Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA₃, GA_4, and GA₇) but also pure GA₄ or GA₇. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA₃, GA₄, and GA₇ explaining the varying GA levels of genetically almost identical mutant strains.     

C and N allocation in soil under ryegrass and alfalfa estimated by 13C and 15N labelling

Background and Aims

Below-ground translocated carbon (C) released as rhizodeposits is an important driver for microbial mobilization of nitrogen (N) for plants. We investigated how a limited substrate supply due to reduced photoassimilation alters the allocation of recently assimilated C in plant and soil pools under legume and non-legume species.

Methods

A non-legume (Lolium perenne) and a legume (Medicago sativa) were labelled with ¹⁵N before the plants were clipped or shaded, and labelled twice with ¹³CO₂ thereafter. Ten days after clipping and shading, the ¹⁵N and ¹³C in shoots, roots, soil, dissolved organic nitrogen (DON) and carbon (DOC) and in microbial biomass, as well as the ¹³C in soil CO₂ were analyzed.

Results

After clipping, about 50 % more ¹³C was allocated to regrowing shoots, resulting in a lower translocation to roots compared to the unclipped control. Clipping also reduced the total soil CO₂ efflux under both species and the ¹³C recovery of soil CO₂ under L. perenne. The ¹⁵N recovery increased in the shoots of M. sativa after clipping, because storage compounds were remobilized from the roots and/or the N uptake from the soil increased. After shading, the assimilated ¹³C was preferentially retained in the shoots of both species. This caused a decreased ¹³C recovery in the roots of M. sativa. Similarly, the total soil CO₂ efflux under M. sativa decreased more than 50 % after shading. The ¹⁵N recovery in plant and soil pools showed that shading has no effect on the N uptake and N remobilization for L. perenne, but, the ¹⁵N recovery increased in the shoot of M. sativa.

Conclusions

The experiment showed that the dominating effect on C and N allocation after clipping is the need of C and N for shoot regrowth, whereas the dominating effect after shading is the reduced substrate supply for growth and respiration. Only slight differences could be observed between L. perenne and M. sativa in the C and N distribution after clipping or shading.     

Enhancing the structural diversity between forest patches—A concept and real-world experiment to study biodiversity,multifunctionality and forest resilience across spatial scales

Intensification of land use by humans has led to a homogenization of landscapes and decreasing resilience of ecosystems globally due to a loss of biodiversity, including the majority of forests. Biodiversity–ecosystem functioning (BEF) research has provided compelling evidence for a positive effect of biodiversity on ecosystem functions and services at the local (α-diversity) scale, but we largely lack empirical evidence on how the loss of between-patch β-diversity affects biodiversity and multifunctionality at the landscape scale (γ-diversity). Here, we present a novel concept and experimental framework for elucidating BEF patterns at α-, β-, and γ-scales in real landscapes at a forest management-relevant scale. We examine this framework using 22 temperate broadleaf production forests, dominated by Fagus sylvatica. In 11 of these forests, we manipulated the structure between forest patches by increasing variation in canopy cover and deadwood. We hypothesized that an increase in landscape heterogeneity would enhance the β-diversity of different trophic levels, as well as the β-functionality of various ecosystem functions. We will develop a new statistical framework for BEF studies extending across scales and incorporating biodiversity measures from taxonomic to functional to phylogenetic diversity using Hill numbers. We will further expand the Hill number concept to multifunctionality allowing the decomposition of γ-multifunctionality into α- and β-components. Combining this analytic framework with our experimental data will allow us to test how an increase in between patch heterogeneity affects biodiversity and multifunctionality across spatial scales and trophic levels to help inform and improve forest resilience under climate change. Such an integrative concept for biodiversity and functionality, including spatial scales and multiple aspects of diversity and multifunctionality as well as physical and environmental structure in forests, will go far beyond the current widely applied approach in forestry to increase resilience of future forests through the manipulation of tree species composition.     

Greenhouse Gas Emissions Quantification and Reduction Efforts in a Rural Municipality

The present article aims to determine the current carbon footprint (CF) of Zernez, a Swiss mountain village, and to identify reduction potentials of greenhouse gas (GHG) emissions. For this purpose, material and energy flows were assessed mainly based on detailed household surveys, interviews, and energy bills, but also by means of other information sources, for example, national statistics, traffic censuses, and literature values. To set up the GHG balance, special attention was paid to the consistent definition of system boundaries by adopting two fundamentally different perspectives: purely geographical accounting (PGA) and the consumption‐based footprint (CBF) method. Each of these two perspectives total approximately 10 tonnes of carbon dioxide equivalents per capita per year. The PGA revealed that 70% of the direct emissions in Zernez are caused by agricultural activities, whereas no consumption area dominated the consumption‐induced CF. For the identification of targeted measures, both perspectives were considered in a complementary manner. The building stock and its underlying energy supply system showed a GHG reduction potential of 80%. The building sector was thus detected as a reasonable first step for the municipality to adopt GHG mitigation strategies. In the case of Zernez, building‐stock‐related measures are predicted to decrease the current CF by 13% (CBF) and 17% (PGA), respectively.     

Evaluation of detection distance‐dependent reflectance spectroscopy for the determination of the sun protection factor using pig ear skin

Determination of sun protection factors (SPFs) is currently an invasive method, which is based on erythema formation (phototest). Here we describe an optical setup and measurement methodology for the determination of SPFs based on diffuse reflectance spectroscopy, which measures UV‐reflectance spectra at 4 distances from the point of illumination. Due to a high spatial variation of the reflectance data, most likely due to inhomogeneities of the sunscreen distribution, data of 50 measurement positions are averaged. A dependence of the measured SPF on detection distance is significant for 3 sunscreens, while being inconclusive for 2 sunscreens due to high inter‐sample variations. Using pig ear skin samples (n=6), the obtained SPF of 5 different commercial sunscreens corresponds to the SPF values of certified test institutes in 3 cases and is lower for 2 sunscreens of the same manufacturer, suggesting a formulation specific reason for the discrepancy. The results demonstrate that the measurement can be performed with a UV dose below the minimal erythema dose. We conclude the method may be considered as a potential noninvasive in vivo alternative to the invasive in vivo phototest, but further tests on different sunscreen formulations are still necessary.

     

A structural analysis of the interaction between ncd tail and tubulin protofilaments

ncd is a minus-end directed, kinesin-like motor, which binds to microtubules with its motor domain and its cargo domain as well. Typical of retrograde motors, the motor domain of ncd locates to the C-terminal end of the polypeptide chain, and hence, the cargo domain constitutes the N-terminal region. To date, several studies have investigated the interaction properties of the motor domain with microtubules, but very few structural data are available about the tail itself or its interaction with microtubules as cargo. Here, we applied cryo-electron microscopy and helical 3D image reconstruction to 15 protofilament microtubules decorated with an ncd tail fragment (N-terminal residues 83-187, named NT6). In our study, the ncd tail shows a behaviour resembling filamentous MAPs such as tau protein, exhibiting a highly flexible structure with no large globular domains. NT6 binds to four different sites on the outer side of microtubules within the proximity of the kinesin motor-binding site. Two of these sites locate within the groove between two neighbouring protofilaments, and appear as strong binding sites, while the other two sites, located at the outer rim, appear to play a secondary role. In addition, the ncd tail fragment induces the formation of large protofilament sheets, suggesting a tail-induced modification of lateral protofilament contacts.     

Expression of prostanoid receptors in human lower segment pregnant myometrium

Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

RNA-Seq analyses reveal the order of tRNA processing events and the maturation of C/D box and CRISPR RNAs in the hyperthermophile Methanopyrus kandleri

The methanogenic archaeon Methanopyrus kandleri grows near the upper temperature limit for life. Genome analyses revealed strategies to adapt to these harsh conditions and elucidated a unique transfer RNA (tRNA) C-to-U editing mechanism at base 8 for 30 different tRNA species. Here, RNA-Seq deep sequencing methodology was combined with computational analyses to characterize the small RNome of this hyperthermophilic organism and to obtain insights into the RNA metabolism at extreme temperatures. A large number of 132 small RNAs were identified that guide RNA modifications, which are expected to stabilize structured RNA molecules. The C/D box guide RNAs were shown to exist as circular RNA molecules. In addition, clustered regularly interspaced short palindromic repeats RNA processing and potential regulatory RNAs were identified. Finally, the identification of tRNA precursors before and after the unique C8-to-U8 editing activity enabled the determination of the order of tRNA processing events with termini truncation preceding intron removal. This order of tRNA maturation follows the compartmentalized tRNA processing order found in Eukaryotes and suggests its conservation during evolution.