• Aims The objective of the study was to characterize variations in proline, arginine, histidine, vegetative storage proteins, and cold-inducible gene expression in overwintering roots of field-grown alfalfa, in response to autumn defoliation, and in relation to spring regrowth and winter survival.
Arctic Mesorhizobium sp. N33 isolated from nodules of Oxytropis arctobia in Canada's eastern Arctic has a growth temperature range from 0°C to 30°C and is a well-known cold-adapted rhizobia. The key molecular mechanisms underlying cold adaptation in Arctic rhizobia remains totally unknown. Since the concentration and contents of metabolites are closely related to stress adaptation, we applied GC-MS and NMR to identify and quantify fatty acids and water soluble compounds possibly related to low temperature acclimation in strain N33. Bacterial cells were grown at three different growing temperatures (4°C, 10°C and 21°C). Cells from 21°C were also cold-exposed to 4°C for different times (2, 4, 8, 60 and 240 minutes). We identified that poly-unsaturated linoleic acids 18∶2 (9, 12) & 18∶2 (6, 9) were more abundant in cells growing at 4 or 10°C, than in cells cultivated at 21°C. The mono-unsaturated phospho/neutral fatty acids myristoleic acid 14∶1(11) were the most significantly overexpressed (45-fold) after 1hour of exposure to 4°C. As reported in the literature, these fatty acids play important roles in cold adaptability by supplying cell membrane fluidity, and by providing energy to cells. Analysis of water-soluble compounds revealed that isobutyrate, sarcosine, threonine and valine were more accumulated during exposure to 4°C. These metabolites might play a role in conferring cold acclimation to strain N33 at 4°C, probably by acting as cryoprotectants. Isobutyrate was highly upregulated (19.4-fold) during growth at 4°C, thus suggesting that this compound is a precursor for the cold-regulated fatty acids modification to low temperature adaptation.
Unseeded annual bluegrass ( Poa annua L.) is an important component of golf greens in many regions of Canada and the United States. Although this turfgrass species has desirable playing attributes, it suffers from susceptibility to environmental and biological stresses including subfreezing temperatures and snow molds. In this study, we compared 29 genotypes collected from golf greens located in Québec and Ontario for their resistance to pink snow mold (SM). Plants were inoculated with Microdochium nivale [(Fries) Samuels & Hallett], causal agent of SM, and incubated under controlled conditions. High levels of variation in SM resistance were detected within the collection and between genotypes. Analysis of the relationship between climatic parameters at the sites of origin and SM susceptibility revealed that level of resistance was positively correlated to the duration of snow cover. Genetic diversity within the Poa collection was estimated using the sequence related amplified polymorphism (SRAP) technique. The UPGMA (unweighted‐pair group method arithmetic average) dendrogram yielded two main clusters that differed markedly in their proportion of SM‐resistant genotypes. Our results show that SM disease is a major selection pressure for the generation of genetic diversity among annual bluegrass biotypes that evolved on golf greens in northern climates. SRAP polymorphisms between bulked genotypes with contrasting resistance to SM were identified and could be used as markers for SM resistance in annual bluegrass.
Mesorhizobium sp. strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation. They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated. These unusual substitutions may contribute to the peculiar host range of N33. Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria. Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.
A glutamyl-tRNA synthetase has been purified to homogeneity from Rhizobium meliloti, using reversed-phase chromatography as the last step. Amino acid sequencing of the amino-terminal region of the enzyme indicates that it contains a single polypeptide, whose molecular weight is about 54 000, as judged by SDS–gel electrophoresis. The primary structures of the amino-terminus region and of an internal peptide obtained by cleavage of the enzyme with CNBr have similarities of 58 and 48% with regions of the glutamyl-tRNA sythetase of Escherichia coli; these are thought to be involved in the binding of ATP and tRNA, respectively. The small amount of glutamyl-tRNA synthetase present in R. meliloti is consistent with the metabolic regulation of the biosynthesis of many aminoacyl-tRNA synthetases.Key words: glutamyl-tRNA synthetase, Rhizobium meliloti, purification, reverse-phase chromatography, amino acid sequence.
During harvest and post‐harvest handling, alfalfa ( Medicago sativa L.) forage undergoes metabolic changes that result in a rapid, significant loss of nutritional quality, especially in protein content. In the present work, the hypothesis was raised that these changes might be initiated by the onset of specific metabolic changes under the control of de novo gene expression. Changes in the population of translatable poly(A) + RNA from alfalfa leaves after harvest were monitored by in vitro translation followed by a two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) analysis of translation products. In a first experiment, which involved plants grown in a growth chamber, the disappearance of 34 of about 120 translation products present before harvest and the appearance of 37 new ones were observed. Comparison of harvest stress with heat shock, cold, and water deficit, revealed that 14 of the 37 translation products increased specifically after harvest, whereas most of the decreasing polypeptides were common to all treatments. In a second experiment, alfalfa was grown and harvested in open field conditions. Among the 40 polypeptides found to increase after harvest, 23 were common with those induced in the first experiment. These results suggest that harvest leads to both specific and non‐specific stress responses within plant cells, that result not only in the disappearance of many mRNA species, but also in the de novo expression of several mRNAs. Further characterization of genes whose expression is specifically induced during post‐harvest could provide tools for the study of post‐harvest metabolism and its modification by genetic engineering.
The environmental impact of transgenic plants has been questioned due to the potential persistence of proteins encoded by transgenes and horizontal gene transfer from the plant to gut microbes. The outcome of the encoded Bt [cry1A(b)] protein and transgene fragments (CaMV-35S, cry1A(b), bar, and bla) was monitored in silage and processed grains of two commercial Bt11 (NK N44-P4 Bt LL and NK N27-M3 Bt LL) and one Bt176 (Elite N09-K9 Bt) corn hybrids. The three Bt-corn hybrids with their non-Bt isolines were field-grown in four replicates. Seven 1.4-kg capacity mini-silos were prepared per plot and then opened after 1, 2, 4, 8, 16, 32, and 64 d. For each hybrid, two 500-kg plastic bag silos were also prepared, sampled after 30 and 198 d, and used in a feeding trial. In the mini-silos, the transgene fragments could no longer be amplified 32 d after ensiling. In the 500-kg plastic bag silos, the transgene fragments were still detectable in the three Bt hybrid silages 30 and 198 d after ensiling. At ensiling, the Bt protein concentration was 2.69, 4.11, and 0.83 µg g -1 DM for the Bt hybrids N44-P4, N27-M3, and N09-K9, respectively. After 64 d of fermentation in the mini-silos, the concentration was 4, 2, and 1% of the initial concentration, respectively, but was 16, 9, and 9% after 198 d in the 500-kg silos. After stringent processing treatments, transgene fragments, except the bla gene fragment, could still be amplified from corn grains. The Bt protein concentration in corn grain of the three Bt hybrids (initially 61, 239, and 21 ng g -1 DM, respectively) decreased by 35, 74, and 67%, respectively, after micronization, by 92, 98, and 89% after extrusion, and by 100% after flaking. After 7 d of feeding Bt corn silage to six dairy cows, Bt protein increased in ruminal digesta with higher concentrations for Bt11 hybrids than the Bt176 hybrid; in all cases, however, concentrations were very low in forage digesta and below detection levels in rumen fluid. Key words: Transgenic maize, silage, processed grains, genetically modified plants, Bacillus thuringiensis endotoxin
