Phosphorylation-Mediated Dynamics of Nitrate Transceptor … · The phosphomimetic NRT1.1T101D form...

Phosphorylation-Mediated Dynamics of NitrateTransceptor NRT1.1 Regulate Auxin Flux and NitrateSignaling in Lateral Root Growth1[OPEN]

Xi Zhang,a,b Yaning Cui,a,b Meng Yu,a,b Bodan Su,a,b Wei Gong,a,b Frantisek Baluska,c George Komis,d

Jozef Šamaj,d Xiaoyi Shan,a,b,2,3 and Jinxing Lina,b,3

aBeijing Advanced Innovation Center for Tree Breeding by Molecular Design, Beijing Forestry University,Beijing 10083, ChinabCollege of Biological Sciences & Biotechnology, Beijing Forestry University, Beijing 10083, ChinacInstitute of Cellular and Molecular Botany, University of Bonn, Bonn, D-53115, GermanydCentre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, PalackyUniversity, Olomouc 78301, Czech Republic

ORCID IDs: 0000-0001-5518-4220 (X.Z.); 0000-0003-4750-2123 (J.Š.); 0000-0003-3755-4659 (X.S.); 0000-0001-9338-1356 (J.L.).

The dual-affinity nitrate transceptor NITRATE TRANSPORTER1.1 (NRT1.1) has two modes of transport and signaling,governed by Thr-101 (T101) phosphorylation. NRT1.1 regulates lateral root (LR) development by modulating nitrate-dependent basipetal auxin export and nitrate-mediated signal transduction. Here, using the Arabidopsis (Arabidopsis thaliana)NRT1.1T101D phosphomimetic and NRT1.1T101A nonphosphorylatable mutants, we found that the phosphorylation state ofNRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylationaffected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membranepartitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed lowlateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediatedendocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LRdevelopment by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca21-ARABIDOPSIS NITRATEREGULATED1 signaling from the endosome.

Nitrate is the primary nitrogen source in most plantsand plants modulate their root system architectureunder high-nitrate (HN) and low-nitrate (LN) con-ditions (Wang et al., 2018b). NITRATE TRANS-PORTER1.1 (NRT1.1), also known as NPF6.3 (Léranet al., 2014), is a dual-affinity nitrate transceptor(transporter/receptor) that localizes on the plasmamembrane (PM) and functions in nitrate-dependent

regulation of root system architecture (Liu et al., 1999;Liu and Tsay, 2003; Ho et al., 2009; Krouk et al., 2010;Léran et al., 2014; Bouguyon et al., 2015), affectingprimary root and lateral root (LR) development throughmultiple pathways (Remans et al., 2006). Notably, thephosphorylation of NRT1.1 at Thr-101 (T101) is crucialfor its transport and sensing functions (Liu et al., 1999;Liu and Tsay, 2003; Ho et al., 2009).

In LR development, NRT1.1 functions in nitrate-dependent auxin transport, regulating auxin accumula-tion and thereby affecting LR growth (Krouk et al., 2010).A signaling network involving Ca21, Ca21-sensor pro-tein kinases (CPKs), and NIN-like proteins (NLPs) isinvolved in nitrate-regulated LR development via reg-ulation of primary transcription (Remans et al., 2006;Krouk, 2017; Liu et al., 2017). Furthermore, Ca21-ARA-BIDOPSIS NITRATE REGULATED1 (ANR1), a tran-scription factor downstream of NRT1.1 and NIN-likeprotein 7 (NLP7), has been implicated in LR elongationunderHN conditions (Gan et al., 2012). Nevertheless, themechanisms by which the phosphorylation state ofNRT1.1 regulates LR development through auxintransport and the Ca21–CPKs–ANR1 signaling pathwayhave remained unknown.

PM microdomains are enriched in sterols, sphingo-lipids, and PM-specific proteins, and are involved in

1This work was supported by the National Natural Science Foun-dation of China (31871424 to X.S., 31530084 to J.L., and 31622005 toX.L.), Introducing Talents of Discipline to Universities (111 ProjectNo. B13007 to J.L.), and the European Regional Development Fund(No. CZ.02.1.01/0.0/0.0/16_019/0000827 to J.Š. and G.K.).

2Author for contact: [email protected] authors.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Author (www.plantphysiol.org) is:Xiaoyi Shan ([email protected]).

J.L. and X.S. conceived the project; X.Z. performed most of theexperiments; Y.C., M.Y., and B.S. assisted in some experiments;W.G. assisted in capturing some confocal microscope images; X.Z.and X.S. analyzed the data and wrote the article; F.B., G.K., and J.Š.revised the article.

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regulating the dynamic behavior and assembly states ofPM proteins (Bücherl et al., 2017; Cui et al., 2018). Theinternalization and intracellular trafficking of PMproteins is regulated via control of PM organizationand initiation of vesicle transport processes in re-sponse to environmental stimuli (Li et al., 2013; Fanet al., 2015). Combining protein labeling techniquesand single-particle tracking (SPT) analysis providesa powerful approach to visualize the dynamic be-haviors of individual protein molecules with highspatiotemporal resolution (Li et al., 2011; Wang et al.,2013a, 2018a). As an advanced single-molecule tech-nique,fluorescence cross-correlation spectroscopy (FCCS)provides quantitative information on molecular in-teractions at the single-molecule and nanosecond-timescale levels (Bacia et al., 2006; Wang et al., 2015;Li et al., 2016).To elucidate the mechanisms underlying the regula-

tion of LR development via phosphorylation ofNRT1.1,we investigated the diffusion dynamics and membranepartitioning of NRT1.1 phosphomimetic and non-phosphorylatable mutants in LR cells, allowing us tolink data collected at the individual molecule level toauxin transport at the PM. We also analyzed the in-tracellular trafficking of these NRT1.1 mutants inLR cells to address the possibility that differentialphosphorylation of NRT1.1 contributes to the modu-lation of NRT1.1-mediated signal transduction duringLR development.

RESULTS

NRT1.1 Phosphorylation Affects Basipetal AuxinTransport and LR Development

To investigate whether the phosphorylation state ofNRT1.1 affects LR development, we measured thedensity of visible LRs (.0.5 mm) on 8-d–old wild type,chl1-5 (an NRT1.1 null mutant), NRT1.1T101A/chl1-5 (anonphosphorylatable T101A mutant, hereafter ab-breviated T101A), and NRT1.1T101D/chl1-5 (a phos-phomimetic T101D mutant, hereafter abbreviated asT101D) Arabidopsis (Arabidopsis thaliana) seedlings(Supplemental Fig. S1A). Among seedlings grown inthe absence of nitrate or under LN conditions (0.2mM), the LR density of T101A seedlings was muchhigher than that of T101D seedlings (Fig. 1, A and B).By contrast, in HN conditions (1 mM), the LR densityof the mutants did not significantly differ from that ofwild-type plants (Fig. 1, A and B).To determine whether the phosphorylation state of

NRT1.1 affects auxin transport, we generated trans-formants of the yeast strain YPH499 expressing emptyvector (CK, pESC-mRuby-URA), NRT1.1, T101A, orT101D (Supplemental Fig. S1, B and C). The T101Dyeast cells showed significantly greater indole-3-aceticacid (IAA) influx than T101A yeast cells by noninvasivemicrotest technology (NMT) analysis (Fig. 1, C and D).A parallel experiment in planta showed that under LN,

LRs of T101D seedlings displayed a 51% increase in[3H]IAA accumulation compared with the T101Aseedlings (Fig. 1E; Supplemental Fig. S1D).To further analyze the effect of NRT1.1 phospho-

rylation state on auxin accumulation in LRs, wecrossed an Arabidopsis line expressing the auxin-inducible DR5::GFP reporter with wild-type, chl1-5,T101A, and T101D plants. The chl1-5 DR5::GFP off-spring exhibited strong fluorescence in LRs in re-sponse to different concentrations of nitrate (SupplementalFig. S2, A–F). Consistent with their low auxin transportcapacity, the T101A plants displayed strongly en-hanced DR5::GFP expression in the primordia andyoung LRs, whether grown in nitrate-free medium orin LN, as compared to the T101D plants (Fig. 1, F andG; Supplemental Fig. S2, C-–F). However, there was nosignificant difference in DR5 activity between theT101A and T101D plants in HN conditions (Fig. 1, Hand I; Supplemental Fig. S2, A–F). These data indicatethat T101A, and by extension, nonphosphorylatedwild-type NRT1.1, enhances LR growth in LN byinhibiting basipetal auxin transport, causing the ac-cumulation of auxin in the tips of LRs.

NRT1.1 Phosphorylation Variants Have DifferentSpatiotemporal PM Dynamics

The spatiotemporal dynamics of PM proteins couldcontrol their biological functions (Kusumi et al., 2012).To gain insight into the effect of NRT1.1 phosphoryl-ation on its dynamic behavior, we generated trans-genic plants expressing a C-terminal GFP fused toNRT1.1, T101D, or T101A under the control of theNRT1.1 native promoter in the chl1-5 mutant back-ground (Supplemental Fig. S3A). Confocal images(after mannitol-induced plasmolysis) revealed GFPsignals mainly on the PM of epidermal cells in LRs(Supplemental Fig. S3, B and C). Gene expression,immunoblot analysis, and LR phenotype assessmentconfirmed that each transgenic line was functional(Supplemental Figs. S3, D and E and S4; SupplementalTable S1).Using variable-angle total internal reflection fluo-

rescence microscopy (VA-TIRFM), we found that un-der LN and HN conditions, spots of T101D-GFPand T101A-GFP localized on the PM and appearedas dispersed diffraction-limited fluorescent spots(Supplemental Fig. S5A). Sequential images showedthat the individual particles stayed on the PM for a fewseconds and then rapidly disappeared (SupplementalFig. S5B; Supplemental Videos S1 and S2). SPT anal-ysis revealed that individual T101D-GFP spots hadmotion trajectories of more than 4 mm within 12 s,whereas T101A-GFP spots were limited to muchshorter motion tracks of 1 mm within 6 s (Fig. 2A).The motion ranges of the spots had a bimodal dis-

tribution, with 91.3% of T101D-GFP spots showinglong-distance motion and 8.7% showing short-distancemotion (G 5 1.05 6 0.03 mm and 0.30 6 0.01 mm,

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Role of NRT1.1 Phosphorylation in LR Development

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respectively; Fig. 2B, upper). For T101A-GFP, the per-centage of spots with long-distance motion (G5 0.8260.03 mm) was lower, 84.1%, and the percentage withshort-distance motion was higher, 15.9% (G 5 0.33 60.01 mm; Fig. 2B, lower; Supplemental Fig. S6A).Moreover, the peak particle velocity of T101A-GFPwas significantly lower than that of T101D-GFP (2.896 0.05 mm/s versus 2.99 6 0.04 mm/s; Fig. 2C;Supplemental Fig. S6B). However, the diffusion coeffi-cient of T101A-GFP was markedly higher than that of

T101D-GFP (G 5 9.10 3 1023 6 0.22 3 1023 mm2/sversus 6.84 3 1023 6 0.33 3 1023 mm2/s; Fig. 2D;Supplemental Fig. S6C).

To determine the relationships among motion range,residence time, and velocity of T101D-GFP and T101A-GFP particles, we performed a bubble-plot analysis.The distribution range of T101D-GFP spots was moreextensive than that of T101A-GFP spots, indicating thatT101D-GFP has a longer lifetime and higher particlevelocity (Fig. 2E). Taken together, these results imply

Figure 1. The phosphorylation state ofNRT1.1 affects nitrate-regulated auxintransport and LR growth under LN condi-tions. A, Phenotype of visible LRs (.0.5mm)in wild-type (WT), chl1-5, T101A, andT101D seedlings grown for 8 d on mediumcontaining different concentrations (0, 0.2,and 1 mM) of NO3

–. Arrowheads indicatevisible LRs. Scale bar 5 1 cm. B, Density ofvisible LRs (.0.5 mm) in wild-type, chl1-5,T101A, and T101D seedlings grown for 8 don medium containing different concentra-tions (0, 0.2 and 1 mmM)) of NO3

–. Data aremeans 6 SD from seven independent ex-periments (n 5 74–91). C and D, Net IAAinflux profile (C) and average net auxin flux(D) in yeast strains expressing empty vector(CK), NRT1.1, T101A, and T101Dmeasuredby NMT assay. Data are means 6 SD (n 510). E, Basipetal [3H]IAA uptake (counts perminute) in LRs of wild-type, T101A, andT101D seedlings grown for 10 d on mediumcontaining 0.2 and 1 mM of NO3

–. Data aremeans6 SE from five independent seedlingsper treatment. F and H, Confocal images ofDR5::GFP and pseudocolor images (blue-green-red palette) showing fluorescence in-tensity in LRs of T101A and T101D seedlingsexposed to 0.2 mM (F) and 1mM (H) of NO3

–:LRs initiating primordia (a); LR primordiabefore emergence (b); newly emerged LRs(c). Scale bars 5 20 mm. G and I, Fluores-cence intensity (a.u., arbitrary units) ofDR5::GFP in LR primordia before emer-gence of T101A and T101D seedlings ex-posed to 0.2 mM (G) and 1 mM (I) of NO3

–.Boxplots represent mean, 25th, and 75thquartiles (n 5 6); whiskers represent 6 SD.Significant differences are denoted by letters(P , 0.05; Duncan multiple-comparisontest) in (B), (D) and (E), and by asterisks(*P, 0.05, ***P , 0.001, ns, not significant;Student’s t test) for differences betweenT101A and T101D in (B), (D), (E), (G), and (I).

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that phosphorylation of NRT1.1 at T101 leads to long-distance motion and fast particle velocity with reduceddiffusion coefficient on the PM of LR cells, whereasnonphosphorylated NRT1.1 has a shorter motion tra-jectory and lower particle velocity with a higher diffu-sion coefficient.The phosphorylation state of NRT1.1 influences its

oligomeric state (Tsay, 2014); therefore, we moni-tored the maximum fluorescence intensity distributionto visualize oligomerization of T101D and T101A.T101D-GFP spots were distributedwith a single peak of

2.055073 105, whereas T101A-GFP spots exhibited twopeaks of 2.64327 3 105 and 3.56615 3 105 by Gaussianfitting, implying that T101D and T101A exist as mon-omers and mixed monomers/dimers, respectively(Supplemental Fig. S6, D and E). Furthermore, a single-molecule subunit counting assaywas used to determinethe accurate monomeric/dimeric states of T101D andT101A, respectively. We analyzed the precise ratio ofindividual T101D-GFP and T101A-GFP cluster pho-tobleaching steps with a progressive idealizationand filtering program (Fig. 2F). Approximately 77% of

Figure 2. NRT1.1 phosphorylation vari-ants have different spatiotemporal PMdynamics in the LR cells. A, Motion tra-jectories of T101D-GFP (0.2mM ofNO3

–)and T101A-GFP (10 mM of NO3

–) at thePM. Left and middle, VA-TIRFM imagesof T101D-GFP and T101A-GFP; greencircles indicate the single particles ofGFP and colorful fold lines indicate themotion trajectories of GFP spots. Right,typical time-lapse trajectories of T101D-GFP (blue lines) and T101A-GFP (pinklines) at indicated time points. Bar 5 2mm. B to D, Distribution of motion range(B), particle velocity (C), and diffusioncoefficients (D) of T101D-GFP (upper)and T101A-GFP (lower) spots at thePM respectively, exposed to 0.2 and10 mM of NO3

–. Results are summationsof 10 independent experiments each(n 5 12,283 for T101D-GFP and 38,560for T101A-GFP in B; n 5 66,398 forT101D-GFP and 49,562 for T101A-GFPin C; n 5 1,181 for T101D-GFPand 3,764 for T101A-GFP in D). E,Bubble plots representing differencesamong T101D-GFP and T101A-GFP formotion ranges, residence time, and par-ticle velocity. Results are summationsof 10 independent experiments each(n 5 66,398 spots for T101D-GFP and49,562 spots for T101A-GFP). F, Selectedtime courses of GFP emissions after back-ground correction showing T101D-GFPone-step bleaching (left) and T101A-GFPtwo-step bleaching (right) at the PM under0.2 and 10 mM of NO3

– treatment respec-tively. Data are means 6 SD from multi-ple normalized traces depicted in gray(n 5 10).






T101D-GFP particles were in monomer form andonly 23% were homodimerized under LN. Notably,the proportion of monomers was lower, 54% and theproportion of dimers was increased to 46% for T101A-GFP under HN (Supplemental Fig. S6F). These resultssuggest that the ratio of dimer to monomer of NRT1.1on the PM of LR cells is substantially changed by thephosphorylation modification on T101, which mightlead to distinct spatiotemporal characters.

Phosphorylation State Affects NRT1.1 Partitioning intoPM Microdomains under Low Nitrate

To assess the relationship between NRT1.1 phos-phorylation state and its localization in membranemicrodomains, we transiently coexpressed NRT1.1-GFP, T101D-GFP, and T101A-GFP with the fluorescentmicrodomain reporter AtRem1.3-mCherry in Nicotianabenthamiana leaf epidermal cells (Supplemental Fig. S7).

Based on Pearson and Manders coefficient analysis,we found moderately lower colocalization betweenNRT1.1-GFP and Rem1.3-mCherry under HN com-pared with LN conditions (Fig. 3, A and B). However,T101D-GFP and AtRem1.3-mCherry exhibited higherPearson and Manders correlation coefficients, com-pared with T101A-GFP and AtRem1.3-mCherry, un-der both HN and LN (Fig. 3, A and B).

To obtain a more dynamic picture of the colocaliza-tion of NRT1.1, T101D, T101A, and AtRem1.3 in epi-dermal cells of LRs, we used Förster resonanceenergy transfer-fluorescence lifetime imaging micros-copy (FRET-FLIM) on the transgenic lines expressingNRT1.1/T101D/T101A-GFPwith AtRem1.3-mCherry(Fig. 3C). In plants coexpressing T101D-GFP withAtRem1.3-mCherry, fluorescence lifetime sharply de-creased and the FRET efficiency (t) increased to 38.7%upon LN treatment and to 18.2% upon HN treatment,in comparison with T101D-GFP alone (Fig. 3D). Incontrast, no obvious changes were observed in the

Figure 3. Phosphorylation state affectsNRT1.1 partitioning into PM micro-domains. A and B, Quantitative coloc-alization analysis for NRT1.1-GFP,T101D-GFP, and T101A-GFP respec-tively, with AtRem1.3-mCherry in epi-dermal leaf cells of N. benthamianaby the Pearson (A) and Manders (B)coefficients. Data are means 6 SD

(n 5 16–21). C, Lifetime maps of the LRcells expressing T101D-GFP andT101A-GFP, with or without AtRem1.3-mCherry exposed to 0.2 and 10 mM ofNO3

–. The pseudocolor scale variedfrom 1.8 ns to 2.8 ns. Scale bar5 5 mm.D, Average fluorescence lifetime andFRET efficiency of T101D-GFP andT101A-GFP in the presence or absenceof AtREM1.3-mCherry. Data are means6 SD (n5 12–16). Significant differencesare denoted by different letters (P ,0.05; Duncan multiple-comparisonstest) in (A), and by asterisks (*P , 0.05,**P, 0.01, ***P, 0.001; Student’s t test)for differences between T101D-GFPandT101A-GFP in (A), (B), and (D).


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fluorescence lifetime of T101A-GFP coexpressed withAtRem1.3-mCherry in comparison with T101A-GFPalone. FRET efficiency between T101A-GFP andAtRem1.3-mCherry was only 0.57% and 2.6% underLN and HN conditions, respectively (Fig. 3D). Col-lectively, these analyses reveal that PM microdomainscontribute to the partitioning of the phosphorylatedform of NRT1.1.

Phosphorylation State Affects Secretion andInternalization of NRT1.1

Most PM proteins undergo intracellular traffickingbetween the PM and endosomal compartments (Wuet al., 2014), and their phosphorylation states are vitalelements in the regulation of this trafficking (Offringaand Huang, 2013). To dissect the effects of phospho-rylation on NRT1.1 secretion in the epidermal cellof LR, we first carried out a fluorescence recov-ery after photobleaching assay with NRT1.1, T101D,and T101A. We divided the LR into three bleachingsectors (top, middle, and bottom). If the sectors showdifferences in recovery, this indicates that the new,unbleached proteins arrive by lateral diffusion; ifthey show no difference, this indicates the proteinsarrive by secretion. Indeed, these sections showed nodifference in recovery rate between the three pro-teins, demonstrating that all three were recruitedmainly through secretion rather than lateral diffusion(Supplemental Fig. S8, A and B). The fluorescencerecovery rate in T101D-GFP seedlings was muchlower than that of T101A-GFP under LN (0.3% forT101D-GFP versus 18.9% for T101A-GFP) or HNconditions (12.7% for T101D-GFP and 49.5% forT101A-GFP; Fig. 4A). Furthermore, brefeldin A(BFA), an inhibitor of vesicle recycling through ADP-ribosylation factor-guanine exchange factors (ARF-GEFs), inhibited fluorescence recovery in T101D-GFPmarkedly, by 69%, but had a much weaker effect onT101A-GFP, causing only a 15% decrease (Fig. 4A,inset; Supplemental Fig. S8C). These results implythat T101D relies on the ARF-GEFs pathway for ef-fective secretion, whereas T101A does not.Next, we dissected the internalization of NRT1.1,

T101D, and T101A in response to different nitrateconcentrations in the epidermal cell of LR. Afterpretreatment with the protein synthesis inhibitorcycloheximide (CHX), the internalization of NRT1.1-GFP, as reflected by the number of fluorescent spotsin the cytoplasm, showed a dose-dependent increaseafter treatment with nitrate at 0.2, 1, and 10 mM

(Fig. 4B; Supplemental Fig. S8D). Notably, T101D-GFP was found at the cell surface and was mostlyabsent from intracellular compartments, whereasT101A-GFP showed substantial accumulation in thecytoplasm (Fig. 4B). Statistical analysis of the inter-nalization spots on the cells of T101A-GFP andT101D-GFP seedlings showed a significantly highernumber of spots in T101A-GFP compared with

T101D-GFP under both LN (0.2 mM) and HN (10 mM)conditions (Supplemental Fig. S8E).In parallel experiments to detect the formation of

BFA-induced compartments, we found that NRT1.1,T101D, and T101A all clearly accumulated in BFAbodies after treatment with BFA only (Fig. 4C). Aftertreatment with BFA and CHX, T101D-GFP signals werestill trapped in BFA compartments, whereas T101A-GFP formed dispersed spots centered around BFAcompartments that were also dyed by FM4-64 (a PM-specific dye), a pattern similar to that seen for NRT1.1-GFP after HN treatment (Fig. 4D; Supplemental Fig.S8E). These observations suggest that the absence ofphosphorylation on NRT1.1 facilitates its internaliza-tion independently of ARF-GEFs.We then implemented TIRFM analysis to determine

the PM residence time of T101D-GFP and T101A-GFP.We used MATLAB (https://www.mathworks.com/)to track 6,696 spots of T101D-GFP and 44,065 spots ofT101A-GFP under LN andHN conditions, respectively.T101D-GFP particles stayed on the PM for ;15 s,whereas T101A-GFP particles had a PM residence timeof only 10 s (Fig. 4E). In accordance with this, normal-ized fluorescence intensity and kymograph testsshowed a significantly longer PM lifetime for T101D-GFP than for T101A-GFP (Fig. 4, F–H). Similarly, theresidence times of T101D-GFP and T101A-GFP on thePM averaged 6.73 s and 5.63 s, respectively (Fig. 4I).To obtain more data on PM residence time of T101D-

GFP and T101A-GFP, we conducted frequency dis-tribution analysis of lifetime. Almost 53% of T101D-GFP spots persisted for more than 5 s on the PM,whereas over 70% of the T101A-GFP spots residedthere for less than 5 s (Fig. 4J). Together, these resultsreveal that the phosphomimetic mutant of NRT1.1undergoes slower constitutive endocytosis, whereasthe nonphosphorylated form is internalized via afaster inducible endocytosis process.

NRT1.1 Phosphorylation State Dictates Endocytic andIntracellular Trafficking Routes

Endocytosis of PM proteins can be generally cate-gorized into clathrin-mediated endocytosis (CME) andclathrin-independent endocytosis (CIE) pathways(Mayor and Pagano, 2007; Fan et al., 2013, 2015). Togain insight into the mechanism of NRT1.1 internali-zation, we characterized NRT1.1 endocytosis in theLR cells under the CLATHRIN H CHAIN2 (CHC2)knockout mutant chc2-1 background. The intracellu-lar accumulation of T101D-GFP and T101A-GFPclusters was significantly inhibited in response toLN and HN treatment, indicating that clathrin is re-quired for NRT1.1 endocytosis (Fig. 5, A and B).Quantification of NRT1.1 spots in endocytic vesi-cles showed that endocytosis was blocked almosttotally, by 88.5%, in T101D-GFP/chc2-1 seedlings,but by only 65.2% in T101A-GFP/chc2-1 seedlings(Supplemental Fig. S9A), implying that the CIE










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pathway may contribute more (22.2%) to the endo-cytosis of T101A than to that of T101D (SupplementalFig. S9B). To further test this, we applied methyl-b-cyclodextrin (MbCD), a well-established sterol-interfering agent that is routinely used to block theCIE pathway, to T101A-GFP seedlings. This resultedin a 17.5% decrease in the average number of endo-cytosis spots (Supplemental Fig. S9, C and D), con-sistent with the earlier results.

We further examined the relationship betweenT101A and the CME andmicrodomain marker proteinsby using dual-color confocal microscopy in transgeniclines coexpressing T101A-GFP and AtCLC-mCherry,AtFlot1-mCherry, or AtRem1.3-mCherry (Fig. 5C).T101A-GFP populations showed good colocalizationwith AtCLC-mCherry and AtFlot1-mCherry (Pearsoncorrelation coefficients (r) 5 0.123 and 0.152, respec-tively) but not with AtRem1.3 (r 5 0.004; Fig. 5D). By

Figure 4. Phosphorylation state ofNRT1.1 influences its exocytosis andendocytosis in the LR cells. A, Fluores-cence recovery curves of the photo-bleached regions in the LR cellsexpressing T101D-GFPand T101A-GFP,exposed to 0.2 and 10 mM of NO3

– withor without BFA pretreatment (n 5 4).Inset, percentage of decreased fluores-cence recovery rate under BFA pretreat-ment. Asterisks indicate significance(***P , 0.001; Student’s t test). B to D,Confocal images of NRT1.1-GFP,T101D-GFP, and T101A-GFP in LRcells respectively, exposed to differentconcentrations (0.2, 1, and 10 mM) ofNO3

– with CHX (B), BFA (C), or CHX1BFA (D) pretreatment. Scale bars 5 10mm. E, Representative time-lapse imageseries of individual T101D-GFP andT101A-GFP spot in LR cells by VA-TIRFM. Images were collected at 3 Hz;for brevity of presentation, one in sixframes (at 2-s intervals) are shown.Scale bar 5 300 nm. F and G, Nor-malized fluorescence intensity (DF/F;n 5 3) of T101D-GFP (F) and T101A-GFP (G) in LR cells, with 6 SD frommultiple events in gray. H, Kymographanalysis of T101A-GFPand T101D-GFPfluorescence in LR cells showing indi-vidual points from yellow rectangles.Scale bar 5 5 s. I, Average lifetime ofT101D-GFPand T101A-GFP clusters inLR cells. Boxplots represent mean, 25thand 75th quartiles. Whiskers representminimum and maximum. n 5 6,697dots are pooled across seven indepen-dent experiments for T101D-GFP, andn5 44,066 dots are pooled across nineindependent experiments for T101A-GFP. Asterisks indicate significant dif-ferences between T101D-GFP andT101A-GFP (***P # 0.0001, Student’st test). J, Frequency distribution analysisof T101D-GFP and T101A-GFP life-times shown in (I).


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FCCS, T101A-GFP showed high relative cross-correlation amplitude with AtCLC-mCherry andAtFlot1-mCherry (r 5 0.88 6 0.001 and 0.81 6 0.082,respectively), whereas the fluorescence of T101A-GFP and AtRem1.3-mCherry gave a low relativecross-correlation amplitude of 0.59 6 0.014 (Fig. 5,E–H). These results indicate that the endocytosis of

T101A occurs via both the CME and CIE pathways,whereas T101D is internalized mainly via the CMEpathway.To gain a better understanding of the intracellular

trafficking routes of NRT1.1, we investigated the in-fluence of the vacuolar H1-ATPase inhibitor con-canamycin A (Conc A) on the subcellular localization

Figure 5. NRT1.1 phosphorylationstate dictates endocytic pathways inLR cells. A, Confocal images show-ing internalization of T101D-GFP andT101A-GFP either in the chc2-1mutantbackground (right) or not in the mutantbackground (left), exposed to 0.2 mM

and 10 mM of NO3– with CHX pre-

treatment. Scale bars 5 10 mm. B,Quantification of internalized spotsshown in (A). Boxplots represent mean,25th, and 75th quartiles. The coloredpoints represent 10 independent ex-periments (n5 254 and 249 for T101D-GFP and T101D-GFP/chc2-1, n 5 75and 86 for T101A-GFPand T101A-GFP/chc2-1, respectively). Whiskers repre-sent 6 SD. Asterisks denote statisticallysignificant differences (***P , 0.001;Student’s t test). C, Confocal imagesshowing colocalization of T101A-GFPwith AtCLC-mCherry, AtFlot1-mCherry,and AtRem1.3-mCherry in LR cells,exposed to 10 mM NO3

–. Scale bar 51 mm. D, Quantitative colocalizationanalysis for the data from (C) by Pearsoncorrelation coefficients. The coloredpoints represent three independent ex-periments (n5 12). Whiskers represent6 SD. Asterisks denote statistically sig-nificant differences (*P , 0.01, ***P ,0.001; ns, not significant; two-tailedStudent’s t test). E to G, FCCS analysisof the fluorescence cross-correlationcurves (G[t]) between T101A-GFP andAtCLC-mCherry (E), AtFlot1-mCherry(F), or AtRem1.3-mCherry (G) in LRcells exposed to 10 mM of NO3

–. H,Average relative cross correlation ofT101A with AtCLC/AtFlot1/AtRem 1.3.Data are means 6 SD (n 5 75) from atleast five independent seedlings pertreatment.





of NRT1.1 in LR cells, T101A-GFP and T101D-GFP.NRT1.1-GFP and T101A-GFP showed vacuolar tar-geting after 6 h of treatment, but not within 2 h(Fig. 6A). Consistent with this, 6 h of darkness also

led to vacuole-like accumulation of NRT1.1 andT101A-GFP signals. T101D-GFP did not localize tovacuoles under any of the treatments tested. In par-allel, we applied the phosphatidylinositol-3-kinase

Figure 6. NRT1.1 phosphorylation state dictates intracellular trafficking routes in LR cells. A, Confocal images of NRT1.1-GFP,T101A-GFP, and T101D-GFP in LR cells, exposed to 10 mM NO3

– with Conc A for 2 h or Conc A, dark and Conc A plusWM for 6h. Scale bar5 20 mm. B, Confocal images and quantitative analysis of colocalization between T101A-GFP with TGN, MVB, andLEmarkers in LR cells respectively, exposed to 10mMNO3

–. Arrowheads and arrows point to T101A-GFP vesicles that colocalizedand did not colocalize with markers, respectively. Scale bars 5 5 mm. Graphs at right show the percentages of T101A-GFP-positive vesicles colocalized with the TGN, MVB, and LE markers respectively (n5 64, 41, 179, and 133, where n is the numberof T101A-GFP-positive vesicles). C, Confocal images of colocalization between T101A-GFP with the PVC and vacuole markersrespectively, exposed to 10 mM of NO3

– for 2 h and 6 h. Arrows point to T101A-GFP-positive vesicles that colocalized withmarkers. Scale bars 5 5 mm. D, model for intracellular trafficking between PM and cytoplasm of NRT1.1 phosphorylation var-iants. Under LN conditions, phosphorylated NRT1.1 presents a constitutive secretion and endocytosis between PM and TGN/EE,which is dependent on the activity of ARF-GEF, whereas under HN conditions, the induced secretion and endocytosis ofdephosphorylated NRT1.1 between PM and MVB/PVC compartments increase, which is largely independent of the activity ofARF-GEFs. The gray and pink/blue numbers represent the unknown and quantitative percentage of secretion/endocytosis in total.The numbers in red indicate increased percentage of secretion and endocytosis in total.


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inhibitor wortmannin (WM) together with Conc A toblock the recycling of vacuolar sorting receptors be-tween the prevacuolar compartment (PVC) andtrans-Golgi network (TGN; Kleine-Vehn et al., 2008).WM strongly reduced vacuolar trafficking ofNRT1.1-GFP and T101A-GFP, and the fluorescentproteins accumulated in the typical Conc A bodies(Fig. 6A).Considering that T101D-GFP did not accumulate in

vacuoles even under Conc A treatment, we assessed thespatial relationship between T101A-GFP and differentfluorescence-tagged endomembrane markers by quan-titative colocalization analysis. After 2 h of treatmentwith HN, T101A-GFP partially colocalized with alltested fluorescent TGN and early endosome (EE)markers with low Pearson correlation coefficients, in-cluding VTI12-mRFP (18.8%), VHA-a1-mRFP (19.5%),and CLC-mCherry (5.7%; Fig. 6B). In contrast, T101A-GFP colocalized with 64.8% of the multivesicular body(MVB) marker AtSNX1-mRFP spots (Fig. 6B). More-over, T101A-GFP signals were well colocalizedwith thePVC and late endosome (LE) markers (RabG3f-mRFPand mRFP-SYP22) and were delimited within the ton-oplast after 2 h and 6 h of Conc A treatment (Fig. 6C).Based on the results of intracellular trafficking ofNRT1.1, we propose a model for the transfer ofunphosphorylated NRT1.1 between the PM and cyto-plasm where unphosphorylated NRT1.1 follows anintracellular trafficking route from the PM to the vac-uole, passing through the MVB and PVC compart-ments, which is largely independent of the TGN andEEs (Fig. 6D).To clarify whether T101A is also degraded by the

proteasome pathway, we treated T101A-GFP seedlingswith the specific 26S proteasome inhibitor MG132.Without MG132, no detectable degradation of T101Awas observed after 15 min of HN treatment; however,there was a 50% reduction in T101A after 4 h of HN(Supplemental Fig. S10A). Notably, treatment withMG132 reduced T101A to 30% after 4 h of HN, reveal-ing that the 26S proteasome is involved in T101A deg-radation. To determine whether NRT1.1 was involvedin initiating autophagy after nitrogen deficiency,we assessed the colocalization of T101D-GFP andthe fluorescent autophagosome marker ATG8-CFP.We observed no colocalization between T101D andATG8 under nitrogen-rich conditions (SupplementalFig. S10B), but a clear colocalization under nitrogen-free conditions, as indicated by line-scan analysis(Supplemental Fig. S10C). These results suggest thatthe proteasome and autophagy pathways providedifferent mechanisms for NRT1.1 degradation indifferent nitrate conditions.

Internalization of NRT1.1 Promotes Nitrate-InducedCa21-ANR1 Signaling and LR Development

Nitrate triggers unique Ca21–CPKs–NLPs signalingin the central nutrient-growth network (Krapp et al.,

2014). As a downstream component of NLP7, ANR1acts as a key regulator of LR proliferation (Mounieret al., 2014). To uncover whether NRT1.1 phosphoryl-ation state affects cytoplasmic calcium concentration([Ca21]cyt), we assessed nitrate-triggered Ca21 signal-ing in the epidermal cell of LR from the various geno-types using Fluo-4 AM dye (Supplemental Fig. S11A).Based on the pseudocolor and kymograph images ofwild-type samples, we proposed 60 s as a suitablelength of time to monitor [Ca21]cyt signaling after ni-trate stimulation (Supplemental Fig. S11, B–E). As de-scribed previously in Riveras et al. (2015), we found thatnitrate specifically stimulated an increased Ca21 sig-nature in wild type but not chl1-5 mutant seedlings(Fig. 7A). Under both LN and HN conditions, T101Aseedlings showed a transient increase in [Ca21]cyt,whereas T101D seedlings showed a reduction (Fig. 7B).In parallel to [Ca21]cyt accumulation, HN-induced ex-pression ofANR1 in LRs was detected in T101A but notin T101D (Fig. 7C). Based on these findings, we con-clude that the nonphosphorylated form of NRT1.1could activate the Ca21–CPKs–NLPs signaling path-way to elevate the expression of ANR1.To address the function of internalization in signaling

mediated by the nonphosphorylated form of NRT1.1,we first perturbed the internalization pathway andlooked for changes in nitrate-triggered [Ca21]cyt sig-naling. When T101A seedlings were treated withMbCD, nitrate-induced [Ca21]cyt accumulation wassignificantly impaired. Similar results were obtained inT101A/chc2-1 seedlings. Nevertheless, both Conc Aand Bafilomycin A1 (Baf A1) treatment promoted[Ca21]cyt influx (Fig. 8, A and B).We thenmonitored theexpression of NRT1.1 and of ANR1 in LRs, the target ofthe nitrate-Ca21 signaling cascade. Clathrin mutationand MbCD treatment reduced the induction of bothgenes, whereas Conc A and Baf A1 treatment upregu-lated the expression level of ANR1 but not NRT1.1,which can be explained by feedback repression actingthrough the accumulation of NRT1.1 (Fig. 8, C–F).Moreover, we found that T101A-GFP/chc2-1 seedlingsshowed limited development of LRs compared withT101A-GFP when grown on HNmedium (Fig. 8, G andH). Therefore, the endocytosis of nonphosphorylatedNRT1.1 appears to be positively associatedwith nitrate-triggered signaling.

DISCUSSION

Nitrate has a strong effect on plant morphogenesis androot system architecture (Wang et al., 2018b). The nitratetransceptor NRT1.1 functions as a dual-affinity trans-porter and nitrate sensor in response to variations in ni-trate concentration by switching between phosphorylatedand dephosphorylated forms (Liu et al., 1999; Liu andTsay, 2003; Ho et al., 2009; Léran et al., 2014). Asidefrom the transceptor function mentioned above, NRT1.1could transport auxin basipetally via a nitrate-dependentpathway, and trigger the Ca21–CPKs–NLPs signaling












pathway to regulate LR development (Krouk et al.,2010; Liu et al., 2017). However, little is known abouthow different NRT1.1 phosphorylation variants maybe associated with the regulation of LR growth. Here,we provide several lines of evidence indicating thatphosphorylation of NRT1.1 at T101 affects NRT1.1-modulated LR development by altering NRT1.1 sub-cellular distribution, PM dynamics, and subcellulartrafficking in LR cells to alter nitrate-dependent auxintransport and nitrate-regulated signaling.

LR plasticity plays a key role in enabling plants toobtain nutrients and survive in complicated sur-roundings (Stoeckle et al., 2018). Nitrate and auxin canhave various functions in plant morphogenesisthrough their regulation of LR growth (Sun et al., 2017;Du and Scheres, 2018). Recently, genetic and physio-logical evidence has suggested that NRT1.1 repressesLR growth by promoting basipetal auxin transport inLRs at LN (Krouk et al., 2010). The model was pre-liminarily explained by the repression of NRT1.1protein accumulation in LR primordia (LRP) through

posttranscriptional regulatory mechanisms by HN(Bouguyon et al., 2016). Furthermore, the point mu-tation of NRT1.1T101A NRT1.1 was shown to inhibit itsauxin transport facilitation, thereby leading to mis-regulation of DR5 activity in LRP and emergence of LRin absence of NO3

2 (Bouguyon et al., 2015). Here, weprovided more in-depth understanding for the role ofNRT1.1 phosphorylation in LR development throughhigh-resolution NMT analysis and LR [3H]IAA uptakeassay in combination with DR5-GFP fluorescence in-tensity quantification.We found that plants producingthe nonphosphorylatable T101A mutant version ofNRT1.1 exhibited increased LR density and auxin ac-cumulation in LRP and new emerged LR resultingfrom reduced basipetal auxin transport in response toNO3

2 limitation (0 and 0.2 mM), whereas plants pro-ducing the phosphomimetic T101D mutant of NRT1.1displayed cessation of LR elongation and auxin accu-mulation due to increased auxin transport (Fig. 1).Given the differences in LR growth between T101Aand T101D plants, we conclude that the switch

Figure 7. Phosphorylation state of NRT1.1 affects nitrate-regulated [Ca21]cyt signaling and ANR1 expression. A, Pseudocolorimages (blue-yellow-red palette) showing [Ca21]cyt signals in LRmeristematic zone of wild-type (WT), chl1-5, T101D, and T101Aseedlings treatedwith different concentrations (0, 0.2, and 10mM) of NO3

– for 60 s. Scale bar5 20mm. B, Quantification analysisof [Ca21]cyt signals (a.u., arbitrary units) from (A). The colored points represent three independent seedlings per treatment(n 5 30–70). Whiskers represent 6 SD. C, Relative ANR1 expression level in LRs of wild-type, chl1-5, T101D, and T101Aseedlings treated with different concentrations (0, 0.2, and 10 mM) of NO3

– for 15 min. Data are means 6 SE from three inde-pendent experiments (n 5 22). In (B) and (C), statistically significant differences are denoted by letters (P , 0.05; Duncanmultiple-comparisons test), and by asterisks for differences between T101D and T101A (*P , 0.05, ***P , 0.001; two-tailedheteroscedastic Student’s t test).


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between the phosphorylation states of NRT1.1 pre-sent on the PM is also involved in the comprehen-sive model of how NRT1.1 acts to modulate LRPdevelopment.

An important determinant for controlling the fun-damental properties of PM proteins is thought to bethe spatiotemporal organization of these proteins.Bouguyon et al. (2015) showed that P492L mutation

Figure 8. LR development and PNR signaling are enhanced by HN-induced endocytosis of NRT1.1. A, Pseudocolor images(blue-yellow-red palette) showing [Ca21]cyt signals in LR meristematic zone of T101A seedlings grown in NO3

–-free medium,exposed to 10 mM of NO3

– with no inhibitors (CK), MbCD, Conc A, or Baf A1 pretreatment, as well as in chc2-1 mutant back-ground. Bar5 20 mm. B, Quantification analysis of [Ca21]cyt signals (a.u., arbitrary units) from (A). The colored points representthree independent seedlings per treatment (n 5 40–60). Whiskers represent 6 SE. Significant differences are denoted by letters(P, 0.05; one-way ANOVAwith Tukeymultiple-comparisons test). C andD, Gene expression level ofNRT1.1 andANR1 (C) andprotein expression level of NRT1.1 (D) in LRs of T101A-GFP and T101A-GFP/chc2-1 seedlings treated with 10 mM of NO3

– for15 min. E and F, Gene expression levels ofNRT1.1 and ANR1 (E) and protein expression level (F) of NRT1.1 in LRs of T101A-GFPseedlings exposed to 0 or 10 mM of NO3

– with no inhibitor, MbCD, Conc A, and Baf A1 (only for E) pretreatment. In (C) and (E),boxplots represent average, 25th and 75th percentiles; colored points represent three independent experiments (n 5 9–24);whiskers represent6 SD. Significant differences are denoted by asterisks (***P, 0.001; Student’s t test). In (D) and (F), stainingwithCoomassie Brilliant Blue serves as the protein loading control. Numbers refer to quantitative results by the software ImageJ. G andH, LR phenotype (G) and density of LRs (H) in T101A-GFPand T101A-GFP/chc2-1 seedlings grownon 10mM of NO3

–medium for8 d. In (G), arrowheads indicate visible LRs. Scale bar 5 1 cm. In (H), boxplots represent average, 25th and 75th percentiles;colored points represent four independent experiments (n 5 38 [green] and 51 [gray]); whiskers represent 6 SD. Significantdifferences are at ***P , 0.001 from Student’s t test.





of NRT1.1 led to repression of LR development bypreventing auxin transport, which might be due to itsmistargeting from PM to the intracellular localiza-tion. Moreover, the mutation of P492L had no sig-nificant influences on primary nitrate response (PNR)and regulation of the genes of clusters 13 and 17(Bouguyon et al., 2015), suggesting the possible roleof NRT1.1 accumulation in intracellular compart-ments might partly contribute to its mediated NO3

2

signaling. These observations provide clues to indi-cate that the subcellular distribution is associatedwith the function of NRT1.1.

Protein phosphorylation is one of the most importantand best-characterized posttranslational modifica-tions. Of first importance, phosphorylation could in-duce the electrostatic and conformational changes ofproteins. For NRT1.1, crystal structure studies re-veal that T101 phosphorylation controls its dimer-ization and/or structural flexibility, thereby affectingthe intrinsic NO3

2 transport capacity (Parker andNewstead, 2014; Sun et al., 2014). Phosphorylationof PM proteins also plays a vital role in a wide rangeof cellular processes including subcellular traffickingand PM dynamics (Offringa and Huang, 2013). Here,we showed that as the active form of NRT1.1 in re-sponse to LN and HN, respectively, phosphorylatedand dephosphorylated, NRT1.1 exhibited the distinctbehavior of LR development (Fig. 1). These data rai-ses the question of whether the phosphorylationstate of NRT1.1 could induce changes in its PM dy-namics and subcellular trafficking, and specificallyaffect NO3

2-dependent auxin transport and NO32

signaling involved in LR development regulation.Thus, an intensive study by single molecular analysiswas performed to provide strong support for ourhypothesis.

Our protein labeling and SPT analysis data indicatedthat T101D clusters had fast lateral mobility with longmotion range, high particle velocity, and reduced dif-fusion coefficient, which may enhance auxin transportefficiency. Conversely, T101A clusters had shorterlifetime and showed more stable behavior with lim-ited motion range, low particle velocity, and increaseddiffusion coefficient, which are favorable for itsdimerization and internalization (Fig. 2, A–E;Supplemental Fig. S6, A–C). The crystal structure ofNRT1.1 indicates that under LN, nonphosphorylatedNRT1.1 forms a homodimer with low structural flex-ibility, and phosphorylation at T101 may decouple thedimer and increase its flexibility (Tsay, 2014). Usingsubunit counting analysis, we found that both T101Aand T101D proteins exhibited the mix of monomericand dimeric status, and the percentage of dimer forT101A was significantly higher than that for T101D,suggesting that the phosphorylation state of NRT1.1exerts significant effect on the interconversion be-tween monomer and dimer state on the PM of LR cells(Fig. 2F; Supplemental Fig. S6F).

Membrane microdomains, sterol- and sphingolipid-enriched regions in the PM, can form platforms for

clustering PM proteins and regulating the spatiotem-poral dynamics of PM proteins (Bücherl et al., 2017).Here, we investigated the spatial relationship betweenphosphorylated and nonphosphorylated NRT1.1 andAtRem1.3, a commonly accepted marker of membranemicrodomains (Demir et al., 2013). In quantitativecolocalization assays, we found higher correlation co-efficients for T101D-GFP with AtRem1.3-mCherry thanfor T101A-GFP with AtRem1.3-mCherry under bothLN and HN (Fig. 3, A and B). Using FRET-FLIM, wefound that the transgenic plants coexpressing T101D-GFP andAtRem1.3-mCherry had a shorter GFP lifetimethan those coexpressing T101A-GFP and AtRem1.3-mCherry (Fig. 3, C and D). Considering previous re-ports that the most highly ordered lipid structures incells of the transition zone correspond with highauxin flux (Mancuso et al., 2007; Zhao et al., 2015;Baluska et al., 2018), we speculate that phosphorylatedNRT1.1 may aggregate into the PM microdomain in aprocess triggered by LN, which might provide an effi-cient means for transporting auxin.

Studies on PM dynamics of plant proteins and spe-cifically on the role of phosphorylation in this processhave only been initiated more recently. Xue et al. (2018)showed that the phosphorylation of blue light receptorphot1 enhanced its interaction with AtRem1.3 andpromoted faster movement on the PM. The distinctbehaviors of NRT1.1 phosphorylation variants, in-cluding dwell time, lateral diffusion, assembly state,and partitioning pattern were found in our study, in-dicating a nonnegligible role of phosphorylation on thePM spatiotemporal dynamics of NRT1.1 (Figs. 2–4).Given our finding of auxin transport activity differencebetween T101A and T101D, it is reasonable to proposethat the spatiotemporal features of NRT1.1 on the PMare important for its auxin transport capacity.

Phosphorylation of PM proteins can regulate theirsorting and subcellular localization, either indirectly byenhancing the exposure of sorting signals throughconformational changes, or directly by enhancing thebinding of regulatory proteins to the phosphorylatedsorting signal itself (Cadena et al., 1994; Nesterov et al.,1995; Moeller et al., 2011). Phosphorylation might affectthe endocytosis of PM proteins, either positively ornegatively (Nguyen et al., 2013; Offringa and Huang,2013). In this investigation, the T101Dmutant showed alonger dwell time and constitutive endomembranetrafficking with slow recycling via a BFA-sensitivepathway, whereas T101A had a much shorter lifetimeand underwent HN-induced internalization that waslargely independent of ARF-GEFs (Fig. 4). These resultsprovide support for the hypothesis that the non-phosphorylated form of T101 facilitates rapid internal-ization of NRT1.1, which may attenuate NRT1.1’scapacity for auxin transport.

A recent study indicates that both CME and CIEpathways are involved in PM protein internalization(Fan et al., 2015). In our study, we found that under LNconditions, almost 88.5% of T101D internalization wasblocked in the chc2 mutant, implying that the CME


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pathway has a dominant role in the endocytosis ofphosphorylated NRT1.1. After short-term HN treat-ment, mutation of CHC2 inhibited T101A endocytosisconsiderably (by ;33.8%), suggesting that the CIEpathway plays an auxiliary, although not trivial, role innonphosphorylated NRT1.1 internalization. We alsofound a high colocalization coefficient and cross cor-relation between T101A-GFP and AtFlot1-mCherry,implying that microdomain-associated endocytosisparticipates in the internalization of T101A as well(Fig. 5; Supplemental Fig. S9, A–D). After prolongedexposure to HN, T101D exhibited no visible vacuolartargeting, whereas T101A followed an endocytic traf-ficking route from the PM to the vacuole, passingmainly through the MVB, PVC, and LE and only par-tially via the TGN and EEs (Fig. 6), which is in goodagreement with the results of other recent studies(Takano et al., 2005; Mbengue et al., 2016; Ortiz-Moreaet al., 2016). Together, our data reveal a dual role ofphosphorylation state in NRT1.1 endocytic pathwaysand intracellular trafficking routes.Phosphorylation-mediated subcellular trafficking in

plant cells was provided by studies on several func-tional proteins such as receptor kinase FLS2 andtransporters PIP2;1 and PINs (Robatzek et al., 2006;Prak et al., 2008; Dhonukshe et al., 2010). From theseevents, specific phosphorylation has been shown to

function in ligand or stress-induced endocytosis andapical recycling for polar targeting. Our findings pro-vide new insights into phosphorylation-regulated PMprotein trafficking in plants. We not only revealed theeffects of phosphorylation on constitutive and HN-induced endocytosis and exocytosis of NRT1.1, butalso on its endocytic pathways as well as intracellulartrafficking routes (Figs. 5 and 6).Receptor-mediated endocytosis is considered an

integral part of signal transduction (Mukhopadhyayand Riezman, 2007; Huang and Chen, 2012; Irannejadet al., 2015; Paez Valencia et al., 2016; Dubeauxand Vert, 2017). However, the phosphorylationstates and endocytic processes of NRT1.1 involvedin nitrate-sensing systems have not been identified.Our results show that nonphosphorylated NRT1.1promotes increases in [Ca21]cyt and ANR1 expres-sion (Fig. 7), supporting the possibility that the T101phosphorylation site of NRT1.1 has a role in fine-tuning nitrate-dependent signaling. In addition, wefound that inhibiting T101A endocytosis decreasednitrate signaling, whereas increased vesicular accu-mulation of T101A increased nitrate signaling(Fig. 8, A–F), confirming that the endosome-localizedNRT1.1 retains signaling activity before recycling ordegradation. Notably, LR growth was greatly re-duced in T101A-GFP/chc2-1 seedlings (Fig. 8, G and

Figure 9. Schematic model of phosphorylation-state–mediated NRT1.1 vesicle cycling and nitrate-dependent signal responsefor LR development. Blue solid arrows represent the direction of endocytosis and pink solid arrows represent the direction ofsecretion or recycling; Blue solid arrows over the PM represent more activity of NRT1.1 that moves down each branch through theindicated pathway. Arrowheads on the black lines represent activation and the vertical crossbars at the ends of lines representinhibition. Dotted lines indicate attenuated responses in LN compared with HN. LN conditions (0.2 mM of NO3

–). HN conditions(10 mM of NO3

–).







H), revealing that clathrin is required for PNR in re-sponse to LR development. Based on these results,we proposed a novel role of endocytosis in regulat-ing nitrate signaling (modeled in Fig. 9): underLN, phosphorylated NRT1.1 proteins undergo slowendocytosis, which contributes to constitutive nitratesignaling, whereas under HN, the induced endocy-tosis of nonphosphorylated NRT1.1 proteins pro-mote their entry into MVBs and/or LEs, whichprobably perpetuates PNR. With recent findings thatintracellular localization of NRT1.1P492L does not af-fect some downstream signaling, our results alsoprovide evidence for the hypothesis that NRT1.1-mediated signaling partly initiates from endosomesrather than PM.

Collectively, the results of this study identify a rolefor the different phosphorylation states of NRT1.1 inits PM spatiotemporal dynamics and subcellular traf-ficking in LR cells and suggest that this modulationmechanism is a novel strategy to control NRT1.1-reg-ulated LR development. Under LN conditions, phos-phorylation of T101 promotes NRT1.1 recruitmentinto functional membrane microdomains at the PM.These actions could facilitate NRT1.1-dependent auxinflux, thus depleting auxin levels in LRP and inhibitingtheir outgrowth. With an increase in nitrate, the non-phosphorylated NRT1.1 displays oligomerization andlow lateral mobility at the PM, and undergoes a faster,inducible endocytosis. These behaviors could promoteLR development by suppressing NRT1.1-auxin trans-port activity on the PM and stimulating Ca21-ANR1signaling from the endosome (Fig. 9).

MATERIALS AND METHODS

Plasmid Constructs

To create transgenic plants expressing NRT1.1, NRT1.1T101D (T101D), andNRT1.1T101A (T101A) fusions with GFP, the 1,751-bp coding region fragmentwas PCR-amplified from the wild-type NRT1.1 complementary DNA (cDNA)and the point mutations T101D and T101A were introduced using the recom-binant PCR technique described in Vaucheret et al. (1990) and Ho et al. (2009).After that, the NRT1.1, T101D, and T101A cDNAs were each subcloned into amodified pCAMBIA 2300-GFP vector under the control of its native promoterregion (;3 kb) to generate the pNRT1.1::NRT1.1, pNRT1.1::T101D, andpNRT1.1::T101A constructs, respectively, and then introduced into the chl1-5mutant plants by using Agrobacterium tumefaciens GV3101 to generate thetransgenic plants expressing NRT1.1-GFP, T101D-GFP, and T101A-GFP(Supplemental Fig. S3A). For expression of NRT1.1, T101A, and T101D fusionswith mRuby in transgenic yeast, NRT1.1, T101A, and T101D cDNA fragmentsamplified by PCR from the pNRT1.1::NRT1.1, pNRT1.1::T101A, andpNRT1.1::T101D-GFP constructs were subcloned into the modified binaryvector pESC-mRuby-URA to create the pESC-NRT1.1-mRuby-URA, pESC-T101A-mRuby-URA, and pESC-T101D-mRuby-URA vectors, respectively,which were then transferred into Saccharomyces cerevisiae strain YPH 499(Supplemental Fig. S1B).

Transgenic Lines

Thewild-type Arabidopsis (Arabidopsis thaliana) accession usedwas Columbia(Col-0). The transgenic Arabidopsis mutant lines NRT1.1T101A (T101A) andNRT1.1T101D (T101D) and the NRT1.1 total deletion mutant chl1-5 were kindlyprovided by Yi-Fang Tasy (Ho et al., 2009; Supplemental Fig. S1A). Thetransgenic line DR5::GFP was kindly provided by Rujin Chen (Laxmi et al.,

2008). Transgenic lines expressing DR5::GFP were obtained by crossing theDR5::GFP line with the chl1-5, T101A, or T101D lines. The chc2-1 mutant seedswere provided by Ji�rí Friml (Ghent University; Kitakura et al., 2011). T101D-GFP/chc2-1 and T101A/chc2-1 were obtained by crossing the T101A-GFP andT101D-GFP lines with chc2-1. The fluorescent reporters AtRem1.3-mCherry andFlot1-mCherry were developed in our laboratory (Wang et al., 2015; Lv et al.,2017). Lines expressing TGN, EE, MVB, LE, PVC, and vacuole markers wereVTI12-mRFP (Zouhar et al., 2009), VHA-a1-mRFP (Dettmer et al., 2006), CLC-mCherry (Wang et al., 2015), AtSNX1-mRFP (Jaillais et al., 2008), mRFP-SYP22(Ebine et al., 2008), and RabG3f-mRFP (Geldner et al., 2009), respectively. Fordual-color imaging, transgenic Arabidopsis plant carrying T101A-GFP werecrossed with AtRem1.3-mCherry, Flot1-mCherry, VTI12-mRFP, VHA-a1-mRFP, CLC-mCherry, AtSNX1-mRFP, mRFP-SYP22, and RabG3f-mRFPplants. The Arabidopsis transgenic line ATG8-CFP (expressing a fusion of theautophagosome marker ATG8 and cyan fluorescent protein [CFP]) was kindlyprovided by Yule Liu (Wang et al., 2013b). Lines expressing NRT1.1-GFP andATG8-CFP were obtained by crossing the ATG8-CFP line with the NRT1.1-GFP line.

Plant Growth Conditions

Arabidopsis seeds were routinely surface-sterilized for 3 min in 4:1 (v/v)85% ethanol:H2O2, plated on basic medium containing 12.5 mM of (NH4)2succinate, 0.5mM of CaSO4, 0.5mM ofMgCl2, 1mM of KH2PO4, 2.5mM ofMES atpH 6.5, 50 mM of NaFeEDTA, 50 mM of H3BO3, 12 mM of MnCl2, 1 mM of CuCl2,1 mM of ZnCl2, and 0.03 mM of NH4MoO4, then chilled at 4°C for 2 d andtransferred to a growth room with a photoperiod of 16 h (light, 100 mmol m22

s21)/8 h (dark) and temperature of 23°C/20°C. After the indicated time ofgrowth (depending on the experiment), the seedlings were shifted to a versionof the same growth medium in which the (NH4)2 succinate was replaced withdifferent concentrations of KNO3 (0, 0.2, 1, and 10 mM; Ho et al., 2009).

IAA Uptake Assay in Yeast

The transformed yeast cells were grown to log phase in synthetic dex-trose dropout media without uracil (SD-URA) and then transferred to syn-thetic galactose dropout media without uracil (SG-URA; SD-URA containing2% [m/v] Gal instead of dextrose) for 24–48 h to induce NRT1.1, T101A, T101D,and mRuby expression, and then harvested by centrifugation and resuspendedin SG-URA. For each assay, 10 mL of resuspended cells were immobilizedon a coverslip for 5 min, washed off with standard medium (0.5 mM of IAA, 2%[m/v] Gal, 0.3 mM of MES, at pH 5.8) to leave a monolayer of attached yeastcells, and then incubated in the standardmedium for 10min. The kinetics of netIAA flux of each cell population were measured by NMT (Beijing Science andTechnology) and analyzed with the program “Mage Flux” (http://www.xuyue.net/mageflux). The experiment was replicated six times for each cate-gory of transformed cell.

IAA Uptake Assay in LRs

Wild-type, T101A, and T101D seedlings were grown on basic mediumcontaining different concentrations of KNO3 (0.2 and 1 mM) for 10 d. Agarblocks of 1 mm in diameter supplemented with 100 nM of tritium-labeled IAA([3H]IAA, cat. no. NET1175; Perkin-Elmer) were placed next to the LR tips(Supplemental Fig. S1D). After incubation for 1.5 h in the dark, agar blockswereremoved. Meanwhile, 0.3 mm of each LR tip was excised and discarded, andthen a 1-cm root segment was excised from the remaining LR and placed into avial containing 2 mL of scintillation fluid. The radioactivity level of the rootsegment was measured with a model no. 1450 MicroBeta Liquid ScintillationCounter (Perkin-Elmer) for 1 min. The experiment was repeated six times foreach category of plant. Six seedlings for each genotype and treatment weredetected and presented.

Confocal Microscopy

Confocal microscopy was done with a model no. TCS SP8 Confocal Mi-croscope (Leica, Germany) fitted with 633 and 1003 oil-immersion objectives.A fluorescence recovery after photobleaching assay of the GFP-labeled seed-lings was performed using a laser (488 nm) for excitation and detecting lightemission at 505–545 nm. The region of interest (ROI) was selected and bleachedfor 15 s with a 488-nm laser at 100% laser power after a prebleach of 3 s. A 2-s


Zhang et al.





http://www.xuyue.net/mageflux

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time interval was used for monitoring fluorescence recovery. The fluorescencerecovery was quantified using ImageJ (1.51w; Collins, 2007). OriginPro v8.5was used for curve fitting (Stevenson, 2011). For the mRuby fluorescence assayin yeast, mRuby was excited using the 563-nm line of an argon laser anddetected from a 581-nm bandpass filter (red). For GFP fusions and Fluo-4-AM-dyed seedlings, an argon laser (488-nm line) was used for excitation and theemission light from 505 to 545 nm (green) was detected with a 633 objective(HCX PL APO 1.4 NA; Leica). Confocal imaging of Arabidopsis LR cellsexpressing GFP with FM4-64 dye treatment was performed with excitation at488 nm. The fluorescence emissions were detected with the spectral detector setBP 505–545 (green) and LP 560–640 (red; Leica). Confocal imaging of Arabi-dopsis LR cells expressing GFPwithmCherry or red fluorescent protein (mRFP)was performed via excitation with 488- and 585-nm light, respectively (multi-track mode) and detection of fluorescence emissions at 505–545 nm (GFP) and600–630 nm (mCherry or mRFP; Leica). Arabidopsis LR cells coexpressingATG8-CFP and NRT1.1-GFP were excited with 436-nm and 488-nm light anddetected with 450–476 (CFP) and 505–545 (GFP).

Fluo-4 AM-Based Ca21 Imaging in LRs

For [Ca21]cyt signal imaging of the LR cells of wild-type, chl1-5, T101A, andT101D seedlings, seedlings were grown on the normal half-strength Murashigeand Skoog (MS) medium for 8 d, transferred to liquid basic medium withoutnitrate for 2 d, then incubated with Fluo-4 AM for 20 h at 4°C in the dark. Afterthree repeats of awash-out with basicmedium followed by 1 h of standing, eachseedling was plated on a slide and covered with a cover glass, and then basalmedium (200 mL) with 0, 0.2, and 10 mM of KNO3 was loaded along one edge ofthe coverslip. A piece of absorbent paper was placed at the opposite edge towick out the buffer. For [Ca21]cyt signal imaging of the effects of inhibitortreatment, the inhibitor was added for 1 h and then the plants were treated with10 mM of KNO3 before imaging. Confocal imaging was acquired using a modelno. SP8 Confocal Microscope (Leica). The fluorescence intensity was deter-mined with the “ROI” function of ImageJ (1.51w) for each seedling. The in-tensity data were exported and processed using the program OriginPro v8.5.The modified rainbow RGB Lookup Table was applied with ImageJ. The im-ages were exported and processed using the software Adobe Illustrator (CC2017).

RNA Isolation and RT-qPCR

For ANR1 expression in LRs, wild-type, chl1-5, T101A, and T101D seedlingswere grown in basic medium at pH 5.8 for 10 d, transferred into fresh liquidbasic medium at pH 5.5 overnight/16 h and then placed for 15 min in the samegrowth medium but with the (NH4)2 succinate replaced with the appropriateconcentration of KNO3 (depending on the experiment). The apical parts (10–15mm)of the plant LRs were separated surgically from the rest of the root system(Remans et al., 2006). For NRT1.1 and ANR1 expression in GFP-labeled trans-genic plants, 7-d–old plants grown on basic medium were harvested and thentransferred to liquid medium of appropriate composition. Total RNA wasextracted from homogenized tissues by using the RNA prep Pure Plant Kit(Tiangen Biotech). Three micrograms of total RNAwere used to prepare cDNAusing a PrimeScript first-Strand cDNA Synthesis Kit (Takara BiomedicalTechnology) by reverse transcription with Moloney-Murine Leukemia Virusreverse transcriptase and oligo(dT)18 primers. Gene expression was determinedby PCR and quantitative real-time PCR with a CFX Connect Real-Time PCRsystem (Bio-Rad) by SYBR Premix Ex Taq II (Tli RNase H Plus; Takara Bio-medical Technology). ACTIN2, CLATHRIN, and UBIQUITIN10 served as thecontrols for RT-qPCR and gene-specific primers are listed in SupplementalTable S1. The experiments were repeated six times, and the 2–DDCT quantifica-tion method was used to evaluate quantitative variation.

Analysis of LR Growth

Seedlings were germinated on half-strength MS medium for 3 d and thentransferred to basic medium containing different concentrations of KNO3 (0, 0.2and 1mM) for an extra 5 d. The root systems were scanned at 500 dpi (model no.EOS 600D; Canon). Root growth parameters including LR length and densitywere analyzed using the software ImageJ. Fifteen seedlings for each genotypeand treatment were measured and presented. The experiment was repeatedseven times.

VA-TIRFM and Fluorescence Image Analysis

VA-TIRFM was performed on an inverted microscope (model no. IX-71;Olympus) with a total internal reflective fluorescence illuminator (model no.IX2-RFAEVA-2; Olympus) and a 1003 oil-immersion objective (numericalaperture5 1.45; Olympus). GFP-labeled proteins in LR epidermal cell from 12-d–old Arabidopsis seedlings were excited with 488-nm laser lines from a diodelaser (Chang-chun New Industries Optoelectronics Technology) and theiremission fluorescence was obtained with a BA510IF (505/50) filter for GFP. Thefluorescence signals were detected with a back-illuminated EMCCD camera(model no. iXonDV8897D–CS0–VP; Andor Technology). Imageswere acquiredwith 100-ms exposure times and a 330-ms time-lapse series of single particles ofT101D-GFP or T101A-GFP, which was taken with up to 100 images per se-quence. For the analysis of VA-TIRFM images, the background was subtractedwith a “rolling-ball radius: 50 pixels,” and pseudo-colors (green) were added.The stand-alone MATLAB Graphical User Interface program (R2014a; https://neurophotonics.ca/software) was then used for SPT according to the methoddescribed by Jaqaman et al. (2008). Kymograph and lifetime analyses wereperformed as described in Eichel et al. (2016). The diffusion coefficient, motionrange and particle velocity analyses were done according to methods describedin Li et al. (2011), Hao et al. (2014) and Lv et al. (2017). The single-particle flu-orescence intensity and the photobleaching steps were calculated according tothemethods described inWang et al. (2015) and Lv et al. (2017). The ProgressiveIdealization and Filtering program was used for step-wise photobleachinganalysis after background subtraction as described in McGuire et al. (2012).

FCCS Analysis

To quantitatively measure the interactions among T101A-GFP and AtCLC-mCherry, AtFlot1-mCherry, and AtRem1.3-mCherry, LR cells of 10-d–oldseedlings expressing T101A-GFP with AtCLC-mCherry/AtFlot1-mCherry/AtRem1.3-mCherrywere detected by FCCS, performed on amodel no. TCS SP5microscope (Leica) as described in Mütze et al. (2011). GFP and mCherry wereexcited with 488-nm and 561-nm laser lines, respectively. Emission filters wereset as BP 505–540 and LP 580 for the green and red channels, respectively. Therelative cross correlation ([Gc(0)]/[Gg(0)]) was calculated by normalizing theamplitude of the cross-correlation function to the amplitude of the autocorre-lation function of GFP as described in Li et al. (2011).

Transient Infiltration of Nicotiana benthamiana Leaves

Leaves of 7-week–oldN. benthamianawere infiltrated with equal amounts ofA. tumefaciens expressing full-length NRT1.1-GFP, T101A-GFP, or T101D-GFPfusion constructs together with AtRem1.3-mCherry, AtFlot1-mCherry, orAtCLC-mCherry with all the possible combinations. After 2 d of inoculation,leaves were harvested and treated with 0.2- and 10-mM nitrate and then pho-tographed using a model no. SP8 Confocal Microscope (Leica).

FRET-FLIM

For FRET-FLIM, seedlings coexpressing AtRem1.3-mCherry and T101A-GFP or T101D-GFP were treated with the appropriate concentration of nitrate.FLIM was performed on an inverted model no. FV1200 microscope (Olympus)equippedwith a model no. picoHarp300 controller (PicoQuant). The excitation at488 nmwas carried out by a picosecond pulsed diode laser at a repetition rate of40 MHz through a water-immersion objective (603, numerical aperture 5 1.2).The emitted lightwas filteredwith a 510/55-nmbandpassfilter and detectedwithan Micro Photon Devices Single Photon Avalanche Diodes detector. Images ofeach selected ROIwere obtainedwith acquisition of at least 50,000 photons. Fromthe fluorescence intensity images, the decay curves were calculated per pixel andfitted with either a mono- or double-exponential decay model using the software“SymphoTime 64” (PicoQuant). The monoexponential model function was ap-plied for donor sampleswith onlyGFP present and the double-exponentialmodelfunction was used for samples containing GFP and mCherry. Data on fluores-cence lifetime (in picoseconds) were obtained and FRET efficiency (%) analysiswas performed using the software Excel 2013 (Microsoft).

Colocalization Analysis

Colocalization analysis of confocal images was performedwith the softwareImageJ. For transient infiltration ofN. benthamiana leaves, NRT1.1-GFP, T101A-






https://neurophotonics.ca/software

https://neurophotonics.ca/software


GFP, and T101D-GFP fluorescence colocalized with AtRem1.3-mCherry werecalculated by using the plug-in “Coloc 2” (https://imagej.net/Coloc_2). Theobtained modified Manders coefficients were interpreted as the fraction ofcolocalization for both channels (i.e. colocalization of AtRem1.3-mCherry withNRT1.1-GFP, T101A-GFP, or T101D-GFP and vice versa; Bücherl et al., 2013).Pearson correlation coefficients were obtained as another measure of colocali-zation (Bücherl et al., 2013, 2017; Demir et al., 2013). The colocalization analysisfor T101A-GFP and AtFlot1-mCherry, AtCLC-mCherry, and AtRem1.3-mCherry in LR cells was carried out as described using ImageJ in Jarsch et al.(2014) and Bücherl et al. (2017) . Endomembrane compartments were excludedfrom the colocalization analysis by ROI selection. First, acquired images were“Mean-Shift” filtered with a radius of 2 pixels. Background was subtractedusing the “Rolling-ball”method with a radius of 20 pixels. ROIs were manuallyselected and the colocalization plug-in “Intensity Correlation Analysis” wasapplied for quantification (Bücherl et al., 2017; the plug-in is available at:http://www.aomf.ca/WCIF.html). For the colocalization analysis betweenT101A-GFP with different endomembrane markers in LR cells, the plug-in“PSC colocalization” in the software ImageJ was used to obtain the Pearsoncorrelation coefficients as colocalization readouts. To determine the percentageof T101A-GFP vesicles colocalizedwith different endomembranemarkers, eachindividual T101A-GFP spot was selected as an individual ROI and colocaliza-tion was analyzed with a threshold of 10, considering the spots for which thePearson correlation value was .0.5 as positively labeled endosomes. Athreshold of 10 was used to avoid noise, and calculations were done by mea-suring the background gray values in the analyzed images.

Drug Treatments

BFA and CHX were obtained from Sigma-Aldrich and dissolved in 100%DMSO to yield 50-mM stock solutions that were diluted to 50 mM with half-strength liquid MS medium for use. A solution of 200 mM of MbCD (Sigma-Aldrich) was prepared in deionized water and then diluted to 10 mM with half-strength liquid MS medium for use. FM4-64 (Invitrogen) was kept as a 5-mM

stock solution and diluted to a 5-mM working solution with half-strength liquidMSmedium. Inhibitors were used at the following concentrations: 2 mM of ConcA (Sigma-Aldrich; with 2 mM of DMSO stock), 33 mM of WM (Sigma-Aldrich;with 33 mM of DMSO stock), 1 mM of Baf A1 (Tocris Biosciences; with 1 mM ofDMSO stock), and 20 mM of MG132 (Sigma-Aldrich; with 20 mM DMSO stock).

Immunoblot Analysis

For immunoblot analyses, total proteinswere extracted fromLRs of 12-d–oldseedlings (100–200 mg). For protein detection, the following antibodies (Abb-kine Scientific) were used: anti-GFP tag rabbit polyclonal antibody (1/5,000)and antiplant actin mouse monoclonal antibody (1/10,000) as primary anti-body, and IPKine HRP Goat Anti-Rabbit IgG HCS (1/10,000) and IPKine HRPGoat Anti-Mouse IgGHCS (1/10,000) as second antibody. The intensity of eachband was detected with ImageJ and normalized with respect to the control.

Accession Numbers

Sequence data from this work can be found in The Arabidopsis InformationResource database under the following accession numbers: AT1G12110(NRT1.1), AT2G14210 (ANR1), AT3G18780 (ACTIN 2), AT4G24550 (CLATH-RIN), and AT4G05320.2 (UBIQUITIN 10).

Supplemental Data

The following materials are available.

Supplemental Figure S1. T101D mutation strengthens the auxin transportactivity of NRT1.1 in LRs.

Supplemental Figure S2. Confocal images and quantitative analysis ofDR5::GFP in LRs.

Supplemental Figure S3. The roots of transgenic NRT1.1-GFP,NRT1.1T101D-GFP, and NRT1.1T101A-GFP plants have normal biologicalfunction and organizational structure.

Supplemental Figure S4. The transgenic NRT1.1-GFP, NRT1.1T101D-GFP,and NRT1.1T101A-GFP plants have corresponding LR phenotypes.

Supplemental Figure S5. Dynamics of T101A-GFP and T101D-GFP onthe PM.

Supplemental Figure S6. Motion parameters of T101A-GFP and T101D-GFP on the PM.

Supplemental Figure S7. The phosphorylation state of NRT1.1 affects itscolocalization with AtRem1.3.

Supplemental Figure S8. Secretion and internalization of NRT1.1 are af-fected by its phosphorylation state.

Supplemental Figure S9. Both CME and CIE pathways are involved in theendocytosis of T101D-GFP and T101A-GFP.

Supplemental Figure S10. The degradation of NRT1.1 is involved in pro-teasome system and autophagy under HN conditions and nitrogen star-vation, respectively.

Supplemental Figure S11. Ca21 signaling disappears within 120 s afternitrate treatment in LR epidermis cells of wild-type Arabidopsis.

Supplemental Table S1. Primer used in this research.

Supplemental Video S1. The movie of dynamics of T101D-GFP spots(green) on the PM under the treatment of 0.2-mM NO3

2 concentration.

Supplemental Video S2. The movie of dynamics of T101A-GFP spots(green) on the PM under the treatment of 10-mM NO3

2 concentration.

ACKNOWLEDGMENTS

We thank Yi-Fang Tsay (Institute of Molecular Biology, Academia Sinica,Taipei 115, Taiwan) for the seeds of T101A, T101D, and chl1-5. We thankWen--Juan Wang and Yan-Li Zhang (Imaging Core Facility of Protein Research Cen-ter for Technology Development, Tsinghua University) for FRET-FLIMtechnical assistance. We thank Qi-Hua He (Peking University Health ScienceCenter) for technical assistance with FCCS. We thank Ming-Hua Tian (Instituteof Genetics and Developmental Biology, Chinese Academy of Science) for iso-tope test technical assistance.

Received March 22, 2019; accepted August 9, 2019; published August 20, 2019.

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Phosphorylation-Mediated Dynamics of Nitrate Transceptor … · The phosphomimetic NRT1.1T101D form...

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