*3.9. Phylogenetic Analysis of the PAL Gene Family*

Of the 37 *TaPAL* genes identified in this study, four *PAL* genes from *Arabidopsis thaliana*, nine *PAL* genes from *Oryza sativa*, and eight *PAL* genes from *Zea mays* were used to construct a maximum-likelihood-approach tree using MEGA X to determine the evolutionary relationships (Figure 7). The resultant phylogenetic tree based on protein sequence similarities divided PAL proteins into four major clades or groups represented in different colors. The first three groups represent the monocots, while the fourth group shows the dicots. Overall group I exhibited 30 *TaPAL* genes (*TaPAL35-37*, *TaPAL1-2*, *TaPAL4-18*, *TaPAL20-23*, *TaPAL25*, *TaPAL27*, *TaPAL29*, TaPAL31, and *TaPAL33-37*) and one *PAL* gene from each rice and maize. Group II possessed five *TaPAL* genes (*TaPAL3*, *TaPAL19*, *TaPAL24*, *TaPAL30*, and *TaPAL32*) that were found to be more closely associated with the genes of *Z. mays* genes as compared to *O. sativa* genes. Moreover, group III illustrated two *TaPAL* genes (*TaPAL28* and *TaPAL26*) that were closely associated with *PAL* genes of *O. sativa* (*OsPAL1*, *OsPAL5*, and *OsPAL6*)

versus that of *Z. mays* (*ZmPAL1*). The *AtPALs* genes which are dicots and made a separate group IV.

**Figure 5.** Regulatory network relationship between the miRNA and their targeted *TaPAL* genes.


**Table 3.** *Cis*-regulatory elements involved in plant growth regulation, stress, and hormonal responses.


**Table 3.** *Cont*.

**Figure 6.** The predicted functional partners of TaPAL29.

**Figure 7.** Comparative phylogenetic tree of PAL genes between *Triticum aestivum* (*Ta*), *Oryza sativa* (*Os*), *Zea maize* (*Zm*), and *Arabidopsis thaliana* (*At*). One thousand replicates were used for bootstrap test and the percentage of replication is presented next to the branches.

To investigate the ancestral relationship of the *PAL* gene family in *T. aestivum* with its ancestral species, the phylogenetic analysis also showed all the *PAL* genes from *Hordeum vulgare*, *Solanum tuberosum*, and *Triticum urartu*. *Hordeum vulgare* was domesticated from its wild relative, *Hordeum spontaneum*, while *Triticum urartu* is the progenitor of tetraploid *Triticum turgidum* and hexaploid *Triticum aestivum*. The ancestral plants had 8, 10, and 11 *PAL* genes, respectively. Common wheat *PAL* genes showed maximum association with *HvPAL* (*H. vulgare*), followed by *TuPAL* (*T. urartu*), as shown in Supplementary Figure S1.

To study the origin and evolutionary relationship of *Triticum aestivum* (*tr*), *Aegilops tauschii* (*ae*), *Triticum turgidum* (*tg*), and *Triticum dicocoides* (*td*) PAL protein sequences, a comparative synteny analysis was conducted (Figure 8 and Supplementary sheet S2). The proteins from four species were closely associated and showed higher similarity in evolutionary correlation analysis. It was noted that *TaPAL* genes on chromosome trchr6D have some evolutionary origins in common wheat with genes on chromosomes td6B, td6A, and ae6D. We identified that 10 genes of *Aegilops tauschii* are duplicated with *TaPAL33*, *TaPAL37*, *TaPAL27*, *TaPAL34*, *TaPAL36*, *TaPAL22*, *TaPAL23*, *TaPAL35*, *TaPAL26*, and *TaPAL30*. Sixteen genes of *Triticum dicocoides* are orthologs with *TaPAL* genes of wheat, and *TaPAL26* is twice duplicated in *PAL* genes of *Triticum dicocoides*. Nineteen orthologous gene pairs of *Triticum turgidum* and wheat were identified. More than two of the orthologous gene pairs of *TaPAL 37*, *TaPAL36*, *TaPAL35*, *TaPAL34*, and *TaPAL26* were identified in *Triticum turgidum*. Seven paralogous pairs of *TaPAL* genes were identified.

**Figure 8.** Evolutionary relationship of *PAL* genes between *Triticum aestivum* (*tr*, red), *Aegilops tauschii* (*ae*, blue), *Triticum turgidum* (*tg*, pink), and *Triticum dicocoides* (*td*, green). White bars represent chromosomes.
