NO‐degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR‐1a gene expression in Mhb1‐overproducing tobacco plants

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Abstract
Tobacco plants overproducing alfalfa class 1 hemoglobin (HOT plants) have been shown to have reduced necrotic symptom development. Here, we show that this altered pathogenic response is linked to a significant increase in the nitric oxide (NO)-affected pathogenesis-related (PR-1a) transcript accumulation in the transgenic plants. Homogenates of HOT transgenic seedlings were also found to have higher NO-scavenging activity than non-transformed ones. The NO-scavenging properties of recombinant alfalfa class1 hemoglobin have been examined. Recombinant Mhb1 (rMhb1) was produced in bacteria and purified using polyethylene glycol (10-25%) fractionation, chromatography on DEAE-Sephacel, and Phenyl Superose columns. After the final purification step, the obtained preparations were near homogeneous and had a molecular weight of 44 kDa determined by size-exclusion chromatography and 23 kDa by SDS-PAGE, indicating that rMhb1 is a dimer. The protein participated in NO-degradation activity with NAD(P)H as a cofactor. After ion-exchange columns, addition of FAD was necessary for exhibiting maximal NO-degradation activity. The NAD(P)H-dependent NO-scavenging activity of rMhb1, which is similar to that of barley hemoglobin, supports a conclusion that both monocot and dicot class 1 hemoglobins can affect cellular NO levels by scavenging NO formed during hypoxia, pathogen attack and other stresses.