as drought, excessive salt, and low temperature stress. Previous studies in rice and *Arabidopsis* demonstrated the involvement of *TPP* genes in various environmental stresses and ABA signaling [32,37,38,40,41,100]. Under ABA treatment, *TaTPP1* and *TaTPP4* exhibited upregulated transcriptions at most of the time points and significant upregulations were observed from 3 to 12 hpt. Moreover, three *TaTPPs* (*TaTPP7*, *TaTPP8* and *TaTPP9*) were

upregulated immediately after ABA treatment and transcriptions were decreased with the duration after ABA treatment and a significant downregulated transcription level was observed for *TaTPP2*, *TaTPP3* and *TaTPP11* at most of the time points after ABA treatment (Figure 9a). During the drought stress treatment, only *TaTPP1* and *TaTPP4* showed significant upregulations at later time post treatment. A slight upregulation or significant downregulation was observed for all other *TaTPPs* after drought stress in wheat (Figure 9b). An obvious significant upregulation was observed for *TaTPP1*, *TaTPP2*, *TaTPP4* and *TaTPP9* at an early stage of salt stress and downregulated transcriptions were observed at the latter stage of salt treatment. A similar expression pattern was also observed for rice *TPP* and *BdTPPC* genes that were upregulated in the first hour under abiotic stress [28,38]. Moreover, *TaTPP7* showed a significant upregulation only at 12 dpt. In contrast, either no changes or significant downregulation was observed for other *TaTPP* members compared to the control (Figure 10). The phenotype of mature *otsB*-overexpressing *Arabidopsis* plants included delayed senescence and decreased anthocyanin accumulation, suggesting that the role of *TPP* may perform a crucial role during leaf senescence in plants [42–45]. Our results showed that most of the *TaTPP* members were slightly or highly induced during leaf senescence, especially *TaTPP1* showed an obvious upregulated transcription (Figure 9c). Overall, these findings suggest that *TaTPPs* might act as an important regulator of wheat abiotic stress and leaf senescence responses and could be good candidate genes for wheat improvement under environmental stimuli. Moreover, protein network prediction revealed that TaTPP proteins possible interact with TaTPS or TaTRE protein which involved in trehalose biosynthesis pathway to accelerate the trehalose biosynthesis process (Figure S5).

Furthermore, we suggested a feasible working model based on *TaTPPs* transcription profiling to illustrate the roles of *TaTPPs* in a range of biological processes in wheat (Figure 11). TPS produces T6P, a phosphorylated intermediate, from UDPG and G6P and then TPP dephosphorylates T6P to produce trehalose in the second phase. Trehalose is then hydrolyzed by an enzyme called TRE to synthesize two molecules of glucose [16]. The expression of *TaTPPs* was induced by both endogenous and exogenous stimuli in this model. These signals were detected by multiple *Cis*-regulatory elements, which then regulated the transcription and functions of *TaTPPs* involved in numerous plant developmental stages and stress situations, affecting plant growth and tolerance mechanisms (Figure 11).

**Figure 11.** A proposed model for *TaTPP* genes functions in various wheat developmental processes and diverse stress conditions. ABA: Abscisic acid; MeJA: Methyl jasmonate; JA: Jasmonic acid; SA: Salicylic acid; ARF: Auxin response factors; MYB: Myeloblastosis; NAC: No apical meristem; TPS: trehalose-6-phosphate synthase; T6P: trehalose-6-phosphate; TPP: trehalose-6-phosphate phosphatase; TRE: trehalase. Yellow boxes indicate carbohydrates and light green boxes indicate proteins.

#### **5. Conclusions**

In conclusion, a relatively comprehensive analysis of the *TaTPP* gene family was performed in this study, which may help to explain the biological activities of TaTPP proteins in developmental processes, stress responses and leaf senescence of wheat. However, our knowledge of their precise biological role is still lacking. Thus, in order to give important insights to help wheat breeders for developing resistant crops cultivars to unfavorable stress conditions, an extensive functional validation study of *TaTPPs* is necessary.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/genes12111652/s1 Figure S1. Schematic illustration of 3-D protein structures and the Ramachandran plot for TaTPP proteins, Figure S2. Schematic representation of protein alignment of all TaTPPs, Figure S3. Schematic representation of TaTPP1-A side chains catalytic triads, Figure S4. Heat map of *TaTPPs* transcriptions in wheat development stages, Figure S5. Protein–protein interaction analysis of TaTPP proteins, Figure S6. Radiation tree of TaTPPs along with TPPs from other species, Table S1. List of primers used for *TaTPPs* qRT-PCR analysis, Table S2. TPP protein sequences identified from wheat genome, Table S3. Number of TPP proteins in different plant species, Table S4. Sequence identity and query cover of TaTPP proteins, Table S5. Pairwise identities and divergence between *TaTPP* genes and details about the duplication of those genes, Table S6. Phylogenetic tree member with their gene ID, Table S7. The synteny relationships of wheat *TPP* genes with different plant species, Table S8. Details of the calculated secondary structure elements TaTPPs by SOPMA, Table S9. Validation of TaTPP protein structures, Table S10. Frequency of all identified *Cis*-Regulatory Elements (CREs) in different *TaTPPs* promoters, Table S11. *Cis*-Regulatory Elements (CREs) with sequences and functions, Table S12: The protein-protein interaction network between TaTPPs and other proteins in wheat.

**Author Contributions:** The present study was conceptualized by D.S. and M.A.I.; bioinformatics analysis and visualization were conducted by M.M.R. (Md Mustafzur Rahman), M.A.I. and M.M.R. (Md Mizanor Rahman); experiments were investigated by M.A.I., X.J., L.S., K.Z., S.W., and H.N.; writing—original draft was prepared by M.A.I. and M.M.R. (Md Mustafzur Rahman); writing reviewed and edited by J.-S.J., M.M.R. (Md Mizanor Rahman), D.S., A.S. and W.Z.; funding was acquired by D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was sponsored by the State Key Laboratory of Sustainable Dryland Agriculture, Shanxi Agricultural University (No. 202002-2).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

