The isolation, preliminary structural studies, and physiological activity of water-soluble polysaccharides from the squeezed berries of the snowball treeViburnum opulus

Water-soluble polysaccharide fractions VO1–VO4 were isolated from the squeezed berries of the snowball tree (Viburnum opulus) by successive extraction with water at various temperatures and pH and with aqueous solutions of ammonium oxalate. These fractions were purified by ion-exchange chromatography on DEAE cellulose, and the homogeneity of the purified polysaccharides was determined by gel filtration on Sephacryl S-500. Acidic polysaccharides close to pectins in their sugar composition were found in all the extracts (fractions VO1-1, VO2-1, VO3-2, and VO4-2). Residues of galacturonic acid, galactose, arabinose, and (to a lesser extent) rhamnose are their main constituents. Neutral polysaccharides composed mainly of galactose and mannose residues were additionally found in fractions extracted with acidified water (pH 4.0) and with aqueous ammonium oxalate solutions. Partial acidic hydrolysis and digestion with pectinase of acidic polysaccharides indicated that their carbohydrate backbone consists of α-1,4-linked residues ofD-galacturonic acid. NMR spectra of acidic polysaccharides (fractions VO3-2 and VO3-3) confirmed this and demonstrated that their side oligosaccharide chains are composed of β-1,4-linked galactopyranose residues and of terminal and 2,5- and 3,5-substituted residues of α-arabinofuranose at a Gal : Ara ratio of 3 : 1. Some polysaccharides fromV. opulus were found to possess an immunostimulating activity: they enhance phagocytosis, in particular, the phagocytic index and the secretion of lysosomal enzymes with peritoneal macrophages. Calcium ions were found to be necessary for the appearance of the stimulating effect of acidic polysaccharides fromV. opulus.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Structure and physiological activity of lemnan, Lemna minor L. pectin

A pectic polysaccharide, lemnan, was isolated from freshly collected duckweed Lemna minor L. Its sugar chain was shown to be mainly composed of the residues of D-galacturonic acid (64%), galactose, arabinose, xylose, and D-apiose, a branched chain sugar. The high content of D-apiose (25%) indicated that lemnan is an apiogalacturonan type pectin similar to zosteran, a pectic polysaccharide from a sea phanerogam of the Zosteraceae family. The results of partial acidic hydrolysis, pectinase digestion, and NMR studies of lemnan demonstrated that its macromolecule contains regions of the linear alpha-1,4-D-galacturonan and branched apiogalacturonan. The side chains of apiogalacturonan were found to be formed of single and 1,5-linked residues of D-apiofuranose attached to 2- and 3-positions of the D-galacturonic acid residues of the apiogalacturonan backbone. Lemnan was shown to exhibit an immunomodulatory effect by activating the system of phagocytosis.     

Structure of tanacetan,a pectic polysaccharide from tansy Tanacetum vulgare L

Tanacetan TVF was found to have a branched structure with a backbone of linear -1,4-D-galacturonan. The ramified regions consist of linear -1,2-L-rhamno--1,4-D-galacturonan as the core. The side chains appear to attach to the 4-position of the L-rhamnopyranose residues. They are present as single -galactopyranose residues or a branching -1,4-galactopyranan bearing 4,6-substituted -D-galactopyranose residues as branched points. In addition, the ramified regions contain side chains of a branched -1,5-arabinofuranan possessing 2,5- and 3,5-substituted -L-arabinofuranose residues as branching points. Some side chains of rhamnogalacturonan appear to be arabinogalactan which contains branched sugar chains of -1,5-arabinofuranan attached to the linear chains of -1,4-galactopyranan by 1,3- and 1,6-linkages. The residues of -L-arabinofuranose seem to occupy the terminal positions of the arabinogalactan side chains.     

Isolation and General Characterization of Polysaccharides from Tansy Tanacetum vulgare L.

Using extraction with 0.75% aqueous ammonium oxalate, the following polysaccharide fractions were isolated: tanacetans TVF, TVS, and TVR from floscules, sprouts, and roots, respectively, of Tanacetum vulgare L., spread throughout the European North of Russia. The sugar chain of tanacetan TVF consists of D-galacturonic acid (61.4%), arabinose (14.7%), galactose (10.2%), and rhamnose (3.7%) as the main constituents as well as xylose, glucose, mannose, apiose, and 2-O-methylxylose in trace amounts. Tanacetans TVS and TVR were shown to differ in the sugar quantitative composition. They contain 67 and 28% galacturonic acid, respectively. A partial acid hydrolysis of the tanacetan TVF gave a polysaccharide fragment TVF1, -1,4-D-galacturonan (GalA 98.2%). Digestion with pectinase (-1,4-D-polygalacturonase) resulted in fragment TVF3, containing residues of arabinose (27.1%) and galactose (17.3%). NMR spectroscopy allowed detection of the terminal residues of -Araf and -Galp as well as of the residues of -Arap substituted in 3,5- and 5-positions. Thus, tanacetan TVF was proved to be a pectic polysaccharide.     

Structural study of bergenan,a pectin from <Emphasis Type="Italic">Bergenia crassifolia</Emphasis>

A pectin polysaccharide named bergenan was isolated from the freshly collected leaves of the leather bergenia Bergenia crassifolia by extraction with an aqueous solution of ammonium oxalate. The main component of its carbohydrate chain was shown to be the residues of D-galacturonic acid (about 80%). In addition, the polysaccharide contains the residues of galactose, arabinose, and rhamnose; their total content is less than 15%. It was shown that the bergenan samples from bergenia leaves collected at different vegetation periods (from July to September) do not substantially differ either in monosaccharide composition or in the viscosity of their aqueous solutions. The results of enzymatic hydrolysis by α-1,4-galacturonase (pectinase), partial acidic hydrolysis, NMR spectroscopy, and methylation with subsequent analysis of the results by GC-MS indicate that the bergenan macromolecule contains the regions of a linear α-1,4-D-galactopyranosyluronan and rhamnogalacturonan-I (RG-I). Galacturonan responds for a greater part of the macromolecule. A considerable amount of its constitutent galacturonic acid residues are present as methyl esters. The side chains in RG-I are attached to the rhamnopyranose residues of the backbone by a 1,4-linkages and are composed of the residues of terminal arabinofuranose and galactopyranose, 1,5-linked α-arabinofuranose, and 1,4- and 1,6-linked β-galactopyranose. The branching points of the side chains of the RG-I molecule are 3,4- and 3,6-di-O-substituted galactose residues.     

Structural studies of arabinogalactan and pectin from Silene vulgaris (M.) G. callus

Arabinogalactan and pectin (named silenan) were isolated from Silene vulgaris (M.) G. callus. Fractionation by ion-exchange chromatography on DEAE-cellulose and digestion with pectinase demonstrated that silenan from S. vulgaris callus (80% of D-galacturonic acid) and silenan from the aerial part of the campion S. vulgaris are similar: both pectins contain a high quantity of homogalacturonan segments. The NMR spectral data and mass spectrometry of the purified polysaccharide and its fragment obtained by Smith degradation confirmed that the core of the arabinogalactan consisted of the different segments of β-1,3-D-galactopyranan. Some of the β-galactopyranose residues of the backbone are branched at O-6. The side chains of the arabinogalactan were shown to contain residues of terminal and 3-O-substituted β-galactopyranose, terminal α-arabinofuranose and α-rhamnopyranose, and 2-O-substituted α-rhamnopyranose. The α-rhamnopyranose residues in the sugar chain appeared to be 2-O-glycosylated by the β-1,4-D-galactopyranosyl uronic acid residues. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 6, pp. 798–807.     

小蔓长春花组织培养快速繁殖中的遗传稳定性

以小蔓长春花茎段为外植体,在含1.5mg·L-1NAA的MS固体培养基上直接生根,多次继代培养、移栽,建立小蔓长春花组织培养快速繁殖体系。利用RAPD技术结合高效液相色谱技术对连续三次继代无菌苗和炼苗后小蔓长春花的遗传稳定性、长春胺含量稳定性进行分析。结果显示,实验选取的20条随机引物共扩增出清晰的46个条带,不同植株扩增获得的条带数目和带型一致,在所检测范围内继代培养和炼苗均未影响小蔓长春花DNA序列。同时HPLC测得相应材料中长春胺含量均在0.12%以上,培养过程中长春胺含量稳定。本实验建立的小蔓长春花组织培养快速繁殖体系遗传稳定、长春胺含量稳定,可用于大量繁殖小蔓长春花无菌植株,为长春胺生产的提供资源。     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Structural Studies on Pectin from Marsh Cinquefoil <Emphasis Type="Italic">Comarum palustre</Emphasis> L.

Pectin with [alpha]D(20) +192 degrees (c 0.1; water), named comaruman, was isolated from marsh cinquefoil Comarum palustre L., which is widespread in the European North. The sugar chain of comaruman contains residues of D-galacturonic acid (64%), D-galactose (13%), L-rhamnose (12%), L-arabinose (6%), and trace amounts of xylose and glucose. Partial acid hydrolysis and digestion with pectinase demonstrated that comaruman composed of the backbone comprised regions of linear alpha-1,4-D-galactopyranosyl uronan interconnected by numerous residues of alpha-1,2-L-rhamnopyranose. In addition to the backbone (core of the macromolecule), ramified regions are involved in comaruman and comprise alpha-2,4-L-rhamno-alpha-4-D-galacturonan with side chains consisting mainly of beta-1,4-linked residues of D-galactopyranose. The ramified region contains additionally residues of 5-O-substituted arabinofuranose and 3- and 6-O-substituted galactopyranose. The present 3,4- and 4,6-di-O-substituted residues of galactopyranose appear to be branching points of the side chains. Some galactopyranose residues were found to occupy the terminal positions of the side chains or appeared to be single sugar residues attached to the side chains. Methylation analysis data indicated that comaruman contains residues of terminal, 3- and 3,4-di-O-substituted galactopyranosyl uronic acid, which appeared to be constituents of the side chains, and the latter represented additionally branching points of the backbone.     

A novel mechanism of abscission in fronds of Lemna minor L. and the effect of silver ions

Lemna minor L. (duckweed) forms colonies through vegetative propagation because mother fronds remain connected for some time with their daughter fronds by stipes. The colony size is controlled by abscission of stipes at a specific preformed abscission zone. Application of silver ions (Ag(+) ) enhances the rate of frond abscission, thus resulting in smaller colonies. The mechanism behind this process has not yet been identified. Silver caused an abscission response that saturated after 7 h of treatment. The half-maximal effective concentration was 0.72 μm Ag(+) for the standard clone, L. minor St. Other clones of the same species show sensitivities that differ by one order of magnitude. Transmission electron microscopy revealed: (i) large numbers of vesicles close to the plasmalemma in cells adjacent to the abscission zone, which proves a vesicular type secretory activity; and (ii) a moderately electron-dense secretion accumulated in the enlarging intercellular spaces, and seemed to flow from the adjacent cells towards the abscission zone. We assume that increasing pressure causes this material to push apart the cells, thereby causing the break in the abscission zone of the stipe. This is a novel mechanism of abscission that has not previously been described. The same mechanism occurs in stipes of both control and Ag(+) -treated samples. Silver ions only accelerate the process leading to abscission of stipes, without affecting the mechanism involved.     

Phytotoxic activity of selected water-soluble metabolites of Fusarium against Lemna minor L. (Duckweed)

Phytotoxicity and inhibitory effects of the fusarial toxins fumonisin B₁ (FB₁) [m.p. 103–105 °C], fusaric acid [m.p. 106–107 °C], butenolide (4-acetamido-4-hydroxy-2-butenoic acid lactone) [116–117 °C], 9, 10-dihydroxyfusaric acid [m.p. 150–155 ° C], and moniliformin on chlorophyll synthesis in the aquatic macrophyte Lemna minor (duckweed) were examined. FB₁ proved to be most active, reducing the growth of L. minor fronds and their ability to synthesize chlorophyll by 53% and 59%, respectively, at 0.7 g/ml. The growth rate of L. minor was reduced 59% by 6.7 g/ml fusaric acid, 62% by 66.7 g/ml butenolide, and 22% by 66.7 g/ml 9,10-dihydroxyfusaric acid. Moniliformin was the least phytotoxic to L. minor, with only a 16% suppression of growth rate and a 54% reduction in chlorophyll at 66.7 g/ml.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Growth rate based dose-response relationships and EC-values of ten heavy metals using the duckweed growth inhibition test (ISO 20079) with Lemna minor L. clone St

The duckweed Lemna minor L. clone St was used to investigate the effect of 10 heavy metals under the standardised test conditions of the ISO protocol 20079. By using growth rates derived from frond number (FN), fresh weight (FW), dry weight (DW), chlorophyll and carotenoid (Car) contents, concentration–response curves for all heavy metals and all growth parameters were classified. In addition, all data were fitted to obtain the inhibitions of growth rates (E_rC_x) at the level of 10%, 20% and 50% (E_rC₁₀, E_rC₂₀ and E_rC₅₀, respectively) then used to evaluate the phytotoxicity of the different heavy metals. On the basis of the E_rC₅₀ values (average ranking of all five growth parameters), the following series of phytotoxicity was detected by using molar concentrations: Ag⁺>Cd²⁺>Hg²⁺>Tl⁺>Cu²⁺>Ni²⁺>Zn²⁺>Co²⁺>Cr(VI)>As(III)>As(V).     

Branching of the galacturonan backbone of comaruman, a pectin from the marsh cinquefoil Comarum palustre L.

Galacturonan, the main constituent of the backbone (core) of the comaruman macromolecule, a pectin from the marsh cinquefoil Comarum palustre L., was obtained on partial acid hydrolysis of the pectin. Using atomic force microscopy and methylation analysis of the galacturonan, the backbone of the comaruman macromolecule was shown to contain branches as side chains consisting of α-1,4-linked residues of D-galactopyranosyl uronic acid attached to the 2-and 3-positions of the galacturonic acid residues of the core, in addition to linear regions of α-1,4-D-galacturonan. A few side chains appear to attach to 2,3-positions of the D-galacturonic acid residues. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 5, pp. 666–671.     

环境因子对奇异虉草和小子虉草种子萌发的影响

在室内条件下考察了环境因子温度、pH、盐分、土壤含水量以及种子埋深对外来入侵植物奇异虉草和小子虉草种子萌发和幼苗生长的影响,并与小麦进行对比研究.结果显示;(1)当温度为5℃、NaCl浓度为0.25 mol/L以及种子埋藏深度达到30 mm时,奇异虉草和小子虉草的种子完全不能萌发,而同等条件下小麦种子的发芽率分别依次为89.33%、53.33%和95.00%.(2)在pH 4.0～10.0和土壤含水量10.0%～25.0%条件下,奇异虉草和小子虉草的种子均能萌发;但pH在4.0和10.0以及土壤含水量低于15%时,其发芽率受到显著抑制;当土壤含水量为10%时奇异虉草和小子虉草的种子发芽率分别为19.33%和16.67%,而小麦种子的萌发完全受到抑制.(3)奇异虉草和小子虉草的幼苗最适生长环境为,温度25℃～30℃、pH为6.0～9.0、NaCl浓度0～0.05 mol/L以及种子埋藏深度为0～5 mm.研究表明温度、盐分和种子埋藏深度是影响奇异虉草和小子虉草种子萌发的关键因素,而偏碱性环境更有利于其种子萌发,其种子萌发对干旱胁迫的耐受性强于小麦.     

不同浓度铜对紫背萍和青萍色素含量及抗氧化酶系统的影响

通过水培试验研究了不同浓度的重金属Cu对紫背萍(Spirodela polyrrhiza)和青萍(Lemna minor)的色素含量和抗氧化酶系统的影响.结果表明,低浓度Cu(0.056 mg·L^-1)的处理下,紫背萍和青萍的叶绿素a、叶绿素b和叶绿素(a+b)的含量均出现不同程度的增加,分别高出其对照11％、46％、22％和8％、15％、11％,而在高浓度Cu(0.18～5.60 mg·L^-1)的处理下,上述色素含量均显著下降,平均下降幅度分别达63％、62％、65%和46％、44％、45％.紫背萍体内丙二醛(MDA)含量为青萍的2.57倍.两种浮萍的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和过氧化物酶(POD)的活性均随Cu浓度的增加而呈先升后降的趋势,紫背萍体内3种酶活性在Cu浓度仅为0.18 mg·L^-1时即达峰值,随后则大幅下降,而青萍体内3种酶活性却在Cu浓度分别升高到0.56、1.0和1.0 mg·L^-1时才达到峰值.可见,在Cu胁迫下,紫背萍受Cu毒害较青萍深,且其体内抗氧化酶系统也较青萍敏感.     

Structural characterization of a highly branched polysaccharide from the seeds of Plantago asiatica L.

In this paper, polysaccharides were extracted from the seeds of Plantago asiatica L. with hot water and separated into three fractions PLP-1 (18.9%), PLP-2 (52.6%) and PLP-3 (28.5%) by Sephacryl™ S-400 HR column chomatography. The main fraction PLP-2's structure was elucidated using oxalic acid hydrolysis, partial acid hydrolysis, methylation, GC, GC-MS, 1D and 2D NMR. PLP-2 was composed of Rha, Ara, Xyl, Man, Glc and Gal, in a molar ratio of 0.05:1.00:1.90:0.05:0.06:0.10. Its uronic acid was GlcA. PLP-2 was highly branched heteroxylan which consisted of a β-1,4-linked Xylp backbone with side chains attached to O-2 or O-3. The side chains consisted of β-T-linked Xylp, α-T-linked Araf, α-T-linked GlcAp, β-Xylp-(1 → 3)-α-Araf and α-Araf-(1 → 3)-β-Xylp, etc. Based on these results, the structure of PLP-2 was proposed.