MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

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.

Epigenetic differences between wild and cultivated grapevines highlight the contribution of DNA methylation during crop domestication.

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.

Genetic analysis of a white-to-red berry skin color reversion and its transcriptomic and metabolic consequences in grapevine (Vitis vinifera cv. 'Moscatel Galego').

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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.

Genetic diversity analysis of cultivated and wild grapevine (Vitis vinifera L.) accessions around the Mediterranean basin and Central Asia.

BACKGROUND: The mountainous region between the Caucasus and China is considered to be the center of domestication for grapevine. Despite the importance of Central Asia in the history of grape growing, information about the extent and distribution of grape genetic variation in this region is limited in comparison to wild and cultivated grapevines from around the Mediterranean basin. The principal goal of this work was to survey the genetic diversity and relationships among wild and cultivated grape germplasm from the Caucasus, Central Asia, and the Mediterranean basin collectively to understand gene flow, possible domestication events and adaptive introgression. RESULTS: A total of 1378 wild and cultivated grapevines collected around the Mediterranean basin and from Central Asia were tested with a set of 20 nuclear SSR markers. Genetic data were analyzed (Cluster analysis, Principal Coordinate Analysis and STRUCTURE) to identify groups, and the results were validated by Nei's genetic distance, pairwise FST analysis and assignment tests. All of these analyses identified three genetic groups: G1, wild accessions from Croatia, France, Italy and Spain; G2, wild accessions from Armenia, Azerbaijan and Georgia; and G3, cultivars from Spain, France, Italy, Georgia, Iran, Pakistan and Turkmenistan, which included a small group of wild accessions from Georgia and Croatia. Wild accessions from Georgia clustered with cultivated grape from the same area (proles pontica), but also with Western Europe (proles occidentalis), supporting Georgia as the ancient center of grapevine domestication. In addition, cluster analysis indicated that Western European wild grapes grouped with cultivated grapes from the same area, suggesting that the cultivated proles occidentalis contributed more to the early development of wine grapes than the wild vines from Eastern Europe. CONCLUSIONS: The analysis of genetic relationships among the tested genotypes provided evidence of genetic relationships between wild and cultivated accessions in the Mediterranean basin and Central Asia. The genetic structure indicated a considerable amount of gene flow, which limited the differentiation between the two subspecies. The results also indicated that grapes with mixed ancestry occur in the regions where wild grapevines were domesticated.

A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines.

BACKGROUND: In Vitis vinifera L., domestication induced a dramatic change in flower morphology: the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism. RESULTS: In V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles. CONCLUSIONS: Hermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool.

Dual domestications and origin of traits in grapevine evolution.

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.

Coastal Wild Grapevine Accession (Vitis vinifera L. ssp. sylvestris) Shows Distinct Late and Early Transcriptome Changes under Salt Stress in Comparison to Commercial Rootstock Richter 110.

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.

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.).

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.

Berry color variation in grapevine as a source of diversity.

Even though it is one of the oldest perennial domesticated fruit crops in the world, grapevine (Vitis vinifera L.) cultivation today is the result of both conventional breeding practices (i.e. hybridizations adopted during the last century) and vegetative propagation. Human-assisted asexual propagation has allowed the maintenance of desired traits but has largely impacted the frequency of spontaneous somatic mutations observed in the field. Consequently, many grapevine fruit attributes to date have been artificially selected, including: fruit yield, compactness, size and composition, the latter being greatly diversified in the pursuit of altering berry skin coloration. The present review provides an overview of various aspects related to grapevine diversity, with a special emphasis on grape berry skin color variation and will discuss the current knowledge of how grape skin color variation is affected by the synthesis of phenolic compounds, particularly anthocyanins and their underlying genetic factors. We hope this knowledge will be useful in supporting the importance of the berry color trait diversity in cultivated grapevines, which is used as basis for selection during breeding programs because of its application for vine growers, winemakers and consumers.

Spontaneous variation regarding grape berry skin color: A comprehensive study of berry development by means of biochemical and molecular markers.

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berry's color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. CHEMICAL COMPOUNDS: Gallic acid (PubChem CID: 370); Caftaric acid (PubChem CID: 6,440,397); Catechin (PubChem CID: 73,160); Epigallocatechin gallate (PubChem CID: 65,064); Quercetin-3-O-galactoside (PubChem CID: 5,281,643); Quercetin-3-O-glucoside (PubChem CID: 25,203,368); Malvidin-3-O-glucoside (PubChem CID: 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167).

Current distribution and characterization of the wild grapevine populations in Andalusia (Spain).

For decades, human activities have gradually destroyed the natural habitats of wild grapevine, Vitis vinifera L. subsp. sylvestris (Gmelin) Hegi, and nowadays this species is endangered in southern Europe. In this paper, 94 populations of this species have been localized and characterized in the Andalusian region in the Iberian Peninsula between 1989 and 2013. Location, ecological aspects, and sanitary characteristics are described. Must properties and in vitro tolerance to calcareous conditions were also checked. The paper also contains a global description of female and male individuals. Two hundred individuals from six river basin populations have been sampled, and their genetic structure analyzed by using 25 nuclear microsatellites loci to investigate the gene diversity of wild grape populations in Andalusia at two levels: total individuals and at river basin populations. Also, the genetic relationship of wild and cultivated accessions has been tested. Wild grapevine is considered the ancestor of the cultivated varieties and should be preserved as this material could be used to start breeding programs of cultivated varieties and also to restore riverbank forests, which constitute one of the worst preserved ecosystems in the area.

Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.

Identification of Vitis vinifera L. grape berry skin color mutants and polyphenolic profile.

A germplasm set of twenty-five grapevine accessions, forming eleven groups of possible berry skin color mutants, were genotyped with twelve microsatellite loci, being eleven of them identified as true color mutants. The polyphenolic profiling of the confirmed mutant cultivars revealed a total of twenty-four polyphenols, comprising non-colored compounds (phenolic acids, flavan-3-ols, flavonols and a stilbene) and anthocyanins. Results showed differences in the contribution of malvidin-3-O-glucoside to the characteristic Pinot Noir anthocyanins profile. Regarding the two Pique-Poul colored variants, the lighter variant was richer than the darker one in all classes of compounds, excepting anthocyanins. In Moscatel Galego Roxo the F3'H pathway seems to be more active than F3'5'H, resulting in higher amounts of cyanidin, precursor of the cyanidin derivatives. As far as we are aware, this is the first time that a relationship between the content of polyphenolic compounds is established in groups of grape berry skin color mutant cultivars.

Dm3 is one member of a large constitutively expressed family of nucleotide binding site-leucine-rich repeat encoding genes.

The major cluster of resistance genes in lettuce cv. Diana contains approximately 32 nucleotide binding site-leucine-rich repeat encoding genes. Previous molecular dissection of this complex region had identified a large gene, RGC2B, as a candidate for encoding the downy mildew resistance gene, Dm3. This article describes genetic and transgenic complementation data that demonstrated RGC2B is necessary and sufficient to confer resistance with Dm3 specificity. Ethylmethanesulphonate was used to induce mutations to downy mildew susceptibility in cv. Diana (Dm1, Dm3, Dm7, and Dm8). Nineteen families were identified with a complete loss of resistance in one of the four resistance specificities. Sequencing revealed a variety of point mutations in RGC2B in the six dm3 mutants. Losses of resistance were due to single changes in amino acid sequence or a change in an intron splice site. These mutations did not cluster in any particular region of RGC2B. A full-length genomic copy of RGC2B was isolated from a lambdaphage library and introduced into two genotypes of lettuce. Transgenics expressing RGC2B exhibited resistance to all isolates expressing Avr3 from a wide range of geographical origins. In a wildtype Dm3-expressing genotype, many of the RGC2 family members are expressed at low levels throughout the plant.

Forest restoration in a fog oasis: evidence indicates need for cultural awareness in constructing the reference.

BACKGROUND: In the Peruvian Coastal Desert, an archipelago of fog oases, locally called lomas, are centers of biodiversity and of past human activity. Fog interception by a tree canopy, dominated by the legume tree tara (Caesalpinia spinosa), enables the occurrence in the Atiquipa lomas (southern Peru) of an environmental island with a diverse flora and high productivity. Although this forest provides essential services to the local population, it has suffered 90% anthropogenic reduction in area. Restoration efforts are now getting under way, including discussion as to the most appropriate reference ecosystem to use. METHODOLOGY/PRINCIPAL FINDINGS: Genetic diversity of tara was studied in the Atiquipa population and over a wide geographical and ecological range. Neither exclusive plastid haplotypes to loma formations nor clear geographical structuring of the genetic diversity was found. Photosynthetic performance and growth of seedlings naturally recruited in remnant patches of loma forest were compared with those of seedlings recruited or planted in the adjacent deforested area. Despite the greater water and nitrogen availability under tree canopy, growth of forest seedlings did not differ from that of those recruited into the deforested area, and was lower than that of planted seedlings. Tara seedlings exhibited tight stomatal control of photosynthesis, and a structural photoprotection by leaflet closure. These drought-avoiding mechanisms did not optimize seedling performance under the conditions produced by forest interception of fog moisture. CONCLUSIONS/SIGNIFICANCE: Both weak geographic partitioning of genetic variation and lack of physiological specialization of seedlings to the forest water regime strongly suggest that tara was introduced to lomas by humans. Therefore, the most diverse fragment of lomas is the result of landscape management and resource use by pre-Columbian cultures. We argue that an appropriate reference ecosystem for ecological restoration of lomas should include sustainable agroforestry practices that emulate the outcomes of ancient uses.

Epigenetic changes detected in micropropagated hop plants.

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques: random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested.

Chloroplast microsatellite polymorphisms in Vitis species.

The use of consensus chloroplast microsatellites primers for dicotyledonous chloroplast genomes revealed the existence of intra and interspecific length variation within the genus Vitis. Three chloroplast microsatellite loci were found to be polymorphic in samples of Vitis vinifera, Vitis berlandieri, Vitis riparia, and Vitis rupestris out of a total of 10 consensus primer pairs tested. These polymorphisms were always due to a variable number of mononucleotide residues within A and (or) T stretches in the amplified regions. Chloroplast microsatellite polymorphisms were used to demonstrate the maternal inheritance of chloroplast in V. vinifera and to characterise the chloroplast haplotypes present in wine grape cultivars of this species grown in Spain and Greece. The different distribution of haplotype frequencies in the two ends of the Mediterranean growth area suggests the existence of independent domestication events for grapevine.