Forward-directed genetic screens are extremely powerful in identifying novel genes involved in a specific biological process, including various chromatin regulatory pathways. However, the traditional ways of genetic mapping are time- and cost-demanding. Recently, the whole process was revolutionized by the development of mapping-by-sequencing (MBS) protocols. In MBS, the causal mutations and their positions within genes are identified directly by whole-genome sequencing and bioinformatics analysis of the bulk of mutant plants selected based on the mutant phenotype from a segregating population. MBS increases precision and economizes the mapping. Here, we describe a general protocol and provide practical tips on how to proceed with the mapping-by-sequencing on the example of Arabidopsis forward-directed genetic screen designed to identify mutants sensitive to a specific type of DNA damage. The described protocol is generally applicable to a wide range of genetic screens in various inbreeding species with a reference genome sequence.

• Klíčová slova
• DNA damage repair, DNA-protein crosslinks, Forward genetics, Genetic mapping, High-throughput sequencing, Mapping-by-sequencing, SNP calling, Zebularine,
• MeSH
• Arabidopsis * genetika   MeSH
• fenotyp MeSH
• genom rostlinný MeSH
• mapování chromozomů * metody   MeSH
• mutace MeSH
• sekvenování celého genomu metody   MeSH
• výpočetní biologie metody   MeSH
• vysoce účinné nukleotidové sekvenování * metody   MeSH
• Publikační typ
• časopisecké články MeSH

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

• Klíčová slova
• DNA methylation modulation, DNA methylation profiling, bisulfite sequencing, crop epigenome, immunological techniques, mass spectrometry, next-generation sequencing,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

DNA-protein cross-links (DPCs) are highly toxic DNA lesions consisting of proteins covalently attached to chromosomal DNA. Unrepaired DPCs physically block DNA replication and transcription. Three DPC repair pathways have been identified in Arabidopsis (Arabidopsis thaliana) to date: the endonucleolytic cleavage of DNA by the structure-specific endonuclease MUS81; proteolytic degradation of the crosslinked protein by the metalloprotease WSS1A; and cleavage of the cross-link phosphodiester bonds by the tyrosyl phosphodiesterases TDP1 and TDP2. Here we describe the evolutionary conserved STRUCTURAL MAINTENANCE OF CHROMOSOMEs SMC5/6 complex as a crucial component involved in DPC repair. We identified multiple alleles of the SMC5/6 complex core subunit gene SMC6B via a forward-directed genetic screen designed to identify the factors involved in the repair of DPCs induced by the cytidine analog zebularine. We monitored plant growth and cell death in response to DPC-inducing chemicals, which revealed that the SMC5/6 complex is essential for the repair of several types of DPCs. Genetic interaction and sensitivity assays showed that the SMC5/6 complex works in parallel to the endonucleolytic and proteolytic pathways. The repair of zebularine-induced DPCs was associated with SMC5/6-dependent SUMOylation of the damage sites. Thus, we present the SMC5/6 complex as an important factor in plant DPC repair.

• MeSH
• Arabidopsis * genetika   metabolismus   MeSH
• DNA metabolismus   MeSH
• oprava DNA genetika   MeSH
• poškození DNA MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny metabolismus   MeSH
• sumoylace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• proteiny buněčného cyklu MeSH
• proteiny MeSH

Epigenetic regulation of gene expression is expected to be an important mechanism behind phenotypic plasticity. Whether epigenetic regulation affects species ecophysiological adaptations to changing climate remains largely unexplored. We compared ecophysiological traits between individuals treated with 5-azaC, assumed to lead to DNA demethylation, with control individuals of a clonal grass originating from and grown under different climates, simulating different directions and magnitudes of climate change. We linked the ecophysiological data to proxies of fitness. Main effects of plant origin and cultivating conditions predicted variation in plant traits, but 5-azaC did not. Effects of 5-azaC interacted with conditions of cultivation and plant origin. The direction of the 5-azaC effects suggests that DNA methylation does not reflect species long-term adaptations to climate of origin and species likely epigenetically adjusted to the conditions experienced during experiment set-up. Ecophysiology translated to proxies of fitness, but the intensity and direction of the relationships were context dependent and affected by 5-azaC. The study suggests that effects of DNA methylation depend on conditions of plant origin and current climate. Direction of 5-azaC effects suggests limited role of epigenetic modifications in long-term adaptation of plants. It rather facilitates fast adaptations to temporal fluctuations of the environment.

• MeSH
• azacytidin farmakologie   MeSH
• epigeneze genetická * MeSH
• klimatické změny MeSH
• lidé MeSH
• metylace DNA * MeSH
• rostlinné geny MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azacytidin MeSH

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA-protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

• MeSH
• Arabidopsis MeSH
• cytidin analogy a deriváty   toxicita   MeSH
• DNA vazebné proteiny genetika   MeSH
• DNA-(cytosin-5-)methyltransferasa genetika   MeSH
• heterochromatin účinky léků   metabolismus   MeSH
• léková rezistence MeSH
• membránové transportní proteiny genetika   MeSH
• mutace MeSH
• mutageny toxicita   MeSH
• proteiny buněčného cyklu genetika   MeSH
• proteiny huseníčku genetika   MeSH
• RNA ribozomální účinky léků   genetika   MeSH
• transkripční faktory genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AT4G05120 protein, Arabidopsis MeSH Prohlížeč
• cytidin MeSH
• DDM1 protein, Arabidopsis MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH
• heterochromatin MeSH
• membránové transportní proteiny MeSH
• MET1 protein, Arabidopsis MeSH Prohlížeč
• mutageny MeSH
• proteiny buněčného cyklu MeSH
• proteiny huseníčku MeSH
• pyrimidin-2-one beta-ribofuranoside MeSH Prohlížeč
• RNA ribozomální MeSH
• RNA, ribosomal, 45S MeSH Prohlížeč
• SMC6B protein, Arabidopsis MeSH Prohlížeč
• transkripční faktory MeSH

Effective microspore embryogenesis (ME) requires substantial modifications in gene expression pattern, followed by changes in the cell proteome and its metabolism. Recent studies have awakened also interest in the role of epigenetic factors in microspore de-differentiation and reprogramming. Therefore, demethylating agent (2.5-10 μM 5-azacytidine, AC) together with low temperature (3 weeks at 4 °C) were used as ME-inducing tiller treatment in two doubled haploid (DH) lines of triticale and its effect was analyzed in respect of anther protein profiles, expression of selected genes (TAPETUM DETERMINANT1 (TaTPD1-like), SOMATIC EMBRYOGENESIS RECEPTOR KINASE 2 (SERK2) and GLUTATHIONE S-TRANSFERASE (GSTF2)) and ME efficiency. Tiller treatment with 5.0 µM AC was the most effective in ME induction; it was associated with (1) suppression of intensive anabolic processes-mainly photosynthesis and light-dependent reactions, (2) transition to effective catabolism and mobilization of carbohydrate reserve to meet the high energy demand of cells during microspore reprograming and (3) effective defense against stress-inducing treatment, i.e. protection of proper folding during protein biosynthesis and effective degradation of dysfunctional or damaged proteins. Additionally, 5.0 µM AC enhanced the expression of all genes previously identified as being associated with embryogenic potential of microspores (TaTPD1-like, SERK and GSTF2).

• MeSH
• azacytidin farmakologie   MeSH
• embryonální vývoj * genetika   MeSH
• proteom * MeSH
• proteomika * metody   MeSH
• regulace genové exprese u rostlin účinky léků   MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• triticale účinky léků   genetika   metabolismus   MeSH
• výpočetní biologie metody   MeSH
• vývoj rostlin účinky léků   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azacytidin MeSH
• proteom * MeSH
• rostlinné proteiny MeSH

Herbicide resistance is broadly recognized as the adaptive evolution of weed populations to the intense selection pressure imposed by the herbicide applications. Here, we tested whether transcriptional gene silencing (TGS) and RNA-directed DNA Methylation (RdDM) pathways modulate resistance to commonly applied herbicides. Using Arabidopsis thaliana wild-type plants exposed to sublethal doses of glyphosate, imazethapyr, and 2,4-D, we found a partial loss of TGS and increased susceptibility to herbicides in six out of 11 tested TGS/RdDM mutants. Mutation in REPRESSOR OF SILENCING 1 (ROS1), that plays an important role in DNA demethylation, leading to strongly increased susceptibility to all applied herbicides, and imazethapyr in particular. Transcriptomic analysis of the imazethapyr-treated wild type and ros1 plants revealed a relation of the herbicide upregulated genes to chemical stimulus, secondary metabolism, stress condition, flavonoid biosynthesis, and epigenetic processes. Hypersensitivity to imazethapyr of the flavonoid biosynthesis component TRANSPARENT TESTA 4 (TT4) mutant plants strongly suggests that ROS1-dependent accumulation of flavonoids is an important mechanism for herbicide stress response in A. thaliana. In summary, our study shows that herbicide treatment affects transcriptional gene silencing pathways and that misregulation of these pathways makes Arabidopsis plants more sensitive to herbicide treatment.

• Klíčová slova
• 2,4-D, ROS1, chromatin mutants, epigenetics, glyphosate, herbicide resistance, imazethapyr,
• MeSH
• acyltransferasy genetika   MeSH
• Arabidopsis účinky léků   genetika   MeSH
• chromatin chemie   MeSH
• demetylace DNA MeSH
• genetická transkripce MeSH
• herbicidy farmakologie   MeSH
• jaderné proteiny genetika   MeSH
• kyselina 2,4-dichlorfenoxyoctová farmakologie   MeSH
• kyseliny nikotinové farmakologie   MeSH
• metylace DNA MeSH
• mutace MeSH
• proteiny huseníčku genetika   MeSH
• regulace genové exprese u rostlin * MeSH
• RNA rostlin genetika   MeSH
• sekvenování transkriptomu MeSH
• umlčování genů * MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acyltransferasy MeSH
• AT5G13930 protein, Arabidopsis MeSH Prohlížeč
• chromatin MeSH
• herbicidy MeSH
• imazethapyr MeSH Prohlížeč
• jaderné proteiny MeSH
• kyselina 2,4-dichlorfenoxyoctová MeSH
• kyseliny nikotinové MeSH
• proteiny huseníčku MeSH
• RNA rostlin MeSH
• ROS1 protein, Arabidopsis MeSH Prohlížeč

The Venus flytrap (Dionaea muscipula J. Ellis) is a carnivorous plant able to synthesize large amounts of phenolic compounds, such as phenylpropanoids, flavonoids, phenolic acids, and 1,4-naphtoquinones. In this study, the first genetic transformation of D. muscipula tissues is presented. Two wild-type Rhizobium rhizogenes strains (LBA 9402 and ATCC 15834) were suitable vector organisms in the transformation process. Transformation led to the formation of teratoma (transformed shoot) cultures with the bacterial rolB gene incorporated into the plant genome in a single copy. Using high-pressure liquid chromatography, we demonstrated that transgenic plants were characterized by an increased quantity of phenolic compounds, including 1,4-naphtoquinone derivative, plumbagin (up to 106.63 mg × g-1 DW), and phenolic acids (including salicylic, caffeic, and ellagic acid), in comparison to non-transformed plants. Moreover, Rhizobium-mediated transformation highly increased the bactericidal properties of teratoma-derived extracts. The antibacterial properties of transformed plants were increased up to 33% against Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli and up to 7% against Pseudomonas aeruginosa. For the first time, we prove the possibility of D. muscipula transformation. Moreover, we propose that transformation may be a valuable tool for enhancing secondary metabolite production in D. muscipula tissue and to increase bactericidal properties against human antibiotic-resistant bacteria. KEY POINTS: • Rhizobium-mediated transformation created Dionaea muscipula teratomas. • Transformed plants had highly increased synthesis of phenolic compounds. • The MBC value was connected with plumbagin and phenolic acid concentrations.

• Klíčová slova
• Phenolic acids, Plumbagin, Rhizobium rhizogenes, Teratomas, Venus flytrap,
• MeSH
• Agrobacterium genetika   MeSH
• antibakteriální látky farmakologie   MeSH
• Droseraceae * MeSH
• fenoly MeSH
• lidé MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky MeSH
• fenoly MeSH