Věra Hadačová

Quantitative determination of starch, amylose, and amylopectin in plant tissues using glass fiber paper

Methods for accurate and rapid determination of starch, amylose, and amylopectin in plant tissues are described. They are based on simplified extraction of starch with 32% perchloric acid and selective retention of the starch-iodine complex on a glass fiber disk (Whatman GF/A). The starch on the disk is dissolved in 0.75 M sulfuric acid and estimated with phenol. For amylose and amylopectin determination the starch on the disk is dissolved in perchloric acid, precipitated with ethanol, and retained on a 10-cm glass fiber strip. Both polysaccharides are separated by a chromatographic procedure involving development of the strip in a mixture of ethanol and dimethyl sulfoxide and in dimethyl sulfoxide. The strip is washed in ethanol and stained with iodine or used for polysaccharide quantitation. As little as 5 micrograms of starch or its components present in different amounts of plant material can be estimated.

Regulation of Glutamate Dehydrogenase and Nitrate Reductase Levels in Excised Pea Roots by Exogeneously Supplied Sugar

Summary The activities of NADH 2 dependent glutamate dehydrogenase (GDH) and nitrate reductase were followed in excised pea roots cultured in liquid medium with or without sucrose. Whilst the persistance of nitrate reductase activity in excised pea roots depends directly on the presence of sugar in the medium and whilst in its absence nitrate reductase activity drops to the initial value before the induction with nitrate, both NADH 9 dependent GDH and NADPH2 dependent GDH activities rise in excised pea roots in the absence of sugar in the medium, and vice versa, their activities are maintained at approximately the same level as in corresponding parts of roots on intact seedlings in the presence of sugar in the medium. The increase in NADH 2 dependent GDH activity is inhibited by actidione which indicates that de novo synthesis of the enzyme is probably responsible for the increase. Glucose and fructose are in their effects equivalent to sucrose. The omission of sugar from the medium causes also a change in the ratio of the activities of individual isoenzymes of NAD + dependent GDH. Physiological concentrations of ammonium and nitrate ions neither influence GDH level in the presence of sugar nor in the absence of it. The above mentioned sugars are apparently involved in the regulation of these enzymes directly and not indirectly by means of their metabolites formed during respiration. This is indicated by the results obtained in the experiments with exogenously supplied metabolites of glycolysis, pentose phosphate cycle and Krebs cycle, which did not show any significant effect on GDH and nitrate reductase levels. The increase in GDH level observed after the addition of sublethal concentrations of some respiratory inhibitors to the medium containing sugar was probably caused by decreased sugar uptake and transport. The regulation of GDH level controlled by sugars is in isolated pea roots independent of some other regulatory mechanisms (controlled by acids, nitrite and hydroxylamine), and vice versa. On the other hand, the results of our experiments indicate that the increase in the level of NADH 2 dependent GDH observed by other authors in the roots of whole intact plants after their exposure to high concentrations of ammonium salts, may be, at least partly, caused indirectly by changes in the level of sugars in these roots.

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.

Comparison of esterase isoenzyme patterns in seeds of someAllium species and in cultivars ofAllium cepa L

Esterase isoenzyme patterns were studied in seeds of 6 cultivars ofAllium cepa L. and of14 species ofAllium, namelyAllium aflatunense B. Fedtsch.,A. altaicum (Pall.) Reyse,A. Cristophii Trautv.,A. fistulosum L.,A. jajlae Vved.,A. Karsianum Fom.,A. nutans L.,A. porrum L. cv. Gigant,A. praemixtum Vved.,A. pskemense Vved.,A. ramosum L.,A.rotundum L.,A. schoenoprasum L.,A. stipitatum Regel. The cultivars differ in their isoenzyme patterns, the cultivar Kaštická stands apart from all the other cultivars, probably due to the high alkalinity of its seed extract. The examined species, arranged according to their mutual similarity of isoenzyme patterns, form several groups corresponding to individual sections of the genus. Our results corroborate the recognizing of the sectionCepa and the subsectionPhyllodolon. The maintaining ofA. jajlae andA. rotundum as well described species fits better with our results, than degrading them to subspecies ofA. scorodoprason.Our results agree in main features with those obtained by the immunological method. Some few differences appear concerning individual species. It is evident that esterase isoenzyme patterns can be used in chemotaxonomy ofAllium on an infrageneric level, and, at least inA. cepa, also on an infraspecific level.

Electrophoretic investigation of proteins in different root zones ofvicia faba L.

The differentiation of tissues is closely connected with the proteosynthesis. One can therfore assume that tissues with different types of cell growth (meristematic or elongation growth) and with different degrees of differentiation are different in their protein composition. In order to compare the protein composition of different plant organs, the method of disc electrophoresis on a polyacrylamide gel has been used by some authors. As compared with other methods used up to now, e. g. isolating proteins on DEAE cellulose or in Sephadex, this method does not need so much material and its resolution ability seems to be higher. It is also quicker and enables the study of several samples simultaneously. Its disadvantage is that proteins can be identified mainly by means of Rf and their quantity, measured from the intensity of staining of individual fractions in the gel, which may be misleading due to different sorption capacity of different proteins (FričFričová 1967). None the less, it is good for comparison of protein composition of individual parts of the plant body.Different methods have been used to compare protein composition of individual growth zones in roots.Barsky,Ivanov andPushakova (1965) used luminiscence microscopy and found that in maize roots it is not possible to find substantial differences by this method.Morgan andReith (1954) arrived at similar conclusions. On the other hand,Steward et al. (1965) andMorris (1966) found qualitative differences in protein composition of different parts of pea roots using acrylamide electrophoresis. The results of the last named authors show considerable discrepancies in details, due perhaps to a different method of extraction (buffer, pH, purifying method).We have used acrylamide gel electrophoresis for investigating proteins in precisely defined growth zones of theVicia faba root.AbstractMetodou akrylamidově elektroforesy nebyly zjištěny v meristematické, prodlužovací zoně a v zoně s ukončeným prodlužovacím růstem buněk podstatné kvalitativní rozdíly v bílkovinném složení. Ani kvantitativní rozdíly nebyly výrazné.Je diskutována shoda většiny bίlkovinných frakcí kořene bobu koňského (Vicie faba) a hrachu (Pisum sativum).AbstractЭлсктрофорезом в акриламиде не было найдсно пиких сушествснных различий в качственном состаяе белков между образцом из меристематической зоны удлинения и образцом из зопы с законченым удлинением клсток корняVicia faba L. Ра3личия в количсствеппом составе не были бостсвсрны. Обсуждается соответствие большинетва Rf белковых фраклий у корня копских бобов (Vicia faba L.) и гороха (pisum sativum)

A contribution to the standardization of methods for the preparation of seed proteins ofAllium cepa L.

The study of certain conditions for the extraction of seed proteins ofAllium cepa revealed that the best extractibility of proteins is obtained by the use of a buffered physiological solution at 20 °C in comparison with TRIS-glycine buffer at 5 °C. Using potassium phosphate buffer with 0.01 M mercaptoethanol and 0.4 M NaCl, an amount of proteins by up to 25 per cent higher passes into solution as compared with the physiological solution, but these extracts are unsuitable for the electrophoretic separation in polyacrylamide gels. The defatting of the seed meal under low temperature did not affect the qualitative composition of the protein complex studied, the addition of a 1 per cent soluble starch to the polyacrylamide gel. improved its resolution.

Use of esterase isoenzymes revealed by gel isoelectric focusing as an aid in chemotaxonomical study of the genusAllium

Eleven species of the genusAllium belonging to the sectionCepa, Phyllodolon, Rhizirideum, Melanocrommyum andAlliun ware investigated as to the presence of esterase isoenzymes by means of the gal isoelectric focusing which shows better resolution than polyaorylamide gel electrophoresis. In all examined species twenty six isoenzymes were found. The individual sections are well characterized by esterase isoenzymes revealed by this method, on the other hand, the differences, as shown by the Jaceard index, between the subganera are insignificant. Investigation of seven cultivars ofAllium cepa shows the isolated position of the cultivar Kaštická. These results are in full agreement with those found with use of the polyacrylamide gel electrophoresis and show the usefulness of gel isoelectric focusing for the solution of the chemotaxonomical problems.

The screening of the enzyme and isoenzyme patterns in seeds ofAllium cepa L.

The screening of enzyme patterns in seeds ofAllium cepa cv. Všetatská revealed the presence of the following enzymes: alcohol dehydrogenase, lactate dehyd ogenase, NAD+- and NADP+-glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase NAD+- and NADP+-malate dehydrogenase, NADH2- and NADPH2-tetrazolium reductase catalase, Superoxide dismutase, acid and alkaline phosphatase, L-leucine aminopeptidase, glutamate dehydrogenase, non-specific esterase, and cholinesterase. Altogether 17 enzymes were detected in onion seeds, nine of which had more than three isoenzymes, NAD+-malate dehydrogenase had 8, and non-specific esterase 9 isoenzymes. The demonstration of cholinesterase and Superoxide dismutase activities is remarkable.

Comparison of seed proteins of some representatives of the genusPisum from the point of view of their relationship comparison by disc electrophoresis

Ten taxa of the genusPisum were examined by disc electrophoresis in gels according to Davis and to Reisfeldet al. For evaluation of band patterns the Jaccard Index was applied.The results in both types of gels show thatPisum abyssinicum and especiallyP. fulvum have biochemically a relatively isolated position.Pisum elatius and its subspeciescaspicum andpalestinicum form a subgroup withP. cinereum;P. sativum var.zeylanicum and cv. Jupiter form another subgroup withP. syriacum.Our results are in good agreement with the results of Przybylskaet al. (with the exception ofP. cinereum) and also with immunoelectrophoretic analyses performed by Turkováet al. (1980), with the same exception.

Serological comparisons of seed proteins of some representatives of the genusAllium

Results obtained by comparison of similarity of seed protein spectra of 15 species of the genusAllium (in 22 samples) by means of immunochemical methods more or less correspond to the contemporary division of this genus. Species belonging to the sectionCepa (according to Vvedenskiy) andPhyllodolon have very similar spectra, which suggests that they either should be allocated to one sectionCepa or to two subsections (Cepa andPhyllodolon). Species belonging to the subgenusRhizirideum considerably differ in seed proteins. The investigated species of the subgenusMelanocrommyum show the most distinct spectrum of all the other analyzed representatives, less marked differences were found in the case of the representatives of the subgenusAllium.