Phytochemistry (v.71, #4)

Roles for glutathione transferases in plant secondary metabolism by David P. Dixon; Mark Skipsey; Robert Edwards (338-350).
A review of the potential diverse catalytic and binding roles of plant glutathione transferases in secondary metabolism and the techniques for identifying these functions.Plant glutathione transferases (GSTs) are classified as enzymes of secondary metabolism, but while their roles in catalysing the conjugation and detoxification of herbicides are well known, their endogenous functions are largely obscure. Thus, while the presence of GST-derived S-glutathionylated xenobiotics have been described in many plants, there is little direct evidence for the accumulation of similarly conjugated natural products, despite the presence of a complex and dichotomous metabolic pathway which processes these reaction products. The conservation in glutathione conjugating and processing pathways, the co-regulation of GSTs with inducible plant secondary metabolism and biochemical studies showing the potential of these enzymes to conjugate reactive natural products are all suggestive of important endogenous functions. As a framework for addressing these enigmatic functions we postulate that either: (a) the natural reaction products of GSTs are unstable and undergo reversible S-glutathionylation; (b) the conjugation products of GSTs are very rapidly processed to derived metabolites; (c) GSTs do not catalyse conventional conjugation reactions but instead use glutathione as a cofactor rather than co-substrate; or (d) GSTs are non-catalytic and function as transporter proteins for secondary metabolites and their unstable intermediates. In this review, we describe how enzyme biochemistry and informatics are providing clues as to GST function allowing for the critical evaluation of each of these hypotheses. We also present evidence for the involvement of GSTs in the synthesis of sulfur-containing secondary metabolites such as volatiles and glucosinolates, and the conjugation, transport and storage of reactive oxylipins, phenolics and flavonoids.
Keywords: Arabidopsis thaliana; Flavonoids; Glucosinolates; Isomerisation; Ligandin; Oxylipins; Unstable intermediates; Intracellular transport; Sulfur metabolism;

Proteomic approaches to study plant–pathogen interactions by B.F. Quirino; E.S. Candido; P.F. Campos; O.L. Franco; R.H. Krüger (351-362).
The proteomics approach is just beginning to be applied to study how plants interact with pathogens. Opportunities and challenges of this approach are reviewed herein.The analysis of plant proteomes has drastically expanded in the last few years. Mass spectrometry technology, stains, software and progress in bioinformatics have made identification of proteins relatively easy. The assignment of proteins to particular organelles and the development of better algorithms to predict sub-cellular localization are examples of how proteomic studies are contributing to plant biology. Protein phosphorylation and degradation are also known to occur during plant defense signaling cascades. Despite the great potential to give contributions to the study of plant–pathogen interactions, only recently has the proteomic approach begun to be applied to this field. Biological variation and complexity in a situation involving two organisms in intimate contact are intrinsic challenges in this area, however, for proteomics studies yet, there is no substitute for in planta studies with pathogens, and ways to address these problems are discussed. Protein identification depends not only on mass spectrometry, but also on the existence of complete genome sequence databases for comparison. Although the number of completely sequenced genomes is constantly growing, only four plants have their genomes completely sequenced. Additionally, there are already a number of pathosystems where both partners in the interaction have genomes fully sequenced and where functional genomics tools are available. It is thus to be expected that great progress in understanding the biology of these pathosystems will be made over the next few years. Cheaper sequencing technologies should make protein identification in non-model species easier and the bottleneck in proteomic research should shift from unambiguous protein identification to determination of protein function.
Keywords: Proteomics; Proteome; Plant; Pathogen; Pathosystem;

A proteinase inhibitor was purified from seeds of Vigna mungo (cv. TAU-1) using ion-exchange and affinity chromatography followed by gel-filtration chromatography. Biochemical characterization indicated that it belonged to Bowman-Birk inhibitor family.A proteinase inhibitor (BgPI) was purified from black gram, Vigna mungo (cv. TAU-1) seeds by using ammonium sulfate fractionation, followed by ion-exchange, affinity and gel-filtration chromatography. BgPI showed a single band in SDS–PAGE under non-reducing condition with an apparent molecular mass of ∼8 kDa correlating to the peak 8041.5 Da in matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum. BgPI existed in different isoinhibitor forms with pI values ranging from 4.3 to 6.0. The internal sequence “SIPPQCHCADIR” of a peak 1453.7 m/z, obtained from MALDI-TOF-TOF showed 100% similarity with Bowman-Birk inhibitor (BBI) family. BgPI exhibited non-competitive-type inhibitory activity against both bovine pancreatic trypsin (K i of 309.8 nM) and chymotrypsin (K i of 10.7 μM), however, with a molar ratio of 1:2 with trypsin. BgPI was stable up to a temperature of 80 °C and active over a wide pH range between 2 and 12. The temperature-induced conformational changes in secondary structure are reversed when BgPI was cooled from 90 to 25 °C. Further, upon reduction with dithiothreitol, BgPI lost both its inhibitory activity as well as secondary structural conformation. Lysine residue(s) present in the reactive site of BgPI play an important role in inhibiting the bovine trypsin activity. The present study provides detailed biochemical characteristic features of a BBI type serine proteinase inhibitor isolated from V. mungo.
Keywords: Vigna mungo; Protein purification; Proteinase inhibitor; Bowman-Birk inhibitor; MALDI-TOF; CD spectroscopy;

A candidate cDNA clone for (−)-limonene-7-hydroxylase from Perilla frutescens by Christopher J.D. Mau; Frank Karp; Michiho Ito; Gisho Honda; Rodney B. Croteau (373-379).
A homology-based screening of a cDNA library made from peltate gland secretory cell mRNA from Perilla frutescens identified a candidate clone with preferential limonene-7-hydroxylase activity, and to a lesser extent, other regiospecific hydroxylations.Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (−)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (−)-limonene. The perilla cDNA was a partial clone of 1550 bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively.
Keywords: Perilla frutescens; Lamiaceae; Monoterpenoid; (−)-Limonene-7-hydroxylase; Perillyl alcohol; Perillaldehyde; Cytochrome P450;

Brassinosteroids control AtEXPA5 gene expression in Arabidopsis thaliana by Chan Ho Park; Tae-Wuk Kim; Seung-Hyun Son; Jung-Yun Hwang; Sang Cheul Lee; Soo Chul Chang; Soo-Hwan Kim; Si Wouk Kim; Seong-Ki Kim (380-387).
Molecular genetic analysis of AtEXPA5-related mutants indicated that AtEXPA5 is controlled by brassinosteroid signaling for normal growth of Arabidopsis thaliana.To elucidate the spatial and temporal roles of EXPANSIN A5 (AtEXPA5) in growth and development of Arabidopsis thaliana, phenotypic alterations in loss-of-function mutants were observed. Seedlings of the null mutant, expA5-1, had shorter roots and hypocotyls than those of wild-type plants under both light and dark conditions. Compared to wild-type plants, the mutants had smaller rosette leaves. AtEXPA5 was dominantly expressed in aerial parts of A. thaliana, especially in the inflorescence stems and flowers. Expression of AtEXPA5 was enhanced by exogenously applied brassinosteroids. AtEXPA5 expression was reduced in a brassinosteroid-deficient mutant (det2) and a signaling mutant (bri1-301), while it was increased in bzr1-1D, a dominant mutant of a brassinosteroid transcription factor. A double mutant, bzr1-1DXexpA5-1, showed reduced growth compared to the bzr1-1D mutant. In addition, the brassinazole resistance of bzr1-1D was impaired in the double mutant. These findings indicate that AtEXPA5 is a growth-regulating gene whose expression is controlled by brassinosteroid signaling downstream of BZR1 in A. thaliana.
Keywords: Arabidopsis thaliana; Cruciferae; Thale cress; Target gene for brassinosteroid signaling; Brassinosteroids; Expansin; AtEXPA5;

Biosynthetic origin of the saw-toothed profile in δ 13C and δ 2Η of n-alkanes and systematic isotopic differences between n-, iso- and anteiso-alkanes in leaf waxes of land plants by Youping Zhou; Kliti Grice; Hilary Stuart-Williams; Graham D. Farquhar; Charles H. Hocart; Hong Lu; Weiguo Liu (388-403).
Low abundance even-carbon-numbered n-alkanes have been identified in higher plant leaf waxes, however, in contrast to odd-carbon-numbered n-alkanes in numerous studies, uncertainties exist as to their biosynthetic precursor. We provide stable carbon and hydrogen isotopic evidence to suggest pyruvate as a precursor and a tentative pathway is presented. We also provide a biochemical explanation for the observed isotopic order of δ 2H n -alkane  >  δ 2H iso -alkane  >  δ 2H anteiso -alkane.The n-fatty acids containing an even number of carbons (ECN-n-FAs) in higher plants are biosynthesised by repetitive addition of a two carbon unit from malonyl-ACP. The n-alkanes containing an odd number of carbon atoms (OCN-n-alkanes) are generally formed by the decarboxylation of ECN-n-FAs, but it is unknown how the less abundant even-carbon-numbered alkanes (ECN-n-alkanes) are biosynthesised in higher plants.There is a distinctive compositional pattern of incorporation of stable carbon (13C) and hydrogen (2H) isotopes in co-existing ECN- and OCN-n-alkanes in leaves of higher plants, such that the OCN n-alkanes are relatively enriched in 13C but relatively depleted in 2H against the ECN-n-alkanes. This is consistent with the OCN-n-fatty acids having a propionate precursor which is derived from reduction of pyruvate. A tentative pathway is presented with propionate produced by enzymatic reduction of pyruvate which is then thio-esterified with CoSH (coenzyme A thiol) in the chloroplast to form the terminal precursor molecule propionyl-CoA. This is then repetitively extended/elongated with the 2-carbon unit from malonyl-ACP to form the long chain OCN-n-fatty acids.The anteiso- and iso-alkanes in Nicotiana tabacum leaf waxes have previously been found to be systematically enriched in 13C compared with the n-alkanes by . This is consistent with the isotopic composition of their putative respective precursors (pyruvate as precursor for n-alkanes, valine for iso-alkanes and isoleucine for anteiso-alkanes). The current study complements that of and looks at the distribution of hydrogen isotopes. The n-alkanes were found to be more enriched in deuterium (2H) than the iso-alkanes which in turn were more enriched than the anteiso-alkanes. We propose therefore that the depletion of 2H in the iso-alkanes, relative to the n-alkanes is the consequence of accepting highly 2H-depleted hydrogen atoms from NADPH during their biosynthesis. The anteiso-alkanes are further depleted again because there are three NADPH-derived hydrogen atoms in their precursor isoleucine, as compared with only one NADPH-derived hydrogen in valine, the precursor of the iso-alkanes.
Keywords: anteiso-Alkanes; iso-Alkanes; iso-Alkanes; Biosynthetic precursors; Stable carbon isotopes; Stable hydrogen isotopes;

Salinity resulted in increased accumulation of choline in both glycinebetaine (GB) accumulating and non-accumulating lines. When grown in the presence of NaCl, GB-non-accumulating lines had increased concentrations of choline and phosphocholine, but not GB. Decreased phosphatidylcholine turnover results in an increase in phosphocholine, which then inhibits the activity of N-phosphoethanolamine methyltransferase. The block in GB synthesis in GB non-accumulating lines does not occur at the initial choline oxidation step, and the previously identified bet1 locus does not appear to be choline monooxygenase. However, the lack of GB synthesis does affect synthesis and turnover of choline moieties in GB non-accumulating lines, which may lead to alterations in overall 1-carbon metabolism in plants.Glycinebetaine (GB) is a compatible solute that is accumulated by some plant species, especially under conditions leading to tissue osmotic stress. Genetic modification for accumulation of GB in an attempt to produce more stress tolerant plants has been a focus for several groups in recent years. However, attempts to increase tissue GB concentrations have been unsuccessful, with many transgenic lines accumulating far lower concentrations than naturally-occurring GB accumulators. A better understanding of the metabolic regulation of GB synthesis is necessary for successful molecular breeding and biotechnology. We utilized previously developed near-isogenic lines for GB accumulation to characterize the biochemical basis for GB deficiency in maize and sorghum. Salinity resulted in increased accumulation of choline in both accumulating and non-accumulating lines. When grown in the presence of NaCl, GB-non-accumulating lines had increased concentrations of choline and phosphocholine, but not GB. Decreased GB synthesis can be explained from the increased concentrations of phosphocholine in planta and the strong inhibition of N-phosphoethanolamine methyltransferase by phosphocholine observed in vitro. The lack of GB accumulation in GB−/− homozygous NILs was not due to the lack of the putative choline monooxygenase (the enzyme responsible for choline oxidation to betaine aldehyde) gene or protein that we describe. The previously identified bet1 locus does not appear to be choline monooxygenase. However, the lack of GB synthesis does affect the synthesis and turnover of choline moieties in GB non-accumulating lines, which may lead to alterations in overall 1-carbon metabolism in plants.
Keywords: Zea mays; Sorghum bicolor; Gramineae; Metabolism; Quaternary ammonium compound; Choline monooxygenase; Betaine aldehyde dehydrogenase; N-phosphoethanolamine methyltransferase; Choline; Glycinebetaine;

Mean δ 13C values and ranges (min/max) of values averaged over three sampling dates for whole leaf tissue (BULK), leaf fatty acids C16:0, C18:2 and C18:3 and leaf monosaccharides arabinose, xylose, galactose and glucose from a range of plants from a cattle-grazed or deer-grazed field.Leaves of 26 grass, herb, shrub and tree species were collected from mesotrophic grasslands to assess natural variability in bulk, fatty acid and monosaccharide δ 13C values under different grazing management (cattle- or deer-grazed) on three sample dates (May, July and October) such that interspecific and spatiotemporal variations in whole leaf tissues and compound-specific δ 13C values could be determined. The total mean leaf bulk δ 13C value for plants was −28.9‰ with a range of values spanning 7.5‰. Significant interspecific variation between bulk leaf δ 13C values was only determined in October (P  = <0.001) when δ 13C values of the leaf tissues from both sites was on average 1.5‰ depleted compared to during July and May. Samples from May were significantly different between fields (P  = 0.03) indicating an effect from deer- or cattle-grazing in young leaves. The average individual monosaccharide δ 13C value was 0.8‰ higher compared with whole leaf tissues. Monosaccharides were the most abundant components of leaf biomass, i.e. arabinose, xylose, mannose, galactose and glucose, and therefore, fluctuations in their individual δ 13C values had a major influence on bulk δ 13C values. An average depletion of ca. 1‰ in the bulk δ 13C values of leaves from the deer-grazed field compared to the cattle-grazed field could be explained by a general depletion of 1.1‰ in glucose δ 13C values, as glucose constituted >50% total leaf monosaccharides. In October, δ 13C values of all monosaccharides varied between species, with significant variation in δ 13C values of mannose and glucose in July, and mannose in May. This provided an explanation for the noted variability in the tissue bulk δ 13C values observed in October 1999. The fatty acids C16:0, C18:2 and C18:3 were highly abundant in all plant species. Fatty acid δ 13C values were lower than those of bulk leaf tissues; average values of −37.4‰ (C16:0), −37.0‰ (C18:2) and −36.5‰ (C18:3) were determined. There was significant interspecific variation in the δ 13C values of all individual fatty acids during October and July, but only for C18:2 in May (P  = <0.05). This indicated that seasonal trends observed in the δ 13C values of individual fatty acids were inherited from the isotopic composition of primary photosynthate. However, although wide diversity in δ 13C values of grassland plants ascribed to grazing management, interspecific and spatiotemporal influences was revealed, significant trends (P  = <0.0001) for fatty acid and monosaccharide δ 13C values: δ 13C16:0  <  δ 13C18:2  <  δ 13C18:3 and δ 13Carabinose  >  δ 13Cxylose  >  δ 13Cglucose  >  δ 13Cgalactose, respectively, previously described, appear consistent across a wide range of species at different times of the year in fields under different grazing regimes.
Keywords: Mesotrophic grassland; Leaves; Gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS); Bulk δ 13C values; Compound-specific δ 13C values; Monosaccharides; Fatty acids;

Cytotoxic triterpenoid saponins from the stem bark of Antonia ovata by Abdulmagid Alabdul Magid; Hélène Bobichon; Nicolas Borie; Nathalie Lalun; Christophe Long; Christian Moretti; Catherine Lavaud (429-434).
Four triterpenoid saponins named antoniosides A–D, were isolated from the stem bark of Antonia ovata (Loganiaceae), along with eleven known compounds. Their structures were established by spectroscopic methods and acid hydrolysis. The cytotoxic activity of isolated saponins was evaluated in vitro against KB cell line.Phytochemical investigation of the MeOH extract of the stem bark of Antonia ovata led to the isolation of four triterpenoid saponins, along with eleven known compounds. Their structures were established by extensive 1D and 2D NMR, as well as HR-MS analysis and acid hydrolysis. All isolated saponins contained the same tetrasaccharide chain O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucopyranosyl-(1 → 3)-O-[β-d-glucopyranosyl-(1 → 2)]-β-d-glucuropyranoside linked to C-3 of esterified derivatives of R1-barrigenol, A1-barrigenol, barringtogenol C, or camelliagenin. Biological evaluation of the compounds against KB cell line revealed a potent cytotoxic activity with IC50 values ranging from 3.1 to 6.6 μM. The known compounds were found to be inactive at 10 μg/ml concentration.
Keywords: Antonia ovata; Loganiaceae; Triterpenoid saponins; R1-barrigenol; A1-barrigenol; Barringtogenol C; Camelliagenin; Cytotoxic activity;

Phenolic compounds and their anti-oxidative properties and protein kinase inhibition from the Chinese mangrove plant Laguncularia racemosa by Cui Shi; Min-Juan Xu; Mirko Bayer; Zhi-Wei Deng; Michael H.G. Kubbutat; Wim Waejen; Peter Proksch; Wen-Han Lin (435-442).
Phenolic compounds, named integracin D (1), (7′R, 8′S, 8S)-8-hydroxyisoguaiacin (3), (2R, 3R) pinobanksin-3-caffeoylate (5) and threo-8S-7-methoxysyringylglycerol (6), were isolated from the Chinese mangrove plant Laguncularia racemosa (L) Gaertn. f. (Combretaceae). Their inhibitory activities against protein kinases, antioxidation, and insecticide were evaluated.Phenolic compounds, named integracin D (1), (7′R, 8′S, 8S)-8-hydroxyisoguaiacin (3), (2R, 3R) pinobanksin-3-caffeoylate (5) and threo-8S-7-methoxysyringylglycerol (6), respectively, were isolated from the Chinese mangrove plant Laguncularia racemosa (L) Gaertn. f. (Combretaceae), together with 23 known phenolic metabolites. Their structures were elucidated on the basis of extensive spectroscopic analyses including that of IR, UV, MS, CD, 1D and 2D NMR spectra as well as by comparison with literature data. Compound 5 showed significant anti-oxidative activity in the DPPH and TEAC free-radical-scavenging assays, while several of the phenolic compounds were tested for protein kinase inhibitory activity in an assay involving 24 different human tumor related protein kinases. Compounds 5, 7, and 23 showed potential inhibition with IC50 values between 2.2 and 3.6 μg/mL toward individual kinases. The ellagic acid derivatives were tested for insecticidal activity.
Keywords: Laguncularia racemosa; Combretaceae; White mangrove; Phenolic compounds; Structure elucidation; Anti-oxidative activity; Protein kinase inhibitors; Insecticidal activity;

Dihydrochalcones: Implication in resistance to oxidative stress and bioactivities against advanced glycation end-products and vasoconstriction by Thomas Dugé de Bernonville; Sylvain Guyot; Jean-Pierre Paulin; Matthieu Gaucher; Laurent Loufrani; Daniel Henrion; Séverine Derbré; David Guilet; Pascal Richomme; James F. Dat; Marie-Noëlle Brisset (443-452).
Sieboldin, a dihydrochalcone purified from leaves of Malus domestica genotype Evereste, was shown to correlate with plant resistance to oxidative stress and to be a promising therapeutic with high antioxidant activity and potential pharmacological effects.Flavonoids are a group of polyphenol compounds with known antioxidant activities. Among them, dihydrochalcones are mainly found in apple leaves (Malus domestica). Glycosylated dihydrochalcones were previously found in large amounts in leaves of two genotypes of Malus with contrasting resistance to fire blight, a bacterial disease caused by Erwinia amylovora. In the present study we demonstrate that soluble polyphenol patterns comprised phloridzin alone or in combination with two additional dihydrochalcones, identified as sieboldin and trilobatin. Presence of sieboldin in young leaves correlated well with a high 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity. Moreover, these leaves displayed enhanced tolerance to paraquat, a photooxidative-stress generating herbicide. Interestingly, phloridzin had a high activity in the oxygen radical absorbance capacity (ORAC) assay, but its presence alone in leaves did not correlate with tolerance to paraquat. In order to further characterise the activity of these compounds, we tested their ability to prevent oxidative-dependent formation of advanced glycation end-products (AGEs) and phenylephrine-induced contraction of isolated rat mesenteric arteries. The antioxidant capacity of sieboldin was clearly demonstrated by showing that this compound (i) prevented vasoconstriction and (ii) inhibited AGEs formation. Both assays provided interesting information concerning a potential use of sieboldin as a therapeutic. Hence, our results strongly argue for a bioactivity of dihydrochalcones as functional antioxidants in the resistance of Malus leaves to oxidative stress. In addition, we demonstrate for the first time that sieboldin is a powerful multipotent antioxidant, effective in preventing physiopathological processes. Further work should aim at demonstrating the potential use of this compound as a therapeutic in treating free radical-involving diseases.
Keywords: Malus domestica; Rosaceae; Antioxidant; Phenylephrine; AGEs; Flavonoids; Dihydrochalcones; Sieboldin; Phloridzin; Trilobatin;

Macromolecular replication during lignin biosynthesis by Yi-ru Chen; Simo Sarkanen (453-462).
It is proposed that, during the final step in lignin biosynthesis, macromolecular primary structure is replicated by the direct action of an antiparallel double-stranded lignin template. Lignol radicals about to undergo coupling interact noncovalently with the aromatic rings of the dimeric substructure being copied in the proximal template strand. The process is controlled by strong intermolecular forces arising from dynamical electron correlation in the interacting π-orbitals.Lignins play a crucial role in the cell-wall architecture of all vascular plants. They are composed of p-hydroxyphenylpropanoid units interconnected through covalent bonds formed during lignol radical coupling between six different pairs of atomic centers. For 50 years, the primary structures of lignins have been thought to be random, but for a number of reasons such an assumption is not tenable. For example, it has been reported that, by simple physicochemical means, the rather recalcitrant lignins in spruce wood can be decisively separated into two fractions containing quite dissimilar biopolymer chains. Thus, a paradigm shift should be imminent, and a detailed working hypothesis for the mechanism of lignin biosynthesis would be invaluable in delineating how the process of macromolecular lignin assembly can be properly investigated. In conjunction with an earlier experimental result, an explicit model for a template dehydropolymerization process has been developed that describes how lignin primary structure is replicated. The strengths of the powerful noncovalent interactions have been calculated that control the transient placement of lignol radicals about to undergo coupling on a double-stranded lignin template. These elementary steps engender, in the growing daughter chain, a primary structure identical to that of the distal template strand. The interactions are governed by dynamical electron correlation in the π-orbitals of each immobilized lignol radical and the complementary aromatic ring in the antiparallel proximal strand. The resulting noncovalent forces are computed to be stronger than those stabilizing GC/CG base pairs in DNA double-helices, but the mechanism of replication is fundamentally different from that of any other biopolymer.
Keywords: Lignin primary structure; Replication; Template polymerization; Density functional theory; Dynamical electron correlation;

Acylated flavonol tetraglycosides from Delphinium gracile by Jesús G. Diaz; Werner Herz (463-468).
Monoacylated quercetin tetraglycosides 13, the diacylated kaempferol tetraglycoside kaempferol-3-O-α-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)-β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside, the latter’s 3-O-{[β-d-xylopyranosyl analog and 4-β-d-glucopyranosyloxy-6-methyl-2H-pyran-2-one were isolated from the aerial parts of Delphinium gracile DC. Structures were established by spectroscopic methods and chemical preparation of derivatives.An ethanol extract of the aerial parts of Delphinium gracile DC. yielded five flavonol glycosides quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-caffeoyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (1), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (2), quercetin-3-O-{[β-d-xylopyranosyl (1 → 3)-4-O-(Z-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranosyl (1 → 2)]}-β-d-glucopyranoside (3), kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (4) kaempferol-3-O-{[β-d-glucopyranosyl (1 → 3)-4-O-(E-p-coumaroyl)-α-l-rhamnopyranosyl (1 → 6)][β-d-glucopyranoside-7-O-(4-O-acetyl)-α-l-rhamnopyranoside (5) in addition to 4-(β-d-glucopyranosyloxy)-6-methyl-2H-pyran-2-one (6) and rutin. Structures were elucidated by spectroscopic methods.
Keywords: Delphinium gracile; Ranunculaceae; Flavonoids; Acylated kaempferol tetraglycosides; Acylated quercetin tetraglycosides;

Aporphine and bisaporphine alkaloids from Aristolochia lagesiana var. intermedia by Marcia L.R. Ferreira; Inara C. de Pascoli; Isabele R. Nascimento; Julio Zukerman-Schpector; Lucia M.X. Lopes (469-478).
Corydines and analogous aporphine alkaloids were isolated from Aristolochia lagesiana var. intermedia, together with three bisaporphine salts. Their structures were determined by chemical derivatizations and spectroscopic analyses.Corydines, isocorydines, and analogous aporphine alkaloids were isolated from the leaves of Aristolochia lagesiana var. intermedia, together with three bisaporphine salts (lagesianines B–D). Their structures were determined by chemical derivatizations and spectroscopic analyses. Lagesianines B and C are the first examples of N–CH2N′ and C-2–O–C-1′ linked dimeric aporphine alkaloids, respectively, while the monomeric units of lagesianine D, which has a carbon skeleton, are linked through C-7–C-5′via an ethane-1,2-diol group (C-7–CHOHCHOH–C-5′).
Keywords: Aristolochia lagesiana var. intermedia; Aristolochiaceae; Alkaloids; Aporphine; Bisaporphine; Alkaloid dimer; Lagesianines; Corydine; Isocorydine; Corydine Nα-oxide;

Flavonoid glycosides of the black locust tree, Robinia pseudoacacia (Leguminosae) by Nigel C. Veitch; Peter C. Elliott; Geoffrey C. Kite; Gwilym P. Lewis (479-486).
The 7-O-β-d-glucuronopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranosides of luteolin (1), apigenin (2), diosmetin (3), and acacetin (8) were isolated from leaves of Robinia pseudoacacia together with two known acacetin triglycosides. Comparative analysis of leaf and flower extracts by LC–UV and LC–MS highlighted a divergence in flavonoid chemistry between flavone 7-O-glycosides and flavonol 3,7-di-O-glycosides, respectively, and differences in glycosylation profiles.Four flavone glycosides isolated from extracts of the leaves of Robinia pseudoacacia (Leguminosae) were characterised by spectroscopic and chemical methods as the 7-O-β-d-glucuronopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranosides of acacetin (5,7-dihydroxy-4′-methoxyflavone), apigenin (5,7,4′-trihydroxyflavone), diosmetin (5,7,3′-trihydroxy-4′-methoxyflavone) and luteolin (5,7,3′,4′-tetrahydroxyflavone). Assignment of glycosidic 1H and 13C resonances in their NMR spectra was facilitated by 2 J HC correlations detected using the H2BC (heteronuclear two-bond correlation) pulse sequence. Spectroscopic analysis of two known triglycosides, acacetin 7-O-β-d-glucopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (previously unrecorded from this species) and acacetin 7-O-β-d-xylopyranosyl-(1 → 2)[α-l-rhamnopyranosyl-(1 → 6)]-β-d-glucopyranoside (‘acacetin trioside’), enabled inconsistencies in the literature relating to these structures to be resolved. Comparison of the flavonoid chemistry of leaves and flowers of R. pseudoacacia using LC–UV and LC–MS showed that flavone 7-O-glycosides, particularly of acacetin, predominated in the former, whereas the latter comprised mainly flavonol 3,7-di-O-glycosides, including several examples new to this species. Tissue dependent differences in flavonoid chemistry were also evident from the glycosylation patterns of the compounds.
Keywords: Robinia pseudoacacia; Leguminosae; Black locust tree; Chemotaxonomy; Flavone triglycosides; Glucuronides; Acacetin; Robinin; NMR; H2BC;