Does a voltage-sensitive outer envelope transport mechanism contributes to the chloroplast iron uptake?

MAIN CONCLUSION: Based on the effects of inorganic salts on chloroplast Fe uptake, the presence of a voltage-dependent step is proposed to play a role in Fe uptake through the outer envelope. Although iron (Fe) plays a crucial role in chloroplast physiology, only few pieces of information are available on the mechanisms of chloroplast Fe acquisition. Here, the effect of inorganic salts on the Fe uptake of intact chloroplasts was tested, assessing Fe and transition metal uptake using bathophenantroline-based spectrophotometric detection and plasma emission-coupled mass spectrometry, respectively. The microenvironment of Fe was studied by Mössbauer spectroscopy. Transition metal cations (Cd2+, Zn2+, and Mn2+) enhanced, whereas oxoanions (NO3-, SO42-, and BO33-) reduced the chloroplast Fe uptake. The effect was insensitive to diuron (DCMU), an inhibitor of chloroplast inner envelope-associated Fe uptake. The inorganic salts affected neither Fe forms in the uptake assay buffer nor those incorporated into the chloroplasts. The significantly lower Zn and Mn uptake compared to that of Fe indicates that different mechanisms/transporters are involved in their acquisition. The enhancing effect of transition metals on chloroplast Fe uptake is likely related to outer envelope-associated processes, since divalent metal cations are known to inhibit Fe2+ transport across the inner envelope. Thus, a voltage-dependent step is proposed to play a role in Fe uptake through the chloroplast outer envelope on the basis of the contrasting effects of transition metal cations and oxoaninons.

Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

Uptake and incorporation of iron in sugar beet chloroplasts.

Chloroplasts contain 80-90% of iron taken up by plant cells. Though some iron transport-related envelope proteins were identified recently, the mechanism of iron uptake into chloroplasts remained unresolved. To shed more light on the process of chloroplast iron uptake, trials were performed with isolated intact chloroplasts of sugar beet (Beta vulgaris). Iron uptake was followed by measuring the iron content of chloroplasts in the form of ferrous-bathophenantroline-disulphonate complex after solubilising the chloroplasts in reducing environment. Ferric citrate was preferred to ferrous citrate as substrate for chloroplasts. Strong dependency of ferric citrate uptake on photosynthetic electron transport activity suggests that ferric chelate reductase uses NADPH, and is localised in the inner envelope membrane. The K(m) for iron uptake from ferric-citrate pool was 14.65 ± 3.13 µM Fe((III))-citrate. The relatively fast incorporation of (57)Fe isotope into Fe-S clusters/heme, detected by Mössbauer spectroscopy, showed the efficiency of the biosynthetic machinery of these cofactors in isolated chloroplasts. The negative correlation between the chloroplast iron concentration and the rate of iron uptake refers to a strong feedback regulation of the uptake.