Evaluating the Potential of Using 5-Azacytidine as an Epimutagen

A number of early flowering lines were induced when 5-azacytidine was applied to germinating flax (Linum usitatissimum L.) seed. The genetics of these lines indicate that the induced changes are epigenetic and probably result from demethylation of the genomic DNA at loci that affect flowering age. Although the growth and development of three stable early flowering lines are altered and the percentage of filled seed was reduced in all three lines compared with controls, measures of seed productivity demonstrated that harvest index was unaffected in two of the lines. In the third, harvest index was lower than normal and both seed set per capsule and seed mass per 100 seed were reduced. Furthermore, six generations after induction this line began to display relatively high levels of polyembryony. The late appearance of this twinning and other aspects related to working with lines induced by 5-azacytidine and using 5-azacytidine as an epimutagen are discussed

Influence of soil type and natural Zn chelates on flax response, tensile properties and soil Zn availability

A greenhouse experiment was conducted on weakly acidic and calcareous soils to evaluate the relative efficiencies of three natural Zn chelates [Zn-aminelignosulphonate (Zn-AML), Zn-polyhydroxyphenylcarboxylate (Zn-PHP) and Zn-S,S-ethylenediaminedisuccinate (Zn-S,S-EDDS)] applied to a crop textile flax (Linum ussitatisimum L.) at application rates of 0, 5 and 10 mg Zn kg−1. In the flax plant, the following parameters were determined: dry matter yield, soluble and total Zn concentrations in leaf and stem, chlorophyll, crude fibre, and tensile properties. For the different soil samples, the following parameters were determined: available Zn (DTPA-AB and Mehlich-3 extractable Zn), easily leachable Zn (BaCl2-extractable Zn), the distribution of Zn fractions, pH and redox potential. On the basis of the use of added Zn by flax, or Zn utilization, it would seem recommendable to apply Zn-S,S-EDDS at the low Zn rate in both soils. In contrast, adding the high Zn rate of this chelate to the weakly acidic soil produced an excessive Zn concentration in the plant, which caused a significant decrease in both dry matter yield and chlorophyll content. Furthermore, assessing available Zn with the DTPA-AB method proved the best way of estimating the level of excess Zn in flax plants. The soluble Zn concentration, which was established with 2-(N-morpholino)ethanesulfonic acid reagent (MES), of plant fresh and dry matter could be used as an alternative way of diagnosing the nutritional status of Zn in flax plants. In this experiment, the highest soil pHs were associated with the lowest redox potentials, which coincided with the smallest amounts of available Zn and water soluble Zn in soil, and the lowest levels of Zn uptake by flax plants

Zymographic assay of plant diamine oxidase on entrapped peroxidase polyacrylamide gel electrophoresis. A study of stability to proteolysis

A zymographic assay of diamine oxidase (DAO, histaminase, EC 1.4.3.6), based on a coupled peroxidase reaction, and its behavior at proteolysis in simulated gastric and intestinal conditions, are described. The DAO activity from a vegetal extract of Lathyrus sativus seedlings was directly determined on sodium dodecyl sulfate polyacrylamide electrophoretic gels containing entrapped horseradish peroxidase, with putrescine as substrate of histaminase and ortho-phenylenediamine as co-substrate of peroxidase. The accumulation of azo-aniline, as peroxidase-catalyzed oxidation product, led to well-defined yellow-brown bands on gels, with intensities corresponding to the enzymatic activity of DAO. After image analysis of gels, a linear dependency of DAO content (Coomassie-stained protein bands) and of its enzymatic activity (zymographic bands) with the concentration of the vegetal extract was obtained. In simulated gastric conditions (pH 1.2, 37 °C), the DAO from the vegetal extract lost its enzymatic activity before 15 min of incubation, either in the presence or absence of pepsin. The protein pattern (Coomassie-stained) revealed that the DAO content from the vegetal extract was kept almost constant in the simulated intestinal fluid (containing pancreatin or not), with a slight diminution in the presence of pancreatic proteases. After 10 h of incubation at 37 °C, the DAO enzymatic activity from the vegetal extract was 44.7% in media without pancreatin and 13.6% in the presence of pancreatin, whereas the purified DAO retained only 4.65% of its initial enzymatic activity in the presence of pancreatin

DNA methylation patterns of Brachypodium distachyon chromosomes and their alteration by 5-azacytidine treatment

Sequential immunolocalisation of 5-methylcytosine (5-MeC) and fluorescence in situ hybridisation with chromosome-specific BAC clones were performed on Brachypodium distachyon mitotic metaphase chromosomes to determine specific DNA methylation patterns of each chromosome in the complement. In the majority of cells examined, chromosomes Bd4 and Bd5, which bear the loci of 5S and 35S ribosomal DNA, respectively, had characteristic 5-MeC patterns. In contrast, the distribution of 5-MeC along the metacentric chromosome pairs Bd1, Bd2 and Bd3 was more variable. There were numerous differences in distribution of methylated sites between homologous chromosomes as well as between chromosome arms. Some chromosome sites, such as pericentromeric regions, were highly methylated in all chromosomes. Additionally, the influence of a hypomethylating agent, 5-azacytidine, on B. distachyon chromosome methylation patterns was confirmed. It was found that some chromosome pairs underwent demethylation more easily than others, but there was no apparent regularity in demethylation of particular chromosome segments

Peroxidase activity and relative mobility at anthesis in flax genotrophs and their F₂ progeny: developmental and genetic effects

The genetic regulation of the environmentally induced heritable difference in peroxidase activity between Durrant's large (L) and small (S) flax genotrophs was examined in leaves from plants ranging in developmental age from 6 days before anthesis to 3 days after. Mean peroxidase activity was higher for S than L and intermediate for the reciprocal F2's from L × S and S × L crosses (F2L × S and F2S × L). However, activity increased with development and, since there were small but significant differences in the average developmental ages of L, S, F2L × S, and F2S × L plants, the effects of development on activity had to be taken into account in examining the F2 activity data for segregation. A regression method was used to remove developmental effects and, underlying these effects, total peroxidase activity appeared to be regulated by a single locus with two alleles and L dominance. Two other dimorphic loci, both described previously, were also examined. One regulates the presence-absence of septa hairs in the seed capsules and the other the relative mobility of anionic peroxidase isozymes. There was no phenotypic linkage between the three segregating parameters. The genetic control of activity appeared to regulate cationic rather than anionic activity. In addition, a relationship between activity and plant height indicated either that peroxidase activity is one of the factors regulating main stem elongation or that the locus regulating peroxidase activity is linked to one of the loci involved in the regulation of plant height.Key words: flax genotrophs, peroxidase, genetic control, development. </jats:p

ACTIVITY AND RELATIVE MOBILITY OF PEROXIDASE ISOENZYMES IN GENOTROPHS AND GENOTYPES OF FLAX (<i>LINUM USITATISSIMUM</i>L.)

Acrylamide gel electrophoresis was used to separate anionic peroxidase isoenzymes in genotypes and genotrophs of flax. Activities and relative mobilities were measured directly from the separations on the gels.The effects of growth of one flax genotype in soil supplemented by either nitrogen, phosphorus and potassium (NPK) or nitrogen and potassium (NK) on subsequent generations of its progeny produced by complete selfing were studied. Both activity and relative mobility of anionic peroxidase isoenzymes displayed effects of fertilizer treatments applied in previous generations. NPK increased the relative mobility of all isoenzymes, while depressing the activity of at least three of them. Successive generations of growth in NPK produced approximately linear increases in relative mobility. Such environmentally induced heritable changes were detectable five generations later.Two other flax genotypes were crossed, and relative mobility and activity of anionic peroxidase isoenzymes were examined in both parents and F2progeny. Between parents, there were differences in relative mobility for two of the four isoenzymes; their F2hybrids showed intermediate mobility for these particular isoenzymes. There were no differences between reciprocal F2hybrids for mobility or activity of any isoenzyme. The parents differed in activity in all four isoenzymes; the F2hybrids displayed dominance towards the lower activity parent for each of the isoenzymes.Total anionic isoenzyme activity was highly correlated with gross peroxidase activity measured prior to electrophoretic separation.</jats:p

ACTIVITY AND RELATIVE MOBILITY OF PEROXIDASE AND ESTERASE ISOZYMES OF FLAX (<i>LINUM USITATISSIMUM</i>) GENOTROPHS. II. F₁HYBRIDS AND NUCLEAR DNA REVERSION TYPES

The effects of growth of one genotype of flax in soil supplemented by either nitrogen, phosphorous and potassium (NPK) or nitrogen and potassium (NK) on its progeny produced by several generations of complete selfing were studied. The two types of progeny produced, genotroph L, induced by NPK and genotroph S induced by NK, and their F1reciprocal hybrids were examined. L and S differed in plant weight and in the activities and relative mobilities of their corresponding anionic peroxidase and esterase isozymes. No reciprocal differences were detected and the mean F1values for all characteristics were intermediate between the parents, with the exception of the peroxidase isozyme relative mobilities which displayed dominance of parent L. The genotrophs were known to exhibit a 16% difference in nuclear DNA content which could be reverted to 0%, without altering the genotroph fresh weight difference, by growing the genotrophs in lower than normal temperatures. Examination of the peroxidase and esterase isozymes of nDNA reverted genotrophs showed that, with one possible exception, there was no accompanying reversion of the activities and relative mobilities of the peroxidase or esterase isozymes.</jats:p