{'fraction_non_alphanumeric': 0.03032036613272311, 'fraction_numerical': 0.016781083142639208, 'mean_word_length': 4.913190529875987, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Reverse Genetics of IRT1, or How to Catch an Iron Transporter and Pin It Down.

We rely on plants as our main dietary source for the essential micronutrient iron. The Earth’s crust is itself very rich in iron (fourth most common element by weight), but much of this iron is not readily bio-available, and can severely limit plant growth and crops yield. Our knowledge of the mechanism employed by plants to extract iron from the soil took a leap forward with the demonstration that the Iron-Regulated Transporter IRT1 was the main route for iron in the root (Vert et al., 2002). Work from the Guerinot laboratory showed in the 1990s that iron assimilation in yeast and the model plant Arabidopsis thaliana were probably similar enough to follow a comparative biology approach. Indeed, a root ferric chelate reductase activity, responsible for the reduction of ferric iron (Fe3+) to ferrous iron (Fe2+), was induced under iron deficiency conditions, just as in yeast (Yi and Guerinot, 1996). The identity of the ferric reductase was later established by two complementary approaches: the cloning of the Arabidopsis FRO2 (Ferric Reduction Oxidase 2) gene with similarity to the yeast ferric reductase, and the map-based cloning of the frd1 mutant, defective in low iron-inducible ferric reductase activity (Robinson et al., 1999). The identification of FRO2/FRD1 offered the hope that dedicated iron transporters might be discovered in a similar fashion. Classical genetics had failed to reveal an obvious transporter. However, the suspected parallels between yeast and Arabidopsis provided another route: complementation of a yeast strain lacking iron transport with Arabidopsis genes. A single putative transporter was thus cloned that exhibited clear selectivity for Fe2+, and was designated IRT1 (for Iron-Regulated Transporter, Eide et al., 1996). Expression of IRT1 was restricted to roots of iron-deficient plants, as expected for an iron transporter. The transporter also exhibited the capacity to take up heavy metals like cobalt and cadmium. The ambition of using Arabidopsis IRT1 to create smarter crops for human nutrition depended on one crucial assumption: that IRT1 could, in fact, function as a Fe2+ transporter in planta. The mutant screen that yielded frd1 did not uncover an irt1 mutant. At the time, collections of insertional mutants were being generated, and DNA pools needed to be screened by PCR to discover if your favorite gene had been tagged. One collection from the University of Madison, Wisconsin, did not hold an insertion near IRT1. This is where the story takes an interesting and inspiring turn: the team of Briat and Curie in Montpellier, France, identified an insertion in IRT1 in the French T-DNA collection generated in Versailles. Rather than competing with Guerinot, who had admittedly claimed “dibs!” on IRT1 when her laboratory cloned the gene, Briat and Curie got in touch with her to combine resources: they had a mutant, she had a good peptide antibody against IRT1. The article itself is a text-book example on how to harness the power of reverse-genetics to determine the role of a gene. The irt1 allele is chlorotic and grows poorly, and eventually dies before it can set seeds, unless watered with excess iron. The iron content of irt1 leaves is about one third of wild-type, and is the consequence of impaired iron uptake by irt1 roots. First-author Grégory Vert, then a graduate student with Briat in Montpellier, and now a group leader in Toulouse, France, remembers this experiment very well. After a long day spent in the radiation room collecting leaves from plants that had been fed radiolabeled 55Fe, he recalls finding his advisor waiting for him at the scintillation counter, and fighting with him over the counter’s printout to see the results: radioactivity was high in iron-deficient wild-type leaves, but low in the irt1 mutant, demonstrating that “irt1-1 lost the capacity to accumulate iron under iron-deficient conditions, which strongly supports the idea that IRT1 is the main iron transporter operating in roots of ironstarved plants”. Two other groups published their own phenotypic characterization of irt1 mutants that same year; one screened a transposon insertion collection by PCR (Henriques et al., 2002), while the other evaluated a T-DNA insertion collection for altered chlorophyll fluorescence parameters (Varotto et al., 2002). A photosynthesis phenotype in irt1 makes intuitive sense, as the chloroplast is the main iron sink in leaves. This observation also stresses the importance of proper plant (micro)nutrition for plant fitness and crop yield. The article by Vert et al. has been cited 812 times since publication (according to Web of Science; Google Scholar Principles of iron uptake in the Arabidopsis root. Soil particles are rich in the poorly soluble ferric iron. Rhizosphere acidification (shown as a yellow to green gradient), supported by the release of protons via the Arabidopsis H+-ATPase AHA2, ameliorates Fe3+ solubility. The ferric reductase FRO2 can then reduce Fe3+ to Fe2+, which is taken up into the plant root by the high-affinity iron transporter IRT1. Plant Cell Advance Publication. Published on April 29, 2019, doi:10.1105/tpc.19.00246