The expression of heterologous Fe (III) phytosiderophore transporter HvYS1 in rice increases Fe uptake, translocation and seed loading and excludes heavy metals by selective Fe transport

Abstract

Many metal transporters in plants are promiscuous, accommodating multiple divalent cations including some which are toxic to humans. Previous attempts to increase the iron (Fe) and zinc (Zn) content of rice endosperm by overexpressing different metal transporters have therefore led unintentionally to the accumulation of copper (Cu), manganese (Mn) and cadmium (Cd). Unlike other metal transporters, barley Yellow Stripe 1 (HvYS1) is specific for Fe. We investigated the mechanistic basis of this preference by constitutively expressing HvYS1 in rice under the control of the maize ubiquitin1 promoter and comparing the mobilization and loading of different metals. Plants expressing HvYS1 showed modest increases in Fe uptake, root-to-shoot translocation, seed accumulation and endosperm loading, but without any change in the uptake and root-to-shoot translocation of Zn, Mn or Cu, confirming the selective transport of Fe. The concentrations of Zn and Mn in the endosperm did not differ significantly between the wild-type and HvYS1 lines, but the transgenic endosperm contained significantly lower concentrations of Cu. Furthermore, the transgenic lines showed a significantly reduced Cd uptake, root-to-shoot translocation and accumulation in the seeds. The underlying mechanism of metal uptake and translocation reflects the down-regulation of promiscuous endogenous metal transporters revealing an internal feedback mechanism that limits seed loading with Fe. This promotes the preferential mobilization and loading of Fe, therefore displacing Cu and Cd in the seed.

Keywords: 2′ deoxymugenic acid; Rice; barley YS1 transporter; iron; metal transporters; toxic metals.

Figure 1

RNA blot analysis showing transgene expression in the leaf tissue of wild‐type (WT) and transgenic lines expressing HvYS1. rRNA: ribosomal RNA; HvYS1: barley yellow stripe 1 transporter.

Figure 2

Concentrations of Fe (μg/g DW) in roots (a), leaves (b), husks (c), unpolished seeds (d) and polished seeds (e) of wild‐type (WT) and T₂ generation transgenic lines expressing HvYS1 (lines 1, 2, 3, 4, 5). Asterisks indicate a statistically significant difference between wild‐type and transgenic plants as determined by Student's t‐test (P < 0.05; n = 6). DW: dry weight. Iron measurements in husk were taken from two representative transgenic lines.

Figure 3

Concentration of 2′‐deoxymugenic acid (DMA) and nicotianamine (NA) (μg/g FW) in roots, leaves and polished seeds of wild‐type (WT) and two selected T₂ generation transgenic lines expressing HvYS1 (lines 1, 2). Asterisks indicate a statistically significant difference between wild‐type and transgenic plants as determined by Student's t‐test (P < 0.05; n = 3). NA levels in the roots were below the detection limit. FW: fresh weight.

Figure 4

Concentrations of Zn (left), Mn (middle) and Cu (right), all in μg metal per g DW, in roots (a), leaves (b), husks (c), unpolished seeds (d) and polished seeds (e) of wild‐type (WT) and two selected T₂ generation transgenic lines expressing HvYS1 (lines 1, 2). Asterisks indicate a statistically significant difference between wild‐type and transgenic plants as determined by Student's t‐test (P < 0.05; n = 6). DW: dry weight.

Figure 5

Concentrations of Cd (top row) and Fe (bottom row), both in μg/g DW, in (a and d) roots, (b and e) leaves and (c and f) unpolished seeds of wild‐type (WT) and T₃ generation transgenic lines expressing HvYS1 (lines 1, 2, 3) supplied with 10 μm CdCl₂. Asterisks indicate a statistically significant difference between wild‐type and transgenic plants as determined by Student's t‐test (P < 0.05; n = 6). DW: dry weight.

Figure 6

The mechanistic basis of selective Fe transport by HvYS1. Heterologous expression of HvYS1 results in the selective uptake, translocation, remobilization and seed loading of Fe. Endogenous Fe homeostasis limits Fe accumulation in seeds to rather modest levels (i.e. twofold) by modulating the expression of endogenous genes controlling Fe uptake (1), remobilization (2) and seed loading (3), but this is sufficient to displace the toxic heavy metals Cd and Cu from the endosperm (4).