*2.4. Gene Structure and Conserved Motif Analysis*

In the Gene Structure Display server program (http://gsds.cbi.pku.edu.cn/) (accessed on 10 March 2022), genomic and CDS sequences of *TaTLP*s genes were used to create an exon/intron map [22]. The conserved motifs in the TUBBY proteins were discovered using the online server MEME 4.11.3 (http://meme-suite.org/tools/meme) (accessed on 10 March 2022) [23].

#### *2.5. Gene Ontology and Cis-Elements Analysis of TUBBY Family Genes*

A 1.5 Kb genomic DNA sequence upstream of each identified *TaTLP* gene's start codon was obtained from the Ensemble Plants database (http://plants.ensemble.org/Triticum\_ aestivum) (accessed on 11 March 2022) using the Ensemble Plants search engine (ATG). The online Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 11 March 2022) database was used to identify cis-regulatory elements for all the TUBBY genes. Ontology analysis of the *TaTLP* protein sequences was performed using the Blast2GO program Ver.2.7.2 (http://www.blast2go.com) (accessed on 11 March 2022), and the groups of GO classification (molecular functions, biological process, and cellular component) were documented.

#### *2.6. RNA Isolation and cDNA Synthesis*

Total RNA was isolated from stress-exposed seedlings at selected time points, including 0 (control) and stress using the RNeasy plant mini kit (Qiagen, Redwood City, CA, USA), as per the manufacturer's instructions. The quantity and quality of isolated RNA were determined by spectrophotometry (Nanodrop 2000, Thermo Fisher Scientific, Waltham, MA, USA) and formaldehyde-based gel electrophoresis, respectively. For cDNA synthesis,1 μg of total RNA was transcribed in 20 μL using Revert Aid First Strand cDNA Synthesis Kit (Fermentas Life Sciences, Waltham, MA, USA) using oligo (dT)primers as per the manufacturer's instructions.
