Fig 1.
Role of the endodermal bypass in radial substrate transport in roots.
(A) Schematic representation of radial transport pathways in roots with (WT, Columbia-0) or without (sgn3 mutant) functional CSs. (B–E) Normalized shoot accumulation as readout for radial transport rates of Sr2+ or 15NH4+ when supplied in the nutrient solution at low (200 μM) or high (4 mM) concentration. Bars represent means ± SD. P values were calculated using Student t test (n = 4 biological replicates). Underlying data can be found in S1 Data. 15NH4+, 15N-labeled ammonium; CS, Casparian strip; DW, dry weight; sgn3, schengen 3; Sr2+, strontium ion; WT, wild-type.
Fig 2.
Quantitative contribution of symplastic and apoplastic pathways to radial transport of ammonium in roots.
(A) Schematic representation of radial transport pathways in roots of tko or tko sgn3 without or with reconstituted expression of AMT1;2 (+1;2) or AMT1;3 (+1;3). (B and C) Normalized shoot accumulation as readout for radial transport rates of NH4+-N at 200 μM (B) or 4 mM 15NH4+ (C). (D and E) Estimated contribution of the ATP or the STP in absence or presence of an EB at 200 μM (D) or 4 mM 15NH4+ (E). Values were calculated by subtracting the background of tko. Bars represent means ± SD (n = 4 biological replicates). Different letters indicate significant differences according to Tukey’s multiple test at p < 0.05. Underlying data can be found in S1 Data. 15NH4+, 15N-labeled ammonium; AMT, ammonium transporter; ATP, apoplastic transport pathway; DW, dry weight; EB, endodermal bypass; N, nitrogen; sgn3, schengen 3; STP, symplastic transport pathway; tko, amt1;1 amt1;2 amt1;3.
Fig 3.
Interaction of the EB with the ATP or STP.
(A) Experimental setup for the transport assay on horizontally split agar plates. Plants were placed in a way that only approximately 10 mm of the apical root zone were in contact with the 15N-containing segment, while shoots were placed on the N-free upper compartment. (B) Shoot DWs, and (C) 15N concentrations in shoots, as readout for radial transport rates. All plants were precultured on one-half MS agar with 1 mM nitrate for 5 d, then on N-free medium for 3 d before transfer to horizontally split agar plates with 4 mM 15NH4+ in the bottom segment. Plants were labeled with 15NH4+ for 6 h. Bars represent means ± SE (n = 6–8 biological replicates). Different letters indicate significant differences according to Tukey’s multiple test at p < 0.05. Underlying data can be found in S1 Data. 15NH4+, 15N-labeled ammonium; ATP, apoplastic transport pathway; DW, dry weight; EB, endodermal bypass; N, nitrogen; one-half MS, half-strength Murashige and Skoog basal salt mixture; STP, symplastic transport pathway.
Fig 4.
Contribution of the ATP or the STP to radial ammonium transport in the presence of a lignin biosynthesis inhibitor.
(A) Tile scans of representative roots, in which the presence of functional CS was verified by PI diffusion. White arrows indicate where PI diffusion was blocked, and inserts highlight PI distribution in selected root zones. Scale bars represent 500 μm in tile scans and 200 μm in inserts. (B) The length of the zone devoid of functional CS was estimated by measuring the length of the PI-penetrating zone. Treatment with PA did not significantly affect root growth rate (C) or shoot fresh weight (D). (E and F) 15N concentrations in shoots as readout for radial transport rates. tko, tko+1;2, and tko+1;3 plants were either exposed to (E) 0.05% DMSO or (F) 10 μM PA for 48 h before exposure of root segments to 4 mM 15NH4+. Precisely 7 mm of the apical root tips were exposed to 15NH4+, since PA treatment inhibited CS formation up to 9 mm (B). After 6 h, shoots were collected for 15N analysis. Bars represent means ± SE. P values were calculated using Student t test (n = 5–10 biological replicates). Underlying data can be found in S1 Data. 15NH4+, 15N-labeled ammonium; ATP, apoplastic transport pathway; CS, Casparian strip; N, nitrogen; PA, piperonylic acid; PI, propidium iodide; STP, symplastic transport pathway; tko, amt1;1 amt1;2 amt1;3.
Fig 5.
Contribution of the AMT1;2-mediated apoplastic and AMT1;3-mediated symplastic transport pathway to N partitioning between roots and shoots.
(A and C) Ratio of 15N concentration in shoots to 15N concentration in roots of tko or tko sgn3 without or with reconstituted expression of AMT1;2 (+1;2) or AMT1;3 (+1;3). (B and D) Estimated contribution of the ATP or STP in the absence or presence of an EB at 200 μM (B) or 4 mM 15NH4+ (D). Values were calculated by subtracting the background of tko. All plants were grown hydroponically for 5 weeks in nutrient solution containing 2 mM nitrate, followed by 3 d of N starvation before transfer to 15NH4+ labeling for 1 h. Shoot-to-root 15N concentration ratio was compared when 15NH4+ was supplied at 200 μM (A) or 4 mM concentration (C). Bars represent means ± SD (n = 4 biological replicates). Different letters indicate significant differences according to Tukey’s multiple test at p < 0.05. Underlying data can be found in S1 Data. AMT, ammonium transporter; ATP, apoplastic transport pathway; EB, endodermal bypass; N, nitrogen; 15NH4+, 15N-labeled ammonium; sgn3, schengen 3; STP, symplastic transport pathway; tko, amt1;1amt1;2amt1;3.
Fig 6.
Longitudinal distribution of AMTs and apoplastic diffusion barriers along the root axis.
(A) Establishment of functional CSs (PI unblocked), patchy suberin, and continuous suberin along the root axis. CS formation started from the 13th elongated cell onward, indicating that the root zone below the 13th elongated cell down to the initiation of xylem cells represents an endodermal bypass even in WT plants. A zone of patchy suberization extended from the 27th elongated cell up to the 67th cell, from which onward suberin deposition became continuous. (B–D) Fluorol yellow staining shows the presence of suberin in endodermal cells of mature (B), middle (C), and young (D) apical root zones. (E–H) Quantitative assessment (E) and confocal images of root tissue expressing proAMT1;2:AMT1;2:GFP in mature (F), middle (G), and young root zones (H). (I–L) Quantitative assessment (I) and confocal images of root tissue expressing proAMT1;3:AMT1;3:GFP in mature (J), middle (K), and young root zones (L). Roots were stained with PI (magenta). Blockage of PI penetration into the apoplastic space of the stele shows the presence of functional CSs. Overlay of PI and GFP gives white signal. Scale bars in D, H, and L represent 50 μm. In B–D, F–H, and J–L, representative images of >15 plants are shown. In A, E, and I, the data represent means ± SD (n ≥ 15 roots). Numbers in brackets are mean values. “Onset of elongation” is defined as the point at which length of an endodermal cell was more than twice its width. Underlying data can be found in S1 Data. AMT, ammonium transporter; Cor., cortex; CS, Casparian strip; End., endodermis; Epi., epidermis; GFP, green fluorescent protein; PI, propidium iodide; WT, wild-type.
Fig 7.
Estimated contribution of the EB, STP, and ATP to radial ammonium movement in different zones along the root of WT plants.
(A) Schematic arrangement of apical root zones expressing different combinations of AMT1;2 and AMT1;3 and apoplastic barriers. (B and C) Relative contribution of the EB as well as the ATP and STP in the high-affinity (B) or low-affinity range (C). The root axis is divided into 4 zones based on longitudinal gradients of CSs and patchy or continuous suberin. Calculations on the relative contribution of individual pathways are based on radial transport rates measured in hydroponic experiments (Fig 2 and S2 Table). The red shaded area indicates synergistic action between the EB and STP. Underlying data can be found in S1 Data. AMT, ammonium transporter; ATP, apoplastic transport pathway; CS, Casparian strip; EB, endodermal bypass; STP, symplastic transport pathway; WT, wild-type.
Fig 8.
Interaction between the EB and the STP in a root zone–dependent 15N-labeling assay.
(A) Experimental design for the assessment of root zone–dependent radial transport pathways. Plants were placed in a way that root segments of different lengths were in contact with 15NH4+, while shoots were placed on the N-free upper compartment. (B) Correlation between “labeled root length” and “labeled elongated cell number.” (C) 15N contents in shoots resulting from exposure of indicated root segments to 15NH4+. All plants were precultured on one-half MS agar with 1 mM nitrate for 5 d, then on N-free medium for 3 d before transfer to horizontally split agar plates with 4 mM 15NH4+ in the bottom segment. Values were normalized to labeled root length. Bars represent means ± SE (n = 6–9 biological replicates). Significant differences are indicated by different letters at p < 0.05 according to Tukey’s test. Underlying data can be found in S1 Data. EB, endodermal bypass; N, nitrogen; 15NH4+, 15N-labeled ammonium, one-half MS, half-strength Murashige and Skoog basal salt mixture; STP, symplastic transport pathway.