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Copper (Cu) is an essential micronutrient for plants, but when present in excess, it induces toxicity. In this study, cuttings of four wine-producing varieties of Vitis vinifera L. were used: ‘Tinta Barroca’, ‘Tinto Cao’, ‘Malvasia Fina’ and ‘Viosinho’. The grapevine cuttings were distributed by hydroponic solutions enriched with different Cu concentrations (1, 10, 25 and 50 μM) plus control. At the end of the experiment, the root growth was evaluated, and individual roots were collected, fixed, and used for histological sections and chromosome spreads preparation. The higher Cu concentrations induced toxicity and inhibited root growth. However, the grapevine varieties responded with the thickening of the root exodermis and endodermis. In the chromosome spreads, normal and abnormal interphase and mitotic cells were observed in all varieties and treatments. The increase of Cu concentration decreased the nucleolar activity, as seen by reducing the nucleolar number and area. It increased the frequency of interphase cells with anomalies (ICA), but it did not influence total soluble protein concentration. The augment of Cu concentration also decreased the mitotic index (MI) and increased the percentage of dividing cells with anomalies (DCA). Different types of chromosomal anomalies in all mitotic phases, treatments and varieties were found. Overall, the white wine varieties, ‘Malvasia Fina’ and ‘Viosinho’, appeared to be more tolerant to the Cu-induced stress because they showed higher root growth and mean MI and lower mean DCA than the red wine varieties.

The use of anti-hail nets on orchards changes the microclimate underneath the net. This might be of great importance in apple growing regions characterized by high radiation levels and hot and dry climates during the summer season. But, depending on the net colour and on the local climatic conditions, the shade promoted triggers different responses by the trees. Grey and black anti-hail nets were applied in an apple orchard (cv. ‘Golden Delicious’) located in Northeast Portugal. Under the nets a lower concentration of glomalin related-soil proteins was observed, along with an improvement on trees water status, stomatal conductance, net photosynthetic rate, total chlorophylls, N, Mg, Fe and Cu concentrations, as well as an increase in mean fruit weight. The major difference between nets was on the photosynthetic efficiency, being higher on black net in sunny days, while grey net performed better under cloudy conditions. The use of netting systems proved to be effective in improving “Golden Delicious” apple trees performance under a Mediterranean climate, mainly when the radiation reaching the plants surpass the tree saturation point for photosynthesis. Therefore, these findings anticipate solutions for current and forecasted negative effects of climate change. This research was funded by the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UIDB/00690/2020) and CITAB (UIDB/04033/2020), as well by the project “Introdução de telas anti-granizo em macieira: Avaliação de efeitos colaterais e impacte económico” PA 54824, funded by the European Agricultural Fund for Rural Development and the Portuguese state under the measure 4.1- Cooperation for the Innovation-Rural Development Program (PRODER). info:eu-repo/semantics/publishedVersion

Climate change is expected to reduce water availability in several wheat (Triticum aestivum L.) growing areas. Drought, during and immediately after sowing can compromise germination, seedling development, and ultimately crop yield. Our study’s objective was to investigate early drought stress resistance and evaluate genetic diversity of four bread wheat cultivars – three modern elite germplasm lines (‘Antequera’, ‘Jordão’ and ‘Roxo’) and an ancient Portuguese cultivar (‘Mestiço’) – at five water potentials, ranging from 0 to –1.5 MPa. Germination was annotated daily during eight days. Dry and fresh weights as well as root and shoot growth were measured. Genetic variability among cultivars was evaluated using start codon targeted (SCoT) markers. Overall, when water potential was lowered, germination rate and seedling growth were delayed. Final germination percentages were only affected by the higher stress intensity. This study reinforces the importance of research into ancient wheat cultivars, such as ‘Mestiço’. This cultivar showed the highest genetic difference when compared with the remaining cultivars. ‘Mestiço’ presented the highest coleoptile size, high vigor index values, and the lowest variation in water content due to stress conditions. Therefore, it may be able to germinate in extremly dry soil and be the least affected by drought stress. Still, due to the comparatively reduced yield of older wheat cultivars, stress tolerance should be researched within elite cultivars. Among these, Jordão revealed particularly good germination and early biomass development characteristics across water potentials, presenting a likely choice for future studies under drought stress conditions.

The Pinus nigra distribution in Portugal is restricted to six allochthonous populations with unknown origin and infraspecific taxonomy. This work intends to evaluate their genetic diversity, structure and relationships, and to infer about their infraspecific taxonomy by comparing molecular patterns produced by inter-simple sequence repeat and Start Codon Targeted markers among Portuguese and foreign samples with known taxonomy and provenance. 127 Portuguese P. nigra individuals were clustered per population. The genetic differentiation was higher within rather than among populations. The pooled molecular data indicated high genetic proximity among the Portuguese and foreign samples of subspecies laricio. However, the separate analysis per marker system demonstrated that two varieties of subspecies laricio (corsicana and calabrica) may have been used in the plantations of the Portuguese P. nigra stands performed in the last century. The genetic characterization and extrapolation of the intraspecific taxonomy of these populations provide useful information for forest management, afforestation and germplasm use.

Seed priming with iron (Fe) and/or zinc (Zn) can overcome the reduced availability of these micronutrients in soils and crops, but suitable dosages should be predetermined. Nucleolus responds to stress, such as cytotoxicity, with alterations perceivable by cytogenetic analyses. This work intends to study how seed priming with Fe and/or Zn affects the nucleolar activity in roots and the total soluble protein content in the flour of bread wheat cv. ‘Jordao’. Seven priming treatments with 0 mg L−1 to 8 mg L−1 of Fe and/or Zn were performed. In all treatments, each metaphase cell presented a maximum of six nucleolar organizer regions positively stained with silver nitrate (Ag-NORs). Also, a maximum number of six nucleoli per nucleus were observed in all treatments, except in the hydroprimed seeds (used as control) that showed a maximum of five nucleoli, probably due to nucleolar fusion. Irregular interphases were frequent in treatments with the highest dosage of micronutrients (8 mg L−1 Fe and/or 8 mg L−1 Zn). The nucleolar area reduced (p

Vitis vinifera L. is one of the most important fruit crops in the world, having thousands of different varieties. This diploid species has 38 small chromosomes that have been characterised with Giemsa staining, fluorochrome banding, silver staining, and fluorescence in situ hybridisation (FISH). Previous works reported that the number of ribosomal DNA (rDNA) loci in V. vinifera is four. In this work, we intend to study the nucleolar activity and to physically map the 45S rDNA loci in seven varieties of V. vinifera using an optimized protocol of silver nitrate staining and sequential FISH, respectively. Nucleoli and mitotic chromosomes of root-tips and leaves of the seven V. vinifera varieties were stained with silver nitrate. The number of nucleoli per interphase cell was scored and statistically analysed. Most of the varieties showed a maximum of three nucleoli per nucleus and three Ag-NORs per metaphase. The variety Bastardo showed a maximum of two nucleoli per nucleus in leaf tissue whereas Pinot Noir presented a maximum of four nucleoli per interphase in roots. However, in both tissues of all varieties, 1 nucleolus per nucleus was most frequent. High-quality chromosome spreads were selected for sequential FISH with the 45S rDNA probe pTa71 which allowed the physical location of four 45S rDNA loci in all varieties.

The Pinus nigra distribution in Portugal is restricted to six allochthonous populations with unknown origin and infraspecific taxonomy. This work intends to evaluate their genetic diversity, structure and relationships, and to infer about their infraspecific taxonomy by comparing molecular patterns produced by inter-simple sequence repeat and Start Codon Targeted markers among Portuguese and foreign samples with known taxonomy and provenance. 127 Portuguese P. nigra individuals were clustered per population. The genetic differentiation was higher within rather than among populations. The pooled molecular data indicated high genetic proximity among the Portuguese and foreign samples of subspecies laricio. However, the separate analysis per marker system demonstrated that two varieties of subspecies laricio (corsicana and calabrica) may have been used in the plantations of the Portuguese P. nigra stands performed in the last century. The genetic characterization and extrapolation of the intraspecific taxonomy of these populations provide useful information for forest management, afforestation and germplasm use. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-019-00649-5) contains supplementary material, which is available to authorized users.

Regarding the foreseeing climate change is reasonable to expect harmful consequences to olive tree (Olea europaea L.), an iconic species of Mediterranean region. Thus, the selection of practices that allow a better drought resistance and recovery capacity needs the immediate attention of scientific community. This study evaluates the strategies adopted by young potted olive trees, subjected to three cycles of drought and rewatering, in the presence of a reflective clay, kaolin (KL). The results demonstrated that KL induced shade-related leaf structural changes and was effective in keeping leaf water status during the most stressful periods. In general, photosynthetic activity of sprayed plants was improved by the alleviation of drought-induced stomatal and non-stomatal limitations. Moreover, during stress imposition sprayed leaves showed reduced oxidative damages, allowing lower investment in antioxidant defences. Furthermore, sprayed plants also had lower nighttime water losses due to inferior nighttime stomatal conductance, and are able to maintain higher respiration rates. Upon rewatering, the shaded effect conferred by KL limited gas exchange restauration, but improved the plants' capacity to restore the metabolic functions. In spite of the induced physiological and biochemical changes, no significant differences were found in whole-plant water use efficiency and plant biomass accumulation, possibly by the attenuation of photosynthesis restauration during the recovery events. In conclusion, the changes induced by KL might be beneficial under severe conditions, as on realistic Mediterranean field environments.

Currently, the biofortification of crops like wheat with micronutrients such as iron (Fe) and zinc (Zn) is extremely important due to the deficiencies of these micronutrients in the human diet and in soils. Agronomic biofortification with Fe and Zn can be done through different exogenous strategies such as soil application, foliar spraying, and seed priming. However, the excess of these micronutrients can be detrimental to the plants. Therefore, in the last decade, a high number of studies focused on the evaluation of their phytotoxic effects to define the best strategies for biofortification of bread wheat. In this study, we investigated the effects of seed priming with different dosages (1 mg L−1 to 8 mg L−1) of Fe and/or Zn in germination, mitosis and yield of bread wheat cv. ‘Jordao’ when compared with control. Overall, our results showed that: micronutrient dosages higher than 4 mg L−1 negatively affect the germination; Fe and/or Zn concentrations higher than 2 mg L−1 significantly decrease the mitotic index and increase the percentage of dividing cells with anomalies; treatments performed with 8 mg L−1 of Fe and/or 8 mg L−1 Zn caused negative effects in germination, mitosis and grain yield. Moreover, seed priming with 2 mg L−1 Fe + 2 mg L−1 Zn has been shown to be non-cytotoxic, ensuring a high rate of germination (80%) and normal dividing cells (90%) as well as improving tillering and grain yield. This work revealed that seed priming with Fe and Zn micronutrients constitutes a useful and alternative approach for the agronomic biofortification of bread wheat.

Pinus sylvestris L. (Scots pine) is the conifer with widest natural distribution area. Portugal constitutes its westernmost limit of distribution. Most of the Portuguese populations were planted but two autochthonous populations were recently ascribed to ‘Serra do Geres’ (NW Portugal), and seem to be well adapted to the temperate climate. However, the ongoing climate changes may compromise their survival. With this study we intend to evaluate the anatomic-physiological and genetic responses of Scots pine from five European provenances (‘Geres’, ‘Puebla de Lillo’, ‘Montes Universales’, Germany and Sweden) to three water availability regimes, in order to determine which one(s) present higher resistance to drought. Individuals from ‘Geres’ presented the highest stability in photosynthetic reactions as well as the better photochemical and metabolic behaviours under drought (T3). Hence, the relative expression ratio of three water stress-responsive genes during drought was lower and gradual in ‘Geres’, compared to all other provenances, followed by Germany. The results achieved in ‘Geres’ and Germany provenances are very interesting since they reflected that the native populations of ‘Geres’ along with the Portuguese Scots pine planted populations with a probable German provenance, have ability and high adaptive potential to respond to situations of water deficit. Moreover, the present genetic and physiological data demonstrated the urgent demand for the conservation of Portuguese Scots pine genetic resources as well as its use in plantation/afforestation of areas where the warming and drought has been affecting the survival of this species.

Portugal constitutes the southwestern end limit of the distribution area of Scots pine in the world. The population of ‘Ribeira das Negras’ (‘Serra do Geres’; NW Portugal) has been considered potentially native and peripheral. This species has 24 chromosomes, hardly distinguishable because of their similar size and shape. Cytogenetic studies are scarce and instabilities were previously reported in peripheral Scots pine populations. Here, we intended to cytogenetically characterize individuals from ‘Ribeira das Negras’ using 14 simple sequence repeats (SSRs) and 45S rDNA sequence (pTa71) as probes, by nondenaturing fluorescence in situ hybridization (ND-FISH) and FISH, respectively. Eight SSRs [(AC)10; (AG)10; (AG)12; (AAG)5; (AAC)5; (GATA)4; (GACA)5 and (GGAT)4] and pTa71 showed hybridization. The (AG)10 probe hybridized on all chromosomes and an ideogram was constructed. Each metaphase cell presented cytogenetic instabilities corroborating ‘Ribeira das Negras’ as a peripheral population. As far as we know, this is the first cytogenetic study in Scots pine using SSRs in FISH experiments. Scots pine (Pinus sylvestris L.) is one of the most widely distributed conifers across Eurasia and an economically important species as source of timber (Manson and Alia 2000). Peripheral populations of Scots pine are susceptible to marginal environments and could present high phenotypic variation as well as cytogenetic irregularities (Muratova 1994). Cytogenetic studies in gymnosperms constitute a hard task due to incomplete cell division synchronization