*4.1. Plant Materials and Growth Conditions*

For stress experiments, hexaploid wheat *Triticum aestivum* cv. C-306 (received from Punjab Agriculture University, Ludhiana) was used. Briefly, seeds were surface sterilized using 1.2% sodium hypochlorite prepared in 10% ethanol and then rinsed twice with autoclaved MQ. The seeds were kept on moist filter paper inside a Petri dish and stratified for 1 day at 4 ◦C in dark condition. Stratified seeds were further kept for germination for six days at room temperature. The remaining seed/endosperm were excised from seedlings at one leaf stage and was shifted to phytaboxes (10–12 seedling/phytabox) containing the Hoagland nutrient media for respective treatments. The standard composition of nutrient media for control includes 6 mM KNO3, 1 mM MgSO4, 2 mM Ca(NO3), 2 mM NH4H22PO4, 20 μM Fe-EDTA, 25 μM H3BO3, 2 μM MnSO4, 0.5 μM CuSO4, 2 μM ZnSO4, 50 μM KCl and 0.5 μM Na2MoO4. The variable concentrations used for treatments were excess Fe (+Fe; 200 μm), Fe starvation (-Fe; 2 μm), Zn deficiency (-ZnSO4; 0 μm), Mn deficiency (-MnSO4; 0 μm), Cu deficiency (-CuSO4; 0 μm), Cadmium stress (+Cd; 50 μm) [40], Cobalt stress (+Co; 50 μm) [41] and Nickel stress (+Ni; 50 μm) [42]. The aerobic condition was provided in hydroponics and the media was replaced every alternate day to avoid any contamination and drastic nutrient depletion. The respective roots and

shoots samples belonging to iron deficient and su fficient plant groups were collected at three and six days after stress (D). For the rest of the treatments, root and shoot, samples were collected on the 15th Day of treatments. All the experiments were performed in a growth chamber under controlled environmental conditions at 22–24 ◦C temperature, 65%–70% humidity, at a photoperiod of 16 h day and 8 h night and 300 nm of light.

### *4.2. Identification of VIT Family and Classification of VTL Genes in Wheat*

For the identification of wheat *VTL* genes, the Ensembl database was used to extract *VIT* family genes (Pfam ID: PF01988) for wheat. The identification was confirmed by bidirectional BLAST analysis. VIT family sequences from Arabidopsis, rice, maize, *Brachypodium* were also extracted using Pfam search. The identity of VIT family genes was further validated by confirming the presence of CCC1-like superfamily domain using NCBI-CDD domain search. CCC1 sequence for *S. cerevisiae* was also retrieved from its genome database. To separate out *VTL* genes from *VIT* genes and for further phylogenetic analysis, all the proteins were aligned through MUSCLE alignment and an unrooted neighbor-joining phylogenetic tree with 1000 bootstrap replicates was constructed with all the retrieved sequences. The tree was constructed through MEGA-7 [43]. Rice vacuolar iron transporter homolog 1–5 from UniProt were used for the nomenclature of the 23 *TaVTL* sequences based on the closest orthologs. The naming of the genes indicates the chromosome number and the sub-genome on which they are present.

### *4.3. Conserved Domains and Motif Detection, Analysis of Gene, Promoter and Protein Structure*

Wheat VIT family genes were searched for conserved domains using NCBI-CDD database [44]. MEME suite v5.1.0 was used for further analysis to identify the common conserved motifs for both VIT and VTL proteins. The maximum number of motifs was set to 15 for MEME analysis. Gene structure for VITs and VTLs was studied using (GSDS) (http://gsds.cbi.pku.edu.cn/) [45] using genomic and CDS sequences. Sub-cellular localization and TM domains were predicted using web-based prediction programs Wolf PSORT and Phobius respectively [46]. For promoter analysis, ~2 Kb promoter elements of the corresponding wheat *VTL* genes were surveyed for the presence of the respective cis-elements. The promoter sequence was obtained for the respective genes using the IWGSC

### *4.4. Total RNA Isolation and cDNA Preparation*

The collected root and shoot samples were ground separately in liquid nitrogen. Total RNA from respective samples was extracted by TRIZOL based method. The extraction was followed by the DNase treatment using Turbo DNAfree kit (Invitrogen, Carlsbad, CA, USA) to remove any genomic DNA contamination in the RNA samples. Subsequently, RNA purity was checked and quantified for the preparation of the cDNA. 2 μg of total RNA was used for cDNA synthesis using superScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The cDNA quality was ascertained by using internal control and was further diluted 20X and used for gene expression studies.

### *4.5. Quantitative-Real Time PCR (qRT-PCR) Expression Analysis*

To perform quantitative real time-PCR (qRT-PCR), forward and reverse primers of *TaVTL* genes were designed and used as listed in Table S5. The primers were designed from the conserved region of the all homoeolog of each gene. For *TaVTL5* the primers were designed from the conserved region of nine sequences, the significant conserved region was not found for remaining four homoeologs (TraesCS2B02G454900, TraesCS2B02G610400, TraesCS2D02G431900, TraesCS2D02G588000). qRT-PCR was performed in 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using 1/20 times dilution of the respective cDNAs. All qRT-PCR reactions were performed using SYBR Green I (QuantiFast ® SYBR ® Green PCR Kit, Qiagen, Hilden, Germany) chemistry and ARF (ADP-Ribosylation Factor: *TaARF1*—AB050957.1) as an internal control [40]. The e fficiency of the qRT-PCR was checked and melt curve analysis was performed for each of the PCR reactions as per the guidelines. Gene expression analyses was carried out with three biological replicates and 2–3 technical replicates. Relative fold expression of genes was determined based on delta-delta CT-method (2ΔΔCT) [47].

### *4.6. RNA-Seq Expression Analysis for VIT Family Genes*

To ge<sup>t</sup> the transcript expression levels for VIT family genes under Fe stress, RNAseq data from SRA project ID SRP189420 were utilized to extract transcript expression values (as FPKM) from control as well as Fe starved wheat root samples using the cu fflinks pipeline. Subsequently, for expression analysis of *VTL* and *VIT* genes in wheat grain tissue developmental time course [48], expression values as Transcripts Per Kilobase Million (TPM) were retrieved from expVIP database [49]. Expression values from both studies were then used to plot heatmaps using MeV software (mev.tm4.org).
