Qualitative analysis and HPLC isolation and identification of procyanidins from Vicia faba

The soluble proanthocyanidins of the coloured seed coats of Vicia faba L. were isolated and separated by solvent partition. The chemical characteristics of the proanthocyanidins were elucidated by total oxidation and partial degradation in the presence of phloroglucinol followed by HPLC analysis. The native extract of proanthocyanidins contained (+)-gallocatechin, (-)-epigallocatechin, (+)-catechin and (-)-epicatechin units. Oligomeric procyanidins were purified by chromatography on Sephadex LH-20 and the accessible compounds were isolated by RP-HPLC using a Licrospher Li 100 Column. The structures of the purified oligomeric procyanidins were elucidated using a procedure involving TLC, UV spectroscopy, ESI-MS and HPLC analysis of the products from the phloroglucinol reaction. The major condensed tannins of Vicia faba comprise six compounds identified as two A-type procyanidin dimers, the procyanidin dimers B1, B2 and B3, and a procyanidin trimer.     

Differential tissue-specific expression of cysteine proteinases forms the basis for the fine-tuned mobilization of storage globulin during and after germination in legume seeds

The temporal and spatial distribution of cysteine proteinases (CPRs) was analyzed immunologically and by in situ hybridization to identify the CPRs involved in the initiation of storage-globulin degradation in embryonic axes and cotyledons of germinating vetch (Vicia sativa L.). At the start of germination several CPRs were found in protein bodies in which they might have been stored in the mature seeds. Cysteine proteinase 1 was predominantly found in organs like the radicle, which first start to grow during germination. Cysteine proteinase 2 was also present at the start of germination but displayed a less-specific histological pattern. Proteinase B was involved in the globulin degradation of vetch cotyledons as well. The histological pattern of CPRs followed the distribution of their corresponding mRNAs. The latter were usually detected earlier than the CPRs but the in situ hybridization signals were histologically not as restricted as the immunosignals. Proteolytic activity started in the radicle of the embryonic axis early during germination. Within 24 h after imbibition it had also spread throughout the whole shoot. At the end of germination, newly synthesized CPRs might have supplemented the early detectable CPRs in the axis. In the cotyledons, only the abaxial epidermis and the procambial strands showed proteinase localization during germination. Both CPR1 and CPR2, as well as the less common proteinase B, might have been present as stored proteinases. Three days after imbibition, proteolytic activity had proceeded from the cotyledonary epidermis towards the vascular strands deeper inside the cotyledons. The histochemical detection of the CPRs was in accordance with the previously described histological pattern of globulin mobilization in germinating vetch [Tiedemann J, et al. (2000)]. A similar link between the distribution of CPRs and globulin degradation was found in germinating seeds of Phaseolus vulgaris L. The coincidence of the histological patterns of globulin breakdown with that of the CPRs indicates that at least CPR1, CPR2 and proteinase B are responsible for bulk globulin mobilization in the seeds of the two legumes. Received: 14 February 2000 / Accepted: 16 August 2000     

Cultivar-Specific Carbohydrate-Binding Properties of Lectins from Broad-Bean Seeds

Lectins were isolated and purified from three broad bean (Vicia faba L.) cultivars differing in the effectiveness of their symbiosis with root nodule bacteria (Rhizobium leguminosarum bv. viciae). From seeds of symbiotically effective cvs. Aushra and Daiva, we isolated only one lectin from each cultivar, whereas two lectins, Yu-1 and Yu-2, were isolated from seeds of symbiotically ineffective cv. Yugeva. Lectins from cvs. Aushra and Daiva were more active than lectins from cv. Yugeva and exhibited similar carbohydrate specificity. Methyl--D-mannopyranoside and trehalose were the most potent inhibitors of their hemagglutination activity. Lectin Yu-1 resembled them in its carbohydrate-binding properties. However, D-mannose, trehalose, and melecitose were its most effective inhibitors. Lectin Yu-2 differed substantially from these lectins. It exhibited an affinity for D-glucuronic acid, D-glucosamine, and 2-deoxy-D-glucose. In addition, it could interact with carbohydrates of the galactose family (2-deoxy-D-galactose, D-galactosamine, and lactose) and also with D-xylose and 2-deoxy-D-talose. Thus, lectins from cvs. Aushra and Daiva and also Yu-1 can be considered D-mannose/D-glucose-specific lectins, whereas Yu-2 lectin exhibited a combined carbohydrate specificity. The affinity of Yu-1 and Yu-2 lectins for their natural receptors, exopolysaccharides and lipopolysaccharides of broad-bean nodule bacteria, was twice as low as that of lectins from cvs. Aushra and Daiva. We believe that properties of seed lectins are an important cultivar-specific trait that determines host-plant (broad beans) specificity during the establishment of legume–rhizobia symbiosis.     

Effects of elevated ultraviolet-B radiation on native and cultivated plants of southern Africa

Seventeen herb, shrub and tree species of commercial and ecological importance in southern Africa were exposed at one location to ultraviolet-B (UV-B, 280-315 nm) radiation approx. 35 % above clear-sky background (control). The aims were to assess how UV-B affects canopy area, dry mass, and some biochemical and morphological properties of leaves, and to investigate whether differences between species are related to growth form of the plants. There was no pattern of response to UV-B related to growth form. Leaves of trees had altered chlorophyll a and b, carotenoid and flavonoid concentrations, but those of shrubs or herbs did not. Non-structural carbohydrates were unaffected. Smaller canopy areas and dry masses were observed under enhanced UV-B, but these were not statistically different among growth forms. There was a general insensitivity of species to elevated UV-B. Only five species had significantly altered leaf biochemical and morphological properties, canopy area and dry mass, the changes differing in magnitude. There was no consistent pattern of change in leaf thickness or biochemical composition with increased UV-B. Correlation analyses did not support the view that growth is less negatively affected in species with thick leaves or in those where leaf thickness increases, or in species with naturally high leaf flavonoid contents or that are able to synthesize additional flavonoids in response to UV-B enhancement. The analyses did not support the hypothesis that growth was inhibited by starch accumulation in leaves under elevated UV-B. However, changes in leaf shape did correlate with canopy area and dry mass, showing the importance of photomorphogenetic changes caused by UV-B which affect species' performance. We conclude that generalizations on plant sensitivity to UV-B based on growth form and functional type could be misleading, and that the great majority of economically important species of the region are likely to be insensitive to future UV-B increases. Notable exceptions include the Colophospermum mopane tree ecotypes chota and leslie and the arable annual Vigna unguiculata, both of which are traditional sources of livelihood to rural African populations and of importance to African industry and agriculture.     

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).     

Screening of the occurrence of copper amine oxidases in Fabaceae plants

Aim of this work was to find the best source for obtaining high amount of copper amine oxidase (EC 1.4.3.6) that can be further used for analytical or industrial applications. The study focused on plant enzymes, because they occur in much higher content in the starting material than the enzymes from other sources, have higher specific activity and are also more thermostable. Presence of the amine oxidase was tested in extracts from 4 to 7-d-old seedlings of thirty-four various Fabaceae plants. Amine oxidases from nine selected plants were purified by general method involving ammonium sulfate fractionation, controlled heat denaturation, and three chromatographic steps. Kinetic properties of the amine oxidases purified were tested with a wide range of substrates and inhibitors and were found to be very similar. Best purification yield, and total and specific activities were obtained for the enzyme from grass pea (Lathyrus sativus) throughout all purification steps. Hence, the grass pea extract was chosen as a suitable candidate for massive production of the amine oxidase. This revised version was published online in July 2006 with corrections to the Cover Date.     

Calcium-potassium selectivity: kinetic analysis of current-voltage relationships of the open, slowly activating channel in the vacuolar membrane of Vicia faba guard-cells

Single-channel current-voltage (IV ) relationships of the open, slowly activating vacuolar (SV) channel of Vicia faba L. were recorded in solutions with different activities of Ca²⁺ and K⁺, and have been analyzed for Ca²⁺/K⁺ selectivity. Two models with one binding site have been examined. A rigid-pore model with a main binding site between two energy barriers (nine free parameters) provides fair fits. Slightly better fits are obtained with an alternative, dynamic-pore model, where the selectivity filter is located between two Mitchellian ion wells of the cytoplasmic and luminal pore sections, and where the selectivity filter alternates the orientation of the binding site between the two faces of the pore (ten free parameters). Using sets of IV-relationships with only Ca²⁺ or only K⁺ as transportable substrates, both models consistently predict open-channel IV-relationships in the presence of both substrates. Fits of both models to the entire ensemble of␣data yield very similar flux-voltage characteristics for␣Ca²⁺ and for K⁺ in experimental conditions, and consistently predict such flux-voltage characteristics over physiologically relevant ranges of voltage and substrate concentrations. In a very general sense, physiological Ca⁺ fluxes through the open SV channel are predominantly inward and about 50 times smaller than K⁺ fluxes. The ions Cl⁻, OH⁻, and H⁺, do not pass the SV channel at significant rates. Kinetic details of the SV channel with respect to binding and passage of Ca²⁺ and K⁺ are discussed on the basis of the consistent results of the reaction-kinetic analysis of the experimental data by the two models. Received: 14 July 1997 / Accepted: 26 September 1997     

黑曲霉3.795glaA基因及其5'调控区的克隆和序列分析

黑曲霉Ｔ２１是由黑曲霉３．７９５经诱变育种获得的糖化酶高产菌株,为阐明其高产的分子机制,由黑曲霉３．７９５克隆了糖化酶结构基因及其５′旁侧序列,并与黑曲霉Ｔ２１的相应序列进行了比较．由黑曲霉３．７９５菌丝体分离染色体ＤＮＡ,Ｓｏｕｔｈｅｒｎ杂交分析表明,糖化酶结构基因位于～２．５ｋｂ的ＥｃｏＲⅠ－ＥｃｏＲⅤ染色体ＤＮＡ片段上,在此ＥｃｏＲⅠ位点上游约１．０ｋｂ处有一ＳａｌⅠ位点．为构建糖化酶结构基因及其５′旁侧序列的基因组文库,该染色体ＤＮＡ分别用ＥｃｏＲⅠ＋ＥｃｏＲⅤ和ＥｃｏＲ＋ＳａｌⅠ消化,琼脂糖凝胶电泳分离并回收长度在１．０ｋｂ左右和２．５ｋｂ左右的ＤＮＡ片段,分别与ｐＵＣ１９载体连接后转化入Ｅ．ｃｏｌｉＤＨ５．用原位杂交方法筛选到了携带糖化酶基因编码区及其１５０５ｂｐ５′旁侧序列的阳性克隆．对克隆片段的ＤＮＡ序列进行了测定并与黑曲霉Ｔ２１的相应序列进行了比较,结果表明,在糖化酶基因编码区及其１５０ｂｐ３′非编码区内,未发现碱基差异,但在－３４０～－１５０５的５′上游区内发生了９个位置的碱基变化,包括缺失、插入和替换．这些结果表明,黑曲霉Ｔ２１与３．７９５的糖化酶产量的差异与其结构基因无关,但可能与其     

箬叶多糖的分离纯化及其理化性质的研究

采用分步提取的方式从中药箬叶中分离得到８种多糖组分：酸性杂多糖ＦＳ、ＦＥ、ＦⅠ,β－Ｄ－葡萄糖醛酸聚糖ＦⅡ和四种半纤维素多糖α－Ｄ－木聚糖ＦⅢ－ａ、ＦⅢ－ｂ、ＦⅣ－ａ及ＦⅣ－ｂ．紫外光谱、红外光谱、凝胶色谱、元素分析等结果表明８种箬叶多糖为纯品．并采用纸层析,气相色谱分析确定其单糖组成．采用高效凝胶渗透色谱ＧＰＣ法测定了４种箬叶多糖ＦＥ、ＦⅠ、ＦⅢ－ａ及ＦⅣ－ａ的重均分子量Ｍｗ、数均分子量Ｍｎ,均为大分子,分子量分布较窄,纯度较高．     

Taxonomic relationships in East AsianVicia species with unijugate leaves based on random amplified polymorphic DNA markers

Random amplified polymorphic DNA(RAPD) markers were investigated to clarify the taxonomic positions ofVicia linearifolia andV. bifolia, and to assess the genomic diversity among the 9 populations ofV. unijuga, each of which represents a geographical variation or infraspecific taxa in southern Korea. These species are characterized by unijugate leaves in East Asia and have been controversial as to infra-or interspecific classification. The polymorphic markers among the populations examined were observed for fifteen decamer primers. The degree of band sharing was used to calculate genetic similarity between populations, and a phenogram using UPGMA cluster analysis was generated based on the Dice similarity coefficient. The taxa studied were divided into two main groups and the populations ofV. unijuga were all grouped together in the phenogram. The genetic similarities ofV. unijuga were very high among the populations and did not show distinctions between the infraspecific taxa, although the populations of Mt. Odae and adjacent areas in eastern Korea were different from others of the species.V. linearifolia fell within the range of the genomic variation among the populations ofV. unijuga, whileV. bifolia was grouped withV. venosa var.cuspidata having multijugate leaves rather thanV. unijuga. The result from studying RAPD markers suggested thatV. linearifolia should be integrated intoV. unijuga and that species with unijugate leaves ofV. bifolia andV. unijuga are polyphyletic.