Targeted regulation of 5-aminolevulinic acid enhances flavonoids, anthocyanins and proanthocyanidins accumulation in Vitis davidii callus.

BACKGROUND: Spine grape (Vitis davidii) is a promising source of high-quality anthocyanins, with vast potential for application in food, pharmaceutical, and cosmetic industries. However, their availability is limited by resource constraints. Plant cell culture has emerged as a valuable approach for anthocyanin production and serves as an ideal model to investigate the regulation of anthocyanin biosynthesis. Elicitors are employed to achieve targeted enhancement of anthocyanin biosynthesis. The present study investigated the impact of 5-aminolevulinic acid (ALA) as an elicitor on the accumulation of anthocyanins and flavonoids during spine grape callus growth. Specifically, we examined the effects of ALA on anthocyanin and its component accumulation in callus, and biosynthetic anthocyanin gene expression. RESULTS: ALA at 25 µg/L increased the biomass of spine grape callus. ALA induction enhanced the levels of flavonoids, anthocyanins and proanthocyanidins in callus, with maximum values reaching 911.11 mg/100 g DW, 604.60 mg/100 g DW, and 5357.00 mg/100 g DW, respectively, after callus culture for 45 days. Notably, those levels were 1.47-, 1.93- and 1.83-fold higher than controls. ALA induction modulated the flavonoid profile, and among 97 differential flavonoid metabolites differing from controls, 77 were upregulated and 20 were downregulated. Six kinds of anthocyanins, namely cyanidin (8), delphinidin (6), peonidin (5), malvidin (4), petunidin (3) and pelargonidin (3), were detected in callus, with peonidin most abundant. Compared with controls, anthocyanin components were increased in ALA-treated callus. The key genes PAL1, PAL2, PAL4, CHI, CHS3, F3'H, F3H, FLS, DFR, UFGT, MYBA1, LDOX, OMT3, GT1 and ACT involved in anthocyanin biosynthesis were upregulated following ALA treatment, resulting in anthocyanin accumulation. CONCLUSION: This study revealed a novel mode of ALA-mediated promotion of plant anthocyanin biosynthesis and accumulation at the cellular level, and a strategy for enhancing anthocyanin content in spine grape callus. The findings advance commercial-scale production of anthocyanins via spine grape callus culture. we also explored the accumulation patterns of flavonoids and anthocyanins under ALA treatment. Augmentation of anthocyanins coincided with elevated expression levels of most genes involved in anthocyanin biosynthesis within spine grape callus following ALA treatment.

Assessment of antioxidant properties in selected pigmented and non-pigmented rice (Oryza sativa L.) germplasm and determination of its association with Rc gene haplotypes.

BACKGROUND: Antioxidant properties of rice provide various health benefits due to its ability to inhibit cellular oxidation. Antioxidant content of rice is known to be linked with the pericarp pigmentation. The Rc gene of rice (Os07g0211500) codes for a basic helix-loop-helix (bHLH) protein, acting as a transcriptional factor in regulating proanthocyanidin biosynthesis. The current study was carried out to evaluate the variation of antioxidant properties in a selected panel of rice accessions and assess the possibility of using haplotypes defined based on the Rc gene to predict pericarp pigmentation and antioxidant content in rice. RESULTS: Thirty-two rice accessions were evaluated for grain pericarp colour and antioxidant properties; total phenolic content (TPC), total flavonoids (TFC), proanthocyanidins (PAC) and radical scavenging activity (RSA). The parameters TPC, TFC and PAC showed significant positive correlation with RSA (r > 0.69; P < 0.01). The study panel showed a wide variation for antioxidant properties and rice accessions such as Sudu Heenati, Deweraddiri, Madathawalu, Masuran, Ld 368, At 311, Kalu Heenati, Bw 272-6B, Pokkali, At 362 and Wanni Dahanala exhibited profound potential with respect to antioxidant properties. Based on three-target sites previously reported as critical for the function of the coded bHLH protein (an A/C SNP at 1,353-bp, a 1-bp insertion/deletion at 1,388-bp, and a 14-bp insertion/deletion at 1,408-1,421-bp positioned in the mRNA corresponding to the exon 6 of rice Rc gene), three haplotypes were defined (H1-H3). Pigmentation of the rice pericarp could be successfully explained based on the defined haplotypes (H1 (C/G/+): red, and H2 (A/G/+) and H3 (C/G/-): white), and the H1 haplotype corresponded to a significantly (P < 0.05) higher TPC, TFC, PAC and RSA compared to the other haplotypes. CONCLUSIONS: The studied rice accessions showed a significant variation with respect to antioxidant properties. Haplotype H1 defined based on the three-target sites in the exon 6 of Rc gene can detect rice accessions with red pigmented pericarp and high antioxidant properties effectively. Hence, its use can be recommended as an alternative to biochemical assays for screening during rice breeding programs.

Sex Differences in the Absorption, Disposition, Metabolism, and Excretion of Grape Seed Proanthocyanidins in Prepubescent Rats.

SCOPE: The absorption, disposition, metabolism, and excretion (ADME) of phenolic compounds are key factors in determining their bioactivity. The group demonstrates that the ADME of a Grape Seed Proanthocyanidin Extract (GSPE) depends on sex in adult rats and specifically, methylated metabolites are only quantified in brain male adult rats. The aim of this study is to determine whether these differences exist before puberty. METHODS AND RESULTS: Prepubescent 4-week-old male and female Wistar rats are administered GSPE at a dose of 1000 mg kg-1. Plasma, liver, mesenteric white adipose tissue (MWAT), brain, and kidneys are extracted excised 2 h after GSPE administration, and the PAs metabolite profile is studied by HPLC-ESI-MS/MS. Moreover, plasma estradiol and brain and liver catechol-O-methyltransferase (COMT) protein levels are also studied. Results showed that there are no differences in plasma and brain among sexes and only differences are observed in liver, MWAT, and kidney with individual metabolites. This agrees with the lack of differences in estradiol and COMT levels among sexes. However, the ADME of PAs metabolites is higher in male rats. CONCLUSIONS: The results demonstrate lack of sex-dependence in metabolite profile in prepubescent rats, suggesting that sex differences in the metabolism of GSPE occur due to puberty.

Functional characterization of TrGSTF15, a glutathione S-transferase gene family member, on the transport and accumulation of anthocyanins and proanthocyanidins in Trifolium repens.

Anthocyanins and proanthocyanidins (PAs) are important secondary metabolites in plants, high contents of which are an important goal for quality breeding of white clover (Trifolium repens). However, the involvement of glutathione S-transferase (GST) in the transport of anthocyanins and PAs remains unexplored in white clover. This study identified 153 different TrGSTs in white clover. At the transcriptional level, compared to other TrGSTFs, TrGSTF10 and TrGSTF15 are highly expressed in the 'Purple' white clover, and they may work with the anthocyanin biosynthesis structural genes CHS and CHI to contribute to pigment buildup in white clover. Subcellular localization confirmed that TrGSTF10 and TrGSTF15 are located in the cytoplasm. Additionally, molecular docking experiments showed that TrGSTF10 and TrGSTF15 have similar binding affinity with two flavonoid monomers. Overexpression of TrGSTF15 complemented the deficiency of anthocyanin coloring and PA accumulation in the Arabidopsis tt19 mutant. The initial findings of this research indicate that TrGSTF15 encodes an important transporter of anthocyanin and PA in white clover, thus providing a new perspective for the further exploration of related transport and regulatory mechanisms.

Characterization of a longan pericarp browning related peroxidase with a focus on its role in proanthocyanidin and lignin polymerization.

The longan pericarp turns brown dramatically after harvesting, but the mechanism is not well understood. In this work, two peroxidases were purified from longan pericarp and found to be identical to the class III peroxidases PRX53-2 and PRX53-3. In vitro, PRX53-2/3 catalyzed the browning of several pericarp abundant proanthocyanidin and lignin monomers, such as (-)-epicatechin (EC), (+)-catechin (CT) and coniferyl alcohol (ConA). PRX53-2 was upregulated and highly-expressed, while PRX53-3 was expressed at low levels after harvesting; thus, PRX53-2 was considered a browning-related gene. The reaction with both proanthocyanidin and lignin presented a greater degree of brown coloration compared to the single substrate reactions. Several procyanidins isomers, EC-ConA and CT-ConA were detected in the double-substrate reaction. These results not only demonstrate that the effects of PRX53-2 on proanthocyanidin and lignin polymerization may be crucial for longan pericarp browning, but also help in developing new strategies or preservatives to delay pericarp browning.

The functions of laccase gene GhLAC15 in fiber colouration and development in brown-colored cotton.

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.

CaLAP1 and CaLAP2 orchestrate anthocyanin biosynthesis in the seed coat of Cicer arietinum.

MAIN CONCLUSION: Our findings shed light on the regulation of anthocyanin and proanthocyanidin biosynthesis in chickpea seed coats. Expression of R2R3-MYB transcription factors CaLAP1 and CaLAP2 enhanced the anthocyanins and proanthocyanidins content in chickpea. The seed coat color is a major economic trait in leguminous crop chickpea (Cicer arietinum). Anthocyanins and proanthocyanidins (PAs) are two classes of flavonoids that mainly contribute to the flower, seed coat and color of Desi chickpea cultivars. Throughout the land plant lineage, the accumulation of anthocyanins and PAs is regulated by MYB and bHLH transcription factors (TFs), which form an MBW (MYB, bHLH, and WD40) complex. Here, we report two R2R3-MYB TFs in chickpea belonging to the anthocyanin-specific subgroup-6, CaLAP1 (Legume Anthocyanin Production 1), and CaLAP2 (Legume Anthocyanin Production 2), which are mainly expressed in the flowers and developmental stages of the seeds. CaLAP1 and CaLAP2 interact with TT8-like CabHLH1 and WD40, forming the MBW complex, and bind to the promoter sequences of anthocyanin- and PA biosynthetic genes CaCHS6, CaDFR2, CaANS, and CaANR, leading to anthocyanins and PA accumulation in the seed coat of chickpea. Moreover, these CaLAPs partially complement the anthocyanin-deficient phenotype in the Arabidopsis thaliana sextuple mutant seedlings. Overexpression of CaLAPs in chickpea resulted in significantly higher expression of anthocyanin and PA biosynthetic genes leading to a darker seed coat color with higher accumulation of anthocyanin and PA. Our findings show that CaLAPs positively modulate anthocyanin and PA content in seed coats, which might influence plant development and resistance to various biotic and abiotic stresses.

Genome-wide identification of R2R3-MYB family in Eriobotrya japonica and functional analysis of EjMYB5 involved in proanthocyanidin biosynthesis.

Loquat (Eriobotrya japonica Lindl.) is a popular fruit and medicinal plant. Proanthocyanidins (PAs), as one of the main types of flavonoids, are the key components of loquat fruit quality and medicinal properties. However, the identification of transcription factors (TFs) involved in PA accumulation in loquat remains limited. R2R3-MYB TFs play key regulatory role in PA accumulation in plants. In this study, 190 R2R3-MYB TFs were identified in loquat genome. Combined with transcriptome data, R2R3-MYB TF EjMYB5 involved in PA accumulation in loquat was isolated. EjMYB5 was transcriptional activator localized to nucleus. Expression of EjMYB5 was closely related to PA accumulation in loquat fruits. Heterogenous overexpression of EjMYB5 in tomato (Solanum lycopersicum) inhibited anthocyanin accumulation and promoted PA accumulation. Additionally, transient overexpression of EjMYB5 in tobacco (Nicotiana benthamiana) leaves promoted PA accumulation by upregulating flavonoid biosynthesis genes (NtDFR, NtANS, and NtLAR). Transcriptome analysis of EjMYB5-overexpressing tomato fruits suggested that EjMYB5 was involved in several biological pathways, including lipid metabolism, MAPK signaling, phenylpropanoid biosynthesis, and flavonoid biosynthesis. Collectively, our findings provided basic data for further analysis the function of R2R3-MYB TFs in loquat, and revealed that EjMYB5 functioned as PA accumulation in loquat.

The R2R3-MYB transcription factor PgTT2 from Panax ginseng interacts with the WD40-repeat protein PgTTG1 during the regulation of proanthocyanidin biosynthesis and the response to salt stress.

Proanthocyanidins (PAs) are flavonoid compounds with important defensive roles in plants. The application of PAs in industries such as the pharmaceutical industry has led to increased interest in enhancing their biosynthesis. In Arabidopsis thaliana, PAs are biosynthesized under the regulation of an R2R3-MYB transcription factor TRANSPARENT TESTA 2 (TT2), which can interact with other proteins, including TRANSPARENT TESTA GLABRA 1 (TTG1), while also regulating a plant's response to abiotic stressors. However, the regulation of PA biosynthesis in the high-value medicinal plant Panax ginseng (ginseng) has not yet been studied. Understanding the mechanism of PAs biosynthesis regulation in ginseng may be helpful in increasing the plant's range of pharmacological applications. This study found that the overexpression of PgTT2 increased PA biosynthesis by an average of 67.3% in ginseng adventitious roots and 50.5% in arabidopsis seeds. Furthermore, transgenic arabidopsis plants overexpressing PgTT2 produced increased reactive oxygen species (ROS) scavenging ability by influencing abscisic acid synthesis and signaling. However, under high salinity stress, seed germination and growth rate of seedlings were decreased. An expression analysis of plants facing salt stress revealed increased transcripts of an ABA biosynthetic gene, NCED3, and ABA signaling genes ABI5 and ABI3. Moreover, the PgTT2 protein showed a direct interaction with PgTTG1 in yeast two-hybrid assays. This study therefore reveals novel information on the transcriptional regulation of PA production in ginseng and shows how PgTT2 influences the ABA response pathway to regulate responses to ROS and salt stress.

The HD-ZIP IV transcription factor GLABRA2 acts as an activator for proanthocyanidin biosynthesis in Medicago truncatula seed coat.

Proanthocyanidins (PAs), a group of flavonoids, are found in leaves, flowers, fruits, and seed coats of many plant species. PAs are primarily composed of epicatechin units in the seed coats of the model legume species, Medicago truncatula. It can be synthesized from two separate pathways, the leucoanthocyanidin reductase (MtLAR) pathway and the anthocyanidin synthase (MtANS) pathway, which produce epicatechin through anthocyanidin reductase (MtANR). These pathways are mainly controlled by the MYB-bHLH-WD40 (MBW) ternary complex. Here, we characterize a class IV homeodomain-leucine zipper (HD-ZIP IV) transcription factor, GLABRA2 (MtGL2), which contributes to PA biosynthesis in the seed coat of M. truncatula. Null mutation of MtGL2 results in dark brown seed coat, which is accompanied by reduced PAs accumulation and increased anthocyanins content. The MtGL2 gene is predominantly expressed in the seed coat during the early stages of seed development. Genetic and molecular analyses indicate that MtGL2 positively regulates PA biosynthesis by directly activating the expression of MtANR. Additionally, our results show that MtGL2 is strongly induced by the MBW activator complexes that are involved in PA biosynthesis. Taken together, our results suggest that MtGL2 acts as a novel positive regulator in PA biosynthesis, expanding the regulatory network and providing insights for genetic engineering of PA production.

Gut microbiota-mediated polyphenol metabolism is restrained by parasitic whipworm infection and associated with altered immune function in mice.

Polyphenols are phytochemicals commonly found in plant-based diets which have demonstrated immunomodulatory and anti-inflammatory properties. However, the interplay between polyphenols and pathogens at mucosal barrier surfaces has not yet been elucidated in detail. Here, we show that proanthocyanidin (PAC) polyphenols interact with gut parasites to influence immune function and gut microbial-derived metabolites in mice. PAC intake inhibited mastocytosis during infection with the small intestinal roundworm Heligmosomoides polygyrus, and altered the host tissue transcriptome at the site of infection with the large intestinal whipworm Trichuris muris, with a notable enhancement of type-1 inflammatory and interferon-driven gene pathways. In the absence of infection, PAC intake promoted the expansion of Turicibacter within the gut microbiota, increased fecal short chain fatty acids, and enriched phenolic metabolites such as phenyl-γ-valerolactones in the cecum. However, these putatively beneficial effects were reduced in PAC-fed mice infected with T. muris, suggesting concomitant parasite infection can attenuate gut microbial-mediated PAC catabolism. Collectively, our results suggest an inter-relationship between a phytonutrient and infection, whereby PAC may augment parasite-induced inflammation (most prominently with the cecum dwelling T. muris), and infection may abrogate the beneficial effects of health-promoting phytochemicals.

Heterogeneity of transport and metabolism of Tormentillae rhizoma constituents across human intestinal epithelium cellular model.

Tormentilla erecta (L.) Raeusch is a widespread plant in Europe and Western Asia. Its rhizomes (Tormentilae rhizoma) are the main ingredient of herbal alcoholic beverages and can be used as a natural preservative in beer production. Apart from its unique taste qualities, therapeutic properties in gastrointestinal tract ailments are attributed to the tincture obtained from Tormentillae rhizoma. The presented research aimed to determine the mutual relationship between the components of Tormentillae tincture, present in popular alcoholic beverages, and intestinal epithelium (Caco-2 cell monolayers). A comprehensive qualitative and quantitative analysis of the tincture was performed, including the determination of condensed and hydrolyzable tannins as well as triterpenoids (UHPLC-DAD-MS/MS). Incubation of the tincture with Caco-2 monolayers has shown that only triterpenes pass through the monolayer, while condensed tannins are mainly bound to the monolayer surface. Ellagic acid derivatives were the only components of the Tormentillae tinctura being metabolized by cell monolayers to the compounds not previously described in the literature, which may be crucial in the treatment of intestinal diseases with inflammatory background.

Effects of UV-B Radiation Exposure on Transgenerational Plasticity in Grain Morphology and Proanthocyanidin Content in Yuanyang Red Rice.

The effect of UV-B radiation exposure on transgenerational plasticity, the phenomenon whereby the parental environment influences both the parent's and the offspring's phenotype, is poorly understood. To investigate the impact of exposing successive generations of rice plants to UV-B radiation on seed morphology and proanthocyanidin content, the local traditional rice variety 'Baijiaolaojing' was planted on terraces in Yuanyang county and subjected to enhanced UV-B radiation treatments. The radiation intensity that caused the maximum phenotypic plasticity (7.5 kJ·m-2) was selected for further study, and the rice crops were cultivated for four successive generations. The results show that in the same generation, enhanced UV-B radiation resulted in significant decreases in grain length, grain width, spike weight, and thousand-grain weight, as well as significant increases in empty grain percentage and proanthocyanidin content, compared with crops grown under natural light conditions. Proanthocyanidin content increased as the number of generations of rice exposed to radiation increased, but in generation G3, it decreased, along with the empty grain ratio. At the same time, biomass, tiller number, and thousand-grain weight increased, and rice growth returned to control levels. When the offspring's radiation memory and growth environment did not match, rice growth was negatively affected, and seed proanthocyanidin content was increased to maintain seed activity. The correlation analysis results show that phenylalanine ammonialyase (PAL), cinnamate-4-hydroxylase (C4H), dihydroflavonol 4-reductase (DFR), and 4-coumarate:CoA ligase (4CL) enzyme activity positively influenced proanthocyanidin content. Overall, UV-B radiation affected transgenerational plasticity in seed morphology and proanthocyanidin content, showing that rice was able to adapt to this stressor if previous generations had been continuously exposed to treatment.

Transcriptome analysis reveals important regulatory factors for condensed tannins synthesis in acorn.

Condensed tannins are widely present in the fruits and seeds of plants and effectively prevent them from being eaten by animals before maturity due to their astringent taste. In addition, condensed tannins are a natural compound with strong antioxidant properties and significant antibacterial effects. Four samples of mature and near-mature Quercus fabri acorns, with the highest and lowest condensed tannin content, were used for genome-based transcriptome sequencing. The KEGG enrichment analysis revealed that the differentially expressed genes (DEGs) were highly enriched in phenylpropanoid biosynthesis and starch and sucrose metabolism. Given that the phenylpropanoid biosynthesis pathway is a crucial step in the synthesis of condensed tannins, we screened for significantly differentially expressed transcription factors and structural genes from the transcriptome data of this pathway and found that the expression levels of four MADS-box, PAL, and 4CL genes were significantly increased in acorns with high condensed tannin content. The quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiment further validated this result. In addition, yeast one-hybrid assay confirmed that three MADS-box transcription factors could bind the promoter of the 4CL gene, thereby regulating gene expression levels. This study utilized transcriptome sequencing to discover new important regulatory factors that can regulate the synthesis of acorn condensed tannins, providing new evidence for MADS-box transcription factors to regulate the synthesis of secondary metabolites in fruits.

Ubiquitin ligase VvPUB26 in grapevine promotes proanthocyanidin synthesis and resistance to powdery mildew.

Proanthocyanidins (PAs) are an important group of flavonoids that contribute to astringency, color, and flavor in grapes (Vitis vinifera) and wines. They also play a crucial role in enhancing plant resistance to various stresses. However, the underlying regulatory mechanism governing PAs biosynthesis, particularly in relation to conferring resistance to powdery mildew, has not been extensively explored. This study focused on identifying a key player in PAs biosynthesis, namely the plant U-box (PUB) E3 ubiquitin ligase VvPUB26. We discovered that overexpression of VvPUB26 in grapes leads to a significant increase in PAs content, whereas interfering with VvPUB26 has the opposite effect. Additionally, our findings demonstrated that overexpression of VvPUB26 in transgenic grapevines enhances defense against powdery mildew while interfering with VvPUB26 results in increased susceptibility to the pathogen. Interestingly, we observed that VvPUB26 interacts with the WRKY transcription factor VvWRKY24, thereby facilitating ubiquitination and degradation processes. Through RNA-Seq analysis, we found that VvWRKY24 primarily participates in secondary metabolites biosynthesis, metabolic pathways, and plant-pathogen interaction. Notably, VvWRKY24 directly interacts with the promoters of dihydroflavonol-4-reductase (DFR) and leucoanthocyanidin reductase (LAR) to inhibit PAs biosynthesis. Meanwhile, VvWRKY24 also influences the expression of MYB transcription factor genes related to PAs synthesis. In conclusion, our results unveil a regulatory module involving VvPUB26-VvWRKY24-VvDFR/VvLAR that plays a fundamental role in governing PAs biosynthesis in grapevines. These findings enhance our understanding of the relationship between PAs biosynthesis and defense mechanisms against powdery mildew.

Senolytic and senomorphic agent procyanidin C1 alleviates structural and functional decline in the aged retina.

Increased cellular senescence burden contributes in part to age-related organ dysfunction and pathologies. In our study, using mouse models of natural aging, we observed structural and functional decline in the aged retina, which was accompanied by the accumulation of senescent cells and senescence-associated secretory phenotype factors. We further validated the senolytic and senomorphic properties of procyanidin C1 (PCC1) both in vitro and in vivo, the long-term treatment of which ameliorated age-related retinal impairment. Through high-throughput single-cell RNA sequencing (scRNA-seq), we comprehensively characterized the retinal landscape after PCC1 administration and deciphered the molecular basis underlying the senescence burden increment and elimination. By exploring the scRNA-seq database of age-related retinal disorders, we revealed the role of cellular senescence and the therapeutic potential of PCC1 in these pathologies. Overall, these results indicate the therapeutic effects of PCC1 on the aged retina and its potential use for treating age-related retinal disorders.

Proanthocyanidins Ameliorate LPS-Inhibited Osteogenesis of PDLSCs by Restoring Lysine Lactylation.

Periodontitis is a bacteria-induced inflammatory disease characterized by the progressive destruction of periodontal supporting tissues. Periodontal ligament stem cells (PDLSCs) are capable of differentiating into osteoblasts, which is an important stem cell source for endogenous periodontal tissue regeneration. Lysine lactylation (Kla) is a novel post-translational modification of proteins that is recently thought to be associated with osteogenic differentiation. Here, we found that lactylation levels are reduced both in the periodontal tissue of rats with periodontitis and lipopolysaccharide (LPS)-stimulated human PDLSCs. Proanthocyanidins were able to promote the osteogenesis of inflamed PDLSCs by restoring lactylation levels. Mechanistically, proanthocyanidins increased lactate production and restored the lactylation levels of PDLSCs, which recovered osteogenesis of inflamed PDLSCs via the Wnt/ß-catenin pathway. These results provide evidence on how epigenetic regulation by pharmacological agents influence the osteogenic phenotype of stem cells and the process of periodontal tissue repair. Our current study highlights the valuable potential of natural product proanthocyanidins in the regenerative engineering of periodontal tissues.

Evaluation of biological activity and prebiotic properties of proanthocyanidins with different degrees of polymerization through simulated digestion and in vitro fermentation by human fecal microbiota.

The bioactive activity of proanthocyanidins (PAs) is closely associated with their degree of polymerization (DP), however, the effects of PAs with different DP on digestion and gut microbiota have remained unclear. To investigate this, we conducted in vitro simulated digestion and colonic fermentation studies on samples of PAs with different DP. The results showed that PAs was influenced by both protein precipitation and enzymolysis, resulting in a decrease in functional activity. PAs with a high DP were more sensitive to the gastrointestinal environment. The significant clustering trend in colonic fermentation verified the reliability of multivariate statistical techniques for screening samples with distinct functional differences. The gut microbiota analysis showed that oligomeric PAs had a stronger promoting effect on beneficial bacteria, while high polymeric PAs had a greater inhibitory effect on harmful bacteria. This study offers new insights into the biological activity and microbiological mechanisms of PAs with different DP.

Integrated Transcriptomic and Proteomic Analysis Identifies Novel Regulatory Genes Associated with Plant Growth Regulator-Induced Astringency in Grape Berries.

Astringency influences the sensory characteristics and flavor quality of table grapes. We tested the astringency sensory attributes of berries and investigated the concentration of flavan-3-ols/proanthocyanidins (PAs) in skins after the application of the plant growth regulators CPPU and GA3 to the flowers and young berries of the "Summer Black" grape. Our results showed that CPPU and GA3 applications increase sensory astringency perception scores and flavan-3-ol/proanthocyanidin concentrations. Using integrated transcriptomic and proteomic analysis, differentially expressed transcripts and proteins associated with growth regulator treatment were identified, including those for flavonoid biosynthesis that contribute to the changes in sensory astringency levels. Transient overexpression of candidate astringency-related regulatory genes in grape leaves revealed that VvWRKY71, in combination with VvMYBPA1 and VvMYC1, could promote the biosynthesis of proanthocyanidins, while overexpression of VvNAC83 reduced the accumulation of proanthocyanidins. However, in transient promoter studies in Nicotiana benthamiana, VvWRKY71 repressed the promoter of VvMYBPA2, while VvNAC83 had no significant effect on the promoter activity of four PA-related genes, and VvMYBPA1 was shown to activate its own promoter. This study provides new insights into the molecular mechanisms of sensory astringency formation induced by plant growth regulators in grape berries.

Non-Mature miRNA-Encoded Micropeptide miPEP166c Stimulates Anthocyanin and Proanthocyanidin Synthesis in Grape Berry Cells.

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.