*2.7. Protein–Protein Interaction*

Protein–protein interactions of wheat were analyzed by using the STRING online server (http://string.embl.de, accessed on 14 September 2021) with the default setting [47].

#### *2.8. Analysis of RNA-Seq Base expression profiling*

Six different wheat varieties (Table 1) were used to analyze *PAL* RNA-seq base expression profiling of the *TaPAL* genes. All the wheat varieties were grown under normal conditions at the National Agricultural Research Centre (NARC), Islamabad, Pakistan. The root samples (each data point pooled from eight plants) were collected as previously described [48,49] from 35-day-old seedlings of all six wheat varieties and were frozen in liquid nitrogen prior to storage at −80 ◦C until use. Total RNA of the aboveprepared samples was isolated using the Gene JET™ Plant RNA Purification Mini Kit (Catalog # K0801). Illumina HiSeq2500 platform was used for paired-end (PE) sequencing of wheat RNA samples. The quality of raw data was checked with the help of FastQC (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/, accessed on 25 June 2021). Trimming of reads (quality scores < 20) was done with the help of the Trimmomatic tool [50]. The HISAT2 (version 2.0.5) (http://ccb.jhu.edu/software/hisat2/faq.shtml, accessed on 28 June 2021) tool with default settings [51] was used for constructing a transcriptome map based on the genome reference of wheat (ftp://ftp.ensemblgenomes.org/pub/release-25 /plants/fasta/triticum\_aestivum/dna/, accessed on 4 July 2021). The transcripts were assembled with String Tie software [52], while the NOIseq package was used to find the expression level of genes and transcripts and to draw the graph of the genes. The NOIseq package [53] was used to calculate the FPKM (fragments per kilobase per million) mapped. The genes with FPKM values greater than one were retained for subsequent analyses.

**Table 1.** Wheat genotypes used for RNA-seq analysis.


The expression levels were also analyzed at different stages in root and shoot tissues in response to abiotic stress (drought, heat, combination of both, Supplementary Sheet S1). The RNA-seq data was retrieved in transcripts per million (TPM) from the expVIP [54] wheat expression browser (http://www.wheat-expression.com/, accessed on 6 August 2021). To check the expression patterns of a given *PAL* gene subjected to abiotic stress, the ratio of the expression under treatment to the control was calculated (ratio ≥ 1 = altered under stress; ratio ≤ 1 = un-altered under stress). Finally, a heatmap was constructed by R package pheatmap (version 1.7) [55].
