Azacitidine (5-AzaC)-treatment and mutations in DNA methylase genes affect embryogenic response and expression of the genes that are involved in somatic embryogenesis in Arabidopsis

Epigenetic processes including DNA methylation play a pivotal role in regulating the genes that control plant development. In contrast to in planta development, the contribution of DNA methylation to the morphogenic processes that are induced in vitro are much less recognised. Hence, in the present study, we analysed the impact of DNA methylation on somatic embryogenesis (SE) that was induced in Arabidopsis. The results demonstrated a decrease in the global DNA methylation level during SE that contrasted with the up-regulation of MET1 and CMT3 DNA methylases and the down-regulation of DNA demethylases (ROS1, DME and DML2). Hence, the global DNA methylation level appears not to correlate with the transcriptional activity of the genes encoding DNA methylases/demethylases, thereby implying the complexity of the regulatory mechanism that controls the DNA methylation status of the SE-epigenome. Moreover, distinct changes in the expression level of the SE-regulatory genes were indicated in the 5-AzaC-treated and DNA methylase mutant cultures. Accordingly, a significant repression of the LEC2, LEC1 and BBM genes was found in the 5-AzaC-treated culture that was incapable of SE induction. In contrast, the distinct up-regulation of these genes was observed in the drm1drm2 and drm1drm2cmt3 mutant cultures with an improved embryogenic response. The modulated expression of DNA methylase genes and the significantly modified embryogenic response of the met1 and drm mutants imply that both the maintenance and the de novo pathway of DNA methylation are engaged in the regulation of SE in Arabidopsis

Hypermethylation of Auxin-Responsive Motifs in the Promoters of the Transcription Factor Genes Accompanies the Somatic Embryogenesis Induction in Arabidopsis

The auxin-induced embryogenic reprogramming of plant somatic cells is associated with extensive modulation of the gene expression in which epigenetic modifications, including DNA methylation, seem to play a crucial role. However, the function of DNA methylation, including the role of auxin in epigenetic regulation of the SE-controlling genes, remains poorly understood. Hence, in the present study, we analysed the expression and methylation of the TF genes that play a critical regulatory role during SE induction (LEC1, LEC2, BBM, WUS and AGL15) in auxin-treated explants of Arabidopsis. The results showed that auxin treatment substantially a ected both the expression and methylation patterns of the SE-involved TF genes in a concentration-dependent manner. The auxin treatment di erentially modulated the methylation of the promoter (P) and gene body (GB) sequences of the SE-involved genes. Relevantly, the SE-e ective auxin treatment (5.0 M of 2,4-D) was associated with the stable hypermethylation of the P regions of the SE-involved genes and a significantly higher methylation of the P than the GB fragments was a characteristic feature of the embryogenic culture. The presence of auxin-responsive (AuxRE) motifs in the hypermethylated P regions suggests that auxin might substantially contribute to the DNA methylation-mediated control of the SE-involved genes

Compound-specific chlorine isotope fractionation in biodegradation of atrazine

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (ΛArthroCl/C = 1.7 ± 0.9 vs. ΛRhodoCl/C = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (ΛArthroCl/N = −1.2 ± 0.7 vs. ΛRhodoCl/N = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = −4.3 ± 1.8 ) were surprisingly large, whereas in hydrolysis (εCl = −1.4 ± 0.6 ) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways