Isoenzymes of lactate dehydrogenase in the root growth zones ofVicia faba L.

The present studies of the LDH isoenzymes have been made as a part of our studies on the respiration systems of the roots ofVicia faba. All the root zones had 5 LDH isoenzymes and 2 antibands, but there were differences in their relative quantity. Cathodic isoenzymes (LDH₅, LDH₄) prevailed in the meristematic zone, whereas the anodic ones (LDH₁) prevailed in the others. The different enzyme activity in individual zones is probably connected with the transition of the anaerobic metabolism in the meristem to the aerobic one in the differentiating parts of the root.     

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 (Fri?Fri?ová 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.     

Contribution to the estimation of malic dehydrogenase isoenzymes in the root growth zones ofVicia faba L.

Highly active NAD-MDH (E.C.1.1.1.37) and low activity of NADP-MDH (E.C.1.1.1.40) were found inVicia faba roots. The NAD-MDH activity is associated with 6 to 12 isoenzymes. The number of isoenzymes is dependent on the extraction (use of Triton X-100etc.) and detection procudures (presence of KCN, phenazine methosulphate). The meristematic zone does not contain one isoenzyme (X) which is present in the other two zones. The meristematic zone, elongation zone and zone with the beginning differentiation differ in their activity of individual isoenzymes.     

Glucose-6-phosphate dehydrogenase isoenzymes in root growth zones ofVicia faba

The specific activity of glucose-6-phosphate dehydrogenase (G-6-PD) in growth zones ofVicia faba roots is increasing with cell maturation and differentiation. Changes in the total activity of G-6-PD are not associated with a change in the number of G-6-PD isoenzymes. Five G-6-PD isoenzymes were found in all root growth zones. Some differences were found in the activity of individual isoenzymes.     

Stimulation of sugar exit from leaf tissues ofVicia faba L.

After removal of the lower epidermis, leaf discs ofVicia faba L. were loaded with either [¹⁴C]sucrose or [³H]3-O-methylglucose (3-O-MeG). The exit of preloaded sucrose was strongly stimulated when sucrose was present in the bathing medium, and the exit of 3-O-MeG was also markedly increased in the presence of 3-O-MeG. This specific stimulation exhibited single saturation dependence on the external concentration of sugar (K _m=9 mM for sucrose, 5 mM for 3-O-MeG), and was sensitive to low temperature, uncouplers and thiol reagents. Sucrose exit was never affected by 3-O-MeG in the bathing medium. Sucrose did not affect the exit of 3-O-MeG in fresh discs, but promoted this exit in discs previously aged for 12 h, indicating partial external hydrolysis of sucrose in the latter tissues. Ageing also dramatically increased the exit of 3-O-MeG induced by 3-O-MeG but had no effect on the exit of sucrose induced by sucrose. The ability of 53 compounds (pentoses, hexoses, hexose-phosphates, polyols, di- and trisaccharides, phenyl- and nitrophenyl-derivatives, sweeteners) to interact with the sucrose carrier and with the hexose carrier was tested. Sucrose, maltose, -phenylglucoside andp-nitrophenyl--glucoside interacted with the sucrose carrier.d-glucose,d-xylose,d-fucose,d-galactose,d-mannose, 3-O-MeG and 2-deoxyglucose interacted with the hexose carrier.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - 3-O-MeG 3-O-methylglucose - PCMBS p-chloromercuribenzenesulphonic acid     

Phloem unloading in developing seeds ofVicia faba L.

Planta - Several characteristics of the process of phloem unloading in the seed coat of developing seeds ofVicia faba L. were investigated. Prior to the procedure of measuring the release of...     

Transfer cell induction in cotyledons ofVicia faba L.

Summary Immediately prior to seed fill, a dermal transfer cell complex, comprised of epidermal and subepidermal cells, differentiates on the abaxial surface of the cotyledons in seed ofVicia faba. Over the period of differentiation of this complex in vivo, the principal sugars of the seed apoplasmic sap change from hexoses, glucose and fructose, to sucrose. Cotyledons were removed from seeds before differentiation of the transfer cell complex and cultured for 6 days on an agar-based medium in the dark with their abaxial surface in contact with a medium containing either 100 mM hexoses (glucose and fructose in equimolar concentrations) or 100 mM sucrose. On both media, cotyledon growth rate was maintained throughout the culture period at, or above, that of in vivo grown cotyledons of equivalent developmental age. When cotyledons were cultured on a medium containing glucose and fructose, epidermal cells of both the ab- and adaxial surfaces developed wall ingrowths on their outer periclinal walls and their cytoplasm became dense, vesicular, and rich in mitochondria. Extensive ingrowth deposition also occurred on walls of the subepidermal cells and several rows of underlying storage cells where they abutted intercellular spaces. This latter ingrowth development was apparent on both cotyledon surfaces, but extended into more of the underlying cell layers on the abaxial surface at the funicular end of the cotyledon. In in vivo grown cotyledons, such ingrowth development is restricted to the subepidermal cells of the abaxial surface. Ingrowth morphology was commensurate with that of transfer cells of in vivo grown cotyledons. In contrast to the observed induction on a medium containing glucose and fructose, cotyledons cultured with sucrose as the sole sugar source exhibited no ingrowth deposition or small wall ingrowths in some abaxial epidermal cells. While the potential sugar signalling mechanism is unknown, this culture system offers an exciting opportunity to explore the molecular biology of transfer cell development.Abbreviations DAA days after anthesis - GC-MS gas chromatography and mass spectrometry - PAR photosynthetically active radiation - RGR relative growth rate - SCM standard culture medium     

Electrophoretic study on peroxidase,indoleacetic acid oxidase,and o-diphenol oxidase fractions in extracts from different growth zones ofvicia faba L. roots

Using electrophoresis in acrylamide gel, fractions of peroxidase, indoleacetic acid oxidase, and o-diphenol oxidase were investigated in extracts from three growth zones ofVicia faba L. roots. Three peroxidase fractions (zones) moving towards the anode were revealed as well as four peroxidase fractions (zones) migrating towards the cathode. Three peroxidase fractions showed detectable indoleacetic acid oxidase activity. The o-diphenol oxidase activity was revealed in all peroxidase fractions moving towards the anode, in those moving towards the cathode the o-diphenol oxidase activity differred according to the substrate used. One fraction with both peroxidase and o-diphenol oxidase activity occurred only in electrophoreograms of extracts from the maturation zone; in this fraction no indoleacetic acid oxidase activity was demonstrable.     

Developmental aspects of long-term callus culture ofVicia faba L.

Summary Vicia faba callus line (VFS 1), isolated from expiants of immature embryo, grew satisfactorily onMurashige andSkoog complete medium with 1.38 M 2,4-D, or with 0.92 M 2,4-D to which 1.0 M kinetin was added. It also grew well on the B 5 modified medium containing 2.3 M 2,4-D and 25.0 M kinetin. On the last of these media the cultures grew more uniformly and without necrosis. They also showed diminishing variation in polyploidy in favour of diploids and corresponding aneuploids (hypodiploids).After being cultured for nearly three years on MS containing 1.38 M 2,4-D, 8–33% of cultures of VFS 1 were able to regenerate roots when transferred to either MS half strength with 5.37 M NAA, or to a medium without 2,4-D, or else to media with the addition of kinetin only (in various concentrations).     

Microtubule dynamics are involved in stomatal movement ofVicia faba L.

Summary To obtain a full picture of microtubule (MT) behavior during the opening and closure of guard cells we have microinjected living guard cells ofVicia faba with fluorescent tubulin, examined fine detail by freeze shattering fixed cells, and used drug treatments to confirm aspects of MT dynamics. Cortical MTs in fully opened guard cells are transversely oriented from the ventral wall to the dorsal wall. When the stomatal aperture was decreased by darkness, these MTs became twisted and patched and broken down into diffuse fragments when stomata were closed. When the closed stomata were opened in response to light, the MTs in guard cells changed from the diffused, transitional pattern back to one in which MTs are transversely oriented from stomatal pore to dorsal wall. This observation indicates a linkage between these MT changes and stomatal movement. To confirm this, we used the MT-stabilizing agent taxol and the MT-depolymerizing herbicide oryzalin and observed their effects on the stomatal aperture and MT dynamics. Both drugs suppressed light-induced stomatal opening and dark-induced closure. MTs are known to be necessary for maintaining the static kidney shape of guard cells; the present data now show that the dynamic properties of polymeric tubulin accompany changes in shape with stomatal movement and may be functionally involved in stomatal movement.     

Influence of exogenous DNA on Ypenyl-treated chromosomes ofVicia faba L.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0·01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.     

A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.

A new method is described for the isolation of large quantities of Vicia faba metaphase chromosomes. Roots were treated with 2.5 mM hydroxyurea for 18 h to accumulate meristem tip cells at the G₁/S interface. After release from the block, the cells re-entered the cell cycle with a high degree of synchrony. A treatment with 2.5 M amiprophos-methyl (APM) was used to accumulate mitotic cells in metaphase. The highest metaphase index (53.9%) was achieved when, 6 h after the release from the hydroxyurea block, the roots were exposed to APM for 4 h. The chromosomes were released from formaldehyde-fixed root tips by chopping with a scalpel in LB01 lysis buffer. Both the quality and the quantity of isolated chromosomes, examined microscopically and by flow cytometry, depended on the extent of the fixation. The best results were achieved after fixation with 6% formaldehyde for 30 min. Under these conditions, 1 · 10⁶ chromosomes were routinely obtained from 30 root tips. The chromosomes were morphologically intact and suitable both for high-resolution chromosome studies and for flow-cytometric analysis and sorting. After the addition of hexylene glycol, the chromosome suspensions could be stored at 4° C for six months without any signs of deterioration.Abbreviations APM amiprophos-methyl - DAPI 4,6-diamidino-2-phenylindole The authors thank Mrs. Jiina Eliáová for her excellent technical assistance and Dr. Slavomir Ondro for the supply of V. faba seeds. A gift sample of APM from the Mobay Corporation (Agricultural Chemicals Division, Kansas City, Mo., USA) is gratefully acknowledged.     

Abscisic-acid contents and concentrations in protoplasts from guard cells and mesophyll cells ofVicia faba L.

The abscisic-acid (ABA) contents of isolated guard-cell protoplasts and mesophyll-cell protoplasts fromVicia faba were determined by high-pressure liquid chromatography followed by gas chromatography. The amounts of ABA found immediately after preparation of the protoplasts varied from 90 to 570 amol per guard-cell protoplast, and from 75 to 100 amol per mesophyll-cell protoplast. These contents correspond to concentrations between 36 and 230 mol per liter in guard-cell protoplasts and between 2.7 and 3.3 mol per liter in mesophyll-cell protoplasts. During exposure of protoplasts to betaine concentrations of 0.3, 0.5, and 0.8 mol·l^-1 at 0° and 20°C for 30 min, ABA contents as well as the fractions of ABA that leaked into the medium remained constant for both protoplast types. There was no evidence for net production of ABA in isolated protoplasts subjected to osmotic stress.Abbreviation ABA abscisic acid     

Rapid method for isolation and purification of protoplasts from epidermal tissue ofVicia faba L. leaves

Summary A method has been developed for isolating and purifying epidermal and guard cell protoplasts (ECPs and GCPs) from leaves ofVicia faba L. This method has three essential characteristics: 1) requires only small quantities of initial plant tissue; 2) is rapid, being based on a two-step enzymatic digestion and purification by discontinuous density gradient centrifugation using Percoll, and 3) gives a high viability of purified protoplasts. The procedure yielded about 6% ECPs and 10% GCPs on the basis of their occurrence on epidermal foliar tissue, the final suspension of protoplasts being 100% pure. Cell viability was assessed by the ability of each protoplast type to accumulate neutral red in their vacuoles. Values of 90% and 95% were obtained for ECPs and GCPs respectively. The complete lack of cell wall after enzymatic treatment was checked at the light microscope level by the absence of Calcofluor fluorescent staining of cellulosic material. Representative counts for purified ECPs and GCPs obtained at the interfaces of 20/30% and 40/80% Percoll gradients were 1.32×10⁵ and 3.7×10⁵ elements/ml, which represents a yield of 930 and 2,200 protoplasts/cm² of epidermal tissue respectively. The integrity of the plasma membrane and organelles after the isolation procedures was confirmed by transmission electron microscopy and by the ability of protoplasts to exclude Evans blue.

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Resumen Se ha desarrollado un método para el aislamiento y purifición de protoplastos de células epidérmicas y células guardianas (PCEs PCGs) de hojas deVicia faba L. Este método posee tres características esenciales: 1) solamente requiere pequeñas cantidades de tejido vegetal inicial; 2) rapidez, en base a una digestión enzimática de sólo dos etapas y centrifugación en gradiente discontinuo de Percoll, y 3) la elevada viabilidad de los protoplastos purificados. Este método permitió obtener ca. 6% de PCEs y 10% de PCGs sobre la base de su ocurrencia en el tejido epidérmico foliar, con una pureza del 100% para las suspensiones finales de protoplastos. Se determinó la viabilidad de cada tipo celular por su habilidad de acumular rojo neutro en sus vacuolas, obteniéndose valores de 90% y 95% para PCEs y PCGs respectivamente. Se determinó la ausencia total de pared celular después del tratamiento enzimático mediante microscopía de fluorescencia con Calcofluor, específico para sustancias celulósicas. El recuento de PCEs y PCGs purificados — obtenidos en las interfases 20/30% y 40/80% del gradiente de Percoll — fue de 1,32×10⁵ y 3,7×10⁵ elementos/ml, lo cual representó un rendimiento de 930 y 2200 protoplastosi cm² de tejido epidérmico respectivamente. La integridad de la membrana plasmática y de las organelas fue confirmada por microscopía electrónica de transmisión y por la habilidad de los protoplastos de excluir azul de Evans.

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Résumé Une méthode a été développée pour l'isolement et la purification de proto-plastes épidermiques (ECPs) et de cellules de garde (GCPs) de feuilles deVicia faba L. Cette méthode présente trois caractéristiques essentielles: 1) Elle ne requiert que de petites quantités du tissu végétal originel; 2) elle est rapide, car elle se base sur une digestion enzymatique en deux étapes et une purification dans un gradient discontinu de densité utilisant le Percoll, et 3) elle fournit une forte proportion de protoplastes prufiés bien vivants. La procédure fournit environ 6% d'ECPs et 10% de GCPs sur la base de leur présence dans le tissue foliaire épidermique et d'une suspension pure à 100% de protoplastes. La viabilité des cellules a été testée par la capacité de chaque type de protoplaste d'accumuler de rouge neutre dans ses vacuoles. On a obtenu des valeurs de 90% et de 95% respectivement pour les ECPs et les GCPs. L'absence totale de paroi cellulaire après le traitement enzymatique a été vérifiée au microscope optique par l'absence de fluorescence après coloration du matériel cellulosique par le calcofluor. Des comptages typiques pour les ECPs et les GCPs purifiés obtenus aux interfaces 20/30% et 40/80% des gradients de Percoll ont donné 1.32×10⁵ et 3.7×10⁵ éléments/ml, ce qui représente des rendements respectifs de 930 et de 2200 protoplastes par cm² de tissue épidermique. L'intégrité de la membrane plasmique et des organites après les procédures d'isolement a été confirmée par microscopie électronique à transmission et par la papacité de protoplastes d'exclure le bleu d'Evans.

     

Oxidation of ethylene by cotyledon extracts from Vicia faba L.

Improved rates of ethylene oxidation by cell-free preparations from cotyledons of Vicia faba L. have been obtained using cryogenic storage techniques and by developing a method for the hydrolysis of ethylene oxide. Gel permeation chromatography showed that a low-molecular-size fraction was required for activity; accordingly, the kinetics of ethylene oxidation in the presence of this fraction were studied. Reduced pyridine nucleotides could substitute for the low-molecular-size fraction. Activity under a nitrogen atmosphere was 60% lower than in air. The need for reduced nicotinamide adenine dinucleotide phosphate (NADPH) and oxygen indicated that the enzyme might be a mixed-function oxidase. Using sufficient NADPH to approach saturation, the apparent Michaelis constant (K _m) for ethylene was 1.94±0.38 · 10^-8 M (aqueous phase), and when ethylene was saturating, the K _m for NADPH was 3.7 · 10^-5 M. Carbon monoxide was found to inhibit by competing with ethylene, and the inhibitor constant was 5.97 · 10^-7 M in solution. In the presence of excess ethylene and NADPH, activity was highest in phosphate-buffered medium pH 7.9. The bulk of the activity was found in a microsomal fraction.Abbreviations Epps N-2-hydroxyethylpiperazine-N-3-propane sulphinic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-porpanediol     

The nitrogen fixing potential ofVicia faba rhizobia (R. leguminosarum) from different agricultural locations

Summary The size and symbiotic effectiveness, withVicia faba, ofRhizobium leguminosarum populations from five locations in southern Britain has been estimated. Population numbers varied from 4.54×10³ to 1.69×10⁵. Nitrogen fixing potential differed by up to 30%. The implications of the results for improving the productivity of field beans are discussed.     

Stomatal responses to ABA and IAA in isolated epidermal strips ofVicia faba L.

Epidermal strips from well-watered faba-bean plants were subjected to a range of abscisic acid (ABA) and indolyl-3-acetic acid (IAA) concentrations (10^-5 to 1 mM) in the presence or absence of CO₂ in light or dark. ABA had inhibitory effect on abaxial stomatal apertures in all the concentrations studied and retained them closed even after addition of KCl (SO and 100 mM) to the incubation medium. It also influenced stomatal responses to CO₂. In the presence of CO₂ apertures were greater than in its absence in light as well as in darkness. This relationship remained unchanged also after addition of KCl. The action of ABA inhibited accumulation of potassium in the guard cells. IAA stimulated stomatal opening and its effect was quite opposite to ABA; in the presence of CO₂ the apertures were smaller than in its absence. IAA, however, was able to inhibit the closing effect of darkness, CO₂, and ABA, and stimulated potassium accumulation in the guard cells. Simultaneous action of ABA+IAA manifested effects of both substances.     

The extent and differences in mitotic activity of the root tip ofVicia faba L.

Mitotic activity does not stop for different meristematic cells of the root apex at the same distance from the initials. The differences are connected with the functional heterogeneity of the apical meristem of the root. The arrangement of vascular bundles,i.e. the alternation of independent xylem and phloem groups, is of major importance. In broad bean roots, the protophloem sieve elements stop dividing first. The centre of the stelei. e. late metaxylem elements stop dividing next. Division in the stele gradually ceases centrifugally, while it ceases centripetally in the peripheral part of the root. The cylindrical region with prolonged cell division includes internal layers of the cortex including endodermis, pericycle and adjoining cells of the stele. Proximally apical meristem is reduced to isolated strands of cells adjacent to the protoxylem poles. Pericycle cells stop dividing last at a distance of approx. 9–10 mm from the initials. The number of the division cycles is limited and is specific for individual cell types. Epidermal and cortical cells divide in broad bean roots transversely approximately seven times, cells of late metaxylem approximately five times. Root apical meristem is an asynchronous cell population with a different duration of the mitotic cycle. We determined local variations in the duration of the mitotic cycle in the apical meristem of broad bean root by means of colchicine-induced polyploidy. The cells of the quiescent centre had the longest mitotic cycle after colchicine treatment. The region of the proper root adjacent to the quiescent centre was mixoploid (2n and 4n). Isolated cells with a long cycle occurred also in the cortex and in the central cylinder. Cells with a division cycle of 18h were found in the root cap, in the epidermis, in the cortex and in the central cylinder. Relatively numerous cells with the shortest division cycle, approx. 12 h, occurred farther of the quiescent centre in the epidermis, in the cortex, in the pericycle, and in adjacent layers of the stele through-out the entire meristematic region. The results derived from the analysis of the apical meristem are discussed in connection with the ontogenesis of different types of cells taking part in the primary structure of the root.