Involvement of DNA polymerase (pol) enzymes in meiotic DNA repair has not been clearly understood in plants. DNA polymerase λ is involved in short patch DNA synthesis and base excision repair in both plants and animals. The presence and activity of the pol λ enzyme was studied in a protein isolated from spikelets during flower development stages of rice (Oryza sativa L.) and maize (Zea mays L.). Western blot analysis showed a 2- to 2.5-fold higher accumulation of pol λ in spikelets than in shoots. Assays of pol and in-gel activity showed the dideoxynucleotide triphosphate sensitive pol λ enzyme in spikelets of both the plants. An enhanced presence of the enzyme and its high activity suggests an active role of pol λ in meiotic recombination during microspore development.

An inhibitor of DNA methylation 5-azacytidine (5A) is a chemical analog of the nucleoside cytidine. This study investigated the influence of 5A-induced DNA hypomethylation on the accumulation of somatic DNA mutations (nucleotide substitutions, indels) in the Actin2 3′ untranslated region, nuclear internal transcribed spacer ITS1-5.8rRNA-ITS2, and the ribulose-1,5-bisphosphate carboxylase/oxygenase gene of Arabidopsis thaliana and analyzed concurrent changes in the expression of DNA methyltransferase and DNA repair genes. The 5A treatment (20 mg per 100 g of soil) decreased DNA methylation, and the detected 5A-induced demethylation was associated with the up-regulation of the DNA methyltransferase genes: chromomethylase AtCMT3, methyltransferase AtMETI, and domains rearranged methyltransferases AtDRM1 and AtDRM2. Cultivation of plants in the presence of 5A led to a considerable increase in the number of single nucleotide substitutions in the analyzed DNA regions of 5A-treated A. thaliana. The 5A treatment significantly increased the transcriptions of 7 DNA repair genes (endonuclease AtARP, DNA demethylases AtDME and AtDML2, DNA glycosylase AtMBD4, DNA damage-binding protein AtDDB1, and photolyases AtUVR2 and AtUVR3) out of the 17 analyzed genes from the base excision repair, nucleotide excision repair, and photoreactivation pathways. However, 5A decreased the transcription of DNA 3′-phosphatase AtZDP, DNA repair protein AtRad23a, mismatch repair proteins AtMsh2 and AtMsh3. It is possible that the changes in the transcription of the DNA repair genes contributed to the detected increase in the number of single nucleotide substitutions that accumulated in the 5A-treated A. thaliana. Taken together, the data indicate that there is an interaction between the processes of DNA methylation and mutation accumulation.

The objective of this study is to understand the role of bark water uptake in refilling of xylem vessels after embolism in Salix matsudana. Further, we determined expression of genes closely related to xylem embolism repair. The isolated branch segments of S. matsudana were soaked in deionized water and after 2 h, the micro-computed tomography (CT) images, volume of xylem sap, osmotic potential of xylem sap, and the content of starch and soluble sugar were measured. At the same time, Illumina high-throughput sequencing (Hi Seq TM) was applied to obtain the xylem transcriptome profiling. The results showed that the soaked branches had 58.86 % fewer embolized vessels than branches before soaking. The volume of xylem sap was increased by 21.51 % in comparison with initial sample and the osmotic potential (Ψs) of xylem sap was decreased by 87.18 %. The content of ions and soluble sugars in the xylem sap significantly increased after soaking. Fourteen genes were clarified and found to have a significant correlation with osmotic potential of xylem sap. Our findings showed that water can enter the xylem vessels through the bark and can repair the xylem embolism in branches. The saccharides played an important role in osmotic potential difference formation and the genes UXL1 (uridine diphosphate-xylose synthase 1), UGDH (uridine diphosphate-glucose 6-dehydrogenase), BGLU40 (β-glucosidase 40), PPE8B (pectinesterase/pectinesterase inhibitor), UG1 (uridine diphosphate-glucuronate 1), and XYL4 (β-D-xylosidase 4)) were the key genes to promote embolized vessels refilling.

The Czech Plant Nucleus Workshop 2021 (CPNW2021) took place during mid-September 2021 in Olomouc, Czech Republic. About 80 researchers and students working in the field of plant nuclear and chromosome biology in the Czech Republic gathered together to present and discuss their current research. The meeting revealed many plant models that are used to study plant genomes and their organization, and also a great diversity of topics including epigenetic regulation of gene expression, genome stability, telomere biology, or sex chromosomes. CPNW2021 provided a broad platform for establishing new research contacts and collaborations. Here, we summarize the main research directions and findings presented at the CPNW2021 meeting.

Paspalum fasciculatum Willd. ex Flüggé grows in mining soils which are Cd- and Pb-contaminated where it exhibits tolerance to Pb and the ability to extract Pb from these soils. To elucidate tolerance mechanisms to Pb-stress, liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to quantify changes in the accumulation of proteins in leaves. We identified 323 proteins involved in primary metabolism and response to biotic or abiotic stresses. Although proteins involved in the processes of photosynthesis and saccharide and energy metabolism presented the greatest amount of down-regulated proteins, the plant was able to maintain photosynthetic functions and obtain energy to sustain the vital balance. P. fasciculatum based their tolerance on increased antioxidant defenses, improving the protection and repair of proteins and transduction signals to coordinate physiological response to Pb-stress. Our results provide important information to understand the tolerance mechanisms in P. fasciculatum and could be important in future molecular studies on the resistance and accumulation of Pb in plants.

Oxidative stress plays an important role in plant ageing and in response to different stresses. Oxidative DNA damage, unless repaired, may have detrimental consequences and increase genetic instability. Therefore, we determined the role of heat-shock induced oxidative stress on induction and repair of DNA damage in relation to oxidative stress tolerance in senescent tobacco plants. One-month-old (young) and three-month-old (senescent) plants were exposed to 42 °C for 2 and 4 h and left to recover at 26 °C for 24 and 72 h. The progression of senescence was characterized by the lower soluble protein and malondialdehyde content compared to young plants. Immediately after the heat shock, an increase in lipid peroxidation and guaiacol peroxidase activity, as well as DNA damage measured by the Comet assay were induced to higher extent in the young plants than in the senescent ones compared to their respective controls. Moreover, after 24-h recovery, the DNA damage further increased in the young plants whereas tendency of DNA repair was observed in the senescent plants. Upon 72-h recovery, no significant differences were noticed in all parameters studied (regardless of plant age) compared to the controls. The random amplified polymorphic DNA (RAPD) analysis confirmed genetic stability of the tobacco plants during the heat-shock exposures as well as the subsequent recovery periods.

The present work examines global transcriptional and limited metabolic profiling to dissect the signalling pathways associated with gibberellin A₃ (GA₃) induced alterations in tobacco (Nicotiana tabacum L.) under field conditions. Our microarray analysis revealed that GA3 foliar application elicited 7 032 differentially expressed genes (DEGs) with 3 507 and 3 525 genes displaying up- and down-regulation, respectively. These DEGs broadly belong to diverse metabolic pathways: GA signalling and homeostasis, ethylene and auxin biosyntheses, saccharide metabolism, glycolysistricarboxylic acid cycle, terpenoid biosynthesis, shikimate-phenylpropanoid pathway, S-adenosyl methionine cycle, fatty acid biosynthesis, cell wall metabolism, oxidative stress, DNA repair, and stress responses. Transcription validation of representative DEGs was confirmed by real-time quantitative PCR. A relative enhancement in cellular acetyl-CoA pool corroborated with a concomitant increase and decrease in transcriptions as well as activities of histone acetyltransferases and histone deacetylases, respectively. Phytochemical analysis shows an enhanced content of total sugars, reducing sugars, total flavonoids, and phenolics with a concomitant decrease of the content of starch, chlorophylls a and b, and carotenoids. Gas chromatography with mass spectrometry analysis revealed the presence of 30 differential metabolites, mostly belonging to terpenoid biosynthesis pathway.

The link between the MtTdp1α (tyrosyl-DNA phosphodiesterase) gene, involved in the repair of DNA topoisomerase I mediated DNA damage, and the plant defense response has been investigated in MtTdp1α-depleted Medicago truncatula transgenic lines obtained by intron-spliced hairpin RNA approach, compared to the control line (CTRL, empty vector). Reduction of cuticle permeability highlighted by chlorophyll efflux assays positively correlated with the level of MtTdp1α gene silencing. The increased cuticle thickness was confirmed by transmission electron microscopy, which revealed an apparent expansion of the epicuticular waxes deposited on the outer surface. RNA-Seq analysis, carried out in the MtTdp1α-depleted plants, revealed the different expression of resistance (R) genes, PAMP (pathogen-associated-molecular pattern) triggered immunity (PTI) genes and transcription factors (TFs) involved in the regulation of the plant defense response.

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.

Immunocytochemical analysis using antibody raised against human H2AX histones phosphorylated at serine 139 (γ-H2AX) demonstrates that root meristem cells of Vicia faba exposed to UV-radiation or incubated with hydroxyurea (HU) reveal discrete foci at the border of the nucleolus and perinucleolar chromatin or scattered over the whole area of cell nucleus. Western blots detected only one protein band at the position expected for the phosphorylated form of H2AX. The dose-effect relationship was demonstrated following treatment with 2.5 and 10 mM HU. Proteins extracted from root meristems incubated for 2 h either with HU and caffeine or with HU and sodium metavanadate showed unchanged amounts of bound γ-H2AX antibodies, as compared to root meristems treated with 2.5 mM HU. Higher quantities of phosphorylated H2AX histones were detected in proteins extracted from roots treated with HU and 2-aminopurine. All treatments were effective in producing evident aberrations of premature mitosis: broken and lagging chromatids, acentric fragments, chromosomal bridges and micronuclei. Our results show that phosphorylation of H2AX at the carboxy-terminal Ser-Gln-Glu sequence is among the earliest responses to double-strand breaks and, presumably, one of the key ATM/ATR-dependent signals indispensable for the repair of spontaneous and induced DNA damage in plant cells.

The effect of five 3-(2-alkoxyphenylcarbamoyloxy)chinuclidium chlorides (alkoxy = butoxy-octyloxy) on survival of a wild-type strain and repair-deficient strains ofChlamydomonas reinhardtii was studied. There was a direct relationship with increased toxic effects in the algal strains as a function of the elongation of the alkyl chain of the alkoxy substituents of the phenylcarbamate acid derivatives. Repairdeficient strains were more sensitive than the wild-type strain. The recombination-deficient strain uvs10 expressed the highest sensitivity to the test agents. This suggests that a gene responsible for recombination repair is involved in an important role in DNA repair of damages induced inC. reinhardtii by the phenylcarbamic esters.

X-rays at doses between 2.5 and 20 Gy were applied to Allium cepa L. bulbs containing either dormant root primordia (before water imbibition) or actively proliferating meristems. Irradiation of the primordia that were enriched in G₀ cells neither delayed proliferation onset nor root sprouting. Under both protocols, irradiation decreased the final length of the roots to about 60 % (at 20 Gy) of that reached by the unirradiated controls. Irradiation of the proliferating meristems increased the mitotic index at some fixation times. This could not be due to a rise in the cell entry into mitosis, as the rate of root growth decreased simultaneously. The increased mitotic index should be the consequence of a delay in the relative time taken by mitosis in the whole cycle time. Lengthened mitosis probably allows the post-replicative repair of most DNA lesions, as the frequency of interphases with micronuclei was higher in the cells which were irradiated when still dormant than in those irradiated when cycling. Thus, the mitotic delays should be the consequence of a checkpoint pathway activated by the presence of DNA damage. This feedback mechanism seems only to develop after cell proliferation is restored.

This paper compares the repair of DNA single strand breaks (ssb) induced by γ-radiation in two strains ofChlamydomonas reinhardtii (137C/+/ and UVS-I) and three lines ofPisum sativum (NN 131, 198, 140) differing in the degree of radioresistance. DNA ssb in cells exposed to γ-rays (50, 100, 200, 500 Gy) were measured by electrophoresis and alkaline unwinding method with subsequent chromatography on hydroxyapatite immediately after irradiation and after 30 min of post-irradiation incubation at 25°C. An increase of double-strand DNA (in%) was found in cells after 30 min post-irradiation incubation.C. reinhardtii strains displayed an equal level of DNA degradation and repair efficiency in the DNA single strand breaks. The radioresistant line N 198 ofP. sativum is characterized by a lower level of induced DNA ssb and higher efficiency of repair of these breaks as compared with less radioresistant lines NN 131 and 140.

Osmotic priming of aged onion seeds with 25% polyethylene glycol-8000 for 5 d resulted in a marked increase in the rate of germination and early seedling growth. Priming reduced electrolyte leakage as well as lipid peroxidation in seeds implying the activation of membrane repair processes. Priming was also associated with increased levels of antioxidants,i.e. ascorbic acid and tocopherols particularly the latter and the activities of catalase and peroxidase involved in the mitigation of oxidative damage. In comparison with the priming of unaged seeds, the aged seeds experienced a diminution of response in terms of changes in the levels of antioxidants and scavenging enzymes.

The protein coding region of theE. coli DNA repair geneada combined with the CaMV 35S promoter has been transferred to tobacco by means ofAgrobacterium tumefaciens Ti plasmid. In transgenic plants having theada gene in a sense orientation, detectable amounts of O⁶-alkylguanine-DNA-alkyltransferase has been found whereas in non-transformed plants this activity is absent. Cell suspension cultures derived from the former plants showed lower sensitivity to the toxic (growth inhibiting) effects of the bifunctional alkylating agent 1-(2-chloroethyl)-1-nitroso-3-(aminomethyl-1,3-diazinylo)-methylurea compared with cell cultures derived from a control non-transformed plant or from transgenic plants harbouring theada gene in an opposite, non-sense orientation.

The main scientific results achieved in individual departments of the Institute of Experimental Botany during 30 years of its existence are briefly summarized. They include methods of studying photosynthesis, ontogenetic changes of photosynthetic characteristics, stress factors affecting photosynthetic activities, photosynthesis of transgenic plants and duringin vitro cultivation, roles of auxins and cytokinins in plant growth and development, development and testing of new plant growth regulators, models of organogenesisin vitro, metabolic and mutagenic activities of phenolic substances, hormonal regulation of flowering, activities of promutagens (nitrosamines, 7,12-dimethylbenzanthracene), model systems of genetic damage, repair synthesis and post-replication repair, developmental pollen biology and biotechnology, extracellular nucleolytic activity of pollen, selection of apple scab immune cultivars of apple tree, chemotaxonomy ofFabaceae andAllium species, selection pressures in embryoids, somatic embryogenesis and nuclear genome changes in plant cell and callus cultures, discoveries of new plant viruses, virus spread and persistence in crops, development of polyclonal and monoclonal antibodies, role of oxidative pentosephosphate cycle in biosynthesis of viral RNA, and virus diseases of forest trees.

The effect of potassium administration in early germination of wheat (Triticum durum L., cv. Appulo) was studied by incubating isolated embryos at different viability levels with 1 mM KC1. The stimulation of germination and growth in aged embryos by K⁺ was accompanied by a significant increase in (³H) leucine incorporation into protein and in phospholipid synthesis as evidenced by TLC analysis of embryo phospholipids extracted after 24 h of imbibition. The metabolic improvement mediated by the cation would in turn repair aging damage occurring at subcellular sites including membranes.

The possibility of using a combination of the mutation process with the induction of the repair processes has been studied to increase the mutation frequencies in algal populations after UV-treatment. From this study it follows that the repair process induced by visible light is much more effective than the dark repair processes in the chlorococcal algae used. In these algae, visible light perhaps does not induce only those repair processes which affect their DNA, but probably also some recovery ones which affect their damaged structures and physiological functions. A suitable combination of the sensitization of algal cells by a DNA-base analogue before UV-treatment and the induction of the light repair and recovery processes resulted in a rather high increase of viable mutations in chlorococcal algae. These findings may be useful in the breeding of chlorococcal algae, which have no possibility of hybridization (except somatic).

The effect of exogenous DNA of syngeneic origin on the course of reparation of meristematic cells ofVicia faba primary roots followed after treatment with monofunctional alkylating agent, ethyl methanesulphonate (EMS), was tested. Time course of alternations in mitotic activity of investigated cellular population and the dynamics of formation of postmetaphase chromosomal aberrations was evaluated. A reparation of damaged cells was significantly supported by syngeneic DNA; its application induced an increased incidence of cellular division already in the early intervals of the repair which was accompanied by concomitant decrease of microscopically detectable rupture in the chromosomes. The study performed on the localization of induced damages occurring in metaphase chromosomes pointed out an increased sensitivity of small chromosomes ofVicia faba to EMS. Similarly, a reparative action of syngeneic DNA was exhibited by significant decrease of aberrations frequency, predominantly in the same chromosomal group. Per cent representation of individual types, not affected by the action of syngeneic DNA, was established by detailed classification of induced aberrations. In both cases, isochromatide breaks were found of greatest predominance.

The sensitization of chlorococcal algae by 5-BdU for the purpose of UV-light mutagenesis was studied. The results obtained were compared with our earlier findings on the sensitization of the same algal strains by 5-BU. No shielding effect of the 5-BdU molecules against UV-light was observed. Probably, the uptake of them from the liquid medium did not result in such excess as compared with the treatment by 5-BU, even if the cells were long enough (24 h) exposed to the concentration of 5-BdU. Likewise, neither stimulating nor inhibiting growth effects on the growing cell colonies were observed after treatment with 5-BdU. The sensitization of the algal cells for UV-light effects was effective in all the experiments. An increased damage of the algal cells by UV-light after sensitization was proved in all the parameters recorded. The frequencies of permanent changes of the cells or their colonies were also increased, but their spectrum did not change significantly. A suitable combination of the 5-BdU sensitization of the cells before their influencing by UV-light and the induction of their repair mechanisms by visible light may decrease the frequencies of the lethal or sublethal damage and increase the frequencies of the useful permanent changes in the characteristics of the chlorococcal algae. The results obtained are discussed from the viewpoint of the regulated mutation process in the breeding of algae.

The mitotic activity of merstematic cells ofVicia faba, the frequency of chromosomal abnormalities and their interchromosomal distribution are evaluated in dependence on the concentration of maleic hydrazide (MH) and on the recovery period. The influence of exogenous DNA of different genetic origin on the course of repair of primary root cells damaged by MH was also studied. Isologous DNA which exhibited a strong repair effect in authors' previous experiments was quite ineffective in the case of maleic hydrazide. Heterologous DNA, on the other hand, had to some extent a parallel effect with MH in breaking down the structural integrity of chromosomes and increased the frequency of aberrations induced by maleic hydrazide in meristematic cells ofVicia faba.

This paper is a study of the effect of exogenous DNA of different genetic origins on the repair of meristematic cells of primary roots ofVicia faba, damaged by 24 hour treatment with 0.01mm solution of Ypenyl. Both kinds of DNA,i.e. isologous and heterologous, stimulated cell proliferation which was decreased by the action of the radiomimetic and influenced both dynamics of production of chromosome aberrations and the interchromosomal distribution of induced damage. While heterologous DNA increased the frequency of aberrations after all recovery periods studied, isologous DNA significantly decreased the number of chromosomal aberrations. Heterologous DNA increased at the same time the relative number of breaks in the group of small chromosomes, while by the action of isologous DNA the number of aberrations related to this group of chromosomes was relatively decreased.

In this paper we compare the influence of heterologous and isologous DNA on the radiation damage repair of primary root meristematic cells ofVicia faba. Roots, irradiated by exposure of 150 r were cultivated at different time intervals either in tap water, or in a solution of heterologous or isologous DNA. In comparing mitotic activity of meristematic cells it was found that both types of DNA studied enhance the recovery of irradiated cells. The frequency of postmetaphase chromosomal aberrations of irradiated cells was influenced also by post-irradiation action of exogenous DNA. While heterologous DNA exhibited synergical effect with radiation in the sense that it increased the post-irradiation incidence of aberrations in all time intervals studied, isologous DNA had a strong repair effect-the application caused a significant decrease of the percentage of post-metaphase aberrations. Both kinds of DNA caused changes in the relation of chromosome to chromatid aberrations; a higher percentage of chromatid aberrations was registered. The study of the distribution of aberrations between large and small chromosomes ofVicia faba showed that the post-irradiation application of heterologous DNA increases damage of small chromosomes while isologous DNA caused an increased repair ability in this chromosomal group.