Iron is abundant in most agricultural systems, however it is also one of the three most limiting nutrients for crop growth. This can be attributed to iron’s low solubility in aerobic and alkaline conditions, rendering it non-bioavailable for plant uptake. With around 1/3 of the world arable land presenting conditions that limit iron solubility, deciphering the plant machinery behind iron uptake and identifying microbial benefits to iron deficiency are of major interest. In this work, 33 endophytic bacterial isolates previously isolated from three regions in Jordan were tested for iron stress alleviation of Arabidopsis plants with the goal of identifying novel interaction mechanisms. On media with a low concentration of bioavailable iron, 11 isolates were found to be beneficial, while 15 isolates behaved in a pathogenic manner, reducing plant growth in both control as well as limited iron conditions. Beneficial isolates were then tested in plant assays with non-bioavailable iron, we concluded that our strains are also beneficial in these conditions. To further characterize the interaction between the beneficial strains and Arabidopsis plants, gene expression was assessed for plants colonized with select strains. The highest expression of iron-deficiency response genes was at day 6. The increase in expression was also met with an increase in colonization of plants at day 6. Mutant studies revealed that the beneficial effect by the bacterial isolates is dependent on the coumarin pathway, with mutants in FERULOYL-COA 6-HYDROXYLASE 1, f6’h1, showing a drastic decrease in fresh weight compared to wild type counterparts. We also discovered the phytohormone abscisic acid as an important contributor to iron stress alleviation by the beneficial isolates. Colonization assays as well as additional mutant studies will be necessary to further assess the effect of f6’h1 mutants on plant-microbe interaction as well as ABA’s role in plants under iron deficient conditions.

Low bioavailability of phosphate (P) due to low concentration and high immobility in soils is a key limiting factor in crop production. Application of excess amounts of P fertilizer is costly and by no means sustainable, as world-wide P resources are finite and running out. To facilitate the breeding of crops adapted to low-input soils, it is essential to understand the consequences of P deficiency. The second messenger calcium (Ca2+) is known to signal in plant development and stress perception, and most recently its direct role in signalling nutrient availability and deficiency has been partially elucidated. The use of Ca2+ as a signal has to be tightly controlled, as Ca2+ easily complexes with P groups and therefore is highly toxic to cellular P metabolism. It is unknown whether Ca2+ signals P availability or whether signalling is altered under P starvation conditions. The aim of this PhD project was to characterise the use of Ca2+ ions, particularly cytosolic free Ca2+ ([Ca2+]cyt), in stress signalling by P-starved roots of the model plant Arabidopsis thaliana. The hypothesis was that under P starvation and a resulting decreased cellular P pool, the use of [Ca2+]cyt may have to be restricted to avoid cytotoxic complexation of Ca2+ with limited P groups. Employing a range of genetically encoded Ca2+ reporters in Arabidopsis, P starvation but not nitrogen starvation was found to strongly dampen the root [Ca2+]cyt increases evoked by mechanical, salt, osmotic, and oxidative stress as well as by extracellular nucleotides. The strongly altered root [Ca2+]cyt response to extracellular nucleotides was shown to manifest itself during seedling development under chronic P deprivation, but could be reversed by P resupply. Fluorescent imaging elucidated that P-starved roots showed a normal [Ca2+]cyt response to extracellular nucleotides at the apex, but a strongly dampened [Ca2+]cyt response in distal parts of the root tip, correlating with high reactive oxygen species (ROS) levels induced by P starvation. Excluding iron, as well as P, rescued the altered [Ca2+]cyt response, and restored ROS levels to those seen under nutrient-replete conditions. P availability was not signalled through [Ca2+]cyt. In another part of this PhD project, a library of 77 putative Ca2+ channel mutants was compiled and screened for aberrant root hair growth under P starvation conditions. No mutant line showed aberrant root hair growth. These results indicate that P starvation strongly affects stress-induced [Ca2+]cyt modulations. The data generated in this thesis further understanding of how plants can integrate nutritional and environmental cues, adding another layer of complexity to the use of Ca2+ as a signal transducer.

Investigation of the relationship between iron deficiency, iron supplementation and susceptibility to infection, was suggested by the author's initial observations of an association of anaemia with serious bacterial infections in infancy in Papua New Guinea. The bulk of previous longitudinal clinical intervention studies in infancy showed beneficial effects of iron supplementation. However, defects of control and design and recording in these studies and contradictory anecdotal reports left the question unresolved. A prospective, placebo-controlled, randomised, double-blind trial of iron prophylaxis (3ml intramuscular iron dextran = 150mg Fe) to two month old infants was carried out on the North Coast of Papua New Guinea where there is high transmission of malaria. A literature review, pilot studies, protocol, demography, geography and laboratory methods developed are described. Findings indicate that the placebo control group became relatively iron deficient over the first year of life and that the iron dextran group had adequate, although not excessive iron stores and a higher mean haemoglobin; however, the prevalence and effects of malaria recorded in the field were higher in the iron dextran group. Analysis of field and hospital infectious morbidity in the trial indicated a deleterious association with iron dextran for all causes including respiratory infections (the main single reason for admission). Total duration of hospitalisation was significantly increased in the iron dextran group. Analysis of other factors showed (1) a higher admission rate associated with low weight-for-height recorded at the start of the trial; (2) a significant positive correlation between birth haemoglobin and hospital morbidity rates; (3) increased malaria rates in primiparous mothers of the cohort infants who received iron infusion during pregnancy; (4) lower relative risk of malaria associated with iron prophylaxis in individuals with alpha thalassaemia, which was found to be highly prevalent in this region. In conclusion, it is suggested that policies of iron supplementation, total dose iron injection and routine presumptive iron therapy for anaemia which are widely in practice in malaria endemic areas should be closely reviewed.