Huazhong Agricultural University

HOST | Huazhong Agricultural University

Local Organizing Committee (LOC)

● Chair

Xuelu Wang, Huazhong Agricultural University, China

● Members

Yangrong Cao, Huazhong Agricultural University, China Sanfeng Chen, China Agricultural University, China Youguo Li, Huazhong Agricultural University, China Xia Li, Huazhong Agricultural University, China Yan Liang, Zhejiang University, China Min Lin, Chinese Academy of Agricultural Sciences, China Jeremy Murray, Chinese Academy of Sciences, China Christian Staehelin, Sun Yat-sen (Zhongshan) University Gary Stacey, University of Missouri, USA Changfu Tian, China Agricultural University, China Ertao Wang, Chinese Academy of Sciences, China Yiping Wang, Peking University, China Fang Xie, Chinese Academy of Sciences, China Zubin Xie, Institute of Soil Sciences, Chinese Academy of Sciences, China Shutong Xu, Huazhong Agricultural University, China Zhongming Zhang, Huazhong Agricultural University, China Zhipeng Zhou, Huazhong Agricultural University, China Zhaohui Zhou, Xiamen University, China

● Secretaries

Shiyong Sun, Huazhong Agricultural University, China Deqiang Duanmu, Huazhong Agricultural University, China Chunjiao Xia, Huazhong Agricultural University, China International Steering Committee (ISC)

Barbara Reinhold-Hurek, University of Bremen, Germany (Chair) María J. Delgado, Consejo Superior de Investigaciones Científicas (CSIC), Spain Ray Dixon, John Innes Centre, UK; Chinese Academy of Sciences, China Sharon R. Long, Stanford University, USA Esperanza Martínez-Romero, Universidad Nacional Autónoma de México, Mexico Kiwamu Minamisawa, Tohoku University, Japan Graham O’Hara, Murdoch University, Australia John W. Peters, Montana State University & Washington State University, USA Luis M. Rubio, Universidad Politécnica de Madrid, Spain Igor A. Tikhonovich , All-Russia Research Institute for Agricultural Microbiology & Saint Petersburg State University, Russia Yiping Wang, Peking University, China

International Scientific Advisory Committee (ISAC)

Wenxin Chen, China Agricultural University, China María J. Delgado , Consejo Superior de Investigaciones Científicas (CSIC), Spain Ray Dixon, John Innes Centre, UK; Chinese Academy of Sciences, China Rene Geurts, Wageningen University, Netherlands Clare Gough, LIPM, Université de Toulouse, INRA, CNRS, France Georgina Hernandez, Universidad Nacional Autónoma de México, Mexico Éva Kondorosi, Hungarian Academy of Sciences, Hungary Min Lin, Chinese Academy of Agricultural Sciences, China Sharon R. Long, Stanford University, USA Veronica Massena Reis, Embrapa Agrobiologia, Brazil Catherine Masson-Boivin, LIPM, Université de Toulouse, INRA, CNRS, France Ulrike Mathesius, Australia National University, Australia Kiwamu Minamisawa, Tohoku University, Japan Katharina Pawlowski, Stockholm University, Sweden Fábio O. Pedrosa, Universidade Federal do Paraná, Brazil John W. Peters, Montana State University & Washington State University, USA Jens Stougaard, Aarhus University, Denmark Simona Radutoiu, Aarhus University, Denmark Barbara Reinhold-Hurek, University of Bremen, Germany Luis M. Rubio, Universidad Politécnica de Madrid, Spain Gary Stacey, University of Missouri, USA Ertao Wang, Chinese Academy of Sciences, China Yiping Wang, Peking University, China Jindong Zhao, Peking University, China PARALLEL SESSION 10

Insights and Pitfalls of the Methodology for Free Radical Research in Legume Nodules: Hemoglobins as a Case Study Manuel Becana1, Maria Carmen Rubio1, Laura Calvo-Begueria1, Jesús I. Martínez2, Deqiang Duanmu3, Carmen

Pérez-Rontomé1

1Estación Experimental de Aula Dei, CSIC, Apartado 13034, 50080 Zaragoza, Spain; 2Instituto de Ciencia de Materiales de

Aragón, Universidad de Zaragoza-CSIC, Pedro Cerbuna 12, 50059 Zaragoza, Spain; 3State Key Laboratory of Agricultural

Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

Corresponding author: [email protected] Keywords: EPR; leghemoglobins; legume nodules; reactive oxygen/nitrogen species

Reactive oxygen (ROS; superoxide radicals and H2O2) and nitrogen (RNS; NO and nitrosothiols) species are of paramount importance in biology. In plants, ROS and RNS are generated in most cellular compartments. At the light microscopy level, the production of superoxide radicals, H2O2 and NO may be detected based on their reactions with nitroblue tetrazolium, diaminobenzidine and

4,5-diaminofluorescein diacetate, respectively. Additional techniques are the HyPer bioprobe for H2O2 and electron paramagnetic resonance (EPR) spectroscopy for free radicals. However, strict controls must be used to ensure specificity, discard interferences and minimize artifacts during tissue extraction. In the rhizobia-legume symbiosis, all three reactive species were detected in the infection threads and in mature and senescent nodules. Leghemoglobins (Lbs) are heme proteins found at millimolar concentrations in the cytoplasm of infected cells, where they transport and deliver O2 to the symbiosomes. We have used two experimental approaches involving Lbs to localize ROS and RNS in nodules and identify potential pitfalls of the methodology. The first approach was to detect the nitrosyl-Lb complex by low temperature EPR of intact soybean nodules formed by wild-type and mutant bradyrhizobia deficient in denitrification enzymes. A comparison was made between the EPR and the fluorescent probe to detect NO. We conclude that bacteroid nitrite and NO reductases are essential to keep nodule NO homeostasis, that NO accumulates in both the inner cortex and infected zone, that Lbs may scavenge NO in vivo, and that NO is artifactually generated in nodule extracts but not in intact nodules. The second approach was to localize ROS in mutant Lotus japonicus nodules completely lacking Lbs. We found a drastic increase in superoxide production caused, at least in part, by induction of NADPH oxidases and changes in superoxide dismutase composition. The mutant nodules also showed an increase of H2O2. Taken together, our observations suggest that Lbs contribute to control the nodule concentrations of ROS and RNS.