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.     

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.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

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.     

Electrophoretic investigation of ribonuclease in roots and leaves ofVicia faba L.

Isozyme patterns and specific activity of ribonuclease (ribonucleate pyridinenucleotido-2′-transferase, E. C. 2.7.7.16) were followed in the extracts of segments from three growth zones of the root and in extracts of young and senescent leaves ofVicia faba L. Electrophoreograms of extracts from all three investigated root zones were identical, in the electrophoreograms of extracts from senescent leaves however one new ribonuclease occurred which could not be detected in the electrophoreograms of extracts from young leaves. Extracts from senescent leaves had higher specific activity of ribonuclease than extracts from young leaves. Extracts from the enlargement zone of the root and those from the maturation zone had a three times higher specific activity of RNase than extracts from the division zone.     

Histogenesis and localization of non-specific esterase in root tip

A procedure was developed for satisfactory freeze-sectioning of root tips. The use of Ca formol-fixed material kept and frozen in Holt's syrup is recommended. The existence and different localization of 2 fractions of non—specific esterase was verified in root tips ofVicia faba. The same results were revealed in fixed and unfixed material. The dynamics ofin situ reaction was followed with respect to optimal incubation time. The results with substrates of different chain length support the existence of 2 fraction of the studied enzyme, none of which, concerning substrate specificity, is a lipase. It follows from the present studies inVicia faba and other species (Cucurbita pepo, Lupinus albus, Pisum sativum Zea mays), that non-specific esterase localization is not directly given by histogenesis.     

The participation of flavine enzymes in the respiration of root zones of the horse bean (Vicia faba L.).

As part of our studies on respiration systems in root zones of the horse bean (Vicia faba L., cv. Chlumecký) the influence of quininehydrochloride and potassium cyanide on respiration was followed. The inhibition of the oxygen uptake by quininehydrochloride was highest in the elongation zone and lowest in the meristematic zone. Thes shows that the flavine enzyme content is lowest in the meristem, highest in the elongation zone. The inhibition of Fe- and Cu-enzymes by KCN increases from the root tip towards the older parts of the root, but is lower than the inhibition by quininehydrochloride. The difference between the intensity of action of both, applied separately, and of the combined effect of both if compared with the action of cyanide alone, is highest in the elongation zone; this shows that relatively higher amounts of non-metallic flavine enzymes participate in this zone. Their relation to the pentose phosphate cycle is discussed.     

Cytoplasmic Components of the Broad Bean Root Tip Cells

The cytoplasmic components of broad bean (Vicia faba L.) root tip cells were studied light- and electronmicroscopically. Using a light microscope, differences were revealed between cells from the cortex and from the central cylinder. For electron microscope studies the cells near the boundary between the mentioned parts of the root tip were selected at a distance of about 2 mm from the initials. The orientation of objects during embedding made possible fairly accurate localization. No peculiar, strikingly osmiophillic bodies were seen, which could be identified without doubt as osmiophillic platelets. It follows that some of the current cytoplasmic components, perhaps more or less altered were described as osmiophillic platelets. After fixation with KMnO₄, in which case the electronmicrographs are most instructive, the leucoplasts show several inclusions, mostly only partially limited against the matrix; it is not clear whether the true membrane is concerned here. The origin of intravacuolar membraneous formations is discussed.     

Effects of salt stress on nitrogenase activity and growth of four legumes

The effects of salt stress on nitrogenase (N2-ase) activity, growth and nitrogen content ofVicia faba (L.),Medicago sativa (L.) Merrill,Glycine max andVigna sinensis (L.) were investigated. Four levels of salinity were applied and salt treatments were imposed on inoculated and N-fertilized plants.M. sativa tolerated mild levels of salinity but higher salt concentrations depressed N₂-ase activity of this species. The other three legumes were considerably affected by salt treatments, and N₂-ase activity was significantly reduced by salinity. Vicia faba, carrying elongate nodules, could restore a partial N₂-ase activity upon recovery from salt stress whereasG. max andV. sinesis, both with spherical nodules, could not regain significant activity when salinity was removed. Salt stress retarded growth of both inoculated and N-fertilized plants. The nitrogen content of both treatments was also affected by salinity and the effect was more severe for inoculated than N-fertilized plants.     

The effect of maleic hydrazide and exogenous DNA on chromosomes ofVicia faba

The mitotic activity of merstematic cells ofVicia faba, the frequency of chromosomal abnormalities and their interchromosomal distribution are evaluated in dependence on the concentration of maleic hydrazide (MH) and on the recovery period. The influence of exogenous DNA of different genetic origin on the course of repair of primary root cells damaged by MH was also studied. Isologous DNA which exhibited a strong repair effect in authors' previous experiments was quite ineffective in the case of maleic hydrazide. Heterologous DNA, on the other hand, had to some extent a parallel effect with MH in breaking down the structural integrity of chromosomes and increased the frequency of aberrations induced by maleic hydrazide in meristematic cells ofVicia faba.     

Formation of GA20 glucosyl conjugates in seedlings ofVicia faba

[³H]GA₂₀ (1)¹, fed toVicia faba seedlings, was converted to [³H]GA₂₀ glucosyl ester (5) and [³H]GA₂₀-13-0-glucoside (6). The GA₂₀ glucosyl ester (5) was identified by HPLC-RC and by GC-MS of GA₂₀-Me formed by transesterification of (5). The [³H]GA₂₀-Me was crystallized to constant specific radioactivity with authentic GA₂₀-Me. On HPLC-RC the GA₂₀-13-0-glucoside (6) was shown to have the same retention time as an authentic sample. Subsequent enzymic hydrolysis gave a product with an HPLC retention time identical to that of authentic GA₂₀ (1).     

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.     

The presence and distribution of α and β glucosidase in root tip

A comparison was made of post and simultaneous azocoupling procedures for β glucosidase localization on unfixed and fixed root tips ofZea mays. Using this object, more detailed studies of β glucosidase distribution were undertaken, concerning the time course of enzyme reaction, its pH dependence, the effect of various buffers, the comparison of several diazonium salts and the use of different naphtholic substrates. The indigogenic reaction was also applied. Attempts were made by means of azocoupling procedures to localize a and β glucosidase in root tips ofCucurbita pepo, Lupinus albus, Piswm sativum andVicia faba in comparison withZea mays. In addition to certain technical problems, the questions of constitutive and adaptive enzymes, sugar distribution and histogenesis versus function are discussed in relation to the presence and distribution of ß glucosidase in the studied objects.     

Cytogenetic effect of radiation products of cytosine: Parabanic and oxaluric acids

The two final products of radiolytic degradation of cytosinei.e. parabanic and oxaluric acids were investigated as regards their cytogenetic effects. Broad bean (Vicia faba L.) root meristem was used as experimental material. While the oxaluric acid was not able to induce chromosomal aberrations to a greater extent, the parabanic acid acts as a clastogenic compound. When applied on a resting broad bean root meristem at concentration from 10^-3 to 10^-2 M it induces chromosome and chromatid aberrations, especially breakages. Their frequency reaches 9–12% at the highest tested concentration. The same concentrations of the parabanic acid increases mitotic index of the first cell generation of primary root.     

Position and extent of the elongation zone in the root tip of the broad beanVicia faba L.

It was found by measuring the length of the cortex cells of the root tips of the broad beanVicia faba L. that the beginning of the elongation zone lies at about 1–2 mm from the initials and its end at about 7–8 mm from the initials. Shrinkage of the object during microtechnical treatment was negligible. The autonomy of the individual tissues of the root tip was taken into account.     

Studies on the rhizosphere mycoflora of broad bean and cotton

Investigations were made on the effect of species of fungi isolated from therhizosphere of broad bean (Vicia faba Linn.) variety Giza I, and cotton (Gossypium barbadense Linn.) variety Giza 47, on plant growth. Broad bean rhizosphere fungi differently affected plant growth.Fusarium moniliforme, Penicillium martensii, Rhizopus stolonifer, andCladosporium sphaerospermum stimulated both root and shoot growth.Aspergillus niger andAlternaria tenuis have an inhibitory effect on plant growth. On the other hand, the rhizosphere fungi of cotton namely,Penicillium cyclopium, Aspergillus terreus, Cephalosporium sp.,Aspergillus niger, Cladosporium sphaerospermum, andFusarium oxysporum stimulated plant growth.     

Seed-Specific Expression of AINTEGUMENTA in Medicago truncatula Led to the Production of Larger Seeds and Improved Seed Germination

The increase of seed size is of great interest in Medicago spp., to improve germination, seedling vigour and, consequently, early forage yield as well as for optimizing seeding techniques and post-seeding management. This study evaluated the effects of the ectopic expression of the AINTEGUMENTA (ANT) cDNA from Arabidopsis thaliana, under the control of the seed-specific USP promoter from Vicia faba, on seed size, germination and seedling growth in barrel medic (Medicago truncatula Gaertn.). All the transgenic T₂ barrel medic lines expressing ANT produced seeds significantly larger than those of control plants. Microscopic analysis on transgenic T₃ mature seeds revealed that cotyledon storage parenchyma cells were significantly larger and contained larger storage vacuoles than those of the untransformed control. Moreover, the percentage of germination was significantly higher and germination was more rapid in transgenic than in control seeds. Our results indicate that the seed-specific expression of ANT in barrel medic led to larger seeds and improved seed germination, and revealed a regulatory role for ANT in controlling seed size development.     

Effect of the herbicide terbutryn on meiosis,yield and mitotic chromosomes in C2 plants ofVicia faba L.

The herbicide terbutryn induced up to 11.3 % chromosomal abnormalities in cells undergoing meiosis inVicia faba. The mean number of pods/plant and the mean number of seeds/pod were considerably reduced, but the average seed dry mass was not or was only slightly affected by this herbicide. In C₂ plants chromosomal abnormalities were observed in few but significant numbers of cells.     

Ethylene-enhanced ethylene oxidation inVicia faba

The alkene oxygenase (AO) of fababean (Vicia faba L.) converts ethylene to ethylene oxide. Treatment of fababeans with 10μl/liter ethylene increases the activity of this enzyme within 2 hours of ethylene treatment. Though other alkenes were taken up by fababean seedlings, ethylene was the most active in inducing AO activity. The ability of ethylene to increase AO was blocked 60% by cycloheximide, an inhibitor of protein synthesis, and 35% by AgNO₃, an inhibitor of ethylene action.     

Maintenance of plant cell membrane integrity and function by the immobilisation of protoplasts in alginate matrices

Isolated protoplasts (Vicia faba) suspended in a mannitol solution over a time period of about 10 d undergo a process of aging characterised by increased ethane formation, decrease in oxygen evolution and CO₂ uptake, pigment degradation and a change in certain enzyme activities (ribulose-1,5-bisphosphate carboxylase and protease). The technique of immobilisation of protoplasts in a cross-linked alginate matrix offers a way to delay the appearance of the indicators mentioned. Various possible mechanisms for the increased stability of protoplasts are discussed.