Excess nitrate induces nodule greening and reduces transcript and protein expression levels of soybean leghaemoglobins

Efficient biological nitrogen fixation (BNF) requires leghaemoglobin (Lb) to modulate oxygen pressure in nodules. Excess N supply severely inhibits BNF through effects on Lb during nodulation. As yet, a systematic identification and characterization of Lb-encoding genes in soybean has not been reported.The effects of N on BNF were studied in soybean plants inoculated with rhizobia and exposed to excess or low N availability in hydroponic cultures. To identify soybean Lb proteins, BLAST searches were performed on the Phytozome website. Bioinformatic analysis of identified GmLbs was then carried out to investigate gene structure, protein homology and phylogenetic relationships. Finally, quantitative real-time PCR was employed to analyse the expression patterns of soybean Lb genes in various tissues and in response to high N availability.Excess N significantly accelerated nodule senescence and the production of green Lb in nodules. In total, seven haemoglobin (Hb) genes were identified from the soybean genome, with these Hb genes readily split into two distinct clades containing predominantly symbiosis-associated or non-symbiotic Hb members. Expression analysis revealed that all of the symbiosis-associated Lbs except GmLb5 were specifically expressed in nodules, while the non-symbiotic GmHbs, GmHb1 and GmHb2, were predominantly expressed in leaves and roots, respectively. Among identified GmLbs, GmLb1-4 are the major Lb genes acting in soybean nodulation, and each one is also significantly suppressed by exposure to excess N.Taken together, the results show that excess N inhibits BNF by reducing nodule formation, Lb concentration and nitrogenase activity. The characteristics of the entire Hb family were analysed, and we found that GmLb1-4 are closely associated with nodule development and N2 fixation. This works forms the basis for further investigations of the role of Lbs in soybean nodulation.