2'-Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions

Abstract

Poaceae plants release 2'-deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil-plant analysis development (SPAD) values after treatment with 3-30 μm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT-PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high-affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.

Keywords: 2′-deoxymugineic acid; Oryza sativa L.; alkali tolerance; iron; nitrate assimilation; nitrate transport; plant growth.

Figure 1

Effects of DMA application and pH on rice seedling growth. Seedlings were germinated in deionized water for 1 week, and grown in nutrient solution containing 0, 0.3, 3, 30, or 150 μm DMA at either pH 5.8 or 8.0 for 2 weeks. All treatments contained equal amounts of Fe. (a) Shoot height. (b) Soil-plant analysis development (SPAD) value. Asterisks indicate significant difference from control (0 μm DMA) at pH 5.8 or 8.0 (**P < 0.01; Student's t-test). Values shown are mean ± standard deviation (SD) (n = 5).

Figure 2

Effect of DMA application on concentrations of various metals in rice shoot tissues. Metal content was analysed in rice seedlings grown under various concentrations of DMA at pH 5.8 or 8.0 for 2 weeks. Three seedlings were analysed for each treatment. All treatments contained equal amounts of Fe. Statistical analysis was conducted using Holm's pairwise t-test or Tukey's honest significant difference (HSD) depending on the P-value obtained in the Bartlett test in R program. ^a,b,cDifferent letters in the same group of bars indicate significant difference (P < 0.05). Values shown are mean ± standard deviation (SD) (n = 5).

Figure 3

Concentrations of various metals in rice root tissues. Experiment was conducted as described in the legend to Figure 2. ^a,b,cDifferent letters in the same group of bars indicate significant difference (P < 0.05). Values shown are mean ± standard deviation (SD) (n = 5).

Figure 4

Uptake and distribution of ⁵⁵Fe in rice seedlings. Two-week-rice seedlings were treated either with 5μM of ⁵⁵Fe-DMA or ⁵⁵Fe-EDTA for 4 h and were subjected to autoradiography. (a, b) Autoradiographs of rice seedlings treated with ⁵⁵Fe-DMA or ⁵⁵Fe-EDTA at pH 5.8 (a) and 8.0 (b). (c, d) Photographs of rice seedlings used for autoradiographic analysis shown in panels (a) and (b), respectively. Scale bars = 4 cm. (e, f) Quantification of ⁵⁵Fe signal intensity in rice shoots using the BAS-5000 system at pH 5.8 (e) and pH 8.0 (f). Control, no chelator added. All treatments contained equal amounts of Fe. Values shown are means ± standard deviation (SD) (n = 5).

Figure 5

Gene expression analysis of DMA-treated rice seedlings. Seedlings treated with 30 μm DMA, 30 μm EDTA, or no chelator (control), at pH 5.8 and 8.0, were analysed. Transcript levels of various genes in shoots and roots are shown in panel (a) (black bars) and (b) (white bars), respectively. Transcript levels of selected genes related to absorption/assimilation of nitrate and Fe were quantified by qRT-PCR. Primers used to amplify NIA1 may also have amplified NIA2 transcripts because of the high sequence similarity between these genes. Expression profiles of other genes are shown in Figure S6. Asterisks indicate significance difference from transcript level of each gene in control (0 μm DMA) at each pH (*P < 0.05; **P < 0.01; Student's t-test). Values shown are mean ± standard deviation (SD) (n = 3).

Figure 6

Enzyme activity of NADH- and NADPH-dependent nitrate reductases. Enzyme activities were determined in shoots of seedlings in different treatments. DMA or EDTA was added to medium to a final concentration of 30 μm, no chelators were added to control. Assays were performed separately to measure NADH- and NADPH-dependent NR activities. Asterisks indicate significant difference to control (no DMA) under each pH (*P < 0.05; **P < 0.01; Student's t-test). Values shown are mean ± standard deviation (SD) (n = 3).