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.

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).

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.

Molecular instability induced by aluminum stress in Plantago species.

Aluminum (Al) is one of the most abundant metals on earth's crust and Al toxicity represents one of the major factors that limit plant growth and productivity in acid soils (with a pH≤5.0). In this study the mutagenic/genotoxic effects of Al were evaluated in roots and leaves of two Plantago, species, Plantago almogravensis and Plantago lagopus, using ISSRs markers. Both species were exposed to 400 µM Al during 7 and 21 days. Ten ISSR primers produced polymorphic bands. In P. almogravensis, a total of 257 and 258 bands in roots and 255 and 265 bands in leaves were produced in the presence and absence of Al, respectively. In P. lagopus were produced 279 and 278 a total bands in roots and 275 and 274 bands in leaves, under the same conditions. The changes in ISSR profiles after Al treatment were considered as gain and/or loss of bands compared with the controls. The results suggest that changes in genomic template stability (GTS) could be detected with ISSR profiles. This molecular marker proved to be a good tool to detect the effects of Al on DNA profiles. It seems that Al did not interfere significantly with DNA integrity in both species but generated less ISSR stability in P. almogravensis than in P. lagopus. The results confirm the tolerance of P. almogravensis and suggest the same behavior of P. lagopus. Although further studies are required for confirmation the Al tolerance behavior of P. lagopus, a potential application for phytoremediation can be also considered due its wide distribution.

Rye oxidative stress under long term Al exposure.

Aluminium (Al) toxicity decreases plant growth. Secale cereale L. is among the most Al-tolerant crop species. In order to study the response to Al-long term exposure, two rye genotypes with different Al sensitivity ('D. Zlote' and 'Riodeva') were exposed to 1.11 and 1.85mM Al and the antioxidant responses were followed for 2 and 3 weeks in roots and leaves. Al toxicity signals, such as a severe decrease in root growth, occurred sooner in 'Riodeva.' The antioxidant response was dependent on the genotype, the organ, Al concentration and the exposure period. Al-exposed roots of 'D. Zlote' showed earlier enhancements of APX, SOD and G-POX activities than those of 'Riodeva.' 'D. Zlote' roots showed stimulation of the AsA-GSH cycle after the second week (when root growth inhibition was less severe), while later (when severe root growth inhibition was observed), oxidation of AsA and GSH pools was observed. In leaves of both genotypes, CAT, SOD and G-POX activities increased with Al exposure. In these leaves, the effect of AsA-GSH was time dependent, with maximum oxidation at the second week, followed by recovery. We confirmed that the oxidation state of AsA and GSH pools is involved in the detoxification of Al-induced oxidative stress. Moreover, our data demonstrate that the production of ROS does not correlate with the Al-induced root growth decrease. Finally, the differences observed over time indicate that long term exposure may provide additional information on rye sensitivity to Al, and contribute to a better understanding of this species' mechanisms of Al tolerance.

Copper induced upregulation of apoptosis related genes in zebrafish (Danio rerio) gill.

Copper (Cu) is an essential micronutrient that, when present in high concentrations, becomes toxic to aquatic organisms. It is known that Cu toxicity may induce apoptotic cell death. However, the precise mechanism and the pathways that are activated, in fish, are still unclear. Thus, this study aimed to assess which apoptotic pathways are triggered by Cu, in zebrafish (Danio rerio) gill, the main target of waterborne pollutants. Fish where exposed to 12.5 and 100 µg/L of Cu during 6, 12, 24 and 48 h. Fish gills were collected to TUNEL assay and mRNA expression analysis of selected genes by real time PCR. An approach to different apoptosis pathways was done selecting p53, caspase-8, caspase-9 and apoptosis inducing factor (AIF) genes. The higher incidence of TUNEL-positive cells, in gill epithelia of the exposed fish, proved that Cu induced apoptosis. The results suggest that different apoptosis pathways are triggered by Cu at different time points of the exposure period, as the increase in transcripts was sequential, instead of simultaneous. Apoptosis seems to be initiated via intrinsic pathway (caspase-9), through p53 activation; then followed by the extrinsic pathway (caspase-8) and finally by the caspase-independent pathway (AIF). A possible model for Cu-induce apoptosis pathways is proposed.

Chloroplast genome diversity in Portuguese grapevine (Vitis vinifera L.) cultivars.

Grapevine chloroplast (cp) DNA diversity was analysed for the first time through amplification and digestion of fragments of the large single copy (LSC) region by polymerase chain reaction-restriction fragment length polymorphism methodology and also by amplification of three microsatellite loci, previously described as polymorphic in grapevine. Thirty-eight grapevine cultivars collected mainly in the North of Portugal, including some neglected cultivars, four international cultivars (Chasselas, Muscat of Alexandria, Muscat of Hamburg and Pinot) and Vitis riparia and Vitis rupestris, were used in this study with the main goal of finding out their cpDNA diversity and compare the obtained results with previously published data on cultivars from other regions to ascertain their possible origin. Two different alleles were found in each of the three cpSSR loci. Allele variants of the three loci combined in a total of three different haplotypes (A, B and D). The most frequent haplotype, A, was previously reported as the most frequent in Iberian Peninsula and Occidental Europe. Haplotype B was unique to Rabigato, Muscat of Alexandria, V. riparia and V. rupestris. This haplotype was previously proposed to be an ancestral haplotype. Twenty-seven fragments of the LSC region of Vitis vinifera cpDNA were amplified and then digested with HinfI and TaqI restriction enzymes. Polymorphisms were found in the trnT-psbC (TC) and orf184-petA (OA) fragments. In the TC fragment, the polymorphism corresponds to a point mutation in a restriction site of TaqI and is only present in all cultivars with cpSSR haplotype D. In the OA fragment, a short deletion exclusive to the Rabigato cultivar was found. In this case, one sequence tagged site-based marker was developed and will be very useful in future phylogenetic and fingerprinting studies in a broader number of cultivars and in wild grapevine populations. Inference about the progenitors of the Touriga Franca cultivar is done. The present work supports and completes its origin as a descendent of the female and male parents, Marufo and Touriga Nacional.

Polymorphism of the storage proteins in Portuguese rye (Secale cereale L.) populations.

Currently, due to the abandonment of traditional agricultural practices and the decline of rye production in Portugal, there is a need to assess the genetic diversity of rye in order to preserve its biodiversity. Furthermore, a greater knowledge of rye secalins is important for rye bread-making quality and other crop breeding purposes. The genetic variation and diversity of storage proteins were estimated for fourteen populations of rye (Secale cereale L.) sampled in northern Portugal. The work showed the high genetic diversity within the Portuguese rye gene-pool as an important source for plant breeding and emphasized the necessity of an integrated resources genetic program to allow a more efficient management and conservation of these resources. The rye populations were compared with 'Picasso' and 'Marder' varieties. Several alleles were identified by the single electrophoretic mobility patterns. We studied a set of 1600 rye seeds, including regional populations and varieties, having observed a total of 24, 5, 21 and 47 alleles for HMW, 75k γ-, 40k γ- and ω-secalins, respectively. The coefficient of similarity within populations is presented using cluster representation. The mean value of genetic variation indices (H) for rye storage proteins was very high in regional populations, ranging from 0.67 to 0.78, while in the varieties ranged from 0.57 to 0.58. Knowledge of the diversity of secalins will increase our understanding of the quality differences between rye varieties, especially considering the relative small number of rye cultivars grown around the world.

Zonal responses of sensitive vs. tolerant wheat roots during Al exposure and recovery.

Aluminium (Al) irreversibly inhibits root growth in sensitive, but not in some tolerant genotypes. To better understand tolerance mechanisms, seedlings from tolerant ('Barbela 7/72' line) and sensitive ('Anahuac') Triticum aestivum L. genotypes were exposed to AlCl(3) 185 µM for: (a) 24 h followed by 48 h without Al (recovery); (b) 72 h of continuous exposure. Three root zones were analyzed (meristematic (MZ), elongation (EZ) and hairy (HZ)) for callose deposition, reserves (starch and lipids) accumulation, endodermis differentiation and tissue architecture. Putative Al-induced genotoxic or cytostatic/mytogenic effects were assessed by flow cytometry in root apices. Tolerant plants accumulated less Al, presented less root damage and a less generalized callose distribution than sensitive ones. Starch and lipid reserves remained constant in tolerant roots but drastically decreased in sensitive ones. Al induced different profiles of endodermis differentiation: differentiation was promoted in EZ and HZ, respectively, in sensitive and tolerant genotypes. No ploidy changes or clastogenicity were observed. However, differences in cell cycle blockage profiles were detected, being less severe in tolerant roots. After Al removal, only the 'Barbela 7/72' line reversed Al-induced effects to values closer to the control, mostly with respect to callose deposition and cell cycle progression. We demonstrate for the first time that: (a) cell cycle progression is differently regulated by Al-tolerant and Al-sensitive genotypes; (b) Al induces callose deposition >3 cm above root apex (in HZ); (c) callose deposition is a transient Al-induced effect in tolerant plants; and (d) in HZ, endodermis differentiation is also stimulated only in tolerant plants, probably functioning in tolerant genotypes as a protective mechanism in addition to callose.

Aluminium long-term stress differently affects photosynthesis in rye genotypes.

The ability of crops to overcome Al toxicity varies among crop species and cultivars. Among the Triticeae genus, rye (Secale cereale) is considered the most Al-tolerant species. In the present work, two rye genotypes differing in Al tolerance ('Riodeva': Al-sensitive and 'Donkowsky Zlote': Al-tolerant) were exposed to 1.11 and 1.85 mM Al during three weeks. Growth, water status and photosynthesis related parameters were assessed. After three weeks of Al exposure, both genotypes presented similar decrease in leaf growth. Al-induced RWC decreased in both genotypes, but was more remarkable in 'Riodeva'. Al toxicity induced a decrease in net photosynthetic rate only after three weeks of exposure. In 'D. Zlote', A decrease was accompanied by stomatal closure, Chl a content and q(p) reduction, but no alterations in RuBisCo or sFBPase activity were observed. In 'Riodeva' plants exposed to 1.11 mM Al, A decrease was accompanied by C(i)/C(a) increase whereas in plants exposed to 1.85 mM Al C(i)/C(a) was not affected. Nevertheless, for both conditions RuBisCo activity decreased. A decrease did not limited glucose accumulation in neither of the rye genotypes. This study revealed that Al-induced earlier damages in the 'Riodeva' genotype, but both genotypes showed long-term high susceptibility to Al. Furthermore, the photosynthetic parameters proved to be a good tool to monitor Al-sensitivity and long-term exposure showed to be crucial to evaluate Al-sensitivity.

Effectiveness of AFLPs and retrotransposon-based markers for the identification of Portuguese grapevine cultivars and clones.

Grapevine germplasm, including 38 of the main Portuguese cultivars and three foreign cultivars, Pinot Noir, Pinot Blanc and Chasselas, used as a reference, and 37 true-to-type clones from the Alvarinho, Arinto, Loureiro, Moscatel Galego Branco, Trajadura and Vinhão cultivars were studied using AFLP and three retrotransposon-based molecular techniques, IRAP, REMAP and SSAP. To study the retrotransposon-based polymorphisms, 18 primers based on the LTR sequences of Tvv1, Gret1 and Vine-1 were used. In the analysis of 41 cultivars, 517 IRAP, REMAP, AFLP and SSAP fragments were obtained, 83% of which were polymorphic. For IRAP, only the Tvv1Fa primer amplified DNA fragments. In the REMAP analysis, the Tvv1Fa-Ms14 primer combination only produced polymorphic bands, and the Vine-1 primers produced mainly ISSR fragments. The highest number of polymorphic fragments was found for AFLP. Both AFLP and SSAP showed a greater capacity for identifying clones, resulting in 15 and 9 clones identified, respectively. Together, all of the techniques allowed for the identification of 54% of the studied clones, which is an important step in solving one of the challenges that viticulture currently faces.