A Metal-binding Member of the Late Embryogenesis Abundant Protein Family Transports Iron in the Phloem ofRicinus communis L.

Claudia Krüger,Oliver Berkowitz,Udo W Stephan,Rüdiger Hell

doi:10.1074/jbc.m201896200

Abstract

The transport of metal micronutrients to developing organs in a plant is mediated primarily by the sieve elements. Ligands are thought to form complexes with the free ions in order to prevent cellular damage, but no binding partners have been unequivocally identified from plants so far. This study has used the phloem-mediated transport of micronutrients during the germination of the castor bean seedling to identify an iron transport protein (ITP). It is demonstrated that essentially all (55)Fe fed to seedlings is associated with the protein fraction of phloem exudate. It is shown that ITP carries iron in vivo and binds additional iron in vitro. ITP was purified to homogeneity from minute amounts of phloem exudate using immobilized metal ion affinity chromatography. It preferentially binds to Fe(3+) but not to Fe(2+) and also complexes Cu(2+), Zn(2+), and Mn(2+) in vitro. The corresponding cDNA of ITP was cloned using internal peptide fragments. The deduced protein of 96 amino acids shows high similarity to the stress-related family of late embryogenesis abundant proteins. Its predicted characteristics and its RNA expression pattern are consistent with a function in metal ion binding. The ITP from Ricinus provides the first identified micronutrient binding partner for phloem-mediated long distance transport in plants and is the first member of the late embryogenesis abundant protein family shown to have such a function.

Highlights

The metal micronutrients iron, zinc, manganese, and copper are imported into plants by specific uptake systems in the plasmalemma of root cells [1]
An iron transport protein has been purified from phloem exudates, and its cDNA has been cloned using Ricinus as a model for phloem transport analysis
The following evidence supports the conclusion that iron transport protein (ITP) is a transport species for iron in the phloem

Summary

EXPERIMENTAL PROCEDURES

Collection of Sieve Tube Exudate and 55Fe Labeling in Vivo—Seedlings of R. communis L. cv. Binding assays of 55Fe with blotted protein were carried out with membranes washed three times for 10 min in TBS buffer (0.02 M Tris, 0.15 M NaCl) of pH 7.0. Protein Purification—The high molecular weight fraction of 1 ml of phloem exudate was recovered from a PD-10 size exclusion column containing Sephadex G-25 M (bed volume, 8.3 ml; Amersham Biosciences) and treated with elution buffer (20 mM HEPES, pH 7.2, and 1 M NaCl). For affinity purification of protein, a HiTrap chelating HP column (bed volume, 1 ml; Amersham Biosciences) containing chelating Sepharose was rinsed with 5 ml of water before the matrix was loaded with ferric iron by the application of 0.5 ml of 0.1 M FeCl3 solution according to the instructions of the supplier. The membranes were exposed to x-ray film, and signals were quantified using a phosphorimaging system (Fuji BAS3000) and AIDA2.1 software (Raytest)

RESULTS

Iron Transport in the Phloem

DISCUSSION