e-ISSN 2231-8542
ISSN 1511-3701

Home / Regular Issue / JTAS Vol. 46 (1) Feb. 2023 / JTAS-2522-2022


In silico Analysis of OsNRT2.3 Reveals OsAMT1.3, OsZIFL9, OsbZIP27, and OsIRT1 as Potential Drought-related Genes During Nitrogen Use Efficiency in Oryza sativa L.

Muhammad-Redha Abdullah-Zawawi, Karwatik Busiri, Syafiqah Johan, Mohammad Asad Ullah and Zamri Zainal

Pertanika Journal of Tropical Agricultural Science, Volume 46, Issue 1, February 2023


Keywords: Drought stress, in silico, nitrogen use efficiency, NRT2.3, Oryza sativa L.

Published on: 22 Febuary 2023

Nitrate transporter (NRT) is responsible for the molecular mechanism of the root nitrate (NO3-) uptake system for plant development. Although several NRT genes are identified and characterised in plants, knowledge of the NRT2 gene family and its nitrogen use efficiency (NUE) function in drought stress has remained elusive in rice. This study conducted an in silico analysis on 20 NRT2 family genes of rice, wheat, soybean, barley, maize, and papaya. Phylogenetic and motifs analysis clustered genes encoding NRT2 proteins into four monophyletic groups, and the motifs of NRT2 genes were significantly conserved for the specific domain of NO3- transmembrane transporter. Interestingly, co-expression analysis revealed that potential drought-related genes were expressed similarly to the functional NUE gene, OsNRT2.3. Furthermore, half of the co-expressed genes were enriched in nitrogen use efficiency (NUE)-related processes, such as transport, stress, macromolecule metabolic pathways, and transcription regulation. Expression pattern analysis of OsNRT2.3 and its co-expressed genes in tissue-specific and nitrogen (N) response led to the discovery of OsAMT1.3, OsZIFL9, OsbZIP27, and OsIRT1 as four strong candidates to participate in drought stress during NO3- uptake system. The co-expression of iron (Fe) uptake genes, OsZIFL9 and OsIRT1, with OsNRT2.3 also suggested a possible interaction of Fe and nitrogen (N) during an increasing amount of Fe, which led to the acidification of rice apoplasts during water deficiency. Together, this study will provide a valuable resource for potential candidate genes that can further investigate their molecular response to drought during NUE.

  • Abdullah‐Zawawi, M. R., Afiqah‐Aleng, N., Ikhwanuddin, M., Sung, Y. Y., Tola, S., Fazhan, H., & Waiho, K. (2021). Recent development in ecdysone receptor of crustaceans: Current knowledge and future applications in crustacean aquaculture. Reviews in Aquaculture, 13(4), 1938-1957.

  • Ai, Z., Wang, G., Liang, C., Liu, H., Zhang, J., Xue, S., & Liu, G. (2017). The effects of nitrogen addition on the uptake and allocation of macro- and micronutrients in Bothriochloa ischaemum on Loess Plateau in China. Frontiers in Plant Science, 8, 1476.

  • Awasthi, S., Chauhan, R., Indoliya, Y., Chauhan, A. S., Mishra, S. K., Agrawal, L., Dwivedi, S., Singh, S. N., Srivastava, S., Singh, P. C., Chauhan, P. S., Chakrabarty, D., Srivastava, S., & Tripathi, R. D. (2021). Microbial consortium mediated growth promotion and arsenic reduction in rice: An integrated transcriptome and proteome profiling. Ecotoxicology and Environmental Safety, 228, 113004.

  • Bailey, T. L., Boden, M., Buske, F. A., Frith, M., Grant, C. E., Clementi, L., Ren, J., Li, W. W., & Noble, W. S. (2009). MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Research, 37(suppl_2), W202–W208.

  • Bao, A., Liang, Z., Zhao, Z., & Cai, H. (2015). Overexpressing of OsAMT1-3, a high affinity ammonium transporter gene, modifies rice growth and carbon-nitrogen metabolic status. International Journal of Molecular Sciences, 16(5), 9037-9063.

  • Cao, P., Jung, K. H., Choi, D., Hwang, D., Zhu, J., & Ronald, P. C. (2012). The Rice Oligonucleotide Array Database: An atlas of rice gene expression. Rice, 5, 17.

  • Chen, J., Qi, T., Hu, Z., Fan, X., Zhu, L., Iqbal, M. F., Yin, X., Xu, G., & Fan, X. (2019). OsNAR2.1 positively regulates drought tolerance and grain yield under drought stress conditions in rice. Frontiers in Plant Science, 10, 197.

  • Coneva, V., Simopoulos, C., Casaretto, J. A., El-Kereamy, A., Guevara, D. R., Cohn, J., Zhu, T., Guo, L., Alexander, D. C., Bi, Y. M., McNicholas, P. D., & Rothstein, S. J. (2014). Metabolic and co-expression network-based analyses associated with nitrate response in rice. BMC Genomics, 15, 1056.

  • Djaman, K., Mel, V., Ametonou, F., El-Namaky, R., Diallo, M., & Koudahe, K. (2018). Effect of nitrogen fertilizer dose and application timing on yield and nitrogen use efficiency of irrigated hybrid rice under semi-arid conditions. Journal of Agricultural Science and Food Research, 9(2), 1000223.

  • Edgar, R. C. (2004). MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research, 32(5), 1792-1797.

  • Fenchel, T., King, G. M., & Blackburn, T. H. (2012). Transport mechanisms. Bacterial Biogeochemistry, 3(10), 35–47.

  • Ferreira, L. M., de Souza, V. M., Tavares, O. C. H., Zonta, E., Santa-Catarina, C., de Souza, S. R., Fernandes, M. S., & Santos, L. A. (2015). OsAMT1.3 expression alters rice ammonium uptake kinetics and root morphology. Plant Biotechnology Reports, 9(4), 221–229.

  • Food and Agriculture Organization of the United Nations. (2017). The future of food and agriculture – Trends and challenges. FAO.

  • Forde, B. G. (2000). Nitrate transporters in plants: Structure, function and regulation. Biochimica et Biophysica Acta-Biomembranes, 1465(1–2), 219–235.

  • García, M. J., Angulo, M., García, C., Lucena, C., Alcántara, E., Pérez-Vicente, R., & Romera, F. J. (2021). Influence of ethylene signaling in the crosstalk between Fe, S, and P deficiency responses in Arabidopsis thaliana. Frontiers in Plant Science, 12, 643585.

  • Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D., & Bairoch, A. (2005). Protein identification and analysis tools on the ExPASy server. In J. M. Walker (Eds.), The proteomics protocols handbook (pp. 571–607). Humana.

  • Hoang, D. T., Hiroo, T., & Yoshinobu, K. (2019). Nitrogen use efficiency and drought tolerant ability of various sugarcane varieties under drought stress at early growth stage. Plant Production Science, 22(2), 250-261.

  • Hossain, M. A., Lee, Y., Cho, J. I., Ahn, C. H., Lee, S. K., Jeon, J. S., Kang, H., Lee, C. H., An, G., & Park, P. B. (2010). The bZIP transcription factor OsABF1 is an ABA responsive element binding factor that enhances abiotic stress signaling in rice. Plant Molecular Biology, 72(4), 557–566.

  • Huang, S., Zhao, C., Zhang, Y., & Wang, C. (2018). Nitrogen use efficiency in rice. In A. Amanullah & S. Fahad (Eds.), Nitrogen in agriculture – Updates. IntechOpen.

  • Hulo, N., Bairoch, A., Bulliard, V., Cerutti, L., De Castro, E., Langendijk-Genevaux, P. S., Pagni, M., & Sigrist, C. J. A. (2006). The PROSITE database. Nucleic Acids Research, 34(suppl_1), D227–D230.

  • Ishimaru, Y., Suzuki, M., Tsukamoto, T., Suzuki, K., Nakazono, M., Kobayashi, T., Wada, Y., Watanabe, S., Matsuhashi, S., Takahashi, M., Nakanishi, H., Mori, S., & Nishizawa, N. K. (2006). Rice plants take up iron as an Fe3+-phytosiderophore and as Fe2+. Plant Journal, 45(3), 335–346.

  • Izawa, T., Foster, R., & Chua, N. H. (1993). Plant bZIP protein DNA binding specificity. Journal of Molecular Biology, 230(4), 1131–1144.

  • Jin, Z., Zhu, Y., Li, X., Dong, Y., & An, Z. (2015). Soil N retention and nitrate leaching in three types of dunes in the Mu Us desert of China. Scientific Reports, 5, 14222.

  • Kabange, N. R., Park, S. Y., Lee, J. Y., Shin, D., Lee, S. M., Kwon, Y., Cha, J. K., Cho, J. H., Duyen, D. Van, Ko, J. M., & Lee, J. H. (2021). New insights into the transcriptional regulation of genes involved in the nitrogen use efficiency under potassium chlorate in rice (Oryza sativa L.). International Journal of Molecular Sciences, 22(4), 2192.

  • Khan, M. R., Khan, I., Ibrar, Z., Léon, J., & Naz, A. A. (2017). Drought-responsive genes expressed predominantly in root tissues are enriched with homotypic cis-regulatory clusters in promoters of major cereal crops. Crop Journal, 5(3), 195–206.

  • Khazanah Research Institute. (2019). The status of the paddy and rice industry in Malaysia. Khazanah Research Institute.

  • Kumar, S., Kumar, S., & Mohapatra, T. (2021). Interaction between macro‐ and micro-nutrients in plants. Frontiers in Plant Science, 12, 665583.

  • Kumar, S., Stecher, G., & Tamura, K. (2016). MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7), 1870–1874.

  • Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P., & Rombauts, S. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Research, 30(1), 325–327.

  • Letunic, I., & Bork, P. (2007). Interactive Tree Of Life (iTOL): An online tool for phylogenetic tree display and annotation. Bioinformatics, 23(1), 127–128.

  • Liang, G., Zhang, H., Li, Y., Pu, M., Yang, Y., Li, C., Lu, C., Xu, P., & Yu, D. (2020). Oryza sativa FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (OsFIT/OsbHLH156) interacts with OsIRO2 to regulate iron homeostasis. Journal of Integrative Plant Biology, 62(5), 668–689.

  • Little, D. Y., Rao, H., Oliva, S., Daniel-Vedele, F., Krapp, A., & Malamy, J. E. (2005). The putative high-affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues. Proceedings of the National Academy of Sciences of the United States of America, 102(38), 13693–13698.

  • Liu, W., Xie, Y., Ma, J., Luo, X., Nie, P., Zuo, Z., Lahrmann, U., Zhao, Q., Zheng, Y., Zhao, Y., Xue, Y., & Ren, J. (2015). IBS: An illustrator for the presentation and visualization of biological sequences. Bioinformatics, 31(20), 3359–3361.

  • Marchler-Bauer, A., Derbyshire, M. K., Gonzales, N. R., Lu, S., Chitsaz, F., Geer, L. Y., Geer, R. C., He, J., Gwadz, M., Hurwitz, D. I., Lanczycki, C. J., Lu, F., Marchler, G. H., Song, J. S., Thanki, N., Wang, Z., Yamashita, R. A., Zhang, D., Zheng, C., & Bryant, S. H. (2015). CDD: NCBI’s conserved domain database. Nucleic Acids Research, 43(D1), D222–D226.

  • Matthews, D. E., Carollo, V. L., Lazo, G. R., & Anderson, O. D. (2003). GrainGenes, the genome database for small-grain crops. Nucleic Acids Research, 31(1), 183–186.

  • Mering, C. Von, Huynen, M., Jaeggi, D., Schmidt, S., Bork, P., & Snel, B. (2003). STRING: A database of predicted functional associations between proteins. Nucleic Acids Research, 31(1), 258–261.

  • Mistry, J., Chuguransky, S., Williams, L., Qureshi, M., Salazar, G. A., Sonnhammer, E. L. L., Tosatto, S. C. E., Paladin, L., Raj, S., Richardson, L. J., Finn, R. D., & Bateman, A. (2021). Pfam: The protein families database in 2021. Nucleic Acids Research, 49(D1), D412–D419.

  • Obara, M., Tamura, W., Ebitani, T., Yano, M., Sato, T., & Yamaya, T. (2010). Fine-mapping of qRL6.1, a major QTL for root length of rice seedlings grown under a wide range of NH4+ concentrations in hydroponic conditions. Theoretical and Applied Genetics, 121, 535-547.

  • Orsel, M., Krapp, A., & Daniel-Vedele, F. (2002). Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression. Plant Physiology, 129(2), 886–896.

  • Proost, S., Van Bel, M., Vaneechoutte, D., Van de Peer, Y., Inzé, D., Mueller-Roeber, B., & Vandepoele, K. (2015). PLAZA 3.0: An access point for plant comparative genomics. Nucleic Acids Research, 43(D1), D974–D981.

  • Qian, X., Shen, Q., Xu, G., Wang, J., & Zhou, M. (2004). Nitrogen form effects on yield and nitrogen uptake of rice crop grown in aerobic soil. Journal of Plant Nutrition, 27(6), 1061–1076.

  • Ranade-Malvi, U. (2011). Interaction of micronutrients with major nutrients with special reference to potassium. Karnataka Journal of Agricultural Sciences, 24(1), 106–109.

  • Rao, I. S., Neeraja, C. N., Srikanth, B., Subrahmanyam, D., Swamy, K. N., Rajesh, K., Vijayalakshmi, P., Kiran, T. V., Sailaja, N., Revathi, P., Rao, P. R., Rao, L. V. S., Surekha, K., Babu, V. R., & Voleti, S. R. (2018). Identification of rice landraces with promising yield and the associated genomic regions under low nitrogen. Scientific Reports, 8, 9200.

  • Remans, T., Nacry, P., Pervent, M., Girin, T., Tillard, P., Lepetit, M., & Gojon, A. (2006). A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis. Plant Physiology, 140(3), 909–921.

  • Safavi-Rizi, V., Uellendahl, K., Öhrlein, B., Safavi‐Rizi, H., & Stöhr, C. (2021). Cross‐stress tolerance: Mild nitrogen (N) deficiency effects on drought stress response of tomato (Solanum lycopersicum L.). Plant‐Environment Interactions, 2(5), 217-228.

  • Sayers, E. W., Beck, J., Bolton, E. E., Bourexis, D., Brister, J. R., Canese, K., Comeau, D. C., Funk, K., Kim, S., Klimke, W., Marchler-Bauer, A., Landrum, M., Lathrop, S., Lu, Z., Madden, T. L., O’Leary, N., Phan, L., Rangwala, S. H., Schneider, V. A., … Sherry, S. T. (2021). Database resources of the National Center for Biotechnology Information. Nucleic Acids Research, 49(D1), D10-D17.

  • Schaeffer, R. D., Kinch, L. N., Liao, Y., & Grishin, N. V. (2016). Classification of proteins with shared motifs and internal repeats in the ECOD database. Protein Science, 25(7), 1188–1203.

  • Shannon, P., Markiel, A., Ozier, O., Baliga, N. S., Wang, J. T., Ramage, D., Amin, N., Schwikowski, B., & Ideker, T. (2003). Cytoscape: A software environment for integrated models of biomolecular interaction networks. Genome Research, 13(11), 2498-504.

  • Sharma, S., Kaur, G., Kumar, A., Meena, V., Kaur, J., & Pandey, A. K. (2019). Overlapping transcriptional expression response of wheat zinc-induced facilitator-like transporters emphasize important role during Fe and Zn stress. BMC Molecular Biology, 20, 22.

  • Singh, B. R., Timsina, Y. N., Lind, O. C., Cagno, S., & Janssens, K. (2018). Zinc and iron concentration as affected by nitrogen fertilization and their localization in wheat grain. Frontiers in Plant Science, 9, 307.

  • Sonoda, Y., Ikeda, A., Saiki, S., Wirén, N. V., Yamaya, T., & Yamaguchi, J. (2003). Distinct expression and function of three ammonium transporter genes (OsAMT1; 1–1; 3) in rice. Plant and Cell Physiology, 44(7), 726-734.

  • Tian, T., Liu, Y., Yan, H., You, Q., Yi, X., Du, Z., Xu, W., & Su, Z. (2017). AgriGO v2.0: A GO analysis toolkit for the agricultural community, 2017 update. Nucleic Acids Research, 45(W1), W122–W129.

  • Tong, J., Walk, T. C., Han, P., Chen, L., Shen, X., Li, Y., Gu, C., Xie, L., Hu, X., Liao, X., & Qin, L. (2020). Genome-wide identification and analysis of high-affinity nitrate transporter 2 (NRT2) family genes in rapeseed (Brassica napus L.) and their responses to various stresses. BMC Plant Biology, 20, 464.

  • Tong, H. H., Chen, L., Li, W. P., Mei, H. W., Xing, Y. Z., Yu, X. Q., Xu, X. Y., Zhang, S. Q., & Luo, L. J. (2011). Identification and characterization of quantitative trait loci for grain yield and its components under different nitrogen fertilization levels in rice (Oryza sativa L.). Molecular Breeding, 28, 495-509.

  • Waese, J., Fan, J., Pasha, A., Yu, H., Fucile, G., Shi, R., Cumming, M., Kelley, L. A., Sternberg, M. J., Krishnakumar, V., Ferlanti, E., Miller, J., Town, C., Stuerzlinger, W., & Provart, N. J. (2017). ePlant: Visualizing and exploring multiple levels of data for hypothesis generation in plant biology. Plant Cell, 29(8), 1806–1821.

  • Wang, H., Yang, Z., Yu, Y., Chen, S., He, Z., Wang, Y., Jiang, L., Wang, G., Yang, C., Liu, B., & Zhang, Z. (2017). Drought enhances nitrogen uptake and assimilation in maize roots. Agronomy Journal, 109(1), 39–46.

  • Wang, M., Zhang, P., Liu, Q., Li, G., Di, D., Xia, G., Kronzucker, H. J., Fang, S., Chu, J., & Shi, W. (2020). TaANR1-TaBG1 and TawAbi5-TaNRT2S/NARs link ABA metabolism and nitrate acquisition in wheat roots. Plant Physiology, 182(3), 1440–1453.

  • Wang, Y. Y., Hsu, P. K., & Tsay, Y. F. (2012). Uptake, allocation and signaling of nitrate. Trends in Plant Science, 17(8), 458–467.

  • Wang, Q., Nian, J., Xie, X., Yu, H., Zhang, J., Bai, J., Dong, G., Hu, J., Bai, B., Chen, L., Xie, Q., Feng, J., Yang, X., Peng, J., Chen, F., Qian, Q., Li, J., & Zuo J. (2018). Genetic variations in ARE1 mediate grain yield by modulating nitrogen utilization in rice. Nature Communications, 9, 735.

  • Waraich, E. A., Ahmad, R., & Ashraf, M. Y. (2011). Role of mineral nutrition in alleviation of drought stress in plants. Australian Journal of Crop Science, 5(6), 764-777

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

  • Wu, J., Wang, C., Zheng, L., Wang, L., Chen, Y., Whelan, J., & Shou, H. (2011). Ethylene is involved in the regulation of iron homeostasis by regulating the expression of iron-acquisition-related genes in Oryza sativa. Journal of Experimental Botany, 62(2), 667–674.

  • Wu, K., Ali, I., Xie, H., Ullah, S., Iqbal, A., Wei, S., He, L., Huang, Q., Wu, X., Cheng, F., & Jiang, L. (2021). Impact of fertilization with reducing in nitrogen and phosphorous application on growth, yield and biomass accumulation of rice (Oryza sativa L.) under a dual cropping system. PeerJ, 9, e11668.

  • Xia, L., Zou, D., Sang, J., Xu, X., Yin, H., Li, M., Wu, S., Hu, S., Hao, L., & Zhang, Z. (2017). Rice Expression Database (RED): An integrated RNA-Seq-derived gene expression database for rice. Journal of Genetics and Genomics, 44(5), 235–241.

  • Xu, G., Fan, X., & Miller, A. J. (2012). Plant nitrogen assimilation and use efficiency. Annual Review of Plant Biology, 63, 153–182.

  • Zhang, X., Liu, H., Zhang, S., Wang, J., & Wei, C. (2019). NH4+-N alleviates iron deficiency in rice seedlings under calcareous conditions. Scientific Reports, 9, 12712.

  • Zhang, Z., Gao, S., & Chu, C. (2020). Improvement of nutrient use efficiency in rice: Current toolbox and future perspectives. Theoretical and Applied Genetics, 133, 1365-1384.

  • Zhao, L., Chen, P., Liu, P., Song, Y., & Zhang, D. (2021). Genetic effects and expression patterns of the nitrate transporter (NRT) gene family in Populus tomentosa. Frontiers in Plant Science, 12, 661635.

ISSN 1511-3701

e-ISSN 2231-8542

Article ID


Download Full Article PDF

Share this article

Related Articles