Sorghum is a crop plant that is grown for seeds, sucrose, forage and biofuel production. In all these applications, lignin is a superfluous component that decreases the efficiency of technological processes. Caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) is an enzyme involved in monolignol synthesis that affects the efficiency of lignification and lignin composition. The sorghum genome harbors one CCoAOMT gene and six closely related CCoAOMT-like genes. The structures of four sorghum CCoAOMT-like enzymes suggest that these proteins might methylate caffeoyl coenzyme A and contribute to monolignol synthesis. In this study, two sorghum genes, CCoAOMT and one CCoAOMT-like, were found to be highly expressed in leaves, stems and immature seeds. The promoters of these genes possess clusters of transcription factor-binding sites specific for lignification, and this suggests that they are important for lignification. Phylogenetic analysis revealed that one sorghum CCoAOMT-like enzyme is closely related to ancestral cyanobacterial CCoAOMT-like proteins. The remaining CCoAOMT-like enzymes, including the one highly expressed in the leaves and stem, are closely related to CCoAOMT. Genes from these two groups possess different, evolutionarily conserved gene structures. The structure of the sorghum CCoAOMT-like protein from the ancestral clade was modeled and differences between enzymes from the two clades were analyzed. These results facilitate a better understanding of the evolution of genes involved in lignification, and provide valuable data for sorghum improvement through traditional breeding or molecular genetic techniques. The findings suggest that CCoAOMT-like genes might be recruited in lignification and raise questions of the frequency of such functional shifts.Electronic supplementary materialThe online version of this article (10.1007/s00438-018-1441-6) contains supplementary material, which is available to authorized users.

Cellular senescence is a tumor-suppressive mechanism blocking cell proliferation in response to stress. However, recent evidence suggests that senescent tumor cells can re-enter the cell cycle to become cancer stem cells, leading to relapse after cancer chemotherapy treatment. Understanding how the senescence reprogramming process is a precursor to cancer stem cell formation is of great medical importance. To study the interplay between senescence, stemness, and cancer, we applied a stem cell medium (SCM) to human embryonic fibroblasts (MRC5 and WI-38) and cancer cell lines (A549 and 293T). MRC5 and WI-38 cells treated with SCM showed symptoms of oxidative stress and became senescent. Transcriptome analysis over a time course of SCM-induced senescence, revealed a developmental process overlapping with the upregulation of genes for growth arrest and the senescence-associated secretory phenotype (SASP). We demonstrate that histone demethylases jumonji domain-containing protein D3 (Jmjd3) and ubiquitously transcribed tetratricopeptide repeat, X chromosome (Utx), which operate by remodeling chromatin structure, are implicated in the senescence reprogramming process to block stem cell formation in fibroblasts. In contrast, A549 and 293T cells cultured in SCM were converted to cancer stem cells that displayed the phenotype of senescence uncoupled from growth arrest. The direct overexpression of DNA methyltransferases (Dnmt1 and Dnmt3A), ten-eleven translocation methylcytosine dioxygenases (Tet1 and Tet3), Jmjd3, and Utx proteins could activate senescence-associated beta-galactosidase (SA-β-gal) activity in 293T cells, suggesting that epigenetic alteration and chromatin remodeling factors trigger the senescence response. Overall, our study suggests that chromatin machinery controlling senescence reprogramming is significant in cancer stem cell formation.

In recent years, the number of metagenomic studies increased significantly. Wide range of factors, including the tremendous community complexity and variability, is contributing to the challenge in reliable microbiome community profiling. Many approaches have been proposed to overcome these problems making hardly possible to compare results of different studies. The significant differences between procedures used in metagenomic research are reflected in a variation of the obtained results. This calls for the need for standardisation of the procedure, to reduce the confounding factors originating from DNA isolation, sequencing and bioinformatics analyses in order to ensure that the differences in microbiome composition are of a true biological origin. Although the best practices for metagenomics studies have been the topic of several publications and the main aim of the International Human Microbiome Standard (IHMS) project, standardisation of the procedure for generating and analysing metagenomic data is still far from being achieved. To highlight the difficulties in the standardisation of metagenomics methods, we thoroughly examined each step of the analysis of the human gut microbiome. We tested the DNA isolation procedure, preparation of NGS libraries for next-generation sequencing, and bioinformatics analysis, aimed at identifying microbial taxa. We showed that the homogenisation time is the leading factor impacting sample diversity, with the recommendation for a shorter homogenisation time (10 min). Ten minutes of homogenisation allows for better reflection of the bacteria gram-positive/gram-negative ratio, and the obtained results are the least heterogenous in terms of beta-diversity of samples microbial composition. Besides increasing the homogenisation time, we observed further potential impact of the library preparation kit on the gut microbiome profiling. Moreover, our analysis revealed that the choice of the library preparation kit influences the reproducibility of the results, which is an important factor that has to be taken into account in every experiment. In this study, a tagmentation-based kit allowed for obtaining the most reproducible results. We also considered the choice of the computational tool for determining the composition of intestinal microbiota, with Kraken2/Bracken pipeline outperforming MetaPhlAn2 in our in silico experiments. The design of an experiment and a detailed establishment of an experimental protocol may have a serious impact on determining the taxonomic profile of the intestinal microbiome community. Results of our experiment can be helpful for a wide range of studies that aim to better understand the role of the gut microbiome, as well as for clinical purposes.

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Transgenic Nicotiana tabacum with cDNA coding for uridine diphosphate glucose and sucrose phosphate phosphatase from Hordeum vulgare expressed from chrysanthemum rubisco small subunit promoter shows high expression of these transgenes in young leaves and low in roots. Although the activity of this promoter decreased during leaves development, even in fully developed leaves it was over 10 times higher than the expression of the native genes of these enzymes. The plants overexpressing the transgenes grew faster and started the generative phase earlier than the control plants, without any disturbances in leaves, flower and seed development. The dry weight of the transgenic plants at the end of the generative phase was slightly higher than in the control group and showed a greater proportion of carbohydrates. The content of lignin, cellulose and hemicellulose was higher in the transgenic plants than in the control plants, and similar differences showed the energy value of these plants. A comparison of the energy value sequestered in the aerial part of these plant showed that the transgenic plants stored up to 18% more energy than the control plants. Mesophyll-specific overexpression of the transgenes showed beneficial effects - faster plants growth and higher accumulation of energy in the transgenic plants than in the controls. This effect was achieved in N. tabacum, a plant without specific storage organs or tissues. The use of a promoter directing expression of transgenes into mesophyll cells allowed to increase the efficiency of the selected metabolic pathway - photosynthesis-dependent sucrose synthesis.