We obtained the wheat genome data used by this study from the Chinese Spring IWGSC RefSeq v1.1 reference genome assembly (https://wheat-urgi.versailles.inra.fr/). First, we used a UNIX pipeline to convert the wheat genome to a local BLAST database. We then used 23 TPP protein sequences from Arabidopsis and rice to execute a BLAST search (BLASTP) with the local blast database, using a cut-off E-value < 1e− 10. After filtering redundant sequences, we analyzed the remaining protein sequences and used the Simple Modular Architecture Research Tool (SMART; http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). to identify the TPP domain. Lastly, we identified 31 TPP domain containing proteins in the most recent wheat genome (Additional file 1: Table S1). Of these, we assigned 11 clusters to various A, B, or D sub-genomes, which we considered to be homoeologous copies of a single TPP gene. Wheat TPP genes were labeled as TaTPPX_ZA, TaTPPX_ZB, or TaTPPX_ZD, and Z denote the location on the wheat chromosome where the gene number (X) is located. The detailed information of TaTPP genes in wheat was listed in Table 1. As shown in Table 1, the identified TaTPP genes in wheat encode proteins ranging from 249 (TaTPP5-2A) to 584 (TaTPP7-3D) amino acids (aa) in length with an average of 386 aa. Furthermore, the computed molecular weights of these TaTPP proteins ranged from 28.66 (TaTPP5-2A) to 96.02 (TaTPP7-3D) kDa. The theoretical pI of the deduced TaTPP proteins ranged from 5.53 (TaTPP1-1B and TaTPP1-1D) to 9.26 (TaTPP10-6A).
Genomic comparison is a quick and easy method of transporting genomic information from a well-studied species to a newly-studied species. We used the genomic position information to locate 31 TaTPP genes over 17 wheat chromosomes, which ranged from 1 to 5 members per chromosome (Table 1; Fig. 2). We used Holub’s method  to identify nine tandem duplication events (TaTPP2-2A/TaTPP3-2A, TaTPP2-2A/TaTPP4-2A, TaTPP3-2A/TaTPP4-2A, TaTPP2-2B/TaTPP3-2B, TaTPP2-2B/TaTPP4-2B, TaTPP3-2B/TaTPP4-2B, TaTPP2-2D/TaTPP3-2D, TaTPP2-2D/TaTPP4-2D, and TaTPP3-2D/TaTPP4-2D) in wheat TPP genes, suggesting that certain TaTPP genes could be produced via gene duplication (Fig. 2). Brachypodium distachyon has a close phylogenetic relationship with wheat, and is considered a model for monocotyledonous angiosperm plants. As such, we performed a synteny analysis between wheat and Brachypodium distachyon TPP genes to explore their relationship. This analysis of wheat and Brachypodium distachyon TPP genes identified 14 pairs of syntenic TPP genes between Brachypodium distachyon and wheat (with E-value < 1e− 5), including 17 TaTPP genes (TaTPP1-1A, TaTPP1-1D, TaTPP3-2A, TaTPP3-2D, TaTPP6-2A, TaTPP6-2D, TaTPP7-3A, TaTPP7-3D, TaTPP8-5A, TaTPP8-5D, TaTPP9-6A, TaTPP9-6D, TaTPP10-6A, and TaTPP10-6D) and seven BdTPP genes (BdTPP1, BdTPP2, BdTPP3, BdTPP4, BdTPP5, BdTPP7, and BdTPP8) (Fig. 2). This suggests that most TPP genes existed before Brachypodium distachyon and wheat diverged.
Subcellular localization of TaTPP proteins in different subfamilies
We further characterized the subcellular localization of four TaTPPs (TaTPP6, TaTPP7, TaTPP9, TaTPP11) that belong to the distinct cluster in the phylogenetic tree shown in Fig. 1. In order to confirm the subcellular localization of these TaTPPs, we developed the 35S::TaTPP6-GFP, 35S::TaTPP7-GFP, 35S::TaTPP9-GFP, and 35S::TaTPP11-GFP transient expression vectors to express TaTPP6-GFP, TaTPP7-GFP, TaTPP9-GFP, and TaTPP11-GFP fusion proteins in wheat protoplasts, with 35S::GFP as positive control. The result was as expected, all four TaTPPs-GFP fusion proteins were located in both cytoplasm and the nucleus (Fig. 5).
Cis -acting regulatory elements in TaTPP promoters
Specific gene expression is primarily regulated by certain promoters, the action of which is mediated by transcription factors via directly binding to cis-acting regulatory elements . As such, analyzing upstream regulatory sequences will contribute to a better understanding of how target genes are regulated, allowing us to assess potential functions . We extracted and scanned ~ 2000 bp of non-coding sequences upstream from the predicted translation start site of each TaTPP gene to fully identify the putative cis-acting regulatory elements. We used the online software tools PlantCARE and PlantRegMap to locate the abundant regulatory cores associated with responses to hormones, stress, and development (Fig. 6; Additional file 5: Table S2).
We observed significantly enriched hormone-related motifs in the majority of the regulatory regions of the TaTPP genes we tested, including abscisic acid (ABRE-element), auxin (TGA-element, AuxRE-core), gibberellin (P-box, GARE-motif and TATC-box), salicylic acid (TCA-element), and methyl jasmonate (TGACG- and CGTCA-motif). Statistical analysis indicated that two kinds of stress-related motifs are involved in abscisic acid and MeJA (methyl jasmonate), which were the most common cis-acting hormone-responsive elements. These elements were found in the promoters of most TaTPP genes, except TaTPP5-2B, TaTPP6-2D, TaTPP7-3D and TaTPP10-6D for ABA response and TaTPP5-2A, TaTPP7-3A, TaTPP8-5B, TaTPP8-5D, TaTPP9-6D, and TaTPP11-7D for MeJA response. Of the 31 TaTPP genes, 17 contained both gibberellin-response elements (P-box, GARE-motif and TATC-box) and auxin-response elements (TGA-element or AuxRE-core). We also found the salicylic acid-responsive TCA-element in the promoters of 11 TaTPP genes (Fig. 6; Additional file 5: Table S2).
Along with hormone-related motifs, we observed stress elements in the TaTPP gene promoters. In particular, elements pertaining to light response were found in all TaTPP gene promoters, including G-box, TCT-motif, I-box, Sp1, and MRE. Regarding drought response, seven TaTPP gene promoters possessed DRE (dehydration-responsive element) or MBS (MYB binding site involved in drought-inducibility) elements. LTR is a low-temperature response element and is a primary component of the motifs related to stress observed in 16 TaTPPs promoters. A WUN-motif wound response element was found in 10 TaTPP genes, while the other seven TaTPPs genes possessed TC-rich repeats, which are cis-acting elements associated with defense and stress responses. Certain cis-elements are involved in the specific expression in organs and tissues or with metabolism, including the role of MBS I in flavonoid biosynthetic genes regulation, the role of motif I in root-specific expression, the role of CAT-box in meristem expression, the role of GCN4 motif in endosperm expression, the role of the RY-element in seed-specific regulation, and the role of O2-site in zein metabolism regulation, and the role of MSA-like in cell cycle regulation (Fig. 6; Additional file 5: Table S2). These results indicated that TaTPP genes might be involved in plant development, multiple hormone and stress responses.
Tissue-specific expression profiles of TaTPP genes
Gene expression is required for the normal growth and development of healthy plants, and, as a result, is highly regulated. Specific patterns of expression of candidate genes indicate potential roles in both growth and development. We used publicly available RNA-seq data to observe these expression patterns in seedling stems, seedling roots, seedling leaves, flag leaves, and during two stages of spike development (5 days and 15 days after head sprouting) and four stages of grain development (5 days, 10 days, 15 days, and 20 days after pollination), allowing us to assess the possible role of TaTPP genes during the growth and development of wheat. We obtained 26 TaTPP gene transcripts (Fig. 7), and could not locate five other TaTPP genes due to low levels of expression or the fact that they could be pseudogenes. Levels of expression vary widely in different tissues of wheat TaTPP genes, and between different tissues in individual TaTPP genes. We observed three homologous genes TaTPP8-5A/B/D that demonstrated widespread expression patterns that were higher in almost all tissues and stages. There are high levels of TaTPP1-1A/B/D expression in both seedling stems and young spikes, while there are high levels of TaTPP2-2A and TaTPP4-2A/D expression in seedling leaves, seedling stems, and grains. Compared with the seedling roots, TaTPP3-2A/D display relatively higher expression in other tissues and stages. There are high levels of TaTPP9-6A/D expression in seedling leaves, roots, spikes, and stems. There are higher levels of TaTPP10-6A/D expression in seedling stems, leaves, and mature spikes, while there is a strong and particular expression of TaTPP2-2D, TaTPP4-2B, and TaTPP11-7A/B/D during grain development, suggesting that these genes could play significant roles during this stage (Fig. 7).
Most homologous genes demonstrate similar patterns of expression during developmental stages, though several clustered expression profiles do not have similar genes, including copies of individual kinds of TaTPP genes from their sub-genomes; some demonstrate opposite expression patterns. For example, TaTPP2-2A is found on chromosome 2A and is preferentially expressed in the seedling leaves and stems, while the homologous TaTPP2-2D gene (located on chromosome 2D) is expressed in these tissues at a lower point. TaTPP10-6A is located on 6A and displays higher levels of expression in mature spikes and seedling stems. The homologous TaTPP10-6B, found on 6B, is expressed preferentially in the seedling leaves, stems, and mature spikes, while homologous genes from 6D are expressed only in the seedling leaves (Fig. 7). This difference in expression profiles between homologous genes from different subgenomes demonstrates that some have acquired new functions or lost old functions following polyploidization during wheat’s evolutionary history.
Expression analysis of TaTPP genes respond to abiotic stresses
Environmental stresses significantly affect the productivity of wheat, making it important to study the wheat genes responsible for stress response in order to increase yields. We used quantitative real-time PCR (qRT-PCR) to assess how TaTPP gene expression responds to continuous ABA, low temperature, and salt stress, allowing us to analyze the role of TaTPP genes that could be associated with plant defense to abiotic stresses. We designed allele pairs from A-, B- and D-subgenomes and tested them together, since the products of their transcription share similar sequences. Each gene we analyzed had a different expression when responding to a minimum of one abiotic stress (Fig. 8). In response to ABA, there were eight up-regulated TaTPPs (TaTPP1, TaTPP3, TaTPP4, TaTPP6, TaTPP7, TaTPP8, TaTPP9, and TaTPP11) and three down-regulated TaTPPs (TaTPP2, TaTPP5, and TaTPP10) in seedling leaves at a minimum of one time point. In response to low-temperature conditions, there were seven up-regulated TaTPPs (TaTPP1, TaTPP3, TaTPP4, TaTPP7, TaTPP8, TaTPP9, and TaTPP11) and four down-regulated TaTPPs (TaTPP2, TaTPP5, TaTPP6, and TaTPP10). In response to salt stress, there were eight up-regulated TaTPPs (TaTPP1, TaTPP3, TaTPP4, TaTPP6, TaTPP7, TaTPP8, TaTPP9, and TaTPP11) and three down-regulated TaTPPs (TaTPP2, TaTPP5, and TaTPP10) (Fig. 8).
To better understand the functions of TaTPP genes in regulating wheat drought response, the expression patterns of 11 TaTPPs were experimentally examined in leaves and roots of 3-week-old drought-stressed wheat seedlings. As illustrated in Fig. 9, a dramatic upregulation of 8 TaTPP genes (TaTPP1, TaTPP2, TaTPP3, TaTPP4, TaTPP5, TaTPP9, TaTPP10, and TaTPP11) were observed in response to drought stress, especially in the leaves. TaTPP7 also showed a slightly up-regulation in leaves and roots after drought stress. The changed expression levels of TaTPP2, TaTPP3 and TaTPP4 in leaves after drought stress were very sharp, with more than 60 folds, indicating that these genes are extremely susceptible to drought stress. Some genes showed very similar expression profiles after drought stress, such as TaTPP3 and TaTPP4 pairs. Some TaTPPs were significantly upregulated after light/early drought stress, such as TaTPP2, TaTPP3, TaTPP4 and TaTPP9 in leaves and TaTPP9 and TaTPP11 in roots, suggesting positive roles of these genes in early drought stress response. Some genes were significantly upregulated after severe stress, such as TaTPP1, TaTPP10 and TaTPP11 in leaves and TaTPP6 and TaTPP8 in roots, suggesting that these genes are important for the plant response to drought stress at a severe level. Upon drought stress, TaTPP5 and TaTPP10 were upregulated in leaves but downregulated in roots (Fig. 9). These data show the potential of some TaTPP genes for enhancing adversity resistant capacity, especially for wheat drought improvement.
Ectopic expression of TaTPP11 in Arabidopsis delayed plant development and enhanced drought tolerance
Alignment of the protein sequences determined the presence of three TaTPP11 homeologs sharing a sequence similarity of approximately 95% (Additional file 6: Figure S4). Additional information regarding the spatiotemporal profile of TaTPP11 expression could contribute to a better understanding of how TaTPP11 functions biologically. In this case, we observed TaTPP11 expression across various tissues and organs of wheat at different stages of development, such as the roots and leaves of seedlings, young panicles, flag leaves, and seeds. Our results demonstrated high levels of TaTPP11 expression in seedling leaves and developing seeds, and low levels of TaTPP11 expression in developing panicles (Fig. 10A). This indicates that TaTPP11 could serve an important purpose as wheat seeds develop.
To better understand how TaTPP11 relates to the development of wheat, we produced 35S::TaTPP11-7D transgenic Arabidopsis lines and assessed their levels of TaTPP11-7D expression to select three independent transgenic lines (OE1, OE2, and OE3) for subsequent analysis (Fig. 10B; Additional file 7: Figure S5). First, we observed the germination of the seeds and found there were no significant differences between transgenic and wild type lines (Fig. 10C, D). Next, we detected the phenotypes of the 35S::TaTPP11-7D transgenic Arabidopsis lines and that of the wild-type over various developmental stages. The TaTPP11-7D transgenic Arabidopsis seedlings grew vegetation for much longer, bolted and flowered later, and had a lower plant height compared to the wild type (Fig. 10E). We also analyzed the organs from both the Arabidopsis wild-type plants and transgenic lines, and did not find any significant differences in the organs of the transgenic and wild type varieties, including in the seeds, flowers, and siliques (Fig. 10E, F).
We next assessed the drought tolerance of the wild type and transgenic plants to identify the role of TaTPP11 in plant drought stress. Both 35S::TaTPP11-7D transgenic plants and wild type plants were grown for three weeks in soil, after which they were not watered for 14 days. The plants were then watered for six days. Afterward, approximately 30% of wild type plants survived, while approximately 80–90% of the transgenic plants survived (Fig. 11A, B). Next, the trehalose contents of the wild type plants and the 35S::TaTPP11-7D transgenic plants were assayed, demonstrating that the trehalose levels in transgenic lines were significantly higher than in wild type plants (Fig. 11C). Additionally, we assessed other cellular processes impacted by drought stress conditions, paying particular attention to substances regulating osmosis. We observed increased levels of proline and soluble sugar, and decreased levels of malonaldehyde under drought stress conditions in transgenic lines (Fig. 11D-I). These results indicate that TaTPP11 overexpression in Arabidopsis might delay the development of plants, but could also increase the drought tolerance of transgenic varieties.