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.
Saxifraga L. is the largest genus in Saxifragaceae and a characteristic component of the herbaceous flora of the temperate and alpine mountains in the Northern Hemisphere. Section Irregulares is a small group of 15-20 species, representing one of the early-diverged lineages in the genus characterized with unique zygomorphic flowers. We used both nuclear internal transcribed spacer and chloroplast DNA regions (psbA-trnH, trnL-F, and matK) to reconstruct its species relationships, estimate divergence times, and infer its historical biogeography. Our phylogenetic results corroborate the monophyly of sect. Irregulares and its sister relationship to sect. Heterisia from North America. The section was well resolved into two lineages corresponding to their morphological features and biogeographic distributions. One represents ser. Stoloniferae including S. stolonifera Curtis and S. nipponica Makino with long-creeping stolons/rhizomes and small petals with spots and the other comprises the remaining taxa (ser. Rufescentes) which lack long-creeping rhizomes. Spots on leaves (abaxially spotted vs. abaxially without spots) and spots on petals (without spots vs. with spots) are inferred to be phylogenetically informative within ser. Rufescentes. Divergence time estimates and ancestral area analysis suggested a western North American origin of sect. Irregulares with migration into East Asia by way of the Bering land bridge in the Middle Oligocene. The development of drying and desertification belt in the late Miocene could have played an important role in the subsequent restriction and separation of the north and south lineages within eastern Asia.
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.
Background Beverages prepared by fermenting plants have a long history of use for medicinal, social, and ritualistic purposes around the world. Socio-linguistic groups throughout China have traditionally used plants as fermentation starters (or koji ) for brewing traditional rice wine. The objective of this study was to evaluate traditional knowledge, diversity, and values regarding plants used as starters for brewing glutinous rice wine in the Dong communities in the Guizhou Province of China, an area of rich biological and cultural diversity. Methods Semi-structured interviews were administered for collecting ethnobotanical data on plants used as starters for brewing glutinous rice wine in Dong communities. Field work was carried out in three communities in Guizhou Province from September 2017 to July 2018. A total of 217 informants were interviewed from the villages. Results A total of 60 plant species were identified to be used as starters for brewing glutinous rice wine, belonging to 58 genera in 36 families. Asteraceae and Rosaceae are the most represented botanical families for use as a fermentation starter for rice wine with 6 species respectively, followed by Lamiaceae (4 species); Asparagaceae, Menispermaceae, and Polygonaceae (3 species respectively); and Lardizabalaceae, Leguminosae, Moraceae, Poaceae, and Rubiaceae (2 species, respectively). The other botanical families were represented by one species each. The species used for fermentation starters consist of herbs (60.0%), shrubs (23.3%), climbers (10.0%), and trees (6.7%). The parts used include the root (21.7%), leaf (20.0%), and the whole plant (16.7%). Findings indicate a significant relationship between knowledge of plants used as fermentation starters with age ( P value < 0.001) and educational status ( P value = 0.004) but not with gender ( P value = 0.179) and occupation ( P value = 0.059). The species that are most used by informants include Pueraria lobata var. montana (Lour.) van der Maesen (UV = 1.74; Leguminosae), Actinidia eriantha Benth. (UV = 1.51; Actinidiaceae), Oryza sativa L. var. glutinosa Matsum (UV = 1.5; Poaceae). Conclusion This study highlights that while most of the Dong informants continue to use a diverse range of plants as a fermentation starter for brewing glutinous rice wine, knowledge of these plants is being lost by the younger generations. Documentation of traditional ethnobotanical knowledge and outreach is thus needed to conserve biocultural diversity in the rural Dong communities in southern China.
This paper aims to avoid the tampering or leakage of sensitive data in a distance education platform. To this end, an intelligent identity authentication model was proposed to realize continuous user authentication, and high security infrastruc-ture was designed for distance education platform based on the proposed model. The experimental evaluation shows that the proposed infrastructure based on pri-vate cloud can greatly improve the security of distance education platform. Suf-fice it to say that the research findings provide new insights into the security of distance education.
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