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The effect of viral infection on glycosidases activity in tobacco plants (Nicotiana tabacum L.)
Kloudová, Kateřina ; Tichá, Marie (referee) ; Ryšlavá, Helena (advisor)
Glycosidases are widely distributed among microorganisms and fungi, in plants and animals, too. They belong to the hydrolase class, which means that they hydrolyze glycosidic linkages in oligosacharides, polysacharides and other glycokonjugates, as for example glycoproteins, glycolipids or some alkaloids. The role of plant glycosidases has not been made clear till now, so that it is a subject of many studies as well as their characterization. The studied functions are the role during seed germination, during fruit ripening and softening and in defence against biotic and abiotic stress, too. Under biotic-stress, plants synthesize so-called pathogenesis-related proteins (PRP). It was found out that chitinases and β-1,3-glucanaces, which are endoglycosidases, act as PRPs in tobacco leaves. The goal of my work was to find out whether exoglycosidases (α- galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-hexosaminidase and α- mannosidase) can also act in defence against biotic stress. I studied the effect of infection in tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) leaves caused by Potato virus Y (PVY) on glycosidases activity. I examined the activity in control and infected tobacco leaves, in both cases the activity of soluble and membrane associated glycosidases was examined. I...
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Study of β-N-acetylhexosaminidase in tobacco plants
Valenta, Robert ; Tichá, Marie (referee) ; Ryšlavá, Helena (advisor)
β-N-acetylhexosaminidases are ubiquitous in all living organisms, but there is little information about these enzymes in plants, especially in leaves. Suggested functions of plant β-N-acetylhexosaminidases are participation in defence responses against fungal and other pathogens and also in degradation of storage glycoproteins. β-N-acetylhexosaminidase from tobacco leaves (Nicotiana tabacum L.) was purified to final specific activity 190 nmol min-1 mg-1 with p-NP-GlcNAc as substrate. Precipitation by ammonium sulphate, gel filtration chromatography on Sephacryl S-300 column and affinity chromatography on Con A- Sepharose column were used. Michaelis constant and maximal reaction rate of β-N- acetylhexosaminidases determined for p-NP-GlcNAc was 0.33 mM and 414 nmol min-1 mg-1 , respectively. The cleavage of diacetylchitobiose catalyzed by β-N-acetylhexosaminidase was studied using capillary electrophoresis. Determined activity of β-N-acetylhexosaminidase for diacetylchitobiose was more than 10-times lower compared to β-N-acetylhexosaminidase activity for p-NP-GlcNAc. These results indicate that chitobiose and probably other chitooligomers are not natural substrates of β-N-acetylhexosaminidase and thus this enzyme is most likely not involved in protection against fungal pathogens. Natural substrate...
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Metabolic changes in tobacco plants grown in the presence of sucrose
Garčeková, Květa ; Ryšlavá, Helena (advisor) ; Folwarczna, Jitka (referee)
Metabolism of plants grown in vitro is affected by the composition of culture medium. Beside essential nutrients culture medium are often composed of sucrose as well. The aim of this study was to find out, if additional source of carbon-sucrose affected activity of phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), NADP-malic enzyme (NADP-ME, EC 1.1.1.40), and pyruvate, phosphate dikinase (PPDK, EC 2.7.9.1). Activities of these enzymes were determined in different parts of tobacco plants (Nicotiana tabacum L., cv. Petit Havana SR1) grown in vitro in culture medium: Murashige-Skoog agar containing sucrose (SR1+S) and in plants grown without sucrose (SR1-S). Fresh weight activity and specific activity of studied enzymes in SR1+S plants were increased. In upper leaves fresh weight activity of PEPC, NADP-ME and PPDK was increased 1.7- times, 1.4-times and 1.9-times, respectively. Also the protein content was higher in SR1+S plants. The results indicate that metabolic pathways catalyzed by these enzymes are in the presence of sucrose more involved. Key words: Nicotiana tabacum, L., NADP-ME, PEPC, PPDK, sucrose
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Influence of sucrose in culture medium on phosphoenolpyruvate carboxylase activity in tobacco leaves
Fiala, Martin ; Ryšlavá, Helena (advisor) ; Hoffmeisterová, Hana (referee)
Plants are under physiological conditions autotrophic organisms that are capable to fix CO2 for the synthesis of carbohydrates. Metabolism of plants may be affected by presence of sucrose in the culture medium. The aim of this study was to determine whether activity of phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) and nitrate reductase (EC 1.7.1.1, NR) were affected by the presence of sucrose in the culture medium. PEPC plays important role in photosynthesis of C4 and CAM plants; in C3 plants PEPC plays an anaplerotic role by replenishing of citric acid cycle intermediates and providing carbon skeletons to support nitrogen assimilation. PEPC activity in plants grown in culture medium with sucrose was about 70 % higher than in plants grown without sucrose. Level of phosphorylation of PEPC was studied by detemnination of PEPC activity after alkaline phosphatase treatment, activation of PEPC by D-glucose-6-phosphate and inhibition of PEPC by L-malate. PEPC activity after dephosphorylation in both groups of plants was decreased at similar level. Also activation of PEPC by D-glucose-6-phosphate and inhibition of PEPC by L-malate was very similar in plants grown with sucrose and without sucrose. NR is a key enzyme in nitrogen fixation. Activity of NR in plants grown with sucrose was about 90 %...
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Effect of drought on the metabolism of tobacco plants
Miedzińska, Lucia ; Ryšlavá, Helena (advisor) ; Vaňková, Radomíra (referee)
EEffffeecctt ooff ddrroouugghhtt oonn tthhee mmeettaabboolliissmm ooff ttaabbaaccccoo ppllaannttss (Nicotiana tabacum L.) Diploma thesis - abstract Lucia Miedzińska, Bc. Work Supervisor: Doc. RNDr. Helena Ryšlavá, CSc. Consultant: RNDr. Veronika Doubnerová, PhD. Drought stress is one of the most frequent form of plant stress, which occurs not only under condition of limited water availability, but also under reduced ability to accept water by roots, for example in salted or cold soils. In this thesis the changes in enzyme activities of: NADP-malic enzyme (EC 1.1.1.40; NADP-ME), phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) and pyruvate, phosphate dikinase (EC 2.7.9.1; PPDK) in tobacco plants (Nicotiana tabacum L., cv. W38) after drought were investigated. Enzyme activities in tobacco leaves were significantly increased during 11 days of stress, PEPC 2-fold, PPDK 3,3- fold and NADP-ME 4-fold compared to control plants. The regulation of NADP-ME and PEPC activities were studied on transcriptional level - by the real-time PCR method and on translational level - immunochemically. The expression of NADP-ME protein and transcription of mRNA for chloroplast NADP-ME isoform were increased, but mRNA for cytosolic isoform was not affected. The protein expression of PEPC was slightly increased, transcription of...
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New types of magnetic sorbents for phosphoprotein analysis
Emmerová, Tereza ; Ryšlavá, Helena (referee) ; Kučerová, Zdenka (advisor)
The method for the study of protein phoshorylation sites was elaborated. This method is based on the IMAC separation of phosphopeptides from protein proteolytic digests using new magnetic sorbents and on their subsequent identification by mass spectrometry (MS). Magnetic non-porous hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) particles prepared by the dispersion polymerization and modified with iminodiacetic acid (IDA) with immobilized Fe(III) and Ga(III) ions were employed for the enrichment of phosphopeptides from the proteolytic digests of two model proteins, porcine pepsin A and bovine α-casein. The optimum conditions for phosphopeptide adsorption and desorption in both cases were investigated and compared. The phosphopeptides separated from both proteolytic digests were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS. For the immunochemical separation of phosphoproteins, protein fraction containing antibodies was obtained from egg yolk of hens immunized with O-phosphoryl-L-serine conjugated to key limpet hemocyanin. Antibodies were purified using affinity chromatography on immobilized α-casein and their presence was proven by MS. Specificity of the obtained antibodies was examined using ELISA tests. Obtained results showed, that specificity...
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