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.

Revealing Further Insights into Astringent Seeds of Chinese Fir by Integrated Metabolomic and Lipidomic Analyses.

Chinese fir (Cunninghamia lanceolata (Lamb.) Hook.) stands as one of the pivotal afforestation tree species and timber resources in southern China. Nevertheless, the occurrence of seed abortion and a notably high proportion of astringent seeds significantly curtail the yield and quality of elite seeds, resulting in substantial economic losses. The development of astringent seeds is accompanied by significant physiological and biochemical alterations. Here, the first combined lipidomic and metabolomic analysis was performed to gain a comprehensive understanding of astringent seed traits. A total of 744 metabolites and 616 lipids were detected, of which 489 differential metabolites and 101 differential lipids were identified. In astringent seeds, most flavonoids and tannins, as well as proline and γ-aminobutyric acid, were more accumulated, along with a notable decrease in lipid unsaturation, indicating oxidative stress in the cells of astringent seeds. Conversely, numerous elemental metabolites were less accumulated, including amino acids and their derivatives, saccharides and alcohols, organic acids and nucleotides and their derivatives. Meanwhile, most lipid subclasses, mainly associated with energy storage (triglyceride and diglyceride) and cell membrane composition (phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine), also exhibited significant reductions. These results reflected a disruption in the cellular system or the occurrence of cell death, causing a reduction in viable cells within astringent seeds. Furthermore, only one lipid subclass, sphingosine phosphate (SoP), was more accumulated in astringent seeds. Additionally, lower accumulation of indole-3-acetic acid and more accumulation of salicylic acid (SA) were also identified in astringent seeds. Both SA and SoP were closely associated with the promotion of programmed cell death in astringent seeds. Collectively, our study revealed significant abnormal changes in phytohormones, lipids and various metabolites in astringent seeds, allowing us to propose a model for the development of astringent seeds in Chinese fir based on existing research and our findings. This work enriches our comprehension of astringent seeds and presents valuable bioindicators for the identification of astringent seeds.

Comparative Metabolomic and Transcriptomic Analyses Reveal Distinct Ascorbic Acid (AsA) Accumulation Patterns between PCA and PCNA Persimmon Developing Fruit.

Persimmon fruit has a high nutritional value and significantly varies between pollination-constant astringent (PCA) and pollination-constant non-astringent (PCNA) persimmons. The astringency type affects sugar, flavonoids, and tannin accumulation and is well known in persimmon fruit. However, the impact of the fruit astringency type on ascorbic acid (AsA) accumulation is limited. In this study, typical PCA varieties ('Huojing' and 'Zhongshi5') and PCNA varieties ('Yohou' and 'Jiro') of persimmon fruit were sampled at four developing stages (S1-S4) to provide valuable information on AsA content variation in PCA and PCNA persimmon. Persimmon fruit is rich in ascorbic acid; the AsA content of the four varieties 'Zhongshi5', 'Huojing', 'Jiro', and 'Youhou' mature fruit reached 104.49, 48.69, 69.69, and 47.48 mg/100 g. Fruit of the same astringency type persimmon showed a similar AsA accumulation pattern. AsA content was significantly higher in PCA than PCNA fruit at S1-S3. The initial KEGG analysis of metabolites showed that galactose metabolism is the major biosynthetic pathway of AsA in persimmon fruit. There were significant differences in galactose pathway-related metabolite content in developing PCA and PCNA fruit, such as Lactose, D-Tagatose, and D-Sorbitol content in PCA being higher than that of PCNA. Combined gene expression and WGCNA analyses showed that the expression of the GME (evm.TU.contig4144.37) gene was higher in PCA-type than in PCNA-type fruit in S1-S3 and exhibited the highest correlation with AsA content (r = 690 **, p < 0.01). Four hub genes, including the DNA methylation gene, methyltransferase gene, F-box, and Actin-like Protein, were identified as potential regulators of the GME gene. These results provide basic information on how astringency types affect AsA accumulation and will provide valuable information for further investigation on AsA content variation in persimmon fruit.

Paper chromatography approach for the assessment of interaction between red wine and whole saliva.

In-mouth interaction of red wine compounds with salivary proteins is a primary event allegedly responsible for eliciting the mouth-feel sensation of astringency. Those interactions have been currently associated with precipitation of salivary protein/polyphenol complexes. However, such single physicochemical evidence for interaction does not account for the complexity of astringency. This study aimed to develop a paper chromatography method to assess interactions between red wine and the salivary protein fraction using stepwise series of red wine/saliva binary mixtures from 100% wine to 100% saliva ("Alpha and Omega series"). Aliquots of each one of the mixtures were spotted on a cellulose membrane to scrutinize independently the distribution areas of wine components (naturally pink-colored) and salivary protein (stained blue in Coomassie Brilliant R-250). This double target detection revealed interactions between saliva and red wine components along most of the quantitative Alpha and Omega series, a point of equivalence corresponding to maximum interactivity for both complex reactants and a non-diffusible sub-fraction of saliva displaying the highest interactivity. The results indicate a novel way to assess quantitatively physicochemical interactions between red wines and human saliva but also provide new lights to approach the identification of molecular salivary structures involved in triggering astringency.

Preferential transport activity of DkDTX5/MATE5 affects the formation of different astringency in persimmon.

Proanthocyanidins (PAs) are specialized metabolites that influence persimmon fruit quality. Normal astringent (A)-type and non-astringent (NA)-type mutants show significant variation in PA accumulation, but the influencing mechanism remains unclear. In this study, among the six identified DTXs/MATEs proteins associated with PA accumulation, we observed that allelic variation and preferential transport by DkDTX5/MATE5 induced variation in PA accumulation for A-type and NA-type fruit. The expression pattern of DkDTX5/MATE5 was correlated with PA accumulation in NA-type fruit. Upregulation and downregulation of DkDTX5/MATE5 promoted and inhibited PA accumulation, respectively, in the NA-type fruit. Interestingly, transporter assays of Xenopus laevis oocytes indicated that DkDTX5/MATE5 preferentially transported the PA precursors catechin, epicatechin, and epicatechin gallate, resulting in their increased ratios relative to the total PAs, which was the main source of variation in PA accumulation between the A-type and NA-type. The allele lacking Ser-84 in DkDTX5/MATE5 was identified as a dominantly expressed gene in the A-type and lost its transport function. Site-directed mutagenesis revealed that DkDTX5/MATE5 binds to PA precursors via Ser-84. These findings clarify the association between the transporter function of DkDTX5/MATE5 and PA variation, and can contribute to the breeding of new cultivars with improved fruit quality.

Transcriptome and Metabolome Reveal Distinct Sugar Accumulation Pattern between PCNA and PCA Mature Persimmon Fruit.

Persimmon (Diospyros kaki) fruit have significant variation between pollination-constant non-astringent (PCNA) and pollination-constant astringent (PCA) persimmons. The astringency type affects not only the soluble tannin concentration but also the accumulation of individual sugars. Thus, we comprehensively investigate the gene expression and metabolite profiles of individual sugars to resolve the formation of flavor differences in PCNA and PCA persimmon fruit. The results showed that soluble sugar, starch content, sucrose synthase, and sucrose invertase were significantly different between PCNA and PCA persimmon fruit. The sucrose and starch metabolism pathway was considerably enriched, and six sugar metabolites involving this pathway were significantly differentially accumulated. In addition, the expression patterns of diferentially expressed genes (such as bglX, eglC, Cel, TPS, SUS, and TREH genes) were significantly correlated with the content of deferentially accumulated metabolites (such as starch, sucrose, and trehalose) in the sucrose and starch metabolism pathway. These results indicated that the sucrose and starch metabolism pathway maintained a central position of sugar metabolism between PCNA and PCA persimmon fruit. Our results provide a theoretical basis for exploring functional genes related to sugar metabolism and provide useful resources for future studies on the flavor differences between PCNA and PCA persimmon fruit.

Artificial saliva precipitation index (ASPI): An efficient evaluation method of wine astringency.

Astringency is one of the most important organoleptic characteristics of red wines, and its intensity evaluation method has been the focus of research in recent years. An artificial saliva system was developed to establish an accurate and reliable evaluation method for the astringency intensity of dry red wines based on saliva precipitation index (SPI). To achieve this, five key protein families, which presented high reactivities and sensitivities in protein-tannin binding reactions, were selected from human whole saliva. The concentrations of the five proteins (proline-rich protein, α-amylase, lactoferrin, lysozyme, and albumin) and pH were optimized using response surface methodology based on the human salivary conditions to simulate the real salivary environment. The artificial saliva precipitation index method was applied to 60 commercial dry red wines and it exhibited a high correlation (CoefASPI = 0.94) with the sensory scores, indicating better performance than the traditional SPI method and other analytical approaches.

Diversity and determinants of bitterness, astringency, and fat content in cultivated Nacional and native Amazonian cocoa accessions from Ecuador.

Cocoa (Theobroma cacao L.) is the only tree that can produce cocoa. Cocoa beans are highly sought after by chocolate makers to produce chocolate. Cocoa can be fine aromatic, characterized by floral and fruity notes, or it can be described as standard cocoa with a more pronounced cocoa aroma and bitterness. In this study, the genetic and biochemical determinants of sensorial notes and nonvolatile compounds related to bitterness, astringency, fat content, and protein content will be investigated in two populations: a cultivated modern Nacional population and a population of cocoa accessions collected recently in the Ecuadorian South Amazonia area of origin of the Nacional ancestral variety. For this purpose, a genome-wide association study (GWAS) was carried out on both populations, with results of biochemical compounds evaluated by near-infrared spectroscopy (NIRS) assays and with sensory evaluations. Twenty areas of associations were detected for sensorial data especially bitterness and astringency. Fifty-three areas of associations were detected linked to nonvolatile compounds. A total of 81 candidate genes could be identified in the areas of the association.

Interaction between Ellagitannins and Salivary Proline-Rich Proteins.

The first contact of tannins with the human body occurs in the mouth, where some of these tannins are known to interact with salivary proteins, in particular with proline-rich proteins (PRPs). These interactions are important at a sensory level, especially for astringency development, but could also affect the biological activities of the tannins. This study gathers information on the relative affinity of the interaction, complex stoichiometry, and tannin molecular epitopes of binding for the interactions between the families of PRPs (bPRPs, gPRPs, and aPRPs) and three representative ellagitannins (castalagin, vescalagin, and punicalagin). These interactions were studied by saturation-tranfer difference NMR and microcalorimetry. The effect of the PRP-ellagitannin interaction on their antioxidant ability was also assessed by ferric reduction antioxidant power (FRAP) assays. The results support a significant interaction between the studied tannins and PRPs with binding affinities in the micromolar range. Punicalagin was always the ellagitannin with higher affinity. aPRPs were the salivary PRPs with higher affinity. Moreover, it was observed that when ellagitannins are present in low concentrations (5-50 µM), as occurs in food, the antioxidant ability of these tannins when complexed with salivary PRPs could be significantly impaired.

Effect of lactic acid bacteria fermentation on tannins removal in Xuan Mugua fruits.

The present study was conducted for developing a new method to reduce the tannins content of Xuan Mugua fruits. Lactic acid bacteria fermentation-like incubation was explored as an efficient method to reduce the astringency of Mugua fruits by reducing tannins content. To ensure the efficient removal of tannins, the fermentation conditions were optimized such as temperature, time, and ratio of bacteria to Mugua. Bacteria were able to reduce 78% of the tannins content in Mugua under the optimized conditions as assessed by the methylcellulose perceptible tannins assay. Meanwhile, the active components including polysaccharides, triterpenes, and antioxidants of Mugua with bacterial incubation were also explored, which showed the significant decrease compared to non-fermented Mugua. However, the trend for flavor components was different. This research provides a natural method to solve the astringency problem of these fruits and it will extend their application in food industry.

Mechanisms of astringency: Structural alteration of the oral mucosal pellicle by dietary tannins and protective effect of bPRPs.

The interaction of tannins with salivary proteins is involved in astringency. This paper focussed on saliva lining oral mucosae, the mucosal pellicle. Using a cell-based model, the impact of two dietary tannins (EgC and EgCG) on the mucosal pellicle structure and properties was investigated by microscopic techniques. The role of basic Proline-Rich-Proteins (bPRPs) in protecting the mucosal pellicle was also evaluated. At low (0.05 mM) tannin concentration, below the sensory detection threshold, the distribution of salivary mucins MUC5B on cells remained unaffected. At 0.5 and 1 mM, MUC5B-tannin aggregates were observed and their size increased with tannin concentration and with galloylation. In addition, 3 mM EgCG resulted in higher friction forces measured by AFM. In presence of bPRPs, the size distribution of aggregates was greatly modified and tended to resemble that of the "no tannin" condition, highlighting that bPRPs have a protective effect against the structural alteration induced by dietary tannins.

Interactions between wine phenolic compounds and human saliva in astringency perception.

Astringency is a complex perceptual phenomenon involving several sensations that are perceived simultaneously. The mechanism leading to these sensations has been thoroughly and controversially discussed in the literature and it is still not well understood since there are many contributing factors. Although we are still far from elucidating the mechanisms whereby astringency develops, the interaction between phenolic compounds and proteins (from saliva, oral mucosa or cells) seems to be most important. This review summarizes the recent trends in the protein-phenol interaction, focusing on the effect of the structure of the phenolic compound on the interaction with salivary proteins and on methodologies based on these interactions to determine astringency.

Effect of Astringent Stimuli on Salivary Protein Interactions Elucidated by Complementary Proteomics Approaches.

The interaction of astringent substances with salivary proteins, which results in protein precipitation, is considered a key event in the molecular mechanism underlying the oral sensation of puckering astringency. As the chemical nature of orally active astringents is diverse and the knowledge of their interactions with salivary proteins rather fragmentary, human whole saliva samples were incubated with suprathreshold and isointensity solutions of the astringent polyphenol (-)-epigallocatechin gallate, the multivalent metal salt iron(III) sulfate, the amino-functionalized polysaccharide chitosan, and the basic protein lysozyme. After separation of the precipitated proteins, the proteins affected by the astringents were identified and relatively quantified for the first time by complementary bottom-up and top-down mass spectrometry-based proteomics approaches. Major salivary target proteins, which may be involved in astringency perception, are reported here for each astringent stimulus.

Astringent Mouthfeel as a Consequence of Lubrication Failure.

Herein, we systematically investigate the origin of astringent mouthfeel when we eat unripe fruits, drink coffee or tea, from the perspective of lubrication by simulating the dynamic weak interaction on the tongue with model protein (mucoprotein, MP) and polyphenolic compounds (tannic acid, TA). Astringency was due to the protein-mediated lubrication failure when encountering polyphenolic molecules that normally exist, for example in unripe fruits, coffee, tea. The underlying molecular mechanism of oral tribology is widely present in nature and enables us to engineer a tongue-like polyacrylamide composite hydrogel that exhibits high TA sensitivity and to develop a scientific strategy for catching slippery fish using TA-containing gloves. These results provide novel and useful insights into the failure of biological boundary lubrication on soft tissue surface with the adsorbed proteins.

Zinc sulfate taste acuity reflects dietary zinc intake in males.

Gauging an individual's response after they taste a solution of zinc sulfate has been proposed as a method of determining nutritional zinc deficiency, a so-called "zinc taste test." Despite the lack of evidence regarding any relationship between dietary zinc intake and zinc sulfate taste acuity, clinicians continue to utilize zinc sulfate taste testing with their patients. Therefore, assessing the relationship between zinc sulfate taste acuity and dietary zinc intake is warranted. This report assessed 363 individuals (77 males, 286 females) for zinc sulfate taste acuity and dietary zinc intake. Zinc sulfate taste acuity was assessed by both the Bryce-Smith & Simpson zinc taste test (BSZTT) and the taste intensity visual analog scale (TIVAS). Dietary intake of zinc was assessed by a zinc-specific food frequency questionnaire (ZnFFQ). Zinc sulfate taste acuity, as measured by the TIVAS, was found to be significantly different between the sexes (U = 8766; p = 0.013). Males averaged a TIVAS score of 21.58 ± 2.52 (Mean ± SEM) whereas females had a TIVAS score averaging 31.49 ± 1.67. No correlations were found between female zinc sulfate taste perception and dietary zinc intake as measured by both the BSZTT (rs = 0.014; p = 0.816) and the TIVAS (rs = 0.025; p = 0.679). Similarly, male zinc intake was not correlated with BSZTT scores (rs = 0.199; p = 0.099). However, zinc sulfate taste acuity, measured by the TIVAS, was significantly correlated with dietary zinc intake in the male population (rs = 0.237; p = 0.048). These findings suggest that zinc sulfate taste acuity measurement may aid in the assessment of zinc nutriture among males.

Astringency: a more stringent definition.

Despite being an everyday sensory experience, the nature of astringency perception is not clear. In this issue of Chemical Senses, Schöbel et al. demonstrate that astringency is a trigeminal sensation in human, and astringents trigger a G protein-coupled pathway in trigeminal ganglion cells in the mouse.

Astringency is a trigeminal sensation that involves the activation of G protein-coupled signaling by phenolic compounds.

Astringency is an everyday sensory experience best described as a dry mouthfeel typically elicited by phenol-rich alimentary products like tea and wine. The neural correlates and cellular mechanisms of astringency perception are still not well understood. We explored taste and astringency perception in human subjects to study the contribution of the taste as well as of the trigeminal sensory system to astringency perception. Subjects with either a lesion or lidocaine anesthesia of the Chorda tympani taste nerve showed no impairment of astringency perception. Only anesthesia of both the lingual taste and trigeminal innervation by inferior alveolar nerve block led to a loss of astringency perception. In an in vitro model of trigeminal ganglion neurons of mice, we studied the cellular mechanisms of astringency perception. Primary mouse trigeminal ganglion neurons showed robust responses to 8 out of 19 monomeric phenolic astringent compounds and 8 polymeric red wine polyphenols in Ca(2+) imaging experiments. The activating substances shared one or several galloyl moieties, whereas substances lacking the moiety did not or only weakly stimulate responses. The responses depended on Ca(2+) influx and voltage-gated Ca(2+) channels, but not on transient receptor potential channels. Responses to the phenolic compound epigallocatechin gallate as well as to a polymeric red wine polyphenol were inhibited by the Gαs inactivator suramin, the adenylate cyclase inhibitor SQ, and the cyclic nucleotide-gated channel inhibitor l-cis-diltiazem and displayed sensitivity to blockers of Ca(2+)-activated Cl(-) channels.

Phenolic composition and mouthfeel characteristics resulting from blending Chilean red wines.

BACKGROUND: The blending of wine is a common practice in winemaking to improve certain characteristics that are appreciated by consumers. The use of some cultivars may contribute phenolic compounds that modify certain characteristics in blended wines, particularly those related to mouthfeel. The aim of this work was to study the effect of Carménère, Merlot and Cabernet Franc on the phenolic composition, proanthocyanidin profile and mouthfeel characteristics of Cabernet Sauvignon blends. RESULTS: Significant differences in chemical composition were observed among the monovarietal wines. Separation using Sep-Pak C18 cartridges revealed differences in the concentration but not in the proportion of various proanthocyanidins. Blending reduced polyphenol concentration differences among the various monovarietal wines. Although no major overall differences were observed after blending the monovarietal wines, this oenological practice produced clear differences in mouthfeel characteristics in such a way that the quality of the perceived astringency was different. CONCLUSION: This study showed that the use of a particular wine variety (Cabernet Sauvignon) in a higher proportion in wine blending produced blends that were less differentiable from the monovarietal wine, owing to a suppression effect, producing an apparent standardization of the wines regarding chemical composition.

Ethylene-responsive transcription factors interact with promoters of ADH and PDC involved in persimmon (Diospyros kaki) fruit de-astringency.

The persimmon fruit is a particularly good model for studying fruit response to hypoxia, in particular, the hypoxia-response ERF (HRE) genes. An anaerobic environment reduces fruit astringency by converting soluble condensed tannins (SCTs) into an insoluble form. Although the physiology of de-astringency has been widely studied, its molecular control is poorly understood. Both CO(2) and ethylene treatments efficiently removed the astringency from 'Mopan' persimmon fruit, as indicated by a decrease in SCTs. Acetaldehyde, the putative agent for causing de-astringency, accumulated during these treatments, as did activities of the key enzymes of acetaldehyde synthesis, alcohol dehydrogenase (ADH), and pyruvate decarboxylase (PDC). Eight DkADH and DkPDC genes were isolated, and three candidates for a role in de-astringency, DkADH1, DkPDC1, and DkPDC2, were characterized by transcriptional analysis in different tissues. The significance of these specific isoforms was confirmed by principal component analysis. Transient expression in leaf tissue showed that DkPDC2 decreased SCTs. Interactions of six hypoxia-responsive ERF genes and target promoters were tested in transient assays. The results indicated that two hypoxia-responsive ERF genes, DkERF9 and DkERF10, were involved in separately regulating the DkPDC2 and DkADH1 promoters. It is proposed that a DkERF-DkADH/DkPDC cascade is involved in regulating persimmon de-astringency.

The role of salivary proteins in the mechanism of astringency.

Understanding astringency has focused on the interaction of tannins with the salivary proline-rich proteins (PRPs), although it remains unclear if other astringents precipitate the PRPs or how this interaction relates to sensory perceptions of astringency. We used 2 approaches to compare how distinct classes of astringent compounds interacted with the salivary PRPs and mucins. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis, we evaluated protein patterns and characterized the salivary proteins present in the supernatants and pellets of pooled saliva assayed with tannin, alum, and hydrochloric acid solutions. Tannins and alum precipitated many of the PRPs, but acid did not. Mucins were precipitated by both the acid and alum, but not by the tannins. From our research, it appears that the precipitation of salivary proteins may be involved in the mechanism of astringency, but the precipitation of PRPs is not requisite for the development of astringency. We also measured mucin and deoxyribonucleic acid content of expectorated solutions of astringents that panelists swished in their mouths to determine if astringency was associated with a loss of oral lubricating films.