Seasonal dynamics of N in two Sphagnum moss species and the underlying peat treated with 15NH415NO3

Uptake of 15N labelled NH4NO3 by two Sphagnum mosses on a raised bog in north east Scotland was measured at different times of the year. In a field experiment, fortnightly additions of NH4NO3 at natural abundance, equivalent to 3 g N m-2 yr-1, were made over 14 months to cores of Sphagnum capillifolium occupying hummocks and S. recurvum colonizing hollows. Pre-harvested cores were treated with 15NH415NO3 two weeks before harvesting and 15N abundance determined for the total N in the moss, inorganic and dissolved organic N (DON) in the moss water and extractable inorganic, organic and microbial N in the underlying peat. The proportion of added 15N taken up by the mosses two weeks after each addition averaged 72% and ranged between 11 and 100%, tending to be least during October when the rising water table reached the surface, particularly for S. recurvum. A small proportion of the 15N was detected in the moss water as NH4+ (0.01%) and as DON (0.03%) and on occasions a large proportion remained unaccounted for. In waters from S. capillifolium, DON was proportional to the amount of inorganic N added, but this was not the case for S. recurvum. Little or no 15N was detected in the underlying peat partly because of the large size and variability of the NH4+, DON and microbial N pools.     

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.     

Enhanced recovery of a secreted recombinant human growth factor using stabilizing additives and by co-expression of human serum albumin in the moss Physcomitrella patens

The production of pharmaceutical proteins in plants provides a valuable alternative to other traditional eukaryotic expression systems from economic and safety perspectives. The moss Physcomitrella patens allows the expression and secretion of complex target proteins into a simple aqueous maintenance medium, which facilitates downstream processing by rendering it less complex. To address the question of whether the addition of protein-stabilizing substances enhances the recovery of a target protein secreted into the culture medium, several additives at different concentrations were tested in a small-scale screening system. Although polyvinylpyrrolidone (PVP) and human serum albumin (HSA) showed a significant impact on protein levels, supplementation of the medium with these substances was accompanied by certain limitations in upstream processes, such as foam formation (HSA), and in downstream processes, such as reduced binding efficiency on chromatography columns (PVP), respectively. In order to reap the benefit of the enhancing effect and to avoid the given negative aspects, we developed a new strategy based on the recombinant expression of HSA in plants that are already capable of expressing a target protein. First, we analysed the expression and secretion of recombinant HSA in transiently and stably transformed wild-type (WT) plants. HSA was then co-expressed in Physcomitrella plants transgenic for human vascular endothelial growth factor (VEGF). Even with high expression levels of recombinant human VEGF (rhVEGF), the co-expression of recombinant HSA (rHSA) resulted in 48%-102% higher recovery of the target protein without concomitant negative effects on the upstream process. This strategy enables the enhanced recovery of target protein and does not require the addition of foreign components directly to the culture medium.     

Cloning and characterization of micro-RNAs from moss

Micro-RNAs (miRNAs) are one class of endogenous tiny RNAs that play important regulatory roles in plant development and responses to external stimuli. To date, miRNAs have been cloned from higher plants such as Arabidopsis, rice and pumpkin, and there is limited information on their identity in lower plants including Bryophytes. Bryophytes are among the oldest groups of land plants among the earth's flora, and are important for our understanding of the transition to life on land. To identify miRNAs that might have played a role early in land plant evolution, we constructed a library of small RNAs from the juvenile gametophyte (protonema) of the moss Physcomitrella patens. Sequence analysis revealed five higher plant miRNA homologues, including three members of the miR319 family, previously shown to be involved in the regulation of leaf morphogenesis, and miR156, which has been suggested to regulate several members of the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family in Arabidopsis. We have cloned PpSBP3, a moss SPL homologue that contains an miR156 complementary site, and demonstrated that its mRNA is cleaved within that site suggesting that it is an miR156 target in moss. Six additional candidate moss miRNAs were identified and shown to be expressed in the gametophyte, some of which were developmentally regulated or upregulated by auxin. Our observations suggest that miRNAs play important regulatory roles in mosses.     

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.     

Current understanding on Villosiclava virens,a unique flower‐infecting fungus causing rice false smut disease

Villosiclava virens (Vv) is an ascomycete fungal pathogen that causes false smut disease in rice. Recent reports have revealed some interesting aspects of the enigmatic pathogen to address the question of why it specifically infects rice flowers and converts a grain into a false smut ball. Comparative and functional genomics have suggested specific adaptation of Vv in the colonization of rice flowers. Anatomical studies have disclosed that Vv specifically infects rice stamen filaments before heading and intercepts seed formation. In addition, Vv can occupy the whole inner space of a spikelet embracing all floral organs and activate the rice grain‐filling network, presumably for nutrient acquisition to support the development of the false smut ball. This profile provides a general overview of the rice false smut pathogen, and summarizes advances in the Vv life cycle, genomics and genetics, and the molecular Vv–rice interaction. Current understandings of the Vv–rice pathosystem indicate that it is a unique and interesting system which can enrich the study of plant–pathogen interactions. Taxonomy: Ustilaginoidea virens is the anamorph form of the pathogen (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Incertae sedis; Order Incertae sedis; Family Incertae sedis; Genus Ustilaginoidea). The teleomorph form is Villosiclava virens (Kingdom Fungi; Phylum Ascomycota; Class Ascomycetes; Subclass Sordariomycetes; Order Hypocreales; Family Clavicipitaceae; Genus Villosiclava). Disease symptoms: The only visible symptom is the replacement of rice grains by ball‐shaped fungal mycelia, namely false smut balls. When maturing, the false smut ball is covered with powdery chlamydospores, and the colour changes to yellowish, yellowish orange, green, olive green and, finally, to greenish black. Sclerotia are often formed on the false smut balls in autumn. Identification and detection: Vv conidia are round to elliptical, measuring 3–5 μm in diameter. Chlamydospores are ornamented with prominent irregularly curved spines, which are 200–500 nm in length. The sclerotia are black, horseshoe‐shaped and irregular oblong or flat, ranging from 2 to 20 mm. Nested polymerase chain reaction (PCR) and quantitative PCR have been developed to specifically detect Vv presence in rice tissues and other biotic and abiotic samples in fields. Host range: Rice is the primary host for Vv. Natural infection by Vv has been found on several paddy field weeds, including Digitaria marginata, Panicum trypheron, Echinochloa crusgalli and Imperata cylindrica. However, the occurrence of infection in these potential alternative hosts is very rare. Life cycle: Vv infects rice spikelets at the late rice booting stage, and produces false smut balls covered with dark‐green chlamydospores. Occasionally, sclerotia form on the surface of false smut balls in late autumn when the temperature fluctuates greatly between day and night. Both chlamydospores and sclerotia may serve as primary infection sources. Rainfall at the rice booting stage is a major environmental factor resulting in epidemics of rice false smut disease. Disease control: The use of fungicides is the major approach for the control of Vv. Several fungicides, such as cuproxat SC, copper oxychloride, tebuconazole, propiconazole, difenoconazole and validamycin, are often applied. However, the employment of resistant rice cultivars and genes has been limited, because of the poor understanding of rice resistance to Vv. Useful websites: Villosiclava virens genome sequence: http://www.ncbi.nlm.nih.gov/Traces/wgs/?val=JHTR01#contigs     

荒漠冻融期积雪变化对生物结皮甲烷通量的影响

温带荒漠积雪覆盖是土壤含水量增加的重要方式,在冻融期带来丰富的水分变化。全球气候变化导致温带荒漠积雪深度时空格局发生改变,从而影响荒漠生态系统温室气体的通量。生物结皮在维持荒漠生态系统结构和功能的稳定具有非常重要的作用,但对环境的变化较为敏感,尤其是降水变化。然而,目前有关冻融期不同积雪条件下生物结皮温室气体通量的研究十分匮乏。CH₄是重要的温室气体,选择古尔班通古特沙漠藓和地衣2种类型生物结皮为研究对象,以裸沙为对照,设置增雪、自然降雪(对照)和除雪三种处理,通过静态箱-气相色谱法,探究CH₄通量在冻融期的变化特征。结果表明：冻融期多数情况下积雪处理和结皮类型对CH₄通量影响不显著。荒漠生物结皮对CH₄整体表现为吸收作用,是荒漠生态系统CH₄重要的“汇”。两种结皮类型及裸沙CH₄的吸收速率呈现为地衣结皮(-10.12μg m^-2 h^-1)>藓结皮(-1.73μg m^-2 h^...     

Associative Cyanobacteria Isolated from the Roots of Epiphytic Orchids

Associative cyanobacteria were isolated from the rhizoplane and velamen of the aerial roots of the epiphytic orchids Acampe papillosa, Phalaenopsis amabilis, and Dendrobium moschatum and from the substrate roots of A. papillosa and D. moschatum. Cyanobacteria were isolated on complete and nitrogen-free variants of BG-11 medium. On all media and in all samples, cyanobacteria of the genus Nostoc predominated. Nostoc, Anabaena, and Calothrixwere isolated from the surface of the A. papillosa aerial roots, whereas the isolates from the substrate roots were Nostoc, Oscillatoria,and representatives of the LPP group (Lyngbia, Phormidium, and Plectonema, incapable of nitrogen fixation). On the D. moschatum substrate roots, Nostoc and LPP group representatives were also found, as well as Fischerella. On the aerial roots of P. amabilis and D. phalaenopsis grown in a greenhouse simulating the climate of moist tropical forest, cyanobacteria were represented by Nostoc, LPP group, and Scytonema in D. phalaenopsis and by Nostoc, Scytonema, Calothrix, Spirulina, Oscillatoria, and the LPP group in P. amabilis. For D. moschatum, the spectra of cyanobacteria populating the substrate root rhizoplane and the substrate (pine bark) were compared. In the parenchyma of the aerial roots of P. amabilis, fungal hyphae and/or their half-degraded remains were detected, which testifies to the presence of mycorrhizal fungi in this plant. This phenomenon is attributed to the presence of a sheath formed by cyanobacteria and serving as a substrate for fungi.