Efficient regeneration of <Emphasis Type="Italic">Sphagnum fallax</Emphasis> from isolated protoplasts

Summary While the in vitro clonal propagation of peat mosses (Sphagnaceae) in bioreactors has been established since the late 1980s, it has never been possible to regenerate Sphagnum species from isolated protoplasts, which is a key step towards the production of closely defined genetically modified clones. The present study describes an efficient protocol for protoplast isolation and regeneration of Sphagnum fallax. Protoplast survival rates of over 50% and regeneration rates of up to 20% were achieved by using excised capitulum buds as starting material and by co-cultivating Sphagnum protoplasts with protoplasts from a chlorophyll-deficient Solanum hybrid clone. Besides the effects of nutrient components and differential osmotic readjustment of the regenerant cell clusters, the interference of unique Sphagnum phenolics, sphagnum acid and hydroxybutenolide, with protoplast isolation efficiency is demonstrated.     

Characterization of a peroxidase from Sphagnum magellanicum

The peroxidase from Sphagnum magellanicum is strongly inhibited by CN^? and N^3? and is rather heat stable. The pH optimum is 5.0. The peroxidase rapidly degrades the common hydroxycinnamic acids and sphagnum acid to non-phenolic products; t-cinnamic acid itself is not attacked. The significance of these reactions is discussed with respect to earlier investigations on the level of cinnamic acids in Sphagnum magellanicum. The peroxidase consists of five acidic and five basic isoenzymes. This band pattern does not change during the colouring of the moss, so that the peroxidase from the green and the red moss is identical.     

Species composition and structure of testate amoebae community in a sphagnum bog at the initial stage of its formation

A total of 42 species and forms have been revealed in the testate amoebae community of a transitional bog at the initial stage of transformation into a typical sphagnum bog. A distinctive features of its species composition is dominance of widespread species Assulina muscorum, Arcella arenaria, Phryganella hemisphaerica, and Euglypha laevis in the absence of common sphagnobionts of the genera Nebela, Hyalosphenia, and Heleopera. Vertical heterogeneity of the community structure is weakly manifested, since dominant species are abundant in all horizons. The highest species richness is characteristic of the marginal community formed at the boundary between a sphagnum quagmire and a reed grass-dominated fen.     

Facilitation vs. competition: Does interspecific interaction affect drought responses in Sphagnum?

The stress-gradient hypothesis (SGH) predicts that the relative importance of competition decreases and facilitation increases with an increase in abiotic stress. In peatlands, Sphagnum faces the threat of drought and differentiates into hummock species (drought-tolerant) and hollow species. Whether interspecific interaction affects the influence of drought on bryophyte composition in peatlands is unknown. We established an experiment by simulating drought and building bryophyte communities with two hummock species (S. palustre and S. capillifolium) and one hollow species (S. fallax). In all three species, drought decreased biomass production, height increment and side-shoot production. Sphagnum stores water in the hyaline cells, and leaf hyaline cell percentage (HCP) in the two hummock species increased with drought while no effect was found in S. fallax, suggesting that adjusting HCP is not an effective response to drought for the hollow species. Morphological traits and carbon and nitrogen contents in hummock species responded more to drought than in the hollow species, indicating a rapid response in phenotypic plasticity is an important strategy to resist drought in the hummock species. The presence of neighboring Sphagnum species, rather than drought, decreased carbon content for all three species. All three bryophytes showed interaction between drought and neighbor in two or more plant traits. Our study, however, did not support SGH, and there were no changes from competition under wet to facilitation under dry treatments in any of the six species combinations. On the contrary, when S. fallax was the target species, a change from facilitation under wet to competition under dry treatments was observed. The results suggest that hummock species can facilitate hollow species in wet environments but they could suppress hollow species under drought conditions by competing for water resources. Both drought and strong competition are the probable reasons why hollow species rarely grow in hummocks.     

Biochemical status of in vitro regenerated Lilium bosniacum and Lilium cattaniae plantlets

Lilium cattaniae (Vis.) Vis. and Lilium bosniacum (G. Beck) Beck ex Fritsch, endemic species of Balkan Dinaric Alps, were micropropagated from seeds collected from their natural habitats. The relationship between peroxidase activity, photosynthetic status and differentiation of Lilium cattaniae and L. bosniacum in vitro was investigated. Peroxidase activity recorded for somatic embryos of Lilium cattaniae obtained on Murashige and Skoog (MS) medium containing 9.05 mM 2.4-dichlorophenoxyacetic acid (2.4-D) and 4.44 mM N6-bezyladenin (BA), was about two times higher than for any other treatment. Photosynthetic status of plantlets obtained through regeneration was explant-specific and generally higher for plantlets regenerated from basal leaf explants than from bulb explants. The accumulation of anthocyanin was detected in some regenerated shoots and more often in plantlets obtained through regeneration from bulb explants. High frequency of somatic embryo formation was recorded for L. cattaniae on MS medium containing 9.05 mM 2.4-D and 4.44 mM BA. The peroxidase activity for L. bosniacum plantlets obtained through regeneration showed treatment-specific elevations. We consider that basal leaf parts are suitable for successful regeneration of these two lilies and that elevation in peroxidase activity is a good indicator of somatic embryogenesis in both lilies.     

Structure of a community of testate amoebae in a sphagnum dominated bog in upper sura flow (Middle Volga Territory)

Fifty nine species of testate amoebae were found in a sphagnum moss bog located in the upper Sura flow (Volga highland). The active diversity in communities of sphagnum moss biotopes was formed by 24 species. Minimal species diversity (3 species) and maximum density of organisms were noted in the driest habitat. In the habitats with the medium humidity levels, the level of species richness was similar (13–16 species) and the total density was determined by redox characteristics of the environment: under the conditions of oxygen deficit, it was two to three times lower than in an oxidized environment. The species composition specificity in sphagnum moss biotopes was determined by the degree of humidity: in the driest habitats, Arcella arenaria predominated; in the biotopes with medium humidity, Nebela tenella and Hyalosphenia elegans; and in the most humid biotopes, Hyalosphenia papilio and Heleopera sphagni. Communities of testate amoebae differed by species composition and integral characteristics at different sphagnum moss horizons: the abundance and species richness increased with depth. The proportion of living individuals in the total number of tests was 35 to 75%.     

Distribution and development of necroticSphagnum patches in two Estonian raised bogs

Sphagnum mosses dominate the plant cover of boreal bogs and accumulate carbon as peat. However, discoloured necroticSphagnum patches are also common in bogs. NecroticSphagnum inhibits peat accumulation, and consequently these areas may sink with respect to their surroundings with healthy mosses and continuing peat accumulation. Therefore, necrotic patches in the moss carpet could have an important role in triggering the succession ofSphagnum communities and the differentiation of bog microtopography. Our main aim was to find out how necroticSphagnum patches are distributed on a microtopographic gradient and amongSphagnum species. Based on these results we discuss the development and likely role of necrotic patches. It was found that necrotic patches occur on all types of bog microforms and contain the most of commonSphagnum species. Necrotic patches were more common and larger in wet hollows. The development of necrotic patches depends on their location on the microtopographic gradient. Necrotic patches on higher microforms usually re-vegetate, whereas those in hollows can result in mud-bottom hollows.     

The modulation of various physiochemical changes in <Emphasis Type="Italic">Bruguiera cylindrica</Emphasis> (L.) Blume affected by high concentrations of NaCl

Bruguiera cylindrica is a major mangrove species in the tropical mangrove ecosystems and it grows in a wide range of salinities without any special features for the excretion of excess salt. Therefore, the adaptation of this mangrove to salinity could be at the physiological and biochemical level. The 3-month-old healthy plantlets of B. cylindrica, raised from propagules were treated with 0 mM, 400 mM, 500 mM and 600 mM NaCl for 20 days under hydroponic culture conditions provided with full strength Hoagland medium. The modulation of various physiochemical changes in B. cylindrica, such as chlorophyll a fluorescence, total chlorophyll content, dry weight, fresh weight and water content, Na⁺ accumulation, oxidation and antioxidation (enzymatic and non-enzymatic) features were studied. Total chlorophyll content showed very minute decrease at 500 mM and 600 mM NaCl treatment for 20 days and the water content percentage was decreased both in leaf and root tissues with increasing concentration. A significant increase of Na⁺ content of plants from 84.505 mM/plant dry weight in the absence of NaCl to 543.38 mM/plant dry weight in plants treated with 600 mM NaCl was recorded. The malondialdehyde and the metabolites content associated with stress tolerance (amino acid, total phenols and proline) showed an increasing pattern with increasing NaCl concentration as compared to the control in both leaf and root tissues but the increase recorded in plantlets subjected to 500 mM was much less, indicating the tolerance potential of this species towards 500 mM NaCl. The significant decrease of sugar content was found only in 600 mM NaCl on 20 days of treatment, showing that the process of sugar synthesis was negatively affected but the same process remains less affected at 500 mM NaCl. A slight reduction in ascorbate and glutathione content and very less increase in carotenoid content were observed at 500 mM and 600 mM NaCl stress. Antioxidant enzymes (APX, GPX, SOD and CAT) showed an enhanced activity in all the treatments and the increased activity was more significant in 600 mM treated plants. The result establishes that B. cylindrica tolerates high NaCl concentration, to the extent of 500 mM NaCl without any major inhibition on photosynthesis and metabolite accumulation. Understanding the modulation of various physiological and biochemical changes of B. cylindrica at high levels of NaCl will help us to know the physiochemical basis of tolerance strategy of this species towards high NaCl.     

Response curves and the environmental limits for peat-forming species in the northern Andes

Peatlands, with organic soils, are usually dominated by one or a few species. Above and belowground ecological processes are regulated by the characteristics of the dominant species in the peat. Understanding how these species relate to climatic or water chemistry gradients will help to predict the fate of those ecosystems under current climate change. The patterns of abundance and occurrence of 12 major peat-forming species from different structural plant groups were quantified along gradients of elevation, precipitation, and water chemistry. The species were distributed in four major structural groups: cushion plants, true mosses, sedges, and Sphagnum mosses. A response curve for each species was built with Generalized Additive Models. Niche breadths, species tolerances, and species optima were estimated using bootstrap resampling. Our results showed that species were limited in their elevational ranges; Sphagnum species were biased toward low to intermediate elevations, sedges, and true mosses to intermediate elevations and cushion plants to very high elevations. Sphagnum species were more likely to occur in sites with low precipitation, while sedges preferred wet sites. Sphagnum species preferred habitats with acidic pH. The species tolerances and optimum distributions are an indication to the vulnerability of the species to changes in their environment. Species with limited tolerances are more vulnerable, such as the narrow elevational distribution of cushion plants or the low pH and narrow tolerances observed for Sphagnum species. Climate and hydrological changes will very possibly affect the distribution of those species forcing changes on ecosystem functioning.     

Sphagnum mosses harbour highly specific bacterial diversity during their whole lifecycle

Knowledge about Sphagnum-associated microbial communities, their structure and their origin is important to understand and maintain climate-relevant Sphagnum-dominated bog ecosystems. We studied bacterial communities of two cosmopolitan Sphagnum species, which are well adapted to different abiotic parameters (Sphagnum magellanicum, which are strongly acidic and ombrotrophic, and Sphagnum fallax, which are weakly acidic and mesotrophic), in three Alpine bogs in Austria by a multifaceted approach. Great differences between bacterial fingerprints of both Sphagna were found independently from the site. This remarkable specificity was confirmed by a cloning and a deep sequencing approach. Besides the common Alphaproteobacteria, we found a discriminative spectrum of bacteria; although Gammaproteobacteria dominated S. magellanicum, S. fallax was mainly colonised by Verrucomicrobia and Planctomycetes. Using this information for fluorescent in situ hybridisation analyses, corresponding colonisation patterns for Alphaproteobacteria and Planctomycetes were detected. Bacterial colonies were found in high abundances inside the dead big hyalocytes, but they were always connected with the living chlorocytes. Using multivariate statistical analysis, the abiotic factors nutrient richness and pH were identified to modulate the composition of Sphagnum-specific bacterial communities. Interestingly, we found that the immense bacterial diversity was transferred via the sporophyte to the gametophyte, which can explain the high specificity of Sphagnum-associated bacteria over long distances. In contrast to higher plants, which acquire their bacteria mainly from the environment, mosses as the phylogenetically oldest land plants maintain their bacterial diversity within the whole lifecycle.     

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Sphagnum re-introduction in degraded peatlands: The effects of aggregation,species identity and water table

In European peatlands which have been drained and cut-over in the past, re-vegetation often stagnates after the return of a species-poor Sphagnum community. Re-introduction of currently absent species may be a useful tool to restore a typical, and more diverse, Sphagnum vegetation and may ultimately improve the functioning of peatland ecosystems, regarding atmospheric carbon sequestration. Yet, the factors controlling the success of re-introduction are unclear. In Ireland and Estonia, we transplanted small and large aggregates of three Sphagnum species into existing vegetation. We recorded changes in cover over a 3-year period, at two water levels (?5 and ?20 cm).Performance of transplanted aggregates of Sphagnum was highly species specific. Hummock species profited at low water tables, whereas hollow species profited at high water tables. But our results indicate that performance and establishment of species was also promoted by increased aggregate size. This mechanism (positive self-association) has earlier been seen in other ecosystems, but our results are the first to show this mechanism in peatlands. Our results do not agree with present management, which is aimed at retaining water on the surface of peat remnants in order to restore a functional and diverse Sphagnum community. More than the water table, aggregate size of the reintroduced species is crucial for species performance, and ultimately for successful peatland restoration.     

The submersed macrophyte communities of adirondack lakes (New York,U.S.A.) of varying degrees of acidity

The submersed macrophyte communities of nine high-altitude oligotrophic lakes in the Adirondack Mountain region of upstate New York were characterized in detail in terms of percent cover of each species. The pH of these clear-water lakes ranged from 6.9 to 4.4 and the acid neutralizing capacity ranged from 91 μeq 1^?1 to ?38 μeq l^?1. The degree of acidity covaried with lower total ion content and nutrient status. The dominant species in the circumneutral lakes and in the acidic lakes were the same, with several Utricularia species being the major component of the bottom cover. Species richness and diversity were lower in the acidic lakes (pH < 5.5). The plants found in the acidic lakes were typical of oligotrophic systems in general, regardless of pH, and similar to those found in lake surveys in Scandinavia and Eastern Canada. Potamogeton confervoides Reichenb., Scirpus subterminalis Torr., Sphagnum spp. and Utricularia geminiscapa Benj. were the only species that were primarily exclusive to the acidic lakes surveyed. In contrast to the results of Scandinavian studies, Sphagnum was found to be a major component of the plant cover in only two of the five acidic lakes.     

Die Sukzession der Testaceen-Assoziationen (Protozoa,Rhizopoda) im rezenten und subfossilen Sphagnum des Obersees bei Lunz (Niederösterreich)

The Succession of Testacea-Associations (Protozoa, Rhizopoda) in Recent and Subfossil Sphagnum from the Obersee near Lunz (Austria). The present paper deals with the Testacean micro-distribution and succession on Sphagnum plants and in a peat profile from the Sphagnum bog of the Obersee near Lunz (Austria). The vertical distribution of living Testacea on Sphagnum plants from the Sphagnum bog of the Obersee near Lunz (Austria) was studied as well as the succession of the Testacean nekrocoenoses in a peat profile (0–100 cm). In the green portion of Sphagnum species possessing Zoochlorellae (Amphitrema flavum, Heleopera sphagni, Hyalosphenia papilio) and Centropyxis aculeata are living. The remaining species are distributed in the lower (dead brown) zone. Active animals were seen to a depth of 18 cm. Some Testacea individuals were able to keep alive again to a depth of 45 cm. Certain species of the Forest Moss Type (Trinema lineare, T. enchelys, Euglypha laevis) dominated in recent Sphagnum and in the peat profile to 12 cm depth. In this time the number of specimens increased. At a depth of 18 cm several typical sediment species of the genus Difflugia (amphora, corona, acuminata, lebes) appeared and the populations of Centropyxis aculeata showed characteristics of sediment-inhabiting individuals (tests covered with mineral particles and vaulted, no spines).     

Salt stress enhanced antioxidant response in callus of three halophytes (Salsola baryosma, Trianthema triquetra, Zygophyllum simplex) of Thar Desert

Effects of salinity on growth, protein content, proline, catalase and antioxidant enzyme activity in callus of three halophytes of the Thar Desert; Salsola baryosma, Trianthema triquetra and Zygophyllum simplex were evaluated. Callus tissues were cultured on Murashige and Skoog’s medium containing different concentrations of NaCl (50, 100 and 200 mM). Increase in dry weight and soluble proteins were observed in the callus exposed to lower salinity (50 and 100 mM NaCl) in all the three species, whereas on the medium containing 200 mM NaCl, significant decrease in these two growth parameters was recorded. Under the salinity stress maximum proline accumulation was found in S. baryosma with parallel increase in soluble sugars. Among the three species, T. triquetra callus showed maximum CAT activity with 50 and 100 mM NaCl treatment, whereas the enzyme activity decreased at 200 mM NaCl treatment in all three species. The antioxidant potential steadily elevated under salt treatment in all the above three species using 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant potential (FRAP) assay. Whereas, superoxide dismutase (SOD) quenching were recorded maximum at low (50 and 100 mM) concentrations in all the three species. However, T. triquetra callus showed maximum total phenolic content (TPC) 15 mg GAE g^?1 with the elevated concentration of NaCl up to 200 mM, and S. baryosma callus showed lower TPC as compared to both species. A significant correlation between antioxidant capacity and TPC was observed indicating that phenolic compounds are the major contributors to the antioxidant potential in these halophyte species. FRAP and DPPH activity of Z. simplex showed maximum correlation (R = 0.992), as compared to other two species. We can conclude that all the three species exhibit a protection mechanism by sustaining growth parameters and antioxidant capacity. Due to high antioxidant property of all these species, the plant extracts may be included in nutraceutical formulations.     

Antioxidative enzymes in two in vitro cultured Salicornia species in response to increasing salinity

The effects of salt stress on dry mass, lipid peroxidation, polyphenol and hydrogen peroxide content and activities of antioxidative enzymes were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under a broad range of NaCl concentrations (0, 100, 200, and 300 mM) on Murashige and Skoog medium for 45 d. Dry mass of both species increased at low (100 mM) salinity but decreased at higher NaCl concentrations. Malondialdehyde (MDA) content decreased at low salinity, whereas increased at 200 and 300 mM NaCl. H₂O₂ content in S. europaea was considerably enhanced by salinity, but it was not significantly affected in S. persica. The salt stress progressively enhanced the polyphenol content in S. persica, whereas in S. europaea, it increased with respect to the control only at higher salinities. In both species, the salinity progressively enhanced the superoxide dismutase (SOD) and peroxidase (POD) activities, whereas the CAT activity was only registered at the low salinity and the APX activity decreaseed in both species. The results indicate that S. persica exhibited a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea.     

东北地区部分大型藓类植物遗传多样性的RAPD研究

采用RAPD标记构建了东北地区20种大型藓类植物的系统关系。从200个随机引物中筛选出清晰且多态性高的7个引物,共产生了82条DNA片段,其中79条谱带具有遗传多态性,约占96.34%,平均每个引物扩增的DNA片段数为11.71条。采用NTSYS-pc数据分析软件,计算Nei氏相似性系数,建立了UPGMA聚类图。结果表明：以遗传相似性系数0.27为界限划分,可将20种苔藓植物分为二大类,即泥炭藓类和真藓类。以遗传相似性系数0.42为界限划分,可将20种苔藓植物划分为四大类群,即泥炭藓类、顶蒴单齿亚类、顶蒴双齿亚类和侧蒴双齿类。从整个聚类图可以看出,泥炭藓类是较原始的类群,而金发藓类是较进化的类群,这一结果和大多数苔藓植物经典分类系统相同。     

Alleviation of osmotic stress in Brassica napus,B. campestris,and B. juncea by ascorbic acid application

The roles of ascorbic acid (AsA, 1 mM) under an osmotic stress [induced by 15 % (m/v) polyethylene glycol, PEG-6000] were investigated by examining morphological and physiological attributes in Brassica species. The osmotic stress reduced the fresh and dry masses, leaf relative water content (RWC), and chlorophyll (Chl) content, whereas increased the proline (Pro), malondialdehyde (MDA), and H₂O₂ content, and lipoxygenase (LOX) activity. The ascorbate content in B. napus, B. campestris, and B. juncea decreased, increased, and remained unaltered, respectively. The dehydroascorbate (DHA) content increased only in B. napus. The AsA/DHA ratio was reduced by the osmotic stress in all the species except B. juncea. The osmotic stress increased the glutathione (GSH) content only in B. juncea, but increased the glutathione disulfide (GSSG) content and decreased the GSH/GSSG ratio in all the species. The osmotic stress increased the activities of ascorbate peroxidase (APX) (except in B. napus), glutathione reductase (GR) (except in B. napus), glutathione S-transferase (GST) (except in B. juncea), and glutathione peroxidase (GPX), and decreased the activities of catalase (CAT) and monodehydroascorbate reductase (MDHAR) (only in B. campestris). The osmotic stress decreased the glyoxalase I (Gly I) and increased glyoxalase II (Gly II) activities. The application of AsA in combination with PEG improved the fresh mass, RWC, and Chl content, whereas decreased the Pro, MDA, and H₂O₂ content in comparison with PEG alone. The AsA addition improved AsA-GSH cycle components and improved the activities of all antioxidant and glyoxalase enzymes in most of the cases. So, exogenous AsA improved physiological adaptation and alleviated oxidative damage under the osmotic stress by improving the antioxidant and glyoxalase systems. According to measured parameters, B. juncea can be recognized as more drought tolerant than B. napus and B. campestris.     

Characterization of yeast groupings in the phyllosphere of <Emphasis Type="Italic">Sphagnum</Emphasis> mosses

Significant differences were revealed in the taxonomic structure of the epiphytic yeast communities formed on Sphagnum mosses and on the leaves of vascular plants. On mosses, low abundance of red yeasts was found (the most typical epiphytes on vascular plant leaves), along with a relatively high content and diversity of nonpigmented dimorphic basidiomycetes related to the order Leucosporidiales. The species composition of epiphytic yeasts from mosses is different from that of both forest and meadow grasses and of the parts of vascular plants submerged in the turf. The specific composition of the Sphagnum mosses yeast community is probably determined by the biochemical characteristics of this environment, rather than by the hydrothermal regime in the turf.