Biotechnological approaches to develop nitrogen-fixing cereals: A review

  • Asma Boujenna Université Abdel Malek Essâadi, Faculté des Sciences, Dept. de Biologie, Av. Sebta, Tétouan 93002,
  • Luis F. Garcia del Moral University of Granada, Faculty of Sciences, Institute of Biotechnology, Dept. of Plant Physiology, Avda. Fuentenueva s/n, 18075 Granada
Keywords: nitrogenase, nif genes, O2 tolerance, plastids, mitochondria, plant growth-promoting rhizobacteria, biological nitrogen fixation


Agricultural yields are often limited by nitrogen (N) availability, especially in countries of the developing world, whereas in industrialized nations the application of chemical N fertilizers has reached unsustainable levels that have resulted in severe environmental consequences. Finding alternatives to inorganic fertilizers is critical for sustainable and secure food production. Although gaseous nitrogen (N2) is abundant in the atmosphere, it cannot be assimilated by most living organisms. Only a selected group of microorganisms termed diazotrophs, have evolved the ability to reduce N2 to generate NH3 in a process known as biological nitrogen fixation (BNF) catalysed by nitrogenase, an oxygen-sensitive enzyme complex. This ability presents an opportunity to improve the nutrition of crop plants, through the introduction into cereal crops of either the N fixing bacteria or the nitrogenase enzyme responsible for N fixation. This review explores three potential approaches to obtain N-fixing cereals: (a) engineering the nitrogenase enzyme to function in plant cells; (b) engineering the legume symbiosis into cereals; and (c) engineering cereals with the capability to associate with N-fixing bacteria.


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Aasfar A, Bargaz A, Yaakoubi K, Hilali A, Bennis I, Zeroual Y, Kadmiri I M, 2021. Nitrogen fixing Azotobacter species as potential soil biological enhancers for crop nutrition and yield stability. Front Microbiol 12: 628379.

Addo MA, Dos Santos PC, 2020. Distribution of nitrogen-fixation genes in prokaryotes containing alternative nitrogenases. Chem BioChem 21: 1749-1759.

Allen RS, Tilbrook K, Warden AC, Campbell PC, Rolland V, Singh SP, Wood CC, 2017. Expression of 16 nitrogenase proteins within the plant mitochondrial matrix. Front Plant Sci 8: 287.

Anas M, Liao F, Verma KK, Sarwar MA, Mahmood A, Chen ZL, et al., 2020. Fate of nitrogen in agriculture and environment: agronomic, eco‑physiological and molecular approaches to improve nitrogen use efficiency. Biol Res 53: 47.

Arnold W, Rump A, Klipp W, Priefer UB, Pühler A, 1988. Nucleotide sequence of a 24,206 base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol 203: 715-738.

Baca BE, Elmerich C, 2007. Microbial production of plant hormones. In: Associative and endophytic nitrogen-fixing bacteria and cyanobacterial associations; Elmerich C, Newton WE (eds), pp: 113-143, Kluwer Acad Publ, Dordrecht.

Batista MB, Dixon R, 2019. Manipulating nitrogen regulation in diazotrophic bacteria for agronomic benefit. Biochem Soc Trans 47: 603-614.

Beatty PH, Good AG, 2011. Future prospects for cereals that fix nitrogen. Science 333: 416-417.

Burén S, Jiang X, López-Torrejón G, Echavarri-Erasun C, Rubio LM, 2017a. Purification and in vitro activity of mitochondria targeted nitrogenase cofactor maturase NifB. Front Plant Sci 8: 1-16.

Burén S, Young EM, Sweeny EA, Lopez-Torrejon G, Veldhuizen M, Voigt CA, Rubio LM, 2017b. Formation of nitrogenase NifDK tetramers in the mitochondria of Saccharomyces cerevisiae. ACS Synth Biol 6: 1043-1055.

Burén S, Rubio LM, 2018. State of the art in eukaryotic nitrogenase engineering. Microbiol Lett 365 fnx274.

Chen YB, Dominic B, Mellon MT, Zehr JP, 1998. Circadian rhythm of nitrogenase gene expression in the diazotrophic filamentous non heterocystous cyanobacterium Trichodesmium sp. strain IMS 101. J Bacteriol 180: 3598-3605.

Cheng Q, Day A, Dowson-Day M, Dixon R, 2005.The Klebsiella pneumoniae nitrogenase Fe protein gene (nifH) functionally substitutes for the chlL gene in Chlamydomonas reinhardtii. Biochem Biophys Res Commun 329: 966-975.

Curatti L, Rubio LM, 2014. Challenges to develop nitrogen-fixing cereals by direct nif-gene transfer. Plant Sci 225: 130-137.

Dénarié J, Debelle F, Prome J-C, 1996. Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis. Ann Rev Biochem 65: 503-535.

Dent DR, Cocking EC, 2017. Establishing symbiotic nitrogen fixation in cereals and other non-legume crops: The greener nitrogen revolution. Agri Food Secur 6: 7.

Dixon RA, Postgate JR, 1971. Transfer of nitrogen-fixation genes by conjugation in Klebsiella pneumoniae. Nature 234: 47-48.

Dixon R, Kahn D, 2004. Genetic regulation of biological nitrogen fixation. Nat Rev Microbiol 2, 621-631.

Dixon R, Cheng Q, Shen GF, Day A, Dowson-Day M, 1997. Nif gene transfer and expression in chloroplasts: Prospects and problems. Plant Soil 194: 193-203.

Dong W, Song Y. 2020. The significance of flavonoids in the process of biological nitrogen fixation. Int J Mol Sci 21: 5926.

Dos Santos PC, Fang Z, Mason SW, Setubal JC, Dixon R, 2012. Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes. BMC Genom 13: 162.

Erisman JW, Galloway JN, Dice NB, Sutton MA, Bleeker A, Grizzetti B, et al., 2015. Nitrogen: too much of a vital resource. Science Brief WWF Netherlands 1-48.

FAO, 2020. The state of food security and nutrition in the world 2020. Transforming food systems for affordable healthy diets. FAO, IFAD, UNICEF, WFP and WHO, Rome.

Flores E, Herrero A, 2010, Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat Rev Microbiol 8: 39-50.

Foley JA, Ramankutty N, Brauman KA, Cassidy ES, Gerber JS, Johnston M, et al., 2011. Solutions for a cultivated planet. Nature 478: 337-342.

Froussart E, Bonneau J, Franche C, Bogusz D, 2016. Recent advances in actinorhizal symbiosis signaling. Plant Mol Biol 90: 613-622.

Fukami J, Cerezini P, Hungria M, 2018. Azospirillum: benefits that go far beyond biological nitrogen fixation. AMB Express 8: 73.

Galloway JN, Cowling EB, 2002. Reactive nitrogen and the world: 200 years of change. Ambio 31: 64-71.

Galloway JN, Townsend AR, Willem J, Erisman JW, Bekunda M, Cai Z, et al., 2008. Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science 320: 889-892.

García del Moral LF, De la Morena I, Ramos JM, 1999. Nitrogen and foliar sulfur interaction in determining grain yield and yield components in barley. J Agron Crop Sci 183: 287-295.

Geddes BA, Ryu MH, Mus F, Garcia Costas A, Peters JW, Voigt CA, Poole P, 2015. Use of plant colonizing bacteria as chassis for transfer of N2-fixation to cereals. Curr Opin Biotechnol 32: 216-222.

Geddes BA, Paramasivan P, Joffrin A, Thompson AL, Christensen K, Jorrin B, et al., 2019. Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria. Nat Commun 10 (1): 3430.

Glendining MJ, Dailey AG, Williams AG, van Evert FK, Goulding KWT, Whitmore AP, 2009. Is it possible to increase the sustainability of arable and ruminant agriculture by reducing inputs? Agr Syst 99: 117-125.

Good A, 2018. Toward nitrogen-fixing plants. Science 359: 869-870.

Gordon DM, Ryder MH, Heinrich K, Murphy PJ, 1996. An experimental test of the rhizopine concept in Rhizobium meliloti. Appl Environ Microbiol 62: 3991-3996.

Goyal RK, Schmidt MA, Hynes MF. 2021. Molecular biology in the improvement of biological nitrogen fixation by Rhizobia and extending the scope to cereals. Microorganisms 9: 125.

Griesmann M, Chang Y, Liu X, Song Y, Haberer G, Crook MB, et al., 2018. Phylogenomics reveals multiple losses of nitrogen-fixing root nodule symbiosis. Science 361: eaat1743.

Hensel G, 2020. Genetic transformation of Triticeae cereals - Summary of almost three decade's development. Biotechnol Adv 40: 107484.

Hu Y, Ribbe MW, 2015. Nitrogenase and homologs. J Biol Inorg Chem 20: 435-445.

Ibañez F, Wall L, Fabra A, 2017. Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots. J Exp Bot 68: 1905-1918.

Ivleva NB, Groat J, Staub JM, Stephens M, 2016. Expression of active subunit of nitrogenase via integration into plant organelle genome. PLoS One 11: e0160951.

Jiang X, Payá-Tormo L, Coroian D, García-Rubio I, Castellanos-Rueda R, Eseverri A, et al., 2021. Exploiting genetic diversity and gene synthesis to identify superior nitrogenase NifH protein variants to engineer N2-fixation in plants. Comm Biol 4: 4.

Ladha JK, Tirol-Padre A, Reddy CK, Cassman KG, SudhirVerma, Powlson DS, et al., 2016. Global nitrogen budgets in cereals: A 50-year assessment for maize, rice, and wheat production systems. Sci Rep 6: 19355.

Larrainzar E, Villar I, Rubio MC, Perez-Rontome C, Huertas R, Sato S, et al., 2020. Hemoglobins in the legume-Rhizobium symbiosis. New Phytol 228: 472-484.

Li Q, Chen S, 2020. Transfer of nitrogen fixation (nif) genes to non-diazotrophic hosts. ChemBioChem 21: 1717-1722.

Li M, Xu J, Gao Z, TianH, Gao Y, Kariman K, 2020. Genetically modified crops are superior in their nitrogen use efficiency-A meta-analysis of three major cereals. Sci Rep 10: 8568.

Liu D, Liberton M, Yu J, Pakrasi HB, Bhattacharyya-Pakrasi M, 2018. Engineering nitrogen fixation activity in an oxygenic phototroph. mBio 9: e01029-18.

Lodwig E, Poole P, 2003. Metabolism of Rhizobium bacteroids. CRC Crit Rev Plant Sci 22: 37-78.

López-Torrejón G, Jiménez-Vicente E, Buesa JM, Hernandez JA, Verma HK, Rubio LM, 2016. Expression of a functional oxygen-labile nitrogenase component in the mitochondrial matrix of aerobically grown yeast. Nat Comm 7: 11426.

Murphy P, Wexler W, Grzemski W, Rao J, Gordon D, 1995. Rhizopines-Their role in symbiosis and competition. Soil Biol Biochem 27: 525-529.

Mus F, Crook MB, Garcia K, Garcia Costas A, Geddes BA, Kouri ED, et al., 2016. Symbiotic nitrogen fixation and the challenges to its extension to nonlegumes. App Environ Microbiol 82: 3695-3710.

Nag P, Shriti S, Das S, 2019. Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes. J App Microbiol 129: 186-198.

Olivares J, Bedmar EJ, Sanjuan J, 2013. Biological nitrogen fixation in the context of global change. Mol Plant-Microbe Interact 26: 486-494.

Oldroyd GED, Dixon R, 2014. Biotechnological solutions to the nitrogen problem. Curr Opin Biotechnol 26: 19-24.

Oldroyd GED, Poole PS, 2019. Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria. Nat Commun 10: 1-11

Oldroyd GED, Murray JD, Poole PS, Downie JA, 2011. The rules of engagement in the legume-rhizobial symbiosis. Annu Rev Genet 45: 119-144.

Ormeño-Orrillo E, Hungria M, Martínez-Romero E, 2013. Dinitrogen-fixing prokaryotes. In: The prokaryotes: prokaryotic physiology and biochemistry; Rosenberg E et al. (eds). pp: 427-451, Springer, Berlin.

Pankievicz VCS, Irving TB, Maia LCS, Ané JM, 2019. Are we there yet? The long walk towards the development of efficient symbiotic associations between nitrogen-fixing bacteria and non-leguminous crops. BMC Biol 17: 99.

Pérez-González A, Kniewel R, Veldhuizen M, Verma HK, Navarro-Rodríguez M, Rubio LM, Caro E, 2017. Adaptation of the GoldenBraid modular cloning system and creation of a toolkit for the expression of heterologous proteins in yeast mitochondria. BMC Biotechnol 17: 80.

Pérez-Montaño F, Alías-Villegas C, Bellogín RA, Del Cerro P, Espuny MR, Jiménez-Guerrero I, et al., 2014. Plant growth promotion in cereal and leguminous agricultural important plants: From microorganism capacities to crop production. Microbiol Res 169: 325-336.

Ramos JM, García del Moral LF, Molina Cano JL, Salamanca P, Roca de Togores F, 1989. Effects of an early application of sulphur or etephon as foliar spray on the growth and yield of spring barley in a Mediterranean environment. J Agron Crop Sci 163: 129 137.

Raymond J, Siefert JL, Staples CR, Blankenship RE, 2004. The natural history of nitrogen fixation. Mol Biol Evol 21: 541-554.

Reinhold-Hurek B, Hurek T, 2011. Living inside plants: bacterial endophytes. Curr Op Plant Biol 14: 435-443.

Richardson AE, Barea JM, McNeill AM, Prigent-Combaret C, 2009. Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms. Plant Soil 321: 111-117.

Rockström J, Steffen W, Noone K, Persson A, Chapin FS 3rd, Lambin EF, et al., 2009. A safe operating space for humanity. Nature 461: 472-475.

Rogers C, Oldroyd GED, 2014. Synthetic biology approaches to engineering the nitrogen symbiosis in cereals. J Exp Bot 65: 1939-1946.

Rolfe BG, Gresshoff PM, 1988. Genetic-analysis of legume nodule initiation. Ann Rev Plant Physiol Plant Mol Biol 39: 297-319.

Rosenblueth M, Martinez-Romero E, 2006. Bacterial endophytes and their interactions with hosts. Mol Plant-Microbe Interact 19: 827-837.

Rosenblueth M, Ormeño-Orrillo E, López-López A, Rogel MA, Reyes-Hernández BJ, Martínez-Romero JC, et al., 2018. Nitrogen fixation in cereals. Front Microbiol 9: 1794.

Ryu MH, Zhang J, Toth T, Khokhani D, Geddes BA, Mus F, et al., 2020. Control of nitrogen fixation in bacteria that associate with cereals. Nat Microbiol 5: 314-330.

Saharan BS, Nehra V, 2011. Plant growth promoting rhizobacteria: a critical review. Life Sci Med Res 21: 1-30.

Santi C, Bogusz D, Franche C, 2013. Biological nitrogen fixation in non-legume plants. Ann Bot 111: 743-767.

Savka MA, Dessaux Y, Oger P, Rossbach S, 2002. Engineering bacterial competitiveness and persistence in the phytosphere. Mol Plant-Microbe Interact 15: 866-874.

Savka MA, Dessaux Y, Gardener BBM, Mondy S, Kohler PRA, Rossbach S, 2013. The "biased rhizosphere" concept and advances in the Omics era to study bacterial competitiveness and persistence in the phytosphere. In: Mol Microb Ecol Rhizosphere 1, pp: 1145-1161. John Wiley & Sons, Ltd.

Scharff LB, Bock R, 2014. Synthetic biology in plastids. Plant J 78: 783-798.

Seefeldt LC, Hoffman BM, Dean DR, 2009. Mechanism of Mo-dependent nitrogenase. Ann Rev Biochem 78: 701-722.

Seefeldt LC, Hoffman BM, Dean, DR, 2012. Electron transfer in nitrogenase catalysis. Curr Opin Chem Biol 16: 19-25.

Sickerman NS, Ribbe MW, Hu Y, 2017. Nitrogenase cofactor assembly: an elemental inventory. Acc Chem Res 50: 2834-2841.

Stokstad E, 2016. The nitrogen fix. Science 353: 1225-1227.

Stoltzfus JR, So R, Malarvithi PP, Ladha JK, de Bruijn FJ, 1997. Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen. Plant Soil 194: 25-36.

Taiz L, Zeiger E, Møller IM, Murphy A, 2015. Plant physiology and development, 6th ed. Sinauer Assoc, Inc. Sunderland, MA, USA 761 p.

Temme K, Zhao DH, Voigt CS, 2012. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc Natl Acad Sci USA 109: 7085-7090.

Townsend AR, Howarth RW, Bazzaz FA, Booth MS, Cleveland CC, Collinge SK, et al., 2003. Human health effects of a changing global nitrogen cycle. Front Ecol Environ 1: 240-246.[0240:HHEOAC]2.0.CO;2

Tsyganova AV, Brewin NJ, Tsyganov VE, 2021. Structure and development of the legume-rhizobial symbiotic interface in infection threads. Cells 10: 1050.

UN-DESA, 2017. World population projected to reach 9.8 billion in 2050, and 11.2 billion in 2100. United Nations Dept of Econ and Soc Aff. [March 6, 2021].

Van Deynze A, Zamora P, Delaux PM, Heitmann C, Jayaraman D, Rajasekar S, et al., 2018. Nitrogen fixation in a landrace of maize is supported by a mucilage associated diazotrophic microbiota. PLoS Biol 16: 1-21.

Van Velzen R, Holmer R, Bu F, Rutten L, Van Zeijl A, Liu W, et al., 2018. Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses. Proc Natl Acad Sci USA 115: e4700-e4709.

Venado RE, Liang J, Marin M, 2020. Rhizobia infection, a journey to the inside of plant cells. In: Regulation of nitrogen-fixing symbioses in legumes. Adv Bot Res 94: 97-118, Frendo P et al. (eds.), Elsevier, The Hague.

How to Cite
BoujennaA., & Garcia del MoralL. F. (2021). Biotechnological approaches to develop nitrogen-fixing cereals: A review. Spanish Journal of Agricultural Research, 19(4), e08R01.
Plant physiology