Structure and Biological Properties of Ribosome-Inactivating Proteins and Lectins from Elder (Sambucus nigra L.) Leaves.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that catalyze the removal of a specific adenine located in the sarcin-ricin loop of the large ribosomal RNA, which leads to the irreversible inhibition of protein synthesis and, consequently, cell death. The case of elderberry (Sambucus nigra L.) is unique, since more than 20 RIPs and related lectins have been isolated and characterized from the flowers, seeds, fruits, and bark of this plant. However, these kinds of proteins have never been isolated from elderberry leaves. In this work, we have purified RIPs and lectins from the leaves of this shrub, studying their main physicochemical characteristics, sequences, and biological properties. In elderberry leaves, we found one type 2 RIP and two related lectins that are specific for galactose, four type 2 RIPs that fail to agglutinate erythrocytes, and one type 1 RIP. Several of these proteins are homologous to others found elsewhere in the plant. The diversity of RIPs and lectins in the different elderberry tissues, and the different biological activities of these proteins, which have a high degree of homology with each other, constitute an excellent source of proteins that are of great interest in diagnostics, experimental therapy, and agriculture.

The Content of Selected Minerals, Bioactive Compounds, and the Antioxidant Properties of the Flowers and Fruit of Selected Cultivars and Wildly Growing Plants of Sambucus nigra L.

This study compared the mineral content and bioactive properties of flowers and fruit coming from wild elderberry plants with those of flowers and fruit harvested from elderberry cultivars grown in an orchard. Elderberry fruit and flowers were analyzed for the content of selected minerals, phenolic compounds, and anthocyanins, as well as for antioxidant activity. Mineral content was determined by the atomic absorption spectrometry method, while antioxidant activity and the content of polyphenols and anthocyanins were determined by spectrophotometric methods. Flowers were found to contain more total ash and much higher content of most of minerals, except magnesium which was present in high concentrations in fruit. Fruit showed significantly higher antioxidant activity than flowers, whereas the total phenolic content varied depending on the growing location or cultivar. The material obtained from selected cultivars growing in an orchard had higher antioxidant activity and polyphenol and anthocyanin content than the material obtained from wild plants. Fruit of the "Haschberg" cultivar and flowers of the "Sampo" cultivar had the best bioactive properties of the studied samples.

Polyphenols pattern and correlation with antioxidant activities of berries extracts from four different populations of Sicilian Sambucus nigra L.

Sambucus nigra L. (Caprifoliaceae) is wide spread in temperate and sub-tropical zones. The consumption of its berries has been associated with health benefits especially for its high content of natural antioxidants such as polyphenols, in particular anthocyanins. In this work we investigated the polyphenolic composition and the in vitro antioxidant activities (ABTS, DPPH, BCB and FRAP-ferrozine (FRAP-FZ) assays) of S. nigra berries, collected in four different Sicilian areas (Italy). Elderberries are considered one of the fruits with highest anthocyanins content, the amount of phenolic compounds, other than anthocyanins, is approximately 1.5 times greater than the latter. The LC-MS analyses have revealed an opposite trend in the polyphenols pattern in Sicilian populations. Moreover, a statistical correlation was found between cyanidin-3-sambubioside-5-glucoside and antioxidant activity evaluated by FRAP-FZ and ABTS assays. In conclusion, Sicilian S. nigra berries are appealing for its antioxidant potential and for its particularly high content of anthocyanins.

Carbohydrate-binding activity of the type-2 ribosome-inactivating protein SNA-I from elderberry (Sambucus nigra) is a determining factor for its insecticidal activity.

In recent years, different classes of proteins have been reported to promote toxic effects when ingested. Type-2 ribosome-inactivating proteins (RIPs) are a group of chimeric proteins built up of an A-chain with RNA N-glycosidase activity and a B-chain with lectin activity. These proteins are thought to play a role in plant protection. Sambucus nigra agglutinin I (SNA-I) is a type-2 RIP, isolated from the bark of elderberry (S. nigra L.). This study demonstrated the insecticidal potency of SNA-I on two Hemipteran insect species using two different methods. An artificial diet supplemented with different concentrations of the purified RIP reduced survival and fecundity of pea aphids Acyrthosiphon pisum. In addition, feeding of tobacco aphids, Myzus nicotianae, on leaves from transfected plants constitutively expressing SNA-I, resulted in a delayed development and reduced adult survival and also the fertility parameters of the surviving aphids were reduced, suggesting that a population of aphids would build up significantly slower on plants expressing SNA-I. Finally, a series of experiments with transgenic lines in which a mutant RIP was expressed, revealed that the carbohydrate-binding activity of SNA-I is necessary for its insecticidal activity. In a first set of mutants, the B-chain was mutated at one position (Asp231DeltaGlu), and in the second set both carbohydrate-binding sites were mutated (Asn48DeltaSer and Asp231DeltaGlu). Mutation of one carbohydrate-binding site strongly reduced the insecticidal activity of SNA-I, whereas mutation of both lectin sites (almost) completely abolished the SNA-I effect on tobacco aphids.

Changes in expansin activity and gene expression during ethylene-promoted leaflet abscission in Sambucus nigra.

During ethylene-promoted leaflet abscission in Sambucus nigra a 7-fold increase in expansin activity was detected specifically in tissues undergoing cell separation, whilst only low levels of activity were seen in adjacent non-abscising tissues. An RT-PCR strategy was used to amplify expansin fragments from a cDNA library generated from mRNA extracted from ethylene-treated leaflet abscission-zone tissue. Two different full-length expansin-encoding cDNAs were isolated with sizes of 1190 bp and 1169 bp and named SniExp2 and SniExp4, respectively. The deduced protein sequences of SniExp2 and SniExp4 exhibited 67% homology and comprised 256 and 249 amino acids, respectively. Both putative proteins contained signal sequences at their N terminal ends, suggesting that they were likely to encode secreted or transmembrane proteins. Northern analyses with probes specific for each gene revealed that transcripts for both of these cDNAs accumulated specifically in abscission zone tissues in response to ethylene treatment, with no expression being apparent in either freshly excised material or non-separating tissues. These data support a role for expansins in ethylene-stimulated abscission and provide evidence that specific family members contribute to the cell separation process that takes place during organ shedding.

Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins.

Although the type-2 ribosome-inactivating proteins (SNA-I, SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are all devoid of rRNA N-glycosylase activity towards plant ribosomes, some of them clearly show polynucleotide-adenosine glycosylase activity towards tobacco mosaic virus RNA. This particular substrate specificity was exploited to further unravel the mechanism underlying the in planta antiviral activity of ribosome-inactivating proteins. Transgenic tobacco (Nicotiana tabacum L. cv Samsun NN) plants expressing the elderberry ribosome-inactivating proteins were generated and challenged with tobacco mosaic virus in order to analyze their antiviral properties. Although some transgenic plants clearly showed antiviral activity, no clear correlation was observed between in planta antiviral activity of transgenic tobacco lines expressing the different ribosome-inactivating proteins and the in vitro polynucleotide-adenosine glycosylase activity of the respective proteins towards tobacco mosaic virus genomic RNA. However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mechanism on the virus. In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins.

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.).

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.

The Sambucus nigra type-2 ribosome-inactivating protein SNA-I' exhibits in planta antiviral activity in transgenic tobacco.

Transgenic tobacco (Samsun NN) plants transformed with a cDNA clone encoding SNA-I' from Sambucus nigra synthesize, and correctly process and assemble, a fully active type-2 ribosome-inactivating protein. Expression of SNA-I' under the control of the 35S cauliflower mosaic virus promoter enhances the plant's resistance against infection with tobacco mosaic virus. In contrast to type-1 ribosome-inactivating proteins, the expression of SNA-I' does not affect the growth and fertility of the transgenic plants and is not accompanied by an increased expression of pathogenesis-related proteins indicating that its antiviral activity most probably differs from that of pokeweed antiviral protein.