Phloem iron remodels root development in response to ammonium as the major nitrogen source

Abstract

Plants use nitrate and ammonium as major nitrogen (N) sources, each affecting root development through different mechanisms. However, the exact signaling pathways involved in root development are poorly understood. Here, we show that, in Arabidopsis thaliana, either disruption of the cell wall-localized ferroxidase LPR2 or a decrease in iron supplementation efficiently alleviates the growth inhibition of primary roots in response to NH₄⁺ as the N source. Further study revealed that, compared with nitrate, ammonium led to excess iron accumulation in the apoplast of phloem in an LPR2-dependent manner. Such an aberrant iron accumulation subsequently causes massive callose deposition in the phloem from a resulting burst of reactive oxygen species, which impairs the function of the phloem. Therefore, ammonium attenuates primary root development by insufficiently allocating sucrose to the growth zone. Our results link phloem iron to root morphology in response to environmental cues.

Fig. 1. LPR2-dependent growth inhibition of primary roots in NH4+ medium.

a Phenotype comparison of Col-0 and the isas, lpr2-1, lpr1-1, and lpr1lpr2 mutants. b Elongation of primary roots. c Phenotype comparison of Col-0, lpr2-1, and complementation lines (COM #7 and COM #15). d Number of meristematic cells. e Number of elongation cells. f Length of the first differentiated cell. g Length of the root growth zone (elongation plus meristem zone). b, d-g Data shown are mean ± SD. P values < 0.05 indicate significant differences (two-way ANOVA with post-hoc Tukey HSD test; n = number of seedlings). Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with 100 µM Fe and analyzed 4 days after seedling transfer. Each experiment was repeated independently at least three times with similar results, and a representative experiment is shown.

Fig. 2. LPR2 is a cell wall ferroxidase and is irreplaceable by LPR1.

a Fluorescence co-localization of LPR2-GFP and propidium iodide in cells of p35S::LPR2-GFP roots. b Fluorescence of LPR2-GFP and FM4-64 in cells of p35S::LPR2-GFP roots after plasmolysis with 0.8 M sorbitol. Four-day-old seedlings were transferred to NH₄⁺ medium with 100 µM Fe, and confocal analyses were performed 4 days after seedling transfer. c Ferroxidase activities of recombinant GST-LPR2. Ferroxidase assay using 1 μg purified GST-LPR2 protein. Pink indicates the Fe²⁺-ferrozine complex. The substrate Fe²⁺ was added in the form of Fe(NH₄)₂(SO₄)₂·6H₂O at an initial concentration of 50 μM. d Fe²⁺ concentration-dependent (0–300 μM) ferroxidase activity of GST-LPR2 protein. Data shown are mean ± SD of three biological replicates. e, f Phenotype of lpr2-1 mutant lines with LPR2 promoter-confined tissue-specific expression of LPR1^CDS and LPR1^genomic. Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with 100 µM Fe and analyzed 4 days after seedling transfer. Two (c, d) or three (a, b, e, f) independent experiments were performed with similar results, and one representative experiment is shown.

Fig. 3. Fe-dependent inhibition of primary root growth in NH4+ medium.

a Primary root elongation of Col-0 seedlings in NO₃⁻-N or NH₄⁺-N source with various doses of Fe. Center line represents mean and bounds of box are SD; whiskers indicate the minimum and maximum values; n = number of seedlings. P values <0.05 indicate significant interactions between N form and Fe dose (two-way ANOVA with post-hoc Tukey HSD test). b NH₄⁺-sensitivity comparison of Col-0, isas, lpr2-1, and complementation line COM#7 in Fe^suff (100 µM) and Fe^low (10 µM) conditions. c Fe deposition indicated by Perls/DAB staining in primary roots. d Close-up view of Fe deposition in root stele of Col-0 seedlings grown in Fe^suff NH₄⁺ medium. Ep epidermis, Co cortex, En endodermis, Pe pericycle, Ph phloem, Xy xylem. e Perls/DAB staining in the phloem of the primary roots of Col-0 seedlings grown in Fe^suff NH₄⁺ medium. Red and green arrows show Fe depositions at lateral cell walls and sieve plates of the phloem, respectively. f Acetone washed-Perls/DAB staining in phloem of the primary roots of Col-0 seedlings grown in Fe^suff NH₄⁺ medium. The Perls/DAB-stained roots were washed with acetone for 3 h. Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with various doses of Fe supply and analyzed 4 days after seedling transfer. Each experiment was repeated independently three times with similar results, and a representative experiment is shown.

Fig. 4. Distribution of LPR2 in roots and its response to NH4+.

a pLPR2::LPR2-YFP expression in primary roots of complementation line COM#7. Roots were counterstained with propidium iodide (purple fluorescence) and analyzed for YFP fluorescence (green). b Radial section (top) and close-up view (bottom) of pLPR2::LPR2-YFP expression in a root of a COM#7 seedling grown in Fe^suff NH₄⁺ medium. c LPR2 expression in Col-0 roots. Relative expression levels were normalized to the geometric mean of expression of UBQ10 and EF1α. d, e Representative gels and relative protein levels in Col-0 roots. Relative LPR2 levels were estimated from the ratio of the signal intensity of LPR2 to that of actin from the same sample. c, e Data shown are mean ± SD of three biological replicates. P values < 0.05 indicate significant differences (two-way ANOVA with post-hoc Tukey HSD test). Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with Fe^suff (100 µM) or Fe^low (10 µM) and analyzed 4 days after seedling transfer. Each experiment was repeated independently three times with similar results, and a representative experiment is shown.

Fig. 5. Fe-dependent primary root growth inhibition by NH4+ is associated with a burst in reactive oxygen species (ROS).

a, b Venn diagram and Gene Ontology enrichment analysis of differentially expressed genes (DEGs) that are up- and downregulated in the pairwise comparisons of Col-0 Fe^low vs. Col-0 Fe^suff and lpr2-1 Fe^suff vs. Col-0 Fe^suff in the NH₄⁺-N source. The size of the circle represents gene numbers, and the color represents the value of P-adjust the was calculated by hypergeometric tests and adjusted for multiple testing using FDR. RNA sequencing was conducted with three biological replicates per line and condition. c ROS visualization in primary roots of Col-0, lpr2-1, and complementation line COM#7 seedlings by H₂DCFDA staining. d Close-up view of H₂DCFDA staining in primary roots of Col-0 seedlings grown in Fe^suff NH₄⁺ medium. e Co-localization of false color representation of H₂O₂ and fluorescence of phloem marker esculin in primary roots of roGFP2-Orp1 seedlings grown in Fe^suff NH₄⁺ medium. f, g Dose-response relationship of the primary root elongation of Col-0 seedlings plotted as a function of the concentration of ROS scavengers. Data shown are mean ± SD. n = number of seedlings. P values < 0.05 indicate significant differences (one-way ANOVA with post-hoc Tukey HSD test). h Images of the effects of 750 µM dimethyl thiourea (DMTU) and 200 µM 4-hydroxy-TEMPO (TEMPO) on Col-0 seedlings. Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with Fe^suff (100 µM) or Fe^low (10 µM) with or without the indicated ROS scavengers. Analyses were performed 4 days after seedling transfer. The experiments (c–h) were repeated independently three times with similar results, and representative data from one experiment are shown.

Fig. 6. Fe-dependent callose deposition in the phloem via reactive oxygen species (ROS) in response to NH4+.

a Callose detection in primary roots of Col-0, lpr2-1, and complementation line COM#7 seedlings by aniline blue staining. b Radial section (top) and close-up view (bottom) of aniline blue staining in primary roots of Col-0 seedlings grown in Fe^suff NH₄⁺ medium. c Fluorescence co-localization of GFP-sporamin and aniline blue staining in pSUC2::GFP-sporamin roots. d ROS scavengers abolished the NH₄⁺-induced callose deposition. Four-day-old seedlings of the indicated genotypes were transferred to NO₃⁻ or NH₄⁺ medium with Fe^suff (100 µM) or Fe^low (10 µM) with or without the indicated ROS scavenger. Each experiment was repeated independently three times with similar results, and a representative experiment is shown.

Fig. 7. Inhibition of phloem action and sucrose complementation of primary root growth in NH4+ medium.

a, b Phloem transport velocity and phloem unloading of esculin. Center line represents mean and bounds of box are SD; whiskers indicate the minimum and maximum values. P values < 0.05 indicate significant differences (two-way ANOVA with post-hoc Tukey HSD test, n = 12 seedlings per line and condition). Four-day-old seedlings of Col-0, lpr2-1, and complementation line COM#7 were transferred to NO₃⁻ or NH₄⁺ medium with Fe^suff (100 µM) or Fe^low (10 µM) and analyzed 4 days after seedling transfer. c Growth response of detached primary roots to localized sucrose supply. Data shown are mean ± SD. P values < 0.05 indicate significant differences (two-way ANOVA with post-hoc Tukey HSD test, n = 16 detached primary roots per line and condition). Inset: Scheme depicting the growth response analyses of detached primary roots to localized sucrose supply. Roots (1 cm) were cut from four-day-old seedlings. The detached roots were then transferred to a vertical two-layer split agar system of either NO₃⁻ or NH₄⁺ medium with Fe^suff or Fe^low. The upper layers were treated with various doses of sucrose, and the lower layers were absent of sucrose. Analyses were performed 3 days after seedling transfer. Each experiment was repeated independently three times similar results, and a representative experiment is shown.

Fig. 8. Schematic model of the mechanism that connects remodeling of root development to NH4+ as the primary N source.

In comparison with NO₃⁻ as the primary N source, NH₄⁺ supplementation as the predominant N source induces excessive Fe deposits in the apoplast of root phloem via a catalytic reaction of Fe²⁺ oxidation to Fe³⁺, which depends on the action of the cell wall-localized ferroxidase LPR2 in root stele. Such an aberrant Fe accumulation then inhibits phloem transport and unloading by triggering a burst of reactive oxygen species, which subsequently induces massive callose deposition. Consequently, sucrose is insufficiently allocated to the root growth zone, which arrests root development.