Ildikó Kerepesi

Frost hardiness depending on carbohydrate changes during cold acclimation in wheat

The effect of cold hardening on the dynamics of frost tolerance and on carbohydrate metabolism was studied in the frost-sensitive Chinese Spring and the frost tolerant Cheyenne genotypes, and in some of the chromosome substitution lines, derived from the crosses of the donor Cheyenne to Chinese Spring. Total water-soluble carbohydrate, glucose, fructose, sucrose and fructan contents were measured in the leaves. Differences in the accumulation of carbohydrates associated with cold tolerance occurred early in response to low temperature. Total water-soluble carbohydrates and total fructan content increased continuously during the cold treatment in all genotypes, resulting in higher contents in tolerant genotypes than in sensitive ones. Their rate of accumulation correlated significantly with the frost tolerance after 19 days of cold treatment. During the cold acclimation, the maximum of fructose accumulation proceeded that of sucrose. Significant correlation was detected between fructose and sucrose content and frost hardiness on the 43rd day of cold treatment. Fructose accumulated to a greater extent in the most tolerant genotypes with a sharp peak on the 35th day of cold hardening, followed by a decrease. In the chromosome substitution lines, the considerable sucrose accumulation started after the 11th day with a maximum on the 43rd day of cold hardening, coinciding with the tolerance test.

The rice Osmyb4 gene enhances tolerance to frost and improves germination under unfavourable conditions in transgenic barley plants

The Osmyb4 rice gene, coding for a transcription factor, proved to be efficient against different abiotic stresses as a trans(cis)gene in several plant species, although the effectiveness was dependent on the host genomic background. Eight barley transgenic lines carrying the rice Osmyb4 gene under the control of the Arabidopsis cold inducible promoter cor15a were produced to test the efficiency of this gene in barley. After a preliminary test, the best performing lines were subjected to freezing at −11°C and −12°C. Frost tolerance was assessed measured the Fv/Fm parameter widely used to indicate the maximum quantum yield of photosystem II photochemistry in the dark adapted state. Three transgenic lines showed significantly increased tolerance. These selected lines were further studied under a complex stress applying cold and hypoxia at germinating stage. In these conditions the three selected transgenic lines outperformed the wild type barley in terms of germination vigour. The transgenic plants also showed a significant modification of their metabolism under cold/hypoxia conditions as demonstrated through the assessment of the activity of key enzymes involved in anoxic stress response. None of the transgenic lines showed dwarfism, just a slight retarded growth. These results provide evidence that the cold dependent expression of Osmyb4 can efficiently improved frost tolerance and germination vigour at low temperature without deleterious effect on plant growth.

Cold acclimation and abscisic acid induced alterations in carbohydrate content in calli of wheat genotypes differing in frost tolerance

The effect of cold and abscisic acid (ABA) treatment on soluble carbohydrate content was compared in callus cultures of wheat genotypes differing in frost tolerance. The effect of 5A chromosome substituted from the frost tolerant <<Cheyenne>> to the sensitive <<Chinese Spring>> on cold-induced carbohydrate accumulation was also determined. Following cold hardening, the increase in sucrose and fructan level in calli of tolerant varieties was significantly higher than those of the sensitive ones. In 5A substitution line higher sucrose and fructan content was detected than in recipient <<Chinese Spring>>. Tendentiously, cold stress caused higher degree of changes in carbohydrate content than the exogenously applied ABA did. Comparing the accumulation pattern of the components of WSC measured in vitro to the previously published in vivo results it can be concluded that in the case of sucrose and fructans it was similar, while for the reducing sugars it was different. The regulatory role of chromosome 5A either in the development of freezing tolerance or carbohydrate accumulation was confirmed in dedifferentiated calli, as well.

Comparison of blood and saliva lactate level after maximum intensity exercise

Several studies have described high correlation of salivary and blood lactate level during exercise. Measuring the effectiveness and intensity of training, lactate concentration in blood, and lately in saliva are used.The aim of our study was to evaluate the correlation between the concentration and timing of salivary and blood lactate level in endurance athletes and non-athletes after a maximal treadmill test, and to identify physiological and biochemical factors affecting these lactate levels.Sixteen volunteers (8 athletes and 8 non-athletes) performed maximal intensity (Astrand) treadmill test. Anthropometric characteristics, body composition and physiological parameters (heart rate, RR-variability) were measured in both studied groups. Blood and whole saliva samples were collected before and 1, 4, 8, 12, 15, 20 min after the exercise test. Lactate level changes were monitored in the two groups and two lactate peaks were registered at different timeperiods in athletes. We found significant correlation between several measured parameters (salivary lactate - total body water, salivary lactate - RR-variability, maximal salivary lactate - maximal heart rate during exercise, salivary- and blood lactate -1 min after exercise test). Stronger correlation was noted between salivary lactate and blood lactate in athletes, than in controls.

H Protein of Bacteriophage 16-3 and RkpM Protein of Sinorhizobium meliloti 41 Are Involved in Phage Adsorption

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.

Genetic Analysis of the rkp-3 Gene Region in Sinorhizobium meliloti 41: rkpY Directs Capsular Polysaccharide Synthesis to KR5 Antigen Production

Rhizobial surface polysaccharides, including capsular polysaccharides (KPS), are involved in symbiotic infection. The rkp-3 locus of Sinorhizobium meliloti 41 is responsible for the production of pseudaminic acid, one of the components of the KR5 antigen, a strain-specific KPS. We have extended the sequence determination and genetic dissection of the rkp-3 region to clarify the structure and function of the rkpY gene and to identify additional rkp genes. Except for rkpY, no other genes were found where mutation affected the KPS structure and symbiosis. These mutants show a unique phenotype producing a low molecular weight polysaccharide (LMW PS). Creating double mutants, we have shown that biosynthesis genes of the KR5 antigen except rkpZ are not necessary for the production of this LMW PS. Polysaccharide analysis of genetically modified strains suggests that rkpY has pleiotropic effects on polysaccharide production. It directs KPS synthesis to the KR5 antigen and influences lipo-oligo 3-deoxy-d-manno-2 octulosonic acid (Kdo) production in S. meliloti 41. In addition, rkpY suppresses the lipo-oligoKdo production when it is introduced into S. meliloti 1021.

Draft genome sequence of an efficient antibiotic-producing industrial strain of Saccharomonospora azurea, SZMC 14600

Although certain rare actinomycetes have been recognized as prolific sources of bioactive natural products, their potential for producing biologically active metabolites still remains unexplored. With the aim of gaining global insights into the genetic background and the metabolic capability of Saccharomonospora azurea SZMC 14600, whole-genome sequencing was performed.

Mapping of Genes Controlling Cold Hardiness on Wheat 5A and its Homologous Chromosomes of Cereals

The analysis of monosomic and substitution lines showed that at least ten of the 21 pairs of chromosomes are involved in the control of frost tolerance and winter hardiness in wheat (Triticum aestivum L.). Of particular importance for adaptation to autumn sowing are the genes for vernalization requirement (Vrn). Major genes influencing frost resistance (Fr1) and vernalization requirement (Vrn1) were localized on the long arm of chromosome 5 A. To map the location of these genes, single chromosome recombinant lines were developed from the cross between substitution lines: Chinese Spring(Cheyenne 5A) (CS(Ch5A)) and CS(Triticum spelta 5A). The RFLP data show that although a close genetic linkage exist between the loci Fr1 and Vrn1, they are, nevertheless, separable. The location of Vrn1 suggests that it is homologous to other spring habit genes in related species, particularly to the Sh2 locus on choromosome 7(5H) of barley and to the Sp1 locus on chromosome 5R of rye. The level of abscisic acid (ABA) in plants increases as a result of water deprivation or low temperatures. Our study on CS(Ch) substitution lines showed that chromosome 5A of the frost resistant Ch increased the ABA content in the frost sensitive CS background following cold treatment. The crown ABA accumulation in several of the 5A recombinant lines subjected to polyethylene glycol induced water stress and revealed a close genetic linkage between both Fr1 and Vrn1 genes and the gene regulating ABA production. Studies on different substitution lines also showed that the 5A chromosome is involved in osmotic stress induced amino acid and polyamine and, moreover, in cold stress induced proline accumulation. A gene regulating cold induced sugar accumulation has also been localized on chromosome 5A. Accordingly, gene(s) involved in os-moregulation could also be localized on the chromosome 5A.

Involvement of Glutathione and Carbohydrate Biosynthesis Moreover COR14B Gene Expression in Wheat Cold Acclimation

The winter-hardiness, including frost tolerance has long been regarded as a trait being under complex multigenic control. However, it would still be possible that major differences in cold-adaptation between species or cultivars depend on allelic differences in a small number of genes as suggested in cold-acclimation in pea and Solanum (Liebefeld et al., 1986; Stone et al., 1993). Since common wheat (Triticum aestivum L.) is a hexaploid its vital genes are replicated. This permitted Sears (1953) to develop series of chromosome substitution lines. By comparing chromosome substitution lines with the parental lines it was possible to determine which chromosomes carry gene locus for freezing tolerance. The analysis of substitution lines showed that at least ten of the 21 pairs of the chromosomes are involved in the control of frost tolerance (Sutka 1981). However, major genes influencing frost tolerance (Fr) and vernalisation requirement (Vrn) were localized on the long arm of 5A and 5D chromosomes (Galiba et al., 1995; Snape et al. 1997). Of particular importance for adaptation to autumn sowing are the genes for vernalisation requirement. Vrn genes determine the needs for cold temperature required for flower development. Recent studies indicated that Vrn1-Fr1 interval on 5A chromosome of wheat has a major effect on freezing tolerance (Storlie et al., 1998) Conservation of gene order (synteny) in Triticeae is well known and this is true for the Vrnl-Fr1 interval studied in barley, rye and Triticum monococcum, as well (reviewed in Galiba et al., 1997a). For example mapping of a quantitative trait loci (QTL) in barley has resulted in the identification of a 21-cM region on chromosome 7 that has a major role in frost tolerance. This region accounted for 32% of the variance in LT50 values and 39–79% of the variance in winter field survival (Hayes et al., 1993).

Proteomic insight into the primycin fermentation process of Saccharomonospora azurea.

Saccharomonospora azurea SZMC 14600 is a member of the family Pseudonocardiaceae exclusively used for industrial scale production of primycin a large 36-membered non-polyene macrolide lactone antibiotic belonging to the polyketide class of natural products. Even though maximum antibiotic yield has been achieved by empirically optimized two-step fermentation process, little is known about the molecular components and mechanisms underlying the efficient antibiotic production. In order to identify differentially expressed proteins (DEPs) between the pre- and main-fermentation stages of primycin, comparative 2D-PAGE experiments were performed. In total, 98 DEP spots were reproducibly detected, out of which four spots were excised from gels, and identified through MALDI-TOF/TOF mass spectrometry. Peptide mass fingerprint analysis revealed peptide matches to HicB antitoxin for the HicAB toxin-antitoxin system (EHK86651), to a nucleoside diphosphate kinase regulator ((Ndk; EHK81899) and two other proteins with unknown function (EHK88946 and EHK86777).