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

AbstractTobacco 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

Improved reliability in production of maize inbred lines by the combination of the R1-navajo marker with flow cytometry or microsatellite genotyping

Doubled haploid (DH) technology is an essential component in producing inbred lines for a competitive maize (Zea maysL.) breeding program. TheR1-navajo(R1-nj) gene provides phenotypic marker that insures only variable reliability for seed selection of haploid embryos. Therefore, in the present study we outline a complex protocol for early stage genome size determination that integrates the phenotypic screening with the flow cytometry of nuclei from root tips and with the use of DNA isolated from seedlings for molecular marker-based genotyping. In a representative experiment with three genotypes, only 59% of the color marker pre-selected seeds were confirmed to be haploid by cytometric analysis of nuclei isolated from root tips. As a novel tool we have identified the UMC1152 SSR marker being polymorphic between the haploid inducer line (K405) and the K4390 hybrid as parents to screen seedlings pre-selected with theR1-navajo marker. Using this molecular marker, alleles characteristic for the inducer K405 line could not be detected in 83% of seedlings previously selected as haploid candidate. Seedlings identified as haploids were exposed to 0.06% colchicine solution for rediploidization. This procedure resulted in doubled haploids with 3% frequency relative to the initial population as it was quantified by the number of mature maize plants with fertile tassel. The described complex approach can support safer identification of haploids at early seedling stage in a hybrid population derived from crossing with a haploid inducer line

Immunodetection of retinoblastoma-related protein and its phosphorylated form in interphase and mitotic alfalfa cells

Plant retinoblastoma-related (RBR) proteins are primarily considered as key regulators of G1/S phase transition, with functional roles in a variety of cellular events during plant growth and organ development. Polyclonal antibody against the C-terminal region of the Arabidopsis RBR1 protein also specifically recognizes the alfalfa 115 kDa MsRBR protein, as shown by the antigen competition assay. The MsRBR protein was detected in all cell cycle phases, with a moderate increase in samples representing G2/M cells. Antibody against the human phospho-pRb peptide (Ser807/811) cross-reacted with the same 115 kDa MsRBR protein and with the in vitro phosphorylated MsRBR protein C-terminal fragment. Phospho-MsRBR protein was low in G1 cells. Its amount increased upon entry into the S phase and remained high during the G2/M phases. Roscovitine treatment abolished the activity of alfalfa MsCDKA1;1 and MsCDKB2;1, and the phospho-MsRBR protein level was significantly decreased in the treated cells. Colchicine block increased the detected levels of both forms of MsRBR protein. Reduced levels of the MsRBR protein in cells at stationary phase or grown in hormone-free medium can be a sign of the division-dependent presence of plant RBR proteins. Immunolocalization of the phospho-MsRBR protein indicated spots of variable number and size in the labelled interphase nuclei and high signal intensity of nuclear granules in prophase. Structures similar to phospho-MsRBR proteins cannot be recognized in later mitotic phases. Based on the presented western blot and immunolocalization data, the possible involvement of RBR proteins in G2/M phase regulation in plant cells is discussed

Phytoglobins and nitric oxide: new partners in an old signalling system in plants

The recent review summarizes the major achievements in discovery of role of phytoglobins in mediation of nitric oxide generated cellular functions in higher plants. Genes encoding non-symbiotic hemoglobins have been cloned from several plant species. The expression pattern of these genes show tissue-specificity that is also under the control of stress factors like hypoxia. The nitric oxide has pivotal role in signalling pathway specifically in hypersensitive reactions and programmed cell death. Production of transgenic tobacco plants overexpressing the alfalfa hemoglobin showed altered necrotic symptoms after treatment with nitric oxide generating compounds or infection by necrotic pathogens. The present review helps to outline the similar relation between hemoglobin and nitric oxide in plants as it was found in animal cells