Iron deficiency-induced secretion of phenolics facilitates the reutilization of root apoplastic iron in red clover

Abstract

Phenolic compounds are frequently reported to be the main components of root exudates in response to iron (Fe) deficiency in Strategy I plants, but relatively little is known about their function. Here, we show that removal of secreted phenolics from the root-bathing solution almost completely inhibited the reutilization of apoplastic Fe in roots of red clover (Trifolium pratense). This resulted in much lower levels of shoot Fe and significantly higher root Fe compared with control and also resulted in leaf chlorosis, suggesting this approach stimulated Fe deficiency. This was supported by the observation that phenolic removal significantly enhanced root ferric chelate reductase activity, which is normally induced by plant Fe deficiency. Furthermore, root proton extrusion, which also is normally increased during Fe deficiency, was found to be higher in plants exposed to the phenolic removal treatment too. These results indicate that Fe deficiency-induced phenolics secretion plays an important role in the reutilization of root apoplastic Fe, and this reutilization is not mediated by proton extrusion or the root ferric chelate reductase. In vitro studies with extracted root cell walls further demonstrate that excreted phenolics efficiently desorbed a significant amount of Fe from cell walls, indicating a direct involvement of phenolics in Fe remobilization. All of these results constitute the first direct experimental evidence, to our knowledge, that Fe deficiency-induced secretion of phenolics by the roots of a dicot species improves plant Fe nutrition by enhancing reutilization of apoplastic Fe, thereby improving Fe nutrition in the shoot.

Figure 1.

The time course of total amounts of phenolic compounds secreted from roots in response to Fe deficiency. Seedlings were grown at 0 (−Fe) and 20 μm FeEDTA (control) with a cycling of 1 d in the nutrient solution and the following day in 0.5 mm CaCl₂ solution. The total phenolics content in 0.5 mm CaCl₂ solution was measured by Folin-Ciocalteu's reagent. Data are means ± sd (n = 3).

Figure 2.

The pump system to remove phenolics from the nutrient solution. The setup includes three parts: (1) the 1-L pot used for plant cultivation; (2) the pump used for circulating nutrient solution; and (3) the resin column (filled with SP825 Sepabeads resin, Mitsubishi Chemical) used for removing the phenolics in nutrient solution that were secreted by roots.

Figure 3.

Effect of phenolic removal on chlorophyll synthesis. A, SPAD readings of the newly formed leaves of the plants treated with (−Fe + pump) or without (−Fe) phenolics removal. The pictures of the plants at 12th (B) and 20th (C) days after phenolics-removal treatment. For the phenolic-removing treatment, the plants were first grown in −Fe solution for 8 d, then one-half of the plants were transferred to the phenolics-removing pump system (−Fe + Pump), and the other half were continuously grown in the −Fe solution (−Fe). Data are means ± sd (n = 5). *, Significant differences (P < 0.05) between two treatments at each time point.

Figure 4.

Effect of phenolics removal on the growth of red clover. The treatments are the same as Figure 2, and the fresh weights were analyzed after 16 d of the treatments. Data are means ± sd (n = 4). *, Significant differences (P < 0.05) between two treatments.

Figure 5.

Role of phenolics secretion in reutilization of root apoplast Fe in red clover. The treatments are the same as Figure 2. Data are means ± sd (n = 4). *, Significant differences (P < 0.05) between two treatments at each time point.

Figure 6.

Effects of the phenolics removal on Fe (A), Mn (B), Cu (C), and Zn (D) contents in red clover roots and shoots. The treatments are the same as Figure 2, and the fresh weights were analyzed after 16 d of the treatments. Data are means ± sd (n = 4). *, Significant differences (P < 0.05) between two treatments.

Figure 7.

In vitro effect of phenolics on desorption of cell wall-bound Fe. A total of 0.050 g of cell wall powder that was prepared from the whole root systems of Fe-sufficient plant was placed in a 2-mL column. The kinetic studies were conducted as described in “Materials and Methods.” At least two independent replicates were conducted, and one set of results was presented.

Figure 8.

Effects of the phenolics removal on Fe deficiency-induced proton extrusion (A) and ferric chelate reductase activity (B) in red clover roots. The treatments are the same as Figure 2. Data are means ± sd (n = 4). *, Significant differences (P < 0.05) between two treatments at each time point.