Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.     

Cytological localization of fast renaturing and satellite DNA sequences inVicia faba

Summary DNA sequences reassociating within a Cot value of 1.8×10^–1 and those producing a light satellite in a CsCl density gradient were isolated fromVicia faba DNA and hybridizedin situ on squashes of roots of the same species. Silver grains were seen to be scattered over both the interphase nuclei and the metaphase chromosomes after hybridization with fast renaturing DNA sequences, indicating these are fairly regularly interspersed in theV. faba genome. Clustered labeling occurred after hybridization with satellite DNA sequences, indicating these are clustered in the genome. The localization of satellite DNA in chromosomes appeared to correspond closely to the position of the bright bands detectable after staining with quinacrine mustard. After hybridization with both DNA probes, labeling intensity over the nuclei of meristematic cells was higher than that over the nuclei of differentiating and/or differentiated cells. These results are discussed in relation to the structure of the cell nucleus, the mechanism of quinacrine banding and to previous data suggesting underrepresentation of nuclear repeated DNA sequences in differentiatingV. faba root cells.     

Cytophotometrical measurement of nuclear DNA content in some coniferous and deciduous trees

Summary The DNA content of nuclei from meristematic root tip cells of five coniferous and one deciduous tree species and, for comparison, ofVicia faba was cytophotometrically determined. The DNA values of diploid nuclei fromGinkgo biloba are approximately a quarter lower than those fromVicia faba. The nuclear DNA values of the other tree species are merely a third to a ninth part of those ofVicia faba. In three tree species, as well as diploid, we have found nuclei of different polyploid level.The reliability of different cytochemical methods, which are used for determination of the nuclear DNA content, is critically analyzed. The DNA values of the investigated tree species are discussed in connection with the evolution of the DNA content in higher plants.Dedicated to Professor Dr. F. Mechelke in honour of his 60th birthday     

Different effects of pretreatment with tritiated thymidine (3H-dTh) on radiation-induced sister chromatid exchanges (SCEs) and micronuclei inVicia faba root tip cells

Primary roots ofVicia faba were grown for 24 h in the presence of tritiated thymidine (1.85–18.5 kBq ml⁻¹) and then irradiated with a dose of 1.5 Gy of⁶⁰Co-gamma- rays. The aim of these experiments was to determine whether low-level endogenous beta-irradiation from incorporated radioactive thymidine could influence the frequencies of sister chromatid exchanges (SCEs) and the numbers of micronuclei induced by subsequent external irradiation with high doses of gamma-rays. The results demonstrated that the pretreatment with³H-dTh had no significant effect on the frequencies of SCEs in gamma-irradiated root tip cells ofVicia faba. In contrast to SCEs, the yields of micronuclei in the³H-dTh pretreated cells were altogether less than the yield induced by gamma-rays alone (protective effects).     

Localization of ribosomal cistrons in metaphase chromosomes of Vicia faba (L.)

Tritiated ribosomal RNA (rRNA) was prepared from the roots of Vicia faba after incubation in ³H-uridine. Separation of the nucleic acids by MAK chromatography yielded fractions of specific activity of 4–5 × 10⁵ dpm/μg. 4 + 5S, 18S and 25S RNA fractions were used for cytological hybridization on squash preparations of Vicia faba root tip meristems. Autoradiographs of the 18S and 25S RNA preparations exhibited a clear labelling in the secondary constriction of the satellite (SAT) chromosomes after exposition times of 28 weeks.     

Localization of telomeres in plant interphase nuclei by in situ hybridization and 3D confocal microscopy

We investigated the three-dimensional (3D) arrangement of telomeres in structurally well preserved, interphase nuclei of Pisum stativum and Vicia faba root tips using in situ hybridization of a probe to telomeric sequences. The probe was labelled with either digoxygenin or biotin and hybridized sequences were detected by immunofluorescence. Three-dimensional data sets were collected by confocal optical microscopy or using a cooled CCD camera. Twelve stacks of optical sections of P. sativum nuclei and nine of V. faba nuclei were studied in detail. Projections through the stacks of optical sections revealed that, in both species, most of the telomeres were adjacent to the nuclear envelope except for a small number next to the nucleolar periphery. In V. faba nuclei, the telomeres were clearly clustered at one pole while in P. sativum there was only a slight tendency for clustering. In V. faba, clusters were found at opposite poles in pairs of sister nuclei rather than at adjacent poles as would be expected if the arrangement at telophase were maintained into interphase.by D. Bazett-Jones     

Nuclear DNA characterization of two species ofVicia:Vicia bithynica L. andVicia narbonensis L.

The speciesVicia bithynica andVicia narbonensis, from the same subgeneric section ofVicia faba, show variations in nuclear DNA content Nuclear DNAs, extracted from root tips of the twoVicia species, were characterized by thermal denaturation, analytical ultracentrifugation and reassociation kinetics. The thermal denaturations of DNA, the number of DNA components reassociating with second order kinetics, the proportion of repeated DNA sequences, the frequency of the repeated DNA classes are reported and compared to previous data onVicia faba DNA. Feulgen absorptions at different thresholds of optical density⁺ of interphase nuclei in cytological preparations of the root meristems ofV. bithynica andV. narbonensis are determined and compared withV. faba analogous determinations. The results, confirming that plant genome is highly flexible, are discussed in relation to other data on the interspecific variations of the nuclear DNA content.     

Cation sensitivity and kinetics of guard-cell potassium channels differ among species

in ward rectifying g uard c ell K ⁺ c hannel, GCKC1_in, from three major crop plants Solanum tuberosum L., Nicotiana tabacum L., and Vicia faba L. Selecting guard cells for our analyses we aimed to test whether K⁺ channels of the same cell type differ among species. The channels shared basic features including voltage-dependence, selectivity and single-channel conductance. They activated at hyperpolarization (V _1/2 ≈ −164 mV) with single channels of 7 pS underlying the whole-cell current. The channel density in S. tuberosum was higher than in V. faba and N. tabacum while the activation and deactivation kinetics were faster in the latter two species. Among different monovalent cations the K⁺ channels discriminated strongly against Na⁺, Li⁺, and Cs⁺. The sensitivity to Cs⁺ was similar for the three species. Extracellular Ca²⁺ blocked the V.␣faba K⁺ channel at concentrations ≥1 mM but only affected its functional homologs in S. tuberosum and N.␣tabacum at higher concentrations and more-negative membrane potentials. Like the differences in Ca²⁺-sensitivity, protoplasts from the three species differed remarkably in their response towards extracellular pH changes. Whereas protons neither altered the open probability nor the kinetic parameters of the V. faba GCKC1_in, in S. tuberosum and N. tabacum this cation affected the voltage-dependent properties strongly. An increase in proton concentration from pH 8.5 to 4.5 shifted the potential of half-maximal open probability to less-negative values with a maximum effect around pH 6.2. The pH modulation of the K⁺ channels could be described assuming a two-state model where the open and closed channel can be protonated. The observed differences in cation-sensitivity and voltage-dependent kinetics between K⁺ channels reflect the diversification of guard-cell channels that may contribute to species-specific variations in the control of stomatal aperture. Received: 19 July 1997 / Accepted: 2 October 1997     

STRANDEDNESS OF VICIA FABA CHROMOSOMES AS REVEALED BY ENZYME DIGESTION STUDIES

Chromosomes and nuclei isolated from neutral formalin-fixed Vicia faba lateral roots were treated with trypsin, pepsin, RNase, or DNase. Only trypsin affected the morphology of the chromosomes and nuclei. The appearance of the chromosomes after trypsin digestion indicated that each chromatid contained four strands that could be seen with an ordinary light microscope. The experiments are interpreted as indicating that mitotic chromosomes of Vicia faba are multistranded and that the linear continuity of the chromosome is dependent on protein.     

Calcium channels and membrane disorders induced by drought stress in Vicia faba plants supplemented with calcium

Vicia faba plants were grown under drought conditions and variously supplemented with calcium. Drought stress markedly inhibited the growth of Vicia faba plants. Ca²⁺ ameliorated to a large extent this inhibition; fresh weight, dry mass, chlorophyll and water contents were variably improved. Membranes were, also, negatively affected by drought stress and percentage leakage was elevated. Concomitantly, the efflux of K⁺ and Ca²⁺ was enhanced by drought but lowered by supplemental Ca²⁺. In addition, membranes of droughted plants were sensitive to the Ca²⁺ channel blockers lanthanum, nifedipine or verapamil more than those of control plants. These blockers significantly increased the efflux of K⁺ and Ca²⁺ as well as percentage leakage particularly in those of droughted plants. The above results indicated that the functioning of the calcium channels was negatively affected when Vicia faba was grown under drought conditions. However, much of the drought-induced disorders including sensitivity towards the applied calcium channel blockers could be ameliorated by supplemental Ca²⁺.     

Constitutive heterochromatin distribution in monocentric and polycentric chromosomes

Monocentric chromosomes of Vicia faba (2n=12) and polycentric chromosomes of Luzula purpurea (2n=6) and L. multiflora (2n=36) were studied by the C-banding technique. C-positive regions exhibited a restricted distribution in V. faba nuclei and were located near the centromeres of the chromosomes. Each chromosome had both a characteristic number and distribution of C-positive regions permitting homologue identification. L. purpurea and L. multiflora C-bands were much more numerous than those of V. faba and were widely distributed throughout both nuclei and chromosomes. Three distinct constitutive heterochromatin distribution patterns were present in L. purpurea metaphase chromosomes permitting homologous chromosome identification. One of three C-band distribution patterns was also evident in L. multiflora chromosomes.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G₂, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.     

Cytological localization of inverted repeated DNA sequences in Vicia faba

Inverted repeated DNA sequences have been isolated from sheared Vicia faba DNA by hydroxylapatite column chromatography, treated with nuclease S₁, tritiated by the nick translation method and hybridized in situ on squashes of Vicia faba root tips. Silver grains appear grouped in a rather limited portion of interphase nuclei and form a sort of band across them. The central regions of metaphase chromosomes are preferentially labeled, labeling being excluded from telomeres, centromeres and secondary constrictions. These results are briefly discussed in relation to those obtained in other species and the functional significance of inverted repeats.     

Heavy meromyosin complexing filaments in the phloem of Vicia faba and Xylosma congestum

Summary Stem sections of Vicia faba L. were incubated with rabbit-muscle heavy meromyosin (HMM) and HMM complexes with phloem filaments (P-protein) were observed with the electron microscope. Treatment of sections of Vicia faba and of Xylosma congestum (Lour.) Merr. with fluorescent HMM resulted in a weak fluorescence of the phloem region. Inasmuch as HMM-binding is believed to be specific for actin-like proteins, it is proposed to classify P-protein as such.     

A simple method for identification of S phase nuclei in Vicia faba root meristems using BrdUrd labeling and indirect immunofluorescence (comparison with 3H-thymidine incorporation)

A modified immunocytochemical method for identification of S-phase cells in root meristems is desribed. The technique combines incorporation of BrdUrd, rapid isolation of cell nuclei and indirect immunocytochemical detection of BrdUrd-labeled areas of chromatin using monoclonal anti-BrdUrd (I) and FITC-conjugated (II) antibodies. The labeling indexes calculated using ³H-thymidine autoradiography and BrdUrd-immunofluorescence in root meristems of Vicia faba subsp. minor were found comparable. However, the increased sensitivity of fluorescent staining reveals an enhanced complexity of visible structure emerging during successive periods of S-phase, allowing for discriminating not only quantitative but also qualitative aspects of nuclear labeling patterns characteristic for specific periods of DNA replication.     

Indirect mitotic nondisjunction in Vicia faba and Chinese hamster cells

The hypothesis of indirect mitotic nondisjunction was tested in plant and mammalian cells. This hypothesis states that micronuclei derived from lagging chromosomes or chromatids are able to perform DNA synthesis and undergo mitotic condensation synchronously with main nuclei. Hence, as chromosomes, they can be moved to spindle poles together with the chromosomes of the main nuclei during mitosis. In that way chromosomes lost as micro-nuclei can be reincorporated in the main nuclei. In order to test this, both Vicia faba meristematic cells and cells of a Chinese hamster line (Cl-1) were treated with low doses of colchicine. Mitotic anomalies, micronuclei and cells with a polyploid or aneuploid karyotype were scored at different fixation times. A detailed analysis was performed on single chromosome misdistributions, as well as on micronuclei and cells with aneuploid karyotypes derived from single chromosome misdistributions. Indirect mitotic nondisjunction was shown to play a primary role in the origin of aneuploid karyotypes in Vicia faba, but not in Cl-1 cells.     

Die photochemische Aktivität von Chloroplasten Aus unterschiedlich mit Kalium ernährten Pflanzen

Zusammenfassung Es wurde der Einflu? des Kaliumern?hrungszustandes auf die Photoreduktion und auf die Photophosphorylierung an aufgebrochene Chloroplasten vonSpinacia oleracea, Vicia faba undHelianthus annuus untersucht. Chloroplasten von Pflanzen mit hoher K-Versorgung zeigten erh?hte Photoreduktions-und Photophosphorylierungsraten. Hierbei wurde die Phosphorylierung st?rker beeinflu?t als die Reduktion. Dementsprechend war das P/2e⁻-Verh?ltnis kaliumarmer Pflanzen erniedrigt. Zugaben von Kaliumchlorid (0–100 mM KCl) zum Inkubationsmedium erh?hten die Photophosphorylierungs-und die Photoreduktionsrate. Dieser Effekt war jedoch unabh?ngig vom Kaliumern?hrungszustand, und die ziemlich niedrigen Phosphorylierungs-und Reduktionsraten Kalium armer Chloroplasten konnten durch Zugaben von Kaliumchlorid nicht auf das Niveau von Chloroplasten mit guter Kaliumversorgung gebracht werden. Es wird angenommen, da? die günstige Wirkung einer guten Kaliumern?hrung auf Photophosphorylierung und Photoreduktion auf gesteigerte Ionenfluxe (Protonen und Kalium) durch Thylakoidmembranen zurückgeht. Weiterhin dürfte Kalium auch die Aktivit?t und die Wirksamkeit der beteiligten Enzyme beeinflu?st haben.

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Summary The influence of the potassium nutritional status upon the photoreduction and photophosphorylation was studied with “broken” chloroplasts ofSpinacia oleracea, Vicia faba andHelianthus annuus. Chloroplasts from plants with a high K supply showed increased rates of photoreduction and photophosphorylation. The latter process was more affected than the photoreduction. Therefore the P/2e⁻ ratio was lower for plants poor in potassium. Additions of potassium chloride (0 to 100 mM) to the incubation medium increased the photophosphorylation and photoreduction rates. But this effect appeared to be independent of the potassium nutrition status, while the rather low rates of photophosphorylation and photoreduction of the chloroplasts poor in potassium could not be overcome by the addition of potassium chloride. It is assumed, that the favorable effect of an adequate potassium nutrition upon photophosphorylation and photoreduction is brought about by enhanced ion fluxes (protons and potassium) through the thylakoid membranes and also by higher activities and efficiencies of enzymes involved

     

Effects of sodium chloride and polyethylene glycol on root-hair infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum

The effects of sodium chloride and polyethylene glycol (PEG) on the interaction between Rhizobium leguminosarum strain 29d and root hairs of field bean (Vicia faba L. cv. Maris Bead) plants were investigated. Two levels each of NaCl (50 and 100 mol·m^–3) and PEG (100 and 200 mol·m^–3) were given at the time of root-hair formation. Scanning electron microscopy showed rhizobial attachment and colonization on root-hair tips. Adhesion of rhizobia in both lateral and polar orientation, sometimes associated with microfibrils, occurred mainly in crooks at the root-hair tips; most of the infections also occurred here. Bacterial colonization and root-hair curling were both reduced by stress treatments. Polyethylene glycol but not NaCl significantly reduced root-hair diameter. The proportion of root hairs containing infection threads was reduced by 30% under NaCl and by 52% under PEG. The structure of some of the root hairs, epidermal and hypodermal cells, as seen by light microscopy in ultrasections, was distorted as a result of NaCl and PEG treatments; cells showed plasmolysis and folded membranes. After three weeks of treatment, both NaCl and PEG inhibited nodule number by about 50% and nodule weight by more than 60%. It is concluded that the root-hair infection process in Vicia faba is impaired by NaCl and PEG treatments and this in turn results in fewer nodules being produced.Abbreviation PEG polyethylene glycol     

Soil and plant resistances to water uptake byVicia faba L.

Summary The hydraulic resistivity ofVicia faba L. roots grown in soil was estimated from steady state measurements of transpiration rate and leaf and soil water potentials. Root and stem axial resistivities, estimated from xylem vessel radii, were negligible. Root radial resistivity was estimated to be 1.3×10¹² sm⁻¹. This root radial resistivity value was used to estimate, root resistance to water uptake for a field crop ofVicia faba. Previously published results were used for root distribution and soil water contents at the drained upper limit (DUL) and the lower limit (LL) of extractable soil water. Soil resistance to water uptake was estimated from single root theory using the steady rate solution. At the DUL, root resistance was about 10⁵ times greater than soil resistance. At the LL, soil resistance exceeded root resistance for depths less than 0.3 m, but for depths greater than this soil resistance was smaller than root resistance. Estimates of possible uptake rates at given leaf water potentials indicated that overall soil resistance had a negligible influence upon uptake, even at the LL. The reliability of this result is examined in detail. It is concluded that over the complete range of extractable soil water contents soil resistanceper se would not have limited water use by this crop. This conclusion may also be valid for a wide range of soil and crop combinations.     

紫茎泽兰叶片水浸液对蚕豆的化感效应

外来入侵植物可以通过淋溶、自然挥发、根系分泌和植株凋落物分解等途径向周围环境释放化感物质,抑制伴生植物的生长、发育。该研究以不同浓度紫茎泽兰(Eupatorium adenophorum)叶片水浸液处理蚕豆(Vicia faba)种子,研究紫茎泽兰叶片水浸液对蚕豆根尖细胞微核、染色体畸变、细胞凋亡、蚕豆幼苗叶片叶绿素和N含量、光合生理特性、生物量的影响。结果表明:(1)紫茎泽兰叶片水浸液处理显著抑制蚕豆根尖的伸长和细胞的有丝分裂,并诱导蚕豆根尖细胞染色体畸变和细胞微核的产生,有丝分裂指数随着叶片水浸液浓度增加而减小,根尖细胞微核率随叶片水浸液浓度增加而增大,高浓度叶片水浸液处理对蚕豆根尖细胞的凋亡及坏死有明显影响。(2)紫茎泽兰叶片水浸液处理引起蚕豆幼苗叶片的叶绿素和N含量显著降低,并导致蚕豆幼苗最大净光合速率和生物量的显著下降。总之,紫茎泽兰叶片水浸液可能引起蚕豆根尖的氧化损伤和抑制根尖的伸长,且叶片水浸液的抑制作用呈现一定的剂量效应。紫茎泽兰叶片水浸液对蚕豆根尖的损伤和抑制作用可能影响了植株对氮素的吸收,进而对蚕豆幼苗光合生理表现以及生物量积累产生显著负面效应。