*2.3. Conserved Domain and Motif Analysis*

All the *VIT* and *VTL* genes were found to have the typical CCC1-like superfamily domains of yeast, which were demonstrated earlier for the iron and manganese transport from the cytosol to vacuole. Motif analysis using MEME webserver suggested that motifs 6, 9, and 10 are VIT specific with exceptions for motif 10 been absent in TaVIT3 and motif 6 absent in TaVIT3-7A. Similarly, motifs 5, 7, 8, 11 to 14 are VTL specific with the exception that motif 5 was absent in TaVTL5-2B\_3 and TaVTL5-2B\_5, where motif 7 was specific for TaVTL5 sequences except in TaVTL5-2A\_3, TaVTL5-2B\_6, TaVTL5-2D\_3. Motif 8 was specific for TaVTL1, 2 and 4. Motif 11 for TaVTL1 and 2. Motifs 12 and 13 were unique for TaVTL2, whereas Motif 14 was present only in TaVTL1 (Figure 3, Table S3).

**Figure 3.** Conserved motifs identified for TaVIT and TaVTL proteins usingMEME suite 5.1.0. The colored rectangles on each sequence represent specific conserved motifs numbered 1 through 14, as depicted by the color codes in the box.

### *2.4. Expression of Wheat VTL Genes under Fe Deficiency and Surplus Condition*

To check the regulation of *VTL* genes at the transcriptional level, the promoters for the wheat *VTL* genes were scanned for the cis-elements responsive for Fe and heavy metals. The analysis revealed the presence of multiple such sequences, including iron-deficiency-responsive element 1 (IDE1), metal response element (MRE), heavy metal responsive element (HMRE) and iron-related bHLH transcription factor 2 (IRO2) binding site (Table S4). In the most abundant category, iron-deficiency-responsive element 1 (IDE1) was predominant. Interestingly, the IRO2 binding site was present only in the regulatory region of *TaVTL2B*/*D*. These observations sugges<sup>t</sup> that *VTL* expression could be regulated by the presence-absence of specific metals including micronutrients such as Fe and Zn.

Previously, *VTL* genes were reported to have differential expression patterns under the changing regimes of Fe and Zn [31]. Therefore, we tested if wheat *VTL* genes could respond at the transcript level when subjected to changing Fe concentration. The expression in roots and shoots of wheat seedlings was measured after subjecting them for three and six days of starvation. Our expression analysis suggests that in roots all the *VTL* genes (*TaVTL1, TaVTL2, TaVTL4* and *TaVTL5*) were downregulated at both the days, whereas, only *TaVTL5* was upregulated at six days of starvation (Figure 4A).

**Figure 4.** Tissue-specific qRT-PCR expression analysis of wheat VTL genes during Fe deficiency (-Fe) and in the control (C) conditions. Wheat seedlings were subjected to Fe deficiency for three and six days represented as -Fe(3D) and -Fe(6D). The controls for the respective time points are represented as C(3D) and C(6D). (**A**) Fold expression analysis was performed in roots and (**B**) in shoots. 2 μg of total RNA for the cDNA preparation. Relative fold expression levels were calculated relative to C(3D). Ct values were normalized using wheat *ARF1* as an internal control. Vertical bars represent the standard deviation. # represents the significant difference at *p* < 0.05 with respect to their respective control treatments.

Similarly, in shoots also all the expression of wheat *VTL* genes was suppressed except for *TaVTL2* that was upregulated only on six days post starvation (Figure 4B). These expression data demonstrate that under Fe deprivation *VTL* gene expression are negatively regulated in wheat seedling. Transcriptomic sequencing data from wheat seedlings after 20 days of Fe starvation (SRP189420) were also used to check expression response upon Fe starvation. Categorically, *TaVTL5* group genes were seen to be upregulated upto 12-fold, with *TaVTL5-2B\_6* showing upregulation of ~60 fold, although the expression was not very high (Figure S3).

Next, we performed the gene expression analysis under the excess Fe regime. This was done to test if wheat *VTL* genes could be potentially involved in detoxification of excess Fe. Interestingly, we observed a significant up-regulation of all the *TaVTL* genes in roots at both the time points. Out of all, *TaVTL4* showed the highest fold gene expression (~100 fold) when compared to its control (Figure 5A).

**Figure 5.** Tissue-specific qRT-PCR expression analysis of wheat VTL genes during Fe surplus (+Fe) and in the control (C) conditions. Wheat seedlings were subjected to Fe surplus for three and six days represented as +Fe(3D) and +Fe(6D). The controls for the respective time points are represented as C(3D) and C(6D). (**A**) Fold expression analysis was performed in roots and (**B**) in shoots. 2 μg of total RNA for the cDNA preparation and relative fold expression levels were calculated relative to C(3D). Ct values were normalized using wheat *ARF1* as an internal control. Vertical bars represent the standard deviation. # represents the significant difference at *p* < 0.05 with respect to their respective control treatments.

*TaVTL2* show very early and high expression response, whereas both *TaVTL1* and *TaVTL5* were highly expressed at six days of treatment. At this time their gene expression level was more than ~14 fold compared to control. In contrast, in shoots most of the wheat *VTL* genes were expressed at the three days of treatment with *TaVTL1* and *TaVTL2* showing the transcript accumulation of 8–14 folds with respect to their control (Figure 5B).

### *2.5. Manganese, Zinc and Copper Deficiency Causes Di*ff*erential Changes in VTL Expression*

Wheat *VTL* genes showed high similarity to previously known *VIT* genes. In addition to Fe, *VIT* genes are known to be affected by the perturbed concentration of Mn [14]. Since many of these cation transporters are known for their reduced substrate specificity [34,35], therefore, expression of wheat *VTL* genes during Zn, Cu and Mn deprivation was also studied (Figure S4A). In general, during the changing regimes of Zn and Mn, wheat *VTL* genes showed specific expression in a tissue-specific manner (Figure 6). *TaVTL2* was the only gene showing enhanced accumulation of its transcript under Zn deficiency in both root and shoot tissue, whereas *TaVTL1* and *TaVTL5* showed high expression in roots only under Zn deficiency (Figure 6A and B). No significant changes in the expression of *TaVTL4* were observed for the studied time point under the changing Zn regime. In contrast, no induction of wheat *VTL* genes was observed in roots under Mn deficiency with respect to its control, whereas, in shoots, *TaVTL2* and *TaVTL4* showed high transcript accumulation (Figure 6A). Under Cu deficiency, all *VTL* genes showed an induced expression in shoots while only two of the genes, including *TaVTL1* and *TaVTL2* were upregulated in roots.

**Figure 6.** Tissue-specific qRT-PCR expression analysis of wheat *VTL* genes during Mn (-Mn), Zn (-Zn) and Cu (-Cu) deficiency with respect to the control (C) conditions. (**A**) Fold expression analysis was performed in roots and (**B**) in shoots. 2 μg of total RNA was used for the cDNA preparation and relative fold expression levels were calculated relative to control tissue (C). The Ct values were normalized using wheat *ARF1* as an internal control. Vertical bars represent the standard deviation. # represents the significant difference at *p* < 0.05 with respect to Control tissue.

### *2.6. Heavy Metal (Ni, Cd and Co) Mediated Expression of VTL Genes*

To check the effect of the heavy metal stress on the gene expression pattern, wheat seedlings were subjected to treatment with Ni, Cd and Co and expression of *VTL* genes was performed. In the treated plants, decreased growth of the shoot and root length was observed, suggesting that heavy metals could affect the plant performance (Figure S4A). In general, the presence of heavy metals led to significant retardation in the growth of roots and shoots, thereby impacting the total plant growth (Figure S4B). Interestingly, none of the wheat *VTL* genes showed enhanced expression in roots after 15 days of heavy metal exposure, but downregulation was observed for *TaVTL1*, *TaVTL4* and *TaVTL5* (Figure 7A). In shoots, only Co stress could influence the gene expression when compared to the control. Only, *TaVTL2*, *TaVTL4* and *TaVTL5* genes were upregulated during the Co stress as compared to control shoot samples (Figure 7B). Altogether, this suggests the metal-specific expression of *VTL* genes in a tissue-specific manner. The previously reported wheat *VIT* genes showed grain specific expression data. Surprisingly, *VTL* genes showed very low or no expression in grains or their tissue parts, suggesting their probable roles in the specific organs of the plants (Figure S5).

**Figure 7.** Tissue-specific qRT-PCR expression analysis of wheat *VTL* genes upon heavy metal treatments. Wheat seedlings were exposed to Ni (+Ni, 50 μm), Cd (+Cd, 50 μm) and Co (+Co, 50 μm). Control seedlings (C) without any exposure to heavy metals were compared with the treated ones. (**A**) Fold expression analysis was performed in roots and (**B**) in shoots. 2 μg of total RNA for the cDNA preparation and relative fold expression levels were calculated relative to control samples. Ct values were normalized using wheat *ARF1* as an internal control. Vertical bars represent the standard deviation. # represents the significant difference at *p* < 0.05 with respect to their respective control treatments.
