Nitrogen Fixation, Free Radicals and Hemoglobins in the Legume-Rhizobia Symbiosis

Manuel Becana Ausejo (PI)

Professor CSIC
Department of Plant Nutrition, Estación Experimental de Aula Dei, CSIC, Zaragoza, Spain





Other group members

Manuel A. Matamoros Galindo

Tenured Scientist CSIC

Maria C. Rubio Luna

Tenured Scientist CSIC

Carmen Pérez Rontomé

Technician PhD CSIC

Irene Villar Rua


Samuel Minguillón Campos


Research lines

  • Legume-rhizobia symbiosis
  • Plant hemoglobins
  • Free radicals, antioxidants and nitro-oxidative stress

Representative publications

  • Villar I, Larrainzar E, Milazzo L, Pérez-Rontomé C, Rubio MC, Smulevich G, Martínez JI, Wilson MT, Reeder B, Huertas R, Abbruzzetti S, Udvardi MK, Becana M (2021) A plant gene encoding one-heme and two-heme hemoglobins with extreme reactivities toward diatomic gases and nitrite. Front Plant Sci 11: 600336 (doi: 10.3389/fpls.2020.600336).
  • Becana M, Yruela I, Sarath G, Catalán P, Hargrove MS (2020) Plant hemoglobins: a journey from unicellular green algae to vascular plants. New Phytol 227: 1618-1635.
  • Larrainzar E, Villar I, Rubio MC, Pérez-Rontomé C, Huertas R, Sato S, Mun JH, Becana M (2020) Hemoglobins in the legume-Rhizobium New Phytol 228: 472-484.
  • Matamoros MA, Cutrona MC, Wienkoop S, Begara-Morales JC, Sandal N, Orera I, Barroso JB, Stougaard J, Becana M (2020) Altered plant and nodule development and protein S-nitrosylation in Lotus japonicus mutants deficient in S-nitrosoglutathione reductases. Plant Cell Physiol 61: 105-117.
  • Wang L, Rubio MC, Xin X, Zhang B, Fan Q, Wang Q, Ning G, Becana M, Duanmu D (2019) CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation. New Phytol 224: 818-832.
  • Fukudome M, Watanabe E, Osuki K, Imaizumi R, Aoki T, Kucho K, Becana M, Uchiumi T (2019) Stably transformed Lotus japonicus plants overexpressing phytoglobin LjGlb1-1 show decreased nitric oxide levels in roots and nodules as well as delayed nodule senescence. Plant Cell Physiol 60: 816-825.
  • Rubio MC, Calvo-Begueria L, Díaz-Mendoza M, Elhiti M, Moore M, Matamoros MA, James EK, Díaz I, Pérez-Rontomé C, Villar I, Calvo V, Hebelstrup KH, Dietz KJ, Becana M (2019) Phytoglobins in the chloroplasts, nuclei and cytoplasm modulate nitric oxide signaling and interact with abscisic acid. Plant J 100: 38-54.
  • Calvo-Begueria L, Rubio MC, Martínez JI, Pérez-Rontomé C, Delgado MJ, Bedmar EJ, Becana M (2018) Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes. J Exp Bot 69: 3703-3714.
  • Matamoros MA, Kim A, Peñuelas M, Ihling C, Griesser E, Hoffmann R, Fedorova M, Frolov A, Becana M (2018) Protein carbonylation and glycation in legume nodules. Plant Physiol 177: 1510-1528.
  • Calvo-Begueria L, Cuypers B, Van Doorslaer S, Abbruzzetti S, Bruno S, Berghmans H, Dewilde S, Ramos J, Viappiani C, Becana M (2017) Characterization of the heme pocket structure and ligand binding kinetics of non-symbiotic hemoglobins from the model legume Lotus japonicus. Front Plant Sci 8: article
  • Matamoros MA, Saiz A, Peñuelas M, Bustos-Sanmamed P, Mulet JM, Barja MV, Rouhier N, Moore M, James EK, Dietz KJ, Becana M (2015) Function of glutathione peroxidases in legume root nodules. J Exp Bot 66: 2979-2990.
  • Sainz M, Calvo-Begueria L, Pérez-Rontomé C, Wienkoop S, Abián J, Staudinger C, Bartesaghi S, Radi R, Becana M (2015) Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism. Plant J 81: 723-735.
  • Matamoros MA, Fernández-García N, Wienkoop S, Loscos J, Saiz A, Becana M (2013) Mitochondria are an early target of oxidative modifications in senescing legume nodules. New Phytol 197: 873-885.
  • Sainz M, Pérez-Rontomé C, Ramos J, Mulet JM, James EK, Bhattacharjee U, Petrich JW, Becana M (2013) Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress. Plant J 76: 875-887.
  • Navascués J, Pérez-Rontomé C, Gay M, Marcos M, Yang F, Walker FA, Desbois A, Abián J, Becana M (2012) Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules. Proc Natl Acad Sci USA 109: 2660-2665.
  • Bustos-Sanmamed P, Tovar-Méndez A, Crespi M, Sato S, Tabata S, Becana M (2011) Regulation of nonsymbiotic and truncated hemoglobin genes of Lotus japonicus in plant organs and in response to nitric oxide and hormones. New Phytol 189: 765-776.
  • Tovar-Méndez A, Matamoros MA, Bustos-Sanmamed P, Dietz KJ, Cejudo FJ, Rouhier N, Sato S, Tabata S, Becana M (2011) Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus. Plant Physiol 156: 1535-1547.


  • Becana M (IP), Rubio MC (co-IP), Matamoros MA, Pérez-Rontomé C, Villar I, Minguillón S, Wienkoop S, Sandal N, Duanmu D (2018-2021) Function of hemoglobins and nitric oxide in the Rhizobium-legume symbiosis. Ministerio de Ciencia e Innovación. AGL2017-85775-R.
  • Becana M (IP), Matamoros MA (co-IP), Pérez-Rontomé C, Calvo-Begueria L, Calvo V, Villar I, Cutrona C, Uchiumi T, Hoffmann R, Viappiani C (2015-2017) Nitrogen fixation by the rhizobia-legume symbiosis: hemoglobins and protein oxidative modifications during nodule development and senescence. Ministerio de Economía y Competitividad. AGL2014-53717-R.
  • Becana M (IP), Rubio MC, Matamoros MA, Ramos J, Pérez-Rontomé C, Saiz A, Sainz M, Calvo-Begueria L, Clemente M, Peñuelas M, Wienkoop S (2012-2014) Signaling by reactive oxygen/nitrogen species and antioxidants in the nitrogen-fixing Rhizobium-legume symbiosis. Ministerio de Economía y Competitividad. AGL2011-24524.
  • Becana M (IP), Rubio MC, Matamoros MA, Ramos J, Pérez-Rontomé C, Navascués J, Tovar-Méndez A, Clemente M, Bustos-Sanmamed P, Saiz A, Sainz M (2009-2011) Symbiotic nitrogen fixation: antioxidants, abiotic stress and senescence in nodules of model and crop legumes. Ministerio de Ciencia e Innovación. AGL2008-01298.
  • Becana (IR, partner 1B), Rubio MC, Matamoros MA, Ramos J, Pérez-Rontomé C, Clemente M (2006-2009) Bridging genomics and agrosystem management: resources for adaptation and sustainable production of forage Lotus species in environmentally-constrained South-American soils. Unión Europea. INCO contract 517617.

Relevant methods

  • Production of recombinant proteins and biochemical characterization of hemoglobins
  • Determination and localization of antioxidants and reactive oxygen and nitrogen species
  • Post-translational modifications of proteins mediated by free radicals

Collaborations with other national and international research groups

  • Stefania Abbruzzetti (Universita di Parma, Italy)
  • Deqiang Duanmu (Huazhong Agricultural University, China)
  • Estíbaliz Larrainzar (Universidad Pública de Navarra, Spain)
  • Jesús I. Martínez (Universidad de Zaragoza, Spain)
  • Brandon Reeder (University of Essex, UK)
  • Niels Sandal and Jens Stougaard (University of Aarhus, Denmark)
  • Toshiki Uchiumi (University of Kagoshima, Japan)
  • Stefanie Wienkoop (University of Vienna, Austria)