Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis

Abstract

In plants, the controlled absorption of soil nutrients by root epidermal cells is critical for growth and development. IRON-REGULATED TRANSPORTER 1 (IRT1) is the main root transporter taking up iron from the soil and is also the main entry route in plants for potentially toxic metals such as manganese, zinc, cobalt, and cadmium. Previous work demonstrated that the IRT1 protein localizes to early endosomes/trans-Golgi network (EE/TGN) and is constitutively endocytosed through a monoubiquitin- and clathrin-dependent mechanism. Here, we show that the availability of secondary non-iron metal substrates of IRT1 (Zn, Mn, and Co) controls the localization of IRT1 between the outer polar domain of the plasma membrane and EE/TGN in root epidermal cells. We also identify FYVE1, a phosphatidylinositol-3-phosphate-binding protein recruited to late endosomes, as an important regulator of IRT1-dependent metal transport and metal homeostasis in plants. FYVE1 controls IRT1 recycling to the plasma membrane and impacts the polar delivery of this transporter to the outer plasma membrane domain. This work establishes a functional link between the dynamics and the lateral polarity of IRT1 and the transport of its substrates, and identifies a molecular mechanism driving polar localization of a cell surface protein in plants.

Keywords: Arabidopsis; PI3P; endocytosis; nutrition; radial transport.

Fig. 1.

IRT1 localization to the outer polar domain in low metal conditions. (A) IRT1 immunolocalization with anti-IRT1 antibodies on root hair cells of irt1-1, irt1-1/35S::IRT1, and irt1-1/35S::IRT1_K154RK179R plants grown 7 d in different metal conditions. MS/2 refers to standard plant growth medium containing metals. Metal depleted conditions correspond to combined absence of Zn, Mn, and Co. (B) Phenotype of wild-type, irt1-1/35S::IRT1, and irt1-1/35S::IRT1_K154RK179R plants grown on standard medium (MS/2), iron- (-Fe), and manganese-depleted media (-Mn). (C) IRT1 immunolocalization with anti-IRT1 antibodies on root epidermal cells of irt1-1, irt1-1/35S::IRT1, tamoxifen-induced DN-HUB1, and irt1-1/35S::IRT1_K154RK179R plants grown 7 d in different metal conditions. DN-HUB1 plants express an inducible dominant-negative clathrin HUB and are defective in CME. Red and green arrows mark the inner and outer plasma membrane domains of root epidermal cells (PM_in and PM_out) used for quantification of polarity profiles (Fig. S1B). (D) Quantification of the outer/inner polarity ratios in root epidermal cells. Data represents the mean ± SE (n = 10). Different letters indicate means that were statistically different by one-way ANOVA and Tukey’s multiple testing method (P < 0.05). Ratio = 1 indicates apolar plasma membrane localization, or nonplasma membrane localization in the case of wild-type plants, ratio < 1 indicates inner localization, and ratio >1 indicates outer localization. (Scale bars: 10 µm.)

Fig. 2.

FYVE1 interacts with IRT1 and PI3P and is recruited to late endosomes. (A) Yeast two-hybrid screening using IRT1 as bait identified FYVE1. The interaction is revealed by the activation of HIS3 transcription and growth on -HIS medium. (B) Coimmunoprecipitation analyses between FYVE1-mCitrine and IRT1 in iron-deficient roots. (C) Lipid binding assays of in vitro transcribed/translated FYVE1 protein. LPA, lysophosphatidic acic; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI3P, PI-3-phosphate; PI3,4P2, PI-3,4-bisphosphate; PI3,4,5P3, PI-3,4,5-triphosphate; PI3,5P2, PI-3,5-bisphosphate; PI4P, PI-4-phosphate; PI4,5P2, PI-4,5-bisphosphate; PI5P, PI-5-phosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. (D) Localization of FYVE1-mCitrine fusion protein in differentiated root cells. (E) Sensitivity of FYVE1-mCitrine trafficking to Wortmannin (Wm) in differentiated root cells. (F and G) Colocalization of FYVE-mCitrine with the late endosomal marker RabF2a-mCherry (F) and 2xFYVE_HRS-mCherry (G) in differentiated root cells. Arrows show an example of colocalization. Scale bar, 10µm. (H) Quantification of FYVE1 colocalization with the late endosomal markers RabF2a and 2xFYVE_HRS. Colocalization of punctate structures was quantified in 10 cells from the F₁ progeny from crosses between parental lines FYVE1-mCitrine and marker lines RabF2a and 2xFYVE_HRS. Data represents the mean ± SE. (Scale bars: 10 µm.)

Fig. 3.

FYVE1 overexpression leads to iron deficiency and impaired transport of IRT1 substrates. (A) Phenotype of wild-type (WT), two independent 35S:FYVE1, and two independent 35S::2xFYVE_HRS transgenic lines grown 5 d in +Fe (Upper) or in -Fe (Lower). (B) Root length of 5-d-old wild-type (WT), 35S:FYVE1, and 35S::2xFYVE_HRS grown with or without Fe. Results are presented as mean ± SD (n = 20). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05). (C) Metal content determined by ICP-MS on roots of 7-d-old plants grown with or without Fe. Results are presented as mean ± SD from three to four batches of 30 seedlings. Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05) for genotypes within a given growth condition (+Fe or -Fe).

Fig. 4.

FYVE1 overexpression leads to apolar IRT1 accumulation at the cell surface. (A) IRT1 protein accumulation in roots of 7-d-old wild-type (WT) and 35S::FYVE1 independent transgenic lines grown in the presence (+) or absence (−) of Fe. Total protein were extracted and analyzed by Western blot with anti-IRT1 antibodies. The nonspecific band indicated with an asterisk serves as a loading control. (B) IRT1 immunolocalization with anti-IRT1 antibodies, in differentiated roots of wild-type (WT) and 35S::FYVE1 plants grown in Fe-deficient conditions. (Scale bars: 10 µm.) Red and green arrows mark the inner and outer plasma membrane domains of root epidermal cells (PM_in and PM_out), respectively. Quantification of polarity profiles across corresponding epidermal cells is shown below each image and represents the best fitted curve from independent immunofluorescence experiments (n = 10). (C) Quantification of the outer/inner polarity ratios in differentiated root epidermal cells. Data represents the mean ± SE (n = 10). Statistical differences were calculated by one-way ANOVA. Different letters indicate means that were statistically different by Tukey’s multiple testing method (P < 0.05). Ratio = 1 indicates apolar localization or nonplasma membrane localization in the case of wild-type plants, ratio < 1 indicates inner localization, and ratio >1 indicates outer localization.