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In vitro characterization of iron-phytosiderophore interaction with maize root plasma membranes: evidences for slow
N von Wirén1, R Gibrat, J F Briat
1Biochimie et Physiologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (URA 2133), Institut National de la Recherche Agronomique et Ecole Nationale Supérieure d'Agronomie, Place Viala, F-34060 Montpellier, France.
Abstract:
As an attempt to characterize iron(III)-phytosiderophore transport across plant membranes in vitro, a rapid filtration approach was set up in which plasma membrane vesicles from maize roots were incubated with 55Fe-labelled deoxymugineic acid (DMA). Fe-DMA, and not Fe-EDTA, could associate with plasma membrane vesicles. The rate of Fe-DMA association decreased with a half time of 15 min. The initial Fe-DMA association rate, estimated from the amount of Fe-DMA associated after 10 min incubation, exhibited a saturation curve as a function of Fe-DMA concentration. This curve could be satisfactorily fitted to the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein). The association rate of Fe-DMA with control liposomes remained negligible and constant in a pH range from 4 to 8, whereas it strongly increased at acidic pH with plasma membrane vesicles. However, the specific association of Fe-DMA to root plasma membrane could not be explained by a vesicle-filling process because: (i) lowering the vesicle volume by decreasing the osmotic potential of the assay medium with sorbitol did not decrease 55(Fe) labelling of the vesicles, (ii) creating inside-out vesicles by a Brij-58 treatment had almost no effect on Fe-DMA association to vesicles, (iii) 55(Fe) labelling is reversible by EDTA and excess free DMA, and (iv) 55(Fe) labelling was the same using plasmalemma vesicles prepared either from wild type maize or from the ys1 maize mutant deficient in iron-phytosiderophore transport. A model is proposed to account for the observed Fe-DMA association as the result of very slow binding kinetics onto membrane proteins. This model was validated by its ability to describe quantitatively both Fe-DMA association as a function of time and of substrate concentration. A prediction of the model was that association of Fe-DMA to plasma membranes might overcome a high activation energy barrier. Indeed, the Arrhenius plot for the association rate constant was linear with an activation energy of 64 kJ mol-1.
Insights
This study investigated iron(III)-phytosiderophore transport in maize roots. Results indicate slow binding kinetics of Fe-DMA to plasma membranes, not a simple vesicle-filling process.
Area of Science:
- Plant Physiology
- Molecular Biology
- Biochemistry
Background:
- Iron is essential for plant growth, and phytosiderophores facilitate its uptake in many plant species.
- Understanding the mechanism of iron-phytosiderophore transport across plant plasma membranes is crucial for improving iron nutrition in crops.
Purpose of the Study:
- To characterize the in vitro transport of iron(III)-phytosiderophore (Fe-DMA) across maize root plasma membranes.
- To elucidate the kinetic and mechanistic aspects of Fe-DMA association with plant membranes.
Main Methods:
- Utilized a rapid filtration approach with 55Fe-labelled deoxymugineic acid (DMA) and maize root plasma membrane vesicles.
- Investigated Fe-DMA association kinetics, saturation, pH dependence, and effects of osmotic potential and vesicle orientation.
- Applied Michaelis-Menten modeling and Arrhenius plot analysis to determine kinetic parameters and activation energy.
Main Results:
- Fe-DMA, but not Fe-EDTA, associated with plasma membrane vesicles, with a half-time of 15 min.
- Fe-DMA association exhibited saturation kinetics, fitting the Michaelis-Menten model (KM=600 nM, Vmax=2 nmol min-1 mg-1 protein).
- Association rate increased at acidic pH and was reversible by EDTA and excess DMA, suggesting specific binding rather than vesicle filling.
Conclusions:
- Fe-DMA association with maize root plasma membranes is best described by slow binding kinetics to membrane proteins, not a vesicle-filling process.
- The transport mechanism involves overcoming a high activation energy barrier (64 kJ mol-1), as indicated by Arrhenius plot analysis.
- This study provides a quantitative model for Fe-DMA association, offering insights into plant iron uptake mechanisms.