ALBERT

Description: Sialic acid acetyl esterase (SIAE) removes acetyl moieties from the hydroxyl groups in position 9 and 4 of sialic acid. Recently, a dispute has been opened on its association to autoimmunity. In order to get new insights on human SIAE biology and to clarify its seemingly contradictory molecular properties, we combined in silico characterization, phylogenetic analysis and homology modeling with cellular studies in COS7 cells. Genomic and phylogenetic analysis revealed that in most tissues only the "long" isoform, originally referred to lysosomal sialic acid esterase, is detected. Using the homology modeling approach, we predicted a model of SIAE 3D structure, which fulfills the topological features of SGNH-hydrolase family. In addition, the model and site-directed mutagenesis experiments allowed the definition of the residues involved in catalysis. SIAE transient expression revealed that the protein is glycosylated and is active in vitro as an esterase with a pH optimum corresponding to 8.4–8.5. Moreover, glycosylation influences the biological activity of the enzyme and is essential for release of SIAE into the culture medium. According to these findings, co-localization experiments demonstrated the presence of SIAE in membranous structures corresponding to endoplasmic reticulum and Golgi complex. Thus, at least in COS7 cells, SIAE behaves as a typical secreted enzyme, subjected to glycosylation and located along the classical secretory route or in the extracellular space. In these environments, the enzyme could act on 9- O -acetylated sialic acid residues, contributing to the fine-tuning of the various functions played by this acidic sugar.

Description: Vaccination against the ubiquitous parasite Toxoplasma gondii would provide the most efficient prevention against toxoplasmosis-related congenital, brain and eye diseases in humans. We investigated the immune response elicited by pathogen-specific glycosylphosphatidylinositol (GPI) glycoconjugates using carbohydrate microarrays in a BALB/c mouse model. We further examined the protective properties of the glycoconjugates in a lethal challenge model using the virulent T. gondii RH strain. Upon immunization, mice raised antibodies that bind to the respective GPIs on carbohydrate microarrays, but were mainly directed against an unspecific GPI epitope including the linker. The observed immune response, though robust, was unable to provide protection in mice when challenged with a lethal dose of viable tachyzoites. We demonstrate that anti-GPI antibodies raised against the here described semi-synthetic glycoconjugates do not confer protective immunity against T. gondii in BALB/c mice.

Description: Carbohydrate antigens are valuable as components of vaccines for bacterial infectious agents and human immunodeficiency virus (HIV), and for generating immunotherapeutics against cancer. The crystal structures of anti-carbohydrate antibodies in complex with antigen reveal the key features of antigen recognition and provide information that can guide the design of vaccines, particularly synthetic ones. This review summarizes structural features of anti-carbohydrate antibodies to over 20 antigens, based on six categories of glyco-antigen: (i) the glycan shield of HIV glycoproteins; (ii) tumor epitopes; (iii) glycolipids and blood group A antigen; (iv) internal epitopes of bacterial lipopolysaccharides; (v) terminal epitopes on polysaccharides and oligosaccharides, including a group of antibodies to Kdo-containing Chlamydia epitopes; and (vi) linear homopolysaccharides.

Description: Sialic acids (SAs) are widely expressed on immune cells and their levels and linkages named as sialylation status vary upon cellular environment changes related to both physiological and pathological processes. In this study, we performed a global profiling of the sialylation status of macrophages and their release of SAs in the cell culture medium by using flow cytometry, confocal microscopy and liquid chromatography tandem mass spectrometry (LC-MS/MS). Both flow cytometry and confocal microscopy results showed that cell surface α-2,3-linked SAs were predominant in the normal culture condition and changed slightly upon treatment with atorvastatin for 24 h, whereas α-2,6-linked SAs were negligible in the normal culture condition but significantly increased after treatment. Meanwhile, the amount of total cellular SAs increased about three times (from 369 ± 29 to 1080 ± 50 ng/mL) upon treatment as determined by the LC-MS/MS method. On the other hand, there was no significant change for secreted free SAs and conjugated SAs in the medium. These results indicated that the cell surface α-2,6 sialylation status of macrophages changes distinctly upon atorvastatin stimulation, which may reflect on the biological functions of the cells.

Description: Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP C , to a misfolded isoform called PrP Sc . Although PrP Sc is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP Sc . Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP Sc deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP C and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a β-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.

Description: The display of cell-surface glycolipids and glycoproteins is essential for the motility, adhesion and colonization of pathogenic bacteria such as Campylobacter jejuni . Recently, the cell-surface display of C. jejuni glycoconjugates has been the focus of considerable attention; however, our understanding of the roles that glycosylation plays in bacteria still pales in comparison with our understanding of mammalian glycosylation. One of the reasons for this is that carbohydrate metabolic labeling, a powerful tool for studying mammalian glycans, is difficult to establish in bacterial systems and has a significantly more limited scope. Herein, we report the development of an alternative strategy that can be used to study bacterial cell-surface glycoconjugates. Galactose oxidase (GalO) is used to generate an aldehyde at C-6 of terminal GalNAc residues of C. jejuni glycans. This newly generated aldehyde can be conjugated with aminooxy-functionalized purification tags or fluorophores. The label can be targeted towards specific glycoconjugates using C. jejuni mutant strains with N -glycan or lipo-oligosaccharides (LOS) assembly defects. GalO-catalyzed labeling of cell-surface glycoproteins with biotin, allowed for the purification and identification of known extracellular N-linked glycoproteins as well as a recently identified O-linked glycan modifying PorA. To expand the scope of the GalO reaction, live-cell fluorescent labeling of C. jejuni was used to compare the levels of surface-exposed LOS to the levels of N-glycosylated, cell-surface proteins. While this study focuses on the GalO-catalyzed labeling of C. jejuni , it can in principle be used to evaluate glycosylation patterns and identify glycoproteins of interest in any bacteria.

Description: Human sialidases (NEUs) catalyze the removal of N -acetyl neuraminic acids from the glycome of the cell and regulate a diverse repertoire of nominal cellular functions, such as cell signaling and adhesion. A greater understanding of their substrate permissivity is of interest in order to discern their physiological functions in disease states and in the design of specific and effective small molecule inhibitors. Towards this, we have synthesized soluble fluorogenic reporters of mammalian sialidase activity bearing unnatural sialic acids commonly incorporated into the cellular glycocalyx via metabolic glycoengineering. We found cell-surface sialidases in Jurkat capable of cleaving unnatural sialic acids with differential activities toward a variety of R groups on neuraminic acid. In addition, we observed modulated structure–activity relationships when cell-surface sialidases were presented glycans with unnatural bulky, hydrophobic or fluorinated moieties incorporated directly via glycoengineering. Our results confirm the importance of cell-surface sialidases in glycoengineering incorporation data. We demonstrate the flexibility of human NEUs toward derivatized sugars and highlight the importance of native glycan presentation to sialidase binding and activity. These results stand to inform not only metabolic glycoengineering efforts but also inhibitor design.

Description: A defect in the assembly of the oligosaccharide donor (Dol-PP-GlcNAc 2 Man 9 Glc 3 ) for N-linked glycosylation causes hypoglycosylation of proteins by the oligosaccharyltransferase (OST). Mammalian cells express two OST complexes that have different catalytic subunits (STT3A or STT3B). We monitored glycosylation of proteins in asparagine-linked glycosylation 6 (ALG6) deficient cell lines that assemble Dol-PP-GlcNAc 2 Man 9 as the largest oligosaccharide donor. Based upon pulse labeling experiments, 30–40% of STT3A-dependent glycosylation sites and 20% of STT3B-dependent sites are skipped in ALG6-congenital disorders of glycosylation fibroblasts supporting previous evidence that the STT3B complex has a relaxed preference for the fully assembled oligosaccharide donor. Glycosylation of STT3B-dependent sites was more severely reduced in the ALG6 deficient MI8-5 cell line. Protein immunoblot analysis and RT–PCR revealed that MI8-5 cells express 2-fold lower levels of STT3B than the parental Chinese hamster ovary cells. The combination of reduced expression of STT3B and the lack of the optimal Dol-PP-GlcNAc 2 Man 9 Glc 3 donor synergize to cause very severe hypoglycosylation of proteins in MI8-5 cells. Thus, differences in OST subunit expression can modify the severity of hypoglycosylation displayed by cells with a primary defect in the dolichol oligosaccharide assembly pathway.

Description: Xenopus laevis (African clawed frog) has two types of proto-type galectins that are similar to mammalian galectin-1 in amino acid sequence. One type, comprising xgalectin-Ia and -Ib, is regarded as being equivalent to galectin-1, and the other type, comprising xgalectin-Va and -Vb, is expected to be a unique galectin subgroup. The latter is considerably abundant in frog skin; however, its biological function remains unclear. We determined the crystal structures of two proto-type galectins, xgalectin-Ib and -Va. The structures showed that both galectins formed a mammalian galectin-1-like homodimer, and furthermore, xgalectin-Va formed a homotetramer. This tetramer structure has not been reported for other galectins. Gel filtration and other experiments indicated that xgalectin-Va was in a dimer–tetramer equilibrium in solution, and lactose binding enhanced the tetramer formation. The residues involved in the dimer–dimer association were conserved in xgalectin-Va and -Vb, and one of the Xenopus (Silurana) tropicalis proto-type galectins, but not in xgalectin-Ia and -Ib, and other galectin-1-equivalent proteins. Xgalectin-Va preferred Galβ1-3GalNAc and not Galβ1-4GlcNAc, while xgalectin-Ib preferred Galβ1-4GlcNAc as well as human galectin-1. Xgalectin-Va/Vb would have diverged from the galectin-1 group with accompanying acquisition of the higher oligomer formation and altered ligand selectivity.

Description: Legionaminic acids (Leg) are bacterial analogs of neuraminic acid, with the same stereochemistry but different substituents at C5, C7 and C9. Hence they may be incorporated into useful analogs of sialoglycoconjugates, and we previously reported two sialyltransferases that could utilize cytidine monophosphate (CMP)-Leg5Ac7Ac for preparation of Leg glycoconjugates, which were resistant to sialidases [Watson DC, Leclerc S, Wakarchuk WW, Young NM. 2011. Enzymatic synthesis and properties of glycoconjugates with legionaminic acid as a replacement for neuraminic acid. Glycobiology . 21:99–108.]. These were the porcine ST3Gal1 and Pasteurella multocida sialyltransferases. We now report two additional sialyltransferases with superior Leg-transferase properties to the previous two. These are (i) a truncated form of a Photobacterium α2,6-sialyltransferase with an Ala-Met mutation in its active site, and (ii) an α2,3-sialyltransferase from Neisseria meningitidis MC58 with a higher transferase activity than the P. multocida enzyme, with either CMP-Neu5Ac or CMP-Leg5Ac7Ac as the donor. These enzymes will enable the production of useful Leg5Ac7Ac glycoconjugate derivatives with either α2,6 or α2,3 linkages and unique biological properties.

Description: The rare N-unsubstituted glucosamine ( $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ ) residues in heparan sulfate (HS) have important biological and pathophysiological roles. Because of their low natural abundance, the use of chemically generated, structurally defined, N-unsubstituted heparin/HS oligosaccharides can greatly contribute to the investigation of their natural role in HS. However, the sequencing of mixtures of chemically generated oligosaccharides presents major challenges due to the difficulties in separating isomers and the available detection methods. In this study, we developed and validated a simple and sensitive method for the sequence analysis of N-unsubstituted heparin/HS oligosaccharides. This protocol involves pH 4 nitrous acid (HNO 2 ) degradation, size-exclusion HPLC and ion-pair reversed-phase liquid chromatography-ion trap/time-of-flight mass spectrometry (IPRP-LC-ITTOF MS). We unexpectedly found that absorbance at 232 nm (normally used for specific detection of C4–C5 unsaturated oligosaccharides) was, in most cases, still sufficiently sensitive to also simultaneously detect saturated oligosaccharides during HPLC, thus simplifying the positional analysis of $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ residues. The IPRP-LC-ITTOF MS system can supply further structural information leading to full sequence determination of the original oligosaccharide. This new methodology has been used to separate and sequence a variety of chemically generated, N-unsubstituted dp6 species containing between 1 and 3 $$\hbox{ GlcN }{{\hbox{ H }}_{3}}^{+}$$ residues per oligosaccharide in different positional combinations. This strategy offers possibilities for the sequencing of natural N-unsubstituted oligosaccharides from HS and should also be applicable, with minor modification, for sequencing at N-sulfated residues using alternative pH 1.5 HNO 2 scission.

Description: A major aspect of carbohydrate-dependent galectin functionality is their cross-linking capacity. Using a cell surface as biorelevant platform for galectin binding and a panel of 40 glycans as sensor part of a fluorescent polyacrylamide neoglycopolymer for profiling galectin reactivity, properties of related proteins can be comparatively analyzed. The group of the chicken galectins (CGs) is an especially suited system toward this end due to its relatively small size, compared with mammalian galectins. The experiments reveal particularly strong reactivity toward N -acetyllactosamine repeats for all tested CGs and shared reactivity of CG-1A and CG-2 to histo-blood group ABH determinants. In cross-species comparison, CG-1B's properties closely resembled those of human galectin-1, as was the case for the galectin-2 (but not galectin-3) ortholog pair. Although binding-site architectures are rather similar, reactivity patterns can well differ.

Description: Glycosaminoglycans (GAG) play a ubiquitous role in tissues and cells. In eukaryotic cells, heparan sulfate (HS) is initially degraded by an endo-β-glucuronidase called heparanase-1 (HPSE). HS oligosaccharides generated by the action of HPSE intensify the activity of signaling molecules, activating inflammatory response, tumor metastasis, and angiogenesis. The aim of the present study was to understand if sulfated GAG could modulate HPSE, since the mechanisms that regulate HPSE have not been completely defined. CHO-K1 cells were treated with 4-methylumbelliferone (4-MU) and sodium chlorate, to promote total inhibition of GAG synthesis, and reduce the sulfation pattern, respectively. The GAG profile of the wild CHO-K1 cells and CHO-745, deficient in xylosyltransferase, was determined after [ 35 S]-sulfate labeling. HPSE expression was determined via real-time quantitative polymerase chain reaction. Total ablation of GAG with 4-MU in CHO-K1 inhibited HPSE expression, while the lack of sulfation had no effect. Interestingly, 4-MU had no effect in CHO-745 cells for these assays. In addition, a different enzyme location was observed in CHO-K1 wild-type cells, which presents HPSE mainly in the extracellular matrix, in comparison with the CHO-745 mutant cells, which is found in the cytoplasm. In view of our results, we can conclude that GAG are essential modulators of HPSE expression and location. Therefore, GAG profile could impact cell behavior mediated by the regulation of HPSE.

Description: Aims This study explores the patterns of niche differentiation in a group of seven closely related columbines (genus Aquilegia , Ranunculaceae) from the Iberian Peninsula. Populations of these columbines are subject to complex patterns of divergent selection across environments, which partly explain the taxonomic structure of the group. This suggests the hypothesis that niche divergence must have occurred along the process of diversification of the group. Methods We used MaxEnt to build environmental niche models of seven subspecies belonging to the three species of Aquilegia present in the Iberian Peninsula. From these models, we compared the environmental niches through two different approaches: ENMtools and multivariate methods. Important Findings MaxEnt distributions conformed closely to the actual distribution of the study taxa. ENMtools methods failed to uncover any clear patterns of niche differentiation or conservatism in Iberian columbines. Multivariate analyses indicate the existence of differentiation along altitudinal gradients and along a gradient of climatic conditions determined by the summer precipitation and temperatures. However, climatic conditions related to winter temperature and precipitation, as well as soil properties, were equally likely to show conservatism or divergence. The complex patterns of niche evolution we found suggest that Iberian Columbines have not been significantly constrained by forces of niche conservatism, so they could respond adaptively to the fast and profound climate changes in the Iberian Peninsula through the glacial cycles of the Pleistocene.

Description: Aims The pattern and driving factors of forest fires are of interest for fire occurrence prediction and forest fire management. The aims of the study were: (i) to describe the history of human-caused fires by season and size of burned area over time; (ii) to identify the spatial patterns of human-caused fires and test for the existence of ‘hotspots’ to determine their exact locations in the Daxing’an Mountains; (iii) to determine the driving factors that determine the spatial distribution and the possibility of human-caused fire occurrence. Methods In this study, K -function and Kernel density estimation were used to analyze the spatial pattern of human-caused fires. The analysis was conducted in S-plus and ArcGIS environments, respectively. The analysis of driving factors was performed in SPSS 19.0 based on a logistic regression model. The variables used to identify factors that influence fire occurrence included vegetation types, meteorological conditions, socioeconomic factors, topography and infrastructure factors, which were extracted and collected through the spatial analysis mode of ArcGIS and from official statistics, respectively. Important Findings The annual number of human-caused fires and the area burnt have declined since 1987 due to the implementation of a forest fire protection act. There were significant spatial heterogeneity and seasonal variations in the distribution of human-caused fires in the Daxing’an Mountains. The heterogeneity was caused by elevation, distance to the nearest railway, forest type and temperature. A logistic regression model was developed to predict the likelihood of human-caused fire occurrence in the Daxing’an Mountains; its global accuracy attained 64.8%. The model was thus comparable to other relevant studies.

Description: Aims Oxidative stress is one of the most important mechanisms in a plant’s reaction to the effects of different stressors; however, its role in plants’ resistance is still poorly understood. The objective of this study is to evaluate an influence of oxidative stress induced by stress factors of different origin—ozone, ultraviolet (UV)-B radiation, drought, cadmium (Cd) and copper (Cu), to growth of spring barley and to check the hypothesis, that intensification of oxidative stress is the main factor of growth depression induced by strong treatments of different stressors; meanwhile, mitigation of oxidative stress determines eustress-induced growth stimulation. Methods A pot experiment was carried out in phytotron chambers with a controlled environment. Spring barley ( Hordeum vulgare L.) plants were exposed to different doses of investigated environmental stress factors (O 3 , UV-B radiation, drought, Cd and Cu), and their effects on shoots growth, accumulation of superoxide (O 2 .– ), intensification of lipid peroxidation and antioxidative protection (superoxide dismutase, glutathione reductase and catalase activities and concentration of carotenoids) were measured. Analysis of variance (ANOVA) with classical eta-squared ( 2 ) values was used to evaluate and to compare the contribution of non-specific oxidative stress and stressor-specific mechanisms on plants growth. Important Findings Low doses of most stressors stimulated antioxidative protection and growth of barley shoots, reduced the concentration of O 2 .– and/or intensity of lipid peroxidation. Whereas an impairment of growth and intensification of oxidative stress as well as a reduction in concentration of carotenoids and further increase in activity of antioxidative enzymes were noticed when the intensity of the stressors was increased. In the cases of ozone and UV-B stress, the effects of oxidative stress on plant growth was mitigated by strong antioxidative protection—highly increased catalase (CAT) and superoxide dismutase (SOD) activities, respectively. In the cases of drought and Cu, relatively strong oxidative stress was the major cause of plant growth depression. Additionally, mitigation of oxidative stress due to increased SOD activity was likely to be one of the main causes of growth stimulation induced by low doses of UV-B, Cd and Cu stress. Possible reasons for O 3 -induced growth stimulation were increased CAT activity and concentration of carotenoids. Generalizing the effects of different stressors, the contribution of non-specific oxidative stress on plant growth was stronger compared with stressor-specific action mechanisms: oxidative stress determined 42% of the changes in plants’ dry biomass, whereas the contribution of stressor-specific mechanisms accounted for 35% of variability in barley growth.

Description: Aims The biochemical defense of lichens against herbivores and its relationship to lichen frequency are poorly understood. Therefore, we tested whether chemical compounds in lichens act as feeding defense or rather as stimulus for snail herbivory among lichens and whether experimental feeding by snails is related to lichen frequency in the field. Methods In a no-choice feeding experiment, we fed 24 lichen species to snails of two taxa from the Clausilidae and Enidae families and compared untreated lichens and lichens with compounds removed by acetone rinsing. Then, we related experimental lichen consumption with the frequency of lichen species among 158 forest plots in the field (Schwäbische Alb, Germany), where we had also sampled snail and lichen species. Important findings In five lichen species, snails preferred treated samples over untreated controls, indicating chemical feeding defense, and vice versa in two species, indicating chemical feeding stimulus. Interestingly, compared with less frequent lichen species, snails consumed more of untreated and less of treated samples of more frequent lichen species. Removing one outlier species resulted in the loss of a significant positive relationship when untreated samples were analyzed separately. However, the interaction between treatment and lichen frequency remained significant when excluding single species or including snail genus instead of taxa, indicating that our results were robust and that lumping the species to two taxa was justified. Our results imply lichen-feeding snails to prefer frequent lichens and avoid less frequent ones because of secondary compound recognition. This supports the idea that consumers adapt to the most abundant food source.

Description: Aims Hydrogeomorphic processes operating at watershed, process zone and site scales influence the distribution of riparian vegetation. However, most studies examining the relationships between hydrogeomorphic processes and riparian vegetation are conducted at site scales. We quantified the relative importance of watershed, process zone and site geomorphic characteristics for predicting riparian plant community types and plant species abundances in four small mountain watersheds in central Nevada, USA. Methods We mapped riparian vegetation types and identified process zones (based on dominant geomorphic process and valley fill material) within the watersheds. We sampled sites in each combination of vegetation type and process zone ( n = 184 sites) and collected data on watershed scale factors, valley and stream geomorphic characteristics and on plant cover of each geomorphic surface. Plant community types were defined by cluster and indicator species analyses of plant cover data, and related to geomorphic variables using ordination analysis (nonmetric multidimensional scaling). Linear mixed effects models were used to predict abundances of indicator species. Important Findings Variables describing position in the watershed (elevation, contributing area) that are related to gradients of temperature, moisture and stream discharge were of primary importance in predicting plant community types. Variables describing local geomorphic setting (valley width, stream gradient, channel sediments, geomorphic surface height) were of secondary importance, but accurately described the geomorphic setting of indicator species. The process zone classification did not include position in the watershed or channel characteristics and only predicted plant community types with unique geomorphic settings. In small mountain watersheds, predicting riparian vegetation distribution requires explicit consideration of scale and geomorphic context within and among watersheds in addition to site variables.

Description: Aims Plant–soil interaction (PSI) has been implicated as a causative mechanism promoting plant invasions, and some mechanisms underlying PSI effects remain unclear. Here, we attempted to address how altered soil microbes and nutrients influence PSI effects. Methods Soil was cultured by an invasive forb Solidago canadensis for two years. We conducted an experiment, in which S. canadensis and Chinese natives were grown either alone or together in control and cultured soils, and determined the growth of S. canadensis and five natives and the competitive ability of S. canadensis . We analyzed the microbial community composition and nutrients of two types of soils. Important Findings Compared to the control soil, the soil cultured by S. canadensis decreased the subsequent growth of S. canadensis and five Chinese natives, as well as the competitive ability of S. canadensis against Chinese natives. Soil microbial community composition was significantly altered due to soil culturing. Total fatty acids, bacteria, Gram-negative bacteria and Gram-positive bacteria had no responses to soil culturing; fungi, aerobic bacteria and fungi/bacteria ratio significantly decreased with soil culturing; anaerobes and Gram-negative/positive bacteria ratio greatly increased with soil culturing. Soil nitrogen (N) dramatically decreased with soil culturing, whereas soil phosphorus (P) was unchanged. These results suggest that negative PSI effects may be linked to decreases in soil fungi, aerobic bacteria and soil N and increases in soil anaerobic bacteria and the ratio of Gram-negative/positive bacteria. Our findings provide an initial indication that S. canadensis– soil interaction alone could exhibit limited contributions to its success in the early stage of invasion.

Description: Aims Nitrogen (N) fertilization and lime addition may affect soil microbial and nematode communities and ecosystem functions through changing environmental conditions, such as soil pH and soil organic carbon. The objectives of this experiment were to examine the impact of N input and liming on soil microbial and nematode communities and to identify the key environmental determinant of community composition in a century-old fertilization and crop rotation experiment. Methods The field experiment consisting of a 3-year crop rotation regime was established in 1911 in southeastern USA. Four treatments, (i) no-input control, (ii) NPK with winter legume, (iii) PK with legume and lime and (iv) NPK with legume and lime, were included in this study. Soil samples collected at the 0–5cm depth were used to determine the bacterial growth rate by the 3 H-thymidine incorporation technique. Incorporation of 13 C into neutral lipids, glycolipids and phospholipid fatty acids (PLFAs) was measured after incubation of soil with 13 C-labeled acetate for 24h. Free-living nematodes in fresh soil were extracted using a density sucrose centrifugal flotation method and identified to trophic group level. Important Findings Liming resulted in a 10-fold increase in bacterial growth rates compared with the no-input control, whereas N fertilization had no significant effect. Multivariate analysis of PLFA profiles showed that soil microbial community composition was different among the four treatments; the difference was primarily driven by soil pH. PLFAs indicative of Gram-negative bacteria covaried with soil pH, but not those of fungi and actinobacteria. Liming enhanced 13 C incorporation into neutral lipids, glycolipids and phospholipids by 2–15 times. In addition, 13 C incorporation into 16:0, 16:19, 18:19, 18:17 and 18:26 were greater than other PLFAs, suggesting that Gram-negative bacteria and fungi were more active and sensitive to simple C input. Bacterivorous nematodes were the dominant trophic group in the soil, but no significant differences in nematode communities were found among the treatments. Our results suggest that soil pH had a greater impact than N fertilization on soil microbial community composition and activity in a crop rotation system including legumes.

Description: Aims In cool-season grasses, systemic and vertically transmitted Epichloë infections often provide a suite of benefits including increased growth, reproduction and competitive abilities. However, these effects of Epichloë endophytes on their hosts often depend upon host and endophyte genotype and environmental factors. Methods Achnatherum robustum (sleepygrass) harbors at least two Epichloë species within natural populations in the Southwest USA. We tested the effects of endophyte infection and species, host population and plant genotype (by experimentally removing the endophyte), and soil moisture (a key limiting factor) on growth and drought stress response of infected A. robustum plants from two populations (Weed and Cloudcroft) in the Sacremento Mountains of New Mexico, USA). Important Findings Although the two populations harbor distinct Epichloë species each with very different chemoprofiles, neither endophyte status (infected vs. uninfected) nor endophyte species affected most growth parameters at 8 or 25 weeks of the experiment, except for leaf length. In high water treatment, infected plants from the Weed population had longer leaf length compared with uninfected plants. In contrast, the population of origin affected all growth parameters, including plant height, leaf number, length and width, tiller number and shoot and root biomass, as well as wilting time. Grasses from the Cloudcroft population generally showed greater growth than grasses from the Weed population. Endophyte infection did affect wilting time, with infection in the Weed population generally reducing time to wilting under low and high water, whereas infection in the Cloudcroft population reduced time to wilting only under high water conditions. Our results suggest that plant population and their associated plant genotypes may play a much larger role in endophyte–host grass interactions in varying environments than previously thought. Asexual Epichloë species may be compatible with only specific host genotypes within populations such that the phenotypic effects due to population may be greater than phenotypic changes influenced by variation in the endophyte.

Description: Glycomics may assist in uncovering the structure–function relationships of protein glycosylation and identify glycoprotein markers in colorectal cancer (CRC) research. Herein, we performed label-free quantitative glycomics on a carbon-liquid chromatography–tandem mass spectrometry-based analytical platform to accurately profile the N-glycosylation changes associated with CRC malignancy. N -Glycome profiling was performed on isolated membrane proteomes of paired tumorigenic and adjacent non-tumorigenic colon tissues from a cohort of five males (62.6 ± 13.1 y.o.) suffering from colorectal adenocarcinoma. The CRC tissues were typed according to their epidermal growth factor receptor (EGFR) status by western blotting and immunohistochemistry. Detailed N -glycan characterization and relative quantitation identified an extensive structural heterogeneity with a total of 91 N -glycans. CRC-specific N-glycosylation phenotypes were observed including an overrepresentation of high mannose, hybrid and paucimannosidic type N -glycans and an under-representation of complex N -glycans ( P 〈 0.05). Sialylation, in particular α2,6-sialylation, was significantly higher in CRC tumors relative to non-tumorigenic tissues, whereas α2,3-sialylation was down-regulated ( P 〈 0.05). CRC stage-specific N-glycosylation was detected by high α2,3-sialylation and low bisecting β1,4-GlcNAcylation and Lewis-type fucosylation in mid-late relative to early stage CRC. Interestingly, a novel link between the EGFR status and the N-glycosylation was identified using hierarchical clustering of the N -glycome profiles. EGFR-specific N -glycan signatures included high bisecting β1,4-GlcNAcylation and low α2,3-sialylation (both P 〈 0.05) relative to EGFR-negative CRC tissues. This is the first study to correlate CRC stage and EGFR status with specific N -glycan features, thus advancing our understanding of the mechanisms causing the biomolecular deregulation associated with CRC.

Description: Fucosylated chondroitin sulfate (FCS) is a glycosaminoglycan found in sea cucumbers. It has a backbone like that of mammalian chondroitin sulfate (4-β- d -GlcA-1-〉3-β- d -GalNAc-1) n but substituted at the 3rd position of the β- d -glururonic acid residues with α-fucose branches. The structure of these branches varies among FCSs extracted from different species of sea cucumbers, as revealed by solution NMR spectroscopy. Some species ( Isostichopus badionotus and Patalus mollis ) contain branches formed by single α-fucose residues but with variable sulfation patterns (2,4-, 3,4- and 4-sulfation). FCS from Ludwigothurea grisea is distinguished because it contains preponderant branches formed by disaccharide units containing non-sulfated and 3-sulfated α-fucose units at the reducing and non-reducing ends, respectively. Despite the structural variability on their α-fucose branches, these FCSs have similar anticoagulant action on assays using purified reagents. They have serpin-dependent and serpin-independent effects. Pharmacological assays using experimental animals showed that the three types of FCSs have similar antithrombotic effect and bleeding tendency. They also activate factor XII on the same range of concentration. Based on these observations, we proposed that only few sulfated α-fucose branches along the FCS chain are enough to assure the binding of this glycosaminoglycan to proteins of the coagulation system. Substitution with additional sulfated α-fucose does not increase further the activity. Overall, the use of FCSs with marked variability on their branches of α-fucose allowed us to establish correlations between structures vs biological effects of these glycosaminoglycans on a more refined basis. It opens new avenues for therapeutic intervention using FCSs.

Description: Enzymes that affect glycoproteins of the human immune system, and thereby modulate defense responses, are abundant among bacterial pathogens. Two endoglycosidases from the human pathogen Streptococcus pyogenes , EndoS and EndoS2, have recently been shown to hydrolyze N-linked glycans of human immunoglobulin G. However, detailed characterization and comparison of the hydrolyzing activities have not been performed. In the present study, we set out to characterize the enzymes by comparing the activities of EndoS and EndoS2 on a selection of therapeutic monoclonal antibodies (mAbs), cetuximab, adalimumab, panitumumab and denosumab. By analyzing the glycans hydrolyzed by EndoS and EndoS2 from the antibodies using matrix-assisted laser desorption ionization time of flight, we found that both the enzymes cleaved complex glycans and that EndoS2 hydrolyzed hybrid and oligomannose structures to a greater extent compared with EndoS. A comparison of ultra-high-performance liquid chromatography (LC) profiles of the glycan pool of cetuximab hydrolyzed with EndoS and EndoS2 showed that EndoS2 hydrolyzed hybrid and oligomannose glycans, whereas these peaks were missing in the EndoS chromatogram. We utilized this difference in glycoform selectivity, in combination with the IdeS protease, and developed a LC separation method to quantify high mannose content in the Fc fragments of the selected mAbs. We conclude that EndoS and EndoS2 hydrolyze different glycoforms from the Fc-glycosylation site on therapeutic mAbs and that this can be used for rapid quantification of high mannose content.

Description: Calnexin (CNX), known as a lectin chaperone located in the endoplasmic reticulum (ER), specifically recognizes G 1 M 9 GN 2 -proteins and facilitates their proper folding with the assistance of ERp57 in mammalian cells. However, it has been left unidentified how CNX works in Aspergillus oryzae , which is a filamentous fungus widely exploited in biotechnology. In this study, we found that a protein disulfide isomerase homolog TigA can bind with A. oryzae CNX (AoCNX), which was revealed to specifically recognize monoglucosylated glycans, similarly to CNX derived from other species, and accelerate the folding of G 1 M 9 GN 2 -ribonuclease (RNase) in vitro. For refolding experiments, a homogeneous monoglucosylated high-mannose-type glycoprotein G 1 M 9 GN 2 -RNase was chemoenzymatically synthesized from G 1 M 9 GN-oxazoline and GN-RNase. Denatured G 1 M 9 GN 2 -RNase was refolded with highest efficiency in the presence of both soluble form of AoCNX and TigA. TigA contains two thioredoxin domains with CGHC motif, mutation analysis of which revealed that the one in N-terminal regions is involved in binding to AoCNX, while the other in catalyzing protein refolding. The results suggested that in glycoprotein folding process of A. oryzae , TigA plays a similar role as ERp57 in mammalian cells, as a partner protein of AoCNX.

Description: Polysialic acid (polySia) is a linear polymer of sialic acid that modifies neural cell adhesion molecule (NCAM) in the vertebrate brain. PolySia is a large and exclusive molecule that functions as a negative regulator of cell–cell interactions. Recently, we demonstrated that polySia can specifically bind fibroblast growth factor 2 (FGF2) and BDNF; however, the protective effects of polySia on the proteolytic cleavage of these proteins remain unknown, although heparin/heparan sulfate has been shown to impair the cleavage of FGF2 by trypsin. Here, we analyzed the protective effects of polySia on the proteolytic cleavage of FGF2 and proBDNF/BDNF. We found that polySia protected intact FGF2 from tryptic activity via the specific binding of extended polySia chains on NCAM to FGF2. Oligo/polySia also functioned to impair the processing of proBDNF by plasmin via binding of oligo/polySia chains on NCAM. In addition, the polySia structure synthesized by mutated polysialyltransferase, ST8SIA2/STX(SNP7), which was previously identified from a schizophrenia patient, was impaired for these functions compared with polySia produced by normal ST8SIA2. Taken together, these data suggest that the protective effects of polySia toward FGF2 and proBDNF may be involved in the regulation of the concentrations of these neurologically active molecules.

Description: Aims Theory suggests that species perform best at intermediate densities, where density-dependent facilitation and antagonism are balanced, but empirical evidence is scarce, particularly in plants. In a self-incompatible perennial herb ( Saussurea nigrescens ), whose recruitment heavily relies on seed output, we test whether both intraspecific facilitation and antagonism significantly affect seed production, resulting in highest seed yield at an intermediate capitulum density. Methods Plots with different S. nigrescens densities were sampled in an Eastern Tibetan meadow during the growing season of 2012 to investigate the relationships between capitulum density and pollinator visitation rate, seed set ratio, parasite ratio, seed damage ratio, and capitulum size. Both simple linear and quadratic models were employed to determine the shape of relationships. Important Findings In line with general theory, hump-shaped relationships of capitulum density versus seed set ratio and number of florets per capitulum indicate intraspecific facilitation in sparse populations, which can be attributed to positive density-dependent pollinator visitation and the amelioration of detrimental physical factors. However, the proportion of seeds damaged by pre-dispersal predators increased monotonically with capitulum density, which may have—in combination with increased intraspecific competition for light and soil nutrients—resulted in density-dependent antagonism. Both positive and negative density-dependent agents acted simultaneously throughout the density range investigated and led to the highest seed yield at intermediate density levels in the Tibetan lotus. More efforts concurrently exploring the two effects are needed to facilitate understanding species abundance and community structure.

Description: Aims Vegetation sampling employing observers is prone to both inter-observer and intra-observer error. Three types of errors are common: (i) overlooking error (i.e. not observing species actually present), (ii) misidentification error (i.e. not correctly identifying species) and (iii) estimation error (i.e. not accurately estimating abundance). I conducted a literature review of 59 articles that provided quantitative estimates or statistical inferences regarding observer error in vegetation studies. Important Findings Almost all studies (92%) that tested for a statistically significant effect of observer error found at least one significant comparison. In surveys of species composition, mean pseudoturnover (the percentage of species overlooked by one observer but not another) was 10–30%. Species misidentification rates were on the order of 5–10%. The mean coefficient of variation (CV) among observers in surveys of vegetation cover was often several hundred % for species with low cover, although CVs of 25–50% were more representative of species with mean covers of 〉50%. A variety of metrics and indices (including commonly used diversity indices) and multivariate data analysis techniques (including ordinations and classifications) were found to be sensitive to observer error. Sources of error commonly include both characteristics of the vegetation (e.g. small size of populations, rarity, morphology, phenology) and attributes of the observers (e.g. mental fatigue, personal biases, differences in experience, physical stress). The use of multiple observers, additional training including active feedback approaches, and continual evaluation and calibration among observers are recommended as strategies to reduce observer error in vegetation surveys.

Description: Aims Understanding relationships between vegetation and environments is of importance for ecosystem restoration and management. However, information on how environments influence the floristic patterns of shrublands is lack, especially in the subtropical China. In this study, we explored how environments regulate species composition of shrublands at landscape scale in mid-subtropical China. Methods We investigated species composition and measured the climate and soil environments for 207 shrubland plots in mid-subtropical China (24°39'–30°08'N, 108°47'–114°15'E). We applied a hierarchical cluster analysis and indicator species analysis based on the Bray–Curtis similarity index to identify the main shrubland types and employed principal coordinate analysis (PCoA) to explore the relationship between floristic composition and environment. Important Findings We identified four shrubland types occurring in different environmental conditions. Montane shrubland, dominated by species suitable for cool climates (e.g. Rhododendron simsii ), were distributed in steep areas at comparatively high altitudes; foothill shrubland, dominated by mesophilous species (e.g. Loropetalum chinense ), were distributed in low mountains and hills; pioneer shrubland, dominated by fast grow and short-life cycles species (e.g. Rhus chinensis ), were distributed at low altitudes with dense population; and finally, limestone shrubland, dominated by calcicole plants (e.g. Coriaria nepalensis ), were distributed in the extensive karst areas. Communities occurring in high pH soils were completely separated from those in low pH soils according to the hierarchical cluster analysis. PCoA ordination associated the four types with distinct edaphic and climatic gradients. Soil pH explained 63.3% of variation in PCoA, followed by soil depth and soil bulk density.

Description: Aims The loss of canopy trees associated with forest decline can greatly influence the species composition and structure of a forest and have major impacts on the ecosystem. We studied the changes in forest composition and structure 1 and 5 years following nearly total canopy mortality on several hundreds of hectares of xeric oak forests in south-central United States. Because the forests were within an ecotonal vegetation type composed of a mosaic of forest, savanna and grassland, we sought to learn whether forest decline areas would recover to forest or change to more open savanna and grassland conditions in the landscape pattern of vegetation. Because low intensity fire shaped the vegetation type, we sought to learn whether fire would keep the decline areas open. Methods The study was conducted in a xeric oak forest in east-central Oklahoma, USA. Randomly located vegetation and regeneration surveys were conducted in decline and non-decline stands 1 and 5 years following nearly total canopy mortality. Diameter at breast height (DBH), regeneration and sprout origin were recorded for all woody species. Important Findings The major canopy species post oak ( Quercus stellata Wangenh.), blackjack oak ( Quercus marilandica Muenchh.) and black hickory ( Carya texana Buckl.) suffered 85–92% mortality; however, minor canopy components experienced limited mortality. Mortality affected all size classes of canopy trees except those below 5cm breast height diameter. There was abundant regeneration of all species and fire seemed to maintain a high level of sprouting. Decline appeared to decrease the relative importance of stump sprouting and increase other types including root sprouts. Decline areas had abundant true seedlings, with stem origin from a root with the same diameter as the stem, which is very unusual for xeric oak forests. Regeneration height in decline areas was twice that of non-decline forests. Our findings suggest that forest decline may lead to: (i) reduced oak dominance and species change in the canopy, (ii) change in reproduction type to increase success of true seedlings and maintain genetic diversity of oaks.

Description: Aims To assess the role of moisture in phenological timing in the mediterranean coastal flora of Baja California, and specifically to assess the role of coastal fog and ocean-derived moisture in plant phenology. Moisture seems to be the primary driver of flowering times and durations at the arid end of the mediterranean-climate region, where rainfall is often sporadic (temperature and day length can be expected to play a much lesser role as they are not growth limiting). We aimed to understand: What factors drive climatic variation between sites? Are there general flowering patterns allowing us to identify phenological categories? Do flowering patterns vary in relation to site-specific weather? and most importantly, does maritime influence on weather affect flowering dynamics in coastal mediterranean ecosystems? Methods The southernmost extent of the California Floristic Province (in Baja California, Mexico) is a biological diversity hotspot of high endemism and conservation value, with two steep moisture gradients: rainfall (N–S) and coastal fogs (W–E), providing an ideal study system. We installed five weather stations across the moisture gradients, recording data hourly. We monitored flowering phenology in the square kilometer surrounding each weather station from 2010 to 2013. About 86 plant taxa were monitored across the five sites, every 6–8 weeks. Averaged climatic data is presented with general trends in flowering, and specific flowering syndromes were observed. Data for flowering intensity across the sites was analyzed using a principal components analysis. Important Findings Data analysis demonstrates a general seasonal pattern in flowering times, but distinct differences in local weather and phenology between the five study sites. Three flowering syndromes are revealed in the flora: (i) water responders or spring bloomers, (ii) day-length responders or fall-blooming taxa and (iii) aseasonal bloomers with no seasonal affinity. The two moisture gradients are the strongest drivers of flowering times. Inland sites showed higher phenological variation than coastal sites where seasonality is dampened by ocean-derived moisture, which extends and buffers perennial plant phenology and is a probable driver of local endemism. Phenological controls vary globally with climate and geography; moisture is the primary driver of phenology in mediterranean climates and fog is an important climatic variable in coastal Mexico.

Description: Aims The aim of the study was to discover what set of variables best explains the transition from warm to mesic forest vegetation. Based on various variables grouped into sets (geomorphological, ecological, structural, soil characteristics and chorological), six models were built and tested by generalized additive mixed models (GAMMs). We assumed that each set of variables has different explanatory power. Our aim was to compare the six different models (sets of variables), to test which model best explains the species turnover in forest communities along the transition between warm and mesic temperate forests and to try to find reasons for the different explanatory power of the models. Methods The research took place in the southern part of the Balkan Peninsula. Field sampling was done according to standard methods. The gradient from warm to mesic forests was defined as the turnover of species and evaluated by projection of samples on the first unconstrained DCA axis. Geomorphological, ecological, structural and soil characteristics, together with chorological sets of variables, were regressed on the turnover of species composition. Based on the five sets of variables, six models were constructed and tested by generalized additive mixed models. Important Findings Ecological conditions best explain the change of forest communities along the gradient; evolution and the development of vegetation reflected in chorotypes are also of high importance; geomorphology and structure seem not to change so dramatically and soil shows the least significant differences of all. Ecological variables are the most important set of variables in the transition between warm and mesic temperate forests but eco-evolutionary dynamics after the Pleistocene should also be taken into consideration.

Description: Aims Increasing evidence worldwide indicates that high mountain regions are not immune to invasion by alien plants. Here, we aimed to address whether three major woody invaders of low-mountain areas are constrained to lower altitudes due to climatic restrictions or just by low propagule pressure. We hypothesize that the increasing climatic harshness towards higher altitudes restricts seedling establishment and survival of these woody invaders and thus their potential for altitudinal expansion. Methods The study was carried out in the Sierras Grandes Mountains, Córdoba, central Argentina (32º50'S, 64º90'W). We addressed the hypothesis with an experimental approach, dissociating the effect of the environmental gradient from that of propagule pressure, by discarding the influence of seed sources. We translocated seeds of Gleditsia triacanthos L. , Ligustrum lucidum W.T. Aiton. and Pyracantha angustifolia (Franch.) C.K.Schneid. along the altitudinal gradient (from 900 to 2700 m asl). Seven sites were selected along the altitudinal gradient, spaced every 200–400 m. Three plots (4 x 4 m) were selected within each altitudinal site and excluded from livestock; 100 seeds of each species were sown within each plot (6300 seeds in total). Seedling emergence and survival was monitored during two growing seasons and soil temperature and moisture was recorded as well. The field experiment was complemented with lab assays. Important Findings Climate along this altitudinal gradient might be least restrictive at mid elevations, as a result of the intersection between opposite soil temperature and moisture gradients. Sown seeds germinated along the whole altitudinal gradient and seedlings successfully established and over-wintered well above their current altitudinal distribution (up to 2200 m for Ligustrum and 2400 m for Gleditsia and Pyracantha ). Additional lab assays confirmed field patterns and gave some insights into contrasting regeneration strategies between these invaders that might help to overcome stochastic environmental constraints in the germination stage. Overall, seedlings of three major woody invaders of low-mountain woodlands can establish and survive at higher elevations than their current distribution. In contrast to natives, they seem to be broad climate tolerators, rather than specialized stress tolerators, capable of germinating and growing across a wide elevational range. While long-term experiments might be necessary to fully address the potentials for altitudinal expansion, out results on early lifecycle stage suggest that the invaders studied here would have mostly a dispersal barrier rather than a climate barrier to establish in the upper belt of the Sierras Grandes.

Description: Aims The diversity–productivity relationship is one of the most critical questions in ecology and can be altered by environmental factors. Hydrological fluctuation affects growth of wetland plants, and such effects vary with plant species. Therefore, we hypothesized that hydrological fluctuation changes effects of species richness on productivity of wetland plant communities. Methods We constructed wetland plant communities consisting of three or six wetland plant species and subjected them to hydrological fluctuation (i.e. gradually changing water level) of two frequencies and two ranges, with unchanged water level as the control. We measured height, root and shoot dry mass of each plant at harvest. Important Findings Hydrological fluctuation significantly decreased biomass of wetland plant communities, which was due to impacts of fluctuation range, but not those of fluctuation frequency. Community biomass was significantly higher when species richness was higher, and such an effect did not depend on hydrological fluctuation. Therefore, hydrological fluctuation can decrease the productivity of wetland plant communities but may not alter the diversity–productivity relationship.

Description: Aims We aimed at determining differences in the leaf spectral signatures of plant species groups growing in habitats along the hydrological gradient of an intermittent wetland and to define leaf traits that explain their variability. We want to contribute to the understanding of the causes for plant spectrum variability at leaf and community levels. Methods We measured leaf reflectance spectra (300–887nm) of representative plant species from different habitats and analyzed spectral differences among species groups. To explain leaf spectra variability within a group, we performed detailed analyses of leaf morphological and biochemical traits in selected species. Important Findings The reflectance spectra of the different species groups differed most in the green, yellow and red spectral ranges. The reflectance spectra of submerged leaves of hydrophytes with simple structures were explained by their biochemical traits (carotenoids), while for more complex aerial leaves, morphological traits were more important. In submerged and natant leaves of amphiphytes, total mesophyll and spongy tissue thickness were the most important traits, and these explained 44% and 47%, respectively, of the spectrum variability of each plant group. In general, the redundancy analysis biplots show that samples of different plant species colonizing the same habitat form separate clusters and are related to the explanatory variables in different ways. The redundancy analysis biplots of helophytes and wet meadow species show clustering of graminoids and dicots into two distinct groups. Leaf encrustation (prickle hair properties and epidermis thickness) is important for graminoids, while leaf thickness and specific leaf area have more important roles in dicots. Our results show that knowledge of the species composition and leaf traits is necessary to interpret the reflectance spectra of such plant communities.

Description: Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae). Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l –1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 . Important Findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica . Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3 . P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.

Description: Aims Woody plant-browser systems represent an understudied facet of herbivory. We subjected four genotypes of trembling aspen to artificial browsing, similar to that of a large mammalian herbivore, and applied deer saliva to clipped and unclipped trees to assess: (i) the effects of artificial browsing on aspen growth and phytochemistry of leaves and stems, (ii) genotypic variation in responses and (iii) potential alterations of responses by mammalian saliva. Methods Potted aspen trees were grown outdoors on the University of Wisconsin-Madison campus. The experiment consisted of a fully-crossed, 2 x 2 x 4 randomized complete block design, with two levels of artificial browsing (unclipped and clipped), two levels of saliva application (no saliva and saliva) and four aspen genotypes. To simulate ungulate browsing damage, we removed the upper 50% of the stem of half of the trees by pinching the stem with needle-nosed pliers and then separating it by tearing. For half of the damaged trees, we immediately swabbed the wound with deer saliva. Trees in the unclipped plus saliva treatment were swabbed with saliva at the 50% height mark. To assess the effects of clipping and saliva application, we harvested all trees after 2 months and measured various growth and chemical properties. Growth measurements included height, vertical growth, mass of leaves, stems and roots, leaf number and area and bud set. Chemical parameters included defensive, nutritional and structural components of both foliage and stems. Important Findings Clipping affected most of the growth parameters measured, decreasing tree height, leaf, stem, root and total tree mass and leaf area. Clipped trees had greater vertical growth, more leaves and higher specific leaf area (SLA) than unclipped trees. Deer saliva had little to no effect on plant growth response to the clipping treatment. Terminal budset was delayed by clipping and varied among genotypes but not in response to saliva application. Clipping also affected most of the phytochemical variables measured, reducing defensive compounds (phenolic glycosides and condensed tannins (CTs)) and nutrients (N), but increasing structural components (cellulose and lignin) in both leaves and stems. Saliva had very little effect on tree chemistry, causing only a slight decrease in the amount of CTs in leaves. In general, leaves contained more defensive compounds and nutrients, but much less cellulose, compared with stems. Genotypes differed for all physical and chemical indices, and in tolerance to damage as measured by vertical growth. In addition, for most of the physical and chemical variables measured, genotype interacted with the clipping treatment, suggesting that in natural stands some genotypes will resist or tolerate browsing better than others, affecting forest genetic composition and ultimately forest dynamics.

Description: Aims In the Core Cape Subregion (CCR), a Mediterranean-climate ecosystem with infertile soils, the legume species Podalyria calyptrata and P. burchellii are in a separate clade to P. leipoldtii and P. myrtillifolia . The closely related species are allopatric, and with the west-east climate gradient and variation in soil nutrient availability in the CCR, it was hypothesized that the two closely related allopatric species would differ in their ecological niche and root:shoot ratio, specific root length (SRL) and organic acid exudation responses to phosphorus (P) supply. Methods With increasing P supply in the glasshouse, we measured plant biomass, leaf nitrogen ([N]), [P], root morphology and release of organic acids. We determined species soil and leaf [N] and [P] and climate in field sites. Important Findings At low P supply, P. calyptrata roots exuded more organic acids than P. burchellii which instead produced roots with a greater SRL, and P. myrtillifolia allocated more biomass to roots than P. leipoldtii . In the field, leaf [P] and climate suggested that P. leipoldtii occupied the most oligotrophic niche followed by P. burchellii and then P. calyptrata and P. myrtillifolia . Closely related allopatric species differed in their mechanisms for P-acquisition and ecological niche, indicating that the environment overrides phylogeny in determining P-acquisition traits for these species, and suggesting that climate regulates nutrient availability, driving distribution and speciation.

Description: Aims Exploring flowering patterns and detecting processes are essential when probing into the nature of reproductive traits during the life history and the interactions among different evolutionary clades. Such patterns are believed to be influenced by many factors, but quantifying these impacts at the community-level remains poorly understood. Methods We investigated the flowering patterns based on long-term herbarium records in a given area from subtropical forest regions in southern China. We obtained 5258 herbarium voucher specimens collected from the Dinghushan National Nature Reserve (DNNR) belonging to 166 families, 943 genera and 2059 species and examined the month when each species was flowering during the period 1920–2007. Important Findings The results showed that plants flowered sequentially almost throughout the whole year, showing the characteristics of subtropical evergreen broad-leaved forests. Flowering spectrums of the entire flora and the four life forms exhibited a clear unimodality that is probably typical of subtropical forest communities. Flowering patterns of the DNNR were positively correlated with mean rainfall, mean air temperature and mean sunshine duration. Median flowering dates of the 38 large species-rich families ranged from early April to late August; 25 families exhibited significant unimodal distribution patterns, whereas the remaining families were unclear or bimodal. Median flowering dates of the 10 most species-rich genera ranged from middle May to later July. While the results are consistent with climatic factors playing a general role in flowering patterns, median flowering dates varied significantly among species-rich families and genera, suggesting that phylogenies could provide specific constraints in subtropical forests.

Description: Aims Most pollinator fig wasps are host plant specific, with each species only breeding in the figs of one fig tree species, but increasing numbers of species are known to be pollinated by more than one fig wasp, and in rare instances host switching can result in Ficus species sharing pollinators. In this study, we examined factors facilitating observed host switching at Xishuangbanna in Southwestern (SW) China, where Ficus squamosa is at the northern edge of its range and lacks the fig wasps that pollinate it elsewhere, and its figs are colonized by a Ceratosolen pollinator that routinely breeds in figs of F. heterostyla . Methods We recorded the habitat preferences of F. squamosa and F. heterostyla at Xishuangbanna, and compared characteristics such as fig size, location and colour at receptive phase. Furthermore, the vegetative and reproductive phenologies in the populations of F. squamosa and F. heterostyla were recorded weekly at Xishuangbanna Tropical Botanical Garden for 1 year. Important Findings Ficus squamosa is a shrub found near fast-flowing rivers, F. heterostyla is a small tree of disturbed forest edges. Although preferring different habitats, they can be found growing close together. Both species have figs located at or near ground level, but they differ in size when pollinated. Fig production in F. squamosa was concentrated in the colder months. F. heterostyla produced more figs in summer but had some throughout the year. The absence of its normal pollinators, in combination with similarly located figs and partially complementary fruiting patterns appear to have facilitated colonization of F. squamosa by the routine pollinator of F. heterostyla . The figs probably also share similar attractant volatiles. This host switching suggests one mechanism whereby fig trees can acquire new pollinators and emphasizes the likely significance of edges of ranges in the genesis of novel fig tree–fig wasp relationships.

Description: Aims Within inflorescences of sexually reproducing hermaphrodites, the production of ovules, fruits and seeds commonly declines from basal (early-opening) to distal (late-opening) flowers, while pollen production remains constant or only changes slightly, with the result that distal (late-opening) flowers become functionally male. However, few empirical studies have specifically examined whether or not changes in allocation to pollen production actually lead to changes in the number of seeds sired, a more direct measure of male fitness. In pseudogamous apomicts, fitness depends on the number of seeds produced; thus, a contrasting pattern of variation in the pollen-to-ovule (P/O) ratio within inflorescences might be expected. Methods We investigated floral sex allocation and reproductive success within racemes of Hosta ventricosa , a pseudogamous apomictic hermaphrodite possessing flowers that open acropetally. We quantified variations in pollen number, ovule number, the P/O ratio and fruit and seed production, from 2007 to 2011, among flowers within racemes of four populations of H. ventricosa in southwest China. Ecological causes for fruit and seed production were evaluated by observing patterns of pollen deposition, flower removal and supplemental pollination. Important Findings Pollen number, ovule number and the P/O ratio declined from basal-to-distal positions in all sampled populations (years). Fruit and seed production decreased distally in most populations (years). Low fruit and seed set of distal flowers was not due to pollen limitation because pollen deposition never declined distally and the low fruit and seed set of distal flowers remained even after supplementary pollination was provided. The flower-removal experiment indicated that inter-fruit competition for resources among flowers was common. The low P/O ratio of distal flowers in H. ventricosa might be favored because they were unable to obtain fitness by donating pollen and siring seeds on other plants. Our study may help to understand the adaptive significance of sex allocation among flowers within inflorescences of sexually reproducing hermaphrodites.

Description: Aims Both high and low densities of macrophyte vegetation can impair its ecosystem service function. Harvesting is often applied to macrophyte vegetation to maintain an appropriate density. Vegetation harvesting has occasionally gone awry and caused catastrophes, such as vegetation disappearance and cyanobacterial dominance in waterways and lakes. Because water depth influences macrophyte density at all life stages, the simultaneous influences of harvesting and water depth should be carefully examined. Thus, this study aims to quantify the effects of differently harvesting Elodea nuttallii on its growth and reproduction at different water depths in field experiments. Methods Four harvest intensities (harvesting E. nuttallii plant heights equal to 25%, 50%, 75% and 100% of the water depth) were applied to E. nuttallii growing at four different water depths (60, 90, 120 and 150cm). Plant length and root length were measured. The node number, root number of each plant and number of floating plants were counted before harvesting. The harvested plant were dried to a constant weight for dry weight determination. Important Findings The rate of increase in the length and shoot number of E. nuttallii varied from –0.012 to 0.440 day –1 and from –0.020 to 0.639 day –1 , respectively. Water depth 〉150cm would limit E. nuttallii growth. Elodea nuttallii responded to increasing water depths and low-intensity harvesting by increasing internodal length and decreasing shoot number. The larger internodal length of E. nuttallii observed in relatively deeper water was also induced by the physical strain generated by its buoyancy as its specific gravity was less than water’s. The physical mechanism of removing the plant canopy by harvesting decreased E. nuttallii buoyancy and prevented floating. Harvesting increased plant production in shallow waters 〈90cm deep. Moreover, it is also necessary to perform three medium-intensity harvests at a water depth of 120cm and one low-intensity harvest or no harvesting at a water depth of 150cm to achieve longer lifetimes and less biomass near the water surface when the plants reach or approach the water surface.

Description: Aims Distyly has been regarded as an adaptation to improve compatible pollination between two floral morphs with reciprocal herkogamy. The hypothesis that the different positions of anthers and stigmas within flowers as well as their reciprocal position between morphs, reduce the probability of self pollination raised by Darwin has been rarely tested. In this study, we measured stigmatic pollen loads in response to reduced reciprocal herkogamy in two Primula species. Methods To see whether reciprocal herkogamy can increase compatible and/or reduce incompatible pollen deposition, thus promoting compatible pollination, we shortened the distance between anthers and stigmas within the flowers by changing the position of the corolla tube, to which the anthers were fused, i.e. reduced herkogamy in natural populations of Primula secundiflora and P. poissonii and quantified stigmatic pollen loads in the field over 2 years. Important Findings In both species, stigmatic pollen loads were significantly higher in the long-styled (L-morph) than in the short-styled morph (S-morph) in both control and manipulated flowers, but percentage of compatible pollen in S-morph were higher. Flowers manipulated to halve the anther–stigma distance showed a similar pattern for 2 years: total pollen grain counts on stigmas did not differ significantly but compatible pollen grains in L- and S-morphs were significantly decreased in both species. The percentage of compatible pollen loads was decreased by 68.7% in P. secundiflora and 65.3% in P. poissonii in L-morphs, while it decreased by 30.6% and 2.9% in S-morphs, respectively. Our manipulation of the relative position of anthers and stigmas in the two distylous species indicated that a lower degree of herkogamy reduced compatible but incompatible pollen transfer was likely to increase. The higher proportion of compatible pollen in the S-morph than in the L-morph in the two Primula species could be attributed to the accessibility of two-level sexual organs, floral orientations and pollinator behaviors. This is a first attempt to manipulate intraflower herkogamy for understanding adaptation of heterostyly, shedding insights into how the reciprocal herkogamy promotes compatible pollination.

Description: Aberrant glycosylation is highly associated with cancer progression. The aim of this study was to compare bifucosylated N -glycans in sera obtained from healthy controls and breast cancer patients, with the goal of identifying a potential indicator for monitoring the recurrence and metastasis of breast cancer. A unique structural pattern of bifucosylated N -glycan, with both core and antennary fucosylation, was identified in breast cancer patients. The spectrum of antennary fucosylation was a composite of the standard spectra of Lewis X and H2, indicating a mixture of the two epitopes. Permethylated N -glycans of the glycoproteins extracted from 91 breast cancer patients and 43 healthy controls were detected using linear ion-trap quadrupole-electrospray ionization mass spectrometry, which appeared to be a highly sensitive and useful approach in the detection and identification of N -glycans. To evaluate MS profile data, several statistical tools were applied, including Student's t -test, partial least squares discriminant analysis and receiver-operating characteristic curve. The results showed that the measurement of bifucosylation degree and CEA levels had an improved diagnostic performance compared with that of CEA alone. We compared the potential of bifucosylated N -glycan as an indicator of breast cancer recurrence with the current clinical biomarkers, i.e., CEA, CA 15-3 and CA125. The result revealed that, compared with CEA, CA 15-3 and CA125, the bifucosylation degree of N -glycans could be a more reliable indicator of breast cancer recurrence.

Description: Colitose, also known as 3,6-dideoxy- l -galactose or 3-deoxy- l -fucose, is one of only five naturally occurring 3,6-dideoxyhexoses. Colitose was found in lipopolysaccharide of a number of infectious bacteria, including Escherichia coli O55 & O111 and Vibrio cholera O22 & O139. To date, no colitosyltransferase (ColT) has been characterized, probably due to the inaccessibility of the sugar donor, GDP-colitose. In this study, starting with chemically prepared colitose, 94.6 mg of GDP-colitose was prepared via a facile and efficient one-pot two-enzyme system involving an l -fucokinase/GDP- l -Fuc pyrophosphorylase and an inorganic pyrophosphatase (EcPpA). WbgN, a putative ColT from E. coli O55:H5 was then cloned, overexpressed, purified and biochemically characterized by using GDP-colitose as a sugar donor. Activity assay and structural identification of the synthetic product clearly demonstrated that wbgN encodes an α1,2-ColT. Biophysical study showed that WbgN does not require metal ion, and is highly active at pH 7.5–9.0. In addition, acceptor specificity study indicated that WbgN exclusively recognizes lacto- N -biose (Galβ1,3-GlcNAc). Most interestingly, it was found that WbgN exhibits similar activity toward GDP- l -Fuc ( k cat / K m = 9.2 min –1 mM –1 ) as that toward GDP-colitose ( k cat / K m = 12 min –1 mM –1 ). Finally, taking advantage of this, type 1 H-antigen was successfully synthesized in preparative scale.

Description: Information about specificity and affinity is critical for use of carbohydrate-binding antibodies. Herein, we evaluated eight monoclonal antibodies to the blood group A (BG-A) antigen. Antibodies 87-G, 9A, HE-10, HE-24, HE-193, HE-195, T36 and Z2A were profiled on a glycan microarray to assess specificity, relative affinity and the influence of glycan density on recognition. Our studies highlight several noteworthy recognition properties. First, most antibodies bound GalNAcα1–3Gal and the BG-A trisaccharide nearly as well as larger BG-A oligosaccharides. Second, several antibodies only bound the BG-A trisaccharide when displayed on certain glycan chains. These first two points indicate that the carrier glycan chains primarily influence selectivity, rather than binding strength. Third, binding of some antibodies was highly dependent on glycan density, illustrating the importance of glycan presentation for recognition. Fourth, some antibodies recognized the tumor-associated Tn antigen, and one antibody only bound the variant composed of a GalNAc-alpha-linked to a serine residue. Collectively, these results provide new insights into the recognition properties of anti-BG-A antibodies.

Description: During malignant transformation, glycosylation is heavily altered compared with healthy tissue due to differential expression of glycosyltransferases, glycosidases and monosaccharide transporters within the cancer microenvironment. One key change of malignant tissue glycosylation is the alteration of sialic acid processing that leads to a general upregulation of sialylated glycans (hypersialylation) on cell surfaces and an increased introduction of the non-human sialic acid N -glycolyl-neuraminic acid (Neu5Gc) instead of N -acetyl-neuraminic acid into cell surface glycans. These changes have been shown to be the result of altered sialyltransferase and sialidase expression. Functionally, cancer-associated hypersialylation appears to directly impact tumor cell interaction with the microenvironment, in particular the modulation of sialic acid-binding lectins on immune cells. Moreover, Neu5Gc expression in human tissues enhances inflammation due to an anti-Neu5Gc immune response, which can potentially influence inflammation-induced cancer and cancer-associated inflammation. In this review, we summarize the changes of sialic acid biology within the malignant microenvironment and the resulting effect on cancer immunity.

Description: In this work, we present the first XOS degrading glycoside hydrolase from Weissella , W Xyn43, a two-domain enzyme from GH43. The gene was amplified from genomic DNA of the XOS utilizing Weissella strain 92, classified under the species-pair Weissella cibaria/W.confusa , and expressed in Escherichia coli . The enzyme is lacking a putative signal peptide and is, from a homology model, shown to be composed of an N-terminal 5-fold β-propeller catalytic domain and a C-terminal β-sandwich domain of unknown function. W Xyn43 hydrolyzed short (1–4)-β- d -xylooligosaccharides, with similar k cat / K M for xylobiose (X 2 ) and xylotriose (X 3 ) and clearly lower efficiency in xylotetraose (X 4 ) conversion. WXyn43 displays the highest reported k cat for conversion of X 3 (900 s –1 at 37°C) and X 4 (770 s –1 ), and k cat for hydrolysis of X 2 (907 s –1 ) is comparable with or greater than the highest previously reported. The purified enzyme adopted a homotetrameric state in solution, while a truncated form with isolated N-terminal catalytic domain adopted a mixture of oligomeric states and lacked detectable activity. The homology model shows that residues from both domains are involved in monomer–monomer hydrogen bonds, while the bonds creating dimer–dimer interactions only involved residues from the N-terminal domain. Docking of X 2 and X 3 in the active site shows interactions corresponding to subsites –1 and +1, while presence of a third subsite is unclear, but interactions between a loop and the reducing-end xylose of X 3 may be present.

Description: The acquisition of mannose 6-phosphate (Man6 P ) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6 P monosaccharide that, through an array-screening approach against a number of phosphorylated N -glycans, is shown to bind mono- and diphosphorylated Man 6 and Man 7 glycans that contain terminal αMan6 P (1 -〉 2)αMan(1 -〉 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man 8 or mono- or diphosphorylated Man 9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6 P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second p K a of Man6 P (p K a = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6 P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ~ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6 P recognition.

Description: The role of aberrant protein modifications in cancer and its diagnosis have emerged as a promising research field. Nonenzymatic glyco-oxidation of proteins under oxidative stress has been associated with carcinogenesis through advanced glycation end products (AGE)–receptors for advanced glycation end products (RAGE) axis. Modified proteins that are immunogenic and stimulate cellular and humoral immune responses are being studied to develop early detection markers of cancer. This study has probed the structural alternations; leading to the formation of adducts and aggregates, in histone H2A upon in vitro modification by methylglyoxal (MG). The immunogenicity of modified histone H2A and its binding with cancer autoantibodies was also assessed. MG induced lysine side chain modifications, blocking of free amino groups and the formation of condensed cross structures in histone H2A; and its effect was inhibited by carbonyl scavengers. It led to the adduct formation and generation of N -epsilon-(carboxyethyl)lysine (CEL) and its decomposition forms as revealed by Matrix-assisted laser desorption ionization–mass spectrometry, high-performance liquid chromatography and LC–MS. MG-H2A showed amorphous aggregate formation under electron microscopy and altered binding with DNA in circular dichroism studies. The modified histone elicited high titer immunogen-specific antibodies in rabbits when compared with the native, thus pointing toward the generation of neo-epitopes in MG-H2A. The autoantibodies derived from cancer patients exhibited enhanced binding with MG-H2A as compared with the native histone in enzyme-linked immunosorbent assay and gel retardation assay. This reflects sharing of epitopes on MG-H2A and histones in cancer patients. The neo-epitopes on H2A may be responsible for induction and elevated levels of antibodies in cancer patients. Thus, MG-H2A may be considered as potential antigenic candidate for auto-immune response in cancer.

Description: E-cadherin is often dysregulated in aggressive lung cancer, the mechanism of which cannot always be explained at the level of transcription. In 66 patients with lung cancer, immunohistochemical staining demonstrated that co-localization of E-cadherin and core fucose by Lens culinaris agglutinin was significantly less extensive in tumor than in nontumor tissue. Through gain and loss of fucosylation experiments in the giant lung carcinoma cell lines 95C and 95D, our results revealed that E-cadherin core fucosylation in 95C cells overexpressing α-1, 6-fucosyltransferase (Fut8) inhibited Fut8-95C cell migration, whereas knockdown of Fut8 in 95D cells enhanced migration of short-interfering RNA-targeting Fut8 (siFut8)-95D cells. The level of active Src (phosphorylated Src [Y416]) was significantly reduced in Fut8-95C cells, but elevated in siFut8-95D cells. In protein complexes immunoprecipitated from Fut8-95C cell lysates with anti-E-cadherin, less phosphorylated Src (Y416) and more β-catenin were observed, but immunoprecipitates from siFut8-95D cells, containing less core fucosylated E-cadherin, contained an elevated level of phospho-Src Y416. In Fut8-95C cells, phosphorylation of Akt (Y315, Y326) and GSK-3β (S9) was significantly reduced, but β-catenin (S37) phosphorylation was enhanced. Expression of N-cadherin and Snail1 was also reduced in Fut8-95C cells, but significantly increased in siFut8-95D cells. Intriguingly, when Src kinase activity was inhibited by treatment of cells with PP2 and SU6656, regulation of N-cadherin, Snail1 and cell migration by E-cadherin core fucosylation was abrogated in both Fut8-95C and siFut8-95D cells. Therefore, posttranslational modification of E-cadherin by less core fucosylation recruited and activated Src, and induced an epithelial–mesenchymal transition-like process in lung cancer cells.

Description: Statins, which specifically inhibit HMG Co-A reductase, the rate-limiting step of cholesterol biosynthesis, are widely prescribed to reduce serum cholesterol and cardiac risk, but many other effects are seen. We now show an effect of these drugs to induce profound changes in the step-wise synthesis of glycosphingolipids (GSLs) in the Golgi. Glucosylceramide (GlcCer) was increased several-fold in all cell lines tested, demonstrating a widespread effect. Additionally, de novo or elevated lactotriaosylceramide (Lc3Cer; GlcNAcβ1-3Galβ1-4GlcCer) synthesis was observed in 70%. Western blot showed that GlcCer synthase (GCS) was elevated by statins, and GCS and Lc3Cer synthase (Lc3S) activities were increased; however, transcript was elevated for Lc3S only. Supplementation with the isoprenoid precursor, geranylgeranyl pyrophosphate (GGPP), a downstream product of HMG Co-A reductase, reversed statin-induced glycosyltransferase and GSL elevation. The Rab geranylgeranyl transferase inhibitor 3-PEHPC, but not specific inhibitors of farnesyl transferase, or geranylgeranyl transferase I, was sufficient to replicate statin-induced GlcCer and Lc3Cer synthesis, supporting a Rab prenylation-dependent mechanism. While total cholesterol was unaffected, the trans-Golgi network (TGN) cholesterol pool was dissipated and medial Golgi GCS partially relocated by statins. GSL-dependent vesicular retrograde transport of Verotoxin and cholera toxin to the Golgi/endoplasmic reticulum were blocked after statin or 3-PEHPC treatment, suggesting aberrant, prenylation-dependent vesicular traffic as a basis of glycosyltransferase increase and GSL remodeling. These in vitro studies indicate a previously unreported link between Rab prenylation and regulation of GCS activity and GlcCer metabolism.

Description: Galectin-3 is a ubiquitous lectin exerting multiple cellular functions such as RNA splicing, protein trafficking and apoptosis. Its expression is positively correlated with the poor prognosis in lung cancer patients. Galectin-3 can promote cancer progression through its effects on cell proliferation, cell survival or cancer metastasis. However, the role of galectin-3 in the regulation of cancer stem-like cells (CSCs) is still unclear. Here, we investigated the hypothesis that galectin-3 might regulate lung CSCs via the EGF receptor (EGFR) signaling pathway. In our study, galectin-3 facilitated EGFR activation and enhanced the sphere formation activity of lung cancer cells. Furthermore, galectin-3 promoted Sox2 expression in an EGFR activation-dependent manner; importantly, forced expression of Sox2 blunted the effect of galectin-3 knockdown on lung cancer sphere formation ability. These results suggest that galectin-3 promotes EGFR activation leading to the upregulation of Sox2 expression and lung CSCs properties. Moreover, we showed that the carbohydrate-binding activity of galectin-3 was important for the regulation of EGFR activation, Sox2 expression and sphere formation. We have recently reported that c-Myc is a transcriptional activator of Sox2. We further found that galectin-3 enhanced c-Myc protein stability leading to increased c-Myc binding to the Sox2 gene promoter. We also examined the effect of the stemness factors, Oct4, Nanog and Sox2 on the expression of galectin-3. We found that Oct4 enhanced galectin-3 expression. Our results together suggest that galectin-3 enhances lung cancer stemness through the EGFR/c-Myc/Sox2 axis; Oct4, in turn, promotes galectin-3 expression, forming a positive regulatory loop in lung CSCs.

Description: β-1,2-Linked mannosides are expressed on numerous cell-wall glycoconjugates of the opportunistic pathogen yeast Candida albicans . Several studies evidenced their implication in the host–pathogen interaction and virulence mechanisms. In the present study, we characterized the in vitro activity of CaBmt3, a β-1,2-mannosyltransferase involved in the elongation of β-1,2-oligomannosides oligomers onto the cell-wall polymannosylated N -glycans. A recombinant soluble enzyme Bmt3p was produced in Pichia pastoris and its enzyme activity was investigated using natural and synthetic oligomannosides as potential acceptor substrates. Bmt3p was shown to exhibit an exquisite enzymatic specificity by adding a single terminal β-mannosyl residue to α-1,2-linked oligomannosides capped by a Manβ1–2Man motif. Furthermore, we demonstrated that the previously identified CaBmt1 and CaBmt3 efficiently act together to generate Manβ1-2Manβ1–2[Manα1–2] n sequence from α-1,2-linked oligomannosides onto exogenous and endogenous substrates.

Description: Aims Deserts are one of the ecosystems most sensitive to global climate change. However, there are few studies examining how changing abiotic and biotic factors under climate change will affect plant species diversity in the temperate deserts of Asia. This study aimed to: (i) characterize species distributions and diversity patterns in an Asian temperate desert; and (ii) to quantify the effects of spatial and environment variables on plant species diversity. Methods We surveyed 61 sites to examine the relationship between plant species diversity and several spatial/environmental variables in the Gurbantunggut Desert. Spatial and environmental variables were used to predict plant species diversity in separate multiple regression and ordination models. Variation in species responses to spatial and environmental conditions was partitioned by combining these variables in a redundancy analysis (RDA) and by creating multivariate regression trees (MRT). Important Findings We found 92 plant species across the 61 sites. Elevation and geographic location were the dominant environmental factors underlying variation in site species richness. A RDA indicated that 93% of the variance in the species–environment relationships was explained by altitude, latitude, longitude, precipitation and slope position. Precipitation and topographic heterogeneity, through their effects on water availability, were more important than soil chemistry in determining the distribution of species. MRT analyses categorized communities into four groups based on latitude, soil pH and elevation, explaining 42.3% of the standardized species variance. Soil pH strongly influenced community composition within homogeneous geographic areas. Our findings suggest that precipitation and topographic heterogeneity, rather than edaphic heterogeneity, are more closely correlated to the number of species and their distributions in the temperate desert.

Description: Aims The introduction of potentially invasive species through ornamental cultivation or for rehabilitation purposes is a serious environmental problem. They cause damage to biodiversity through loss, increased mortality or ‘ in situ ’ selection phenomena in natural flora. Spartium junceum is a Mediterranean shrub that is not native in most areas of the Iberian Peninsula, although it is extensively grown for the rehabilitation of roadsides. We have investigated the effect on the native vegetation of an old S. junceum (Fabaceae) plantation in a conservation area in the centre of the Iberian Peninsula: the Cuenca Alta del Manzanares nature reserve in Madrid. Methods We compared S. junceum stands with the native nanophanerophytic Cistus ladanifer community at different ecosystem properties: soil properties, temporal soil seed bank contents, standing vegetation and net primary production of annual grasslands growing in these shrublands. Important Findings The results highlighted S. junceum ’s ability to become established in the new environment (marginal areas of the nature reserve) and ensure its successful growth. This is more apparent in northern and eastern exposures where this formation contacts with the core of the best conserved native vegetation in the nature reserve. Soils under Spartium showed a higher nitrogen content, indicating its capacity—in common with other legumes—to fix N, and conferring an advantage over Cistus , which is N-limited. Other soil nutrients such as phosphorus, magnesium and calcium and water availability are also higher in Spartium soils than in Cistus . Phosphorus is usually a constraint for N-fixers, but in our study, it is the most significant soil variable in both shrub formations and is important to the success of Spartium . Water availability is a key factor for Mediterranean vegetation, and particularly in autumn when soils are recharged. The Spartium formation is able to retain water as its growth produces a closer canopy than Cistus , thereby preventing water evaporation and contributing to the success of this species. Perennials are more frequent in the standing vegetation than in the seed bank, whereas therophytes are similar. The standing vegetation has therophytes and chamaephytes as the predominant growth forms in Spartium sites, and hemicryptophytes and phanerophytes in Cistus . Therophytes are dominant in Spartium and Cistus seed banks, although the first formation has more species. Spartium has a higher number of hemicryptophytes and Cistus is higher in phanerophytes. Northern and eastern aspects show significant differences in richness—with a predominance of annual weed species in Spartium —and in above-ground net primary production, probably as a consequence of the nutrients present in the soils. Ruderal annual species under Spartium ( Bromus tectorum , Chenopodium album ) have higher germination rates in the greenhouse than in the standing vegetation, suggesting they are at potential risk if environmental conditions change.

Description: Aims Positive interactions are defined as non-trophic interactions where at least one of the interacting species is benefited in terms of fitness and the other remains unaffected. Nevertheless, the bidirectional feedbacks between species may be positive, neutral or negative. Thus, if facilitated species induce negative effects on their ‘nurses’, the assumed definition of positive interactions could be reconsidered. Methods We assessed if ecological interactions between cushions of Azorella madreporica and their facilitated species are positive. Specifically, we tested if cover of facilitated species has any costs for cushion plants from an ecophysiological perspective, and if these costs increase with the amount of cover of facilitated species. In addition, through pathway analysis and correlations, we assessed if cover and richness of facilitated species have a direct and/or indirect effect on the fitness of cushion plants. Important Findings We found that facilitated plant species induced a significant cost for their nurses (cushion plants), and this cost increases with cover of the facilitated species. Additionally, the facilitated species exert a strong direct negative effect on the cushion’s fitness and a moderate indirect negative cost evident through the nutrient status and physiological performance of cushion plants. We thus contribute evidence that positive interactions between high mountain cushion plants of central Chile and their ‘facilitated’ species may be an artifact more than a fact, especially when bidirectional effects are considered; contrasting with the majority of studies that document only one side of the interaction.

Description: Aims Plants of similar life forms and closely related species have been observed to create similar types of plant–soil feedbacks (PSFs). However, investigations of the consistency of PSFs within species have not yielded clear results. For example, it has been reported that species create different types of PSFs in their native and introduced ranges. The aim of this project is to examine if four species create similar PSF types from soils collected from widely distributed areas within their introduced range. The soil for this project was collected from three areas in western North America. With this design, we aim to determine species- and site-specific ability to create PSFs and if the type of PSF created is consistent in all soil from all three collection areas. The species examined are Agropyron cristatum , Centaurea solstitialis, Poa pratensis and Taeniatherum caput-medusae. Methods We used three-field collected soils (from northern Nevada, western Montana and eastern Montana) in a two-phase greenhouse experiment to quantify the type of PSFs created by four invasive species. The first phase was a conditioning phase wherein each invasive species created species-specific changes to the soil. The second phase of the experiment was the response phase wherein both the conditioning species and a native phytometer were grown in the conditioned soil and in unconditioned (control) soil. The final aboveground biomass was used to evaluate the effect of conditioning and to determine the type of PSF created by each invasive species. Important Findings Our results suggest that three of our four study species did show consistency in relation to PSF. Two species A. cristatum and T. caput-medusae consistently created PSF types that benefit conspecifics more than heterospecifics (and thus are ‘invasive’ PSF types) and P. pratensis consistently exhibited no, or ‘neutral’, feedbacks. The fourth species ( C. solstitialis ) was inconsistent: in one soil, no feedback was created; in other soil, an invasive PSF was created and in the last soil, a feedback that relatively benefited the native phytometer was created. Thus, PSFs appear to uniformly contribute to the success of two species ( A. cristatum and T. caput-medusae ) but not C. solstitialis nor P. pratensis.

Description: The epidermal growth factor (EGF)-like repeat is a common, evolutionarily conserved motif found in secreted proteins and the extracellular domain of transmembrane proteins. EGF repeats harbor six cysteine residues which form three disulfide bonds and help generate the three-dimensional structure of the EGF repeat. A subset of EGF repeats harbor consensus sequences for the addition of one or more specific O -glycans, which are initiated by O -glucose, O -fucose or O - N -acetylglucosamine. These glycans are relatively rare compared to mucin-type O -glycans. However, genetic experiments in model organisms and cell-based assays indicate that at least some of the glycosyltransferases involved in the addition of O -glycans to EGF repeats play important roles in animal development. These studies, combined with state-of-the-art biochemical and structural biology experiments have started to provide an in-depth picture of how these glycans regulate the function of the proteins to which they are linked. In this review, we will discuss the biological roles assigned to EGF repeat O -glycans and the corresponding glycosyltransferases. Since Notch receptors are the best studied proteins with biologically-relevant O -glycans on EGF repeats, a significant part of this review is devoted to the role of these glycans in the regulation of the Notch signaling pathway. We also discuss recently identified proteins other than Notch which depend on EGF repeat glycans to function properly. Several glycosyltransferases involved in the addition or elongation of O -glycans on EGF repeats are mutated in human diseases. Therefore, mechanistic understanding of the functional roles of these carbohydrate modifications is of interest from both basic science and translational perspectives.

Description: The sialyl Lewis a and x (sLe a/x ) antigens frequently displayed on the surface of tumor cells are involved in metastasis. Their synthesis has been attributed to altered expression of selective glycosyltransferases. Identification of these glycosyltransferases and the glycoproteins that carry these carbohydrate antigens should help advance our understanding of selectin-mediated cancer metastasis. In this study, quantitative real-time polymerase chain reaction analysis coupled with in situ proximity ligation assay and small interference RNA treatment shows involvement of β3galactosyltransferase-V in the synthesis of MUC16-associated sLe a in H292 cells. Also, α3fucosyltransferase-V, which is absent in BEAS-2B human immortalized bronchial epithelial cells and A549 lung carcinoma cells, participates in the synthesis of MUC1-associated sLe x in CFT1 human immortalized bronchial epithelial cells and H292 lung carcinoma cells. Neither selectin ligand is found on MUC1 in BEAS-2B and A549 cells. Knockdown of either enzyme suppresses migration, and selectin tethering and rolling properties of H292 cells under dynamic flow as determined by wound healing and parallel plate flow chamber assays, respectively. These results provide insights into how the synthesis of mucin-associated selectin ligands and the metastatic properties of cancer cells can be regulated by selective glycosyltransferases that work on mucins. They may help develop novel anticancer drugs.

Description: Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of d -glucuronic acid (GlcA) and 4,6- O -disulfated N -acetyl- d -galactosamine (GalNAc) residues (E-unit) in the amount of ~60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen. These antibodies recognized CSE but not other CSs isomers or dermatan sulfate (DS). We evaluated the reactivities of the antibodies to 6- O -sulfated CSA (6S-CSA) and DS (6S-DS) that possessed ~60% of GalNAc (4S, 6S) moieties in their structures. Neither of the antibodies reacted with 6S-DS. The antibodies strictly distinguished the structural difference of GlcA and l -iduronic acid in the polysaccharide. Binding affinities of the antibodies were determined by a surface plasmon resonance assay using CSE and 6S-CSA. The binding affinities were strongly associated with the molecular weight of CSE and the E-unit content of 6S-CSA. Moreover, we demonstrated that the antibodies are applicable to histochemical analysis. In conclusion, the new anti-CSE monoclonal antibodies specifically recognize the E-unit of CSE. The antibodies will become useful tools for the investigation of the biological and pathological significance of CSE.

Description: D-Galactose-binding lectin from the octocoral, Sinularia lochmodes (SLL-2), distributes densely on the cell surface of microalgae, Symbiodinium sp., an endosymbiotic dinoflagellate of the coral, and is also shown to be a chemical cue that transforms dinoflagellate into a non-motile (coccoid) symbiotic state. SLL-2 binds with high affinity to the Forssman antigen ( N -acetylgalactosamine(GalNAc)α1-3GalNAcβ1-3Galα1-4Galβ1-4Glc-ceramide), and the presence of Forssman antigen-like sugar on the surface of Symbiodinium CS-156 cells was previously confirmed. Here we report the crystal structures of SLL-2 and its GalNAc complex as the first crystal structures of a lectin involved in the symbiosis between coral and dinoflagellate. N -Linked sugar chains and a galactose derivative binding site common to H-type lectins were observed in each monomer of the hexameric SLL-2 crystal structure. In addition, unique sugar-binding site-like regions were identified at the top and bottom of the hexameric SLL-2 structure. These structural features suggest a possible binding mode between SLL-2 and Forssman antigen-like pentasaccharide.

Description: Aims Small plant populations may be more likely to suffer more severe pollen limitation due to the lower number of potential mates or suitable pollinators. For invasive species, this phenomenon may be more common when an invading population colonizes a new habitat. Here, we investigated whether pollen limitation occurs in invasive populations of Solanum rostratum during its invasion from North America to China and evaluated the patterns between pollen limitation and population size. Methods Pollen addition experiments were performed on six invasive populations of S. rostratum . By comparing fruit set and seed production with open pollination treatment, we calculated the index of pollen limitation and regressed it to population size and density. Important Findings Among the six sampled invasive populations of S. rostratum , the fruit set and seed production per fruit were 0.346±0.014 and 52.38±9.29, respectively, with open pollination treatment and 0.572±0.022 and 56.28±10.79, respectively, with pollen addition treatment. Compared with open pollination, pollen addition significantly increased fruit set and seed production by 65.3 and 7.4%, respectively. The standardized index of pollen limitation ranged from 0.022 to 0.125, with an average of 0.065, suggesting that invasive populations of S. rostratum do suffer from pollen limitation. The index of pollen limitation was negatively correlated with population size, which is consistent with the pattern that smaller populations suffer from more severe pollen limitation.

Description: Aims Floral longevity, the duration that a flower remains open and functional, varies greatly among species. Variation in floral longevity has been considered to be optimal strategy for resource allocation under different ecological conditions, mainly determined by the rates of pollination and cost of flower maintenance. However, it is unclear whether an intrinsic factor, floral sexual investment, constrains evolution of floral longevity. The theoretical model also predicts that dichogamy favors long-lived flowers, but empirical studies to test this prediction remain unexplored. Methods To examine the effect of floral sexual investment on floral longevity, we measured flower size together with pollen and ovule production in 37 sympatric flowering plants in a natural community. The duration of the female and male phase in 21 protandrous species and floral longevity of the other 16 adichogamous species were documented in the field. Important Findings Floral longevity varied from 1 day to 15 days, while pollen number per flower varied from 643 to 710880 and ovule number per flower from 1 to 426 in the 37 species. Flower size was correlated with pollen production as well as ovule production. Floral longevity was positively related to pollen production but not to ovule production. Consistent with the prediction that dichogamy favors long-lived flowers, we found the floral longevity of protandrous species was significantly longer than that of adichogamous species. In the protandrous species, pollen production per flower was observed to be positively related to male duration, while ovule production was not related to female duration. Our analyses of variation in floral longevity and sexual investment among different species suggest that the floral sexual investment could be an intrinsic factor contributing to the selected floral longevity, particularly the male phase, and that high pollen production could potentially increase pollen removal, i.e. male productive success.

Description: Aims The hypothesis of predator satiation has been proposed to explain mast fruiting in various flowering plants. It considers that the simultaneous production of large numbers of seeds by a plant population reduces the risk of seed predation for each individual. Orchids produce huge numbers of seeds per fruit and rarely experience seed predation. It remains unclear which factors may affect fluctuating fruit production in orchids, which generally suffer a widespread pollen limitation. To explore the temporal pattern of fruiting and potential factors related to fluctuation in fruit production, we investigated reproductive success of a long-spurred orchid ( Habenaria glaucifolia ) in an alpine meadow with thousands of individuals over 8 years. Methods To estimate reproductive success, pollinator observation was conducted by day and at night, and pollinia removal and receipt were recorded in the field population for 8 years. To examine whether fruit set and seed set are pollen limited, we conducted supplementary pollination experiments and compared fruit set, seed set and pollinia movement of open-pollinated flowers from 2011 to 2013. We measured lengths of spurs and pollinator proboscises, and nectar volume and concentration, to identify potential pollinators. Important Findings Hawkmoths were seen to be effective pollinators for H. glaucifolia in 3 years, whereas in the remaining 5 years no pollinators were observed, and consequently pollinia were rarely transferred. Numerous pollinia movements were observed in 2012, 2013 and 2014 (pollinia removal: 48, 59 and 85%; pollinia receipt 51, 70 and 80%), and correspondingly fruit set was significantly higher in 2012 and 2013 (59 and 46%) than in 2011 (25%). It was fruit set, rather than seed set, that was pollen limited in this orchid in the 3 years, in that supplementary pollination increased fruit set but did not increase seed set per fruit compared to natural. Three species of hawkmoths had proboscis lengths that matched the spur length of H. glaucifolia . Fruit set in this long spurred orchid depends on the activity of long-tongued hawkmoths, resulting in significant temporal variation in fruit production. Mast fruiting in this alpine orchid could be attributed to a ‘sit and wait’ strategy, awaiting an abundance of effective pollinators.

Description: Aims Sex allocation in plants is often plastic, enabling individuals to adjust to variable environments. However, the predicted male-biased sex allocation in response to low resource conditions has rarely been experimentally tested in hermaphroditic plants. In particular, it is unknown whether distal flowers in linear inflorescences show a larger shift to male allocation relative to basal flowers when resources are reduced. In this study, we measure position-dependent plasticity of floral sex allocation within racemes of Aconitum gymnandrum in response to reduced resource availability. Methods Using a defoliation treatment in the field applied to potted plants from a nested half-sibling design, we examined the effects of the treatment, flower position, family and their interactions. Important Findings Allocation to male function increased with more distal flower position, while female allocation either did not change with position or declined at the most distal flowers. Defoliation significantly reduced the mass of both the androecium and gynoecium, but not anther number or carpel number. Gynoecial mass declined more strongly with defoliation than did androecial mass, resulting in a significant increase in the androecium/gynoecium ratio as predicted by sex allocation theory. Plastic responses of androecium mass and gynoecium mass were affected by flower position, with less mass lost in basal flowers, but similar plastic magnitude in both sexual traits across flower position lead to consistent variation in the androecium/gynoecium ratio along the inflorescence. A significant treatment*paternal family interaction for the androecium/gynoecium ratio is evidence for additive genetic variation for plastic floral sex allocation, which means that further evolution of allocation can occur.

Description: Aims When sympatric flowering plant species in a natural community share pollinators, study of plant–plant interactions via interspecific pollen transfer (IPT) is essential for understanding species coexistence. However, little is known about the extent of IPT between interactive species and its causes. Methods To explore how sympatric flowering plants sharing pollinators minimize deleterious effects of IPT, we investigated the pollination ecology of two endemic species, Salvia przewalskii and Delphinium yuanum , in an alpine meadow in the Hengduan Mountains, southwest China. We quantified conspecific and interspecific visits by shared bumblebee pollinators, amounts of pollen placed on different body sites of the pollinators and stigmatic pollen loads on open-pollinated flowers. To examine whether IPT affects female fitness, we measured pollen germination and seed production in the two species in an artificial pollination experiment. Important Findings One bumblebee species, Bombus trifasciatus , was found to be the sole effective pollinator for the two coflowering species. Pollination experiments indicated that deposition of heterospecific pollen could significantly decrease seed set in both species. Experiments showed that S. przewalskii pollen could germinate well on stigmas of D. yuanum , inhibiting conspecific pollen germination in D. yuanum . However, seed set was not lower under open pollination than under cross-pollination within species, suggesting that no female fitness loss was caused by IPT. In foraging bouts with pollinator switches, switches from S. przewalskii to D. yuanum were relatively more frequent (8.27%) than the converse (1.72%). However, IPT from S. przewalskii to D. yuanum accounted for only 1.82% of total stigmatic pollen loads while the reverse IPT to S. przewalskii was 8.70%, indicating that more switches of bumblebees to D. yuanum did not result in higher IPT. By contrast, selection for reduced IPT to S. przewalskii would limit pollinator switches from D. yuanum . We found that a bumblebee generally carried pollen grains from both species but the two species differed in the position of pollen placement on the bumblebee’s body; S. przewalskii ’ s pollen was concentrated on the dorsal thorax while D. yuanum ’ s pollen was concentrated ventrally on the head. This differential pollen placement along with pollinator fidelity largely reduced IPT between the two species with a shared pollinator.

Description: Aims Studying the ecological significance of highly specialized morphological traits evolved by alpine plants could help us to understand the adaptation and evolution of these plants under severe alpine environment. We explored the adaptive significance of woolly and overlapping leaves for reproduction in Eriophyton wallichii , a perennial herb native to the subnival belt of the Himalaya-Hengduan Mountains. Methods We examined whether the trichomes could influence the leaf wettability, temperature and leaf reflectance spectra in the lab. And we investigated the thermal benefits of the woolly and overlapping leaves for flowers and fruits in the field. Pollen viability and seed germination were also examined in the lab to assess whether these leaves enhance reproductive fitness. Important Findings Our results showed that dense trichomes impart good water repellency, absorption of solar radiation and accumulation of leaf heat. The woolly and overlapping leaves increased the interior temperature of flowers and fruits to an optimal level on sunny hours, but prevented them from overheating when transient intense solar radiation occurs. This kept optimal temperatures in plants’ reproductive organs, thus promoting the development of pollen and seed in alpine environment.

Description: Aims It is generally accepted that visual displays and floral scent play important roles in communication between flowering plants and their pollinators. However, the relative role of visual and olfactory cues in pollinator attraction is largely unknown. In this study, we determined the roles of both types of cue in attracting pollinators to Cornus capitata , a medium sized tree with each capitulum surrounded by four large, white, petaloid bracts. Methods Pollinator observations and pollination experiments were conducted in a natural population; the inflorescences’ visual and olfactory signals were characterized by spectral and chemical analyses; the responses of pollinators to visual and olfactory cues were tested using dual-choice behavioural bioassays; the relative roles of visual and olfactory cues in pollinator attraction were tested by comparing the responses of pollinators to inflorescences subjected to three experimental treatments (intact, all bracts removed, and capitulum removed) within the natural population. Important Findings For fruit set, C. capitata is entirely dependent on pollinators, with a bee, Anthophora sp., being the main pollinator. Bracts present high colour distance and green contrast against the leaves. Twelve volatile compounds in the floral scent were detected, most of which have previously been reported to be attractive to a broad spectrum of bee species. Behavioural bioassays showed that both, visual cues alone and olfactory cues alone, are attractive to pollinating bees. However, visual cues alone attracted significantly more approaches than olfactory cues alone, while olfactory cues alone elicited a significantly higher landing percentage than visual cues alone. The finding suggests that, in the C. capitata – Anthophora sp. interaction, visual cues are mainly used for location from long distances, while olfactory cues mainly aid landing from short distances. Our results indicate that different modalities of floral cues should be considered together to understand fully the communication between flowering plant and pollinators.

Description: Aims Flowering time has been suggested to be an important adaptive trait during the dispersal of invasive species, and identifying the molecular mechanisms underlying flowering time may provide insight into the local adaptation during the process of invasion. Here, we conducted a preliminary exploration on the genetic basis of the differentiation of flowering time in Ambrosia artemisiifolia . Methods Using relative real-time fluorescent quantitative polymerase chain reaction, we investigated the expression levels of eight flowering-related genes, including AP1 , FT , SOC1 , CRY2 , FKF1 , GI , CO2 and SPY , in leaves and flowers at different time points in individuals from northern Beijing and southern Wuhan populations that exhibit significant differences in flowering times to identify any rhythmic changes in gene expression and their association with differential flowering times. Important Findings The differentiation of flowering time in the A. artemisiifolia populations was closely associated with five genes involved in flowering pathways. The floral pathway integrators FT and SOC1 and floral meristem identity gene AP1 exhibited increased expression during flowering. The photoreceptor CRY2 in the light-dependent pathway and the SPY gene in the gibberellin pathway displayed specific expression patterns over time. In earlier-flowering Beijing plants, CRY2 expression was lower and SPY expression was higher than in Wuhan plants. The expression patterns of these five genes suggest a molecular basis for the differentiation of flowering time in A. artemisiifolia .

Description: Aims Hybridization usually leads to gene introgression between related species in hybrid zones, associated with complex patterns of morphological variation. Nevertheless, previous studies have tended to ignore the effects of geographic variation in hybridization rates on species taxonomy. This study aims to investigate the variation of morphological traits between two sympatric and taxonomically confused oak species, Quercus liaotungensis and Q. mongolica , and reveal the effects of hybridization rates on morphological traits and the taxonomic boundary. Methods We used seven microsatellite loci to evaluate species status and measured 15 morphological traits in 26 trees in the recent hybrid zone between Q. liaotungensis and Q. mongolica , and we characterized the differences between the two oak species and their hybrids for the investigated traits. Important Findings Molecular analyses indicated that 74% of 78 sampled maternal trees were hybrids between Q. liaotungensis and Q. mongolica although the observed morphological variation suggested that they had remained distinct species. Across all of the differentiated leaf and reproductive traits, the hybrids expressed patterns similar to Q. liaotungensis , which may suggest dominant expression of parental characters. These results are consistent with our expectation that hybrids will be difficult to distinguish from parental species in a recent hybrid zone.

Description: Aims The calyx, the outermost whorl of a flower (usually green), has been considered to function to protect flowers. In some species, however, calyces are colorful and retained during seed development. Limonium species have been exploited as cut flower crops because the calyces persist for several months after the corolla has closed. To explore the adaptive significance of the persistent calyx in a desert plant Limonium leptolobum , we ask whether persistence of calyces can enhance pollinator attraction by enlarging floral displays, increasing reproductive success in this self-incompatible species. Methods The yellow flower of L. leptolobum lasted 1–2 days but its white, membranous calyx extended fully after the corolla closed, and persisted for over 2 months in the field, making hundreds of ‘showy flowers’ on one individual. To examine the ecological function of calyces, we test the pollinator attraction hypothesis. In an experimental population, we compared the difference in visit frequency and visitor behavior between intact inflorescences and inflorescences with their calyces removed on the same individual plants. Important findings In four experimental plots four types of floral visitors were observed including bees, butterflies, syrphid flies and day-flying moths. No significant preference was observed between calyx-free and intact inflorescences for both first arrivals and total visit frequency of all types of floral visitors, indicating that the persistence of calyces did not make plants more attractive to potential pollinators. The pollinator attraction hypothesis for the showy calyces was not supported by the current data. Whether the calyx in this desert plant helps seed development or has other functions needs further study.

Description: Aims Floral nectar plays a vital role in plant reproductive success by attracting pollinators. Nectar traits of a flower can depend directly on plant characteristics other than environmental factors and exhibit extensive flower- and plant-level variations. Studies on nectar traits frequently focused on intraplant variation for dichogamous plants, but few have paid attention to both intra- and interplant nectar variations in relation to plant characteristics. Revealing within- and among-plant variation and its relative magnitude is important for our understanding of how pollinator-mediated selection can act on nectar traits and evolution of nectar traits. Methods Through investigating protandrous Aconitum gymnandrum populations at the Alpine Meadows and Wetland Ecosystems Research Station of Lanzhou University, we examined the relationships between nectar production per flower and plant characteristics (e.g. flower position within inflorescences, floral sexual phases, flowering time, inflorescence size and floral attractive traits). Important Findings A. gymnandrum exhibited a declining gradient in the nectar volume along inflorescences, with more nectar in basal flowers than distal ones. Protandrous flowers of A. gymnandrum did not show gender-biased nectar production while the nectar volume varied with different stages of floral sexual phases. The significant correlation between the first flowering date of individuals and the mean nectar volume per flower was positive in 2013, but became negative in 2014, suggesting complex effects of biotic and abiotic factors. The mean nectar volume per flower was not related to inflorescence size (the number of total flowers per plant). Furthermore, nectar production was weakly associated with floral attractive traits (the petal width and the galea height), even if the effect of flowering time of individuals was removed, suggesting that the honesty of floral traits as signals of nectar reward for pollinators is not stable in this species.

Description: Aims Exploring the characteristics and function of a flower color polymorphism contributes to our understanding of floral evolution in angiosperms. However, little information is available on stigma color polymorphisms in flowering plants despite their important functional role in plant reproduction. Methods We studied a stigma color polymorphism at the individual level in Acer pictum subsp. mono (aka A. mono ) by investigating stigma-color morph proportion and comparing stigma performance and components of female fitness (pollen adhesion, pollen germination, fruit set, seed set and fruit weight) between the two morphs (red and greenish-yellow stigma) within natural populations at Dongling Mountain in the north of China. In the flowering period, we conducted hand pollination in natural populations and then made microscopic observations using the aniline blue fluorescence method. Important Findings Individuals in the studied populations did not change their stigma color between years, and flowers produced by a single tree were uniform in their stigma color. This strongly suggests that stigma color variation is genetically controlled. The percentage of the red stigma flowers with germinated pollen grains was significantly higher than that of the greenish-yellow stigma flowers when hand pollination was conducted in the early flowering period, but not so in the late flowering period. There was no significant difference in the percentage of flowers with pollen adhesion to the stigma between the two morphs. Fruit set of the red morph was significantly higher than that of the greenish-yellow morph. Our findings suggest that the red morph may be more advantageous for pollen germination and fruit set than the greenish-yellow morph, which may provide a functional explanation for the high incidence of the red stigma morph in the studied populations. Alternative explanations for the stigma color polymorphism are discussed to stimulate further work.

Description: Aims In heterantherous plants, ‘division of labor’ among structurally different stamens, i.e. pollinating and feeding functions, has been thought to reduce the evolutionary conflict of relying on pollen both as the carrier of male gametes and as the food for pollinators. The key to successful division of labor among different sets of stamens is the size match between stamens and legitimate pollinators, which results in the precise deposition of pollen onto specific locations on pollinator’s body and facilitates cross pollination. However, the potential impact of small illegitimate insects that are ubiquitous during the pollination process on the plant reproduction in heterantherous species has been largely neglected in previous studies and never been demonstrated experimentally. Methods Here, we investigated the functions of three different types of stamens in Commelina communis . The pollinator visitation, pollen removal and deposition were compared among flowers with different types of anthers emasculated at two natural populations. Moreover, the mating systems of C. communis in wild populations were estimated using microsatellite markers. Important Findings Our data showed that the main floral visitors for C. communis at the two studied populations were small illegitimate bees rather than legitimate pollinators, accounting for 77.5 and 92.2% of total flower visits, respectively. Flower manipulations in C. communis demonstrated that the two types of brightly yellow stamens separately functioned as ‘deceptive attraction’ and ‘feeding’ functions. Although the brown inconspicuous stamens of C. communis with the largest amount of fertile pollen had the potential function in offering pollen for cross pollination, the high ratio of illegitimate visitation by small bees significantly affected the dispersal and deposition of pollen from the pollinating anthers, and subsequently decreased the levels of outcrossing ( t m = 0.23–0.32) in wild populations. Our work further confirmed that the size match between pollinators and the floral morphology is the prerequisite to successfully fulfill the functional differentiation among different sets of stamens in heterantherous plants. Local high ratio of illegitimate visitation by size unmatched insects could significantly weaken the potential functions of heteranthery, affecting the dispersal and deposition of functional pollen in heterantherous plants and further the whole mating systems.

Description: Protein glycosylation is one of the most significant post-translation modifications and plays a critical role in various biological functions. Haptoglobin (Hp) is one of the acute-phase response proteins secreted by liver. Its glycosylation could be analyzed by many analytical techniques qualitatively and quantitatively. The glycosylation alterations of Hp are reported to be associated with different kinds of diseases. The main glycosylation alterations of Hp in cancer appear to be the presence of aberrantly fucosylated and sialylated structures as well as increased branching. In this mini review, we provided a brief overview of Hp structure and biological function, discussed its glycosylation alterations in different cancers, and described the existing technologies for analyzing glycosylation site and glycan of Hp. Given the importance of Hp glycosylation, its unknown and unclear biological complexity and significances, Hp glycosylation has become a major target in cancer research. Development of sensitive and specific detection of Hp glycosylation including large-scale validation may be significant steps forward to its clinical application.

Description: Keratan sulfate (KS) was isolated from chicken egg white in amounts corresponding to ~0.06 wt% (dry weight). This KS had a weight-average molecular weight of ~36–41 kDa with a polydispersity of ~1.3. The primary repeating unit present in chicken egg white KS was -〉4) β- N -acetyl-6- O -sulfo- d -glucosamine (1 -〉 3) β- d -galactose (1-〉 with some 6- O -sulfo galactose residues present. This KS was somewhat resistant to depolymerization using keratanase 1 but could be depolymerized efficiently through the use of reactive oxygen species generated using copper (II) and hydrogen peroxide. Of particular interest was the presence of substantial amounts of 2,8- and 2,9-linked N -acetylneuraminic acid residues in the form of oligosialic acid terminating the non-reducing ends of the KS chains. Most of the KS appears to be N -linked to a protein core as evidenced by its sensitivity to PNGase F.

Description: UDP- N -acetylglucosamine (UDP-GlcNAc) is a glucose metabolite with pivotal functions as a key substrate for the synthesis of glycoconjugates like hyaluronan, and as a metabolic sensor that controls cell functions through O-GlcNAc modification of intracellular proteins. However, little is known about the regulation of hexosamine biosynthesis that controls UDP-GlcNAc content. Four enzymes can catalyze the crucial starting point of the pathway, conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P): glutamine-fructose-6-phosphate aminotransferases (GFAT1 and 2) and glucosamine-6-phosphate deaminases (GNPDA1 and 2). Using siRNA silencing, we studied the contributions of these enzymes to UDP-GlcNAc content and hyaluronan synthesis in human keratinocytes. Depletion of GFAT1 reduced the cellular pool of UDP-GlcNAc and hyaluronan synthesis, while simultaneous blocking of both GNPDA1 and GDPDA2 exerted opposite effects, indicating that in standard culture conditions keratinocyte GNPDAs mainly catalyzed the reaction from GlcN6P back to Fru6P. However, when hexosamine biosynthesis was blocked by GFAT1 siRNA, the effect by GNPDAs was reversed, now catalyzing Fru6P towards GlcN6P, likely in an attempt to maintain UDP-GlcNAc content. Silencing of these enzymes also changed the gene expression of related enzymes: GNPDA1 siRNA induced GFAT2 which was hardly measurable in these cells under standard culture conditions, GNPDA2 siRNA increased GFAT1, and GFAT1 siRNA increased the expression of hyaluronan synthase 2 (HAS2). Silencing of GFAT1 stimulated GNPDA1 and GDPDA2, and inhibited cell migration. The multiple delicate adjustments of these reactions demonstrate the importance of hexosamine biosynthesis in cellular homeostasis, known to be deranged in diseases like diabetes and cancer.

Description: The balance between neutrophil serine proteases (NSPs) and protease inhibitors (PIs) in the lung is a critical determinant for a number of chronic inflammatory lung diseases such as chronic obstructive pulmonary disease, cystic fibrosis and acute lung injury. During activation at inflammatory sites, excessive release of NSPs such as human neutrophil elastase (HNE), proteinase 3 (Pr3) and cathepsin G (CatG), leads to destruction of the lung matrix and continued propagation of acute inflammation. Under normal conditions, PIs counteract these effects by inactivating NSPs; however, in chronic inflammatory lung diseases, there are insufficient amounts of PIs to mitigate damage. Therapeutic strategies are needed to modulate excessive NSP activity for the clinical management of chronic inflammatory lung diseases. In the study reported here, a panel of N- arylacyl O - sulfonated aminoglycosides was screened to identify inhibitors of the NSPs. Dose-dependent inhibitors for each individual serine protease were identified. Select compounds were found to inhibit multiple NSPs, including one lead structure that is shown to inhibit all three NSPs. Two lead compounds identified during the screen for each individual NSP were further characterized as partial mixed inhibitors of CatG. Concentration-dependent inhibition of protease-mediated detachment of lung epithelial cells is demonstrated.

Description: Galectin-12 is a member of an animal lectin family with affinity for β-galactosides and containing consensus amino acid sequences. Here, we found that galectin-12 was expressed in macrophages and thus aimed to determine how galectin-12 affects inflammation and macrophage polarization and activation. The ablation of galectin-12 did not affect bone marrow cells to differentiate into macrophages, but reduced phagocytic activity against Escherichia coli and lowered the secretion of nitric oxide. The ablation of galectin-12 also resulted in the polarization of macrophages into the M2 direction, as indicated by increases in the levels of M2 markers, namely, resistin-like β (FIZZ1) and chitinase 3-like 3 (Ym1), as well as a reduction in the expression levels of a number of M1 pro-inflammatory cytokines. We found that the diminished expression of pro-inflammatory cytokines in macrophages resulting from galectin-12 deletion was due to reduced activation of IKKα/β, Akt and ERK, which in turn caused decreased activation of NF-B and activator protein 1. The activation of STAT3 was much higher in Gal12 –/– macrophages activated by lipopolysaccharide, which was correlated with higher levels of IL-10. Adipocytes showed higher insulin sensitivity when treated with Gal12 –/– macrophage-conditioned media than those treated with Gal12 +/+ macrophages. We conclude galectin-12 negatively regulates macrophage polarization into the M2 population, resulting in enhanced inflammatory responses and also in turn causing decreased insulin sensitivity in adipocytes. This has implications in the treatment of a wide spectrum of metabolic disorders.

Description: The enzyme UDP- N -acetylglucosamine: α- d -mannoside β-1-6 N -acetylglucosaminyltransferase V (GnT-V) catalyzes the transfer of GlcNAc from the UDP-GlcNAc donor to the α-1-6-linked mannose of the trimannosyl core structure of glycoproteins to produce the β-1-6-linked branching of N-linked oligosaccharides. β-1-6-GlcNAc-branched N -glycans are associated with cancer growth and metastasis. Therefore, the inhibition of GnT-V represents a key target for anti-cancer drug development. However, the development of potent and specific inhibitors of GnT-V is hampered by the lack of information on the three-dimensional structure of the enzyme and on the binding characteristics of its substrates. Here we present the first 3D structure of GnT-V as a result of homology modeling. Various alignment methods, docking the donor and acceptor substrates, and molecular dynamics simulation were used to construct seven homology models of GnT-V and characterize the binding of its substrates. The best homology model is consistent with available experimental data. The three-dimensional model, the structure of the enzyme catalytic site and binding information obtained for the donor and acceptor can be useful in studies of the catalytic mechanism and design of inhibitors of GnT-V.

Description: Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8–2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-β-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry.

Description: N-Glycosylation is a post-translational modification performed in all three domains of life. In the halophilic archaea Haloferax volcanii , glycoproteins such as the S-layer glycoprotein are modified by an N-linked pentasaccharide assembled by a series of Agl (archaeal glycosylation) proteins. In the present study, mass spectrometry (MS) and nuclear magnetic resonance spectroscopy were used to define the structure of this glycan attached to at least four of the seven putative S-layer glycoprotein N-glycosylation sites, namely Asn-13, Asn-83, Asn-274 and Asn-279. Such approaches detected a trisaccharide corresponding to glucuronic acid (GlcA)-β1,4-GlcA-β1,4-glucose-β1-Asn, a tetrasaccharide corresponding to methyl- O -4-GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, and a pentasaccharide corresponding to hexose-1,2-[methyl- O -4-]GlcA-β-1,4-galacturonic acid-α1,4-GlcA-β1,4-glucose-β1-Asn, with previous MS and radiolabeling experiments showing the hexose at the non-reducing end of the pentasaccharide to be mannose. The present analysis thus corrects the earlier assignment of the penultimate sugar as a methyl ester of a hexuronic acid, instead revealing this sugar to be a methylated GlcA. The assignments made here are in good agreement with what was already known of the Hfx. volcanii N-glycosylation pathway from previous genetic and biochemical efforts while providing new insight into the process.

Description: Defining how a glycan-binding protein (GBP) specifically selects its cognate glycan from among the ensemble of glycans within the cellular glycome is an area of intense study. Powerful insight into recognition mechanisms can be gained from 3D structures of GBPs complexed to glycans; however, such structures remain difficult to obtain experimentally. Here an automated 3D structure generation technique, called computational carbohydrate grafting, is combined with the wealth of specificity information available from glycan array screening. Integration of the array data with modeling and crystallography allows generation of putative co-complex structures that can be objectively assessed and iteratively altered until a high level of agreement with experiment is achieved. Given an accurate model of the co-complexes, grafting is also able to discern which binding determinants are active when multiple potential determinants are present within a glycan. In some cases, induced fit in the protein or glycan was necessary to explain the observed specificity, while in other examples a revised definition of the minimal binding determinants was required. When applied to a collection of 10 GBP–glycan complexes, for which crystallographic and array data have been reported, grafting provided a structural rationalization for the binding specificity of 〉90% of 1223 arrayed glycans. A webtool that enables researchers to perform computational carbohydrate grafting is available at www.glycam.org/gr (accessed 03 March 2016).

Description: Glycans are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells' physiological state, but also regulate cellular communication and responses by engaging receptors on neighboring cells and in the extracellular matrix. The assembly of simple monosaccharide building blocks into linear or branched oligo- and polysaccharides gives rise to a large repertoire of diverse glycan structures. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell-surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organized network with specialized functions, called the glycocalyx. Nanoscale glycomaterials, which can mimic the various components of the glycocalyx, have been instrumental in revealing how the presentation of glycans can influence their biological functions. In this review, we wish to highlight some recent developments in this area, while placing emphasis on the applications of glycomaterials providing new insights into the mechanisms through which glycans mediate cellular functions.

Description: Hepatocellular carcinoma (HCC) is a malignant tumor originating in the liver. Previous studies have indicated that O-GlcNAc transferase (OGT) and histone deacetylase-1 (HDAC1) play important roles in the pathogenesis of HCC. In the present study, we investigated the physical link between OGT and HDAC1. The O-GlcNAcylation of HDAC1 is overexpressed in HCC. We found that HDAC1 has two major sites of O-GlcNAcylation in its histone deacetylase domain. HDAC1 O-GlcNAcylation increases the activated phosphorylation of HDAC1, which enhances its enzyme activity. HDAC1 O-GlcNAc mutants promote the p21 transcription regulation through affecting the acetylation levels of histones from chromosome, and then influence the proliferation of HCC cells. We also found that mutants of O-GlcNAcylation site of HDAC1 affect invasion and migration of HepG2 cells. E-cadherin level is highly up-regulated in HDAC1 O-GlcNAc mutant-treated liver cancer cells, which inhibit the occurrence and development of HCC. Our findings suggest that OGT promotes the O-GlcNAc modification of HDAC1in the development of HCC. Therefore, inhibiting O-GlcNAcylation of HDAC1 may repress the progression of HCC.

Description: Glycosaminoglycans represent a class of linear anionic periodic polysaccharides, which play a key role in a variety of biological processes in the extracellular matrix via interactions with their protein targets. Computationally, glycosaminoglycans are very challenging due to their high flexibility, periodicity and electrostatics-driven nature of the interactions with their protein counterparts. In this work, we carry out a detailed computational characterization of the interactions in protein–glycosaminoglycan complexes from the Protein Data Bank (PDB), which are split into two subsets accounting for their intrinsic nature: non-enzymatic-protein–glycosaminoglycan and enzyme-glycosaminoglycan complexes. We apply molecular dynamics to analyze the differences in these two subsets in terms of flexibility, retainment of the native interactions in the simulations, free energy components of binding and contributions of protein residue types to glycosaminoglycan binding. Furthermore, we systematically demonstrate that protein electrostatic potential calculations, previously found to be successful for glycosaminoglycan binding sites prediction for individual systems, are in general very useful for proposing protein surface regions as putative glycosaminoglycan binding sites, which can be further used for local docking calculations with these particular polysaccharides. Finally, the performance of six different docking programs (Autodock 3, Autodock Vina, MOE, eHiTS, FlexX and Glide), some of which proved to perform well for particular protein–glycosaminoglycan complexes in previous work, is evaluated on the complete protein–glycosaminoglycan data set from the PDB. This work contributes to widen our knowledge of protein–glycosaminoglycan molecular recognition and could be useful to steer a choice of the strategies to be applied in theoretical studies of these systems.

Description: Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1–111) and the conserved carbohydrate recognition domain (CRD, residues 112–250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete 13 C/ 15 N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1–50 and 51–107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded β-sheet behind the canonical carbohydrate-binding 6-stranded β-sheet of the S-face) and NT in full-length galectin-3, with the sequence P 23 GAW 26 ...P 37 GASYPGAY 45 defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for β-galactosides.

Description: GDP- l -fucose functions as a biological donor for fucosyltransferases, which are required for the catalysis of l -fucose to various acceptor molecules including oligosaccharides, glycoproteins and glycolipids. Mortierella alpina is one of the highest lipid-producing fungi and can biosynthesis GDP- l -fucose in the de novo pathway. Analysis of the M. alpina genome suggests that there is a gene encoding l -fucokinase (FUK) for the conversion of fucose to l -fucose-1-phosphate in the GDP- l -fucose salvage pathway, which has never been found in fungi before. This gene was characterized to explore its role in GDP- l -fucose synthesis. The yield of GDP- l -fucose is relatively higher in lipid accumulation phase (0.096 mg per g cell) than that in cell multiplication phase (0.074 mg per g cell) of M. alpina . Additionally, the transcript level of FUK is up regulated by nitrogen exhaustion when M. alpina starts to accumulate lipid, highlights the functional significance of FUK in the GDP- l -fucose biosynthesis in M. alpina . Gene encoding FUK was expressed heterologously in Escherichia coli and the resulting protein was purified to homogeneity. The product of FUK reaction was analyzed by liquid chromatography and mass spectrometry. Kinetic parameters and other properties of FUK were investigated. Comparative analyses between the FUK protein and other homologous proteins were performed. To our knowledge, this study is the first to report a comprehensive characterization of FUK in a fungus. Mortierella alpina could be used as an alternative source for the production of GDP- l -fucose.