The large diversity of grapevine cultivars includes genotypes more tolerant to water deficit than others. Widely distributed cultivars, like Merlot, are more sensitive to water deprivation than local cultivars like Callet, which are more adapted to water deficit due to their Mediterranean origin. Despite their tolerance, adaptation to water deficit influenced by grafting in rootstocks like 110 Richter is key to facing drought in vineyards, defining the scion-rootstock relationship. To understand these differences, we explored transcriptomic, metabolic, hormonal and physiological responses under three levels of water deficit (mild, high, and extreme), using 110 Richter as the rootstock in both cultivars. Results revealed that sensitivity to abscisic acid (ABA) is essential for water deficit tolerance in the aerial part, guiding root responses. Callet/110 Richter activates more gene expression patterns in response to ABA, reducing water loss compared to Merlot/110 Richter in both aerial and root parts. This modulation in Callet/110 Richter involves regulating metabolic pathways to increase cell turgor, reducing photosynthesis, and producing molecules like polyphenols or flavonoids to respond to oxidative stress. In contrast, Merlot/110 Richter shows a lack of specific response, especially in the roots, indicating less resilience to water stress. Therefore, selecting genotypes more sensitive to ABA and their interaction with rootstocks is key for managing vineyards in future climate change scenarios.

The domestication process in grapevines has facilitated the fixation of desired traits. Nowadays, vegetative propagation through cuttings enables easier preservation of these genotypes compared to sexual reproduction. Nonetheless, even with vegetative propagation, various phenotypes are often present within the same vineyard due to the accumulation of somatic mutations. These mutations are not the sole factors influencing phenotype. Alongside somatic variations, epigenetic variation has been proposed as a pivotal player in regulating phenotypic variability acquired during domestication. The emergence of these epialleles might have significantly influenced grapevine domestication over time. This study aims to investigate the impact of domestication on methylation patterns in cultivated grapevines. Reduced-representation bisulfite sequencing was conducted on 18 cultivated and wild accessions. Results revealed that cultivated grapevines exhibited higher methylation levels than their wild counterparts. Differential Methylation Analysis between wild and cultivated grapevines identified a total of 9955 differentially methylated cytosines, of which 78% were hypermethylated in cultivated grapevines. Functional analysis shows that core methylated genes (consistently methylated in both wild and cultivated accessions) are associated with stress response and terpenoid/isoprenoid metabolic processes. Meanwhile, genes with differential methylation are linked to protein targeting to the peroxisome, ethylene regulation, histone modifications, and defense response. Collectively, our results highlight the significant roles that epialleles may have played throughout the domestication history of grapevines.

Aedes mosquito-borne diseases remain a significant global health threat, necessitating effective control strategies. This study introduces monoterpenes-based nanohydrogels for potential use as repellents against Aedes aegypti, the primary dengue vector worldwide. We formulated hydrogels using cymene- and myrcene-based nanoemulsions with different polymers: chitosan, carboxymethylcellulose (CMC), and carbopol®. Our evaluations of rheological, texture, and bioadhesive properties identified CMC hydrogel as the most promising gelling agent for topical application, exhibiting sustained monoterpene release over 12 h with low skin permeation and high retention in the stratum corneum. Myrcene-loaded CMC hydrogel achieved a 57% feeding deterrence compared to 47% with cymene hydrogel in the mosquito membrane-feeding model. Molecular docking studies revealed interactions between myrcene and an essential amino acid (Ile116) in the Ae. aegypti odorant-binding protein 22 (AeOBP22), corroborating its higher repellent efficacy. These findings suggest that myrcene-loaded CMC hydrogels offer a promising, minimally invasive strategy for personal protection against Ae. aegypti and warrant further investigation to optimize monoterpene concentrations for vector control.

Drought caused by climate change has a dramatic incidence on the vineyard. Despite employing specific rootstocks tolerant to drought like 110 Richter, the vineyard continues to experience various losses, revealing the importance of the scion cultivar in the adaptation to drought stress. In this regard, Merlot, a widely cultivated grapevine, exhibited reduced drought tolerance compared to less cultivated varieties like Callet, a local cultivar originating from the Balearic Islands that demonstrated greater resilience to drought. Therefore, understanding the drought stress response in both cultivars and the cross-talk between scion and rootstock is key to unveiling possible differences that could affect to the adaptation to drought in vineyard. Plants from both cultivars grafted in the tolerant rootstock, 110 Richter, underwent different drought stages. For each stage, samples from leaves and roots were analyzed at metabolic, hormonal, physiological and transcriptomic level. The results revealed differences at most levels, increasing the production of osmolytes and antioxidant molecules involved in response to drought stress in Callet. However, hormonal analysis showed similar ABA production in both cultivars, indicating lower sensitivity to ABA in the case of Merlot compared to Callet. Moreover, the transcriptomic analysis revealed a modulation of genes involved in response to ABA and miRNA in leaves and roots of Callet, whereas in Merlot was mostly absent in roots, evidencing a poor cross-talk between Merlot and rootstock and increasing the value of the correct combination scion/rootstock for the vineyard adaptation to climate change.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

Increase in soil salinity, driven by climate change, is a widespread constrain for viticulture across several regions, including the Mediterranean basin. The implementation of salt-tolerant varieties is sought after to reduce the negative impact of salinity in grape production. An accession of wild grapevine (Vitis vinifera L. ssp. sylvestris), named AS1B, found on the coastline of Asturias (Spain), could be of interest toward the achievement of salt-tolerant varieties, as it demonstrated the ability to survive and grow under high levels of salinity. In the present study, AS1B is compared against widely cultivated commercial rootstock Richter 110, regarding their survival capabilities, and transcriptomic profiles analysis allowed us to identify the genes by employing RNA-seq and gene ontology analyses under increasing salinity and validate (via RT-qPCR) seven salinity-stress-induced genes. The results suggest contrasting transcriptomic responses between AS1B and Richter 110. AS1B is more responsive to a milder increase in salinity and builds up specific mechanisms of tolerance over a sustained salt stress, while Richter 110 maintains a constitutive expression until high and prolonged saline inputs, when it mainly shows responses to osmotic stress. The genetic basis of AS1B's strategy to confront salinity could be valuable in cultivar breeding programs, to expand the current range of salt-tolerant rootstocks, aiming to improve the adaptation of viticulture against climate change.

The presence of naturally-occurring color mutants in plants has permitted the identification of many regulatory genes implicated in the synthesis of discrete metabolic compounds, mostly anthocyanins and carotenoids. Conversely, transcription factors that coordinate more than one specialized metabolic pathway seem challenging to screen from a forward genetics’ perspective. We explored the relationship between different branches of the phenylpropanoid and isoprenoid pathways while examining an infrequent berry skin color variegation in grapevine. Red and white berry skin sections were compared at the genetic, transcriptomic and metabolomic levels showing that, as in most cultivated white grape varieties, the uncolored skin section convened the non-functional alleles of the anthocyanin regulators MYBA1 and MYBA2, explaining the lack of pigments. In contrast, light-responsive flavonols and monoterpenes increased in anthocyanin-depleted areas. We disclosed an enrichment of the flavonol, terpene and carotenoid pathways among up-regulated genes from white-skin sections, accompanied by increased expressions of flavonol regulators and the still uncharacterized MYB24 gene. We used DAP-seq to examine the in vitro binding of affinity-purified MYB24 protein to genomic DNA and demonstrated its binding in the promoter regions of terpene (22) and carotenoid (6) genes, in addition to more than 30 photosynthesis and light-response genes, including the flavonol-regulator HY5 homologue (HYH). We confirmed the activation of TPS35 and HYH promoter:luciferase reporters in the presence of MYB24 and the grape bHLH MYC2, all of which correlate in their higher expression in white skin variegated sections. The integration of several datasets allowed to define a list of high confidence targets, suggesting MYB24 as a modulator of light responses including the synthesis of flavonoids (flavonols) and isoprenoids (terpenes, and putatively carotenoids). The correspondence between MYB24 and monoterpenes in all conditions surveyed implies that this regulatory network is broadly triggered towards berry ripening, and that the absence of anthocyanin sunscreens accelerates its activation most likely in a dose-dependent manner due to increased radiation exposure.

Vitis vinifera L. can be divided into two subspecies, V. vinifera subsp. vinifera, the cultivated grapevine, and its wild ancestor, V. vinifera subsp. sylvestris. Three flower types have been described: hermaphrodite and female in some varieties of vinifera, and male or female flowers in sylvestris. We have conducted an expression analysis of the functional genes candidate to sex determination in the newly defined sex locus described by Picq et al (2014) using four flower types. The candidate gene Ethylene overproducer-1 (ETO1) localized in the sex locus region and which inhibits the enzyme activity of the enzyme ACS (1-aminocyclopropane-1-carboxylic acid synthase) was showed highly significantly different expression pattern according to the sex flower. Other genes studied in the sex locus do not reveal significant different expression patterns. For genes located outside of the sex locus, only the SAUR (Small auxin up RNAs) protein and the ACS gene showed different expression among sex flowers. Therefore, as ETO1 is only expressed in female and hermaphrodite flowers, it could be a good candidate for the recessive female fertility mutation and ACS copy could be implied in the reaction cascade leading to the inhibition of stamens in female flowers. However, the ETO1 only negatively interacts with type 2 ACS and our ACS phylogeny analysis confirmed that the VviACS copy is not type 2. Therefore, it is unlikely that there is such molecular interaction in grapevine. Another hypothesis could be that the molecular mechanisms that regulated the activity of VviACS2 are induced by the VvETO1 protein regulating the activity of both families of ACS type I and type 2. The last gene showing differential expression according to sex is the SAUR protein. This gene consists in early auxin response genes family playing key role in hormonal and environmental signals. Our results pointed out that one gene (ETO1) inside of the flower sex locus region and two genes (ACS, SAUR) located outside of the sex locus region, could be considered as putative candidate genes for the control of sexual traits in grapevine.

Somatic mutations occurring within meristems of vegetative propagation material have had a major role in increasing the genetic diversity of the domesticated grapevine (Vitis vinifera subsp. vinifera). The most well studied somatic variation in this species is the one affecting fruit pigmentation, leading to a plethora of different berry skin colors. Color depletion and reversion are often observed in the field. In this study we analyzed the origin of a novel white-to-red skin color reversion and studied its possible metabolic and transcriptomic consequences on cv. ‘Muscat à Petits Grains Blancs’ (synonym cv. ‘Moscatel Galego Branco’), a member of the large family of Muscats. The mild red-skinned variant (cv. ‘Muscat à Petits Grains Rouge’, synonym cv. ‘Moscatel Galego Roxo’), characterized by a preferential accumulation of di-hydroxylated anthocyanins, showed in heterozygosis a partially-excised Gret1 retrotransposon in the promoter region of the MYBA1 anthocyanin regulator, while MYBA2 was still in homozygosis for its non-functional allele. Through metabolic (anthocyanin, resveratrol and piceid quantifications) and transcriptomic (RNA-Seq) analyses, we show that within a near-isogenic background, the transcriptomic consequences of color reversion are largely associated to diminished light/UV-B responses probably as a consequence of the augment of metabolic sunscreens (i.e. anthocyanins). We propose that the reduced activity of the flavonoid tri-hydroxylated sub-branch and decreased anthocyanin synthesis and modification (e.g. methylation and acylation) are the potential causes for the mild red-skinned coloration in the pigmented revertant. The observed positive relation between anthocyanins and stilbenes could be attributable to an increased influx of phenylpropanoid intermediaries due to the replenished activity of MYBA1, an effect yet to be demonstrated in other somatic variants.

•‘Vinhão’ has black-bluish skin color, highly appreciated for wine production.•First insights into the genetics behind the color feature of the cv. ‘Vinhão’.•MYBA genes play a major role on the berry skin color of the cv. ‘Vinhão’.•Berry color locus of cv. ‘Vinhão’ carries the most ancestral haplotype (Hap C-N).•The involvement of key anthocyanin genes was revealed, mainly from veraison onwards.

ABSTRACTAnthocyanins are flavonoids responsible for the color of berries in skin-pigmented grapevine (Vitis vinifera L.). Due to the widely adopted vegetative propagation of this species, somatic mutations occurring in meristematic cell layers can be fixed and passed into the rest of the plant when cloned. In this study we focused on the transcriptomic and metabolic differences between two color somatic variants. Using microscopic, metabolic and mRNA profiling analyses we compared the table grape cultivar (cv.) ‘Red Globe’ (RG, with purplish berry skin) and cv. ‘Chimenti Globe’ (CG, with a contrasting reddish berry skin color). As expected, significant differences were found in the composition of flavonoids and other phenylpropanoids, but also in their upstream precursors’ shikimate and phenylalanine. Among primary metabolites, sugar phosphates related with sucrose biosynthesis were less accumulated in cv. ‘CG’. The red-skinned cv. ‘CG’ only contained di-hydroxylated anthocyanins (i.e. peonidin and cyanidin) while the tri-hydroxylated derivatives malvidin, delphinidin and petunidin were absent, in correlation to the reddish cv. ‘CG’ skin coloration. Transcriptomic analysis showed alteration in flavonoid metabolism and terpenoid pathways and in primary metabolism such as sugar content. Eleven flavonoid 3’5’-hydroxylase gene copies were down-regulated in cv. ‘CG’. This family of cytochrome P450 oxidoreductases are key in the biosynthesis of tri-hydroxylated anthocyanins. Many transcription factors appeared down-regulated in cv. ‘CG’ in correlation to the metabolic and transcriptomic changes observed. The use of molecular markers and its confirmation with our RNA-seq data showed the exclusive presence of the null MYBA2 white allele (i.e. homozygous in both L1 and L2 layers) in the two somatic variants. Therefore, the differences in MYBA1 expression seem sufficient for the skin pigmentation differences and the changes in MYBA target gene expression in cv. ‘Chimenti Globe’.

Additional file 9. List of primers used for molecular analyses.

Additional file 11. FPKM values of the F3’5’H and F3’H gene families in cv. ‘Moscatel Galego’ and cv. ‘Red Globe’ skin color somatic variants. FPKM values were calculated from A) multiple-mapped and B) filtered reads and are displayed for each biological replicate.

The transcriptional and biochemical changes during berry development of the grapevine Vitis vinifera L. subsp. sativa have been well characterized, but little is known regarding wild grapevine berries (V. vinifera subsp. sylvestris). We analyzed 126 female wild accessions to measure the content of phenolic compounds in the berries. Different biochemical profiles were observed in the wild berries. We selected two extreme biochemical profiles between one wild accession and the cultivated grapevine `Monastrell' at two developmental stages (veraison and full ripening). Transcriptomic analysis was performed to evaluate the gene expression patterns and associate them with the phenolic profiles. Some genes of the phenylpropanoid pathway were differentially expressed between wild and cultivated berries, and this was related to the different biochemical profiles.

BACKGROUND: Anthocyanins are the main pigments in red grapes, and have been detected in samples of wild grapevine berries. OBJECTIVE: Evaluation of whether the phenolic composition of those plants differs from that presented by cultivated grapes, and assessing if some wild genotypes present some genetic characters of interest related to color accumulation during grape maturation. METHODS: The anthocyanin composition of 75 wild grapevines from different Iberian natural populations was studied by HPLC. The anthocyanins fingerprint of those genotypes was determined, and data were analysed by applying several statistical procedures. RESULTS: Genotypes under study showed a considerable variability in their anthocyanin fingerprints, being possible to distinguish two great families and several great phenotypic groups. CONCLUSIONS: The anthocyanin fingerprint of wild grapevines differs among genotypes collected in different geographical regions, suggesting that the evolution of the biosynthetic pathway leading to anthocyanins is affected by the climatic conditions of different environments.

BACKGROUND: During grapevine domestication somatic variation has been used as a source of diversity for clonal selection. OBJECTIVE: This work provides additional information on the molecular mechanisms responsible for berry skin color reversion on a subset of somatic variants for berry skin color never investigated before. METHODS: The berry color locus and its surrounding genomic region were genetically characterized through a layer-specific approach, which has already been proven to be a successful method to decipher the molecular mechanisms responsible for color reversions on somatic variants. RESULTS: Deletions of different extent and positions were detected among less-pigmented/unpigmented variants derived from a pigmented wild-type. These deletions affected only the inner cell layer in the less pigmented variants and both cell layers in the unpigmented variants. Regarding the pigmented variants derived from an unpigmented wild-type, only one group was distinguished by the Gret1 retrotransposon partial excision from the VvMybA1 promoter. Moreover, within this latter group, VvMybA2 showed an important role regarding the phenotypic variation, through the recovery of the functional G allele. CONCLUSIONS: This investigation focused on the berry color locus using somatic variants for berry skin color, promotes a better understanding of the evolutionary events behind their origin and variability, opening an opportunity for their use in the genetic improvement of varieties.

Phenolic compounds are a group of natural products that play an important role in the quality of wines. Most phenolic compounds present in wine are derived from those contained in grapes and extracted from skins, seeds, and pulp during the initial steps of winemaking. Among them, anthocyanins and flavonols are involved in the color of red wines as pigments or copigments and also as precursors of polymeric pigments after reaction with other phenols. Biosynthesis of those phenolics in grapes is regulated by different genes; thus, each grape genotype presents a characteristic phenolic fingerprint, which is modulated by different environmental conditions. In this chapter, the anthocyanins and flavonols composition of different genotypes of wild grapes preserved at El Encin Germplasm Bank has been examined in detail. Wild grapevines are a remarkable genetic resource that may be used in breeding programs to improve the phenolic composition of cultivated grapes and, hence, the quality of red wines.