Phytochemistry (v.71, #5-6)

Vitamin B6: Killing two birds with one stone? by Sutton Mooney; Hanjo Hellmann (495-501).
Pyridoxal 5′-phosphate is one of six common B6 vitamers that broadly functions as an enzymatic co-factor in amino acid, carbohydrate, and fatty acid metabolism.Vitamin B6 comprises a group of compounds that are involved in a surprisingly high diversity of biochemical reactions. Actually, most of these reactions are co-catalyzed by a single B6 vitamer, pyridoxal 5′-phosphate, making it a crucial and versatile co-factor in many metabolic processes in the cell. In addition, it has been demonstrated in recent years that vitamin B6 has a second important function by being an effective antioxidant. Because of these two characteristics the vitamin is an interesting compound to study in plants. This review provides a brief overview and update on such important aspects like vitamin B6-dependent enzymes and known biosynthetic pathways in plants, phenotypes of plant mutants affected in vitamin B6 biosynthesis, and the potential benefits of modifying vitamin B6 content in plants.
Keywords: Vitamin B6; Pyridoxine; PLP; Co-factor; Antioxidant;

Misincorporation of the proline homologue Aze (azetidine-2-carboxylic acid) into proteins results in severe distortion of the polypeptide chain at every site of substitution.We have evaluated the effects of the proline homologue Aze (1) (azetidine-2-carboxylic acid) on growth of Escherichia coli strains used to over-express recombinant forms of murine myelin basic protein (rmMBP), and on the degree of misincorporation. Addition of Aze to minimal media resulted in severe diminution of growth rate, but rmMBP could still be produced and purified. Mass spectrometry indicated that a detectable proportion of the rmMBP produced had incorporated Aze instead of proline (Pro), to a maximum of three of eleven possible sites. Molecular modelling of a proline-rich region of rmMBP illustrated that the misincorporation of Aze at any site would cause a severe bend in the polypeptide chain, and that multiple Pro → Aze substitutions would completely disrupt a poly-proline type II structure that has been conjectured to be functionally significant.
Keywords: Myelin basic protein; Intrinsically disordered protein; Multiple sclerosis; Poly-proline II; Aze (azetidine-2-carboxylic acid);

Flavone synthase II (CYP93B16) from soybean (Glycine max L.) by Judith Fliegmann; Katarina Furtwängler; Georg Malterer; Corrado Cantarello; Göde Schüler; Jürgen Ebel; Axel Mithöfer (508-514).
The enzyme responsible for the inducible accumulation of 7,4′-dihydroxyflavone in soybean cell cultures was identified as a flavone synthase of type II, and classified as CYP93B16. The biochemical characterization of the recombinant protein indicated a direct and stereoselective formation of the flavone from the flavanone substrate.Flavonoids are a very diverse group of plant secondary metabolites with a wide array of activities in plants, as well as in nutrition and health. All flavonoids are derived from a limited number of flavanone intermediates, which serve as substrates for a variety of enzyme activities, enabling the generation of diversity in flavonoid structures. Flavonoids can be characteristic metabolites, like isoflavonoids for legumes. Others, like flavones, occur in nearly all plants. Interestingly, there exist two fundamentally different enzymatic systems able to directly generate flavones from flavanones, flavone synthase (FNS) I and II. We describe an inducible flavone synthase activity from soybean (Glycine max) cell cultures, generating 7,4′-dihydroxyflavone (DHF), which we classified as FNS II. The corresponding full-length cDNA (CYP93B16) was isolated using known FNS II sequences from other plants. Functional expression in yeast allowed the detailed biochemical characterization of the catalytic activity of FNS II. A direct conversion of flavanones such as liquiritigenin, naringenin, and eriodictyol into the corresponding flavones DHF, apigenin and luteolin, respectively, was demonstrated. The enzymatic reaction of FNS II was stereoselective, favouring the (S)- over the (R)-enantiomer. Phylogenetic analyses of the subfamily of plant CYP93B enzymes indicate the evolution of a gene encoding a flavone synthase which originally catalyzed the direct conversion of flavanones into flavones, via early gene duplication into a less efficient enzyme with an altered catalytic mechanism. Ultimately, this allowed the evolution of the legume-specific isoflavonoid synthase activity.
Keywords: Glycine max L.; Leguminosae; Enzyme characterization; Enantiomer selectivity; Flavonoids; Flavone synthase II; 7,4′-Dihydroxyflavone; Liquiritigenin; CYP93B16; Coronalon; 12-Oxo-phytodienoic acid; JA, Jasmonic acid;

Structure–function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana by Miguel A. Mazorra-Manzano; Takuji Tanaka; Derek R. Dee; Rickey Y. Yada (515-523).
Interactions of the plant specific insert and histidine residues may explain the broad pH stability (pH 3–8) of recombinant aspartic proteinase A1 from Arabidopsis thaliana.Aspartic proteinases (APs) are involved in several physiological processes in plants, including protein processing, senescence, and stress response and share many structural and functional features with mammalian and microbial APs. The heterodimeric aspartic proteinase A1 from Arabidopsis thaliana (AtAP A1) was the first acid protease identified in this model plant, however, little information exists regarding its structure function characteristics. Circular dichroism analysis indicated that recombinant AtAP A1 contained an higher α-helical content than most APs which was attributed to the presence of a sequence known as the plant specific insert in the mature enzyme. rAtAP A1 was stable over a broad pH range (pH 3–8) with the highest stability at pH 5–6, where 70–80% of the activity was retained after 1 month at 37 °C. Using calorimetry, a melting point of 79.6 °C was observed at pH 5.3. Cleavage profiles of insulin β-chain indicated that the enzyme exhibited a higher specificity as compared to other plant APs, with a high preference for the Leu15–Tyr16 peptide bond. Molecular modeling of AtAP A1 indicated that exposed histidine residues and their interaction with nearby charged groups may explain the pH stability of rAtAP A1.
Keywords: Arabidopsis thaliana; Cruciferae; Plant aspartic proteinase; Substrate specificity; Thermal stability; pH stability; Plant specific insert; Proteases;

Biochemical comparison of two proteolytic enzymes from Carica candamarcensis: Structural motifs underlying resistance to cystatin inhibition by Marco Túlio R. Gomes; Henrique A. Ribeiro; Miriam T.P. Lopes; Fanny Guzman; Carlos E. Salas (524-530).
While CMS1MS2 displays the highest enzyme activity, the mitogenic proteinase CMS2MS2 is barely inhibited by cystatin. It is proposed that Arg180 is responsible for cystatin resistance.The lattices of Carica candamarcensis and Carica papaya, members of the Caricaceae family, contain isoforms of cysteine proteinases that help protect these plants against injury. In a prior study, we fractionated 14 discrete proteinaceous components from C. candamarcensis, two of them displaying mitogenic activity in mammalian cells. In this study, we compared the kinetic parameters of one of the mitogenic proteinases (CMS2MS2) with one of the isoforms displaying the highest enzyme activity of this group (CMS1MS2). Both enzymes display a similar Km value with either BAPNA (Benzoyl-Arg-pNA) or PFLPNA (Pyr-Phe-Leu-pNA), but the kcat of CMS1MS2 is about 14-fold higher for BAPNA and 129-fold higher with PFLPNA. While both enzymes are inhibited by E-64 and iodoacetamide, chicken cystatin fully inhibits CMS1MS2, but scarcely affects activity of CMS2MS2. Based on the structure of these proteins and other enzymes from the Caricaceae family whose structures have been resolved, it is proposed that Arg180 located in the cleft at the active site in CMS2MS2 is responsible for its resistance to cystatin.
Keywords: Carica candamarcensis; Caricaceae; Cystatin; Cysteine proteinases; Latex;

Consequences of antisense down-regulation of a lignification-specific peroxidase on leaf and vascular tissue in tobacco lines demonstrating enhanced enzymic saccharification by Bahram Kavousi; Arsalan Daudi; Charis M. Cook; Jean-Paul Joseleau; Katia Ruel; Alessandra Devoto; G. Paul Bolwell; Kristopher A. Blee (531-542).
Peroxidase down-regulated tobacco plants were profiled for effects on cell walls, photosynthetic capacity and improved saccharification efficiency. Growth and potential industrial utility were not compromised by morphological changes.Tobacco plants expressing an antisense construct for a cationic peroxidase, which down-regulated lignin content at the presumed level of polymerisation, have been further analysed. T1 plants were derived from a large-scale screen of T0 mutant lines, previously published, which identified lines demonstrating consistent lignin down-regulation. Of these, line 1074 which had the most robust changes in lignin distribution through several generations was shown to have accompanying down-regulation of transcription of most lignin biosynthesis genes, except cinnamoyl-CoA reductase. The consistent 20% reduction in lignin was not accompanied by significant gross changes in vascular polysaccharide content and composition, despite a modest up-regulation of transcripts of genes involved in cellulose and hemicellulose synthesis. Morphologically, 1074 plants have under-developed xylem with both fibers and vessels having thin cell walls and limited secondary wall thickening with an abnormal S2 layer. However, they were not compromised in overall growth. Nevertheless, these and other lines showed improved potential industrial utility through a threefold increase in enzymic saccharification efficiency compared with wild-type (wt). Therefore, they were profiled for further un-intended effects of transgenesis that might compromise their value for industrial or biofuel processes. Other phenotypic changes included increased leaf thickness and bifurcation at the tip of the leaf. wt-Plants had smaller chloroplasts and higher stomatal numbers than mutants. Transgenic lines also showed a variable leaf pigment distribution with light-green areas that contained measurably less chlorophyll a, b, and carotenoids. Changes in epidermal pavement cells of mutant lines were also observed after exposure to various chemicals, while wt leaves retained their structural integrity. Despite these changes, the mutant plants grew and were viable indicating that lignification patterns can be manipulated considerably through targeting polymerisation without serious deleterious effects.
Keywords: Tobacco; Nicotiana tabacum; Peroxidase; Antisense; Cell wall; Saccharification; Photosynthesis;

Domain swapping between the cold shock domain proteins (CSDPs) 1 and 2 from Arabidopsis thaliana demonstrates that a specific modular arrangement of the cold shock domain and the zinc finger domain establishes both RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced chilling tolerance in plants as well as in bacteria.Among the four cold shock domain proteins (CSDPs) identified in Arabidopsis thaliana, it has recently been shown that CSDP1 harboring seven CCHC-type zinc fingers, but not CSDP2 harboring two CCHC-type zinc fingers, function as a RNA chaperone during cold adaptation. However, the structural features relevant to this differing RNA chaperone activity between CSDP1 and CSDP2 remain largely unknown. To determine which structural features are necessary for the RNA chaperone activity of the CSDPs, the importance of the N-terminal cold shock domain (CSD) and the C-terminal zinc finger glycine-rich domains of CSDP1 and CSDP2 were assessed. The results of sequence motif-swapping and deletion experiments showed that, although the CSD itself harbored RNA chaperone activity, the number and length of the zinc finger glycine-rich domains of CSDPs were crucial to the full activity of the RNA chaperones. The C-terminal domain itself of CSDP1, harboring seven CCHC-type zinc fingers, also has RNA chaperone activity. The RNA chaperone activity and nuclei acid-binding property of the native and chimeric proteins were closely correlated with each other. Collectively, these results indicate that a specific modular arrangement of the CSD and the zinc finger domain determines both the RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced cold tolerance in plants as well as in bacteria.
Keywords: Arabidopsis thaliana; Cruciferae; Cold adaptation; Cold shock protein; Posttranscriptional regulation; RNA chaperone;

Cytosolic APx knockdown indicates an ambiguous redox responses in rice by Sílvia B. Rosa; Andréia Caverzan; Felipe K. Teixeira; Fernanda Lazzarotto; Joaquim A.G. Silveira; Sérgio Luiz Ferreira-Silva; João Abreu-Neto; Rogério Margis; Márcia Margis-Pinheiro (548-558).
Ascorbate peroxidases convert H2O2 into H2O, using ascorbate as an electron donor. Rice transgenic plants silenced for cytosolic Apx1 and Apx2 genes were obtained. The double silencing of cytosolic OsApx genes induced compensatory antioxidant mechanisms while single knockdown of these genes resulted in the impairment of normal plant development.Ascorbate peroxidases (APX, EC are class I heme-peroxidases, which catalyze the conversion of H2O2 into H2O, using ascorbate as a specific electron donor. Previously, the presence of eight Apx genes was identified in the nuclear genome of rice (Oryza sativa), encoding isoforms that are located in different sub-cellular compartments. Herein, the generation of rice transgenic plants silenced for either both or each one of the cytosolic Apx1 and Apx2 genes was carried out in order to investigate the importance of cytosolic Apx isoforms on plant development and on plant stress responses. Transgenic double Apx1/2-silenced plants exhibited normal development, even though these plants showed a global reduction of Apx activity which strongly impacts the whole antioxidant system regulation. Apx1/2-silenced plants also showed increased H2O2 accumulation under control and stress situations and presented higher tolerance to toxic concentration of aluminum when compared to wild type plants. On the other hand, silencing OsApx1 and OsApx2 genes individually resulted in strong effect on plant development producing semi-dwarf phenotype. These results suggested that the double silencing of cytosolic OsApx genes induced compensatory antioxidant mechanisms in rice while single knockdown of these genes did not, which resulted in the impairing of normal plant development.
Keywords: Oryza sativa; Gramineaea; Rice; Apx; Ascorbate peroxidase; Antioxidant; Redox; H2O2 scavenging; RNAi;

The distribution of flavan-3-ols in various parts of Camellia sinensis seedlings was investigated along with the expression of genes encoding enzymes associated with the biosynthesis of flavan-3-ols and related phenolic compounds. The main biosynthetic pathways leading to (−)-epigallocatechin-3-O-gallate and (−)-epicatechin-3-O-gallate biosynthetic pathways in tea leaves are proposed.The distribution of phenolic compounds in young and developing leaves, stems, main and lateral roots and cotyledons of 8-week-old tea (Camellia sinensis) seedlings was investigated using HPLC-MS2. Fourteen compounds, flavan-3-ols, chlorogenic acids, and kaempferol-O-glycosides, were identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern. The major phenolics were (−)-epigallocatechin-3-O-gallate and (−)-epicatechin-3-O-gallate, located principally in the green parts of the seedlings. Considerable amounts of radioactivity from [ring-14C]phenylalanine were incorporated in (−)-epicatechin, (−)-epigallocatechin, (−)-epicatechin-3-O-gallate and (−)-epigallocatechin-3-O-gallate, by tissues of young and developing leaves and stems. Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis, CHS, CHI, F3H, F35H, DFR, ANS, ANR and LAR was investigated. Transcripts of all genes, except LAR, were more abundant in leaves and stems than in roots and cotyledons. No significant difference was found in the amount of transcript of LAR. These findings indicate that in tea seedlings flavan-3-ols are produced by a naringenin-chalcone → naringenin → dihydrokaempferol pathway. Dihydrokaempferol is a branch point in the synthesis of (−)-epigallocatechin-3-O-gallate and other flavan-3-ols which can be formed by routes beginning with either a flavonoid 3′-hydroxylase mediated conversion of the flavonol to dihydroquercetin or a flavonoid 3′,5′-hydroxylase-catalysed conversion to dihydromyricetin with subsequent steps involving sequential reactions catalysed by dihydroflavanol 4-reductase, anthocyanidin synthase, anthocyanidin reductase and flavan-3-ol gallate synthase.
Keywords: Camellia sinensis; Theaceae; Tea seedlings; Flavan-3-ols (aka catechins); (−)-Epigallocatechin gallate; Distribution; Biosynthesis; Gene expression;

Biological variation of Vanilla planifolia leaf metabolome by Tony Lionel Palama; Isabelle Fock; Young Hae Choi; Robert Verpoorte; Hippolyte Kodja (567-573).
The effects of the leaf age, day-time, season and accession on the metabolome was carried out on Vanilla planifolia leaves using 1H NMR spectrometry and multivariate data analysis techniques.The metabolomic analysis of Vanilla planifolia leaves collected at different developmental stages was carried out using 1H-nuclear magnetic resonance (NMR) spectroscopy and multivariate data analysis in order to evaluate their variation. Ontogenic changes of the metabolome were considered since leaves of different ages were collected at two different times of the day and in two different seasons. Principal component analysis (PCA) and partial least square modeling discriminate analysis (PLS-DA) of 1H NMR data provided a clear separation according to leaf age, time of the day and season of collection. Young leaves were found to have higher levels of glucose, bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-isopropyltartrate (glucoside A) and bis[4-(β-d-glucopyranosyloxy)-benzyl]-2-(2-butyl)-tartrate (glucoside B), whereas older leaves had more sucrose, acetic acid, homocitric acid and malic acid. Results obtained from PLS-DA analysis showed that leaves collected in March 2008 had higher levels of glucosides A and B as compared to those collected in August 2007. However, the relative standard deviation (RSD) exhibited by the individual values of glucosides A and B showed that those compounds vary more according to their developmental stage (50%) than to the time of day or the season in which they were collected (19%). Although morphological variations of the V. planifolia accessions were observed, no clear separation of the accessions was determined from the analysis of the NMR spectra. The results obtained in this study, show that this method based on the use of 1H NMR spectroscopy in combination with multivariate analysis has a great potential for further applications in the study of vanilla leaf metabolome.
Keywords: Vanilla planifolia; Orchidaceae; Metabolomic; NMR spectroscopy; CAM plants; Malic acid; Phenolic glucosides;

Survey of volatile oxylipins and their biosynthetic precursors in bryophytes by Emmanuel Croisier; Martin Rempt; Georg Pohnert (574-580).
A screening of 23 moss species revealed that these bryophytes release a surprising diversity of C5, C6, C8 and C9 volatile oxylipins upon tissue disruption. Precursors of these oxylipins are both, C18 and C20 fatty acids.Oxylipins are metabolites which are derived from the oxidative fragmentation of polyunsaturated fatty acids. These metabolites play central roles in plant hormonal regulation and defense. Here we survey the production of volatile oxylipins in bryophytes and report the production of a high structural variety of C5, C6, C8 and C9 volatiles of mosses. In liverworts and hornworts oxylipin production was not as pronounced as in the 23 screened mosses. A biosynthetic investigation revealed that both, C18 and C20 fatty acids serve as precursors for the volatile oxylipins that are mainly produced after mechanical wounding of the green tissue of mosses.
Keywords: Dicranum scoparium; Mosses; Liverworts; Volatiles; Oxylipins; Fatty acids; Lipoxygenases; Hydroperoxide lyases; GC–MS;

Analyses of non-polar and polar metabolites from Thellungiella salsuginea subjected to various forms of stress led to elucidation of the chemical structures of five constitutive metabolites and identification of a broad range of polar metabolites identical to those of other crucifers. The phytoalexins wasalexins A and B were consistently elicited by biotic and abiotic stresses.The metabolite profiles of the model crucifer Thellungiella salsuginea (salt cress) ecotype Shandong subjected to various biotic and abiotic stresses were analyzed using HPLC-DAD-ESI-MS. Two different cruciferous microbial pathogens, Albugo candida, a biotrophic oomycete, and Leptosphaeria maculans, a necrotrophic fungus, elicited formation of the phytoalexins wasalexins A and B without causing visual damage on inoculated leaves. Analyses of non-polar and polar metabolites led to elucidation of the chemical structures of five metabolites: 4′-O-(E)-sinapoyl-7-methoxyisovitexin-2″-O-β-d-glucopyranoside, 4′-O-(E)-sinapoylisovitexin-2″-O-β-d-glucopyranoside, 4-O-β-d-glucopyranosyl-7-hydroxymatairesinol, 5′-O-β-d-glucopyranosyldihydroneoascorbigen and 3-O-β-d-glucopyranosylthiane. 3-O-β-d-glucopyranosylthiane, an unique metabolite for which we suggest the name glucosalsuginin, is proposed to derive from the glucosinolate glucoberteroin. In addition, the identification of a broad range of polar metabolites identical to those of other crucifers was carried out. Quantification of several metabolites over a period of eight days showed that concentrations of the polar phytoanticipin 4-methoxyglucobrassicin increased substantially in leaves irradiated with UV light (λ max 254 nm) relative to control leaves, but not in leaves subjected to other stresses.
Keywords: Albugo candida; Brassicaceae; Crucifer; Glucosalsuginin; Leptosphaeria maculans; Phytoalexin; Salt cress; UV stress; Thellungiella halophila; T. salsuginea; Thiane; Wasalexins;

Dual metabolomics: A novel approach to understanding plant–pathogen interactions by J. William Allwood; Andrew Clarke; Royston Goodacre; Luis A.J. Mur (590-597).
A co-culture method is proposed for the dual metabolomic profiling of both Arabidopsis and bacterial pathogens which will facilitate the modelling of the interaction of both partners.One of the most well-characterised plant pathogenic interactions involves Arabidopsis thaliana and the bacteria Pseudomonas syringae pathovar tomato (Pst). The standard Pst inoculation procedure involves infiltration of large populations of bacteria into plant leaves which means that metabolite changes cannot be readily assigned to the host or pathogen. A plant cell–pathogen co-culture based approach has been developed where the plant and pathogen cells are separated after 12 h of co-culture via differential filtering and centrifugation. Fourier transform infrared (FT-IR) spectroscopy was employed to assess the intracellular metabolomes (metabolic fingerprints) of both host and pathogen and their extruded (extracellular) metabolites (metabolic footprints) under conditions relevant to disease and resistance. We propose that this system will enable the metabolomic profiling of the separated host and pathogen (i.e. ‘dual metabolomics’) and will facilitate the modelling of reciprocal responses.
Keywords: Dual metabolomics; Arabidopsis thaliana; Pseudomonas syringae pv. tomato; Fourier transform infrared (FT-IR) spectroscopy;

Constituents and secondary metabolite natural products in fresh and deteriorated cassava roots by Soad A.L. Bayoumi; Michael G. Rowan; John R. Beeching; Ian S. Blagbrough (598-604).
Hydroxycoumarins from cassava roots: scopoletin, scopolin, esculetin, and esculin.A phytochemical analysis of cassava (Manihot esculenta Crantz) fresh roots and roots suffering from post-harvest physiological deterioration (PPD) has been carried out. The first isolation and identification of galactosyl diacylglycerides from fresh cassava roots is reported, as well as β-carotene, linamarin, and β-sitosterol glucopyranoside. The hydroxycoumarin scopoletin and its glucoside scopolin were identified from cassava roots during PPD, as well as trace quantities of esculetin and its glucoside esculin. There is no isoscopoletin in cassava roots during PPD.
Keywords: Galactosyl diacylglyceride; Hydroxycoumarin; Linamarin; Manihot esculenta Crantz; family Euphorbiaceae; Post-harvest physiological deterioration; Scopoletin; β-Sitosterol glucopyranoside; Spectroscopy;

Synergetic effects of nitrogen depletion, temperature, and light on the content of phenolic compounds and gene expression in leaves of tomato by Trond Løvdal; Kristine M. Olsen; Rune Slimestad; Michel Verheul; Cathrine Lillo (605-613).
Phenolics are valued for improving pathogen-resistance and other quality properties in plants. We show how their accumulation may be enhanced in tomato by non-transgenic means, and how flavonoid-associated genes are affected by various abiotic factors.Tomato plants (Solanum lycopersicum, cv. Suzanne) were subjected to complete nutrient solution or a solution without nitrogen (N), and placed at different temperatures and light conditions to test the effects of environment on flavonoids and caffeoyl derivatives and related gene expression. N depletion during 4–8 days resulted in enhanced levels of flavonoids and caffeoyl derivatives. Anthocyanins showed pronounced increased levels when lowering the growth temperature from 24 °C to 18 °C or 12 °C. Flavonol levels increased when the light intensity was increased from 100 μmol m−2  s−1 PAR to 200 μmol m−2  s−1 PAR. Synergistic effects of the various environmental factors were observed. The increase in content of quercetin derivatives in response to low temperatures was only found under conditions of N depletion, and especially at the higher light intensity. Expression of structural genes in the phenylpropanoid and flavonoid pathways, PAL (phenylalanine ammonia lyase), CHS (chalcone synthase), F3H (flavanone 3-hydroxylase), and FLS (flavonol synthase) increased in response to N depletion, in agreement with a corresponding increase in flavonoid and caffeoyl content. Expression of these structural genes generally also increased in response to lower temperatures. As indicated through expression studies and correlation analysis, effects of N depletion were apparently mediated through the overall regulators of the pathway the MYB transcription factor ANT1 (ANTHOCYANIN 1) and SlJAF13 (a bHLH transcription factor orthologue of petunia JAF13 and maize RED genes). A PAL gene (PAL6) was identified, and correlation analysis was compatible with PAL6 being an actively expressed gene with function in flavonoid synthesis.
Keywords: Solanum lycopersicum; Tomato; Nitrogen; Temperature; Flavonoids; Anthocyanins; Kaempferol; Rutin; ANT1; Phenylalanine ammonia lyase;

Cadmium activates Arabidopsis MPK3 and MPK6 via accumulation of reactive oxygen species by Xiao-Min Liu; Kyung Eun Kim; Kang-Chang Kim; Xuan Canh Nguyen; Hay Ju Han; Mi Soon Jung; Ho Soo Kim; Sun Ho Kim; Hyeong Cheol Park; Dae-Jin Yun; Woo Sik Chung (614-618).
Cd activates the mitogen-activated protein kinases, MPK3 and MPK6, in Arabidopsis, with the Cd sensing pathway using build-up of ROS to trigger activation of the MPKs.Cadmium (Cd) is a non-essential toxic heavy metal that influences normal growth and development of plants. However, the molecular mechanisms by which plants recognize and respond to Cd remain poorly understood. We show that, in Arabidopsis, Cd activates the mitogen-activated protein kinases, MPK3 and MPK6, in a dose-dependent manner. Following treatment with Cd, these two MAPKs exhibited much higher activity in the roots than in the leaves, and pre-treatment with the reactive oxygen species (ROS) scavenger, glutathione, effectively inhibited their activation. These results suggest that the Cd sensing signaling pathway uses a build-up of ROS to trigger activation of Arabidopsis MPK3 and MPK6.
Keywords: Arabidopsis; Cruciferae; Cadmium; Heavy metal; MAPK; Reactive oxygen species; Signal transduction;

Induction of acid phosphatase transcripts, protein and enzymatic activity by simulated herbivory of hybrid poplar by Vasko Veljanovski; Ian T. Major; Joseph J. Patton; Eric Bol; Stephanie Louvet; Barbara J. Hawkins; C. Peter Constabel (619-626).
The enzymatic activity and protein levels of a pest defense-related enzyme of hybrid poplar, acid phosphatase, are shown to be inducible by tissue damage.Herbivory and wounding upregulate a large suite of defense genes in hybrid poplar leaves. A strongly wound- and herbivore-induced gene with high similarity to Arabidopsis vegetative storage proteins (VSPs) and acid phosphatase (AP) was identified among genes strongly expressed during the poplar herbivore defense response. Phylogenetic analysis showed that the putative poplar acid phosphatase (PtdAP1) gene is part of an eight-member AP gene family in poplar, and is most closely related to a functionally characterized soybean nodule AP. Unlike the other poplar APs, PtdAP1 is expressed in variety of tissues, as observed in an analysis of EST data. Following wounding, the gene shows an expression profile similar to other known poplar defense genes such as protease inhibitors, chitinase, and polyphenol oxidase. Significantly, we show for the first time that subsequent to the wound-induction of PtdAP1 transcripts, AP protein and activity increase in extracts of leaves and other tissues. Although its mechanism of action is as yet unknown, these results suggest in hybrid poplar PtdAP1 is likely a component of the defense response against leaf-eating herbivores.
Keywords: Hybrid poplar; Populus trichocarpa; P. deltoides; Salicaceae; Plant defense; Plant-herbivore interactions; Systemic defense response;

Chemotaxonomy of Gonospermum and related genera by Jorge Triana; José Luis Eiroa; Juan José Ortega; Francisco León; Ignacio Brouard; Juan Carlos Hernández; Francisco Estévez; Jaime Bermejo (627-634).
Sesquiterpenes lactones isolated from Gonospermum and Lugoa, as well as endemic Canarian species of Tanacetum show a close relationship, and feature highly oxidized eudesmane or germacrane lactones. The presence of these types of lactones supports the proposal to group them in a monophyletic group in the Canary Islands.Aerial parts of Gonospermum fruticosum collected at several locations in the Canary Islands afforded, in addition to known compounds, four sesquiterpene alcohols related to costol and a sesquiterpene lactone, whose structures were established on the basis of their spectroscopic data and chemical transformations. Except for Gonospermum species collected on the island of Tenerife, those collected on the island of El Hierro and, in a previous study those from La Gomera, contain sesquiterpene lactones that can be used as chemotaxonomic markers confirming the inclusion of Gonospermum, Lugoa, and species of Tanacetum endemic to the Canary Islands in a genus that does not support the monophyly of Gonosperminae.
Keywords: Gonospermum fruticosum; Gonosperminae; Asteraceae; Chemotaxonomy; Sesquiterpene lactones; Sesquiterpene alcohols;

Bioactivity-guided isolation of cytotoxic sesquiterpenes of Rolandra fruticosa by Li Pan; Daniel D. Lantvit; Soedarsono Riswan; Leonardus B.S. Kardono; Hee-Byung Chai; Esperanza J. Carcache de Blanco; Norman R. Farnsworth; Djaja Doel Soejarto; Steven M. Swanson; A. Douglas Kinghorn (635-640).
Bioassay-guided fractionation of a methanol extract of Rolandra fruticosa led to the isolation of seven germacrane-type derived sesquiterpenes, including two previously unknown compounds, 13-methoxyisorolandrolide (1), and 2α,13-diacetoxy-4α-hydroxy-8α-isobutyloyloxybourbonen-12,6α-olide (2). All isolates were evaluated for their cytotoxicity using the HT-29 cell line. Compounds 3, 5, 6 were also tested in an enzyme-based ELISA NF-κB (p65) inhibition assay. 13-Acetoxyrolandrolide (3) was investigated in an in vivo hollow fiber assay.Cytotoxicity-guided fractionation of a methanol extract of the leaves and twigs of Rolandra fruticosa using the HT-29 human colon cancer cell line led to the isolation of seven sesquiterpene lactones, including the hitherto unknown isorolandrolide, 13-methoxyisorolandrolide (1), and bourbonenolide, 2α,13-diacetoxy-4α-hydroxy-8α-isobutyroyloxybourbonen-12,6α-olide (2), as well as five known compounds, 13-acetoxyrolandrolide (3), 8-desacyl-13-acetoxyrolandrolide-8-O-tiglate (4), 2-epi-glaucolide E (5), 2α,13-diacetoxy-4α-hydroxy-8α-methacryloyloxybourbonen-12,6α-olide (6), and 2α,13-diacetoxy-4α-hydroxy-8α-tigloyloxybourbonen-12,6α-olide (7). The structures of the two sesquiterpenes were elucidated on the basis of spectroscopic methods. All isolates were evaluated for their cytotoxicity using the HT-29 cell line, and only 13-acetoxyrolandrolide (3) was found to possess a potent inhibitory effect against this cell line. Compounds 3, 5 and 6 were also tested in a NF-κB (p65) inhibition assay, and 3 was assessed in an in vivo hollow fiber assay.
Keywords: Rolandra fruticosa (Asteraceae); Germacrane-type sesquiterpene lactones; Isorolandrolide; Rolandrolide; Glaucolide; Bourbonenolide; Cytotoxicity; HT-29 human colon cancer cells; NF-κB (p65) inhibition assay; In vivo hollow fiber assay;

Isolation and evaluation of kaempferol glycosides from the fern Neocheiropteris palmatopedata by Jian-Hong Yang; Tamara P. Kondratyuk; Laura E. Marler; Xi Qiu; Yongsoo Choi; Hongmei Cao; Rui Yu; Megan Sturdy; Scott Pegan; Ying Liu; Li-Qin Wang; Andrew D. Mesecar; Richard B. Van Breemen; John M. Pezzuto; Harry H.S. Fong; Ye-Gao Chen; Hong-Jie Zhang (641-647).
Six kaempferol glycosides including three hitherto unknown compounds (1–3) were isolated from the roots of the fern Neocheiropteris palmatopedata. Compounds 1 and 2 were determined to possess an unusual sugar moiety containing a 4,4-dimethyl-3-oxo-butoxy substituent group, respectively. The isolates were evaluated for their cancer chemopreventive potential based on their ability to inhibit tumor necrosis factor-α-induced induced NF-κB, nitric oxide (NO) production, aromatase, quinone reductase 2 (QR2) and COX-1/-2 activities.Kaempferol glycosides, named palmatosides A (1), B (2) and C (3), together with three known kaempferol glycosides, multiflorins A (4) and B (5), and afzelin (6), were isolated from the roots of the fern Neocheiropteris palmatopedata. Palmatosides A (1) and B (2) each possessed an unusual sugar moiety containing a 4,4-dimethyl-3-oxo-butoxy substituent group. The isolated compounds were evaluated for their cancer chemopreventive potential based on their ability to inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activity, nitric oxide (NO) production, aromatase, quinone reductase 2 (QR2) and COX-1/-2 activities. Palmatosides B (2) and C (3) inhibited TNF-α-induced NF-κB activity with IC50 values of 15.7 and 24.1 μM, respectively; multiflorin A (4) inhibited aromatase enzyme with an IC50 value of 15.5 μM; afzelin (6) showed 68.3% inhibition against QR2 at a concentration of 11.5 μg/ml; palmatoside A (1) showed 52% inhibition against COX-1 enzyme at a concentration of 10 μg/ml; and multiflorin B (5) showed 52% inhibition against nitric oxide production at a concentration of 20 μg/ml. In addition, compounds 36 were shown to bind QR2 enzyme using LC–MS ultrafiltration binding assay.
Keywords: Neocheiropteris palmatopedata; Polypodiaceae; Flavone; Kaempferol glycosides; Palmatosides; Cancer chemoprevention; NF-κB; NO; QR2; Aromatase; COX-1; COX-2;

Toxic isolectins from the mushroom Boletus venenatus by Masashi Horibe; Yuka Kobayashi; Hideo Dohra; Tatsuya Morita; Takeomi Murata; Taichi Usui; Sachiko Nakamura-Tsuruta; Masugu Kamei; Jun Hirabayashi; Masanori Matsuura; Mina Yamada; Yoko Saikawa; Kimiko Hashimoto; Masaya Nakata; Hirokazu Kawagishi (648-657).
Ingestion of the mushroom Boletus venenatus causes a gastrointestinal syndrome, such as diarrhea. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom. BVL ingestion resulted in fetal toxicity to mice and also caused diarrhea.Ingestion of the toxic mushroom Boletus venenatus causes a severe gastrointestinal syndrome, such as nausea, repetitive vomiting, diarrhea, and stomachache. A family of isolectins (B. venenatus lectins, BVLs) was isolated as the toxic principles from the mushroom by successive 80% ammonium sulfate-precipitation, Super Q anion-exchange chromatography, and TSK-gel G3000SW gel filtration. Although BVLs showed a single band on SDS–PAGE, they were further divided into eight isolectins (BVL-1 to -8) by BioAssist Q anion-exchange chromatography. All the isolectins showed lectin activity and had very similar molecular weights as detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. Among them, BVL-1 and -3 were further characterized with their complete amino acid sequences of 99 amino acids determined and found to be identical to each other. In the hemagglutination inhibition assay, both proteins failed to bind to any mono- or oligo-saccharides tested and showed the same sugar-binding specificity to glycoproteins. Among the glycoproteins examined, asialo-fetuin was the strongest inhibitor. The sugar-binding specificity of each isolectin was also analyzed by using frontal affinity chromatography and surface plasmon resonance analysis, indicating that they recognized N-linked sugar chains, especially Galβ1 → 4GlcNAcβ1 → 4Manβ1 → 4GlcNAcβ1 → 4GlcNAc (Type II) residues in N-linked sugar chains. BVLs ingestion resulted in fatal toxicity in mice upon intraperitoneal administration and caused diarrhea upon oral administration in rats.
Keywords: Boletus venenatus; Boletaceae; Mushroom; Purification; Lectin; Lethal toxicity; Diarrhea;

Triterpenoid saponins from Astragalus wiedemannianus Fischer by Emre Polat; Erdal Bedir; Angela Perrone; Sonia Piacente; Ozgen Alankus-Caliskan (658-662).
Three cycloartane type glycosides, together with eight known compounds were isolated from the roots of Astragalus wiedemannianus. The presence of an arabinose moiety on the acyclic side chain of cycloartanes is reported for the first time.Three cycloartane-type triterpene glycosides were isolated from Astragalus wiedemannianus together with eight known secondary metabolites namely cycloastragenol, cycloascauloside B, astragaloside IV, astragaloside VIII, brachyoside B, astragaloside II, astrachrysoside A, and astrasieversianin X. The structures were established mainly by a combination of 1D and 2D-NMR techniques as 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl]-25-O-β-D-glucopyranosyl-20(R),24(S)-epoxy-3β,6α,16β,25-tetrahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-(4’-O-acetoxy)-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane, 3-O-[α-L-rhamnopyranosyl-(1 → 2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O-α-L-arabinopyranosyl-16-O-acetoxy-3β,6α,16β,24(S),25-pentahydroxycycloartane. To the best of our knowledge, the presence of an arabinose moiety on the acyclic side chain of cycloartanes is reported for the first time.
Keywords: Astragalus wiedemannianus; Leguminosae; Saponin; Cycloartane;

Triterpene glycosides from Agrostemma gracilis by Omer Koz; Erdal Bedir; Milena Masullo; Ozgen Alankus-Caliskan; Sonia Piacente (663-668).
Four triterpene saponins, agrostemmoside A–D were isolated from the methanol extract of Agrostemma gracilis. Their structures were determined by a combination of one- and two-dimensional NMR techniques, and mass spectrometry.Four triterpene saponins, agrostemmosides A–D were isolated from the methanol extract of Agrostemma gracilis. The structures of the compounds were determined as 3-O-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-α-l-rhamnopyranosyl-(1 → 2)-β-d-xylopyranosyloleanolic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester, 3-O-β-d-xylopyranosylechinocystic acid 28-O-β-d-glucopyranosyl-(1 → 2)-[β-d-xylopyranosyl-(1 → 6)]-β-d-glucopyranosyl-(1 → 6)-β-d-glucopyranosyl ester by a combination of one- and two-dimensional NMR techniques, and mass spectrometry. To the best of our knowledge this is the first phytochemical report on A. gracilis, and echinocystic acid saponins were encountered for the first time in Caryophyllaceae family.
Keywords: Agrostemma gracilis; Caryophyllaceae; Triterpene glycosides; Saponins; Echinocystic acid;

Euphane triterpenoids of Cassipourea lanceolata from the Madagascar rainforest by Yanpeng Hou; Shugeng Cao; Peggy J. Brodie; James S. Miller; Chris Birkinshaw; Mamisoa N. Andrianjafy; Rabodo Andriantsiferana; Vincent E. Rasamison; Karen TenDyke; Yongchun Shen; Edward M. Suh; David G.I. Kingston (669-674).
A Madagascar collection of the leaves and fruit of Cassipourea lanceolata Tul. led to the isolation of three euphane triterpenoids 13. The three compounds showed weak antiproliferative activities against the A2780 human ovarian cancer cell line, with IC50 values of 25, 25 and 32 μM, respectively.Fractionation of an ethanol extract of a Madagascar collection of the leaves and fruit of Cassipourea lanceolata Tul. led to the isolation of three euphane triterpenoids 13. The 1H and 13C NMR spectra of all compounds were fully assigned using a combination of 2D NMR experiments, including COSY, TOCSY, HSQC (HMQC), HMBC and ROESY sequences. The three compounds showed weak antiproliferative activities against the A2780 human ovarian cancer cell line, with IC50 values of 25, 25 and 32 μM, respectively.
Keywords: Cassipourea lanceolata; Rhizophoraceae; Euphanes; Triterpenoids; NMR; Antiproliferative activity;

Microtropiosides A–F: ent-Labdane diterpenoid glucosides from the leaves of Microtropis japonica (Celastraceae) by Yuka Koyama; Katsuyoshi Matsunami; Hideaki Otsuka; Takakazu Shinzato; Yoshio Takeda (675-681).
From the leaves of Microtropis japonica, collected in the Okinawa islands, six ent-labdane glucosides, named microtropiosides A–F, were isolated. The structures of the compounds were elucidated by a combination of spectroscopic analyses, application of the β-d-glucopyranosylation-induced shift-trend rule in the 13C NMR spectroscopy and the modified Mosher’s method.From a 1-BuOH-soluble fraction of a MeOH extract of the leaves of Microtropis japonica, collected in the Okinawa islands, six ent-labdane glucosides, named microtropiosides A–F, were isolated together with one known acyclic sesquiterpene glucoside. Their structures were elucidated by a combination of spectroscopic analyses, and their absolute configurations determined by application of the β-d-glucopyranosylation-induced shift-trend rule in 13C NMR spectroscopy and the modified Mosher’s method.
Keywords: Microtropis japonica; Celastraceae; ent-Labdane glucoside; Microtropioside;

Chemical constituents from Saussurea cordifolia by Xu-Wen Li; Zi-Tao Guo; Yun Zhao; Zheng Zhao; Jin-Feng Hu (682-687).
C10-Acetylenic glycosides (1–4), and a lignan (5), together with 31 known compounds, were isolated from whole plants of Saussurea cordifolia . Selected compounds were evaluated for their cytotoxicity against MCF-7 human breast cancer cells using the MTT assay.Thirty-six naturally occurring compounds, including four C10-acetylenic glycosides and a lignan, were isolated from the whole plants of Saussurea cordifolia. Their structures were elucidated by means of spectroscopic and chemical methods to be 4,6-decadiyne-1-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (1), 4,6-decadiyne-1-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside (2), (8E)-decaene-4, 6-diyn-1-O-α-l-rhamnopyranosyl-(1 → 6)-β-d-glucopyranoside (3), (8Z)-decaene-4,6-diyn-1-O-β-d-apiofuranosyl-(1 → 6)-β-d-glucopyranoside (4), and (2R, 3S, 4S)-4-(4-hydroxy-3-methoxybenzyl)-2-(5-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-tetrahydrofuran-3-ol (5).
Keywords: Saussurea cordifolia; Compositae; Acetylenic glycoside; Lignan;

A C14-polyacetylenic glucoside with an α-pyrone moiety and four C10-polyacetylenic glucosides from Mediasia macrophylla by Shin-ichiro Kurimoto; Mamoru Okasaka; Yoshiki Kashiwada; Olimjon K. Kodzhimatov; Yoshihisa Takaishi (688-692).
Polyacetylenic glucosides (15) were isolated from the MeOH extract of Mediasia macrophylla. Their structures were established by spectroscopic analyses. Compounds 24 were the first examples of C10-polyacetylenic glucosides found in the family Umbelliferae, while compound 1 was a unique polyacetylenic glucoside possessing an α-pyrone moiety.Polyacetylenic glucosides (15) were isolated from the MeOH extract of Mediasia macrophylla, and their structures were established by spectroscopic analyses. Compounds 24 were the first examples of C10-polyacetylenic glucosides found in the family Umbelliferae, while compound 1 was a unique polyacetylenic glucoside possessing an α-pyrone moiety.
Keywords: Mediasia macrophylla; Umbelliferae; Polyacetylene; α-Pyrone;

Gynostemosides A–E, megastigmane glycosides from Gynostemma pentaphyllum by Zhen Zhang; Wei Zhang; Yan-Ping Ji; Yun Zhao; Chuan-Gui Wang; Jin-Feng Hu (693-700).
Megastigmane glycosides (15) were isolated from the whole plants of Gynostemma pentaphyllum. Their absolute structures were elucidated by means of spectroscopic methods including 2D NMR, HR-ESIMS, and circular dichroism (CD), as well as by chemical transformations.Megastigmane glycosides (15) together with seven (612) related known compounds were isolated from the whole plants of Gynostemma pentaphyllum. The structures were elucidated by means of spectroscopic methods, including 2D NMR, HR-ESIMS, and circular dichroism (CD), as well as chemical transformations to be (3R, 4R, 5S, 6S, 7E)-3,4,6-trihydroxymegastigmane-7-en-9-one-3-O-β-d-glucopyranoside (gynostemoside A, 1), (3S, 4S, 5R, 6R, 7E, 9R)-3,4,6,9-tetrahydroxymegastigmane-7-en-3-O-β-d-glucopyranoside (gynostemoside B, 2), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-9-O-β-d-glucopyranoside (gynostemoside C, 3), (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-3-O-β-d-glucopyranoside (gynostemoside D, 4), and (3S, 4S, 5S, 6S, 7E, 9R)-3,4,9-trihydroxymegastigmane-7-en-4-O-β-d-glucopyranoside (gynostemoside E, 5), respectively.
Keywords: Gynostemma pentaphyllum; Cucurbitaceae; Megastigmane glycosides; Absolute configuration;

by Stephen C. Fry (701-702).