*3.7. Promoter Analysis*

The promoter sequence is known as a regulatory element that controls gene expression and regulation [7–9]. The promoters are also called *cis*-acting regulatory DNA elements. Their location can be retrieved from the PLACE database (Table 3). Three regulatory elements, TCA-element, CGTAC-motif, and ABRE (abscisic acid or ABA responses), were identified for *TaPAL* genes. The TCA-element, CGTAC-motif, and ABRE-motifs are associated with SA responses, MeJA, and ABA, respectively.

Additionally, the expression of the *TaPAL* gene family is closely related to light, which was confirmed by the presence of MRE light-responsive element, G-Box, GT1-motif, AEbox, ATC-motif, C-box, CAG-motif, I-box, Sp1, Box 4, and ACE on some member *TaPAL* gene families. The putative TATA box was present on the upstream sequences from the start codon ATG on all *TaPAL* genes. Moreover, *TaPAL* gene promoters also contained several phytohormone-responsive elements, including ABRE, AuxRE (auxin-response elements), and GARE (gibberellin (GA) responses). The promoter of *TaPAL* genes also contained MBS (drought induction) and LTR repetitive sequences (cold stress) related to stress-response regulatory elements. *TaPAL21*, *TaPAL35*, *TaPAL8*, *TaPAL5*, *TaPAL16*, *TaPAL15*, *TaPAL28*, *TaPAL32*, *TaPAL26*, *TaPAL25*, *TaPAL18*, *TaPAL27*, *TaPAL36*, *TaPAL34*, and *TaPAL12* contained a single copy of MBS *cis*-regulatory element, while *TaPAL22* contained two

copies of MBS *cis*-regulatory element. It was also observed that only *TaPAL37*, *TaPAL29*, *TaPAL34*, *TaPAL35*, *TaPAL31*, *TaPAL24*, *TaPAL28*, *TaPAL3*, and *TaPAL19* contained the LTR *cis*-regulatory element. Additionally, the upstream regulatory sequences of the *TaPAL34* and *TaPAL16* genes contained the TC-rich repeat, which is related to the defense mechanism. These results suggested that the *TaPAL* gene family members may play an important role in the survival of plants under various environmental stresses. These *cis*-regulatory elements (promoters) receive stimuli from the environment via complex mechanisms and induce gene expression and regulation in response to various abiotic and biological stresses.

**Figure 4.** GO analysis of *PAL* genes. The data show (**A**) biological processes, (**B**) molecular processes, and (**C**) cellular processes.

### *3.8. Protein–Protein Interaction of TaPAL*

The TaPAL protein predicted analysis showed an array of other proteins which coregulate with TaPAL29 (Traes\_1BS\_BD86C90A7.1) (Figure 6). Arogenate dehydratase (Traes\_5BL\_7B0ED7548.1), which is a key enzyme involved in synthesis of L-phenylalanine from L-arogenate, showed interaction with our reference gene. The bit-score of 0.895 showed that optimum interaction with our reference gene TaPAL29, which is a member of the *TaPAL* genes, while rest of the gene was uncharacterized.
