(-)-Epicatechin gallate accumulates in foamy macrophages in human atherosclerotic aorta: implication in the anti-atherosclerotic actions of tea catechins

The localization and target sites of tea catechins underlying their biological activity including anti-atherosclerotic activity have not yet been fully understood. To identify the target sites of catechins in vivo, we have developed a novel monoclonal antibody (mAb5A3) specific for (−)-epicatechin-3-gallate (ECg), one of the major tea catechins. The immunoreactive materials with mAb5A3 were detected in the human atherosclerotic lesions but not in the normal aorta, and were specifically localized in the macrophage-derived foam cells. In vitro experiments using macrophage-like cell lines also showed the significant accumulation of ECg in the cells. We also demonstrated that ECg could suppress the gene expression of a scavenger receptor CD36, a key molecule for foam cell formation, in macrophage cells. These results, for the first time, showed the target site of a tea component ECg in the aorta and might provide a mechanism for the anti-atherosclerotic actions of the catechins.     

Comparison of Proanthocyanidins and Related Compounds in Leaves and Leaf-Derived Cell Cultures of Ginkgo bioloba L., Pseudotsuga menziesii Franco, and Ribes sanguineum Pursh

Proanthocyanidins, flavan-3-ols, and their flavanoid precursors in leaves and leaf-derived callus and cell suspension cultures have been isolated and analyzed by high performance liquid chromatography with C₁₈ columns, paper chromatography, and by chemical and spectrophotometric methods. Cultures of Ginkgo biloba and Pseudotsuga menziesii (Douglas-fir) produced much greater amounts of proanthocyanidins than leaves per milligram dry weight. In cultures, however, the prodelphinidin component relative to that of procyanidins decreased; this was most pronounced in Pseudotsuga. In contrast, callus cultures of Ribes sanguineum accumulated proanthocyanidins in amounts about equal to those in intact leaves per milligram dry weight and the prodelphinidin content remained high. Although Ginkgo and Ribes leaves contained major amounts of flavan-3-ols and dimers with the 2,3-cis-stereochemistry, their cultures tended to synthesize 2,3-trans-isomers instead. Glycosides of flavanone and 3-hydroxyflavanone precursors accumulated in medium to high amounts on a dry weight basis in leaves and cultures of Ribes and Pseudotsuga, and the 3′-glycosidic linkage predominated when the latter species was cultured with 2,4-dichlorophenoxyacetic acid rather than naphthaleneacetic acid.     

Enhanced oxidation of flavan-3-ols and proanthocyanidin accumulation in water-stressed tea plants

(-)-Epicatechin (EC) and (-)-epigallocatechin gallate (EGCG), two major tea flavan-3-ols, have received attention in food science and biomedicine because of their potent antioxidant properties. In plants, flavan-3-ols serve as proanthocyanidin (PA) building blocks, and although both monomeric flavan-3-ols and PAs show antioxidant activity in vitro, their antioxidant function in vivo remains unclear. In the present study, EC quinone (ECQ) and EGCG quinone (EGCGQ), the oxidation products of EC and EGCG, increased up to 100- and 30-fold, respectively, in tea plants exposed to 19 days of water deficit. Oxidation of EC and EGCG preceded PAs accumulation in leaves, which increased from 35 to 53 mg gDW(-1) after 26 days of water deficit. Aside from the role monomeric flavan-3-ols may play in PAs biosynthesis, formation of ECQ and EGCGQ strongly negatively correlated with the extent of lipid peroxidation in leaves, thus supporting a protective role for these compounds in drought-stressed plants. Besides demonstrating flavonoid accumulation in drought-stressed tea plants, we show for the first time that EC and EGCG are oxidized to their respective quinones in plants in vivo.     

Heterogeneity of the interflavanyl bond in proanthocyanidins from natural sources lacking C-4 (C-ring) deoxy flavonoid nucleophiles

The proanthocyanidin pool in the floral kingdom usually involves the presence of carbon-carbon bonds linking predominantly flavan-3-ol constituent moieties. Such an ensemble of flavan-3-ol units originates via electrophilic aromatic substitution of flavan-4-yl carbocations (or their equivalents) derived from flavan-4-ols and/or flavan-3,4-diols and the nucleophilic centers of the m-oxygenated A-rings of flavan-3-ol nucleophiles. In the absence of these potent flavan-3-ol nucleophiles with their aptitude for the formation of carbon-carbon bonds, alternative centers emerge as participants in interflavanyl bond formation. Such a phenomenon is demonstrated for the distribution of various profisetinidin-, prorobinetinidin-, proguibourtinidin-, promelacacinidin- and proteracacinidin-type pro- and leuco-anthocyanidins in several southern hemisphere heartwood species.     

Challenges and complexity of functionality evaluation of flavan-3-ol derivatives

Flavan-3-ol derivatives are common plant-derived bioactive compounds. In particular, (–)-epigallocatechin-3-O-gallate shows various moderate biological activities without severe toxicity, and its health-promoting effects have been widely studied because it is a main ingredient in green tea and is commercially available at low cost. Although various biologically active flavan-3-ol derivatives are present as minor constituents in plants as well as in green tea, their biological activities have yet to be revealed, mainly due to their relative unavailability. Here, I outline the major factors contributing to the complexity of functionality studies of flavan-3-ol derivatives, including proanthocyanidins and oligomeric flavan-3-ols. I emphasize the importance of conducting structure-activity relationship studies using synthesized flavan-3-ol derivatives that are difficult to obtain from plant extracts in pure form to overcome this challenge. Further discovery of these minor constituents showing strong biological activities is expected to produce useful information for the development of functional health foods.     

Intermittent exposure to traces of green leaf volatiles triggers the production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in exposed plants

Intermittent exposure during a period of 3 weeks of undamaged Arabidopsis plants to trace amounts of volatiles emitted by freshly damaged Arabidopsis plants resulted in an increase of subsequent artificial-damage-induced production of (Z)-3-hexen-1-yl acetate and (Z)-3-hexen-1-ol in the exposed Arabidopsis plants when compared with Arabidopsis plants exposed to undamaged Arabidopsis plant volatiles (control plants). We previously showed that (Z)-3-hexen-1-yl acetate attracts a parasitic wasp, Cotesia glomerata. Thus, the induced production of this volatile explained our previously reported finding that, when artificially damaged, the exposed plants were more attractive to C. glomerata than control plants.     

Gastrophysa polygoni herbivory on Rumex confertus: single leaf VOC induction and dose dependent herbivore attraction/repellence to individual compounds

We report large induction (>65_fold increases) of volatile organic compounds (VOCs) emitted from a single leaf of the invasive weed mossy sorrel, Rumex confertus Willd. (Polygonaceae), by herbivory of the dock leaf beetle, Gastrophysa polygoni L. (Coleoptera: Chrysomelidae). The R. confertus VOC blend induced by G. polygoni herbivory included two green leaf volatiles ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate) and three terpenes (linalool, ß-caryophyllene, (E)-ß-farnesene). Uninjured leaves produced small constitutive amounts of the GLVs and barely detectable amounts of the terpenes. A Y-tube olfactometer bioassay revealed that both sexes of adult G. polygoni were attracted to (Z)-3-hexenal and (Z)-3-hexen-1-yl acetate at a concentration of 300 ng h⁻¹. No significant G. polygoni attraction or repellence was detected for any VOC at other concentrations (60 and 1500 ng h⁻¹). Yet, G. polygoni males and females were significantly repelled by (or avoided) at the highest test concentration (7500 ng h⁻¹) of both GLVs and (E)-ß-farnesene. Mated male and female G. polygoni might be attracted to injured R. confertus leaves, but might avoid R. confertus when VOC concentrations (especially the terpene (E)-ß-farnesene) suggest high overall plant injury from conspecifics, G. viridula, or high infestations of other herbivores that release (E)-ß-farnesene (e.g., aphids). Tests in the future will need to examine G. polygoni responses to VOCs emitted directly from uninjured (constitutive) and injured (induced) R. confertus, and examine whether R. confertus VOC induction concentrations increase with greater tissue removal on a single leaf and/or the number of leaves with feeding injury.     

Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria

The proanthocyanidin polymer fractions of the leaves of the forage legume Dorycnium rectum were analysed by acid catalysis with benzyl mercaptan, NMR and ES-MS. The results showed that D. rectum differs from other temperate proanthocyanidin-containing forage legumes in that the range of polymers extends up to very high degrees of polymerisation. Three fractions were characterised as low, medium, and high molecular weight proanthocyanidin fractions with mean degree of polymerisations of 10.3, 41 and 127, respectively. Epigallocatechin was the most abundant extension unit and the terminating flavan-3-ols comprised largely catechin and gallocatechin units in equal proportions. Formation of thiolyated dimer products showed the interflavan-linkages of the lower molecular weight proanthocyanidins to be predominantly C4-->C8 with a small amount of C4-->C6. ES-MS spectra distinguished lower from higher polymeric proanthocyanidins from M2- to M8(2)-. The antibacterial activity of proanthocyanidin fractions against pure cultures of microbes selected from the ruminal population to represent fibre degrading, proteolytic and hyper ammonia producing bacteria in broth culture was evaluated. The activity of proanthocyanidin fractions against Clostridium aminophilum, Butyrivibrio fibrisolvens and Clostridium proteoclasticum was significantly dependent on their structure but not so against Ruminococcus albus and Peptostreptococcus anaerobius. The latter observation was unique in that they were sensitive to all proanthocyanidin fractions evaluated, even at the lowest concentration (100 microg/ml). The results suggest the effects of the extractable proanthocyanidins on rumen microbes should be considered when evaluating an alternative proanthocyanidin-containing forage source for ruminants, such as D. rectum.     

Phytochemistry of the mopane,Colophospermum mopane

The polyphenolic pool of the heartwood of the mopane, Colophospermum mopane Kirk ex J. Leonard, exhibits extreme diversity and complexity. It comprises a variety of monomeric flavonoids, e.g. flavan-3-ols, flavan-3,4-diols including the mopanols and peltogynols, flavonols, dimeric proanthocyanidins, e.g. proguibourtinidins, profisetinidins, promopanidins, propeltogynidins, and a variety of profisetinidin-type triflavanoids. The di- and tri-meric proanthocyanidins are accompanied by several functionalized tetrahydropyrano- and hexahydrodipyrano-chromenes (phlobatannins) that originate from the bi- and tri-flavanoids, respectively, via rearrangement of the pyran heterocycle(s). Owing to the predominance of the 5-deoxy (A-ring) flavan-3-ols, the chain terminating moieties in the biosynthesis of oligo- and poly-meric proanthocyanidins, the di- and tri-meric analogs also exhibit diversity as far as interflavanyl bonding positions are concerned. Such heterogeneity results from the reduced nucleophilicity of the A-rings of 5-deoxy flavan-3-ols, compared to the A-rings of the 5-oxy analogs (catechins), hence permitting alternative centers to participate in proanthocyanidin formation. Biomimetic-type syntheses were extensively utilized to unequivocally establish constitution and absolute stereochemistry of both the conventional and pyran ring rearranged-type di- and tri-meric compounds. Comprehension of the intricate mechanistic and stereochemical course of the pyran ring rearrangement reactions also contributed significantly to unambiguous structure elucidations. The aerial parts of the mopane are rich in essential oils that comprise mainly alpha-pinene and limonene, which are presumably responsible for the strong turpentine odor of the pods. The leaves also contain significant concentrations of beta-sitosterol and stigmasterol which are apparently the source of sterols in various organs of the mopane moth, Gonimbrasia belina. Three diterpenes, dihydrogrindelic acid, labd-13E-en-15-oate and dihydrogrindelaldehyde are present in the bark and seeds, the latter compound exhibiting significant cytotoxicity against a human breast cancer cell line.     

Metabolomic Analysis Using Ultra-Performance Liquid Chromatography-Quadrupole-Time of Flight Mass Spectrometry (UPLC-Q-TOF MS) Uncovers the Effects of Light Intensity and Temperature under Shading Treatments on the Metabolites in Tea

To investigate the effect of light intensity and temperature on the biosynthesis and accumulation of quality-related metabolites, field grown tea plants were shaded by Black Net and Nano-insulating Film (with additional 2–4°C cooling effect) with un-shaded plants as a control. Young shoots were subjected to UPLC-Q-TOF MS followed by multivariate statistical analysis. Most flavonoid metabolites (mainly flavan-3-ols, flavonols and their glycosides) decreased significantly in the shading treatments, while the contents of chlorophyll, β-carotene, neoxanthin and free amino acids, caffeine, benzoic acid derivatives and phenylpropanoids increased. Comparison between two shading treatments indicated that the lower temperature under Nano shading decreased flavonols and their glycosides but increased accumulation of flavan-3-ols and proanthocyanidins. The comparison also showed a greater effect of temperature on galloylation of catechins than light intensity. Taken together, there might be competition for substrates between the up- and down-stream branches of the phenylpropanoid/flavonoid pathway, which was influenced by light intensity and temperature.     

Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight

Transgenic apple plants (Malus × domestica cv. ‘Holsteiner Cox’) overexpressing the Leaf Colour (Lc) gene from maize (Zea mays) exhibit strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins). Greenhouse plants investigated in this study exhibit altered phenotypes with regard to growth habit and resistance traits. Lc-transgenic plants show reduced size, transversal gravitropism of lateral shoots, reduced trichome development, and frequently reduced shoot diameter and abnormal leaf development with fused leaves. Such phenotypes seem to be in accordance with a direct or an indirect effect on polar-auxin-transport in the transgenic plants. Furthermore, leaves often develop necrotic lesions resembling hypersensitive response lesions. In tests, higher resistance against fire blight (caused by the bacterium Erwinia amylovora) and against scab (caused by the fungus Venturia inaequalis) is observed. These phenotypes are discussed with respect to the underlying altered physiology of the Lc-transgenic plants. The results are expected to be considered in apple breeding strategies.     

Physiological responses to flooding and light in two tree species native to the Amazonian floodplains

In Amazonian floodplains, plant survival is determined by adaptations and growth strategies to effectively capture sunlight and endure extended periods of waterlogging. By measuring gas exchange, quantum efficiency of photosystem 2 (PSII), and growth parameters, we investigated the combined effects of flooding gradients and light on two common evergreen floodplain tree species, the light-tolerant Cecropia latiloba and the shade-tolerant Pouteria glomerata. Individual plants were subjected to different combinations of light and flooding intensity in short-term and long-term experiments. Plants of C. latiloba lost all their leaves under total submersion treatments (plants flooded to apex and with reduced irradiance) and showed highest maximum assimilation rates (A_max) in not flooded, high light treatments (6.1 μmol CO₂ m⁻² s⁻¹). Individuals of P. glomerata showed similar patterns, with A_max increasing from 1.9 μmol CO₂ m⁻² s⁻¹ under total flooding to 7.1 μmol CO₂ m⁻² s⁻¹ in not flooded, high light treatments. During the long-term flooding experiment, quantum efficiency of PSII (F_v/F_m) of C. latiloba was not affected by partial flooding. In contrast, in P. glomerata F_v/F_m decreased to values below 0.73 after 120 days of total flooding. Moreover, total submergence led P. glomerata to reduce significantly light saturation point (LSP), as compared to C. latiloba. For both species morphological adjustments to long-term flooding, such as the production of adventitious roots, resulted in reduced total biomass, relative growth rate (RGR) and leaf mass ratio (LMR). Growth increase in C. latiloba seemed to be more limited by low-light than by flooding. Therefore, the predominant occurrence of this species is in open areas with high light intensities and high levels of inundation. In P. glomerata flooding induced high reductions of growth and photosynthesis, whereas light was not limiting. This species is more abundant in positions where irradiance is reduced and periods of submergence are slightly modest. We could show that the physiological requirements are directly responsible for the flooding (C. latiloba) and shade (P. glomerata) tolerance of the two species, which explains their local distribution in Amazonian floodplain forests.     

Molecular cloning and functional characterization of the anthocyanidin reductase gene from Vitis bellula

Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (?)-catechin and (?)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K _m , K _cat, V _max, and K _cat/K _m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols and PAs in the leaves of V. bellula.     

Root morphological and proteomic responses to growth restriction in maize plants supplied with sufficient N

The primary objective of this study was to better understand how root morphological alteration stimulates N uptake in maize plants after root growth restriction, by investigating the changes in length and number of lateral roots, ¹⁵NO₃⁻ influx, the expression level of the low-affinity Nitrate transporter ZmNrt1.1, and proteomic composition of primary roots. Maize seedlings were hydroponically cultured with three different types of root systems: an intact root system, embryonic roots only, or primary roots only. In spite of sufficient N supply, root growth restriction stimulated compensatory growth of remaining roots, as indicated by the increased lateral root number and root density. On the other hand, there was no significant difference in ¹⁵NO₃⁻ influx between control and primary root plants; neither in ZmNrt1.1 expression levels in primary roots of different treatments. Our data suggested that increased N uptake by maize seedlings experiencing root growth restriction is attributed to root morphological adaptation, rather than explained by the variation in N uptake activity. Eight proteins were differentially accumulated in embryonic and primary root plants compared to control plants. These differentially accumulated proteins were closely related to signal transduction and increased root growth.     

Salinity stress constrains photosynthesis in Fraxinus ornus more when growing in partial shading than in full sunlight: consequences for the antioxidant defence system

Background and Aims

A major challenge in plant ecophysiology is understanding the effects of multiple sub-optimal environmental conditions on plant performance. In most Mediterranean areas soil salinity builds up during the summer because of low availability of soil water coupled with hot temperatures. Although sunlight and soil salinity may strongly interact in determining a plant''s performance, this has received relatively little attention.

Methods

Two-year-old seedlings of Fraxinus ornus were grown outdoors in pots during a Mediterranean summer in either 45 % (shaded plants) or 100 % (sun plants) sunlight irradiance and were supplied with either deionized water or deionized water plus 75 mm NaCl. Morpho-anatomical traits, water and ionic relations, gas exchange and photosystem II performance, concentrations of individual carotenoids, activity of antioxidant enzymes, concentrations of ascorbic acid and individual polyphenols were measured in leaves. Leaf oxidative stress and damage were estimated by in vivo analysis of stable free radicals and ultrastructural analyses.

Key Results

Leaf concentrations of potentially toxic ions did not markedly differ in shaded or sun plants in response to salinity. Leaves of sun plants displayed superior water use efficiency compared with leaves of shaded plants, irrespective of salinity treatment, and had both better stomatal control and higher CO₂ carboxylation efficiency than leaves of shaded plants. In the salt-treated groups, the adverse effects of excess midday irradiance were greater in shade than in sun plants. The activity of enzymes responsible for detoxifying hydrogen peroxide decreased in shaded plants and increased in sun plants as a result of salinity stress. In contrast, the activity of guaiacol peroxidase and the concentration of phenylpropanoids increased steeply in response to salinity in shaded plants but were unaffected in sun plants.

Conclusions

It is concluded that salinity may constrain the performance of plants growing under partial shading more severely than that of plants growing under full sun during summer. The results suggest co-ordination within the antioxidant defence network aimed at detoxifying salt-induced generation of reactive oxygen species.