Nitrate deficiency induces differential endocytosis in roots through NRT1.1

ABSTRACT

Roots grow asymmetrically, sometimes helically, around their growth direction likely to facilitate environmental sensing. We recently demonstrated that nitrate deficiency induces root coiling on horizontal surface through nitrate transporter/sensor NRT1.1 and PIN2- and AUX-mediated polar auxin transport. Here, we show that nitrate deficiency or NRT1.1 loss-of-function induces differential distribution of PIN2 between the future concave and concave sides in root epidermal cells. Treatment with pharmacological drugs suggests that enhanced endocytosis at the future convex side leads to reduced plasma membrane (PM) association of PIN2. A reduction of PIN2 at the PM would maintain a low auxin response to further enhance endocytosis at the convex side, leading to root coiling.

KEYWORDS:

• Nitrate
• asymmetric root growth
• endocytosis
• auxin
• BFA

Plant roots grow asymmetrically, sometimes helically, around their growth direction likely to facilitate environmental sensing and to increase plant fitness. Asymmetric root growth (ARG) is best studied in root responses to gravity and mechanical stresses. Gravitropism enables deep rooting for more efficient nutrient absorption while thigmotropism allows roots to be away from obstacles and therefore to avoid harsh growth conditions. However, asymmetry of root growth was also observed at the absence of gravity or touch,¹ a behavior named circumnutation, i.e. the helical movement that all plant organs produce around the growth direction.^2–4

ARG is developmentally regulated.^5,6 ARG in general involves microtubule (MT) reorganization. Mutations at MT plus end-binding proteins EB1⁷ and SKU6/SPIRAL1,⁸ or variants of α-tubulin,⁹ cause ARG. By contrast, several genetic factors, including two seven-transmembrane proteins, the MILDEW RESISTANCE LOCUS O (MLO) family members,^10,11 and the heterotrimeric G proteins XLG3 and AGB1,¹² are involved in ARG in a gravity- or touch-independent way. ARG requires polar auxin transport,^1,5,10–13 most importantly through the activities of auxin carriers, such as PIN-FORMED2 (PIN2) and AUXIN RESISTANT 1 (AUX1).^14–20

We recently showed that nitrate deficiency resulted in a strong ARG in Arabidopsis. Nitrate deficiency caused root coiling on horizontal plates, which is inhibited by functional loss of PIN2 and AUX1. In addition, suppression of ARG by nitrate is mediated by the nitrate transporter/sensor NRT1.1.²¹

Because either nitrate deficiency or NRT1.1 loss-of-function resulted in an asymmetric distribution of PIN2 at the PM, i.e. lower signals at the convex than at the concave,²¹ we wondered whether it was a result of asymmetrically enhanced endocytosis of PIN2, as reported previously during gravitropic responses.^14,22,23 To test this hypothesis, we analyzed PIN2:GFP plants by using confocal laser scanning fluorescence microscopy (CLSM) and pharmacological treatments. BRASSINOSTEROID INSENSITIVE1:YFP (BRI1:YFP) plants was also included as a control since BRI1 is constitutively internalized without showing asymmetry at the PM.²⁴ Brefeldin A (BFA) is a fungal toxin that inhibits exocytic trafficking and causes aggregation of endosomes.²⁵ Cycloheximide (CHX), an inhibitor of de novo protein synthesis, was applied (50 µM) during BFA treatment (50 µM) to exclude the influence of the newly synthesized proteins. After being transferred to horizontally placed medium, PIN2:GFP showed a significantly reduced intensity at the PM of the future convex on 0 N medium (Supplemental Methods), compared with that on 7 N medium (Figure 1a, c). By contrast, there was no significant difference of BRI1:YFP intensity at the PM in all samples (Figure 1b, e). Treatment of BFA resulted in the accumulation of PIN2:GFP into so-called “BFA compartments” (Figure 1a), i.e. cytoplasmic aggregates containing both trans-Golgi network/early endosomes and the Golgi.²⁵ The intensity ratio of PIN2:GFP between BFA compartment-trapped and PM-associated was significantly increased at the future convex side compared with that at the future concave on 0 N medium but not 7 N medium (Figure 1d), suggesting asymmetric endocytosis upon nitrate deficiency. The differential effect of nitrate deficiency on endocytosis is not specific on PIN2 because the intensity ratio of BRI1:YFP between BFA compartment-trapped and PM-associated was also significantly increased at the future convex side compared with that at the future concave on 0 N medium but not 7 N medium (figure 1f).

Figure 1. Nitrate deficiency induces differential dynamic distribution of PIN2 in horizontally placed roots.

(A‐B) CLSM of a PIN2:GFP (A) or BRI1:YFP (B) root at elongation zone. Roots of 4 DAG seedlings were transferred to 7 N or 0 N medium for 2 days and then treated with 50µM CHX for 30 min, and then stained with FM4‐64 before imaging (DMSO), or roots of 4 DAG seedlings were transferred to 7 N or 0 N medium for 2 days and then treated with 50 µM CHX for 30 min, pulse‐labeled with FM4‐64, and then incubated with 50µM CHX/50 µM BFA for 45 min before imaging (BFA). The GFP channel images are displayed in pseudocolors, covering the full range of measured values within each dataset. cc, concave; cv, convex. Arrowheads point at the PM of root epidermal cells; arrows at BFA compartments. (C‐F) Quantification of fluorescence intensity at the PM (C, E) or intensity ratio between BFA compartments and the PM (D, F) of PIN2:GFP (C,D) or BRI1:YFP (E, F). a.u., arbitrary fluorescence unit. Results are means ± standard deviation (SD). In total, 30‐40 cells from four independent experiments involving 15 roots in each genetic background or treatment were analyzed. P values between concave (cc) and convex (cv) are shown in the bar graphs (Unpaired t‐test). Bars = 10 μm.

Functional loss of NRT1.1 caused the same differential endocytosis on PIN2 as that caused by nitrate deficiency (Figure 2). Examination of PIN2 distribution at the PM in PIN2:GFP;chl1-5 plants on 7 N medium showed that PM-association of PIN2 was reduced at the future concave side (Figure 2a, c). Upon BFA treatment, significantly higher ratio between BFA compartment-associated and PM-associated PIN2 signals was detected at the future concave side (Figure 2b, d), suggesting differential endocytosis in root epidermal cells by NRT1.1 loss-of-function.

Figure 2. Chl1‐5 shows differential dynamic distribution of PIN2 in horizontally placed roots.

(A‐B) CLSM or a PIN2:GFP;chl1‐5 root upon DMSO (A) or BFA (B) treatment. The GFP channel images are displayed in pseudocolors, covering the full range of measured values within each dataset. cc, concave; cv, convex. Arrowheads point at the PM of root epidermal cells; arrows at BFA compartments. (C‐D) Quantification of fluorescence intensity at the PM (C) or intensity ratio between BFA compartments and the PM (D) of PIN2:GFP;chl1‐5. a.u., arbitrary fluorescence unit. Results are means ± SD. In total, 30‐40 cells from four independent experiments involving 15 roots in each genetic background or treatment were analyzed. P values between concave (cc) and convex (cv) are shown in the bar graphs (Unpaired t‐test). Bars = 10 μm.

We show that nitrate deficiency or NRT1.1 loss-of-function resulted in enhanced internalization of PIN2, indicative of enhanced endocytosis at the future convex side. The asymmetry of PIN2 endocytosis is likely resulted from a general reduction of endocytosis at the future convex side^26,27 because the dynamic localization of BRI1:YFP were also affected by nitrate deficiency. Auxin inhibits endocytosis,²³ and thus the future concave side with higher auxin responses²¹ would have reduced endocytosis, leading to PIN2 asymmetry. On the other hand, the asymmetric distribution of PIN2 would further stabilize the established auxin asymmetry and thus maintain differential endocytosis between the two sides, leading to root coiling.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Prof. Yong Wang for chl1-5. This work was supported by Natural Science Foundation of China (31771558 and 31970332 to S.L.).

Supplementary material

The supplemental data for this article can be accessed here.

Additional information

Funding

This work was supported by the National Natural Science Foundation of China [31970332]; National Natural Science Foundation of China [31771558].