Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1)

Abstract

IRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot mobilization of Fe. We show that the irt1-2 (pam42) mutant over-accumulates Fe in roots, most prominently in the cortex of the differentiation zone, when compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. In the transgenic Arabidopsis lines, short-term root Fe uptake rates and secondary substrate Mn accumulation resemble those of irt1-2, suggesting that these plants remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve iron nutrition and the nutritional quality of agricultural crops.

Involvement of Arabidopsis Multi-Copper Oxidase-Encoding LACCASE12 in Root-to-Shoot Iron Partitioning: A Novel Example of Copper-Iron Crosstalk

“…On control medium, there was a pronounced increase in LAC12 transcript levels in the irt1 mutant compared to the wild-type (Figure 1). This was consistent with a regulation of LAC12 transcript levels in dependence on plant physiological Fe status, similar to the regulation of a number of other Fe deficiency-responsive genes in roots of the irt1 mutant (Quintana et al, 2021).…”

“…Under Fe deficiency, Mn concentrations in both roots and shoots of wild-type and lac12 mutant plants are higher than in Fe-sufficient conditions, in which the increase of root Mn concentration in lac12 mutants were significantly higher when compared to wild-type plants. This result could be related to an increase of IRT1 protein levels, which is well known to transport Mn as well (Eide et al, 1996;Rogers et al, 2000), as is known to happen in Fe-deficient wild-type plants (Quintana et al, 2021). The accumulation of Zn under Fe deficiency has been previously reported in WT plants (Buckhout et al, 2009) and could be due to a low specificity of IRT1, which is transcriptionally induced under Fe-deficient conditions (Supplementary Figure 6) (Vert et al, 2002).…”

“…The irt1 mutant (pam42) was obtained from Prof. Dr. Dario Leister, LMU Munich (DE) (Varotto et al, 2002). spl7-2 and frd3-7 mutants were previously used in our lab (Bernal et al, 2012;Quintana et al, 2021).…”

“…Equal amounts of RNA (1 µg) were used as a template for cDNA synthesis (RevertAid First Strand cDNA Synthesis Kit, Thermo Fisher Scientific) with oligo-dT primers following manufacturer s instructions. RT-qPCR was performed on a LightCycler480 (Roche) in a 10-µL reaction mixture containing 16 ng of cDNA, each primer at 0.25 µM and 5 µL of 2X GoTaq qPCR Master Mix (Promega) as described (Quintana et al, 2021). Reaction efficiencies (RE) for each PCR reaction were determined with the LinRegPCR program, version 2016.0 (Ruijter et al, 2009).…”

“…To examine if LAC12 expression was regulated under Fe deficiency, we cultivated wild-type (WT) and irt1 seedlings under Fe-deficient and Fe-sufficient (control) conditions for 10-d, subsequent to a 5-d pre-cultivation period on Fesufficient medium (Figure 1; Quintana et al, 2021). The IRON REGULATED TRANSPORTER1 (irt1) mutant defective in root high-affinity Fe uptake was included as a control genotype exhibiting constitutive activation of the Fe deficiency responses (Connolly et al, 2002;Henriques et al, 2002;Varotto et al, 2002;Vert et al, 2002;Kobayashi and Nishizawa, 2012).…”