*3.10. In Silico Expression Profile Analysis of PAL Gene Family in Six Genotypes of Wheat*

Gene expression analysis helps to probe the potential role and functions of a gene family [57]. Comparative gene expression analysis was used to elucidate the physiological function of different *PAL* gene family members. The in-silico expression profiling was done on the roots of different wheat genotypes (Figure 9). For this purpose, RNA-seq-normalized data were analyzed and based on FPKM values, a heatmap was constructed for diverse *TaPAL* genes. The expression of *TaPAL* genes was variable in different wheat genotypes. *TaPAL35*, *TaPAL31*, *TaPAL23*, *TaPAL22*, *TaPAL8*, *TaPAL5*, and *TaPAL6* were only expressed in Local White. Very low expression of *TaPAL10*, *TaPAL30*, *TaPAL32*, *TaPAL3*, and *TaPAL28* were recorded in all wheat genotypes. Nonetheless, they may have tissue-specific expression, such as in seeds, or their expression may be induced only by certain environmental stresses. *TaPAL11*, *TaPAL14*, *TaPAL12*, *TaPAL34*, *TaPAL4*, *TaPAL21*, *TaPAL19*, *TaPAL24*, and *TaPAL36* were highly expressed in UZ-11-CWA-8. Similarly, *TaPAL27*, *TaPAL16*, *TaPAL9*, and *TaPAL15* were highly expressed in Chakwal-50. Overall, *TaPAL* genes showed a higher expression

pattern in roots of drought-tolerant genotypes as compared to drought-sensitive genotypes of wheat.

**Figure 9.** Heatmap of expression pattern of *TaPAL* genes in roots of different wheat genotypes based on FPKM values. Cluster grouping shown as in Figure 7. Color scheme showing the intensity of expression (blue, low expression; red, high expression, and Z score was used).

### *3.11. Expression of PAL Gene Family under Abiotic Stress*

The expression of *TaPAL* genes under abiotic stresses such as drought (DH), heat stress (HS), and phosphorous deficiency (PS) at various stages and in various tissues were explored (Figure 10 and Supplementary Sheet S3). The expression levels of *TaPAL37*, *TaPAL36*, *TaPAL35*, *TaPAL33*, *TaPAL29*, *TaPAL25*, *TaPAL24*, *TaPAL17*, *TaPAL14*, *TaPAL11*, *TaPAL7*, *TaPAL3*, and *TaPAL4* were upregulated in roots. Similarly, the same trend was shown by *TaPAL10*, *TaPAL27*, *TaPAL15*, *TaPAL6*, and *TaPAL21* under drought stress, while *TaPAL23*, *TaPAL20*, *TaPAL19*, *TaPAL2*, and *TaPAL13* were regulated non-significantly under all conditions.

**Figure 10.** Expression profile of *TaPAL* genes under abiotic stresses. Cluster grouping shown as in Figure 7. Expression values are represented by color scale. Red and blue colors represent the gene expression of up-regulated and down-regulated genes, respectively; yellow shows expression was unregulated. Details are presented in Supplementary Sheet S1.
