High Levels Of Proanthocyanidins Research Articles

Metabolite and Transcriptome Profiles of Proanthocyanidin Biosynthesis in the Development of Litchi Fruit.

The fruit of Litchi chinensis contains high levels of proanthocyanidins (PAs) in the pericarp. These substances can serve as substrates of laccase-mediated rapid pericarp browning after the fruit is harvested. In this study, we found that the major PAs in litchi pericarp were (-)-epicatechin (EC) and several procyanidins (PCs), primarily PC A2, B2, and B1, and the EC and the PC content decreased with the development of the fruit. RNA-seq analysis showed that 43 early and late structure genes related to flavonoid/PA biosynthesis were expressed in the pericarp, including five ANTHOCYANIDIN REDUCTASE (ANR), two LEUCOANTHOCYANIDIN REDUCTASE (LAR), and two ANTHOCYANIDIN SYNTHASE (ANS) genes functioning in the PA biosynthesis branch of the flavonoid pathway. Among these nine PA biosynthesis-related genes, ANR1a, LAR1/2, and ANS1 were highly positively correlated with changes in the EC/PC content, suggesting that they are the key PA biosynthesis-related genes. Several transcription factor (TF) genes, including MYB, bHLH, WRKY, and AP2 family members, were found to be highly correlated with ANR1a, LAR1/2, and ANS1, and their relevant binding elements were detected in the promoters of these target genes, strongly suggesting that these TF genes may play regulatory roles in PA biosynthesis. In summary, this study identified the candidate key structure and regulatory genes in PA biosynthesis in litchi pericarp, which will assist in understanding the accumulation of high levels of browning-related PA substances in the pericarp.

Proanthocyanidins Alleviate Cadmium Stress in Industrial Hemp (Cannabis sativa L.).

Industrial hemp (Cannabis sativa L.), an annual herbaceous cash crop, is widely used for the remediation of heavy metal-contaminated soils due to its short growth cycle, high tolerance, high biomass, and lack of susceptibility to transfer heavy metals into the human food chain. In this study, a significant increase in proanthocyanidins was found in Yunnan hemp no. 1 after cadmium stress. Proanthocyanidins are presumed to be a key secondary metabolite for cadmium stress mitigation. Therefore, to investigate the effect of proanthocyanidins on industrial hemp under cadmium stress, four experimental treatments were set up: normal environment, cadmium stress, proanthocyanidin treatment, and cadmium stress after pretreatment with proanthocyanidins. The phenotypes from the different treatments were compared. The experimental results showed that pretreatment with proanthocyanidins significantly alleviated cadmium toxicity in industrial hemp. The transcriptome and metabolome of industrial hemp were evaluated in the different treatments. Proanthocyanidin treatment and cadmium stress in industrial hemp mainly affected gene expression in metabolic pathways associated with glutathione metabolism, phenylpropanoids, and photosynthesis, which in turn altered the metabolite content in metabolic pathways of phenylalanine, vitamin metabolism, and carotenoid synthesis. The combined transcriptomic and metabolomic analysis revealed that proanthocyanidins mitigated cadmium toxicity by enhancing photosynthesis, secondary metabolite synthesis, and antioxidant synthesis. In addition, exogenous proanthocyanidins and cadmium ions acted simultaneously on EDS1 to induce the production of large amounts of salicylic acid in the plant. Finally, overexpression of CsANR and CsLAR, key genes for proanthocyanidins synthesis in industrial hemp, was established in Arabidopsis plants. The corresponding plants were subjected to cadmium stress, and the results showed that CsLAR transgenic plants were more tolerant to cadmium than the CsANR transgenic and wild-type Arabidopsis plants. The results showed that salicylic acid and jasmonic acid were increased in Arabidopsis overexpressing CsLAR compared to AT wild-type Arabidopsis, and levels of secondary metabolites were significantly higher in Arabidopsis overexpressing CsLAR than in AT wild-type Arabidopsis. These results revealed how proanthocyanidins alleviated cadmium stress and laid the foundation for breeding industrial hemp varieties with higher levels of proanthocyanidins and greater tolerance.

Responses of Lotus corniculatus to environmental change 3: The sensitivity of phenolic accumulation to growth temperature and light intensity and effects on tissue digestibility

Growth temperature and light intensity are major drivers of phenolic accumulation in Lotus corniculatus resulting in major changes in carbon partitioning which significantly affects tissue digestibility and forage quality. The response of plant growth, phenolic accumulation and tissue digestibility to light and temperature was determined in clonal plants of three genotypes of Lotus corniculatus (birdsfoot trefoil) cv Leo, with low, intermediate or high levels of proanthocyanidins (condensed tannins). Plants were grown from 10 °C to 30 °C, or at light intensities from 20 to 500 µm m-2 s-1. Plants grown at 25 °C had the highest growth rate and highest digestibility, whereas the maximum tannin concentration was found in plants grown at 15 °C. Approximately linear increases in leaf flavonol glycoside levels were found with increasing growth temperature in the low tannin genotype. Tannin hydroxylation increased with increasing growth temperature but decreased with increasing light intensity. The major leaf flavonols were kaempferol glycosides of which kaempferol-3-glucoside and kaempferol-3,7-dirhamnoside were the major components. Increases in both tannin and total flavonol concentrations in leaves were linearly related to light intensity and were preceded by a specific increase in the transcript level of a non-legume type chalcone isomerase. Changes in growth temperature and light intensity, therefore, result in major changes in the partitioning of carbon into phenolics, which significantly affects tissue digestibility and nutritional quality with a high correlation between tannin concentration and leaf digestibility.

CRISPR/Cas9-mediated functional recovery of the recessive rc allele to develop red rice.

SummaryRed rice contains high levels of proanthocyanidins and anthocyanins, which have been recognized as health‐promoting nutrients. The red coloration of rice grains is controlled by two complementary genes, Rc and Rd. The RcRd genotype produces red pericarp in wild species Oryza rufipogon, whereas most cultivated rice varieties produce white grains resulted from a 14‐bp frame‐shift deletion in the seventh exon of the Rc gene. In the present study, we developed a CRISPR/Cas9‐mediated method to functionally restore the recessive rc allele through reverting the 14‐bp frame‐shift deletion to in‐frame mutations in which the deletions were in multiples of three bases, and successfully converted three elite white pericarp rice varieties into red ones. Rice seeds from T1 in‐frame Rc lines were measured for proanthocyanidins and anthocyanidins, and high accumulation levels of proanthocyanidins and anthocyanidins were observed in red grains from the mutants. Moreover, there was no significant difference between wild‐type and in‐frame Rc mutants in major agronomic traits, indicating that restoration of Rc function had no negative effect on important agronomic traits in rice. Given that most white pericarp rice varieties are resulted from the 14‐bp deletion in Rc, it is conceivable that our method could be applied to most white pericarp rice varieties and would greatly accelerate the breeding of new red rice varieties with elite agronomic traits. In addition, our study demonstrates an effective approach to restore recessive frame‐shift alleles for crop improvement.

Proanthocyanidins in seed coat tegmen and endospermic cap inhibit seed germination in Sapium sebiferum.

Sapium sebiferum, an ornamental and bio-energetic plant, is propagated by seed. Its seed coat contains germination inhibitors and takes a long time to stratify for germination. In this study, we discovered that the S. sebiferum seed coat (especially the tegmen) and endospermic cap (ESC) contained high levels of proanthocyanidins (PAs). Seed coat and ESC removal induced seed germination, whereas exogenous application with seed coat extract (SCE) or PAs significantly inhibited this process, suggesting that PAs in the seed coat played a major role in regulating seed germination in S. sebiferum. We further investigated how SCE affected the expression of the seed-germination-related genes. The results showed that treatment with SCE upregulated the transcription level of the dormancy-related gene, gibberellins (GAs) suppressing genes, abscisic acid (ABA) biosynthesis and signalling genes. SCE decreased the transcript levels of ABA catabolic genes, GAs biosynthesis genes, reactive oxygen species genes and nitrates-signalling genes. Exogenous application of nordihydroguaiaretic acid, gibberellic acid, hydrogen peroxide and potassium nitrate recovered seed germination in seed-coat-extract supplemented medium. In this study, we highlighted the role of PAs, and their interactions with the other germination regulators, in the regulation of seed dormancy in S. sebiferum.

The R2R3-MYB TT2b and the bHLH TT8 genes are the major regulators of proanthocyanidin biosynthesis in the leaves of Lotus species

By exploiting interspecific hybrids and their progeny, we identified key regulatory and transporter genes intimately related to proanthocyanidin biosynthesis in leaves of Lotus spp. Proanthocyanidins (PAs), known as condensed tannins, are polymeric flavonoids enriching forage legumes of key nutritional value to prevent bloating in ruminant animals. Unfortunately, major forage legumes such as alfalfa and clovers lack PAs in edible tissues. Therefore, engineering the PA trait in herbage of forage legumes is paramount to improve both ecological and economical sustainability of cattle production system. Progresses on the understanding of genetic determinants controlling PA biosynthesis and accumulation have been mainly made studying mutants of Arabidopsis, Medicago truncatula and Lotus japonicus, model species unable to synthesize PAs in the leaves. Here, we exploited interspecific hybrids between Lotus corniculatus, with high levels of PAs in the leaves, and Lotus tenuis, with no PAs in these organs, and relative F2 progeny, to identify among candidate PA regulators and transporters the genes mainly affecting this trait. We found that the levels of leaf PAs significantly correlate with the expression of MATE1, the putative transporter of glycosylated PA monomers, and, among the candidate regulatory genes, with the expression of the MYB genes TT2a, TT2b and MYB14 and the bHLH gene TT8. The expression levels of TT2b and TT8 also correlated with those of all key structural genes of the PA pathways investigated, MATE1 included. Our study unveils a different involvement of the three Lotus TT2 paralogs to the PA trait and highlights differences in the regulation of this trait in our Lotus genotypes with respect to model species. This information opens new avenues for breeding bloat safe forage legumes.

Isolation and characterization of genes encoding leucoanthocyanidin reductase (FeLAR) and anthocyanidin reductase (FeANR) in buckwheat (Fagopyrum esculentum)

Proanthocyanidins (PAs) are a major group of flavonoids synthesized via the phenylpropanoid biosynthesis pathway, however the pathway has not been fully characterized in buckwheat. Anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR) are involved in the last steps of PA biosynthesis. To isolate the genes for these enzymes from buckwheat we performed PCR using degenerate primers and obtained cDNAs of ANR and LAR, which we designated FeANR and FeLAR1. A search for homologs in a buckwheat genome database with both sequences returned two more LAR sequences, designated FeLAR2 and FeLAR3. Linkage analysis with an F2 segregating population indicated that the three LAR loci were not genetically linked. We detected high levels of PAs in roots and cotyledons of buckwheat seedlings and in buds and flowers of mature plants. FeANR and FeLAR1–3 were expressed in most organs but had different expression patterns. Our findings would be useful for breeding and further analysis of PA synthesis and its regulation in buckwheat.

Antifungal activity of mango peel and seed extracts against clinically pathogenic and food spoilage yeasts

The antioxidant and antifungal (antiyeast) properties of mango (Mangifera indica) peel and seed by-products were investigated. Nine extracts were obtained using three cultivars and two extraction methods. Significant differences between cultivars and extraction methods were detected in their bioactive compounds and antioxidant activity. The antifungal property was determined using agar diffusion and broth micro-dilution assays against 18 yeast species of the genera Candida, Dekkera, Hanseniaspora, Lodderomyces, Metschnikowia, Pichia, Schizosaccharomyces, Saccharomycodes and Zygosaccharomyces. All mango extracts showed antifungal activity. The minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC) values were lower for seed than for peel extracts. MICs and MFCs ranged from values <0.1 to 5 and 5 to >30 mgGAE/mL, respectively. The multivariate analysis showed a relationship between antifungal activity, the capacity to inhibit lipid peroxidation and total phenol content. These properties were associated with high levels of proanthocyanidins, gallates and gallotannins in the extracts.

Survey of antioxidant capacity and phenolic composition of blueberry, blackberry, and strawberry in Nanjing

Berries are a good source of natural antioxidants. In the present study, the total antioxidant capacity and phenolic composition of three berry fruits (blueberry, blackberry, and strawberry) cultivated in Nanjing were investigated. Blueberry, with a Trolox equivalent antioxidant capacity (TEAC) value of 14.98 mmol Trolox/100 g dry weight (DW), exhibited the strongest total antioxidant capacity using both the 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) methods. Blueberry also had the highest total phenolic content (TPC, 9.44 mg gallic acid/g DW), total flavonoid content (TFC, 36.08 mg rutin/g DW), and total anthocyanidin content (TAC, 24.38 mg catechin/g DW). A preliminary analysis using high performance liquid chromatography (HPLC) showed that the blueberry, blackberry, and strawberry samples tested contained a range of phenolic acids (including gallic acid, protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, ellagic acid, and cinnamic acid) and various types of flavonoids (flavone: luteolin; flavonols: rutin, myricetin, quercetrin, and quercetin; flavanols: gallocatechin, epigallocatechin, catechin, and catechin gallate; anthocyanidins: malvidin-3-galactoside, malvidin-3-glucoside, and cyanidin). In particular, the blueberries had high levels of proanthocyanidins and anthocyanidins, which might be responsible for their strong antioxidant activities. These results indicate a potential market role for berries (especially blueberries) as a functional food ingredient or nutraceutical.

Modulation of Helicobacter pylori colonization with cranberry juice and Lactobacillus johnsonii La1 in children

Objective Probiotics and cranberry have been shown to inhibit Helicobacter pylori in vitro owing to bacteriocin production and high levels of proanthocyanidins, respectively. These effects have been confirmed in clinical trials with H. pylori–positive subjects. The aim of this study was to evaluate whether regular intake of cranberry juice and the probiotic Lactobacillus johnsonii La1 (La1) may result in an additive or synergistic inhibition of H. pylori in colonized children. Methods A multicentric, randomized, controlled, double-blind trial was carried out in 295 asymptomatic children (6–16 y of age) who tested positive for H. pylori by 13C-urea breath test (UBT). Subjects were allocated in four groups: cranberry juice/La1 (CB/La1), placebo juice/La1 (La1), cranberry juice/heat-killed La1 (CB), and placebo juice/heat-killed La1 (control). Cranberry juice (200 mL) and La1 product (80 mL) were given daily for 3 wk, after which a second UBT was carried out. A third UBT was done after a 1-mo washout in those children who tested negative in the second UBT. Results Two hundred seventy-one children completed the treatment period (dropout 8.1%). Helicobacter pylori eradication rates significantly differed in the four groups: 1.5% in the control group compared with 14.9%, 16.9%, and 22.9% in the La1, CB, and CB/La1 groups, respectively ( P < 0.01); the latter group showed a slight but not significant increase when compared with the other treated groups. The third UBT was carried out only in 19 of the 38 children who tested negative in the second UBT and H. pylori was detected in 80% of them. Conclusion These results suggest that regular intake of cranberry juice or La1 may be useful in the management of asymptomatic children colonized by H. pylori; however, no synergistic inhibitory effects on H. pylori colonization were observed when both foodstuffs were simultaneously consumed.

Proanthocyanidins (condensed tannins) and the use of leaves from Sesbania sesban and Sesbania goetzei as protein supplements

Three accessions of Sesbania sesban (10865, 15019 and 15036) and one accession of Sesbania goetzei (15007) were used as protein supplements for rams fed maize ( Zea mays) stover in a growth trial and a digestion trial which investigated the effects of proanthocyanidins (PAs) on protein digestion. Levels of soluble and insoluble PAs were highest in accessions 15036 and 15007, intermediate in 15019 and lowest in 10865. In a 70 days growth trial, 60 rams were fed these accessions of sesbania which provided three levels of crude protein (25, 50, and 75 g day −1). Intakes of maize stover by rams fed accessions 10865 and 15019 were significantly higher ( P < 0.05) than rams fed 15036 and 15007. Rate of gain for rams fed accessions 10865 and 15019 were significantly higher ( P < 0.01) than for rams fed accessions 15036 and 15007. In the digestion trial, 12 rams were fed the four sesbania accessions and three levels of sesbania in a replicated 3 × 3 Latin square design. Intake of maize stover decreased as the level of sesbania increased for rams fed accessions 15036 and 15007. High levels of PAs depressed the digestibility of neutral-detergent fiber (NDF) which decreased the intake of maize stover. The digestion coefficients for acid-detergent lignin (ADL) were negative for accessions 15019, 15036, and 15007. The digestion coefficients for neutral-detergent insoluble N (NDIN) were negative for rams fed accessions 15036 and 15007. Negative digestion coefficients resulted from more fecal excretion of these fractions than were consumed. PAs may form detergent insoluble complexes in the digestive tract. Rams fed accessions 15036 and 15007 had the highest excretion of total fecal N and fecal NDIN and the lowest urinary N. Rams fed accession 10865 had the highest urinary N excretion and the lowest excretion of total fecal N and fecal NDIN. Rams fed accession 15019 had the highest N retention. The intermediate level of PAs in accession 15019 may have beneficial effects on protein digestion. High levels of PAs in accessions 15036 and 15007 may have detrimental effects. Forage N had nutritionally non-uniform digestion in accessions 15019, 15036, and 15007. Fecal excretion of NDIN was significantly correlated to intake of soluble PAs ( r = 0.941) and insoluble PAs ( r = 0.789). The formation of detergent insoluble complexes in the digestive tract violate principles of detergent analysis but this deviation from ideal behavior can be used to determine the effects of PAs on protein digestion.

Gravimetric determination of soluble phenolics including tannins from leaves by precipitation with trivalent ytterbium

AbstractA method for the gravimetric determination of soluble phenolics based on precipitation with trivalent ytterbium is described. Solutions of ytterbium‐precipitated phenolics can be prepared in order to study u.v. absorbance, colorimetric assays for phenolics, protein precipitation and cellulase inhibition. The method was applied to mature leaves of 10 East African browse species in the diets of goats and camels. There was a large range in ytterbium‐precipitated phenolics (Yb‐ppt), from 50% dry matter (DM) in Acacia nilotica to less than 15% in Acacia brevispica and Cadaba farinosa. Results from u.v. absorbance and the Folin‐Ciocalteu assay on solutions of ytterbium‐precipitated phenolics indicated that ytterbium precipitates many types of phenolics including proanthocyanidins. The vanillin‐HCl and n‐butanol‐HCl assays for proanthocyanidins separated the samples into two groups with high or low levels. The group with high levels of proanthocyanidins had greater protein precipitation capacity and cellulase inhibition at similar amounts of Yb‐ppt. The results indicate that the method may be useful in nutritional studies involving browse and other forages. Gravimetric analysis avoids problems associated with colorimetric assays because it does not rely on the use of standards. The preparation of solutions of Yb‐precipitated phenolics allows direct comparison between amount and nutritional effects such as protein precipitation and enzyme inhibition.