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Finding the iron sensing protein in crop plants

Periodic Reporting for period 1 - FeSensor (Finding the iron sensing protein in crop plants)

Reporting period: 2015-05-01 to 2017-04-30

Iron (Fe) is needed for all living organisms. Iron deficiency anaemia is the most widespread nutritional disease in humans and Fe is a limiting factor for crop growth on many arable soils. However, in excess, Fe can cause serious oxidative damage and induce cell death. Therefore, uptake and accumulation need to be tightly regulated and cells must be able to sense Fe levels. The Fe sensing mechanism is well characterized for bacteria, yeast and mammals, but it is still not known in plants. This gap in our knowledge limits the possibilities to manipulate iron levels in edible parts of the plant without causing toxicity symptoms in the rest of the plant, a major goal for biofortification efforts.
The overall aim of the project is to unravel the iron sensing mechanism in plants. In a previous study I discovered two genes that may be involved in signalling the iron status of the plant cell. In the project proposal, the genes were provisionally named IRS1 and IRS2, for Iron-Regulated Sensor, but during the project they were renamed BTSL1 and BTSL2 for BRUTUS-Like 1 and 2, based on homology to a gene called BRUTUS that was previously described in the model plant Arabidopsis. The BTSL proteins have several putative Fe binding domains, known as hemerythrin domains, and a RING Zn-finger domain that is likely to mediate protein-protein interactions. The objectives of the project were (i) to test the Fe binding properties of the hemerythrin domains of BTSL1 and BTSL2; (ii) characterize Arabidopsis plants lacking functional BTSL1/2 for iron homeostasis phenotypes; (iii) to screen for mutants disrupted in Fe homeostasis.
The results showed that BTSL1 and BTSL2 are able to bind Fe through their hemerythrin domains. Double knock-out lines of BTSL1/BTSL2 failed to regulate Fe uptake, leading to an excess of iron in the plant. A collection of mutants to screen for defects in Fe homeostasis has been produced and interesting lines are currently being selected for further characterization. The main conclusion of this Marie Sklodowska Curie action is that I have identified two proteins, BTSL1 and BTSL2, that play an important role in the Fe sensing mechanism of dicotyledonous plants.
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.
The understanding of the Fe sensing process in plants has been greatly improved as a result of the Marie Sklodowska Curie action. New genes involved in Fe sensing and Fe homeostasis have been annotated and described, and new knowledge about the molecular mechanism has been acquired, which allows us to manipulate the Fe sensing process in plants and increase the Fe content of staple crops. This will have a great impact in society, since Fe deficiency anaemia is the most common nutritional deficiency in humans, especially in women and children.

The results have been presented in one national and one international conference as an oral presentation. The project led to two new collaborations with laboratories in Beijing and Dusseldorf. A manuscript is about to be submitted to The Plant Cell, a journal with a high impact factor, and another is being prepared. A review article on iron homeostasis in plants has been accepted for publication by the journal Metallomics. During the action the fellow has successfully supervised three summer students, two lab attendants and one final year student, gaining experience in project and people management. The fellow and supervisor have successfully applied for additional funding for the project to the Biotechnology and Biological Sciences Research Council (BBSRC) and for a small grant to collaborate with Prof Hong-Qing Ling in Beijing.

The action has also provided contacts with industry. The fellow visited OMEX, a fertilizer company based in East Anglia, and Hutchinsons, a company that provides agronomic advice to farmers and a range of agronomic products.
Luciferase activity in Arabidopsis seedlings in response to iron deficiency