Inherited phenotype instability of inflorescence and floral organ development in homeotic barley double mutants and its specific modification by auxin inhibitors and 2,4-D.

BACKGROUND AND AIMS: Barley (Hordeum vulgare) double mutants Hv-Hd/tw2, formed by hybridization, are characterized by inherited phenotypic instability and by several new features, such as bract/leaf-like structures, long naked gaps in the spike, and a wide spectrum of variations in the basic and ectopic flowers, which are absent in single mutants. Several of these features resemble those of mutations in auxin distribution, and thus the aim of this study was to determine whether auxin imbalances are related to phenotypic variations and instability. The effects of auxin inhibitors and 2,4-D (2,4-dichlorophenoxyacetic acid) on variation in basic and ectopic flowers were therefore examined, together with the effects of 2,4-D on spike structure. METHODS: The character of phenotypic instability and the effects of auxin inhibitors and 2,4-D were compared in callus cultures and intact plants of single homeotic Hv-tw2 and Hv-Hooded/Kap (in the BKn3 gene) mutants and alternative double mutant lines: offspring from individual plants in distal hybrid generations (F9-F10) that all had the same BKn3 allele as determined by DNA sequencing. For intact plants, two auxin inhibitors, 9-hydroxyfluorene-9-carboxylic acid (HFCA) and p-chlorophenoxyisobutyric acid (PCIB), were used. KEY RESULTS: Callus growth and flower/spike structures of the Hv-tw2 mutant differed in their responses to HFCA and PCIB. An increase in normal basic flowers after exposure to auxin inhibitors and a decrease in their frequencies caused by 2,4-D were observed, and there were also modifications in the spectra of ectopic flowers, especially those with sexual organs, but the effects depended on the genotype. Exposure to 2,4-D decreased the frequency of short gaps and lodicule transformations in Hv-tw2 and of long naked gaps in double mutants. CONCLUSIONS: The effects of auxin inhibitors and 2,4-D suggest that ectopic auxin maxima or deficiencies arise in various regions of the inflorescence/flower primordia. Based on the phenotypic instability observed, definite trends in the development of ectopic flower structures may be detected, from insignificant outgrowths on awns to flowers with sterile organs. Phenotypically unstable barley double mutants provide a highly promising genetic system for the investigation of gene expression modules and trend orders.

Influence of cobalt uptake by Vicia faba seeds on chlorophyll morphosis induction, SOD polymorphism, and DNA methylation.

Vicia faba plants show polymorphism to cobalt (Co) excess, expressed by a different degree of chlorophyll morphosis (CM)-from normally green (N) to yellow (Y) seedlings. For superoxide dismutase (SOD), the high V. faba polymorphism was revealed and increased by Co stress. Epigenetic mechanisms may be involved in both phenomena. For such reasons, we investigated the effect of 5-azacytosine (AzaC) and Na butyrate (NaBut) on CM induction, SOD polymorphism, and DNA methylation-demethylation events in Co(NO(3) )(2) affected plants, without or with AzaC or NaBut. CMs were induced after treatment of seeds for 8 h with 7.5 mM Co(NO(3) )(2) plus 12 h with H(2) O or 8 h with H(2) O plus 12 h with Co(NO(3) )(2) . In the same order AzaC and NaBut were applied in concentrations equimolar to Co(NO(3) )(2) . SOD isoforms were investigated electrophoretically, and for DNA methylation-demethylation events the Aina [Aina et al. (2004) Physiol Plant 121:472-480] system was applied upon using the random amplified polymorphic DNA (RAPD) method employing restrictases MspI and HpaII. The effect of AzaC and NaBut on CM induction in combination with Co was unclear. Posttreatment with Co was more effective than Co-pretreatment. SOD polymorphism was significantly strengthened by NaBut. Detection of DNA methylation-demethylation events depended on the primers used for RAPD analysis. With AP5 and MP4 primers, DNA demethylation was observed in N-seedlings after exposure to Co, AzaC or NaBut applied separately. With primer A6, only DNA methylation events were determined in N-seedlings from seeds exposed to Co or Co-AzaC, and in Y-seedlings after Co-AzaC or Co-NaBut treatment. UPGMA grouping of the results showed that all N-seedlings comprised one common cluster after Co exposure, independently of treatment combinations (Co alone, Co with AzaC, Co with NaBut). On the contrary, no significant differences were determined in SOD polymorphism among the most resistant N-seedlings and the most severely affected Y-seedlings.

Genetic Structure of Native Blue Honeysuckle Populations in the Western and Eastern Eurasian Ranges.

Blue honeysuckle (Lonicera caerulea L.) is a promising berry crop producing edible early-ripening berries with a valuable chemical composition. We evaluated the genetic diversity of native L. caerulea populations from the western (Baltic states) and eastern (the Russian Far East and Japan) edges of the Eurasian range using inter-simple sequence repeat (ISSR) and chloroplast DNA (psbA-trnH and trnL-trnF) markers. The genetic relationships of populations and genotypes were analyzed using principal coordinate and cluster analyses (neighbor joining and Bayesian clustering). Sampling was carried out in two disjunct areas of this circumpolar species and the analyses showed clustering of individuals and populations according to geographic origin. The analysis of genetic structure based on ISSR markers showed that the studied populations of L. caerulea were highly differentiated. However, sequence analysis of two chloroplast DNA (cpDNA) regions revealed no phylogeographic structure among the populations. We also found that the eastern populations of blue honeysuckle had significantly greater genetic diversity parameters than the populations from the Baltic region. This finding correlates with the endangered status of blue honeysuckle in the Baltic states.

Soil-surface genotoxicity of military and urban territories in Lithuania, as revealed by Tradescantia bioassays.

The soil surface is a major natural system that accumulates pollutants and allows researchers to disclose the history and the present state of contamination of an area with toxic pollutants. This conclusion is based on the comparison of genotoxicity of aqueous extracts and DMSO extracts of topsoil from military territories in various locations left behind after the retreat of the Soviet Army from Lithuania, and several sites in the city of Vilnius, characterized by different history and current traffic intensity. The specific character of the soil-surface contamination was shown in a series of Tradescantia micronucleus (Trad-MN) and stamen-hair mutation (Trad-SHM) bioassays and tests. The most effective ones were the Trad-MN and, unexpectedly, the branched-hair tests. A preliminary result of the study is the somaclonal variation of individual Tradescantia plants revealed by the RAPD method of DNA analysis. A comparison of aqueous extracts and DMSO extracts of the soil showed the permanent character of the mobile forms of genotoxic pollutants in the soil surface, despite the fact that several military territories were already closed 20 years ago.

Genetic Diversity of Aquatic Ranunculus (Batrachium, Ranunculaceae) in One River Basin Caused by Hybridization.

Aquatic Ranunculus (sect. Batrachium) include homophyllous and heterophyllous plants. The development of floating leaves may be induced by genetic mechanisms or/and environmental conditions and this fact complicates the morphologically based identification of species. DNA-based studies provide the opportunity to expand the knowledge of this complicated group. We studied heterophyllous Ranunculus with well-developed capillary and intermediate leaves and visually homophyllous plants with capillary leaves from a single river basin, with the aim to evaluate their genetic polymorphism and taxonomic status-whether the plants with well-developed and weakly expressed intermediate leaves belong to different forms (taxa) or if they just express morphological variation of one or two taxa in a specific, very variable river environment. The studied heterophyllous and homophyllous plants from different rivers showed high genetic differentiation and a low level of genetic diversity within these groups. The molecular analysis did not reveal any inter simple sequence repeat (ISSR) polymorphism associated with the development of intermediate leaves. An analysis of nuclear ribosomal internal transcribed spacers ITS1-2 sequences revealed several ribotypes, which indicated the genetic heterogeneity of studied plants and indirectly confirmed the hybrid origin of some of them. Sterile plants originated from crossing of R. circinatus and R. penicillatus were discovered in the Skroblus River; however, identification of the parental species was impeded by the polymorphism detected. For this reason, cytological studies were performed and allowed confirmation of the hybrid origin of these plants.

Cyto/genotoxicological evaluation of hot spots of soil pollution using Allium bioassays in relation to geochemistry.

Soil from industrial and landfill sites affected by anthropogenic activity was screened for implicit negative effects in an Allium test-system in relation to geochemistry. The concentrations of 15 elements were compared to the ecotoxicologically-based soil guideline values. Admitted geoindices were used to classify test-soils according to risk/hazard categories. Test-soils were screened for the possible deleterious effects in common onion (Allium cepa L.) by employing a test battery of cytogenetic bioassays (root growth inhibition, mitotic activity, frequency of chromosome aberrations and micronuclei, and cell death rate) complemented with two assays of molecular DNA markers, random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR). Soil from industrial sites was more severely polluted and more cytotoxic for onions compared to soil from landfill sites. However, the cyto/genotoxic outcome of soil exposure in A. cepa was the same for all test-soils; the detrimental effects were observed in onions treated with every test-soil. Thus, test-soils could not be classified as non- and genotoxic, although certain of them had permissible contamination levels. The chromosome aberration frequency and cell death rates were consistent with the intensity of soil contamination, contrary to the micronuclei rate, which was independent of the soil risk/hazard level. Despite a relationship between risk (RI) and total soil contamination (Z) geoindices, both indices correlated with a different Allium cyto/genotoxicity endpoint, although the Z index was preferred over the RI index as being more informative in correlation analysis. Allium bioassays complemented each other by depicting different aspects of exposure to toxic substances, and determination of cyto/genotoxicity in a battery of different bioassays is important in the risk assessment of ecologically dangerous soils, and an application of a test battery is strongly advised.

Evaluation of the Inter- and Intrahospital Spread of Multidrug Resistant Gram-Negative Bacteria in Lithuanian Hospitals.

Spread of multidrug-resistant pathogenic bacteria became one of the greatest threats in healthcare worldwide. It is generally accepted that both inter- and intrahospital transmissions of these bacteria contribute significantly to this problem. The purpose of the current study was the evaluation of the inter- and intrahospital spread of multidrug resistant Gram-negative pathogenic bacteria in Lithuania. Clinical isolates of Acinetobacter sp., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were subjected for the screening for extended spectrum ß-lactamase, carbapenemase, as well as plasmid-mediated AmpC ß-lactamase genes. BOX-PCR genotyping was used for the genotyping of these isolates. Our results show that all four pathogens are involved in the intra- and/or interhospital dissemination between the Lithuanian healthcare institutions. The level of transmissions differed between pathogens, and the worst situation was detected for Acinetobacter sp. followed by E. coli. In almost all cases, transmissible strains had at least one gene conferring ß-lactam resistance, thereby contributing to the dissemination of the resistance determinants in and between Lithuanian hospitals. Our study clearly demonstrated that immediate actions, more effective strategy, and surveillance are needed to confine and prevent further spread of multidrug resistant Gram-negative pathogenic bacteria in Lithuanian healthcare institutions.

Response of Tradescantia plants to oxidative stress induced by heavy metal pollution of soils from industrial areas.

Numerous investigations have demonstrated that even soil in which concentrations of individual elements do not exceed permissible limits can cause harmful effects in living organisms. In the present study, polluted-soil-induced oxidative stress was evaluated using Tradescantia clone 4430, which is widely used for genotoxicity evaluations, employing biochemical (superoxide dismutase (SOD), contents of ascorbic acid (AA), carotenoids (Car), hydrogen peroxide (H2O2), chlorophyll (Chl) a/b ratio), and molecular (RAPD and differential display (DD-PCR)) markers after long-term exposure. The activity (staining intensity) of SOD isoforms in Tradescantia leaves was higher in plants grown in all heavy-metal-polluted test soils compared to the control. No direct link between the soil pollution category and the contents of AA, Car, Chl a/b in Tradescantia leaves was revealed, but the concentration of H2O2 was shown to be a sensitive biochemical indicator that may appropriately reflect the soil contamination level. Both short-term (treatment of cuttings with H2O extracts of soil) and long-term (0.5 and 1.0 year) exposure increased MN frequencies, but the coincidence of the MN induction and the soil pollution level was observed only in some cases of long-term exposure. Soil (geno)toxin-induced polymorphism in the RAPD profile was determined with two primers in plants after long-term exposure to soils of an extremely hazard category. Transcript profiling of plants after long-term cultivation in test soils using DD-PCR showed that the majority of differentially expressed transcript-derived fragments (TDFs) were homologous to genes directly or indirectly participating in photosynthesis, the abiotic stress response, and signal transduction cascades.

Metal bioaccumulation and mutagenesis in a Tradescantia clone following long-term exposure to soils from urban industrial areas and closed landfills.

Soil mutagens, particularly metals, may persist long after the source of pollution has been removed, representing a hazard to plants, animals, and humans in or near contaminated areas. Often, due to urban growth, previous land uses may be forgotten and hazards overlooked. We exposed Tradescantia clone #4430 plants to soil from two industrial areas (with different former uses) and two urban waste landfills in the city of Vilnius, all of which were long disused. Two modes of exposure were used: long-term exposure of growing plants in test soils for 0.5 or 1.0y, and short-term exposure of cuttings to water and dimethyl sulfoxide (DMSO) soil extracts. An increased frequency of micronuclei (MN) was observed with both modes of exposure. The concentrations of 24 metals and other elements were analyzed in the test soils and in above-ground plant parts, under both exposure modes, and the concentration coefficients (Cc) for various elements, the total contamination index (Zs) for soils and plants, and the bioaccumulation factor (BAF) for plants were calculated. These measurements allow a comparison of the contamination levels of soils and plants with equalized values. Metal accumulation levels in plants and soils showed significant differences, providing a better understanding of the genotoxicity of soils from closed landfills and highlighting the need to determine the concentrations of metals and other genotoxicants in plants in relation to genotoxicity.

Use of Tradescantia clone 4430 for direct long-term soil mutagenicity studies.

Soil mutagens permanently and directly affect terrestrial plants, soil microorganisms and invertebrates and indirectly impact vertebrate and human populations. However, the dynamic response to soil mutagens under conditions of long-term exposure has been studied insufficiently. The clonal nature of Tradescantia #4430 and the well-developed associated test systems for evaluation of genotoxicity allow investigation of the permanent effects of direct exposure to the soil not only on plants of the same genotype, but even with the same individual plants, as was done in the present study. Twenty soil samples of different origin were studied in Tradescantia after 0.5, 1 and 2 years of direct exposure to the soil, in order to examine the induction of pink cells (PC), colorless cells (CC) and branched hairs (BH). Additionally, after 0.5 years, micronucleus (MN) formation was assessed, and after 1 year, the variation of random amplified polymorphic DNA (RAPD) markers was evaluated. Nine soil samples were of urban origin, and the others came from different soils from former military grounds. The results of the 0.5-year exposure were compared with the effects of aqueous/DMSO extracts tested in previous work. The long-term exposure period allowed not only a better differentiation of the soils within territories, but also permitted detection of differences between soils of urban and military origin. The results also depended on the test used to evaluate genotoxicity. The 'burst' phenomenon, consisting of a sudden increase of BH variation and, especially, of CC, was observed only in tests of samples from several military sites, mainly after a 2-year exposure. MN formation was less frequent after 0.5 years of exposure compared with the results from aqueous extracts, and this difference was highly significant. The variation of the RAPD markers in a significant portion of the soil samples coincided with a higher level of variation according to other tests (PC, CC, BH), but predicting the genotoxicity of the soils based on their elemental soil composition at the end of the experiment was impossible. At several points where a 'burst' was observed, the concentration of a number of elements was lower. A cumulative effect is hypothesized to explain these observations. Tradescantia clone #4430 is the most suitable system for such studies.

The genetic diversity of barley cultivars from the Baltic States and Belarus, as determined by RAMPs.

A Random Amplified Microsatellite Polymorphism (RAMP) analysis was carried out on 30 barley cultivars from the Baltic states and Belarus. Seven primer combinations produced 60 polymorphic DNA fragments ranging in size from 54 b to 400 b. A Genetic Distance coefficient (GDxy) matrix was generated and a dendrogram constructed using cluster analysis of the unweighted pair-group method of arithmetic averages (UPGMA). The genetic distance between cultivars ranged from 0.067 to 0.714. The results were compared with available pedigree information. The dendrogram did not indicate any clear pattern of division among the barley cultivars based on geographic origin.