**4. Discussion**

Wheat is the main crop for half of the world's population. Wheat faces various types of biotic and abiotic stresses. It has been suggested that *phenylalanine ammonia-lyase (PAL)* genes are essential for plant growth, development, adaptation, and mitigation responses to various environmental and pathogens stresses by producing secondary metabolites regulating plant growth response [11,58,59]. Phenylpropanoids are plant-based organic compounds, which are produced from the amino acids phenylalanine and tyrosine. PAL serves as the first enzyme in the phenylpropanoid pathway and in flavonoid biosynthesis that catalyzes the deamination of phenylalanine [1,24,45,60,61]. Recently, these enzymes have been reported by many researchers in different crops, including *Juglans regia* [62], *Citrus reticulata* [63], *Citrullus lanatus* [64], and *Medicago truncatula* [65]. This study was an investigation of *PAL* in wheat.

The *PAL* family is a very large, multigene family. The family includes ten putative members in maize [66], four members in *Arabidopsis* [19] and tobacco [67], and more than 20 copies in tomato and potato [68]. In the present study, we demonstrated that common wheat (*Triticum aestivum*) has 37 genes of the *PAL* family, a significantly higher number than the above-mentioned species. However, the increase and decrease of *PAL* genes present among species (*Z. mays*, *A. thaliana*, and *O. sativa)* is random [6]. Our results showed that the number of *PAL* genes in *T. aestivum* far exceeds the four *AtPALs*, seven *OsPALs*, twelve *JrPALs*, and six *ZmPALs*, suggesting that whole-genome duplication, small-scale segmental duplications, local tandem duplications, or a combination of these duplication events may have caused this expansion in *T. aestivum* [7,69,70]. The duplicated *PAL* genes in this study were mapped to 11 chromosomes (Figure 1). This diversity of chromosomal distribution indicates that these genes have diverse function. The duplication events might have caused the expansion and dispersion of *PAL* genes giving rise to potential sources of functional variability in common wheat. Gene duplication events may have caused the significant increase in *PAL* genes in *T. aestivum*, as stated in recent studies on different species [16,42,65]. The isolation and identification of *PAL* genes in *T. aestivum* is critical because of their importance in adaption and stress resistance [1,71,72]. The activity of *PAL* genes in response to cold stress of *Juglans regia* (walnut) suggested that the *PAL* gene family in *T. aestivum* is also involved in providing resistance against cold, drought, salt, and disease [70,72]. Similarly, this study also indicates that the expression of *TaPAL* genes is higher in drought-tolerant wheat genotypes as compared to sensitive genotypes. Furthermore, we also checked the subcellular location of TaPAL. Our results showed that the 37 PAL genes are localized to the cytoplasm [62,73,74].

Conserved motifs referred to a part of proteins that is functionally important. The motifs were selected from the PLACE database and conservation patterns were retrieved from MEME suite (Figure 2 and Table 3) and UGENE depicted that the protein structure of the *PAL*-gene family has been highly conserved. The *PAL*-gene family, including *Z. mays*, *A. thaliana*, *O. sativa*, *H. vulgare*, and *T. urartu* plant species, contained all the conserved domains indicating that the *PAL*-gene family remained highly conserved during evolution and took long-term speciation and duplication events to evolve; thus, the results demonstrated its importance in antiretroviral effects. It was evident that the key domain is phenyl ammonium lyase/aromatic lyase, which exists in all families and ancestral species, suggesting a structural similarity between proteins of the *PAL* gene family.

The intron–exon gene structure gives clues for gene evolution [10]. In parallel to the gene number, the structure of the *TaPAL* genes in *Triticum aestivum* has experienced developmental/evolutionary modifications. Out of 37 *TaPAL* genes, ten *TaPAL* genes (*TaPAL3*, *TaPAL5*, *TaPAL8*, *TaPAL18*, *TaPAL19*, *TaPAL24*, *TaPAL27*, *TaPAL35*, *TaPAL36*, and *TaPAL37*) have no intron in their coding regions, two of the *TaPAL* genes (*TaPAL25*, and *TaPAL13*) are interrupted by two introns in their ORFs, while 25 *TaPAL* genes have one intron in their ORFs (Figure 3). Recent studies stated that the duplicated genes showed structural divergence, which is very prevalent in the generation of functionally distinct paralogs. This structural divergence has played a key role in the evolution of duplicated genes compared to non-duplicated genes [69]. The *PAL-*gene structural-data analysis showed a significant variation in the evolution of the *PAL* family of common wheat, walnut, and poplar.

For the functional prediction of *TaPAL* genes we did the GO enrichment analysis (Figure 4). In silico prediction indicated that *TaPAL* genes were involved in numerous developmental processes by regulating biological processes (BPs), molecular processes (MPs), and cellular process (CPs), and showed response against environmental stresses. Many previous studies also reported that microRNAs respond to stress stimuli through regulation of gene expression [42]. *TaPAL* is highly expressed in roots as compared to shoot tissues against abiotic stress. The miRNAs tae-miR1119, tae-miR398, tae-miR444a, tae-miR444b, and tae-miR9664-3p targeting *TaPAL29* have high expression in root tissues (Figures 5 and 10). Previously it has been reported that plant miRNAs play a role in response to environmental stress. In bread wheat under the drought stress, different miRNAs such as miR159, and miR395 were found to be differentiated [75]. Similarly, *VM-* *milR37* plays role in pathogenicity through regulation of the *VmGPX* gene [76]. In another study, miR164 regulated the salinity tolerance in maize [77]. We also checked the protein– protein interaction of TaPAL29 with other co-regulated proteins. Results showed that arogenate dehydratase belongs to the class lyases and is a key enzyme that catalyzes the reaction of L-arogenate into L-phenylalanine [78] and shows interaction with the TaPAL29 (Figure 6).

Phylogenetic analysis, both with ancestral and family species, proposed that the evolution trajectories are like family species (*Z. mays*, *A. thaliana*, and *O. sativa)* and suggested that the *PAL* gene family converge to a single ancestor. This ancestor might be involved in the evolution of plants with respect to adaptation and resistance. Previously it has been reported that during the evolution of PAL, lineage-specific duplication (to promote the diversity of multi-gene families) occurs in *Arabidopsis* and other species [79]. The close paralogs of each PAL gene clustered together phylogenetically into clades in *T. aestivum*, *A. thaliana*, *O. sativa*, and *Z. mays* (Figure 7). In contrast, the PALs from *T. aestivum* and *Z. mays* clustered together along with some of the *O. sativa* genes (*OsPAL1*, *OsPAL5*, *OsPAL6*, and *OsPAL8*), indicating that the expansion of the common wheat PAL gene family might have occurred after the divergence of eurosids I and eurosids II (approximately 100 million years ago) which was reported by [62,80]. Based on phylogenetic analysis, our 37 *TaPAL* genes were separated into three different groups as in tea plant (*Camellia sinensis)* [79] and in other woody plants (*Juglans regia* L., *Salix babylonica*, *Ornithogalum saundersiae*, and *Populus trichocarpa*) they cluster into two groups [18,21,42,81]. TaPALs showed no expansion events as in *Cucumis sativus* [26]. The *PAL* gene family has significant similarities and dissimilarities among various plant species, i.e., *ZmPAL3-5* and *OsPAL2-4*. Among *TaPAL* genes, *TaPAL13*, *TaPAL31*, *TaPAL36*, and *TaPAL37* showed a slight difference in sequence as compared to other 33 *PAL* genes of *T. aestivum* (common wheat), which indicated an 80% similarity score in syntenic analysis. This relationship demonstrated that PALs with comparable evolutionary status might play a similar role in plant development, which enabled us to examine the elements of PALs from different families such as Poaceae via utilizing a comparative genomic approach.

*PAL* gene is strictly involved in controlling the pre- and post-transcriptional stages, which is considered a doorway to the initiation of the phenylpropanoid pathway. Differential expression patterns for *PAL* genes in higher plants was observed. Moreover, the *PAL* genes in common wheat (*T. aestivum*) show distinct patterns of expression in roots. The genes *TaPAL11*, *TaPAL14*, *TaPAL12*, *TaPAL29*, *TaPAL20*, *TaPAL7*, *TaPAL1*, *TaPAL2*, *TaPAL9*, *TaPAL15,* and *TaPAL16* exhibited high expression levels in roots of drought-tolerant genotypes as compared to drought-susceptible genotypes (Figure 9). These variations in expression level were attributed to the differences in proteins and gene structures, as shown in Figures 2 and 3. The *PAL* family genes showed diverse expression patterns, which indicated that a complex regulation of the PAL-mediated phenylpropanoid pathways existed during the development of drought-tolerant and drought-sensitive wheat genotypes (Figure 9). A similar expression pattern of the *PAL* gene family has also been reported in walnut and barrel clover [62,82]. *Cis*-regulatory elements are also present upstream of the TaPALs (Table 3). Some of the TaPALs from the same evolutionary cluster co-express under stress conditions. This might be due to the presence of *Cis* elements [16]. Similarly, *GdPAL5* is also reported to be an auxin producer which activates plant defense mechanisms during the abiotic stress [83]. Different gene family members usually display abundance disparities in different tissues or under distinct stresses [84].

To overcome the problem of changing climatic conditions of abiotic stress including heat and drought stress on wheat, there is a need to explore the transcriptome profile of this gene family. This study used transcriptomic information of various tissues, at various stages, as shown in Figure 10. The transcript levels of *TaPAL37*, *TaPAL36*, *TaPAL35*, *TaPAL33*, *TaPAL29*, *TaPAL25*, *TaPAL24*, *TaPAL17*, *TaPAL14*, *TaPAL11*, *TaPAL7*, *TaPAL3*, and *TaPAL4* were upregulated in roots. The expression levels of *TaPAL* genes were consistent with previous studies, showing that expression of *TaPAL* genes is higher in roots as compared to

other tissues of plants such as *Hordeum vulagare* [85], *Solanum tuberosum* [86], *Arabidopsis thaliana* [19], and *Juglans regia* [87]. The higher expression of the *TaPAL* gene family in drought-tolerant genotypes as compared to drought-sensitive genotypes may be due to high level of lignification, which is part of normal root development [88]. Furthermore, publicly available transcriptomic data which we used was validated by qRT-PCR [89,90].
