| Summary: | Zinc (Zn) is an essential micronutrient for plants and animals, and Zn deficiency is a widespread problem for agricultural production. Although many studies have been performed on biofortification of staple crops with Zn, few studies have focused on forages. Here, the molecular mechanisms of Zn transport in alfalfa (<i>Medicago sativa</i> L.) were investigated following foliar Zn applications. Zinc uptake and redistribution between shoot and root were determined following application of six Zn doses to leaves. Twelve putative genes encoding proteins involved in Zn transport (<i>MsZIP1-7</i>, <i>MsZIF1</i>, <i>MsMTP1</i>, <i>MsYSL1</i>, <i>MsHMA4</i>, and <i>MsNAS1</i>) were identified and changes in their expression following Zn application were quantified using newly designed RT-qPCR assays. These assays are the first designed specifically for alfalfa and resulted in being more efficient than the ones already available for <i>Medicago truncatula</i> (i.e., <i>MtZIP1-7</i> and <i>MtMTP1</i>). Shoot and root Zn concentration was increased following foliar Zn applications ≥ 0.1 mg plant<sup>−1</sup>. Increased expression of <i>MsZIP2</i>, <i>MsHMA4</i>, and <i>MsNAS1</i> in shoots, and of <i>MsZIP2</i> and <i>MsHMA4</i> in roots was observed with the largest Zn dose (10 mg Zn plant<sup>−1</sup>). By contrast, <i>MsZIP3</i> was downregulated in shoots at Zn doses ≥ 0.1 mg plant<sup>−1</sup>. Three functional gene modules, involved in Zn uptake by cells, vacuolar Zn sequestration, and Zn redistribution within the plant, were identified. These results will inform genetic engineering strategies aimed at increasing the efficiency of crop Zn biofortification.
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