Is auxin involved in the induction of genetic instability in barley homeotic double mutants?

MAIN CONCLUSION: The triggers of genetic instability in barley homeotic double mutants are tweaky spike -type mutations associated with an auxin imbalance in separate spike phytomeres. Barley homeotic tweaky spike;Hooded (tw;Hd) double mutants are characterized by an inherited instability of spike and flower development, which is absent in the single parental constituents. The aim of the present study was to show that the trigger of genetic instability in the double mutants is the tw mutations, which are associated with an auxin imbalance in the developing spikes. Their pleiotropic effects on genes related to spike/flower development may cause the genetic instability of double mutants. The study of four double-mutant groups composed of different mutant alleles showed that the instability arose only if the mutant allele tw was a constituent of the double mutants. Application of auxin inhibitors and 2,4-dichlorophenoxyacetic acid (2,4-D) demonstrated the relationship of the instability of the double mutants and the phenotype of the tw mutants to auxin imbalance. 2,4-D induced phenocopies of the tw mutation in wild-type plants and rescued the phenotypes of three allelic tw mutants. The differential display (dd-PCR) method allowed the identification of several putative candidate genes in tw that may be responsible for the initiation of instability in the double mutants by pleiotropic variations of their expression in the tw mutant associated with auxin imbalance in the developing spikes. The results of the present study linked the genetic instability of homeotic double mutants with an auxin imbalance caused by one of the constituents (tw). The genetic instability of the double mutants in relation to auxin imbalance was studied for the first time. A matrocliny on instability expression was also observed.

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.

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.

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.

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.

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.