2.1. Identification and annotation of wheat TaELPs family members
Arabidopsis and rice ELP proteins were used as reference sequences to search the wheat genome database. After HMM and smart analysis, 18 wheat ELP genes (Table 1 and Table S1) were finally identified and named according to their physical positions on the chromosome. The members of the wheat TaELPs family were further annotated by gene ID, position and open reading frame (ORF) length, and protein physicochemical properties. The ORFs of TaELPs ranged from 753 to 3978 bp and the protein length ranged from 250 to 1325 amino acids. The molecular weights of TaELPs ranged from 27.05 to 147.31 KDa, and according to the predicted isoelectric point (PI) values, the PI ranged from 5.30 to 8.97, of which 6 genes were found to be basic (> 7) and 12 genes were found to be acidic (< 7) [35].
Table 1
Detailed annotations of the TaELPs in wheat.
Gene Name | Gene ID | Splice | PC | ORF | Chromosome Location | Introns | Exons | Length | M.W. | PI | Instability | Aliphatic Index | GRAVY | SL Prediction |
Variant | Chr | Strand | Start | End | Chr Length | (aa) | (KDa) | Index |
TaELP1-A | TraesCS1A02G104700.1 | 1 | II | 2511 | 1A | reverse | 100,291,939 | 100,297,147 | 594,102,056 | 10 | 11 | 836 | 91.40 | 6.26 | 44.07 | 84.67 | -0.093 | cytosol |
TaELP1-B | TraesCS1B02G116100.1 | 1 | II | 2508 | 1B | reverse | 136,647,720 | 136,652,235 | 689,851,870 | 9 | 10 | 835 | 91.36 | 6.23 | 42.10 | 86.05 | -0.090 | cytosol |
TaELP1-D | TraesCS1D02G096900.1 | 2 | II | 2535 | 1D | reverse | 83,724,571 | 83,729,227 | 495,453,186 | 9 | 10 | 844 | 92.51 | 6.25 | 43.44 | 83.98 | -0.085 | nucleus |
TaELP2-A | TraesCS2A02G203700.1 | 1 | III | 3978 | 2A | reverse | 179,670,464 | 179,675,987 | 780,798,557 | 5 | 6 | 1325 | 147.31 | 5.60 | 44.21 | 90.59 | -0.163 | cytosol, nucleus, plasma membrane |
TaELP2-B | TraesCS2B02G231000.1 | 1 | III | 3975 | 2B | reverse | 227,082,327 | 227,087,872 | 801,256,715 | 5 | 6 | 1324 | 147.14 | 5.52 | 44.05 | 90.95 | -0.161 | cytosol, nucleus, plasma membrane |
TaELP2-D | TraesCS2D02G212000.1 | 1 | III | 3978 | 2D | forward | 170,443,244 | 170,448,462 | 651,852,609 | 5 | 6 | 1325 | 147.17 | 5.57 | 42.54 | 90.60 | -0.158 | cytosol, nucleus, plasma membrane |
TaELP3-A | TraesCS2A02G320900.1 | 1 | I | 1710 | 2A | forward | 550,539,215 | 550,542,666 | 780,798,557 | 8 | 9 | 569 | 63.57 | 8.88 | 35.59 | 85.55 | -0.310 | cytosol |
TaELP3-B | TraesCS2B02G361800.1 | 1 | I | 1710 | 2B | reverse | 514,861,001 | 514,865,480 | 801,256,715 | 9 | 10 | 569 | 63.61 | 8.88 | 35.55 | 85.89 | -0.307 | cytosol |
TaELP3-D | TraesCS2D02G341600.1 | 1 | I | 1710 | 2D | forward | 436,369,113 | 436,372,717 | 651,852,609 | 8 | 9 | 569 | 63.58 | 8.88 | 35.55 | 85.55 | -0.312 | cytosol |
TaELP4-A | TraesCS4A02G045700.1 | 1 | V | 1155 | 4A | forward | 37,776,004 | 37,782,022 | 744,588,157 | 9 | 10 | 384 | 42.72 | 5.36 | 51.44 | 84.32 | -0.397 | cytosol |
TaELP4-B | TraesCS4B02G259300.1 | 1 | V | 1155 | 4B | forward | 526,726,516 | 526,729,679 | 673,617,499 | 9 | 10 | 384 | 42.71 | 5.38 | 53.49 | 83.05 | -0.413 | cytosol |
TaELP4-D | TraesCS4D02G259200.1 | 1 | V | 1155 | 4D | reverse | 428,719,059 | 428,727,934 | 509,857,067 | 9 | 10 | 384 | 42.71 | 5.30 | 52.06 | 85.34 | -0.390 | cytosol |
TaELP5-A | TraesCS4A02G105200.1 | 1 | VI | 759 | 4A | forward | 119,080,643 | 119,083,199 | 744,588,157 | 4 | 5 | 252 | 27.05 | 5.97 | 31.37 | 101.83 | 0.193 | cytosol |
TaELP5-B | TraesCS4B02G198800.1 | 1 | VI | 753 | 4B | reverse | 427,496,766 | 427,499,136 | 673,617,499 | 5 | 6 | 250 | 27.08 | 5.98 | 33.83 | 99.52 | 0.159 | cytosol |
TaELP5-D | TraesCS4D02G199700.1 | 1 | VI | 765 | 4D | reverse | 346,452,075 | 346,454,270 | 509,857,067 | 4 | 5 | 254 | 27.24 | 5.91 | 35.06 | 97.56 | 0.163 | cytosol |
TaELP6-A | TraesCS7A02G522900.2 | 2 | IV | 1152 | 7A | forward | 705,684,956 | 705,687,644 | 736,706,236 | 7 | 8 | 383 | 41.20 | 8.69 | 53.26 | 80.84 | -0.252 | plastid |
TaELP6-B | TraesCS7B02G439900.1 | 1 | IV | 1158 | 7B | forward | 705,251,306 | 705,254,066 | 750,620,385 | 7 | 8 | 385 | 41.29 | 8.97 | 50.04 | 78.88 | -0.250 | plastid |
TaELP6-D | TraesCS7D02G512100.1 | 2 | IV | 1155 | 7D | forward | 613,867,768 | 613,870,602 | 638,686,055 | 7 | 8 | 384 | 41.18 | 8.69 | 47.94 | 79.35 | -0.252 | plastid |
PC, Phylogenetic clade; ORF, Open Reading Frame; No, Number; bp, Base pair; Chr, Chromosome; aa, Amino Acid; M.W., MolecularWeight; Pi, Iso electric point; GRAVY, Grand average of hydropathy, SL,Subcellular Localization. |
The aliphatic amino acid index and instability index were calculated. The aliphatic amino acid index ranged from 79.35 to 101.83 and the instability index ranged from 31.37 to 53.49. The high aliphatic amino acid index of the protein sequence indicates that it can play a role in a wide temperature range, while the instability index indicates whether the protein is stable or unstable[35]. Among them, 6 genes were stable (instability index < 40), and the remaining TaELPs genes were unstable (instability index > 40)[36]. The calculated hydropathic index (GRAVY) of TaELPs ranges from − 0.085 to 0.193, indicating that they were hydrophilic and can better interact with water [36]. Subcellular localization prediction of TaELPs genes showed that most TaELPs family members were localized in the cytoplasm, and 3 genes (TaELP6-A, TaELP6-B, TaELP6-D) were localized in the plastid and (TaELP2-A, TaELP2-B, TaELP2-D) were located in the cytoplasm, nucleus, and plasma membrane respectively.
2.2. Chromosomal distribution and gene duplication of wheat TaELPs genes
Eighteen TaELPs genes of wheat were located on 12 wheat chromosomes (Fig. 2A and Table 1). TaELPs genes were evenly distributed in A, B, and D subgenomes, each subgenome contained 6 TaELPs genes (Fig. 2B). 2A, 2B, 2D, 4A, 4B, and 4D all contained 2 genes, while 1A, 1B, 1D, 7A, 7B, and 7D all contained 1 gene (Fig. 2C). No TaELPs genes were found on chromosomes 3, 5, and 6, suggesting that TaELPs family genes were unevenly distributed throughout the chromosomal of wheat.
Gene duplication analysis showed that there were 18 pairs of ELP paralogous genes in the wheat genome (Fig. 2D, Table S2), all of which were derived from segmental duplication and located at conserved positions in segmental duplication regions on different chromosomes, indicating that segmental duplication in play an important role in the quantitative expansion of wheat ELP genes [37]. Two segmental duplications occurred on chromosomes 2A, 2B, 2D, 4A, 4B, and 4D, and one segmental duplication occurred on chromosomes 1A, 1B, 1D, 7A, 7B, and 7D (Fig. 1D, Table S2). It is further speculated what kind of selection the wheat ELP gene has undergone in the evolutionary process. We calculated the nonsynonymous mutation rate (Ka), synonymous mutation rate (Ks) and the ratio of nonsynonymous mutation rate (Ka) to synonymous mutation rate (Ks) (Ka/Ks) (Table S2). The value of Ka/Ks = 1 denotes that genes experienced a neutral selection; <1 suggests a purifying or negative selection, and > 1 indicates a positive selection [38]. The Ka/Ks values of the 18 pairs of ELP paralogous genes were all less than 1, suggesting that the TaELPs genes all underwent purification selection after fragment duplication, and the divergence time ranged from 1.83 to 8.53 million years ago (MYA). In conclusion, these results indicate the conserved evolution of TaELPs genes.
2.3. Phylogenetic and cluster analysis of wheat TaELPs
To further understand the evolutionary relationship and phylogeny of TaELPs and ELPs in other plant species, we constructed a phylogenetic tree of ELP protein sequences from seven plant species (Fig. 2, Table S3) by neighbor-joining (NJ). Phylogenetic tree results indicated (Fig. 3) that ELP proteins were divided into 6 clades. Among them, clade I was the largest, containing 11 members. Clade II to Clade VI each contained 9 members. Each clade contained both monocotyledonous and dicotyledonous ELP proteins, indicating that the structural features of ELP proteins evolved before the separation of monocotyledonous and dicotyledonous plants. Within each clade, wheat ELP proteins were more distantly related to Arabidopsis thaliana and Solanum lycopersicum; wheat ELP proteins were closely related to Zea mays, Hordeum vulgare, Brachypodium distachyon, and Oryza sativa, indicating that these species were highly conserved in protein sequences and had similar functions, which can further study the close relationship with wheat.
The 18 wheat ELP proteins were evenly divided into 6 clades, each of which contained A, B, and D subgenomes, and the protein sequences were clustered together in a phylogenetic tree. We compared the protein sequence similarity of the A, B, and D subgenomes of the same group, and the results showed that the similarity was more than 95% (Table S4). Studies have shown that the protein sequence similarity and identity of gene duplications exceed 70% and 75%, respectively [39]. By analyzing the protein sequence and the constructed phylogenetic tree (Fig. 3, Table S4), it was further confirmed that there is a gene duplication event in the wheat TaELPs family genes.
2.4. Orthologous analysis of wheat TaELPs genes
To study the evolutionary relationship of the wheat TaELPs gene family, McscanX software was used to visualize the results of collinearity analysis. We selected the dicotyledonous plants (Arabidopsis and G. max), monocotyledonous plants (O. sativa) and wheat relatives (Brachypodium distachyon、Triticum dicoccoides and Aegilops tauschii) to identify orthologous gene pairs of the wheat ELP genes (Fig. 4, Table S2). We identified a total of 56 orthologous gene pairs of ELP genes (Table S2). No ELP orthologous gene pairs were observed between Arabidopsis and wheat (At-Ta), and only 3 ELP orthologous gene pairs were found between G. max and wheat (Gm-Ta). Thirteen ELP orthologous gene pairs were found between O.sativa and wheat (Os-Ta). We also found that there are 13, 10 and 17 orthologs of ELPs genes between wheat relatives Brachypodium distachyon、Triticum dicoccoides and Aegilops tauschii (Bd-Ta, Td-Ta and Aet-Ta) and wheat. These results suggest that ELP genes in wheat are distantly related to those in dicotyledonous species and are most closely associated with those in Aegilops tauschii, which might be because Aegilops tauschii are widely considered to be the D-genome ancestor of wheat [33, 40]. The Ka/Ks ratio indicates the selection pressure of plant genes and can be used to diagnose the evolutionary form of the sequence [41]. Thus, we calculated the Ka, Ks, Ka/Ks and T values of all orthologous gene pairs in wheat to further investigate the evolutionary trends of the ELP gene family (Table S2). The results showed that the Ka/Ks ratios of all orthologous genes (Ta-Gm, Ta-Os, Ta-Bd, Ta-Td, and Ta-Aet) were less than 1, suggesting that purification selection plays a dominant role in the evolutionary trend of the ELP gene family. According to the divergence time T value calculated from the Ks value, we found that the divergence time of the orthologous genes (Ta-Gm, Ta-Os, Ta-Bd, Ta-Td, and Ta-Aet) was different, among which the orthologous genes (Ta-Gm) had the longest divergence time and had the shortest divergence time with Aegilops tauschii. In conclusion, TaELPs genes in wheat may have evolved from orthologous genes of other plant species or closely related plants.
2.5. Gene structure and conserved motif analysis of TaELPs genes
The phylogenetic tree constructed based on the protein sequences of the members of the wheat TaELPs gene family showed that the members of the TaELPs gene family were divided into three groups (GroupA, GroupB and GroupC), and the results were visualized by combining the gene structure and conserved motifs (Fig. 4). The gene structure analysis of wheat TaELPs found that introns ranged from 4–10, and exons ranged from 5–11. A maximum of 11 exons were found in the TaELP1-A, while a minimum of five introns were found in the TaELP5-A and TaELP5-D. In the TaELPs members of GroupA, GroupB and GroupC, the exon-intron numbers of genes were relatively close, and the exon-intron structure of most genes was relatively conservative. For example, in Group A, the number of introns and exons of TaELPs genes were mostly 9 and 10, and the number of untranslated regions (UTRs) was relatively close.
To further understand the structural diversity of wheat ELPs, we submitted the protein sequences of 18 TaELPs genes to the MEME5.4.1 online website and predicted 10 conserved motifs (Fig. 5B). The results showed that the number of conserved motifs ranged from 3 to 9. All TaELPs gene family members contained motif 2, while TaELP4-A, TaELP4-B, and TaELP4-D lacked motif 1. Motif 5 was unique to TaELP3-A, TaELP3-B, and TaELP3-D. The same group of ELP proteins contained similar motifs, and they may also have similarities in gene function. For example, Motif 2, Motif 8, Motif 4, Motif 10 and Motif 7 were included in Group A. The differences in the types and numbers of conserved motifs in wheat ELP proteins reflected the structural diversity of these proteins, indicating that they might have different biological functions.
2.6. Protein conservation domain and 3-D protein structure analysis of TaELPs gene
Pfam database was utilized to find the important component domains of TaELPs proteins [42]. The conserved domains of TaELPs were shown in Fig. 6. TaELP1-A, TaELP1-B, and TaELP1-D contain four WD40(WD domain, G-beta repeat) protein domains; TaELP4-A, TaELP4-B and TaELP4-D contain 1 Elong_Iki1 (Elongator subunit Iki1) domain; TaELP2-A, TaELP2-B and TaELP2-D contain 1 IKI3 domain; TaELP6-A, TaELP6-B, and TaELP6-D contain 1 PAXNEB domain; TaELP3-A, TaELP3-B and TaELP3-D contain a catalytic domain of S-adenosylmethionine (Radical SAM superfamily) and a histone acetyltransferase (Acetyltransferase (GNAT) family) domain; TaELP5-A, TaELP5-B and TaELP5-D all contain an ELP6 (Elongator complex 6) domain, In addition, an Elong_Iki1 (Elongator subunit Iki1) domain was found in TaELP5-B and TaELP5-D.
We used SWISS-MODEL to further identify 3-D models of TaELPs proteins[35, 43] and the 3-D structure reveals a few key residues linked to biological processes or intended outcomes, (Figure S1). For TaELP1-A, TaELP1-B and TaELP1-D proteins, 3-D structures were analyzed using the template "6qk7.1.B", a template that describes Elongator complex protein 2. TaELP2-A, TaELP2-B and TaELP2-D proteins, 3-D structures were analyzed using the "6qk7.1.A" template, a template describing the Elongator complex protein 1. TaELP3-A, TaELP3-B, and TaELP3-D Protein, the 3-D structure was analyzed using the "6qk7.1.C" template, a description of Elongator complex protein 3, and identified as containing two ligands (1 x 5AD and 1 x SF4), of which, 5AD (5'-DEOXYADENOSINE)9 residues within 4Å and 4 PLIP interactions, SF4(IRON/SULFUR CLUSTER)8 residues within 4Å and 3 PLIP interactions. TaELP4-A, TaELP4-B and TaELP4-D, 3-D structures were analyzed using the"4a8j.1.B" template, "4a8j.1.B" template was a description ELONGATOR COMPLEX PROTEIN 5; TaELP5-A used "4ejs.1.C" template to analyze 3-D structure, TaELP5-B and TaELP5-D proteins, used "4wia.1.A" to analyze 3-D structure; TaELP6-A,TaELP6-B and TaELP6-D proteins, used The "4a8j.1.A" template analyzes 3-D structures. The Residues in the favored region of the Ramachandran plots generated by all TaELPs ranged from 86.39% to 94.32; the Residues in the outlier region ranged from 0.44–6.27%; Coverage of most TaELPs was above 80%, only TaELP4-A, TaELP4- B and TaELP4-D protein coverage was 58% (Table S5).
In addition, we also used SOPMA to calculate the secondary structure elements of the protein sequence (Table S6), the results showed that the TaELPs protein α-helix (Alpha helix) ranged from 13.52–44.46%; β-turn (Beta turn) ranged from 3.39–10.24%; Random coil ranged from 30.31–49.94%; Extended strand ranged from 9.66–32.30%.
2.7. Cis-acting element regulation (CARE) analysis of wheat TaELPs genes
A total of 91 different CAREs were identified by analyzing the upstream 2000bp promoter region of the TaELPs gene, mainly investigating abiotic stress and defense-related hormone response elements. All of the identified CAREs were divided into five groups according to their known functions (Fig. 7B, Table S7). Group I contained 48 environmental stress-related CAREs. Among them, 14 different types of abiotic stress response elements, one cis-element involved in low-temperature response (LTR), one cis-acting element (DRE core) regulating cold and dehydration response gene expression, three anaerobic-induced Essential cis-regulatory elements (ARE, GC-motif, plant_AP-2-like), one MYB binding site (MBS) associated with drought induction, and eight stress response-related response elements (such as MYC, as-1, Unnamed__1, WRE3, etc.) (Fig. 7A, Table S7/S8). 4 cis-acting elements related to wounding and pathogen response (box S, TC-rich repeats, W box, CCAAT-box), the rest were light-responsive elements of different types, such as 3-AF1 binding site, AE-box, Box II, GT1-motif, chs-CMA1a, etc.
Group II contained hormone response-related CAREs. There was a total of 13 different types of CAREs that regulate hormone response, such as cis-acting elements involved in abscisic acid response (ABRE, ABRE2), cis-regulatory elements involved in MeJA response (CGGTA-motif, TGACG-motif), cis-regulatory elements (AuxRR-core, TGA-element) involved in auxin response, and cis-acting elements (TCA-element, TGACG-motif) related to salicylic acid response, etc. Group III contained four core cis-acting elements, among which, CAAT-box and TATA-box appear most frequently in all TaELPs genes, indicating that they play an important role in transcription initiation. TATA-box (including TATA and ATTATA-box) and CAAT-box cis-elements are promoter-associated elements that function at the initiation of transcription [35]. Group IV was plant growth and development-related CAREs, including cis-elements involved in seed-specific expression (AAGAA-motif, RY-element), cis-acting elements involved in cell cycle regulation (MSA-like), and meristem expression associated cis-regulatory elements (CAT-box) and several other CAREs associated with cell division. Group V was a small number of CAREs with unknown functions, which are also commonly found in the promoter sequences of TaELPs genes, indicating that they may also be involved in the regulatory mechanism of TaELPs genes on the environment (Fig. 7A, 7B, Table S7).
In conclusion, TaELPs genes may be involved in the regulation of the above environmental stress-related, phytohormone responses, and cell growth and development. These transcription factors CAREs play an important role to induce transcription of TaELPs.
2.8. TaELPs expression pattern prediction analysis
To further explore the expression patterns of TaELPs genes in different tissues, developmental stages, and abiotic stresses in wheat, we retrieved all wheat mRNA transcription data from the wheat expression database and visualized TPM values with a heat map (Fig. 8). The results showed that TaELP3-A, TaELP3-B, TaELP3-D, and TaELP5-D were expressed at high levels in the tissues of seedlings, roots, stems, leaves, and inflorescences at various stages. In addition, we found that TaELP5-D was up-regulated in flag leaves with the prolongation of post-flowering time, while TaELP3-A, TaELP3-B, and TaELP3-D were gradually down-regulated in flag leaves. Therefore, we speculated that TaELPs genes may be related to senescence; However, TaELP1-D and TaELP6-A had lower expression levels in all different tissues and developmental stages of wheat. TaELP4-A、TaELP4-B and TaELP4-D were found to be at higher levels expressed in the root, stem, leaf, and spike tissues. TaELP1-A, TaELP1-B, TaELP2-A, TaELP2-B, TaELP2-D, TaELP5-A, TaELP5-B, TaELP6-B, and TaELP6-D had tissue expression specificity in the stem, milk grain stage、stem 1 cm spike、leaf, seven leaf stage and roots, three-leaf stage.
TaELP3-A, TaELP3-B, TaELP3-D, and TaELP5-D had the most obvious up-regulation of gene expression after drought treatment 6h (Figure S2); similarly, the gene expression was up-regulated most obviously after heat treatment 6h; After drought and heat stress treatment, gene expression was slightly down-regulated. TaELP3-A, TaELP3-B, and TaELP3-D had higher expression in low-temperature stress. In salt stress, TaELP3-A, TaELP3-B, and TaELP3-D were all up-regulated to varying degrees and showed a downward trend as a whole; TaELP4-A, TaELP4-B, and TaELP4-D had the highest up-regulated expression after salt stress 48h and showed a trend of upward. TaELP5-D showed very low expression in different treatment times of salt stress; the expression patterns of TaELP2-B and TaELP1-D genes showed an upward trend and the up-regulated expression was most obvious at salt stress 48h; The gene expression of other TaELPs genes were slightly up-regulated or down-regulated in different treatment times of salt stress (Figure S2).
2.9. Expression pattern validation analysis of TaELPs
Further understand the potential response mechanism of wheat TaELPs gene family in tolerance to abiotic stress, hormone response, and leaf senescence, we detected the transcription patterns of all TaELPs genes in abiotic stresses (drought, salt, and dark treatment), hormones treatments (IAA, SA, ABA), and during leaf senescence. Under drought treatment, TaELP2 exhibited down-regulated transcriptions at most of the time points. The expression of other TaELPs genes was upregulated to varying degrees at different times of drought treatment. Among them, TaELP3, TaELP1, and TaELP4 were significantly upregulated, And TaELP3 significant upregulation was observed after 6h and 72h of drought treatment, The overall trend of TaELP1was upregulated, and significant upregulation was observed from 24h to 48h. The expression of TaELP4 was most significantly upregulated after 12h of drought treatment (Fig. 9).
Under salt stress, TaELP3, TaELP1, and TaELP6 were slightly up-regulated or down-regulated compared with the control (0h); TaELP4 was significantly down-regulated under salt stress 6h, 12h, 24h, and 48h, but after 72h of treatment, The expression was significantly up-regulated immediately; The expression of TaELP2 and TaELP5 was up-regulated, TaELP2 were significantly upregulated from 48h to 72h, And TaELP5 significant upregulation was observed after 6h and 72h of salt treatment (Fig. 9).To explore the induced expression patterns of all members of the TaELPs gene family in plant growth and development, we performed dark treatments at different times (Fig. 9). The results showed that the expression of all TaELPs genes was up-regulated to varying degrees in the early or late stage of dark treatment. For example, the expression pattern of the TaELP2 gene showed an up-regulated trend; TaELP3, TaELP1, TaELP4, TaELP6 genes showed an up-regulated trend from rising to decline; TaELP5 significant upregulation was observed after 72h of dark treatment (Fig. 9).
Under IAA treatment, the expression patterns of most TaELPs genes were down-regulated, and only TaELP3 and TaELP5 genes were up-regulated. TaELP3 was most significantly up-regulated after 48h of IAA treatment; TaELP5 was up-regulated immediately after 6h of IAA treatment and the expression level reached a peak and then showed a downward trend (Fig. 10A). Under SA treatment, it was observed that only TaELP5 exhibited low expression levels at all time treatment stages and other TaELPs genes were up-regulated to varying degrees. For example, TaELP2 was significantly up-regulated in the early and late stages of SA treatment; the gene expression patterns of TaELP4 and TaELP6 were significantly up-regulated after 12h of SA treatment; TaELP3 and TaELP1 significant upregulation was observed after 24h of SA treatment (Fig. 10B).
Under ABA treatment, the relative expression levels of all TaELPs genes were significantly different (Fig. 10C). TaELP6 showed a significant upregulation at all-time treatment stages compared to the control. TaELP1, TaELP2, TaELP4, and TaELP5 showed an overall upward trend, and the gene expression was up-regulated most significantly after 72h of treatment. Moreover, TaELP3 showed a significant upregulation only at ABA treatment 6h compared to the control (Fig. 10C).
During leaf senescence, all TaELPs genes were up-regulated to varying degrees in the late senescence (Fig. 9). The up-regulation trend of TaELP2 and TaELP6 is the same, and the overall showed a trend from decline to rise, TaELP2 was most significantly expressed at 30 days after flowering, followed by TaELP6 at 10 days after flowering; TaELP5 was significantly up-regulated at 10 days and 19 days after flowering; TaELP1, TaELP3, and TaELP4 were up-regulated in the same trend. Overall, members of the wheat TaELPs gene family play important regulatory roles in abiotic stresses, hormones, and leaf senescence.
2.10. Prediction of protein-protein interactions of wheat ELPs
To study the interaction between wheat TaELPs and other proteins, a network was constructed using the STRING database (Figure S3, Table S9). Based on the predicted results, we observed that TaELP1, TaELP2, TaELP3 and TaELP6 had protein interactions with a Chromatin associated protein KTI12 (Traes_5BL_92F800E16.1, Traes_5BL_D8ECD483D.2 and Traes_5DL_A9A62BF38.1) and a Diphthamide biosynthesis protein 3 (Traes_7BL_69CD9E49D.2, Traes_7DL_EF5C1F9EA.1). In addition, TaELP3 had protein interactions with Traes_2AS_03ED0D137.1, Traes_2BS_E0BE8F2D1.1 and Traes_2DS_E75C5D4AC.1 which encodes WD40 repeat-containing proteins. No protein interacting with TaELP4 and TaELP5 was found.
WD repeats proteins are widely present in eukaryotes and are involved in various cellular behavioral and physiological regulations, such as signal transduction, activation of transcriptional activity, cell growth and development, and control of apoptosis. The presence of WD40 domains or repeated WD40 motifs can act as a scaffold for protein-protein or protein-DNA assembly, play an important role in protein interactions and can act as a mediator of transient interactions between other proteins [44]. Besides, chromatin-associated protein KTI12 was found to interact with the Elongator complex (ELP) in the process of RNA polymerase II promoting transcription elongation [45].