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 of NRT1.1 plays a key role in NRT1.1 function during LR development. Single-particle tracking revealed that phosphorylation affected NRT1.1 spatiotemporal dynamics. The phosphomimetic NRT1.1T101D form showed fast lateral mobility and membrane partitioning that facilitated auxin flux under low-nitrate conditions. By contrast, nonphosphorylatable NRT1.1T101A showed low lateral mobility and oligomerized at the plasma membrane (PM), where it induced endocytosis via the clathrin-mediated endocytosis and microdomain-mediated endocytosis pathways under high-nitrate conditions. These behaviors promoted LR development by suppressing NRT1.1-controlled auxin transport on the PM and stimulating Ca2+-ARABIDOPSIS NITRATE REGULATED1 signaling from the endosome.

Beijing Advanced Innovation Center for Tree Breeding by Molecular Design Beijing Forestry University Beijing 10083 China

Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Olomouc 78301 Czech Republic

College of Biological Sciences and Biotechnology Beijing Forestry University Beijing 10083 China

Institute of Cellular and Molecular Botany University of Bonn Bonn D 53115 Germany

See more in PubMed

Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3: 83–89 PubMed

Baluška F, Strnad M, Mancuso S (2018) Substantial evidence for auxin secretory vesicles. Plant Physiol 176: 2586–2587 PubMed PMC

Bouguyon E, Brun F, Meynard D, Kubeš M, Pervent M, Léran S, Lacombe B, Krouk G, Guiderdoni E, Zažímalová E, et al. (2015) Multiple mechanisms of nitrate sensing by Arabidopsis nitrate transceptor NRT1.1. Nat Plants 1: 15015. PubMed

Bouguyon E, Perrine-Walker F, Pervent M, Rochette J, Cuesta C, Benkova E, Martinière A, Bach L, Krouk G, Gojon A, Nacry P (2016) Nitrate controls root development through posttranscriptional regulation of the NRT1.1/NPF6.3 transporter/Sensor. Plant Physiol 172: 1237–1248 PubMed PMC

Bücherl CA, Jarsch IK, Schudoma C, Segonzac C, Mbengue M, Robatzek S, MacLean D, Ott T, Zipfel C (2017) Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains. eLife 6: e25114. PubMed PMC

Bücherl CA, van Esse GW, Kruis A, Luchtenberg J, Westphal AH, Aker J, van Hoek A, Albrecht C, Borst JW, de Vries SC (2013) Visualization of BRI1 and BAK1(SERK3) membrane receptor heterooligomers during brassinosteroid signaling. Plant Physiol 162: 1911–1925 PubMed PMC

Cadena DL, Chan CL, Gill GN (1994) The intracellular tyrosine kinase domain of the epidermal growth factor receptor undergoes a conformational change upon autophosphorylation. J Biol Chem 269: 260–265 PubMed

Collins TJ. (2007) ImageJ for microscopy. Biotechniques 43(1, Suppl): 25–30 PubMed

Cui Y, Li X, Yu M, Li R, Fan L, Zhu Y, Lin J (2018) Sterols regulate endocytic pathways during flg22-induced defense responses in Arabidopsis. Development 145: dev165688. PubMed

Demir F, Horntrich C, Blachutzik JO, Scherzer S, Reinders Y, Kierszniowska S, Schulze WX, Harms GS, Hedrich R, Geiger D, Kreuzer I (2013) Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proc Natl Acad Sci USA 110: 8296–8301 PubMed PMC

Dettmer J, Hong-Hermesdorf A, Stierhof YD, Schumacher K (2006) Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell 18: 715–730 PubMed PMC

Du Y, Scheres B (2018) Lateral root formation and the multiple roles of auxin. J Exp Bot 69: 155–167 PubMed

Dubeaux G, Vert G (2017) Zooming into plant ubiquitin-mediated endocytosis. Curr Opin Plant Biol 40: 56–62 PubMed

Dhonukshe P, Huang F, Galvan-Ampudia CS, Mähönen AP, Kleine-Vehn J, Xu J, Quint A, Prasad K, Friml J, Scheres B, Offringa R (2010) Plasma membrane-bound AGC3 kinases phosphorylate PIN auxin carriers at TPRXS(N/S) motifs to direct apical PIN recycling. Development 137: 3245–3255 PubMed

Ebine K, Okatani Y, Uemura T, Goh T, Shoda K, Niihama M, Morita MT, Spitzer C, Otegui MS, Nakano A, Ueda T (2008) A SNARE complex unique to seed plants is required for protein storage vacuole biogenesis and seed development of Arabidopsis thaliana. Plant Cell 20: 3006–3021 PubMed PMC

Eichel K, Jullié D, von Zastrow M (2016) β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation. Nat Cell Biol 18: 303–310 PubMed PMC

Fan L, Hao H, Xue Y, Zhang L, Song K, Ding Z, Botella MA, Wang H, Lin J (2013) Dynamic analysis of Arabidopsis AP2 σ subunit reveals a key role in clathrin-mediated endocytosis and plant development. Development 140: 3826–3837 PubMed

Fan L, Li R, Pan J, Ding Z, Lin J (2015) Endocytosis and its regulation in plants. Trends Plant Sci 20: 388–397 PubMed

Gan Y, Bernreiter A, Filleur S, Abram B, Forde BG (2012) Overexpressing the ANR1 MADS-box gene in transgenic plants provides new insights into its role in the nitrate regulation of root development. Plant Cell Physiol 53: 1003–1016 PubMed

Geldner N, Dénervaud-Tendon V, Hyman DL, Mayer U, Stierhof YD, Chory J (2009) Rapid, combinatorial analysis of membrane compartments in intact plants with a multicolor marker set. Plant J 59: 169–178 PubMed PMC

Hao H, Fan L, Chen T, Li R, Li X, He Q, Botella MA, Lin J (2014) Clathrin and membrane microdomains cooperatively regulate RbohD dynamics and activity in Arabidopsis. Plant Cell 26: 1729–1745 PubMed PMC

Ho CH, Lin SH, Hu HC, Tsay YF (2009) CHL1 functions as a nitrate sensor in plants. Cell 138: 1184–1194 PubMed

Huang F, Chen YG (2012) Regulation of TGF-β receptor activity. Cell Biosci 2: 9. PubMed PMC

Irannejad R, Tsvetanova NG, Lobingier BT, von Zastrow M (2015) Effects of endocytosis on receptor-mediated signaling. Curr Opin Cell Biol 35: 137–143 PubMed PMC

Jaillais Y, Fobis-Loisy I, Miège C, Gaude T (2008) Evidence for a sorting endosome in Arabidopsis root cells. Plant J 53: 237–247 PubMed

Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5: 695–702 PubMed PMC

Jarsch IK, Konrad SS, Stratil TF, Urbanus SL, Szymanski W, Braun P, Braun KH, Ott T (2014) Plasma membranes are subcompartmentalized into a plethora of coexisting and diverse microdomains in Arabidopsis and Nicotiana benthamiana. Plant Cell 26: 1698–1711 PubMed PMC

Kitakura S, Vanneste S, Robert S, Löfke C, Teichmann T, Tanaka H, Friml J (2011) Clathrin mediates endocytosis and polar distribution of PIN auxin transporters in Arabidopsis. Plant Cell 23: 1920–1931 PubMed PMC

Kleine-Vehn J, Leitner J, Zwiewka M, Sauer M, Abas L, Luschnig C, Friml J (2008) Differential degradation of PIN2 auxin efflux carrier by retromer-dependent vacuolar targeting. Proc Natl Acad Sci USA 105: 17812–17817 PubMed PMC

Krapp A, David LC, Chardin C, Girin T, Marmagne A, Leprince A-S, Chaillou S, Ferrario-Méry S, Meyer C, Daniel-Vedele F (2014) Nitrate transport and signalling in Arabidopsis. J Exp Bot 65: 789–798 PubMed

Krouk G. (2017) Nitrate signalling: Calcium bridges the nitrate gap. Nat Plants 3: 17095. PubMed

Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, Hoyerova K, Tillard P, Leon S, Ljung K, Zazimalova E, Benkova E, et al. (2010) Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Dev Cell 18: 927–937 PubMed

Kusumi A, Fujiwara TK, Chadda R, Xie M, Tsunoyama TA, Kalay Z, Kasai RS, Suzuki KG (2012) Dynamic organizing principles of the plasma membrane that regulate signal transduction: commemorating the fortieth anniversary of Singer and Nicolson’s fluid-mosaic model. Annu Rev Cell Dev Biol 28: 215–250 PubMed

Laxmi A, Pan J, Morsy M, Chen R (2008) Light plays an essential role in intracellular distribution of auxin efflux carrier PIN2 in Arabidopsis thaliana. PLoS One 3: e1510. PubMed PMC

Léran S, Varala K, Boyer JC, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B, Gassmann W, Geiger D, et al. (2014) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci 19: 5–9 PubMed

Li X, Luu DT, Maurel C, Lin J (2013) Probing plasma membrane dynamics at the single-molecule level. Trends Plant Sci 18: 617–624 PubMed

Li X, Wang X, Yang Y, Li R, He Q, Fang X, Luu DT, Maurel C, Lin J (2011) Single-molecule analysis of PIP2;1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation. Plant Cell 23: 3780–3797 PubMed PMC

Li X, Xing J, Qiu Z, He Q, Lin J (2016) Quantification of membrane protein dynamics and interactions in plant cells by fluorescence correlation spectroscopy. Mol Plant 9: 1229–1239 PubMed

Liu KH, Huang CY, Tsay YF (1999) CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11: 865–874 PubMed PMC

Liu KH, Niu Y, Konishi M, Wu Y, Du H, Sun Chung H, Li L, Boudsocq M, McCormack M, Maekawa S, Ishida T, Zhang C, et al. (2017) Discovery of nitrate–CPK–NLP signalling in central nutrient-growth networks. Nature 545: 311–316 PubMed PMC

Liu KH, Tsay YF (2003) Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J 22: 1005–1013 PubMed PMC

Lv X, Jing Y, Xiao J, Zhang Y, Zhu Y, Julian R, Lin J (2017) Membrane microdomains and the cytoskeleton constrain AtHIR1 dynamics and facilitate the formation of an AtHIR1-associated immune complex. Plant J 90: 3–16 PubMed

Mancuso S, Marras AM, Mugnai S, Schlicht M, Zársky V, Li G, Song L, Xue HW, Baluška F (2007) Phospholipase dzeta2 drives vesicular secretion of auxin for its polar cell-cell transport in the transition zone of the root apex. Plant Signal Behav 2: 240–244 PubMed PMC

Mayor S, Pagano RE (2007) Pathways of clathrin-independent endocytosis. Nat Rev Mol Cell Biol 8: 603–612 PubMed

Mbengue M, Bourdais G, Gervasi F, Beck M, Zhou J, Spallek T, Bartels S, Boller T, Ueda T, Kuhn H, Robatzek S (2016) Clathrin-dependent endocytosis is required for immunity mediated by pattern recognition receptor kinases. Proc Natl Acad Sci USA 113: 11034–11039 PubMed PMC

McGuire H, Aurousseau MRP, Bowie D, Blunck R (2012) Automating single subunit counting of membrane proteins in mammalian cells. J Biol Chem 287: 35912–35921 PubMed PMC

Moeller HB, Olesen ET, Fenton RA (2011) Regulation of the water channel aquaporin-2 by posttranslational modification. Am J Physiol Renal Physiol 300: F1062–F1073 PubMed

Mounier E, Pervent M, Ljung K, Gojon A, Nacry P (2014) Auxin-mediated nitrate signalling by NRT1.1 participates in the adaptive response of Arabidopsis root architecture to the spatial heterogeneity of nitrate availability. Plant Cell Environ 37: 162–174 PubMed

Mukhopadhyay D, Riezman H (2007) Proteasome-independent functions of ubiquitin in endocytosis and signaling. Science 315: 201–205 PubMed

Mütze J, Ohrt T, Schwille P (2011) Fluorescence correlation spectroscopy in vivo. Laser Photonics Rev 5: 52–67

Nesterov A, Wiley HS, Gill GN (1995) Ligand-induced endocytosis of epidermal growth factor receptors that are defective in binding adaptor proteins. Proc Natl Acad Sci USA 92: 8719–8723 PubMed PMC

Nguyen LK, Kolch W, Kholodenko BN (2013) When ubiquitination meets phosphorylation: a systems biology perspective of EGFR/MAPK signalling. Cell Commun Signal 11: 52. PubMed PMC

Offringa R, Huang F (2013) Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells. J Integr Plant Biol 55: 789–808 PubMed

Ortiz-Morea FA, Savatin DV, Dejonghe W, Kumar R, Luo Y, Adamowski M, Van den Begin J, Dressano K, Pereira de Oliveira G, Zhao X, et al. (2016) Danger-associated peptide signaling in Arabidopsis requires clathrin. Proc Natl Acad Sci USA 113: 11028–11033 PubMed PMC

Paez Valencia J, Goodman K, Otegui MS (2016) Endocytosis and endosomal trafficking in plants. Annu Rev Plant Biol 67: 309–335 PubMed

Parker JL, Newstead S (2014) Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature 507: 68–72 PubMed PMC

Prak S, Hem S, Boudet J, Viennois G, Sommerer N, Rossignol M, Maurel C, Santoni V (2008) Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP2;1 in response to salt stress. Mol Cell Proteomics 7: 1019–1030 PubMed

Remans T, Nacry P, Pervent M, Filleur S, Diatloff E, Mounier E, Tillard P, Forde BG, Gojon A (2006) The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches. Proc Natl Acad Sci USA 103: 19206–19211 PubMed PMC

Riveras E, Alvarez JM, Vidal EA, Oses C, Vega A, Gutiérrez RA (2015) The calcium ion is a second messenger in the nitrate signaling pathway of Arabidopsis. Plant Physiol 169: 1397–1404 PubMed PMC

Robatzek S, Chinchilla D, Boller T (2006) Ligand-induced endocytosis of the pattern recognition receptor FLS2 in Arabidopsis. Genes Dev 20: 537–542 PubMed PMC

Stevenson KJ. (2011) Review of OriginPro 8.5. J Am Chem Soc 133: 5621

Stoeckle D, Thellmann M, Vermeer JE (2018) Breakout-lateral root emergence in Arabidopsis thaliana. Curr Opin Plant Biol 41: 67–72 PubMed

Sun CH, Yu JQ, Hu DG (2017) Nitrate: A crucial signal during lateral roots development. Front Plant Sci 8: 485. PubMed PMC

Sun J, Bankston JR, Payandeh J, Hinds TR, Zagotta WN, Zheng N (2014) Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature 507: 73–77 PubMed PMC

Takano J, Miwa K, Yuan L, von Wirén N, Fujiwara T (2005) Endocytosis and degradation of BOR1, a boron transporter of Arabidopsis thaliana, regulated by boron availability. Proc Natl Acad Sci USA 102: 12276–12281 PubMed PMC

Tsay YF. (2014) Plant science: How to switch affinity. Nature 507: 44–45 PubMed

Vaucheret H, Chabaud M, Kronenberger J, Caboche M (1990) Functional complementation of tobacco and Nicotiana plumbaginifolia nitrate reductase deficient mutants by transformation with wild-type alleles of the tobacco structural genes. Mol Gen Genet 220: 468–474

Wang L, Li H, Lv X, Chen T, Li R, Xue Y, Jiang J, Jin B, Baluška F, Šamaj J, Wang X, Lin J (2015) Spatiotemporal dynamics of the BRI1 receptor and its regulation by membrane microdomains in living Arabidopsis cells. Mol Plant 8: 1334–1349 PubMed

Wang L, Xue Y, Xing J, Song K, Lin J (2018a) Exploring the spatiotemporal organization of membrane proteins in living plant cells. Annu Rev Plant Biol 69: 525–551 PubMed

Wang Q, Zhao Y, Luo W, Li R, He Q, Fang X, Michele RD, Ast C, von Wirén N, Lin J (2013a) Single-particle analysis reveals shutoff control of the Arabidopsis ammonium transporter AMT1;3 by clustering and internalization. Proc Natl Acad Sci USA 110: 13204–13209 PubMed PMC

Wang Y, Yu B, Zhao J, Guo J, Li Y, Han S, Huang L, Du Y, Hong Y, Tang D, Liu Y (2013b) Autophagy contributes to leaf starch degradation. Plant Cell 25: 1383–1399 PubMed PMC

Wang YY, Cheng YH, Chen KE, Tsay YF (2018b) Nitrate transport, signaling, and use efficiency. Annu Rev Plant Biol 69: 85–122 PubMed

Wu LG, Hamid E, Shin W, Chiang HC (2014) Exocytosis and endocytosis: modes, functions, and coupling mechanisms. Annu Rev Physiol 76: 301–331 PubMed PMC

Xue Y, Xing J, Wan Y, Lv X, Fan L, Zhang Y, Song K, Wang L, Wang X, Deng X, Baluška F, Christie JM, et al. (2018) Arabidopsis blue light receptor phototropin 1 undergoes blue light-induced activation in membrane microdomains. Mol Plant 11: 846–859 PubMed

Zhao X, Zhang X, Qu Y, Li R, Baluška F, Wan Y (2015) Mapping of membrane lipid order in root apex zones of Arabidopsis thaliana. Front Plant Sci 6: 1151. PubMed PMC

Zouhar J, Rojo E, Bassham DC (2009) AtVPS45 is a positive regulator of the SYP41/SYP61/VTI12 SNARE complex involved in trafficking of vacuolar cargo. Plant Physiol 149: 1668–1678 PubMed PMC

Imaging plant cells and organs with light-sheet and super-resolution microscopy