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Cloning and Molecular Characterization of CcNRT2.1/CcNAR2, a Putative Inducible High Affinity Nitrate Transport System in Capsicum chinense Jacq. Roots

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Abstract

NRT2 family of nitrate transporters normally require a partner protein, NAR2 (nitrate assimilation related protein), to transport nitrate to a high-affinity (high affinity nitrate transport system, HATS), although its role is still not well understood. In this study, the CcNRT2.1 and CcNAR2 cDNAs from of Capsicum chinense were cloned and characterized in terms of their structure, phylogeny, and their organ-specific transcriptional regulation, and by ligh/dark cycle, N and sucrose. CcNRT2.1 (putative molecular mass 57.5 kDa) and CcNAR2 (22.66 kDa) retained the characteristic domains of NRT2 and NAR2 family, respectively. CcNRT2.1 is expressed exclusively in the root, and both western blot and protein immunolocalization, demonstrated its exclusive location in root epidermal and cortical cells. CcNAR2 is expressed mainly in the root, but also in fruits of 25 days post-anthesis (DPA). The presence of two protein bands (~60 and ~80 kDa) detected in the western blot, suggested that CcNRT2.1 monomer and CcNRT2.1/CcNAR2 heterodimer work together for nitrate transport function in roots. Both genes were induced by NO3−, NH4+ and glutamate, and CcNRT2.1 was induced by sucrose. The co-expression of both genes in the specific zone of the root (2–4 cm from the apex) in which the high-affinity nitrate transport occurs (evaluated by root NO3− endogenous content, extracellular pH alkalinization, and H+ net flux by MIFE technique), strongly supports that CcNRT2.1/CcNAR2 belong to a HATS, with a functional similarity to AtNRT2.1/AtNAR2.1 of Arabidopsis. These are the first components of a HATS characterized in the Capsicum genus.

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Abbreviations

DPA:

Days post-anthesis

HATS:

High-affinity transport system

HTM:

Transmembrane helix

LATS:

Low-affinity transport system

LIX:

Liquid ion exchanger

LR:

Lateral root

MFS:

Major facilitator family

NAR2:

Nitrate assimilation related protein 2

NNP:

Nitrate/nitrite porter

NRT1/PTR:

Nitrate transporter 1/peptide transporter family

NRT2:

Nitrate transporter 2

PR:

Primary root

References

  • Bowman JL et al (2017) Insights into land plant evolution garnered from the Marchantia polymorpha genome. Cell 171(2):287–304

    CAS  PubMed  Google Scholar 

  • Cawse PA (1967) The determination of nitrate in soil solutions by ultraviolet spectrophotometry. Analyst 92(1094):311–315

    CAS  Google Scholar 

  • Celis-Arámburo TJ, Carrillo-Pech M, Castro-Concha LA, Miranda-Ham ML, Martínez-Estévez M, Echevarría-Machado I (2011) Exogenous nitrate induces root branching and inhibits primary root growth in Capsicum chinense Jacq. Plant Physiol Biochem 49(12):1456–1464

    Google Scholar 

  • Cerezo M, Tillard P, Filleur S, Muños S, Daniel-Vedele F, Gojon A (2001) Major alterations of the regulation of root NO3− uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis. Plant Physiol 127(1):262–271

    CAS  PubMed  PubMed Central  Google Scholar 

  • Charrier A, Bérard JB, Bougaran G, Carrier G, Lukomska E, Schreiber N, Fournier F, Charrier AF, Rouxel C, Garnier M, Cadoret JP, Saint-Jean B (2015) High-affinity nitrate/nitrite transporter genes (Nrt2) in Tisochrysis lutea: identification and expression analyses reveal some interesting specificities of Haptophyta microalgae. Physiol Plant 154(4):572–590

    CAS  PubMed  Google Scholar 

  • Chen X, Yao Q, Gao X, Jiang C, Harberd NP, Fu X (2016) Shoot-to-root mobile transcription factor HY5 coordinates plant carbon and nitrogen acquisition. Curr Biol 26(5):640–646

    CAS  PubMed  Google Scholar 

  • Crawford NM, Forde BG (2002) Molecular and developmental biology of inorganic nitrogen nutrition. Arab B 1:e0011

    Google Scholar 

  • Dang S, Sun L, Huang Y, Lu F, Liu Y, Gong H, Wang J, Yan N (2010) Structure of a fucose transporter in an outward-open conformation. Nature 467:734

    CAS  PubMed  Google Scholar 

  • Dechorgnat J, Nguyen CT, Armengaud P, Jossier M, Diatloff E, Filleur S, Daniel-Vedele F (2011) From the soil to the seeds: the long journey of nitrate in plants. J Exp Bot 62(4):1349–1359

    CAS  PubMed  Google Scholar 

  • dos Santos TB, Lima JE, Felicio MS, Soares JDM, Domingues DS (2017) Genome-wide identification, classification and transcriptional analysis of nitate and ammonium transporters in Coffea. Genet Mol Biol 40(1 suppl 1):346–359

    PubMed  PubMed Central  Google Scholar 

  • Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39(4):783–791

    PubMed  Google Scholar 

  • Feng H, Yan M, Fan X, Li B, Shen Q, Miller AJ, Xu G (2011) Spatial expression and regulation of rice high-affinity nitrate transporters by nitrogen and carbon status. J Exp Bot 62(7):2319–2332

    CAS  PubMed  Google Scholar 

  • Fernández E, Galván A (2007) Inorganic nitrogen assimilation in Chlamydomonas. J Exp Bot 58(9):2279–2287

    PubMed  Google Scholar 

  • Filleur S, Daniel-Vedele F (1999) Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display. Planta 207(3):461–469

    CAS  PubMed  Google Scholar 

  • Filleur S, Dorbe MF, Cerezo M, Orsel M, Granier F, Gojon A, Daniel-Vedele F (2001) An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake. FEBS Lett 489(2–3):220–224

    CAS  PubMed  Google Scholar 

  • Forde BG (2000) Nitrate transporters in plants: structure, function and regulation. Biochim Biophys Acta Biomembr 1465(1):219–235

    CAS  Google Scholar 

  • Fraisier V, Gojon A, Tillard P, Daniel-Vedele F (2000) Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source. Plant J 23(4):489–496

    CAS  PubMed  Google Scholar 

  • Fu Y, Yi H, Bao J, Gong J (2015) LeNRT2.3 functions in nitrate acquisition and long-distance transport in tomato. FEBS Lett 589(10):1072–1079

    CAS  PubMed  Google Scholar 

  • Galván A, Fernández E (2001) Eukaryotic nitrate and nitrite transporters. Cell Mol Life Sci C 58(2):225–233

    Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50(1):151–158

    CAS  PubMed  Google Scholar 

  • González T, Espadas G, Villanueva L, Gutiérrez L, Contreras F (2006) Análisis de la morfología del fruto de chile habanero (Capsicum chinense Jacq.) de Yucatán. III Reunión Estatal de Investigación Agropecuaria Forestal y Pesca. 19–21 enero 2006. Mérida, Yucatán

  • Gu C, Song A, Zhang X, Wang H, Li T, Chen Y, Jiang J, Chen F, Chen S (2016) Cloning of chrysanthemum high affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1. Sci Rep 6:23462

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gu C, Zhang X, Jiang J, Guan Z, Zhao S, Fang W, Liao Y, Chen S, Chen F (2014) Chrysanthemum CmNAR2 interacts with CmNRT2 in the control of nitrate uptake. Sci Rep 4:5833

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ishikawa S, Ito Y, Sato Y, Fukaya Y, Takahashi M, Morikawa H, Ohtake N, Ohyama T, Sueyoshi K (2009) Two-component high-affinity nitrate transport system in barley: membrane localization, protein expression in roots and a direct protein-protein interaction. Plant Biotechnol 26(2):197–205

    CAS  Google Scholar 

  • Kawachi T, Sunaga Y, Ebato M, Hatanaka T, Harada H (2006) Repression of nitrate uptake by replacement of Asp105 by asparagine in AtNRT3.1 in Arabidopsis thaliana L. Plant Cell Physiol 47(10):1437–1441

    CAS  PubMed  Google Scholar 

  • Kotur Z, Glass ADM (2015) A 150 kDa plasma membrane complex of AtNRT2.5 and AtNAR2.1 is the major contributor to constitutive high-affinity nitrate influx in Arabidopsis thaliana. Plant Cell Environ 38(8):1490–1502

    CAS  PubMed  Google Scholar 

  • Kotur Z, Mackenzie N, Ramesh S, Tyerman SD, Kaiser BN, Glass ADM (2012) Nitrate transport capacity of the Arabidopsis thaliana NRT2 family members and their interactions with AtNAR2.1. New Phytol 194(3):724–731

    CAS  PubMed  Google Scholar 

  • Knapp S, Bohs L, Nee M, Spooner DM (2004) Solanaceae—a model for linking genomics with biodiversity. Comp Funct Genomics 5(3):285–291

    CAS  PubMed  PubMed Central  Google Scholar 

  • Krapp A, Fraisier V, Scheible WR, Quesada A, Gojon A, Stitt M, Caboche M, Daniel-Vedele F (1998) Expression studies of Nrt2:1Np, a putative high-affinity nitrate transporter: evidence for its role in nitrate uptake. Plant J 14(6):723–731

    CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680

    CAS  PubMed  Google Scholar 

  • Lam HM, Coschigano KT, Oliveira IC, Melo-Oliveira R, Coruzzi GM (1996) The molecular-genetics of nitrogen assimilation into amino acids in higher plants. Annu Rev Plant Physiol Plant Mol Biol 47(1):569–593

    CAS  PubMed  Google Scholar 

  • Lejay L, Tillard P, Lepetit M, Fd O, Filleur S, Daniel-Vedele F, Gojon A (1999) Molecular and functional regulation of two NO3− uptake systems by N- and C-status of Arabidopsis plants. Plant J 18(5):509–519

    CAS  PubMed  Google Scholar 

  • Léran S et al (2013) A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plant Sci 19(1):5–9

    PubMed  Google Scholar 

  • Li W, Wan Y, Okamoto M, Crawford NM, Siddiqi MY, Glass ADM (2007) Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster. Plant Physiol 143(1):425–433

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu LH, Fan TF, Shi DX, Li CJ, He MJ, Chen YY, Zhang L, Yang C, Cheng XY, Chen X, Li D, Sun YC (2018) Coding-sequence identification and transcriptional profiling of nine AMTs and four NRTs from tobacco revealed their differential regulation by developmental stages, nitrogen nutrition, and photoperiod. Front Plant Sci 9:210

    PubMed  PubMed Central  Google Scholar 

  • Liu X, Huang D, Tao J, Miller AJ, Fan X, Xu G (2014) Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport. New Phytol 204(1):74–80

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lupini A, Mercati F, Araniti F, Miller AJ, Sunseri F, Abenavoli MR (2016) NAR2.1/NRT2.1 functional interaction with NO3− and H+ fluxes in high-affinity nitrate transport in maize root regions. Plant Physiol Biochem 102:107–114

    CAS  PubMed  Google Scholar 

  • Machida S, Yuan YA (2013) Crystal structure of Arabidopsis thaliana dawdle forkhead-associated domain reveals a conserved phospho-threonine recognition cleft for dicer-like 1 binding. Mol Plant 6(4):1290–1300

    CAS  PubMed  Google Scholar 

  • Marois E, Van den Ackerveken G, Bonas U (2002) The Xanthomonas type III effector protein AvrBs3 modulates plant gene expression and induces cell hypertrophy in the susceptible host. Mol Plant-Microbe Interact 15(7):637–646

    CAS  PubMed  Google Scholar 

  • Martín Y, González YV, Cabrera E, Rodríguez C, Siverio JM (2011) Npr1 Ser/Thr protein kinase links nitrogen source quality and carbon availability with the yeast nitrate transporter (Ynt1) levels. J Biol Chem 286(31):27225–27235

    PubMed  PubMed Central  Google Scholar 

  • Medina-Lara F, Echevarría-Machado I, Pacheco-Arjona R, Ruiz-Lau N, Guzmán-Antonio A, Martinez-Estevez M (2008) Influence of nitrogen and potassium fertilization on fruiting and capsaicin content in habanero pepper (Capsicum chinense Jacq.). HortScience 43(5):1549–1554

    Google Scholar 

  • Monforte-González M, Guzmán-Antonio A, Uuh-Chim F, Vázquez-Flota F (2010) Capsaicin accumulation is related to nitrate content in placentas of habanero peppers (Capsicum chinense Jacq.). J Sci Food Agric 90(5):764–768

    PubMed  Google Scholar 

  • Montanini B, Viscomi AR, Bolchi A, Martin Y, Siverio JM, Balestrini R, Bonfante P, Ottonello S (2006) Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete. Biochem J 394(Pt 1):125–134

    CAS  PubMed  PubMed Central  Google Scholar 

  • Morère-Le Paven MC, Viau L, Hamon A, Vandecasteele C, Pellizzaro A, Bourdin C, Laffont C, Lapied B, Lepetit M, Frugier F, Legros C, Limami AM (2011) Characterization of a dual-affinity nitrate transporter MtNRT1.3 in the model legume Medicago truncatula. J Exp Bot 62(15):5595–5605

    PubMed  Google Scholar 

  • Morris ER, Chevalier D, Walker JC (2006) DAWDLE, a forkhead-associated domain gene, regulates multiple aspects of plant development. Plant Physiol 141(3):932–941

    CAS  PubMed  PubMed Central  Google Scholar 

  • Navarro FJ, Machín F, Martín Y, Siverio JM (2006) Down-regulation of eukaryotic nitrate transporter by nitrogen-dependent ubiquitinylation. J Biol Chem 281(19):13268–13274

    CAS  PubMed  Google Scholar 

  • Navarro FJ, Martín Y, Siverio JM (2008) Phosphorylation of the yeast nitrate transporter Ynt1 is essential for delivery to the plasma membrane during nitrogen limitation. J Biol Chem 283(45):31208–31217

    CAS  PubMed  PubMed Central  Google Scholar 

  • Navarro MT, Guerra E, Fernández E, Galván A (2000) Nitrite reductase mutants as an approach to understanding nitrate assimilation in Chlamydomonas reinhardtii. Plant Physiol 122(1):283–290

    CAS  PubMed  PubMed Central  Google Scholar 

  • O’Brien JA, Vega A, Bouguyon E, Krouk G, Gojon A, Coruzzi G, Gutiérrez RA (2016) Nitrate transport, sensing, and responses in plants. Mol Plant 9(6):837–856

    PubMed  Google Scholar 

  • Okamoto M, Kumar A, Li W, Wang Y, Siddiqi MY, Crawford NM, Glass ADM (2006) High-affinity nitrate transport in roots of Arabidopsis depends on expression of the NAR2-like gene AtNRT3.1. Plant Physiol 140(3):1036–1046

    CAS  PubMed  PubMed Central  Google Scholar 

  • Orsel M, Chopin F, Leleu O, Smit SJ, Krapp A, Daniel-Vedele F, Miller AJ (2007) Nitrate signaling and the two component high affinity uptake system in Arabidopsis. Plant Signal Behav 2(4):260–262

    PubMed  PubMed Central  Google Scholar 

  • Orsel M, Chopin F, Leleu O, Smith SJ, Krapp A, Daniel-Vedele F, Miller AJ (2006) Characterization of a two-component high-affinity nitrate uptake system in Arabidopsis. Physiology and protein-protein interaction. Plant Physiol 142(3):1304–1317

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pao SS, Paulsen IT, Saier JMH (1998) Major facilitator superfamily. Microbiol Mol Biol Rev 62(1):1–34

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pellizzaro A, Clochard T, Plamchet E, Limami AM, Morére-Le Paven MC (2015) Idetification and molecular characterization of Medicago truncatula NRT2 and NAR2 families. Physiol Plant 154(2):256–269

    CAS  PubMed  Google Scholar 

  • Quesada A, Galvan A, Fernandez E (1994) Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J 5(3):407–419

    CAS  PubMed  Google Scholar 

  • Quesada A, Krapp A, Trueman LJ, Daniel-Vedele F, Fernández E, Forde GB, Caboche M (1997) PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high-affinity nitrate transporters of the crnA family. Plant Mol Biol 34(2):256–274

    Google Scholar 

  • Santiago-Antonio G, Lizama-Gasca MG, Carrillo-Pech M, Echevarría-Machado I (2014) Natural variation in response to nitrate starvation among varieties of habanero pepper (Capsicum chinense Jacq.). Aust J Crop Sci 8(4):523–535

    CAS  Google Scholar 

  • Sestili F, Rouphael Y, Cardarelli M, Pucci A, Bonini P, Canaguier R, Colla G (2018) Protein hydrolysate stimulates growth in tomato coupled with N-dependent gene expression involved in N assimilation. Front Plant Sci 9:1233

    PubMed  PubMed Central  Google Scholar 

  • Shabala S, Shabala L, Bose J, Cuin T, Newman I (2013) Ion flux measurements using the MIFE technique. In: Maathuis F (ed) Plant mineral nutrients. Methods in molecular biology (methods and protocols), vol 953. Humana Press, Totowa, NJ

    Google Scholar 

  • Solcan N, Kwok J, Fowler PW, Cameron AD, Drew D, Iwata S, Newstead S (2012) Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J 31(16):3411–3421

    CAS  PubMed  PubMed Central  Google Scholar 

  • Soria-Fregoso MJ, Trejo Rivero JA, Tun Suarez JM, Teran-Saldivar R (2002) Paquete tecnológico para la producción de chile habanero (Capsicum chinense Jacq.). Instituto Tecnológico de Mérida 2:3–74

  • 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

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729

    CAS  PubMed  PubMed Central  Google Scholar 

  • Taulemesse F, Le Gouis J, Gouache D, Gibon Y, Allard V (2015) Post-flowering nitrate uptake in wheat is controlled by N status at flowering, with a putative major role of root nitrate transporter NRT2.1. PLoS ONE 10(3):e0120291

    PubMed  PubMed Central  Google Scholar 

  • Tong Y, Zhou JJ, Li Z, Miller AJ (2005) A two-component high-affinity nitrate uptake system in barley. Plant J 41(3):442–450

    CAS  PubMed  Google Scholar 

  • Tsay YF, Chiu CC, Tsai CB, Ho CH, Hsu PK (2007) Nitrate transporters and peptide transporters. FEBS Lett 581(12):2290–2300

    CAS  PubMed  Google Scholar 

  • Tsirigos KD, Peters C, Shu N, Käll L, Elofsson A (2015) The TOPCONS web server for consensus prediction of membrane protein topology and signal peptides. Nucleic Acids Res 43:(W1)W401–W407

    Google Scholar 

  • Tsujimoto R, Yamazaki H, Maeda S, Omata T (2007) Distinct roles of nitrate and nitrite in regulation of expression of the nitrate transport genes in the moss Physcomitrella patens. Plant Cell Physiol 48(3):484–497

    CAS  PubMed  Google Scholar 

  • Unkles SE, Wang R, Wang Y, Glass ADM, Crawford NM, Kinghorn JR (2004) Nitrate reductase activity is required for nitrate uptake into fungal but not plant cells. J Biol Chem 279(27):28182–28186

    CAS  PubMed  Google Scholar 

  • Wang YY, Hsu PK, Tsay YF (2012) Uptake, allocation and signaling of nitrate. Trends Plant Sci 17(8):458–467

    CAS  PubMed  Google Scholar 

  • Wei J, Zheng Y, Feng H, Qu H, Fan X, Yamaji N, Feng Ma J, Xu G (2018) OsNRT2.4 encodes a dual-affinity nitrate transporter and functions in nitrate-regulated root growth and nitrate distribution in rice. J Exp Bot 69(5):1095–1107

    CAS  PubMed  Google Scholar 

  • Wirth J, Chopin F, Santoni V, Viennois G, Tillard P, Krapp A, Lejay L, Daniel-Vedele F, Gojon A (2007) Regulation of root nitrate uptake at the NRT2.1 protein level in Arabidopsis thaliana. J Biol Chem 282(32):23541–23552

    CAS  PubMed  Google Scholar 

  • Yan M, Fan X, Feng H, Miller AJ, Shen Q, Xu G (2011) Rice OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a nitrate transporters to provide uptake over high and low concentration ranges. Plant Cell Environ 34(8):1360–1372

    CAS  PubMed  Google Scholar 

  • Yong Z, Kotur Z, Glass ADM (2010) Characterization of an intact two-component high-affinity nitrate transporter from Arabidopsis roots. Plant J 63(5):739–748

    CAS  PubMed  Google Scholar 

  • Zepeda-Jazo I, Pottosin II (2018) Methods related to polyamine control of cation transport across plant membranes. In: Alcázar R, Tiburcio AF (eds) Polyamines: methods and protocols (Methods in Molecular Biology), vol 1694. New York, Humana Press, pp 257–276 ISBN 978-1-4939-7397-2

    Google Scholar 

  • Zhuo D, Okamoto M, Vidmar JJ, Glass ADM (1999) Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana. Plant J 17(5):563–568

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Consejo Nacional de Ciencia y Tecnología, CONACYT (Project 169041), and CONACYT fellowships to M. G. L. G. (242990) and C. A. E. M. (242989). We thank Ángela Ku González for their kind contribution in work, with confocal microscopy. We thank Federico García Laines for their support in the fruit development experiment for genetic expression. We thank Germán Carnevali Fernández-Concha for his support in the phylogenetic analysis of CcNRT2.1 and CCNAR2 proteins.

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  1. Unidad de Bioquímica y Biología Molecular de Plantas, Centro de Investigación Científica de Yucatán, Calle 43, #130, x 32 and 34, CP 97205, Mérida, Yucatán, Mexico

    Marta G. Lizama-Gasca, Georgina Estrada-Tapia, Camilo A. Escalante-Magaña, Manuel Martínez-Estévez, Fátima Medina-Lara & Ileana Echevarría-Machado

  2. Departamento de Genómica Alimentaria, Universidad de la Ciénaga, Sahuayo, 59103, Michoacán de Ocampo, CP, Mexico

    Isaac Zepeda-Jazo

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Fig. S1

a cDNA CcNRT2 sequence. In underline are primer sequences used in this work and their names (upper). b Protein CcNRT2 sequence: in red cylinders and numbered are the hydrophobic amino acid segments presumed to form membrane-spanning regions. The protein kinase C recognition motifs (S/T-X-R/K) are overlined. The inverted triangles indicate the two arginine residues corresponding to those required for nitrate transport by an NRT2 homolog (NRTA) of Aspergillus nidulans. The amino acid signatures conserved in the NNP family proteins and NRT2 proteins are highlighted and italics, and cian box, respectively. Red letters indicate the consensus sites of phosphorylation. In bold and underline are probable amino acid residues for the interaction with Nar2 protein in homolog of Arabidopsis and barley. c cDNA CcNAR2 sequence. In underline are primer sequences used in this work and their names (upper). d CcNAR2 sequence: in red cylinder are the hydrophobic amino acid segment presumed to form membrane-spanning region. In yellow box are the amino acids that form the signal peptide. The amino acid signatures conserved in the NAR2 family proteins are highlighted and italics, and purple box. The Forkhead-associated (FHA) domain profile is in a cyan box. Four β (β1- β4) strand in this domain are shown by cyan arrows. In bold, italic, and underline are probable amino acid residues for the interaction with NRT2 protein in homolog of Arabidopsis and barley. The recognition motifs of protein kinase C (S/T-X-R/K) are found with a line at the top. The recognition residues of Casein kinase II (CK2) are in red. The recognition residues for N-glycosylation and N-myristoylation are in blue and pink, respectively. (PNG 1229 kb)

High resolution image (TIF 354403 kb)

Fig. S2

Predicted transmembrane regions for A) CcNRT2.1 and B) CcNAR2. The prediction of transmembrane helices was made with the bioinformatic program TOPCONS (Tsirigos et al. 2015) (http://topcons.cbr.su.se). (PNG 116 kb)

High resolution image (TIF 1734 kb)

Fig. S3

In silico CcNRT2-CcNAR2 interaction. (A) CcNRT2 interaction with different protein. StuNRT2 protein of S. tuberosum was used as a reference by STRING 10 program. (B) CcNAR2 interaction with different protein. SlNAR3.2 protein of S. lycopersicum was used as a reference by STRING 10 program. (PNG 894 kb)

High resolution image (TIF 4781 kb)

Fig. S4

a Extracellular pH analysis by using the Oregon Green® 488 fluorophore. Roots exposed to 0.5 mM KNO3 were used to evaluate the nitrate uptake, indirectly following the alkalinization of the medium with the pH-sensitive Oregon Green® 488 fluorophore. Figure shown a representative image (n = 3 roots). Roots in absence of nitrate were used as control (-NO3−). b Autofluorescence of C. chinense root. Root zones according to the distances from the root apex: Z1 (0–20 mm), Z2 (21–41 mm), and Z3 (42–62 mm). (PNG 614 kb)

High resolution image (TIF 168779 kb)

Supplemental table S1

Post-translational modifications of CcNRT2.1 and CcNAR2. (XLSX 623 kb)

Supplemental table S2

Accession numbers of NRT2 proteins extracted from GenBank and used for the phylogenetic analysis. (XLSX 16 kb)

Supplemental table S3

Accession numbers of NAR2 proteins extracted from GenBank and used for the phylogenetic analysis. (XLSX 13 kb)

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Lizama-Gasca, M.G., Estrada-Tapia, G., Escalante-Magaña, C.A. et al. Cloning and Molecular Characterization of CcNRT2.1/CcNAR2, a Putative Inducible High Affinity Nitrate Transport System in Capsicum chinense Jacq. Roots. Tropical Plant Biol. 13, 73–90 (2020). https://doi.org/10.1007/s12042-019-09248-w

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