Impact of an extended endodermal bypass in sgn3 for radial element transport in roots

The sgn3 mutant was used to dissect the apoplastic route into an ATP, which entails an obligatory transporter-mediated step at the plasma membrane of the endodermis (Fig 1A, blue), and an endodermal bypass (Fig 1A, purple), allowing for unhindered diffusion across the endodermal layer. To quantify radial transport, we first exposed N-deficient plants to ¹⁵NH₄⁺ and compared ¹⁵N accumulation in shoots to that in xylem exudates and found closely related patterns of ¹⁵N accumulation (S1 Fig). Since NH₄⁺ uptake rates and the expression of AMTs increase during daytime and decrease in the dark [17], we assessed shoot accumulation rates during the light period, when transpiration is high and root pressure low, thereby suppressing the effect of root pressure on xylem transport rates. In order to favor transpiration as the major driving force for radial transport of water and nutrients, we calculated radial transport rates based on tracer accumulation in shoots rather than in xylem saps. Referring tracer accumulation in shoots to root dry weight and time (“normalized shoot accumulation”) thus integrates the rates of radial transport and of xylem loading in roots. Since AMT2;1, a critical transporter for root-to-shoot NH₄⁺ translocation [13], was present in all tested lines, we assumed no significant changes in xylem loading and hence considered “normalized shoot accumulation” as a readout for radial substrate transport rates in roots.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g001

We then verified the relevance of an extended endodermal bypass in sgn3 by exposing hydroponically grown wild-type (WT) and sgn3 mutant plants to strontium ion (Sr²⁺), which, similar to calcium ion (Ca²⁺), is transported to shoots mainly via the ATP [18–21]. Indeed, compared to wild type, normalized shoot accumulation of Sr²⁺ was approximately 3- or 20-fold higher in sgn3 at low or high external Sr²⁺, respectively (Fig 1B and 1C). In roots, Sr²⁺ accumulation increased comparatively little (S2A and S2B Fig), indicating an extraordinary impact of the endodermal bypass generated in the sgn3 mutant to radial transport and delivery of Sr²⁺ to the shoot. In the case of NH₄⁺, the impact of the endodermal bypass on shoot or root accumulation of NH₄⁺ was insignificant (Fig 1D and 1E, S2C and S2D Fig), suggesting that the action of the whole set of dedicated NH₄⁺ transporters in WT and sgn3 plants dominated over the contribution of an endodermal bypass. Thus, the relevance of a purely apoplastic pathway in the form of an extended endodermal bypass for radial element transport depends on the substrate and the presence of transporter-mediated radial pathways.

In agreement with an earlier study showing that phenotypical changes in sgn3 plants are highly dependent on the prevailing environmental conditions [9], this mutant had significantly less root and shoot biomass than the wild type (S3A and S3C Fig). Potential pleiotropic effects arising from the sgn3 mutation were assessed by verifying the relation between biomass and normalized shoot accumulation of NH₄⁺ in esb1 and myb36, 2 other CS-defective mutants [4,5]. Both mutants showed a similar decrease in shoot and root dry weight to sgn3 (S3A and S3C Fig), but their normalized shoot accumulation of ¹⁵N was 20%–25% lower and went along with increased root accumulation of ¹⁵N (S3B and S3D Fig). Thus, we concluded that radial transport rates of NH₄⁺ were not primarily affected by plant biomass but rather by the properties of existing apoplastic barriers. Unlike sgn3, the CS defects of esb1 and myb36 are partially compensated for by a stronger and earlier suberization of endodermal cells [4,5], which makes endodermal transporters inaccessible for their substrates [21]. Thus, the decreased biomass of sgn3 is not specific to the sgn3 mutation but most likely the consequence of a leaky CS. To compensate for the differences in root dry weight, we normalized all shoot accumulation rates to root dry weight, as practiced in other transport studies [22, 23].

Quantitative contribution of different pathways to radial ammonium transport in roots

To quantify the contribution of individual pathways to radial NH₄⁺ transport, we introgressed the sgn3 mutation into the triple amt1;1 amt1;2 amt1;3 (tko) knockout line, which has only 5%–10% of the wild type capacity for high-affinity NH₄⁺ uptake [12]. The obtained quadruple knockout (tko sgn3) had an extended endodermal bypass (Fig 2A) and smaller rosette leaves than tko but no visible symptoms of nutrient deficiency (S4 Fig). The presence of such endodermal bypass in a root devoid of the 3 major NH₄⁺ uptake transporters increased significantly the normalized shoot accumulation of NH₄⁺-derived N but only at high external supply (tko versus tko sgn3; Fig 2B and 2C). Although alternative uptake pathways for NH₃ or NH₄⁺ likely exist in roots, the absence of a significant difference between wild type and sgn3 (Fig 1D and 1E) suggests that the increased normalized shoot accumulation of tko sgn3 relative to tko plants at high external ammonium (Fig 2C) is most likely due to the extended endodermal bypass rather than the action of low-affinity transport pathways.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g002

To compare the contribution of the endodermal bypass with that of the ATP or STP, we generated tko lines with reconstituted expression of either endodermal AMT1;2 (tko+1;2), thus installing the end point for the ATP at the endodermis, or epidermal AMT1;3 (tko+1;3), thereby establishing an early entry into the STP (Fig 2A). At low external ¹⁵NH₄⁺, symplastic transport via AMT1;3 alone conferred slightly higher normalized shoot accumulation for the tracer than the AMT1;2-dependent ATP (Fig 2B and 2D). At the same time, ¹⁵N accumulation in roots was significantly higher in tko+1;3 than in tko+1;2 (S5A and S5B Fig). However, at elevated substrate levels, the difference between tko+1;3 and tko+1;2 to tko reversed (Fig 2C), and the estimated contribution of the AMT1;2-dependent ATP to shoot ¹⁵NH₄⁺ accumulation became twice as high as that through the STP (Fig 2E). Notably, the higher normalized shoot accumulation attributed to the ATP was independent of ¹⁵N accumulation in roots (S5C and S5D Fig), indicating that root and shoot accumulation of NH₄⁺-derived N were uncoupled. The radial transport capacity of AMT1;1, AMT1;2, and AMT1;3 together, as reflected by the difference of normalized shoot accumulation between wild type and tko (Figs 1D, 1E, 2B and 2C), was somewhat lower than the sum of the 2 individual capacities conferred by AMT1;2 and AMT1;3 (Fig 2D and 2E). This was not unexpected with regard to the fact that AMT1;1 and AMT1;3 can individually compensate for the lacking uptake capacity of each other because the formation of a heterotrimeric complex and concomitant posttranslational down-regulation of interacting monomers was no longer possible [12, 24]. This was also the reason why the contribution of AMT1;1 could be disregarded here.

In conclusion, at low external substrate concentrations, the contribution of the STP to radial transport of NH₄⁺-N prevails over the ATP, whereas at millimolar NH₄⁺ supply, the ATP mediated by AMT1;2 confers an approximately 2-fold higher capacity than that of the symplastic route. These results represent, to our knowledge, the first quantitative comparison of individual radial transport pathways for any nutrient.

Interaction between the endodermal bypass and the symplastic or apoplastic transport pathway

To investigate the cross-talk between radial transport pathways, we hypothesized that the presence of an extended endodermal bypass will decrease radial ¹⁵NH₄⁺ transport via the apoplastic or symplastic pathway because tracer reaching the stele may leak out in the absence of a functional CS. Indeed, shoot ¹⁵NH₄⁺ accumulation via the AMT1;2-dependent apoplastic pathway in the presence of an endodermal bypass (i.e., tko sgn3+1;2 versus tko sgn3) revealed similar or significantly lower ¹⁵NH₄⁺ accumulation relative to the contribution of the ATP alone (Fig 2B and 2C). These results indicated that AMT1;2-mediated ¹⁵NH₄⁺ transport across the endodermis was compromised by concomitant NH₄⁺ efflux through an endodermal bypass. This observation highlights that an intact CS improves the efficiency of the apoplastic transport route by limiting apoplastic backflow out of the vasculature. Unexpectedly, the interaction between an endodermal bypass and the STP was opposite: normalized shoot accumulation of ¹⁵NH₄⁺ via AMT1;3 was significantly higher in the tko sgn3 background (tko sgn3+1;3 versus tko sgn3) than in the tko background (tko +1;3 versus tko) (Fig 2B and 2C). Importantly, the estimated contribution of the STP in the presence of an endodermal bypass is higher than either of these transport routes alone, indicating a synergistic interaction between the STP and the endodermal bypass (Fig 2D and 2E). Presumably, part of the NH₄⁺ transported via the symplastic route was exported into the apoplast during the radial move and profited from unhindered apoplastic diffusion at the endodermis to reach the vascular system. The opposite contribution of AMT1;2 and AMT1;3 in the tko sgn3 background was not due to altered gene expression, as the expression of AMTs was not significantly affected by the sgn3 mutation (S6 Fig). Moreover, we supplied sufficient amounts of potassium (K) in our hydroponic solution to prevent latent K deficiency in sgn3 [9]. With this measure, sgn3 mutants showed no symptoms of nutrient deficiency, and despite slight variations in the accumulation of other mineral elements, all of these were in a usual physiological range and far away from critical deficiency or toxicity levels [25] (S4A and S7 Figs, S1 Table). Thus, our elemental analysis supported that the results with the sgn3 mutant were largely independent of mineral element disorders or potential interactions between K⁺ and NH₄⁺ at the level of uptake or xylem loading.

To verify the opposite interaction between an extended endodermal bypass and the 2 radial transport pathways in an independent growth system, we carried out a transport assay on horizontally split agar plates, in which ¹⁵NH₄⁺ was supplemented only to the lower agar segment (Fig 3A). Roots of N-deficient plants were placed across the trench, separating the upper and lower agar compartments so that only approximately 10 mm of the apical root zone were in contact with the ¹⁵N-containing segment. At this developmental stage, the shoot biomass between tko lines and tko sgn3 lines was almost indistinguishable (Fig 3B). As labeled root segments were too small to measure root dry weights, we determined ¹⁵N concentrations in shoots as readout for radial transport of ¹⁵NH₄⁺-N. Shoot ¹⁵N concentrations were higher in tko sgn3+1;3 plants than in tko+1;3, thus confirming the synergistic interaction between an endodermal bypass and the STP. On the other hand, we observed a slightly decreased contribution of AMT1;2 in the tko sgn3 background (tko sgn3+1;2 versus tko+1;2; Fig 3C), further supporting that an endodermal bypass antagonizes the ATP.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g003

To further validate our findings independently of the sgn3 mutation, we treated WT plants with the lignin biosynthesis inhibitor piperonylic acid (PA), which blocks CS formation and hence partially mimics the CS defects obtained by mutating SGN3 [3]. Since the PA effect is confined to newly grown root portions, we carried out the experiment on agar plates and limited the PA treatment to 48 h. Within this period, the zone of unrestricted penetration of the apoplastic tracer propidium iodide (PI) greatly expanded, as the endodermal bypass was now open up to >8 mm from the root tip (Fig 4A and 4B). Notably, this short-term disturbance of CS formation in PA-treated roots did not significantly compromise root growth or shoot biomass formation (Fig 4C and 4D). We then exposed the apical 7 mm of the root tip to ¹⁵NH₄⁺ in order to determine the contribution of PA-dependent endodermal bypass. Shoot ¹⁵N concentrations were strongly suppressed by PA (Fig 4E and 4F), indicating an inhibitory side effect of PA on root physiology. Nevertheless, when compared to tko, the endodermal bypass created by PA decreased shoot ¹⁵N accumulation in tko+1;2 further but increased shoot ¹⁵N accumulation in tko+1;3, indicating a synergistic interaction (Fig 4E and 4F). Thus, 3 independent approaches provided evidence for the opposite interaction of the endodermal bypass with either the ATP or STP in roots.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g004

Distinct roles of AMTs in ammonium partitioning between roots and shoots

The overall dominant contribution of the STP to radial nutrient transport raised the question of the biological significance of the ATP. We thus compared root-to-shoot translocation in our mutant lines by calculating shoot-to-root ¹⁵N concentration ratios. At micromolar NH₄⁺ supply, AMT1;2 conferred in the tko background a slightly higher increase in ¹⁵N translocation to shoots than AMT1;3, while in the presence of an endodermal bypass, AMT1;3 delivered the most N to shoots, reflecting the synergistic interaction with the endodermal bypass (Fig 5A and 5B). At millimolar supply, shoot N provision profited most from NH₄⁺ delivered by the AMT1;2-dependent apoplastic pathway or from NH₄⁺ entering the vasculature via the endodermal bypass (Fig 5C). Without the presence of an endodermal bypass, the AMT1;3-dependent STP alone made no contribution to ¹⁵N allocation to shoots (Fig 5D). We thus concluded that the AMT1;2-mediated ATP favors partitioning of NH₄⁺-N to the shoots and contributes to shoot N provision particularly at elevated external supply.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g005

Relative contribution of individual pathways to radial NH₄⁺ transport along the root axis

Since the occurrence of CSs and suberin lamellae show distinct developmental gradients along the root axis [3,26], we compared their localization with that of the 2 investigated AMTs. The root zone below the 13th elongated cell down to the initiation of xylem cells, which is devoid of a functional CS or suberin deposition, represents an endodermal bypass even in WT plants (Fig 6A and S8 Fig). AMT1;2 was absent from the root tip and present at the endodermis from the 11th to the 60th elongated cell (Fig 6E–6H and S8A–s8D Fig). Notably, from the 30th cell onward, this transporter was also detected in cortical cells, while shifting completely to the cortex from the 60th cell onward. This shift in cell type–specific expression coincided with suberin formation and indicated that the presence of suberin in endodermal cells influenced cell type–specific AMT1;2 localization (Fig 6A–6E and S8A–S8D Fig). By contrast, AMT1;3 expression started from the very root tip in epidermal cells and expanded toward the cortex from the 26th cell onward (Fig 6I–6L and S8E–S8H Fig).

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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.

https://doi.org/10.1371/journal.pbio.2006024.g006

Regarding the establishment of functional radial transport pathways, the localization pattern of the 2 NH₄⁺ transporters and the apoplastic barriers allowed differentiating 4 zones along the axis of an Arabidopsis root (Fig 7A). We then projected our estimates for the contribution of the individual radial pathways along the longitudinal axis of a WT root to build a model that estimates the relative contribution of different transport pathways to radial NH₄⁺ transport in roots. In the high-affinity range, for which up to 80% of radial NH₄⁺ transport depends on AMTs, the EB does not contribute to radial NH₄⁺ transport along the whole root axis (Figs 2B and 7B, and S2 Table). In “zone 1,” radial transport is completely dominated by the AMT1;3-dependent STP, which profits from the synergistic action of the EB. Once AMT1;2 is expressed, its contribution is rather modest (approximately 16%), as it is negatively affected by the presence of an EB. When the CS becomes established (zone 2), the STP and ATP contribute by 45% and 34%, respectively (Figs 2B and 7B, and S2 Table). With progressing suberization of the endodermis (zone 3 + zone 4), the estimated contribution of the ATP ceases and is taken over by a combined pathway, which is defined by the expression of both transporters in cortical cells mediating uptake of apoplastically transported NH₄⁺ across the epidermal layer into the symplastic route.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g007

In the low-affinity range, the overall contribution of the AMT- and sgn3-dependent pathways is only 45%–65%, as NH₄⁺ is transported additionally by not yet fully characterized low-affinity transporters (Figs 2C and 7C, and S2 Table). In this case, the endodermal bypass contributes by 22% to radial NH₄⁺ transport in “zone 1.” AMT1;3-mediated symplastic transport, in turn, contributes by 29%, albeit in a synergistic manner, with the endodermal bypass. In contrast, the contribution of AMT1;2 is strongly decreased by the endodermal bypass (Fig 2C). In “zone 2,” AMT1;2-dependent apoplastic transport dominates radial NH₄⁺ transport, with 30% over the 15% of AMT1;3-dependent symplastic transport. In “zone 3,” the contribution of the ATP decreases due to suberization of endodermal cells, and in “zone 4,” the contribution was taken over by a combined pathway.

To validate our model, we exposed apical root tips of different length to a ¹⁵NH₄⁺-containing agar plate compartment and correlated root lengths touching the tracer with counted elongated cell numbers (Fig 8A and 8B). Samples were grouped into 4 classes, corresponding to the 4 zones with distinct combinations of transport pathways along the root axis (Fig 7A). With this procedure, radial transport of ¹⁵NH₄⁺ was integrated over longitudinal root segments with different sets of radial transport pathways. Longitudinal gradients of CS or suberin were neither affected by the sgn3 mutation nor by AMT1;3 expression (S9 Fig). Shoot ¹⁵N accumulation after 4 h of labeling was related to root length and taken as readout for the radial transport rate of each root zone (Fig 8C). In all lines, the highest radial transport rate was detected in the most apical part. The contribution of the STP (tko+1;3 versus tko) to radial transport, here represented by shoot accumulation of ¹⁵NH₄⁺, was particularly significant in “zone 1” (<5 mm), where CS are not yet formed (Figs 7A and 8C). The endodermal bypass alone (tko sgn3 versus tko) had no significant impact on shoot ¹⁵N accumulation. The comparison between tko+1;3 and tko sgn3+1;3 showed that loss of a functional CS increased AMT1;3-mediated symplastic transport capacity, especially in “zone 3” and “zone 4” (Fig 8C), further indicating a synergistic effect between the STP and the EB.

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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.

https://doi.org/10.1371/journal.pbio.2006024.g008

Discussion

While the contribution of the ATP and STP to radial transport of nutrients through the root tissue has been adopted as a general principle in classical textbooks [6,7], the quantitative share between these 2 pathways for radial transport of any nutrient has remained elusive. Here, we took advantage of the differential cell type–specific expression of AMT1–type transporters in combination with the CS-defective mutant sgn3 to dissect radial transport pathways for NH₄⁺ and to determine their quantitative contribution. Thereby, we discovered previously unanticipated interactions between radial transport pathways and differential roles of ATP and STP for nutrient partitioning between roots and shoots.

A prerequisite for the dissection of radial transport pathways is the availability of membrane transport proteins mediating either early substrate passage via the epidermis into the symplastic continuum or completing the apoplastic transport by transmembrane passage into endodermal cells. Unlike for most other nutrients, such transporters were known for NH₄⁺ and previously characterized for their contribution to root uptake [12,16]. Assessing their contribution to radial NH₄⁺ transport required a longer period of exposure to the tracer and revealed a higher capacity of the AMT1;3-mediated symplastic route at low external NH₄⁺ supply but a higher capacity of the AMT1;2-mediated apoplastic route at elevated supply (Fig 2B–2E). Although AMT1;2 is characterized by lower substrate affinity, i.e., 234 μM relative to 61 μM for AMT1;3 [12], better adapted biochemical transport properties to higher apoplastic NH₄⁺ concentrations alone cannot explain its superior contribution to shoot NH₄⁺ accumulation (Fig 5). AMT1;2-dependent NH₄⁺ transfer to shoots was uncoupled from NH₄⁺ accumulation in roots (Fig 2E and S5D Fig) because apoplastically transported NH₄⁺ circumvents retention by root cells in favor of direct movement to the stele. Thus, the ATP may function as a “fast track” for nutrient delivery to the shoot. The reason for its greater importance at elevated external substrate supplies may lie in a lower number of low-affinity NH₄⁺ transporters competing with AMT1;2 for NH₄⁺ transport across the endodermis, whereas low-affinity NH₄⁺ transporters in epidermal cells may be more abundant, masking the contribution of AMT1;3. At least this holds true for AMT2;1, which has been shown to localize in the epidermis and cortex of N-deficient roots, where it confers approximately 10%–25% of the low-affinity uptake capacity [13]. A supposedly differential contribution of these poorly characterized low-affinity transport systems for NH₄⁺ in different cell types may restrict direct comparison of AMT1;2- versus AMT1;3-mediated radial transport capacities. In the high-affinity range, >90% of the NH₄⁺ uptake capacity in roots relies on AMT1;1, AMT1;2, and AMT1;3 [12]. Respecting the compensatory increase in AMT1;3 capacity in the absence of AMT1;1 [12,24], quantitative estimates of the share between apoplastic and symplastic pathways to radial transport in the high-affinity range are unlikely to be affected by other transport systems.

It is important to note that the contribution of an extended endodermal bypass might change according to growth conditions, particularly to substrate concentration, root pressure, or transpiration rates. Therefore, we assessed ¹⁵N shoot accumulation during the light phase, when transpiration is high and root pressure is low, thereby suppressing the effect of root pressure on xylem transport rates. Such conditions allowed investigating the contribution of AMT1;2- and AMT1;3-dependent pathways in presence or absence of an extended EB more directly and at a higher resolution. In our experimental conditions, the ATP reached highest capacity when CSs were intact, and this capacity was as high as that mediated by the EB alone (Fig 2C). We therefore conclude that the membrane transport steps required for xylem loading, i.e., NH₄⁺ transfer to pericycle cells and subsequent export to xylem vessels, are not limiting for root radial transport. Hence, a quantitative comparison between the ATP and STP for NH₄⁺, as addressed by our approach, appears valid.

Apart from determining the contribution of individual pathways to radial NH₄⁺ transport, we also focused on their interplay. This question is of particular biological relevance due to the longitudinal gradients of apoplastic barrier formation, which generate a suite of possible radial pathway interactions. At the primary root axis, an interplay between all 3 pathways is confined to a small zone, in which epidermal AMT1;3 is coexpressed with endodermal AMT1;2, while CS or suberin are not yet formed (Figs 6 and 7). In the sgn3 mutant, the largely expanded zone with an endodermal bypass enhanced radial transport, as shown by higher normalized shoot accumulation of the apoplastic tracer Sr²⁺ (Fig 1B and 1C), whereas EB of NH₄⁺ was outcompeted by AMT-mediated radial transport pathways (Figs 1D–1E and 2B–2C). Surprisingly, we found that the EB has an opposing role on the 2 radial transport pathways, which we confirmed in 3 independent methodological approaches, i.e., by a) examining tko and tko sgn3 lines with reconstituted expression of AMT1;2 or AMT1;3 in 5-week-old hydroponically grown plants (Fig 2), b) in 8-d-old agar-grown plants (Fig 3), or c) by circumventing the use of the sgn3 background with the help of a lignin biosynthesis inhibitor (Fig 4). A compromising action of the EB on the ATP occurs when NH₄⁺ leaking out through a defective CS must be retrieved by AMT1;2, i.e., by the same transporter that is already engaged in the apoplastic transport route. Although the net contribution of an open EB was 0 at low external NH₄⁺ supply, it promoted shoot ¹⁵N accumulation via the AMT1;3-dependent symplastic route (Fig 2D). Presumably, part of NH₄⁺ transported via the symplastic route was exported into the apoplast during the radial move and reached the vascular system by profiting from unhindered apoplastic diffusion through the endodermal cell layer. NH₄⁺ efflux has been extensively characterized in physiological studies and shown to be dominated by the export of NH₃ [27, 28]. Currently, it is not clear which membrane transporters mediate NH₃ export from root cells, but export across the plasma membrane may be facilitated through NH₃-transporting aquaporins, as shown for tonoplast intrinsic proteins [29] or through AMT2;1, which alters its localization toward inner root cells under NH₄⁺ nutrition [13] and is able to permeate NH₃ [30]. Such synergistic interplay between the STP and the EB may be regarded as experimental evidence for the so-called coupled transcellular transport pathway [1]. This third transport pathway has been postulated to couple repeated steps of symplastic and apoplastic transport based on the polarized localization of importers and exporters, as shown for silicon or boron [31–33].

The relative contribution of the individual radial pathways as well as their interplay is subject to root zonation and the formation of apoplastic barriers. As radial NH₄⁺ transport via the AMT1;2-mediated ATP was decreased by an extended endodermal bypass in the most apical root zone and progressively sealed by suberin deposition in a shootward direction, an efficient ATP was restricted to a zone between 13 and approximately 67 cells above the first elongated root cell (Figs 6 and 7). Thus, the “fast track” for NH₄⁺ delivery to the shoot is already in place as soon as the primary root explores new nutrient-rich soil layers and establishes an almost equal share of NH₄⁺ partitioning between the shoot and the roots, since NH₄⁺ provision to roots depends mainly on the STP (Fig 5). Since elevated substrate supply can push the relative share of NH₄⁺ in favor of the shoot, apoplastic NH₄⁺ transport may finally contribute to enhanced shoot growth under ample N supply. From a general perspective, this principle appears advantageous during root foraging, when soil nutrients are vertically translocated and accumulate in deeper soil layers [34]. However, it is noteworthy that the apoplastic transport route is highly sensitive to environmental conditions. Abiotic stress factors, including high salt or K deficiency, accelerate the suberization of endodermal cells via abscisic acid (ABA) and can restrict the zone of substrate-accessible endodermal transporters to less than 50% [35]. This may also be the reason why the suberin-enriched mutants esb1 and myb36 showed lower normalized shoot accumulation of ¹⁵NH₄⁺ (S3 Fig). Endodermal transporters directly impact the nutrient composition of the shoot and, in turn, may be subject to straight control by the nutritional status of the shoot. Thus, characterizing transport processes across the endodermal plasma membrane in both directions and quantifying the contribution of the ATP versus the STP is highly relevant. Such knowledge might help specify breeding targets in plants, enabling more selective nutrient translocation to shoots, e.g., to meet the increasing N demand of shoots growing under elevated carbon dioxide (CO₂), decreasing shoot accumulation of xenobiotics, or fortifying seeds with essential mineral elements.

Generation of multiple mutant lines

tko plants (amt1;1amt1;2amt1;3) were obtained from a selfed F2 population after backcrossing a homozygous amt1;1-1amt1;3-1amt2;1-1amt1;2–1 quadruple insertion line [12] to WT Col-0. tko and sgn3;3 (in Col-0 background, SALK_043282) [9] were crossed and selfed to obtain an F2 population. Within the F2 population, homozygous lines of tko+AMT1;2 (tko+1;2), tko+AMT1;3 (tko+1;3), tko sgn3, tko sgn3+AMT1;2 (tko sgn3+1;2), and tko sgn3+AMT1;3 (tko sgn3+1;3) were selected by PCR. For details of primers for genotyping, see S3 Table. Homozygous tko and sgn3;3 from this population served as reference lines. All homozygous mutant lines were selfed, and seeds from F3 generations were used in all experiments. In this work, 1 biological replicate represents a sample consisting of 2 plants from the same line or treatment (except for xylem sap, for which 4 plants were combined as 1 sample). The number of biological replicates indicated in the figure legends represents the number of independent biological replicates (originating from same line/treatment but different pot or agar plate).

Measurement of normalized shoot accumulation rates in Arabidopsis thaliana

Plants were grown hydroponically for 5 weeks, as previously described [12,16]. Nitrate was used as the sole N source for preculture in all experiments. The climate chamber had a day–night regime of 10 h (22 °C)/14 h (18 °C), 200 μmol m⁻² s⁻¹ light intensity, and 70% humidity. Prior to labeling, plants were precultured for 3 d on an N-free nutrient solution. Just before ¹⁵N or Sr²⁺ labeling, plant roots were rinsed in 1 mM CaSO₄ for 1 min to remove nutrients from the apoplast and then transferred to the N-free solution containing different concentrations of (¹⁵NH₄)₂SO₄ (98 atom% ¹⁵N) or SrCl₂. After 1 h, roots were washed again in 1 mM CaSO₄ for 1 min to remove tracers from the apoplast. Shoots and roots were harvested, freeze dried, and ground to fine powder. To harvest xylem sap samples, plants were decapitated below the rosette, and xylem sap from 4 plants was combined in 1 sample. ¹⁵N concentrations were determined by isotope-ratio mass spectrometry (DELTAplus XP, Thermo-Finnigan). Other mineral elements were determined by high-resolution inductively coupled plasma mass spectrometry (Element 2, Thermo Fisher Scientific). Sr was determined by high-resolution inductively coupled plasma mass spectrometry (ICP-MS, Element 2, Thermo Fisher Scientific). Normalized shoot accumulation was calculated by normalizing total Sr²⁺ or ¹⁵N shoot content to root dry weight and labeling time. The formula used is as follows: ¹⁵N accumulation rate in shoot (or xylem exudate) = (sample ¹⁵N abundance − natural ¹⁵N abundance) × total N concentration × shoot (or xylem exudate) dry weight/(¹⁵N molecular weight × ¹⁵N purity × labeled time × root dry weight). Natural ¹⁵N abundance was obtained by measuring unlabeled leaf material. For harvest, 2 plants were taken as 1 sample in 4 replicates for each data point. Normalized shoot accumulation in different lines was compared in 3 independent experiments.

Microscopy, histology, and quantitative analysis

Construction of promoterAMT1;2:ORF:GFP and promoterAMT1;3:ORF:GFP lines were described previously [12,16]. Plants were precultured on a one-half Murashige and Skoog (MS) medium containing 1 mM NO₃^- as the sole N source, 2.5 mM 2-(N-morpholino) ethanesulfonic acid (MES) (pH 5.7), 1% sucrose, and 0.8% Difco agar (Becton Dickinson). Five-d-old seedlings were transferred to the N-free one-half MS medium for 3 d. N-deficient plants were used for visualization of GFP and root barriers. For visualizing propidium iodide (PI) penetration, plants were stained with PI (10 μg mL^-1) for 10 min in the dark [3,36]. PI-dependent red fluorescence and AMT:GFP–dependent green fluorescence in different root zones were observed with a confocal laser scanning microscope Zeiss LSM 780 (Carl Zeiss). Excitation/emission wavelengths of 488 nm/490–540 nm and 561 nm/650–710 nm were used for detection of GFP signals and PI signals, respectively. Cross sections from each zone were reconstructed from z-stacks of longitudinal confocal sections. For visualizing suberin, plants were stained with Fluorol Yellow 088, as described previously [3], and observed with a conventional light microscope (Zeiss Axio Imager M2, Carl Zeiss) using a GFP filter. For quantification, “onset of elongation” was defined as the zone where an endodermal cell length was more than twice its width [3]. More than 20 plants were counted. Experiments were repeated at least 3 times.

¹⁵N-labeling assay for root zone–dependent radial transport

Arabidopsis seeds were surface sterilized and precultured on one-half MS medium containing 1 mM NO₃^- as the sole N source, 2.5 mM MES (pH 5.7), 1% sucrose, and 0.8% Difco agar (Becton Dickinson). Five-d-old seedlings were transferred to one-half MS medium without N and supplemented with 2.5 mM MES (pH 5.7), 1% sucrose, and 0.8% Difco agar. After 3 d, plants with similar root size were transferred to horizontally split agar plates containing the ¹⁵N tracer in the bottom segment. To prepare the ¹⁵N-labeled agar plates, 50 ml one-half MS medium without N, supplemented with 2.5 mM MES (pH 5.7) and 1% Difco agar, was spread on the agar in a 12-cm square Petri dish. No sucrose was added in order to slow down the root growth. A trench of 5-mm width was cut in the solidified agar to avoid ¹⁵N diffusion between the upper and lower agar segments. (¹⁵NH₄)₂SO₄ (98 atom% ¹⁵N) stock solution was spread on the lower agar segment to reach a final ¹⁵NH₄⁺ concentration of 4 mM. Petri dishes were placed in vertical orientation in a growth cabinet at a day–night regime of 10 h (22 °C)/14 h (19 °C) and a light intensity of 120 μmol m^-2 s^-1 during the day period. In each treatment, 10 plants were placed on the agar plate in a way that approximately 2 mm, 5 mm, 10 mm, or 15 mm of the apical primary root zone touched the ¹⁵N-containing bottom agar segment, while the shoot was placed on the upper segment (-N agar). Eight-d-old seedlings were used for this ¹⁵N transport assay to avoid interference from lateral roots and to make sure that only a predefined root portion was in contact with the tracer. For each line, 30 plants were placed on 3 independent plates to normalize the variation on different agar plates. After 4 h of incubation, shoots were separated at the hypocotyl and dried separately in tin capsules. To measure the shoot ¹⁵N concentration in each plant, whole shoots (about 40 μg dry weight) were mixed with BBOT isotope reference standard (atom percent: 0.366, N percent: 6.5, HEKAtech GmbH) in order to reach the detection limit for total N. Shoot ¹⁵N concentration was determined by isotope-ratio mass spectrometry (DELTAplus XP, Thermo-Finnigan). Labeled root segments were cut off, mounted on slides, and scanned by an Epson Expression 10000XL scanner (Seiko, Epson). Root lengths were determined by Smartroot software V4.126, and the number of cells from the start of cell elongation onward was counted under differential interference contrast using a conventional light microscope (Zeiss Axio Imager M2, Carl Zeiss). Since the roots grew nearly 2 mm during this 4-h period, final labeled root length was grouped into classes of <5 mm, <10 mm, <15 mm, and <20 mm. For each root length class, 6–9 plants from 3 agar plates were analyzed to yield 1 data point.

Lignin inhibitor assay

Col-0 plants were precultured on one-half MS agar with 1 mM NO₃^- for 5 d and then transferred for 3 d to agar plates without N. During the last 48 h of growth on the N-free medium, part of the plants were transferred to the same medium containing either 0.05% DMSO (control) or 10 μM of the lignin biosynthesis inhibitor piperonylic acid (PA). To verify the presence of functional CS, roots were stained with PI (10 μg mL^-1) for 10 min in the dark. For shoot ¹⁵N analysis, tko, tko+1;2, and tko+1;3 plants were precultured as described above and then either exposed to 0.05% DMSO or 10 μM PA for 48 h before transferring to horizontally split agar plates. Since PA treatment inhibited CS formation up to 9 mm, precisely 7 mm of the apical root tips were exposed to 4 mM ¹⁵NH₄⁺. After 6 h, shoots were collected for ¹⁵N analysis, as described above.

Real-time quantitative PCR

Total RNA was extracted using the QIAzol Lysis reagent (Qiagen) following the manufacturer’s instructions. Prior to cDNA synthesis, samples were treated with DNase (Thermo Fisher Scientific). Reverse transcription was performed using SuperScript II (Thermo Fisher Scientific) reverse transcriptase and Oligo(dT)12-18. Real-time PCR was performed using a Mastercycler ep realplex (Eppendorf) and QuantiTect SYBR Green qPCR mix (Qiagen) using the primers listed in S3 Table. Relative transcript levels were calculated according to Vandesompele and colleagues (2002) [37].