Transcriptional Regulation of Genes Involved in Zinc Uptake, Sequestration and Redistribution Following Foliar Zinc Application to Medicago sativa

Abstract

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 (Medicago sativa 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 (MsZIP1-7, MsZIF1, MsMTP1, MsYSL1, MsHMA4, and MsNAS1) 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 Medicago truncatula (i.e., MtZIP1-7 and MtMTP1). Shoot and root Zn concentration was increased following foliar Zn applications ≥ 0.1 mg plant^-1. Increased expression of MsZIP2, MsHMA4, and MsNAS1 in shoots, and of MsZIP2 and MsHMA4 in roots was observed with the largest Zn dose (10 mg Zn plant^-1). By contrast, MsZIP3 was downregulated in shoots at Zn doses ≥ 0.1 mg plant^-1. 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.

Keywords: ZIP transporters; heavy metal transporters (HMA); metal tolerance protein (MTP); nicotianamine; yellow stripe-like protein (YSL); zinc-induced facilitators (ZIF).

Figure 1

Suggested model for roles of putative genes encoding proteins involved in Zn transport- related processes. The sites of action in the plant (i.e., root cytoplasm, rc; root vacuole, rv; xylem and apoplast, X/A; phloem, P; leaf cytoplasm, lc; leaf vacuole, lv) and the element (E) fluxes (K_1–13) are reported. The concentration of the element is indicated in each site [E]. The scheme synthetizes information across studies in various plants. Gene abbreviations: ZIP, Zrt-/Irt-like protein; NAS, nicotianamine synthase; ZIF, zinc-induced facilitator; MTP, metal transporter protein; HMA, P1B-type heavy metal ATPase; YSL, yellow stripe like protein; ZIP? indicates a generic ZIP; free diffusion: diffusion through leaf epidermis; stomata: absorption through stomata. Plant abbreviations: Mt, Medicago truncatula; At, Arabidopsis thaliana; Os, Oryza sativa. References: [29,30,41,46,47,48,49,50,51,52,53,54].

Figure 2

Zinc concentration (a) and content (b) in shoots and roots of alfalfa after the application of Zn to leaves. The Zn doses were 0, 0.01, 0.1, 0.5, 1, or 10 mg Zn plant⁻¹. Means ± standard error of three replicates are shown. Differences among the applied Zn doses were tested separately for shoot and root by one-way analysis of variance. Different letters denote significant differences in Zn concentrations in shoots and roots independently, according to Tukey-B honestly test (p < 0.05).

Figure 3

Relative expression of transmembrane Zn transporter genes after leaf Zn application to alfalfa. The Zn doses were 0, 0.1, 1, or 10 mg Zn plant⁻¹. Means ± standard error of three replicates are shown. The expression levels were calculated relative to reference genes (MsACT-101 for shoot and MsEF1-α for root) and to the control (0 mg Zn plant⁻¹). The broken line denotes the threshold between up- and downregulation relative to the control. Differences in the expressions of each gene after different Zn doses were tested separately for shoot and root by one-way analysis of variance. Different letters denote significant differences among Zn doses, according to Tukey-B test (p < 0.05).

Figure 4

Relative expression of genes related to Zn transport processes after leaf Zn application to alfalfa. The Zn doses were 0, 0.1, 1, or 10 mg Zn plant⁻¹. Means ± standard error of three replicates are shown. The expression levels were calculated relative to reference genes (MsACT-101 for shoot and MsEF1-α for root) and to the control (0 mg Zn plant⁻¹). The broken line denotes the threshold between up- and downregulation relative to the control. Differences in the expression of each gene at the different Zn doses were tested separately for shoot and root by one-way analysis of variance. Different letters denote significant differences among Zn doses, according to Tukey-B test (p< 0.05).

Figure 5

Heatmaps reporting correlations between expression of genes related to Zn transport after foliar Zn application. Gene expression is calculated as the difference between 0.1, 1, or 10 mg Zn plant⁻¹ and a control of 0 mg Zn plant⁻¹. The similarity in the degree of correlation in fold-change of gene expression to Zn application relative to the control was based on the average linkage clustering of the Pearson correlations (r). In the clustering trees, the genes are indicated in brown for roots and in green for shoots, while the ranks of correlations of the heatmap are indicated by color intensity (r 0 to 1: from low to strong intensity of green). Seven genes encoding transmembrane Zn transporter (MsZIP1-7) (a); four genes encoding cellular Zn transporters (including vacuolar transporters) (MsZIF1, MsHMA4, MsYSL1, and MsMTP1) and a gene encoding a nicotianamine synthase (MsNAS1) (b).