SummaryTransgenic alfalfa root cultures expressing sense and antisense barley hemoglobin transcripts were examined under varying levels of atmospheric oxygen. Root cultures overexpressing the hemoglobin gene (Hb ) maintained root growth when placed under 3% oxygen, whereas control cultures or cultures underexpressing hemoglobin (Hb À ) experienced 30±70% declines in growth under the same conditions. ATP levels and ATP/ADP ratios for Hb lines did not signi®cantly differ in 40 and 3% oxygen, whereas the ATP levels and ATP/ADP ratios in control and Hb À lines were signi®cantly lower under 3% oxygen. Large increases in the production of nitric oxide (NO) were measured in root cultures grown under hypoxic conditions compared to aerobic conditions. The amount of NO accumulated in an Hb À line was 2.5-fold higher than that in the Hb line. Treatment of transgenic root lines under 40% oxygen with NO resulted in signi®cant declines in the ATP levels and ATP/ADP ratio of an Hb À line and the control line, with no signi®cant change in an Hb line. The root cell structure of an Hb À line showed evidence of cell breakdown under hypoxic growth, whereas an Hb line had no evidence of cell breakdown under similar growth conditions. These results lead us to hypothesize that NO is involved in the response of plants to hypoxia and that hemoglobin modulates the levels of NO in the hypoxic cell.
Transgenic alfalfa ( Medicago sativa L.) root cultures expressing sense and antisense barley ( Hordeum vulgare L.) hemoglobin were examined for their ability to metabolize NO. Extracts from lines overexpressing hemoglobin had approximately twice the NO conversion rate of either control or antisense lines under normoxic conditions. Only the control line showed a significant increase in the rate of NO degradation when placed under anaerobic conditions. The decline in NO was dependent on the presence of reduced pyridine nucleotide, with the NADH-dependent rate being about 2.5 times faster than the NADPH-dependent rate. Most of the activity was found in the cytosolic fraction of the extracts, while only small amounts were found in the cell wall, mitochondria, and 105,000- g membrane fraction. The NADH-dependent NO conversion exhibited a broad pH optimum in the range 7-8 and a strong affinity to NADH and NADPH ( K(m) 3 microM for both). It was sensitive to diphenylene iodonium, an inhibitor of flavoproteins. The activity was strongly reduced by applying antibodies raised against recombinant barley hemoglobin. Extracts of Escherichia coli overexpressing barley hemoglobin showed a 4-fold higher rate of NO metabolism as compared to non-transformed cells. The NADH/NAD and NADPH/NADP ratios were higher in lines underexpressing hemoglobin, indicating that the presence of hemoglobin has an effect on these ratios. They were increased under hypoxia and antimycin A treatment. Alfalfa root extracts exhibited methemoglobin reductase activity, using either cytochrome c or recombinant barley hemoglobin as substrates. There was a correspondence between NO degradation and nitrate formation. The activity was eluted from a Superose 12 column as a single peak with molecular weight of 35+/-4 kDa, which corresponds to the size of the hemoglobin dimer. The results are consistent with an NO dioxygenase-like activity, with hemoglobin acting in concert with a flavoprotein, to metabolize NO to nitrate utilizing NADH as the electron donor.
Plastid lipid-associated proteins, also termed fibrillin/CDSP34 proteins, are known to accumulate in fibrillar-type chromoplasts such as those of ripening pepper fruit, and in leaf chloroplasts from Solanaceae plants under abiotic stress conditions. It is shown here that treatments generating active oxygen species (high light combined with low temperature, gamma irradiation or methyl viologen treatment) result in potato CDSP34 gene induction and protein accumulation in leaves. Using transgenic tomato plants containing the pepper fibrillin promoter, a significant increase in promoter activity in leaves subjected to biotic stress, namely bacterial infections, was observed. In WT, a higher level of the endogenous fibrillin/CDSP34 protein is also observed after infection by E. chrysanthemi strain 3739. In addition to stress-related induction, a progressive increase in the fibrillin promoter activity is noticed during ageing in various tomato photosynthetic tissues and this increase correlates with a higher abundance of the endogenous protein in WT leaves. It is proposed that a mechanism related to oxidative events plays an essential role in the regulation of fibrillin/CDSP34 genes during stress and also during development. Using a biolistic transient expression assay, the pepper fibrillin promoter is found to be active in various dicot species, but not in monocots. Further, substantially increased levels of fibrillin/ CDSP34 proteins are shown in various dicotyledonous and monocotyledonous plants in response to water deficit.
A novel halophilic fermentative bacterium has been isolated from the black sediment below a gypsum crust and a microbial m a t in hypersaline ponds of Mediterranean salterns. Morphologically, physiologically and genetically this organism belongs to the genus Haloanaerobacter. Haloanaerobacter strain SG 3903T (T = type strain) is composed of non-sporulating long flexible rods with peritrichous flagella, able to grow in the salinity range of 5-30% NaCI, with an optimum a t 14-15%. The strain grows by fermenting carbohydrates or by using the Stickland reaction with either serine or H, as electron donors and glycine-betaine as acceptor, which is reduced to trimethylamine. The two species described so far in the genus Haloanaerobacter are not capable of Stickland reaction with glycine-betaine + serine; however, Haloanaerobacter chitinovorans can use glycine-betaine with H, as electron donor. Strain SG 3903T thus represents the first described strain in the genus Haloanaerobacter capable of the Stickland reaction with two amino acids. Although strain SG 3903T showed 67 O/ O DNA-DNA relatedness to H. chitinovorans, it is physiologically sufficiently different from the two described species to be considered as a new species which has been named Haloanaerobacter salinarius sp. nov.
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