Iron and Zinc in the Embryo and Endosperm of Rice (Oryza sativa L.) Seeds in Contrasting 2'-Deoxymugineic Acid/Nicotianamine Scenarios

Abstract

Iron and Zn deficiencies are worldwide nutritional disorders that can be alleviated by increasing the metal concentration of rice (Oryza sativa L.) grains via bio-fortification approaches. The overproduction of the metal chelator nicotianamine (NA) is among the most effective ones, but it is still unclear whether this is due to the enrichment in NA itself and/or the concomitant enrichment in the NA derivative 2'-deoxymugineic acid (DMA). The endosperm is the most commonly consumed portion of the rice grain and mediates the transfer of nutrients from vegetative tissues to the metal rich embryo. The impact of contrasting levels of DMA and NA on the metal distribution in the embryo and endosperm of rice seeds has been assessed using wild-type rice and six different transgenic lines overexpressing nicotianamine synthase (OsNAS1) and/or barley nicotianamine amino transferase (HvNAATb). These transgenic lines outlined three different DMA/NA scenarios: (i) in a first scenario, an enhanced NA level (via overexpression of OsNAS1) would not be fully depleted because of a limited capacity to use NA for DMA synthesis (lack of -or low- expression of HvNAATb), and results in consistent enrichments in NA, DMA, Fe and Zn in the endosperm and NA, DMA and Fe in the embryo; (ii) in a second scenario, an enhanced NA level (via overexpression of OsNAS1) would be depleted by an enhanced capacity to use NA for DMA synthesis (via expression of HvNAATb), and results in enrichments only for DMA and Fe, both in the endosperm and embryo, and (iii) in a third scenario, the lack of sufficient NA replenishment would limit DMA synthesis, in spite of the enhanced capacity to use NA for this purpose (via expression of HvNAATb), and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. Also, quantitative LA-ICP-MS metal map images of the embryo structures show that the first and second scenarios altered local distributions of Fe, and to a lesser extent of Zn. The roles of DMA/NA levels in the transport of Fe and Zn within the embryo are thoroughly discussed.

Keywords: laser ablation; ligands; mass spectrometry; metals; rice; seeds.

FIGURE 1

RNA blot analysis showing transgene expression in the leaf tissue of wild-type (WT) and six independent transgenic lines, two expressing OsNAS1 (lines N1 and N2), two expressing HvNAATb (lines D1 and D2) and two co-expressing OsNAS1 and HvNAATb (lines ND1 and ND2). rRNA, ribosomal RNA. Plants were grown under nutrient-sufficient conditions, and total RNA was isolated from flag leaves at physiological maturity.

FIGURE 2

Nicotianamine (NA) and 2′-deoxymugineic acid (DMA) concentrations (in μg g^-1 FW) in the embryo and endosperm of WT rice and six different transgenic lines, two expressing OsNAS1 (lines N1 and N2), two expressing HvNAATb (lines D1 and D2) and two co-expressing OsNAS1 and HvNAATb (lines ND1 and ND2). Plants (WT and T₃ transgenic lines) were grown under nutrient-sufficient conditions, and the WT and T₄ seeds were harvested at physiological maturity. Asterisks indicate significant differences between WT and transgenic plants as determined by Student’s t-test (P < 0.05). Values shown are means ± SE, n = 3–4. BLQ: below limit of quantification.

FIGURE 3

Micronutrient metal concentrations (μg g^-1 DW) in embryo and endosperm of WT rice and six different transgenic lines, two expressing OsNAS1 (lines N1 and N2), two expressing HvNAATb (lines D1 and D2) and two co-expressing OsNAS1 and HvNAATb (lines ND1 and ND2). Plants (WT and T₃ transgenic lines) were grown under nutrient-sufficient conditions. WT and T₄ seeds were harvested at physiological maturity. Asterisks indicate significant differences between WT and transgenic plants as determined by Student’s t-test (P < 0.05). Values shown are means ± SE, n = 3. BLQ: below limit of quantification.

FIGURE 4

Images of longitudinal dorso-ventral sections of a rice seed. Optical image describing the different structures: AL, aleurone layer; LP, leaf primordium; RP, root primordium; SC, scutellum; SE, starchy endosperm; EP, epithelium (A). Perl’s Prussian blue staining of WT rice seeds and three different transgenic lines, one expressing OsNAS1 (line N1), one expressing HvNAATb (line D2) and one co-overexpressing OsNAS1 and HvNAATb (line ND2) (B). Plants (WT and T₃ transgenic lines) were grown under nutrient-sufficient conditions. WT and T₄ seeds were harvested at physiological maturity.

FIGURE 5

Quantitative images obtained by LA-ICP-MS for Fe, Zn, Mn, Cu, P, and S distribution in the embryo and neighboring endosperm tissues for WT rice and six different transgenic lines, two overexpressing OsNAS1 (lines N1 and N2), two expressing HvNAATb (lines D1 and D2) and two co-expressing OsNAS1 and HvNAATb (lines ND1 and ND2). Plants (WT and T₃ transgenic lines) were grown under nutrient-sufficient conditions, and the WT and T₄ seeds were harvested at physiological maturity and dehusked. 60 μm-thick seed sections were used for LA-ICP-MS analysis. Elemental images were obtained processing LA-ICP-MS data with the software Origin^®. Color scales represent the concentrations for each element, with the lowest ones in dark blue and the highest ones in red. Depending on the element, the scale bar indicates the elemental concentrations in μg g^-1 (Fe, Zn, Mn, and Cu) and in mass fraction percentage (S and P). For a given element, the same scale was used in all genotypes. Pictures shown in the first row are optical images of the sections subjected to LA-ICP-MS analysis. AL, aleurone layer; LP, leaf primordium; RP, root primordium; SC, scutellum; SE, starchy endosperm; EP, epithelium.

FIGURE 6

High-resolution quantitative images obtained by LA-ICP-MS of Fe and Zn in the embryo and neighboring endosperm tissues of WT rice and three different transgenic lines, one expressing OsNAS1 (line N2), one expressing HvNAATb (line D2) and one co-expressing OsNAS1 and HvNAATb (line ND1). Quantitative images of Fe and Zn distributions were obtained processing LA-ICP-MS data with the software ImageJ^®. The scale bar indicates the elemental concentrations in μg g^-1, with the maximum in red and the minimum in dark blue. The concentration scale for each image is different. AL, aleurone layer; LP, leaf primordium; RP, root primordium; SC, scutellum; SE, starchy endosperm; EP, epithelium.

FIGURE 7

Simplified biosynthesis pathway of nicotianamine (NA) and 2′-deoxymugineic acid (DMA) (A) and a schematic representation of the accumulation and distribution of Fe and Zn between the embryo and endosperm of rice seeds in WT and in the three DMA/NA scenarios studied (B). Scenario 1 occurs (e.g., in N1, N2 and ND1) when an enhanced NA level is not fully depleted because of a limited capacity to use NA for DMA synthesis, resulting in consistent enrichments in NA, DMA, Fe, and Zn in the endosperm and NA, DMA and Fe in the embryo. Scenario 2 occurs (e.g., in ND2) when an enhanced NA level is depleted by an enhanced capacity use NA for DMA synthesis, resulting in enrichments only for DMA and Fe, both in the endosperm and embryo. Scenario 3 occurs (e.g., in D1 and D2) when the lack of sufficient NA replenishment limits DMA synthesis, in spite of the enhanced capacity to use NA for this purpose, and results in decreases in NA, variable changes in DMA and moderate decreases in Fe in the embryo and endosperm. NAS, nicotianamine synthase; NAAT, nicotianamine aminotransferase.