[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: To determine the content of huperzine A in the root, stem and leaf of Huperzia serrata from western Hunan, and analyze the relationship between the distribution of huperzine A and the expression of lysine decarboxylase gene in different parts of huperzia serrata.
The content of huperzine A in the root, stem and leaf of Huperzia serrata was determinated by HPLC, of which the chromatographic column was Diamonsil C18 (250 mm x 4.6 mm,5 microm), mobile phase was methanol-0.08 mol/L CH3COONH4 with flow rate at 1.0 mL/min, column temperature at 25 degrees C and detection wavelength at 308 nm. The expression of lysine decarboxylase gene in different parts of Huperzia serrata was analyzed by semi-quantitative RT-PCR, the beta-actin gene was used as internal reference.
Huperzine A had good linear relationships within the range of 0.5 - 10.0 microg/mL, with the recovery rate of 102% and RSD 0.32%. The content of huperzine A in the root,stem and leaf of huperzia serrata was (118.35 +/- 0.77) microg/g and (411.09 +/- 2. 47) microg/g, (562.15 +/- 2.86) microg/g, respectively. Semi-quantitative RT-PCR results showed that lysine decarboxylase gene had nearly identical expression in the root, stem and leaf of Huperzia serrate.
The content of huperzine A changes with different parts of Huperzia serrata, lysine decarboxylase might be not the key enzyme to regulate the biosynthesis of huperzine A.
Zhong yao cai = Zhongyaocai = Journal of Chinese medicinal materials 03/2013; 36(3):361-4.
[Show abstract][Hide abstract] ABSTRACT: Proanthocyanidins (PAs) are fundamental nutritional metabolites in different types of grape products consumed by human beings. Although the biosynthesis of PAs in berry of Vitis vinifera has gained intensive investigations, the understanding of PAs in other Vitis species is limited. In this study, we report PA formation and characterization of gene expression involved in PA biosynthesis in leaves of V. bellula, a wild edible grape species native to south and south-west China. Leaves are collected at five developmental stages defined by sizes ranging from 0.5 to 5 cm in length. Analyses of thin layer chromatography (TLC) and high performance liquid chromatography-photodiode array detector (HPLC-PAD) show the formation of (+)-catechin, (-)-epicatechin, (+)-gallocatechin and (-)-epigallocatechin during the entire development of leaves. Analyses of butanol-HCl boiling cleavage coupled with spectrometry measurement at 550 nm show a temporal trend of extractable PA levels, which is characterized by an increase from 0.5 cm to 1.5 cm long leaves followed by a decrease in late stages. TLC and HPLC-PAD analyses identify cyanidin, delphinidin and pelargonidin produced from the cleavage of PAs in the butanol-HCl boiling, showing that the foliage PAs of V. bellula include three different types of extension units. Four cDNAs, which encode VbANR, VbDFR, VbLAR1 and VbLAR2, respectively, are cloned from young leaves. The expression patterns of VbANR and VbLAR2 but not VbLAR1 and VbDFR follow a similar trend as the accumulation patterns of PAs. Two cDNAs encoding VbMYBPA1 and VbMYB5a, the homologs of which have been demonstrated to regulate the expression of both ANR and LAR in V. vinifera, are also cloned and their expression profiles are similar to those of VbANR and VbLAR2. In contrast, the expression profiles of MYBA1 and 2 homologs involved in anthocyanin biosynthesis are different from those of VbANR and VbLAR2. Our data show that both ANR and LAR branches are involved in PA biosynthesis in leaves of V. bellula.
[Show abstract][Hide abstract] ABSTRACT: Proanthocyanidins (PAs) are oligomers or polymers of plant flavan-3-ols and are important to plant adaptation in extreme environmental conditions. The characterization of anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) has demonstrated the different biogenesis of four stereo-configurations of flavan-3-ols. It is important to understand whether ANR and the ANR pathway widely occur in the plant kingdom. Here, we report an integrated approach to demonstrate the ANR pathway in plants. This includes different methods to extract native ANR from different tissues of eight angiosperm plants (Lotus corniculatus, Desmodium uncinatum, Medicago sativa, Hordeum vulgare, Vitis vinifera, Vitis bellula, Parthenocissus heterophylla, and Cerasus serrulata) and one fern plant (Dryopteris pycnopteroides), a general enzymatic analysis approach to demonstrate the ANR activity, high-performance liquid chromatography-based fingerprinting to demonstrate (-)-epicatechin and other flavan-3-ol molecules, and phytochemical analysis of PAs. Results demonstrate that in addition to leaves of M. sativa, tissues of other eight plants contain an active ANR pathway. Particularly, the leaves, flowers and pods of D. uncinatum, which is a model plant to study LAR and the LAR pathways, are demonstrated to express an active ANR pathway. This finding suggests that the ANR pathway involves PA biosynthesis in D. uncinatum. In addition, a sequence BLAST analysis reveals that ANR homologs have been sequenced in plants from both gymnosperms and angiosperms. These data show that the ANR pathway to PA biosynthesis occurs in both seed and seedless vascular plants.
[Show abstract][Hide abstract] ABSTRACT: A Gram-stain-positive, endospore-forming, motile, catalase- and oxidase-positive, aerobic, rod-shaped bacterium, designated strain JSM 081008(T), was isolated from non-saline forest soil in China. Strain JSM 081008(T) was able to grow with 0-20% (w/v) NaCl, at pH 6.0-10.5 and at 10-45 degrees C; optimum growth was observed with 2-5% (w/v) NaCl, at pH 7.0-8.0 and at 30-35 degrees C. The peptidoglycan type was A1alpha linked directly through L-Lys. The major cellular fatty acids (>10% of the total) were anteiso-C15:0, iso-C15:0, anteiso-C17:0 and C16:0. The predominant respiratory quinone was menaquinone 7 and the genomic DNA G + C content of the strain was 42.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 081008(T) should be assigned to the genus Jeotgalibacillus and was related most closely to the type strains of Jeotgalibacillus alimentarius (sequence similarity 99.4%) and Jeotgalibacillus salarius (97.0%), followed by Jeotgalibacillus campisalis (95.4%) and Jeotgalibacillus marinus (95.2%). The combination of phylogenetic analysis, DNA-DNA relatedness values, phenotypic characteristics and chemotaxonomic data supports the view that strain JSM 081008(T) represents a novel species of the genus Jeotgalibacillus, for which the name Jeotgalibacillus soli sp. nov. is proposed. The type strain is JSM 081008(T) (=DSM 22174(T) = KCTC 13528(T)). An emended description of the genus Jeotgalibacillus is also presented.
Antonie van Leeuwenhoek 10/2010; 98(3):415-21. DOI:10.1007/s10482-010-9455-z · 1.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A Gram-positive, endospore-forming, catalase- and oxidase-positive, motile, rod-shaped, aerobic bacterium, designated strain JSM 079157(T), was isolated from surface seawater off the coastline of Naozhou Island in South China Sea. The organism was able to grow with 1-15% (w/v) total salts (optimum, 4-7%), and at pH 6.0-10.0 (optimum, pH 7.5) and 10-45 degrees C (optimum, 30 degrees C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7, and the polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The major cellular fatty acids were anteiso-C(15:0) (45.1%) and anteiso-C(17:0) (16.2%), and the DNA G + C content was 39.5 mol%. A phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 079157(T) should be assigned to the genus Virgibacillus, being related most closely to the type strains of Virgibacillus litoralis (97.4% sequence similarity), Virgibacillus necropolis (97.3%) and Virgibacillus carmonensis (97.1%). These four strains formed a distinct subcluster in the phylogenetic tree. The levels of DNA-DNA relatedness between the new isolate and the type strains of V. litoralis, V. necropolis and V. carmonensis were 30.4, 19.3 and 12.6%, respectively. The results of the phylogenetic analysis, combined with DNA-DNA relatedness data, phenotypic characteristics and chemotaxonomic information, support the suggestion that strain JSM 079157(T) represents a new species of the genus Virgibacillus, for which the name Virgibacillus zhanjiangensis sp. nov. is proposed. The type strain is JSM 079157(T) (=DSM 21084(T) = KCTC 13227(T)).
Antonie van Leeuwenhoek 09/2009; 96(4):645-52. DOI:10.1007/s10482-009-9381-0 · 1.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A moderately halophilic, Gram-positive, catalase- and oxidase-positive, rod-shaped, aerobic bacterium, designated strain JSM 071077(T), was isolated from a subterranean brine sample collected from a salt mine in Hunan Province, China. Cells were motile by means of peritrichous flagella and formed ellipsoidal endospores lying in subterminal swollen sporangia. Strain JSM 071077(T) was able to grow with 2-25% (w/v) total salts (optimum, 5-10%), at pH 6.0-10.0 (optimum, pH 7.5) and 10-40 degrees C (optimum, 25-30 degrees C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The predominant menaquinone was MK-7, and the major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0) and iso-C(15:0). The genomic DNA G+C content was 41.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons revealed that strain JSM 071077(T) should be assigned to the genus Halobacillus, being related most closely to the type strain of Halobacillus naozhouensis (98.8% sequence similarity), and the two strains formed a distinct subline in the neighbor-joining, minimum-evolution and maximum-parsimony phylogenetic trees. The sequence similarities between the novel isolate and the type strains of other recognized Halobacillus species ranged from 97.6% (with Halobacillus alkaliphilus) to 95.2% (with Halobacillus kuroshimensis). The results of the phylogenetic analyses, combined with DNA-DNA relatedness data, phenotypic characteristics and chemotaxonomic information, support that strain JSM 071077(T) represents a new species of the genus Halobacillus, for which the name Halobacillus hunanensis sp. nov. is proposed. The type strain is JSM 071077(T) (=DSM 21184(T) = KCTC 13235(T)).
Antonie van Leeuwenhoek 08/2009; 96(4):497-504. DOI:10.1007/s10482-009-9365-0 · 1.81 Impact Factor