Olinda Pinto-Carnide

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.

Determination of relationships among autochthonous grapevine varieties (Vitis vinifera L.) in the Northwest of the Iberian Peninsula by using microsatellite markers

Fifty six grapevine varieties traditionally grown in the Northwest region of the Iberian Peninsula were analysed for six microsatellite loci, in order to determine the relationships among them as well as the plant material that should be collected and preserved in germplasm banks. Previous morphological and molecular results were taken into account for assessment of the existing synonymies among accessions from different European countries. Percent distribution of the main alleles was calculated. Multivariate analysis was carried out and similarities among the studied material were described and commented.

Aluminum tolerance variability in rye and wheat Portuguese germplasm

Regional rye populations and wheat cultivars/lines were screened for aluminum tolerance using the hematoxylin staining method. Portuguese regional rye populations showed better tolerance than the Polish cv. Dank. Zlote, used as tolerant tester. In the group of bread wheats, EPM 305/81, a Barbela reselection, was the most tolerant genotype with the same behaviour as the cv. BH 1146, a tolerant tester. In a study with lines selected from a local Barbela landrace, aluminum tolerance variability was detected. Some lines were as tolerant, or higher, as wheat tester. As Portuguese rye populations and the Barbela wheat landrace have grown for centuries on an acid soil region, the data supports the idea that natural biotic or abiotic stresses associated to man selection, lead to the adaptation of genotypes to specific regional conditions and, in this case, to acid soils where aluminum toxicity occurs.

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.

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.

The effect of spikelet position on the success frequencies of wheat haploid production using the maize cross system

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize.

Molecular instability induced by aluminum stress in Plantago species.

Aluminum (Al) is one of the most abundant metals on earths 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.

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 berrys 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).

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.

Aluminum Phytotoxicity: Physiological Approaches and Tolerance

Abstract The increase of aluminum (Al) bioavailability in the soil seriously compromises plant growth and crop production. Al phytotoxicity highly depends on the interactions between Al and root cells, apoplast and symplast, which are influenced by genotype and environmental conditions (e.g., soil pH, nutrients availability). Within physiological parameters used to assess Al phytotoxicity, root elongation is widely known, either by cell wall extensibility and cell cycle impairment. Besides, recent data gave new insight on the relations between Al and nutrients, Al allocation in apoplast/symplast, cell oxidative stress homeostasis, cell differentiation, and photosynthesis. We also discuss some of the recent advances on plant tolerance including genetic mechanisms for Al exclusion, where organic acids play a crucial role, and mechanisms involved in Al compartmentation and tolerance. Important advances on the interaction of Al with genes regulating these physiological events are also highlighted.