Flower angle favors pollen export efficiency in the snowdrop Galanthus nivalis (Linnaeus, 1753) but not in the lesser celandine Ficaria verna (Huds, 1762).

Flower angle is crucially important for accurate pollination and flower protection against abiotic factors. Evolutionary factors shaping floral traits are particularly strong for bilaterally symmetric flowers because these flowers require more pollination accuracy than radially symmetrical flowers. We experimentally investigated the flower angle in the snowdrop's (Galanthus nivalis) radially symmetrical, early-blooming downward flowers. Bumblebees were able to gather significantly more pollen grains from downward flowers than from upward flowers, but female traits (fertility in the field) seem unaffected by flower angle. Similar experiments with radially symmetrical, later flowering Lesser celandine (Ficaria verna) upward flowers showed no differences in bees' abilities to gather pollen in upward vs downward-facing flowers. The downward angle of snowdrop flowers is an adaptation that increases the ability of insects to collect more pollen grains under unfavorable early spring weather conditions when pollinators are scarce.

Common snowdrop as a climate change bioindicator in Czechia.

The phenological response to climate change differs among species. We examined the beginning of flowering of the common snowdrop (Galanthus nivalis) in connection with meteorological variables in Czechia in the period 1923-2021. The long-term series were analyzed from phenological and meteorological stations of the Czech Hydrometeorological Institute (CHMI). Temporal and spatial evaluation (using Geographic Information System) in timing of beginning of flowering (BBCH 61) of G. nivalis was investigated under urban and rural settings. Furthermore, the detailed analysis of selected meteorological variables to onset of G. nivalis flowering was performed. Moreover, the trends (using Mann-Kendall test) and Pearson's correlation coefficients between phenological phase and meteorological variable were calculated. The main finding of this study was that the trend of the beginning of flowering of the common snowdrop during the studied period (1923-2021) is negative, and it varies in urban and rural environments. The results showed most significant acceleration of the beginning of flowering of G. nivalis by - 0.20 day year-1 in urban area and by - 0.11 day year-1 in rural area. Above that, a major turning point occurred between 1987 and 1988 (both, in phenological observations and meteorological variables), and the variability of the beginning of flowering is significantly higher in the second period 1988-2021. On top of, the study proved that the beginning of flowering of G. nivalis closely correlated with number of days with snow cover above 1 cm (December-March) at both types of stations (urban and rural), and with mean air temperature in February, maximum air temperature in January, and minimum air temperature in March. The Mann-Kendall test showed a reduction in the number of days with snow cover above 1 cm (December-March) during 99 years period at Klatovy station (a long-term time series) by - 0.06 day year-1, i.e., by - 5.94 days per the whole period. Conversely, air temperatures increase (maximum and minimum air temperature by 0.03 °C year-1 (2.97 °C per the whole period) and average air temperature by 0.02 °C year-1 (1.98 °C per the whole period)). Thus, our results indicate significant changes in the beginning of flowering of G. nivalis in Czechia as a consequence of climate change.

The snowdrop, wellspring of galanthamine : A brief descriptive and scientific history.

A history of the common snowdrop, Galanthus nivalis, is set out in folkloric fashion. Its religious connection is evident as well as the inspiration it arouses in artists and in ordinary persons. It is intended to shed light on the initial works relating to the snowdrop's alkaloid, galanthamine, performed by Soviet and Bulgarian scientists, which lie buried away and generally remain unknown. A brief history and nature of alkaloids continues with the emphasis placed on galanthamine. Heretofore hidden uses of snowdrops in traditional (folk) medicine are unveiled. An anticholinesterase with central and peripheral activity, feasible clinical indications with galanthamine are described. Certain disorders for which galanthamine has been deemed beneficial are presented for their historical value. Ongoing research regarding drugs and chemicals developed from the snowdrop and galanthamine is mentioned.

Functional significance of flower orientation and green marks on tepals in the snowdrop Galanthus nivalis (Linnaeus, 1753).

Flower shapes, colors, sizes and fragrances are shaped mostly for pollinator attraction. Flower phenotypes are, however, subjected to conflicting selection directed by both pollinators and non-pollinating agents. We investigated flower attractiveness to a model pollinator in the snowdrop (Galanthus nivalis L.) under laboratory conditions. Naïve bumblebees (Bombus terrestris L.) showed strong, innate preferences for experimentally altered upward positioned flowers, suggesting that the natural, downward orientation did not evolve to attract pollinators. Experimentally treated green marks on inner tepals decreased pollinator attraction compared with flowers expressing intact marks, suggesting that green marks serve to guide/attract pollinators. Attractiveness of green marks was significantly compromised by flower orientation; green marks were attractive only for untreated downward-oriented flowers, but they did not improve the attractiveness of upward-oriented flowers. Our results suggest that downward flowers in snowdrop evolved under conflicting selection directed by biotic and abiotic factors, and that green marks on inner tepals could evolve later to enhance flower attractiveness.

Purification of GNA-Related Lectins from Natural Sources.

The Galanthus nivalis lectin, abbreviated as GNA, is the model protein for a large group of mannose-binding lectins. Here, we describe the purification of GNA starting from dry bulbs. Using a combination of ion exchange chromatography and affinity chromatography on mannose-Sepharose, a highly pure preparation of GNA can be obtained.

New lectin ligands: Testing of Amadori rearrangement products with a series of mannoside-specific lectins.

1-(N-Phenyl)amino-1-deoxy-α-D-manno-hept-2-ulose (2) and two multivalent BSA-based structures 7 and 8, d-manno-configured C-glycosyl-type compounds derived from an Amadori rearrangement, were evaluated as ligands for mannoside-specific lectins of various sources. The determination of the concentration corresponding to 50% of inhibition (IC50) is described. Multivalency turned out to effectively influence ligand selectivity and lectin binding.

Isolation, Synthesis, and Semisynthesis of Amaryllidaceae Constituents from Narcissus and Galanthus sp.: De Novo Total Synthesis of 2- epi-Narciclasine.

An efficient protocol for the isolation of narciclasine from common Amaryllidaceae bulbs, separation from haemanthamine, and the occurrence of a trace alkaloid, 2- epi-narciclasine, are reported. Attempts to convert natural narciclasine to its C-2 epimer by Mitsunobu inversion or oxidation/reduction sequences were compromised by rearrangement and aromatization processes, through which a synthesis of the alkaloid narciprimine was achieved. The methylation of the 7-hydroxy group of natural narciclasine followed by protection of the 3,4-diol function and oxidation/reduction sequence provided the target C-2 epimer. A de novo chemoenzymatic synthesis of 2- epi-narciclasine from m-dibromobenzene is also described. Haemanthamine and narciprimine were readily detected in the crude extracts of Narcissus and Galanthus bulbs containing narciclasine, and the occurrence of 2- epi-narciclasine as a trace natural product in Galanthus sp. is reported for the first time.

Expression of the Galanthus nivalis agglutinin (GNA) gene in transgenic potato plants confers resistance to aphids.

Aphids, the largest group of sap-sucking pests, cause significant yield losses in agricultural crops worldwide every year. The massive use of pesticides to combat this pest causes severe damage to the environment, putting in risk the human health. In this study, transgenic potato plants expressing Galanthus nivalis agglutinin (GNA) gene were developed using CaMV 35S and ST-LS1 promoters generating six transgenic lines (35S1-35S3 and ST1-ST3 corresponding to the first and second promoter, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that the GNA gene was expressed in leaves, stems and roots of transgenic plants under the control of the CaMV 35S promoter, while it was only expressed in leaves and stems under the control of the ST-LS1 promoter. The levels of aphid mortality after 5 days of the inoculation in the assessed transgenic lines ranged from 20 to 53.3%. The range of the aphid population in transgenic plants 15 days after inoculation was between 17.0±1.43 (ST2) and 36.6±0.99 (35S3) aphids per plant, which corresponds to 24.9-53.5% of the aphid population in non-transformed plants. The results of our study suggest that GNA expressed in transgenic potato plants confers a potential tolerance to aphid attack, which appears to be an alternative against the use of pesticides in the future.

[Reducing viral load in life-threatening viral diseases using snowdrops]. / Schneeglöckchen zur Senkung der Viruslast bei lebensbedrohlichen Viruserkrankungen.

In the case of life-threatening viral diseases, viral load is associated with mortality. A new and innovative therapeutic approach is the reduction of viral load by extracorporeal elimination without simultaneously weakening the immune system by removing specific antibodies. Basis of this therapy is a modified plasma filter coated with a lectin derived from the snowdrop.

Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities.

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

Formal Synthesis of Gracilamine Using Rh(I)-Catalyzed [3 + 2 + 1] Cycloaddition of 1-Yne-Vinylcyclopropanes and CO.

Reported here is a formal synthesis of gracilamine using Rh(I)-catalyzed [3 + 2 + 1] reaction of yne-VCP (±)-4 and CO. The key reaction gave the cycloadduct (±)-trans-3 with the A-B-C core structure of gracilamine. This advanced intermediate was further transformed to Gao's intermediate (±)-2 via regular transformations to realize the formal synthesis of gracilamine. The present strategy was used to accomplish the asymmetric formal synthesis of gracilamine using chiral substrate (+)-4.

Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops.

Sensitivity of transmitted and founder human immunodeficiency virus type 1 envelopes to carbohydrate-binding agents griffithsin, cyanovirin-N and Galanthus nivalis agglutinin.

Human immunodeficiency virus type 1 (HIV-1) transmission often results from infection by a single transmitted/founder (T/F) virus. Here, we investigated the sensitivity of T/F HIV-1 envelope glycoproteins (Envs) to microbicide candidate carbohydrate-binding agents (CBAs) griffithsin (GRFT), cyanovirin-N (CV-N) and Galanthus nivalis agglutinin (GNA), showing that T/F Envs demonstrated different sensitivity to CBAs, with IC50 values ranging from 0.006 ± 0.0003 to >10 nM for GRFT, from 0.6 ± 0.2 to 28.9 ± 2.9 nM for CV-N and from 1.3 ± 0.2 to >500 nM for GNA. We further revealed that deglycosylation at position 295 or 448 decreased the sensitivity of T/F Env to GRFT, and at 339 to both CV-N and GNA. Mutation of all the three glcyans rendered a CBA-sensitive T/F Env largely resistant to GRFT, indicating that the sensitivity of T/F Env to GRFT is mainly determined by glycans at 295, 339 and 448. Our study identified specific T/F Env residues associated with CBA sensitivity.

Quantititative determination of lycorine and galanthamine in Galanthus trojanus and G. cilicicus by HPLC-DAD.

Lycorine and galanthamine have various biological activities. A reliable HPLC method coupled with DAD detection was developed and validated for the determination of galanthamine and lycorine in Galanthus trojanus and G. cilicicus. A simple method for the extraction of the alkaloids in low-mass plant samples was employed utilizing columns pre-packed with diatomaceous earth (Extrelut). This method was applied to the aerial parts and bulbs of G. trojanus and G. cilicicus (Amaryllidaceae) collected during the flowering season. The chromatographic separation was performed using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:92.5:7.5) applied at a flow rate of 1 mL min(-1) and using a diode array detector. Validation procedures showed that the method was specific, accurate and precise. The highest amount of lycorine (0.012%) was detected in the bulbs of G. trojanus collected from Can (Canakkale), whereas the aerial parts of this species collected from Bayramiç (Canakkale) was not found to contain this alkaloid. In G. cilicicus samples, lycorine was only determined in the bulbs, giving yields of 0.004%; galanthamine yields were between 0.015-0.016%, but none of the G. trojanus samples contained this latter alkaloid.

Novel biopesticide based on a spider venom peptide shows no adverse effects on honeybees.

Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.

Growth regulator requirement for in vitro embryogenic cultures of snowdrop (Galanthus nivalis L.) suitable for germplasm preservation.

In this study, we report on the production of bulb scale-derived tissue cultures capable of efficient shoot and plant regeneration in three genotypes of snowdrop (Galanthus nivalis L., Amaryllidaceae), a protected ornamental plant. For culture line A, high auxin and low cytokinin concentration is required for callus production and plant regeneration. The type of auxin is of key importance: α-naphthaleneacetic acid (NAA) in combination with indole-3-acetic acid (IAA) at concentrations of 2 mg L-1 or 2-10 mg L-1 NAA with 1 mg L-1 N6-benzyladenine (BA), a cytokinin on full-strength media are required for regeneration. Cultures showing regeneration were embryogenic. When lines B and C were induced and maintained with 2 mg L-1 NAA and 1 mg L-1 BA, they produced mature bulblets with shoots, without roots. Line A produced immature bulblets with shoots under the above culture condition. Amplified Fragment Length Polymorphism (AFLP) analysis showed that (i) genetic differences between line A and its bulb explants were not significant, therefore these tissue cultures are suitable for germplasm preservation, and (ii) different morphogenetic responses of lines A, B and C originated from genetic differences. Culture line A is suitable for field-growing, cultivation and germplasm preservation of G. nivalis and for the production of Amaryllidaceae alkaloids.

Snowdrops falling slowly into place: an improved phylogeny for Galanthus (Amaryllidaceae).

Snowdrops (Galanthus, 20 spp.; Amaryllidaceae) are cherished garden plants and the world's most traded wild-sourced ornamental bulb genus. Despite their popularity and economic importance, species delimitation is problematic and the infrageneric classification uncertain. We present a molecular phylogenetic study of Galanthus with the aim of resolving these issues and to better understand the evolution within the genus. Sequences of nuclear encoded nrITS, and plastid encoded matK, trnLF, ndhF, and psbK-psbI, for all currently recognised species and two naturally occurring putative hybrids, were analysed using maximum parsimony and Bayesian inference. Phylogenetic analysis of Galanthus, based on nuclear ITS sequences, provides a well-resolved topology, including seven well-supported named clades (platyphyllus, trojanus, ikariae, elwesii, nivalis, woronowii, and alpinus), and five major clades (A-E). The recovered ITS topology is in accordance with the geographical distribution of Galanthus species. The combined plastid data set provided far less resolution than that of ITS, with generally lower levels of statistical support, and one case of significant incongruence with the ITS dataset (involving G. gracilis). Phylogenetic network and hybridization analyses identified several possible hybridization events but these are more likely to be due to the result of a lack of resolution in the plastid dataset. The putative natural hybrid, G. ×valentinei nothosubsp. subplicatus, is supported by our data and analyses, whereas a hybrid origin for G. ×allenii is not. ITS and plastid data indicated that some Galanthus species are in need of taxonomic recircumscription.

Combined biochemical and cytological analysis of membrane trafficking using lectins.

We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein ß-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.

GC-MS investigation of Amaryllidaceae alkaloids in Galanthus xvalentinei nothosubsp. subplicatus.

A GC-MS analysis of alkaloids in the aerial parts and bulbs of Galanthus xvalentinei nothosubsp. subplicatus was performed for the first time. Totally, twenty-six alkaloids were identified, of which tazettine and galanthindole were the major ones. Acetylcholinesterase inhibitory activity of the alkaloidal extracts was determined using modified in vitro Ellman's method. Significant anticholinesterase activity was observed in the tested samples (bulbs: IC50 = 21.3 microg/mL, aerial parts: IC50 = 16.3 microg/mL).