Application of polymeric dimethylaminoethyl methacrylate based carriers of plasmid DNA for genetic transformation of Ceratodon purpureus moss

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.

Gene-rich UV sex chromosomes harbor conserved regulators of sexual development

Nonrecombining sex chromosomes, like the mammalian Y, often lose genes and accumulate transposable elements, a process termed degeneration. The correlation between suppressed recombination and degeneration is clear in animal XY systems, but the absence of recombination is confounded with other asymmetries between the X and Y. In contrast, UV sex chromosomes, like those found in bryophytes, experience symmetrical population genetic conditions. Here, we generate nearly gapless female and male chromosome-scale reference genomes of the moss Ceratodon purpureus to test for degeneration in the bryophyte UV sex chromosomes. We show that the moss sex chromosomes evolved over 300 million years ago and expanded via two chromosomal fusions. Although the sex chromosomes exhibit weaker purifying selection than autosomes, we find that suppressed recombination alone is insufficient to drive degeneration. Instead, the U and V sex chromosomes harbor thousands of broadly expressed genes, including numerous key regulators of sexual development across land plants.

Formation of adaptive reactions of Ceratodon purpureus (Hedw.) Brid. via the participation of chlorophylase and nitrogen oxide metabolites

Changes in chlorophylase activity and the content of nitric oxide metabolites in adaptive reactions of gametophyte of the cosmopolitan moss Ceratodon purpureus (Hedw.) Brid under different temperature regime, water deficit, high insolation on the dump of the mine “Nadiia” of Chervonohrad mining district of Lviv region have been studied. It was established that unfavorable conditions for the existence of plants due to lack of moisture, high surface temperature of the substrate and high insolation are created in the summer months on the dumps of coal mines. Due to the degradation of the photosynthetic apparatus in stressful conditions of technogenic-disturbed areas in chloroplasts of C. purpureus, the total content of chlorophylls was 294–413 μg/g of dry matter mass. In the gametophyte of moss in the localities on the terrace of the dump with a high insolation of 100 thousand lux., it was determined higher chlorophylase activity in 1.3 and 2.2 times, compared with the base localities (55 thousand lux.) and the top localities (70 thousand lux.), respectively. Under the influence of significant water stress, which was induced by a 10% solution of polyethylene glycol, a decrease in both the content of photosynthesis pigments and chlorophylase activity was determined compared to the control. The highest content of nitric oxide metabolites was determined on the terrace of dump due to their accumulation under the influence of significant insolation and temperature. The results of experimental studies of the effects of hypo-, hyperthermic and water stresses indicate the time-dependent changes in the content of nitric oxide metabolites in moss cells in the post-stress period. The increase in the NO2- content in the period of post-stress reaction indicates its important role in the adaptation of gametophyte of moss under the influence of abiotic stressors.

Syntaxonomy of plant communitiesat the quarriesafter the cuttingsof lichen and green mosspine forests(middle taiga subzone of the European North-East of Russia)

Plant communities of sandy (quarry 3 — 62°05ʹ26ʺ N, 48°43ʹ56ʺ E; 6 — 61°57ʹ35ʺ N, 50°36ʹ22ʺ E) and sandy loam (1 — 62°06ʹ28ʺ N, 48°48ʹ48ʺ E; 2 — 62°06ʹ19ʺ N, 48°48ʹ42ʺ E; 4 — 62°04ʹ36ʺ N, 48°34ʹ55ʺ E; 8d — 61°44ʹ00ʺ N, 50°39ʹ05ʺ E) quarries and the surrounding lichen and green-moss pine forests quarries are charaterized. The research has been performed in the middle taiga subzone of the European North-East of Russia. Floristic classification (Braun-Blanquet, 1964) was made for 91 relevés. The Ellenberg ecological values (Ellenberg, 1974) were used to assess moisture (F), acidity (R) and nitrogen content (N). Ordination was made by NMS approach using ExcelToR software. Plant communities of renewing and young pine stands at the quarries as well as of surrounding middle aged and mature stands are assigned to the alliance Cladonio stellaris–Pinion sylvestris Kielland-Lund ex Ermakov et Morozova 2011 order Pinetalia sylvestris Oberdorfer 1957 class Vaccinio-Piceetea Br.-Bl. in Br.-Bl. et al. 1939 (Ermakov, Morozova, 2011) while communities of meadow succession stage to the union Cynosurion cristati Tx. 1947 order Arrhenatheretalia elatioris class Molinio-Arrhenatheretea Tx. 1937 (Mirkin, Naumova, 2012). Five associations, two subassociations, one variants, two subvarants and one community are established within two classes (among these, two associations. one variants, two subvariants and one community are new). Community Polytrichum piliferum (Table 3, rel. 1–7). DS: Ceratodon purpureus, Cladonia subulata, C. fimbriata, Niphotrichum canescens, Polytrichum piliferum, Salix acutifolia. These are pioneer communities at the second decade stage of succession on sandy substrate. (quarry 3) with recovering Pinus sylvestris tree layer, absence of herb-dwarf shrub layer and. presence of pioneer mosses (Polytrichum piliferum, Ceratodon purpureus), algal crusts, and lichen thalli of Cladonia, Peltigera, and Stereocaulon. Number of species — 32 (13–20). Ass. Polytricho piliferi–Pinetum sylvestris ass. nov. hoc loco (Table 3, rel. 8–30; nomenclature type (holotypus hoc loco) — rel. 18; Fig. 4). DS: Ceratodon purpureus, Cladonia gracilis ssp. turbinata, C. fimbriata, C. subulata, C. verticillata, Niphotrichum canescens, Peltigera malacea, Polytrichum piliferum, Salix acutifolia, Stereocaulon tomentosum. These are renewing pine forests at 3rd and 5th decades of succession with ground cover dominated by pioneer mosses and lichens in sandy quarries 3 and 6, and in the most drained habitats of sandy loam quarries 1, 2, 4 (slopes). Pine canopy density is 0.1–0.3, its height — 3–6 m. Herb-dwarf shrub layer is scarce (1– 10 %), that of moss-lichen is of 20–80 %. Number of species — 111 (27–45). Subass. Deschampsio–Agrostietum tenuis typicum Turubanova 1986. D S : Agrostis tenuis, Deschampsia cespitosa, Galium mollugo, Hieracium umbellatum, Leucanthemum vulgare, Pimpinella saxifraga, Stellaria graminea. These are communities of short meadow succesion stage at the sandy loam quarries 1, 2, 4, 8d. Unlike meadows of abandoned lands and pastures, described by L. P. Turubanova (1986) as subass. Deschampsio–Agrostietum tenuis typicum, the communities on technogenic habitats have lower abundance of meadow species and higher diversity of synanthropic and xerophylous species, as well as tree invasion. This is a reason to establish variant Calamagrostis epigeios (Table 4, Fig. 5) within the subass. typicum. Its DS: Betula pendula/pubescens, Calamagrostis epigeios, Ceratodon purpureus, Chamaenerion angustifolium, Erigeron acris, Pinus sylvestris, Tussilago farfara. Multispecies herb layer is mainly formed by meadow species and weeds (20–80 %). PC of moss layer — 1–50 %. Number of species — 103 (30–41). Duration of meadow stage is responsible for differences between subvariants Amoria hybrida and Trifolium medium. Ass. Polytricho juniperini–Pinetum sylvestris ass. nov. hoc loco (Table 5, nomenclature type (holotypus hoc loco) — rel. 9; Fig. 6). D S : Agrostis tenuis, Amoria repens, Brachythecium salebrosum, Orthilia secunda, Peltigera didactyla, Polytrichum juniperinum, Pyrola media, Trifolium medium, Vicia sepium. These communities are young pine stands at the third decade of succession on sandy loam in quarries 1, 2, 4, 8d. Canopy density of pines — 0.4–0.8, height — 4–8 m. There is a lot of species typical for meadow succession stage in scarce herb layer (5–40 %). as well as an occurrence of forest dwarf shrubs, and few species indicative to ass. Linnaeo borealis–Pinetum sylvestris. Moss-lichen layer (10–60 %) is formed by pioneer species and forest mosses. Number of species — 137 (27–67). NMS-ordination (Fig. 7) and data on substrate preferences of syntaxa as well as information about the duration of recovery process is the reason to built a scheme of vegetation succession in quarries in place of reduced pine trees after the cutting of lichen and green moss pine forests (Fig. 8). Series 1. Community Polytrichum piliferum (F 4.1, N 3.5, R 3.8) → ass. Polytricho piliferi–Pinetum sylvestris (F 4.1, N 3.5, R 4.1) → ??? → subass. Cladonio arbusculae–Pinetum sylvestris typicum (F 3.8, N 2.5, R 2.4). Series 2. Subvar. Amoria hybrida var. Calamagrostis epigeios subass. Deschampsio–Agrostietum tenuis typicum (F 4.4, N 4.8, R 5.4) → subvar. Trifolium medium var. Calamagrostis epigeios subass. Deschampsio–Agrostietum tenuis typicum (F 4.7, N 4.6, R 4.9) → асс. Polytricho juniperini–Pinetum sylvestris (F 4.5, N 3.9, R 4.3) → ??? → ass. Linnaeo borealis–Pinetum sylvestris (F 4.3, N 3.1, R 2.8). Additional studies are necessary to reveal the duration of succession period and intermediate stages of transition from ass. Polytricho piliferi–Pinetum sylvestris and Polytricho juniperini–Pinetum sylvestris to ass. Cladonio arbusculae–Pinetum sylvestris and Linnaeo borealis–Pinetum sylvestris. Thus, the restoration of lichen pine forests of subass. Cladonio arbusculae–Pinetum sylvestris typicum at sandy quarries passes through the stage of pine forests of ass. Polytricho piliferi–Pinetum sylvestris, which is preceded by pioneer vegetation of сommunity Polytrichum piliferum. The restoration of green moss pine forests (ass. Linnaeo borealis–Pinetum sylvestris) at sandy loam quarries passes through the stage of young pine stands (ass. Polytricho juniperini–Pinetum sylvestris), which is preceded by meadows of subvariantas Amoria hybrida and Trifolium medium var. Calamagrostis epigeios subass. Deschampsio–Agrostietum tenuis typicum.

Identification of biofilm composition covering lime-based materials

This paper aims at identification of biofilms composition taken from lime and lime-cement plasters. The samples of plasters were exposed to natural weathering for more than three years to weather conditions of Prague as a representative of Central European continental climate. After biofilms sampling, they were cultivated using specific nutrient solutions and analysed using various types of microscopes. It was found, that the plasters were covered mostly by green algae (Hematococcus pluvialis, Klebsormidium sp., Trebouxiophyceae, Chlorophyceae and Chlorellaceae), cyanobacteria (Nostoc, Anabaena, Leptolyngbya, Phormidium, Plectonema, Synechococcales and Oscillatoriale), moulds (Aspergillus niger, Alternaria) and moss (Ceratodon purpureus). Possible influence of these organisms on hygrothermal behaviour of the base materials is subsequently discussed.

Lamprospora sylvatica (Pyronemataceae), a new bryophilous ascomycete on Dicranum montanum

Lamprospora sylvatica is described as a new species based on finds from Ukraine, Slovakia, Germany and Norway. It is characterised by the combination of the following features: pinkish, orange to reddish-orange apothecia with a fimbriate margin, globose ascospores with more or less regular areolate ornamentation, infecting strong rhizoids of Dicranum montanum with an infectious structure consisting of a one-celled appressorium surrounded by a multi-layered cluster of thick-walled cells and haustorium within the rhizoids. The apothecia were always found on rotten wood, which is an unusual habitat for hosts of bryophilous Pezizales. The new species is compared to similar taxa morphologically and by means of DNA sequencing. In the phylogenetic analysis based on LSU and ITS regions, L. sylvatica forms a well-supported clade close to L. feurichiana (on Ceratodon purpureus), L. kristiansenii (also on C. purpureus) and L. campylopodis (on Campylopus spp.).