NO-degradation by alfalfa class 1 hemoglobin (Mhb1): a possible link to PR-1a gene expression in Mhb1-overproducing tobacco plants.
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.
Seregélyes
Csaba
C
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.
Igamberdiev
Abir U
AU
Maassen
Anna
A
Hennig
Jacek
J
Dudits
Dénes
D
Hill
Robert D
RD
D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000455:Medicago sativa / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D030821:Plants, Genetically Modified / Q000378:metabolism; D044224:Pseudomonas syringae / Q000472:pathogenicity; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D017931:DNA Primers; D018744:DNA, Plant; D006454:Hemoglobins; D010940:Plant Proteins; D011994:Recombinant Proteins; D009569:Nitric Oxide