A class 1 hemoglobin gene from Alnus firma functions in symbiotic and nonsymbiotic tissues to detoxify nitric oxide

Actinorhizal symbiosis is as important in biological nitrogen fixation as legume-rhizobium symbiosis in the global nitrogen cycle. To understand the function of hemoglobin (Hb) in actinorhizal symbiosis, we characterized a Hb of Alnus firma, AfHb1. A cDNA that encodes nonsymbiotic Hb (nonsym-Hb) was isolated from a cDNA library of A. firma nodules probed with LjHb1, a nonsym-Hb of Lotus japonicus. No homolog of symbiotic Hb (sym-Hb) could be identified by screening in the cDNA library or by polymerase chain reaction (PCR) using degenerate primers for other sym-Hb genes. The deduced amino acid sequence of AfHb1 showed 92% sequence similarity with a class 1 nonsym-Hb of Casuarina glauca. Quantitative reverse transcriptase-PCR analysis showed that AfHb1 was expressed strongly in the nodules and enhanced expression was detected under cold stress but not under hypoxia or osmotic stress. Moreover, AfHb1 was strongly induced by the application of nitric oxide (NO) donors, and the application of a NO scavenger suppressed the effect of NO donors. Acetylene reduction was strongly inhibited by the addition of NO donors. AfHb1 may support the nitrogen fixation ability of members of the genus Frankia as a NO scavenger.