FRD3 controls iron localization in Arabidopsis

Abstract

The frd3 mutant of Arabidopsis exhibits constitutive expression of its iron uptake responses and is chlorotic. These phenotypes are consistent with defects either in iron deficiency signaling or in iron translocation and localization. Here we present several experiments demonstrating that a functional FRD3 gene is necessary for correct iron localization in both the root and shoot of Arabidopsis plants. Reciprocal grafting experiments with frd3 and wild-type Arabidopsis plants reveal that the phenotype of a grafted plant is determined by the genotype of the root, not by the genotype of the shoot. This indicates that FRD3 function is root-specific and points to a role for FRD3 in delivering iron to the shoot in a usable form. When grown under certain conditions, frd3 mutant plants overaccumulate iron in their shoot tissues. However, we demonstrate by direct measurement of iron levels in shoot protoplasts that intracellular iron levels in frd3 are only about one-half the levels in wild type. Histochemical staining for iron reveals that frd3 mutants accumulate high levels of ferric iron in their root vascular cylinder, the same tissues in which the FRD3 gene is expressed. Taken together, these results clearly indicate a role for FRD3 in iron localization in Arabidopsis. Specifically, FRD3 is likely to function in root xylem loading of an iron chelator or other factor necessary for efficient iron uptake out of the xylem or apoplastic space and into leaf cells.

Figure 1.

The frd3 phenotype is controlled by root genotype in reciprocal grafts. Four-day-old seedlings were grafted, allowed to recover for 2 weeks, and then transferred to iron-sufficient media for 3 d. Root ferric chelate reductase activity (A) and chlorophyll content (B) were measured spectrophotometrically. Ferritin protein was measured by immunoblot using anti-ferritin antibodies (C). Labels are shoot genotype/root genotype.

Figure 2.

frd3 mutant roots can appropriately regulate their iron deficiency responses in the absence of a shoot signal. Seedlings had the shoot portion removed prior to transfer to iron-sufficient (+Fe) or iron-deficient (−Fe) media with added Suc. The ferrozine method was used to quantitate root ferric chelate reductase 5 d after transfer. This experiment was repeated three times; a representative experiment is shown.

Figure 3.

Protoplasts from frd3 mutant shoots contain less iron than wild-type protoplasts. Protoplasts were isolated from 2-week-old wild-type and frd3 protoplasts and subjected to elemental analysis. This experiment was performed three times; representative results are shown.

Figure 4.

Iron accumulates in the vascular cylinder of frd3 mutant roots. Five-day-old seedlings were fixed in paraformaldehyde and stained with Perls' stain to visualize ferric iron. Roots were then imbedded and 70-μm sections cut. A, frd3-1 Intact roots; B, wild-type intact roots; C, frd3-1 cross section; D, wild-type cross section. E, Immunoblot showing ferritin protein levels in roots of 2-week-old plants grown under iron-sufficient or deficient conditions for 3 d prior to harvest.

Figure 5.

FRD3 is expressed in the root pericycle and vascular cylinder. A, Confocal fluorescence section of a root expressing FRD3-GFP fusion protein. B, GFP image from A overlaid with a transmitted light image of the same root. C, Optical cross section of a root expressing FRD3-GFP fusion protein. D, Optical cross section of a similar root stained with the cell wall dye FM-143. E, Optical cross section of a wild-type root showing epidermal autofluorescence. F, Immunofluorescence of FRD3-FLAG. G, Immunofluorescence control of a root not expressing the FLAG epitope.