**3. Discussion**

Fluctuation in the nutrient availability in the soil results in suitable adaptations by the plants. In general, plants rely on different physiological and molecular processes to minimize nutrient stress [36]. In this regard, MFS gene family plays an important role to provide the tolerance as well as mobilization of important minerals, including micronutrient translocation to the foliar parts including seeds [19]. In this study, the characterization of VTL was done in the hexaploid wheat. Our data reinforce the importance of *VTL* genes for their roles during metal homeostasis and substantiated them as a good candidate for micronutrient biofortification in cereal crops such as wheat and rice.

MFS family has been widely explored for its role as metal transporters and providing the necessary support for multiple functions in plants [37]. Previously, five *VTL* genes were reported in *Arabidopsis* and rice for this sub-class. Our study in wheat resulted in the identification of a maximum number of *VTL* genes from any crop plants. The high number of genes is due to the presence of multiple homoeologous and occurrence of the duplication of multiple wheat *VTL* genes. Interestingly, chromosome 2 having the highest number of wheat *VTL* genes has been linked with multiple quantitative trait loci (QTL) for the high grain Fe and Zn content [38]. Further dissection is required in this direction to identify if any of the wheat VTL could be linked with the loading of micronutrient in grains. Based on our expression analysis and the support from the previous studies, it could be suggested that *VTL* genes could also be involved in providing the tolerance to high levels of Fe and Zn in the soils [31]. In fact, the predicted localization data indicate that VTL could be localized at either the plasma membrane or the vacuolar membrane (Table S2). AtVTL1 was reported to be localized in the vacuolar membrane and others been associated with the plasma membrane [31]. Our PSORT analysis suggests that most of the wheat VTL proteins are localized in the vacuolar membrane, thus making them a suitable candidate

for sequestering micronutrients such as Fe and Zn. AtVTL1 also rescued Δccc1 function in yeas<sup>t</sup> by catalysing Fe uptake [31]. Vacuoles are the prime sites for the sequestration of micronutrients such as Fe. Whether, any of these predicted vacuolar TaVTL proteins could perform similar function as TaVIT2 for Fe biofortification needs to be studied [14].

The substrate specificity of the metal transporters is a major bottleneck to achieve high Fe and Zn in grains. Manipulating the specificity of these metal transporters to enrich the Fe and Zn remains the major challenge [23,34,39]. Therefore, studying the expression pattern of *VTL* genes in the presence of heavy metals could provide preliminary clues for employing such strategies. Consequently, the study was undertaken to see the influence of other metals like Ni, Cd and Co. The expression of wheat *VTL* genes in roots and shoots suggested an interesting phenomenon, where no significant changes in the expression of their transcript was observed when exposed to either Ni or Cd. In contrast, only Co was able to induce the expression of *TaVTL1*, *TaVTL2*, *TaVTL4* and *TaVTL5* in shoots only (Figure 7B). These data sugges<sup>t</sup> the controlled expression of wheat *VTL* genes in a tissue specific manner. Additionally, besides Fe homeostasis, the vacuolar transporters are also linked with the impaired activity of Zn and Mn transport [39]. In our study only, *TaVTL2* was significantly induced by the Mn deficiency in shoots (Figure 6B). No such effects were observed in roots wherein, all the quantified wheat VTL showed downregulation under Mn deficiency (Figure 6A). Interestingly, *TaVTL1* and *TaVTL2* showed upregulation in roots under Zn deficiency (Figure 6A). The tissue dependent expression patterns of wheat *VTL* genes under the changing regimes of the metal exposure was observed. It has been observed that *VTL* genes from *Arabidopsis* showed transcriptional changes in response to Fe, Zn and Mn [31]. Based on the previous work and our results it could be suggested that regulation of the *VTL* genes at the transcript level could be conserved. Infact, wheat *VIT* genes can also transport Mn and Fe [14]. This sugges<sup>t</sup> that *VTL*/*VIT* genes could be regulated only by Fe but also by other metals like Mn and Zn. Additionally, our gene expression data also corelate with the presence of multiple cis-elements in the promoter of wheat *VTL* genes. This indicated that primarily *VTL* genes could be involved during metal homeostasis related responses. Coupled with the localization information, it is possible that few wheat VTL proteins could sequester metals in an organelle specific manner.

Herein, a detailed inventory, structure and expression characterization of wheat *VTL* genes was performed. The expression analysis and analysis for the cis-elements in the promoters of wheat *VTL* genes implicated for their role in metals homeostasis including in Fe and Zn. Overall, the work presented here provide an important framework for identifying the molecular and physiological functions in bread wheat.
