Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera.
ABSTRACT: Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.
Project description:Exposure to solar radiation is a determining factor of grape composition. Flavonol synthesis is upregulated by solar radiation leaving a fingerprint on flavonol profile. This study aimed to test the factors affecting flavonol accumulation and profile and their potential as an indicator to assess the overall exposure of red wine grape berry to solar radiation. We performed three experiments to study the response of flavonol accumulation and profile to (1) three different solar radiation exclusion treatments during berry development; (2) canopy porosity and leaf area index (LAI); and (3) spatial variability of water status, vigor and ripening and cultural practices in commercial vineyards. Results showed a strong relationship between global radiation, inverse dormant pruning weights or canopy porosity (inversely proportional to LAI) and % kaempferol or % quercetin. Furthermore, the increase in concentration of the above two flavonols was associated with a reduction of % myricetin. Total flavonol content, % kaempferol, % quercetin, and % myricetin had significant correlations with inverse dormant pruning weights, but these were less sensitive to over-ripening or water deficits. Flavonol profile was associated to site hydrology (wetness index) through changes in vigor, and to LAI; and responded to shoot thinning or fruit-zone leaf removal. These results support the reliability of the flavonol profile as an assessment parameter for studies aiming to discuss canopy architecture or the effect of solar radiation on grapevine berries.
Project description:The influence of growing season (winter vs. summer) on the flavonoid accumulation and composition was studied in the skins of three grape cultivars for two consecutive years under a two-crop-a-year viticulture practice in Southwest China. The total anthocyanin, flavonol and flavan-3-ol contents in winter berry skins were significantly higher than those in summer berry skins for 'Kyoho' and 'Muscat Hamburg'. Reversely, the content of anthocyanin in 'NW196' winter berry was lower than summer berry. However, the percentage of diglycosylated, trihydroxylated, methylated, and acylated anthocyanins, trihydroxylated and methylated flavonols, and flavan-3-ol polymers were higher in the summer berry skins than the winter berry skins among all the three grape cultivars. Winter climatic conditions were favorable to flavonoid accumulation for the non native grapes 'Kyoho' and 'Muscat Hamburg', while the summer climatic conditions were beneficial to anthocyanin accumulation for 'NW196' that has 50% genetic background from a local wild grape species Vitis quinquangularis. These seasonal variations of flavonoid accumulations and compositions in the grape skins were primarily contributed by different climatic factors, such as temperature, solar radiation, and rainfall.
Project description:The evolution of flavonoids under altered temperature and light conditions in the fruit zone was followed in Cabernet Sauvignon (Vitis vinifera L.) grapes during ripening. The study was conducted over two consecutive seasons in 2010/2011 and 2011/2012 comprising two main treatments in which the light quantity was manipulated in the bunch zone: (1) standard (STD) with no lateral shoot or leaf removal and (2) leaf removal west (LRW) treatment with leaf removal on the western side of the bunch zone. Furthermore, the light quality was altered by installing ultraviolet B-suppression sheets within the bunch zone in both seasons. Tannin evolution was dependent on the prevailing light quality/quantity and temperatures during berry development in a particular season. Grape seed tannin accumulation coincided with seed development and commenced at the early stages of berry development. Seed proanthocyanidin composition was not influenced by the treatments. The largest impact on proanthocyanidin accumulation and structure in the skin was due to seasonal variations highlighting the complex interaction between light quality and/or quantity across the two growing seasons and eventually the complex interaction with temperature. Flavonol accumulation was significantly influenced by the light quality, which is known to be the main abiotic driver of flavonol biosynthesis regulation. Anthocyanin concentration and content were largely dependent on the temperature and light quality in a particular season. Anthocyanin composition was altered by the season rather than the treatment.
Project description:To investigate the effect of light intensity and temperature on the biosynthesis and accumulation of quality-related metabolites, field grown tea plants were shaded by Black Net and Nano-insulating Film (with additional 2-4°C cooling effect) with un-shaded plants as a control. Young shoots were subjected to UPLC-Q-TOF MS followed by multivariate statistical analysis. Most flavonoid metabolites (mainly flavan-3-ols, flavonols and their glycosides) decreased significantly in the shading treatments, while the contents of chlorophyll, ?-carotene, neoxanthin and free amino acids, caffeine, benzoic acid derivatives and phenylpropanoids increased. Comparison between two shading treatments indicated that the lower temperature under Nano shading decreased flavonols and their glycosides but increased accumulation of flavan-3-ols and proanthocyanidins. The comparison also showed a greater effect of temperature on galloylation of catechins than light intensity. Taken together, there might be competition for substrates between the up- and down-stream branches of the phenylpropanoid/flavonoid pathway, which was influenced by light intensity and temperature.
Project description:Blackberries (<i>Rubus</i> spp.) are among the high added value food products relevant for human health due to the increasing evidence of the beneficial effects of polyphenols, which are very abundant in these fruits. Interestingly, these compounds also play a role on plant physiology, being especially relevant their role in plant defense against biotic and abiotic stress. Hence, we hypothesize that since blackberry fruits have high amounts of flavonols and anthocyanins, leaves would also have high amounts of these compounds, and can be studied as a source of active molecules; furthermore, leaf synthesis would support their high contents in fruits. To explore this hypothesis, the present study reports a <i>de novo</i> transcriptome analysis on field grown blackberry leaves and fruits at the same time point, to establish the metabolic relationship of these compounds in both organs. Transcripts were aligned against <i>Fragaria vesca</i> genome, and genes were identified and annotated in different databases; tissue expression pattern showed 20,463 genes common to leaves and fruits, while 6,604 genes were significantly overexpressed only in fruits, while another 6,599 genes were significantly overexpressed in leaves, among which flavonol-anthocyanin transporter genes were present. Bioactives characterization indicated that total phenolics in leaves were three-fold, and flavonols were six-fold than in fruits, while concentration of anthocyanins was higher in fruits; HPLC-MS analysis indicated different composition in leaves and fruits, with cyanidin-3-glucoside as the only common compound identified. Next, RT-qPCR of the core genes in the flavonol anthocyanin pathway and regulatory MYB genes were carried out. Interestingly, genes in the flavonol-anthocyanin pathway and flavonol-transport families were overexpressed in leaves, consistent with the higher bioactive levels. On the other hand, transcription factors were overexpressed in fruits anticipating an active anthocyanin biosynthesis upon ripening. This suggests that, in addition to the biosynthesis taking place in the fruits during ripening, translocation of flavonols from leaves to fruits contributes to the high amounts of bioactives starting to accumulate in fruits.
Project description:Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways-VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT-were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.
Project description:BACKGROUND: The selective removal of grapevine leaves around berry clusters can improve the quality of ripening fruits by influencing parameters such as the berry sugar and anthocyanin content at harvest. The outcome depends strongly on the timing of defoliation, which influences the source-sink balance and the modified microclimate surrounding the berries. We removed the basal leaves from Vitis vinifera L. cv Sangiovese shoots at the pre-bloom and veraison stages, and investigated responses such as shoot growth, fruit morphology and composition compared to untreated controls. Moreover, we performed a genome-wide expression analysis to explore the impact of these defoliation treatments on berry transcriptome. RESULTS: We found that pre-bloom defoliation improved berry quality traits such as sugar and anthocyanin content, whereas defoliation at veraison had a detrimental effect, e.g. less anthocyanin and higher incidence of sunburn damage. Genome-wide expression analysis during berry ripening revealed that defoliation at either stage resulted in major transcriptome reprogramming, which slightly delayed the onset of ripening. However, a closer investigation of individual gene expression profiles identified genes that were specifically modulated by defoliation at each stage, reflecting the uncoupling of metabolic processes such as flavonoid biosynthesis, cell wall and stress metabolism, from the general ripening program. CONCLUSIONS: The specific transcriptional modifications we observed following defoliation at different time points allow the identification of the developmental or metabolic processes affected in berries thus deepening the knowledge of the mechanisms by which these agronomical practices impact the final berry ripening traits.
Project description:Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs). However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated 12 structural genes and two putative transcription factors (TFs) in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C, and icariin) decreased slightly or dramatically in two lines of Epimedium sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1), together with a bHLH TF (EsGL3) and WD40 protein (EsTTG1), were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase) in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering, and molecular breeding studies of Epimedium species.
Project description:Flavonoids are secondary metabolites that play important roles in plant physiology. Despite numerous studies examined the effects of available carbon (C) or nitrogen (N) on flavonoid biosynthesis, the mechanism of C/N interactive effects on flavonoid metabolism is still unclear. In this study, we analyzed the composition of flavonoids and the expression levels of flavonoid-related genes in leaves and calli of crabapple (Malus sp.) cultivars with different leaf colors grown on media with different C/N ratios. Our results show that high C/N ratios induce anthocyanin pigmentation in leaves of the ever-red cultivar 'Royalty' and the spring-red cultivar 'Prairifire,' as well as in three types of calli derived from the ever-green cultivar 'Spring Snow,' but not in the leaves of the ever-green cultivar 'Flame.' This phenomenon therefore correlated with anthocyanin content in these different samples. In addition, high C/N ratios in the growth media resulted in an increase in the concentration of flavones and flavonols in the leaves of the three crabapple cultivars. The transcript levels of the general flavonoid pathway genes [from chalcone synthase (CHS) to uridine diphosphat-glucose: flavonoid 3-O-glycosyltransferase (UFGT) and flavonol synthase (FLS)] increased in response to high C/N ratios, and this in turn was correlated with the concentration of anthocyanins, flavones and flavonols in the leaves and calli. Expression of the late flavonoid/anthocyanin biosynthetic genes, anthocyanidin synthase (ANS), UFGT and FLS in particular, was more strongly influenced by C/N ratios than other structural genes, and the increased expression of the structural genes under high C/N ratios coincided with a coordinated increase in transcript levels of a MYB transcription factor, MYB10. These results are likely to be useful for future generation of plants with an optimized flavonoid/anthocyanin content or desirable organ coloration.
Project description:BACKGROUND: Flavonoids such as anthocyanins, flavonols and proanthocyanidins, play a central role in fruit colour, flavour and health attributes. In peach and nectarine (Prunus persica) these compounds vary during fruit growth and ripening. Flavonoids are produced by a well studied pathway which is transcriptionally regulated by members of the MYB and bHLH transcription factor families. We have isolated nectarine flavonoid regulating genes and examined their expression patterns, which suggests a critical role in the regulation of flavonoid biosynthesis. RESULTS: In nectarine, expression of the genes encoding enzymes of the flavonoid pathway correlated with the concentration of proanthocyanidins, which strongly increases at mid-development. In contrast, the only gene which showed a similar pattern to anthocyanin concentration was UDP-glucose-flavonoid-3-O-glucosyltransferase (UFGT), which was high at the beginning and end of fruit growth, remaining low during the other developmental stages. Expression of flavonol synthase (FLS1) correlated with flavonol levels, both temporally and in a tissue specific manner. The pattern of UFGT gene expression may be explained by the involvement of different transcription factors, which up-regulate flavonoid biosynthesis (MYB10, MYB123, and bHLH3), or repress (MYB111 and MYB16) the transcription of the biosynthetic genes. The expression of a potential proanthocyanidin-regulating transcription factor, MYBPA1, corresponded with proanthocyanidin levels. Functional assays of these transcription factors were used to test the specificity for flavonoid regulation. CONCLUSIONS: MYB10 positively regulates the promoters of UFGT and dihydroflavonol 4-reductase (DFR) but not leucoanthocyanidin reductase (LAR). In contrast, MYBPA1 trans-activates the promoters of DFR and LAR, but not UFGT. This suggests exclusive roles of anthocyanin regulation by MYB10 and proanthocyanidin regulation by MYBPA1. Further, these transcription factors appeared to be responsive to both developmental and environmental stimuli.