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.

Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip.

Dendrimers were fitted out with up to eight mannose moieties by "click" chemistry. They were subsequently attached to aluminum oxide chips via a spacer that was linked to the dendrimer core; this resulted in a microarray of glycodendrimers. Binding of the glycodendrimers to the fluorescent lectins ConA and GNA was observable in real time. In a single experiment it was possible to observe the multivalency enhancement or cluster effect in the binding event. This effect was small for ConA, in agreement with its widely spaced binding sites, whereas it was large for GNA, with its twelve much more closely spaced binding sites. The dendrimer-fitted chip represents a valuable screening tool for multivalency effects. Furthermore kinetic and thermodynamic data on binding events can be deduced. Inhibition experiments are also possible with the system as was shown for ConA with alpha-methyl mannose as the inhibitor.

Localization and topogenesis studies of cytoplasmic and vacuolar homologs of the Galanthus nivalis agglutinin.

The Galanthus nivalis agglutinin (GNA) is synthesized as a preproprotein. To corroborate the role of the different targeting peptides in the topogenesis of GNA and related proteins, different constructs were made whereby both the complete original GNA gene and different truncated sequences were coupled to the enhanced green fluorescent protein (EGFP). In addition, a GNA ortholog from rice that lacks the signal peptide and C-terminal propeptide sequence was fused to EGFP. These fusion constructs were expressed in tobacco BY-2 cells and their localization analyzed by confocal fluorescence microscopy. We observed that the processed preproprotein of GNA was directed towards the vacuolar compartment, whereas both the truncated forms of GNA corresponding to the mature lectin polypeptide and the rice ortholog of GNA were located in the nucleus and the cytoplasm. It can be concluded, therefore, that removal of the C-terminal propeptide and the signal peptide is sufficient to change the subcellular targeting of a normally vacuolar protein to the nuclear/cytoplasmic compartment of the BY-2 cells. These findings support the proposed hypothesis that cytoplasmic/nuclear GNA-like proteins and their vacuolar homologs are evolutionarily related and that the classical GNA-related lectins might have evolved from cytoplasmic orthologs through an evolutionary event involving the insertion of a signal peptide and a C-terminal propeptide.

Intraspecific variability in the alkaloid metabolism of Galanthus elwesii.

Alkaloid pattern of individuals from 16 Bulgarian Galanthus elwesii populations was investigated by GC/MS and TLC. Twenty-one Amaryllidaceae alkaloids were detected and 14 of them were identified. Crinane type alkaloids, haemanthamine or crinine, dominated alkaloid metabolism in most of the populations. With exception of one population, where the separate individuals showed variable alkaloid profiles (dominated by crinine or haemanthamine) the individuals of the rest of populations have identical and characteristic alkaloid profiles. Some populations showed remarkable differences in respect to their alkaloid pattern-type of biosynthesis, main alkaloids and number of alkaloids. Populations dominated by galanthamine type alkaloids were found as well. These data demonstrate that like the morphological features, the alkaloid metabolism of G. elwesii is also variable.

Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM).

The cuticle of terrestrial vascular plants and some bryophytes is covered with a complex mixture of lipids, usually called epicuticular waxes. Self-assembly processes of wax molecules lead to crystalline three-dimensional micro- and nanostructures that emerge from an underlying wax film. This paper presents the first AFM study on wax regeneration on the surfaces of living plants and the very early stages of wax crystal formation at the molecular level. Wax formation was analysed on the leaves of Euphorbia lathyris, Galanthus nivalis, and Ipheion uniflorum. Immediately after wax removal, regeneration of a wax film began, consisting of individual layers of, typically, 3-5 nm thickness. Subsequently, several different stages of crystal growth could be distinguished, and different patterns of wax regeneration as well as considerable variation in regeneration speed were found.

Large-scale production and purification of recombinant Galanthus nivalis agglutinin (GNA) expressed in the methylotrophic yeast Pichia pastoris.

The gene coding for agglutinin from Galanthus nivalis (GNA) was expressed in, and secreted by, the methylotrophic yeast, Pichia pastoris. Transformants of P. pastoris were selected and a process to produce and purify gram quantities of recombinant GNA was developed. GNA was secreted at approximately 80 mg l(-1) at the 200 1 scale and was purified to 95% homogeneity using hydrophobic interaction chromatography. The recombinant protein was similar to the protein synthesised in plant with respect to structure and biological activity.

Galanthamine distribution in Bulgarian Galanthus spp.

Sixteen populations of Galanthus elwesii Hook.fil and five populations of Galanthus nivalis L. growing in Bulgaria were investigated for the presence of galanthamine by TLC and GC-MS. Between 3 and 11 alkaloids were detected by TLC in the total alkaloid fractions. Galanthamine was found in 2 populations of G. elwesii.