*3.6. TaTLPs Cis-Regulatory Elements*

According to the results of the in-silico analyses of the *TaTLP*-genes, the upstream region of the *TaTLP* genes contained 13 hormonal, stress, and growth responsive cisregulatory elements, six of which were responsive to hormones. Seven of these elements were responsive to stress and growth-related changes (Table 3). The hormone-responsive cis-elements were ABRE, which participates in the abscisic acid responsiveness, TCA (cisacting element involved in salicylic acid responsiveness), TATC-Box (gibberellin-responsive element), AuxRR-Core (auxin-responsive element), CGTCA (cis-acting regulatory element involved in the MeJA-responsiveness), and TGACG (Cis-acting regulatory element involved in the MeJA-responsiveness). The stress and growth responsive cis-elements were ARE (stimulate mRNA decay), ACE (cis-acting element involved in light responsiveness), G-Box (cis-acting element involved in light responsiveness), LTR (Long-terminal repeat), CAT-Box (Cis-acting element involved in meristem development), O2-Site (Cis-acting regulatory element involved in zein metabolism regulation), and MSA-Like (Cis-acting regulatory element involved in the cell cycle). The presence of these cis-elements in the promoter region of *TaTLP* genes indicates that they regulate gene expression in response to various environmental stimuli at various stages of development.

**Table 3.** Analysis of the diverse types and numbers of cis-acting regulatory elements involved in growth, development, stress, and hormonal response.


*3.7. Expression Analysis of TaTLP Genes in Different Wheat Cultivars in Response to Fusarium graminum Stress*

The gene expression pattern in different wheat cultivars subjected to *Fusarium graminum* stress was drawn on the heatmap (Figure 5). The *TaTLP7, TaTLP22, TaTLP26, TaTLP27, TaTLP35,* and *TaTLP36* showed dominant expression in all cultivars compared to other *TLPs* genes, whereas *TaTLP9, TaTLP12, TaTLP20,* and *TaTLP39* showed dominant expression in annonng0771 and zhongmai66 cultivars as compared to the control and sumai3. The *TaTLP6* showed low expression levels in annonng0771 and zhongmai66 cultivars compared to the

control and sumai3. The *TaTLP*6 showed lower expression levels for cultivars annonng0771 and zhongmai66 than the control and sumai3 cultivar, which showed low expression levels. Similarly, *TaTLP15* and *TaTLP30* showed lower expression levels for cultivar zhongmai66, and in comparison, the control, sumai3, and annonng0771 showed higher expression levels*. TaTLP10, TaTLP37,* and *TaTLP24* displayed higher expression levels for annonng0771 and zhongmai66 cultivars, and lower expression levels were recorded for the control and sumai3. The *TaTLP1, and TaTLP2,* showed lower expression levels in sumai3 than the control, annonng0771, and zhongmai66 cultivars. The *TaTLP4* and *TaTLP31* showed high expression levels in the control and sumai3 compared to annonng0771 and zhongmai66 cultivars. Furthermore, *TaTLP8, TaTLP23, TaTLP29, TaTLP3, TaTLP25, TaTLP38, TaTLP13* and *TaTLP28* displayed high expression levels as compared to *TaTLP17, TaTLP18, TaTLP5, TaTLP16, TaTLP33, TaTLP32, TaTLP40, TaTLP19, TaTLP11, TaTLP21, TaTLP14*, and *TaTLP*34 which displayed low expression levels, except for *TaTLP19, TaTLP11*, and *TaTLP21* which showed higher expression levels for annong0711 cultivar under *Fusarium graminum* stress.

**Figure 5.** Heatmap representing expression analysis of *TaTLP* genes in different wheat cultivars subjected to *Fusarium graminum* stress.

#### *3.8. Expression Analysis of TaTLP Genes in Wheat in Response to Different Temperatures*

The gene expression pattern in response to temperature stress was drawn on the heatmap (Figure 6). *TaTLP*10, *TaTLP*15, *TaTLP*28, *TaTLP*2 *TaTLP*34, *TaTLP*24, *TaTLP*26, *TaTLP*16, *TaTLP*30, *TaTLP*7, *TaTLP*40, *TaTLP*20, *TaTLP*31, *TaTLP*38, *TaTLP*22, *TaTLP*14, and *TaTLP*17 displayed a high expression level under temperatures of 20 and 30 ◦C, whereas *TaTLP*24 and *TaTLP*22 showed the highest expression levels in comparison to other *TaTLP* genes. Similarly, *TaTLP*25 and *TaTLP*39 showed higher expression levels at 30 ◦C than under 20 or 40 ◦C.

The role of *TaTLP* proteins in response to temperature stress is of great interest. To reveal the potential role of *TaTLP* genes in response to temperature stress, we used qRT-PCR to detect *TaTLP* expression levels for 0, 3, and 6 h stress at 40 ◦C (Figure 7). *TaTLP* genes were found to respond positively to temperature stress. For example, *TaTLP1, TaTLP 2, TaTLP6, TaTLP8,* and *TaTLP15* expression increased at 3 and 6 h temperature stress, indicating that these genes are stress responsive in wheat. Further, *TaTLP3, TaTLP12, TaTLP16,* and *TaTLP32* expression significantly decreased under high-temperature stress. *TaTLP4, TaTLP5, TaTLP10. TaTLP17, TaTLP34,* and *TaTLP36* expression decreased under

3 h temperature stress, and, as temperature stress increased, the expression levels of these genes significantly increased.

**Figure 6.** Heatmap representing expression analysis of *TaTLP* genes in wheat subjected to different temperatures.

**Figure 7.** Expression analysis of *TaTLP* genes under high temperature at different time points.

#### *3.9. Expression Analysis of TaTLP Genes in Wheat Subjected to Hormonal Treatment*

The heatmap analysis showed different expression patterns under various hormonal treatments. *TaTLP*11, *TaTLP*2, *TaTLP*9, *TaTLP*12, *TaTLP*34, *TaTLP*13, *TaTLP*35, *TaTLP*30, *TaTLP*22, *TaTLP*23, *TaTLP*29, *TaTLP*10, *TaTLP*4, *TaTLP*37 *TaTLP*3 *TaTLP*19, *TaTLP*38, and *TaTLP*17 showed high levels of expression in comparison to other *TATLP* genes under control (CK) and a similar expression patterns were observed for ABA (abscisic acid) treatment, whereas *TaTLP*11, *TaTLP*2, *TaTLP*9, and *TaTLP*34 showed the highest expression level under CK and ABA treatments (Figure 8). Furthermore, *TaTLP*40, *TaTLP*11, *TaTLP*2, *TaTLP*9, *TaTLP*12, *TaTLP*34, *TaTLP*13, *TaTLP*35, *TaTLP*30, *TaTLP*22, *TaTLP*23, *TaTLP*3 *TaTLP*19, *TaTLP*38, *TaTLP*17, *TaTLP*1, and *TaTLP*27 showed a high expression level under GA (gibberellic acid) treatment as compared to other *TLP* genes. The expression pattern of *TaTLP* genes in response to the combination of ABA and FG (*Fusarium graminum*) treatment showed a similar expression pattern as that of GA treatment. The expression pattern of *TaTLP* genes in response to the combination of GA and FG was different; for example, *TaTLP*16, *TaTLP*25, *TaTLP*7, *TaTLP*15, *TaTLP*11, *TaTLP*12, *TaTLP*34, *TaTLP*13, *TaTLP*35, *TaTLP*22, *TaTLP*23, *TaTLP*37, *TaTLP*19, *TaTLP*38, *TaTLP*17, *TaTLP*1, and *TaTLP*27 displayed high expression levels as compared to other *TLP* genes.

**Figure 8.** Heatmap represents expression analysis of *TaTLP* genes in wheat subjected to hormonal treatment and hormones + *Fusarium graminum.* ABA (abscisic acid), GA (gibberellic acid), ABA+FG (abscisic acid + *Fusarium graminum*)*,* GA + FG (gibberellic acid + *Fusarium graminum*).

#### *3.10. Expression Analysis of TaTLP Genes in Wheat in Response to Iron Deficiency Stress*

The heatmap analysis showed varying expression patterns under iron deficiency conditions in roots and leaf tissues, as shown in Figure 9. *TaTLP7, TaTLP17, TaTLP24, TaTLP16, TaTLP22, TaTLP2, TaTLP7, TaTLP710, TaTLP7,14, TaTLP720, TaTLP32, TaTLP35*, and *TaTLP39* displayed the highest expressions levels as compared to other *TLP* genes in root-control and root low-Fe conditions, whereas *TaTLP*26 showed high expression as compared to root-control. The remaining *TLP* genes' expression levels were unaffected under root low-Fe conditions. Similarly, *TaTLP7, TaTLP17, TaTLP24, TaTLP16, TaTLP22, TaTLP10, TaTLP14,* and *TaTLP20* showed high expression levels as compared to other *TLP*Ss, but similar expression levels between leaf-control and leaf low-Fe conditions. *TaTLP*34 showed a high expression level under the leaf low-Fe condition, whereas *TaTLP*40 and

*TaTLP*1 showed a high expression level in leaf-control compared to the leaf low-Fe condition. The remaining *TLP* gene expression levels were unaffected under leaf low-Fe conditions.

**Figure 9.** Heatmap representing expression analysis of *TaTLP* genes in wheat under iron deficiency stress.
