Phytochemistry (v.66, #24)

Author Index (XXVI-XXXVIII).

Author Index (II).

Keyword Index (XXXIX-LII).

Sugar beet (Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall by Marie-Christine Ralet; Gwénaëlle André-Leroux; Bernard Quéméner; Jean-François Thibault (2800-2814).
After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between arabinan and galactan side chains through diferulic bridges.Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after 18O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (β-1 → 4-linked-dimer), Ara (α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.
Keywords: Amaranthaceae (Chenopodiaceae); Ferulic acid; Dehydrodimer; Dehydrodiferulate; Pectin; Arabinan; Galactan; Conformational minimum;

Factors affecting oligomerization status of UDP-glucose pyrophosphorylase by Leszek A. Kleczkowski; Françoise Martz; Malgorzata Wilczynska (2815-2821).
Certain buffers and molecular crowding conditions had strong effects on oligomerization status of purified recombinant barley UDP-glucose pyrophosphorylase (UGPase). This in turn could affect the activity of the protein which is active only as monomer. The data are discussed with respect to molecular determinants of structure/function properties of UGPase.UDP-glucose pyrophosphorylase (UGPase) is involved in the production of UDP-glucose, a key precursor to polysaccharide synthesis in all organisms. UGPase activity has recently been proposed to be regulated by oligomerization, with monomer as the active species. In the present study, we investigated factors affecting oligomerization status of the enzyme, using purified recombinant barley UGPase. Incubation of wild-type (wt) UGPase with phosphate or Tris buffers promoted oligomerization, whereas Mops and Hepes completely dissociated the oligomers to monomers (the active form). Similar buffer effects were observed for KK127-128LL and C99S mutants of UGPase; however, the buffers had a relatively small effect on the oligomerization status of the LIV135-137NIN mutant, impaired in deoligomerization ability and showing only 6–9% activity of the wt. Buffer composition had no effect on UGPase activity at UGPase protein concentrations below ca. 20 ng/ml. However, at higher protein concentration the activity in Tris, but not Mops nor Hepes, underestimated the amount of the enzyme. The data suggest that oligomerization status of UGPase can be controlled by subtle changes in an immediate environment (buffers) and by protein dilution. The evidence is discussed in relation to our recent model of UGPase structure/function, and with respect to earlier reports on the oligomeric integrity/activity of UGPases from eukaryotic tissues.
Keywords: Cellulose; Protein oligomerization; Sucrose; UDP-glucose synthesis;

Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant by Kazuhiro Ohta; Ryo Moriguchi; Koki Kanahama; Shohei Yamaki; Yoshinori Kanayama (2822-2828).
A NAD-dependent sorbitol dehydrogenase (SDH)-like cDNA was cloned from tomato (Lycopersicon esculentum Mill.) to provide molecular evidence of SDH in non-Rosaceae species that do not synthesize sorbitol as the primary photosynthetic product.The enzyme NAD-dependent sorbitol dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants.Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of sorbitol and fructose with NAD (H). SDL showed highest activity for sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the K m values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.
Keywords: Lycopersicon esculentum Mill.; Solanaceae; Tomato; cDNA cloning; Recombinant protein expression; Antisense transformation; Sorbitol dehydrogenase; Carbohydrate; Sorbitol; Fructose;

Flavonoids in flowers of 16 Kalanchoë blossfeldiana varieties by Allan Holm Nielsen; Carl Erik Olsen; Birger Lindberg Møller (2829-2835).
Kalanchoë blossfeldiana flowers were found to contain 3,5-O-β-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin, along with at least 7 distinct flavonol glycosides based on quercetin or kaempferol, including quercetin 3-(2″-O-β-d-glucopyranosyl-α-l-rhamnopyranoside), a flavonol previously found in Ginkgo biloba. Based on flavonoid findings, approaches for molecular breeding towards blue flower colour are discussed. Kalanchoë blossfeldiana varieties with orange, pink, red and magenta flowers were found to contain 3,5-O-β-d-diglucosides of pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. Pink, red and magenta varieties contained relatively high amounts of quercetin based flavonols. Four distinct quercetin flavonols were identified, namely quercetin 3-O-β-d-glucoside and three that were quercetin 3-O-α-l-rhamnoside based, with either glucose, xylose or arabinose attached to position 2 of the rhamnose. In addition, the presence of at least three kaempferol based diglycosides was suggested from LC–MS analyses. Orange varieties contained very low amounts of flavonol co-pigments and of delphinidin derivatives. The flower extracts of the varieties ‘Diva’ (magenta) and ‘Molly’ (red) had identical anthocyanin ratios but differed significantly in flavonol content. The magenta variety contained four times as much quercetin relative to anthocyanidin as the red variety. This difference was mainly due to a larger content of quercetin 3-O-(2″-O-β-d-glucopyranosyl-α-l-rhamnopyranoside). Based on pigment and co-pigment analyses, approaches for molecular breeding towards blue flower colour are discussed.
Keywords: Kalanchoë blossfeldiana; Crassulaceae; Flavonoids; Pelargonidin; Cyanidin; Peonidin; Delphinidin; Petunidin; Malvidin; Quercetin; Kaempferol; Rhamnose; Glucose; Arabinose; Xylose;

Chemical variation within and among six northern willow species by Tommi Nyman; Riitta Julkunen-Tiitto (2836-2843).
Concentrations of phenolic compounds were measured in six Salix species. Multivariate analyses show that interspecific chemical variation exceeds the variability within species, but among-species similarities depend on data analysis methods and the chemical class under study. The implications for herbivory and chemosystematics are discussed.Plant tissues typically contain a diverse complement of secondary metabolites that serve as protection against various biotic and abiotic hazards. Chemical similarities are commonly used to infer phylogenetic relationships among plant taxa, but the studies are typically based on the mean concentration of each compound in each study species, thus overlooking within-species variability. In order to investigate patterns of intra- and interspecific chemical variation in plants, we measured the concentrations of condensed tannins and 36 other phenolic compounds in 120 leaf samples representing six northern Salix species. Multivariate clustering and ordination analyses of the data show that: (1) Despite considerable within-species variation in chemical profiles, intraspecific variability is on average lower than the variation among species. (2) Interspecific similarities are sensitive to the data analysis methods used, and different chemical classes produce partly contradictory results. (3) Compounds within each biosynthetic class tend to behave in a correlated manner and, consequently, overall chemical similarities are weakly correlated with the phylogeny of the studied species. The conclusion is that chemical data are poorly suited for phylogenetic inference, unless methods for data analysis are improved to take into account the biosynthetic routes by which the compounds are produced.
Keywords: Salix; Willows; HPLC; Multivariate analysis; Chemosystematics; Herbivory; Phenolics; Salicylates; Condensed tannins;

Cytotoxic clerodane diterpenes from Glossocarya calcicola by Heidi L. Rasikari; David N. Leach; Peter G. Waterman; Robert N. Spooner-Hart; Albert H. Basta; Linda K. Banbury; Kelly M. Winter; Paul I. Forster (2844-2850).
Three novel compounds were isolated from Glossocarya calcicola Domin. Calcicolin-A (1), was characterised as (rel)-10βH-trans-12ξ-(2-methylbut-2(E)-enoyl)-1β-(isobutanoyl)-6α,13ξ-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide. Calcicolin-B (2) and –C (3) possessed the same skeletal structure but differed in the C-1 esterifying group. In 2, the C-1 group becomes 2-methylbut-2(E)-enoic acid and in 3 it becomes 2-methylbutanoic acid. Compounds 2 and 3 showed greatest cytotoxic activities against insect and mammalian cell lines.Three clerodane diterpenes were isolated and identified from leaf extract of Glossocarya calcicola. Compound 1 has been characterised as (rel)-10βH-trans-12ξ-(2-methylbut-2(E)-enoyl)-1β-(isobutanoyl)-6α,13ξ-dihydroxyclerodan-4(20),8(18)-dien-7,15-dione-15,16-oxide, to which we have assigned the trivial name calcicolin-A. The other two compounds had the same skeletal structure and C-12 substituent but in compound 2, the C-1 esterifying group becomes 2-methylbut-2(E)-enoic acid and in 3 it becomes 2-methylbutanoic acid. Although anti-insect activity was not observed for G. calcicola, cytotoxicity against insect and human carcinoma cell lines was detected.
Keywords: Glossocarya calcicola; Lamiaceae; Cytotoxicity; Insect cell lines; Mammalian cell lines; Tetranychus urticae; Plutella xylostella; Clerodane diterpenes; Calcicolin;

Antifungal activities of Thymus eriocalyx and Thymus X-porlock essential oils were inhibitory to Aspergillus parasiticus growth and its aflatoxin production. The oils brought about irreversible damage to cell wall, cell membrane, and cellular organelles of the fungus. Low concentrations of essential oils could act as food preservatives.The essential oils from Thymus eriocalyx and Thymus X-porlock obtained by hydrodistillation were analyzed by GC/MS. The major components of T. eriocalyx and T. X-porlock oils were thymol (63.8, 31.7%), β-phellandrene (13.30, 38.7%), cis-sabinene hydroxide (8.1, 9.6%), 1,8-cineole (2, 1.7%), and β-pinene (1.31, 2%), respectively. Antifungal activities of the oils were studied with special reference to the inhibition of Aspergillus parasiticus growth and aflatoxin production. Minimal inhibitory (MIC) and minimal fungicidal (MFC) concentrations of the oils were determined. Static effects of the above oils against A. parasiticus were at 250 ppm and lethal effects of T. eriocalyx and T. X-porlock were 500 and 1000 ppm of the oils, respectively. Aflatoxin production was inhibited at 250 ppm of both oils with that of T. eriocalyx being stronger inhibitor. Transmission electron microscopy (TEM) of A. parasiticus exposed to MIC level (250 ppm) of the oils showed irreversible damage to cell wall, cell membrane, and cellular organelles. It is concluded that the essential oils could be safely used as preservative materials on some kinds of foods at low concentrations to protect them from fungal infections.
Keywords: Thymus eriocalyx; Thymus X-porlock; Aspergillus parasiticus; Essential oils; Aflatoxin; Ultrastructure;

Tricalysiosides H–O: Ent-kaurane glucosides from the leaves of Tricalysia dubia by Dong-Hui He; Katsuyoshi Matsunami; Hideaki Otsuka; Takakazu Shinzato; Mitsunori Aramoto; Masahiko Bando; Yoshio Takeda (2857-2864).
From the leaves of Tricalysia dubia, eight ent-kaurane glucosides were isolated. The structure of tricalysioside H (1) was established by X-ray crystallography and those of tricalysiosides I–O (28) were elucidated by analysis of spectroscopic evidence.Eight ent-kaurane glucosides, named tricalysiosides H–O (18), were isolated from Tricalysia dubia. Tricalysioside H (1) possessed a hydroxyl group at the 1-position, to which the glucose moiety was attached. The structure was first elucidated by means of spectroscopic data analysis and finally confirmed by X-ray crystallography. Since acid hydrolysis of 1 gave d-glucose, the aglycone was proved to have an enantio-kaurane type skeleton. The structures of tricalysiosides I–O (28) were mainly elucidated from analysis of spectroscopic evidence.
Keywords: Tricalysia dubia; Rubiaceae; Ent-kaurane glucoside; Tricalysiosides H–O; X-ray analysis;