Os03g0216700

The rice gene Os03g0216700 was reported as OsFRDL1 in 2004 .

Annotated Information

Function

• The results suggested that OsFRDL1 encodes a transporter for citrate at the plasma membrane of pericycle cells, which is required for efficient translocation of Fe3+ as a citrate complex from the roots to the shoots. To confirm whether OsFRDL1 has activity to transport citrate, the researchers expressed OsFRDL1 in Xenopus oocytes. The efflux activity for citrate was significantly higher in the oocytes injected with OsFRDL1 cRNA than in oocytes injected with water (Fig. 1). This result indicates that OsFRDL1 is able to transport citrate out of the cells..

Figure 1. Efflux transport activity for citrate in Xenopus oocytes. OsFRDL1 cRNA or water was injected into Xenopus oocytes. After 1-d cultivation, the oocytes were injected with 14C-labeled citrate. The release of 14C-labeled citrate from the oocytes was determined 2 h later. Data as means 6 SD (n = 3). .

• OsFRDL1 is not responsible for Al-induced secretion of citrate from the rice roots.

Evolution

• OsFRDL1 (Os03g0216700) was initially isolated as a homolog of AtFRD3 (Fig. 2). In the rice genome, there are six close homolog genes of AtFRD3, HvAACT1, and SbMATE (Fig. 2)

Figure 2. Phylogenetic relationship of OsFRDL1-like proteins in different plant species. The amino acid sequences were aligned by ClustalW. .

Mutation

• In this study, to examine the function and role of OsFRDL1 in rice, the researchers obtained two independent Tos-17 insertion lines; ND8025 (ND) and NC2637 (NC) for this gene. The OsFRDL1 was knocked out in the ND line, but knocked down in the NC line. When the wild-type rice and two Tos-17 insertion lines were grown at 10 mM Fe (as FeSO4), no visible difference was observed among the three lines (Fig. 3A). However, at 0.2 mM Fe, chlorosis was observed in the newly expanded leaves of two Tos-17 lines, but not in the wild type (Fig. 3B). The Fe3+ precipitation in the roots was investigated at 10 mM Fe with Perls blue staining (Green and Rogers, 2004). Fe precipitation was observed in the epidermal cells of the roots of all three lines (Fig. 3, C–E), which was not observed in Arabidopsis roots (Green and Rogers, 2004). This distinct precipitation in rice is attributed to secreted oxygen from rice roots, which oxidizes ferrous iron (Fe2+) into insoluble ferric iron (Fe3+) on the root surface (Horiguchi, 1995). However, there was no difference in the epidermal staining among three lines (Fig. 3, C–E). In contrast, heavy staining was observed in the central vascular part of the knockout line, ND (Fig. 3, D and G), but not in the wild-type line (Fig. 3, C and F). In the knockdown line NC, staining was also observed in the central vascular part (Fig. 3, E and H), although the intensity was not as strong as that of ND.

Figure 3. Phenotype of Tos-17 insertion lines of OsFRDL1. .

• The researchers compared the Fe concentration in the roots and shoots between the wild-type rice and the knockout line (ND). The concentration of Fe in the shoots was significantly lower in the knockout line than in the wild-type rice at either Fe concentration (Fig. 4A). By contrast, the concentration of Fe in the roots was 2 times higher in the knockout line than in the wild-type line at 0.2 mM Fe (Fig. 4B). At 10 mM Fe, the root Fe increased to an extremely high concentration, probably due to precipitation of Fe on the root epidermal layer (Fig. 4C). These results indicate that knockout of OsFRDL1 causes accumulation of Fe in the roots and decreased Fe concentration in the shoots.

Figure 4. Fe concentration in the shoots (A) and roots (B and C). .

• The researchers compared the concentration of Fe and citrate in the xylem sap between ND and wild type at different Fe concentrations in the external solution. The concentration of citrate in the xylem sap of ND was less than one-half that of wild type at either external Fe concentration (Fig. 5A). However, there was no significant difference in malate concentration in the xylem sap between ND and wild type (Fig. 5B). The total Fe concentration in the xylem sap was significantly lower in ND than in wild type (Fig. 5C). In a separate experiment with 10 mM Fe, speciation analysis showed that there was no difference in Fe2+ concentration in the xylem sap between ND and wild type, but the concentration of Fe3+ in ND xylem sap was less than one-half that in wild type (Fig. 5D).

Figure 5. Organic acids and Fe in the xylem sap. .

Expression Pattern

• The expression of OsFRDL1 was examined with quantitative reverse transcription (RT)-PCR. OsFRDL1 was mainly expressed in the roots, but not in the shoots (Fig. 6A). A time course experiment showed that the expression in the roots was not affected by Fe deficiency (Fig. 6, A and B), in contrast to IRT1, which expression was greatly increased with development of Fe deficiency (Fig. 6C). Interestingly, the expression of OsFRDL1 was higher in the mature root zone than in the root tip (Fig. 6D). This result is in agreement with Fe precipitation and development of xylem.

Figure 6. Expression pattern of OsFRDL1. A, Expression of OsFRDL1 mRNA in the roots and shoots of rice grown in a nutrient solution with or without Fe. .

Subcellular localization

• The localization of OsFRDL1 was examined with an anti-OsFRDL1 antibody. Immunostaining showed that OsFRDL1 was localized at pericycle cells (Fig. 7, A and B). This result is consistent with OsFRDL1 promoter GUS staining (Inoue et al., 2004). No signal was observed in the knockout line (Fig. 7C), indicating the high specificity of the anti-OsFRDL1 antibody. The subcellular localization of OsFRDL1 was found to be localized on the plasma membrane by using heterologous expression in the onion (Allium cepa) epidermal cells (data not shown), which was the same as that reported previously.

Figure 7. Tissue-specific localization of OsFRDL1 in rice root. A and B, Immunostaining with anti-OsFRDL1 antibody in the roots (20 mm from the tip) of wild-type rice grown in a nutrient solution containing 10 mM FeSO4. C, Immunostaining in the roots of knockout line (ND). Scale bars = 100 mm. .

Labs working on this gene

• Research Institute for Bioresources, Okayama University, Kurashiki 710–0046, Japan