Not available / text

[Truncated abstract] Nitrogen fixation is a feature of the symbiotic association between legumes and rhizobia, which occurs within the symbiosomes of root nodules and involves the conversion of atmospheric N2 to ammonia to be used by the plant in exchange for carbon compounds. Exchange of other nutrients is controlled by plant-synthesised proteins on the symbiosome membrane. Iron is a component of symbiotically important proteins, so is essential for nitrogen fixation. Low soil iron leads to decreased plant yields, whilst in other environments plants may accumulate iron to toxic levels. Knowledge of iron acquisition, transport and storage mechanisms is important to elucidate the role of iron transporters in the maintenance of iron homeostasis in the plant. This study provides evidence that iron has a profound effect in the Bradyrhizobium japonicum-Glycine max symbiosis on the development of the nodule, and on the development of the symbiotic soybean plant itself. cDNAs encoding four putative iron transporters in soybean; GmDmt1, GmYSL1, GmCCC1;1 and GmCCC1;2, were identified, isolated and characterised in this study. GmDmt1 is localised to the symbiosome membrane. Expression of GmDmt1 occurs in nodules, roots and leaves and increases in response to iron starvation. GmDmt1 rescues growth and enhances 55Fe(II) uptake in the iron transport deficient yeast strain fet3fet4, with uptake following Michaelis-Menten kinetics, resembling the situation in isolated symbiosomes. Competition experiments using fet3fet4 indicated that GmDmt1 is able to transport other divalent cations, including zinc, copper and manganese, and is also able to complement a zinc transport deficient yeast mutant. ... These results suggest the divalent metal transporter GmDmt1, the putative iron chelate transporter GmYSL1 and the putative vacuolar iron transporters GmCCC1;1 and GmCCC1;2 act together to maintain iron homeostasis in symbiotic soybeans. The possible interactions and regulation of these proteins and their roles in the acquisition, transport and utilisation of iron in symbiotic soybeans are discussed.

Abstract not available

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. In mammals since no controlled means of eliminating unwanted iron has evolved, body iron balance is maintained by alterations in dietary iron intake. This occurs in the duodenum where most dietary iron is absorbed. Absorption involves at least two steps, uptake of iron from the intestinal lumen and then its transport into the body, processes that occur at the apical and basal membranes of enterocytes, respectively. In chapter one of this thesis the background information relevant to iron absorption is described. Despite numerous studies, the role of these proteins in iron absorption remains unclear, partly because many studies have reported them in non-enterocyte cell lines where the expression of the proteins involved in iron absorption is unlikely and therefore the physiological significance of the findings uncertain. Therefore, the study of iron absorption would value from additional cell lines of intestinal origin being used, preferably derived from a species used to comprehensively study this process in vivo, namely the rat. Validation of such a model would enable comparisons to be made from a molecular level to its relevance in the whole organism. In chapter 3 of this thesis, the rat intestinal cell line 6 (IEC-6) was examined as a model of intestinal iron transport. IEC-6 cells expressed many of the proteins involved in iron absorption, but not the ferrireductase Dcytb, sucrase or αvβ3 integrin. In addition, in IEC-6 cells the expression of the apical transporter divalent metal transporter 1 (DMT1), the iron storage protein ferritin, the uptake of Fe(II) and Fe(III) were regulated by cellular iron stores as is seen in vivo. This suggests that IEC-6 cells are of a lower villus enterocyte phenotype. Presented in chapter 4 is the study of the uptake of iron from Fe(II):ascorbate and Fe(III):citrate by IEC-6 cells in the presence of a blocking antibody to the putative basolateral transporter ferroportin1 and of colchicine and vinblastine, different pHs, and over-expression of DMT1. It was shown that optimal Fe(II) uptake required a low extracellular pH and was dependent on DMT1. Uptake of Fe(III) functioned optimally at a neutral pH, did not require surface ferrireduction, and was increased during over-expression of DMT1. These observations suggest that intravesicular ferrireduction takes place before transport of Fe(II) to the cytoplasm by DMT1. This pathway was not blocked by a functional antibody against αvβ3 integrin but was inhibited by competition with unlabeled iron citrate or citrate alone. Surprisingly, a functional antibody against ferroportin1 had no effect on efflux but significantly reduced (p<0.05) uptake of Fe(II) by 40-50% and Fe(III) by 90%, indicating two separate pathways for the uptake of iron from Fe(II)-ascorbate and from Fe(III)-citrate in IEC-6 cells. Presented in chapter 5 is the development and validation of a technique for the removal of freshly isolated enterocytes from the rat duodenum and their use to study iron transport processes that enabled comparisons to be made between these cells, IEC-6 cells and the human enterocyte cell line Caco-2 cells. In chapter 6 a blocking antibody to ferroportin1 was shown to inhibit uptake of Fe(II) but not release of iron in freshly isolated duodenal enterocytes from rats and Caco-2 cells supporting the findings obtained with IEC-6 cells described in chapter 4. Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane indicating its expression at the microvillus membrane. Confirming this, ferroportin1 was shown along the microvillus membrane of Caco-2 cells, in enriched microvillus membrane preparations and in enterocytes of duodenum tissue of rats where it co-localised with lactase. The significant findings to emerge from this thesis are that the IEC-6 cell is a valid model to study iron absorption producing results consistent with those found in freshly isolated enterocytes and in human enterocyte-like cells. In particular, ferroportin1 functions in the uptake of iron at the apical membrane possibly by modulating surface binding of Fe(II) to DMT1 or the activity of DMT1. In addition to this in Fe(II) uptake from Fe(III) ferroportin1 may also affect the number of Fe(III): citrate binding sites. Preliminary studies further characterizing the function of ferroportin1 at the apical membrane and at intracellular sites of IEC-6 cells along with integration of these data are discussed in chapter 7.