Paralogs and mutants show that one DMA synthase functions in iron homeostasis in rice

Abstract

Rice (Oryza sativa) secretes 2'-deoxymugineic acid (DMA) to acquire insoluble iron (Fe) from the rhizosphere. In rice, DMA is synthesized by DMA synthase 1 (OsDMAS1), a member of the aldo-keto reductase super family. We screened OsDMAS1 paralogs for DMA synthesis. None of these paralogs displayed in vitro DMA synthesis activity, suggesting that rice only harbors one functional DMAS. We further characterized OsDMAS1 mutant plants. We failed to screen homozygous knock-out plants (dmas-1), so we characterized DMAS knock-down plants (dmas-kd1 and dmas-kd2). Under Fe-deficient conditions, dmas-kd1 plants were more chlorotic compared to the wild-type (WT) plants, and the expression of OsNAS3, OsYSL2, OsIRT1, and OsIRO2 was significantly up-regulated in the dmas-kd1 mutant, indicating that metal homeostasis was significantly disturbed. The secretion of DMA in dmas-kd1 was not significantly reduced. The dmas-kd1 plants accumulated less Fe in their roots compared to WT plants when grown with 10 μM FeSO4. The dmas-kd1 plants accumulated more Zn in their roots compared to WT plants under Fe-deficient, Fe-EDTA, and FeSO4 conditions. In both dehusked rice seeds (brown rice) and polished rice, no differences were observed for Fe, Cu, or Mn accumulation, whereas dmas-kd1 seeds significantly accumulated more Zn in brown rice. Our data suggests that rice only harbors one functional gene for DMA synthesis. In addition, the knock-down of OsDMAS1 significantly up-regulates the genes involved in Fe uptake and homeostasis.

Keywords: Aldo-keto reductase; DMA; Oryza sativa; deoxymugineic acid synthase; iron; iron deficiency; phytosiderophores; zinc..

Fig. 1.

OsDMAS1 paralogs do not process in vitro DMAS activity. (a) Phylogeny of OsDMAS1 paralogs. (b) Substrate-binding site of graminaceous DMASs and selected paralogs of OsDMAS1. (c) In vitro DMAS enzyme activity of HvDMAS1, OsDMAS1, and four paralogs of OsDMAS1.

Fig. 2.

Characterization of OsDMAS1 knock-down (dmas-kd1) plants. (a) Integration of T-DNA in the promoter of OsDMAS1. White boxes represent exons, while black boxes show 3′ and 5′ untranslated regions. (b) Confirmation of integration of T-DNA and the homozygous status of dmas-kd1; primer positions are shown in (a). (c) Expression of OsDMAS1 in WT and dmas-kd1. Different letters indicate statistically different values according to a Student–Newman–Keuls (SNK) test (P<0.05; n=4).

Fig. 3.

Changes in the expression of Fe homeostasis-related genes in WT and dmas-kd1 plants. Changes in the relative expression of (a) OsNAS1, (b) OsNAS2, (c) OsNAS3, (d) OsNAAT1, (e) OsTOM1, (f) OsYSL15, (g) OsYSL2, (h) OsIRT1, and (i) OsIRO2 in Fe-deficient roots of dmas-kd1 compared to the WT. The values are normalized to the expression of OsActin1. Different letters indicate values that are statistically different from each other according to a Student–Newman–Keuls (SNK) test (P<0.05; n=3).

Fig. 4.

Morphological characteristics of WT and dmas-kd1 plants grown under Fe-deficient conditions. (a) Root length, (b) root fresh weight, (c) shoot length, (d) shoot fresh weight, (e) chlorophyll content (SPAD meter value), and (f) DMA secretion from roots of WT and dmas-kd1 plants. Different letters indicate values that are statistically different from each other according to a Student–Newman–Keuls (SNK) test (P<0.05; n=4, except f where n=6). (g) Phenotype of hydroponically grown 4-week-old WT and dmas-kd1 plants.

Fig. 5.

Metal concentrations of WT and dmas-kd1 plants grown under Fe-deficient (–Fe), Fe-EDTA (100 mM Fe-EDTA), or FeSO₄ (10 μM FeSO₄) conditions. (a–d) Shoots: (a) Fe, (b) Cu, (c) Zn, and (d) Mn. (e–i) Roots: (e, f Fe, (g) Cu, (h) Zn, and (i) Mn. For Fe, (f) is an enlarged view of the first two treatments in (e). Different letters indicate values that are statistically different from each other according to a Student–Newman–Keuls (SNK) test (P<0.05; n=4).

Fig. 6.

Seed metal concentrations of WT and dmas-kd1 plants for (a–d) brown rice (dehusked rice seeds) and (e–h) polished rice seeds. (a, e) Fe, (b, f) Cu, (c, g) Zn, and (d, h) Mn. Different letters indicate values that are statistically different from each other according to a Student–Newman–Keuls (SNK) test (P<0.05; n=4).