Abstract
The expression patterns of the NRT2 genes have been well described; however, the role of OsNRT2.4 in root growth is not well known. In this study, we thus aimed at investigating the role of high-affinity NO3− transport OsNRT2.4 in root growth modulation. Through the amiRNA-mediated gene silencing technique, we successfully obtained osnrt2.4 knockdown lines to study the role of OsNRT2.4 on root growth under low nitrate conditions. We performed real-time PCR analysis to investigate the relative gene expression level in root and shoot, soluble metabolites, and measurement of root system. Knockdown of OsNRT2.4 decreased rice growth. The comparison with wild-type (WT) plants showed that (i) knockdown of OsNRT2.4 inhibited root formation under low NO3− supply; (ii) we demonstrated that the mutant lines had significantly increased NO3− uptake than WT plants when grown in different nitrate supplies; (iii) osnrt2.4 knockdown lines showed an alteration in nitrogen metabolism, and this affected the root growth; and (iv) the downregulation of OsNRT2.4 enhanced the expression of gene response of low external NO3− concentrations. Herein we provide new insights in OsNRT2.4 functions. Our data demonstrated that OsNRT2.4 plays a role in root growth, nitrogen metabolic pathway and probably have functions in nitrate transport from root to shoot under low nitrate availability in rice.
Similar content being viewed by others
Availability of data and material
Not applicable.
Code availability
WinRhizo Arabdopsis software (2012b).
References
Araki R, Hasegawa H (2006) Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction. Breed Sci 56:295–302. https://doi.org/10.1270/jsbbs.56.295
Baldrich P, San Segundo B (2016) MicroRNAs in rice innate immunity. Rice 9:6. https://doi.org/10.1186/s12284-016-0078-5
Bao A, Zhao Z, Ding G, Shi L, Xu F, Cai H (2014) Accumulated expression level of cytosolic Glutamine Synthetase 1 gene (OsGS1;1 or OsGS1;2) alter plant development and the carbon-nitrogen metabolic status in rice. PLoS ONE 9:e95581. https://doi.org/10.1371/journal.pone.0095581
Bernard SM, Habash DZ (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol 182:608–620. https://doi.org/10.1111/j.1469-8137.2009.02823.x
Bloom AJ (2015) The increasing importance of distinguishing among plant nitrogen sources. Curr Opin Plant Biol 25:10–16. https://doi.org/10.1016/j.pbi.2015.03.002
Cai C, Wang J-Y, Zhu Y-G, Shen Q-R, Li B, Tong Y-P, Li Z-S (2008) Gene structure and expression of the high-affinity nitrate transport system in rice roots. J Integr Plant Biol 50:443–451. https://doi.org/10.1111/j.1744-7909.2008.00642.x
Chopin F, Orsel M, Dorbe M-F, Chardon F, Truong H-N, Miller AJ, Krapp A, Daniel-Vedele F (2007) The arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds. Plant Cell 19:1590–1602. https://doi.org/10.1105/tpc.107.050542
Crawford NM (1995) Nitrate: nutrient and signal for plant growth. Plant Cell 7:859–868. https://doi.org/10.1105/tpc.7.7.859
Cren M, Hirel B (1999) Glutamine synthetase in higher plants: regulation of gene and protein expression from the organ to the cell. Plant Cell Physiol 40:1187–1193. https://doi.org/10.1093/oxfordjournals.pcp.a029506
Fan X, Naz M, Fan X, Xuan W, Miller AJ, Xu G (2017) Plant nitrate transporters: from gene function to application. J Exp Bot 68:2463–2475. https://doi.org/10.1093/jxb/erx011
FAO (2017) Food and Agriculture Organization of the United Nations. FAOSTAT database: agriculture production. Available via http://www.fao.org/faostat/en/#home.
Felker P (1977) Micro determination of nitrogen in seed protein extracts with the salicylate-dichloroisocyanurate color reaction. Anal Chem 49:1080. https://doi.org/10.1021/ac50015a053
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 62:2319–2332. https://doi.org/10.1093/jxb/erq403
Fernandes MS (1983) N-carriers, light and temperature influences on the free amino acid pool composition of rice plants. Turrialba 33:297-301. Available via http://orton.catie.ac.cr/repdoc/A0782e/A0782e03.html
Fernandes MS (1991) Effects of environmental stress on the relationship of free amino-N to fresh weight of rice plants. J Plant Nutr 14:1151–1164. https://doi.org/10.1080/01904169109364274
Fernandes MS, Rossiello ROP (1995) Mineral nitrogen in plant physiology and plant nutrition. Crit Rev Plant Sci 14:111–148 https://doi.org/10.1080/07352689509701924
Forde B, Lorenzo H (2001) The nutritional control of root development. Plant Soil 232:51–68. https://doi.org/10.1023/A:1010329902165
Gao J, Liu J, Li B, Li Z (2001) Isolation and purification of functional total RNA from blue-grained wheat endosperm tissues containing high levels of starches and flavonoids. Plant Mol Biol Rep 19:185–186. https://doi.org/10.1007/BF02772163
Gruber BD, Giehl RFH, Friedel S, von Wirén N (2013) Plasticity of the Arabidopsis root system under nutrient deficiencies. Plant Physiol 163:161–179. https://doi.org/10.1104/pp.113.218453
Hoagland DR, Arnon DI (1950) The water-culture method for growing plants without soil. Available via CAB Direct. https://www.cabdirect.org/cabdirect/abstract/19500302257
Holsters M, de Waele D, Depicker A, Messens E, Montagu M, Schell J (1978) Transfection and transformation of Agrobacterium tumefaciens. Molec Gen Genet 163:181–187. https://doi.org/10.1007/BF00267408
Huang S, Chen S, Liang Z, Zhang C, Yan M, Chen J, Xu G, Fan X, Zhang Y (2015) Knockdown of the partner protein OsNAR2.1 for high-affinity nitrate transport represses lateral root formation in a nitrate-dependent manner. Sci Rep 5:18192. https://doi.org/10.1038/srep18192
Huarancca Reyes T, Scartazza A, Pompeiano A, Ciurli A, Lu Y, Guglielminetti L, Yamaguchi J (2018) Nitrate reductase modulation in response to changes in C/N balance and nitrogen source in arabidopsis. Plant Cell Physiol 59:1248–1254. https://doi.org/10.1093/pcp/pcy065
Jain M, Nijhawan A, Tyagi AK, Khurana JP (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem Biophys Res Commun 345:646–651. https://doi.org/10.1016/j.bbrc.2006.04.140
Jiao X, Lyu Y, Wu X, Li H, Cheng L, Zhang C, Yuan L, Jiang R, Jiang B, Rengel Z, Zhang F, Davies W, Shen J (2016) Grain production versus resource and environmental costs: towards increasing sustainability of nutrient use in China. J Exp Bot 67:4935–4949. https://doi.org/10.1093/jxb/erw282
Kiba T, Krapp A (2016) Plant nitrogen acquisition under low availability: regulation of uptake and root architecture. Plant Cell Physiol 57:707–714. https://doi.org/10.1093/pcp/pcw052
Koevoets IT, Venema JH, Elzenga JTM, Testerink C (2016) Roots withstanding their environment: exploiting root system architecture responses to abiotic stress to improve crop tolerance. Front Plant Sci 7:1335–1354. https://doi.org/10.3389/fpls.2016.01335
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. https://doi.org/10.1093/jxb/eru001
Lam-Sánchez A, Santos JE, Takamura K, Treptow RMO, Oliveira JED (1994) Estudos Nutricionais com arroz (Oryza sativa, L.). Alim Nutr 5:37-48. Available via https://agris.fao.org/agris-search/search.do?recordID=DJ2012066293
Lezhneva L, Kiba T, Feria-Bourrellier A-B, Lafouge F, Boutet-Mercey S, Zoufan P, Sakakibara H, Daniel-Vedele F, Krapp A (2014). The Arabidopsis nitrate transporter NRT2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants. Plant J 80:230-241. https://doi.org/10.1111/tpj.12626
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
López-Bucio J, Cruz-Ramírez A, Herrera-Estrella L (2003) The role of nutrient availability in regulating root architecture. Curr Opin Plant Biol 6:280–287. https://doi.org/10.1016/S1369-5266(03)00035-9
Martin A, Lee J, Kichey T, Gerentes D, Zivy M, Tatout C, Dubois F, Balliau T, Valot B, Davanture M, Tercé-Laforgue T, Quilleré I, Coque M, Gallais A, Gonzalez-Moro M-B, Bethencourt L, Habash DZ, Lea PJ, Charcosset A et al (2006) Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell 18:3252–3274. https://doi.org/10.1105/tpc.106.042689
Miller AJ, Fan X, Orsel M, Smith SJ, Wells DM (2007) Nitrate transport and signalling. J Exp Bot 58:2297–2306. https://doi.org/10.1093/jxb/erm066
Miranda KM, Espey MG, Wink DA (2001) A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 5:62–71. https://doi.org/10.1006/niox.2000.0319
Nacry P, Bouguyon E, Gojon A (2013) Nitrogen acquisition by roots: physiological and developmental mechanisms ensuring plant adaptation to a fluctuating resource. Plant Soil 370:1–29. https://doi.org/10.1007/s11104-013-1645-9
Naz M, Luo B, Guo X, Li B, Chen J, Fan X (2019) Overexpression of nitrate transporter OsNRT2.1 enhances nitrate-dependent root elongation. Genes 10:290. https://doi.org/10.3390/genes10040290
Okamoto M, Vidmar JJ, Glass ADM (2003) Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision. Plant Cell Physiol 44:304-317. 10.1093/pcp/pcg036
Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690. https://doi.org/10.1111/j.1365-313X.2007.03328.x
Prinsi B, Espen L (2015) Mineral nitrogen sources differently affect root glutamine synthetase isoforms and amino acid balance among organs in maize. BMC Plant Biol 15:96. https://doi.org/10.1186/s12870-015-0482-9
RAS (2009) Regras para análise de sementes. Ministério da Agricultura, Pecuária e Abastecimento. 1st edn. Mapa/ACS, p 399. Available via MAPA. https://www.gov.br/agricultura/pt-br/assuntos/insumos-agropecuarios/arquivos-publicacoes-insumos/2946_regras_analise__sementes.pdf.
Ravazzolo L, Trevisan S, Forestan C, Varotto S, Sut S, Dall’Acqua S, Malagoli M, Quaggiotti S (2020) Nitrate and ammonium affect the overall maize response to nitrogen availability by triggering specific and common transcriptional signatures in roots. Int J Mol Sci 21:686. https://doi.org/10.3390/ijms21020686
Sahoo KK, Tripathi AK, Pareek A, Sopory SK, Pareek SLS (2011) An improved protocol for efficient transformation and regeneration of diverse indica rice cultivars. Plant Methods 7:49–59. https://doi.org/10.1186/1746-4811-7-49
Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York
Santos LA, Bucher CA, Souza SR, Fernandes MS (2009) Effects of nitrogen stress on proton-pumping and nitrogen metabolism in rice. J Plant Nutr 32:549–564. https://doi.org/10.1080/01904160802714953
Souza SR, Stark EMLM, Fernandes MS, Magalhaes JR (1999) Effects of supplemental nitrogen on nitrogen-assimilation enzymes, free amino nitrogen, soluble sugars, and crude protein of rice. Comm Soil Sci Plant Anal 30:711–724. https://doi.org/10.1080/00103629909370240
Sustainable Development Goals – SDG (2015). Available via https://sdgs.un.org/goals.
Tabuchi M, Sugiyama K, Ishiyama K, Inoue ST, Takahashi H, Yamaya T (2005) Severe reduction in growth rate and grain filling of rice mutants lacking OsGS1;1, a cytosolic glutamine synthetase 1;1. Plant J 42:641–651. https://doi.org/10.1111/j.1365-313X.2005.02406.x
Tang Z, Fan X, Li Q, Feng H, Miller AJ, Shen Q, Xu G (2012) Knockdown of a rice stelar nitrate transporter alters long-distance translocation but not root influx. Plant Physiol 160:2052–2063. https://doi.org/10.1104/pp.112.204461
Tegeder M, Masclaux-Daubresse C (2018) Source and sink mechanisms of nitrogen transport and use. New Phytol 217:35–53. https://doi.org/10.1111/nph.14876
Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka H (2006) Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J 47:969–976. https://doi.org/10.1111/j.1365-313X.2006.02836.x
Tracy SR, Nagel KA, Postma JA, Fassbender H, Wasson A, Watt M (2020) Crop improvement from phenotyping roots: highlights reveal expanding opportunities. Trends Plant Sci 25:105–118. https://doi.org/10.1016/j.tplants.2019.10.015
Trevisan S, Manoli A, Quaggiotti S (2014) NO signaling is a key component of the root growth response to nitrate in Zea mays L. Plant Signal Behav 9(6):e28290. https://doi.org/10.4161/psb.28290
Warthmann N, Chen H, Ossowski S, Weigel D, Herve P (2008) Highly Specific gene silencing by artificial miRNAs in rice. Plos One 3:e1829. https://doi.org/10.1371/journal.pone.0001829
Wei J, Zheng Y, Feng H, Qu H, Fan X, Yamaji N, Ma JF, 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 69:1095–1107. https://doi.org/10.1093/jxb/erx486
Yan H, Deng X, Cao Y, Huang J, Ma L, Zhao B (2011a) A novel approach for the construction of plant amiRNA expression vectors. J Biotechnol 151:9–14. https://doi.org/10.1016/j.jbiotec.2010.10.078
Yan M, Fan X, Feng H, Miller AJ, Shen Q, Xu G (2011b) 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 34:1360–1372. https://doi.org/10.1111/j.1365-3040.2011.02335.x
Yanagisawa S (2014) Transcription factors involved in controlling the expression of nitrate reductase genes in higher plants. Plant Sci 229:167–171. https://doi.org/10.1016/j.plantsci.2014.09.006
Yemm EW, Cocking EC (1955) The determination of amino-acid with ninhydrin. Analyst 80:209–213. https://doi.org/10.1039/AN9558000209
Yemm EW, Willis AJ (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57:508–514. https://doi.org/10.1042/bj0570508
Zhang H, Forde BG (2000) Regulation of Arabidopsis root development by nitrate availability. J Exp Bot 51:51–59. https://doi.org/10.1093/jexbot/51.342.51
Zhang Z, Xiong S, Wei Y, Meng X, Wang X, Ma X (2017) The role of glutamine synthetase isozymes in enhancing nitrogen use efficiency of N-efficient winter wheat. Sci Rep. https://doi.org/10.1038/s41598-017-01071-1
Zhang Z, Gao S, Chu C (2020) Improvement of nutrient use efficiency in rice: current toolbox and future perspectives. Theor Appl Genet 133:1365–1384. https://doi.org/10.1007/s00122-019-03527-6
Acknowledgements
We would like to thank Sonia Regina de Souza (in memoriam) for their substantial assistance and useful discussions on this research project. We thank the undergraduate students from the scientific initiation program for their help with the experiments, and the funding agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (Finance Code 001) and the National Council for Scientific and Technological Development (CNPq), and the Graduate Program in Agronomy - Soil Science (PPGA-CS) at Federal Rural University of Rio de Janeiro (UFRRJ). We thank the anonymous reviewers for their rigorous work to improve this paper.
Funding
This research was funded in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (Finance Code 001) and the National Council for Scientific and Technological Development (CNPq).
Author information
Authors and Affiliations
Contributions
This article is a joint effort by several authors. A.F.F.S. designed the experiments, obtained funding, performed the data analysis, and drafted the manuscript with contributions of authors. L.N.A. and R.P.R. performed part of the experiment, measurements and provided assistance for data analysis. M.S.F. and L.A.S. contributed to research plan and experiment support. S.R.S (in memoriam) supervised all experiments and together with C.A.B conceived the research plan and project design. The authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
ESM 1
(PDF 112 kb)
Rights and permissions
About this article
Cite this article
Souza, A.F.F., Bucher, C.A., Arruda, L.N. et al. Knockdown of OsNRT2.4 modulates root morphology and alters nitrogen metabolism in response to low nitrate availability in rice. Mol Breeding 42, 5 (2022). https://doi.org/10.1007/s11032-021-01273-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11032-021-01273-6