Evaluation of constitutive iron reductase (AtFRO2) expression on mineral accumulation and distribution in soybean (Glycine max. L).

Marta W Vasconcelos,Michael A Grusak,Thomas E Clemente

doi:10.3389/fpls.2014.00112

Abstract

Iron is an important micronutrient in human and plant nutrition. Adequate iron nutrition during crop production is central for assuring appropriate iron concentrations in the harvestable organs, for human food or animal feed. The whole-plant movement of iron involves several processes, including the reduction of ferric to ferrous iron at several locations throughout the plant, prior to transmembrane trafficking of ferrous iron. In this study, soybean plants that constitutively expressed the AtFRO2 iron reductase gene were analyzed for leaf iron reductase activity, as well as the effect of this transgene’s expression on root, leaf, pod wall, and seed mineral concentrations. High Fe supply, in combination with the constitutive expression of AtFRO2, resulted in significantly higher concentrations of different minerals in roots (K, P, Zn, Ca, Ni, Mg, and Mo), pod walls (Fe, K, P, Cu, and Ni), leaves (Fe, P, Cu, Ca, Ni, and Mg) and seeds (Fe, Zn, Cu, and Ni). Leaf and pod wall iron concentrations increased as much as 500% in transgenic plants, while seed iron concentrations only increased by 10%, suggesting that factors other than leaf and pod wall reductase activity were limiting the translocation of iron to seeds. Protoplasts isolated from transgenic leaves had three-fold higher reductase activity than controls. Expression levels of the iron storage protein, ferritin, were higher in the transgenic leaves than in wild-type, suggesting that the excess iron may be stored as ferritin in the leaves and therefore unavailable for phloem loading and delivery to the seeds. Also, citrate and malate levels in the roots and leaves of transgenic plants were significantly higher than in wild-type, suggesting that organic acid production could be related to the increased accumulation of minerals in roots, leaves, and pod walls, but not in the seeds. All together, these results suggest a more ubiquitous role for the iron reductase in whole-plant mineral accumulation and distribution.

Highlights

Iron is one of the most important micronutrients in human and plant nutrition, and a suitable level of iron nutrition in plants is vital to providing adequate concentrations of this mineral in the harvestable plant organs for human food or animal feed
PLANT ORGAN WEIGHT Fourteen days after transfer to the hydroponics solution, WT and 392-3 plants appeared similar to each other, in terms of plant size and leaf color, at 100 μM Fe(III)-EDDHA (Figure 1). Both WT and 392-3 plants grown in the complete absence of iron were already showing severe signs of chlorosis (Figure 1); this treatment had to be discontinued for the analysis that involved growing the plants to full maturity (FM)
The lowest Fe supplies (10 and 32 μM) resulted in significant differences in root dry weight (DW) between the WT and 392-3 line, while no differences were seen between genotypes in the roots at the highest Fe supply of 100 μM Fe (III)-EDDHA (Figure 2)

Summary

Introduction

Iron is one of the most important micronutrients in human and plant nutrition, and a suitable level of iron nutrition in plants is vital to providing adequate concentrations of this mineral in the harvestable plant organs for human food or animal feed. Researchers have been interested in creating plant foods that are nutrient-dense (to guarantee“nutrient security”) in iron and other minerals (Carvalho and Vasconcelos, 2013), using an approach referred to as biofortification. In soybean, most biofortification strategies have aimed at increasing sulfur amino acids (Dinkins et al, 2001) and vitamins, such as α-tocopherol (Dwiyanti et al, 2011), and not at increasing mineral concentrations. To create crop plants with more minerals, researchers must boost their mobilization and uptake from the soil, improve their movement to the edible portions of the plant and enhance their storage in those tissues. It is essential to understand the relevant contributions of the mineral transport system throughout the plant as well as the regulatory system that controls it

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