Objective 1: Biochemical characterization of BTSL1 and BTSL2 with respect to metal binding.
Firstly, I carried out in silico analysis of the amino acid sequences of BTSL1, BTSL2 and BRUTUS (BTS). The proteins share a common domain organization, with an N-terminal domain comprising two to three hemerythrin-like domains, and a C-terminal domain formed by several Zn-fingers and RING-Zn finger domains in tandem.
Secondly, to express the protein domains in E. coli, I decided to focus on BTSL1 because BTSL1 and BTSL2 are very similar in sequence. The amino acid sequence was divided in segments according to the predicted domains and secondary structure. The individual hemerythrin domains were soluble and could be purified by affinity chromatography. All 3 domains were capable of binding 2 Fe ions. The third domain gave the highest yield of recombinant protein and was used to raise antibodies. The C-terminal domain was insoluble despite trying many different expression conditions, probably due to its high cysteine content. As a consequence, we could not measure metal binding to this domain, which is expected to bind 9 zinc ions based on homology to mammalian Pirh2. Gel bands were used to raise antibodies.
Objective 2: Analyse mutants in BTSL1 and BTSL2 for Fe homeostasis defects.
Three single insertion lines for BTSL1 (btsl1-1, btsl1-2 and btsl1-3) and one for BTSL2 (btsl2) were isolated and a double mutant line was produced (btsl double mutant). Single insertion lines behaved as wildtype plants in all the conditions tested, suggesting redundancy of both genes. However, double knockout lines showed clear phenotypes on medium without Fe or with excess Fe. Real time PCR, enzyme activity and protein measurements showed that the double knockout line fails in controlling Fe uptake and has altered levels of the master transcriptional regulator FIT. A model is proposed where BTSL protein stability is controlled by Fe binding to the N-terminal hemerythrin domains and protein stabilization triggers the degradation of part of the Fe deficiency signalling cascade.
Objective 3: Screen for mutants disrupted in Fe homeostasis.
In order to find new genes in the Fe signalling cascade, first I made reporter constructs by fusing well known Fe responsive promotors to the LUCIFERASE gene from fireflies. The constructs were transformed into the model plant Arabidopsis, and the response to Fe deficiency was tested to select the reporter with the best signal-to-noise ratio. Seedlings that were grown on Fe-deficient medium showed strong expression of luciferase, as measured by bioluminescence. The bioluminescence was turned off within 6 hours when Fe was resupplied. Next, seeds from a homozygous reporter plants were mutagenized with a chemical mutagen and grown up. We have so far screened 250 individual lines out of 5000, and have selected 47 lines with altered bioluminescence in response to Fe levels in the medium.
Data obtained has been presented as oral communication in two conferences: the Mini-symposium on The Role of Crops in Providing Micronutrients (Fe, Zn, Se) for Human Health, held in Grasmere in May 2016; and the 18th International Symposium on Iron Nutrition and Interaction in Plants, held in Madrid in June 2016. Results are currently being prepared as a manuscript for the scientific journal The Plant Cell.
