The developmental and iron nutritional pattern of PIC1 and NiCo does not support their interdependent and exclusive collaboration in chloroplast iron transport in Brassica napus.

MAIN CONCLUSION: The accumulation of NiCo following the termination of the accumulation of iron in chloroplast suggests that NiCo is not solely involved in iron uptake processes of chloroplasts. Chloroplast iron (Fe) uptake is thought to be operated by a complex containing permease in chloroplast 1 (PIC1) and nickel-cobalt transporter (NiCo) proteins, whereas the role of other Fe homeostasis-related transporters such as multiple antibiotic resistance protein 1 (MAR1) is less characterized. Although pieces of information exist on the regulation of chloroplast Fe uptake, including the effect of plant Fe homeostasis, the whole system has not been revealed in detail yet. Thus, we aimed to follow leaf development-scale changes in the chloroplast Fe uptake components PIC1, NiCo and MAR1 under deficient, optimal and supraoptimal Fe nutrition using Brassica napus as model. Fe deficiency decreased both the photosynthetic activity and the Fe content of plastids. Supraoptimal Fe nutrition caused neither Fe accumulation in chloroplasts nor any toxic effects, thus only fully saturated the need for Fe in the leaves. In parallel with the increasing Fe supply of plants and ageing of the leaves, the expression of BnPIC1 was tendentiously repressed. Though transcript and protein amount of BnNiCo tendentiously increased during leaf development, it was even markedly upregulated in ageing leaves. The relative transcript amount of BnMAR1 increased mainly in ageing leaves facing Fe deficiency. Taken together chloroplast physiology, Fe content and transcript amount data, the exclusive participation of NiCo in the chloroplast Fe uptake is not supported. Saturation of the Fe requirement of chloroplasts seems to be linked to the delay of decomposing the photosynthetic apparatus and keeping chloroplast Fe homeostasis in a rather constant status together with a supressed Fe uptake machinery.

Chloroplasts preferentially take up ferric-citrate over iron-nicotianamine complexes in Brassica napus.

MAIN CONCLUSION: Fe uptake machinery of chloroplasts prefers to utilise Fe(III)-citrate over Fe-nicotianamine complexes. Iron uptake in chloroplasts is a process of prime importance. Although a few members of their iron transport machinery were identified, the substrate preference of the system is still unknown. Intact chloroplasts of oilseed rape (Brassica napus) were purified and subjected to iron uptake studies using natural and artificial iron complexes. Fe-nicotianamine (NA) complexes were characterised by 5 K, 5 T Mössbauer spectrometry. Expression of components of the chloroplast Fe uptake machinery was also studied. Fe(III)-NA contained a minor paramagnetic Fe(II) component (ca. 9%), a paramagnetic Fe(III) component exhibiting dimeric or oligomeric structure (ca. 20%), and a Fe(III) complex, likely being a monomeric structure, which undergoes slow electronic relaxation at 5 K (ca. 61%). Fe(II)-NA contained more than one similar chemical Fe(II) environment with no sign of Fe(III) components. Chloroplasts preferred Fe(III)-citrate compared to Fe(III)-NA and Fe(II)-NA, but also to Fe(III)-EDTA and Fe(III)-o,o'EDDHA, and the Km value was lower for Fe(III)-citrate than for the Fe-NA complexes. Only the uptake of Fe(III)-citrate was light-dependent. Regarding the components of the chloroplast Fe uptake system, only genes of the reduction-based Fe uptake system showed high expression. Chloroplasts more effectively utilize Fe(III)-citrate, but hardly Fe-NA complexes in Fe uptake.

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.

Revisiting the iron pools in cucumber roots: identification and localization.

MAIN CONCLUSION: Fe deficiency responses in Strategy I causes a shift from the formation of partially removable hydrous ferric oxide on the root surface to the accumulation of Fe-citrate in the xylem. Iron may accumulate in various chemical forms during its uptake and assimilation in roots. The permanent and transient Fe microenvironments formed during these processes in cucumber which takes up Fe in a reduction based process (Strategy I) have been investigated. The identification of Fe microenvironments was carried out with (57)Fe Mössbauer spectroscopy and immunoblotting, whereas reductive washing and high-resolution microscopy was applied for the localization. In plants supplied with (57)Fe(III)-citrate, a transient presence of Fe-carboxylates in removable forms and the accumulation of partly removable, amorphous hydrous ferric oxide/hydroxyde have been identified in the apoplast and on the root surface, respectively. The latter may at least partly be the consequence of bacterial activity at the root surface. Ferritin accumulation did not occur at optimal Fe supply. Under Fe deficiency, highly soluble ferrous hexaaqua complex is transiently formed along with the accumulation of Fe-carboxylates, likely Fe-citrate. As (57)Fe-citrate is non-removable from the root samples of Fe deficient plants, the major site of accumulation is suggested to be the root xylem. Reductive washing results in another ferrous microenvironment remaining in the root apoplast, the Fe(II)-bipyridyl complex, which accounts for ~30 % of the total Fe content of the root samples treated for 10 min and rinsed with CaSO4 solution. When (57)Fe(III)-EDTA or (57)Fe(III)-EDDHA was applied as Fe-source higher soluble ferrous Fe accumulation was accompanied by a lower total Fe content, confirming that chelates are more efficient in maintaining soluble Fe in the medium while less stable natural complexes as Fe-citrate may perform better in Fe accumulation.