**1. Introduction**

Successful micronutrient biofortification of crops through biotechnology requires detailed knowledge of complex homeostatic mechanisms that tightly regulate the micronutrient concentrations in plants. Iron (Fe) is one of the important micronutrients that is involved in multiple important cellular and physiological processes in plants [1–3]. Some of the important functions include its importance in photosynthesis, nitrogen fixation and respiration [4,5]. Although Fe may be present in the soil, ye<sup>t</sup> due to alkaline rhizospheric conditions or unfavorable circumstances, it is not being efficiently taken up by plants [6–9]. Moreover, the Fe is mobilized through a multistep process that overcomes transport bottlenecks and eventually is loaded in the developing grains [10–13]. Researchers worldwide are utilizing multiple approaches to either enrich Fe in grains or their storage with enhanced bioavailability [14–17]. To improve Fe content in cereal grains, multiple transporters and chelators have been targeted through multiple molecular approaches [14,15,18]. A number of additional micronutrient transporters have been identified, those are good candidates for micronutrient biofortification, including transporters belonging to the major facilitator superfamily (MFS) gene family [19,20]. Limited evidences are available that have performed molecular characterization of wheat genes or gene families those which are specifically involved in Fe and Zinc (Zn) homeostasis. Recent reports are emerging for the identification of few functional gene families belonging to, yellow stripe like transporters [21], nicotianamine synthase (NAS), deoxymugineic acid synthase (DMAS) [22], ye<sup>t</sup> many genes families remained to be characterized in hexaploid wheat. Similarly, other wheat genes including Zinc–Induced Facilitator-Like Family (ZIFL) transporters have been characterized for their role in mobilizing the uptake of micronutrient such as Fe and Zn [23]. Most of these gene families are highly upregulated in roots subjected to Fe starvation conditions [24,25]. These works identify some of the important candidate genes as an important resource to strategize approaches for micronutrient biofortification in wheat [26].

Fe storage in seeds gets compartmentalized in major subcellular organelles including chloroplasts and vacuoles. For example, 95% of the iron is stored in vacuoles in the *Arabidopsis* seeds [27]. Vacuoles are an important site for Fe mobilization wherein, they are bound to various chelators like phytic acid, nicotianamine and other organic acids etc. Therefore, uptake of Fe into vacuoles could be an alternate strategy to enhance total micronutrient content with a minimized tradeo ff for its toxicity in the tissue. To design such strategy, the role of vacuolar transporters needs to be addressed and exploited [14,28]. Previously, vacuolar iron transporters (VIT) were shown to be play an important role in maintaining Fe in the optimal physiological range and prevent cellular toxicity [14]. *VIT* genes from multiple plant species have been characterized and assessed for their ability to enhance Fe content in cereal crops [15]. These *VIT* genes show high homology with a small family of nodulin like protein containing a CCC-1 (Ca<sup>2</sup>+-Sensitive Cross Complementer) like domain with yeas<sup>t</sup> CCC1p1 [29]. CCC-1 like the domain was initially discovered in yeas<sup>t</sup> encoded for the vacuolar iron transporter in yeast. Furthermore, mutant *ccc1* cells show increased sensitivity to external iron [27,30] *AtVIT1* is one of the early characterized genes showing the presence of CCC-1 like domain and transport of iron to vacuoles [27].

Utilizing the bioinformatics resources, subsequent studies led to the identification of many vacuolar iron transporters-like (VTL) proteins from di fferent plant species. Model species, *Arabidopsis* genome encodes five VTL proteins and overexpression of the few genes have shown increased Fe content in seeds. AtVIT1 protein can transport iron into the vacuoles to counter the toxicity and support the seedling development under enhanced iron conditions [29,31].

Wheat is an important crop that is consumed in many developing countries, including India and is therefore being targeted for trait improvement for nutritional quality. Therefore, the characterization of vacuolar transporters in an important crop such as wheat becomes a prerequisite to address the global issue of biofortification. In the current work genome-wide identification of wheat *VTL* genes was performed. Further, expression studies during di fferent regimes of Fe, Zn and multiple heavy metals was done to gain insight for the regulation of wheat *VTL* genes in a tissue-specific manner.
