Properties of α-galactosidase II2 from Vicia faba seeds

-Galactosidase II² (MW 43 390) from resting Vicia faba L. seeds had been shown to possess d-glucose/d-mannose-specific lectin activity. Inhibition studies with monosaccharides and an examination of the effects of heat and pH on the catalytic and lectin activities of the enzyme indicate that the enzyme substrate and the lectin haptens bind at different sites on the protein. d-Mannosebinding has been investigated by equilibrium dialysis and spectrophotometrically. Both methods yield K_a values of approx. 3·10³ M^-1 for the interaction and there would appear to be two mannosebinding sites per molecule of enzyme protein. The lectin properties of V. faba -galactosidase II² have been discussed in relation to both V. faba lectin (favin) and other legume -galactosidases.Abbreviations con A concanavalin A - CM-cellulose carboxymethyl cellulose - MW molecular weight - PNPG p-nitrophenyl -d-galactoside - SDS sodium dodecyl sulphate - PAGE polyacrylamide-gel electrophoresis     

Purification and properties of α-galactosidases from Vicia faba seeds

Two forms of alpha-galactosidase, I and II, exist in Vicia faba seeds and these have been purified 3660- and 337-fold respectively. They behaved as homogeneous preparations when examined by ultracentrifugation, disc electrophoresis and gel filtration. The apparent molecular weights of enzymes I and II, as determined by gel filtration, were 209000 and 38000 respectively. The carbohydrate contents of enzymes I and II were 25% and 2.8% respectively, and the enzymes differed in their aromatic amino acid compositions. Enzyme I was split into six inactive subunits in the presence of 6m-urea. alpha-Galactosidases I and II showed different pH optima and K(m) and V(max.) values with p-nitrophenyl alpha-d-galactoside and raffinose as substrates, and also differed in their thermal stabilities.     

Substrate specificity and kinetic properties of α-galactosidases from Vicia faba

1. The hydrolysis of a variety of galactosides and other glycosides by alpha-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl alpha-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on K(m) and V(max.) values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.     

Phosphorus deficiency increases nodule phytase activity of faba bean–rhizobia symbiosis

The effect of phosphorus deficiency on growth, nodulation and phytase activity was studied in glasshouse for four symbioses involving two faba bean cultivars, namely Aguadulce (AG) and Alfia (AL), and two local rhizobial isolates, namely RhF1 and RhF2. The P deficiency was applied by adding 25 µmol of Pi plant^?1 week^?1 to nutrient solution, whereas the sufficient control received 125 µmol plant^?1 week^?1. At flowering stage, the plants were harvested for assessment of growth and nodulation, P and N contents in organs as well as activities of phytase and phosphatases in nodules. The latter were highly stimulated by P deficiency, particularly for AL–RhF1 symbiosis for which shoot growth and P content were not affected by P deficiency. Using in situ RT-PCR, the expression of a plant histidine acid phytase HAP gene was detected in the nodule cortex under P deficiency. It is concluded that high nodule phytase activity constitutes a mechanism for faba bean plants to adapt their nitrogen fixation to P deficiency.     

Fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Steady-state ⁸⁶Rb⁺ fluxes and contents were measured in stomatal guard cells of Vicia faba L., using isolated epidermal strips in which all cells other than the guard cells had been killed by a brief ultrasound treatment. Flux experiments were carried out for a range of stomatal apertures, achieved by incubation in light or dark, or on solutions containing different concentrations of RbCl (1–30 mM). At pH 5.5 the efflux curve of ⁸⁶Rb⁺ could be fitted to the sum of two exponential terms and it was possible to calculate compartmental contents and fluxes. The data indicate that steady-state stomatal opening is achieved by regulation of both influx and efflux. Salt accumulation was not always adequate to contribute the observed osmotic requirement for opening, and this may indicate that some other solute accounts for the discrepancy.Abbreviations and Symbols A amplitude - k rate constant - Mes 2-(N-morpholino)-ethanesulphonic acid - Q_T, Q_C, Q_V total, cytoplasmic, vacuolar chemical content - Q^* tracer content - Ø_P, Ø_l plasmalemma, tonoplast fluxes This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

蚕豆(Vicia faba)M染色体DNA复制顺序的研究

真核细胞中,染色体DNA由许多复制子组成。一个复制子所需的复制时间短于S期时长,这意味着染色体DNA的各个复制子并非同时复制,而是按某种先后次序进行复制的。许多学者以人和动物细胞为材料进行了有关染色体DNA复制顺序的研究,但是关于植物染色体DNA复制顺序的研究报道还不很多。植物根端分生组织细胞在周期时长方面差异很大。Evans曾经研究过蚕豆根端细胞染     

Is low rooting density of faba beans a cause of high residual nitrate content of soil at harvest ?

It was the aim of this study was to evaluate the hypothesis that low rooting density of faba beans is the major reason for the comparable low depletion of N_min-nitrogen from the rooted soil volume during the vegetation period. Therefore a simulation study was carried out using data from a two-year field experiment with faba beans and the reference crop oats. Since the nitrate dynamics in the soil is closely coupled with the water budget, the model simulated also the water uptake by plants, movement and content in the soil applying a numerical solution of the Richard's equation. The nitrogen budget part of the model includes calculation of vertical nitrate movement in the soil, mineralisation of nitrate from organic matter and nitrate uptake by the crop. Vertical nitrate movement was simulated with the convection-dispersion equation. Mineralisation was computed from a simple first order kinetic approach using only one fraction of mineralisable organic matter. Nitrate uptake was assumed to be determined either by the nitrogen demand of the crop, which was estimated from a logistic growth equation that was fitted to measured data of N-accumulation, or by the maximum nitrate transport rate towards the root surface. The latter was computed from a steady state solution of the diffusion - mass flow equation for cylindrical co-ordinates.For oats the model calculated a maximum nitrate transport rate towards roots that was quite close to the measured N-uptake of that crop. For faba beans, however, the calculated maximum nitrate transport towards roots was much lower than total N-uptake and lower than for oats. Consequently, simulated N_min-contents below faba beans were during the growing season about 20-30 kg N ha^–1 higher than below oats. This difference matches quite close with the observed differences between the two crops. Therefore it was concluded that low nitrate uptake resulting from low rooting density is the main reason for higher residual nitrate contents below faba beans at harvest time.     

Binding of 4-methyl umbelliferyl-α-D-glucoPyranoside to Vicia faba lectin: Fluorescence-quenching studies

On binding toVicia faba lectin, the fluorescence of 4-methylumbelliferyl-α-D-glucoPyranoside was quantitatively quenched showing that the interaction of 4-methylumbelliferyl-α-D-glucoPyranoside took Place in a binding environment. The binding of the fluorescent sugar was saccharide sPecific as evidenced by the reversal of 4-methylumbelliferyl-α-D-glucoPyranoside fluorescence quenching by D-fructose. The association constant,K _a, values for the 4-methylumbelliferyl-α-D-glucoPyranoside was determined by comPetition study emPloying reversal of fluorescence quenching of 4-methylumbelliferyl-α-D-glucoPyranoside by D-fructose. TheK _a value obtained for D-fructose was 1.07 ±0.03 X 10⁴ M^-1 and for 4-methylumbelliferyl-α-D-glucoPyranoside was 1.60 ±0.05 X 10⁴ M^-1 at 15°C. TheK _a values of 2.51 ±0.06 X 10⁴M^-1, l.26 ±0.02 X 10⁴ M^-1 and 0.56 ±0.01 X 10⁴M^-1, resPectively at 10°, 20° and 30°C were obtained from the ChiPman equation. The relative fluorescence quenching, ΔF _a, at infinite concentration of the free saccharide sites ofVicia faba lectin [P′] was 93.5% at 30°C and the binding constant for 4-methylumbelliferyl-α-D-glucoPyranoside lectin interaction as derived by Yank and Hanaguchi equation was 0.63 ±0.01 X 10⁴M^-1.     

Ethylene biosynthesis in isolated vacuoles of Vicia faba L. — requirement for membrane integrity

The ability of vacuoles prepared from V. faba leaves to convert 1-aminocyclopropane-1-carboxylic acid to C₂H₄ was destroyed when vacuoles were lysed by passage through a hypodermic needle, freezing and thawing, osmotic shock, treatment with ethanol or with a detergent. Ethylene synthesis in the vacuolar fraction was also inhibited by the uncouplers carbonyl cyanide m-chlorophenyl hydrazone and dinitrophenol and by the ionophores valinomycin, nigericin, and A23187. Ethylene formation increased with increasing pH of the incubation medium over the pH range of 5.0–7.5. These observations support the hypothesis that C₂H₄ biosynthesis in vacuolar preparations is dependent on membrane integrity, possibly because of the requirement for a transmembrane ion gradient.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Microwave-induced stimulation of l-DOPA,phenolics and antioxidant activity in fava bean (Vicia faba) for Parkinson’s diet

Fava bean sprouts are rich in levo-dihydroxy phenylalanine (l-DOPA), the precursor of dopamine, and are being investigated for use in the management of Parkinson’s disease. The phytopharmaceutical value was improved during germination by a microwave treatment of the seeds, the phenolic content of the germinated sprouts increasing 700% and l-DOPA content by 59% compared to control. A higher antioxidant activity that was observed correlated with total phenolics and l-DOPA contents. The glucose-6-phosphate dehydrogenase activity peaked on the seventh day of germination with a concurrent increase in phenolics indicating enhanced mobilization of carbohydrates. A higher guaiacol peroxidase activity was observed indicating an increased polymerization of phenolics. The elevated superoxide dismutase activity was proportional to the stimulation of antioxidant activity. The major implication from this study is that microwave treatment can significantly stimulate the phenolic antioxidant activity and Parkinson’s relevant l-DOPA content of fava beans sprouts.     

Effects of light/dark and calcium-channel drugs on fluxes of 86Rb+ in “isolated” guard cells of Vicia faba L.

Experiments on ⁸⁶Rb⁺ fluxes were used to identify the changes in ionic state induced by transferring stomata from light to darkness. Guard-cell fluxes and contents were measured on isolated epidermal strips from Vicia faba L. in which all cells other than the guard cells had been killed by ultrasonic disruption. Closure of stomata in response to darkness was achieved by a large, transient stimulation of ⁸⁶Rb⁺ efflux from the guard cells, combined with a reduction of ion transfer from cytoplasm to vacuole, but there was little significant change in influx. Removal of blue but not red light appeared to trigger the flux responses associated with darkening. Attempts to inhibit the closing response (using methoxyverapamil, nifedipine and bepridil, compounds possessing activity against the calcium channels of animal cells) were mostly unsuccessful and the significance of this result is discussed.Abbreviations and Symbols A amplitude - K rate constant - Mes 2-(N-morpholino)ethanesulfonic acid - Q ^* tracer content - s specific activity - R rate of trace loss - Q _T, Q _C, Q _V total, cytoplasmic and vacuolar contents - flux This work was supported by a Research Studentship from the Science and Engineering Research Council. I thank Professor E.A.C. MacRobbie for much helpful discussion and advice.     

The crucial elements of the ‘last step’ of programmed cell death induced by kinetin in root cortex of V. faba ssp. minor seedlings

Key message

Kinetin-induced programmed cell death, manifested by condensation, degradation and methylation of DNA and fluctuation of kinase activities and ATP levels, is an autolytic and root cortex cell-specific process.

Abstract

The last step of programmed cell death (PCD) induced by kinetin in the root cortex of V. faba ssp. minor seedlings was explained using morphologic (nuclear chromatin/aggregation) and metabolic (DNA degradation, DNA methylation and kinases activity) analyses. This step involves: (1) decrease in nuclear DNA content, (2) increase in the number of 4′,6-diamidino-2-phenylindole (DAPI)-stained chromocenters, and decrease in chromomycin A₃ (CMA₃)-stained chromocenters, (3) increase in fluorescence intensity of CMA₃-stained chromocenters, (4) condensation of DAPI-stained and loosening of CMA₃-stained chromatin, (5) fluctuation of the level of DNA methylation, (6) fluctuation of activities of exo-/endonucleolytic Zn²⁺ and Ca²⁺/Mg²⁺-dependent nucleases, (7) changes in H1 and core histone kinase activities and (8) decrease in cellular ATP amount. These results confirmed that kinetin-induced PCD was a specific process. Additionally, based on data presented in this paper (DNA condensation and ATP depletion) and previous studies [increase in vacuole, increase in amount of cytosolic calcium ions, ROS production and cytosol acidification “in Byczkowska et al. (Protoplasma 250:121–128, 2013)”], we propose that the process resembles autolytic type of cell death, the most common type of death during development of plants. Lastly, the observations also suggested that regulation of these processes might be under control of epigenetic (methylation/phosphorylation) mechanisms.     

Rhythmisches Kernwachstum während der mitotischen Interphase vonVicia faba

Zusammenfassung Das Kernwachstum zwischen zwei Mitosezyklen geht im Wurzelmeristem vonVicia faba diskontinuierlich vor sich. Nach volumetrischstatistischen Untersuchungen ordnen sich nämlich die Kernvolumina zwei deutlich getrennten Größenkategorien zu und den Übergang vermittelt eine nur relativ geringe Anzahl von Kernen; dies bedeutet, daß die posttelophasische Größe relativ lange beibehalten wird, daß darauf eine kurze Wachstumsperiode und dann im praeprophasischen Zustand ein abermaliger Wachstumsstillstand erfolgt. Die mittleren Volumina von posttelophasischen und praeprophasischen Kernen verhalten sich wie 11,82 (nach früheren Untersuchungen beiRhoeo wie 11,79). Diese Art des Wachstums stellt vermutlich ein weit verbreitetes biologisches Grundphänomen dar; das sprunghafte Wachstum hängt offenbar mit einer rasch erfolgenden Reproduktion von Chromosomensubstanz zusammen. Dies steht in gutem Einklang mit den Resultaten derjenigen Autoren, die eine sprunghafte DNS-Vermehrung finden. Scheinbar abweichende Fälle lassen sich aus nachweisbar oder vermutlich unzureichender Methodik erklären. Bei Protisten dürften ähnliche, jedoch von anderen Erscheinungen überlagerte Gesetzmäßigkeiten bestehen (S. 595).     

蚕豆(Vicia faba)有丝分裂和减数分裂过程中细胞板形成的细胞化学研究

本文以蚕豆(Vicia faba)的体细胞和花粉母细胞为材料,对细胞板形成过程进行了细胞学和细胞化学分析。有丝分裂的后期末或末期初,两组子染色体间的纺锤体区改组成成膜体。在成膜体内许多小颗粒在赤道面融合成细胞板。随成膜体的扩展细胞板横过细胞而最终形成。用PAS反应染色时,体细胞的成膜体显现粉红色,而细胞板呈现很强的多糖反应。体细胞中期的纺锤体被甲基绿-派洛宁染成鲜红色而与周围细胞质有明显区别。成膜体RNA很多,细胞板也含丰富的RNA。在减数分裂I的中期和后期纺锤体的形成是正常的,但在分裂末期不形成成膜体。早末期在纺锤体的赤道面上可以看到一种象似细胞板形成初期阶段的膜状结构。它只出现在纺锤体的赤道面上,但不能离心地扩展,并终于在第一次减数分裂末消失。这个结构对多糖和RNA呈负染色反应。作者认为,减数分裂I的细胞板不能完成其发育,至少部分地是由于没有多糖和RNA。在减数分裂Ⅱ的中期和后期形成纺锤体,但不出现成膜体。四个子核组成后,它们互相间再形成纺锤体,总共形成六个纺锤体。在每个纺锤体的赤道面上形成的细胞板对多糖和RNA呈现正的染色反应。本文对其他作者以前描述过的形成细胞板的小颗粒或小泡的化学本质,以及细胞板形成的机制做了简短的讨论。     

Sharing N resources in the early growth of rapeseed intercropped with faba bean: does N transfer matter?

Background and aims

Legume-brassica intercrops may help to reduce N fertilizer input. We tested whether (i) intercropping with faba bean can improve N status of rapeseed, and (ii) root complementarity and/or N transfer is involved in such performance.

Methods

Pre-germinated rapeseed and faba bean were grown either together or in monospecific rhizotrons (2 plants per rhizotron). Root growth was recorded. N rhizodeposition of the crops and N transferred between species were assessed using a ¹⁵N stem-labelling method.

Results

Intercropped rapeseeds accumulated 20 % higher amounts of N per plant than monocultures. Up to 32 days after sowing, root distribution in the rhizotrons was favourable to physical sharing of the soil N: 64 % of faba bean root length was located in the upper part, as 70 % was in the lower part for rapeseed. At late flowering of the faba bean (52 days after sowing), N rhizodeposition of the two crops were similar and reached 8 to 9 % of the plant N. N transferred from the faba bean to the rapeseed was similar to that transferred from the rapeseed to the faba bean.

Conclusions

Niche complementarity benefits more intercropped rapeseed than net N fluxes between species in the early growth.     

Where in Europe should we look for sources of the cutaneous trematode Collyriclum faba infections in migrating birds?

Cutaneous cysts with trematodes of Collyriclum faba have been found in birds during their spring and post-breeding migrations in the Czech Republic. During spring migrations, C. faba was found in one dunnock Prunella modularis, two European robins Erithacus rubecula, three common nightingales Luscinia megarhynchos, one song thrush Turdus philomelos and one great reed warbler Acrocephalus arundinaceus. During post-breeding migration, the same parasite was found in one garden warbler Sylvia borin, one whitehroat S. communis, three goldcrests Regulus regulus and one Eurasian treecreeper Certhia familiaris. The newly identified hosts of C. faba are dunnock, common nightingale, song thrush, great reed warbler and Eurasian treecreeper. Geographical areas of the birds' infection were identified from an analysis of reports on ringed birds of the same species, the time necessary for the development of cutaneous cysts with C. faba and the time of their survival, and hitherto known geographical areas of endemic occurrence of C. faba. It is presumed that birds trapped during spring migrations were infected in some montane and submontane regions in south-western Europe (the Alps, the Apennines). Birds infected during autumn migration or post-breeding vagrancy could have been infected in the Central European Carpathians, the region of C. faba endemic occurrence. For migrating birds, the impact of C. faba infections has not been hitherto assessed.     

Involvement of phytochelatins in NiCl2-triggered protection against induction of chromatid aberrations by TEM and MH in Vicia faba root tip meristems?

Conditioning treatment of Vicia faba root tip meristem cells with NiCl2 prior to challenge treatment with triethylenemelamine (TEM) or maleic hydrazide (MH) triggered protective functions against both these clastogens, i.e., resulted in a significantly reduced yield of metaphases with chromatid aberrations. Protection was prevented by pretreatment with buthionine sulfoximine (BSI), an inhibitor of the synthesis of plant phytochelatins (PCs), indicating that the NiCl2-triggered PC synthesis may be involved in the protective functions induced by NiCl2 conditioning treatment. BSI (instead of NiCl2) conditioning treatment triggered protection against MH but not against TEM.