Critical level of radiation damage of root apical meristem and mechanisms for its recovery in Pisum sativum L

The dose dependencies of growth and cytogenetical values have been built to determine the critical level of root apical meristem damage induced by cute irradiation in the range from 2 to 20 Gr. We have analyzed the frequencies of aberrant anaphases and the aberration distribution per cell, on the one hand, and the growth of biomass, the survival and regeneration of the root meristem, on the other hand. The critical level of damage to the stem apical meristem and root of seedlings was defined as 44-48% of aberrant anaphase. Exceeding of this level leads to the launch of suicidal program through induction of multiaberrant damages and interphase cell death. It appears that competition of clones of non-aberrant cells, the cells bearing 1 and 2 damages and multiaberrant cells plays the primary role in the mechanisms of recovery. The regeneration provides full or partial restoration of the main root apical meristem. However these local processes are insufficient to restore morphogenesis and survival of seedlings in excess of the critical level damage.     

Structural architectonics of apical root meristems in coherence with the quantitative assessment of its damage by radiation

The dose dependencies of the aberrant anaphases frequency in the root meristem in 48 hours after irradiation in the range of doses of 4-10 Gy is characterized by threshold and plateau at 33% aberrant anaphase. The plateau indicates the activation of the recovery processes. Topology of cell rows in the primary meristem of the dose to 8 Gy are conserved and recovered damages. New cell rows are formed by local cell pools in the distal meristem, pericycle cells and subepidermy. It grows by intrusive character displacing the rows of damaged cells. Apparently the competition between clones of normal and aberrant cells plays the primary role in the mechanisms of recovery. Resulting to competition the promotion of aberrant cells to the extension zone is slowed down or blocked. So critical level of damage of the root apical meristem was defined about 50% of aberrant anaphase. Exceeding of this level leads to lethal consequence for meristem and it is accompanied by the inclusion of more radical process of restoration through regeneration. Regeneration leads to complete replacement of the apex tissues including the extension zone.     

Structural architectonics of the root apical meristem in connection with a quantitative evaluation of its radiation damage

The dose dependences of the frequency of chromosome aberrations in root meristems of pea germinants after their 48-h irradiation in a dose range from 4 to 8 Gy is nonlinear. The plateau of the curve indicates the activation of recovery processes. With the dose rise, the meristem size decreases, while the frequency of cellular inactivation, clustering disorders, and deformations of cell rows in the meristem and extension zone increase. However, the topology of cell rows at the 33% level of aberrations is mostly maintained. Conservation and recovery of the topology of cell rows are performed through repopulation and substitution of damaged cells and rows on its basis. The growth character of new cell rows in damaged tissue is intrusive. The promotion of aberrant cells to the extension zone is slowed down or blocked by the interruption of symplastic growth. In new cellular subpopulations, chromosome mutagenesis continues, and the effectiveness of recovery processes is in many respects defined by the competition between normal and aberrant cells and also their clones. The limits of the recovery potential of root apex are restricted by “critical mass” of the proliferating pool and the degree of damage to the extension zone. At a level of 50%, aberrant anatelophases meristem recovery triggers a more radical mechanism through regeneration, which leads to the complete substitution of apex tissues, including the extension zone.     

Cytogenetic effects of 241Am in the Allium test

The purpose of this study was to elucidate the genome damage induced by 241Am-irradiation using different parameters of cytogenetic evaluation in Allium-test. The root tip cells test-system for the cytogenetic effects studying was used. 241AmCl3 of different concentrations was used (1.5 x 10(-9)-1.5 x 10(-7) g/l). Water solution-to-plant transfer factor for 241Am was found to be 0.18 +/- 0.04. The internal doses of 241Am accumulated during germination were 0.37-37.00 cGy. The impact of 241Am-irradiation was evaluated on the mitotic index (MI), the yield of aberrant anaphases (AA), the distribution of chromosome aberrations number in cells and the average level of lesion of aberrant cell (LAC). Probably all these parameters are differ in sensitivity to damage factor, but only some changes in MI was revealed. It is supposed, that the absence of any changes in the distribution of chromosome aberrations number in cells and the average level of LAC in 241Am-irradiated cells confirm the absence of significant 241Am-impact on chromosomes, as the alpha-irradiation should cause significant damages in chromosomes. Although solutions of 241Am were high-concentrated, the seedlings didn't accumulate high internal doses. It appears the distribution of 241Am is a significantly heterogeneous hence it is possible the absorbed doses in nuclei can't reach the level necessary for revealing of cytogenetic effects.     

Comparative analysis of thermotolerance of sunflower chlorophyll mutants

The influence of high temperatures on sunflower chlorophyll mutants was studied. The tolerance criteria were the level of chromosome aberrations and the mitotic index in the root apical meristem of seedlings, the level of nucleus-free cells in the epidermis of cotyledonous leaves, and the intensity of chlorophyll accumulation after the action of heat shock (HS). In addition, the frequency of plants with an altered content of pigments in M1 and M2 was analyzed. The results indicated that the plastomic mutant en-chlorina-5 is more tolerant to temperature stress as compared to other sunflower lines.     

Aberrant and multiaberrant (rogue) cells in peripheral lymphocytes of Hodgkin's lymphoma patients after chemotherapy

We analyzed spontaneous chromosome lesions in peripheral lymphocytes cultured from Hodgkin's lymphoma (HL) patients before and after cytostatic chemotherapy. The mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20+/-0.58 per 100 metaphases) than in non-treated HL patients (4.80+/-0.54), and in non-treated patients than in healthy subjects (2.12+/-0.13). In lymphocytes of HL patients, who received chemotherapy, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (or rogue) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. Rogue cells were found in 15 out of 18 chemotherapeutically treated HL patients (in total, 60 rogue cells per 5,568 scored cells), whereas in 30 non-treated patients only 1 rogue cell was found (per 4,988 scored cells). No correlation was found between the yield of rogue cells and the aberration frequency in ordinary aberrant cells. Aberration spectra (ratios of chromatid- to chromosome-type aberrations and of breaks to exchanges) were essentially different in ordinary aberrant and multiaberrant cells. These data, as well as analysis of cellular distributions of aberrations, implied independent induction of chromosome damage in ordinary aberrant and rogue cells. Analysis of aberration patterns in diploid and polyploid rogue metaphases belonging to the first, second, and third in vitro division indicated that rogue cells could be formed both in vivo and in vitro, and could survive at least two rounds of in vitro replication, given blocked chromosome segregation. These results suggested that formation of rogue cells, unlike ordinary aberrant cells, was triggered by events other than direct DNA and/or chromosome lesions. A hypothesis regarding disrupted apoptosis as a candidate mechanism for rogue cell formation seems to be most suitable for interpretation of our data. Cultured lymphocytes of chemotherapeutically treated HL patients may represent a model system for further examination of the multiaberrancy phenomenon.     

Aluminum induces chromosome aberrations in wheat root meristem cells]

The yield and pattern of chromosome structure aberrations in wheat seedlings treated with aluminum nitrate and aluminum sulfate at various concentrations have been determined by the anaphase method. Aluminum has a genotoxic effect causing genome, chromatid, and chromosome aberrations in apical root meristem cells. The relationship between the total yield of structural mutations and the aluminum concentration follows a bell-shaped curve. The mutagenic activity of aluminum nitrate peaks at 10(-3) mg/ml, which is twice as high as the permissible concentration limit (PCL) of aluminum in potable water. The maximum of the mutagenic activity of aluminum sulfate is observed at 5 x 10(-4) mg/ml, i.e., one PCL. Tap water boiled for 2 h in an aluminum vessel has virtually no genotoxic effect on wheat cells.     

Genetic consequences of irradiation in a scots pine <Emphasis Type="Italic">Pinus sylvestris</Emphasis> L. population

The genetic consequences of irradiation were studied in a Scots pine population from a region contaminated as a result of the Chernobyl meltdown. Mutations of isozyme loci were not detected in seeds collected from trees of the first post-meltdown generation in 2004. The frequency of cells with chromosome aberrations in the root meristem of seedlings grown from the seeds did not differ from the control level. A deviation from the expected ratio 1: 1 was observed for some isozyme alleles in endosperms of seeds obtained from heterozygous trees.     

Cellular and tissue mechanisms of recovery processes in Hordeum distichum L. under irradiation

Ionizing and UV-B irradiation of barley seedlings increased the number of chromosome aberrations in vegetative and generative meristems and disturbances in microsporo- and microgametogenesis. Other irradiation-induced changes included enhanced cytomixis and an increase in the number of pathologies in meiosis and tetrads and in the polymorphism and disturbances in pollen grains. Damages induced by different irradiation types differ primarily quantitatively. The dynamics of occurrence of chromosome aberrations in the root meristem reciprocally depended on the irradiation dose. Damages induced by low UV doses were detected throughout the ontogeny of plants. Increased irradiation doses activated cytolytic processes during premeiotic interphase, meiosis, and tetrad formation, as well as after the end of gametogenesis. Exposure to maximum doses of UV radiation and moderate doses of γ-radiation restored the fertility of grain pollens. Microsporocytes, microspores, and pollen grain components undergo degeneration primarily via apoptosis. This type of cell death is apparently autonomous, being induced by the cell population itself. The results of this study confirmed the hypothesis on the positive role of cell competition in the recovery processes and adaptation of plants to mutagens.     

Aluminum Induces Chromosome Aberrations in Cells of Wheat Root Meristem

The yield and pattern of chromosome structure aberrations in wheat seedlings treated with aluminum nitrate and aluminum sulfate at various concentrations have been determined by the anaphase method. Aluminum has a genotoxic effect causing genome, chromatid, and chromosome aberrations in apical root meristem cells. The relationship between the total yield of structural mutations and the aluminum concentration follows a bell-shaped curve. The mutagenic activity of aluminum nitrate peaks at 10^–3mg/ml, which is twice as high as the permissible concentration limit (PCL) of aluminum in potable water. The maximum of the mutagenic activity of aluminum sulfate is observed at 5 × 10^–4mg/ml, i.e., one PCL. Tap water boiled for 2 h in an aluminum vessel has virtually no genotoxic effect on wheat cells.     

The number of aberrations in aberrant cells as a parameter of chromosomal instability. 1. Characterization of dose dependency

Analysis of chromosome instability (CI) is of great importance in view of pollution of the environment by genotoxic factors. Frequency of aberrant cells, spectrum of chromosome aberrations, damages of aberrant cell and distribution of aberrations in the cells are the most conventional parameters of CI. We have carried out the comparative analysis of the frequency of aberrant cells and the dynamics of aberrant cell damages induced by different mutagenic factors (alpha-irradiation from 241Am, gamma-irradiation from 60Co and tioTEPA) in Allium-test. This comparative analysis denotes that the studied parameters have different dynamics characterizing different mechanisms of CI in Allium cepa L.     

Apical meristem exhaustion during determinate primary root growth in the moots koom 1 mutant of Arabidopsis thaliana

An indeterminate developmental program allows plant organs to grow continuously by maintaining functional meristems over time. The molecular mechanisms involved in the maintenance of the root apical meristem are not completely understood. We have identified a new Arabidopsis thaliana mutant named moots koom 1 (mko1) that showed complete root apical meristem exhaustion of the primary root by 9?days post-germination. MKO1 is essential for maintenance of root cell proliferation. In the mutant, cell division is uncoupled from cell growth in the region corresponding to the root apical meristem. We established the sequence of cellular events that lead to meristem exhaustion in this mutant. Interestingly, the SCR and WOX5 promoters were active in the mko1 quiescent center at all developmental stages. However, during meristem exhaustion, the mutant root tip showed defects in starch accumulation in the columella and changes in auxin response pattern. Therefore, contrary to many described mutants, the determinate growth in mko1 seedlings does not appear to be a consequence of incorrect establishment or affected maintenance of the quiescent center but rather of cell proliferation defects both in stem cell niche and in the rest of the apical meristem. Our results support a model whereby the MKO1 gene plays an important role in the maintenance of the root apical meristem proliferative capacity and indeterminate root growth, which apparently acts independently of the SCR/SHR and WOX5 regulatory pathways.     

Regeneration mechanisms of ontogenetic radioadaptation in plants

Experimental data were obtained that in pea seedlings modified by decapitation of main root had increased radioresistance (radioadaptation), fixed by various parameters of growth activity of lateral roots, and decreased ability to repair sublethal damages, detected by method of acute gamma-irradiation dose fractionation. These facts both with enlargement of dose dependence shoulder in lateral roots of decapitated seedlings led to conclusion that main role in such mechanism of radioadaptation effect of decapitation belongs to supercellular processes such as repopulation and regeneration. Conclusion was confirmed by the additional comparative investigations of cyto- and histological parameters of apical meristems of intact (control) and decapitated (experiment) lateral roots. It was shown, that the decapitated seedlings had increased mitotic activity of apical meristems of lateral roots and total volume of their meristematic zone. So at the moment of application of irradiation in the test-dose decapitated variant had significantly more meristematic cells of certain size that allowed biological object to form necessary (critical) amount of elements for valid or more complete postradiation recovery.     

Study of external low irradiation dose effects on induction of chromosome aberrations in Pisum sativum root tip meristem

The effects of low doses of ionizing radiation have been a matter of important debate over the last few years. The point of discussion concerns the validity of the linear dose-response extrapolation for low doses, used by international organizations, to establish radio-protection norms. Here, we contributed to this discussion by investigating the induction of chromosome aberrations by low to moderate doses ranging from 0 to 10 Gy in root meristem cells of 6-day-old Pisum plantlets. After acute irradiation of plantlets by a (60)Co source, the percentage of root tip meristem cells displaying chromosome aberrations was estimated immediately after irradiation and after 20 h recovery time. The dose-effect curves show non-linear responses, especially in the low dose range (0- 1 Gy), which is of particular interest. After 20 h of recovery, a steep increase of aberrations was observed for cells exposed to 0.4 Gy, followed by a plateau for doses until 1 Gy. There was an irradiation effect on plant growth during the first and second generations, showing the persistence of cell division anomalies as a long term effect of acute irradiation. This result suggests the induction of a genomic instability.Our results, in agreement with some obtained in animals, show rather non-linear dose-effect responses, with notably higher biological effects of low doses than expected.     

The auxin-insensitive bodenlos mutation affects primary root formation and apical-basal patterning in the Arabidopsis embryo

In Arabidopsis embryogenesis, the primary root meristem originates from descendants of both the apical and the basal daughter cell of the zygote. We have isolated a mutant of a new gene named BODENLOS (BDL) in which the primary root meristem is not formed whereas post-embryonic roots develop and bdl seedlings give rise to fertile adult plants. Some bdl seedlings lacked not only the root but also the hypocotyl, thus resembling monopteros (mp) seedlings. In addition, bdl seedlings were insensitive to the auxin analogue 2,4-D, as determined by comparison with auxin resistant1 (axr1) seedlings. bdl embryos deviated from normal development as early as the two-cell stage at which the apical daughter cell of the zygote had divided horizontally instead of vertically. Subsequently, the uppermost derivative of the basal daughter cell, which is normally destined to become the hypophysis, divided abnormally and failed to generate the quiescent centre of the root meristem and the central root cap. We also analysed double mutants. bdl mp embryos closely resembled the two single mutants, bdl and mp, at early stages, while bdl mp seedlings essentially consisted of hypocotyl but did form primary leaves. bdl axr1 embryos approached the mp phenotype at later stages, and bdl axr1 seedlings resembled mp seedlings. Our results suggest that BDL is involved in auxin-mediated processes of apical-basal patterning in the Arabidopsis embryo.     

Protective effect of wheat germ agglutinin on the course of mitosis in the roots of <Emphasis Type="Italic">Triticum aestivum</Emphasis> seedlings exposed to cadmium

Effect of pretreatment with 28 nM wheat germ agglutinin (WGA) on cell divisions in the root apical meristem of 4-day-old seedlings of wheat (Triticum aestivum L.), distribution of cells among mitotic phases, cadmium-induced disruptions of normal progression through mitosis, and activity of nucleolar organizer regions (NOR) of the chromosomes was studied after 7-h-long exposure to 1 mM cadmium acetate. Pretreatment with WGA has a pronounced protective effect on divisions of root meristem cells exposed to cadmium. Progression of the cells through mitotic phases was normalized, abnormal mitoses became much less numerous, and the share of binuclear cells decreased. Activity of NOR remained at the control level that much depended on the ability of WGA to prevent reduction in cytokinin content under cadmium stress.     

Adventitious shoot formation in decapitated dicotyledonous seedlings starts with regeneration of abnormal leaves from cells not located in a shoot apical meristem

Regeneration of new shoots in plant tissue culture is often associated with appearance of abnormally shaped leaves. We used the adventitious shoot regeneration response induced by decapitation (removal of all preformed shoot apical meristems, leaving a single cotyledon) of greenhouse-grown cotyledon-stage seedlings to test the hypothesis that such abnormal leaf formation is a normal regeneration progression following wounding and is not conditioned by tissue culture. To understand why shoot regeneration starts with defective organogenesis, the regeneration response was characterized by morphology and scanning electron and light microscopy in decapitated cotyledon-stage Cucurbita pepo seedlings. Several leaf primordia were observed to regenerate prior to differentiation of a de novo shoot apical meristem from dividing cells on the wound surface. Early regenerating primordia have a greatly distorted structure with dramatically altered dorsoventrality. Aberrant leaf morphogenesis in C. pepo gradually disappears as leaves eventually originate from a de novo adventitious shoot apical meristem, recovering normal phyllotaxis. Similarly, following comparable decapitation of seedlings from a number of families (Chenopodiaceae, Compositae, Convolvulaceae, Cucurbitaceae, Cruciferae, Fabaceae, Malvaceae, Papaveraceae, and Solanaceae) of several dicotyledonous clades (Ranunculales, Caryophyllales, Asterids, and Rosids), stems are regenerated bearing abnormal leaves; the normal leaf shape is gradually recovered. Some of the transient leaf developmental defects observed are similar to responses to mutations in leaf shape or shoot apical meristem function. Many species temporarily express this leaf development pathway, which is manifest in exceptional circumstances such as during recovery from excision of all preformed shoot meristems of a seedling.     

Effects of Temperature, Moisture, and Oxygen on the Induction of Chromosome Damage in Seeds of Barley, Broad Beans, and Peas during Storage

The effects of temperature, seed moisture content, and oxygenlevel on the production of chromosome aberrations during seedstorage have been investigated. It has been found that an increasein any of these factors increases the rate of loss of seed viabilityand that any treatment which leads to a loss of viability alsoleads to an accumulation of aberrant cells in the embryo. Undermost storage conditions, irrespective of the combination offactors which leads to loss of viability or the rate at whichviability is lost, the relationship between percentage viabilityand mean frequency of aberrant cells in the surviving seed populationis always the same. Under very severe storage conditions, whichresult in half-viability periods of about a week or less, however,the relationship is altered so that for any given percentageviability the mean frequency of aberrant cells in the survivingseeds is less than is typical of more normal storage conditions.In all treatments (except the most severe) the curve showingmean frequency of aberrant cells in surviving seeds againsttime eventually became asymptotic to a critical value peculiarto the species. It is suggested that these results are compatible with the hypothesisthat under most storage conditions death of the embryo is theresult of the accumulation of nuclear damage which is reflectedin chromosome breakage; it is emphasized, however, that thevisible chromosome breakage itself is probably not criticalto the seed's survival. When the nuclear damage has reacheda critical level, of which the frequency of aberrant cells isan index, the seed is no longer capable of germinating. Undervery severe storage conditions, additional factors appear tobe involved. An effort was made to test the frequently suggested hypothesisthat chromosome aberrations produced during the ageing of seedsare the result of the accumulation of automutagenic substances.Attempts to demonstrate the presence of mutagenic substancesin water-, ethanol-, and ether-extracts of fresh and aged seedsgave negative results. Aqueous extracts showed antimitotic andgermination inhibitory activity, but this was the same fromboth aged and fresh seeds.     

Effects of chronic irradiation of Scots pine (Pinus sylvestris L.) in Chernobyl exclusion zone

The quantitative "dose rate-effect" dependences are presented for the cytogenetic damages in the seedlings and apical meristem of Scots pine growing in the Chernobyl zone. The specific patterns of dynamics of formation of the morphological effects in the studied species are considered for the conditions of the internal and external chronic irradiation. The correlation dependencies are established for the irradiation effects appearing at the morphological and cell level. The assumption concerning the mechanism of the morphological changes formation is done.     

Modification of the Effect of Nitrosomethylurea on Sunflower Seedlings by Heat Shock

Separate and combined action of nitrosomethylurea (NMU) and heat shock (HS) on germination of seeds of the inbred line 3629 and derived nuclear and plastome mutant lines, n-chlorina-1 and en-chlorina-5, respectively, were studied. NMU at a concentration of 0.015%, as well as HS (40°C), had no effect on the spontaneous level of chromosome aberrations in root meristem of sunflower seedlings. However, at a concentration of 0.03%, the mutagen considerably increased the frequency of chromosome rearrangements. Pretreatment with heat for 30 min enhanced the cytogenetic effect of NMU (0.03%) on line 3629 seedlings. In the nuclear mutant, the reaction of root meristematic cells did not depend on the additional heat treatment. The combined action of HS and NMU at early germination stages had no effect on the survival of line 3629 plants and plastome mutant en-chlorina-5; the survival of n-chlorina-1 plants decreased. The highest frequency of M₁plants with chlorophyllic abnormalities (green revertants, lethals, and variegated forms) was observed in n-chlorina-1 line.